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==== Front Ann Allergy Asthma Immunol Ann Allergy Asthma Immunol Annals of Allergy, Asthma & Immunology 1081-1206 1534-4436 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. S1081-1206(22)01713-6 10.1016/j.anai.2022.09.001 Editorial Is there value in coronavirus disease 2019 vaccine and vaccine excipient skin testing or split dosing? Greenhawt Matthew MD, MBA, MSc Section of Allergy and Immunology, Food Challenge and Research Unit, Children's Hospital Colorado, University of Colorado School of Medicine, Aurora, Colorado 1 12 2022 12 2022 1 12 2022 129 6 667668 24 8 2022 1 9 2022 2 9 2022 © 2022 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. 2022 American College of Allergy, Asthma & Immunology Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcA new feature in Annals is to have editorials that specifically provide a high-yield perspective of the take-home message from our monthly pieces, in addition to providing an insider's glimpse of why the editors felt the content was important to publish. This is the first of such series, and it is overdue that we discuss coronavirus disease 2019 (COVID-19) vaccination. It took only 8 months after the emergence of an unprecedented global pandemic to develop mRNA and adenovirus-vector vaccines to help prevent contraction of the novel severe acute respiratory syndrome coronavirus 2, and less than 24 hours after their initial public availability for the first reported allergic reactions to occur.1 However, in the subsequent 18 months, there have been significant gains in understanding that the risk of initial and subsequent dose-allergic reactions to these vaccines is very low, and that they can be safely administered to both persons with preexisting polyethylene glycol (PEG) allergy or persons who reacted to their initial dose.1 , 2 The last few issues of Annals have had a focus on vaccine allergy, with comprehensive reviews of both general vaccine allergy and specific issues with COVID-19 vaccination,3 , 4 and multiple reports of COVID-19 vaccine and vaccine excipient testing of persons with both preexisting PEG allergy and suspected immediate or delayed mRNA COVID-19 vaccine allergy from ALMuhizi et al.4 Otani et al.5 St. Claire et al.6 Van Meerbeke et al.7 and Pitlick et al.8 , 9 These reports have highlighted that PEG testing is rarely truly positive (eg, poor sensitivity), and that testing is of limited utility for persons with either suspected risk factors for COVID-19 vaccine reactions or persons who have had a previous immediate reaction to COVID-19 vaccine in terms of predicting outcomes of vaccination (or revaccination). Moreover, here is a deeper thought on the topic to consider. Given the lack of conclusive evidence for COVID-19 vaccine reactions either being immunoglobulin (Ig) E-mediated or, specifically, involving anti-PEG IgE, it may also be appropriate to question the contextual accuracy of what the “true negative” IgE-based test actually represents, as a real-world example of a type III error (eg, the answer is correct but reached through wrong methods). In sum, testing has not provided any evidence of utility to identify persons at risk, has not proven necessary for safely vaccinating or revaccinating persons, and begs the question as to why the field should continue to test.1 , 2 , 7, 5, 8, 9, 6 It is simply time to stop this as a routine practice, similar to the approach that has evolved with other vaccine allergy, such as influenza. Another hot topic that the recent Annals COVID-19 reports have examined is that of the use of split dosing for persons considered at risk because of a previous immediate reaction, or at risk because of potential vaccine excipient allergy. As Dr Kelso detailed in his recent review, although split dosing is a recognized approach to vaccine allergy, the necessity of this approach compared with administering a single, full dose, in terms of split dosing having enhanced safety and efficacy has not been shown.2 Split dosing is a well-accepted approach that both clinicians and patients find reassuring, which allows vaccination to move forward where it otherwise may have been deferred. ALMuhizi et al.4 Van Meerbeke et al.7 St. Claire et al.6 and Pitlick et al.8 , 9 all demonstrate such usefulness within their clinical populations. Although some have raised theoretical questions regarding comparative immunologic responses with single vs split dosing, the study by Van Meerbeke et al.7 is among several reports that show evidence to reassure the clinician this antibody response appears equal. It just remains enigmatic why one would choose a split dose over a single dose. Until there is an updated vaccine allergy practice parameter, both testing to vaccine or vaccine excipient and split dosing will remain unresolved issues with questionable net utility as recommended approaches in the context of medical necessity, patient comfort, and the logistic practicality of deliberately using a more labor-intensive dosing strategy. The editors felt these referenced publications were important to the readership, to help provide options in helping patients who are seeking guidance for either initial or subsequent COVID-19 vaccination. It is our hope that the reviews and content matter of the recent vaccine theme issue, combined with the original reports, have provided rationale and data which the clinician can base future decisions, pending the publication of future, more definitive guidelines. The evidence is more compelling to dismiss the utility of routinely testing individuals with known PEG allergy to mRNA COVID-19 vaccines, or testing individuals to PEG or mRNA vaccine in risk assessment before subsequent vaccination. Although meta-analysis for COVID-19 vaccination may reveal no compelling reason or medical necessity for split vs single dosing based on safety, logic may actually provide an equally compelling rationale to still consider doing so based on patient preference if there is no other way to convince a patient that vaccination is safe.1 Unfortunately, it appears there will be an ongoing need for somewhat frequent booster dosing of COVID-19 vaccines, which means this issue of allergic reactions or management of patients with history of allergy to the vaccine or vaccine excipient remains relevant to the practicing allergist. Disclosures: Dr Greenhawt is a consultant for Aquestive; is a member of physician and medical advisory boards for DBV Technologies, Sanofi/Regeneron, Nutricia, Novartis, Acquestive, Allergy Therapeutics, AstraZeneca, ALK-Abello, and Prota, with all activity unrelated to vaccines and vaccine development or COVID-19 treatment; is an unpaid member of the scientific advisory council for the National Peanut Board and medical advisory board of the International Food Protein Induced Enterocolitis Syndrome Association; is a member of the Brighton Collaboration Criteria Vaccine Anaphylaxis 2.0 working group; is the senior associate editor for the Annals of Allergy, Asthma & Immunology, and is a member of the Joint Taskforce on Allergy Practice Parameters. He has received honoraria for lectures from IMsci, Med Learning Group, RMEI Medical Education LLC, and multiple state and local allergy societies. He received past research support ending in 2020 from the Agency for Healthcare Quality and Research (K08-HS024599). Funding: Dr Greenhawt has no funding sources to report. ==== Refs References 1 Greenhawt M Abrams EM Shaker M Chu DK Khan D Akin C The risk of allergic reaction to SARS-CoV-2 vaccines and recommended evaluation and management: a systematic review, meta-analysis, GRADE assessment, and international consensus approach J Allergy Clin Immunol Pract 9 10 2021 3546 3567 34153517 2 Kelso JM. The adverse reactions to vaccines practice parameter 10 years on-what have we learned? Ann Allergy Asthma Immunol 29 1 2022 35 39 3 Copaescu AM Rosa Duque JS Phillips EJ What have we learned about the allergenicity and adverse reactions associated with the severe acute respiratory syndrome coronavirus 2 vaccines: one year later Ann Allergy Asthma Immunol 129 1 2022 40 51 35390476 4 ALMuhizi F Fein M Gabrielli S Gilbert L Tsoukas C Ben-Shoshan M Allergic reactions to the coronavirus disease 2019 vaccine (ARCOV) study: the McGill University Health Centre experience Ann Allergy Asthma Immunol 129 2022 182-188.e1 5 Otani IM Tsao LR Tang M. Coronavirus disease 2019 vaccine administration in patients with reported reactions to polyethylene glycol- and polysorbate-containing therapeutics Ann Allergy Asthma Immunol 129 1 2022 88-94.e1 6 St Clair BD Hoffman DL McClenathan B Banks T Lee RU. Outcomes of allergic-type reactions after messenger RNA coronavirus disease 2019 vaccination at 3 military medical centers Ann Allergy Asthma Immunol 129 2 2022 248 249 35598884 7 Van Meerbeke SW Fajt ML Marini RV Domsic RT Petrov AA. Antibody response to graded dosing of coronavirus disease 2019 messenger RNA vaccines after allergic reaction to first dose Ann Allergy Asthma Immunol 129 3 2022 373 374 35595005 8 Pitlick MM Sitek AN D'Netto ME Dages KN Chiarella SE Gonzalez-Estrada A Utility and futility of skin testing to address concerns surrounding messenger RNA coronavirus disease 2019 vaccine reactions Ann Allergy Asthma Immunol 128 2 2022 153 160 34798275 9 Pitlick MM Gonzalez-Estrada A Park MA. Graded coronavirus disease 2019 vaccine administration: a safe alternative to vaccine avoidance Ann Allergy Asthma Immunol 128 6 2022 731 733 35257875	36464396	PMC9712058	NO-CC CODE	2022-12-02 23:21:32	no	Ann Allergy Asthma Immunol. 2022 Dec 1; 129(6):667-668	utf-8	Ann Allergy Asthma Immunol	2,022	10.1016/j.anai.2022.09.001	oa_other
==== Front Ann Allergy Asthma Immunol Ann Allergy Asthma Immunol Annals of Allergy, Asthma & Immunology 1081-1206 1534-4436 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. S1081-1206(22)01773-2 10.1016/j.anai.2022.09.023 Correspondence Split-dosing of coronavirus disease 2019 vaccines Mungmunpuntipantip Rujittika PhD *# Wiwanitkit Viroj MD †‡§ ⁎ Private Academic Consultant, Bangkok, Thailand † Department of Community Medicine, DY Patil Medical College, Pune, India ‡ Faculty of Medicine, University of Niš, Niš, Serbia § Department of Eastern Medicine, Government College University, Faisalabad, Pakistan # Corresponding author 1 12 2022 12 2022 1 12 2022 129 6 799799 12 9 2022 13 9 2022 15 9 2022 © 2022 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. 2022 American College of Allergy, Asthma & Immunology Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcTo the Editor, We would like to share ideas on the article titled “Split-Dosing of COVID-19 Vaccines Provides Non-Inferior Antibody Responsiveness to Conventional Vaccine Dosing.”1 Split-dosing of coronavirus disease 2019 (COVID-19) vaccines is still an option that has been found to be just as effective as traditional dosing at promoting antibody response, according to Musa et al.1 The effectiveness of the COVID-19 vaccination has been reported to be influenced by a variety of circumstances. Different administration and dosage methods could play a role. Patients who use prescription drugs or have underlying medical conditions may have different sensitivities to immunizations than the typical, healthy vaccine receiver. We can all agree that it will be beneficial to administer the COVID-19 vaccine. Based on past immunizations, the findings of the investigation imply that the COVID-19 vaccination has a protective effect. The rather frequent antecedent COVID-19 without symptoms is another problem that could get worse.2 It is clear that a history of COVID-19 infection is not reliably indicated by a history of coronavirus disease infection.2 In addition, a suitable set of antibody assays was used to ascertain the COVID-19 prevaccination immunologic status.1 To rule out a previous, asymptomatic COVID-19 infection, it is normal to skip testing. If people receive routine assessments, they might learn more about their underlying immunologic issues. It is good that the study by Musa et al measured prevaccination titers.1 This is a strong point of the current study because most of the comparable, earlier studies that are accessible fail to consider the individuals’ history of COVID-19 infection and how this may affect the future immune response to vaccination. The effects of the COVID-19 vaccination can be foreseen more precisely if participants’ preexisting immune statuses are consistently evaluated. When assessing the effectiveness or safety of the vaccination, this is a crucial factor to consider. Numerous studies have shown the effectiveness, safety, or clinical relationship of the COVID-19 vaccine, even though there is frequently little information available regarding prevaccination immunologic or health status, and the possibility of confounding with nonsymptomatic COVID-19 is not entirely ruled out. Consequently, the scientific evidence from the current investigation can at least address the potential confounding effect of COVID-19, which could undermine the validity of the immunologic assessment parameters. Disclosures: The authors have no conflicts of interest to report. Funding: The authors have no funding sources to report. ==== Refs References 1 Musa A Wood M Rorie A May SM Graaff JV Poole JA Split dosing of coronavirus disease 2019 vaccines provides noninferior antibody responsiveness to conventional vaccine dosing Ann Allergy Asthma Immunol 129 6 2022 794 796 2 Joob B Wiwanitkit V. Letter to the editor: coronavirus disease 2019 (COVID-19), infectivity, and the incubation period J Prev Med Public Health 53 2 2020 70 32268458	36464398	PMC9712059	NO-CC CODE	2022-12-02 23:21:32	no	Ann Allergy Asthma Immunol. 2022 Dec 1; 129(6):799	utf-8	Ann Allergy Asthma Immunol	2,022	10.1016/j.anai.2022.09.023	oa_other
==== Front Ann Allergy Asthma Immunol Ann Allergy Asthma Immunol Annals of Allergy, Asthma & Immunology 1081-1206 1534-4436 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. S1081-1206(22)01720-3 10.1016/j.anai.2022.09.007 Editorial The known knowns, known unknowns, and unknown unknowns of surveys and sleep Shaker Marcus MD, MS, FAAP, FACAAI, FAAAAI *†# Moore-Clingenpeel Melissa MA, MAS ‡ ⁎ Section of Allergy and Immunology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire † Dartmouth Geisel School of Medicine, Hanover, New Hampshire ‡ CorEvitas, LLC, Waltham, Massachusetts # Corresponding author 1 12 2022 12 2022 1 12 2022 129 6 669670 4 9 2022 6 9 2022 © 2022 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. 2022 American College of Allergy, Asthma & Immunology Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcThe known knowns, known unknowns, and unknown unknowns were infamously popularized by Donald Rumsfeld in 2002.1 These often-overlooked epistemological constructs are useful considerations when approaching survey data because the quality of scientific evidence generated by survey research is closely tied to the ability to make inference from a sample to a larger population. In this issue of the Annals of Allergy, Asthma & Immunology, Khan and Marshall2 report on behalf of the Respiratory and Sleep-Related Breathing Disorders Committee of the American College of Allergy, Asthma & Immunology (ACAAI). The authors developed and distributed a questionnaire intended to evaluate the awareness and assessment of sleep disorders by ACAAI members; though this was an ad hoc survey instrument, developed by a small group of the authors, without clear reliability or validity testing, and was based on an unspecified literature base. Some variation in approaches of respondents was noted in the survey; however, of the ACAAI membership surveyed, only 102 valid questionnaires were received, for a response rate of just 3.3%. None of these traits are particularly uncommon within allergy membership research. Still, it is worthwhile to consider these findings under a “Rumsfeldian” paradigm of knowledge theory.1 What are the known knowns? The survey responses do provide data to consider. Sleep is important, and many of us do not get enough of it, with the Centers for Disease Control and Prevention reporting one-third of adults and three-quarters of adolescents have insufficient sleep.2 The impact on sleep can carry over to waking hours, and throughout allergy clinics, sleep can be affected by pruritus associated with atopic dermatitis or chronic spontaneous urticaria, nasal obstruction from allergic or nonallergic rhinitis, asthma, and sleep apnea.2 Khan and Marshall report that ACAAI members who respond to surveys about sleep often assess for sleep disorders in their adult patients. What do allergists who respond to surveys about sleep think? Nearly half of the respondents had been in practice for more than 20 years, and more than 60% discussed treating obstructive sleep apnea.2 Furthermore, more than 40% of the respondents caring for adult patients ordered sleep studies. The authors observed potential variation in assessing sleep quality (both adult and pediatric patients), with this group of motivated allergists-immunologists more likely to ask about sleep in asthma than in allergic rhinitis, atopic dermatitis, or urticaria. Allergists were least likely to ask about sleep in patients with food allergy. The authors also investigated treatment patterns, finding 98% of these allergists-immunologists recommended an intranasal corticosteroid at least occasionally for adults who snore. Nearly 10% of the respondents recommended first-generation antihistamines almost all the time for adults with chronic urticaria, atopic dermatitis, and contact dermatitis, with 73% recommending first-generation antihistamines at least occasionally to children. The frequent use of first-generation antihistamines raises potential concern.3 , 4 What are the known unknowns? The success of any survey in understanding behaviors, feelings, and preferences of larger groups relies on careful design to confirm that respondents understand the questions asked and validation to ensure accuracy and reliability of the survey instrument. Survey design begins with the research question, which is translated into carefully crafted survey questions, often grouped into overarching domains. Although beyond the scope of this editorial, key aspects of questionnaire design include following best practices for question and response type format, consulting with content experts to ensure item relevance and coverage with respect to the research question, including items that address inherent and anticipated limitations of the study design, conducting cognitive interviews to evaluate uniform understanding of the questions, and pilot testing in the planned mode of delivery.5 , 6 These validation steps are important because they provide reassurance that the instrument being used is measuring important items as intended and that responses are reproducible; it is not clear that all of these validation steps were undertaken in the survey reported by Khan and Marshall. Oddly, reliability and validity of survey instruments, although important, are often underreported in many published surveys.7, 8, 9 Apart from question and content validity, bias may also arise from imbalance between the survey sample and the larger population of interest. Despite best efforts, selection bias can arise in any voluntary survey, because individuals interested in a particular subject (and those who feel strongly about a topic) may be more likely to respond to a survey invitation and answer all questions to completion. For example, it is a plausible assumption that individuals who engage in a survey about sleep may have more interest in the topic, which could lead to an overestimation of awareness of the problem. In the survey reported by Khan and Marshall, only 3.3% of those surveyed completed the survey. The feelings and practice approaches of the other 96.7% of the ACAAI member population remain uncharacterized, and such a low response rate can be a “deadly blow to both reliability and validity.”10 A response rate below 5% is very low, and rates should be closer to 60% for most research to be confident in generalizability.10 To some extent, the low response is not surprising—surveys were received over the course of only 9 days, and only a single mode of delivery was used. It is not clear whether more than 1 attempt was made to contact the participants to encourage survey completion. What are the unknown unknowns? This group of unknowns is by definition not understood, and when these unknowns become characterized, they are no longer unknown unknowns. Still, a close approximation of this group relates to how we may improve response rates. Surveys can be administered in a variety of ways, and the method of administration can dramatically influence response rates. For example, e-mailed surveys without a follow-up request may garner a response rate of only 25%.10 However, a multipronged approach to survey that includes mailed and e-mailed surveys, multiple contacts, and an internet-based response element may achieve response rates upward of 70%.10 Of course, mandated surveys, such as those implemented by state boards of medicine, can have unmatched response rates; however, the 'carrot and the stick' approach is often not available to health care researchers and is distasteful to many. In any final analysis, it must be acknowledged that the perfect need not be the enemy of the good. A novel idea or potentially important finding embedded within a study with minor or even major limitations may still offer important insights. Khan and Marshall have provided a glimpse into the practice styles of the small minority of allergists-immunologists who respond to sleep surveys with several important messages that may be expanded on in more robustly–designed, future studies. Although sleep impairments are widely recognized in asthma, it is possible that recognition of sleep difficulties in atopic dermatitis and chronic urticaria could be improved. Second, a greater appreciation of the risks and benefits of first-generation antihistamine use may be needed.3 , 4 Lastly, our field as a whole may benefit from strategies most often used in market research and the social sciences to improve survey response rates among our members as we work to improve the care that we deliver every day. Disclosures: Dr Shaker serves on the editorial board of The Journal of Allergy and Clinical Immunology: In Practice, is an associate editor of the Annals of Allergy, Asthma & Immunology, is a member of the Joint Task Force on Practice Parameters, and has participated in research that has received funding from DBV. Ms Moore-Clingenpeel is employed by CorEvitas, LLC, and is a biostatistics editor of the Annals of Allergy, Asthma & Immunology. Funding: The authors have no funding sources to report. ==== Refs References 1 Shaker M Mauger D. Applying the clinical literature to a science of uncertainty and an art of probability J Allergy Clin Immunol Pract 9 12 2021 4233 4234 34893186 2 Khan FS Marshall ZW Assessment of sleep disturbance in patients with atopic conditions Ann Allergy Asthma Immunol 129 6 2022 796 798 3 Wolfson AR, Wong D, Abrams EM, Waserman S, Sussman GL. Diphenhydramine: time to move on? [e-pub ahead of print]. J Allergy Clin Immunol Pract. 10.1016/j.jaip.2022.07.018, accessed September 3, 2022. 4 Shaker M Ramsey A. Primum non nocere J Allergy Clin Immunol Pract 2022 In press 5 Dillman DA Smyth JD Christian LM. Internet, Phone, Mail, and Mixed-Mode Surveys: the Tailored Design Method 2014 John Wiley & Sons, Inc Hoboken, NJ 6 Payne SLB. The Art of Asking Questions: Studies in Public Opinion 3 2014 United States: Princeton University Press 7 Pagano MB Dunbar NM Tinmouth A Apelseth TO Lozano M Cohn CS A methodological review of the quality of reporting of surveys in transfusion medicine Transfusion 58 11 2018 2720 2727 30260483 8 Pitt SC Schwartz TA Chu D. AAPOR reporting guidelines for survey studies JAMA Surg 156 8 2021 785 786 33825811 9 Story DA Gin V Ranong V Poustie S Jones D Trials ANZCA Inconsistent survey reporting in anesthesia journals Anesth Analg 113 3 2011 591 595 21778334 10 Fincham JE. Response rates and responsiveness for surveys, standards, and the journal Am J Pharm Educ 72 2 2008 43 18483608	36464397	PMC9712060	NO-CC CODE	2022-12-03 23:19:55	no	Ann Allergy Asthma Immunol. 2022 Dec 1; 129(6):669-670	utf-8	Ann Allergy Asthma Immunol	2,022	10.1016/j.anai.2022.09.007	oa_other
==== Front Public Health Public Health Public Health 0033-3506 1476-5616 The Authors. Published by Elsevier Ltd on behalf of The Royal Society for Public Health. S0033-3506(22)00332-8 10.1016/j.puhe.2022.11.016 Original Research Patient-reported health outcomes of SARS-COV-2 tested patients presenting to emergency departments: a propensity-score matched prospective cohort study Bola Rajan BSc a Sutherland Jason PhD b Murphy Rachel A. PhD ac Leeies Murdoch MD, MSc de Grant Lars MD, PhD fg Hayward Jake MD, MPH h Archambault Patrick MD, MSc i Graves Lorraine j Rose Tamara j Hohl Corinne MD, MHSc kl∗ a School of Population and Public Health, University of British Columbia, Vancouver, BC, Canada b Centre for Health Services and Policy Research, University of British Columbia, Vancouver, BC, Canada c Cancer Control Research, BC Cancer, Vancouver, BC, Canada d Department of Emergency Medicine, University of Manitoba, Winnipeg, MB, Canada e Section of Critical Care Medicine, University of Manitoba, Winnipeg, MB, Canada f Department of Emergency Medicine, McGill University, Montreal, QC, Canada g Emergency Department, Jewish General Hospital, Montreal, QC, Canada h Department of Emergency Medicine, University of Alberta, AB, Canada i Université Laval, Department of Family Medicine and Emergency Medicine, QC, Canada j Patient Partner, Canadian COVID-19 Emergency Department Rapid Response Network Patient Engagement Committee k Department of Emergency Medicine, University of British Columbia, Vancouver, BC, Canada l Emergency Department, Vancouver General Hospital, Vancouver, BC, Canada ∗ Corresponding author. , 828 W 12th Ave, Vancouver, Canada. 1 12 2022 1 12 2022 20 7 2022 21 11 2022 23 11 2022 © 2022 The Authors. Published by Elsevier Ltd on behalf of The Royal Society for Public Health. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objective To compare the long-term physical and mental health outcomes of matched severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) positive and negative patients controlling for seasonal effects. Study Design Retrospective cohort study. Methods This study enrolled patients presenting to emergency departments participating in the Canadian COVID-19 Emergency Department Rapid Response Network. We enrolled consecutive eligible consenting patients who presented between March 1st 2020 and July 14th 2021 and were tested for SARS-COV-2. Research assistants randomly selected four site and date-matched SARS-COV-2 negative controls for every SARS-COV-2 positive patient and interviewed them at least 30 days after discharge. We used propensity scores to match patients by baseline characteristics and used linear regression to compare Veterans RAND 12-item physical (PCS) and mental health component scores (MCS), with higher scores indicating better self-reported health. Results We included 1,170 SARS-COV-2 positive patients and 3,716 test-negative controls. The adjusted mean difference for PCS was 0·50 (95%CI: -0·36, 1·36) and -1·01 (95%CI: -1·91, -0·11) for MCS. Severe disease was strongly associated with worse PCS (β=-7·4; 95%CI: -9·8, -5·1), while prior mental health illness was strongly associated with worse MCS (β=-5·4; 95%CI: -6·3, -4·5). Conclusion Physical health, assessed by PCS, was similar between matched SARS-COV-2 positive and negative patients, while mental health, assessed by MCS, was worse during a time when the public experienced barriers to care. These results may inform the development and prioritization of support programs for patients. ==== Body pmc	0	PMC9712064	NO-CC CODE	2022-12-05 23:15:28	no	Public Health. 2022 Dec 1; doi: 10.1016/j.puhe.2022.11.016	utf-8	Public Health	2,022	10.1016/j.puhe.2022.11.016	oa_other
==== Front Heart Lung Heart Lung Heart & Lung 0147-9563 1527-3288 Elsevier Inc. S0147-9563(22)00286-2 10.1016/j.hrtlng.2022.11.017 Article Comment on “Evaluation of pulmonary function and exercise capacity after COVID-19 pneumonia” Sajid Samar Dow University of Health Sciences, Karachi, Sindh Pakistan 1 12 2022 1 12 2022 23 11 2022 29 11 2022 © 2022 Elsevier Inc. All rights reserved. 2022 Elsevier Inc. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcI have read with great interest the manuscript titled “Evaluation of pulmonary function and exercise capacity after COVID-19 pneumonia” by Okan S et al.1 and would like to commend them for their brilliant efforts. However, there are a few additional points that I would like to highlight, and which would have enhanced the quality of this study. The study is cross sectional in design and lacks a control group. This characteristic prevents us from comparing the lung functions of affected individuals with those of healthy individuals. Secondly because of the limited sample size and single-center study design, it is harder to extrapolate the results to a population with a wider range of demographics. Regarding the results, they may not be applicable to a population that has received vaccinations because the study was conducted during an earlier stage of the pandemic when vaccines were scarce. Additionally, because the study was conducted on COVID-19 patients who were hospitalized during an acute infection, it is possible that the findings may not apply to COVID-19 patients who were not admitted. Furthermore, the lack of baseline pulmonary function test results before illness makes it difficult to make a comparison with the results after the illness. It is impossible to determine the true impact caused by the infection because the study did not mention the patients' prior lung function. For instance, this study by Lewis KL et al.2 shows that there is no difference in PFT before and after COVID-19 infection in non-critical patients. In addition to that, the patients' radiological outcomes are also unknown, and PFT or 6MWT comparisons have not been done with them. Another point is that even though patients with COPD or ILD were excluded, underlying asymptomatic lung disease or other ailments that impact lung function cannot be ruled out, regardless of how rare they may be. Moreover, lung capacity and volume were not assessed. It has been demonstrated that reduced DLCO, followed by TLC and FVC, are the most prevalent pulmonary function abnormalities in the study of COVID-19-infected patients.3 Finally, non-voluntary methods such as transdiaphragmatic pressure and diaphragm ultrasound should also be included in an appropriate investigation of pulmonary function and exercise capacity. Declaration of Competing Interest None Acknowledgement None ==== Refs References 1 Okan S. Okan F. Duran Yücesoy F Evaluation of pulmonary function and exercise capacity after COVID-19 pneumonia Heart Lung 54 2022 1 6 10.1016/j.hrtlng.2022.03.004 Jul-Aug Epub 2022 Mar 11 35305515 2 Lewis K.L. Helgeson S.A. Tatari M.M. Mallea J.M. Baig H.Z. Patel N.M. COVID-19 and the effects on pulmonary function following infection: a retrospective analysis EClinicalMedicine 2021 101079 10.1016/j.eclinm.2021.101079 Sep; 39 3 Hui D.S. Joynt G.M. Wong K.T. Gomersall C.D. Li T.S. Antonio G. Impact of severe acute respiratory syndrome (SARS) on pulmonary function, functional capacity, and quality of life in a cohort of survivors Thorax 60 5 2005 401 409 15860716	36494216	PMC9712065	NO-CC CODE	2022-12-06 23:15:40	no	Heart Lung. 2022 Dec 1; doi: 10.1016/j.hrtlng.2022.11.017	utf-8	Heart Lung	2,022	10.1016/j.hrtlng.2022.11.017	oa_other
==== Front Can J Ophthalmol Can J Ophthalmol Canadian Journal of Ophthalmology. Journal Canadien D'Ophtalmologie 0008-4182 1715-3360 Canadian Ophthalmological Society. Published by Elsevier Inc. S0008-4182(22)00369-6 10.1016/j.jcjo.2022.11.019 Original Article Effect of visual impairment on depression and anxiety during the COVID-19 pandemic in the United States Sekimitsu Sayuri * Shweikh Yusrah †‡ Zebardast Nazlee †1 ⁎ Tufts University School of Medicine, Boston, Mass. † Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Mass. ‡ Sussex Eye Hospital, University Hospitals Sussex NHS Foundation Trust, Sussex, United Kingdom 1 Correspondence to: Nazlee Zebardast, MD, Massachusetts Eye and Ear Hospital, 243 Charles Street, Boston, MA 02144. 1 12 2022 1 12 2022 17 7 2022 26 11 2022 © 2022 Canadian Ophthalmological Society. Published by Elsevier Inc. All rights reserved. 2022 Canadian Ophthalmological Society Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Purpose To evaluate associations between visual impairment, anxiety, and depression during the COVID-19 pandemic in the United States. Design Retrospective cross-sectional design. Methods This study included a cohort of U.S. adults enrolled in the National Institutes of Health All of Us Research Program. Individuals who were blind/visually impaired (BVI) were identified via Systematized Nomenclature of Medicine (SNONMED) codes and compared with a control cohort. Prevalences of baseline, new, and worsened depression and anxiety, as defined by SNONMED codes and medication use, were compared between the 2 groups. Anxiety and depression during the COVID-19 pandemic were evaluated using the Generalized Anxiety Disorder 7 and the Patient Health Questionnaire 9 surveys, respectively. Results A total of 324,915 participants (7526 BVI individuals and 317,389 control individuals) were included. BVI individuals had higher prevalences of baseline anxiety and depression (50.4% vs 28.7%; p < 0.001), new anxiety and depression (0.98% vs 0.66%; p < 0.001), and worsened anxiety and depression throughout the COVID-19 pandemic (0.19% vs 0.07%; p < 0.001) compared with control individuals. Being BVI was significantly associated with baseline and worsened anxiety and depression after controlling for age, sex, race, ethnicity, and comorbidity (adjusted odds ratio [aOR] = 1.61; 95% CI, 1.46–1.78 and aOR = 2.07; 95% CI, 1.03–4.13). Similarly, being BVI was associated with a 2.07 point increase on the Generalized Anxiety Disorder 7 survey (adjusted β = 2.07; 95% CI, 1.32–3.27) and a 2.96 point increase on the Patient Health Questionnaire 9 survey (adjusted β = 2.96; 95% CI, 1.64–5.36). Conclusions These findings indicate that BVI individuals were disproportionately affected by anxiety and depression at baseline and throughout the pandemic, highlighting an important need to promote access to mental health services among this population. ==== Body pmcThe response to the COVID-19 pandemic, including the government-enforced quarantine, the suspension of nonessential activities, and restrictions on social gatherings, has significantly affected the way people interact.1 , 2 Previous research demonstrates the detrimental effects of shelter-in-place orders for all individuals but particularly for older people, who may experience greater barriers to technology-based social interactions. Those with visual impairment also have been identified as vulnerable to isolation because of their unique challenges, including difficulties in accessing care or using video-based technology.3 Among people with visual impairment, loneliness during the COVID-19 pandemic has been found to be more prevalent compared with individuals without visual impairment.4 Prior studies focused on the mental health effect of the COVID-19 pandemic for people with visual impairment are few in number, include relatively small sample sizes, and are limited in diversity among studied groups.4 , 5 The All of Us Research Program currently provides the largest and most diverse American research database, with >50% of participants belonging to racial and ethnic minorities.6 In this study, we aim to evaluate the associations between visual impairment, depression, and anxiety during the COVID-19 pandemic. The All of Us data set confers the unprecedented advantages of a large, multisite sample and the ability to explore associations within historically understudied racial and ethnic groups. Chief among our aims is to investigate whether or not minority groups are disproportionately affected, given the known increased prevalence and severity of COVID-19 in minority racial groups7 and documented disparities observed for a wide range of health outcomes.8 Improving our understanding of the longitudinal COVID-19 experience of people with visual impairment is an important step toward addressing inequality if individuals with visual impairment suffer disproportionately, which highlights a broader need to promote equality in access to care and mental health services.2 Methods Study sample All of Us. The National Institutes of Health All of Us Research Program (AoU) data set was used for analysis.5 AoU is a national database emphasizing enrolment of participants traditionally underrepresented in biomedical research. The data set contains sociodemographic information, electronic health record information (including diagnoses and drug prescriptions), and survey data for 331,360 participants as of November 29, 2021. The institutional review boards for AoU have approved all study procedures. All participants provided written informed consent. Further details can be found at the AoU Research Program Protocol (allofus.nih.gov/about/all-us-research-program-protocol). Blind/visual impairment cases and controls. Participants who are blind/visually impaired (BVI) were identified if they met 1 of the following criteria: (i) Systematized Nomenclature of Medicine (SNOMED) diagnosis codes for “blindness/low vision” (SNOMED 4023110); (ii) responded “yes” to survey question, “Are you blind or have serious difficulty seeing, even when wearing glasses?”; and (iii) responded “yes” to survey question, “Has a doctor ever told you that you have blindness?”. Participants who served as control individuals were identified if they met all the following criteria: (i) no SNOMED codes for “blindness/low vision” (SNOMED 4023110) or “eye/vision finding” (SNOMED 4038502); (ii) responded “no” to survey question, “Are you blind or have serious difficulty seeing, even when wearing glasses?”; and (iii) responded “no” to survey question, “Has a doctor ever told you that you have: blindness?”. Previous history of COVID-19. Participants with a previous history of COVID-19 infection were identified if they met the following criteria: (i) SNOMED diagnosis code for “COVID-19” (SNOMED 37311061) or (ii) responded “yes” to survey question, “Do you think you have had COVID-19?”. Outcomes: anxiety and depression. Anxiety and depression were measured in several ways to better understand the effect of COVID-19 on mood disorder outcomes. Baseline anxiety/depression was defined as a diagnosis for anxiety (SNOMED 441542) or depression (SNOMED 440383) or a prescription for anxiolytics or antidepressants prior to March 30, 2020. New anxiety/depression was defined as a diagnosis for anxiety or depression or a prescription for anxiolytics or antidepressants after March 30, 2020, without baseline anxiety/depression. Worsened anxiety/depression was defined as a new SNOMED code for anxiety or depression in individuals with baseline anxiety/depression. COVID-19 Participant Experience (COPE) is a survey administered by AoU starting in May 2020 to participants to better understand the effect of COVID-19 on participant mental and physical health. The survey included questions on social distancing experiences, vaccine perceptions, COVID-19-related effects, and anxiety and mood. At time of the analysis, >100,000 unique participants had completed this survey at least once. Symptoms related to anxiety were assessed with the Generalized Anxiety Disorder 7 (GAD-7) survey, a 7-question survey that corresponds to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria for anxiety in the past 2 weeks.9 Questions assessing frequency of anxiety symptoms are answered on a 4-point scale from “Not at all” (0 points) to “Several days” (1 point), “More than half the days” (2 points), and “Nearly every day” (4 points). The points are summed, and a total score greater than the cut-off of 10 indicates severe anxiety. Symptoms related to depression were assessed with the Patient Health Questionnaire 9 (PHQ-9) survey, a 9-question survey that corresponds to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria for depression in the past 2 weeks.10 Questions assessing frequency of depressive symptoms are answered on a 4-point scale, from “Not at all” (0 points) to “Several days” (1 point), “More than half the days” (2 points), and “Nearly every day” (4 points). The points are summed, and a total score greater than the cut-off of 10 indicates severe depression. Statistical analysis Analyses were performed using RStudio (Posit, PBC, Boston, Mass.) on a cloud-based Jupyter Notebook environment provided by All of Us. Descriptive statistics were calculated for demographic characteristics. Differences in prevalence of outcomes across groups were compared using χ 2 tests. Univariate and multivariate logistic regression models controlled for age, sex, race, and ethnicity, and a modified Charlson comorbidity index11 was used to evaluate the associations between BVI with baseline, new, and worsened anxiety and depression. Univariate and multivariate mixed-effects logistic regression models controlling for the same covariates as above were used to evaluate the association between BVI with COPE-defined anxiety and depression and to control for participants who completed the survey more than once. Similarly, univariate and multivariate mixed-effects linear regression models controlling for the same covariates as above were used to evaluate the association between BVI with GAD-7 and PHQ-9 survey scores. Values of p <0.05 were considered statistically significant. Results Cohort characteristics A total of 324,915 participants (7526 BVI individuals and 317,389 control individuals) were included in the analyses (Table 1 ). Those who were blind and visually impaired were, on average, older (58.8 ± 15.4 years vs 54.1 ± 16.8 years; p < 0.001), with a greater proportion of males (40.2% vs 38.1%; p < 0.001), black individuals (29.4% vs 20.8%; p < 0.001), and Hispanic or Latino individuals (26.2% vs 18.2%; p < 0.001). BVI individuals also had a higher comorbidity index (2.54 ± 2.29 vs 2.30 ± 2.05; p < 0.001) and were more likely to have previously contracted COVID-19 (17.4% vs 11.5%; p < 0.001).Table 1 Cohort characteristics. Table 1Characteristic Control, n = 317,389 Blind/visually impaired, n = 7526 p Value Age, y, n (%) 54.1 (16.8) 58.8 (15.4) <0.001 Sex Male 38.1% 40.2% <0.001 Female 60.1% 58.1% Other 1.18% 1.93% Race Asian 3.41% 1.63% <0.001 Black 20.8% 29.4% White 54.3% 39.1% Other 21.4% 29.9% Ethnicity Hispanic or Latino 18.2% 26.2% <0.001 Non-Hispanic or Latino 78.9% 70.3% Other 2.9% 3.53% Comorbidity index 2.30 (2.05) 2.54 (2.29) <0.001 COVID-19 Contracted COVID-19 11.5% 17.4% <0.001 Did not contract COVID-19 88.5% 82.6% Baseline, new, and worsened anxiety and depression BVI individuals had a significantly higher prevalence of baseline anxiety and depression than control individuals (50.4% vs 28.7%; p < 0.001; Fig. 1 A). Individuals who contracted COVID-19 were more likely to have baseline anxiety and depression; among individuals who contracted COVID-19, 69.4% of BVI individuals had baseline anxiety and depression compared with 37.9% of non-BVI individuals (p < 0.001). In multivariate regression models, being BVI was associated with 1.61 times greater odds of having baseline anxiety and depression (odds ratio [OR] = 2.51; 95% CI, 2.41–2.64; p < 0.001), adjusted odds ratio [aOR] = 1.61; 95% CI, 1.46–1.78).Fig. 1 Association of blindness and visual impairment with COVID-19 pandemic anxiety and depression outcomes: (A) baseline depression and anxiety; (B) new depression and anxiety; (C) worsened depression and anxiety; (D) COPE anxiety (GAD-7); (E) COPE depression (PHQ-9). Fig 1 Similarly, BVI individuals had a higher prevalence of new anxiety and depression during the pandemic; 0.98% of BVI individuals had new anxiety and depression compared with 0.66% of non-BVI individuals (p < 0.001; Fig. 1 B). This was true among individuals who contracted COVID-19. Among individuals who contracted COVID-19, 3.73% of BVI individuals had new anxiety and depression compared with 1.62% of non-BVI individuals (p = 0.014). In an unadjusted model, being BVI was associated with 1.50 times greater odds of having new anxiety and depression, though this relationship was not significant after controlling for age, sex, race, ethnicity, and comorbidity (OR = 1.50; 95% CI, 1.18–1.89; aOR = 1.15; 95% CI, 0.81–1.64). Finally, there was a low prevalence of worsened anxiety and depression throughout the pandemic for both BVI individuals and control individuals. BVI individuals had a higher prevalence of worsened anxiety and depression during the pandemic compared with control individuals (0.19% vs 0.07%; p < 0.001; Fig. 1 C). Among individuals who contracted COVID-19, 0.75% of BVI individuals had worsened anxiety and depression compared with 0.20% of non-BVI individuals, though this result did not reach significance (p = 0.112). In multivariate regression models, being BVI was associated with 2.07 times greater odds of having worsened anxiety and depression (OR = 2.69; 95% CI, 1.56–4.61; aOR = 2.07; 95% CI, 1.03–4.13). COPE survey Of the 324,915 participants included in this analysis, 100,938 participants (1244 BVI individuals and 99,694 control individuals) had responded to the COPE survey. Of BVI individuals, 13.8% had anxiety (GAD-7) on the COPE survey compared with 10.6% of non-BVI individuals (p < 0.001; Fig. 1 D). Individuals who contracted COVID-19 were more likely to have anxiety (GAD-7). Among individuals who contracted COVID-19, 21.4% of BVI individuals had anxiety compared with 16.4% of non-BVI individuals, though this difference was not significant (p = 0.23). In multivariate regression models, being BVI was associated with 1.05 times greater odds of having anxiety (OR = 1.04; 95% CI, 1.01–1.06; aOR = 1.05; 95% CI, 1.02–1.09). Additionally, being BVI was associated with an increase of 2.07 points on the GAD-7 scale, after adjusting for the above covariates, compared with control individuals (β = 1.50; 95% CI, 1.11–2.01; adjusted β = 2.07; 95% CI, 1.32–3.27). Similarly, 20.8% of BVI individuals had depression (PHQ-9) on the COPE survey compared with 18.4% of non-BVI individuals (p = 0.03; Fig. 1 E). Individuals who contracted COVID-19 were more likely to have depression (PHQ-9). Among individuals who contracted COVID-19, 27.1% of BVI individuals had depression compared with 24.4% of non-BVI individuals, though this difference was not significant (p = 0.629). In multivariate regression models, being BVI was associated with 1.05 times greater odds of having depression (OR = 1.03; 95% CI, 1.00–1.06; aOR = 1.05; 95% CI, 1.01–1.10). Additionally, being BVI was associated with an increase of 2.96 points on the PHQ-9 scale, after adjusting for the above covariates, compared with control individuals (β = 1.71; 95% CI, 1.17–2.52; adjusted β = 2.96; 95% CI, 1.64–5.36). Effect of race and ethnicity Our analysis included a high proportion of nonwhite and (or) Hispanic or Latino individuals (Table 1). White individuals who were BVI had higher rates of baseline anxiety and depression (58.0%) than black individuals (44.5%) and Asian individuals (27.6%). Similarly, non-Hispanic or Latino BVI individuals had higher rates of baseline anxiety than Hispanic or Latino BVI individuals (52.0% vs 45.9%). However, in all race/ethnicity groups, BVI individuals had higher rates of baseline anxiety and depression (Table 2 ). Black BVI individuals had the highest rates of new anxiety and depression (1.09%), closely followed by white BVI individuals (1.05%). All race/ethnicity groups except for Asian individuals had higher rates of new anxiety/depression among BVI individuals (Table 3 ). For worsened anxiety/depression, white BVI individuals had the highest rates (0.31%), followed by black BVI individuals (0.09%). All race/ethnicity groups except for Asian individuals had higher rates of worsened anxiety/depression among BVI individuals (Table 4 ).Table 2 Baseline anxiety and depression among racial and ethnic subgroups. Table 2Race/ethnicity Blind/visually impaired Control p Value Race White 58.0% 31.1% <0.001 Black 44.5% 27.6% <0.001 Asian 38.2% 13.6% <0.001 Ethnicity Hispanic or Latino 45.9% 25.4% <0.001 Non-Hispanic or Latino 52.0% 29.4% <0.001 Table 3 New anxiety and depression among racial and ethnic subgroups. Table 3Race/ethnicity Blind/visually impaired Control p Value Race White 1.05% 0.65% 0.009 Black 1.09% 0.70% 0.045 Asian 0.00% 0.53% 1.00 Ethnicity Hispanic or Latino 0.96% 0.70% 0.223 Non-Hispanic or Latino 1.00% 0.65% 0.002 Table 4 Worsened anxiety and depression among racial and ethnic subgroups. Table 4Race/ethnicity Blind/visually impaired Control p Value Race White 0.31% 0.07% <0.001 Black 0.09% 0.08% 0.686 Asian 0% 0.06% 1.00 Ethnicity Hispanic or Latino 0.20% 0.07% 0.065 Non-Hispanic or Latino 0.19% 0.07% 0.005 Of the 100,938 participants who responded to the COPE survey, 89,478 were white (1066 BVI individuals), 5296 were black (82 BVI individuals), 2803 were Asian (13 BVI individuals), and the remainder were categorized as “Other.” A total of 95,064 individuals were non-Hispanic or Latino (1154 BVI individuals), 6273 were Hispanic or Latino (121 BVI individuals), and the remainder were categorized as “Other.” White BVI individuals had the highest rates of anxiety (GAD-7; 13.5%), followed by black individuals (10.7%) and Asian individuals (0%). Hispanic or Latino individuals had higher rates of anxiety (GAD-7) than non-Hispanic or Latino BVI individuals (21.6% vs 13.0%). With the exception of Asian individuals, all race/ethnicity groups had higher rates of anxiety among BVI individuals, though this was only statistically significant for white and non-Hispanic or Latino individuals (Table 5 ). Similarly, White BVI individuals had the highest rates of depression (PHQ-9; 19.9%), followed by black individuals (19.7%) and Asian individuals (0%). Hispanic or Latino individuals had higher rates of depression (PHQ-9) than non-Hispanic or Latino BVI individuals (29.9% vs 19.8%). All BVI individuals of different races/ethnicities had higher rates of depression except Asian individuals, though this did not reach statistical significance in subgroup analysis (Table 6 ).Table 5 COPE survey GAD-7 anxiety among racial and ethnic subgroups. Table 5Race/etnicity Blind/visually impaired Control p Value Race White 13.5% 10.0% <0.001 Black 10.7% 12.9% 0.697 Asian 0% 12.1% 0.381 Ethnicity Hispanic or Latino 21.6% 16.2% 0.154 Non-Hispanic or Latino 13.0% 10.2% 0.003 GAD-7, Generalized Anxiety Disorder 7 Table 6 COPE survey PHQ-9 depression among racial and ethnic subgroups. Table 6Race/ethnicity Blind/visually impaired Control p Value Race White 19.9% 17.8% 0.090 Black 19.7% 19.6% 1.00 Asian 0% 19.2% 0.139 Ethnicity Hispanic or Latino 29.9% 24.8% 0.252 Non-Hispanic or Latino 19.8% 18.0% 0.127 PHQ-9, Patient Health Questionnaire 9 Discussion In this large study of All of Us participants, we found a higher likelihood of electronic health record–defined baseline, new, and worsened anxiety/depression during the COVID-19 pandemic among individuals who are blind and (or) visually impaired. We also demonstrate higher levels of self-reported anxious and depressive symptoms during the COVID-19 pandemic among individuals who are blind and (or) visually impaired. Together these findings indicate that individuals with visual impairment were suffering disproportionately throughout the pandemic and highlight an important need to promote access to mental health services among this population. We found that more than 1 in 2 BVI individuals had anxiety or depression compared with 1 in 4 control individuals. Our results are in agreement with previous studies reporting higher rates of anxiety and depression among individuals with visual impairment. In a large meta-analysis, Parravano et al. 12 found that 1 in 4 patients with visual impairment were affected by depression. Ulhaq et al.13 reported a 31.2% pooled prevalence of anxiety symptoms among patients with ophthalmic diseases and a twofold increase in the prevalence of anxiety symptoms and disorders compared with healthy control individuals. Similarly, Heesterbeek et al. 14 reported that the cumulative incidence of subthreshold (e.g., early) depression and anxiety in older adults with visual impairment was twice as high as in normally sighted peers. There are several hypotheses for what may cause higher rates of mental health disorders among individuals who are visually impaired. These include difficulties adjusting to loss of functional independence and physical mobility,15 restrictions on driving,16 economic burden, and hardship,17 , 18 among others. In a study using semistructured interviews, van Munster et al.19 found that visually impaired individuals often had limited knowledge about mental health, potentially due to lower ability to obtain processable information and thus lower mental health literacy.19 They also found that health care providers did not always provide resources for BVI individuals to access mental health services; indeed, only 25% of ophthalmic and low-vision service workers provide depression screening.20 It may come as no surprise that this study found higher rates of new and worsened anxiety and depression, as well as higher scores on the GAD-7 and PHQ-9 surveys, throughout the COVID-19 pandemic among individuals who are blind or visually impaired. The early days of the COVID-19 pandemic were met with a series of responses including lockdowns and social restrictions to limit the spread of disease. Higher rates of anxiety and depression throughout the pandemic in the general population have been widely reported globally.21, 22, 23, 24 Individuals who are BVI are at higher risk of depression and anxiety and were disproportionately affected by the pandemic. There may be several factors at play, including increased loneliness (potentially due to difficulties transitioning to virtual social interactions) 4 and difficulties adhering to social distancing and hygiene protocols to prevent COVID-19 transmission.5 Furthermore, access to appropriate health care services may have decreased throughout the COVID-19 pandemic; one study found that only 59.3% of patients completed all scheduled eye care visits during the pandemic compared with 82.0% of patients before the pandemic. These missed eye care visits may have provided an opportunity for earlier mental health screening and referral to evaluate and treat anxiety and depression.25 Our study also found that BVI individuals who contracted COVID-19 were more likely to have higher rates of both baseline and new anxiety and depression than non-BVI individuals. This finding may demonstrate a cyclic relationship where BVI individuals are both more susceptible to anxiety and depression after a COVID-19 infection and more susceptible to COVID-19 infection when they have concomitant anxiety and depression. Reasons for increased susceptibility to anxiety and depression after a COVID-19 infection have been discussed previously and may include increased loneliness and difficulties adhering to hygiene protocols. Increased susceptibility to COVID-19 infection in individuals who have anxiety or depression has been demonstrated previously; it is posited that mental health conditions at large have been linked to elevated risk of other comorbidities and decreased levels of self-care, thereby increasing the risk of COVID-19 infection.26 Our findings show that this risk is disproportionately high in individuals who are blind and visually impaired. Finally, our study found that in all races and ethnicities, individuals who are BVI had higher rates of baseline as well as new and worsened anxiety and depression during the pandemic (with the exception of Asian individuals). We also report higher rates of all anxiety and depression outcomes among white BVI populations compared with black and Asian individuals. Prior research has demonstrated higher levels of anxiety and depression among white individuals.27 , 28 This may be due in part to disparities in health care access in addition to social stigma regarding mental health in certain cultures. Disparities in access to mental health care have been well documented, with white individuals receiving higher levels of mental health care, outpatient care, and psychotropic medication prescriptions compared with their black and Hispanic counterparts.29 Other potential explanations of disparity in diagnosis rates may include a strong sense of ethnic identity and greater religious and community support among minority groups.30 However, studies also have found that though black individuals may have lower rates of depression, those who are diagnosed with depression tend to have more severe and chronic disease, underscoring the importance of adequately connecting individuals with appropriate medical services and care.30 While Hispanic or Latino BVI individuals had lower rates of baseline, new, and worsened anxiety and depression than non-Hispanic or Latino individuals, they had higher rates of COPE survey anxiety and depression. This may reflect a higher level of unmet mental health needs during the pandemic that were not formally diagnosed or pharmacologically treated among Hispanic or Latino BVI individuals. This study has many notable strengths. We use a large and comprehensive national database to investigate the association of visual impairment with rates of anxiety and depression. Because of the high diversity of the AoU research database, we were also able to investigate the rates of anxiety and depression among historically understudied racial and ethnic groups. However, our study is also subject to some limitations. First, our definitions of BVI, anxiety, and depression relied on the use of available electronic health record data, specifically diagnosis codes, and medication use, which may not always be precise. Second, our data set did not have sufficient sample size of PHQ-9 and GAD-7 scores among understudied racial and ethnic groups, particularly Asian individuals; such data may allow for identification of anxious and depressive symptoms among individuals who may not have access to health care resources and thus may not be represented in the electronic health record. Further research in historically understudied visually impaired populations may better identify and quantify differences in mental health outcomes among these populations. Finally, while the AoU research database confers a large sample size, we were unable to assess the relationship of individual ophthalmologic diseases (e.g., glaucoma, age-related macular degeneration) to COVID-19-related anxiety and depression due to an insufficient sample of individuals with these ophthalmologic diseases and COVID-19 survey responses. Furthermore, the AoU database does not include measures of visual impairment severity levels, so we were unable to analyze whether the degree of visual impairment had an influence on anxiety and depression levels. In summary, here we demonstrate that BVI individuals are disproportionately affected by anxiety and depression at baseline and throughout the COVID-19 pandemic compared with their non–visually impaired peers. These findings underscore the importance of equity in access to care and mental health services for individuals with visual disabilities. Further research about how to best support mental health outcomes in visually impaired individuals as the COVID-19 pandemic continues should be prioritized. Footnotes and Disclosure The authors have no proprietary or commercial interest in any materials discussed in this article. Funding This work was supported in part by a National Institutes of Health (NIH) K23 Career Development Award (K23EY032634) (N.Z.), NIH R21 Exploratory/Developmental Research Grant Award (1R21EY032953), and Research to Prevent Blindness Career Development Award (N.Z.). ==== Refs References 1 Brooks SK Webster RK Smith LE The psychological impact of quarantine and how to reduce it: rapid review of the evidence Lancet 395 2020 912 920 32112714 2 Razai MS Oakeshott P Kankam H Galea S Stokes-Lampard H. Mitigating the psychological effects of social isolation during the COVID-19 pandemic BMJ 369 2020 m1904 32439691 3 Ting DSJ Krause S Said DG Dua HS. Psychosocial impact of COVID-19 pandemic lockdown on people living with eye diseases in the UK Eye (Lond) 35 2021 2064 2066 32778740 4 Heinze N Hussain SF Castle CL Godier-McBard LR Kempapidis T Gomes RSM. The long-term impact of the COVID-19 pandemic on loneliness in people living with disability and visual impairment Front Public Health 9 2021 738304 5 Shalaby WS Odayappan A Venkatesh R The impact of COVID-19 on individuals across the spectrum of visual impairment Am J Ophthalmol 227 2021 53 65 33781768 6 All of Us Research Program InvestigatorsDenny JC Rutter JL The “All of Us” Research Program N Engl J Med 381 2019 668 676 31412182 7 Lopez L Hart LH Katz MH. Racial and ethnic health disparities related to COVID-19 JAMA 325 2021 719 720 33480972 8 Weinstein JN Geller A Negussie Y Baciu A Communities in action: Pathways to health equity 2017 National Academies Press Washington, DC 9 Newman MG Zuellig AR Kachin KE Preliminary reliability and validity of the generalized anxiety disorder questionnaire IV: a revised self-report diagnostic measure of generalized anxiety disorder Behav Ther 33 2002 215 233 10 Levis B Benedetti A Thombs BD Depression Screening Data (DEPRESSD) Collaboration. Accuracy of Patient Health Questionnaire-9 (PHQ-9) for screening to detect major depression: individual participant data meta-analysis BMJ 365 2019 l1476 30967483 11 Quan H Li B Couris CM Updating and validating the Charlson Comorbidity Index and score for risk adjustment in hospital discharge abstracts using data from 6 countries Am J Epidemiol 173 2011 676 682 21330339 12 Parravano M Petri D Maurutto E Association between visual impairment and depression in patients attending eye clinics: a meta-analysis JAMA Ophthalmol 139 2021 753 761 34042966 13 Ulhaq ZS Soraya GV Dewi NA Wulandari LR. The prevalence of anxiety symptoms and disorders among ophthalmic disease patients Ther Adv Ophthalmol 14 2022 25158414221090100 14 Heesterbeek TJ van der Aa HPA van Rens GHMB Twisk JWR van Nispen RMA. The incidence and predictors of depressive and anxiety symptoms in older adults with vision impairment: a longitudinal prospective cohort study Ophthalmic Physiol Opt 37 2017 385 398 28516509 15 Court H McLean G Guthrie B Mercer SW Smith DJ. Visual impairment is associated with physical and mental comorbidities in older adults: a cross-sectional study BMC Med 12 2014 181 25603915 16 Keay L Munoz B Turano KA Visual and cognitive deficits predict stopping or restricting driving: the Salisbury Eye Evaluation Driving Study (SEEDS) Invest Ophthalmol Vis Sci 50 2009 107 113 18719088 17 Demmin DL Silverstein SM. Visual impairment and mental health: unmet needs and treatment options Clin Ophthalmol 14 2020 4229 4251 33299297 18 Jin Y Lin D Dai ML Economic hardship, ocular complications, and poor self-reported visual function are predictors of mental problems in patients with uveitis Ocul Immunol Inflamm 29 2021 1045 1055 32657648 19 van Munster EPJ van der Aa HPA Verstraten P van Nispen RMA. Barriers and facilitators to recognize and discuss depression and anxiety experienced by adults with vision impairment or blindness: a qualitative study BMC Health Serv Res 21 2021 749 34320953 20 Rees G Fenwick EK Keeffe JE Mellor D Lamoureux EL. Detection of depression in patients with low vision Optom Vis Sci 86 2009 1328 1336 19844188 21 Pashazadeh Kan F Raoofi S Rafiei S A systematic review of the prevalence of anxiety among the general population during the COVID-19 pandemic J Affect Disord 293 2021 391 398 34246947 22 Salari N Hosseinian-Far A Jalali R Prevalence of stress, anxiety, depression among the general population during the COVID-19 pandemic: a systematic review and meta-analysis Global Health 16 2020 57 32631403 23 Shi L Lu ZA Que JY Prevalence of and risk factors associated with mental health symptoms among the general population in China during the coronavirus disease 2019 pandemic JAMA Netw Open 3 2020 e2014053 24 Castaldelli-Maia JM Marziali ME Lu Z Martins SS. Investigating the effect of national government physical distancing measures on depression and anxiety during the COVID-19 pandemic through meta-analysis and meta-regression Psychol Med 51 2021 881 893 33648613 25 Brant AR Pershing S Hess O The impact of COVID-19 on missed ophthalmology clinic visits Clin Ophthalmol 15 2021 4645 4657 34916776 26 Wang Y Yang Y Ren L Shao Y Tao W jian Dai X Preexisting mental disorders increase the risk of COVID-19 infection and associated mortality Front Public Health 9 2021 684112 27 Blanco C Rubio J Wall M Wang S Jiu CJ Kendler KS. Risk factors for anxiety disorders: common and specific effects in a national sample Depress Anxiety 31 2014 756 764 24577934 28 Himle JA Baser RE Taylor RJ Campbell RD Jackson JS. Anxiety disorders among African Americans, blacks of Caribbean descent, and non-Hispanic whites in the United States J Anxiety Disord 23 2009 578 590 19231131 29 Cook BL Trinh NH Li Z Hou SSY Progovac AM. Trends in racial-ethnic disparities in access to mental health care, 2004–2012 Psychiatr Serv 68 2017 9 16 27476805 30 Bailey R Mokonogho J Kumar A. Racial and ethnic differences in depression: current perspectives Neuropsychiatr Dis Treat 15 2019 603 609 30863081	0	PMC9712066	NO-CC CODE	2022-12-15 23:17:57	no	Can J Ophthalmol. 2022 Dec 1; doi: 10.1016/j.jcjo.2022.11.019	utf-8	Can J Ophthalmol	2,022	10.1016/j.jcjo.2022.11.019	oa_other
==== Front Patterns (N Y) Patterns (N Y) Patterns 2666-3899 Elsevier S2666-3899(22)00299-9 10.1016/j.patter.2022.100659 100659 Descriptor Comprehensively identifying Long Covid articles with human-in-the-loop machine learning Leaman Robert 1 Islamaj Rezarta 1 Allot Alexis 1 Chen Qingyu 1 Wilbur W. John 1 Lu Zhiyong 12∗ 1 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health 8600 Rockville Pike, Bethesda, MD, 20894, USA ∗ Corresponding author 2 Lead contact 1 12 2022 1 12 2022 10065929 7 2022 19 9 2022 17 11 2022 . 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. A significant percentage of COVID-19 survivors experience ongoing multisystemic symptoms that often affect daily living, a condition known as Long Covid or post-acute-sequelae of SARS-CoV-2 infection. However, identifying scientific articles relevant to Long Covid is challenging since there is no standardized or consensus terminology. We developed an iterative human-in-the-loop machine learning framework combining data programming with active learning into a robust ensemble model, demonstrating higher specificity and considerably higher sensitivity than other methods. Analysis of the Long Covid collection shows that (1) most Long Covid articles do not refer to Long Covid by any name (2) when the condition is named, the name used most frequently in the literature is Long Covid, and (3) Long Covid is associated with disorders in a wide variety of body systems. The Long Covid collection is updated weekly and is searchable online at the LitCovid portal: https://www.ncbi.nlm.nih.gov/research/coronavirus/docsum?filters=e_condition.LongCovid Graphical abstract A significant percentage of COVID-19 survivors experience Long Covid: ongoing multisystemic symptoms that often affect daily living. Querying for relevant scientific articles is difficult before consensus builds for standardized terminology; we therefore identified Long Covid articles using an iterative human-in-the-loop machine learning approach. Analysis of the ∼9,000 articles in the Long Covid collection shows that most do not mention it by name and emphasizes that Long Covid is associated with disorders in a wide variety of body systems. Keywords Long Covid post-acute sequelae of SARS-CoV-2 infection COVID-19 text classification machine learning weak supervision data programming active learning natural language processing ==== Body pmcThe bigger picture Long Covid causes ongoing multisystemic symptoms in a substantial percentage of COVID-19 survivors and lacks specific treatments. Locating articles that refer to novel entities such as Long Covid is generally challenging since keyword searches suffer from limited results and low accuracy without broadly supported terminology. We developed an iterative human-in-the-loop framework to comprehensively identify articles relevant to Long Covid. Our framework integrates multiple classifiers with complementary views and varying accuracy into a single model that reliably predicts the relevance of each article to Long Covid and its priority for manual annotation. We show that most articles relevant to Long Covid do not name the condition and are missed by keyword search. We present and analyze a comprehensive collection of Long Covid articles in LitCovid, which we believe will help accelerate research into this pressing public health issue. Data Science Maturity DSML 2: Proof of concept: Data science output has been formulated, implemented, and tested for one domain/problem.	36471749	PMC9712067	NO-CC CODE	2022-12-02 23:21:32	no	Patterns (N Y). 2022 Dec 1;:100659	utf-8	Patterns (N Y)	2,022	10.1016/j.patter.2022.100659	oa_other
==== Front Ann Dermatol Venereol Ann Dermatol Venereol Annales De Dermatologie et De Venereologie 0151-9638 0151-9638 Elsevier Masson SAS. S0151-9638(22)00079-5 10.1016/j.annder.2022.08.002 Letter to the Editor A rare case of bilateral ear perforation due to excessive and prolonged mask wearing in a patient with coronaphobia Kaikati Jerome ⁎1 Abou Khater Jad 1 Merhy Reine 1 Stephan Farid Department of Dermatology, Hôtel Dieu de France Hospital, Faculty of Medicine, Saint-Joseph University, Beirut, Lebanon ⁎ Corresponding author. 1 Contributed equally to this work and are considered first co-authors. 1 12 2022 1 12 2022 9 1 2022 30 8 2022 © 2022 Elsevier Masson SAS. All rights reserved. 2022 Elsevier Masson SAS Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Keywords COVID-19 Face mask Pressure injury ==== Body pmcThe spread of Coronavirus disease 2019 (COVID-19) and its mortality rate exacerbated mental disorders throughout the world [1]. Studies reported consequences of COVID-19-related anxiety at the political, economic, social and individual levels [2]. However, somatic repercussions are a possible manifestation of advanced coronaphobia. Herein we report the case of a healthy 30-year-old man who presented at the dermatology department for bilateral gradual deep erosion of the helix reaching the fossa triangularis of both ears (Fig. 1, Fig. 2 ). The lesions were not painful. He had been using standard double disposable surgical masks made of nonwoven polypropylene secured with elastic loops over the ears for at least 18 hours per day since the onset of the pandemic in Lebanon on March 2020. He developed COVID-19-related anxiety, which worsened as time went by. He denied having any previous episodes of ear infection, any attitude of self-harm, or any intrusion of any foreign bodies into his external ears. He did not express any suicidal ideation or attempts. Besides prolonged mask use, he developed such obsessive–compulsive behaviors as washing his hands continuously for one and a half hours several times per day. The patient was referred to the psychiatry department for management of his mental health issues, and to the plastic surgery department for ear reconstruction. The patient was diagnosed with coronaphobia (more recently called COVID Stress Syndrome) [3].Fig. 1 Left ear. Fig. 2 Right ear. The ear has a specific anatomy that increases vulnerability to pressure injury [4]. The postauricular area is in fact anatomically unique as it overlies muscle, tendon and bone medially, and cartilage laterally. Furthermore, it is the site of the thinnest skin on the face, making it vulnerable to pressure injury [4]. Based on the National Pressure Injury Advisory Panel (NPIAP), the bilateral lesions of our patient may be classified as a stage 4 pressure injury [5]. Medical device-related pressure injuries (MDPRI), including those sustained through the ear-loops of masks, have been a concern during this pandemic [4], [6]. Constant pressure on the skin and, consequently, on the auricular cartilage, appears to lead to considerable loss of skin and tissue [5]. COVID Stress Syndrome is a newly described entity considered a pandemic-related adjustment disorder. Diagnosis is based on five inter-correlated elements: fear of COVID-19 infection or of contact with objects contaminated with the virus, fear of socio-economic impact of the pandemic, fear of strangers while considering them to be infected, compulsive pandemic-related checking, and pandemic-related traumatic stress symptoms [3]. A diagnosis of COVID Stress Syndrome should only be made after ruling out obsessive–compulsive disorder or any other disorders that might potentially account for the symptoms [3]. On another note, numerous dermatoses caused by face-mask wearing during the COVID-19 pandemic have been seen, namely acne, rosacea, seborrheic dermatitis, perioral dermatitis, and impetigo. Not only did mask-wearing cause exacerbation of preexisting facial dermatosis, but it also increased incidence thereof [7]. However, to the best of our knowledge, this is the first reported case of bilateral ear perforation linked to the use of masks with ear-loops. Protection of skin beneath the mask straps and evaluation of the tension of the straps are certainly valid preventive strategies [4]. By identifying at-risk patients (including those with mental health issues) and following the recommendations for prevention of MDRPI, it is usually possible to prevent pressure injuries before they occur [4], [6]. Disclosure of interest The authors declare that they have no conflicts of interest. Funding source None. ==== Refs References 1 Garcovich S. Bersani F.S. Chiricozzi A. Mass quarantine measures in the time of COVID-19 pandemic: psychosocial implications for chronic skin conditions and a call for qualitative studies J Eur Acad Dermatol Venereol 34 2020 e293 e294 32330329 2 Heiat M. Heiat F. Halaji M. Phobia and fear of COVID-19: origins, complications and management, a narrative review Ann Ig Med Prev E Comunita 33 2021 360 370 3 Taylor S. COVID Stress Syndrome: Clinical and nosological considerations Curr Psychiatry Rep 23 2021 19 33660068 4 Levine J.M. Ayello E.A. Persaud B. Spinner R. Medical device-related pressure injury to the ear from a mask Adv Skin Wound Care 34 7 2021 380 383 34125728 5 Cuddigan J. NPIAP Position Statements on Preventing Injury with N95 Masks. 2020;5. 6 Mukhtar M. Novel techniques for wearing an ear-looped mask to reduce pressure on the ear J Am Acad Dermatol 83 2020 e333 e334 32702460 7 Olisova O.Y. Teplyuk N.P. Grekova E.V. Dermatoses caused by face mask wearing during the COVID-19 pandemic J Eur Acad Dermatol Venereol 35 2021 e738 e741 34310757	36462940	PMC9712068	NO-CC CODE	2022-12-02 23:21:32	no	Ann Dermatol Venereol. 2022 Dec 1; doi: 10.1016/j.annder.2022.08.002	utf-8	Ann Dermatol Venereol	2,022	10.1016/j.annder.2022.08.002	oa_other
==== Front J Pediatr Health Care J Pediatr Health Care Journal of Pediatric Health Care 0891-5245 1532-656X Published by Elsevier Inc. on behalf of National Association of Pediatric Nurse Practitioners. S0891-5245(22)00347-9 10.1016/j.pedhc.2022.11.013 Article Prolonged Fever: Kawasaki Disease in a Pediatric Patient with COVID-19 Manion Amy Becker PhD, APRN, CPNP-PC ⁎ Lubelchek Alison MSN, RN Bensko Leanne MD, FAAP Associate Professor, Rush University ⁎ Corresponding author: Dr. Amy Becker Manion, Rush University - College of Nursing, United States 1 12 2022 1 12 2022 Copyright © 2022 Published by Elsevier Inc. on behalf of National Association of Pediatric Nurse Practitioners. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Kawasaki Disease is the leading cause of acquired heart disease. The cardiac clincial features seen with Kawasaki Disease requires diagnosis and treatment within 10 days of symptoms in order to decrease risk of complications. This case report examines the complexity of prolonged fever in a pediatric patient with a positive test for Sars-CoV-2 and meeting the criteria for Kawasaki Disease. Keywords COVID-19 Kawasaki Disease Infectious Disease Cardiac Findings Prolonged Fever ==== Body pmcIntroduction Fever is a common symptom seen in illness affecting children and adolescents. In general, fever is one of the body's main immune response reactions to infection suggesting that fever can be beneficial and self-limiting. Often parents will seek medical attention when their child develops a fever. Fever in children accounts for up to 30% of visits to both primary care and emergency departments (Urbane, 2019). COVID-19 is a viral infection and like most viral infections fever is a frequent symptom. In 2022, approximately 6.8 million children have tested positive for COVID-19 thus increasing the reported total of children who have tested COVID-19 positive in the United States since the beginning of the pandemic to nearly 14.7 million (American Academy of Pediatrics [AAP], 2022). Children, under 18, represent 18.5% of the total number of COVID-19 cases and make up 22.2% of the US total population (Centers for Disease Control and Prevention [CDC], 2022). Although mortality from COVID-19 is rare in the pediatric population, 1,462 children, age 18 and under have died from the virus (CDC, 2022b). The incubation period for COVID-19 ranges from 2 to 14 days with an average of 6 days (CDC, 2020). Potential signs and symptoms of COVID-19 in children can include fever, cough, nasal congestion, headache, sore throat, fatigue, myalgia, shortness of breath, difficulty breathing, decreased appetite, abdominal pain, diarrhea, nausea, and vomiting (CDC, 2020). The most common symptoms seen in children with COVID-19 are cough and fever (CDC, 2020). Yet, some children who test positive for COVID-19 may be asymptomatic. Although children with COVID-19 are at lower risk of severe illness compared to adults, children can still develop severe illness and complications from the virus (CDC, 2020). In addition, children under 1 year of age and those with underlying medical conditions including obesity, diabetes, asthma, and cystic fibrosis are at increased risk of more severe illness from COVID-19 (CDC, 2020). The signs and symptoms of COVID-19 can mimic other illness such as influenza, rhinovirus, seasonal allergies, pneumonia, mononucleosis, and streptococcal pharyngitis making accurate diagnosing challenging. Though children infected with COVID-19 are less likely to require hospitalization, approximately 1 in 3 children hospitalized with COVID-19 need intensive care treatment, which is similar to the rate seen in adults (CDC, 2020; Kim, 2020). This case report examines the complexity of prolonged fever in a pediatric patient with a positive test for Sars-CoV-2 and meeting the criteria for Kawasaki disease. Case Presentation A 30-month-old girl presented to the primary care clinic accompanied by her parents and 5-year-old sister. The patient had a history of fever for 4 days with 39.4 °C reported at home that morning. The patient's sibling presented with mild rhinorrhea and tested positive for Sars-CoV-2 at the clinic visit. Her temperature in clinic at time of visit was 38.1 °C and her other vital signs were age appropriate. The patient was awake and alert with slight irritability. Her physical exam showed mild clear rhinorrhea. Her oral mucosa was pink and moist. Her sclerae was white with no eye discharge. Tongue was normal appearance. The tympanic membranes were normal appearing. She had no adenopathy. She had no signs of respiratory distress. Her breath sounds were clear to auscultation with no stridor, retractions or nasal flaring. Her abdomen was soft, non-tender with audible bowel sounds. Skin was warm, dry and intact without rashes or lesions. Despite her sibling testing positive, the patient tested negative for Sars-CoV-2 on rapid COVID test as well as rapid influenza and rapid strep testing. A strep culture was sent which resulted as negative. Patient was sent home with guidance for fever management and to return to the clinic if fever did not resolve in 24 hours. Fever continued, therefore the patient returned to clinic for re-examination on day 6 of fever. On representation to clinic, her temperature was down to 37.6 °C. The child's mother reported that the patient started to develop dry cracked lips on day 5 of fever. In addition, she had injected sclerae bilaterally without discharge. A macular diffuse blotchy non pruritic rash was observed scattered to her body including her face that had started on day 5. In clinic, she tested positive for Sars-CoV-2 on rapid testing and negative for Influenza A & B on rapid testing. Physical Exam Her vital signs included a temperature of 37.6 °C, pulse of 106 beats/minute, and respiratory rate of 24 breaths/minute. On physical exam, the patient had dry cracked lips. Her sclerae was injected bilaterally with no discharge. The tympanic membranes were normal in appearance with normal landmarks and cone of light. She had no adenopathy. She had mild rhinorrhea. Her lungs were clear to auscultation with no signs of respiratory distress. Her heart sounds were clear with no murmur. Her abdomen was soft and non-tender. She had mild swelling of both hands. She had a macular blotchy rash to her body and face. She was awake and alert with noted increased irritability from her previous exam. Hospital Course The provider consulted an infectious disease expert with parental permission and shared pictures of the rash and cracked lips through the electronic medical record system. It was determined that although the patient was positive for Sars-CoV-2, in addition she met diagnostic criteria for Kawasaki disease. For further evaluation and care the patient was admitted to the local children's hospital. Upon admission, inflammatory markers were elevated with an erythrocyte sedimentation rate (ESR) 94 mm/hr (0-20 mm/hr) and C-reactive protein (CRP) 5.8 mg/dL (0-0.8 mg/dL). The patient was positive on PCR for Sars-CoV-2 and rhinovirus/enterovirus. Echocardiogram (ECHO) showed normal coronary artery origins and proximal courses, normal-size right and left ventricles, normal cardiac valve morphology and function, with no evidence of pulmonary hypertension or pericardial effusion. The patient had swelling of her hands and feet on day two of hospital admission. The infectious disease consultant confirmed the diagnosis of Kawasaki disease. On day 2 of hospitalization, IVIG was administered, and the patient was started on low dose aspirin 0.5 tablets (40.5 mg) orally once a day (Sakulchit, 2017). The patient was afebrile on day 3 of hospitalization. Her CRP was 4.8 mg/dL on day 3 and 3.9 mg/dL on day 4 of hospital course. Her rash faded and she started to have peeling between interweb spaces of hands. Her lips became less red and cracked over the 4-day hospital stay. The patient was discharged on day 4 on low dose aspirin for 6 weeks and scheduled for follow up with infectious disease team, repeat ECHO and CRP were scheduled for one week post discharge. The patient was seen at primary care clinic two days after hospital discharge. At this time the patient remained afebrile. Her parents reported improvement of her cracked lips and her physical examination was otherwise unremarkable. Six days after hospital discharge the patient represented with fever of 38.5 °C. Her mother reported she had flushed cheeks and edematous fingers. Her mother contacted the infectious disease provider, and she was readmitted. The patient's physical exam revealed mild nasal congestion and cracked lips; there was no cough, eye redness or discharge, or rash other than flushed cheeks and peeling skin near her fingernails. Her CRP had increased to 4.5 mg/dL (3.9 mg/dL at discharge) and patient had a new anemia and thrombocytosis. Her serum iron was 18 mcg/dL (45 – 143 mcg/dL), total Iron binding capacity 258 mcg/dL (280 – 400 mcg/dL), % saturation 7 (7 – 53%), and Transferrin 184 mg/dL (152-345 mg/dL). Her COVID PCR was negative for Sars-CoV-2. The patient was given a second dose of IVIG. A repeat ECHO revealed. The left anterior descending artery (LAD) was mildly dilated with a z-score of 3.0 and demonstrated luminal irregularity. The z-score is a classification system that uses the standard deviation (SD) of a value to compare to normal distribution and is used to measure coronary artery involvement. A z-score < 2.0 SD is considered normal, whereas a z-score between 2.5 to < 5.0 is considered a small aneurysm of the coronary arteries (Horl, 202). Due to a LAD z-score of 3.0 the patient met the high-risk criteria for Kawasaki disease. She clinically improved after an overnight dose of IVIG and appeared less fatigued. She still had edematous digits and peeling palms. Management Due to her cardiac changes, high-risk KD protocol and intravenous steroids were started, methylprednisolone 2 mg/kg daily for 5 days, followed by an oral steroid taper (Sakulchit, 2017). On day 3 of hospitalization CRP was had decreased to 2.2 mg/dL (ref range & units 0 – 0.8 mg/dL) and by day 4 CRP had decreased further to 1.0 mg/dL. A repeat ECHO was done on day 4 of hospitalization which showed the LAD was no longer dilated. She was discharged on day 5 of hospitalization with CRP 0.6 mg/dL. Discharge medications included aspirin 40.5 mg (half a tablet) by mouth daily and famotidine 8mg/ mL suspension 0.6 mL (0.5 mg/kg/dose) twice a day while on steroid taper (Sakulchit, 2017). Repeat CRP 6 days post-discharge remained normal at < 0.3 mg/dL and repeat ECHO was normal. The patient was weaned off steroids and her aspirin was discontinued 4 weeks following second hospitalization. Her follow up plan included annual visits with Infectious Disease team per high-risk Kawasaki protocol and for routine primary care. Due to receiving IVIG any administration of live vaccines was deferred for 11 months (American Academy of Pediatrics, 2018). The patient received her first COVID vaccine and quadrivalent inactivated influenza vaccine at the primary care clinic 3 months after hospitalization. Discussion Kawasaki disease (KD) is the leading cause of acquired heart disease and the second most common vasculitis in children (John & Brady, 2020). KD is also known as mucocutaneous lymph node syndrome because of its association with cervical lymph node enlargement and involvement of mucus membranes of the mouth, eyes, and throat (John & Brady, 2020). As this case demonstrates, the main symptom of KD is acute prolonged persistent fever. Fever is a common symptom with viral infections and in most cases tends to resolve by 3 to 5 days. When fever is persistent and prolonged other steps are required to determine the underlying cause. With KD the fever is typically high, 102 F or higher, which adds to the degree of concern and further contributes to parental anxiety about their child's illness. The exact etiology of KD remains unknown. It was first identified in Japan in the 1960s and is now seen worldwide. Children between the ages of 6 months to 5 years of age are the most susceptible to KD, with the peak incidence seen in children between 6 and 24 months (Sakulchit, 2017). Although the exact cause of KD is idiopathic, cases do peak in the late winter and early spring months. This case occurred during the late spring/early summer. Japan has the highest incidence of KD cases. Whether this is from an increased awareness of the disease in its country of origin or to a genetic component is uncertain. In the United States, children of Asian/Pacific Islander descent have the highest rate of hospitalization for KD which stresses the need for further investigation of this health disparity (John & Brady, 2020). The patient in this case report met this norm. KD is a diagnosis of exclusion which adds an additional challenge. Consequently, diagnosis is based on a specific criterion along with supporting diagnostic testing. The criteria to meet the diagnosis for KD includes prolonged fever for 5 days or more, and the presentation of at least four out of five additional symptoms which include bilateral conjunctival injection, changes of the lips and oral cavity, unilateral cervical lymphadenopathy, rash, and swelling of hands or feet. Lab investigations are not diagnostic, but rather are useful to help rule in another diagnosis (John & Brady, 2020). Standard diagnostic testing should include CBC with differential and platelet count, CRP, ESR, and comprehensive metabolic profile. Typical laboratory findings include elevated inflammatory markers, both CRP and ESR, as well as neutrophilia with bands and elevated platelets (John & Brady, 2020). The diagnostic lab results in this case report support the diagnosis of KD. The patient had elevated inflammatory markers. Both CRP and ESR were elevated upon hospital admission. As shown in Table 1 , the CRP values were monitored throughout both hospitalizations as well as follow up visits, demonstrating a downward trend as the patient improved. The clinical improvement observed plus the eventual return of the CRP value to normal range further demonstrates the accuracy of the diagnosis of KD in this patient.Table 1 C-Reactive Protein and Days of Illness Table 1Hospitalization Course CRP (ref range 0 – 0.8 mg/dL) Day of illness Hospitalization Day 1 (fever x 6 days) 5.8 mg/dL 6 Hospital Day 3 (afebrile) 4.8 mg/dL 8 Hospital Day 4 (discharged) 3.9 mg/dL 9 Re-admission to Hospital Day 1 (Fever x 2 days) 4.5 mg/dL 15 Day 3 (afebrile) 2.2 mg/dL 17 Day 4 1.0 mg/dL 18 Day 5 (discharge) 0.6 mg/dL 19 Follow up visit < 0.3 mg/dL 25 Due to the ongoing COVID-19 pandemic the use of PCR testing has become more prevalent both to assist in diagnosing and for infection control measures especially for patients requiring hospitalization. This case report is no exception to this new standard. The patient tested positive on PCR for Sars-CoV-2 and rhinovirus/enterovirus. COVID-19 symptoms mimic other illnesses and symptoms such as fever, conjunctival injection and even rash have been reported in children with COVID-19 infection (Danthuluri & Grant, 2020). Other symptoms that can be exhibited with KD and with COVID-19 include abdominal pain, diarrhea, irritability, arthralgia, and vomiting. In this case report the patient showed signs of irritability. This can be difficult to ascertain without caregiver input since children can be fearful of examination and may be extremely irritable while the provider is close by. In this case, both the parents and the provider noted irritability both at home and in the clinic. The similarities in clinical symptoms of KD and COVID-19 are further complicated by the emergence of multisystem inflammatory syndrome in children (MIS-C). MIS-C is considered a late immune response to Sars-CoV-2 infection. Most children with MIS-C present with abdominal pain, diarrhea and vomiting and a history of COVID-19 exposure or positive Sars-CoV-2 PCR test within four weeks of onset of symptoms (Waseem, 2022). Like KD, multisystem inflammatory syndrome in children does not possess definitive diagnostic tests. Elevated inflammatory markers (CRP and ESR) are evident in both KD and MIS-C. In MIS-C platelet counts drop as opposed to KD which typically shows a rise in platelet counts after day 5 of illness (Waseem, 2022). Both KD and MIS-C are associated with coronary artery changes. However, MIS-C patients are more likely (50%) to initially present in shock as opposed to KD patients (5%) (Waseem, 2022). The patient in this case report did not test positive for Sars-CoV-2 until day 6 of illness, therefore MIS-C was not a high concern despite elevated inflammatory markers. The cardiac clinical features seen with KD is the main reason why it is imperative for diagnosis and treatment to begin expeditiously within 10 days of symptom onset. Due to this risk, one of the main goals of treatment is to prevent coronary artery aneurysm (CAA). In the acute phase 15% to 25% of untreated children and <5% of treated children can develop CAA (de La Harpe, 2019). Initiating IVIG therapy and acetylsalicylic acid (ASA) are crucial. ASA has an antiplatelet effect and anti-inflammatory properties. After initiating high-dose ASA, treatment is tapered over a 6-to-8-week period (John & Brady, 2020). ASA is discontinued if the ECHO is normal. If coronary artery abnormalities develop or do not resolve, ASA or other antiplatelet therapy is used indefinitely (John & Brady, 2020). For this case report, the patient was started on IVIG and ASA within 7 days of symptoms and had mild cardiac changes on ECHO which fortunately resolved. Long term prognosis for children with KD is excellent. The disease tends to be self-limiting, and complications are greatly reduced if therapy is started within 10 days of initial symptoms. Possible complications include congestive heart failure, myocardial infarction, myocarditis, pericarditis, and recurrence of the disease (de La Harpe, 2019). Conclusion As throughout the COVID-19 pandemic, we need to remain cognizant of other causes of fever in children. Kawasaki disease was first described by Tomisaku Kawasaki, a Japanese pediatrician, in 1967 and continues to be a leading cause of acquired heart disease and in rare incidences can be fatal (Green, 2020). This case report is a reminder of the complexity of fever in children and the importance of careful consideration of all possible causes to arrive at the appropriate diagnosis and treatment. References American Academy of Pediatrics. (2018) Red book: Report of the Committee on Infectious Diseases (31st ed.) American Academy of Pediatrics, Elk Grove Village, IL. American Academy of Pediatrics. (2022). Children and COVID-19: State-level data report. https://www.aap.org/en/pages/2019-novel-coronavirus-covid-19-infections/children-and-covid-19-state-level-data-report/ Centers for Disease Control & Prevention. (2020). COVID-19: Information for pediatric healthcare providers. https://www.cdc.gov/coronavirus/2019-ncov/hcp/pediatric-hcp.html#symptoms-severity Center for Disease Control & Prevention. (2022). COVID Data Tracker: Pediatric data. https://covid.cdc.gov/covid-data-tracker/#pediatric-data Center for Disease Control & Prevention. (2022b). Provisional COVID-19 Deaths: Focus on ages 0-18 years. https://data.cdc.gov/NCHS/Provisional-COVID-19-Deaths-Focus-on-Ages-0-18-Yea/nr4s-juj3 Danthuluri, V., & Grant, M. B. (2020). Update and recommendations for ocular manifestations of COVID-19 in adults and children: A narrative review. Ophthalmology and Therapy, 9(4), 853–875. https://doi.org/10.1007/s40123-020-00310-5 de La Harpe, M., di Bernardo, S., Hofer, M., & Sekarski, N. (2019). Thirty years of Kawasaki Disease: A single-center study at the University Hospital of Lausanne. Frontiers in Pediatrics, 7, 11. https://doi.org/10.3389/fped.2019.00011 Green, A. (2020). Tomisaku Kawasaki. The Lancet, 396(10244). https://doi.org/10.1016/S0140-6736(20)31492-6 Horal, M., Michel, H., Doring, S., et al. (2021). Value of serial echocardiography in diagnosing Kawasaki's disease. European Journal of Pediatrics, 180(2), 387-395. https://doi.org/10.1007/s00431-020-03752-y John, R.M & Brady, M.A. (2020). Atopic, rheumatic, and immunodeficiency disorders. In D. Maaks, N. Starr, M. Brady, N. Gaylord, N. Driessnack, & K. Duderstadt (Eds.), Burns’ Pediatric Primary Care (pp. 560-561). Mosby. Kim L, Whitaker M, O'Halloran A, et al. (2020). Hospitalization rates and characteristics of children aged <18 years hospitalized with laboratory-confirmed COVID-19 — COVID-NET, 14 States, March 1–July 25, 2020. Morbidity and Mortality Weekly Report, 69:1081–1088. http://dx.doi.org/10.15585/mmwr.mm6932e3 Sakulchit, T., Benseler, S.M. & Goldman, R.D. (2017). Acetylsalicylic acid for children with Kawasaki disease. Canadian Family Physician, 63(8), 607-609. Urbane, U. N., Likopa, Z., Gardovska, D., & Pavare, J. (2019). Beliefs, practices and health care seeking behavior of parents regarding fever in children. Medicina (Kaunas, Lithuania), 55(7), 398. https://doi.org/10.3390/medicina55070398 Waseem, M., Shariff, M. A., Lim, C. A., Nunez, J., Narayanan, N., Patel, K., & Tay, E. T. (2022). Multisystem Inflammatory Syndrome in Children. The Western Journal of Emergency Medicine, 23(4), 505–513. https://doi.org/10.5811/westjem.2022.3.55325 Declaration of Competing Interest No conflicts of interest to declare.	0	PMC9712069	NO-CC CODE	2022-12-10 23:15:25	no	J Pediatr Health Care. 2022 Dec 1; doi: 10.1016/j.pedhc.2022.11.013	utf-8	J Pediatr Health Care	2,022	10.1016/j.pedhc.2022.11.013	oa_other
==== Front Int Dent J Int Dent J International Dental Journal 0020-6539 1875-595X The Authors. Published by Elsevier Inc. on behalf of FDI World Dental Federation. S0020-6539(22)00275-1 10.1016/j.identj.2022.11.019 Scientific Research Report Association of peri-implant health status with corona virus disease-19 (COVID-19) AlAhmari Fatemah 1⁎ Preethanath Reghunathan S 2 Divakar Darshan Devang 34 Ali Dena 5 1 Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia 2 Department of Preventive Dental Sciences, College of Dentistry, Jazan University, P.O Box 114, Jazan 45142, Saudi Arabia 3 Department of Oral Medicine and Radiology, Sharavathi Dental College and Hospital, Shivamogga, 577204, Karnataka, India 4 Department of Oral Medicine and Radiology, Faculty of Dentistry, Levy Mwanawasa Medical University (LMMU), Ministry of Health, Lusaka 10101, Zambia 5 Department of General Dental Practice, Faculty of Dentistry, Kuwait University, P. O. Box 24923, Safat 13110, Kuwait ⁎ Correspondence: Dr. Fatemah AlAhmari. Department of Periodontics and Community Dentistry, College of Dentistry, King Saud University, Riyadh, Saudi Arabia 1 12 2022 1 12 2022 24 9 2022 22 11 2022 26 11 2022 © 2022 The Authors. Published by Elsevier Inc. on behalf of FDI World Dental Federation. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objective To evaluate peri-implant health status in patients infected with corona-virus-disease-19 (COVID-19) and controls (individuals without COVID-19). Methods This observational investigation was performed in adults with (test group) and without (control group) COVID-19 infection having at least one functional dental implant. Self-reported education status and daily frequency of toothbrushing and flossing was determined. A questionnaire was administered to record demographic data, brushing/flossing frequency and education status. Periodontal examination comprised of full-mouth assessment of clinical attachment loss (CAL), plaque index (PI), probing depth (PD), and gingival index (GI). Peri-implant PD, modified PI (mPI), and modified gingival index (mGI) was recorded. Loss of marginal and crestal bone around teeth and implants was also measured. Significance-level was established for P<0.05. Results Seventy-three (41 males) and 71 (44 males) individuals were included in the test and control-groups, respectively. Average age of patients and controls were 44.6±5.2 and 40.1±3.1 years, respectively. University-level education was attained by 52 (75.3%) and 50 (70.4%) individuals in test and control groups, respectively. Seventy (95.9%) and 68 (95.7%) patients and controls were brushing twice a day. Once daily interproximal flossing was reported by 44 (60.3%) and 48 (67.6%) individuals in test- and control-groups. All participants had healthy periodontal and peri-implant tissues. Conclusion The study indicates that there are no short-term adverse effects on the peri implant tissues due to acute COVID-19 infection, and further well controlled longitudinal studies are needed to evaluate the long terms effect of the infection on these tissues. Keywords Coronavirus disease COVID-19 Clinical attachment loss Dental implant Probing depth ==== Body pmcIntroduction The corona-virus-disease-19 (COVID-19) is an infectious illness of the respiratory system caused by novel coronavirus, severe acute respiratory syndrome Coronavirus-2 (SARS CoV 2).1 The virus originated from Wuhan, China in December 2019 and rapidly invaded over 216 countries infecting numerous individuals.1 Initially, high mortality rates were observed in patients infected with SARS CoV 2 particularly in immunocompromised patients, such as those with diabetes mellitus (DM) and cardiovascular diseases (CVD).2 , 3 Common symptoms of COVID-19 comprise fatigue, respiratory fatigue, distress, nasal-congestion and runny-nose, anosmia, hypogeusia or dysgeusia, diarrhea, nausea/vomiting, ocular symptoms and abdominal pain.4 A limited number of studies5 , 6 have shown that COVID-19 is linked with periodontitis. According to Anand et al.5 odds ratios for gingival and plaque indices (GI and PI, respectively), probing depth (PD) ≥4 mm and clinical attachment loss (CAL) ≥3mm are higher among patients with COVID-19 (test-group) compared with individuals who are not infected with COVID-19 (control-group). Results from a recent questionnaire-based investigation also showed that gingivitis and halitosis are commonly manifested in patients with COVID-19 than controls.7 Dental implants are fixed appliances that are often used to restore mastication and orofacial esthetics.8 , 9 Presence of pathogenic bacteria in subgingival oral biofilm (OB) is often linked with the etiopathogenesis of peri-implant diseases;10 however, studies11, 12, 13 have shown that subgingival viral load can also play a role in this regard. There is dearth of clinical investigations that have assessed the influence of COVID-19 on peri-implant tissues; however, it has been proposed that COVID-19 may negatively affect osseointegration by depleting angiotensin-converting-enzyme-2 (ACE2) expressed in osteoblasts, osteoclasts and epithelial cells of respiratory system.12 , 14 , 15 According to Block MS15 implant failures are more often manifested in patients with COVID-19 than their corresponding controls. Between March and December 2022, the author encountered implant failures after placement in five patients and 80% of these were in patients with COVID-19.15 It is therefore hypothesized that peri-implant soft tissue inflammatory parameters (modified plaque index [mPI], modified gingival index [mGI], and PD) are poorer in patients with COVID-19 than controls. Moreover, it is also speculated that crestal bone loss (CBL) is increased in patients infected with COVID-19 than controls. The aim was to investigate the peri-implant health in patients infected with COVID-19 and controls (individuals without COVID-19). Materials and methods Ethical approval The study was done in accordance with the guidelines of the Declaration of Helsinki as revised in the year 2013. Ethics council of the Sharavathi Dental College and Hospital, Karnataka, India reviewed and approved the study protocol. Participation was voluntary and signing a consent form was mandatory. Withdrawal did not bear consequences in any form. Study design and location A case-control observational study was conducted between September 2020 and January 2021 at the Sharavathi Dental College, Karnataka India. Potential participants were recruited from the COVID-19 clinics set at outpatient department of the Sharavathi Dental College, Karnataka, India. Groups “Test-group” comprised of patients who had tested positive for COVID-19 infection at the day of their recruitment in the present study. “Control-group” encompassed individuals who tested negative for COVID-19 infection. Identification of COVID-19 was done via real time reverse transcription polymerase chain reaction (RT-PCR). Eligibility criteria Inclusion criteria: (a) individuals at least 18 years old; (b) Patients diagnosed with COVID-19 with RT-PCR (test-group); (c) individuals that were negative to COVID-19 testing via RT-PCR (control-group); (d) individuals with at least one dental implant in function in either jaw. Individuals were excluded in case they (a) were using nicotinic products in any form; (b) if their medical records showed previous diagnosis of systemic diseases such as DM, CVD, renal diseases, oral or/and systemic cancer, hepatic disorders and other viral infections such as HIV/AIDS; (e) were pregnant and/or nursing; (f) pharmaceutical prescriptions such as antibiotics, steroids, anti-inflammatory medications and/or bisphosphonates within the past 60 days and (g) implants supporting complex rehabilitations such as hybrid dentures and bridges. Severely traumatized teeth were considered missing. Unloaded implants and supernumerary, grossly carious and wisdom teeth were not assessed. Periodontal and peri-implant health Individuals with mean PD and CAL of < 3mm and less than 10% sites with gingival bleeding were categorized as having a healthy periodontal status.16 , 17 Individuals that presented with no clinical signs of peri-implant inflammation (such as gingival redness, bleeding and/or suppuration) and had a mean CBL < 2mm were classified as having a healthy peri-implant status.17 , 18 Questionnaire and dental records A questionnaire gathered the following data: (a) age, (b) gender, (c) education status, (d) daily toothbrushing frequency, (e) daily interproximal flossing frequency, (f) visit dentist and/or dental hygienist. Individuals that reported to have graduated from a school (up to grade 10), college (two additional years of education after school) or university (attending and graduation from a university) as their highest level of attained education were categorized as having “school-level”, “college-level” or “university-level” education, correspondingly.19 Dental records of patients were assessed by one investigator and following data was retrieved: (a) number of implants, (b) jaw location of implant, (c) duration in function, (d) loading protocol (immediate, early or delayed), (e) depth of placement (crestal or subcrestal), (f) implant diameter and length, (g) surface characteristics, (h) implant abutment connection (platform-switching) and (i) prosthesis retention (cement or screw retention). All participants were clinically assessed for symptoms of COVID-19 (such as fever, cough, fatigue, shortness of breath and loss of smell and taste);20 and information pertaining to immunization was retrieved via medical records. Clinical and radiographic investigations Periodontal examination comprising of PI21, GI22, CAL23; and PD24 was performed on all teeth by one investigator (kappa score 0.85). Peri-implant clinical investigations comprising of modified PI (mPI) and modified gingival index (mGI) were carried out by one examiner (Kappa score 0.88). A plastic and graded probe was used to record periodontal and peri-implant PD (Hu-Friedly, Chicago, IL, USA). Using long-cone paralleling technique25 , 26 digital bitewing x-rays were taken by a calibrated investigator (Kappa score 0.88) who also measured CBL on mesial and distal surfaces of implants and marginal bone loss (MBL) on mesial and distal surfaces of teeth. The MBL and CBL were gauged as the vertical distance extending from 2mm below the cemento-enamel junction and implant-abutment connection to alveolar crest.27 , 28 Sample-size estimation Power analysis (PA) or prior sample-size estimation was done on data from a pilot investigation in which, 15 and 15 individuals were included in the test- and control-groups. A computer program (nQuery Advisor/5; Statistical-Solutions, Saugus. MA, USA) was used for PA. It was estimated that in order to attain a power of 88% and detect a 3- and 3-mm difference in CAL and peri-implant PD in the test and control-groups, respectively, at least 69 participants would be needed in each arm (alpha error 5%). Statistical analyses Statistical comparisons for periodontal and peri-implant parameters were done using Student's t-test (IBM Corporation, SPSS, version 20, Armonk, NY, USA) using an alpha error of 0.05. The variables were expressed in terms of mean and standard deviation. Correlation between periodontal and peri-implant parameters and age, gender, duration of implants in function and jaw location was assessed using the Pearson's correlation coefficient and multivariate logistic regression. A probability value < 0.05 was ranked “statistically significant”. Results Patient recruitment Of the 208 individuals initially invited, 64 (41 males and 23 females) declined participation. Refusal to signing the consent form was the sole reason for exclusion of these individuals. All individuals in the test-group displayed signs and symptomatic of COVID-19 infection and were diagnosed at the day of their recruitment into the present investigation. As per medical records, none of the participants in the test- and control groups had been vaccinated against COVID-19 prior to inclusion in the present study. Collectively, 144 individuals (73 and 71 in test and control groups, respectively) agreed to sign the consent form and were included in the present study. In the test and control groups, 41 (56.2%) and 44 (62%) individuals, respectively were males. The mean age of individuals in the test and control group was 44.6 ± 5.2 and 40.1 ± 3.1 years, respectively. In the test and control-group 75.3% and 70.4% patients, respectively reported to have graduated from a university. Toothbrushing twice daily was reported by 70 (95.9%) and 68 (95.7%) individuals in test- and control-groups, respectively. Once daily interproximal flossing was reported by 44 (60.3%) and 48 (67.6%) individuals in test- and control-groups. In both groups, patients reported to have had dental prophylaxis done within the past 24 weeks (Table 1 ).Table 1 The study groups Table 1Parameters Test-group Control-group Patients 73 71 Males and females 41 males and 32 females 44 males and 27 females Mean age 44.6 ± 5.2 years 40.1 ± 3.1 years University level education 52 (75.3%) 50 (70.4%) College level education 21 (24.7%) 21 (29.6%) Toothbrushing twice daily 70 (95.9%) 68 (95.7%) Flossing once daily 44 (60.3%) 48 (67.6%) Last visit to dentist/hygienist 4.05 ± 0.6 months 5.1 ± 0.7 months Implants and related inflammatory parameters In test and control groups, implants were functional since 4.8 ± 0.5 and 4.5 ± 0.4 years, respectively. All implants were single-unit, and were placed at bone level in healed non-grafted sites; and had lengths and diameters ranging between 4.1 and 4.8 mm and 11 and 14 mm, correspondingly. Location of implants in the jaws is shown in Table 2 . There was no significant difference in MBL PD, PI, GI, and CAL among patients in both groups. There was no significant difference in mPI, mGI, peri-implant PD and CBL in both groups (Table 3 ).Table 2 Implant positioning Table 2Parameters Test-group Control-group Implants (n) 91 97 Jaw location Posterior maxilla (%) 42 (46.2%) 40 (41,2%) Posterior mandible (%) 40 (43.9%) 44 (45.4%) Anterior maxilla (%) 6 (6.6%) 8 (8.2%) Anterior mandible (%) 3 (3.3%) 5 (5.3%) Prosthesis retention Cement-retained 51 (56%) 55 (56.7%) Screw-retained 40 (44%) 42 (43.3%) Duration in function 4.8 ± 0.5 years 4.5 ± 0.4 years Posterior region: implants located in regions of missing premolars and/or molars Anterior region: implants located in regions of missing incisors and/or canines Table 3 Periodontal and peri-implant status Table 3Parameters Periodontal status Peri-implant status Test-group Control-group Test-group Control-group Plaque index 0.3 ± 0.007* 0.2 ± 0.0002 0.3 ± 0.001‖ 0.2 ± 0.002 Gingival index 0.2 ± 0.003† 0.2 ± 0.001 0.2 ± 0.003¶ 0.3 ± 0.002 Clinical attachment loss 0.3 ± 0.02 mm‡ 0.2 ± 0.03 mm NA NA Probing depth 1.4 ± 0.2 mm§ 1.2 ± 0.07 mm 1.1 ± 0.04 mm 0.9 ± 0.02 mm Marginal bone loss (mesial surface) 0.5 ± 0.05 mm 0.4 ± 0.04 mm NA NA Marginal bone loss (distal surface) 0.4 ± 0.03 mm 0.4 ± 0.04 mm NA NA Crestal bone loss (mesial surface) NA NA 0.2 ± 0.001 mm# 0.2 ± 0.001 mm Crestal bone loss (distal surface) NA NA 0.1 ± 0.002 mm⁎⁎ 0.2 ± 0.002 mm NA: Not applicable mm: millimeters ⁎ Compared with periodontal status in the control-group (P>0.05) † Compared with periodontal status in the control-group (P>0.05) ‡ Compared with periodontal status in the control-group (P>0.05) § Compared with periodontal status in the control-group (P>0.05) ‖ Compared with peri-implant status in the control-group (P>0.05) ¶ Compared with peri-implant status in the control-group (P>0.05) # Compared with peri-implant status in the control-group (P>0.05) ⁎⁎ Compared with peri-implant status in the control-group (P>0.05) Logistic regression analysis No correlation existed between peri-implant and periodontal clinicoradiographic parameters and age, gender, ES, implant jaw location, prosthesis retention and duration of implants in function in both groups (data not shown). Discussion The precise pathogenesis of oral diseases (periodontitis and peri-implant diseases) remains unclear; however, preliminary data has shown that the SARS-CoV-2 uses it spike protein (trans-membrane serine protease) to bind to CD147 to infect human cells including those of the oral mucosa.29 , 30 Moreover, recently Gupta et al.31 identified the SARS-CoV-2 in the gingival crevicular fluid (GCF) which, increases the likelihood of periodontal diseases. Based on the results by Gupta et al.31 it is hypothesized the peri-implant sulcular fluid also harbors the SARS-CoV-2 in susceptible patients. To date, two case-control studies5 , 6 have shown that periodontal inflammation is more often manifested in patients that are positive for COVID-19 than controls (individuals without COVID-19). Based on the limited available evidence, authors of the present investigation hypothesized that peri-implant diseases, peri-implant mucositis and peri-implantitis, occur more often in patients that are positive for COVID-19 than controls. Surprisingly, current results showed that all patients in test and control groups had healthy periodontal and peri-implant tissue statues as defined in the section of materials and methods. Moreover, no differences in periodontal and peri-implant clinical and radiographic parameters were observed at that point in time. This is contradictory to results by Anand et al.5 and Marouf et al.6; however, there are a number of factors that need to be taken into consideration. In the study by Anand et al.5 oral hygiene maintenance (OHM) was being performed twice daily by nearly 14% and 44% individuals in the test and control-groups, respectively. In the study by Marouf et al.6, domestic OHM protocols remained uninvestigated. In contrast, in the present investigation at least 95% of the study population reported that they were brushing two times a day. Moreover, data pertaining to daily flossing (DF) was not reported by Anand et al.5 We gathered self-reported information pertaining to DF and as per our results, at least 60% of the population under investigation was performing interproximal flossing 1x a day. Furthermore, in the present case-control study we asked participants about their most recent visit to an oral healthcare provider. It is plausible that all participants had visited a dental facility for prophylaxis-related treatment within the past six months. Studies28 , 32 have confirmed that an underprivileged education and socioeconomic status is a risk factor for periodontal inflammatory diseases such as periodontitis. In the present study, at least 70% participants in test and control groups reported that they had graduated from a university. These results demonstrate that the population under investigation was health literate, were possibly from high-income strata and were taking adequate measures to maintain a stable and healthy OHS. The authors would however like to clarify that there were no stringent criteria to induce only well-education individuals belonging to high-income strata in the present study. It is therefore speculated that the severity of periodontal and peri-implant diseases are worse in financially underprivileged and poorly educated individuals with COVID-19 compared with their respective counterparts. Further studies are needed to test this hypothesis. Anand et al.5 investigated the relationship between periodontitis and poor oral hygiene and COVID-19. From the authors perspective, this statement is perplexing as poor OHS is an independent risk-factor of oral inflammatory conditions including periodontitis and peri-implant diseases.25 , 28 , 33 , 34 Moreover, habitual use of nicotinic products such as cigarettes, CVD and impaired glycemic levels (often manifested in patients with DM) are classical risk factors of periodontal and peri-implant diseases.25 , 35, 36, 37 Interestingly, in the study by Anand et al.5 tobacco-smokers and patients with DM were also included.26 , 38, 39, 40 The mere clarification that Anand et al.5 provided for including smokers and immunosuppressed individuals in the study population was “these risk factors were not significantly different between the groups”. However, the authors of the present study suggest that data should be adjusted for potential risk-factors prior to subjecting it to quantitative and/or qualitative evaluation. In this context, we excluded self-reported tobacco-smokers and patients with systemic diseases from our patient population. Thus, it is indistinct whether periodontal inflammatory signs in the study by Anand et al.5 were directly linked with COVID-19, or as a consequence of tobacco-product usage or systemic health status (DM). The authors of the current study suggest that OHS, social history and systemic health of patients with and without COVID-19 should also be taken into consideration as potential factors infuriating an inflammatory response in susceptible populations. In a recent systematic review Qi et al.41 proposed that maintenance of periodontal health and oral hygiene is essential for patients infected with COVID-19. The authors of the present study support this statement as none of the individuals in the test- and control-groups displayed signs and symptoms of periodontal and peri-implant diseases. These individuals were stringently maintaining OHS and were routinely visiting their oral healthcare providers. Therefore, we propose that routine OHM not only helps minimize the risk of periodontal diseases but also minimizes the risk of peri-implant diseases in patients infected with COVID-19 and controls. Anand et al.5 stated “it is essential to maintain periodontal health and good oral hygiene as an important measure for COVID-19 prevention”. The authors perceive that the statement by Anand et al.5 be cautiously interpreted as to date, there is no indexed scientific evidence that has confirmed that maintenance of oral hygiene and periodontal status helps “prevent” COVID-19. A limitation of the present study is that microbiological assessment such as identification of pathogenic microbes in subgingival OB was not performed. Moreover, other experimental investigations including evaluation of inflammatory cytokines in biological fluids (BF) such as GCF, whole saliva, serum and peri-implant sulcular fluid. Further studies are needed to assess the impact of COVID-19 infection on subgingival microbiota and cytokine profile in BF. It is also worth mentioning that the primary objective of collecting information pertaining to implant surgery and prosthesis retention was to assure that standardized protocols were using by providers who had performed implant therapy to minimize the potential risk of bias in relation to peri-implant clinicoradiographic parameters. Nevertheless, the results showed no correlation between implant jaw location, prosthesis retention and duration of implants in function in relation to peri-implant parameters in the population under investigation. Furthermore, based on the currently available limited evidence it is demanding to estimate a minimum time frame in which, COVID-19 could potentially spark signs and symptoms of periodontal/peri-implant inflammation. This warrants additional longitudinal studies. The authors believe that a thorough understanding of all probable manifestations, including oral diseases, in patients with COVID-19 is critical as new, viral variants are presently emerging with mysterious future impacts. Conclusion The study indicates that there are no short-term adverse effects on the peri implant tissues due to acute COVID-19 infection, and further well controlled longitudinal studies are needed to evaluate the long terms effect of the infection on these tissues. AUTHOR CONTRIBUTIONS Fatemah AlAhmari and Darshan Devang Divakar designed the study; Darshan Devang Divakar performed the clinical and statistical analyses; Reghunathan S Preethanath performed the radiologic investigations and wrote the results; Fatemah AlAhmari, Dena Ali and Darshan Devang Divakar interpreted the results. Fatemah AlAhmari, Reghunathan S Preethanath, Dena Ali and Darshan Devang Divakar drafted the manuscript and revised it prior to submission. CONFLICT OF INTEREST AND FINANCIAL DISCLOSURE The authors declare that they have no conflict of interest; and there was no external source of funding for the present study. ACKNOWLEDGEMENT The authors thank the Deanship of Scientific Research at King Saud University for supporting this research project. 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==== Front Technol Forecast Soc Change Technol Forecast Soc Change Technological Forecasting and Social Change 0040-1625 0040-1625 Elsevier Inc. S0040-1625(22)00768-5 10.1016/j.techfore.2022.122247 122247 Article Innovation in manufacturing SMEs during the COVID-19 pandemic: How does environmental dynamism reinforce employee proactive behavior? Huang Yi-Fen a⁎ Lin Hung-Chun b Lee Hsu-Mei c a Department of Business Administration, Dayeh University, 168 University Road, Dacun, Changhua, Taiwan, ROC b College of Management, Dayeh University, 168 University Road, Dacun, Changhua, Taiwan, ROC c Department of International Business Management, Dayeh University, 168 University Road, Dacun, Changhua, Taiwan, ROC ⁎ Corresponding author. 1 12 2022 1 12 2022 1222471 2 2022 19 11 2022 27 11 2022 © 2022 Elsevier Inc. All rights reserved. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. In a turbulent environment such as during the COVID-19 pandemic crisis, employee proactive behavior is imperative for innovation initiatives in small- and medium-sized enterprises (SMEs). We ask whether and how turbulent environments motivate employees to proactively engage in innovative behavior. This study argues that employees' perceptions of environmental dynamism reinforce employee proactive innovation behavior. Using a sample comprising 262 innovative employees from 40 manufacturing SMEs in Taiwan, this study tests a moderated-mediation model in which environmental dynamism is expected to increase the indirect effect of creative self-efficacy on employee innovative behavior through knowledge acquisition. The results confirm the mediating role of knowledge acquisition and the positive moderating effect of environmental dynamism. This study sheds light on the issue of employee proactive behavior in response to changing environments. Keywords Creative self-efficacy Knowledge acquisition Environmental dynamism Employee innovative behavior ==== Body pmc1 Introduction The COVID-19 pandemic disrupted the supply chains of the manufacturing sector on a large scale. Small- and medium-sized enterprises (SMEs) were the firms most affected, as they struggled with the short supply of materials and parts, logistics setbacks, and demand fluctuations (Cai and Luo, 2020). As suppliers and partners, manufacturing SMEs are deeply embedded in an industrial system that is conditioned by the market (Ahmed et al., 2022). Creativity and innovation are vital abilities SMEs must have in order to pivot and change in response to uncertain situations (Ratten, 2020). There must also be close interactions among the different supply chain partners to ensure resilience (Thukral, 2021). Consequently, SME employees face the challenge of helping their firm achieve resilience through creative collaboration in the industrial ecosystem. The issue of employees' responses to change has been debated by theories and research on traits (Bateman and Crant, 1993; Mubarak et al., 2021), behavior (Åmo and Kolvereid, 2005; Parker et al., 2010), and change management (Caldwell and Liu, 2011; Caldwell, 2013). However, the investigations on how employees deal with problems are mostly limited to the context of organizational planned changes (e.g., Caldwell, 2013; Lee et al., 2019). Research on investigating how employees react to external unexpected turbulence still needs more attention. Recently, a few studies have tried to understand how environmental dynamism shapes human behavior in organizations (i.e., Surty and Scheepers, 2020; Lin, 2021). Surty and Scheepers (2020) found that environmental dynamism has a slight significant strengthening effect on the relationship between leadership practices and employee response to change. Lin (2021) found ethical leadership influences team initiatives in highly dynamic work environments. While these studies confirm the moderating role of environmental dynamism on employee behavioral change, they focus on team-level antecedents (i.e., leadership) rather than individual elements. Employee proactivity, defined as the self-starting and change-oriented action of employees in organizations (Parker et al., 2010; Grant and Ashford, 2008), has been identified as a potential driver of workplace innovation (Lee et al., 2019). Innovative work in manufacturing SMEs mainly relies on employees in technical teams (Hervas-Oliver et al., 2021), because they are on the front line of production and have intimate knowledge of process inefficiencies (Unsworth and Parker, 2003). Such knowledge enables them to identify areas of development and perform innovative behavior at work (Unsworth and Parker, 2003). Scholars have recognized that proactive behavior is a consequence of individual motivation in a particular context (Parker et al., 2010). Motivation factors that represent individual psychological states such as role breadth self-efficacy (Parker, 1998; Tierney and Farmer, 2002), felt responsibility for change (Morrison and Phelps, 1999), and change readiness (Rusly et al., 2015), have been demonstrated as antecedents of employee proactive behavior. Following this logic, we focus on creative self-efficacy, which is employees' belief in their own creative ability, and argue that this factor triggers employee proactive learning behavior toward innovation. Unlike large companies that gain a competitive advantage from cost efficiencies, manufacturing SMEs generally focus on differentiation strategies such as diversified product offerings and flexible customer responses (Terziovski, 2010). SMEs also encounter various obstacles to innovation, such as insufficient financial capital, limited management skills, and a lack of technical information and know-how (Ferreira et al., 2014; Thukral, 2021). Based on their strategic orientation and capability constraints, SMEs rarely innovate by themselves; instead, they rely on external sources to strengthen their internal innovation (Lin et al., 2016). The nature of embeddedness of manufacturing SMEs highlights the critical role of collaboration and information exchange activities within a supply chain network. Manufacturing SMEs' inability for solo innovation further impacts their adoption of open innovation and has led to the emerging introduction of supply chain digitalization when faced with turbulence (Madhavan et al., 2022). For example, the turbulence caused by the recent pandemic accelerated SMEs' transference to manufacturing systems that are automated, autonomous, and intelligent (Cai and Luo, 2020). The progress arising from digital transformation creates better connectivity as well as effective communication among supply chain partners (Ahmed et al., 2022). Accordingly, engaging in knowledge acquisition might be a key mechanism through which employees of manufacturing SMEs gain innovative capabilities. As stated above, whether and how environmental dynamism stimulates employees' proactive behavior to initiate innovation has become a critical question. Our contribution stems from the examination of this issue. This study investigates two personal capabilities of employees, i.e., creative self-efficacy and knowledge acquisition, and aims to discover how these two factors influence employee innovative behavior in response to external turbulence. Accordingly, this study draws on social cognitive theory to apply a theoretical framework to explain individual behavior based on interactions with personal factors as well as the external environment. By additionally including the employee proactivity perspective, this study argues that employees in manufacturing SMEs who have higher creative self-efficacy are more likely to embark on collaborative knowledge acquisition and consequently perform more innovative behavior. That is, knowledge acquisition mediates the impact of creative self-efficacy on employee innovative behavior. We also propose that this mediation relationship will be reinforced by the level of dynamism that exists in the environment. The proposed model is depicted in Fig. 1 .Fig. 1 Research framework. Fig. 1 Our research sample consists of 262 employees from 40 manufacturing SMEs in Taiwan. The empirical results show overall support for our predictions. The present study sheds light on the determinants and mechanism of employees' responses to changing environments and makes the following contributions. First, the confirmed mediating relationship reveals that knowledge acquisition is a necessary mechanism by which employees in manufacturing SMEs access their innovation capabilities. This finding is unique because we consider the collaborative nature of innovation work in manufacturing SMEs, thereby deriving a suggestion for managers to build a workplace that encourages and supports interfirm interpersonal knowledge acquisition. Second, the significant positive moderating effect of environmental dynamism indicates that highly creative self-efficacy employees have a greater tendency to respond to external dynamism and initiate innovative learning activities. We believe this is a pioneer finding that provides novel knowledge about the moderating role of environmental dynamism on proactive behavior at the individual level. Third, this research focuses on the innovative staff in manufacturing SMEs and explores their behavior in a workplace that is influenced by the complex changes at both the industrial and macro levels. Our research setting thus provides an insightful viewpoint that past studies have rarely investigated. 2 Literature review and hypotheses development 2.1 Social cognitive theory Social cognitive theory, proposed by Bandura (1986), provides a theoretical framework to identify individual behavior based on a triadic structure between cognitive factors, environmental factors, and human behavior. The theory considers both internal and external influential factors in human actions by embracing the cognitive process of self-regulation as well as recognizing the importance of environmental determinants corresponding to behavior (Cai et al., 2022). According to the theory, cognitive factors affect individual behavior while social and environmental factors alter human beliefs (Kim and Chai, 2022). In this current study, we consider creative self-efficacy as a cognitive factor that influences the innovative behavior of employees. Environmental dynamism is the external determinant affecting people's beliefs and actions. We further propose that knowledge acquisition, which represents a collaborative learning effort, is a necessary action and vehicle for employees to learn to innovate in the context of manufacturing SMEs. 2.2 Creative self-efficacy and innovative behavior Creative self-efficacy refers to a person's self-judgment of their capacity to pursue a creative goal (Tierney and Farmer, 2002), which is derived from the self-efficacy theory of behavioral change proposed by Bandura (1977). Self-efficacy is defined as the “beliefs in one's capabilities to organize and execute the courses of action required to produce given attainments” (Bandura, 1997, p. 3). In the research line on employee proactivity, scholars have demonstrated that employees who feel capable of performing tasks are more likely to present self-starting behavior to deal with change (Parker, 1998; Fuller et al., 2018; Grosser et al., 2017). Employee proactivity has been found to predict several individual outcomes, including creativity and innovative behavior (Zhang et al., 2012), as well as to be vital to the promotion of innovative change (Lee et al., 2019). Innovative behavior is defined as “an initiative from employees concerning the introduction of new processes, new products, new markets or combinations of such into the organization” (Åmo and Kolvereid, 2005, p. 8). Innovation at the individual level begins with identifying a problem and coming up with a solution (Dhar, 2016). Such behavior consists of generating ideas (i.e., creativity), seeking support (e.g., knowledge acquisition), and implementing ideas (i.e., new product or process) (Hoang et al., 2022). Many studies have examined multi-level factors that influence the innovative behavior of employees, for example, personal traits (Ng and Lucianetti, 2016) and demand (Kwon and Kim, 2020), team-level leadership style, and organizational climate (James et al., 2008; Martins and Terblanche, 2003; Dhar, 2016). Based on the claims of the employee proactivity perspective and social cognitive theory, human behavior is influenced by the individual's internal cognitive state and external environmental factors. We therefore focus on the motivation factor of individual-level cognitive belief, which is creative self-efficacy. Through the lens of social cognitive theory, scholars have suggested that elevated self-efficacy leads to the sustaining of effort that is linked to innovative behavior (Tierney and Farmer, 2002). Based on the concept that self-efficacy is a cognitive condition that influences human behavior (Bandura, 1977), individuals' perceptions of the essential knowledge, skill, and ability required for specific creative performance are key motivating factors of their behavior in idea generation, dissemination, and implementation (Tierney and Farmer, 2002; Fino and Sun, 2022). As stated above, self-efficacy is a necessary condition for the discovery of new knowledge and creative productivity (Tierney and Farmer, 2002). The literature on creative self-efficacy has demonstrated its significant association with employees' creativity (Tierney and Farmer, 2002; Al Wali et al., 2022). Previous evidence has shown that employees possessing high levels of creative self-efficacy derive satisfaction from innovative pursuits and exhibit greater extraordinary tenacity when encountering challenges inherent to innovational performance (Grosser et al., 2017; Newman et al., 2018; Tierney and Farmer, 2011). 2.3 The mediating role of knowledge acquisition Knowledge acquisition refers to an employee's ability to identify and acquire new knowledge from inside the organization or from external sources (Jiang and Chen, 2018). Acquiring knowledge is a purposeful process that has become an increasingly important strategy for individuals seeking to improve their innovation capability. Searching within personal networks or industrial environments, creative employees can capture valuable knowledge and know-how (Perry-Smith and Shalley, 2003), find solutions to solve challenging tasks (Jiang and Chen, 2018), and inspire innovative outcomes (Xie et al., 2018). The association between knowledge acquisition and positive innovation performance is evidenced in the literature (Papa et al., 2018), both in the case of young firms and SMEs (Chuang et al., 2016; Naldi and Davidsson, 2014; Huang and Liu, 2019) and large companies (Norman, 2004). Regarding the antecedents of knowledge acquisition, existing studies have mostly focused on intrafirm and inter-firm factors such as human resource management practices (Papa et al., 2018), partner attributes (Norman, 2004), and firms' absorptive capacities (Van Wijk et al., 2008). Little attention has been paid to understanding what stimulates individuals' involvement in the knowledge acquisition process. Individuals represent key players because they are knowledge sources and recipients. As a result, it is reasonable to consider that their willingness and abilities might be critical in initiating collective knowledge activities. Following the logic of the social cognitive theory, understanding whether and how individual-level cognitive factors affect individuals' actions to acquire knowledge can be insightful (Rusly et al., 2015). Rusly et al. (2015) confirmed the impact of an individual's change readiness on knowledge acquisition. This result offers a hint that knowledge acquisition is a mechanism that leads change-oriented individuals toward innovative outcomes. Accordingly, this study argues that knowledge acquisition plays a mediating role between creative self-efficacy and employee innovative behavior. Small enterprises that are capable of adapting change are considered resilient (Kuckertz et al., 2020). Regularly, a manufacturing SME is seen in the context of an industrial supply chain. Hence, SME resilience, which is key for crisis survival, is argued to be derived from the resilience of an ecosystem (Thukral, 2021). For SMEs, innovation capability generally refers to quick responses to changes in clients' demands through flexibility and agility (Huang and Lu, 2020). This is mainly driven by employees in technical teams (Unsworth and Parker, 2003). Kim and Chai (2022) pointed out the influential role of employees' self-efficacy on the implementation of cooperation, coordination, and communication activities that can lead to supply chain resilience. This is because when creative employees feel capable of making constructive changes, they will search for requisite knowledge on their own accord. In addition, during the recent pandemic, many studies observed that SMEs tried to manage the dislocation of the supply chain using modern technology (Chatterjee et al., 2022). The crisis thereby accelerated a paradigm shift based on ongoing digitization and interconnection in the manufacturing sector (Chen, 2020). By applying technologies of big data analytics, the Internet of Things, digital platforms, and cloud computing, manufacturing supply chains evolved into tightly connected innovation ecosystems that help their members live together (Ahmed et al., 2022; Cenamor et al., 2019). Under such a scenario, knowledge acquisition by means of collaboration within and across company boundaries is essential for employees in manufacturing SMEs to pursue innovation. Collective teamwork activities enable employees to access tacit knowledge (Del Giudice et al., 2019), dialogue on complex issues, and learn from coaching (Hooijberg and Watkins, 2021). As a result, knowledge flows that stimulate the intellectual interest of employees are generated (Lai et al., 2015), which further influences employees' engagement in innovation. As such, this study suggests a mediating role of knowledge acquisition as below:Hypothesis 1 For employees in manufacturing SMEs, knowledge acquisition has a mediating effect on the relationship between creative self-efficacy and innovative behavior. 2.4 The moderating role of environmental dynamism Environmental dynamism refers to the level of unpredictability and instability in a firm's environment (Chan et al., 2016). It is commonly accepted as a profound force that can strongly influence not only organizational capabilities and innovation outcomes (Xiao et al., 2020) but the agility and resilience of the industrial open innovation system (Akgul, 2015). Although happening in external contexts, employees' perceptions of workplace dynamism reflect their inability to predict the direction in which their work may change (Waldman et al., 2001), where uncertainty may cause their behaviors to change (Surty and Scheepers, 2020). Therefore, it is necessary to further explore how environmental dynamism influences employees' actions of searching for and developing solutions in response to an unstable work environment. Most extant literature has applied environmental dynamism as a moderator that needs to be considered in organizational contingencies (Zhang and Zhu, 2021; Ahmed et al., 2022; Do et al., 2022). Limited insight has been provided regarding human aspects in the context of a highly dynamic environment. Ahmed et al. (2022) asked the question of how a changing environment affects a firm's obtainment of intellectual capital, including human capital. The results support their prediction of the negative moderating effect of environmental dynamism on a firm's capability and human capital. Although this work did not link to employee behavior, the results imply that unpredictable conditions can change people's choices in work. Another research, by Lin (2021), focused on team-level proactive actions and confirmed the moderating role of environmental dynamism on the relationship between leadership and team initiative, where proactive initiatives were found to be more likely to occur in highly dynamic environments. Regarding work behavior at the individual level, the study by Surty and Scheepers (2020) indicated that environmental dynamism has a strengthening effect on the relationship between leadership practices and employees' response to change. Their findings specifically show that environmental dynamism can encourage employees' change-oriented actions. Compared to large businesses, SMEs have been hardest hit by COVID-19. The manufacturing sector still suffers from supply chain disruption, demand has diminished, there is a raw materials shortage and a severe transportation disruption, and so on (Shafi et al., 2020). Most SMEs have been unable to adjust sufficiently to the unpredictable changes and have thus suffered losses (Ahmed et al., 2022). Such a highly turbulent event is expected to heighten employee awareness and stress in response to the changing environment (Shao et al., 2021; Steinbach et al., 2021; Zhong et al., 2021). In reaction, highly creative self-efficacy employees who feel capable of overcoming obstacles and uncertainties would start their actions of problem-solving (Tantawy et al., 2021). In the context of the manufacturing sector, the nature of external uncertainty is present in terms of fast-changing technologies, varying customer preferences, and fluctuating demand and supply of materials (Hou et al., 2019; Huang and Lu, 2020). In dealing with these problems employees in SMEs have been required to embark on collaborative teamwork across the supply chain. Therefore, for employees of manufacturing SMEs, innovation is a consequence of collaborative learning in an industrial network wherein knowledge is acquired, shared, and combined (Jiang and Chen, 2018). In brief, under highly volatile environments, risky situations push employees who believe in their creative ability to initiate a process of knowledge acquisition, where accessed knowledge further aids employees to engage in innovation activities. Considering the above arguments, our second hypothesis is:Hypothesis 2 The mediating effect of creative self-efficacy on innovative behavior through knowledge acquisition is stronger when employees in manufacturing SMEs sense greater environmental dynamism. 3 Methodology 3.1 Data collection and sample This study conducts a survey targeting innovation staff in Taiwan manufacturing SMEs due to three reasons. First, the economy in Taiwan is known as an SME-dominant structure (Lee and Jioe, 2017); specifically, SMEs account for 98 % of entrepreneurs and employ 71 % of the nation's workforce (Chen et al., 2021). In the manufacturing sector, most SMEs are key suppliers to large companies, responsible for much of the innovation and productivity in the business community (Wu and Chiu, 2016). Second, the manufacturing sector in Taiwan is featured by industrial clustering and is considered a business ecosystem. Small entrepreneurial companies usually form a network of center–satellite systems in which many center factories and cooperative factories cluster together (Chen et al., 2021). The operation of such interconnected systems aids technical knowledge spillover effects (Del Giudice et al., 2019). Third, manufacturing SMEs in Taiwan have invested in industry 4.0 and digitalization (Chen, 2020). Based on these reasons, we believe sampling from Taiwanese manufacturing SMEs is appropriate to test our arguments because these firms are highly embedded in the innovation system and urgently need to adapt to industry and market changes. As our research aims to investigate the innovative behavior of employees, we choose workers in the production and R&D divisions who are responsible for technological processes and product innovation in manufacturing SMEs. We further adopt the governmental definition of SMEs in Taiwan, i.e., enterprises with no more than 200 employees and paid-in capital of less than NT$100 million. We use the list of manufacturing SMEs provided by the Taiwan Ministry of Economic Affairs. We first randomly send 1000 invitations by email to the leaders of targeted divisions in companies on the list, along with a letter explanting the purpose of our study. This step receives 92 companies expressing interest in participating in the study. Next, a questionnaire is sent to these firms. To reduce the issue of potential common method variance, we follow previous studies and send questionaries in two waves within a two-week interval (Liu et al., 2017; Wu et al., 2010). The first wave, which includes 225 questionnaires sent to 45 willing companies, results in 138 responses, 125 of which are valid. The second wave, which includes 235 questionnaires sent to 47 companies, yields 137 valid responses out of the 158 questionnaires returned. Taken together, our survey obtains 262 valid responses from 40 manufacturing SMEs. Among the respondents, 52 % are male, the average age is 38.7, and 67.6 % hold a bachelor's degree or higher. 3.2 Measures This study uses the five-point Likert scale to measure all variables; in the scale, one represents strongly disagree and five represents strongly agree. Following Hou et al. (2019), we translate the English scales into Chinese. To verify the translation, a bilingual expert translates from English to Chinese and then back to English to ensure the quality of the conversation. The measurements of variables are explained below. 3.2.1 Employee innovative behavior The dependent variable is a self-reporting assessment based on the respondents' subjective perceptions of their actions within the workplace (Mitchell et al., 2021). A six-item scale adopted from Dhar (2016) is used: “At work, I come up with innovative and creative notions”; “At work, I try to propose my own creative ideas and convince others”; “At work, I seek new methods, or techniques”; “At work, I provide a suitable plan for developing new ideas”; “At work, I try to secure the funding and resources needed to implement innovations”; and “Overall, I consider myself a creative employee of my organization”. The Cronbach's alpha for the scale is 0.71. 3.2.2 Creative self-efficacy We measure this independent variable using a three-item scale developed by Tierney and Farmer (2002). The items are “I have confidence in my ability to solve problems creatively”; “I feel that I am good at generating novel ideas”; and “I have a knack for further developing the ideas of others”. The Cronbach's alpha for the scale is 0.84. 3.2.3 Knowledge acquisition To measure the knowledge acquisition ability of employees, we draw on Jiang and Chen's (2018) team knowledge acquisition scale. The four items are adapted as “I usually scan the environment inside and outside my organization for knowledge about the market”; “I usually scan the environment inside and outside my organization for technical knowledge”; “I usually seek ideas/expertise from people inside and outside my organization to perform tasks”; and “I usually seek feedback about my work from people outside my team and organization”. The Cronbach's alpha for the scale is 0.75. 3.2.4 Environmental dynamism The environmental dynamism measure reflects employees' subjective assessments of market change (Mitchell et al., 2021). This variable is measured using a five-item scale developed by Jansen et al. (2009). The items include “Recently, environmental changes in our local market are intense”; “Recently, our clients regularly ask for new products and services”; “Recently, changes are taking place continuously in our local market”; and “Recently, the volumes of products and services to be delivered change fast and often in our market”. The Cronbach's alpha for the scale is 0.71. 3.2.5 Control variables This study includes the employees' education, gender, and age as control variables. These variables could affect the employees' efficacy to initiate organizational change (Fuller et al., 2006) and could impact their work performance (Rofcanin et al., 2021). 3.3 Common method bias and non-response bias We test for the possibility of common method bias (CMB) by using Harmen's single-factor test. The total variance extracted by one factor is 22.32 %, which is less than the recommended threshold of 50 % (Podsakoff and Organ, 1986), indicating that CMB is not a major concern in this study. Furthermore, to control for errors in respondent selection, we evaluate non-response bias by testing for significant differences between the two waves of respondents. Performing t-tests on the variables of age and gender, the results indicate that early and late respondents have no significant differences. This suggests that there is no concern regarding non-response bias. 4 Results 4.1 Validity, reliability, and correlations Table 1 presents the scale reliability and validity analysis results. Regarding the reliability of scales, the values of composite reliability (CR) range from 0.77 to 0.92, which are larger than the threshold of 0.6, and the values of Cronbach's α range from 0.71 to 0.84, passing the α < 0.7 criteria (Hair et al., 2018). Next, convergent validity is determined using confirmatory factor analysis. The factor loading values of all the items range between 0.55 and 0.85, exceeding the acceptable threshold of 0.5. Furthermore, the average variance extraction (AVE) of all constructs is better than the AVE > 0.5 cutoff (Hair et al., 2018). In addition, considering evaluating discriminant validity, the square roots of the AVE of all the variables exceed the correlations between the focal variable and other variables in Table 2 ; thus, the discriminant validity of variables is supported. Overall, the results indicate that our constructs have acceptable validity and reliability.Table 1 Results of reliability and validity test. Table 1Constructs and items Loadings Creative self-efficacy (AVE = 0.53, CR = 0.77, α = 0.84) 1. I have confidence in my ability to solve problems creatively. 2. I feel that I am good at generating novel ideas. 3. I have a knack for further developing the ideas of others. 0.79 0.70 0.68 Knowledge acquisition (AVE = 0.61, CR = 0.86, α = 0.75) 1. I usually scan the environment inside and outside my organization for knowledge about the market. 2. I usually scan the environment inside and outside my organization for technical knowledge. 3. I usually seek ideas/expertise from people inside and outside my organization to perform tasks. 4. I usually seek feedback about my work from people outside my team and organization. 0.81 0.74 0.83 0.75 Environmental dynamism (AVE = 0.62, CR = 0.87, α = 0.71) 1. Recently, environmental changes in our local market are intense. 2. Recently, our clients regularly ask for new products and services. 3. Recently, changes are taking place continuously in our local market. 4. Recently, the volumes of products and services to be delivered change fast and often in our market. 0.81 0.77 0.79 0.78 Employee innovative behavior (AVE = 0.66, CR = 0.92, α = 0.71) 1. At work, I come up with innovative and creative notions. 2. At work, I try to propose my own creative ideas and convince others. 3. At work, I seek new techniques, methods, or techniques. 4. At work, I provide a suitable plan for developing new ideas. 5. At work, I try to secure the funding and resources needed to implement innovations. 6. Overall, I consider myself a creative employee of my organization. 0.55 0.84 0.85 0.85 0.94 0.79 Note: AVE = average variance extracted, CR = composite reliability, α = Cronbach's alpha. Table 2 Descriptive statistics and correlations. Table 2 Variables Mean S.D. 1 2 3 4 5 6 VIF 1 Employee innovative behavior 4.03 0.68 0.81 2 Creative self-efficacy 3.88 0.84 0.335 ⁎⁎⁎ 0.73 2.03 3 Knowledge acquisition 4.05 0.74 0.524 ⁎⁎⁎ 0.144 ⁎ 0.78 1.07 4 Environmental dynamism 4.10 0.74 0.203 ⁎⁎ 0.409 ⁎⁎⁎ 0.161 ⁎⁎ 0.79 1.24 5 Gender 1.48 0.50 −0.090 −0.667 ⁎⁎⁎ 0.025 −0.323 ⁎⁎⁎ 1.87 6 Age 38.70 5.74 −0.007 −0.090 0.003 0.042 0.045 1.02 7 Education 1.95 0.77 0.059 0.068 0.008 0.009 −0.066 0.010 1.01 Note: n = 262, Bold numbers on the diagonal line are the square root values of the AVE for each variable. ⁎⁎⁎ Significant at p < 0.001 level. ⁎⁎ Significant at p < 0.01 level. ⁎ Significant at p < 0.05 level. Table 2 provides the means, standard deviations, and correlations of the variables. We additionally conduct collinearity diagnostic tests on all variables. The results reveal that all the variance inflation factor (VIF) values range from 1.01 to 2.03, well below the standard cutoff of 10. Thus, multicollinearity is not a concern in our study. 4.2 Regression analysis Table 3 displays the results of the ordinary least squares regression analysis. This study employs a three-step regression to test the mediation effect (Baron and Kenny, 1986); Models 1–3 show the three steps. In Model 1, creative self-efficacy has a significant positive impact on innovative behavior (β = 0.50, p < 0.001). In Model 2, creative self-efficacy has a significant positive influence on knowledge acquisition (β = 0.29, p < 0.001). In Model 3, controlling for knowledge acquisition, the relationship between creative self-efficacy and innovative behavior remains positively significant (β = 0.34, p < 0.001). Regarding the influence of creative self-efficacy on innovative behavior, the direct effect in Model 1 is larger than the indirect effect in Model 3 (0.50 > 0.34). Taken together, the combination of Models 1–3 satisfies Baron and Kenny's (1986) mediator test. Furthermore, the Sobel test yields a value of 3.34, which exceeds the critical value of ±1.96, thus confirming knowledge acquisition is a mediator between creative self-efficacy and innovative behavior. The mediation effect predicted in Hypothesis 1 is thereby supported. In addition, we test the moderated mediation effect by using the first-stage conditional process model (Hayes and Rockwood, 2020). Results in Model 4 show the interaction term has a significant positive moderating effect (β = 0.44, p < 0.01), providing support for our prediction in Hypothesis 2.Table 3 Results of moderated mediation analysis. Table 3 Model 1 Model 2 Model 3 Model 4 Dependent variable→ Innovative behavior Knowledge acquisition Innovative behavior Innovative behavior Coef. SE Coef. SE Coef. SE Coef. SE (Constant) 1.79 ⁎⁎⁎ 0.48 2.47 ⁎⁎⁎ 0.55 1.62 ⁎⁎⁎ 0.35 1.89 ⁎⁎⁎ 0.46 Gender 0.24 ⁎⁎ 0.11 0.22 ⁎⁎ 0.12 0.06 0.07 0.03 0.07 Age 0.03 0.01 0.02 0.08 0.00 0.01 0.01 0.01 Education 0.04 0.05 0.00 0.06 0.02 0.03 0.03 0.03 Creative self-efficacy 0.50 ⁎⁎⁎ 0.06 0.29 ⁎⁎⁎ 0.07 0.34 ⁎⁎⁎ 0.05 0.07 0.07 Knowledge acquisition 0.26 ⁎⁎⁎ 0.05 0.21 ⁎⁎⁎ 0.05 Environmental dynamism 0.02 0.10 Creative self-efficacy ∗ environmental dynamism 0.44 ⁎⁎ 0.02 Model R2 0.15 0.05 0.38 0.39 F-value 11.01 ⁎⁎⁎ 3.20 ⁎ 31.28 ⁎⁎⁎ 23.27 ⁎⁎⁎ Coef. = standardized coefficient; SE = standard error. n = 262 observations. ⁎⁎⁎ Significant at p < 0.001 level. ⁎⁎ Significant at p < 0.01 level. ⁎ Significant at p < 0.05 level. This study further verifies the moderated mediation effects using the PROCESS macro and bootstrapping methods (Hayes, 2015). With creative self-efficacy (CSE) as the independent variable, environmental dynamism (ED) as the moderator, knowledge acquisition (KA) as the mediator, and innovative behavior (IB) as the dependent variable, Model 7 of the PROCESS tool is used to access the first-stage conditional process model (Hayes and Rockwood, 2020). The results are presented in Table 4 . When the moderator is at its low level (ED = 3.36), the indirect effect of 0.030 shows no significance due to the confidence interval including zero (95 % confidence interval from −0.079 to 0.115). At the middle level of the moderator (ED = 4.10), the indirect effect of 0.125 is significant (95 % confidence interval from 0.044 to 0.206). Similarly, when the value of the moderator comes to a high level (ED = 4.84), the indirect effect of 0.221 is significant (95 % confidence interval from 0.106 to 0.344). In sum, the indirect effect of creative self-efficacy on innovative behavior through knowledge acquisition increases as the value of the moderator (i.e., environmental dynamism) increases. Overall, the index of moderated mediation, which is a test of the moderation of the indirect effect by the moderator (Hayes and Rockwood, 2020), presents a slope of 0.129 and is significant because the confidence interval does not include zero (95 % confidence interval from 0.046 to 0.237). Therefore, our claim in Hypothesis 2 regarding the moderated mediation effect is confirmed. The conditional indirect effects (Table 4) indicate that environmental dynamism moderates the indirect effect of CSE → KA → IB, however, the indirect effect holds only at the middle and high levels of environmental dynamism. The estimated moderating effect is depicted in conceptual form in Fig. 2 , in which the slopes of the creative self-efficacy–knowledge acquisition relationship increases as the level of environmental dynamism rises, indicating a positive moderating effect of environmental dynamism.Table 4 Indirect effect of creative self-efficacy on innovative behavior through knowledge acquisition moderated by environmental dynamism. Table 4Conditional indirect effects (PROCESS Model 7) Moderator: ED, Mediator: KA, Indirect effect: CSE → KA → IB Condition of moderator Value of moderator Effect Standard error 95 % confidence interval Low 3.36 0.030 0.048 −0.079, 0.115 Middle 4.10 0.125 0.041 0.044, 0.206 High 4.84 0.221 0.061 0.106, 0.344 Index of moderated mediation: 0.129 0.048 0.046, 0.237 Note: CSE = creative self-efficacy, KA = knowledge acquisition, IB = innovative behavior, ED = environmental dynamism. Bootstrap analysis based on 10,000 replications, n = 262 observations. Fig. 2 The moderating effect of environmental dynamism on the relationship between creative self-efficacy and knowledge acquisition. Note: CSE = creative self-efficacy, KA = knowledge acquisition, ED = environmental dynamism. Fig. 2 4.3 Supplementary analysis To test the robustness of our results, this study conducts two supplementary analyses. First, as the 262 observations are nested by 40 firms, we examine whether our findings vary across firms by applying a firm dummy. The results remain unchanged. Second, the predicted moderating effect of environmental dynamism might also influence the relationship between knowledge acquisition and innovative behavior. Accordingly, we test the moderated mediation effect of the second-stage conditional process model (Hayes and Rockwood, 2020). The evaluation of conditional indirect effects is accessed by Model 14 of the PROCESS tool. The interaction term of knowledge acquisition and environmental dynamism shows no significance (β = 0.09, p = 0.195), and the index of moderated mediation presents a slope of 0.025 and is not significant as the confidence interval includes zero (95 % confidence interval from −0.066 to 0.006). Thus, our results indicate that the moderated mediation effect does not exist in the second-stage conditional process model. 5 Discussion and conclusion This study considers employees' proactive behavioral response to environmental turbulence, in the context of manufacturing SMEs. The purpose of this study is to understand how employees' personal capabilities influence their proactive behavior in dealing with intense uncertainty. Drawing on social cognitive theory, this study theorizes and tests a mediated moderation model illustrating how creative self-efficacy and knowledge acquisition influence employees' innovative behavior under conditions of varying environmental dynamism. We test the model using 262 samples collected from manufacturing SMEs in Taiwan. The results indicate that, first, knowledge acquisition mediates the relationship between creative self-efficacy and innovative behavior. Second, environmental dynamism moderates the mediation relationship; i.e., employees' innovative behavior that is motivated by creative self-efficacy and knowledge acquisition is reinforced by higher levels of dynamism. 5.1 Discussion and theoretical contributions Our findings are novel and insightful to the literature in several ways. First, the supported Hypothesis 1 confirms the mediating role of knowledge acquisition. This result supports the literature on organizational learning that treats knowledge acquisition as a process of innovation (Huber, 1991; Norman, 2004; Jiang and Chen, 2018; Duong et al., 2022). Different from the past interest in learning at the firm and inter-firm levels (e.g., Norman, 2004; Huang and Liu, 2019; Papa et al., 2018), our findings offer complementary insights by extending the concept into individual learning behavior. Although the influence of creative self-efficacy and knowledge acquisition on individual innovation has been proposed in prior research (Newman et al., 2018; Xie et al., 2018), our examination of the knowledge acquisition process further links to the consequence of innovation behavior. In other words, the supported mediation relationship highlights that knowledge acquisition is a crucial mechanism for employees with high levels of creative self-efficacy to achieve innovation, especially for employees of manufacturing SMEs who face a more interconnected industrial ecosystem. Second, the supported moderated mediation effect suggests that, as the level of environmental dynamism increases, creative self-efficacy employees are more likely to conduct knowledge acquisition activities that will lead to innovative behavior. This result is consistent with the social cognitive theory (Bandura, 1986), in that personal, environmental, and behavioral determinants have a dynamic, bi-directional interaction with each other (Cai et al., 2022). Our findings present unanimous support for the strengthening role of environmental dynamism on employees' change-oriented initiatives (Surty and Scheepers, 2020; Lin, 2021). Our findings thus advance the current understanding of employee behavior in response to external change. The focus on external environmental turbulence, such as the COVID-19 pandemic, therefore, benefits the change management literature (Caldwell and Liu, 2011; Caldwell, 2013). Our work answers a call from Ford (2009) to extend the boundary of change research from organizational planned changes (Caldwell, 2013) to the macro-level of external environmental changes. Third, this study enriches the research lens of employee proactivity, which claims that proactive behavior is an important driver of innovation (Lee et al., 2019). Our results show that a highly dynamic environment could motivate employees with creative self-efficacy to start to learn through knowledge acquisition (i.e., the supported first-stage conditional process model in Table 4), while it could fail to inspire employees with a strong knowledge acquisition capability to engage in innovative behavior (i.e., the insignificant second-stage conditional process model in the supplementary analysis). These results indicate that creative self-efficacy drives employees' proactive innovation, which is insightful because it echoes the findings of Lee et al. (2019) and confirms that employees' proactive behavior is a consequence of their cognitive state. Fourth, this study contributes to the debate concerning managing the transitioning workplace and turbulent market environment that manufacturing SMEs face (Cenamor et al., 2019; Chen, 2020; Chen et al., 2021; Melnyk et al., 2021; Ahmed et al., 2022). The sector of manufacturing SMEs in Taiwan plays an essential role in global manufacturing systems, especially in the information technology supply chain (Huang and Lu, 2020). During the COVID-19 crisis, the movement to restructure industrial innovation systems (e.g., industry 4.0) opened new opportunities for manufacturing SMEs to adapt to changes and improve resilience at the supply chain level (Chen, 2020). In this study, we claim that the nature of collaborative teamwork in the manufacturing system highlights the indispensable process of knowledge acquisition. We find that the mediation of creative self-efficacy influences innovative behavior through knowledge acquisition and exhibits a stronger relationship as the external environment turns dynamic. The results indicate that, for employees in manufacturing SMEs, the importance of conducting knowledge acquisition and learning activities increases as the turbulence of the market environment increases. In sum, our findings show that manufacturing SMEs' innovation management should emphasize nurturing employees' proactive initiatives and facilitating the process of knowledge learning when designing a resilient organization. 5.2 Managerial implications Our findings lead to several managerial implications. First, the findings suggest that managers who aim at facilitating the emergence of innovative behavior in the workplace should increase human capital and focus on creative self-efficacy. Because our findings confirm that it is the personal belief in their creative ability that motivates employees to take actions toward learning and innovation, and that a higher level of environmental dynamism reinforces this motivation. We suggest obtaining people with creative self-efficacy could help increase manufacturing SMEs' initiatives in change-relevant innovation activities during a dynamic time. In addition, managers could consider using human resource practices to encourage proactive employee initiatives, such as providing rewards for pertinent behaviors and subsequent achievements (Lee et al., 2019). Second, knowledge acquisition is a critical mechanism identified in this study as the process through which self-efficacy employees learn to innovate. Accordingly, managers of innovative work in manufacturing SMEs are suggested to consider developing supportive work environments and building information exchange systems to assist knowledge flows among interpersonal, intergroup, and inter-organizational collaborative activities. The post-pandemic future of teamwork has shifted to a combination of virtual coordination and in-person collaboration. To facilitate collaboration and innovation, managers in innovative workplaces are suggested to support work that cannot be done effectively by virtual means, such as integrative work in teams, building relationships and networking, and having difficult conversations (Hooijberg and Watkins, 2021). 5.3 Limitations and future directions Our study has several limitations that may offer some suggestions for future research. The first is the restricted generalizability of our findings. Our sampling from manufacturing SMEs in Taiwan limits the generalizability of our findings to other contexts. Taiwan has built a complete supply chain of electronic components (Huang and Lu, 2020), which demands urgent transformation to smart factories and supply chain digitalization under the threat of disasters. Manufacturing SMEs in Taiwan are concentrated around high-technology production and are featured by industrial clustering. Therefore, we choose manufacturing SMEs in Taiwan, because they are suitable for testing the multiple changes that happen in industry and external environments. However, the two hypotheses of our moderated mediating model may also apply to larger firms and firms in other industries if innovation is a key aspect of their business recovery and resilience. Therefore, we suggest future studies examine employee self-starting innovation behavior in different contexts, such as big firms or SMEs in different industries or country contexts. Second, this study discusses only the moderating effect of environmental dynamism on the relationships between creative self-efficacy, knowledge acquisition, and employee innovative behavior. It is valuable to investigate potential influencing factors related to how employee behavior is impacted by changes in various levels of an individual's environment. We suggest future studies consider other moderators, for example, culture and nationality at the macro-level, industrial competitiveness at the meso-level, organizational culture or strategic actions at the firm level, or innovation climate or leadership at the team level. Third, as our data is cross-sectional, we suggest collecting longitudinal data to observe the evolution of employee behavior, and how the antecedents and consequences interact with the level of change. The transformation of the innovative workplace will continue in the post-pandemic era; longitudinal data can offer better insight regarding what drives employees to show proactive behavior and how the mechanism operates in inspiring employees to make a change. 5.4 Conclusion Combining the employee proactivity perspective and social cognitive theory, this study proposes a moderated-mediation model and examines it using data collected from manufacturing SMEs in Taiwan. The results identify knowledge acquisition as a crucial mediator, through which proactive employees in manufacturing SMEs learn to achieve innovative work. Our work also verifies the positive moderating effect of environmental dynamism. That is, as the level of environmental dynamism increases, employees with creative self-efficacy are more likely to sense the challenging tasks and initiate reactions to acquire essential knowledge, which leads to more innovative behavior at work. In conclusion, the findings reveal a clear picture of employees' proactive innovative behavior in response to environmental changes. CRediT authorship contribution statement Yi-Fen Huang: Conceptualization, Methodology, Software, Writing - Original draft, Visualization, Writing - Reviewing and Editing. Hung-Chun Lin: Data curation, preparation, Investigation. Hsu-Mei Lee: Writing - Reviewing and Editing. Uncited reference Van Auken et al., 2021 Yi-Fen Huang is a professor at the Department of Business Administration, Dayeh University, Taiwan. She received her Ph.D. in Management from National Cheng Kung University, Taiwan. Her current research interests include R&D internationalization, supply chain flexibility, innovation strategy, and transgenerational entrepreneurship. She has published papers in Research Policy, Technovation, Journal of Business Research, International Journal of Physical Distribution & Logistics Management, Sustainable Production and Consumption, and other journals. Hung-Chun Lin is a doctoral student at the College of Management, Dayeh University, Taiwan. His current research interest is on leadership and management issues in the innovative workplace. Hsu-Mei Lee is an associate professor at the Department of International Business Management, Dayeh University, Taiwan. She received her Ph.D. in Management from National Central University, Taiwan. Her research interests are in organizational behavior and human resource management fields. Data availability The authors do not have permission to share data. ==== Refs References Ahmed A. Bhatti S.H. Gölgeci I. Arslan A. 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Herman H.M. Schwarz G. Nielsen I. The effects of employees' creative self-efficacy on innovative behavior: the role of entrepreneurial leadership J. Bus. Res. 89 2018 1 9 Ng T.W. Lucianetti L. Within-individual increases in innovative behavior and creative, persuasion, and change self-efficacy over time: a social–cognitive theory perspective J. Appl. Psychol. 101 1 2016 14 26052714 Norman P.M. Knowledge acquisition, knowledge loss, and satisfaction in high technology alliances J. Bus. Res. 57 6 2004 610 619 Papa A. Dezi L. Gregori G.L. Mueller J. Miglietta N. Improving innovation performance through knowledge acquisition: the moderating role of employee retention and human resource management practices J. Knowl. Manag. 24 3 2018 589 605 Parker S.K. Enhancing role breadth self-efficacy: the roles of job enrichment and other organizational interventions J. Appl. Psychol. 83 1998 835 852 9885197 Parker S.K. Bindl U.K. Strauss K. Making things happen: a model of proactive motivation J. Manag. 36 2010 827 856 Perry-Smith J.E. Shalley C.E. The social side of creativity: a static and dynamic social network perspective Acad. Manag. Rev. 28 1 2003 89 106 Podsakoff P.M. Organ D.W. Self-reports in organizational research: problems and prospects J. Manag. 12 4 1986 531 544 Ratten V. Coronavirus and international business: an entrepreneurial ecosystem perspective Thunderbird Int. Bus. Rev. 62 5 2020 629 634 Rofcanin Y. Las Heras M. Bosch M.J. Berber A. Mughal F. Ozturk M. Servant leadership and family supportiveness: looking into employees' work and family outcomes J. Bus. Res. 128 2021 70 82 Rusly F.H. Sun P.Y.T. Corner J.L. Change readiness: creating understanding and capability for the knowledge acquisition process J. Knowl. Manag. 19 6 2015 1204 1223 Shafi M. Liu J. Ren W. Impact of COVID-19 pandemic on micro, small, and medium-sized enterprises operating in Pakistan Res.Glob. 2 2020 100018 Shao R. Shi Z. Zhang D. Social media and emotional burnout regulation during the COVID-19 pandemic: multilevel approach J. Med. Internet Res. 23 3 2021 e27015 Steinbach A.L. Kautz J. Korsgaard M.A. Caring for their own: how firm actions to protect essential workers and CEO benevolence influenced stakeholder sentiment during the COVID-19 pandemic J. Appl. Psychol. 106 6 2021 811 34138588 Surty S. Scheepers C.B. Moderating effect of environmental dynamism on leadership practices and employees'response to change in South Africa Manag. Res. Rev. 43 70 2020 787 810 Tantawy M. Herbert K. McNally J.J. Mengel T. Piperopoulos P. Foord D. Bringing creativity back to entrepreneurship education: creative self-efficacy, creative process engagement, and entrepreneurial intentions J. Bus. Ventur. Insights 15 2021 e00239 Terziovski M. Innovation practice and its performance implications in small and medium enterprises (SMEs) in the manufacturing sector: a resource-based view Strateg. Manag. J. 31 8 2010 892 902 Thukral E. COVID-19: small and medium enterprises challenges and responses with creativity, innovation, and entrepreneurship Strateg. Chang. 30 2 2021 153 158 Tierney P. Farmer S.M. Creative self-efficacy: its potential antecedents and relationship to creative performance Acad. Manag. J. 45 6 2002 1137 1148 Tierney P. Farmer S.M. Creative self-efficacy development and creative performance over time J. Appl. Psychol. 96 2 2011 277 20954756 Unsworth K.L. Parker S.K. Proactivity and Innovation: Promoting a New Workforce for the New Workplace. The New Workplace: A Guide to The Human Impact of Modern Working Practices 2003 175 196 Van Auken H.E. Ardakani M.F. Carraher S. Avorgani R.K. Innovation among entrepreneurial SMEs during the COVID-19 crisis in Iran Small Bus.Int.Rev. 5 2 2021 e395 Van Wijk R. Jansen J.J. Lyles M.A. Inter-and intra-organizational knowledge transfer: a meta-analytic review and assessment of its antecedents and consequences J. Manag. Stud. 45 4 2008 830 853 Waldman D.A. Ramirez G.G. House R.J. Puranam P. Does leadership matter? CEO leadership attributes and profitability under conditions of perceived environmental uncertainty Acad. Manag. J. 44 1 2001 134 143 Wu J.B. Tsui A.S. Kinicki A.J. Consequences of differentiated leadership in groups Acad. Manag. J. 53 1 2010 90 106 Wu W.K. Chiu S.W. The impact of Guanxi positioning on the quality of manufacturer–retailer channel relationships: evidence from Taiwanese SMEs J. Bus. Res. 69 9 2016 3398 3405 Xiao X. Tian Q. Mao H. How the interaction of big data analytics capabilities and digital platform capabilities affects service innovation: a dynamic capabilities view IEEE Access 8 2020 18778 18796 Xie X. Wang L. Zeng S. Inter-organizational knowledge acquisition and firms' radical innovation: a moderated mediation analysis J. Bus. Res. 90 2018 295 306 Zhang F. Zhu L. Social media strategic capability, organizational unlearning, and disruptive innovation of SMEs: the moderating roles of TMT heterogeneity and environmental dynamism J. Bus. Res. 133 2021 183 193 Zhang Z. Wang M.O. Shi J. Leader-follower congruence in proactive personality and work outcomes: the mediating role of leader-member exchange Acad. Manag. J. 55 1 2012 111 130 Zhong R. Paluch R.M. Shum V. Zatzick C.D. Robinson S.L. Hot, cold, or both? A person-centered perspective on death awareness during the COVID-19 pandemic J. Appl. Psychol. 106 6 2021 839 34138590	36471723	PMC9712071	NO-CC CODE	2022-12-12 23:20:50	no	Technol Forecast Soc Change. 2023 Feb 1; 187:122247	utf-8	Technol Forecast Soc Change	2,022	10.1016/j.techfore.2022.122247	oa_other
==== Front An Pediatr (Engl Ed) An Pediatr (Engl Ed) Anales De Pediatria 2341-2879 Asociación Española de Pediatría. Published by Elsevier España, S.L.U. S2341-2879(22)00236-8 10.1016/j.anpede.2022.10.006 Scientific Letter Intentional self-poisoning increase in the emergency department in Spain during the COVID-19 pandemic☆ Incremento de las intoxicaciones con fin suicida en los servicios de urgencias en España durante la pandemia COVID-19Azkunaga Beatriz ⁎ Echarte Patricia Zumalde Ane Mintegi Santiago Grupo de Trabajo de Intoxicaciones de la Sociedad Española de Urgencias de Pediatría1 Servicio de Urgencias de Pediatría, Instituto de Investigación Sanitaria Biocruces Bizkaia, Hospital Universitario Cruces, Universidad del País Vasco, Barakaldo, Vizcaya, Spain ⁎ Corresponding author. 1 The members of the Working Group on Poisonings of the Sociedad Española de Urgencias de Pediatría are presented in Appendix A. 1 12 2022 1 12 2022 © 2022 Asociación Española de Pediatría. Published by Elsevier España, S.L.U. 2022 Asociación Española de Pediatría Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcDear Editor, Self-harm and suicide are important public health problems in adolescence, and suicide is the second leading cause of death in youth worldwide.1 The method used most commonly in suicide attempts is intentional poisoning with medicines available in the home. These youth frequently have psychiatric disorders.2 Most of the visits made to paediatric emergency departments (PEDs) due to poisoning correspond to young children who have ingested toxic substances unintentionally.3 Suicidal intent accounts for approximately 14% of poisonings managed in PEDs worldwide4 and 7% in Spain,3 chiefly from age 12 years. An alarm was raised during the coronavirus disease 2019 (COVID-19) pandemic of an increase in mental health disorders in both adults and the paediatric age group.5 However, to our knowledge, the impact of the pandemic on the volume of PED visits due to self-poisoning with suicidal intent in Spain has not yet been analysed at the national level. The aim of our study was to assess the impact of the pandemic on visits to PEDs in Spain due to self-poisoning with suicidal intent. We conducted a study based on a prospective register of the poisonings documented in 43 Spanish PEDs that participate in the Toxicology Surveillance Observatory of the Sociedad Española de Urgencias de Pediatría (Spanish Society of Paediatric Emergency Medicine) between 2014 and 2021. We collected data on visits due to exposure to potentially toxic substances on the 13th, 14th and 15th of each month in the period under study. The methods of the register have already been described in a previous issue of this journal.6 For the purpose of the study, the prepandemic period ranged from January 2014 to February 2020 and the pandemic period from March 2020 to December 2021. The participating hospitals are listed in Appendix A. We conducted the statistical analysis with the software IBM SPSS Statistics for Windows, version 23.0 (IBM Corp.; Armonk, NY, USA). We summarised quantitative data as mean and standard deviation, and categorical data as percentages. We compared quantitative variables with the Student t test and categorical variables with the χ2 and Fisher exact tests. We considered P values of less than 0.05 statistically significant. The study was approved by the Clinical Research Ethics Committee of the Basque Country. During the period under study, there were 836 188 care episodes in the 43 PEDs. Of this total, 1909 were for exposure to potentially toxic substances, in 199 (10.4%) intentional exposure with suicidal intent. In the prepandemic period, there were 114 documented cases of self-poisoning with suicidal intent (7.5% of visits due to toxic substance exposure) compared to 85 (22.2%) during the pandemic (P < .01). In the prepandemic period, participating PEDs documented 1 case of self-poisoning with suicidal intent every 2 days compared to every 0.8 days in the pandemic. In the 2 years of the pandemic, the proportion of intentional poisonings with suicidal intent rose from 12.4% in 2020 to 28.2% in 2021. We did not find significant changes in the characteristics of cases of self-poisoning with suicidal intent between the prepandemic and pandemic periods, save for more frequent contact with prehospital emergency care services. We also found no differences in how these cases were managed in the PED setting, save for a more frequent administration of antidotes during the pandemic, and N-acetylcysteine was the most frequently used antidote in both periods. The majority of the cases corresponded to female patients and ingestion of medicines, chiefly benzodiazepines and analgesics, with one third of cases being of combined drug intoxication and more than half resulting in hospital admission. None of the patients died (Table 1 ).Table 1 Characteristics of cases of self-poisoning with suicidal intent managed in the prepandemic and pandemic periods. Table 1 Prepandemic n = 114 Pandemic n = 85 P Sex (n = 196): female 96 (85%) 74 (89.2%) NS Age <14 years 36 (31.6%) 33 (38.8%) NS Setting (n = 170): home 86 (86%) 66 (94.3%) NS First attempt (n = 184): no 46 (43.4%) 37 (47.4%) NS Substance: drug 104 (91.2%) 80 (94.1%) NS Analgesics/antipyretics 44 (38.6%) 40 (47.1%) Paracetamol 32 (28%) 28 (32.9%) Benzodiazepines 39 (34.2%) 32 (37.6%) Combined drug intoxication 42 (36.8%) 30 (35.3%) Previous contact with emergency department (n = 193) 21 (19.4%) 28 (32.9%) <.05 Accompanying person (n = 190): parent 93 (83.8%) 67 (84.8%) NS Transport vehicle (n = 191): ambulance 40 (36.4%) 35 (43.2%) NS Symptoms: yes 64 (56.1%) 57 (67.1%) NS Diagnostic tests (n = 196) 87 (76.3%) 73 (89%) <.05 Treatment in emergency department 59 (51.8%) 58 (68.2%) Decontamination 35 (30.7%) 30 (35.3%) NS Antidote 9 (7.9%) 16 (19%) <.05 Hospital admission 52 (45.6%) 48 (56.5%) NS Prepandemic period: January 2014 to February 2020. Pandemic period: March 2020 to December 2021. NS, not significant. Our study found a significant increase in cases of paediatric self-poisoning with suicidal intent in Spanish PEDs during the COVID-19 pandemic, a finding that would be consistent with an increase in mental health disorders during the pandemic. Previous publications had already warned of a global mental health crisis in youth that started long before the pandemic,1 and the pandemic may have made it more evident. One of the potential limitations of the study could be the difficulty documenting cases in the pandemic, especially at the beginning, due to the current health care and social circumstances. In fact, in the early stages of the pandemic there was a marked overall decrease in PED visits. Our study demonstrates the need to take urgent measures to promote, protect and care for the mental health of children and adolescents, taking into account that the prevention of self-harm and suicide requires both universal aimed at youth in general and specific measures targeting high-risk groups.1 Appendix A Members of the Working Group on Poisonings of the Sociedad Española de Urgencias de Pediatría Hospital Universitario (H.U.) Tajo: García-Vao C.; Hospital (H.) Quirón Bizkaia: Oliver P; H.U. Arnau de Vilanova: Pociello N; H.U. Basurto: Humayor J.; H. Cabueñes: Rodriguez P, García A; H.U. Carlos Haya: Oliva S.; Complejo Hospitalario (C.H.) Navarra: Palacios M, Clerigué N.; C.H. de Jaén: Campo T.; Complejo Asistencial Universitario de Léon: Andrés AG, Muñiz M; Centro de Salud Parc Taulí: Baena I, Comalrena de Sobregrau C; H.U. Cruces: Echarte P, Zumalde A; H.U. Doce de Octubre: Mesa S.; H.U. Donostia: Muñoz JA.; H.U. Dr. Peset: Rodriguez A.; H. Francesc de Borja: Angelats CM, Sequi JM, Villaplana I.; Fundació Sant Hospital de la Seu d’Urgell: Astete J.; H.U. Fundación Alcorcón: Barasoain A.; H.U. Gregorio Marañón: Vázquez P.; H. Infanta Cristina: Rodriguez MD.; H. Infanta Elena: Gómez C, Nuñez T.; H. Terrassa: Pinyot M.; H. Laredo: Jorda A, Canduela V.; H. Mendaro: Herrero L.; H.U. Mutua Terrassa: Pizzá A.; H.U. Niño Jesús: Molina JC.; H. Materno-Infantil Las Palmas de Gran Canaria: Mangione L. H. Materno-Infantil Badajoz: Hurtado P; Complejo Asistencial de Ávila: García E; H. Montepríncipe, H. Sanchinarro, H. Torrelodones, H. Puerta del Sur: Lalinde M.; H. Príncipe de Asturias: García MA; H.U. Puerta de Hierro: Benito C, Armero P.; H. Rey Juan Carlos: Sabrido G; H.U. San Agustín: Melguizo MC.; H.U. Río Hortega: Benito H; H. Sant Joan de Dèu, Xarxa hospitalaria i universitaria de Manresa, Fundació Althaia: Botifoll E, Lobato Z; H.U. Sant Joan de Déu: Martínez Sánchez L, Luaces C, Algarrada L.; H San Pedro: Martínez L.; H.U. Salamanca: López J; H. Virgen de la Salud: Aquino E.; H. Zumarraga: Pérez A; H.U. Lozano Blesa: Lanuza R; H.U. Politécnico La Fe: Señer R; H. Mataró: Baena J; H.U. Marqués de Valdecilla, H. Sierrallana: Peñalba A; H. Gernika-Lumo: Alday A.; H. Miguel Servet: Campos C.; H.U. Son Espases: López V.; H. Son Llatzer: Vidal C.; C. Asistencia Soria: Muñoz N.; H. Infantil La Paz: De Miguel B.; Hospital C. U. Valencia: Khodayar P, H.U. Central de Asturias: Alonso MA, H. Severo Ochoa: Angulo AM. ☆ Previous presentation: oral communication at the XXVI Meeting of the Sociedad Española de Urgencias de Pediatría; June 16–18, 2022, Pamplona, Spain. ==== Refs References 1 Hawton K. Saunders K.E. O’Connor R.C. Self-harm and suicide in adolescents Lancet 379 2012 2373 2382 22726518 2 Findik O.T.P. Erdoğdu A.B. Fadiloğlu E. Arman A.R. Emergency department visits for non-suicidal self-harm, suicidal ideation, and suicide attempts in children and adolescents Child Psychiatry Hum Dev 53 2022 289 299 33523341 3 Santiago P. Bilbao N. Martinez-Indart L. Mintegi S. Azkunaga B. the Intoxications Working Group of the Spanish Society of Pediatric Emergencies Epidemiology of acute pediatric poisonings in Spain: a prospective multicenter study from the Spanish Society of Pediatric Emergency Medicine Eur J Emerg Med 27 2020 284 289 31855890 4 Gonzalez-Urdiales P. Kuppermann N. Dalziel S.R. Prego J. Benito J. Mintegi M. Pediatric intentional self-poisoning evaluated in the emergency department Pediatr Emer Care 37 2021 e1631 e1636 5 Santomauro D.F. Mantilla A.M. Shadid J. Zheng P. Ashbaugh C. Pigott D.M. Global prevalence and burden of depressive an anxiety disorders in 204 countries and territories in 2020 due to the COVID-19 pandemic Lancet 398 2021 1700 1712 34634250 6 Azkunaga B. Mintegi S. Salmón N. Acedo Y. del Arco L. Grupo de Trabajo de intoxicaciones de la SEUP Intoxicaciones en menores de 7 años en España. Aspectos de mejora, prevención y tratamiento An Pediatr (Barc) 78 2013 255 260	36496312	PMC9712072	NO-CC CODE	2022-12-07 23:16:36	no	An Pediatr (Engl Ed). 2022 Dec 1; doi: 10.1016/j.anpede.2022.10.006	utf-8	An Pediatr (Engl Ed)	2,022	10.1016/j.anpede.2022.10.006	oa_other
==== Front Me´decine De Catastrophe, Urgences Collectives 1279-8479 1279-8479 Published by Elsevier Masson SAS on behalf of Société Française de Médecine de Catastrophe. S1279-8479(22)00315-9 10.1016/j.pxur.2022.11.008 Dossier COVID-19, oxygène et incendies dans les établissements de soins COVID-19, oxygen and fires in healthcare facilitiesNoto René Président d’honneur de la SFMC 2, boulevard Pasteur, 07400 le Teil 1 12 2022 1 12 2022 © 2022 Published by Elsevier Masson SAS on behalf of Société Française de Médecine de Catastrophe. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmc	0	PMC9712073	NO-CC CODE	2022-12-12 23:20:48	no	2022 Dec 1; doi: 10.1016/j.pxur.2022.11.008	utf-8	null	null	null	oa_other
==== Front Cell Rep Cell Rep Cell Reports 2211-1247 Cell Press S2211-1247(22)01737-5 10.1016/j.celrep.2022.111845 111845 Article Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents Cao Yunlong 12#∗∗∗∗∗ Jian Fanchong 13# Zhang Zhiying 4# Yisimayi Ayijiang 14# Hao Xiaohua 5# Bao Linlin 6# Yuan Fei 7 Yu Yuanling 2 Du Shuo 4 Wang Jing 14 Xiao Tianhe 18 Song Weiliang 14 Zhang Ying 4 Liu Pulan 4 An Ran 2 Wang Peng 2 Wang Yao 2 Yang Sijie 19 Niu Xiao 13 Zhang Yuhang 7 Gu Qingqing 2 Shao Fei 2 Hu Yaling 10 Yin Weidong 10 Zheng Aihua 7 Wang Youchun 211 Qin Chuan 6∗∗∗∗ Jin Ronghua 5∗∗∗ Xiao Junyu 24912∗∗ Xie Xiaoliang Sunney 12∗ 1 Biomedical Pioneering Innovation Center (BIOPIC), Peking University, Beijing, P.R. China 2 Changping Laboratory, Beijing, P.R. China 3 College of Chemistry and Molecular Engineering, Peking University, Beijing, P.R. China 4 School of Life Sciences, Peking University, Beijing, P.R. China 5 Beijing Ditan Hospital, Capital Medical University, Beijing, P.R. China 6 Key Laboratory of Human Disease Comparative Medicine, Chinese Ministry of Health, Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, P.R. China 7 State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, P.R. China 8 Joint Graduate Program of Peking-Tsinghua-NIBS, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, P.R. China 9 Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, P.R. China 10 Sinovac Biotech Ltd., Beijing, P.R. China 11 Division of HIV/AIDS and Sex-transmitted Virus Vaccines, Institute for Biological Product Control, National Institutes for Food and Drug Control (NIFDC), Beijing, P.R. China 12 Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, P.R. China ∗∗∗∗∗ Corresponding author Yunlong Cao ∗∗∗∗ Corresponding author Chuan Qin ∗∗∗ Corresponding author Ronghua Jin ∗∗ Corresponding author Junyu Xiao ∗ Corresponding author Xiaoliang Sunney Xie # These authors contributed equally. 1 12 2022 1 12 2022 11184521 9 2022 13 11 2022 23 11 2022 © 2022. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. SARS-CoV-2 Omicron sublineages have escaped most RBD-targeting therapeutic neutralizing antibodies (NAbs), which proves the previous NAb drug screening strategies deficient against the fast-evolving SARS-CoV-2. Better broad NAb drug candidate selection methods are needed. Here, we describe a rational approach for identifying RBD-targeting broad SARS-CoV-2 NAb cocktails. Based on high-throughput epitope determination, we propose that broad NAb drugs should target non-immunodominant RBD epitopes to avoid herd immunity-directed escape mutations. Also, their interacting antigen residues should focus on sarbecovirus conserved sites and associate with critical viral functions, making the antibody-escaping mutations less likely to appear. Following the criteria, a featured non-competing antibody cocktail, SA55+SA58, is identified from a large collection of broad sarbecovirus NAbs isolated from SARS-CoV-2-vaccinated SARS convalescents. SA55+SA58 potently neutralizes ACE2-utilizing sarbecoviruses, including circulating Omicron variants, and could serve as broad SARS-CoV-2 prophylactics to offer long-term protection, especially for individuals who are immunocompromised or with high-risk comorbidities. Graphical abstract Cao et al. describe a strategy to identify broad sarbecovirus-neutralizing antibody cocktail that would be hard to escape for future SARS-CoV-2 variants using high-throughput epitope mapping. The resulting antibody cocktail named SA55+SA58 exhibits high neutralizing potency and breadth against ACE2-utilizing sarbecoviruses and efficiently protects mice from BA.1 and BA.5 infection. ==== Body pmcLead Contact: Xiaoliang Sunney Xie ([email protected])	36493787	PMC9712074	NO-CC CODE	2022-12-08 23:16:14	no	Cell Rep. 2022 Dec 1;:111845	utf-8	Cell Rep	2,022	10.1016/j.celrep.2022.111845	oa_other
==== Front Vaccine Vaccine Vaccine 0264-410X 1873-2518 Elsevier Science S0264-410X(22)01493-1 10.1016/j.vaccine.2022.11.069 Article Surveillance of COVID-19 vaccine safety among elderly persons aged 65 years and older Wong Hui-Lee a Tworkoski Ellen b Ke Zhou Cindy a Hu Mao b Thompson Deborah a Lufkin Bradley b Do Rose a Feinberg Laurie b Chillarige Yoganand b Dimova Rositsa a Lloyd Patricia C. a MaCurdy Thomas bc Forshee Richard A. a Kelman Jeffrey A. d Shoaibi Azadeh a Anderson Steven A. a⁎ a US Food and Drug Administration, Silver Spring, MD, USA b Acumen LLC, Burlingame, CA, USA c Department of Economics, Stanford University, Stanford, CA, USA d Centers for Medicare & Medicaid Services, Washington, DC, USA ⁎ Corresponding author. 1 12 2022 1 12 2022 6 5 2022 23 11 2022 28 11 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Background Monitoring safety outcomes following COVID-19 vaccination is critical for understanding vaccine safety especially when used in key populations such as elderly persons age 65 years and older who can benefit greatly from vaccination. We present new findings from a nationally representative early warning system that may expand the safety knowledge base to further public trust and inform decision making on vaccine safety by government agencies, healthcare providers, interested stakeholders, and the public. Methods We evaluated 14 outcomes of interest following COVID-19 vaccination using the US Centers for Medicare & Medicaid Services (CMS) data covering 30,712,101 elderly persons. The CMS data from December 11, 2020 through Jan 15, 2022 included 17,411,342 COVID-19 vaccinees who received a total of 34,639,937 doses. We conducted weekly sequential testing and generated rate ratios (RR) of observed outcome rates compared to historical (or expected) rates prior to COVID-19 vaccination. Findings Four outcomes met the threshold for a statistical signal following BNT162b2 vaccination including pulmonary embolism (PE; RR = 1.54), acute myocardial infarction (AMI; RR = 1.42), disseminated intravascular coagulation (DIC; RR = 1.91), and immune thrombocytopenia (ITP; RR = 1.44). After further evaluation, only the RR for PE still met the statistical threshold for a signal; however, the RRs for AMI, DIC, and ITP no longer did. No statistical signals were identified following vaccination with either the mRNA-1273 or Ad26 COV2.S vaccines. Interpretation This early warning system is the first to identify temporal associations for PE, AMI, DIC, and ITP following BNT162b2 vaccination in the elderly. Because an early warning system does not prove that the vaccines cause these outcomes, more robust epidemiologic studies with adjustment for confounding, including age and nursing home residency, are underway to further evaluate these signals. FDA strongly believes the potential benefits of COVID-19 vaccination outweigh the potential risks of COVID-19 infection. ==== Body pmc1 Introduction The US Food and Drug Administration (FDA) is monitoring the safety of four vaccines for Coronavirus Disease 2019 (COVID-19) currently available in the US. These include the licensed Pfizer BioNTech vaccine (Comirnaty) for persons 12 years and older and authorized under emergency use authorization (EUA) for those 6 months and older years (BNT162b2), the licensed Moderna vaccine (Spikevax) for persons 18 years and older and authorized Moderna (mRNA-1273) for 6 months and older, Janssen (Ad26.COV2.S) vaccines for persons 18 years and older, and the Novavax (NVX-CoV2373) vaccine authorized for persons 12 years and older. Pre-authorization clinical studies provided useful information on the safety of COVID-19 vaccines, but they have limitations such as sample size and follow-up that may be addressed in post-authorization safety studies in large healthcare databases. Accordingly, FDA and the Centers for Medicare & Medicaid Services (CMS) are using the Medicare health insurance database, covering more than 25 million elderly persons aged 65 years and older, to conduct near real-time safety monitoring of 14 outcomes on a weekly basis following COVID-19 vaccine (BNT162b2, mRNA-1273, or Ad26.COV2.S) administration. The US elderly population, including persons in Long-Term Care Facilities or nursing homes [1], were disproportionately affected by COVID-19, as they were among the first US populations to be infected. Because they suffered a higher rate of infection, serious disease, and severe outcomes including death [2], they were among the first groups recommended by the Advisory Committee on Immunization Practices to receive the vaccine [3]. Available information concerning the safety of the vaccine in elderly persons is limited. However, the near real-time surveillance method used by FDA and CMS continues to expand the available knowledge base and further advances our understanding of the safety profile of these new COVID-19 vaccines. The FDA-CMS near real-time active surveillance program complements other FDA and US Government vaccine safety surveillance systems by rapidly detecting safety concerns that may not have been voluntarily reported to passive surveillance systems such as Vaccine Adverse Event Reporting System. Routinely used by FDA in safety surveillance for annual influenza vaccines in the past decade, this method is designed to be sensitive enough to quickly screen safety signals for further evaluation in robust epidemiologic studies [4], [5]. This rapid screening method performs hypothesis testing, sequentially, in a prospective manner as the vaccine data accrue to detect potential safety signals earlier in the course of surveillance, but signals must be further evaluated in more robust studies with confounding adjustment. However, results detected by near real-time surveillance do not establish a causal association between the outcomes and vaccination because of the method has limited adjustments for confounding. In this report, we summarize the results of weekly sequential testing analyses for 14 outcomes where formal testing was initiated. We also describe how the identified signals were evaluated and the epidemiological studies currently underway will provide more robust adjustment for confounding determine if any are true signals. 2 Methods 2.1 Data sources We used the US Medicare Fee-for-Service (FFS) Parts A (inpatient services) and B (outpatient care) claims and enrollment data to define inclusion criteria, exposures, outcomes, and patient characteristics. The number of 65 + beneficiaries with at least 1 day of Medicare FFS enrollment in the study period is 30,712,101. The Minimum Data Set identified nursing home residency status. 2.2 Study period and population The study included Medicare FFS beneficiaries aged 65 years or older who received a COVID-19 vaccine since December 11, 2020. To be included, individuals needed to be enrolled on the vaccination date, and continuously enrolled during an outcome-specific pre-vaccination clean window [6]. 2.3 Exposure and follow-up Exposure was defined as receipt of a BNT162b2, mRNA-1273, or Ad26 COV2.S COVID-19 vaccination, identified using brand and dose-specific Current Procedural Terminology / Healthcare Common Procedure Coding System codes [7] (Table S1). The primary analysis included all observed doses by brand. Dose-specific analyses are described in supplemental materials. Follow-up included all time in the prespecified risk windows; the first-dose risk window was censored at the time of the second-dose vaccination. 2.4 Outcomes The list of 14 outcomes, pre-vaccination clean windows, and post-vaccination risk windows are detailed in Table S2. Claims-based outcome algorithms were developed based on literature review and in consultation with clinical experts [8]. 2.5 Vaccine safety surveillance Weekly vaccine uptake was monitored by brand. The near real-time surveillance compared the observed number of each outcome in the COVID-19 vaccinated population to an expected number based on the background rate of the outcome in a similar COVID-19 unvaccinated population prior to the pandemic, adjusted for the delay in claims processing and standardized by nursing home residency status, age, sex, and race. We calculated annual background rates within the strata of the standardized variables, where possible, during 2017–2019 (pre-COVID-19) and peri-COVID-19 (January 1, 2020–December 10, 2020) [9] (Table S3). If annual rates in the historical period differed substantially from each other, we selected the minimum rate as a more conservative approach. Otherwise, the median annual rate was selected. 2.6 Statistical analysis Poisson Maximized Sequential Probability Ratio Test (PMaxSPRT) was used to detect increased outcome risk following vaccination compared to a historical baseline for 14 outcomes [10], [11], [12] (Table S3). Weekly sequential testing for each outcome commenced when a minimum of three cases accrued. One-tailed tests were used, with a null hypothesis that the observed rate was no greater than the historical comparator beyond a prespecified test margin with an overall alpha of 1 %. The test margin was selected for each outcome based on expert guidance to avoid minimal risk increases that were unlikely to be clinically relevant. The alpha level was selected to address a large number of tests. A statistical signal occurred if the log likelihood ratio exceeded the critical value, or a threshold for determining whether the result was likely to occur due to chance. Additional details are provided in Table S3 and the study protocol [6]. 2.7 Signal evaluation Prespecified signal evaluation analyses were conducted after a statistical signal was observed. Data quality was checked to rule out database errors or changes in event observation as potential sources of the signal. Sensitivity analyses evaluated whether the increase in risk was observed with alternate expected rates in sequential testing – (i) rates from calendar months in the historical period corresponding to those in the study period to address monthly variations in the background rates, and (ii) rates among a subset of Medicare beneficiaries with influenza vaccination in the past year to assess differential healthcare utilization. For pulmonary embolism (PE), additional ad hoc analyses were conducted with (i) the outcome limited to the inpatient setting and (ii) rates from November 1–December 11, 2020 to address changes in rates in the peri-COVID period as alternate expected rates. Signal characterization assessed the cases and the potential elevated risk. Analyses included (i) identifying clusters in the risk window following dose 1 through temporal scans, (ii) estimating relative risks within demographic strata, (iii) assessing distribution of care settings and diagnosis codes in observation and historical periods, (iv) comparing patient characteristics among vaccinated and overall elderly populations, and (v) reviewing patterns of reimbursement codes by clinicians on a random sample of up to 100 cases per outcome to contextualize the medical histories of patients. 2.8 Statistical software All analyses were conducted using R 4.0.3 (R Foundation for Statistical Computing, Vienna, Austria), SAS v. 9.4 (SAS Institute Inc., Cary, NC, United States), and SaTScan v9.6 (Martin Kulldorff, Boston, MA, United States). 2.9 Ethical considerations This surveillance activity was conducted as part of the FDA public health surveillance mandate. 3 Results 3.1 Descriptive statistics of the COVID-19 vaccinated population From December 11, 2020 through January 15, 2022, 17,088,796 BNT162b2, 16,898,376 mRNA-1273, and 634,019 Ad26 COV2.S (34,621,191 total including vaccination days with multiple products) COVID-19 vaccine doses were found among 30,712,101 eligible individuals in the study period. The population of BNT162b2 vaccinees had a slightly higher proportion of older individuals (85 years and older), nursing home residents, and individuals residing in urban areas compared to the general elderly Medicare population (Table 1 ; Fig. 1 ). mRNA-1273 vaccinees exhibited similar characteristics to the general Medicare population, while the population receiving the Ad26 COV2.S vaccine was younger and had fewer nursing home residents.Table 1 Characteristics ofBNT162b2, mRNA-1273, and Ad26 COV2.S Vaccine Doses Administered among Adults Aged 65 years and Older in Medicarea, Dec 11, 2020 to January 15, 2022. Patient Characteristic General 65 + FFSb mRNA-1273c BNT162b2 Ad26 COV2.S SMD - Comparison with General FFSc mRNA-1273 BNT162b2 Ad26 COV2.S Total 25,390,578 15,761,718 15,896,042 576,698 Nursing Home Residency Status Nursing Home Resident 578,908 (2.3) 324,991 (2.06) 711,437 (4.48) 12,948 (2.25) 0.02 0.12 0.00 Non-Nursing Home Resident 24,811,670 (97.7) 15,436,727 (97.94) 15,184,605 (95.52) 563,750 (97.75) 0.02 0.12 0.00 Age (years) 65–74 13,661,915 (53.8) 8,333,648 (52.87) 8,080,719 (50.83) 347,687 (60.29) 0.02 0.06 0.13 75–84 8,288,448 (32.6) 5,369,646 (34.07) 5,383,100 (33.86) 166,011 (28.79) 0.03 0.03 0.08 85+ 3,440,215 (13.5) 2,058,424 (13.06) 2,432,223 (15.30) 63,000 (10.92) 0.01 0.05 0.08 Sex Female 14,191,641 (55.9) 9,010,306 (57.17) 9,324,988 (58.66) 319,812 (55.46) 0.03 0.06 0.01 Male 11,198,935 (44.1) 6,751,412 (42.83) 6,571,054 (41.34) 256,886 (44.54) 0.03 0.06 0.01 Race/Ethnicity Asian 529,362 (2.1) 320,757 (2.04) 341,530 (2.15) 10,113 (1.75) 0.00 0.00 0.03 Black 1,728,808 (6.8) 764,033 (4.85) 898,467 (5.65) 33,219 (5.76) 0.08 0.05 0.04 Hispanic 398,426 (1.6) 146,371 (0.93) 162,556 (1.02) 6,578 (1.14) 0.06 0.05 0.04 Alaskan Native/Native American 117,539 (0.5) 64,557 (0.41) 67,878 (0.43) 1,421 (0.25) 0.01 0.01 0.04 White 21,584,886 (85.0) 13,776,628 (87.41) 13,714,047 (86.27) 504,406 (87.46) 0.07 0.04 0.07 Other 448,630 (1.8) 282,212 (1.79) 299,951 (1.89) 7,555 (1.31) 0.00 0.01 0.04 Missing/Unknown 582,927 (2.3) 407,160 (2.58) 411,613 (2.59) 13,406 (2.32) 0.02 0.02 0.00 Urban/Rural Urban 19,415,683 (76.5) 11,855,600 (75.22) 1,346,1164 (84.68) 436,146 (75.63) 0.03 0.21 0.02 Rural 5,811,513 (22.9) 3,822,143 (24.25) 2,359,912 (14.85) 138,341 (23.99) 0.03 0.21 0.03 Missing/Unknown 163,382 (0.6) 83,975 (0.53) 74,966 (0.47) 2,211 (0.38) 0.01 0.02 0.03 a Individuals included in this table were required to have 365 days of continuous enrollment prior to vaccination date in order to accurately capture medical history. Therefore, the total vaccine doses for this subpopulation is smaller than the study population of Medicare FFS 65 + study population reported in the Results section. b Characteristics of the general Medicare FFS population aged 65 years and older were assessed as of 12/10/2020. c SMD with values greater than 0.1 are presented in bold text and indicate covariates with larger imbalances between populations Abbreviations: FFS, Fee-for-Service; SMD, standardized mean difference; AMI, acute myocardial infarction; COPD, chronic obstructive pulmonary disease; COVID-19, coronavirus disease 2019. Fig. 1 Cumulative COVID-19 Vaccine Doses, by Age and Sex, in Adults Aged 65 Years and Older in the Medicare Shared Systems Data, by Vaccine Brand, Dec 11, 2020 to January 15, 2022. Comparing data cuts through April 24, 2021 (Table S6), March 13, 2021 (Table S7) and February 27, 2021 (Table S8), the populations differed for those who were vaccinated from mid December 2020 to February 2021 versus the later months. BNT162b2 vaccinees experienced more hospitalizations in the prior year and higher proportions of underlying medical conditions (e.g., Charlson comorbidity index greater than 0), when compared to the overall elderly Medicare, mRNA-1273, or Ad26 COV2.S populations. 3.2 PMaxSPRT sequential testing results All outcomes with primary analyses for PMaxSPRT testing met the prespecified criteria for initiation of analyses (Table S3). No statistical signals were identified following vaccination with either the mRNA-1273 or Ad26 COV2.S vaccines. AMI (RR = 1.42), PE (RR = 1.54), disseminated intravascular coagulation (DIC; RR = 1.91), and immune thrombocytopenia (ITP; RR = 1.44) following BNT162b2 vaccination met the statistical threshold for a signal (Table 2 ). Dose-specific results can be seen in Tables S4-5.Table 2 Summary of Sequential Testing Results in Adults Aged 65 years and Older in the Medicare Shared Systems Database for Any Dose by Vaccine Brand, Dec 11, 2020 to January 15, 2022. Outcomes, by Vaccine Brand Vaccine Brand Observed Person Time (Days) Number of Doses Number of Observed Outcomes (as of 01/15/2022)a Relative Risk of Observed vs Expected (Any Dose; 01/15/2022)b Signal Identified c Acute Myocardial Infarction BNT162b2 402,380,589 15,747,074 13,293 0.97 Yes − 2/27/2021 (RR = 1.42) mRNA-1273 423,244,936 15,628,229 12,909 0.94 No Ad26 COV2.S 13,156,815 570,434 548 1.30 No Deep Vein Thrombosis BNT162b2 402,057,021 15,600,926 12,871 0.88 No mRNA-1273 424,775,904 15,507,286 11,342 0.78 No Ad26 COV2.S 13,126,819 567,190 480 1.07 No Pulmonary Embolism BNT162b2 404,173,653 15,684,098 9,443 1.15 Yes – 2/27/2021 (RR = 1.54) mRNA-1273 426,520,976 15,571,477 8,996 1.08 No Ad26 COV2.S 13,184,317 569,676 346 1.34 No Disseminated Intravascular Coagulation BNT162b2 405,993,894 15,891,008 355 0.92 Yes – 3/13/2021 (RR = 1.91) mRNA-1273 426,696,226 15,757,044 303 0.80 No Ad26 COV2.S 13,297,825 576,495 14 1.11 No Non-hemorrhagic Stroke BNT162b2 403,683,932 15,799,026 7,882 0.85 No mRNA-1273 424,648,679 15,680,668 7,646 0.83 No Ad26 COV2.S 13,214,072 572,972 297 1.06 No Hemorrhagic Stroke BNT162b2 405,580,548 15,874,528 2,128 0.96 No mRNA-1273 426,350,816 15,744,161 1,958 0.88 No Ad26 COV2.S 13,283,534 575,871 77 1.12 No Immune Thrombocytopenia BNT162b2 554,443,925 15,857,949 1,670 1.26 Yes – 4/24/2021 (RR = 1.44) mRNA-1273 573,697,153 15,725,918 1,526 1.14 No Ad26 COV2.S 19,848,219 575,565 62 1.34 No Myocarditis/Pericarditis BNT162b2 555,100,577 15,876,623 1,415 1.10 No mRNA-1273 574,346,394 15,743,573 1,259 0.97 No Ad26 COV2.S 19,865,485 576,075 48 1.04 No Guillain-Barre Syndrome BNT162b2 532,555,069 15,892,318 69 1.14 No mRNA-1273 536,703,703 15,758,009 53 0.86 No Ad26 COV2.S 19,582,979 576,548 3.85 No Bell’s Palsy BNT162b2 575,124,693 16,453,363 3,618 1.12 No mRNA-1273 594,439,852 16,299,826 3,505 1.07 No Ad26 COV2.S 20,810,662 603,113 133 1.13 No Encephalomyelitis/Encephalitis BNT162b2 561,415,207 16,479,530 153 1.21 No mRNA-1273 572,296,487 16,324,225 120 0.98 No Ad26 COV2.S 20,648,928 604,024 1.68 No Transverse Myelitis BNT162b2 541,656,398 15,892,229 66 1.36 No mRNA-1273 552,699,497 15,757,865 48 1.00 No Ad26 COV2.S 19,700,796 576,539 1.10 No Narcolepsy BNT162b2 555,189,780 15,879,173 623 1.15 No mRNA-1273 574,433,145 15,745,902 537 0.98 No Ad26 COV2.S 19,864,965 576,089 23 1.15 No Appendicitis BNT162b2 548,982,786 15,880,177 1,180 1.06 No mRNA-1273 564,851,514 15,745,889 1,190 1.04 No Ad26 COV2.S 19,785,242 576,111 37 0.86 No a indicates small cell size counts with fewer than 11. b The relative risk incorporates adjustments for observation delay as well as standardization by nursing home residency, sex, age, and race when indicated by in Table 3. 3.3 Signal evaluation None of the prespecified data quality assurance checks, including claims duplication and unusual variability in claim accrual, raised data quality concerns (Table S9). Primary findings for signal robustness and signal characterization analyses are summarized in Table 3 . Adjustment for monthly variation in the background rates resulted in statistically non-significant associations for AMI, DIC, and ITP following BNT162b2 vaccination. With background rates from the flu-vaccinated population as the historical comparator, DIC and ITP no longer met the signal threshold, while signals for AMI (RR = 1.41) and PE (RR = 1.48) remained. When rates during the peri-COVID period were used as the historical comparator, PE and DIC no longer met the signal threshold. We conducted an additional ad hoc sensitivity analysis for PE. When PE events were restricted to the inpatient setting, the statistical signal remained (RR = 2.17).Table 3 Summary of Characterization of Associations for Acute Myocardial Infarction, Pulmonary Embolism, Disseminated Intravascular Coagulation, and Immune Thrombocytopenia identified via Diagnosis Codes after BNT162b2 vaccination in Medicare Shared Systems Database. Type of Evaluation Evaluation Conducted Summary of Findings Interpretation Signal Robustness PMaxSPRT with secular trends adjustment RR no longer statistically significant for AMI, DIC, and ITP. RR reduced but remained statistically significant for PE Potential confounding for AMI, DIC, and ITP PMaxSPRT with expected rates from influenza vaccinated population RR no longer statistically significant for DIC and ITP. RR remained statistically significant for AMI and PE Potential confounding for DIC and ITP PMaxSPRT with expected rates from peri-COVID-19 time, for PE, DIC, and ITP RR no longer statistically significant for PE and DIC. RR increased and remained statistically significant for ITP Potential confounding for PE, DIC PMaxSPRT with cases in the inpatient setting only, for PE outcome only RR increased and remained statistically significant for PE PE signal robust to inpatient-only definition Signal Characterization Summarized covariate distributions of vaccinated individuals compared to the general Medicare FFS population aged 65+ BNT162b2 vaccinated individuals had a higher proportion of nursing home residents and older individuals than the general elder population throughout the study period. This was not seen for mRNA-1273 or Ad26 COV2.S Potential confounding specific to the BNT162b2 vaccinated population Reviewed profiles of diagnoses and procedures on claims one year prior to and following case date for 100 sampled cases with clinical experts Outcome-specific risk factors seen for 41–96 % of sampled cases, depending on outcome. Outcome-specific treatments or diagnostics only observed for 18–53 % of sampled cases, depending on outcome Requires further investigation Summarized frequency of individual codes and claim settings used to flag outcomes in the historical and observation periods No differences between periods observed for DIC or ITP. A higher proportion of Type II AMI codes and non-primary inpatient diagnoses was observed in the observation period compared to the historical period for AMI. A higher proportion of subsegmental PE codes was observed in the observation period. Additionally, PE case counts in Nov and Dec 2020 were 20–40 % higher than in historical period (i.e., 2019) Requires further investigation Summarized frequency of prior COVID-19 diagnosis Rates of prior COVID-19 diagnoses were higher among BNT162b2 vaccinated individuals than the general population. Rates of prior COVID-19 diagnoses were higher among individuals with an AESI than among all vaccinated individuals. Yes, for all outcomes for which COVID-19 is a risk factor Performed temporal scans to identify clusters of cases post-vaccination No statistically significant cluster for PE, ITP, or DIC. Statistically significant cluster identified from 1 to 17 days following the first dose of BNT162b2 for AMI No specific narrow clusters of high risk identified for AMI, DIC, PE, or ITP Characterized relative risks by patient strata (nursing home residency status, sex, age, race, comorbidities status) Larger RR in the nursing home residents compared to the non-nursing home residents for AMI (1.53 vs 1.34), PE (1.66 vs 1.47), and DIC (2.72 vs 1.28). Larger RR in males compared to females for ITP (1.68 vs 1.28). For AMI, PE, and DIC elevated risk may be concentrated in the nursing home population Clinical subject matter experts reviewed claims-based diagnoses and procedures for selected patients from one year prior through one year after the dates of sampled outcome events. They found outcome-specific comorbidities were present in 41 %, 45 %, 95 %, and 66 % of AMI, PE, DIC, and ITP cases, respectively. Additionally, only a single diagnosis without other mention of the outcome occurred in 26 %, 37 %, 61 %, and 49 % of cases for AMI, PE, DIC, and ITP, respectively. Outcome-specific treatments or diagnostics were observed in 34 %, 53 %, 40 %, and 18 % of AMI, PE, DIC, and ITP cases, respectively. Evaluation of claims-based diagnosis codes showed differences in coding patterns for AMI and PE between the study period and the historical period used to calculate expected rates (Table 3). Type II AMI codes, which are indicative of a mismatch between myocardial oxygen supply and demand (as opposed to acute coronary thrombosis), were more common in outcomes identified during the study period (46 %) than in the historical period (28 %). Additionally, for both AMI and PE outcomes, a higher proportion of inpatient claims-based codes occurred in a non-primary diagnosis position in the study period. No differences in coding patterns between the study period and historical period were noted for either ITP or DIC. Finally, temporal scans conducted at the time of signal did not identify any clustering of cases within the post-vaccination risk window for PE, DIC, or ITP following the first dose of BNT162b2. A scan for clusters 1–18 days long identified a statistically significant cluster of 1–17 days for AMI; however, the observed rate of events in the cluster was only 4 % higher than if events had been evenly distributed in the risk window (Fig. 2 ). Another scan for clusters 1–10 days long that also accounted for censoring due to end of follow-up did not identify a statistically significant cluster for AMI (Fig. 2).Fig. 2 Distribution of Days to Diagnosis of Acute Myocardial Infarction, Pulmonary Embolism, Disseminated Intravascular Coagulation, or Immune Thrombocytopenia in 20 days after BNT162b2 Vaccination (First Dose) in Adults Aged 65 Years and Older, the Medicare Shared Systems Database. 4 Discussion Our early warning safety system is the first to identify-four new statistical signals for modestly elevated risks (RR less than 2) of four serious outcomes of AMI, PE, DIC, and ITP following BNT162b2 vaccination. This FDA and CMS COVID-19 vaccine safety study is one of the largest studies of elderly persons aged 65 years and above including approximately 34 million doses administered to more than 17 million Medicare insured persons. Our surveillance monitoring did not detect statistical signals for the mRNA-1273and Ad26 COV2.S vaccines for any of the 14 monitored outcomes. The statistical signals of four serious outcomes are not necessarily causal and may be due to factors potentially unrelated to vaccination. Additional analyses indicated that the potential association was less than twice the historical rates and may be associated with factors not accounted for in the near real-time surveillance methods. For example, the elderly Medicare population that received the BNT162b2 vaccine differed from other elderly COVID-19 vaccinated populations, including a preponderance of nursing home residents and populations with a higher comorbidity burden. These demographic and medical differences were not fully accounted for, since expected rates were only standardized to a subset of characteristics – age, sex, race, and nursing home residency status. Further, the AMI, DIC, and ITP signals were not robust when additional baseline rates were evaluated, while the PE signal might be explained by differences in rates between the pre-COVID-19 and peri-COVID-19 periods. In addition, the clinical assessment of patterns of reimbursement codes indicated that a substantial fraction had pre-existing outcome-specific comorbidities and risk factors, and that some outcomes may be due to follow-up care to an existing condition preceding the vaccination. Our study has several strengths. This is the largest study of a population of more than 25 million elderly persons who are vulnerable to COVID-19 infections and complications- including residents of long-term care facilities. By using the large Medicare nationwide database with longitudinal linkage of vaccination, health services, and demographic information for millions of elderly persons, we can detect even small increases in the relative risk of rare outcomes for multiple vaccines that may not be captured in pre-authorization clinical trials. In addition, this near real-time surveillance benefits from the experience and knowledge obtained during more than a dozen years of successful collaboration between FDA and CMS conducting vaccine safety analyses using the Medicare database [13], including near real-time surveillance analyses for Guillain-Barré syndrome after influenza vaccination [14], [15], [16]. Furthermore, the weekly data updates and analyses allow for signal detection across 14 outcomes using near-real time monitoring. This further expands our knowledge of COVID-19 vaccine safety for informing timely regulatory action, if warranted as well as decision-making by healthcare providers, patients and the general public. We acknowledge our analysis has limitations. The near real-time analysis did not adjust for underlying risk factors such as comorbidities among recipients in the early vaccination campaign leading to falsely positive or negative signals. Furthermore, the early warning system may falsely identify a signal (false positive) or signals because of the high number of statistical tests performed or possible misspecification of parameters. Conversely, true safety signals (false negatives) may be missed due to mispecified parameters in the analyses. Diagnosis billing codes in claims data may underestimate or overestimate certain clinical conditions because of reimbursement priorities. We also note that results of this near real-time surveillance in elderly persons may not be generalizable to those younger than 65 years and adults who are uninsured or received only commercial health insurance. To address several of these limitations we are conducting further epidemiological studies along with medical record review to adjudicate outcomes identified by claims-based definitions. In conclusion, we demonstrate that this FDA-CMS early warning safety system is working to rapidly identify potential new and important safety concerns following COVID-19 vaccination for consideration and to support potential decision-making by regulatory and public health authorities, healthcare professionals and the general public. Our new findings of statistical signals for four important outcomes for the BNT162b2 vaccine should be interpreted cautiously because the early warning system does not prove that vaccines cause the safety outcomes. FDA strongly believes the potential benefits of COVID-19 vaccination outweigh the potential risks of COVID-19 infection. Per FDA communication of these findings, FDA is currently not taking any regulatory actions based on these signal detection activities because these signals are still under investigation and require more robust study. The FDA active surveillance systems, including the CMS partnership, are a major part of a larger US federal surveillance effort to increase knowledge of COVID-19 vaccine safety to support decision-making that further protects public health during the pandemic. Conflict of Interest Statement Co-authors from U.S. Food and Drug Administration, Acumen LLC, and 4Centers for Medicare & Medicaid Servicesdeclared no conflicts of interests. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A Supplementary material The following are the Supplementary data to this article:Supplementary data 1 Data availability Related documents can be made available by contacting the corresponding author. De-identified participant data will not be shared without approval from the data partners. Acknowledgements We thank Joyce Obidi, PhD, Kristin “Kristine” Sepúlveda, MBA and Tainya C. Clarke, PhD, MPH, MSc for program management and J. Rosser Matthews, PhD, MPP, MPH for writing support. We would like to acknowledge John Hornberger, MD, MS, Nirmal Choradia, MD, Marna Bogan, RN, CPC, Susan Siford, PharmD, MBA, Christina Jessee, RN, CPC for their clinical and coding expertise. Additionally, we acknowledge Anchi Lo, MS, MPH, Jing Wang, BA, Yue Wu, MS, Zhiruo Wan, MSE, Shanlai Shangguan, MPH, Rowan McEvoy, BS, Arnstein Lindaas, MA, and Chianti Shi, MS for providing statistical programming support, and Yixin Jiao, MPP, Manzi Ngaiza, MPH, and Ellie Smith, BS for providing writing support. We acknowledge Michael Sklar, PhD, Lorene Nelson, PhD, MS, Julia Simard, ScD, SM, and Steve Goodman, MD, MHS, PhD for their epidemiologic expertise. Finally, we acknowledge Ivair Silva, PhD for his methodological expertise and development of the ‘Sequential’ R package used to conduct analyses in this manuscript. Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2022.11.069. ==== Refs References 1 Izurieta H.S. Graham D.J. Jiao Y. Hu M. Lu Y. Wu Y. 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Near real-time surveillance for Guillain-Barre syndrome after influenza vaccination among the Medicare population, 2010/11 to 2013/14 Vaccine 35 2017 2986 2992 28449973 15 Arya D.P. Said M.A. Izurieta H.S. Perez-Vilar S. Zinderman C. Wernecke M. Surveillance for Guillain-Barre syndrome after 2015–2016 and 2016–2017 influenza vaccination of Medicare beneficiaries Vaccine 37 2019 6543 6549 31515146 16 Perez-Vilar S. Wernecke M. Arya D. Lo A.C. Lufkin B. Hu M. Surveillance for Guillain-Barre syndrome after influenza vaccination among U.S. Medicare beneficiaries during the 2017–2018 season Vaccine 37 2019 3856 3865 31122853	36496287	PMC9712075	NO-CC CODE	2022-12-07 23:16:36	no	Vaccine. 2022 Dec 1; doi: 10.1016/j.vaccine.2022.11.069	utf-8	Vaccine	2,022	10.1016/j.vaccine.2022.11.069	oa_other
==== Front J Voice J Voice Journal of Voice 0892-1997 1873-4588 Published by Elsevier Inc. on behalf of The Voice Foundation. S0892-1997(22)00384-8 10.1016/j.jvoice.2022.11.034 Article Dysphonia Severity Index and Consensus Auditory-Perceptual Evaluation of Voice Outcomes, and Their Relation in Hospitalized Patients with COVID-19 Aghadoost Samira 1⁎ Molazeinal Yasamin 2 Khoddami Seyyedeh Maryam 1 Shokuhifar Ghazaal 3 Dabirmoghaddam Payman 4 Saffari Maryam 5 1 Department of Speech Therapy, School of Rehabilitation, Tehran University of Medical Sciences, Tehran, Iran 2 MSc at School of Rehabilitation, Tehran University of Medical Sciences, Tehran, Iran 3 Department of audiology, University of Social Welfare and Rehabilitation, Tehran, Iran 4 Otolaryngology Research Center, Tehran University of Medical Sciences, Tehran, Iran 5 Department of radiology, faculty of medicine, Kashan University of Medical Sciences, Tehran, Iran ⁎ Corresponding author: 1 12 2022 1 12 2022 28 11 2022 © 2022 Published by Elsevier Inc. on behalf of The Voice Foundation. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objectives : This study aimed to compare the results of the Dysphonia Severity Index (DSI) and Consensus Auditory-Perceptual Evaluation of Voice (CAPE-V) between patients hospitalized with COVID-19 and healthy subjects, as well as to investigate the correlation between DSI and CAPE-V. Study design : Cross-sectional survey. Material and methods : Eighty subjects, 40 COVID-19 patients (with a mean age of 41.2± 5.41) and 40 healthy subjects (with a mean age of 44.50± 3.50) participated in this study. Assessments included the DSI for aerodynamic-acoustic measurement and the Persian version of CAPE-V for evaluating auditory-perceptual voice quality. Data were analyzed by means of the independent t-test and Pearson correlation at the 5% significance level. Results : The results showed COVID-19 patients got significantly lower score in DSI compared to healthy subjects (P < 0.05). Moreover, the patients with COVID-19 had higher scores in all categories of voice production (severity, roughness, loudness, pitch, strain and breathiness) than the healthy group (P < 0.05). Comparing the result of the two voice assessments in each group revealed that there was a greater negative significant correlation in the diseased group (r p: -0.68, p: 0.001) than in the healthy group (r p: -0.37, p: 0.049). Conclusion : Hospitalized COVID-19 patients experience deviations in the voice quality and acoustic-aerodynamic features of their voice. Also, the results of this study showed the patient group had higher perceptual dysphonia and lower voice quality compared to the healthy group. Further studies are recommended to determine the relationship between objective and subjective voice evaluation in patients with COVID-19 after recovery. Keywords COVID-19 dysphonia Dysphonia severity index (DSI) Consensus Auditory-Perceptual Evaluation of Voice (CAPE-V) Voice ==== Body pmcIntroduction COVID-19 is a high-risk infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-21 ) which has been affecting the world since 2019. It has been becoming one of the most challenging problems the world has faced in the recent decade1, 2, 3. Among all of the COVID-19 symptoms, 57.4% of infected patients experience destructive effects on structures that are involved in voice production such as the lung, larynx, nose, and throat4, 5, 6. According to previous studies, voice complaints especially dysphonia can be present in about 43.7% of COVID-19 patients; this prevalence was reported at 71.4% in hospitalized patients7 , 8. Optimal voice requires precise coordination and interaction between all subsystems of phonation, including the power system, voice source, and sound resonator (Herbst, 2017)9, 10, 11, 12. COVID-19 can cause voice disorders in three possible ways13 , 14. The first theory is about the direct entry of the SARS-COV-2 pathogens into the larynx tissue and connecting to a specific receptor which can lead to tissue inflammation15, 16, 17. The second theory minds the patients who need to be intubated due to COVID-19 and the injury and irritation of vocal folds after intubation is one of the consequences8. The last theory states that the viral neuropathies that have been seen in patients infected with COVID-19 that impact both the recurrent laryngeal nerve and the superior laryngeal nerve, so as a result dysphonia can be expected18. So that due to the mentioned hypothesizes, COVID-19 can cause direct damage to the vocal tract, larynx, and vocal cords, and leading to changes in their functioning5 , 10 , 15, 16, 17, 18. As a result, dysphonia, limitation in loudness, pitch, harshness, and breathiness occur; therefore, voice quality deteriorates in COVID-19 patients5 , 10. The impact of COVID-19 on the respiratory system can include lung infection, pneumonia, and reduced respiratory volume, as well as acute respiratory distress syndrome19 , 20. Changes in lung function secondary to respiratory tract infections commonly seen in patients with COVID-19 can lead to reduced respiratory support for voice and speech; This impact on voice production is recognized in mild-to-severe cases of COVID-19 and is evidenced by the voice assessment outcomes performed in this and similar studies21, 22, 23. For instance, inadequate airflow increased aperiodicity, and signal perturbation are examples of these affected aspects24. Voice production and subsequent vocal quality are influenced by many factors25 , 26. Neurological, medical, and structural deviations in the vocal tract, larynx and respiratory function, inappropriate use of voice, and also psychological issues are the factors that can cause voice disorder27. The mentioned factors are evaluated with the diagnostic assessments such as interview, medical examination, self-evaluation, auditory perceptual assessments, aerodynamic-acoustic, and vocal folds’ functional evaluation27. In studies related to COVID-19 the voice of the patients assessed with different voice evaluations; such as auditory-perceptual assessments (CAPE-V2 and GRBAS3 )24 , 28 , 29, self-report voice questionnaires such as VTD4 and VHI-105 28 , 30, aerodynamic assessments(S/Z ratio and MPT6 )31, and acoustic voice analyses like shimmer, HNR, H1H2, CPP, and jitter(12, 32). The use of complimentary voice assessments, e.g., auditory-perceptual and acoustic-aerodynamic measurements, improves the quality of clinical information and allows for a more comprehensive treatment plan33 , 34. The Dysphonia Severity Index (DSI7 ) is a popular objective voice assessment tool among voice therapists because it measures multiple parameters of voice production.35, 36, 37. On the other hand, the auditory-perceptual assessment tools are known as the commonly methods that are done quickly and are available such as the Consensus Auditory-Perceptual Evaluation of Voice (CAPE-V) or GRBAS26 , 38. These voice assessments are auditory-perceptual subjective judgments of voice quality based on the opinion of voice therapists38. CAPE-V seems to be more sensitive to small changes compared to the GRBAS39 , 40. However, rater's experience is a crucial element for increasing accuracy of auditory voice assessments. Consequently, the use of objective instruments such as multi-parametric voice formulas is suggested to cover the limitations of auditory-perceptual evaluation of voice38 , 41. Some studies confirmed the correlation between objective with subjective voice assessments in patients with voice disorders and healthy speakers35 , 42, 43, 44 The previous studies revealed a significant difference between various aspects of voice quality in COVID-19 patients and healthy people10 , 12 , 24 , 30. All of them have shown that the disease could damage various dimensions of voice after recovering from the COVID-19 infection10 , 32 , 45, 46, 47, but few studies were conducted during the bedridden period of these patients in the hospital24 , 28 , 29. In this study, we intend to examine the effect of COVID-19 infection on the different aspects of voice by implementing DSI and CAPE-V in patients during their bedridden time in the hospital. Then, we would determine DSI and CAPE-V in the COVID-19 patients versus healthy subjects. This is the first study to investigate the correlation between subjective (CAPE-V) and objective voice analysis (DSI) in hospitalized patients with COVID-19 infection that can hold a remarkable contribution to the various voice assessments utilized for these patients. MATERIALS AND METHODS Ethical consideration This study was a cross-sectional analytic study. All participants completed the consent form before data collection. The study was approved by the Ethics Committee affiliated with the Rehabilitation Faculty at Tehran University of Medical Sciences (IR.TUMS.FNM.REC.1399.169). Participants Eighty subjects, 40 patients with positive laboratory-confirmed COVID-19 (with a mean age of 41.2± 5.41), and 40 healthy subjects (with a mean age of 44.50± 3.50) participated in the present study. Each group had 27 men and 13 women. The inclusion criteria for the subjects were as follows: adults who have no previous history of voice disorders, no history of respiratory disorders, and no history of allergy, hearing loss, endocrine diseases, neurologic disorders, and addiction. Moreover, getting a COVID-19 diagnosis (mild to moderate) in the patients' group and negative results for healthy subjects was required. Individuals who were not willing to participate in voice assessment, females who had menstrual periods, and patients with severe COVID-19 who could not cooperate because they were in the ICU at the time of the study were excluded. COVID-19-positive patients in this study were those who were admitted to the Shahid Beheshti and Amiralam hospitals in Iran. Patients were evaluated after being in the hospital for at least three to five days (with a mean of 4.3±1.8 days) and being able to participate in the study. The current study only included patients with mild to moderate COVID-19. To be more exact, the clinical symptoms of these patients vary from mild complications, negative pneumonia signs in imaging, to vocal tract infection, pneumonia symptoms, and fever in moderate patients48. Also, the COVID-19 patients did not experience failure in respiration; so, they did not need mechanical ventilation. A PCR and a chest computed tomographic (CT8 ) scans are typical tests for diagnosing COVID-19, according to the World Health Organization's (WHO) guidelines. The CT scan is usually the first diagnosis technique that is performed for patients suspicious of COVID-19 is performed for and a positive PCR result validated the COVID-19 diagnosis following the CT scan. All of the COVID-19 participants had positive PCR test after getting CT results suspicious of COVID-19. Healthy subjects matched according to age and gender were volunteers from the staff of Amiralam hospital and Tehran University of medical sciences. Procedure and Assessments In the first, demographic data and medical history were gathered by interview. After detecting subjects with inclusion criteria in the COVID-19 group and also in the healthy group, voice sampling was done in a quiet room with a noise level lower than 40 dB49. We used a Zoom H5 microphone (frequency response 5 Hz to 20 kHz) for voice sampling (24 bits, and 44.1 kHz sampling rate). The microphone was placed at a distance of 5 to 10 cm at a 45° angle from the patient's mouth. The necessary explanation for data gathering was explained orally and the sampling procedure was performed considering health protocols to avoid SARS-COV2 transmission. Before the initiation of recording the voice samples, an examiner that was an SLP modeled the tasks for the subjects. Finally, the recorded voice samples, which were collected for analyses of auditory-perceptual, acoustic, and aerodynamic assessments, were transferred to a laptop (Acer Aspire E 14). These data were analyzed with Praat software (version 6.0.39). Acoustic-aerodynamic assessment Another voice sampling was performed for evaluating the acoustic and aerodynamic measures to determine the DSI score35. DSI is a multi-parametric voice assessment that can be an indicator of voice quality as it is affected by acoustic and aerodynamic characteristics. After analyzing the voice samples, acoustic and aerodynamic features including the highest frequency9 (Hz), lowest intensity10 (dB), MPT (seconds), and jitter (%) were extracted according to the instructions of the DSI scale. All acoustic and aerodynamic measures were assessed and analyzed with the Praat software version 6.0.3950. To perform all of these analyses, each task was performed three times and the best result was selected and reported by Praat software. For obtaining MPT, the patients were asked to produce /a/ continually after a deep inhalation for as long as possible in the sitting position as a part of the aerodynamic examination. For calculating F0-High, we explain the instructions to the patients; to produce /a/ sustainably by raising the frequency from normal to as high as possible without changing the intensity of their voice for 5 seconds. The patients performed the maneuver, and then we recorded the voice sample and analyzed them. Finally, for I-Low and Jitter, we first ask them to produce sustained vowel /a/ as comfortable and soft as possible for 5 seconds. After recording the samples, the first second of every sample was extracted and the middle 3 seconds of each voice sample were analyzed with Praat software. The best result of each acoustic and aerodynamic assessment in three trials of a patient was placed in the DSI formula (DSI= 0.13 × MPT + (0.0053 × F0-High) - (0.26 × I-Low) - (1.18 × Jitter %) + 12.4) and ultimately the overall score was determined. According to the study by Wuyts et al. (2000) the score of DSI is between +5 to -5 and the score of +5 represents the normal voice and -5 determines severe dysphonia35 , 51 Auditory-perceptual assessment In this study, an auditory-perceptual assessment of voice was performed using the Persian version of CAPE-V11 52. This questionnaire is a perceptual clinician-based judgment of voice quality. According to the CAPE-Vp protocol, three tasks including producing sustained vowels /a/ and /i/ for 3 to 5 seconds, reading six valid sentences, and narrative speech were assessed52. The audio files were gathered and transferred to the laptop. The files were randomized and then sent to two experienced voice therapists with more than 7 years of experience in voice therapy and familiar with CAPE-Vp scoring, who determined CAPE-Vp evaluation points in total and subtest of them blindly. The raters gave 6 scores from 0 to 100 for CAPE-Vp subtests including overall severity, roughness, loudness, pitch, strain, and breathiness of voice quality. A score of 0 indicates normal voice, and the level of dysphonia increases as the CAPE-Vp points rise (1-9 mild dysphonia, 10-59 moderate dysphonia, 60-100 severe dysphonia)24. The evaluators marked a dot on a one-hundred-millimeter horizontal line, the closer the mark was to the end of the line and 100, the more severe the dysphonia was. The raters gave scores to each participant in a quiet room using headphones set at a comfortable volume level of 40 dB hearing level. As we noted above, scoring in this section was done blindly. After determining the scores of all parts of CAPE-Vp by two evaluators; 25% of the samples were randomized and sent to themselves again to score, and in this way, we calculated the intra-rater reliability for each evaluator separately. This process was done to ensure the accuracy of the auditory perceptual evaluation data and the reliability of the auditory-perceptual assessment. In the end, the results of the evaluator who had the highest intra-rater reliability index were selected as the final score of the CAPE-Vp assessment (r: 0.86%). The results were analyzed by Pearson's correlation coefficient in SPSS software. Statistical analysis In this study, descriptive statistical analysis has utilized for the determination of the frequency, standard deviation, and mean of the variables. To evaluate the normal distribution of the variables the Kolmogorov-Smirnov test‌ was used. An independent Sample T-Test was used to compare the results of CAPE-V and DSI between the two groups. Pearson correlation coefficient was used for determining of correlation between the total score of DSI and CAPE-Vp in both groups. The SPSS software (version 22; SPSS Inc, Chicago, IL) was used for statistical analysis and the level of significance was set at p ≤ 0.05 with a 95% interval confidence which is considered statistically significant. Results In this study, we assessed eighty subjects (with a mean age of 42.76± 4.88 years) in two groups including COVID-19 and healthy subjects. Also, the Kolmogorov-Smirnov test demonstrated the normal distribution of data in two groups (p> 0.05). The demographic data are presented in Table 1 .TABLE 1 Demographic Characteristics of the Participants in Patients with COVID-19 (n= 40) and Healthy Subjects (n= 40). TABLE 1 Age Gender Mean SD Frequency Percent Male Female Male Female Patients with COVID-19 41.2 5.41 27 13 67.5% 32.5% Healthy subjects 44.50 3.50 27 13 67.5% 32.5% Pvalue P< 0.05 P< 0.05 ⁎ Independent Sample t-test Considering Table 1, Participants were matched in the age and sex in both groups by an independent t-test. Furthermore, the analysis results of the DSI score and its components in the two groups of this study are shown in table 3 in detail and compared between COVID-19 patients and healthy subjects. In table 2 the mean± SD of the total score of DSI was 0.40±0.94 in COVID-19 patients and 3.65±0.86 in healthy subjects. As presented in Table 3 , the mean scores of the F0-high, MPT, and DSI were lower in the COVID-19 patients than in the healthy group. Also, I-low and jitter were higher in the COVID-19 patients than in healthy subjects (p≤0.00). As we see, there was a significant difference between the participants of the two groups regarding the DSI and its components (p≤0.00).TABLE 2 Comparison of DSI Score and its Components Between Patients with COVID-19 (n= 40) and Healthy subjects (n= 40). TABLE 2Self-assessment scores Groups Mean ± SD Pvalue Acoustic and Aerodynamic Components (DSI) MPT patients with COVID-19 9.90±2.54 0.00* healthy subjects 17.24±3.92 F0-high patients with COVID-19 345.82±46.33 0.00* healthy subjects 420.45±50.07 I-low patients with COVID-19 55.55±3.26 0.00* healthy subjects 49.90±1.90 Jitter patients with COVID-19 0.56±0.21 0.00* healthy subjects 0.19±0.12 DSI patients with COVID-19 -0.40±0.94 0.00* healthy subjects 3.65±0.86 DSI: Dysphonia Severity Index; SD: Standard Deviation. MPT: Maximum Phonation Time. ⁎ Independent Sample T-test. TABLE 3 Comparison of the CAPE_Vp and its Components Between Patients with COVID-19 (n: 40) and Healthy Subjects (n: 40). TABLE 3Self-assessment scores Groups Mean ± SD Pvalue CAPE-Vp Component Roughness patients with COVID-19 40.90±12.54 0.00 healthy subjects 17.24±3.92 Breathiness patients with COVID-19 49.82±19.33 0.00* healthy subjects 15.45±5.07 Strain patients with COVID-19 35.15±18.26 0.00* healthy subjects 9.91±3.90 Pitch patients with COVID-19 37.56±18.21 0.00* healthy subjects 18.19±6.12 Loudness patients with COVID-19 24.90±12.54 0.00* healthy subjects 4.24±2.92 Overall Severity of patients with COVID-19 62.7±17.94 0.00* healthy subjects 24.65±6.86 CAPE-V: Consensus Auditory-Perceptual Evaluation of Voice; SD: Standard Deviation., numbers (%) ⁎ Independent Sample T-test. The results of subtests and total score of CAPE-Vp assessment were analyzed by independent sample T-test and reported in Table 3 in two groups separately. The result of Table 3, showed that the overall score of CAPE-Vp in COVID-19 patients and healthy participants were 62.7±17.94 and 24.65±6.86 (mean± SD) respectively. In conclusion, as shown in Table 2, there was a significant difference in the results of the overall CAPE_Vp score and its subtests including roughness, loudness, pitch, strain, and breathiness between the two groups of patients with COVID-19 and healthy individuals (p<0.005). Finally, the correlation between the total scores of DSI and CAPE-Vp was assessed in two groups separately. Pearson correlation analysis revealed a significant correlation between the DSI and severity of CAPE-Vp in the COVID-19 infected group (r Pearson= -0.68, p= 0.001); on the contrary, the correlation in the healthy group between DSI score and total score of CAPE-Vp was relatively lower (r Pearson= -0.37, p= 0.049). Discussion In this study, we investigated the effect of COVID-19 on acoustic and perceptual voice assessment with the DSI formula and CAPE-Vp scale. Also, we intended to determine the correlation between these objective and subjective voice assessments in COVID- 19 patients and healthy subjects. After analyzing the tests’ results it was evident that SARS-COV-2 affected DSI and CAPE-Vp scores notably in the patients’ group in comparison to the healthy group. Moreover, a remarkable correlation was illustrated between the total scores of DSI and CAPE-Vp in both groups of study, although the COVID-19 group had a greater negative relationship comparing the healthy group. In this study, the DSI formula was used as a multi-parametric measurement of acoustic and aerodynamic features of voice. As mentioned before, this multi-parametric measurement is a score that represents objective voice quality as it combines acoustic and aerodynamic parameters35 , 53. This study showed that the total score of DSI and its components (MPT, F0-high, I-low, jitter) were harmed statistically significant (P ≤ 0.05) during the infection period in the COVID-19 inpatient group (DSI: -0.40±0.94) in contrast of the healthy group (DSI: 3.65±0.86) (P ≤ 0.05). To be more precise due to SARS-COV-2 infection, unlike the healthy group, in the diseased group I-low and jitter increased, MPT duration and the highest F0 decreased. As we mentioned, jitter was lower in the patients' group (0.56±0.21) compared with the healthy ones (0.19±0.12) and this may be explained based on the hypothesis of inflammation, uneven weighing, incomplete glottis closure, and deterioration of vocal folds' tissue leading to aperiodicity and irregularity in vocal fold vibration after recurrent coughs and virus tissue entry12 , 54. MPT duration in the COVID-19 patients (9.90±2.54 Sec) was shorter than healthy group (17.24±3.92 Sec) because of the reduced respiratory support, and also may be due to disturbed glottal closure after possible larynx inflammation, and insufficient coordination between respiratory and phonatory subsystems of speech12 , 19 , 20, and maybe because of the hypothesis regarding nerve damage and sensory neuropathy caused by COVID-1955. It seems that inadequate control of the larynx and airflow pressure and increased glottal muscle tension during vocalization due to the COVID-19 infection leads to free edge alteration and finally changes vibratory rates of vocal folds or decreased F0-high (345.82±46.33 in patients and 420.45±50.07 in healthy people)12 , 32. Furthermore, we observed that I-low was greater in the patient group (55.55±3.26), and it can be due to increased glottal resistance that necessitates more pressure to initiate vocalization than healthy subjects (49.90±1.90)35. Almost similar assessments were reported by the other studies assessing other aerodynamic and acoustic features in COVID-19 patients such as S/Z ratio, MPT, shimmer, HNR, H1H2, CPP12 , and jitter10 , 12 , 24 , 54. Based on the study of Asiaee12 et al. the results of acoustic assessments such as jitter, shimmer, and MPT were significantly different between the COVID-19 and the healthy subjects12. Also, the same results were reported by Saki et al.in evaluating MPT in two groups of COVID-19 and healthy ones24. Moreover, Shahpasand et al. mentioned in their poster presentation that there were pathological values for DSI in COVID-19 patients but there wasn't any published data54. Contrary to our results in this study, Gölaç et al. did not find any difference in the acoustic assessments' results such as HNR, jitter, and shimmer; the logical explanation of these results includes the time of sampling in the Gölaç study10. It seems that these diverse results are due to that patients in Gölaç study were assessed about 8 months after recovery and not during the bedridden time; however, our results were in line with the MPT values10. Ultimately, it appears that discoordination between the lung as a source of airflow and the larynx as a vocalization box for voice production can increase muscle tension in the larynx and affect each parameter of DSI and consequently the total score of DSI. CAPE-Vp is a perceptual clinician-based judgment questionnaire of voice quality that determines the severity of dysphonia perceptually. In this study, the total score and subtests scores of CAPE-V were affected and deteriorated significantly due to COVID-19 in the patient group compared to the healthy group. The worsened voice quality in auditory-perceptual assessment in these patients might be probably due to the changes after tissue inflammation of the larynx52. Based on Lee et al. neuropathy in vague nerve, specifically recurrent laryngeal nerve can cause recurrent cough which is in line with COVID-19 hypothesis mentioned before56. Frequent coughing in COVID-19 patients irritates vocal folds and leads to tension and hard closure of the folds, therefore dysphonia is not unexpected12 , 18. Based on the given reasons, it is evidently demonstrated in the severity score, for it shows the severity and grade of dysphonia in these patients. Moreover, due to the insufficient air pressure and insufficient respiratory capacity caused by COVID-19, and alteration in vocal folds’ vibrating pattern and closure12 , 30 , 57, patients vocalize with more strain, roughness, and breathiness; and also their voice loudness and pitch deteriorate30. Moreover, another reason for the findings of this study can be the fact that patients were assessed during the acute phase of COVID-19 infection who were hospitalized in the ward. Our results were in line with former studies done by Saki et al. and Tahir et al. regarding CAPE-Vp results in assessing the voice of COVID-19 patients24 , 30. In another study by Tohidast et al. (2020), the voice quality of COVID patients was evaluated by the GRBAS scale and the impact of Covid-19 on these aspects of the patient's voice was also declared to be statistically significant28 that was parallel with our study. Moreover, the correlation between DSI as an objective evaluation scale and CAPE-Vp as a subjective evaluation tool in each group showed there was a notable relationship between the total scores of CAPE-Vp and DSI in the COVID-19- group; although, the correlation between them in the healthy group was lower than patients’ group. These voice assessment tools including DSI and CAPE-Vp are generally used for distinguishing dysphonic and healthy individuals58. Both DSI and CAPE-Vp reflect the severity of dysphonia based on an objective and subjective manner respectively58 , 59. As mentioned by Nemr et al. patients who have dysphonia and get lower scores in voice quality assessments, also tend to have significantly lower scores in DSI60. It seems, in COVID-19 dysphonic patients a significant correlation between the measures depicts that the DSI can be a good predictor of auditory-perceptual voice quality evaluated by the CAPE-Vp scale44. These findings can show that although acoustic-aerodynamic measurements (DSI) and auditory-perceptual assessments (CAPE-Vp) evaluate different voice dimensions, they can be related and support the results each other in COVID-19 patients and healthy subjects. Deeming that voice is a multidimensional phenomenon, it is not surprising that the various aspects of voice evaluations especially in dysphonic patients confirm each other. Some studies have assessed the correlation between these two voice assessment tools, but as far as we searched none of them were performed on COVID-19 patients, and there wasn't any data comparing these assessments in COVID-1942 , 44. In fact, former articles that were done by Ataee et al. and Nemr et al. assessed the relation between DSI and CAPE-Vp and revealed consistent results with ours regarding the overall scores in other dysphonic patients42 , 44. This study had some limitations. Firstly, it was performed during the acute time of the pandemic era which limited further investigations such as laryngo-video-stroboscopy, so we had no data about the structure or function of the vocal cords of the subjects. Secondly, transferring the patients to a quiet and appropriate room for recording voice samples was difficult due to the limitations of distancing protocols. Data gathering was performed during 3 to 5 days after hospitalization. Therefore, defining the exact phase of the disease was not feasible. Furthermore, to perform a thorough investigation, Inflammation and sensory neuropathy can only be evaluated by direct imaging or tissue sampling, and an EEG respectively; which we could not perform in the study. Moreover, it is suggested that these limitations should be addressed in future research on these patients after recovery from COVID-19 infection and later stages as follow-up with different assessment tools to determine the quality of voice. Conclusion This study suggests that hospitalized COVID-19 patients experience difficulties in their voice. They experience more roughness, breathiness and strain, and inappropriate pitch and loudness during their infection period. Also, the aerodynamic and acoustic feature of their voice deteriorates during the disease and should be considered during assessments. Finally, the DSI scale can be used for assessing aerodynamic and acoustic parameters and predicting the overall quality of voice and it can reflect the severity of dysphonia based on an objective manner. Acknowledgments We would like to appreciate the participants of the study both COVID-19 patients and healthy individuals who helped us perform this study. 1 Severe Acute Respiratory Syndrome Coronavirus 2 2 Consensus Auditory-Perceptual Evaluation of Voice 3 Grade, Roughness, Breathiness, Asthenia, Strain 4 Vocal Tract Discomfort 5 Voice Handicap Index-10 6 Maximum Phonation Time 7 Dysphonia Severity Index 8 Computed Tomographic 9 F0-High 10 I-Low 11 CAPE-Vp 12 Cepstral Peak Prominence ==== Refs References 1 Chikhalkar B Gosain D Gaikwad S Deshmukh R. Assessment of National Early Warning Score 2 as a Tool to Predict the Outcome of COVID-19 Patients on Admission Cureus 14 1 2022 e21164 2 Singhal T. A Review of Coronavirus Disease-2019 (COVID-19) Indian J Pediatr 87 4 2020 281 286 32166607 3 Wu Z McGoogan JM. 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==== Front J Environ Manage J Environ Manage Journal of Environmental Management 0301-4797 1095-8630 Elsevier Ltd. S0301-4797(22)02484-7 10.1016/j.jenvman.2022.116911 116911 Article Predictive modeling and analysis of air quality – Visualizing before and during COVID-19 scenarios Persis Jinil a∗ Ben Amar Amine b a Indian Institute of Management (IIM), Kozhikode, Kerala, India b Excelia Business School, La Rochelle, France ∗ Corresponding author. 1 12 2022 1 2 2023 1 12 2022 327 116911116911 23 5 2021 26 9 2022 26 11 2022 © 2022 Elsevier Ltd. All rights reserved. 2022 Elsevier Ltd Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Quality air to breathe is the basic necessity for an individual and in recent times, emission from various sources caused by human activities has resulted in substantial degradation in the air quality. This work focuses to study the inadvertent effect of COVID-19 lockdown on air pollution. Pollutants' concentration before- and during- COVID-19 lockdown is captured to understand the variation in air quality. Firstly, multi-pollutant profiling using hierarchical cluster analysis of pollutants' concentration is performed that highlights the differences in the cluster compositions between before- and during-lockdown time periods. Results show that the particulate matter (PM10 and PM2.5) in air that formed the primary cluster before lock-down, came down to close similarity with other clusters during lockdown. Secondly, predicting air quality index (AQI) based on the forecasts of pollutants' concentration is performed using neural networks, support vector machine, decision tree, random forest, and boosting algorithms. The best-fitted models representing AQI is identified separately for before- and during-lockdown time periods based on its predictive power. While deterministic method reactively evaluates present AQI when current pollutants' concentration at a particular time and place are known, this study uses the best fitted data-driven model to determine future AQIs based on the forecasts of pollutant's concentration accurately (overall RMSE<0.1 for before lockdown scenario and <0.3 for during lockdown scenario). The study contributes to visualize the variation in pollutants' concentrations between the two scenarios. The results show that the reduced economic activities during lockdown period had led to the drop in concentration of PM10 and PM2.5 by 27% and 50% on an average. The findings of this study have practical and societal implications and serve as a reference mechanism for policymakers and governing bodies to revise their actions plans for regulating individual air pollutants in the atmospheric air. Keywords COVID-19 Air pollution Air quality index Machine learning ==== Body pmc1 Introduction Rapid industrialization and urbanization in low- and middle-income countries have led to increased air pollution causing environmental degradation and health hazards (Balietti et al., 2022). Combustion of fossil fuels, industrial emissions, and natural calamities are potential causes of air pollution (Fromer et al., 2019; Jiang and Yu, 2020). Human activities contribute much to air pollution. PM2.5, PM10 (grade of particulate matter having diameter less than or equal to 10, 5, 2.5, 1 μm is shown in subscript) in cities are emitted predominantly from vehicles (X. Ma et al., 2020; Mukherjee et al., 2020; Yang et al., 2020). Pollutants like carbon monoxide (CO), hydrocarbon (HC), Nitrogen oxides (NOX), PM2.5, PM1.0 and volatile organic compounds (VOCs) are extensively present in business zones of urban area and NOX, PM2.5 and PM1.0 are highly present in air in the industrial zones (Song et al., 2019). Further, diffusion and persistence of these pollutants in air is greatly affected by meteorological factors such as wind speed, direction, turbulence, stability, humidity, temperature, radiation and rainfall (Hewson, 1956). Pollutants in air when inhaled causes serious health hazards and give rise to ecological disturbances against which eco-friendly industrial practices, agricultural practices, plant designs and control technologies are suggested by many researchers (Jiang and Yu, 2020; Liu et al., 2020; Wang and Lu, 2020; Xiong et al., 2020; C. Zhang et al., 2020; Zhuang et al., 2020). As part of preventive and control measures to combat air pollution, regulations for vehicles, industries, and power plants are enforced; alternative fuels and zero-emission vehicles are introduced (Bakır et al., 2022; Guo et al., 2020; Sahu et al., 2021; Saz-Salazar et al., 2020; Yadav et al., 2019). (Perman and Stern, 2003) argues that the environmental improvement in developed countries is due, at least in part, to the fairly strict environmental regulations. However, such regulations are not in line with the priorities of the low- and middle-income countries that specialize in natural resources and low-skilled labor (Grossman and Krueger, 1995; Tsurumi and Managi, 2010). have highlighted the economic determinants of environmental quality since large-scale economic activities, which exacerbates industrial combustion, result in high emission volume of VOC and PM in the atmospheric air. India rigorously focus on their mission toward “clean air” to tackle the nexus between economic development and environmental degradation (Sahu et al., 2021). The effect of globalization on air pollution are alarmingly felt in Indian cities and India has six out of ten most polluted cities in the world (as per 2021 IQAir records). For instance, World Health Organization (WHO) has already indicated that Delhi has exceeded the maximum PM10 limit by ten times in 2011 itself and the residents face lot of health difficulties due to polluted air. Hence, there is a need to control air pollution caused by individual pollutants at its source and regulate air quality to build a healthy nation. While preventive measures through regulations and policies have been focused on, reactive strategies too are adopted conditionally based on the current value of the air quality index (AQI) which is estimated from the pollutants’ concentration in the air sensed at an instant of time (Tan et al., 2021). When AQI drops alarmingly down, factories adopt mitigation plan to clean the air through the use of filters and purifiers (Jiang and Yu, 2020; Minet et al., 2018; Vijayan et al., 2015). However, appropriate proactive strategies help prevent or control pollution that would be possible if AQI is predicted accurately, and further, the situation can be safely handled with necessary precautions and preparedness which is the focus of this study (Qiu et al., 2020; Ying Wang et al., 2020). According to NASA (2020), during the COVID-19 lockdown, AQI level was found to be lesser than normal and pollutants’ concentration such as nitrogen dioxide in the atmospheric air was detected to be less (Dang and Trinh, 2021). elucidates the overall reduction in concentration of nitrogen dioxide and PM2.5 globally during lockdown and suggests to adopt reduced mobility as much as possible further to sustain these improved air conditions in future. It is noteworthy that forecasts made by the existing climatic models that assumed business as usual scenario on pollution during COVID-19 lockdown was very much deviating from the actuals. Hence there is a need to reinvestigate the variation in the air quality levels along with its associated determinants (Streiff, 2020). Air quality levels across India were in the downtrend till 2019, however, in 2020, many cities in India recorded improved air quality (According to Statista report 2021) (Bakır et al., 2022). To control the spread of novel coronavirus disease in early 2020 (COVID-19), social distancing and lockdown were enforced that curbed all non-essential and non-emergency human outdoor activities. During this period, it is found that emission of air pollutants has drastically reduced in atmospheric air and eventually air quality substantially increased in many Indian states due to the suspended industrial activities and reduced vehicular movements (Elsaid et al., 2021; Pal et al., 2021). Motivated by this, the authors focused on the following research question. What significant differences COVID-19 lockdown has influenced on the air pollutants’ concentration and in turn on atmospheric air quality? The authors selected prime locations in India where air pollution is generally reported to be alarmingly high. Pollution control board of both central and state governments (CPCB, SPCB) in India is keen about national air quality index. Existing method determines present AQI at a place by capturing the concentration of individual pollutants in unit volume of atmospheric air using appropriate sensors and uses them to evaluate AQI only if at least three pollutants' concentration is available that must include either PM2.5 or PM10 or both. Pollutants' concentration is sensed every hour and AQI values are so computed. This study attempts to determine future AQI values using time-series analysis of pollutants' concentration. In order to model air quality depicting both before and during COVID-19 lockdown scenarios, timeseries data of pollutants' concentrations in selected regions is collected for both the time periods and analyzed through machine learning approaches. Using unsupervised learning approach, multi-pollutant clusters are first formed for these regions for both the scenarios which can reveal variation in pollutants' concentration. Further, using supervised learning approach, data-driven models are developed to foresee air quality at a particular place. We explore the use of Linear regression modeling (LM), support vector regression (SVR), artificial neural network (ANN), decision tree (DT), random forest (RF) and extreme gradient boosting tree (XGB) to determine future AQI values with the forecasts of pollutants' concentrations and the best-fitted model with the highest predictive performance is selected for each region. Understanding the variation in pollutants’ concentrations individually before- and during-lockdown scenarios in a particular region not only help policymakers to frame an appropriate pollutant-specific control strategy, but also, in determining future AQI values allowing to adopt appropriate mitigation plan. 2 Related work Literature evidences show that predicting individual pollutants using learning methods help mitigating them by addressing various emission and non-emission factors associated with them. Forecasts of PM2.5 and NOx concentrations individually under known meteorological and traffic information are obtained accurately using random forest algorithm with high predictive power (Z. Li et al., 2020). Timeseries analysis of O3, SO2, CO, NO2 concentrations using adaptive neuro-fuzzy inference system is performed and the prediction accuracy is found to be higher than the traditional regression methods (Zeinalnezhad et al., 2020). The concentration of O3 is estimated using extreme gradient boosting algorithm and the performance of this algorithm is found to be high when compared with back propagation neural network, generalized regression neural network, Elman neural network, extreme learning model, linear regression, and random forest algorithm with respect to root mean square error and mean prediction error (R. Li et al., 2020). CH4, CO and VOCs are the prime antecedents of O3 and predicting O3 concentration in atmospheric air is very vital to track and control climate change and global warming effects. This is performed using a long short term memory (LSTM) based hybrid deep learning model and the prediction accuracy is found to be high when compared with the baseline models (H. W. Wang et al., 2020). Shallow multi-output and deep multi-output LSTM network is used to forecast pollutants such as PM2.5, PM10 and NOX with high accuracy that yielded low loss function values (Zhou et al., 2019). The concentration of PM2.5 under the influence of industry and weather related factors is predicted using gradient boosted decision trees with high precision accuracy compared to conventional methods (Lee et al., 2020). The concentration of PM2.5 is forecasted based on meteorological and other factors using deep belief network and spatial lag models that helped urban planning (Yuan et al., 2019). The daily concentrations of PM2.5 are determined using partial differential equation model in which the parameters of the equation are determined using Nelder-Mead simplex local optimization method however the computational complexity of large scale problem instances is high (Yufang Wang et al., 2020). Air pollution at a particular place and time is quantitatively assessed using a commonly used comprehensive measure, air quality index (AQI) that is determined based on the proportion of various contaminants present in the air (Tan et al., 2021). Literature evidence shows that AQI can be forecasted accurately through data-driven models (Kumar and Goyal, 2011; Lee et al., 2020; Liu and Chen, 2020; Song and Fu, 2020; Zhou et al., 2019). The influence of PM2.5, PM10, SO2, NO2, CO and O3 concentrations on AQI and their inter-relationships are studied during various meteorological seasons using regression analysis (Q. Zhang et al., 2020) and the concentrations are forecasted for a short-term using machine learning and optimization models (S. Chen et al., 2019). AQI is forecasted using hybrid model of radial basis function neural network, ensemble empirical mode decomposition and autoregressive integrated moving average methods by capturing the influence of PM2.5, PM10, SO2, NO2, CO and O3 and other meteorological factors (Song and Fu, 2020). AQI is predicted using a hybrid method that comprised of binary grey wolf optimization-based feature reduction, discrete wavelet packet transform-based decomposition, extreme learning machine and adaptive boosting-based prediction model which is compared with other prediction models such as artificial neural network (ANN) and support vector regression (SVR). The proposed model surpasses ANN and SVR models in terms of robustness, interpretability and adaptability along with accuracy (Liu and Chen, 2020). The relationship of AQI with meteorological, economic, energy, demographic, mobility, and environmental aspects in a region is studied and multi-variate modeling using extreme gradient boosting decision tree is performed. The experimental study shows high accurate results when compared with other machine learning algorithms (J. Ma et al., 2020). The timeseries analysis of concentrations of PM2.5, PM10, SO2, NO2, CO and O3 is performed using variational mode decomposition and extreme learning machine to predict AQI in which the parameters of the model are determined using multi-objective grasshopper optimization algorithm and further fuzzy based air quality levels are identified (Ying Wang et al., 2020). However, pattern of AQI and pollutants’ concentration are affected by several events that cause changes in the environment and in recent times COVID-19 has brought variations in air quality in many cities. The credibility of the forecasts made by the prediction models is to be examined as pollution data evolves. Moreover, forecasts of AQI values made available in public domain during COVID-19 lockdown showed high deviation (forecast error) from actual due to reduced economic activities (Vasudevan et al., 2021), Therefore, there is a need to develop event-based prediction models for accurate AQI forecasts. Clustering and prediction models using machine learning together could yield multi-pollutant profiling and forecasts of AQI which could help in planning environmental missions, controlling emissions and restoring environmental health. However, such studies are scarcely reported in the literature. 2.1 COVID-19 pandemic and air pollution A novel coronavirus brought the world to a grinding halt turning up to be a massive global pandemic of this era. Air pollution and its effect on the spread and progression of infection was also one among the major concerns especially in cities (Harvard, 2020) (Becchetti et al., 2022). argued that quality of air has relationship with the spread of novel coronavirus and stringent lockdown measures enabled highly polluted area to mitigate spread and mortality effectively. As a preventive and control measure against this pandemic, lockdown was imposed and in all commercial establishments, only essential and very limited services were permitted to operate. Most countries enforced restrictions on travel and imposed mandatory lockdown which led to negative industrial growth in 2020 all around the world, however, it seems that the negative economic impact of the COVID-19 pandemic implies a positive environmental impact (Sahraei et al., 2021). During pandemic, while Governments’ policies, aimed at curbing the virus spread amongst the population, the economic recession deepened and consequently hugely affected the global consumption of fossil fuels and positively influenced the environmental ecosystem and subsequently the air quality (Dang and Trinh, 2021; Tibrewal and Venkataraman, 2022). This highlights the importance of using zero-emission vehicles, alternate fuels and eco-friendly transportation and transshipment practices to sustain this improved air quality further (Rizova et al., 2020). Indeed, NASA reported many evidences indicating the rejuvenation of environment initiated by lockdown measures (Streiff, 2020). (Le Quéré et al., 2020) identified that the global CO2 emission fell by 17% by early April 2020 relative to the 2019 average level. The effect of COVID-19 pandemic on air quality has been studied by Chinese researchers that has opened up new avenues to combat air pollution in China (Magazzino et al., 2021). Environment-friendly industry practices, logistic practices and green mobility initiatives have to be focused rigorously to sustain air quality (Caspersen and Navrud, 2021; Gonzalez et al., 2022). However, more studies are needed to sustain the improved environmental conditions that prevailed during COVID-19 lockdown without affecting the economic and social aspects. 3 Materials and methods The present study focuses on evaluating atmospheric air quality based on the air pollutants such as the PM2.5, PM10, NO, NO2, NOX, NH3, CO, SO2, O3, Benzene, Toluene, Xylene. The study was conducted for selected regions in three Indian states namely Delhi, Telangana and West Bengal. Delhi being highly commercial and highly polluted and Telangana being a newly-found state with huge influx of population having more economical activities and West Bengal being a pivot point for all the economic activities in the eastern region and highly committed towards sustainability are chosen as representative regions to carry out this study. The analysis of air quality before- and during COVID-19 lockdown was carried out for these selected regions to capture the variation in the trends of pollutants' concentration. A framework depicting the research work is shown in Fig. 1 . Timeseries data of pollutants' concentration before- and during-lockdown time periods is taken from public repositories. The analysis is carried out in two phases (1) Multi-pollutant profiling for different stations is carried out from which clusters of pollutants based on the similarity in concentration in atmospheric air at a particular region are made using Ward's algorithm. (2) Predicting the air quality index capturing the trend of various pollutants existing at different stations. Timeseries forecasts of pollutants' concentrations are used to compute future AQI values.Fig. 1 Research framework. Fig. 1 3.1 Data The concentration of PM2.5, PM10, NO, NO2, NOX, NH3, CO, SO2, O3, Benzene, Toluene and Xylene along with air quality index (AQI) is obtained from online websites published by Central Pollution Control Board (CPCB), Ministry of Environment, Forests and Climate change, Government of India.1 Here we use nitrogen oxides individually as well as in combination as there is a high chance of reaction of NO with O3 and conversion of NO to NO2 (Mazzeo et al., 2005). The timeseries data is available for major stations present in every city in every state. For the purpose of experimental modeling, this study considered nine stations present in Delhi, Hyderabad and West Bengal. Timeseries data of pollutants’ concentration and AQI values before and after April 1, 2020 (Hourly records of March and April) is considered to better understand the variation in air quality before and during COVID-19 lockdown respectively. 3.2 Multi-pollutant profiling using cluster analysis Multi-pollutant profiles are used to study the health effects of distinct pollutant clusters formed based on the concentration of different pollutants present in a particular region (Zanobetti et al., 2014). However, many research studies focused on developing city clusters based on the concentrations of a particular pollutant to frame policies and regulations. Based on the concentration of PM2.5, cities in China are clustered taking high dimensional monitoring data evaluating spatial-temporal clustering of PM2.5 concentration (Z. Chen et al., 2019; Yufang Wang et al., 2020). k-means clustering of dataset collected in Beijing is performed to impart high similarity within clusters that is found to further enhance forecasting accuracy (S. Chen et al., 2019). In this study, hierarchical cluster analysis using Ward's method (Amorim, 2015) is used to cluster the pollutants by determining the Euclidean distance between a pair of data points. Initially, the number of clusters is set equal to the number of pollutants and the required number of clusters is also set. Then the pair of clusters (Ci,Cj) that has minimum cost function is iteratively merged based on the following equation till desired number of clusters is reached.(1) ward(Ci,Cj)=n(Ci)×n(Cj)n(Ci)+n(Cj)d(cCi,cCj) where cCi denotes the centroid of cluster Ci and n(Ci) denotes the cardinality of cluster Ci. By applying ward's method, multi-pollutant profiles of different regions can be obtained and their effects on human health in those regions can be studied. 3.3 Predictive modeling of air quality The effect of pollutants' concentration on air quality is first studied using linear regression method (LM). Linear timeseries models assume linear relationships between the dependent and independent variables (Zeinalnezhad et al., 2020). Learning-based non-linear timeseries analysis is also carried out in this study to explore the possible non-linear relationships among the determinants of air quality (Du et al., 2019; R. Li et al., 2020; J. Ma et al., 2020; Zeinalnezhad et al., 2020). Existing studies show that regression trees constructed using Decision Tree (DT) and its ensembles such as Random Decision Trees or Random Forest (RF) and Extreme gradient boosting trees (XGB) are able to fit a robust regression model (Ayoubloo et al., 2011). Tree based models capture the pattern of data over time and effectively predict the future values. The rules on the decision nodes are made with the selection of independent variables in the order of their significance during the training phase in such a way that the seen instances can be accurately fitted with the established rule. After sufficient training, the decision rules are finetuned and the regression tree will eventually become ready to handle unseen instances. Random forest employs simultaneous learning through multiple decision trees and generalizes more quickly especially in hard-to-predict problems. XGB is a boosted tree involving ensemble learning to incorporate dominant relationships among predictors iteratively. Literature also shows evidence that artificial neural network (ANN) and its ensembles are widely used for predictive modeling of air quality (Ayoubloo et al., 2011; Yilmaz and Kaynar, 2011). ANN consists of input, hidden and output neurons that are grouped in layers such that the entire network architecture can be trained to activate appropriate neurons to determine a desired output given an input signal (Song and Fu, 2020). The number of hidden neurons and the number of hidden layers must be experimentally identified as hyper-parameters. There are connections between the layers and the strength of a connection is given as a weight value. The network develops an input-output fitted function iteratively during the training phase with seen instances. The activation function is also chosen experimentally. For each training instance, the error obtained from the deviation between the predicted and actual values is determined and fed back to the input layer through backpropagation and gradient descent method is used to minimize error. The gradient descent method adjusts the weights by an amount that is proportional to the partial derivative of the error function that is back propagated based on a learning rate parameter. This helps to avoid convergence to the saddle (min-max) point and the gradient vector of the approximated error function is obtained through the layers of the network. Literature presents support vector regression (SVR) as another powerful tool that maps the non-linearly related predictors to generalize unseen instances with outstanding performance (Kavousi-fard et al., 2014). Radial basis function incorporated in a Gaussian kernel to model air quality with seen instances of pollutants' concentration during training phase iteratively minimizes the loss function (Smola and Olkapf, 2004). The kernel attempts to determine a statistical fit in the training phase in the predictor space with minimum loss at which the deviation between the actual and obtained values are less than a small tolerance error. A non-linear mapping transforms the input vector onto predictor space performed by kernels and forms mapped support vectors. The variation in pollutants due to COVID-19 and its related events has to be intensely investigated. In this study, every station is investigated using these learning methods to fit regression models for predicting AQI before and during COVID-19 lockdown and the best fitted model is selected which can address COVID-19-induced change in the relationship of pollutants. The predicted pollutants’ concentration using the best fitted models further is used to determine national air quality index based on the guidelines of CPCB. 3.3.1 Data-driven modeling of air quality Best fitted models to determine AQI as a function of its pollutants is to be identified for each region based on the data collected in this study. Linear and non-linear timeseries analysis using support vector regression (SVR), artificial neural network (ANN), decision tree (DT), random forest (RF), and extreme gradient boosting (XGB) tree methods are performed and their prediction error and computational time complexity are obtained for each station before and during COVID-19 lockdown. The forecasts of pollutants' concentration yielded by each model is compared with the actuals in the testing phase and the best fitted models are selected which can be further deployed to forecast AQI accurately in each region (9 stations considered in this study). The timeseries data of pollutants' concentration collected before and after the onset of COVID-19 in these regions are classified into training and test dataset. The training dataset is used to construct the timeseries models to forecast the pollutants’ concentration in each region. During the training process, the models exhibit deviation between actual and predicted from which root mean squared error is derived that is most often fed back to the model for finetuning the model parameters iteratively. After sufficient training, the model is tested on its prediction ability with the test data and again root mean squared error is determined. The training error and testing error are evaluated based on the deviation of estimated AQI from actual AQI values to analyze the generalization capability of the machine learning regressors in fitting the models (Refer to Table 1, Table 2 ).Table 1 Training and test root mean square errors for different stations before COVID-19 lockdown. Table 1Regressors LM SVR ANN DT RF XGB LM SVR ANN DT RF XGB Station ID Training RMSE Test RMSE WB007 0.089 0.062 0.068 0.290 0.192 0.001 0.097 0.110 0.138 0.115 0.106 0.104 WB009 0.062 0.045 0.051 0.296 0.111 0.001 0.090 0.152 0.087 0.104 0.112 0.093 TG001 0.074 0.054 0.063 0.120 0.005 0.001 0.075 0.111 0.078 0.090 0.081 0.079 TG002 0.042 0.037 0.037 0.107 0.043 0.001 0.032 0.034 0.044 0.039 0.031 0.032 TG003 0.070 0.048 0.060 0.201 0.067 0.001 0.066 0.087 0.115 0.103 0.089 0.074 TG004 0.062 0.041 0.047 0.141 0.045 0.001 0.048 0.069 0.046 0.059 0.049 0.049 TG006 0.069 0.047 0.055 0.149 0.052 0.001 0.075 0.076 0.074 0.073 0.068 0.073 DL001 0.066 0.035 0.044 0.218 0.083 0.001 0.096 0.085 0.071 0.071 0.068 0.073 DL019 0.063 0.034 0.043 0.192 0.061 0.001 0.063 0.101 0.077 0.084 0.074 0.072 Table 2 Training and test root mean square errors for different stations during COVID-19 lockdown. Table 2Regressors LM SVR ANN DT RF XGB LM SVR ANN DT RF XGB Station ID Training RMSE Test RMSE WB007 0.0402 0.0416 0.0184 0.26838 0.60922 0.0005 0.2154 0.1356 0.2744 0.1365 0.1791 0.148 WB009 0.0476 0.0272 0.0122 0.23662 0.72568 0.0005 0.1524 0.1165 0.1155 0.1274 0.1355 0.1309 TG001 0.073 0.0594 0.0127 0.72681 0.89294 0.0006 0.7857 0.2508 0.3379 0.1628 0.1584 0.1186 TG002 0.0599 0.0656 0.0137 0.50273 0.97631 0.0006 0.1885 0.155 0.1637 0.1961 0.1903 0.225 TG003 0.1043 0.0725 0.0395 0.22301 0.4974 0.0005 0.1759 0.1869 0.2741 0.3148 0.2156 0.2678 TG004 0.0502 0.0497 0.0131 0.19409 0.90768 0.0006 0.2827 0.2308 0.1748 0.116 0.1605 0.1354 TG006 0.1288 0.0876 0.0159 0.24902 1.07484 0.0006 0.5023 0.304 0.4581 0.2869 0.2346 0.2222 DL001 0.0625 0.0439 0.0163 0.25895 0.7138 0.0005 0.5676 0.1221 0.2654 0.249 0.1562 0.1696 DL019 0.0818 0.122 0.0153 0.24066 0.97345 0.0005 0.1671 0.1046 0.4122 0.0957 0.0909 0.1152 Most of the models exhibit a high root mean squared error in the testing phase than that in the training phase, especially in the during-lockdown scenario. This is because the models could not capture the complex patterns in the data and are unable to represent the problem with an exact function similar to an actual or target function and hence exhibiting poor generalization capability. The learners have different ability to derive the approximated function and often suffer from underfitting or overfitting issues. The models get fitted to the best approximate function possible with the seen instances and have a deviation between the actual and predicted when confronted with unseen instances. However, the difference in errors during training and testing phases is more in during-lockdown models as it is obvious that the pandemic outbreak created turbulence in air pollutants’ concentration that gradually stabilized with time. It can also be observed that the prediction error is high in linear models in most of the cases as there are non-linear relationships in the pollutants which are addressed by the machine learning methods to a great extent. Also, prediction accuracy is found to substantially increase when machine learning models are applied especially in the during-lockdown scenario. To choose an appropriate machine learner, in addition to error, the convergence speed of the regressors is also captured and the execution time in training the models is obtained and presented in Table 3 .Table 3 Execution time. Table 3Regressors LM SVR ANN DT RF XGB LM SVR ANN DT RF XGB Station ID Before COVID Outbreak During COVID Outbreak WB007 0.006 0.017 0.024 0.002 0.128 0.181 0.000 0.008 0.008 0.008 0.008 0.095 WB009 0.000 0.016 0.032 0.008 0.199 0.186 0.000 0.011 0.012 0.006 0.008 0.121 TG001 0.005 0.048 0.069 0.014 7.086 0.730 0.017 0.026 0.018 0.016 0.023 0.127 TG002 0.000 0.053 0.079 0.016 1.037 0.461 0.001 0.713 0.010 0.016 0.019 0.129 TG003 0.006 0.063 0.267 0.016 0.767 0.452 0.000 0.000 0.016 0.000 0.033 0.136 TG004 0.010 0.115 0.105 0.290 1.315 0.592 0.000 0.008 0.016 0.008 0.015 0.135 TG006 0.024 0.104 0.095 0.016 1.774 0.820 0.000 0.008 0.016 0.005 0.018 0.142 DL001 0.000 0.016 0.048 0.008 0.311 0.200 0.000 0.016 0.012 0.008 0.016 0.128 DL019 0.100 0.038 0.072 0.007 0.555 0.333 0.005 0.009 0.008 0.000 0.016 0.122 It can be observed that the computational time during the training phase in ensemble methods such as random forest and boosting learners is high when compared to other methods. The resultant best fitted models were chosen for each station after modeling are presented in Table 4 .Table 4 Best fitted models for AQI prediction. Table 4Station ID During-COVID AQI Prediction model Pre-COVID AQI Prediction model WB007 SVR LM WB009 ANN LM TG001 XGB LM TG002 SVR RF TG003 LM LM TG004 DT ANN TG006 XGB RF DL001 SVR RF DL019 RF LM The best fitted prediction model for AQI in each region for before- and during- COVID-19 lockdown is selected based on the prediction accuracy and computational time taken for constructing the models. These models are then further used to analytically determine the significance of the pollutants and the impact of COVID-19 lockdown measures on the environmental pollution in selected nine regions in India. 4 Results and discussions In this study, the influence of COVID-19 lockdown on various air pollutants and air quality in various regions of India is analyzed using clustering and prediction methods. The best fitted models identified for each region in the previous section are further used to determine AQI based on the pollutants’ concentration before and during COVID-19 lockdown. The multi-pollutant profiles existing during COVID and before the COVID outbreak in each station are obtained using hierarchical cluster analysis using Ward's method and presented in Fig. 2, Fig. 3 . The major air pollutants considered in this study viz, PM2.5, PM10, NO, NO2, NOX, NH3, CO, SO2, O3, Benzene, Toluene, and Xylene are clustered to obtain maximum of five multi-pollutant profiles for every station based on their similarity in variation.Fig. 2 Pre-COVID Clusters of pollutants. Fig. 2 Fig. 3 During-COVID clusters of pollutants. Fig. 3 It can be observed that the pollutants are differently clustered before and during COVID-19 lockdown in all the stations. Delhi is the most polluted city in India and one of the top five polluted cities in the world. In the station, DL001, it could be observed that the concentration of PM10 in atmospheric air is found to be a persistent pollutant affecting the air quality both pre-and during COVID-19 lockdown. It is a unique pollutant and its concentration is found to decrease as the height of the reference point from ground level increases and has high retention in the air for a longer duration (Q. Zhang et al., 2020). The pollutants CO, benzene, xylene, toluene, and NO form the next important cluster before COVID-19 which during COVID-19 has been significantly shown to reduce effect on air quality. The prime source of Benzene, Toluene, and Xylene (BTX) pollutants is emission from the combustion of fuels in automobiles and industries. Since during COVID-19 outbreak, the lockdown of all non-essential activities has drastically reduced emissions and hence these pollutants became less significant. In the station, DL019, before the outbreak of this pandemic, PM2.5 and PM10 formed the main cluster which persisted to influence air pollution followed by the cluster composed of NO, NO2 and cluster composed of SO2, CO, Benzene, and Xylene, and cluster composed of NH3, O3, Toluene. However, after the outbreak of the pandemic, the multi-pollutant profiles have altered their compositions nevertheless PM10 remains to be the main threat due to its prolonged endurance. The effect of NO, CO, and BTX turned relatively significant during a pandemic. In both stations in Delhi, the prime contributor of air pollution remained significant though has reduced effect after reducing the unnecessary movements in the city and hence measures should be taken to reduce particulate matter in the air. PM10 is the most significant cluster in stations, TG001 and TG004 located in Telangana and its intensity is lessened during the pandemic outbreak, and [NO, T, CO, Benzene, Xylene] and [Benzene, CO, Xylene] multi-pollutant profiles dominate the air pollution during the pandemic period. In TG002, [NO, CO] is the prominent cluster however during the lockdown period, the [CO, Xylene] cluster became relatively significant in which CO is persistently prominent. In TG003, [Benzene, CO, Xylene] cluster loses its significance to [NH3, NO2, NOX] during the lockdown and in TG006, [SO2, Xylene, CO, Benzene] cluster reduced its influence, and the [Toluene, Benzene, CO and Xylene] cluster became relatively prominent during the lockdown in which CO and BTX pollutants are persistently significant. Generally in Hyderabad, CO, nitrous, and BTX pollutants are prominent which are primarily due to extensive transport and industrial activities (S.K. et al., 2013). The state pollution board projects above 5700 deaths in Hyderabad due to air pollution.2 The multi-pollutant profiles obtained by this study would be instrumental in developing strategies to combat air pollution. In West Bengal, the scenario is different. [Benzene and Toluene] is the prominent cluster that lost its intensity and the [CO, Xylene] cluster became prominent during the pandemic period in the station, WB007. In WB008, PM10 is the persistent pollutant even after reducing unnecessary movements in the lockdown period and the concentration of O3 has turned up relatively significant. In WB009, the [PM2.5, PM10, O3] cluster turned up significant during the pandemic outbreak whereas the [NO, NO2, O3] multi-pollutant profile was formed with a similar effect in both the scenarios. Commonly observed multi-pollutant profiles are [PM2.5, O3], [NH3, SO2], [CO, Benzene, Xylene] which are found to affect air quality. In this study, a representative list of stations is considered to derive the pollutant clusters which could be extended to all other stations also. This study also proves the significance of the combined effect of these profiles on air quality in all the stations. The variation of the pollutants in air is drawn out from the predictions and presented in Fig. 4, Fig. 5 .Fig. 4 Impact of air pollutants on AQI before the COVID outbreak. Fig. 4 Fig. 5 Impact of air pollutants on AQI during COVID. Fig. 5 It is well known that the outbreak of novel coronavirus disease and its spread has very rapid affected and lockdown regulations are announced and restricted movement is enforced across India. This has drastically reduced emissions from industries and vehicles thereby air pollution has been reduced considerably and a revival of atmospheric air took place. To understand AQI variation in these stations, the mean forecasts of concentrations of these pollutants before and during COVID-19 obtained from the best-fitted prediction models during the testing phase are plotted in Fig. 4, Fig. 5. Generally, pollution due to particulate matter is high in all the regions. However, during COVID lockdown, the concentration of these pollutants including PM in atmospheric air has drastically reduced. The effects of PM2.5, PM10, and BTX in atmospheric air continues to be high-priority pollutants even during the lockdown in Delhi however the concentrations of these pollutants were fairly reduced. NH3, Benzene, and Toluene levels are high during COVID-19 lockdown but comparatively lesser than before lockdown scenario. In Hyderabad, though air pollutants' weakening effect is felt during the lockdown, NO2 and SO2 continue to show importance during COVID-19 lockdown which also formed a prime multi-pollutant cluster in the stations present in Hyderabad. Similarly, after the lockdown there is a drastic reduction in air pollutants in atmospheric air in many places of West Bengal. However, NH3, CO, SO2, and O3 are relatively significant pollutants to be persistently addressed even after reduced activities. However, the dominant air pollutants in West Bengal have turned out to be NH3, SO2, and O3 in zone WB007 and NH3, SO2, CO, NO, and PM2.5 in WB009. The effect of O3 is more in West Bengal due to climatic conditions. Similar foresights about future pollutants’ concentration enable handling these zones with appropriate pollutant specific proactive and preventive measures to mitigate pollution in a timely manner. Linear timeseries analysis yielded high prediction error compared to ensemble learning-based non-linear timeseries analysis of pollutants with better accuracy of forecasts made on AQI. The findings from the analysis (Refer to Table 1, Table 2, Table 3) show that the performance of prediction through non-linear timeseries modeling is high. Hyper-parameter tuning and selection of kernel and activation functions are carefully performed experimentally. The prediction error for different stations using the selected models is minimized by 9% for the before COVID-19 scenario and 50% in during-COVID-19 lockdown compared with that exhibited by linear regression. Especially in COVID-19 scenario, the uncertain changes in the release of air pollutants in the environment have been well addressed by the selected models. For all the regions under consideration, the concentration of particulate matter has reduced due to the COVID-19 outbreak and subsequent lockdown. Emissions from vehicles have greatly reduced and shut-down enforcement of industries during this period had influenced this drastic change. 5 Deployment and limitations This study demonstrated predictive modeling of air quality index using the past pollutants' concentration data. The timeseries data of pollutants' concentration is obtained from the official website of CPCB, India. This data is used in this study to train the prediction models for making forecasts of pollutants' concentration and the best fit model obtained during the test phase is further used to evaluate National air quality index as per the guidelines of CPCB. National air quality index is updated on hourly basis in official application developed by CPCB with a latency. However, this study helps evaluate future values based on the forecasts of pollutants’ concentration obtained from the prediction models. The prediction modeling and selection process has to be often performed at regular intervals or whenever there is an event or whichever is the earliest. In this study, we have presented two scenarios, viz., models before and during- COVID lockdown since the lockdown period in India has witnessed substantial air quality improvement due to reduced economic activities. The impact of events induced by meteorological factors, nature-induced factors, perceptual and situational factors along with pollutants also affect air quality and hence has to be incorporated in the model time-to-time. The deployment model is presented in Fig. 6 .Fig. 6 Deployment model. Fig. 6 The findings of this study are limited to the stations chosen in Delhi, Hyderabad, and Kolkata. The data for this study is obtained through public repositories and hence we had to tackle incompleteness and distortion to a larger extent. The deployment model is scalable to other stations with sufficient dataset. AQI prediction made in this study is based on pollutants’ concentration assuming consistent meteorological conditions (during March–April 2020). 6 Implications The present study used a learning approach to forecast air quality using timeseries analysis of pollutants’ concentration in a particular region. It has significant implications. 6.1 Theoretical contributions This study offers a significant contribution to theory. The study uses event-driven timeseries data of pollutants' concentration for machine learning analysis to predict future values that is used further to determine future AQI values. In reality, pollution data is unbounded and evolving with numerous events in the environment. The trend in timeseries of pollutants' concentration captured analytically often gets distorted due to these events and increase the prediction error. Therefore, this study performs prediction modeling of pollutants’ concentration for the scenarios, viz., before and during COVID lockdown separately which forecasts AQI accurately during these periods. The best fitted models selected for these scenarios for each geographical region also elucidate the variation in the air quality in both the time periods. 6.2 Practical implications Collaborative governance helps effective control of air pollution (Wang and Zhao, 2021). Several attempts are made by the government to provide real-time awareness of pollution status in a particular area that paves way for control and mitigation. The present study attempts to provide prior information about air quality that enables prevention and control. This study developed an evaluation mechanism for AQI accurately through learning-based modeling that can be adopted in industrial and traffic zones to understand the trend in air quality. The variation of pollutants' concentration observed in the COVID-19 lockdown phase allows researchers to analyze the pollutants' concentration before and after the COVID-19 outbreak which can potentially lead to practical solutions and sustainable business practices to retain the improved air conditions further. However, there is a need to detect any event that would bring significant variation in pollutants’ concentration in real-time and the prediction model should be evolving in nature to maintain its predictive performance during these changes which is more challenging and requires more data. It is even more challenging to address these variations in the predictive models if such variations are temporary. This study clearly shows the changes in the concentration of air pollutants during the COVID-19 lockdown and their impact on improved air quality levels. It also highlights those pollutants that can be mitigated by addressing fuel combustion and industrial emission properly. Further, the amount of data generated by monitoring air pollution is vast and highly unstructured. Therefore, the present study serves as the walkthrough to the pollution management team of every city in developing countries to adopt the proposed model (Fig. 1) for evaluating the air quality and introduce reforms in the regulations. 6.3 Societal implications The results of this study have important societal implications. The prevalence of the pollutants in the air in selected regions in India is ranked before and during the COVID-19 lockdown to capture variation in the air quality. The presence of PM2.5 and NOX in the air at Alipur in Delhi has got reduced by 89% and 91% respectively during the COVID-19 lockdown which helped people inhale the air of better quality. PM2.5, CO, NH3, NO, and O3 reduced by 33.5%, 38%, 28%, 13.5%, and 51% respectively in the Mandir Marg region of Delhi during the COVID-19 lockdown. The reduction in the concentration of PM2.5 and nitrogen oxides during the COVID-19 lockdown in Delhi helped the environment rejuvenate which in turn offered better air for the society. Further, a lot of pharmaceutical companies are located in these industrial zones that emit harmful pollutants like PM, ozone, BTX, Sulfur oxides, and carbon monoxide, and proper source management and air filtration methods should be adopted to avoid potential health hazards. Sulfur dioxide and carbon monoxide in the air lead to acid rain which is a major threat to farmers and residents living in this area. In the state of Telangana, in the Bollaram industrial area, PM2.5, O3, NOX, NH3, benzene, toluene, and xylene are observed to be reduced by 64.5%, 99.9%, 27%, 68%, 99.7%, 99.6%, 39% respectively, in the Patancheru industrial zone, PM10, NO, O3, Toluene got reduced by 96%, 78%, 74%, 52% respectively and in Pashamylaram industrial zone, it was observed that the pollutants, PM10, CO, Ozone, BTX reduced completely and PM2.5 reduced by 75%. In the university zone, CO and BTX reduced by 82%, 86%, 81%, and 76% respectively due to reduced vehicle emissions. In West Bengal, the Ballygunge locality of South Kolkata recorded much-improved air quality and shows a reduction of PM2.5, PM10, NO, NO2, NOX, Toluene, and Xylene by 96%, 87%, 75%, 90%, 92%, 94%, 78% respectively. Pollutants such as PM2.5, PM10, NO2, CO reduce by 48%, 69%, 77% respectively in Fort William area in Kolkata city. The major contributor to air pollution in Kolkata city is vehicular emission and during the COVID-19 lockdown, the pollutants' concentration dropped due to travel restrictions. The results of this study enable policymakers to regulate air pollution effectively by detecting the variation in air quality occurring from time to time that brings a positive impact on social life. Improved air quality level is possible during COVID-19 lockdown due to public commitment and people across the three states followed restrictions to the possible extent which in turn improved the air quality level and decreased the pollutants’ concentration in the different stations. 6.4 Economic implications In the field of environmental economics, the Environmental Kuznets Curve (EKC) suggests that, in the short-run, as the economy shifts towards development, the environment worsens, but, in the long run, as the society becomes aware of the social cost of this negative externality, it introduces, through government regulations, changes in environmental standards and policies and reinvests part of its income to improve the environment and restore the ecosystem. In line with this, many studies support the Environmental Kuznets Curve hypothesis for India, for example (Kanjilal and Ghosh, 2013; Usman and Jahanger, 2021), and our results suggest that pollution mitigation in India is not attributed to a slowdown in economic growth, but regulatory efforts to limit pollution as well as the country will to ensure the energy transition necessary for sustainable development. Indeed, aware of the social cost of this negative externality, the Indian society has introduced, through regulations, changes in environmental standards and policies and has recently started to reinvest part of its revenues to improve the environment and restore the ecosystem. Specifically, the main environmental laws in India include (a) the Wild Life Act (1972), (b) the Water Act (1974), (c) the Forest Act (1980), (d) the Air Act (1981), (e) the EP Act (1986) and (f) the National Green Tribunal (NGT) Act. Moreover, from its inception, the NGT ordered the CPCB and the SPCB, which are the Indian environmental regulatory authorities, to strictly enforce the Comprehensive Environmental Pollution Index (CEPI) criteria which were initiated in 2009 (and updated in 2016) to categorize polluting industries as well as polluted industrial areas. In 2019, the NGT monitored the enforcement of the CEPI criteria by the CPCB and the SPCB. To foster the environment rejuvenation instigated by COVID -19 lockdown and to internalize the negative externality, the Indian environmental regulatory authorities must now start thinking not only about seeking compensation from polluting industries (e.g. via a Pigovian Tax, a lump-sum tax, and/or restricting the amount of pollution …), but above all about doing so in an optimal way to improve social welfare. 6.5 Implications for policies Understanding current trend in air quality and its associated pollutants’ is instrumental for developing various mitigation plans and control strategies. Ministry of Housing & Urban Affairs of the Indian government launched the National clean air program in 2019 with a mission to achieve a reduction in PM concentration by 20–30% by 2024 from the concentration observed in 2017 in cities which include Delhi, Kolkata, and Hyderabad. Under this, city-specific action plans need to be devised to reduce emissions, strengthen the monitoring networks, bring mobility and industry policies to regulate air pollution, and create public awareness. Since the major source of air pollution in Delhi and Kolkata is vehicular emissions, strict compliance to exhaust emission standards is enforced and all vehicles must possess valid Pollution under control (PUC) certificates. The mass rapid transport system is introduced for convenient conveyance to reduce traffic. Under a clean fuel program, governments also strictly monitor the quality of petrol and diesel supplied to these places to control harmful emissions during combustion. Particulate filters in diesel vehicles are mandated and the supply of adulterated fuels is curbed. Initiatives toward electric-only vehicles are also being developed by the government to establish zero-emission zones in the cities. Small-, medium- and large-scale pharma industries are under strict monitoring by the pollution control board and safe discharge of emissions is enforced. During the lockdown, the decrease in harmful pollutants in the air has unraveled the potential activities causing large-scale pollution in these cities. It was understood that more than 50% of air pollution is from vehicles and in the case of Hyderabad, emissions from pharma industries also play a major role. The pollution control action plans of these cities need to be revised based on the lessons learned from the pandemic. Further, the COVID-19 pandemic has brought potential initiatives for public well-being such as, no-car road and proving cycling path along streets that are sustainable to the environment and reduce the carbon footprint. Public awareness about everyday air quality and lifestyle modifications will take these initiatives forward for better wellbeing. 7 Conclusions The work focuses on evaluating air pollution and pollutants' impact before and during COVID-19 lockdown. The present study contributes to research, by evaluating Air Quality Index (AQI) for the pre-and during the COVID-19 lockdown time periods. The concentrations of pollutants such as PM2.5, PM10, NO, NO2, NOX, NH3, CO, SO2, O3, Benzene, Toluene, and Xylene along with air quality index (AQI) are obtained as time-series data that is made publicly available for major stations present in every city and states in India. This study considered nine representative stations covering Delhi, Hyderabad, and Kolkata. The pollutant clusters derived from cluster analysis based on their similarity in concentration levels highlight their combined impact on air quality across different stations. This study intends to make a comparison of multi-pollutant clusters formed before and during COVID-19 lockdown to show the effect of reduced human outdoor activities during COVID-19 due to lockdown. Although literature evidences regarding the prediction models for forecasting AQI are existing, the deviation of forecasts made by these models from actuals during the COVID-19 lockdown was alarmingly high, and hence revised models have to be developed. In this study, we performed statistical modeling of time series data collected before and during COVID-19 lockdown at selected regions to capture the change in trends of pollutants' concentration and subsequently AQI. The selected models representing AQI in these regions that resulted from machine learning analysis showed minimal prediction errors and high accuracy. The data used is comprised of hourly data points collected by the pollution monitoring systems installed in these stations. Smart pollution monitoring systems incorporating the Internet of Things, machine learning, and cloud computing technologies integrated into pollution monitoring platforms can be developed in the future to sense these variations in pollutants’ concentration. Real-time Affordable Multi-Pollutant (RAMP) sensor packages can be used to capture the data effectively and seamlessly. The results of clustering and prediction modeling play a vital role in the visualization of insights in such platforms. Further, the google mobility data can also be integrated with these systems to assign events such as the closure of restaurants, shopping centers, theme parks, cafés, pandemic, rainfall and cinemas with variations in air quality. Furthermore, it would be interesting to have future studies using time-frequency econometric tools to examine the environmental impact of the very recent Indian government policies during the COVID-19 aiming at flattering the epidemiological curve, as well as the relationship between economic activity and pollution in India. Credit author statement Jinil Persis: Conceptualization, Methodology, Analysis, Results, Writing, Amine Ben Amar: Writing, Methodology. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper Data availability Data is available in online repositories 1 https://app.cpcbccr.com/AQI_India/. 2 https://www.iqair.com/india/telangana/hyderabad. ==== Refs References Amorim R.C. de Feature relevance in ward's hierarchical clustering using the Lp norm J. Classif. 32 2015 46 62 10.1007/s00357 S K. G A.W. 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==== Front The Egyptian Journal of Remote Sensing and Space Sciences 1110-9823 2090-2476 National Authority of Remote Sensing & Space Science. Published by Elsevier B.V. S1110-9823(22)00098-9 10.1016/j.ejrs.2022.11.007 Research Paper Use of an E2SFCA method to assess healthcare resources in Jordan during COVID-19 pandemic Al-Omari Aslam a⁎ Shatnawi Nawras b Al-Mashaqbeh Alia cd a Department of Civil Engineering, Jordan University of Science and Technology (JUST), Irbid 3030, Jordan b Surveying and Geomatics Engineering Department, Al-Balqa Applied University (BAU), Al-Salt 19117, Jordan c Department of Civil Engineering, Jordan University of Science and Technology (JUST), Irbid 3030, Jordan d Department of Surveying Engineering, Al al-Bayt University (AABU), Mafraq 25113, Jordan ⁎ Corresponding author. 1 12 2022 12 2022 1 12 2022 25 4 10571068 20 1 2022 17 10 2022 21 11 2022 © 2022 National Authority of Remote Sensing & Space Science. Published by Elsevier B.V. 2022 National Authority of Remote Sensing & Space Science Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Healthcare spatial accessibility requires a better understanding and evaluation, especially during pandemic outbreaks like the recent COVID-19 pandemic. The main goal of this study is to measure and assess community-level spatial accessibility in Amman city to various COVID-19 related healthcare resources that could provide any urgent medical care for suspected or confirmed COVID-19 cases. To address this aim, the Enhanced 2-step floating catchment area (E2SFCA) method combined with several geospatial techniques were performed. The main E2SFCA results show the differences in the capacities and spatial accessibility of health facilities within Amman city, as well as how the variations are captured at different regions. The resulted spatial accessibility scores were presented in interactive Geo-spatial maps, analyzed, and compared for several health resources in public, private, and educational hospitals. The current research findings stated that although there are enough healthcare facilities to service almost the entire city, inappropriate health facility distribution, rather than a lack of resources, has resulted in coverage gaps in some areas. The center zones had been fully serviced, or perhaps over-served, by a large number of facilities. The other zones, on the contrary, were partially served or were even underserved by a certain number of resources. Keywords Spatial accessibility FCA 2SFCA E2SFCA GIS Healthcare facility COVID-19 ==== Body pmc1 Introduction The term “accessibility” relates to the ease with which urban possibilities such as work, education, and healthcare may be accessed (Hansen, 1959). It is the product of potential opportunities that incorporate regional population distribution (demand), facilities (supply), and transportation services (Geurs and van Wee, 2004). Healthcare accessibility is a major study topic in the field of accessibility measurements and analyses, as it has significant theoretical and policy implications (Wang, 2012, Zhao et al., 2020). Healthcare accessibility requires a better understanding and evaluation, especially during pandemic outbreaks like the recent Coronavirus pandemic (known as COVID-19 pandemic). Good access to hospitals leads to greater utilization of healthcare resources and contributes to improved health system performance and quality of life, specifically for patients with chronic diseases and for economically vulnerable individuals such as low wage earners, young children, and women (Hare and Barcus, 2007, Zhao et al., 2020). COVID-19 is a SARS-CoV-2 virus disease that was first reported in Wuhan, China in early December 2019 (Wang et al., 2020). This virus is zoonotic and, in extreme cases, causes symptoms close to those of common influenza, including runny nose, sore throat, cough, fever, and trouble breathing. Additionally, the spread has accelerated to the point where it has reached a large number of countries throughout the world. Given the great level of influence, it may have on developing countries, where health and hospital capability is smaller than that of developed countries, COVID-19 has been declared as a pandemic (Escobar et al., 2020). In a number of countries, COVID-19 has overloaded healthcare resources (e.g. healthcare staff, testing resources, hospital beds, and ventilators). The increased demand for healthcare services has increased or worsened health inequalities in healthcare access (Kang et al., 2020). For this reason, diagnosis of areas of cities with less access to health services and facilities and the identification of hospitals that may suffer from overloaded demand for admissions is essential for healthcare planners (Pereira et al., 2020). The use of GIS in different fields makes it possible to plan and analyze the influence of a wide variety of social services and strategies for locational planning. One of the key reasons is that GIS offers an outstanding platform for the analysis and representation of virtually infinite spatial conditions. GIS offers several tools for the storage, management, processing, interpretation, and presentation of geographical data that play an important role in health care planning. This is particularly relevant in the context of the assessment of access to healthcare services as a means of planning for enhanced provision of services. A variety of studies that focused on measures of access to services have been produced. Some of these studies are Tanser et al., 2006, Higgs, 2004, and Lovett et al., 2002, Shatnawi et al., 2022. Most of these studies have shown that GIS has provided useful tools for measuring geographical access to services and analyzing inequalities in access as well as those patterned along social and economic lines. Lovett et al. (2002) attempted to measure accessibility to surgeries by public and private transport in East Anglia; information from patient registries was combined in GIS with information of general practitioner surgery locations, road network features, bus routes, and community transport facilities. The research was able to show the potential integration of patient registries and GIS in accessibility measurements, but it has been advised not to neglect the practical challenges in utilizing these data sources and techniques. Bagheri et al. (2005) used a network analyst to establish the best route in the Otago region of New Zealand, based on the shortest time from residential areas to primary health facilities. The study found that in order to enhance the measurement and assessment of the accessibility of primary healthcare services consideration should be given to non-spatial factors such as deprivation indexes, ethnicity, education, gender, age, income, housing, and transportation modes and then to combining spatial and non-spatial in one frame. Right now, with the rapid development of the GIS environment, the floating catchment area (FCA) approaches have become easier to implement and to interpret. The techniques are effective and efficient health accessibility assessment methods for health care facility managers and policymakers to identify poor areas and assign scarce resources to those areas in need (Zhan et al., 2011). Luo and Wang (2003) merged two GIS-based accessibility measures into a single system and used the methods to examine spatial accessibility to primary health care in the ten-county Chicago region. The authors also investigated the sensitivity of results by experimenting with different threshold travel times and travel friction coefficients in the FCA system and the gravity model. The techniques could assist the US Department of Health and Human Services and state health departments in better identifying Health Professional Shortage Areas. The original FCA metric called the two-step floating catchment area (2SFCA) method was established by Luo and Wang (2003). Measuring accessibility levels in sequential steps is the standard rationale underlying FCA methods. The first step is to measure each health facility's provider-to-population ratio (PPR) as a ratio between its service supply (e.g. number of beds) and the population's potential service demand that falls within a certain catchment area. The second step is to measure each population center's accessibility levels by aggregating the PPR of each healthcare provider accessible from each population center (Pereira et al., 2020). The application of FCA metrics has primarily been for health care services, such as primary care (Bauer and Groneberg, 2016), and tertiary care (Wan et al., 2012). However, spatial accessibility of various non-health-related applications has also been assessed using FCA metrics, such as libraries (Higgs et al., 2013) and recreation areas (Langford et al., 2018). This application diversity underlines the robustness and utility of the FCA metrics for several applications to consider spatial accessibility (Bryant and Delamater, 2019). Luo and Qi (2009) recently presented the enhanced Two-Step Floating Catchment Area (E2SFCA) approach as an improvement of the 2SFCA method. Several travel time zones for each catchment are created using the ArcGIS Network Analyst to distinguish accessibility within a catchment and assigned to different weights according to the Gaussian function (Zhan et al., 2011). The E2SFCA approach has been used in various studies to measure and assess the spatial accessibility of healthcare services (Zhan et al., 2011, Hu et al., 2013, Luo et al., 2018, Zhu et al., 2019, Shah et al., 2020). For example, Luo and Qi (2009) used the E2SFCA as an improvement to the 2SFCA approach for calculating spatial accessibility, the results found that when the E2SFCA approach was used to assess the spatial accessibility to primary care physicians in a study area in northern Illinois, it revealed a spatial accessibility trend that is more compatible with intuition and delineates more spatially explicit health professional shortage areas. It was also simple to implement in GIS and interpret. A study by Escobar et al. (2020) used the E2SFCA model to evaluate the existing ICU supply per thousand people in the Manizales-Villamaría Metropolitan Area, considering the current hospital capacity and the average travel time to the hospital network. The results obtained by the E2SFCA method were compared with those obtained by analysis of minimum roads and their correlation with the economic conditions of the studied areas and allowed identifying the areas that would be neglected regarding different scenarios, which is a necessary stage in developing a successful prevention programs. In Chicago and Illinois, Kang et al. (2020) rapidly evaluated the spatial accessibility of ICU beds and ventilators for the population at risk and COVID-19 patients. The rapid calculation was made possible by using a parallel computing strategy based on cyberGIS to overcome the computational intensity of the E2SFCA approach (cyber geographic information science and systems). The study compared COVID19 patients' spatial accessibility measures to those of the population at risk to assess which geographic areas needed additional healthcare services to enhance access. The findings also identified places where a COVID19-induced shortage of healthcare services could occur. The research also identified vulnerable populations living in Chicago neighborhoods with limited spatial accessibility. There is still a research gap in the literature when it comes to examining the spatial access to healthcare facilities during the COVID-19 outbreak. Until the time this study was completed, it was found that during the COVID-19 pandemic, a few researchers have employed some methods of FCA family to measure and evaluate the spatial access to some health care resources related to the treatment or the examination of confirmed or suspected COVID-19 cases, but so far there has been no study that takes into account all the basic health resources that any country can exploit to face and response to this pandemic such as (medical staff, general beds, ICU beds, ventilators, and PCR devices). Also, most of these studies take only one type of health system component such as public hospitals. This study fills these gaps by measuring and assessing the community-level spatial accessibility for various related COVID–19 healthcare resources using the E2SFCA method for the following three types of health facilities: public, private, and educational hospitals. The difference between the three types of hospitals is the medical insurance for each type. Private hospitals have special insurance, while public and educational hospitals have government insurance, and the responsible authority for each type is different, since the public hospitals are affiliated with the Jordanian Ministry of Health, while private hospitals are affiliated with the Association of Private Hospitals and educational Hospitals have an independent administration, so accessibility to each type has been calculated individually to help these entities responsible for each of the three types improve their health services. Also, this research study makes comparisons between the accessibility scores resulting from these hospitals to determine whether there is any shortage of any health care resources in each hospital, to recognize which hospitals might face the greatest overload of admissions, and will thus need additional funding to increase capacity. In addition to what was mentioned earlier, there is no application for any FCA family methods in all Arabian regions whether for health or other forms of services. Besides, as there are no studies in Jordan measuring and evaluating the spatial accessibility of healthcare services at the community level specifically during the COVID-19 pandemic, this research will apply the E2SFCA method for a health system within a region where it has never been used before. 2 Study area and data description Amman city, the capital of Jordan, was chosen to implement the proposed methodology in this study. The total population and area of Amman are about 4.43 million residents and 7,579 square kilometers, respectively, with a population density of 584.6 people per square kilometer (DoS, 2019). In this study, only habited areas were taken (area = 2572 square kilometers) while desert and uninhabited areas were excluded (area = 5007 square kilometers). The city suffers from a naturally increasing urban population. In addition to accepting many war refugees, with an annual Jordanian population growth of 5.3 % and 18 % of forced migration and refugees, (DoS, 2017). Amman is governed by Greater Amman Municipality (GAM) and it contains 13 sub-districts as shown in Fig. 1 . Some required statistics about these sub-districts are also presented in Table 1 .Fig. 1 Study area; Amman City and its sub-districts. Table 1 Statistics of Amman’s sub-districts. ID Sub-District Area (km2) Environmental structure Population counts Population density (per km2) 1 Al-Jam’ah 119.6 Urban 842,180 7041.3 2 Jizeh 1091.2 Suburban 117,915 108.1 3 Um Rsas 209.3 Suburban 15,665 74.8 4 Alquaismeh 120.9 Urban 659,570 5456.8 5 Mowaqqar 369.7 Suburban 54,060 146.2 6 Rojom Shami 68.4 Suburban 41,450 606.4 7 Sahab 178.1 Urban 191,800 1076.8 8 Um El-Basatien 23.8 Suburban 22,090 927.8 9 Hosban 49.0 Urban 35,250 719.8 10 Na’oor 68.0 Suburban 89,420 1314.9 11 Wadi Essier 120.6 Suburban 415,860 3448.8 12 Amman 49.2 Urban 968,940 19707.6 13 Marka 104.3 Urban 1,082,300 10379.4 Since Jordan recognized settlements with a population of 5000 people or more as urban centers after 1994. In this study, the environmental features in the governorate of Amman were evaluated using this classification (all settlements centers with more than 5000 settlers in 2020 were classified as urban, while settlements with fewer than that were classified as “suburban areas”) (Gharaybeh, 2015). According to this definition, there were 6 urban and 7 non-urban sub-districts in the study area as shown in Table 1. The city of Amman can be taken as a good representative of the study of accessibility to healthcare resources due to the clear contrasts within the area in terms of growth levels, population densities, socio-economic profiles, and the provision of primary health facilities. Although several studies showed a positive correlation between population density and COVID-19 infectious rate such as (Bhadra et al., 2021, Carozzi et al., 2020, Hamidi et al., 2020), the current research was performed based on the assumption that the target demographic element is the entire population of the region, which was considered to be those citizens who are most likely to use any of the health care facilities regardless of its type and whether they have insurance or not. Furthermore; the number of confirmed cases in all types of hospitals were not available and accessible during the pandemic due to unknown and privacy concerns. The prevalence of the Delta variation of COVID-19 in the general population was highest in areas with the highest population density. Population density and absolute population size were shown to have a highly substantial positive correlation. To stop the spread of COVID-19, stricter and more comprehensive means of control should be implemented in densely populated regions (Md Iderus et al., 2022). As a consequence of a number of studies that were carried out in Malaysia, Turkey, and England; more COVID-19 case numbers and incidence in regions with a higher population density (Tammes, 2020, Coşkun et al., 2021, Aw et al., 2021). The current study gives credibility to the research that implies COVID-19 instances tend to rise in regions that are characterized by high population densities and high population sizes. In this paper, various data resources were used. Table 2 summarizes all of the fundamental data obtained for the study, along with a description of its type, source, and acquisition date. During the year 2020, there were 49 hospitals in Amman city; 3 public, 45 private, and 1 educational hospital. The medical resources statistics for public and private hospitals with the original data for medical resources in the educational hospital are summarized in Table 3 .Table 2 Description of the basic collected data. Data Description Data type Data source Acquisition Date Public hospitals attributes Excel file Jordanian Ministry of Health 2021 Private Hospitals attributes Excel file Private Hospitals Association 2020 Educational hospitals attributes Excel file Jordan University hospital 2021 Hospital coordinates Shapefile Greater Amman Municipality 2018 Streets network Shapefile Greater Amman Municipality 2018 Amman administrative boundaries Shapefile Greater Amman Municipality 2018 Population counts Excel file Department of Statistics 2020 Table 3 Hospitals descriptive statistics. ID Statistic Doctors Nurses General beds ICU beds Ventilators PCR devices 1. Public Hospitals Statistics 1 Minimum 150 178 213 8 20 0 2 Maximum 947 1509 1785 80 135 9 3 Sum 1366 1940 2447 165 190 11 4 Mean 455 646 815 55 63.3 3.7 5 Standard Deviation 351.0 610.5 692.2 33.3 51.0 3.8 2. Private Hospitals Statistics 1 Minimum 1 3 15 1 0 0 2 Maximum 240 900 350 36 30 4 3 Sum 1922 4637 4418 408 318 20 4 Mean 42.7 103 98.2 9 7 0.4 5 Standard Deviation 53.7 148.9 86.0 9.1 7.3 0.9 3. Educational Hospital (Jordan University Hospital) 1 Number of 812 936 618 16 60 1 3 Methodology This paper involves four basic steps to quantify and assess the community-level spatial accessibility for Amman sub-districts to various healthcare resources during COVID-19 pandemic. This study was conducted at the sub-district level and did not take lower levels such as communities or neighborhoods due to the lack of detailed information on the components and the characteristics of its transport network that are necessary to conduct the accessibility studies using E2SFCA method. The spatial accessibility for every sub-district were calculated based on the weighted population centroid which represent the point where the population are concentered in each sub-district. In the first step, multiple GIS database preparation tasks were performed such as data collection, construction, and preprocessing tasks. The output of this step was the production of several thematic maps with accurate and reliable databases in building the project and conducting many analyses by using this project. The coordinate systems were unified so that all the resulted maps match each other. The Universal Transverse Mercator (UTM) Zone 37 N projection is fixed as the projection system for the datasets. In the second step, the travel times between the sub-district's weighted population centroids and each healthcare facility were calculated using the Origin-Destination (O-D) cost matrix function of the ArcGIS Network Analyst. In the next step, the E2SFCA method was applied to obtain the accessibility scores at the sub-district level to identify the areas with high and low levels of accessibility to different healthcare resources for the three types of hospitals in Amman. In this paper, the E2SFCA method is used among all methods of FCA family for the following reasons: The E2SFCA metric can be used and evaluated because it needs an applicable collection of data to measure: the attractiveness of healthcare facilities (supply), the demand of the population for healthcare facilities, and the travel impedance between the locations of the service center and the demand point. Also this metric in it formulation has applicable underlying principles: a single travel mode, threshold of an invariant distance of catchment sizes, and total population as potential demand. Furthermore, the method can be easily implemented in ArcGIS 10.8.1 using procedures that use a sequence of “join” operations, the “sum” and “field calculation” operations are used in the 2SFCA mother method. Ultimately, the resulted spatial accessibility scores were presented in interactive Geo-spatial maps, compared, and then analyzed for the public, private, and educational hospitals. Fig. 2 shows the general methodology used in this study starting from the design phase of databases to the stage of producing the final FCA maps and conducting various analyses using these maps.Fig. 2 General methodology of the study. The effect of doctors' level of service in each hospital type on doctors' spatial accessibility scores in each sub-district has been examined using a one-way ANOVA test, the resulted level of services (Rj), and the spatial accessibility scores (SAI). One-way analysis of variance (ANOVA) test examines the means of two or more independent groups to check statistically if the related population means differ significantly. In the ANOVA test, there are two types of variables; the dependent variable and the independent variable. The independent variable is usually referred to as the grouping variable or factor. The dependent variable needs to be a continuous variable while the independent variable needs to be a categorical one. The assumptions for the ANOVA test that are related to the dependent variable must be met. These assumptions are the homogeneity of variance (equal variance) assumption and the normality (the distribution of dependent variable is normally distributed) assumption. In this paper, the ANOVA test has been performed where the independent variable is the total doctors' level of service in each hospital type presented by Rj_D with three levels of service (4.720, 6.108, and 2.873, in public, private, and educational hospitals, respectively), and the dependent variable is the doctors' spatial accessibility in each sub-district. Prior to that, the assumption of homogeneity of variance was utilized using Levene’s statistic. Also, the assumption of normality tests was verified using Kolmogorov-Smirnov and Shapiro-Wilk tests for SAI_D values. When the ANOVA results show statistical significance, the post hoc analysis test should be performed to determine where statistically significant differences exist. Post hoc analysis tests are statistical tests that are performed to determine where statistically significant differences exist. In post hoc analysis, each group mean is compared to all other group means. There are several methods for performing post hoc analyses, but the most frequent and well accepted approach is the Tukey method of multiple comparisons (TMC). The E2SFCA approach uses a two-step search procedure to operate. As shown in Eq. (1), the supply-to-demand ratio Rj is calculated by dividing the capacity of a facility Sj (the supply) by the total population Pi (the demand) within a threshold distance d0 (the catchment size). This step assesses the supply's capacity to meet the needs of the entire population within its service radius. The accessibility index (Ai) for the demand point i is determined in the second step as a sum of the Rj values obtained in the first step, as shown in Eq. (2). E2SFCA incorporates a distance decay function within the catchments of supply and demand, which counts as a function of distance for differences in accessibility within the catchments. To each supply and demand pair, weights Wij calculated from the distance decay function are added, so that closer pairs are more likely to interact than those that are further apart (Bryant and Delamater, 2019).(1) Rj=Sj∑i∈[dij<d0]PiWij (2) Ai=∑j∈[dij<d0]RjWij The catchment size (d0) in this study for all different types of medical facilities for driving traffic mode was estimated by the average travel time from all communities to health facilities and set at 15 min for urban neighborhoods, 30 min for suburban neighborhoods, and another threshold of 60 min will be set to cover remote regions. In this study, the travel time thresholds were defined as 15, 30, and 60 min, respectively. For the first and second criteria, the average travel time required in major population centers to access different health services in Amman city was used to determine the appropriate value. In contrast, the longest time threshold was set in order to cover the most remote areas and to ensure compliance with the principle of the golden hour, which states that health outcomes are adversely affected if treatment is not obtained immediately after a devastating injury happens within the first hour of the injury (Zhu et al., 2019). The distance decay function f(dij) should be specified first before the application of the E2SFCA metric as it controls the conversion of measured travel impedance (travel time) (dij) to weight values (Wij). Several functions of distance decay exist. The Gaussian decay function was adopted among others in this study since it is more sensitive to distance changes, as indicated in Eq. (3) (Wu et al., 2020).(3) fdij=e-12dijd02-e-121-e-12 4 Results and discussions The results of the GIS databases preparation stage that was performed to prepare the basic input maps for E2SFCA measurements are shown in Fig. 3 . E2SFCA main outcomes such as the weighted supply-to-demand ratio (Rj) for each health resource available in each health facility and sub-districts spatial accessibility maps demonstrating the difference in the capacities and the spatial accessibility of health facilities within Amman city and how the variations are captured at the various geographies.Fig. 3 (a) Distribution of public, private, and educational hospitals, (b) Amman sub-districts weighted population centroids map, and (c) road network map. The health facility's capacity to meet the needs of the entire population within its service radius is assessed using the resulted Rj values. For any health resource, the higher the Rj value, the better the level of service. Rj statistics for hospital types per every ten thousand residents are presented in Table 4 . For public hospitals, Dr. Jamil Al-Totanji hospital has the lowest Rj for all health care resources except for nurses for which the lowest value was in Prince Hamza hospital. The highest Rj values for all health resources were in Al Bashir hospital except for ICU beds for which the highest value was in Prince Hamza hospital. For the private hospitals, on the other hand, the lowest Rj value for all health resources was in the Middle East Hospital. The highest value for doctors, nurses, and ICU beds were in King Hussein Cancer Center and Foundation private hospital. For general beds, ventilators, and PCR devices the maximum Rj values were in Islamic hospital, Al-Takhassosi Hospital, and Farah Hospital, respectively. For the educational hospital; the Jordan University Hospital, it was noted that it has maximum Rj for nurses and minimum value for PCR devices compared to all other studied hospitals.Table 4 Rj statistics for hospital types per every ten thousand residents. ID Statistic Doctors Nurses General beds ICU beds ventilators PCR devices 1. Public hospitals 1 Minimum 8.21 6.70 11.66 0.44 1.10 0.00 2 Maximum 28.86 46.00 54.41 3.01 4.12 0.27 3 Mean 15.73 22.18 27.65 1.93 2.18 0.12 4 Standard Deviation 9.32 17.09 19.04 1.10 1.37 0.12 2. Private hospitals: 1 Minimum 0.03 0.10 0.51 0.03 0.00 0.00 2 Maximum 7.39 31.83 10.71 1.27 0.91 0.12 3 Mean 1.36 3.32 3.13 0.29 0.23 0.01 4 Standard Deviation 1.71 5.07 2.76 0.29 0.23 0.03 3. Educational hospital (Jordan University Hospital) 1 Rj 28.73 33.11 21.86 0.57 2.12 0.04 4. All types of hospitals 1 Minimum 0.03 0.10 0.51 0.03 0.00 0.00 2 Maximum 28.87 46.00 54.41 3.01 4.12 0.27 3 Mean 2.80 5.08 5.01 0.40 0.38 0.02 4 Standard Deviation 5.82 8.85 8.34 0.55 0.67 0.05 The mean values of Rj for General beds, ICU beds, ventilators, and PCR devices resources were higher in public hospitals followed by educational hospital and then private hospitals. On the other hand, the higher Rj values for doctors and nurses were in educational hospitals followed by public, and then private hospitals. It was also noted that the lowest mean values of Rj for all health resources were in private hospitals although it has a higher overall total asset value and more staff compared to public and educational hospitals, this may be explained by the concentration of the private hospitals in the city center and inner urban areas which have higher demands compared to other areas. To examine the effect of doctors' level of service in each hospital type on doctors' spatial accessibility in each sub-district, a one-way ANOVA test was performed with three different total doctors' levels of service presented by the following three Rj_D levels 4.720, 6.108, and 2.873, in public, private, and educational hospitals, respectively. Rj_D is defined as the total doctors' level of service in each hospital type. In this test, the Rj_D was considered as the independent factor, while the doctors' spatial accessibility index (SAI_D) in each sub-district was considered as the dependent variable. Prior to that, the homogeneity of variance using Levene’s statistic was utilized showing equal variances for SAI_D values since p-value > 0.05 (Table 5 ). Also the normality tests using Kolmogorov-Smirnov and Shapiro-Wilk tests for SAI_D values were performed as another assumption for ANOVA analysis. Table 6 shows that the SAI_D values are normally distributed since the p-value > 0.05 in both tests.Table 5 Test of Homogeneity of Variances for SAI_D for each sub-district. Levene Statistic df1 df2 Sig. Based on Mean 1.872 2 36 0.168 Based on Median 1.727 2 36 0.192 Based on Median and with adjusted df 1.727 2 29.626 0.195 Based on trimmed mean 1.837 2 36 0.174 Table 6 Tests of Normality for SAI_D for each sub-district. Total Rj_D for each hospital type Kolmogorov-Smirnov Shapiro-Wilk Statistic df Sig. Statistic df Sig. 2.873 0.166 13 0.200* 0.935 13 0.396 4.720 0.099 13 0.200* 0.962 13 0.787 6.108 0.125 13 0.200* 0.963 13 0.797 * This is a lower bound of the true significance. The doctors’ level of service has a statistically significant effect on the spatial accessibility values (p-value < 0.05) as shown from the ANOVA test results (Table 7 ). Since ANOVA analysis yielded significant results, it is usually followed by the multiple compassions part to locate where this significant effect is within the three levels of doctors level of service. Post hoc test results, shown in Table 8 , demonstrated that the farther Rj_D levels are from one another the more likely it is to have statistically significant effect and vice versa.Table 7 One-way ANOVA test results. Sum of Squares df Mean Square F Sig. Between Groups 22.073 2 11.036 9.355 0.001 Within Groups 42.469 36 1.180 Total 64.542 38 Table 8 Tukey honest significant difference (HSD) / post hoc test for Rj_D for each hospital type (Multiple comparisons). Total Rj_D for one hospital type Total Rj_D for the other hospital types Mean Difference Std. Error Sig. 95 % Confidence Interval Lower Bound Upper Bound 2.873 4.720 −1.143* 0.426 0.029 −2.180 −0.102 6.108 −1.823* 0.426 0.000 −2.860 −0.782 4.720 2.873 1.143* 0.426 0.029 0.102 2.184 6.108 −0.680 0.426 0.260 −1.722 0.361 6.108 2.873 1.823* 0.426 0.000 0.782 2.865 4.720 0.680 0.426 0.260 −0.361 1.722 *The mean difference is significant at the 0.05 level. Spatial accessibility maps are presented in Fig. 4 . Sub-districts with a low spatial accessibility index and limited access to health services are shown in a light shade of brown, whereas sub-districts with high access are shown in a darker shade. Analysis of these maps reveals that the spatial access to all health resources in the three types of hospitals is more limited to urban communities than to those whose neighborhoods are in suburban areas. The access scores have decreased from the central sub-districts (Amman, Marka, and Al-Jama’ah) to the peripheral sub-districts such as Jizeh and Um Rsas sub-districts.Fig. 4 Spatial Accessibility in all hospitals based on the number of (a) Doctors, (b) Nurses, (c) General Beds, (d) ICU Beds, (e) Ventilators, and (f) PCR devices. In Amman, health facilities are mostly concentrated in the city center and surrounding areas. A substantial spatial mismatch exists between healthcare resources and underserved communities. The concentrated population had a relatively high demand for healthcare services in the central urban area with more medical facilities, while the population in the suburban areas was sparse but has fewer health care facilities options. Road networks have also been shown to have a more remarkable impact on spatial accessibility. In regions that have high road connectivity such as urban areas, access scores are high while in suburban areas with fewer options for health care facilities the access scores are low. In the meantime, Amman, like other developing-country megacities, continues to grow in the face of increasing urbanization. The current urban sprawl is exacerbating the spatial disparity faced in terms of access to medical services, and urban hospitals were unable to accommodate the oversaturated need for medical care; if accessibility in these areas is to be decreased with time, action must be taken. The highest mean values for spatial accessibility scores were in private hospitals followed by public hospitals, and then in educational hospital which may be explained by the existence of a large number of private hospitals which have a higher overall total asset value compared to the other types of hospitals as shown in Table 9 .Table 9 E2SFCA Spatial Accessibility scores statistics. Spatial Accessibility to E2SFCA Statistics Min Max Mean STDEV Sum Doctors Public 0.866 3.622 2.374 0.837 30.864 Private 0.515 5.125 3.054 1.365 39.708 Educational 0.038 2.865 1.231 0.839 16.005 All 1.418 10.599 6.660 2.772 86.574 Nurses Public 1.277 5.102 3.410 1.147 44.324 Private 1.124 12.902 7.368 3.387 95.78 Educational 0.043 3.302 1.419 0.967 18.447 All 2.445 19.612 12.196 5.117 158.375 Beds Public 1.510 6.559 4.195 1.561 54.541 Private 1.256 11.580 7.050 3.067 91.654 Educational 0.029 2.180 0.937 0.638 0.315 All 2.795 19.283 12.183 4.861 158.375 ICU Beds Public 0.093 0.490 0.277 0.116 3.602 Private 0.114 1.082 0.650 0.283 8.452 Educational 0.001 0.056 0.024 0.017 0.315 All 0.208 1.545 0.952 0.388 12.369 Ventilator Public 0.120 0.504 0.329 0.117 4.281 Private 0.093 0.830 0.510 0.217 6.629 Educational 0.003 0.212 0.091 0.062 1.183 All 0.216 1.449 0.930 0.365 12.091 PCR Public 0.006 0.031 0.018 0.008 0.232 Private 0.006 0.052 0.032 0.014 0.418 Educational 0.000 0.004 0.002 0.001 0.021 All 0.012 0.086 0.052 0.021 0.671 The shortage in most health care resources for all types of hospitals occurred more in suburban areas than in urban areas. Um Rsas, Jizeh, and Na’oor sub-districts are the neediest for all health care resources as they receive very low services from all public, private, and educational hospitals. Although the population density in these areas is low, their peripheral location away from the city center and non-proximity to health care facilities, their poor roads connectivity compared to urban areas, all these reasons would make the spatial access to health resources in these areas low. 5 Conclusions and future work In this paper, the E2SFCA method was adopted to quantify and assess community-level spatial accessibility to various related COVID-19 healthcare resources in public, private, and educational hospitals in Amman. The results of this method revealed that although the number of healthcare facilities available is adequate to serve almost the entire city, improper health facilities distribution rather than resources deficiency have resulted in coverage gaps in some areas. The central zones were completely served or even served by a redundant number of facilities, while other zones were only partially served or were even underserved by certain resources. The supply market for medical services in Amman was already dominated by private hospitals, and there was a strong geographic accessibility gap between private, public, and educational health care resources. Amman's health care system should pay more attention to eliminating disparities in spatial accessibility of health facilities and enhancing people's health. Both the government and the private sector should recognize this spatial disparity when improving current ones and when establishing new hospitals, ensuring that hospitals are situated in locations that are readily accessible to people from all areas. Using the number of doctors as an example of a health resource, the resulting doctors' level of services scores (Rj_D) and doctors' spatial accessibility scores (SAI_D) as well as some statistical tests such as ANOVA and post hoc tests were used to demonstrate that the doctors' spatial accessibility in each sub-district was shown to be significantly affected by their level of service in each hospital type and to specify the location of this major influence among different degrees of doctors' level of service. Post hoc test findings show that the greater the range between Rj_D levels, the greater the effect, and vice versa. Several limitations of this study need to be addressed. First, the disparity in medical needs is neglected. In reality, there are numerous medical needs for different populations. More focus is required regarding women, children, and the elderly with higher healthcare need, particularly in areas with lower accessibility to medical facilities. Second, car driving was the only means of transport included in this study because of the lack of comprehensive data on other means of transport. So, the accessibility, especially for low-income community residents who are generally transit-dependent, is usually overestimated when estimating hospital accessibility based on driving alone. Third, the study did not take into account all the components of the health system in Amman, such as military hospitals and new field hospitals dedicated to the treatment of Covid-19 cases due to data unavailability and privacy concerns. It took into account hospitals within the borders of Amman capital only, and it was not possible to take hospitals in neighboring cities because of the lack of information about demand, supply, and traffic impedance in these cities which are necessary and basic to apply the E2SFCA method. In fact, people in the nearby cities (like Al-Salt) are expected to use the health facilities of the capital Amman not vice versa. Moreover, it was not possible to move between Jordanian cities during the crisis, where mobility was only within the province's borders. Fourth, The study was performed based on the assumption that the target demographic element (healthcare demand) is the entire population of the region, which was considered to be those citizens who are most likely to use any of the health care facilities regardless of its type and whether they have insurance or not. The number of confirmed cases in all types of hospitals was not available and accessible during the pandemic due to some privacy and security concerns and other undetermined reasons. Last but not least, this study was conducted at the sub-district level and did not take lower levels such as communities or neighborhoods due to the lack of detailed information on the components and the characteristics of its transport network that are necessary to conduct the accessibility studies using E2SFCA method. The paper also reveals that the use of gravity models combined with geospatial techniques in Jordan to analyze the provision of health services and establish facility plans leads to changes in governance and equal provision of services, but the scarcity of comprehensive databases remains a bottleneck for more rigorous research, so this constraint should be taken into account by policymakers and health service planners. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Peer review under responsibility of National Authority for RemoteSensing and Space Sciences. ==== Refs References Aw S.B. Teh B.T. Ling G.H.T. Leng P.C. Chan W.H. Ahmad M.H. The COVID-19 pandemic situation in Malaysia: lessons learned from the perspective of population density Int. J. Environ. Res. Public Health 18 2021 6566 34207205 Bagheri, N., Benwell, G. 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==== Front Advances in Oral and Maxillofacial Surgery 2667-1476 2667-1476 Published by Elsevier Ltd on behalf of British Association of Oral and Maxillofacial Surgeons. S2667-1476(22)00133-9 10.1016/j.adoms.2022.100383 100383 Article MRONJ Mimicking post COVID-19 mucormycosis; A diagnostic dilemma Kudva Adarsh Saha Mehul ∗ G Srikanth Shah Arun Department of Oral and Maxillofacial Surgery, Manipal College of Dental Sciences, Mahe University, Manipal, India ∗ Corresponding author. 1 12 2022 1 12 2022 10038324 11 2022 28 11 2022 © 2022 Published by Elsevier Ltd on behalf of British Association of Oral and Maxillofacial Surgeons. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Introduction The aim of this report is to document a rare case of medication related osteonecrosis of the jaw (MRONJ), secondary to a commonly prescribed medication for the prevention of post Coronavirus Disease 2019 (COVID-19) lung fibrosis. Case report A 33-year-old male reported with the typical clinical and radiological features of mucormycosis of the upper jaw post COVID-19 infection. On detailed evaluation of history and laboratory investigations, we ruled out mucormycosis. However, he was started on Nintedanib (tyrosine kinase inhibitor, TKI) for the prevention of lung fibrosis. In the absence of other predisposing factors and negative laboratory findings for mucormycosis, we arrived at a diagnosis of MRONJ secondary to Nintedanib. Conclusion Nintedanib must be considered as a causative factor for MRONJ; especially in the current COVID-19 scenario. ==== Body pmc1 Introduction MRONJ is an adverse drug reaction, entailing progressive bone destruction in the maxillofacial region [1]. MRONJ has the following clinical criteria: ongoing or antecedent treatment with antiangiogenic or antiresorptive drugs, no history of radiation or metastasis to the jaw and exposed bone or intraoral/extraoral fistula persisting for more than 8 weeks [2]. 2 Case Report A 33-year-old male reported with pus discharge and mobility of upper front tooth for 6 months (Fig. 1 ). He had tested positive for COVID-19 8 month back. As part of COVID-19 treatment, he was initiated on short-term steroid therapy; Inj. Dexamethasone 8mg over a tapered dose for 1 week and medication to prevent PF; T. Nintedanib 150mg twice a day for 1 month. The patient was a known diabetic, was on oral hypoglycemics but his diabetic parameters were not deranged.Fig. 1 Clinical picture at the time of presentation; sinus opening in relation to 12 & 13. Fig. 1 Extra-oral examination revealed tenderness in the supra-labial region. Intra-oral examination revealed a patent sinus opening at the buccal vestibular depth of 12, 13 region with pus discharge. Mycology staining and culture revealed no fungal component. Contrast CT showed focal erosions of hard palate and body of maxilla; suggestive of osteomyelitis (Fig. 2 ).Fig. 2 Contract CT showing erosions of the hard palate. Fig. 2 A provisional diagnosis of MRONJ secondary to Nintedanib was made. En-bloc resection of upper anterior alveolus and thorough debridement was done. The bone was necrotic (Fig. 3 ). Final histopathological examination revealed necrotic trabecular bone devoid of osteoblastic rimming and no fungal component. Final diagnosis of MRONJ secondary to Nintedanib was made as the patient fulfilled the criteria by AAOMS.Fig. 3 Intra-operative image showing necrotic bone. Fig. 3 3 Discussion The effect of COVID-19 on the pulmonary system has been severe, often seen as acute respiratory distress syndrome and PF. PF is seen in 44.9% COVID-19 survivors [3]. It is associated with reduced diffusing capacity of lungs and pulmonary interstitial damage. The medical community is trying to manage post COVID-19 PF with various drugs, vaccines, technologies [4]. One such drug is Nintedanib (6-methoxycarbonyl-substituted indolinone) [5,6]. Nintedanib is an intracellular inhibitor of tyrosine kinases (TKI) [7]. It was originally designed as an antiangiogenic drug that would target and inhibit tyrosine kinase receptors; fibroblast growth factor receptor (FGFR)-1, vascular endothelial growth factor receptor (VEGFR)-2 and platelet-derived growth factor receptor (PDGFR)-α and β, for cancer treatment [8]. Nintedanib has been extensively explored in the treatment of idiopathic pulmonary fibrosis (IPF). It significantly improved the forced vital capacity in patients with fibrotic changes in lungs [6]. Due to the success against IPF, clinical trials have been carried out to study its effectiveness in treating post COVID-19 PF. Its anti-fibrotic and anti-inflammatory activity reduces lung inflammation and improves the diffusion capacity [9]. The most frequent adverse effect of Nintedanib is gastrointestinal problems - diarrhoea being the most common [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10]]. Other associated problems are bleeding problems, elevation of liver enzymes and very rarely myocardial infarction (<2% cases) [7]. However, no adverse event of Nintedanib-induced osteonecrosis of jaw has been reported in literature so far. Although our patient gave a history of COVID-19, he received only a short duration of corticosteroid therapy. Being diabetic, he had borderline sugar levels. The negative fungal cultures ruled out mucormycosis. He was not on antiresorptive medications and gave no history of radiation therapy. He was however started on Nintedanib for the prevention of post COVID-19 PF. Therefore, since the cause of osteonecrosis of the jaw could not be attributed to any of the above factors, it is reasonable to conclude that this was a variant of MRONJ induced by Nintedanib (TKI). And, to our knowledge, this is the first such case reported in literature. 4 Conclusion TKI such as Nintedanib should be considered as a causative agent for osteonecrosis of the jaw in the absence of other obvious predisposing factors. Its use has increased in recent times due to the high incidence of post COVID-19 PF. Therefore, Nintedanib must be administered after a thorough consideration of risk factors. Written patient consent was obtained before publishing this report. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ==== Refs References 1 Rosella D. Papi P. Giardino R. Cicalini E. Piccoli L. Pompa G. Medication-related osteonecrosis of the jaw: clinical and practical guidelines J Int Soc Prev Community Dent 6 2 2016 Mar 97 27114946 2 Mian M. Sreedharan S. Kumar R. Osteonecrosis of the jaws associated with protein kinase inhibitors: a systematic review Oral Maxillofac Surg 25 2 2021 Jun 149 158 33037971 3 Amin B.J. Kakamad F.H. Ahmed G.S. Ahmed S.F. Abdulla B.A. Mikael T.M. Salih R.Q. Salh A.M. Hussein D.A. Post COVID-19 pulmonary fibrosis; a meta-analysis study Annals of Medicine and Surgery 2022 Apr 6 103590 4 Bazdyrev E. Rusina P. Panova M. Novikov F. Grishagin I. Nebolsin V. Lung fibrosis after COVID-19: treatment prospects Pharmaceuticals 14 8 2021 Aug 17 807 34451904 5 Rodríguez-Portal J.A. Efficacy and safety of nintedanib for the treatment of idiopathic pulmonary fibrosis: an update Drugs R 18 1 2018 Mar 19 25 6 Flaherty K.R. Wells A.U. Cottin V. Devaraj A. Walsh S.L. Inoue Y. Richeldi L. Kolb M. Tetzlaff K. Stowasser S. Coeck C. Nintedanib in progressive fibrosing interstitial lung diseases N Engl J Med 381 18 2019 Oct 31 1718 1727 31566307 7 Richeldi L. Du Bois R.M. Raghu G. Azuma A. Brown K.K. Costabel U. Cottin V. Flaherty K.R. Hansell D.M. Inoue Y. Kim D.S. Efficacy and safety of nintedanib in idiopathic pulmonary fibrosis N Engl J Med 370 22 2014 May 29 2071 2082 24836310 8 Wollin L. Maillet I. Quesniaux V. Holweg A. Ryffel B. Antifibrotic and anti-inflammatory activity of the tyrosine kinase inhibitor nintedanib in experimental models of lung fibrosis J Pharmacol Exp Therapeut 349 2 2014 May 1 209 220 9 Wollin L. Wex E. Pautsch A. Schnapp G. Hostettler K.E. Stowasser S. Kolb M. Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis Eur Respir J 45 5 2015 May 1 1434 1445 25745043 10 Submission PM-2014-01847-1-4 extract from the clinical evaluation report for ofev/vargatef Nintedanib esilate DRAFT V1.	0	PMC9712138	NO-CC CODE	2022-12-02 23:21:35	no	2022 Dec 1;:100383	utf-8	null	null	null	oa_other
==== Front iScience iScience iScience 2589-0042 Elsevier S2589-0042(22)01975-7 10.1016/j.isci.2022.105702 105702 Review In vitro and in vivo models for monkeypox Rosa Rafael Borges 12 Ferreira de Castro Emilene 2 Vieira da Silva Murilo 2 Paiva Ferreira Denise Caroline 2 Jardim Ana Carolina Gomes 3 Santos Igor Andrade 3 Marinho Mikaela dos Santos 3 Ferreira França Flávia Batista 4 Pena Lindomar José 1∗ 1 Department of Virology and Experimental Therapy (LAVITE), Aggeu Magalhães Institute (IAM), Oswaldo Cruz Foundation (Fiocruz), Recife 50740-465, Brazil 2 Rodents Animal Facilities Complex, Federal University of Uberlândia (REBIR-UFU), Uberlândia 38400-902, Brazil 3 Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia 38405-302, Brazil 4 Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia 38405-318, Brazil ∗ Corresponding author 1 12 2022 20 1 2023 1 12 2022 26 1 105702105702 © 2022. 2022 Elsevier has created a Monkeypox Information Center (https://www.elsevier.com/connect/monkeypox-information-center) in response to the declared public health emergency of international concern, with free information in English on the monkeypox virus. The Monkeypox Information Center is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its monkeypox related research that is available on the Monkeypox Information Center - including this research content - immediately available in publicly funded repositories, with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the Monkeypox Information Center remains active. The emergence and rapid spread outside of monkeypox virus (MPXV) to non-endemic areas has led to another global health emergency in the midst of the COVID-19 pandemic. The scientific community has sought to rapidly develop in vitro and in vivo models that could be applied in research with MPXV. In vitro models include two-dimensional (2D) cultures of immortalized cell lines or primary cells and three-dimensional (3D) cultures. In vitro models are considered cost-effective and can be done in highly controlled conditions; however, they do not always resemble physiological conditions. In this way, several in vivo models are being characterized to meet the growing demand for new studies related to MPXV. In this review, we summarize the main MPXV models that have already been developed and discuss how they can contribute to advance the understanding of its pathogenesis, replication, and transmission, as well as identifying antivirals to treat infected patients. Graphical abstract Virology Subject areas Virology ==== Body pmcIntroduction The etiologic agent of zoonotic monkeypox disease (MPX) is the monkeypox virus (MPXV). Belonging to the family Poxviridae and the genus Orthopoxvirus, this virus was isolated for the first time in 1958 from vesico-pustular lesions of infected cynomolgus monkeys kept for research.1 The virus can be divided into two viral clades: the Congo Basin (clade I) and the West African (clade II). The Congo Basin viruses are more virulent, with human case fatality rates during outbreaks in parts of Africa estimated to be around 10%. Although monkeypox is so named because researchers first detected it in monkeys, the virus is believed to be transmitted by wild animals such as rodents or infected people.2 These animals can transmit the disease to humans and secondarily the disease is limited through person-to-person transmission.3 Patients affected by the disease may initially present with fever, headache, back pain, asthenia, myalgia, rash, and lymphadenopathy. Lymphadenopathy is a distinctive feature of the disease that can be used to differentiate it from other infections that have similar clinical presentation at onset (chickenpox, measles, smallpox).3 In more severe cases, monkeypox complications can cause pneumonitis, encephalitis, vision hazard keratitis, and secondary bacterial infections.4 , 5 Previous studies show that mortality rates vary substantially and are vulnerable to case finding bias.6 Historically, the case fatality rate of monkeypox ranges from 0% to 11%, with young children being the most vulnerable group. In the recent epidemics, the case fatality ratio has varied from 3% to 6%.3 Since the first human monkeypox infection was reported in 1970, most outbreaks have been confined to central Africa. The first significant outbreak outside Africa occurred in 2003 in the U.S. and was epidemiologically linked to imported exotic pets (Gambian pouched rats and dormice) from Ghana that spread the virus to pet prairie dogs and from them to humans.7 In recent years, both travel-related and non-travel-related cases have been reported outside of West and Central Africa, which are considered endemic areas. Europe, North America, Australia, and Asia had records of the disease.8 However, the number of cases registered outside endemic areas has increased considerably. As of November 7, 2022, more than 78 thousand cases of monkeypox with 43 fatalities have been reported in a total of 109 countries (102 non-endemic and 7 endemic countries).9 On July 23, 2022, World Health Organization (WHO) declared the current monkeypox epidemic represents a Public Health Emergency of International Concern (PHEIC).10 Since its discovery to date, several research models have already been developed mimicking the pathophysiology of the disease. In this review, we summarize the main models in vitro (2D and 3D cell culture) and in vivo developed to study monkeypox biology and discuss its advantages, disadvantages, and how these models can contribute to coping with the disease. In vitro models In order to study viral infections, tools rather than the natural host are needed due to the impact and lethality of the disease caused by viruses.11 As a solution, Renato Dulbecco and Marguerite Vogt proposed the use of cell lineages in viral research in late 1950, which revolutionized how knowledge is acquired by facilitating the study of viral infection and its replication kinetics.12 , 13 It also resulted in the evolution of the previous time-consumption drug discovery process to an optimized and high selective one,14 added to the alignment with the ethical desire for reducing animal use.15 In this context, it is possible to hypothesize the importance of knowing the most suitable cell culture model to be applied in the studying of the MPXV pathogenesis or to the screening and identification of active molecules that might be capitalized into the clinical treatment of the disease. The in vitro assays have shown that the cells Vero, Vero 76, Vero E6, LLC-MK2, BSC-40, BSC-1, PEK, HEP-2, HeLa, MA-104, HFF, and Balb/3T3 clone A31 are susceptible and capable of maintaining the MPXV replication, resulting in a high titer stock of virus, added of interested applicability into antiviral identification. The cell culture models, their characteristics, as well as their advantages and disadvantages are detailed below and summarized in Table 1 .Table 1 Cells models used in the study of MPXV Cell/tissue type Virus strain Viral cultivation Main applications Reference Vero Congo-8 virus; VARV-UK-60/Ind-3a, MPXV-Copenhagen/Z79-I-005, VACV- Copenhagen, CPXV-Grishak, and ECTV-K-1 Cells were cultured in RPMI-1640 or MEM supplemented with 2–10% fetal bovine serum, at 37°C in a 5% CO2 atmosphere. Some authors mentioned the use of antibiotics and antimycotics Virus isolation, plaque-forming assay, virus amplification, infection characterization, evaluation of host responses, and evaluation of antiviral activity (Marennikova et al.16; Rogers et al.17) PEK, and HEP-2 Congo-8 virus; VARV-UK-60, MPXV-Copenhagen/Liberia-1/Liberia-2/V-70 1 266 a Cell susceptibility (Marennikova et al.16) LLC-MK2 MPXV-V79-1-005-Scab/Katako Kombe and MPXV-GFP Cells were cultured in RPMI-1640 or OPTI-MEM-I supplemented with 2-10% fetal bovine serum, with antibiotics and antimycotics, at 37°C in a 5% CO2 atmosphere. Cells were infected with an MOI of 0.001, 0.01, 0.1, or 1 and incubated for 1–8 days Plaque-forming assay, neutral red uptake assay, MTT assay, virus amplification, virus isolation, infection characterization, evaluation of host responses, evaluation of antiviral activity, and siRNA transfection (Baker et al.18; Alkhalil et al.19) Vero 76 MPXV-V79-1-005-Scab, VACV-Elstree/Copenhagen, CPXV-Marina/Brighton, and CMLV-Somalia Cells were cultured in RPMI-1640 supplemented with 2 or 10% fetal bovine serum or Eagle’s EMEM with Hanks’ salts and 5% fetal calf serum, supplemented with antibiotics and antimycotics, at 37°C in a 5% CO2 atmosphere. Cells were infected with an MOI of 0.01–1 or with 100 plaque-forming units (p.f.u) of virus/well and incubated for 3–6 days Plaque-forming assay, neutral red uptake assay, virus amplification, virus isolation, infection characterization, evaluation of host responses, and evaluation of antiviral activity (Baker et al.18; Yang and Schneller,20; Smee et al.,21) Vero E6 MPXV-V79-1-005-Scab, MPXV-GFP-tdTR, Katako Kombe and MPXV-GFP Cells were cultured in RPMI-1640, EMEM, or DMEM supplemented with 2%–10% fetal bovine serum, with antibiotics and antimycotics, at 37°C in a 5% CO2 atmosphere. Cells were infected with an MOI of 0.1 or 5 and incubated for 1–5 days Plaque-forming assay, neutral red uptake assay, virus amplification, virus isolation, infection characterization, evaluation of host responses, evaluation of antiviral activity, and transfection (Baker et al.18; Johnston et al.22; Alkhalil et al.19) MA-104 MPXV-GFP-tdTR/Z79-I-005 Cells were cultured in MEM supplemented with 10% fetal bovine serum, with antibiotics and antimycotics, at 37°C in a 5% CO2 atmosphere. Cells were infected with an MOI of 0.01 or 5 and incubated for 24 h Infection characterization, evaluation of host responses, evaluation of antiviral activity (Johnston et al.22) HeLa MPXV-GFP-tdTR/Z79-I-005 CPXV-Brighton Red, VACV-WR-GFP/NLS/WR-A4-YFP/LUC and AKMV Cells were cultured in DMEM supplemented with 2%–10% fetal bovine serum or calf serum, at 37°C in a 5% CO2 atmosphere. Some authors mentioned the use of antibiotics and antimycotics. Cells were infected with an MOI of 0.01 or 5 and incubated for 16–24 h MPXV isolation, plaque-forming assay, infection characterization, evaluation of host responses, evaluation of antiviral activity, immunofluorescent cell staining, transfection, IFN I response analysis (Magnus et al.1; Johnston et al.22; Priyamvada et al.23; Fernández de Marco et al.24) Balb/3T3 clone A31 MPXV-Z79-I005, VARV-Copenhagen, CPXV-Brighton, and CMLV-Somalia Cells were cultured in medium supplemented with 2% serum and infected with 100 plaque-forming units (p.f.u) of virus/well and incubated for 3–6 days Plaque-forming assay, neutral red uptake assay, infection characterization, evaluation of host responses, evaluation of antiviral activity (Smee et al.21) BSC-40 MPXV-V79-1-005-Scab)/V70-I-266/V78-I-3945/V81-I-179/V77-I-823/V1979-I-005/2003-RCG-358/2003-USA-039/2003-USA-044/RCG2003-RCG-358, VARV-SOM77-ali/NEP73-175/BSH74-sol/SUD47-juba/SLN68-258/BRZ66-39, CPXV-Brighton Red, VACV-WR-GFP/NLS/WR-A4-YFP/LUC, and AKMV Cells were cultured at 37°C in a 5% CO2 atmosphere, in Opti-MEM (Invitrogen) or DMEM/RPMI-1640 supplemented with 2%–10% fetal bovine serum, antibiotics, and antimycotics. Cells were infected with an MOI of 0.1 or 10 PFU/cell and incubated for 2–5 days Plaque-forming assay, neutral red uptake assay, virus amplification, infection characterization, evaluation of host responses, evaluation of antiviral activity, transfection (Baker et al.18; Smith et al.25; Priyamvada et al.23; Fernández de Marco et al.24; Fogg et al.26) BSC-1 CPXV-Brighton/GER_1990_2/GER_1991_3/GER_2002_MKY/CPXV-Br (ATCC VR-302)/CPXV-GFP, MPXV-Z76/Z79-I-005/MSF6 B16R, VACV -WR (ATCC VR-1354)/IHD-J/MVA (ATCC VR-1508)/WR B18, VARV-BSH1975 B17R DMEM, RPMI supplemented with 2%–10% fetal bovine Serum or with 5%–10% fetal calf serum. Cells were infected with an MOI of 0.01 or 1 Plaque-forming assay, infection characterization, analysis of the virus-cell fusion process, evaluation of host responses, evaluation of antiviral activity, and yield reduction assay (Altmann et al.27,28; Bengali et al.29; Fernández de Marco et al.24) A549 MPXV 2003-USA-044/RCG2003-RCG-358 DMEM with 5%–10% fetal calf serum Transfection, IFN-III response analysis (Fernández de Marco et al.24) RK 13 MPXV WR-7-61 Cells were maintained in MEM supplemented with 5% fetal bovine serum, at 37°C with 5% CO2 Plaque-forming assay (Arndt et al.30) a Not reported; VARV: variola virus; MPXV: monkeypox virus; VACV: vaccinia virus; CPXV: cowpox virus; ECTV: ectromelia virus; CMLV: camelpox virus; AKMV: Akhmeta virus; MOI: MOI; p.f.u: plaque formation unit. Cell lineages directly employed in the MPXV study MPXV was isolated in non-human primates in 1958 from pustules present on cynomolgus monkeys.1 For this, HeLa, monkey kidney, and human amnion cells were incubated with the supernatant of the emulsified pustules. The presence of cytopathic effect was observed 2–3 days after infection. The three types of infected cells were characterized by presenting alterations in their morphological structure.1 In humans the MPXV was first isolated from a human host in 1970 in the Democratic Republic of the Congo by material collected from skin lesions.16 The isolation and characterization of the cytopathic effect were performed by infecting four immortalized cell lines Vero (African green monkey kidney), PEK (pig embryonic kidney cells), and HEP-2 (Homo sapiens epithelial carcinoma cells) with a 10-fold TCID50 serial dilution of MPXV and analyzed up to 7 days post-infection (d.p.i.). In Vero cells, the MPXV formed larger plates than the variola virus with a pattern of internal structure characteristic of other MPXV isolated from monkeys. In addition, when comparing all cell lines, Vero cells were more sensitive to MPXV when compared with HEP-2, and PEK, producing higher titers (about 107 to 108 TCID50). The MPXV can be distinguished from variola and vaccinia viruses in PEK cells by its lower replication and the lack of hemadsorption phenomena. In the attempt to identify antiviral molecules capable of inhibiting the MPXV replication cycle as well as understanding its replication mechanism, several methodologies were employed throughout the literature. Baker and coworkers used Vero 76 (African green monkey kidney), Vero E6 (African green monkey kidney deficient in type I interferon [IFN-I]), LLC-MK2 (Rhesus monkey kidney cells), and BSC-40 cells. The BSC-40 is a continuous cell line derived from BSC-1 cells (Vervet monkey kidney cell)18 , 31 and is suitable for amplification and isolation of large viruses such as the poxviruses due to their resemblance with the natural host. Therefore, this cell lineage was used by the authors for inoculation and amplification for 5 days and collected by centrifugation associated with freeze-thaw cycles. As for the title of the MPXV, Vero E6 cells were employed because it is deficient in an IFN-I pathway and might favor viral replication.32 The protocol used by the authors was based on incubating the virus with cells for 2 h, adding a medium with carboxymethyl cellulose (CMC), analyzed for up to 3 or 5 days p.i. through fixation with crystal violet staining solution (1.3 mg/mL crystal violet, 5% ethanol, 30% formalin), and plaques counted visually. For antiviral assays, the authors employed the neutral red uptake assay, an absorbance technique to assess cell viability where healthy, uninfected cells will take up neutral red dye through pinocytosis, whereas cells infected with a cytopathic virus will not. The Vero 76 and LLC-MK2 cell lines were infected with MOI of 0.01, 0.1, and 1 in the presence of serial dilutions of each one of the 24 compounds analyzed in 96 well plates. The incubation period was related to the presence of CPE in virus-infected and untreated cell control, and the assessment of the potential compounds by IC50 values was better visualized in 5-day incubation and MOI of 0.1. Alternatively, the plaque-forming assay was also used, and the MPXV took 4–5 days to produce plaques of similar size in Vero 76, Vero E6, or BSC-40 cells, whereas other poxviruses produced small plaques after 5–6 days with lower cytopathic effect. Following the technical protocol of plaque reduction assay, Rogers and collaborators (2008) evaluated the effect of silver nanoparticles against MPXV, however employing only the Vero cells in 12 well plates. The cells were infected with 50 p.f.u/well for 1 h and further treated with a serial dilution of the compound diluted into the medium with methylcellulose for 72 h.17 In another study, BSC-40 cells were also infected with MPXV V70-I-266 (Sierra Leone) to propagation finality, but also for the antiviral screening protocols. The cells were seeded in 96 well plates, infected with MOI of 0.1 for 1 h, removed, and replaced with ST-246 in 8 concentrations. After 3 days, the plate was stained with 2x crystal violet, and absorbance was measured at 570 nm, where the intensity of treatment was compared with the virus-infected control.25 Alternatively, the plaque reduction assay was performed by infecting BSC-40 cells in six-well plates with 25 p.f.u for 1 h, added of media with CMC with the compound in five concentrations for 3 days, fixed with 10% formalin, and submitted to immunohistochemistry polyclonal rabbit anti-variola virus antibody and goat anti-rabbit immunoglobulin G-horseradish peroxidase conjugate. The effect of the compound ST-246 was observed by the protection of reducing the CPE, which was seen even in low concentrations (0.015 μM). The BSC-40 was also employed by the authors in further studies to confirm and evaluate results encountered in vivo and in clinical trials to find viral titer, evaluate drug resistance, and perform serological analysis following the same protocols cited earlier.33 , 34 Differently, other authors employed the original BSC-1 cell line instead of the BSC-40 with interesting results. The BSC-1 cells seeded in 24 well plates were infected with 100 PFU for 1 h, overlaid with agarose 2% in the presence or absence of EB peptide (NH2- RRKKAAVALLPAVLLALLAP-COOH) to assess the reduction effect on plaque formation. To this, BSC-1 cells were infected at an MOI of 0.01 with the MPXV or with an inoculum containing the virus pre-treated with EB. Three days post-infection, cells were harvested by scraping, lysed by repeated cycles of freezing and thawing, and tittered on BSC-1 cells. As a result, the MXPV infectivity was significantly inhibited by the EB peptide, mainly in its attachment stage.27 To confirm, the authors based on the fact orthopoxvirus cores are only accessible to antibodies after being released into the cytoplasm, which allows the differentiation between attached and entered viruses using virion-specific or core-specific antibodies, respectively.35 To examine whether EB was disrupting virus attachment or entry into the cells, the number of attached and entered viruses per cell for 100 cells in the absence or presence of EB at 100 μM was quantified in a fluorescence microscope. Samples for attachment staining were fixed with 4% paraformaldehyde, quenched for 5 min with 100 mM glycine, blocked with 10% FBS in PBS, and stained with 1:200 anti-VACPXV antibody. Even if the compound is not capitalized in the clinic, it possesses the greatest potential as a novel in vitro tool to study the poorly characterized early steps in infection with poxviruses. Further studies conducted by Altmann and collaborators also employed the BSC-1 cells in the assays with MPXV.28 Because the MXN is an intercalating agent and inhibits topoisomerase II function resulting in impaired cellular DNA replication and RNA synthesis,36 the authors suggested the possibility of inhibiting MPXV replication. Interestingly, to identify the activity of the MXN, a growth curve with MPXV at an MOI of 3 in the presence of the mitoxantrone (MXN) at 1 μM was generated. The supernatant was collected in time intervals of 1, 2, 6, 12, 24, and 36 h post-infection (h.p.i.). The authors also used an MPXV containing a GFP marker with MOI 1 in the presence of the serial dilutions of the MXN, which resulted in inhibition of MPXV replication with a low EC50 (0.25 μM). In addition, Bengali et al. (2012) used BSC-1 cells to analyze and compare characteristics involved in the cellular invasion of some orthopoxviruses strains in vitro, including MPXV. For this, MPXV expressing luciferase was constructed, and its expression was measured during the entry of the virus into the cell, using a luminometer. The authors observed that MPXV utilizes a low pH-dependent endocytic pathway during cell invasion.29 Johnston and coworkers took a different approach and employed several cell lineages: Vero-E6, HeLa, and MA-104. The Vero E6 cells were infected with MPXV and then transfected with a DNA plasmid containing the GFP from the early viral synthetic E/L promoter and tandem dimer Tomato Red (TR). The assay resulted in the production of MPXV-GFP-tdTR. After harvesting, the virus was amplified in MA-104 cells. These are non-human primate cells isolated from a pure cell population of African green monkey kidneys, which are also derived from the MPXV’s natural host. Further, the HeLa cells, naturally non-producing IFN-β cells37 , 38 were pre-treated with IFN-β in serial dilutions and then infected with the MPXV-GFP-tdTR in high (5) or low (0.01) MOIs. The effect was assessed by titration in a plaque reduction assay and resulted in the identification that MPXV is susceptible to IFN-β, even in low concentrations.22 In an attempt to identify antiviral molecules capable of inhibiting the MPXV replication cycle, Alkhalil and coworkers evaluated the RNAi pathway as a new approach in drug discovery against poxviruses. For antiviral screening, siRNAs targeted against various monkeypox viral proteins were developed and transfected into LLC-MK2 cells. The morphology of the transfected cells was analyzed with phase-contrast light microscopy after transfection, and no signs of cytotoxicity were observed in cells treated with the siRNA pools. The antiviral properties of the most potent siRNA constructs were characterized by transfecting LLC-MK2 cells with a single serial dilution of each construct at six concentrations between 40 and 1.25 nM. Later, the transfected cells were infected with 100 p.f.u/well of MPXV, and viral replication was examined at 48 h.p.i. The siA6-a siRNA was able to inhibit completely the MPXV infection at concentrations of ≥20 nM. The antiviral effects of siA6-a target the virus directly, silencing gene expression or interfering with vDNA replication without disrupting host cell biology. The study of chemical modification and the development of a siRNA delivery system are necessary before the evaluation of siA6-a in animal models.19 Yang and Schneller (2005) investigated the antiviral potential of S-adenosyl-L-hemocosteine (AdoHcy) hydrolase inhibitors, important for interrupting methylation reaction processes essential for viral replication. The experiments were performed using Vero 76 cells, MPXV, and 5′Homoaristeromycin inhibitor. The authors observed that the use of 5′homoaristeromycin 0.12 μg/mL showed satisfactory results against MPXV replication.20 To assess the antiviral activity of ribavirin and mycophenolic acid, Vero 76 and Balb/3T3 clone A31 cells were infected with MPXV (Zaire strain). The reduction effect on plaque formation was observed from cells infected with 100 plaque-forming units (p.f.u) of virus per well incubated for 1.5–2 h. Antivirals were incubated for 6 days, and plate sizes were analyzed.21 In addition, Priyamvada et al. (2021) used HeLa cells to test the antiviral effects of methylene blue derivatives against orthopoxviruses, including the MPXV WA clade. For the plaque reduction assay, HeLa cells were incubated with MPXV and treated with PAV-164 derived from PAV-866, a methylene blue analog, for 24 h. PAV-164 demonstrated efficiency in inhibiting viral replication at concentrations of 5 μM and 1.6 μM.23 It has been reported that poxvirus evades the immune response through the expression of secreted interferon (IFN) decoy receptors such as IFNα/β-binding protein (IFNα/βBP). These proteins prevent the interaction of IFN with cell receptors. Thus, de Marco et al. (2009) investigated the expression of IFNα/βBP in MPXV, using BSC-1 cells. For this analysis, BSC-1 cells were infected with RCG or USA MPXV strains, and after 40 h of incubation, their supernatant was collected and analyzed using the western blotting technique. It was observed that MPXV secretes proteins with type I IFN inhibitory activity, which leads to a failure in the antiviral response and disease progression. Furthermore, the antiviral activity induced by hIFNα-2b, hIFNα-A, or hIFNβ was inhibited.24 In addition, Arndt et al. (2015) observed that in RK13 (rabbit kidney cells) and BSC-40 cells treated with IFN-α A/D and infected with MPXV, viral replication was not affected. Similar result was found in cells treated and infected with VACV.30 BSC-40 cells were used in a study to investigate the neutralizing potential of monkey sera previously immunized with recombinant VACV protein. The results showed that MPXV incubated with monkey serum were neutralized and had their ability to spread prevented.26 From what has been presented here, it is possible to suggest that MPXV can infect several mammalian cell lineages independently of being a primate or non-primate host. This agrees with previous data described in literature because poxviruses do not need specific receptors for attaching and entering the cells,39 as they can interact with glycosaminoglycans or components of the extracellular matrix,40 , 41 , 42 , 43 and thus cytokine receptors may favor the infection.39 In this context, poxviruses can enter permissive and restrictive cells, but what will differentiate the establishment of infection will be the presence of machinery that can control downstream intracellular events to favor or abort viral replication. It was demonstrated by Marennikova and coworkers that HEP-2 and PEK cells produced low viral titer when compared with Vero cells, confirming that those would not be a good fit for viral isolation, amplification, or research purposes.16 What is more interesting is that a tissue-cell lineage is permissible and has the machinery needed for viral replication, which can result in a good fit for in vitro infection; however, in normal conditions, the vertebrate species might not be considered a permissive host.39 Therefore, the appropriated cell choice will be dependent on the purpose and/or hypothesis to be evaluated. Given the continuous accelerated evolution of MPXV, new studies with the currently circulating strains will contribute to understanding of viral infection mechanism. In vivo models What is expected of an ideal animal model is that it can develop the viral infection in a similar way to what occurs in humans, taking into account the pathogenesis of the disease and the clinical signs presented by patients. It is possible to elucidate virus biology and the mechanisms of infection using in vivo approaches; such findings are important for the definition of a suitable animal model for the development of studies aimed at the search for new preventive and therapeutic therapies. Although each animal species has some limitations as to inoculation route, dose, and age, in vivo experimental studies have demonstrated a wide variety of species capable of efficiently modeling the cycle of infection caused by MPXV.44 , 45 The main animal models already used in monkeypox studies will be detailed below, and their main characteristics are summarized in Tables 2, 3, 4, 5, and 6 . Because some of these studies were done with past MPXV strains, these data should be interpreted with caution in the context of emerging viral lineages and strains.Table 2 Mice used in the study of MPXV Animal species Strain Age Viral strain Route of infection/dose Major findings Reference Mouse (Mus muscullus) a Adults and 2-day-old Egg passage material of the monkey agent Intracerebral and intranasal/agent diluted 104 Adult mice inoculated intracerebrally showed signs of encephalitis followed by 100% lethality. Two-day-old infant mice inoculated intranasally showed 100% lethality (Magnus et al.1) C57BL/6, SCID, DBA, A/Ncr, C3HeJ, IFN-γR−/−, BALB/c, IFN-α/βR−/−, 129 stat1−/−, C57BL/6 stat1−/− 6- to 12-week-old MPXV-ZAI-79 Intranasal/a and footpad 102–104 p.f.u/mL Footpad inoculation in adult mice was not efficient in infecting strains characterized as not responding to IFN-1 or type-2-dependent signaling pathways, on the other hand, infection occurred satisfactorily in C57BL/6 stat1−/− and 129 stat1−/− mice. In contrast, the immunodeficient SCID strain was susceptible to an intranasal MPXV infection, as were C57BL/− and 129 stat1−/− 129 stat1−/−. Overall, high mortality was a feature observed in STAT1-deficient mice, in addition to weight loss and viral presence in internal organs (Stabenow et al.46) CAST/EiJ, C57BL/6, B6.129S7-IFNγ, BALB/c a MPXV-Z79-I-005 and MPXV-Z79-CB2 Intranasal102–106 p.f.u/mL MPXV replicated in the lungs to previous titers from other sites in CAST/EiJ mice. Surprisingly, lung titers in dose-infected BALB/c mice were similar to titers in CAST/EiJ mice, although all mice survived. Animals without IFN-γ, treated by gravity, or left by weight presented an upright posture and became moribund. In contrast, similar disease symptoms were delayed and markedly less pronounced in the animals that received IFN-γ, and these animals fully recovered. Inoculation of IFN in CAST/EiJ mice led to protection against MPXV. In addition, C57BL/6 mice with inactivation of the IFN gene or the IFN receptor gene are more sensitive to the disease (Earl et al.47) a 2-, 8-, 12-, and 15-day-old Copenhagen Intraperitoneal and intranasal/1,2x106 p.f.u/mL, footpad/6x102 p.f.u/mL and oral/a When inoculated intraperitoneally, all animals showed similar symptoms and occurred as a result of infection, whereas 50% died infected via the footpad and 40% died orally. Sequential evaluations showed that the virus can be found in the blood, lungs, liver, spleen, and kidneys, with considerable amounts of virus detected in the lungs and other organs in the acute phase of the disease (Marennikova and Seluhina,48) BALB/c and SCID 3- to 4-week-old MPXV-2003-USA-044 and MPXV-Congo-Luc+ Intraperitoneal/a In BALB/c mice, the luminescent signal had the highest peaks between 96 and 120 h. Greater replication and faster dissemination were observed mainly to organs of the peritoneal cavity, with eventual dissemination to axillary lymph. In SCID mice, a more intense luminescent light was observed after 96 h. It was spread to organs and tissues in the regions of the abdominal, thoracic, and axillary lymph nodes, in addition to showing visible signs in the tail, feet, and nasal region. Biophotonic images also revealed the tropism of MPXV for ovarian tissues (Osorio et al.49) CAST/EiJ and BALB 9-week-old MPXV-z06 and MPXV-z79-CB2 Intranasal/a High luminescence in the nasal area with a peak between 7 and 12 days after infection was observed in the animals. With the recombinant strain MPXV-z79-CB2, CAST/EiJ mice infected intranasally showed lethargy, hunched posture, ruffled hair, and severe weight loss (Earl et al.50) ICR 8- to 10-day-old MPXV-Z79-I-005 Intranasal/104–105 p.f.u/mL Mice by MPXV accumulations of nasal, lung, and brain pathogens. The presence and replication in primary target cells and traditional observations of MPXV (mononuclear phagocytic cells and tract epitheliocytes) are present, as well as some other cell types (endothelial cells, reticular cells, connective tissue cells) were also observed (Sergeev et al.51) 129S1/SvlmJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, CAST/EiJ (WD), DBA/2J, FVB/NJ, SJL/J, SPRET/EiJ (WD), AKR/J, C57 L/J, C58/J, MOLF/EiJ (WD), NOD/ShiLtJ, NZB/BINJ, PERA/EiJ(WD), PL/J, SM/J, SWR/J, BUB/BnJ, C57BL/10J, C57BLKS/J, CBA/J, CZECHII/EiJ (WD), LP/J, RIIIS/J, WSB/EiJ (WD), BTBR T+ tf/J, C57BR/cdJ, CE/J, I/LnJ, MA/MyJ, NON/ShiLtJ, NZW/LacJ, PWK/PhJ (WD), SEA/GnJ and BALB/c 4- to 8- week-old and 5- to 11-month- old MPXV-Z79-I-005 and MPXV-USA-2003-044 Intranasal and intraperitoneal/102–106 p.f.u/mL Thirty-eight inbred mouse strains were tested for MPXV susceptibility. Three strains were developed, from which CAST/ were developed. CAST/EiJ exhibit weight loss, morbidity, and death in a dose-dependent manner, whereas there were no deaths of BALB/c mice at high doses. Both routes of inoculation resulted in replication in the spleen (Americo et al.52) CAST/EiJ, C57BL/6, B6:129X1-Il15ratm1Ama/J and B6-129X1 9- to 13-week-old MPXV-z06 Intraperitoneal/a Administration of IL-15 to CAST mice transiently increased NK and CD8+ T cells that could express IFN-γ, indicating that progenitor cells were able to respond to cytokines. However, the number of NK cells rapidly decreased, indicating a defect in their homeostasis. In addition, antibodies to interferon-γ abrogated the protection by activated NK cells. Thus, the inherent susceptibility of CAST mice to orthopoxviruses may be explained by a low level of natural killer (NK) cells (Earl et al.53) C57BL/6J, CAST/EiJ, MOLF/EiJ, C58/J, NZW/Lacj, CASA/Rkj and BALB/c a MPXV-Z79-CB2 and MPXV-Z79-005 Intranasal/104–106 p.f.u/mL NZW/Lac and C58 mice exhibited more weight loss than other classical inbred strains, but all survived intranasal MPXV challenges. Mice from three naturally derived strains, in addition to CAST, exhibited severe weight loss and died or were euthanized (Earl et al.54) C57BL/6 and BALB/c 6- to 7-week-old MPXV-2003-044 and MPXV-2003-358 Footpad and intranasal/ 105 p.f.u/mL Mice inoculated on the footpad with the Congo Basin strain showed clinical signs of the disease, with BALB/c mice showing greater edema compared with C57BL/6 mice. One mouse of the BALB/c strain showed weight loss, whereas no mouse of the C57/BL6 strain showed this clinical sign. When inoculation took place intranasally, weight loss was observed in both strains of mice. On the other hand, mice inoculated on the footpad with MPXV from West Africa showed only mild swelling at the inoculation site. None of the mice in the group inoculated with intranasal MPXV from West Africa developed any obvious signs of morbidity (Hutson et al.44) CAST/EiJ mice 4- to 6-week-old 2022 MPXV isolate (SP2833) Inhalation/104 or 106 PFU Although the virus replicated efficiently in the respiratory tract, mice did not succumb to the infection (Warner et al.55) a Not reported; p.f.u: plaque formation unit; mL: milliliter; MPXV: monkeypox virus. Table 3 Non-human primates used in the study of MPXV Animal specie Age Viral strain Route of infection/dose Major findings Reference Cynomolgus (Macaca fascicularis) a Virus strain isolated from pustules of naturally infected animals Intradermally/a The infected animal developed a local pustule surrounded by edema 7 days after inoculation. Body temperature increased slightly between the 5th and 9th day after inoculation, but no propagation of the eruption occurred (Magnus et al.1) a MPXV- Z79-005 Intravenous/5x106 and 5x107 p.f.u/mL Unimmunized monkeys became seriously ill with fever and weight loss, with between 575 and 820 skin lesions. There were fewer lesions in 65–140 immunized monkeys each, and they were generally smaller and atypical, developed less synchronously, and healed quickly (Fogg et al.26) a Strain recovered from naturally infected monkey (Strain 1744) Intravenous, intradermally and subcutaneous/a Acute disease was observed, characterized by marked facial swelling that extends to the cervical region. Severe difficulty in breathing, papular eruptions throughout the body, ulcerative lesions of the oral mucous membrane, and generalized lymphadenopathy (Prier and Sauer,56; Prier et al.57) a Strain recovered from the blood of an infected monkey (Strain 10,001) Intramuscular/10−1–10−6 After inoculation of graduated doses of MPVX, the clinical features emerging from the infection at each dilution were visibly indistinguishable. The mortality rate reached 30%, and a more intense rash was observed in these animals, followed by progressive disease, prostration, hypothermia, and collapse. Severe illness lasted from 4 to 11 days; during this time the animals became progressively dehydrated and lost weight. In some cases, there was evidence of pyoderma (Wenner et al.58) a a Intravenous, intramuscular, intradermally or intradermally combined and conjunctival routes/a The main events during the course of the infection were fever and cutaneous and oral mucosal lesions. Some animals developed a generalized smallpox rash, whereas in others only a few scattered pustules were seen (Wenner et al.59) Immature a Intramuscular/a The skin lesions progressed rapidly through the papular and vesicular stages. The pustular phase was of short duration with formation of hard crusts. Pustules were observed on the scrotum of males, lesions on the oral mucosa, on the amygdala with histological characteristics similar to those described for the skin and lymph nodes. Generalized lymphadenopathy developed during the first week and lasted until the 3rd week of the disease; there was moderate enlargement of the spleen, histological and cytological changes in a combination of diffuse and follicular hyperplasia, reticular lymphoblast proliferation (Wenner et al.60) 6.2 ± 0.7 year-old MPXV- Z79-005 Intravenous/5x107 p.f.u/mL MPXV infection caused a vesiculopustular rash, fever, lymphadenopathy, splenomegaly, pulmonary edema, increased white blood cells, and the death of some animals (Huggins et al.61) 3- to 6-year-old MPXV- Z79-005 Intravenous/5x107 p.f.u/mL Infected animals developed severe clinical syndromes consisting of anorexia, extremely low temperature, shock, disseminated intravascular coagulation, or respiratory distress leading to death. Severe pathology was characterized by generalized smallpox lesions on the skin and oral mucosa, notable lymphadenopathy and splenomegaly, and pulmonary congestion/edema with gross lesions (Wei et al.62) a MPXV- Z79-005 Intravenous/5x107 p.f.u/mL All infected and untreated animals presented skin lesions observed first in the mouth and head that spread to the rest of the body, progressive signs of the disease that led to their death (Jordan et al.63) a MPXV- Z79-005 Intravenous/5x107 p.f.u/mL Although the PCR data suggested a low viremia, all immunized animals remained clinically well, whereas the non-immunized animals became extremely ill with fever, weight loss, and decreased activity in addition to having a large number of smallpox skin lesions that exhibited a pustular progression (Earl et al.64) a MPXV- Z79-005 Intravenous/5x106 and 5x107 p.f.u/mL A large number of skin lesions appeared in control animals in the first few days, coinciding with high viremia, but disappeared in 4 out of 6 animals. Four untreated animals had severe illness requiring euthanasia. Although treated animals had a high lesion count, the viral load was much lower than that of unvaccinated animals (Earl et al.65) a MPXV- Z79-005 Intravenous/1 mL 2x107 p.f.u/mL Clinical symptoms and post-challenge survival, lesion counts, and viral loads were analyzed. Unvaccinated animals exhibited typical signs of the disease, depression, lethargy, pustule lesions, and high peaks of viral load after challenge (Buchman et al.66) Adults MPXV- Z79-005 Intravenous/5x107 p.f.u/mL Even the vaccinated animals showed signs of the disease. Untreated animals showed increased body temperature, weight loss, and a high number of skin lesions succumbing to the infection (Denzler et al.67) 2- to 8-year-old MPXV- Z79-005 Intravenous/1,65x107 and 5,4x107 p.f.u/mL After the lethal challenge with MPXV, the unvaccinated control animals succumbed to monkeypox 11 days after the challenge, numerous lesions were observed throughout the body and a weak primary immune response when compared with the treated animals (Russo et al.68) a a Exposure to aerosols/a The first sign of infection after exposure was a rise in temperature. From the 4th or 5th day after exposure, cough, runny nose, apathy, and loss of appetite were observed. Mild rash with papules or pustules limited to face and hands is observed. Histologically, ulcerative bronchiolitis, bronchitis, and peribronchitis were observed; in addition, fibrinous necrosis was found in the bronchial walls, peribronchial lymphoid tissues, and bronchopulmonary lymph nodes (Hahon and McGavran,69) 2-4 years-old MPXV- Z79-005 Exposure to aerosols/2,6x105 p.f.u/mL Untreated animals showed severe infection, weight decline, progressive depression, dyspnea, and nasal discharge. Skin lesions, first appearing on day 6 after challenge, live virus, and viral DNA were detected in the throats of all challenged animals. Most of the tissues analyzed were positive for the virus, and there were histological changes consistent with focal acute necrotizing bronchitis and bronchopneumonia; focal, fibrinous necrotizing alveolitis often accompanied by edema; and acute focal vasculitis, sometimes accompanied by thrombosis and perivascular edema (Hatch et al.70) Youth to adults MPXV- Z79-005 Exposure to aerosols/a The animals presented exanthema, enanthema, mild anorexia, fever, cough, nasal secretion, cutaneous, oral, and gastrointestinal lesions. Dyspnea, depression, severe anorexia, and signs of weakness were evident in all animals. Clinical signs progressed to the animals' natural death or euthanasia (Zaucha et al.71) a MPXV- Z79-005 Intratracheal/3,42×106, 8,37×106 and 3,53×107 p.f.u/mL Animals challenged with the lowest dose had fever, weight loss, pustular skin lesions, and lymphadenopathy. Animals exposed to the 8.37 × 106 p.f.u dose showed similar clear signs but with some animals dying and animals exposed to the higher dose had a similar disease course but with an accelerated progression to death (Goff et al.72) 2- to 4-year-old MPXV-MSF#6 (Isolated human) Intratracheal/107 p.f.u i/5 mL Severe morbidity, extensive skin lesions, dyspnea, and low saturation were observed in all control animals. Treated animals showed mild morbidity despite the typical signs of the disease (Stittelaar et al.73) a MPXV- Z79-005 Exposure to aerosols/3×104, 1×105, 3×105 and 9×105 p.f.u/mL Lesions appeared 6 days after exposure in two animals at the highest dose. One animal succumbed with no apparent injury and a single animal had severe injuries. Another animal that died presented histopathologically with pneumonia and intranuclear inclusion bodies consistent with smallpox infection. Discoloration of the lung and enlargement of the bronchial lymph nodes were also observed (Barnewall et al.74) a MPXV- Z79-005 Exposure to aerosols/4×104, 1×105, 4×105 and 1×106 p.f.u/mL Clinical signs observed were fever, decreased appetite and activity, drowsiness, depression, inguinal and axillary lymphadenopathy, and macules that progressed to pustules. Necrotizing lesions were also present on the skin, gastrointestinal tract, mucosal surfaces, and gonads. The histological findings of animals sacrificed during acute illness were similar to those previously reported for aerosolized MPXV (Nalca et al.75) 2- to 7-year-old MPXV- Z79-005 Exposure to aerosols/1×105 p.f.u/mL Challenged animals showed typical signs of the disease, fever, weight loss, papules and pustules, weight loss, and viremia detected in the first day’s post infection. When treated, they have a greater survival with rapid improvement of clinical signs (Russo et al.76) 2-year-old MPXV- Z79-005 Exposure to aerosols/1×105 p.f.u/mL Typical signs of the disease were observed, which included increased weight loss, dyspnea, anorexia and a spike in body temperature. Viral RNA was also identified in body tissues such as lungs, spleen, amygdala, tongue, kidney, heart, cerebrospinal fluid, and mediastinal lymph nodes. Histological changes were restricted to the respiratory bronchioles in the lungs (Tree et al.77) a MPXV- Z79-005 Intravenous/5x104 to 5x107 p.f.u/mL and Intrabronchial/5x104 to 5x106 p.f.u/mL Both routes of inoculation resulted in a rapid spread of the virus; typical signs of the disease from moderate to severe were observed. Disease progression was twice as rapid for animals infected via the intravenous route compared with those infected via the intrabronchial route. All challenged animals resolved the infection (Johnson et al.78) Rhesus (Macaca mullata) a Virus strain isolated from pustules of naturally infected animals Intradermally/a After inoculation none of the challenged animals developed any signs of disease (Magnus et al.1) a Strain recovered from naturally infected monkey (Strain 1744) Intravenous, Intradermally and Subcutaneous/a Intravenous infection resulted in generalized rashes, the subcutaneous one produced a similar granuloma, and the intracutaneous one caused local lesions without spreading to other parts of the body (Prier and Sauer,56; Prier et al.57) a a Intramuscular/a Signs of typical disease in cynomolgus were much less pronounced in rhesus; however, rash, dermal lesions, and short-lived exanthema were observed. None of the animals looked remarkably sick, and none specifically died of the disease, but all developed antibodies against the virus (Wenner et al.59) 2-5 years-old MPXV- Z79-005 Intravenous/1,65x107 and 5,4x107 p.f.u/mL Lethally challenged with MPXV, the animals showed the typical clinical signs of the disease, skin lesions, strong anamnestic responses to the challenge, and a weak primary immune response when compared with treated animals. Treated animals were protected from lethal challenge with MPXV (Russo et al.68) 4- to 9-year old MPXV- Z79-005 Intrabronchial/2×105 p.f.u/mL The challenge resulted in the appearance of numerous disseminated lesions, initially as pustules on the skin and oral mucosa and then progressing from pustular stages to crusts. The animals also developed coughing, symptoms of labored breathing, and fever (Estep et al.79) Sagui (Callithrix jacchus) Adults MPXV- Z79-005 Intravenous/2,4x10,7 9,5 x 105, 7,8 x 104, 5x10,3 510 or 48 p.f.u/mL All animals developed a similar disease course and died or were sacrificed. The different doses also showed different clinical progression, mainly the temporal onset of the disease and the phenotypic presentation of rash. In general, all animals had a skin rash, significant lymphadenopathy, and pronounced lethargy, some of which needed to be euthanized (Mucker et al.80) Adults MPXV- Z79-005 Intranasal/100, 1000, 5000 p.f.u/mL The disease progressed in all animals; the animals became increasingly unresponsive and lay down until they were sacrificed or succumbed to the disease. Other signs included light sensitivity, swelling, runny nose, swelling around the eyes, skin lesions, papules, petechiae, and crusts. They also showed an increase in white blood cells, a decrease in platelets, lymphocytes, and very high neutrophils (Mucker et al.81) a Not reported; p.f.u: plaque formation unit; mL: milliliter; MPXV: Monkeypox virus. Table 4 Prairie dogs used in the study of MPXV Animal specie Age Viral strain Route of infection/dose Major findings Reference Prairie dog (Cynomys ludovicianus) 3-year-old MPXV ROC-2003-358 Intranasal/105 p.f.u/mL Infected and untreated group: facial swelling, nasal discharge, nasal crust, bloody nose, inappetence, weight loss, pustular lesions, petechial rash, mouth breathing. The treated groups: prophylaxis (0) and post-infection (3) did not present symptoms of the disease, whereas the therapeutic group (after the appearance of lesions) presented facial swelling, nasal discharge, nasal crust, bloody nose, lack of appetite, weight loss, pustular lesions (Smith et al.33) Adults MPXV-USA-2003-044 Intraperitoneal/10x1,5 p.f.u/mL and intranasal/10x6.1 p.f.u/mL Animals infected by the intraperitoneal route died approximately 8–11 days after infection and no mucosal or cutaneous lesions were observed. Via the intranasal route, 60% died similarly to the animals in the intraperitoneal group, and the survivors had vesicular lesions on the lips and tongue, along with nasal congestion and mucopurulent nasal secretion, but recovered (Xiao et al.82) 3-year-old MPXV-USA-2003-044 and MPXV-ROC-2003-358 Intranasal/104, 105, and 106 p.f.u/mL Clinical signs were dose dependent, ranging from inappetence, facial edema, forced breathing, nasal pus, crusty nose, nasal blood, swollen paws, crusted lesion on the face, and death (Hutson et al.83) 2-year-old MPXV-USA-2003-044 and MPXV-ROC-2003-358 Intranasal and intradermal by scarification/104,5 p.f.u/mL The animals presented skin lesions on the head, limbs, and trunk. Animals infected with the Congo strain showed a greater increase in temperature and a tendency to lose weight compared with the African strain (Hutson et al.84) 2- to 4- year-old MPXV-USA-2003-044 Intranasal/8,8x105 p.f.u/mL It was observed that animals from all groups began to show clinical signs about 8 days after infection; among these signs are inappetence, decreased activity with recumbency and reluctance to move, as well as skin lesions; in addition, some animals needed to be euthanized due to clinical condition. The survival rate of the animals varied according to the time of initiation of treatment; the earlier it was started, the greater the effectiveness (Hutson et al.85) 2-year-old MPXV-USA-2003-044 Intranasal/5x104 p.f.u/mL (actual dose confirmed by 5.9x104 p.f.u/10 μL of WT and 4.3x104 p.f.u/10 μL of luc+ MPXV) West African (WA) MPXV can be visualized using in vivo imaging in the nose, lymph nodes, intestines, heart, lung, kidneys, and liver at day 6 post-infection. On day 9, lesions became visible on the skin and, in some cases, the spleen. After day 9 post-infection, the luminescent signal representing replication either increased, indicating a progression to what would be a fatal infection, or decreased as the infection resolved (Weiner et al.86) 20-month-old MPXV-USA-2003-044 Intranasal/9x103 p.f.u/mL Sharing contaminated bedding led to all healthy animals developing the disease. In the group in which healthy animals were in contact with the challenged animal, clinical signs of the disease also developed. All four animals that were experimentally challenged recovered from the infection. The euthanized animals had severe diarrhea and countless skin lesions (Hutson et al.87) 2-3-years-old MPXV-USA-2003-044 and MPXV-ROC-2003-358 Intranasal/6x103 and 5x103 p.f.u/mL Animals challenged with the West African strain developed skin lesions, crusty noses, dehydration, and inappetence. In the group challenged with Congo Basin, all animals showed inappetence, dehydration, nasal congestion, pus/blood in the mouth, breathing difficulties, facial edema, pus in the genitals, and swollen paws, in addition to skin lesions (Hutson et al.88) 10-month-old MPXV-USA-2003-044 and MPXV-ROC-2003-358 Intranasal/8x103 p.f.u/mL Animals infected with the Congo Basin (CB) strain had virus recovered from the nasal mucosa, oropharyngeal lymph nodes, and spleen in animals challenged with on day 4 and in animals challenged with the West African (WA) strain on day 6. For In both groups, primary viremia was observed from days 6–9 to day 17. The CB strain spread faster and accumulated at higher levels, causing greater morbidity in the animals when compared with the WA strain. The viral antigen was abundant in all organs tested, except for the brain. Splenocytes were labeled positive for apoptosis more often than hepatocytes in both groups (Hutson et al.89) Adults MPXV-ROC-2003-358 Intranasal/4,3x106 and 2,25x104 p.f.u/mL The incubation period presented by prairie dogs were similar to that observed in humans with systemic orthopoxvirus infection. Regarding the clinical signs observed in the animals, the occurrence of inappetence and weight loss, as well as cutaneous and mucosal lesions, varying in degree of intensity, stands out (Shannon Keckler et al.90) Not reported; p.f.u: plaque formation unit; mL: milliliter; MPXV: monkeypox virus. Table 5 Squirrels used in the study of MPXV Animal specie Age Viral strain Route of infection/dose Major findings Reference Squirrel (S. tridecemlineatus) Adults MPXV-USA-2003-044 Intraperitoneal/105,1 and intranasal/106,1 p.f.u/mL The infection caused severe and fatal disease in all animals in both inoculation routes. High rate of viral load and wide distribution of the virus through the organs. The squirrels were lethargic and anorexic within 4 or 5 days, with death within 9 days. No apparent skin lesions were observed; however, severe liver and spleen lesions were seen in both groups; in addition, necrosis of peribronchial lymphoid tissue and lymph nodes from other sites was also observed in the group challenged by the intranasal route (Tesh et al.91) a MPXV-ZAI-1979-005 and MPXV-USA-2003-044 Subcutaneous/3,7x104 and 1,8x104 p.f.u/mL Clinical symptoms were earlier and more severe in animals that received strain Z79 and mortality in these animals occurred between days 6 and 11; on the other hand, despite presenting a milder symptomatology, death in animals that received strain US03 occurred in a very similar period. Animals infected with the Z79 virus strain showed consistently higher viral titer in blood and lung tissue compared with those infected with US03 virus alone, but it was also possible to identify virus titers in the spleen and liver of these animals (Sbrana et al.92) a MPXV-ZAI-1979-005 Subcutaneous/106,3 p.f.u/mL Infected and untreated animals show signs of disease on day 4 post-infection, and all died between 6 and 9 days post-infection. The animals had a high viral load; however, none of the challenged and treated squirrels developed detectable viremia (Sbrana et al.93) Squirrel (Marmota bobak) 1–2-year-old MPXV-ZAI-1979-005 Subcutaneous/2.6, 4.1, 5.6, 7.1 log10 p.f.u/mL and intranasal/1.8, 0.2, 2.2, 3.7, 4.2, 5.0, 6.6 and 7.8 log10 p.f.u/mL Virus was recovered from nasal mucosa, oropharyngeal lymph nodes, and spleen in animals challenged by MPXV Congo Basin (CB) on day 4 and in animals challenged with the West African (WA) strain on day 6. For both groups, viremia primary was seen from days 6–9 through day 17. Although the histopathology and immunohistochemistry findings were similar, CB MPXV spread faster and accumulated to higher levels, causing greater morbidity in animals when compared to WA MPXV. Two animals that succumbed to the disease showed abundant viral antigen in all organs tested, except the brain. Interestingly, splenocytes were labeled positive for apoptosis more often than hepatocytes in both MPXV groups (Sergeev et al.94) Squirrel (Funisciurus anerythrus) a Congo Basin MPXV/Luc+ Intranasal and intradermal/106 p.f.u/mL MPXV infection in these animals caused moderate to severe morbidity and mortality, with clinical signs including smallpox lesions on the skin, eyes, mouth, and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, rapid systemic spread, and long periods of viral shedding. The sentinel animal showed clinical signs of infection including increased respiratory rate, nasal discharge and mouth lesions, respiratory problems, weight loss, and severe lethargy (Falendysz et al.95) a Not reported; p.f.u: plaque formation unit; mL: milliliter; MPXV: Monkeypox virus. Table 6 Rabbits used in the study of MPXV Animal species Age Viral strain Route of infection/dose Major findings Reference Rabbit (Oryctolagus cuniculus) Adults and 2-day -old Serial dilutions of virus recovered from infected monkeys Intradermal, scarification, and intracutaneous/a Intradermal inoculation led to severe hemorrhagic reactions, but unlike what is observed with the vaccinia virus, pustules followed by necrosis were also observed. Lesions and pustules were also observed by scarification. Infection was fatal for two-day-old rabbits after scarification or intracutaneous inoculation (Magnus et al.1) a a Scarification, intravenous, intradermally, and subcutaneous/a Scarification produced confluent lesions according to the concentration used. The virus was also infectious by the intravenous, intradermal, and subcutaneous routes. The intradermal rout caused after pulse lesions followed by secondary pustules and the intravenous route resulted in generalized disease although the animals recovered (Prier and Sauer,56; Prier et al.57) Adults a Intradermally/a Intradermal infection led to hemorrhagic-necrotic lesion in rabbits (Gispen and Brand-Saathof,96) Adults and 2-day-old Copenhagen and MPXV-6-7255 Intracerebral and intradermally/a All strains caused hemorrhagic necrotic skin lesions. Intracerebral infections in adult and 2-day-old rabbits were fatal with the development of adult meningoencephalitis (Gispen et al.97) Rabbit (Oryctolagus cuniculus domesticus) 10-day-old Copenhagen Intravenous/107 p.f.u and scarification/105 and 106 p.f.u/0, 1 mL. Oral route/1,4 x 106; 107; 108; 109 p.f.u/2 mL. Animals inoculated with MPXV generally had a generalized process of fever, conjunctivitis, rhinitis, rash on the skin and mucous membranes, and weight loss. Papules also appeared that developed into pustules and in some cases became hemorrhagic, with younger animals being more susceptible than adults (Marennikova and Seluhina,48) a Not reported; p.f.u: plaque formation unit; mL: milliliter; MPXV: monkeypox virus. Murines The mouse (Mus musculus) has been a widely used model since the discovery of MPXV. The pathogenesis of the disease has been modeled largely from animal studies, with mice initially used in the evaluation of ectromelia infection and to make kinetic observations of the spread of some viruses.98 Initial studies were devoted to investigating the susceptibility of mice to MPXV. In these tests, adult mice were inoculated intracerebrally with MPXV recovered from infected monkeys. After infection, the animals showed signs of encephalitis followed by 100% lethality. The brains of the animals were collected, processed, and the supernatant reinoculated into a new group of animals, and all these succumbed to infection. A group of two-day-old suckling mice was also evaluated in this study. The animals were inoculated intranasally. Both the virus passed into monkeys, and the virus passed into mice killed 100% of the animals.1 Mortality rates related to contemporary strains of MPXV are low compared with data with past strains.99 Lethal models certainly helped to elucidate the mechanisms that lead patients to death and contributed to the prophylactic measures that are available. It should also be considered that the current mortality rate may be underestimated considering the lack of adequate surveillance in some countries, in addition to the possibility of new, more virulent variants as the virus circulates. For all these possibilities, lethal models may contribute to further investigations on MPXV. Still, there is a need to develop new non-lethal characterized models for studies of current circulating strains. Recently, Warner and coworkers demonstrated that CAST/EiJ mice, a wild-derived inbred strain, does not succumb to infection with the 2022 circulating strain following intranasal exposure, although they are highly susceptive to clade 1 and 2 MPXV and developed fatal disease upon challenge.55 The subcutaneous paw pad inoculation route in adult mice was not efficient in infecting the A129, C57BL/6, DBA, A/Ncr, C3HeJ, IFNγR−/−, and IFN-α/βR−/− strains. In animals characterized as not responding to IFN-1-dependent or type 2 signaling pathways, on the other hand, infection occurred satisfactorily induced by strain line 1 in C57BL/6 stat1−/− and 129 stat1−/− mice. Twenty-five percent death was observed in female C57BL/6 stat1−/− mice 21 days post-infection, whereas 50% death was observed in male mice 12 days post-infection. When infected at the higher dose (4,700 p.f.u), all animals died within 9 days post-infection showing high viral titers in the lung. The immunodeficient SCID (severe combined immunodeficient) strain was susceptible to an intranasal MPXV infection, as were C57BL/6 and 129 stat1−/−, which are due to their failure to respond to STAT1 induced by IFN type 1 and 2 pathway signaling. Overall, high mortality was a feature observed in STAT1-deficient mice, in addition to weight loss and viral presence in internal organs.46 Stabenow et al. (2010) showed a gender effect on MPXV-induced mortality in STAT1-deficient C57BL/6 mouse, with males showing higher mortality rate than females. New studies are needed to assess the role of sex in monkeypox susceptibility and may elucidate mechanisms of host-pathogen interaction. The importance of IFN in protection against MPXV infection has also been demonstrated by intranasal administration of the cytokine in CAST/EiJ mice, which led to protection against MPXV. In addition, C57BL/6 mice with inactivation of the IFN gene or the IFN receptor gene show increased sensitivity to the disease.47 Interferon knockout mice are historically known as suitable models in studies of viral infections. Many viruses have infection mechanisms that can be easily interrupted by the natural defense of mice with an intact immune system; this can be considered one of the reasons why the characterization of immunocompetent models for studies on the development of antivirals and vaccines is still a difficult task. For this reason, knockout models and/or immunocompetent mice at an age when the immune system is still immature are well accepted. However, the use of these models increases the chances that the tests were underestimated due to the absence of the antiviral defense of the model. To address the limitation of using immunocompetent mice and to allow assessment of susceptibility and the course of infection in this model, S. S. Marennikova and E. M. Seluhina, inoculated 1-, 2-, 8-, 12-, and 15-day-old mice with the Copenhagen strain of MPXV. The 8-day-old animals showed clinical signs such as asthenia and loss of appetite when inoculated intraperitoneally or intranasally. Besides these signs, the animals inoculated via plantar cushion showed edema in the foot. In these inoculation routes, all animals died as a result of infection, whereas the intradermal route resulted in the death of 50% of the inoculated animals. Mice inoculated orally became flabby and lost their appetite, leading to a 40% mortality rate in the infected group. For 12-day-old mice, mortality occurred in only 14% of cases. On the other hand, 100% mortality was observed in 15-day-old animals after intranasal inoculation. Sequential evaluations showed that the virus could be found in the blood, lungs, liver, spleen, and kidneys, with a considerable amount of virus detected in the lungs and other organs in the acute phase of the disease.48 The fact that young mice succumb to the disease caused by MPXV suggests the possibility of using this model for viral adaptation, which would allow the development of an immunocompetent adult mouse capable of modeling the disease. Serial passage studies of MPXV in young mice with intact immune system could result in selection of an adapted strain that would cause disease in adult animals, considering the high rate of viral mutation and adaptation by natural selection. These studies will be useful for the development of challenge models for antivirals and vaccines testing using contemporary strain of MPXV. The progression of infection caused by MPXV was also compared in mice with an intact immune system and in immunodeficient mice using viruses with MPXV engineered to express luminescent markers. Viral infection of Balb/c (immunocompetent) and SCID (immunodeficient) mice was monitored using biophoton imaging. In BALB/c mice, the luminescent signal was visualized in the first 24 h extending to 96–120 h, where higher peaks were detected. Higher replication and faster dissemination were observed mainly to organs in the peritoneal cavity, with eventual dissemination to axillary lymph. After 240 h, the animals were able to clear the infection. Balb/c mice have an immune response skewed toward a Th2 profile that not only negatively regulates the secretion of Th1 cytokines but also inhibits and counteracts the activating actions of IFNγ and tumor necrosis factor alpha (TNF-α), deactivating the transcription and translation of several genes in macrophages. Furthermore, interleukin-10 (IL-10) acts by negatively controlling the immune response in general and mainly the inflammatory response and tissue damage.100 , 101 Therefore, although they are immunocompetent mice, it is possible to observe the infection cycle with the appearance of some disease phenotypes. In SCID mice, luminescence indicative of MPXV infection was also visible at 24 h and limited to the peritoneal cavity. A more intense luminescent light was observed, and after 96 h it had already spread to other organs and tissues in the abdominal, thoracic, and axillary lymph node regions, in addition to showing visible signs in the tail, feet, and nasal region when the evaluation reached 264 h. At 168 h after inoculation the animals died, biophotonics images also revealed the tropism of MPXV for ovarian tissues.49 A study by Duggal and collaborators demonstrated the presence of MPXV in the interstitial cells and seminiferous tubules of the testes, as well as in the lumen of the epididymis, which are the sites of sperm production and maturation, in non-human primates.102 These findings, together with the work of Osorio et al. (2009), support the potential sexual transmission of MPXV. New studies addressing the questions are urgently needed. Studies carried out with Zika virus, a virus known to be sexually transmitted, can help as a basis for investigations for MPXV. Viral load should be quantified in vaginal washes and semen from male testes to have a better understanding of viral tropism for cells of the genitourinary system. Once this potential has been identified, crossovers between infected and healthy mice should be performed to determine the possibility and rates of sexual transmission. Vertical transmission also deserves attention. Murine models may also help to discover whether contemporary strains of MPXV can infect fetuses and what the consequences of infection to the offspring are. Also using bioluminescence imaging, Earl et al. found high luminescence in the nasal area peaking between 7 and 12 days post-infection, moving on to the lung. Using the recombinant strain MPXV-z79-CB2, the study also demonstrated that CAST/EiJ mice infected intranasally showed lethargy, arched posture, raised hairs, and severe weight loss.50 Intact 8- to 10-day-old male and female ICR mice were also challenged intranasally with MPXV showing no lethality; however, after day 7 post-infection, clinical signs of disease such as purulent conjunctivitis, blepharitis, and raised hairs were observed, which disappeared after 11–13 days.51 The CAST/EiJ mouse also proved to be an efficient model of MPXV infection, among 37 mouse strains evaluated. In addition to CAST/Eij, MOLF/EiJ and PERA/EiJ also succumbed to the disease. Females between 5 and 11 weeks of age inoculated intranasally showed weight loss and lethality of 100%, 75%, and 40%, respectively. CAST/EiJ mice showed greater sensitivity to MPXV when infected intraperitoneally. Both routes of inoculation resulted in MPXV replication in the lung, spleen, and liver of the infected animals.52 Intranasal infection in CAST/EiJ mice also led to viral replication in lungs and dissemination to other organs (liver, spleen, kidneys, and brain).47 The inherent susceptibility of CAST/EiJ mice can be explained by a low level of NK cells.53 Crossing of CAST/EiJ mice with C57BL/6 or BALB/c was performed to investigate whether resistance or sensitivity to MPXV is a dominant factor. The F1 progeny was relatively resistant to MPXV. However, there was a sex difference; some crossbred male mice succumbed to the disease, whereas all females survived.54 Mice were also models used in evaluations of phylogenetically distinct strains of MPXV. Hutson and coworkers infected 6- to 7-week-old mice subcutaneously in the footpad or intranasally with a dose of 10.5 West African or Congo Basin MPXV strain. Mice inoculated in the footpad with the Congo Basin strain showed edema in the inoculation region, which led to impaired locomotion of some animals, and BALB/c mice had greater edema compared with C57BL/6 mice. BALB/c mice showed weight loss, whereas no mice from the C57/BL6 strain showed this clinical sign. In general, 13 days after infection, the animals had already recovered from the disease. When inoculation took place intranasally, weight loss was observed in both strains of mice. Although weight loss has been verified, no obvious signs of morbidity (ie, lesions) were observed, and all mice survived. On the other hand, mice inoculated in the footpad with MPXV from West Africa showed mild swelling in the inoculation site. None of the animals lost weight over the course of the study or developed some lesion, and all animals in this group survived the infection. None of the mice in the group inoculated with intranasal MPXV from West Africa developed any obvious signs of morbidity.103 Non-human primate models Reports of MXPV infection in non-human primates (NHPs) have been described in several studies, most of which were carried out in cynomolgus (Macaca fascicularis) and rhesus (M. mulatta) monkeys,1 , 56 but there are studies with Macaca philippinensis 104 and Callithrix jacchus.80 , 81 MPXV was first identified and isolated in 1958 in Copenhagen, Denmark, following the observation of two non-fatal outbreaks of a disease in Asian macaques (mainly M. fascicularis) that had come from Singapore for research of polio vaccines. The infected animals presented generalized petechial eruption in the skin that quickly evolved to a maculopapular eruption, with lesions observed throughout the body, particularly abundant and developed on the palms of the hands and soles of the feet.1 Magnus et al. (1959), after isolating the virus from these animals, intradermally inoculated the palm of M. fascicularis with 0.2 mL of tissue culture material from the pustules, which developed a local pustule surrounded by edema 7 days after inoculation and elevation of body temperature between the fifth and the ninth day, but without propagation of the rash. In 1960, Prier et al. confirmed the findings of Magnus et al. by performing challenges through the intradermal, subcutaneous, and intravenous routes and demonstrated again the susceptibility of cynomolgus monkeys to MXPV infection. Intravenous inoculation resulted in generalized eruptions; the subcutaneous one developed a granuloma and the intracutaneous one generated only local lesions.56 , 57 , 105 Subsequently, a series of experiments with non-human primate models were conducted. In the first experiment, immature cynomolgus monkeys (M. fascicularis) were inoculated by the intravenous, intramuscular, intradermal, or intradermal combined and conjunctival routes, and in the second experiment the monkeys were inoculated by the intramuscular route. Assessed daily, the animals developed a typical papular rash observed throughout the body, buccal mucosa, and soft palate.58 , 59 , 60 Cynomolgus monkeys infected with MPXV intravenously developed a uniformly lethal disease and had lesions like what is seen in human infection. After challenge, the animals developed a generalized vesiculopustular eruption including fever, elevated white blood cell count, lymphadenopathy, splenomegaly, and pulmonary edema that led to death between 7 and 15 days after infection.61 Severe pathology characterized by disseminated lesions, lymphadenopathy, pulmonary edema, and splenomegaly was also observed in vaccine studies performed.62 In a drug trial in cynomolgus monkeys, similar results were found; untreated infected animals had disease progression presenting the main phenotypes and a high mortality rate.63 In human smallpox vaccine research, non-human primates are often challenged for MPXV, and cynomolgus are the most used animal model. In the studies carried out, the animals were infected by the intravenous route; those previously immunized were healthy without the appearance of lesions or with the appearance of few and small lesions of rapid healing, unlike the non-immunized monkeys (control), which became seriously ill with depression, lethargy, fever, weight loss, high number of lesions, high viremia, lymphadenopathy, even death.26 , 64 , 65 , 66 , 67 , 68 In studies carried out with aerosolized virus, these animals showed clinical signs of the disease and elevation of body temperature, cough, coryza, anorexia, eruptions, papules, and deaths in addition to histological changes such as focal acute necrotizing bronchitis and bronchopneumonia; focal, fibrinous necrotizing alveolitis often accompanied by edema; and acute focal vasculitis, sometimes accompanied by thrombosis and perivascular edema.69 , 70 Animals also showed exanthema and enanthema, depression, and anorexia in addition to weakness and progression to natural death or euthanasia.71 Because of the efficiency of MPXV infection by aerosol exposure, new studies were carried out. In one of the studies, the virus was deposited directly in the tracheal carina of the animals, whereas in others, aerosol exposure systems were used for infection. In general, cynomolgus of both sexes with different ages and weights succumb to the disease and develop similar signs such as decreased appetite and activity, elevation of body temperature, respiratory stress followed by bronchopneumonia, vesiculopustular lesions, crusted skin lesions, oral ulcers, enlarged and proliferative peripheral lymph nodes, and necrotizing or ulcerative lesions in the esophagus, stomach, and urinary bladder. In addition, some animals still presented subpleural hemorrhage and testicular hemorrhage, reaching death. In all animals, the presence of MPXV was identified in blood, tissues, and mucosal smears.72 , 73 , 74 , 75 , 76 , 77 A comparison of the disease course after an intravenous and intrabronchial inoculation in cynomolgus monkeys was performed using serial doses of MPXV. In both inoculation routes, a classic smallpox-like disease was observed. Animals infected by the intravenous route showed fever, appearance of lesions, peak viremia, and viral shedding in nasal and oral smears while intrabronchial exposure beyond typical signs led to the development of pneumonia and increased disease progression. The development of cutaneous lesions in relation to viremia also demonstrated that both routes of inoculation resulted in a rapid spread of the virus to surrounding tissues, resulting in moderate to severe lesional disease.78 As identified in cynomolgus, Magnus et al. (1959) also observed a generalized picture of the disease in rhesus monkeys. These animals showed the same signs, petechial eruption with evolution to maculopapular eruption and lesions throughout the body, but when performing the same challenge intradermally inoculating the palm of two rhesus monkeys with 0.2 mL of tissue culture material from the pustules, the animals did not develop any signs of the disease.1 Rhesus monkeys challenged by inoculating the virus by intradermal, subcutaneous, and intravenous routes resulted in generalized and/or local rashes and granulomas.56 , 57 , 105 In intramuscular inoculations, after daily evaluation, it was observed that the animals developed less evident cutaneous eruptions along the body and oral mucosa and short-lived exanthema. Although apparently healthy, all animals developed antibodies against the virus.59 When inoculated intravenously, rhesus monkeys challenged with MPXV were shown to be highly susceptible to the disease. The animals presented a severe condition with all the typical signs and progressive death. Viral titers were detected in the blood of all animals.68 To understand the differences in pathogenicity of Congo Basin (clade I) and the West African (clade II) viruses, Estep and collaborators deleted the MPXV inhibitor of complement enzymes (MOPICE) from the MPXV-Zaire strain, which is not expressed by viruses of the West African clade and infected rhesus monkeys (n = 4) with wild type and a virus lacing MOPICE through the intrabronchial route. Typical signs of the disease were observed in both groups and, in general, the disease manifestations associated with both viruses were similar. However, infection with the recombinant MPXV lacking MOPICE resulted in lethal disease in one animal, and all animals in the wild type group survived infection. Analysis of viral loads in infected animals showed similar patterns between bronchial alveolar lavage and whole blood samples, but viral load levels were higher in animals infected with the virus lacking, suggesting that this protein is not the sole virulence factor of clade I viruses.79 Another animal model of non-human primates is the marmoset (C. jacchus). Adult males infected intravenously through the tail vein, or through the saphenous vein, develop the normal course of the disease caused by MPXV and died 15 days post-infection. Marmosets that receive higher doses show definable clinical signs already on the second day post-infection in addition to decreased activity. Rash, significant lymphadenopathy and pronounced lethargy were also observed in marmosets experimentally infected with MPXV. In contrast, lymphadenopathy and rash in the lower-dose infected group were not observed. Lesions in this group were much more discrete and were flat, well-defined lesions and never progressed through the typical stages of the disease. In addition, later examinations showed high viremia, decreased platelets, and an abbreviated acute phase, reflecting early type hemorrhagic smallpox.80 New experiments by Mucker et al. (2018) with adult male and female marmosets infected via the intranasal route were performed. All animals showed signs of disease and were euthanized or succumbed to the disease 15 days after exposure. The only animal that was exposed to a dose of 5,000 p.f.u had dyspnea, a runny nose, and a temperature rise of about 0.5°C above the pre-exposure temperature. Animals that received doses of 1,000 p.f.u had different degrees of dyspnea. In general, all animals, regardless of the dose received, showed other signs such as sensitivity to light, swelling around the eyes, some signs of photophobia, and skin lesions. Subsequent immunological evaluations revealed an increase in white blood cells and a decrease in platelet counts, in addition to the presence of the virus in the oral cavity of all animals from the 15th day after infection, and the animal with the highest dose was identified as having the virus on the sixth day post-infection.81 The evolutionary kinship of non-human primates with humans makes them effective models for comparative studies. A great example of this is the fact that in the studies carried out so far, mice do not present one of the main characteristics of the disease caused by MPXV—the formation of pustules, which is seen only in non-human primates. Prairie dogs Prairie dogs (PD) (Cynomys ludovicianus) are highly susceptible to monkeypox virus (MPXV) infection, and the pathogenesis and severity of the disease vary according to the route of infection, being 100% fatal by the intraperitoneal route, whereas the intranasal route had a mortality of 60%,82 being the main route of choice for further studies. This animal species is an important model for the study of virulence factors, pathogenesis, transmission routes, and elimination of the MPXV virus, in view of its ability to mimic the way the disease occurs in humans, including the different manifestations caused by different strains of the virus.83 , 84 It has also been a widely used model for testing new prophylactic and therapeutic measures.33 , 85 A study using two groups of animals, males and females approximately 2 years old, inoculated intranasally with the West African strain, helped to understand the pathogenesis of the disease, and showed that both showed similar symptoms, despite the difference when different doses were used. The results showed that West African MPXV could be visualized using in vivo imaging in the nose, lymph nodes, intestines, heart, lung, kidneys, and liver as early as day six post-infection. By day nine, it was possible to visualize the skin lesions and, in some cases, also the spleen. After day nine, the luminescent signal representing MPXV replication increased in some animals, indicating a progression to what would be a fatal infection, and decreased in others as the infection resolved. The use of recombinant MPXV luc+ allowed for a greater understanding of how MPXV spreads throughout the body in prairie dogs during the course of infection.86 Studies on the transmission routes using West African and Congo Basin strains showed that infected animals developed the disease and started shedding the virus at 6 and 10 dpi, through oral, nasal, ocular, and fecal secretions, and that contamination of healthy animals occurred either by sharing bedding, fomites and physical contact, or by respiratory secretion.84 , 87 Respiratory transmissibility was questioned in a later study, according to which no healthy animals were infected by the West African MPXV carrier and only 25% of healthy animals became infected from the Congo Basin MPXV carrier, indicating that transmission respiration appears to be less efficient than contact as a transmission mechanism within this model.88 It is noteworthy that the course of the disease was similar regardless of the transmission route, including weight loss, the development of disseminated skin lesions, and antibody production, among others.87 In these studies, the age of the animals ranged from 20 to 36 months, and the viral inoculation doses used were 5x103 96, 9x103 87 and 104.5 84 p.f.u, demonstrating the ability of the virus to develop disease even at lower doses. Of note, the Congo Basin strain proved to be more pathogenic than the West African strain.84 Still on the study that compared the pathogenicity of the Congo Basin strain with that of West Africa, it is noteworthy that the clinical symptoms observed in animals challenged with West African MPXV and that developed the infection (3 out of 5) included skin lesions, crusty nose, dehydration, and inappetence. In the group challenged with Congo Basin MPXV, all animals developed the infection and presented inappetence, dehydration, nasal congestion, pus/blood in the mouth, breathing difficulties, facial edema, pus in the genitals, and swollen paws, in addition to skin lesions. Of this group, 3 of the 5 infected animals were euthanized due to the severity of the degree of morbidity presented, showing once again that Congo Basin MPXV is more pathogenic than West African MPXV.88 A study carried out to verify the effectiveness of the antiviral ST-246 in prairie dogs aged approximately 3 years showed that the infected and untreated group had a mortality rate of 75%, in contrast to all animals in the groups treated prophylactically at day 0 and at 3 days post-infection showed no symptoms of the disease and survived, whereas the therapeutic group, although treated after the appearance of lesions, also resulted in a survival rate of 100%. The clinical symptoms observed in the animals that developed the disease were inappetence, facial swelling, nasal discharge, congestion, weight loss, and, to a lesser extent, mucosal and cutaneous lesions in different degrees of severity. The results of this study suggest that this anti-orthopoxvirus compound proved effective for prophylactic treatment, although some viruses were recovered from treated animals, suggesting the possibility of resistant strains. The study was performed with adult animals, inoculated intranasally with the strain from the Congo Basin.33 In order to better understand the differences in the pathogenesis of MPXV strains, groups of 10-month-old prairie dogs were infected intranasally with the Congo Basin (CB) strain and the West African MPXV strain (WA), and tissues were collected on days 2, 4, 6, 9, 12, 17, and 24 post-infections. Samples were evaluated for the presence of viruses and macro and microscopic lesions. Virus was recovered from nasal mucosa, oropharyngeal lymph nodes, and spleen in CB-challenged animals on day 4 and in WA-challenged animals on day 6. For both groups, primary viremia was seen on days 6–9 through day 17. Although the histopathology and immunohistochemistry findings were similar, CB MPXV spread faster and accumulated to higher levels, causing greater morbidity in animals when compared with WA MPXV. Two animals that succumbed to the disease showed abundant viral antigen in all organs tested, except the brain. Interestingly, splenocytes were labeled positive for apoptosis more often than hepatocytes in both MPXV groups. These findings allow further characterization of differences between the pathogenesis of MPXV strains, including the identification of important sites during early viral replication and cellular response to viral infection. Clinical manifestations, when they occurred, were similar in both groups and included inappetence, maculopapular skin lesions, nasal discharge, and respiratory depression under anesthetic effect.89 Subsequently, a study was carried out using smallpox vaccines post-monkeypox infection. Dryvax, ACAM2000, and IMVAMUNE vaccines were tested in 2 groups of adult animals infected with the Congo Basin strain intranasally at different doses. The study found that, for high doses of infection, none of the vaccines showed efficacy, culminating in the death of all individuals in the groups tested, but for lower doses of infection, vaccine efficacy is related to the interval of post-infection application and the vaccine applied. The incubation period presented by prairie dogs was like that observed in humans with systemic orthopoxvirus infection. In general, the animals showed prodromal symptoms, such as inappetence and weight loss, in addition to an inflammatory reaction and cutaneous and mucosal lesions such as smallpox. The incubation period presented by prairie dogs was like that observed in humans with systemic orthopoxvirus infection. In general, the animals showed prodromal symptoms, such as inappetence and weight loss, in addition to an inflammatory reaction and cutaneous and mucosal lesions similar to smallpox.90 Recently, another study was carried out to test the effectiveness of Brincidofovir (BCV) against monkeypox virus. Adult animals infected with the West African strain intranasally were used. The results showed that the plasma exposure to BCV observed in prairie dogs after 20 or 5 mg/kg was 2–4 times lower than the exposure parameters observed in mice (ectromelia) and rabbits (cotopor) given the same doses. Our findings suggest potentially suboptimal exposure; however, higher doses have been shown to be toxic to prairie dogs. It is possible that the lower plasma exposure is due to the drug metabolization pathway by prairie dogs. It was observed that animals from all groups began to show clinical signs about 8 days after infection; the signs observed were inappetence, decreased activity with recumbency and reluctance to move, as well as cutaneous lesions, which varied in intensity between the animals from each group. Some animals needed to be euthanized due to the clinical condition. The survival rate of the animals varied according to the time of initiation of treatment; the earlier it was started, the greater the effectiveness.85 Although prairie dogs show a satisfactory result in modeling MPXV infection; these animals are not considered conventional experimental models. The use of unconventional animals implies specialized labor, differentiated facilities, and compliance with specific environmental legislation for this type of species. Compared with conventional laboratory species, these animals do not have well-characterized genome sequence, neither hematological and biochemical profile, and immunological reagents are very scarce. Squirrels The use of ground squirrels (Sciuridae) in studies related to MPXV were considered when natural infections began to be identified in the species, evidencing its importance for the epidemiology of the disease. In addition, the data found suggested that the species has the potential to become a great experimental model aimed at understanding the pathophysiology caused by MPXV and for testing therapeutic and prophylactic countermeasures,98 such as antiviral medications and vaccines. In 2004, a study was carried out in order to study the effects of MPXV infection in adult squirrels of the species S. tridecemlineatus. Animals were infected with the MPX 2003 strain by the intraperitoneal and intranasal routes with the dose of 106 p.f.u/mL. The infection caused severe and fatal illness in all animals in both groups, suggesting that these animals are highly susceptible to the virus. The viral load and the wide distribution of the virus through the organs suggest that the infection was systemic. The squirrels were lethargic and anorexic within 4 or 5 days, with death occurring within 9 days. No apparent skin lesions were observed; however, severe liver and spleen lesions were seen in both groups. In addition, necrosis of peribronchial lymphoid tissue and lymph nodes from other sites were also observed in the group challenged by the intranasal route. It is noteworthy that the pathological features of MPXV in S. tridecemlineatus were similar to severe smallpox virus infection in humans.91 In 2007, another study using the same species of squirrel was performed comparing the pathogenic effects of two strains of MPXV, MPX Z79 and MPX US03, inoculated subcutaneously at doses of 3.7 × 104 p.f.u/mL and 1 0.8 × 104 p.f.u/mL US03, respectively. The research showed that clinical symptoms were earlier and more severe in animals that received the Z79 strain and that mortality in these animals occurred between days 6 and 11; on the other hand, despite presenting a milder symptomatology, death in animals that received the US03 strain occurred in a very similar period, between days 7 and 11 after infection. Animals infected with the Z79 MPX virus strain showed consistently higher viral titer in blood and lung tissue compared with those infected with US03 virus alone, but it was also possible to identify virus titers in the spleen and liver of these animals. Therefore, the results indicate that the US03 virus, belonging to the West African MPX virus family, was less virulent than the Central African MPX virus strains.93 In the same year, squirrels (S. tridecemlineatus) were used to investigate the efficacy of the antipoxvirus compound ST-246, after infection by the strain MPX-ZAI-1979-005, inoculated by the subcutaneous route. The results showed that all ground squirrels inoculated with the MPX virus and treated with ST-246 at 0, 24, 48, or 72 h after the challenge survived and none of these animals showed any apparent clinical symptoms. However, 33% of the animals that received treatment from 96 h after infection died; the others survived although some of them showed symptoms of the disease. In contrast, all animals infected with the MPX virus and treated with placebo showed clinical symptoms and died. All animals inoculated with MPXV and treated with placebo had a high MPXV load. However, none of the squirrels challenged with MPX virus and treated with ST-246 at 0, 24, 48, and 72 h post-infection developed detectable viremia. Squirrels in the ST-246-96h group that were humanely killed had low-titer viremia. None of the animals in the placebo or ST-246-0h, ST-246-24h, ST-246-48h, and ST-246-72h treatment groups developed detectable antibodies to the MPX virus. However, the two squirrels in the ST-246-96h group that became ill and recovered had detectable MPX virus antibody titers at the end of the experiment.92 Subsequently, squirrels of the Marmota bobak species were inoculated with the V79-1-005 strain by either subcutaneous or intranasal routes, at different doses, to verify the infective dose (ID50) and the lethal dose (LD50), as well as the effects of antiviral drugsNIOC-14 and ST-246. The study demonstrated that all doses of subcutaneous inoculation of the pathogen caused evident clinical symptoms, such as hyperthermia, skin rash on the body and mucous membranes, serous and purulent rhinitis, conjunctivitis, and incoordination, among others, culminating in the death of all infected animals. Clinical manifestations were similar in both groups. However, the skin and mucosal eruptions were milder and only 41.67% of the sick animals died. In the surviving animals, after the disappearance of clinical signs, scars were formed at the sites of eruptive elements, like smallpox. It is noteworthy that no sign of the disease was recorded in animals from experimental groups treated with NIOC-14 and ST-246 during the entire observation period. In addition, sufficiently high MPXV antibody titers were detected in the neutralization reaction in all animals in the experimental groups and surviving animals in the control group.94 In 2017, a study aimed at characterizing the infection by the MPXV/Congo/Luc+ strain in squirrels (Funisciurus anerythrus) was carried out. Animals were inoculated by intranasal and intradermal routes at a dose of 1x106 p.f.u/mL. MPXV infection caused moderate to severe morbidity and mortality of 75% in the intranasal group and 50% in the intradermal group, with clinical signs including lesions on the skin, eyes, mouth, and nose, as well as dyspnea and profuse nasal discharge. Both intranasal and intradermal exposure induced high levels of viremia, rapid systemic spread, and long periods of viral shedding. Housed in the same room, albeit in a different cage, was the sentinel animal, which showed clinical signs of MPXV infection, including increased respiratory rate, nasal discharge, and oral lesions and needed to be euthanized due to respiratory problems, weight loss, and lethargy. This study suggested that African rope squirrels develop severe pathology when infected with MPXV and that they shed large amounts of virus, evidencing their role as a source of transmission of MPXV to humans and other animals in MPXV endemic regions.95 Similar to prairie dogs, squirrels should be considered sparingly a model for MPXV as they are also unconventional species. In addition, there are countries where access to these animals is restricted. Rabbits Rabbits (Oryctolagus cuniculus) are generally resistant to infection caused by MPXV as adults, except in albino rabbits by inoculations performed by the intracerebral route or by scarification. In contrast, animals up to 10 days of age can be easily infected by other routes of infection considering the absence of a robust immune system at this stage of life. Intradermally, for example, hemorrhagic conditions, the appearance of pustules, and lesions have already been reported.1 , 48 , 96 Intracerebral infections in adult and 2-day-old rabbits were fatal with the development of adult meningoencephalitis.97 Infection by scarification was fatal for 2-day-old rabbits. Albino adults had fever and lesions, but recovered from the disease.1 , 48 Intravenous infection was more efficient in causing severe disease in adult rabbits when compared with the subcutaneous route. When infected subcutaneously, the animals presented generalized smallpox lesions, whereas in the intravenous route, an acute disease was observed with the presentation of papules that later became pustules followed by crusts on the skin and mucous membranes. Although the disease was more severe, the animals recovered.1 , 56 , 57 Oral infection in 10-day-old rabbits resulted in generalized disease and lesions on the skin, ears, and lips, whereas intranasal infection resulted in weight loss in animals. In both infections the animals died. In adult animals, no clinical signs of the disease were observed.48 The intravenous route seems promising for the development of a non-lethal adult model in rabbits. Considering the low lethality of the new circulating strains of MPXV, this animal model has the potential to become a cost-effective option, because it is an animal widely disseminated in animal experimentation units. Depending on the scientific question to be answered, other animal model options should be considered, as adult rabbits, except for the intravenous route, do not present satisfactory answers, requiring the use of animals with an incomplete immune system and/or inoculation routes with greater complexity. Others animal models Guinea pigs (Cavia porcellus) infected via the footpad showed swelling and edema at the inoculation site with the development of a granulomatous lesion. The animals showed antibody titers 14 days after infection. No viral titers were found in collected organs. By scarification, a mild inflammatory reaction occurred on the second day after infection and the development of discrete papules on the fourth day, followed by crusting and subsequent healing. Other routes of infection (intravenous, intracerebral, subcutaneous, intraperitoneal, intradermal, and oral) were evaluated, but the animals did not develop the disease.1 , 48 , 56 , 57 , 106 Similar to guinea pigs, hamsters (Cricetinae) were also not susceptible to attempts at infection by different routes of inoculation by MPXV, although intraperitoneal and intracardiac infection of the virus was detected in the lungs, liver, and spleen of the animals 7 days after infection. Focal lesions were observed only in the liver, kidney, and brain. The main manifestations were perivascular lymphocytic infiltrate and damage to the endothelial structure of blood vessels.48 , 107 Intravenous inoculation or scarification did not lead to overt in clinical disease in white rats (Rattus sp.) to. However, in newborn rats (1–3 days old) infection reached 100% mortality with virus replication in the lungs and liver after intranasal challenge.48 Cotton rats (Sigmodon sp.) can be infected intranasally, and these animals develop generalized disease that led the animals to develop difficulty breathing, cough, rhinitis, conjunctivitis, and progressive weight loss, which led to 50% mortality in the experimental group. High concentrations of virus were detected in blood and organs.108 The rodent Kellen’s dormice (Graphiurus kelleni) challenged with MPXV had 100% mortality. In general, the animals showed weight loss, dehydration, conjunctivitis, rhinitis, and lymphadenopathy. Necrosis in the submandibular lymph nodes, spleen, and thymus; hepatocellular necrosis; and hemorrhage in the lungs, stomach, and small intestine were also observed.109 Chickens (Gallus Gallus) aged 3 to 4 days or 4 to 8 weeks are permissible to infection when inoculated intradermally but develop mild disease phenotypes. Once infected with MPXV, they present vesicles heal 10 days after infection. The same does not occur in adult animals.1 , 106 Although the findings using guinea pigs, hamsters, rats, Kellen’s dormice, and chickens as an experimental model have been limited, further research using the contemporary strain of MPXV may change what we know about infection in these species and therefore further studies should be considered. Recently, Seang and colleagues described a case of possible human-dog transmission of human MPXV of B.1 lineage, demonstrating the potential of the virus to perform host-jumps. The dog had no contact with other animals, but co-slept with the infected tutors.110 Concluding remarks The number of monkeypox cases has increased outside endemic areas putting the world on alert. However, great discoveries have already occurred and through them it was possible to develop new in vivo and in vitro models capable of mimicking aspects of viral biology and the pathology caused by the disease. Using the models described herein, the characterization of different viral strains and the development of vaccines and antiviral therapies has been successfully achieved. In experimental studies, the most adequate models must provide reliable results that can be extrapolated to humans. Given the rapid evolution that MPXV has undergone over the past few years, care should be taken in translating the findings produced with historical strains to contemporary ones. Many animal models have been shown to be susceptible to infection and develop clinical disease with varying degrees of severity, including cases of lethality. Each model has its own applicability for virus and disease studies. The choice of model is linked to the question to which one wants to answer, and each species will have an adequate morphological or physiological particularity to contribute to the experimental response. Mice and rabbits, for example, are readily available, easy to handle, have low maintenance costs, and have a widely known genome, unlike unconventional laboratory species such as prairie dogs and squirrels. However, translating knowledge from rodent studies to the clinic can be a challenge. Non-human primates are genetically closer models to humans, but handling these animals can be costly, in addition to having a longer life cycle, which makes the development of the model difficult. The in vitro and in vivo models developed so far can contribute to study several aspects of the disease and mechanism of virus-host interactions. These models will be key to the development of new drugs and therapies capable of blocking the advance of MPXV, as the virus is spreading around the world (Figure 1 ).Figure 1 Cellular and animal models for monkeypox studies In vitro models (PEK, HEP-2, LLC, MK2, Vero, MA-104, HeLa, BALB3T3, Clone A31, BSC-40, BSC-1, A549, and RK13 cells) and in vivo models (mice, rabbits, prairie dogs, non-human primates, and squirrels) are the basis for the elucidation the biology of new viral strains and for the discovery of new drugs, treatments, and vaccines. Created with Mindthegraph.com. Acknowledgments The authors thank the postgraduate program in Biosciences and Biotechnology in Health at the Instituto Aggeu Magalhães (10.13039/501100006507 FIOCRUZ - PE) for the institutional assistance and support. Dr. Pena's lab is supported by grants APQ-0560-2.12/19 and 441030/2020-3 from the 10.13039/501100006162 Pernambuco State Foundation for Science and Technology (FACEPE) and 10.13039/501100003593 National Council for Scientific and Technological Development (CNPq), respectively. ACGJ is grateful to CAPES (Coordination for the Improvement of Higher Education Personnel)—Brazil, Prevention and Combat of Outbreaks, Endemics, Epidemics and Pandemics-Finance Code #88881.506794/2020-01, and to FAPEMIG (Minas Gerais Research Foundation APQ-03385-18 and APQ-01487-22). MSM thanks FAPEMIG for the scholarship # 12152. IAS thanks CNPq for the scholarship # 142495/2020-4 and CAPES. Print scholarship # 88887.700246/2022-00. Author contributions R.B.R., E.F.d.C., D.C.P.V., I.A.S., M.d.S.M., and F.B.F.F participated in the search for articles to be reviewed and carried out the writing of the review. R.B.R. designated graphical abstract. L.J.P. participated in the supervision. 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==== Front J Am Pharm Assoc (2003) J Am Pharm Assoc (2003) Journal of the American Pharmacists Association 1544-3191 1544-3450 Published by Elsevier Inc. on behalf of the American Pharmacists Association. S1544-3191(22)00388-0 10.1016/j.japh.2022.11.008 Article COVID-19 test and treat strategy: A precedent-setting opportunity for us as societal leaders Gentles CDR Andrew PharmD, MPH, BCPS 1∗ 1 U.S. Public Health Service (USPHS), American Pharmacists Association (APhA) Trustee-at-Large ∗ U.S. Public Health Service (USPHS), American Pharmacists Association (APhA) Trustee-at-Large 1 12 2022 1 12 2022 18 11 2022 18 11 2022 © 2022 Published by Elsevier Inc. on behalf of the American Pharmacists Association. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmc	0	PMC9712140	NO-CC CODE	2022-12-02 23:21:35	no	J Am Pharm Assoc (2003). 2022 Dec 1; doi: 10.1016/j.japh.2022.11.008	utf-8	J Am Pharm Assoc (2003)	2,022	10.1016/j.japh.2022.11.008	oa_other
==== Front Sex Reprod Healthc Sex Reprod Healthc Sexual & Reproductive Healthcare 1877-5756 1877-5764 Elsevier B.V. S1877-5756(22)00111-2 10.1016/j.srhc.2022.100805 100805 Article Exploring the psychosocial impact of the Covid-19 pandemic on women’s perinatal experiences and wellbeing: a qualitative study Mari Federica Capasso Miriam ⁎ Caso Daniela Department of Humanities, University of Naples Napoli Federico II ⁎ Corresponding author. 1 12 2022 1 12 2022 1008054 8 2022 3 11 2022 28 11 2022 © 2022 Elsevier B.V. All rights reserved. 2022 Elsevier B.V. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objective While many studies have investigated the clinical impact of the Covid-19 pandemic on pregnant women’s mental health, little attention has been paid to the exploration of women’s experiences during the perinatal period from a psychosocial perspective in the Italian context. Thus, the present study aimed to explore the psychosocial changes associated with the pandemic in the perinatal context. Methods Twenty-one Italian women who gave birth between March and November 2020 took part in this research by participating in semi-structured interviews, exploring their childbirth experiences. Our data were analysed using a Grounded Theory approach. Results Our findings revealed the enhanced importance of social support as a protective factor against uncertainties, which strongly characterised all phases of the perinatal period during the pandemic. Such uncertainties were mainly linked to the discontinuity in intrapartum care, as well as to concerns of being infected with Covid-19 combined with other pregnancy-specific worries. The main sources of social support were represented by loved ones – most of all partners – along with health care staff and peer networks. Conclusions Our findings suggest the importance of implementing evidence-based policies and interventions to improve women’s wellbeing in the perinatal period during the pandemic, as well as of guaranteeing intrapartum care continuity and the presence of social support. Keywords intrapartum care perinatal experiences Covid-19 pandemic social support psychosocial wellbeing grounded theory ==== Body pmcIntroduction After the first recognised case of Covid-19 in December 2019 in Wuhan, the coronavirus outbreak quickly impacted the whole world, leading the World Health Organization to declare it a pandemic [1]. During the first months of 2020, Italy became the first European country to be affected by this disease, with 210.717 confirmed cases on 3 May 2020 [2]. Besides the well-documented impact on the lifestyle, habits, and physical and mental health of the general population [3], the pandemic has also profoundly changed maternity care procedures in most European countries [4], including Italy [2], with multiple negative consequences for women’s wellbeing [5]. First, the lack of scientific information about Covid-19 and the challenging situation that hospitals were facing during the first wave of the pandemic led to some restrictions, such as the limitation of face-to-face medical appointments [4], [6]. Moreover, in several EU countries, many hospitals adopted the exclusion of the patient’s “companion of choice” from delivery rooms as a measure to contain the risk of contagion [4], [6], [7]. Other issues that pregnant women faced due to the emergency included the cancellation of medical appointments [8], the impossibility of attending antenatal classes [6], the duty to wear a face mask during the delivery and the mother-child immediate separation [7]. In Italy, where the main caregivers for pregnant women are gynaecologists and obstetricians [9] (whose tasks correspond to the ones of a midwife [10]), the first phase of the pandemic was characterised by serious concerns about safer practices for pregnant women and new-borns [11]. Specifically, although the Italian Ministry of Health issued guidelines for pregnancy and delivery [2], intrapartum care still struggled to ensure continuity all over the nation; for example, some hospitals reduced antenatal appointments and suspended certain services (e.g., the epidural) due to the lack of anaesthesiologists, who had been reassigned to Covid-19 intensive care units [4]. Moreover, in some cases, decisions have been taken (e.g., routine separations of the mother and child immediately post-birth, access restrictions to patients’ companions, etc.) that may have jeopardised maternity care [11]. From a psychosocial point of view [12], women who experienced the perinatal period during Covid-19 could be considered an at-risk population due to the lack of social support [13] that, along with the difficulty of accessing care, may have exacerbated pregnancy-specific stress [14]. On the other hand, social support was already considered a protective factor for the wellbeing of pregnant women, both before and during Covid-19 [15], [16], as it helps to cope with psychosocial stressors with a “buffer” effect: this means that the impact of social support becomes more powerful when the stressor is stronger [15]. For these reasons, it is important to understand how women, especially in the Italian context, have experienced these changes and how Covid-19 has impacted their wellbeing during the perinatal period, by examining the phase that goes from the pregnancy to the postnatal period. Out of several studies examining the impact of the Covid-19 pandemic on women’s physical and mental health during the perinatal period [17], [18], [19], to our knowledge, most of them adopted a clinical perspective, aimed at identifying the main symptomatologic manifestations resulting from the Covid-19 situation [20]. On the other hand, few qualitative researches [13], [21], [22], [23] have explored in-depth how women in the perinatal period experienced the psychosocial changes imposed by the pandemic while also taking into account the already demanding path from pregnancy to postpartum. Therefore, the present study aimed to investigate how the first and second wave of the Covid-19 pandemic changed the perinatal experience in the Italian context, considering not only all the related phases (i.e., pregnancy, birth, and postpartum period) but also the experience of the intrapartum clinical pathway, and potential risk and protective factors, such as the presence or absence of social support, or the advent of new types of maternal stressors (e.g., Covid-19 related changes) from a psychosocial perspective. Method Design This study followed the Grounded Theory Methodology, a systematic inductive method aimed at producing theoretical explanations of social processes based on qualitative data [24]. A Constructivist Grounded Theory approach [25] was chosen due to the centrality it ascribes to the interpersonal relationship between researcher and participants. This type of qualitative analysis seemed to be particularly suitable for the purpose of our investigation for at least two reasons: first, the issue (i.e., the impact of the pandemic on the perinatal period from a psychosocial point of view) has not yet been explored in-depth, so the lack of a solid theoretical background, and the consequent explorative approach, made this methodology particularly indicated; second, the characteristics of the population under investigation and the sensitivity of the topics addressed during the interviews needed an empathic context in which participants could feel supported and understood. Sampling and recruitment In line with the Grounded Theory Methodology, purposive sampling was used to recruit women who gave birth in the first stage of the Covid-19 pandemic, specifically between 11 February 2020, when the first cases of Covid-19 were recognised in Italy, and November 2020, during the second wave of the pandemic. Subsequently, theoretical sampling was used to supplement the data and reach saturation. Participants were recruited through Facebook groups dedicated to the themes of pregnancy and childbirth, inviting their members to participate in a study about pregnancy during the pandemic. The total sample included 21 Italian women between 23 and 41 years old (M = 32.6; SD = 4.3) who gave birth between March and November 2020. No respondent tested positive for Covid-19 at the time of admission to the hospital, thus all mothers gave birth in a first-level hospital. Interviews were conducted between one and nine months after childbirth (M = 5.5; SD = 2.9). The participants’ characteristics are summarised in Table 1 .Table 1 Participants’ characteristics. Characteristics Frequency % Age 18-30 6 28.6 >30 15 71.4 Italian region of residence Northern Italy 12 57.1 Central Italy 4 19.1 Southern Italy and Islands 5 23.8 Current marital status In a romantic relationship 8 38.1 Married 13 61.9 Date of delivery March-May 2020 9 42.9 June-August 2020 5 23.8 September-November 2020 7 33.3 Number of children 1 13 61.9 2 7 33.3 3 1 4.8 Delivery modality Vaginal birth 12 57.1 Caesarean section 9 42.9 Presence of a companion during labour* Yes 5 38.5 No 8 61.5 Presence of a companion during delivery Yes 11 52.4 No 10 47.6 Satisfaction with childbirth Not at all satisfied 1 4.8 Not very satisfied 0 0 Neutral 3 14.3 Somewhat satisfied 12 57.1 Very satisfied 5 23.8 *Note. This data was calculated only on women who went into labour (N = 13), since the remaining participants (N = 8) had a planned C-section. Data collection We conducted 21 semi-structured interviews between November 2020 and January 2021. The interviews were guided by a script designed to explore the characteristics of the respondents’ pregnancy and childbirth during the pandemic, the quality of their hospital experience, and their life after delivery, although the majority of questions were guided by the participants’ own words and willingness to discuss the topics proposed more in depth. Some examples of questions that were asked are “Can you please tell me about how you experienced your pregnancy, considering the context of the pandemic?”; “Can you please tell me about your labour and delivery?”; “What was your experience while in the hospital?”; “How did you experience your homecoming after the birth?”. Before conducting the interviews, a brief personal data sheet was administered to the participants in order to record the sample characteristics and childbirth satisfaction (measured on a scale from 1 = “not at all satisfied” to 5 = “very satisfied”). According to the participants’ own preferences, the data were collected by telephone or video call to ensure the maximum level of comfort and confidence. Before each interview, the informed consent form was read to the participants, and they gave their consent both at the beginning and at the end of each interview. Verbal, rather than written consent, was chosen due to the pandemic context, which would have made it more difficult to obtain written consent. Furthermore, considering the qualitative nature of the research, verbal consent was a further modality to preserve anonymity. The length of the interviews was between 20 and 45 minutes. Data analysis After each interview, the collected data were transcribed verbatim; no sensitive information was recorded in order to maintain the participants’ confidentiality. The first phase, after the transcription, consisted of repeated readings of the interviews. The data were analysed using a three-step process. The first step began with open coding based on a descriptive level [26]. In this phase, 492 codes were identified, including some “in vivo codes” to capture the participants’ meaning and point of view. Then, the second step was focused coding, based on a conceptual level [26]. During this phase, 12 categories were recognised. The last step was theoretical coding, characterised by the maximum level of abstraction [26], in which 5 macro-categories were found and linked to each other and to the core category. Field notes and memos were written during data analysis to define emerging categories and relationships between codes and categories, following The Constant Comparative Method of Qualitative Analysis [27]. Although the authors differ in age and academic status, they share the same background in social psychology applied to health. The first author (FM) is a trainee psychologist, the second author (MC) is a psychologist and a PhD student, and the last author (DC) is a professor and has a PhD in Social and Health Psychology. Starting from the awareness of this shared perspective, the constant dialogue within the group made it possible to reflect on the possible impact of previous theoretical knowledge on the interpretation of the data, so that the coding remained anchored to the data as much as possible. The whole process was characterised by a constant discussion between authors of eventual disagreements or diverse interpretations of the data, to triangulate the results and ensure inter-rater reliability. Moreover, group discussions were a chance to further reflect on the eventual influence of pre-existing knowledge, attitudes, and beliefs on the analysis. The entire analysis process was conducted using Atlas.ti 8. SRQR criteria were used to reporting the results [28]. Ethical considerations This study has received the ethical approval of the university committee. Theoretical sampling and theoretical saturation were used to ensure qualitative representation. Leading ethical standards included respect for people, assuming that individuals should be treated as autonomous and capable of deciding for themselves, and beneficence, defined as the duty to avoid harm and maximise possible benefits [29]. To ensure the respect of these principles, we recruited participants according to their willingness, and audio-recorded the interviews only with their consent, allowing the possibility to withdraw from the study at any time and to contact us after the interview for any further information. In order to preserve the anonymity of the participants, only their voices have been recorded even when interviews were conducted by video call. The interviews and their transcription were conducted by only one author (FM), who encrypted the sensitive information, with the aim of further ensuring the participants’ anonymity. Results The findings indicate that the main issue of giving birth during a pandemic was the uncertainty of the period, which seemed to be counterbalanced in some cases by the certainty of the loved ones’ support or, in other situations, emphasised by the loneliness associated with their absence. Therefore, our core category is “The certainty of the other in the uncertainty of the world”. The changes that occurred due to pregnancy and childbirth, added to the ones associated with the pandemic, were seen as a double upheaval in which healthcare personnel and loved ones, when present, were considered the main points of reference. The results also highlighted the importance of finding support in those who experienced the same situation and avoiding the intrusion of those who could not provide real support. In the next section, our five macro-categories are summarised and discussed. Figure 1 illustrates our findings through a conceptual model of the psychosocial changes of giving birth during the Covid-19 pandemic. To preserve the participants’ anonymity, we reported, after each quotation, only the first letter of the interviewees’ name and the date of childbirth.Figure 1 The certainty of the other in the uncertainty of the world: a conceptual model of the psychosocial changes of giving birth in the Covid-19 pandemic. Giving birth in a pandemic as a double upheaval The first macro-category concerns the double upheaval of giving birth during the Covid-19 pandemic. Our findings suggest that this upheaval was associated with the many changes imposed firstly by pregnancy and childbirth, which mainly represented desired changes, and secondly by the pandemic, which instead was considered to be a forced change. This macro-category involves the representation of Covid-19 uncertainty in a mother’s eyes, an issue that impacted the participant’s experiences, making Covid-19 a limit and an exacerbation of difficulties during the perinatal period. Such conditions led to experiencing several negative emotions, often faced by “looking at the bright side”. Covid-19 uncertainty in a mother’s eyes From the participants’ perspectives, giving birth during a pandemic was a challenging process characterised by concerns about the spread of the outbreak. In many cases, the idea that being infected is partly a matter of chance seemed to lead to a perception of uncertainty, especially in the early stages of the pandemic. Indeed, the higher risk of contagion characterising the first wave (March-May 2020) was associated, in our participants, with a sense of panic, enhanced by the rapid changes in security measures, the overabundance of media information, and low levels of perceived control over protecting themselves. The second wave of the pandemic (which began in mid-September 2020) was instead characterised by a higher sense of security associated with better knowledge of the virus and a sense of control of the contagion.“It was at the very beginning of the pandemic, so we were all really panicked. There were no masks, and you didn’t really know what was going on […]. Now, in hindsight, it’s been nine months, so we know that if you walk around with your high-protection mask, gloves, etc., you are at least as protected as possible, and that healthcare workers test you for Covid, so you’re protected, but at the beginning, we didn’t know anything so […] I was in an absolute panic.” A., gave birth in August 2020 Covid-19 as a limit and exacerbation of difficulties during the perinatal period Covid-19 appears to have made the participants’ experiences of the perinatal period harder because of the concurrence of several negative factors, first of all, due to the practical limitations imposed by the emergency. The pandemic also represented an exacerbation of difficulties during pregnancy, childbirth, and post-partum, because Covid-19 was perceived as a further problem to worry about, along with all the other concerns normally related to perinatal experiences. Another frequently reported problem was the struggle to breastfeed due to the lack of information and practical support from an expert figure.“I was prepared to try to breastfeed her, but there was no way, probably it would’ve been better if it happened at another time where you could request some obstetrician, someone who would even come to help.” V., gave birth in April 2020 Among the main issues, there was the inability to take moments for oneself during the postpartum period, along with a general difficulty in accepting the disruption of lifestyle imposed simultaneously by lockdown, pregnancy, and birth.“There was nothing normal anymore. [...] I was already doing something that changed my life with the birth [...]. And the birth of a child throws you into another world, then you think ‘I sought it, I wanted it, it’s a beautiful thing... but I didn’t ask for this other upheaval’. I mean. It was just an attempt to find some boundaries, and I couldn’t find them because everything was different from how I had left it.” V., gave birth in April 2020 The negative emotions associated with giving birth during a pandemic The uncertainty of the period was linked to harder birth experiences, described by negative emotions such as guilt for giving birth in a challenging moment. Other negative emotions reported were a sense of panic and anxiety, exacerbated by a lack of distractions, the absence of loved ones during the experience, and concerns for the impact of the pandemic on child development.“Because you feel guilty and think: ‘Wow, I gave birth to him kind of at a bad time’. It’s like you feel guilty for giving birth to him in such a difficult time. You couldn’t even go on a simple walk. Since he was born in the summer, I already had the idea to go for walks with him, even though he was small. Instead, now it’s clear that he is not used to going out, […] when he comes home, he’s more serene. But unfortunately... it would have been nice to take walks, since it was summer, but unfortunately, we were forced at home... It was hard because that way you can relax, but if you stay at home 24/7, you become a little nervous.” L., gave birth in July 2020 Looking at the bright side One of the main adaptive coping strategies was to look at the bright side of the situation: during pregnancy, the quarantine represented a moment to rest, characterised by a positive atmosphere.“At the beginning, the first impact with the pandemic wasn’t good at all. Anyway, we were able to move on. We went through the lockdown with a smile, mindful that this little baby was coming.” S., gave birth in October 2020 During the postpartum, looking at the bright side meant considering the quarantine as a chance to better enjoy the first moments with the new-born, stay with family and process the birth in privacy. From the participants’ point of view, quarantine was also a precious opportunity for fathers, as they were able to be a constant and supportive presence for mothers, participating in the parenting life. Lastly, childbirth represented a reason to consider 2020 better than other people did.“My family really feels this domestic dimension so much… right now we are all at home, my husband and I wake up, and every morning we bathe [our child] together, but who can currently afford this luxury? So yes, the situation is dramatic, it’s sad, but from this point of view, there is a positive aspect for me.” L., gave birth in October 2020 Health professionals as a point of reference In the second macro-category, the uncertainties of intrapartum care are described in the light of the relationship with health professionals. Despite the difficulties and discontinuities in the care pathway, the healthcare staff was the mothers’ main point of reference, allowing them to overcome the challenges they had to face. The uncertainties of intrapartum care The main uncertainties of intrapartum care included the access to medical examinations and the cancellation of antenatal classes, experienced as a missed opportunity to learn how to handle the fear of childbirth and the postpartum period, especially in cases of first pregnancy. Although in-person antenatal classes would have been preferred to the online ones, their presence was still perceived as better than their complete absence, which instead put women in a condition of uncertainty and anxiety. When antenatal classes were unavailable, obstetricians helped the women prepare themselves for the birth. Moreover, the regulations introduced to counter the emergency were perceived as limiting in relation to intrapartum care, sometimes preventing the timeliness of necessary treatments and the freedom of choice of pregnant women.“I couldn’t do the nuchal translucency screening, the one that’s done to know whether the baby has Down Syndrome, because the appointment coincided exactly with the start of the first lockdown. I knew it was a very important medical examination for the baby, and I suffered a lot for not having done it. You never know until you find yourself in that situation, and I don’t know how I would have reacted. If there had been any anomalies I would’ve had, let’s say, the possibility to choose, but without that exam I couldn’t have it.” G., gave birth in October 2020 In some cases, the women had to deal with the closing of the hospital of their choice, and they had to search, a few days before childbirth, for a new one that could take care of them, with the uncertainty of being accepted at the moment of birth due to the high number of requests.“I had all the pregnancy check-ups done at the same hospital, so the medical staff knew me and my pregnancy history... A week before the planned date for delivery, the hospital closed as it turned into a Covid facility, so I found myself looking for a new hospital. It hasn’t been easy... Even though there were quite a few hospitals in my area, most were closed, except for the General Hospital and the one where I finally went for the delivery. Of course, since they were the only two wards available, all the pregnant women went there, and when I called, I was told: ‘Look, I am not sure whether we can examine you. If you think you are ready to give birth, just come in through the ER’. I mean, I understand that, but it was still a situation to keep a close eye on.” G., gave birth in March 2020 The uncertainty of intrapartum care due to the closing of several hospitals has taken on the meaning of an interruption of the relationship with health professionals, who were considered highly valuable and necessary by the women interviewed. This situation, added to the obligation to be alone in the hospital and the need for human warmth, further enhanced by the pandemic, led to the perception of the hospital as a place of tension and loneliness, characterised by an unsettling atmosphere and described, in some cases, as a trap. Trust and empathy for a good relationship with health professionals Despite the pandemic, most women experienced a significant and empathic relationship with health professionals, especially obstetricians. The latter were the most cited figure among health professionals and appear to be the main source of support.“All obstetricians were great, both during and after childbirth. Being alone with the child, I could not handle everything, and as soon as you called them, they immediately came to the rescue, also because they said ‘Since it’s a peculiar period and you don’t even have a husband here to help you, we more than ever must be close to you, much more than before’ because we were basically alone.” M., gave birth in March 2020 Loneliness and absence The third macro-category concerns one of the main issues reported in the interviews: the theme of loneliness, referring to the loved ones’ absence during the perinatal period due to their exclusion, and in particular to the absence of the partner. The unmet need to share The absence of loved ones appeared to strongly influence the emotional aspect of the experience, leading to some important consequences such as the lack of emotional and practical support and the feeling of profound loneliness, especially during hospitalisation and postpartum.“It’s a moment charged with emotions, worries, fears, and pain. It’s a unique moment, and in hindsight it’s a beautiful one, but you need someone to share it with... In our hospital, nobody was allowed to enter, so I spent my days mostly alone. You know, it’s tough. Besides, you experience frequent hormone imbalances, and you are not in your right mind during those days, and you feel like crying… but who do you cry with?” A., gave birth in August 2020 Participants often spontaneously reported what was the worst experience for them to go through alone: some of them described the hospitalisation as the worst aspect, others the physical difficulties, labour and delivery, and the medical examinations. More specifically, the missed chances to share some of the key experiences related to pregnancy (e.g., ultrasound scans, the discovery of the child’s sex, the first purchases for the unborn, etc.) with loved ones were associated with the perception of not having fully lived them. The main strategies used to fight loneliness included making video calls with loved ones, trying to distract oneself by thinking about something else, or preparing oneself in advance to be alone. Exclusion of the partner as a missed chance to share feelings and responsibilities The feeling of loneliness was associated with the partner’s absence, which was harder to bear during labour and delivery. His absence, which in hindsight made the memories of childbirth worse, assumed many significant meanings, such as the impossibility to benefit from his practical and emotional support, leading to the impossibility of sharing the emotions associated with childbirth. In addition, the partner’s practical exclusion led to the perception of his marginalisation on the level of emotions and decision-making, conveying the message that his role is secondary and ultimately leaving women alone to assume all responsibilities.“Going to pregnancy check-ups together used to be ‘us’ time... Actually, when I was pregnant with my first child, after the check-ups, we used to go out and have dinner by ourselves. This time, not being able to do this routine, my husband joked, saying: ‘I don’t know this baby because I’ve never seen her’. Also, when they told me the baby was a girl, I had to tell him, but it wasn’t the same. You know, when you get the ultrasounds… It’s nice, it’s an emotional moment, hearing baby’s heartbeat and, also – banally or maybe not so – just talking to the doctor during a complicated pregnancy… I mean, I was handling that situation all by myself.” A., gave birth in August 2020 The certainty of the presence of loved ones While women who experienced their loved ones’ absence reported a sense of loneliness, in those who had the chance to experience their presence prevailed feelings of gratitude and the certainty of receiving support. The luck of being able to share with loved ones The chance to share one’s own experience with loved ones was generally interpreted as a fortune, more so in comparison with other women who could not benefit from such an opportunity.“Luckily, I gave birth at a time when you could access the hospital because I heard of so many mothers who had to face everything alone, poor women.” D., gave birth in March 2020 The presence of loved ones was experienced as a gesture of affection towards the pregnant woman, which positively influenced the recovery from childbirth and its memory. The reassuring and essential presence of someone who knows their needs was, for our participants, a source of huge emotional support.“My parents were outside the ward when the baby was born, so I was aware that, no matter what, there was someone waiting for me.” N., gave birth in August 2020 Partner involvement and parental engagement Most of the time, the partner’s presence represented fundamental emotional and practical support, especially for pain management. In some cases, the partner played an active role in the birthing process, especially in couples that reported a representation of childbirth as a matter that concerns both members of the couple.“The fact that my husband was there was the expression of how much this was and still is the idea of a common project.” L., gave birth in October 2020 One of the main tasks for partners was to be the woman’s voice when she was unable to express herself due to pain or scant consideration by the healthcare staff, protecting her from eventual obstetric violence.“Already during the pregnancy, I told him: ‘If you see that I can’t even breathe – because this is what happened to me with my first childbirth... I was alone, and I wasn’t able to express my needs, so I was completely at the mercy of the medics that under those circumstances can behave very well or maybe not so - you have to be my voice if I can’t get it out, so you need to know what I want to be done to me or not... you must be there and give me a hand’.” L., gave birth in October 2020 Sharing the birth only with those who can understand Besides those who are permanently configured as loved ones in the life of our participants (e.g., partners and parents), experiences related to the perinatal period are usually also shared with other supporting figures. It is interesting to note that the decision to share the experiences related to the birth seemed to depend on the specific ability of the other to support and empathise with the new mother. This ability, from our participants’ point of view, is particularly owned by the peer group, even in an online context, rather than by other people such as distant relatives or acquaintances. For this reason, the fifth and last macro-category consists of two categories: the first is related to peer sharing, whereas the second includes the feelings experienced in relation to the intrusion of those who cannot provide real support. Sharing the birth with those who have experienced the same situation Sharing the experience of becoming a mother in the pandemic era has made the peer group particularly suitable for seeking and providing emotional support.“Fortunately, I did the prep course online, as they say... to make a virtue of necessity. So we adjusted, but online communication wasn’t bad at all. […] We’re still talking about the babies: ‘Is he crying? What’s he doing?’” E., gave birth in October 2020 Therefore, comparison, socialisation, and sharing with other mothers were key aspects of the experience. Participants addressed the peer group especially during the first pregnancy or in case of negative childbirth experiences. The main chance to create a peer sharing group was represented by the online antenatal classes, attended in some cases more to socialise than to gather information. Furthermore, peer support online groups (e.g., Facebook groups) were used to compensate for the lack of in-presence socialisation, as well as to look for emotional and informational support and share negative birthing experiences.“On Facebook, I found a group where I felt a little at home because they described their childbirth, with similar situations to mine, or worse. Not that it gave me relief, but I did not feel like I was the only one.” V., gave birth in April 2020 Avoiding the intrusion of those who cannot provide real support Sharing the birth event with those who cannot provide real support represented, for our participants, a source of negative emotions. A suitable example of such a situation is offered by the typical Italian custom of visiting the new mother immediately after childbirth. Such visits frequently involve not only loved ones but also distant relatives and friends, who often offer nothing more than apparent support. Interestingly, almost all women interviewed agreed that they did not want to receive these visits: indeed, they were considered to be an intrusion into the delicate period of reorganisation of family life with the newcomer. This was also due to the poorly tolerated condescension in the visitors’ comments, which increased the already great tension experienced by the mothers.“For me, [the lockdown] was a godsend. I lived it well because I never wanted people at home... I used to say: ‘If you really want to visit me, come to the hospital and try not to dawdle there.’ So, honestly, I was very happy, despite this unfortunate situation, because I could enjoy the time with my daughter. There was no one there to tell me what to do and how, and it was best for me.” F., gave birth in March 2020 For the majority, the pandemic and the quarantine were valid reasons to relieve themselves of this duty, reserving the possibility to receive guests only in the case of close relatives who could provide solid emotional and instrumental support.“I appreciated the fact that there were no visits, and I think we should keep the stop to visits except for those who can help you out.” G., gave birth in March 2020 Discussion The current study explored the experiences of Italian women who gave birth during the pandemic, and how the changes imposed by Covid-19 impacted their psychosocial wellbeing. Using a Grounded Theory approach, we highlighted that the main problem experienced by women in the perinatal period during the pandemic was uncertainty, which seems to be a problem not specific to the Italian context, as the same issue has also been highlighted by other studies carried out in different countries [13], [21], [30]. In the present study, the aforementioned uncertainty was mainly related to the many changes imposed by the pandemic and childbearing, which occurred at the same time, and specifically related to the limitations in intrapartum care (e.g., the cancellation of antenatal classes and medical appointments, the closing of hospitals…) that worsened the difficulties of the perinatal period, limiting freedom of choice and continuity in intrapartum care. Such conditions led to experiencing several negative emotions related to the great fear of getting infected, infecting the child, and not receiving adequate or timely intrapartum services. The fear of contagion seemed to enhance the uncertainty of the experience of becoming a mother in a pandemic, as also shown by Atmuri et al. [13]. Moreover, when speaking of negative feelings, another key emotion in the narratives collected was that of guilt, consistent with Chivers et al., [31]: in the present study, this feeling was often associated with the experience of giving birth to a child in an emergency situation. In such a situation, “looking at the bright side” was a common strategy to cope with this negative affective state. In this frame of uncertainty, our results highlighted that social support is a crucial resource for psychosocial wellbeing, as was also well-highlighted by previous studies that considered the experience of becoming a mother during the Covid-19 pandemic [13], [32]. Presumably, its significance could also be further increased in the context of the pandemic, in which social support constituted a protective factor for mental wellbeing during pregnancy [16]. Our findings show that social support was mainly sought in partners and loved ones (e.g., parents, close relatives), in the healthcare staff, and in the peer network. In particular, women who could not benefit from the support of loved ones experienced loneliness, while those who had this opportunity felt a great sense of gratitude. Moreover, the presence or absence of the other takes on different meanings: the presence of a “companion of choice” represented both protection from eventual mistreatment and the possibility of sharing responsibilities, as also demonstrated by some other studies on this issue [14], [30], [33]; vice versa, their absence translated into the feeling of being at the mercy of the healthcare workers. This result is particularly significant when considering that the advent of the pandemic has increased the risk for obstetric violence [34]: in this perspective, the presence of a companion of choice could be considered an even more valuable protective factor against this threat. Furthermore, consistent with the literature [35], we found that being accompanied during intrapartum care was more than a chance to be supported: in some cases, partners, in particular, were considered in all respects as active actors in the birthing process. Regarding the healthcare staff, most women looked for a point of reference in healthcare professionals, but only in those who were perceived as willing to offer it: our results highlighted especially the critical role of the obstetricians, hypothetically enhanced by the increased need for human warmth, a central issue in our interviewees’ stories. From this perspective, the patient-obstetrician relationship was always evaluated as good, with the obstetricians representing an important point of reference, in line with Fumagalli et al. [23], who demonstrated that social support from the healthcare personnel, especially obstetricians, was a valid source of physical and psychosocial wellbeing. Peer support was instead mainly sought through online platforms (e.g., Facebook groups), which can represent both important sources of information and meaningful means for sharing negative perinatal experiences, especially during the pandemic. These observations are in line with Charvat et al. [36], who highlighted the important role of online peer support groups in providing social support during pregnancy in the pandemic. Specifically, our participants used such online peer support groups as a source of emotional and informational support, especially to share their experience with people who they thought could understand them, due to the fact that they were facing the same situation. The support received from peers seems to have partially compensated the participants’ (especially first-time mothers) concern for the cancellation of antenatal classes. When possible, online antenatal classes were provided, but the attendance of in-person antenatal classes would have been preferred. This finding is in line with the results of Atmuri et al. [13], who showed that, during Covid-19, in-person educational activities were preferred by pregnant women to online ones. Although the emergency phase has now passed, understanding risks and protective factors that define the balance of women’s psychosocial wellbeing during the perinatal period remains an issue of fundamental importance, in order to provide evidence-based data that can guide better management of epidemics and pandemic situations in the future. Limitations There are several limitations to this study. First, our findings referred in particular to the experiences that occurred between March and November 2020, when pregnancy regulations and procedures in response to the Covid-19 emergency were still unclear [2]. Indeed, in the early stages of the pandemic, the main concerns were related to the lack of knowledge about Covid-19 prevention and treatments, and about the possible negative impact of the virus on pregnant women’s conditions [4]. Currently, women’s concerns and needs may have changed in the light of the clearer hospital procedures, although, in Italy, the latter may still partially vary from hospital to hospital [2]. Moreover, from a psychological point of view, it is plausible to imagine that women’s fears - at least those related to the effects of the virus – may have been allayed due to the introduction of the vaccine. However, this hypothesis is still to be explored, given the widespread Covid-19 vaccine hesitancy affecting many groups of the population [37]. Future directions Although the emergency phase has been overcome and the pandemic situation is relatively under control also due to the beginning of the vaccination campaign [38], we are still a long way from pre-pandemic conditions. Since our results are focused on a specific stage of the pandemic, it is important to understand if and how the key experiences and psychosocial changes reported by our participants (i.e., the feeling of uncertainty and the need for social support) are still present late in the pandemic and what their eventual impact would be on mothers’ psychosocial wellbeing. However, our results highlight the potential of online resources as a tool for social support (i.e., Facebook peer groups and online antenatal classes); thus, this research could represent the starting point for future studies to test the efficacy of health promotion interventions based on this kind of social support. Moreover, future studies would benefit from further investigating the impact of the pandemic on specific perinatal experiences that emerged from our participants’ interviews, such as the intention to breastfeed, which can be linked to several health benefits for mothers and new-borns [39]. Finally, it can be of further interest to understand how fathers experienced being excluded from delivery rooms and intrapartum care in general. Conclusions The present study showed that the Covid-19 pandemic affected the psychosocial wellbeing of Italian women in the perinatal period in many ways. One of the main issues was the discontinuity in intrapartum care, which filled the perinatal period with uncertainties and negative emotions. In this situation, a crucial factor was represented by the presence or absence of social support, sought in loved ones, health professionals, and peer groups, in line with several studies on the same subject [13], [36]. In general, we could conclude that the lack of social support was related to a more stressful perinatal experience, which suggests the importance of paying attention to its decrease during the pandemic [40]. On the other hand, its presence was one of the main protective factors against negative mental health outcomes [32]. Considering in particular the partners’ involvement, sharing the experiences of the perinatal period meant much more than being supported: it represented, above all, a chance to share responsibilities [13], [30]. These results can inform evidence-based policies and interventions aimed at promoting women’s psychosocial wellbeing in the perinatal period – policies that should also consider social support as a central element. Interventions should focus primarily on the power of peer groups and health care personnel, which were essential sources of psychosocial support, in addition to loved ones. Furthermore, policies and health actions should be implemented to guarantee continuity in intrapartum care, particularly concerning the presence of loved ones during key perinatal experiences. Disclosure of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declarations Grant support information No funding involved. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the Ethical Committee of Psychological Research of the Department of Humanities of the University of Naples Federico II and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Disclosure of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability statement As a result of the qualitative nature of the study, participants did not agree with the publication of data. Therefore, full transcripts are not available. ==== Refs References 1 W.H.O. WHO. WHO Timeline - COVID-19. 11 March 2020 2020:19. 2 Giusti A, Zambri F, Marchetti F, Corsi E, J. P, Sampaolo L, et al. Indicazioni ad interim per gravidanza, parto, allattamento e cura dei piccolissimi di 0-2 anni in risposta all’emergenza COVID-19. Aggiornamento Rapp. ISS COVID-19, vol. n, 2021. 3 Caso D. Guidetti M. Capasso M. Cavazza N. Finally, the chance to eat healthily: Longitudinal study about food consumption during and after the first COVID-19 lockdown in Italy Food Qual Prefer 95 2022 104275 10.1016/j.foodqual.2021.104275 4 Coxon K. Turienzo C.F. Kweekel L. 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Leitao S. Olander E.K. Pope J. Matvienko-Sikar K. The impact of COVID-19 on pregnant womens’ experiences and perceptions of antenatal maternity care, social support, and stress-reduction strategies Women Birth 35 2022 307 316 10.1016/j.wombi.2021.04.013 33994134 15 Milgrom J. Hirshler Y. Reece J. Holt C. Gemmill A.W. E. db, Social Support-A Protective Factor for Depressed Perinatal Women? Int J Environ Res Public Health 16 2019 1426 10.3390/ijerph16081426Montgomery 31010090 16 Grumi S. Provenzi L. Accorsi P. Biasucci G. Cavallini A. Decembrino L. Depression and anxiety in mothers who were pregnant during the COVID-19 outbreak in Northern Italy: the role of pandemic-related emotional stress and perceived social support. Front Psychiatry 2021 12 10.3389/fpsyt.2021.716488 33413249 17 Ravaldi C. Ricca V. Wilson A. Homer C. Vannacci A. Previous psychopathology predicted severe COVID-19 concern, anxiety, and PTSD symptoms in pregnant women during “lockdown” in Italy Arch Womens Ment Health 23 2020 783 786 10.1007/s00737-020-01086-0 33215247 18 Wall S. Dempsey M. The effect of COVID-19 lockdowns on women’s perinatal mental health: a systematic review Women Birth 2022 10.1016/j.wombi.2022.06.005 19 Chaves C. Marchena C. Palacios B. Salgado A. Duque A. Effects of the COVID-19 pandemic on perinatal mental health in Spain: Positive and negative outcomes Women Birth J Aust Coll Midwives 35 2022 254 261 10.1016/j.wombi.2021.01.007 20 Kotlar B. Gerson E. Petrillo S. Langer A. Tiemeier H. The impact of the COVID-19 pandemic on maternal and perinatal health: a scoping review Reprod Health 18 2021 10 10.1186/s12978-021-01070-6 33461593 21 Kolker S. Biringer A. Bytautas J. Blumenfeld H. Kukan S. Carroll J.C. Pregnant during the COVID-19 pandemic: an exploration of patients’ lived experiences BMC Pregnancy Childbirth 21 2021 851 10.1186/s12884-021-04337-9 34972506 22 Vermeulen J. Bilsen J. Buyl R. Smedt D. Gucciardo L. Faron G. Women’s experiences with being pregnant and becoming a new mother during the COVID-19 pandemic Sex Reprod Healthc 32 2022 100728 10.1016/j.srhc.2022.100728 23 Fumagalli S. Ornaghi S. Borrelli S. Vergani P. Nespoli A. The experiences of childbearing women who tested positive to COVID-19 during the pandemic in northern Italy Women Birth 35 2022 242 253 10.1016/j.wombi.2021.01.001 33451929 24 Glaser B.G. Strauss A.L. Discovery of Grounded Theory: Strategies for Qualitative Research 2017 Routledge New York 25 Charmaz K. Constructing grounded theory: A practical guide through qualitative analysis Sage 2014 26 Tarozzi M. Che cos’e la grounded theory 2008 Carocci Roma 27 Glaser B.G. The constant comparative method of qualitative analysis Soc Probl 12 1965 436 445 10.1525/sp.1965.12.4.03a00070 28 O’Brien B.C. Harris I.B. Beckman T.J. Reed D.A. Cook D.A. Standards for Reporting Qualitative Research: A Synthesis of Recommendations Acad Med 89 2014 1245 1251 10.1097/ACM.0000000000000388 24979285 29 Swiss Academy of Medical Sciences (SAMS). Research with human subjects. 2nd edition. Bern: 2015. 30 Keating N.E. Dempsey B. Corcoran S. McAuliffe F.M. Lalor J. Higgins M.F. Women’s experience of pregnancy and birth during the COVID-19 pandemic: a qualitative study Ir J Med Sci 2021 1 8 10.1007/s11845-021-02862-2 31 Chivers B.R. Garad R.M. Boyle J.A. Skouteris H. Teede H.J. Harrison C.L. Perinatal Distress During COVID-19: Thematic Analysis of an Online Parenting Forum J Med Internet Res 22 2020 e22002 32857707 32 Lebel C. MacKinnon A. Bagshawe M. Tomfohr-Madsen L. Giesbrecht G. Elevated depression and anxiety symptoms among pregnant individuals during the COVID-19 pandemic J Affect Disord 277 2020 5 13 10.1016/j.jad.2020.07.126 32777604 33 Sweet L. Wilson A.N. Bradfield Z. Hauck Y. Kuliukas L. Homer C.S.E. Childbearing women’s experiences of the maternity care system in Australia during the first wave of the COVID-19 pandemic Women Birth 35 2022 223 231 10.1016/j.wombi.2021.08.010 34535423 34 Sadler M. Leiva G. Olza I. COVID-19 as a risk factor for obstetric violence Sex Reprod Health Matters 28 2020 1785379 10.1080/26410397.2020.1785379 32552522 35 Altshuler A.L. Ojanen-Goldsmith A. Blumenthal P.D. Freedman L.R. Going through it together“: Being accompanied by loved ones during birth and abortion Soc Sci Med 284 2021 114234 10.1016/j.socscimed.2021.114234 36 Charvat E. Horstman H.K. Jordan E. Leverenz A. Okafor B. Navigating pregnancy during the COVID-19 pandemic: The role of social support in communicated narrative sense-making J Fam Commun 21 2021 167 185 10.1080/15267431.2021.1932503 37 Capasso M. Caso D. Conner M. Anticipating pride or regret? Effects of anticipated affect focused persuasive messages on intention to get vaccinated against COVID-19 Soc Sci Med 289 2021 114416 10.1016/j.socscimed.2021.114416 38 Ministero della Salute. Report Vaccini Anti COVID-19. n.d. 39 Grano C. Fernandes M. Conner M. Predicting intention and maintenance of breastfeeding up to 2-years after birth in primiparous and multiparous women Psychol Health 2022 1 17 10.1080/08870446.2021.2025374 40 Zhou J. Havens K.L. Starnes C.P. Pickering T.A. Brito N.H. Hendrix C.L. 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==== Front Thromb Res Thromb Res Thrombosis Research 0049-3848 1879-2472 Elsevier Ltd. S0049-3848(22)00475-3 10.1016/j.thromres.2022.11.026 Letter to the Editors-in-Chief Serial fibrin monomer and D-dimer plasma levels measurements can capture thrombotic complications in critically ill COVID-19 patients: A prospective observational study Hardy M. ab⁎ Michaux I. c Bulpa P. c Schonau B. d Nicolay B. b de Maistre E. e Godon A. f Lecompte T. gh Mullier F. a a Université catholique de Louvain, CHU UCL Namur, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Science (NARILIS), Hematology Laboratory, Namur, Belgium b CHU UCL Namur, Anesthesiology Department, Namur, Belgium c Université catholique de Louvain, CHU UCL Namur, Intensive Care Medicine Department, Namur, Belgium d CHU UCL Namur, Vascular Medicine Department, Namur, Belgium e Service d'Hématologie Biologique, Unité d'hémostase, CHU Dijon, Dijon, France f Department of Anesthesiology and Critical Care, Grenoble Alpes University Hospital, Grenoble, France g University of Namur, Pharmacy Department, Namur, Belgium h Université de Lorraine, Nancy, France ⁎ Corresponding author at: Université catholique de Louvain, CHU UCL Namur, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Science (NARILIS), Hematology Laboratory, Namur, Belgium. 1 12 2022 1 2023 1 12 2022 221 6972 29 8 2022 22 11 2022 28 11 2022 © 2022 Elsevier Ltd. All rights reserved. 2022 Elsevier Ltd Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Keywords Fibrin monomers D-dimer Thrombosis Longitudinal Intensive care unit COVID-19 ==== Body pmcVenous thromboembolism (VTE) is a frequent and dreaded complication in the intensive care unit (ICU), albeit often unrecognized, since its diagnosis is challenging in this setting. The use of biomarkers could be valuable for the differential diagnosis of VTE, and more broadly of thrombotic events. D-dimer plasma level measurement belongs to the exclusion approach for suspected VTE in the outpatient setting, but is poorly effective in the inpatient setting [1]. Moreover, in critically ill patients, D-dimer levels are often far above the threshold for exclusion of VTE (e.g., 500 ng/mL), with very variable levels depending on the individual and the clinical context; the determination of a diagnostic threshold is therefore complicated. The measurement of fibrin monomers (FMs) in plasma has been suggested in this setting, but still lacks firm validation [2], [3]. FMs are produced as a result of the action of thrombin on fibrinogen. They are highly reactive molecules that assemble to form protofibrils but can also associate with two fibrinogen molecules, which limits their molecular mass so that they remain soluble, and thus accessible to measurement in plasma [2]; at the same time such assembly prevents permeation of the extravascular space [4]. Such a specific marker of intravascular thrombin generation could overcome the limitations of D-dimer. We previously reported a pilot study about the potential usefulness of longitudinal FMs measurements for the identification of thrombosis in critically ill coronavirus disease 2019 (COVID-19) patients, who represent a convenience cohort at high thrombotic risk [5]. We found that, in contrast to D-dimer, FMs plasma levels were mostly normal, but a sharp increase of them, the detection of which requires close monitoring, could be observed in presence of substantial intravascular thrombin generation leading to clinically significant fibrin deposits: VTE, arterial thrombosis, disseminated intravascular coagulation (DIC), clotting in an extracorporeal circuit. Those preliminary results deserved to be verified more robustly. Hence we conducted a prospective, observational, monocenter study after approval by the ethics committee (NUB: B0392020000031). We included patients admitted consecutively to the ICU for severe COVID-19 (requiring either high-flow oxygen administration via nasal cannula or tracheal intubation) between October 1, 2020, and May 31, 2021. Patients were excluded if the ICU admission was for any reasons other than severe COVID-19, if the duration of ICU stay was less than seven days, or when citrated plasma samples were not available at least every two days. Leftover plasma from citrated blood samples of included patients drawn at the request of the clinicians was frozen at −80 °C after double centrifugation (1500 g, 15 min, 20 °C). D-dimer and FMs were measured using the STA-Liatest D-Di Plus (results expressed as fibrinogen equivalent units) and STA-Liatest FM reagents, respectively, and the automated STA-R-Max 2 system (Stago). Patients were managed according to local standards of care. During the study period, all patients received thromboprophylaxis according to the GIHP proposals [6] and corticosteroids (dexamethasone 6 mg once daily for 10 days). Doppler ultrasound of the lower limbs and computed tomography pulmonary angiogram (CTPA) were performed within 72 h of ICU admission. The primary outcome was defined as any documented incident arterial or venous thrombotic event (i.e., compression ultrasonography for DVT or superficial vein thrombosis (SVT), CTPA, or echocardiography for PE, cerebral CT or MRI for stroke), or clotting in an extracorporeal circuit; thereafter collectively referred to as thrombotic events (TEs). DVT and PE diagnosed during the systematic admission workup were not included in the analysis. DIC was diagnosed according to the ISTH criteria. Logistic mixed models were used to assess the association between the occurrence of a TE and D-dimer and FMs levels. Patients were censored after the TE or after 30 days of ICU stay. Models were fit by maximum likelihood and inference was performed by likelihood ratio tests. Alpha was set at 0.05, and all tests were two-sided. We included 69 patients in the study (Fig. 1 ). During 1552 patients/days of follow-up a total of 1101 plasma samples were assayed. Median age was 63 years (IQR, 58–69) and median ICU stay was 21 days (IQR, 14–31) (Table S1). Twelve patients were on ECMO at some time-points of their ICU stay and 19 eventually died. Seventeen patients presented an asymptomatic DVT at admission screening. Over the ICU stay, 19 patients developed a TE, including 16 VTE (three PE with DVT, 11 isolated DVT, two SVT), one DIC, one myocardial infarction and one ECMO oxygenator clotting (Table S2). No patients experienced a recurrence of a TE.Fig. 1 Patients flow diagram. ICU, intensive care unit; SARS-CoV-2, severe acute respiratory syndrome corona virus 2; RT-PCR, reverse transcriptase polymerase chain reaction; COVID-19, coronavirus disease 2019. Fig. 1 Median plasma D-dimer and FMs levels outside of TEs were 1900 ng/mL (IQR, 1225–2750) and 3.6 μg/mL (IQR, 3.0–4.6), respectively. The time-courses of D-dimer and FMs levels from ten days before the TE to ten days after are shown in Fig. 2 . Both D-dimer and FMs levels were associated with TEs (p < 0.001 for both). For example, an increase of 5000 ng/mL in D-dimer levels or an increase of 50 μg/mL in FMs levels were associated with an increased odd of TE of 3.9 (95 % CI, 1.8–8.4) or 5.6 (95 % CI, 2.7–12.0), respectively.Fig. 2 Time-related changes in D-dimer and fibrin monomers levels around the day of the incident thrombotic event (19 patients). Day zero is defined as the day of the thrombotic event (either the beginning of clinical suspicion if the event was symptomatic, or the day of diagnosis if not). The median (IQR) D-dimer or fibrin monomer levels of the 19 patients who experienced an incident thrombotic event during their ICU stay are shown in the figure. The dashed lines correspond to the 500 ng/mL threshold for D-dimer and to the upper limit of the normal range (6 μg/mL) for fibrin monomers. Fig. 2 Those results evidenced that serial, closely monitored D-dimer or FMs plasma levels in critically ill COVID-19 patients can point to the occurrence of thrombotic events, and is in line with the results of our pilot study [5]. The advantage of FMs is that levels outside of TEs were close to the reference values in the majority of patients, making the definition of an alert threshold simpler than for D-dimer, the levels of which were much more elevated and variable. Indeed, plasma D-dimer can originate from both intravascular and extravascular fibrin deposition and breakdown, since their molecular mass can be small enough to permit transits between both compartments. In contrast, the source FMs, which have a higher molecular mass, is probably restricted to the intravascular compartment, and FMs are thus more specific to (intravascular) thrombotic disorders [7]. On the other hand, FM levels return to normal more rapidly (average half-lives of 2–6 h vs. >8 h for D-dimer) [4], especially if the patient is on anticoagulant therapy [8], [9]. This can lead to missing the peak when blood collection is not concomitant with or close to the acute phase of thrombosis and intravascular thrombin generation, i.e. during a potentially short window. Indeed, FMs levels were increased only for 24–48 h in some patients we studied; the implementation of enhanced-dose thromboprophylaxis and the rapid initiation of therapeutic anticoagulation after identification of a TE might have played a role in such transience. Regarding elevated D-dimer or FMs levels without identified TEs, potential causes could include unidentified asymptomatic TEs (clinical relevance of which is then questionable), inadequately anticoagulated patients, or preanalytical artifacts. Regarding FMs, most studies agree on their ability to identify incident TEs in both the out and inpatient settings [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]. Interestingly, the studies suggesting better accuracy of FMs over D-dimer for thrombosis diagnosis were those that iteratively measured both biomarkers around the TE [8], [9], [12], [22], [24]. In the COVID-19 setting, two studies failed to identify an association between FMs levels at admission and the occurrence of VTE [26], [27]; this is however not surprising as the biomarker would only increase when the thrombotic process begins. Two other studies, which serially measured both FRMs along the ICU stay of COVID-19 patients, concluded that both biomarkers could be suitable to identify TEs [5], [28]. The strength of the study resides in the very regular and close monitoring of the two biomarkers (at least one measurement every 48 h) and the total number of samples analyzed (>1000), enabling a close evaluation of their time-courses and an analysis of the data with a robust longitudinal statistical method. However, the number of patients remains low. Precise dating of TEs was not always possible; in fact, for three patients, D-dimer and FMs peaks preceded the thrombosis diagnosis and both biomarkers levels had already returned to baseline at diagnosis. Third, we were unable to implement routine and systematic screening for thrombosis during ICU stay. Asymptomatic thrombosis could have been missed that would explain some elevations in D-dimer or FMs. Finally, the study included only COVID-19 patients due to convenience (high incidence of TEs) and results deserve to be extended in a more general population of patients at high thrombotic risk. To conclude, both D-dimer and FMs are capable of capturing incident TEs. D-dimer measurement has the drawback of being frequently elevated in inpatients, the determination of a diagnostic threshold being complicated in this setting. Consideration of their time-related changes (i.e., important increase within a few days) is therefore more appropriate. FMs have the advantage of being specific to thrombin generation within the intravascular compartment, resulting in levels close to normal values (i.e., 6 μg/mL) in patients who do not develop TEs. However, FMs have a shorter half-life than D-dimer, sometimes being increased for only 24 h, with an ensuing risk of missing their peak in case of measurement outside the very acute phase of intravascular thrombin generation. Notwithstanding, their usefulness in the work-up of a suspected TE in high-risk critically ill patients should be further studied. Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MH, IM, PB, BS and BN declare no conflict of interest. EDM and TL report institutional fees from Stago. AG reports personal fees from BMS-Pfizer and Sanofi for conference attendance, outside the submitted work. FM reports institutional fees from Stago, Werfen, Nodia, Roche Sysmex and Bayer. He also reports speaker fees from Boehringer-Ingelheim, Bayer Healthcare, Bristol-Myers Squibb-Pfizer, Stago, Sysmex and Aspen all outside the submitted work. Appendix A Supplementary data Supplementary tables Image 1 Acknowledgements The authors want to thank the Mont-Godinne Foundation for funding this project. The authors also thank the support platform for statistical methods and calculations (SMCS) from the Université catholique de Louvain (UCL) for their assistance with the statistical analysis. Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.thromres.2022.11.026. ==== Refs References 1 Brotman D.J. Segal J.B. Jani J.T. Petty B.G. Kickler T.S. Limitations of D-dimer testing in unselected inpatients with suspected venous thromboembolism Am. J. Med. 114 4 2003 276 282 12681454 2 Dempfle C.E. The use of soluble fibrin in evaluating the acute and chronic hypercoagulable state Thromb. Haemost. 82 2 1999 673 683 10605767 3 Refaai M.A. Riley P. Mardovina T. Bell P.D. The clinical significance of fibrin monomers Thromb. Haemost. 118 11 2018 1856 1866 30312978 4 Shainoff J.R. DiBello P.M. The circulatory half-lives of alpha-profibrin and alpha-fibrin monomer, and comparisons with other fibrin(ogen) derivatives Thromb. Haemost. 89 1 2003 48 52 12540953 5 Hardy M. Michaux I. Dive A. Lecompte T. Mullier F. 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==== Front Telematics and Informatics Reports 2772-5030 2772-5030 The Author(s). Published by Elsevier B.V. S2772-5030(22)00031-7 10.1016/j.teler.2022.100033 100033 Article Factors Impacting Patient Perspectives on Telehealth and Remote Healthcare during COVID-19: A Mixed Methods Study Alhussein Manal Sultan Liu Xiang Michelle ⁎ Marymount University ⁎ Corresponding author: Xiang Liu, 2807 N. Glebe Rd., Arlington, VA 22207, U.S.A. 1 12 2022 1 12 2022 100033© 2022 The Authors 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. A rapid transformation to telehealth was a necessary precaution during the pandemic. This research sought a better understanding of factors impacting patients’ decisions on choosing telehealth. A mixed methods study was conducted with a sample of 276 patients. An online survey was used to collect qualitative and quantitative data. This study found that technical issues were the most significant factor preventing patients from using telehealth, among other factors such as waiting time and privacy concern. The results inform policy-makers and healthcare providers on how to optimize telehealth. The limitations of this study and future research directions are also discussed. Keywords Telehealth COVID-19 technical issues patients healthcare mixed methods ==== Body pmcIntroduction Coronaviruses are an infectious disease caused by SARS-CoV-2 and different from the common cold to cause severe respiratory illness. The patient might develop severe symptoms depending on the patient's medical history and health condition. Elderly patients and patients with chronic diseases such as diabetes, hypertension, or heart problems are considered high-risk patients who might develop the disease in an intensive manner (World Health Organization, 2022). The social distances were recommended during the outbreak of COVID-19 to slow the spread of the viruses as a public health emergency policy [13, 29]. Many industries, including healthcare, have prompted the use of information and communication technology (ICT) due to the pandemic. Telehealth has served as an alternative approach to enable and facilitate remote communications between patients, healthcare professionals, and caregivers to mitigate virus spread to vulnerable and high-risk populations. Because of the travel restriction during quarantine, telehealth is considered an effective solution [18]. Telehealth is a tool that delivers healthcare services and electronic information remotely from a distance using telecommunication modalities and advanced technology [10]. Telehealth uses can be categorized into four areas: (1) live video conferences that include both video or audio between patients and healthcare providers; (2) storing and forwarding the electronic data such as patient history, images, or test results, (3) remote patient monitoring (RPM) that allows patient to record personal health and medical data electronically such as blood pressure or blood sugar being reviewed laterally by the provider in another location, (4) m-health (mobile health) area that allows delivering healthcare services through mobile devices such as smartphones, medical devices, or tablets [4]. This study aims to gain a better understanding of the patient perspectives on telehealth in order to improve and optimize the healthcare quality delivered via telehealth. Furthermore, it highlights the major obstacles and problems faced by the patients during telehealth appointments that might affect quality of healthcare services received from a patient perspective. It also sheds light on the other advanced modalities that support delivering healthcare from a distance using telecommunication technology. Background Telehealth-supported Technology and Wearable Sensing Clinicians must gather correct medical information such as vital signs remotely from the patient for proper diagnoses using different modalities such as home scales, fitness watches, mobile apps, or wearable devices. Using digital devices or wearable sensing devices prompted the adoption of telehealth and ensured patient convenience and trust in receiving healthcare via telehealth. Wearable devices can deliver an accurate, noninvasive, and continuous assessment of patient health parameters [11]. Furthermore, RPM and digital technology enabled many personalized approaches that fit patient needs. For example, RPM helps detect immediate deterioration of a patient's condition and reduces unnecessary emergency room visits [3]. Telehealth, home care, and remote monitoring are great combinations that bring healthcare to a higher level by providing home monitoring services to patients in their place [23]. Purposes of the Study Prior studies investigated whether racial and socioeconomic disparities can be translated as barriers to use and access to telehealth (e.g., [33]). Due to limitations on the dataset and study design, little detail about telehealth barriers was provided. Therefore, there is a pressing need to further examine patients' perspectives on telehealth and gain more insights. Furthermore, such focus can help determine patients’ preferred visit types to receive healthcare services in the future and who would be best suited for telehealth. Another major purpose of our study is to highlight the gaps and barriers in telehealth in order to reduce digital disparities and support physician and patient engagement. It was noted that improving healthcare quality via telehealth requires consulting professionals for cost-benefit analysis and skills assessment for those who were transferred rapidly from face-to-face to the telehealth platform [6]. Despite telehealth has an influence on managing chronic disease and improving patient care, involvement from both patients’ and clinicians’ perspectives is crucial for enhancing telehealth infrastructure and service quality [28]. Several studies have investigated the implementation of telehealth platforms during the pandemic (Olszewski et al., 2022; [25]). Multiple factors were identified to pose significant challenges, including cost, data privacy, the digital divide, and training programs on the adoption and utilization of telehealth services. Our study aims to test on some of those factors using a different, primary data set. The ultimate goal is to gain insights on patient perspectives and the factors impacting their decisions to adopt telehealth or not. The result can better inform policy makers, healthcare providers, and other related parties to build a solid infrastructure collectively for remote healthcare services with optimized quality. Factors Influencing Patient Perspectives on Telehealth The shutdown began in the United States as a prudent policy for COVID-19 forcing many organizations to work remotely. Healthcare providers have to use an alternative mechanism to deliver effective and safe services to patients from a distance. However, the capability to provide optimized remote healthcare compared to the traditional, in-person visits is impacted by a variety of factors. A mixed-methods study [21] conducted to investigate patient perceptions of neurology telehealth visits during COVID-19 found that patient experiences were positively associated with time, cost, and effective interactions with the healthcare professionals. In addition, challenges and barriers to enabling telehealth services can affect a patient's decision on future selection for the type of visit to receive healthcare services. Factors such as accessibility, mitigating barriers, effective technology, and training programs were crucial in facilitating the utilization of telehealth [25]. However, clinicians' and patients’ perspectives toward telehealth can vary depending on facilities' accessibility and barriers. As the COVID-19 crisis accelerated the adoption and utilization of telehealth, understanding patients’ needs and opinions becomes more and more important for healthcare providers to provide optimal contactless care to the patients. Shaverdian et al. [27] investigated patient experiences and perspectives on the quality of the care received, their satisfaction levels, and care optimization in radiation oncology that used telehealth to deliver healthcare services. The authors found that approximately 88% of the surveyed patients understood the treatment plan, and 87% expressed trust that the treatment plan would be better or not differ from in-person visits. Pooni et al. [22] used a mixed-methods survey to evaluate patients, providers, and clinicians' perspectives on telehealth for subspecialties clinics for pediatric patients. The study concluded that telehealth and telemedicine were the safest communication tools for patients. Another study found that 77% of patients were satisfied with telehealth services, and 51% preferred to select telehealth for a future visit. The patients agreed to use telehealth as an effective and convenient tool to receive necessary care [21]. In summary, perspectives and feedback from various stakeholders including patients, caregivers, and healthcare providers help in shaping the framework of telehealth to optimize the services and ensure participants’ satisfaction. However, patients with chronic diseases, mental health patients, psychiatric clinic patients, or patients who need medical consultations are not necessarily benefited equally from telehealth, depending on types of specialty clinics and care received. Hötker et al. [14] found that patients using telehealth for consultation mainly focused on dermatological (46%), surgical (24%), and internal (22%) conditions. Other studies showed a positive impact on patients who received telehealth services to manage their health conditions, such as controlling blood pressure, ophthalmology clinics, or substance use disorder treatment clinics [5, 17, 20]. Methodology The Mixed Methods Design The authors conducted this inquiry through a mixed methods approach. A mixed-methods approach is a combination of quantitative and qualitative data analysis in a single study [30]. A quantitative survey instrument was used to collect responses for close-ended questions for quantitative data analysis. On the other hand, open-ended questions that were designed without predetermined responses formed qualitative data. A mixed-methods study was chosen because of its strength of drawing on both quantitative and qualitative research to complement and minimize limitations of both approaches. Quantitative data and qualitative data combined allow a more in-depth, complete understanding of the research phenomenon. Further, the information system (IS) field embodies a constantly changing environment that poses challenges for researchers to gain insights from new contexts using the existing theories and perspectives, therefore, mixed-methods designs are particularly useful and has been employed by researchers to tackle various emerging technology phenomena (e.g., [32, 35]; Califf, Sarker, & Sarker, 2020). A merge of qualitative and quantitative data in the form of a convergent mixed methods offers a multi-angle lens to explore the research questions. In this single-phase approach, both types of data were collected from a questionnaire survey simultaneously, allowing the researchers to integrate and analyze the overall results in one research setting [9]. The authors conducted data analysis with a sequential design. Specifically, the hypotheses developed for the quantitative study were built from the results of the qualitative study [35]. Figure 1 illustrates the convergent mixed methods design used in this study.Figure 1 Convergent Mixed Methods Design Figure 1 Ethics The Institution Review Board (IRB) at the authors’ University reviewed and approved this research according to the 45CFR46.101(b)(2): (2) Tests, Surveys, Interviews. Data Collection: Study Population and Sample Size This study was conducted based on a convenience sampling approach. The online survey was posted on social media channels such as LinkedIn and Facebook to collect data. The participant who received the URL link were able to access and fill out the online questionnaire. Participants have the right to give consents and proceed to take the survey; or disagree and exit the webpage without negative consequences. The data collection period was lasted between January 24th, 2022 and April 30th, 2022. The research population includes any patient aged eighteen and above who experienced telehealth in the United States and agreed to share their experiences with telehealth. The total sample size was 276, and all responses that did not meet the research criteria were excluded, resulting in 216 usable responses. Qualitative Study The Coding Process In order to conduct a qualitative descriptive study of the textural responses to the open-ended questions in the survey, a content analysis method was used in this study since it is a commonly used method in healthcare research to identify the representation of certain words, themes, or concepts in open-ended questions, interviews, and conversations [8]. A conceptual content analysis is used in this study. This type of content analysis consists of a systematic coding and categorizing approach to determine trends and patterns of words, their frequency, and relationships in the text [31]. 25% of the participants filled out the open-ended question and specified the obstacles and problems that interrupted them during telehealth appointments. The analysis was conducted using Microsoft Excel. The researchers developed a coding process for critical data using trend words, text, or statements and categorized them into groups. Each category was grouped into a specific theme, and frequencies were calculated for each domain theme. Figure 2 illustrates the coding process used by the authors to assess the qualitative data and its detailed explanation was included in Appendix 2.Figure 2 Example of Coding Process of the Qualitative Data of the content analysis Figure 2 Interpretation of Qualitative Data The authors of this study thoroughly reviewed all of the textual responses independently, resolved differences in coding as a team, and completed a test for interrater reliability (Cohen's κ = 0.91). Three themes were identified as the factors that can impact the level of the effectiveness of healthcare services delivered via telehealth, including waiting time, technical issues, and time spent with patients to provide detailed instructions or explanations. The sub-themes such as medical issues, language and lack of instructions, connectivity, video or audio problems, and other general technical matters are also identified. Themes and sub-themes were categorized based on the obstacles that disrupted the users while receiving telehealth services from the healthcare provider. The frequencies of the patients’ comments for each theme were calculated by dividing the sum of the comments of each theme over the sum of the total of observation comments. The data represented that technical problems were the biggest issue impacting the quality of healthcare services via telehealth. Technical problems were categorized into different sub-themes: connectivity and internet accessibility were the most frequently cited issues faced users during their telehealth appointments with approximately 36% of the total responses to the open-ended questions; 23% of the received comments indicated that frozen images during a video call or audio cutting in and out significantly disrupted the telehealth experiences, including 16% of other general issues such as broken links, problematic patch updates, and unexpected system errors. Further, 5% of the users commented on the platform usability provided by the healthcare providers, such as difficulty in figuring out how to manipulate it and its being not as smooth as familiar software. Another critical matter is time since 20% of the respondents complained about the matter such as long waiting time to receive the healthcare, conflicts with other appointments scheduled, or a difficulty of explaining the pain site or lack of instructions due to short time of the appointment or ineffective communication between doctors and patients. In addition, the unavailability of a translator caused trouble for about 7% of the respondents. The results confirmed that those critical factors impact the degree of effectiveness of healthcare services delivered via telehealth to patients, as summarized in Table 1 .Table 1 Obstacles Patients Facing during Telehealth Appointments Table 1Qualitative results Example quote Frequency, n (%) Time Spent with Patients Medical issues “Medicines out of my pocket are too expensive.” 5% “Explain place pain and details symptoms.” “Transforming to a specialist.” Language and lack of instructions “Language issue.” 8% “Figuring out where to find the link.” “I had one problem when receiving the link; I couldn't find the link. the link was supposed to be on my telehealth page; however, I should've filled in some documents that I wasn't aware of, which caused me to get delayed”. “Not having the required application in the device.” Waiting Time “Waiting for a long time to talk to an available doctor.” 7% “They called me at 5:00 am while sleeping”. “Interfere with another appointment.” “Not able to know if the practitioner is aware that his patient is waiting.” Technical Issues Connectivity Included several comments about (Poor connection, Wi-Fi issues, Connectivity, and network issues, Internet freezes for seconds, Access issues, and Disconnected and Weak signal) 36% Video and Audio issues “The video wasn't working, so we transferred to a phone call.” “Volume and microphone interference” 23% Several comments: “video was not clear, Image/audio lagging, and Sound issues.” General Technical problems “Updates, Technical issues, or System problems.” 16% “Link not working.” “Error page would pop in.” “Frozen screen, delays, or lags.” Usability “It took some time to turn on the mic from the settings.” 5% “The use of third-party websites instead of using popular viable applications.” The researchers went through the similar review process deployed above for coding the obstacles that patients faced during telehealth appointments and completed a test for interrater reliability (Cohen's κ = 0.95). Nine themes emerged, including an example from participant quotes, illustrated in Table 2 .Table 2 Feedback from The Participants about Telehealth Services During the Pandemic Table 2Qualitative results Example quote Frequency, n (%) Vital signs tools 9% “Vital signs can be hard to measure, and physical touch from the health provider is important.” “If the patient has the appropriate tools to check his condition, using telehealth would be better than visiting the hospital.” Specialty Clinics 13% “I think telehealth is effective for endocrinology/metabolic diseases.” “Telehealth is a suitable choice for only some types of care that can be delivered via online call.” “I think Telehealth is more efficient for seasonal colds, allergies, etc.” Privacy Issues 8% “My only problem with Telehealth is that I do not feel comfortable sharing my information.” “I don't trust technology. I feel it is disturbing my privacy.” Cost 8% “The cost of telehealth appointments shouldn't be so expensive.” “Telehealth is a useless tool, and I am supposed that they charge the same as a physical appointment.” General Issues 13% “Other than simply follow-up visits, telehealth can never replace in-person healthcare services. Even exposure to covid-19 should never be an excuse for continuing telehealth because I believe we're sacrificing other essential aspects of healthcare in general.” “Telehealth would never provide decent and ethical standards for patients.” “Sometimes it's essential to have a clinical exam which telemedicine can't provide.” Future Selection 8% “I prefer an In-person meeting with my doctor.” “It really depends on the health conditions. In general, I would prefer face-to-face visits all the time except for Minor issues.” Satisfy with the telehealth 25% “I wish all hospitals would offer this service as an option for the patient.” “I love receiving services through Telehealth when available.” “I think telehealth is a wonderful addition to healthcare.” “Great experience using telehealth during the pandemic.” “Telehealth encourages me to communicate more often with my doctor when I don't worry about commute or scheduling issues.” “Telehealth is a fast service to ask your doctor.” Dissatisfied with the services 8% “I once called my health insurance provider to set a telehealth appointment, which was urgent as I had some issues related to chest pain. The provider spent almost an hour and a half discussing the issue with me and collecting information from me. Then they told me we would set an urgent appointment for you, and you should see the virtual doctor in a day or two. Ironically, I haven't received any call from a doctor whatsoever until now. This is why I am so dissatisfied with Telehealth.” “My experience was abysmal with telehealth; it has low-tech quality and bugs.” Suggestions and concerns 8% “I would like to add chat conversations, too, because I had one like this, and it was provided with pictures of the problem from me.” “I am wondering about COVID-19; as a patient who needs medical care, where do I go if hospitals have coronavirus? Some people may visit the hospital because of Corona, while others do not come due to Corona. In this case, will going to the hospitals solve the problem, or will it increase it?” Gathering accurate and up-to-date vital signs from patients is crucial. However, some patients still face difficulty measuring and using new modalities that could help them provide correct health data remotely, such as electronic scales or fitness trackers. These results explained the importance of understanding patient needs and supporting them with precise explanations and training programs to promote and extend telehealth use to a further extent. In addition, a participant commented that some types of specialty clinics that provided services via telehealth benefited them more than others, such as common colds, allergies, or chronic diseases. That means the remote health services may not be suitable for patients whose cases require medical surgery or other hospital interventions such as lab tests or X-rays. Even though patients mainly showed great satisfaction with telehealth services since it facilitates communication with the doctors at any time they need compared to in-person visits, some patients were disappointed about the services because of lower quality of care received or weak platforms that cause technical problems such as software bugs. Furthermore, privacy issues such as the trust to share sensitive information were a critical concern. Although healthcare providers have used applications that comply with Health Insurance Portability and Accountability Act (HIPAA) regulations and provide end-to-end encryptions, some patients have fears and concerns about sharing their data via new technology. In addition to general issues such as the need for clinical examinations that required in-person visits, the cost of telehealth services was a concern for some patients since they paid the same price as if they had a face-to-face appointment in clinics or hospitals. Those patients might still need to talk and go for checkup with their doctors in person to feel more confident paying for the services. Therefore, increasing the awareness of telehealth services and encouraging users to keep selecting it for future visits can effectively change their vision of telehealth and preferred way of receiving healthcare services. Hypotheses Development Three recurrent themes were extracted from the qualitative data analysis as illustrated in the above section. Informed by the qualitative study results along with a comprehensive literature review, the authors developed three hypotheses summarized as follows. In order to improve and expand telehealth services, various factors need to be considered including patient and clinician experience, efficient telehealth scheduling, availability of telehealth access for both patient and clinician, and telehealth infrastructure [26]. Technical issues were listed as one of the concerning challenges resulting in a significant difference in telehealth adoptions [16]. Healthcare providers that employ easy-to-use telehealth applications and robust telehealth platforms are well positioned to yield better patient experience on telehealth and higher likelihood of using telehealth services again. Thus, we hypothesize: Hypothesis 1 The technical error or obstacles patients face during their telehealth appointment significantly impact their desire to select Telehealth for future visits. Ahmad et al. [2] surveyed patient perspective on telemedicine during the pandemic through an online survey. The result revealed that despite the fact that more patients chose to use telehealth compared to the number before the pandemic, majority patients preferred in-person visits for their next appointments. The main cited reason of such preference lies in the relatively longer waiting time associated with telehealth during the early stage of pandemic outbreak. Therefore, healthcare providers can take measures to decrease patients’ waiting time of receiving care services as an incentive for patients to use telehealth. Waiting time can be a significant factor impacting a patient's decision to select telehealth. Thus, we hypothesize: Hypothesis 2 Waiting time to receive Telehealth significantly impacts patient satisfaction with the services. Factors such as data privacy, emotional factors, and technology culture driving the consultations can influence caregivers' and users’ willingness to use telehealth [7]. For example, Gordon et al. [12] emphasized the importance of effective communication with patients during clinical video telehealth appointments to address and answer all patients’ questions as the essential factor influencing their satisfaction with telehealth visits. Thus, spending enough time with the patient during the telehealth appointment is critical to ensure they understand treatment plans and drug prescriptions, including answering all medical concerns to satisfy the patients with high-quality care delivered. Thus, we hypothesize: Hypothesis 3 Time spent with the patient significantly impacts patient satisfaction with the services. Quantitative Study Data analysis visualization tools such as Excel and Tableau were used to generate descriptive statistics. Pearson Correlation statistics were used to examine the relationships between independent and dependent variables. The research hypotheses were tested and their statistical significance was established respectively through SPSS software. Survey Instrument This study used a questionnaire design for the quantitative data based on multiple previously developed and tested study instruments including Patient Satisfaction with Telemedicine [15], Telemedicine Satisfaction Questionnaire [36] and Telemedicine Perceptions and Satisfaction Questionnaire [1] with necessary adjustments to align with the context. Using the well-established and previously-tested survey instruments allows the researchers to obtain high validity of the questionnaire. Data collected on patients' knowledge, experience, and perspectives are used to outline the strengths and weaknesses of telehealth services. Data was collected through a questionnaire survey which included multiple-choices and open-ended questions. Transformation of the research data from categorical to numeric were applied to achieve an accurate quantitative analysis (see Appendix 1 for more details). Open-ended questions were used to identify various kinds of problems that disrupted patients during their appointments and to solicit their detailed feedback about the healthcare services delivered remotely. Measures Sociodemographic variables include gender (male, female), age group (18-24, 25-34, 35-44, 45-65, N/A), race (white, black or African America, Asian America, American Indian/Alaska Native, Native, N/A), annual income ($25,000 - $34,999, $35,000 - $49,999, $50,000 - $74,999, $75,000 - $99,999, >$100,000, N/A). Other measures include the type of specialty clinics (dermatology or skin condition, follow-up such as post-surgical, pregnancy, or chronic diseases, online consultation, refill or prescription management, test results such as lab test or X-rays, urgent care such as coughs and colds, other), vital signs tools (at home, use patient monitor devices or remote fitness such as Apple Watch or Fit-bit, visit the nearest clinic/ pharmacy, other). The Pearson Correlation was selected to test the relationship between the dependent variables (DV) and independent variables (IV) to gain insights about the research phenomenon. The IVs for the Pearson Correlation that tests the research hypotheses are the frequency of coming across obstacles or problems (all the time, most of the time, rare, none at all, other), waiting time (less than 10 minutes, 10-15 minutes, 15-20 minutes, >20 minutes, other), and time spent with the patient (less than 10 minutes, 10-15 minutes, 15-20 minutes, >20 minutes, other) based on the following survey questions: “Have you faced obstacles such as technical problems during your appointment?”; “How long have you been waiting for your provider to provide medical care?”; and “How long has your healthcare professional spent with you to discuss your medical condition?”. The dependent variables are the desire of the participants to select telehealth services in the future (all the time, most of the time, rare, none at all, other) and the satisfaction level about telehealth services (very satisfied, satisfied, natural, dissatisfied, very dissatisfied, other) based on the questions: “How often will you select Telehealth for future visits?”; “How do you describe your satisfaction with the healthcare services you received via Telehealth?”. The detailed measurements of variables are included in Appendix 1. Descriptive Statistics The total number of valid data points is two hundred and nineteen (n = 219) since some of the responses did not meet the research criteria and were excluded as the following:• Fifty-one responses belonged to patients who experienced telehealth other than in the U.S.; • Two participants never used telehealth; • Four participants did not complete the survey. The sample distribution was analyzed by conducting a descriptive and bivariate analysis. Table 3 represents the distribution of the sample characteristics, type of specialty clinics, and tools used to measure vital signs. There are no significant differences among the sex of the participant who experienced telehealth during the crisis, and 77.17% of them were Caucasians. Most participants aged 25-34 years old, approximately 25.11% and 37.90%, have about $25,000 - $49,999 annual income. The data were transformed from categorical data to numeric to enable estimating the mean and standard deviation. For instance, the age group rated from one to four, starting from (18-24 years old). The annual income ranked from one to five, beginning with the lowest value ($25,000 - $34,999). A descriptive statistic was established via Microsoft Excel, as described in Table 4 . The difference in the average number of participants’ ages is approximately 0.80 (SD=0.80) from the mean (2.18). Also, the difference in the average number of the participant's annual income is about 1.50 (SD=1.50) from the mean (1.95). Appendix 1 records the mapping between the categorical variables and their corresponding numerical values ranges.Table 3 Sample Characteristics Distribution Table 3Variable Frequency n(%) Sample size, N 219 Gender Male 101(46.12%) Female 115(52.51%) N/A 3(1.37%) Age Group 18-24 36(16.44%) 25-34 114(52.05%) 35-44 55(25.11%) 45-65 12(5.48) N/A 2(0.91%) Race White 169(77.17%) Black or African America 9(4.11%) Asian America 14(6.39%) American Indian/Alaska native 0(0%) Native 7(3.20%) N/A 20(9.13%) Annual Income $25,000 - $34,999 83(37.90%) $35,000 - $49,999 83(37.90%) $50,000 - $74,999 51(23.29%) $75,000 - $99,999 22(10.05%) >$100,000 10(4.57%) N/A 27(12.33%) Type of Specialty clinics Dermatology or skin condition 25(11.42%) Follow-up such as post-surgical, pregnancy, or chronic diseases 29(13.24%) Online consultation 78(35.62%) Refill or prescription management 15(6.85%) Test results such as lab test or X-rays 32(14.61%) Urgent care such as coughs and colds 20(9.13%) Other 20(9.13%) Vital signs tools used to measure At home 70(31.96%) Use patient monitor devices or remote fitness such as Apple watch or Fit-bit 12(5.48%) Visit the nearest clinic/ pharmacy 79(36.07%) Other 58(26.48%) Table 4 Age and Annual Income Descriptive Statistics Table 4Variable Mean Standard deviation Age group 2.18 0.80 Annual income 1.95 1.50 Determining which specialty clinics can provide adequate care for patients from a distance is critical to improving care quality delivered via telehealth. Patients who need consultation, follow-up, refill, and medication management do not necessarily require hospital tests or other surgical interventions. The research data showed that about 36% of the participants used telehealth for online consultation purposes and 15% to check their test results such as lab tests. 9% of them also used telehealth for other specialty clinics such as mental health, sleep disorder, rehabilitations, physiatrist, or multi-purposes (See Figure 3 ). Focusing on which specialty clinic benefitted patients more via telehealth can lead to continuity of providing this type of service even after the pandemic to save patient and provider time and reduce traveling costs. For instance, online consultation can provide patients initial evaluation of their health conditions and the suspected treatment plan, including its fees. Since online consultation does not require the patient to be present, the patient can later select whether to start a treatment plan or not, even if they are living in another country.Figure 3 Percentage of the types of Specialty Clinics, Participants Used During their Appointment Figure 3 Additionally, advanced technology supports delivering healthcare via telehealth, so the patient can use this tool to measure vital signs or other required data. 32% of the participants measured their vital signs at home; however, 5% used new technology such as wearable fitness devices and RPM Devices. 27% of participants did not need to provide vital signs measurements since the doctors did not request that data in some specialty clinics such as dermatology or mental clinics or the required data were already available in the hospital system (Figure 4 ). The majority of the participant have walked to the nearest clinics or pharmacies to provide an accurate vital sign measurement requested by doctors. Thus, telehealth and advanced modalities can support delivering effective care to a patient remotely.Figure 4 Percentage of Tools Used to Measure Vital Signs Figure 4 Pearson Correlation Coefficient Analysis The correlation coefficient, also called Pearson's correlation (Pearson's R), is used to measure the strength of the association between independent and dependent variables, and R-value can be any value between (-1 and 1) [24]. In this study, SPSS software was used to test the research hypotheses and the significance of each hypothesis was compared to alpha which is equal to 0.05 or 0.01 based on the degree of freedom: (df)=n-2. The obstacles or problems patients faced during the telehealth appointment were significantly associated with their desire to select telehealth for future visits; which is illustrated in Table 5 . Understanding the significant issues that impact patients to receive adequate care remotely is crucial to improving healthcare delivery via telehealth. For instance, if patients have low broadband access or connectivity issues that prevent them from communicating effectively with the healthcare provider, they will probably prefer to select in-person for the next visit.Table 5 Hypotheses Result Summary Table 5Hypothesis P-Value SE R-square Adjusted R F Significancy Result 1 0.019 0.86 0.03 0.02 5.63 Significant Relationship Reject the null hypothesis 2 <0.000 1.04 0.06 0.06 14.52 Significant Relationship Reject the null hypothesis 3 0.808 1.07 0.00 -0.00 0.06 Insignificant Relationship Fail to reject the null hypothesis In addition, the data in the survey categorize from four to one for the waiting time as discussed previously, where a higher value represented less waiting time since less time indicates the best value. The satisfaction level falls into five categories from five (very satisfied) to one (very dissatisfied) with the services delivered via telehealth. Table 5 describes the positive significance of the association between waiting time to receive healthcare services and patients' satisfaction level with telehealth. That means the fewer times patients wait to talk to healthcare professionals such as physicians or nurses, the more satisfied patients are with the services they receive remotely. On the other hand, the time duration that doctors or nurses spent with the patient during telehealth appointments is not significantly associated with their satisfaction level with the services (See Table 5). Hypothesis 3 was rejected. This may imply that doctors and nurses communicated effectively with the patients and answered their questions with a clear and concise manner. Therefore, patients were satisfied about the care provided and explanations obtained from the healthcare providers regarding their health conditions and treatment plans. In those cases, spending fewer minutes can be more effective than a long hour if all patients' questions and fears are answered and relieved. Table 5 represented a summary of the hypotheses significancy results. Discussion Principal Findings Telehealth is an effective tool that facilitates patient communication with doctors and nurses during the pandemic from a distance. It also allows the healthcare professionals to communicate remotely with their patients or with each other, especially doctors or nurses in quarantine. Optimizing and improving healthcare quality require a better understanding of patients' perspectives on telehealth services. Therefore, the primary purposes of this research include defining major factors that impact patients’ decision to select telehealth services for future visits and identifying new modalities that support delivering healthcare from a distance. A mixed-methods study was conducted with the ultimate goal of informing policymakers, researchers, and healthcare leaders to develop an effective and sufficient framework for optimizing healthcare delivered remotely. We assumed that telehealth services were equally distributed for both genders. The demographic data showed no significant differences in gender between males and females who can access and benefit from telehealth services. The availability of technology and access to telehealth in urban cities allowed the users to access the services. Several studies in the literature showed that most participants were satisfied with telehealth and preferred telehealth for future visits [21, 27]. Our study found that 68% of patients were delighted and confident with the healthcare services received via telehealth. 42% of the responses will select remote healthcare in the future, and 47% who rarely used telehealth services before will consider telehealth for a future visit provisional on their health conditions and reasons for appointments. In addition, 25% of the comments conveyed that telehealth is a fast and valuable tool allowing them to communicate with their doctor without excuses such as rescheduling due to lack of transportation. Our data results corroborated that when healthcare providers care about their patients, answer their questions, and alleviate their fears and concerns about their health conditions, patients will be delighted to receive healthcare remotely. Factors Impacting Patient Decision on Future Visit Type The following discussions enumerate several major factors that impact patients’ selection of future healthcare related visit types. Technical and Privacy issues A prior study investigated the opportunities and barriers of telemedicine found that access to the services and data privacy were obstacles contrasted with its chances of being effectively used [19]. A quantitative data analysis in our study confirmed a significant relationship between obstacles patients faced and their willingness to select telehealth for future visits. Moreover, our qualitative data analysis and results represented technical issues as the most crucial problem faced by the participants, such as connectivity errors and video and audio lags. In addition, usability and data privacy were a concern for others since some users could not turn on the mic, manipulate the settings, or feel uncomfortable sharing their sensitive information via telehealth. Therefore, the authors suggested that training programs or video tutorials should be created and provided to the patients before they use telehealth, which can effectively optimize the services. Another recommendation is to use a hospital platform because it is supposed to be HIPAA regulation compliant. It offers certain degree of protection and assurance that patient data is kept available and secure. Furthermore, accessibility and connectivity in urban cities were still listed as a concerning obstacle as some users complained about those issues. Therefore, implementing a user-friendly and useful platform with alternatives such as a phone call in case of disconnection can ensure continuity in delivering healthcare remotely. Time and Cost Effective communication, time, and cost significantly impact patient perspectives on telehealth [7]. Cost of living, traveling, and transportation can be added to the price of health services for patients who need to see their doctors face-to-face. These fees can be easily multiplied for patients who may have a need for regular appointments such as chronic disease [6]. However, most of the participants who used telehealth in our study were patients with low annual income. The survey results of our study showed that the price of telehealth services is not found to be an obstacle for patients to use telehealth since it bears with the same cost as in-person visits. However, the cost for in-person visits may increase if patients live far away from hospitals and need to pay extra for tickets, petrol, hotels, or transportation. Another study commented on encouraging patients to use telehealth by decreasing waiting time for them to receive health care from the caregiver [2]. Similarly, we found a significant relationship between waiting time and patient satisfaction level with telehealth; the longer patients wait to receive healthcare, the more disappointed they feel about the services. In addition, qualitative data from the survey further disclosed that waiting time is a significant issue that impacted participants' decision about receiving health care services remotely. For example, waiting for a long time to talk to the doctor or nurses significantly reduced the likelihood of patients choosing telehealth as their future healthcare delivery mode. However, the third hypothesis in this paper that tested the relationship between time spent by healthcare providers with patients during telehealth appointment and patients’ satisfaction level with telehealth turned out to be insignificant. One explanation for failing to reject this null hypothesis is that effective communication between caregivers and patients such as caregivers answering patients’ questions and resolving any ambiguities about patients’ health status are key factors to satisfy patients no matter how long an appointment lasts. Therefore, a proper scheduling time with a sufficient gap between patients’ appointments is suggested to ensure no conflict between patients’ daily appointments and doctors' schedules, which allows patients to receive healthcare services on the exact booked time. Type of specialty clinics, Telehealth Tools, and wearable sensing Telehealth benefited patients for certain types of specialty clinics more than others requiring hospital mediation procedures such as surgery [5, 14, 17, 20]. Our study showed that most of the participants had used telehealth for online consultation. Results from other studies vary between follow-up appointments, dermatology, check test results, refill medication, or urgent cases. This variation promotes the researchers to identify the type of specialty clinics since telehealth might be used even after the pandemic as an effective way to deliver contactless healthcare from a distance. We suggest continuing telehealth for different specialty clinics and encourage policymakers, researchers, and healthcare providers to cooperate to optimize health care delivered via telehealth after the pandemic. Telehealth has adopted multiple modalities and new technologies supporting healthcare providers in gathering accurate patient data such as fitness watches, RPM, home scales, and mobile apps [11]. Even though some patients faced difficulties collecting their vital signs, quantitative data gathered from our study showed most of the participants have walked to the nearest clinics, and others have measured their vital signs at home by using the RPM technology. Practices such as introducing patients to use various modalities and supporting them through educational programs can further promote telehealth adoption and usability. Trustworthiness In general, reliability of a study refers to the ability to reproduce a consistent finding if the study were repeated by other authors using the same data collection methods and analytic procedures [9]. In terms of qualitative reliability, the authors ensured that their approach was consistent with other researchers that worked on similar projects. The reliability of the survey instruments refers to the consistency or repeatability of that instrument. The authors created survey instruments based on the prior studies published in multiple credible, peer-reviewed journals [15, 36], therefore, the current paper's instrument assumes the reliability of the existing instruments and relies on it. The consideration on validity of a convergent mixed methods approach is based on establishing both quantitative validity and qualitative validity [9]. The authors ensured quantitative validity by mitigating threats to internal validity and external validity. All survey instrument constructs are based on the prior literature in order to assure internal validity. External validity might be compromised when researchers draw incorrect inferences from a specific set of sample data to another population or settings. We restricted our claims about patient groups to which the results cannot be generalized. In terms of qualitative validity, we checked for the accuracy of the findings by employing certain procedures such as triangulating different data source and using a rich description to convey the findings. A mixed-methods study conducted over a relatively large sample is considered a strength of this study. Qualitative data provides a better explanation of the results and enables us to gain insights that the quantitative data cannot generate. Concurrently, a coding process was applied to ensure the trustworthiness of the qualitative research data (Refer to the Appendix 2 for more details). The major limitation lies in the generalizability of our findings due to the fact that most of the participants were middle-aged patients. We did not seek perspectives from the elderly, patients with mobility issues or chronic disease, or healthcare professionals such as nurses and doctors. In addition, not all participants answered the open-ended questions in the survey; thus, our result might vary in case of extensive qualitative data applied. Conclusion Optimization and delivering qualified and effective healthcare services are core principles for healthcare organizations. This paper investigated the patients' knowledge about telehealth and their insights into significant factors that impacted their decisions to choose telehealth in the future. It also highlighted the different modalities that supported delivering healthcare remotely and the types of specialty clinics that benefited patients from a distance compared to others that required hospital procedures. Future studies may focus on how new technologies can help elderly patients use telehealth effectively. Values and quality of telehealth services from both patients and healthcare providers’ perspectives for chronic diseases clinics compared to in-person visits are another important area to investigate. Uncited References [34] Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Manal Sultan Alhussein: Ms. Alhussein is a full-time doctoral student at George Mason University. She earned a master degree in Master of Science in Information Technology specialized in Healthcare Informatics from Marymount University. She earned a graduate certificate in Digital Health from Marymount University. She completed the online Echo MasterClass Echocardiography Course endorsed by Medical University of Vienna and the Austrian Society of Cardiology. She worked under Ministry of Health in Saudi Arabia as a Radiology Technologist Supervisor. Xiang Michelle Liu: Dr. Liu joined Marymount University in 2008 and is currently a full professor of Information Technology and Cybersecurity at School of Technology and Innovation. She received her doctoral degree in Information Systems from Boston University and master degree in MIS from Tianjin Polytechnic University in China. Co-authoring with her colleagues, she has won five Best Paper Awards in different national and international conferences during her tenure at Marymount University. She is a Certified Electronic Records Management Practitioner (ERMp) and a Certified Cyber Intelligence Investigator (CCII). Appendix 1 Quantitative Data – Transforming Categorical Data to Numeric Survey Section and Q: No. Survey Question Survey Options Value Note Section: 2 Q: 5 Age Group 18-24 1 25-34 2 35-44 3 45-65 4 >65 5 Other 0 Section: 2 Q: 6 Annual Income $25,000 - $34,999 1 $35,000 - $49,999 2 $50,000 - $74,999 3 $75,000 - $99,999 4 >$100,000 5 Other 0 Section: 3 Q: 10 10- How long have you been waiting for your provider to provide medical care? (IV) Less than 10 minutes 4 Less time is the best 10-15 minutes 3 15-20 minutes 2 >20 minutes 1 Other 0 Section: 3 Q: 11 11- How long has your healthcare professional spent with you to discuss your medical condition? (IV) Less than 10 minutes 1 Enough time is the best 10-15 minutes 2 15-20 minutes 3 >20 minutes 4 Other 0 Section: 3 Q: 13 13- Have you faced obstacles such as technical problems during your appointment? (IV) All the time 1 The low technical errors, the best services Most of the time 2 Rare 3 None at all 4 Other 0 Section: 3Q: 17 17- How often will you select Telehealth when you need healthcare? (DV) All the time 4 Most of the time 3 Rare 2 None at all 1 Other 0 Section: 3 Q: 18 18- How do you describe your satisfaction with the healthcare services you received via Telehealth? (DV) Very satisfy 5 Satisfy 4 Natural 3 Dissatisfied 2 Very dissatisfied 1 Other 0 Note: IV: Independent Variable; DV: Dependent Variable. Appendix 2 Qualitative Data Coding on the Obstacles and Problems Faced by the Patients During Telehealth Appointment Themes Sub-Themes Category Codes Examples Medical issues Medical Medicine “Medicines out of my pocket are too expensive.” Pain “Explain place pain and details symptoms.” Symptoms “Transforming to a specialist.” Time issue Time Time “Waiting for a long time to talk to an available doctor.” Waiting for long Time “They called me at 5:00 am while sleeping”. Hours “Interfere with another appointment.” “Not able to know if the practitioner is aware that his patient is waiting.” Language and lack of instructions Language Language “Language issue.” Instructions Translator “Figuring out where to find the link.” Figure-out “I had one problem when receiving the link; I couldn't find the link. the link was supposed to be on my telehealth page; however, I should've filled in some documents that I wasn't aware of, which caused me to get delayed”. Find link Was not aware Required application “Not having the required application in the device.” Usability Usability Search “It took some time to turn on the mic from the settings.” Use Turn on “The use of third-party websites instead of using popular viable applications.” Technical Issues Connectivity Connection issues Connect Included several comments about (Poor connection, Wi-Fi issues, Connectivity, and network issues, Internet freezes for seconds, Access issues, and Disconnected and Weak signal) Video and Audio issues Connection Video/Audio issues Disconnect General Technical problems General Tech. Wi-Fi Weak signal “The video wasn't working, so we transferred to a phone call.” Internet Video “Volume and microphone interference” Audio Mic Several comments: “video was not clear, Image/audio lagging, and Sound issues.” Voice Sound “Updates, Technical issues, or System problems.” Image Updates Technical “Link not working.” Not working “Error page would pop in.” “Frozen screen, delays, or lags.” Error Frozen Delayed Lag Qualitative Data Coding for the Feedback Provided by the Patients Themes Category Codes Examples Vital signs tools Vital signs measuring tools Vital signs “Vital signs can be hard to measure, and physical touch from the health provider is important.” Measure Tool “If the patient has the appropriate tools to check his condition, using telehealth would be better than visiting the hospital.” Specialty Clinics Specialty clinics Disease “I think telehealth is effective for endocrinology/metabolic diseases.” Chronic Diseases Clinic “Telehealth is a suitable choice for only some types of care that can be delivered via online call.” Effective for Type Efficient for “I think Telehealth is more efficient for seasonal colds, allergies, etc.” Privacy Issues Privacy Not comfortable “My only problem with Telehealth is that I do not feel comfortable sharing my information.” Trust “I don't trust technology. I feel it is disturbing my privacy.” Privacy Cost Cost Expensive “The cost of telehealth appointments shouldn't be so expensive.” Expenses Charge “Telehealth is a useless tool, and I am supposed that they charge the same as a physical appointment.” Cost General Issues General Comments General “Other than simply follow-up visits, telehealth can never replace in-person healthcare services. Even exposure to covid-19 should never be an excuse for continuing telehealth because I believe we're sacrificing other essential aspects of healthcare in general.” Standards It is essential to have “Telehealth would never provide decent and ethical standards for patients.” “Sometimes it's essential to have a clinical exam which telemedicine can't provide.” Future Selection Type of Visit Preferred Prefer “I prefer an In-person meeting with my doctor.” In-Person visit Depends on Face-to-Face visit “It really depends on the health conditions. In general, I would prefer face-to-face visits all the time except for Minor issues.” Online visit Satisfy with the telehealth Satisfaction Like “I wish all hospitals would offer this service as an option for the patient.” Love Wonderful “I love receiving services through Telehealth when available.” All hospital would offer “I think telehealth is a wonderful addition to healthcare.” Great experience “Great experience using telehealth during the pandemic.” Encourage me “Telehealth encourages me to communicate more often with my doctor when I don't worry about commute or scheduling issues.” Fast “Telehealth is a fast service to ask your doctor.” Dissatisfied with the services Dissatisfaction Dissatisfied “I once called my health insurance provider to set a telehealth appointment, which was urgent as I had some issues related to chest pain. The provider spent almost an hour and a half discussing the issue with me and collecting information from me. Then they told me we would set an urgent appointment for you, and you should see the virtual doctor in a day or two. Ironically, I haven't received any call from a doctor whatsoever until now. This is why I am so dissatisfied with Telehealth.” Abysmal Low-tech quality “My experience was abysmal with telehealth; it has low-tech quality and bugs.” Suggestions and concerns Suggestions and Concerns Add “I would like to add chat conversations, too, because I had one like this, and it was provided with pictures of the problem from me.” Provide Wonder “I am wondering about COVID-19; as a patient who needs medical care, where do I go if hospitals have coronavirus? Some people may visit the hospital because of Corona, while others do not come due to Corona. In this case, will going to the hospitals solve the problem, or will it increase it?” Data Availability The data that has been used is confidential. ==== Refs References 1 Abdulwahab S.A. Zedan H.S. 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==== Front Antiviral Res Antiviral Res Antiviral Research 0166-3542 1872-9096 The Authors. Published by Elsevier B.V. S0166-3542(22)00247-9 10.1016/j.antiviral.2022.105478 105478 Article RBM24 inhibits the translation of SARS-CoV-2 polyproteins by targeting the 5ʹ-untranslated region Yao Yongxuan ab Sun Hao bc Chen Yingshan bc Tian Lingqian bc Huang Dan bc Liu Canyu bc Zhou Yuan b Wang Yun b Wen Zhe a Yang Bo ab∗∗ Chen Xinwen bd∗ Pei Rongjuan b∗∗∗ a Joint Center of Translational Precision Medicine, Guangzhou Institute of Pediatrics, Guangzhou Women and Children Medical Center, Guangzhou, 510623, China b State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China c University of Chinese Academy of Sciences, Beijing, 100049, China d Guangzhou Laboratory, Guangzhou, 510320, China ∗ Corresponding author. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China. ∗∗ Corresponding author. Joint Center of Translational Precision Medicine, Guangzhou Institute of Pediatrics, Guangzhou Women and Children Medical Center, Guangzhou, 510623, China. ∗∗∗ Corresponding author. 1 12 2022 1 12 2022 10547817 9 2022 26 11 2022 30 11 2022 © 2022 The Authors. Published by Elsevier B.V. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. SARS-CoV-2 is a betacoronavirus with single-stranded positive-sense RNA, which is a serious global threat to human health. Understanding the molecular mechanism of viral replication is crucial for the development of antiviral drugs. The synthesis of viral polyproteins is a crucial step in viral progression. The synthesis of viral polyproteins in coronaviruses is regulated by the 5ʹ-untranslated region (UTR); however, the detailed regulatory mechanism needs further investigation. The present study demonstrated that the RNA binding protein, RBM24, interacts with the RNA genome of SARS-CoV-2 via its RNA recognition submotifs (RNPs). The findings revealed that RBM24 recognizes and binds to the GUGUG element at stem-loop 4 (SL4) in the 5ʹ-UTR of SARS-CoV-2. The interaction between RBM24 and 5ʹ-UTR prevents 80S ribosome assembly, which in turn inhibits polyproteins translation and the replication of SARS-CoV-2. Notably, other RNA viruses, including SARS-CoV, MERS-CoV, Ebolavirus, rhinovirus, West Nile virus, Zika virus, Japanese encephalitis virus, yellow fever virus, hepatitis C virus, and human immunodeficiency virus-1 also contain one or several G(U/C/A)GUG sequences in the 5ʹ-UTR, which is also targeted by RBM24. In conclusion, the present study demonstrated that RBM24 functions by interacting with the 5ʹ-UTR of SARS-CoV-2 RNA, and elucidated that RBM24 could be a host restriction factor for SARS-CoV-2 and other RNA viruses. Keywords SARS-CoV-2 RBM24 Polyproteins Translation RNA viruses ==== Body pmc1 Introduction SARS-CoV-2 belongs to the Betacoronavirus genus, which contain a positive-sense single-stranded RNA ((+)ssRNA) genome with a 5ʹ-cap and a poly(A) tail in the 3ʹ-untranslated region (UTR). Host cell entry is mediated via the spike (S) protein of SARS-CoV-2, which attaches to the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell surface (Zhou et al., 2020), following by triggering membrane fusion, viral entry, and infection. Then the uncoated genomic RNA is translated into pp1a and pp1ab polyproteins, which are then cleaved into a total of 16 nonstructural proteins (Nsps) and subsequently assembled into the transcription/replication machinery. The replication and transcription complex assembled by Nsps and virus-induced double-membrane vesicles (DMVs) replicates and synthesizes a nested set of subgenomic RNA species by genomic transcription, which encoded structural proteins and some accessory proteins. The newly formed virus particles subsequently undergo assembly, budding, and release into the extracellular compartment (Ashour et al., 2020; Mohamadian et al., 2021). The RNA genome of SARS-CoV-2 is less than 30 kb in length and includes 14 open reading frames (ORFs) that encode Nsps, structural proteins and other accessory proteins (Krichel et al., 2020; Naqvi et al., 2020). ORF1a/b is translated into the pp1a (Nsp1–11) or pp1ab (Nsp1–16) polyproteins, which undergo proteolytic cleavage by the proteases such as PLpro and 3CLpro to generate a total of 16 Nsps (Kadam et al., 2021). The polyproteins are essential for the translation of structural proteins (Mohamadian et al., 2021). The 5ʹ-end of the SARS-CoV-2 genome contains a leader sequence and an UTR with several stem-loop structures that are necessary for the translation, replication, and transcription of viral RNA. As the RNA genome of SARS-CoV-2 is capped at the 5ʹ-end, it is suggested that the initiation of translation is canonical and cap-dependent. Based on the experimental SHAPE structure of the 5ʹ-UTR of mouse hepatitis virus (MHV) (Yang and Leibowitz, 2015), the epsilon (ε) structure of hepatitis B virus (HBV) (Yao et al., 2018), and the secondary structure of the 5ʹ-UTR of SARS-CoV-2, it is assumed that the 5ʹ-UTR potentially forms a translation initiation complex in proximity of the 5ʹ-cap. RNA structures in proximity of the 5ʹ-cap are known to regulate RNA translation (Babendure et al., 2006; Pestova and Kolupaeva, 2002). Previous structural studies on the 5ʹ-UTRs of other coronaviruses have demonstrated the presence of hairpin structures in the proximity of the cap structure, and the efficiency of translational initiation is significantly modulated by these secondary structures (Babendure et al., 2006). Miao et al. have proved the 5ʹ-UTR of SARS-CoV-2 is highly structured with five simple hairpin structures, similar with other coronaviruses. Among the stem-loops (SLs), the SL2 hairpin domain is expected to be involved in the replication complex formation, the SL3 hairpin is known to encompass the leader TRS (TRS-L) sequence, the SL4 has a long hairpin and is a relatively stable, and the SL5 has a more complex four-way junction structure (Miao et al., 2021). SARS-CoV-2 relies on host RNA-binding proteins (RBPs) for the infection progression, multiple RBPs have been identified involving in SARS-CoV-2 RNA-host protein interaction (such as PABPC1, DDX3X, EIF4B, etc.) by comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) (Flynn et al., 2021). RBPs contain RNA recognition motifs (RRMs) and bind to single or double-strand regions of RNA molecules, and play key roles in the post-transcriptional regulation of RNA. Host RBPs can be recruited for viral replication (Li and Nagy, 2011). RNA-binding protein 24 (RBM24) with two RNA recognition sub-motifs (RNP1 and RNP2) is necessary for cardiovascular development and myogenesis (Grifone et al., 2014; Jiang et al., 2014; Zhang et al., 2016). RBM24 can post-transcriptionally regulate the stability of the members of the p53 transcription factor family (Jiang et al., 2014; Xu et al., 2014), which suggests that RBM24 plays a crucial role in RNA biology. Our previous studies have indicated that RBM24 can modulate the translation and replication of HBV and hepatitis C virus (HCV) by targeting the secondary structure of the viral 5ʹ-UTRs (Cao et al., 2018; Yao et al., 2018). RBM24 is characterized by its single and invariant RRM domain, which binds to the G(U/C/A)GUG element or stem-loop regions (Kuwasako et al., 2014; Yao et al., 2019). The sequence analyses shown that the “GUGUG” elements were located at SL4 and SL5 in the 5ʹ-UTR, implying that RBM24 could be involved in the translation and replication of SARS-CoV-2 polyproteins by targeting the 5ʹ-UTR. In order to test this hypothesis, we investigated the function of RBM24 in the translation of polyproteins and replication of SARS-CoV-2. The data presented in this study demonstrate that RBM24 inhibits the translation of polyproteins and replication of SARS-CoV-2 via its RNA-binding domains. The findings demonstrated that RBM24 specifically binds to the SL4 GUGUG element in the 5ʹ-UTR of SARS-CoV-2, and subsequently inhibits the formation of the 80S translation initiation complex. We also observed that RBM24 targets and inhibits the translation of viral RNA in several RNA viruses containing the G(U/C/A)GUG sequence in the 5ʹ-UTR. The findings therefore demonstrate that RBM24 may serve as a novel host limiting factor in SARS-CoV-2 and other RNA viruses. 2 Results 2.1 RBM24 inhibits the replication of SARS-CoV-2 Our previous study demonstrated that RBM24 directly binds to the 5ʹ-UTR of HCV RNA (Cao et al., 2018) and the 5ʹ-ε stem-loop of HBV pgRNA via RNA structure-dependent manner (Yao et al., 2018, 2019), and subsequently inhibits viral replication and translation. We therefore assume that RBM24 may be involved in regulating SARS-CoV-2 replication by interfering with translation of viral proteins. In order to evaluate our hypothesis, we determined the functions of RBM24 in SARS-CoV-2. The expression of Nsp3 was determined as a measure of polyprotein expression. As depicted in Fig. 1 , the expression of SARS-CoV-2 Nsp3 protein, N protein, and RNA increased following RBM24 knockdown, and correspondingly decreased following RBM24 overexpression in both H1299-ACE2 (Fig. 1B and C) and A549-ACE2 cells (Fig. 1D and E). In order to further confirm the antiviral effect of RBM24, the infectious titers were measured. As shown in Fig. 1F, overexpression of RBM24 leaded to a decrease in infectious titers in supernatant. Taken together, these results indicated that RBM24 inhibited the replication of SARS-CoV-2.Fig. 1 RBM24 inhibits the replication of SARS-CoV-2. (A) Schematic illustration of the experimental protocol. H1299-ACE2 cells or A549-ACE2 cells were seeded in 24-well plates at −1 day post transfection (dpt), transfected at 0 dpt, infected with SARS-CoV-2 at 1 dpt, and harvested at 2 dpt. (B–C) H1299-ACE2 cells were transfected with siNC, siRBM24, pcDNA3.1, or pRBM24, and infected with SARS-CoV-2 at MOI of 0.1. The protein and RNA samples were harvested from the cell lysates. (B) The expression of the Nsp3 and N proteins of SARS-CoV-2 and RBM24 was detected by Western blotting, using actin as the loading control. (C) SARS-CoV-2 RNA was detected by real-time PCR, and the levels were normalized to that of actin RNA; the levels of SARS-CoV-2 RNA in the siNC and pcDNA3.1 groups were considered to be 1. (D–E) A549-ACE2 cells were transfected with siNC, siRBM24, pcDNA3.1, or pRBM24, and infected with SARS-CoV-2 at MOI of 0.1. The protein and RNA samples were harvested from the cell lysates. (D) The Nsp3 and N proteins of SARS-CoV-2 and RBM24 were detected by Western blotting, using actin as the loading control. (E) SARS-CoV-2 RNA was detected by real-time PCR, and the levels were normalized to that of actin RNA; the levels of SARS-CoV-2 RNA in the siNC and pcDNA3.1 groups were considered as 1. (F) H1299-ACE2 cells were transfected with pcDNA3.1 or pRBM24 and infected with SARS-CoV-2. The supernatants were harvested at 16 dpi and subjected to the plaque assay using VeroE6 cells (left panel), then the infectious titers were counted (right panel). Fig. 1 2.2 The RNP domains of RBM24 are essential for the inhibition of SARS-CoV-2 RBM24 mainly functions by binding to RNA via its two putative RNA-binding sub-domains, RNP1 and RNP2 (Yao et al., 2018). In order to verify whether the RNP domains are critical for the inhibitory effects of RBM24 on SARS-CoV-2, H1299-ACE2 cells were transfected with wild-type or truncated forms of RBM24 following infection with SARS-CoV-2. The results demonstrated that the levels of Nsp3 protein, N protein, and viral RNA were significantly reduced only following transfection with the wild-type RBM24; however, their levels remained unaltered following transfection with truncated RBM24 (Fig. 2 A and B). These results confirmed that the RNP1 and RNP2 domains are essential for the inhibitory effect of RBM24 on SARS-CoV-2.Fig. 2 The RNP domains of RBM24 are essential for the inhibitory effect of SARS-CoV-2. (A–B) H1299-ACE2 cells were transfected with pHA-RBM24, pHA-tag, pHA-RBM24ΔRNP1/2, pHA-RBM24ΔRNP1, or pHA-RBM24ΔRNP2, and infected with SARS-CoV-2 at MOI of 0.1. The protein and RNA samples were subsequently harvested from the cell lysates. (A) The expression of the Nsp3 and N proteins of SARS-CoV-2 and RBM24 was detected by Western blotting, using actin as the loading control. (B) SARS-CoV-2 RNA was detected by real-time PCR, and the RNA levels were normalized to that of actin RNA; the levels of SARS-CoV-2 RNA in the pHA-tag group were regarded as 1. Fig. 2 2.3 RBM24 directly binds to the 5ʹ-UTR of SARS-CoV-2 RNA As both RNP1 and RNP2 are RNA recognition domains and truncated RBM24 lacking the RNP1 and RNP2 domains has no influence on the replication of SARS-CoV-2, we speculated that RBM24 may interact with SARS-CoV-2 RNA via the RNP domains. The RNA-IP assay revealed that only wild-type RBM24 co-immunoprecipitated with SARS-CoV-2 RNA (Fig. 3 A), suggesting that RBM24 interacts with SARS-CoV-2 RNA, and the interaction is primarily mediated via the RNP domains.Fig. 3 RBM24 directly binds to the 5′-UTR of SARS-CoV-2 RNA. (A) H1299-ACE2 cells were transfected with pHA-RBM24, pHA-tag, pHA-RBM24ΔRNP1/2, pHA-RBM24ΔRNP1, or pHA-RBM24ΔRNP2, and infected with SARS-CoV-2 at MOI of 0.1. The RNA samples were subsequently harvested from the cell lysates. The input or co-immunoprecipitated RNA of SARS-CoV-2 was detected by real-time PCR. The relative levels of the co-immunoprecipitated RNA of SARS-CoV-2 was normalized to that of the input RNA. (B) Schematic illustration of the 5′-UTR of SARS-CoV-2. (C) HEK293T cells were transfected with pRBM24 and the cell lysates were harvested at 48 hpt. The expression of input RBM24 and actin was detected by Western blotting. (D) Lysates of cells (400 μg) transfected with pRBM24 or 50 pM of rhRBM24 were incubated with 2 μg of biotin-labeled HBV ε, biotin-labeled 5′-UTR of SARS-CoV-2, or yeast tRNA. Unlabeled 5′-UTR RNA (0×, 10×, and 20×) was added in excess for competing with RBM24 in binding to the biotin-labeled 5′-UTR fragment, followed by pulldown with streptavidin beads and assessment by Western blotting. (E) HEK293T cells were transfected with pHA-RBM24, pHA-tag, pHA-RBM24ΔRNP1/2, pHA-RBM24ΔRNP1, or pHA-RBM24ΔRNP2. The cell lysates were harvested at 48 hpt, and 400 μg of cell lysates were incubated with 2 μg of the biotin-labeled 5′-UTR fragments of SARS-CoV-2 RNA, followed by pulldown assays with streptavidin beads and assessment with Western blotting. Fig. 3 RBM24 interacts with RNA in a structure- or sequence-dependent manner. SARS-CoV-2 contains a stable 5′UTR structure which also contains the “GUGUG” element in SL4 and SL5 (Fig. 3B, black asterisks) (Miao et al., 2021). We further determined the role of RBM24 in binding to the 5ʹ-UTR. RBM24 overexpression lysates or purified recombinant RBM24 protein was incubated with the biotin-labeled 5ʹ-UTRs of SARS-CoV-2 RNA. The RNA-protein complex pulled down using streptavidin beads was detected by Western blotting. In the absence or presence of other cellular proteins, RBM24 was capable of binding to the ε element of HBV (Fig. 3D, lane 2, positive control) and the 5ʹ-UTR of SARS-CoV-2 (Fig. 3D, lane 3). RBM24 incubated with yeast tRNA was used as the negative control (Fig. 3D, lane 1). In order to confirm the binding specificity between RBM24 and the 5ʹ-UTR of SARS-CoV-2, the binding of RBM24 to the biotin-labeled 5ʹ-UTR of SARS-CoV-2 was competitively inhibited by adding an excess of the unlabeled 5ʹ-UTR probe of SARS-CoV-2. The results further confirmed that RBM24 specifically binds to the 5ʹ-UTR of SARS-CoV-2 (Fig. 3D, lanes 4–6). The findings revealed that both RNP1 and RNP2 were essential for the binding of RBM24 to SARS-CoV-2 RNA (Fig. 3A). We further verified whether the RNP domains are essential for the binding of RBM24 to the 5ʹ-UTR of SARS-CoV-2. As depicted in Fig. 3E, only wild-type RBM24 was pulled down by the biotin-labeled 5ʹ-UTR fragments. Truncated RBM24 with deletions in any of the RNP domains were not pulled down by the 5ʹ-UTR fragments, indicating that the both the RNP domains, RNP1 and RNP2, are essential for the binding of RBM24 to the 5ʹ-UTR. 2.4 RBM24 binds to the GUGUG sequence in the 5ʹ-UTR of SARS-CoV-2 The biotin-labeled 5ʹ-UTRs of truncated RNAs, namely, SL1+SL2, SL3+SL4, SL4.5+SL5d, and SL5a-c, were synthesized based on the structure of the 5ʹ-UTR of SARS-CoV-2 (Fig. 4 A). RBM24 lysates or purified recombinant RBM24 were incubated with the wild-type or truncated 5ʹ-UTRs, and subjected to the pulldown assay. As depicted in Fig. 4B, the binding ability of RBM24 to SL1+SL2, SL4.5+SL5d, and SL5a-c were reduced compared to that of the full length 5ʹ-UTR (Fig. 4B, lanes 3, 4, 6, and 7). However, SL3+SL4 displayed RBM24 binding activity (Fig. 4B, lane 5), indicating that SL3+SL4 may play a key role in the binding of RBM24 in the absence or presence of other cellular proteins.Fig. 4 RBM24 binds to the GUGUG sequence in the 5′-UTR of SARS-CoV-2. (A) Schematic illustration of the structure of the 5′-UTR of SARS-CoV-2. (B) The biotin-labeled SL1+SL2, SL3+SL4, SL4.5+SL5d, and SL5a-c truncated 5′-UTRs were constructed based on the structure of the 5′-UTR of SARS-CoV-2. Lysates of cells (400 μg) transfected with pRBM24 or 50 pM of rhRBM24 were incubated with 2 μg of yeast tRNA, biotin-labeled 5′-UTR fragments, or its truncated fragments, followed by pulldown with streptavidin beads and assessment by Western blotting. (C) The biotin-labeled SL3, SL4, 5′-UTRΔSL3+SL4, 5′-UTRΔSL3, and 5′-UTRΔSL4 truncated UTRs were constructed based on the structure of the 5′-UTR of SARS-CoV-2. Lysates of cells (400 μg) transfected with pRBM24 or 50 pM of rhRBM24 were incubated with 2 μg of yeast tRNA, biotin-labeled 5′-UTR fragments, or its truncated fragments, followed by pulldown with streptavidin beads and assessment by Western blotting. (D) Schematic illustration of the wild-type SL4 and mutated SL4 (SL4 mut). The GUGUG sequence was mutated to GAGAG and the CACGCA complementary sequence was mutated to CTCGCA in the SL4 mut mutant. (E) The biotin-labeled SL4 and truncated SL4 mut were constructed based on the structure of the 5′-UTR of SARS-CoV-2. Lysates of cells (400 μg) transfected with pRBM24 or 50 pM of rhRBM24 were incubated with 2 μg of yeast tRNA, biotin-labeled 5′-UTR, SL4, or SL4 mut fragment, followed by pulldown with streptavidin beads and assessment by Western blotting. Fig. 4 The substructural domain of SL3+SL4 was subsequently mapped and we observed that SL4 but not SL3 co-precipitated with RBM24 (Fig. 4C, lanes 3–4). Based on this finding, biotin-labeled 5ʹ-UTRs with deletions of SL3+SL4, SL3, or SL4 were constructed. The truncated 5ʹ-UTR with SL3 deletion was pulled down with RBM24, while there were no clear signals for the truncated 5ʹ-UTRs with deletions of SL3+SL4 or SL4 (Fig. 4C, lanes 5–7), indicating that the SL4 domain in the 5ʹ-UTR is critical for the binding of RBM24 to the 5ʹ-UTR. The GUGUG element is present in the SL4 domain, and to further confirm whether the GUGUG element in the 5ʹ-UTR of RNA is critical for the binding of RBM24, we synthesized wild-type and mutated SL4 RNAs (substitution at 2U→A, 3U→A, and pairing bases 2A→T), and evaluated their binding potential (Fig. 4D). The findings revealed that the binding capability of RBM24 to mutated SL4 was significantly reduced compared to the binding ability of RBM24 to the wild-type 5ʹ-UTR and SL4 RNA (Fig. 4E). We also identified that the SL5b stem-loop contains a GUGUG element; however, SL5b was incapable of binding to RBM24 (Fig. 4B). This finding demonstrated that the GUGUG element in SL4 in the 5ʹ-UTR of SARS-CoV-2 is critical for the binding of RBM24 to the 5ʹ-UTR. 2.5 RBM24 inhibits mRNA translation via the 5ʹ-UTR by inhibiting 80S ribosome assembly The 5ʹ-UTR fragment of SARS-CoV-2 is essential for the translational initiation of ORF1a/b (Schubert et al., 2020). RBM24 knockdown apparently enhanced the expression of Nsp3, while the overexpression of RBM24 suppressed Nsp3 expression (Fig. 1B and D). It has been reported that RBM24 inhibits the translation of HBV and HCV by binding to the viral 5ʹ-UTRs (Cao et al., 2018; Yao et al., 2018). These results implied that RBM24 inhibited the translation of polyproteins in SARS-CoV-2 by targeting the 5ʹ-UTR. In order to validate this speculation, the 5ʹ-UTR sequence of SARS-CoV-2 was inserted upstream of the luciferase ORF for generating the pSARS-CoV-2 5ʹ-UTR-luc (pSARS2 5′-UTR-luc) plasmid, which was subsequently co-transfected with pcDNA3.1 or pRBM24. The relative luciferase activity (RLA) was normalized with that of pRBM24 to pcDNA3.1. Compared with the vector plasmid pluc, the insertion of the 5ʹ-UTR of SARS-CoV-2 and the 5ʹ-UTR of HBV significantly reduced the RLA (Fig. 5 B). In order to further verify the effect of RBM24 on the translation of SARS-CoV-2 polyproteins, pluc, pSARS-CoV-2 5ʹ-UTR-luc, and pHBV-5ʹUTR-luc transcripts were generated in vitro and incubated with rhRBM24 or BSA in rabbit reticulocyte lysate (RRL). We observed that RBM24 significantly reduced the RLA following the insertion of the 5ʹ-UTR of SARS-CoV-2 and the 5ʹ-UTR of HBV (Fig. 5C). These results suggested that RBM24 could inhibit the translation of polyproteins by interacting with the 5ʹ-UTR of SARS-CoV-2.Fig. 5 RBM24 inhibits the translation of mRNA via the 5′-UTR by preventing 80S ribosome assembly. (A) Schematic illustration of the constructed pluc, pHBV-ε-luc, and pSARS2-5′-UTR-luc plasmids. (B) HEK293T cells were co-transfected with pluc, pHBV-ε-luc, or pSARS2-5′-UTR-luc, along with pcDNA3.1 or pRBM24. Luciferase activity was determined using a Steady-Glo® luciferase assay system. The values of RLA were calculated and normalized with those of the pRBM24 to pcDNA3.1 groups. (C) The luc, HBV-ε-luc, and SARS2-5′-UTR-luc RNAs were transcribed in vitro, and incubated with rhRBM24 or BSA in RRL, and the luciferase activity was determined using a Steady-Glo® luciferase assay system. The values of RLA were calculated and normalized with those of the rhRBM24 to BSA groups. (D) The biotin-labeled 5′-UTR fragments of RNA were incubated with rhRBM24 or BSA in RRL for 15 min at 30 °C. The ribosomal complexes were separated by sucrose density gradient ultracentrifugation. The distribution of biotinylated-RNA fragments was detected by dot-blot assay using streptavidin-HRP and analyzed by densitometry. Fig. 5 Ribosome assembly assay was performed in an RRL system for elucidating the mechanism underlying the inhibitory effect of RBM24 on the 5ʹ-UTR-mediated translation of SARS-CoV-2. The formation of the 80S ribosome was observed in the control group that was incubated with BSA; however, the intensity of the 80S peak was significantly decreased in the presence of rhRBM24, and the majority of RNAs were retained in the 40/48S peaks (Fig. 5D). These findings indicated that the inhibitory effect of the RBM24–5ʹ-UTR interaction on the translation of polyproteins was attributed to the inhibition of 80S ribosome assembly on SARS-CoV-2 RNA. 2.6 Broad-spectrum inhibition of viral translation by RBM24 The G(U/C/A)GUG sequence is widely found in the 5ʹ-UTRs of RNA viruses, including SARS-CoV-2, SARS, MERS, Ebola, rhinovirus, West Nile virus, Zika, JEV, yellow fever virus, HCV, and HIV-1 (Fig. 6 A). RBM24 inhibits the replication of HBV (Yao et al., 2018), HCV (Cao et al., 2018), and SARS-CoV-2 (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5) by inhibiting the translation of viral proteins, which implies that RBM24 may act as an antiviral effector protein. In order to validate this speculation, 11 kinds of viral 5ʹ-UTRs (Fig. 6A) were inserted upstream of the luciferase ORF for generating the pSARS2 5ʹ-UTR-luc, pSARS 5ʹ-UTR-luc, pMERS 5ʹ-UTR-luc, pEbola 5ʹ-UTR-luc, prhinovirus 5ʹ-UTR-luc, pWest Nile virus 5ʹ-UTR-luc, pZika 5ʹ-UTR-luc, pJEV 5ʹ-UTR-luc, pyellow fever virus 5ʹ-UTR-luc, pHCV 5ʹ-UTR-luc, and pHIV-1 5ʹ-UTR-luc recombinant plasmids, which were subsequently co-transfected with pcDNA3.1 or pRBM24. The RLA was normalized with that of pRBM24 to pcDNA3.1 and compared with that of the vector plasmid pluc. The results demonstrated that all the viral 5ʹ-UTRs significantly reduced the RLA in HEK293T (Fig. 6B) and Huh7 cells (Fig. 6C). These findings implied that RBM24 inhibited viral translation by targeting with the viral 5ʹ-UTR, and may act as a broad-spectrum antiviral effector for RNA viruses containing the G(U/C/A)GUG element at the 5ʹ-UTR. The efficacy of RBM24 as a host restriction factor of SARS-CoV-2 is presently being investigated.Fig. 6 Broad-spectrum inhibition of viral translation by RBM24. (A) Schematic illustration of the viral 5′-UTR sequences; the specific G(U/C/A)GUG binding sequence of RBM24 has been highlighted. (B–C) The sequences of the viral 5′-UTRs were inserted at the 5′-end of the ORF of luciferase in pluc, to generate the pSARS2 5′-UTR-luc, pSARS 5′-UTR-luc, pMERS 5′-UTR-luc, pEbola 5′-UTR-luc, prhinovirus 5′-UTR-luc, pWest Nile virus 5′-UTR-luc, pZika 5′-UTR-luc, pJEV 5′-UTR-luc, pyellow fever virus 5′-UTR-luc, pHCV 5′-UTR-luc, and pHIV 5′-UTR-luc recombinant vectors. The viral 5′-UTR luciferase plasmids were co-transfected with pRBM24 or pcDNA3.1 into HEK293T cells (B) or Huh7 cells (C). Luciferase activity was determined using a Steady-Glo® luciferase assay system. The values of RLA values were normalized with those of the pRBM24 to pcDNA3.1 groups; the RLA values of the pluc group were considered to be 1. Fig. 6 3 Discussion Following its release into the cytoplasm, the uncoated genomic RNA of SARS-CoV-2 is first used as a template for the synthesis of the polyproteins, pp1a and pp1ab, following which the synthesized polyproteins mediate the translation of structural proteins and the replication of viral RNA (Ashour et al., 2020; Mohamadian et al., 2021). The detailed mechanism of polyprotein translation remains to be elucidated. In this study, we identified a novel cellular factor, RBM24, which inhibits the translation of SARS-CoV-2 polyproteins by targeting the 5ʹ-UTR. RBM24 directly interacts with the GUGUG element in the SL4 stem-loop in the 5ʹ-UTR of SARS-CoV-2, inhibits 80S ribosome assembly, and suppresses the efficiency of polyproteins translation. RBM24 represents a limiting factor in the replication of SARS-CoV-2, and may also act as a broad-spectrum antiviral effector protein in other RNA viruses containing the G(U/C/A)GUG element in the 5ʹ-UTR. Coronaviruses have evolved specialized mechanisms for hijacking the host gene expression machinery and utilizes the cellular resources of the host for regulating the translation of viral proteins. The 5ʹ- and 3ʹ-UTRs contain cis-acting sequences that are functionally important for RNA-RNA interactions and for the binding of viral RNA and cellular proteins during RNA replication (Satija and Lal, 2007; Yang and Leibowitz, 2015). In particular, the 5ʹ-UTR of SARS-CoV-2 also efficiently promotes translational initiation (Schubert et al., 2020). The pp1a and pp1ab polyproteins are translated from SARS-CoV-2 RNA and subsequently cleaved into Nsp1–16 (Kadam et al., 2021). The results of this study demonstrated that the RBM24 can directly bind to the 5ʹ-UTR of SARS-CoV-2 (Fig. 3D) to inhibit the translation of SARS-CoV-2 RNA into Nsp3 (Fig. 1B and D). Altogether, the findings demonstrated that RBM24 inhibits the translation of polyproteins (pp1a and pp1ab). The viral proteins, especially Nsp3, Nsp9, Nsp10, Nsp15, and Nsp16 play critical roles in viral replication (Mohamadian et al., 2021), and we also observed that the replication of SARS-CoV-2 were significantly decreased when RBM24 was overexpressed (Fig. 1A–F). It is postulated that the translation of SARS-CoV-2 RNA into polyproteins is initiated by ribosomal scanning, in which the 43S-preinitiation complex binds near the 5′-cap and scans in the 3ʹ-direction until it encounters the AUG codon. The 60S subunit then joins to form a complete 80S ribosome, which is followed by translational initiation (Sonenberg and Hinnebusch, 2009). RBM24 is a strong inhibitor of viral translation and binds to the 5ʹ-UTR of SARS-CoV-2 to inhibit 80S ribosome assembly, which subsequently reduces the pool of available ribosomes that can engage in translation (Fig. 5D). The results of the in vivo and in vitro assays demonstrated that RBM24 overexpression inhibited the translation of luciferase initiated by the 5ʹ-UTR of SARS-CoV-2 (Fig. 5B and C). Furthermore, the ribosome assembly assay revealed that RBM24 did not affect the binding of the 40S subunit to the mRNA containing the 5ʹ-UTR of SARS-CoV-2; however, RBM24 affected the formation of the 80S ribosome assembly complex on mRNAs containing the 5ʹ-UTR of SARS-CoV-2 (Fig. 5D). These findings led us to speculate that RBM24 possibly binds to the 5ʹ-UTR of SARS-CoV-2 to prevent the 40S ribosomal subunit in scanning for the AUG codon, which leads to the failure of assembly of the 80S ribosome, and subsequently inhibits polyproteins translation. The hypothesis was supported by the fact that RBM24 also hampers ribosome assembly during protein translation in HBV and HCV by interacting with the 5ʹ-UTR of HBV and 5ʹ-UTR of HCV, respectively (Cao et al., 2018; Yao et al., 2018). In a previous report neither silencing nor overexpression of RBM24 causes changes in the total protein level (Zhang et al., 2018). And our previous results of WST-1 analysis showed that the down-regulation of RBM24 did not affect cell viability (Yao et al., 2018). Therefore, the observed antiviral effects of RBM24 were probably due to direct inhibition on SARS-CoV-2 translation. However, we could not totally exclude that RBM24 may affect other cellular proteins and indirectly regulates SARS-CoV-2 translation. We also notice the limitations of relatively artificial binding and translation in vitro assays, further assays such as in vivo translation and RNA fluorescence in situ hybridization (FISH) should be performed in the further. For the function of regulating virus replication, RBM24 is more like a switch than a host limiting factor. On the one hand, RBM24 inhibits the translation of HBV and HCV, and on the other hand, enhances the replication of the HBV and HCV genome (Cao et al., 2018; Yao et al., 2018, 2019, 2022). Whether or not RBM24 works as switch modulates the protein translation and RNA replication during other virus infection is an opening question now. Though our results suggest RBM24 as an anti-SARS-CoV-2 target, the detail mechanism of RBM24 in virus replication needs more deep investigation. RBM24 was supposed to be a potential therapeutic target for cancer treatment (Shi, 2022), And different efforts such as small molecule inhibitors, siRNA, peptides and CRISPR-based therapies had been made to target RBP functions for cancer therapy (Mehta et al., 2022). However, how to deliver the drugs to specific tissues with minimal cytotoxicity to normal cells is the main question, which is the same for the antivirus purpose. A possible strategy is to block the interaction of RBM24 with its viral specific partner. RBM24 is characterized by its single and invariant RRM domain, which specifically binds to RNA via a G(U/C/A)GUG sequence-dependent or RNA structure-dependent manner (Kuwasako et al., 2014; Yao et al., 2019). The SARS-CoV-2 RNA contains a 5ʹ-UTR with several stem-loop structures (Manfredonia et al., 2020) and the GUGUG element in SL4 (Fig. 3, Fig. 4D). RBM24 binds to the ε element in the pgRNA of HBV via a structure-dependent manner (Yao et al., 2018, 2019), while in SARS-CoV-2, RBM24 recognizes the 5ʹ-UTR in a sequence-dependent manner, via the GUGUG sequence in SL4 (Fig. 4E). It's still unclear why the “GUGUG”element in SL5b cannot bind to RBM24 (Fig. 4B). Other sequences than the core sequence may also be important for RBM24 binding. More likely, in addition to the sequence, the secondary or even higher structure determines the binding of RBM24 and viral genome. The G(U/C/A)GUG element is present the 5ʹ-UTRs of numerous RNA viruses, including SARS-CoV-2, SARS-CoV, MERS-CoV, Ebola virus, rhinovirus, West Nile virus, Zika virus, Japanese encephalitis virus, yellow fever virus, HCV, and HIV-1 (Fig. 6A). We further evaluated whether RBM24 affects the translation of these 11 viruses by comparing the luciferase activity to that of the vector luciferase. The results demonstrated that RBM24 may suppress the translational efficiency of the luciferase ORF following the insertion of these viral 5ʹ-UTRs (Fig. 6B and C). However, these results haven't shown directly that RBM24 inhibits the translation of these viruses, but suggested that RBM24 may have a common antiviral activity against RNA viruses by inhibiting viral translation. Thus, this hypothesis needs to be evaluated using infection systems. Also, it was not possible to rule out whether RBM24 has inhibitory effects on other viruses that lacking the G(U/C/A)GUG element, and further studies are necessary in this regard. Altogether, the present study demonstrated that the RBM24 host factor, with RNA-binding activity, targets the GUGUG element in the SL4 stem-loop in the 5ʹ-UTR of SARS-CoV-2 to inhibit the translation of polyproteins and suppress the replication of SARS-CoV-2. The findings also indicated that RBM24 may have a common antiviral activity against several RNA viruses in general. Inducing RBM24 overexprssion in vivo or delivering RBM24 to the target site may be the novel antiviral therapies. Taken together, the results provide insights into understanding the mechanism of replication of SARS-CoV-2 and aid in the development of novel antiviral targets for RNA viruses. 4 Materials and methods 4.1 Cell culture and transfection The H1299-ACE2 cells used in this study were a generous gift from Prof. Peng Li, and were cultured in Gibco RP Basic MI 1640 medium (C11875500BT; Gibco). The stable A549-ACE2 cell line expressing human the ACE2 receptor was generated from A549 cells and cultured in Dulbecco's modified Eagle's medium (DMEM, 12100-046; Gibco). Huh7 (YB–H3110; ATCC) and HEK293T cells (HEMCL-032; ATCC) were also cultured in DMEM. The culture media (1640 Basic medium and DMEM) were supplemented with 10% fetal bovine serum (FBS, 10099-141; Gibco) and 100 U/ml penicillin-streptomycin (15140-122; Gibco), and the cells were cultured at 37 °C in an atmosphere of 5% CO2. 4.2 Plasmids The plasmids expressing the wild-type RBM24 protein, namely, pRBM24 and pHA-RBM24, as well as the plasmids expressing the truncated RBM24 protein, including pHA-RBM24ΔRNP1/2, pHA-RBM24ΔRNP1, and pHA-RBM24ΔRNP2, were used as previously described (Yao et al., 2018). In addition, pluc and pHBV-TR-luc (pTR-luc) were used as previously described (Yao et al., 2018). The fragments containing the viral 5ʹ-UTR sequences were synthesized and cloned into pluc, which was located at the 5ʹ-end of the ORF of luciferase, to generate the pSARS2 5ʹ-UTR-luc, pSARS 5ʹ-UTR-luc, pMERS 5ʹ-UTR-luc, pEbola 5ʹ-UTR-luc, prhinovirus 5ʹ-UTR-luc, pWest Nile virus 5ʹ-UTR-luc, pZika 5ʹ-UTR-luc, pJEV 5ʹ-UTR-luc, pyellow fever virus 5ʹ-UTR-luc, pHCV 5ʹ-UTR-luc, and pHIV 5ʹ-UTR-luc recombinant vectors. 4.3 Transfection and infection Plasmids and siRNAs were transfected into H1299-ACE2, A549-ACE2 and Huh7 cells using Lipofectamine® 3000 Reagent (L3000-015; Invitrogen™) or into HEK293T cells using Lipofectamine® 2000 Reagent (11668-019; Invitrogen™) according to the manufacturer's instructions. The cells in the control group were transfected with control siRNA (siNC, SI03650318; QIAGEN), while the cells in the experimental group were transfected with RBM24 siRNA (siRBM24, SI03030195; QIAGEN). For SARS-CoV-2 infection, the cells were infected with the viral preparation at a multiplicity of infection (MOI) of 0.1, and the cells were harvested 24 hours post infection (hpi). Briefly, the infected cells were lysed in TRIzol (15596018; Invitrogen) and the RNA was extracted according to the manufacturer's instructions. The RNA was amplified by real-time polymerase chain reaction (PCR) using the primers for actin (forward: 5ʹ-TGGAATCCTGTGGCATCCATGAAAC-3ʹ; reverse: 3ʹ-TAAAACGCAGCTCAGTAACAGTCCG-5ʹ) and the N protein of SARS-CoV-2 (forward: 5ʹ-GGGGAACTTCTCCTGCTAGAAT-3ʹ; reverse: 3ʹ-CAGACATTTTGCTCTCAAGCTG-5ʹ). The relative N RNA levels were normalized with the actin RNA. The cells were lysed in 2X Laemmli buffer and subjected to Western blotting. 4.4 The plaque assay Viral supernatants were 5-fold serially diluted in DMEM, 200 μl of each serially diluted supernatant was added to the VeroE6 cells. After 1 hour, the inoculums were removed and the 500 μl of 2% Methyl cellulose medium overlay was added to each well for 4 days at 37 °C, 5% CO2. The cells were then fixed with formalin and stained with crystal violet. The number of plaques were counted and the viral titers were expressed in the logarithm of plaque-forming units (PFU) per milliliter. 4.5 Western blotting Western blotting was performed as previously described (18). The following antibodies were used for Western blotting: anti-actin (66009-1; ProteinTech), anti-RBM24 (ab94567; Abcam), rabbit anti-HA (3724S; Cell Signaling Technology), rabbit anti-Nsp3 (GTX135614; GeneTex), rabbit anti-SARS-CoV-2 nucleocapsid (anti-N) (28769-1-AP; ProteinTech), mouse anti-Flag (F1804; Sigma), streptavidin HRP (CT353; U-cytech), anti-mouse secondary antibodies (115-035-146; Jackson), and anti-rabbit secondary antibodies (111-035-003; Jackson). 4.6 RNA immunoprecipitation (RIP) assays RIP assays were performed using the previously described method (Cao et al., 2018; Keene et al., 2006). Briefly, H1299-ACE2 cells were transfected with pHA-tag, pHA-RBM24, pHA-ΔRNP1/2, pHA-ΔRNP1, or pHA-ΔRNP2, and infected with SARS-CoV-2 at MOI of 0.1, following which the cell lysates were harvested at 48 hpi. Then, 800 μg of the cell lysates containing the total protein was incubated with protein G precoated with mouse anti-HA (H9658; Sigma) at 30 °C for 4 h. After washing 5 times with washing buffer, the precipitated RNA and 5% of the total RNA (input RNA) extracted from the cell lysates were detected by real-time PCR. The relative level of the precipitated RNA of SARS-CoV-2 was normalized to that of the input RNA. 4.7 Streptavidin pulldown assay The streptavidin pulldown assay was performed as previously described (Cao et al., 2018; Yang et al., 2012), and the recombinant human RBM24 (rhRBM24) was prepared by prokaryotic expression and purification as previous reported (Cao et al., 2018). Briefly, the biotinylated-RNA fragments were dissolved in RNA folding buffer (20 mM HEPES (pH 7.6), 100 mM KCl, and 5 mM MgCl2) and folded by incubation at 70 °C for 2 min. Then 400 μg of the total protein and 50 pmol of purified rhRBM24 were separately incubated with 2 μg of biotinylated-RNA fragments, 30 μg of yeast tRNA (AM7119; Ambion), and 40 U of RNasin at 30 °C for 30 min. Dynabeads M-280 Streptavidin (11205D; Invitrogen) was resuspended with 300 μL of the RNA binding buffer (100 mM KCl, 20 mM HEPES (pH 7.6), 5 mM MgCl2, 10% glycerol, 0.1% IGEPAL®, 1 mM DTT, and 400 μM RVC). The aforementioned protein-RNA mixtures were subsequently added to the beads and incubated at 30 °C for 2 h. The beads were washed 6 times with RNA binding buffer, and subjected to Western blotting. 4.8 Luciferase reporter assay The luciferase reporter assay was performed as previously described (Cao et al., 2014). Briefly, HEK293T or Huh7 cells were co-transfected with the indicated plasmids. The activity of firefly luciferase was measured at 48 hours post transfection (hpt) with a luciferase reporter assay system (E2940; Promega). All the reporter assays were repeated at least three times. 4.9 In vitro translation and ribosome assembly assay The capped RNAs from pluc, pHBV-TR-luc, and pSARS2-5′UTR-luc were generated by in vitro transcription using a mMESSAGE mMACHINE® Kit (AM1344; Invitrogen). Biotin-11-UTP (AM8450; Invitrogen) was added to the reaction mixture for producing biotin-labeled capped RNAs. The in vitro translation and ribosome assembly assays were performed as previously described (Locker et al., 2007). Briefly, the template RNAs and recombinant human RBM24 protein (rhRBM24) (Cao et al., 2018) or a non-specific control protein (BSA), were incubated in an RRL (L4960; Promega)-based translation reaction system. The luciferase activity assay was subsequently performed using a Steady-Glo® luciferase assay system (E2520; Promega) (Cao et al., 2014). For the ribosome assembly assay, 1 μg of the biotin-labeled 5ʹ-UTRs of SARS-CoV-2 RNA and 12.5 pmol of rhRBM24 or BSA were added to an RRL-based ribosome assembly mixture. The mixtures were incubated for 15 min at 30 °C and subjected to 10–40% sucrose density gradient ultracentrifugation. The gradients were fractionated into 21 fractions and blotted onto an Amersham Hybond™-N+ membrane with a Whatman® Minifold® I 96 well dot-blot array system. The membrane was crosslinked with a HL-2000 Hybrilinker, and the blotted biotinylated-RNA was detected with Streptavidin-HRP (CT353; U-Cytech). 4.10 Statistical analyses The data were analyzed using two-tailed unpaired t-test. Statistical significance was set at * (p < 0.05), (p < 0.01), or * (p < 0.001). Funding This work was supported by the 10.13039/501100001809 National Natural Science Foundation of China (grant number 31900138, to Y. Y.), the 10.13039/501100002858 China Postdoctoral Science Foundation (grant number 2019M662851, to Y. Y.), and the National Basic Research Priorities Program of China (grant number 2018YFA0507201, to X. C.). Declaration of competing interest The authors declare that there is no conflict of interest in the publication of this study. Data availability Data will be made available on request. Acknowledgments The authors would like to acknowledge Dr. Ding Gao, Ms. Anna Du, and Ms. Juan Min of The Core Facility and Technical Support of Wuhan Institute of Virology for their valuable technical support. ==== Refs References Ashour H.M. Elkhatib W.F. Rahman M.M. Elshabrawy H.A. Insights into the recent 2019 novel coronavirus (SARS-CoV-2) in light of past human coronavirus outbreaks Pathogens 9 2020 Babendure J.R. Babendure J.L. Ding J.H. Tsien R.Y. Control of mammalian translation by mRNA structure near caps RNA 12 2006 851 861 16540693 Cao H. Zhao K. Yao Y. Guo J. Gao X. Yang Q. Guo M. Zhu W. Wang Y. Wu C. Chen J. Zhou Y. Hu X. Lu M. Chen X. Pei R. RNA binding protein 24 regulates the translation and replication of hepatitis C virus Protein Cell 9 2018 930 944 29380205 Cao H. Zhu W. Han Q. Pei R. Chen X. Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient Virol. Sin. 29 2014 61 70 24452538 Flynn R.A. Belk J.A. Qi Y. Yasumoto Y. Wei J. Alfajaro M.M. Shi Q. Mumbach M.R. Limaye A. DeWeirdt P.C. Schmitz C.O. Parker K.R. Woo E. Chang H.Y. Horvath T.L. Carette J.E. Bertozzi C.R. Wilen C.B. Satpathy A.T. Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions Cell 184 2021 2394 2411 e2316 33743211 Grifone R. Xie X. Bourgeois A. Saquet A. Duprez D. Shi D.L. The RNA-binding protein Rbm24 is transiently expressed in myoblasts and is required for myogenic differentiation during vertebrate development Mech. Dev. 134 2014 1 15 25217815 Jiang Y.Q. Zhang M. Qian Y.J. Xu E.S. Zhang J. Chen X.B. Rbm24, an RNA-binding protein and a target of p53, regulates p21 expression via mRNA stability J. Biol. Chem. 289 2014 3164 3175 24356969 Kadam S.B. Sukhramani G.S. Bishnoi P. Pable A.A. Barvkar V.T. SARS-CoV-2, the pandemic coronavirus: molecular and structural insights J. Basic Microbiol. 61 2021 180 202 33460172 Keene J.D. Komisarow J.M. Friedersdorf M.B. RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts Nat. Protoc. 1 2006 302 307 17406249 Krichel B. Falke S. Hilgenfeld R. Redecke L. Uetrecht C. Processing of the SARS-CoV pp1a/ab nsp7-10 region Biochem. J. 477 2020 1009 1019 32083638 Kuwasako K. Takahashi M. Unzai S. Tsuda K. Yoshikawa S. He F. Kobayashi N. Guntert P. Shirouzu M. Ito T. Tanaka A. Yokoyama S. Hagiwara M. Kuroyanagi H. Muto Y. RBFOX and SUP-12 sandwich a G base to cooperatively regulate tissue-specific splicing Nat. Struct. Mol. Biol. 21 2014 778 786 25132178 Li Z. Nagy P.D. Diverse roles of host RNA binding proteins in RNA virus replication RNA Biol. 8 2011 305 315 21505273 Locker N. Easton L.E. Lukavsky P.J. HCV and CSFV IRES domain II mediate eIF2 release during 80S ribosome assembly EMBO J. 26 2007 795 805 17255934 Manfredonia I. Nithin C. Ponce-Salvatierra A. Ghosh P. Wirecki T.K. Marinus T. Ogando N.S. Snijder E.J. van Hemert M.J. Bujnicki J.M. Incarnato D. Genome-wide mapping of SARS-CoV-2 RNA structures identifies therapeutically-relevant elements Nucleic Acids Res. 48 2020 12436 12452 33166999 Mehta M. Raguraman R. Ramesh R. Munshi A. RNA binding proteins (RBPs) and their role in DNA damage and radiation response in cancer Adv. Drug Deliv. Rev. 191 2022 114569 Miao Z. Tidu A. Eriani G. Martin F. Secondary structure of the SARS-CoV-2 5'-UTR RNA Biol. 18 2021 447 456 32965173 Mohamadian M. Chiti H. Shoghli A. Biglari S. Parsamanesh N. Esmaeilzadeh A. COVID-19: Virology, biology and novel laboratory diagnosis J. Gene Med. 23 2021 e3303 Naqvi A.A.T. Fatima K. Mohammad T. Fatima U. Singh I.K. Singh A. Atif S.M. Hariprasad G. Hasan G.M. Hassan M.I. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: structural genomics approach Biochim. Biophys. Acta, Mol. Basis Dis. 1866 2020 165878 Pestova T.V. Kolupaeva V.G. The roles of individual eukaryotic translation initiation factors in ribosomal scanning and initiation codon selection Genes Dev. 16 2002 2906 2922 12435632 Satija N. Lal S.K. The molecular biology of SARS coronavirus Ann. N. Y. Acad. Sci. 1102 2007 26 38 17470909 Schubert K. Karousis E.D. Jomaa A. Scaiola A. Echeverria B. Gurzeler L.A. Leibundgut M. Thiel V. Muhlemann O. Ban N. SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation Nat. Struct. Mol. Biol. 27 2020 959 966 32908316 Shi D.L. RBM24 in the post-transcriptional regulation of cancer progression: anti-tumor or pro-tumor activity? Cancers 14 2022 Sonenberg N. Hinnebusch A.G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets Cell 136 2009 731 745 19239892 Xu E.S. Zhang J. Zhang M. Jiang Y.Q. Cho S.J. Chen X.B. RNA-binding protein RBM24 regulates p63 expression via mRNA stability Mol. Cancer Res. 12 2014 359 369 24375645 Yang D. Leibowitz J.L. The structure and functions of coronavirus genomic 3' and 5' ends Virus Res. 206 2015 120 133 25736566 Yang F. Bi J. Xue X. Zheng L. Zhi K. Hua J. Fang G. Up-regulated long non-coding RNA H19 contributes to proliferation of gastric cancer cells FEBS J. 279 2012 3159 3165 22776265 Yao Y. Yang B. Cao H. Zhao K. Yuan Y. Chen Y. Zhang Z. Wang Y. Pei R. Chen J. Hu X. Zhou Y. Lu M. Wu C. Chen X. RBM24 stabilizes hepatitis B virus pregenomic RNA but inhibits core protein translation by targeting the terminal redundancy sequence Emerg. Microb. Infect. 7 2018 86 Yao Y. Yang B. Chen Y. Huang D. Liu C. Sun H. Hu X. Zhou Y. Wang Y. Chen J. Pei R. Wen Z. Chen X. RNA-Binding motif protein 38 (RBM38) mediates HBV pgRNA packaging into the nucleocapsid Antivir. Res. 198 2022 105249 Yao Y. Yang B. Chen Y. Wang H. Hu X. Zhou Y. Gao X. Lu M. Niu J. Wen Z. Wu C. Chen X. RNA-binding motif protein 24 (RBM24) is involved in pregenomic RNA packaging by mediating interaction between hepatitis B virus polymerase and the epsilon element J. Virol. 93 2019 Zhang M. Zhang Y. Xu E. Mohibi S. de Anda D.M. Jiang Y. Zhang J. Chen X. Rbm24, a target of p53, is necessary for proper expression of p53 and heart development Cell Death Differ. 25 2018 1118 1130 29358667 Zhang T. Lin Y. Liu J. Zhang Z.G. Fu W. Guo L.Y. Pan L. Kong X. Zhang M.K. Lu Y.H. Huang Z.R. Xie Q. Li W.H. Xu X.Q. Rbm24 regulates alternative splicing switch in embryonic stem cell cardiac lineage differentiation Stem Cell. 34 2016 1776 1789 Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. Liu M.Q. Chen Y. Shen X.R. Wang X. Zheng X.S. Zhao K. Chen Q.J. Deng F. Liu L.L. Yan B. Zhan F.X. Wang Y.Y. Xiao G.F. Shi Z.L. A pneumonia outbreak associated with a new coronavirus of probable bat origin Nature 579 2020 270 273 32015507	36464077	PMC9712144	NO-CC CODE	2022-12-05 23:15:33	no	Antiviral Res. 2023 Jan 1; 209:105478	utf-8	Antiviral Res	2,022	10.1016/j.antiviral.2022.105478	oa_other
==== Front Enferm Infecc Microbiol Clin (Engl Ed) Enferm Infecc Microbiol Clin (Engl Ed) Enfermedades Infecciosas Y Microbiologia Clinica (English Ed.) 2529-993X Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. S2529-993X(22)00241-6 10.1016/j.eimce.2021.08.016 Scientific Letter SARS-CoV-2 and Legionella pneumophila coinfection Coinfección por SARS-CoV-2 y Legionella pneumophilaArgemí Gemma a⁎ Somoza María a Andrés Marta b Llunell Antonia a a Servicio de Neumología, Hospital de Terrassa (Consorci Sanitari de Terrassa), Terrassa, Spain b Unidad de Enfermedades Infecciosas, Servicio de Medicina Interna, Hospital de Terrassa (Consorci Sanitari de Terrassa), Terrassa, Spain ⁎ Corresponding author. 1 12 2022 12 2022 1 12 2022 40 10 578579 © 2021 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved. 2021 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcDear Editor, Factors associated with a poorer prognosis of COVID-19 are advanced age, male sex, comorbidities, and laboratory parameters such as lymphopenia and elevated inflammatory markers. Bacterial coinfections have also been associated with a worse prognosis.1 Coinfection with Legionella pneumophila has been scarcely reported,2, 3, 4, 5 and to the best of our knowledge no cases have been described in Spain. We present a case of community-acquired coinfection by L. pneumophila and SARS-CoV-2 admitted to our hospital. This is a 35-year-old male patient, non-smoker and with no medical history of interest, who works as a butane cylinder deliverer. He arrived to the emergency room on November 15, 2020 with a 10-day history of fever, myalgia, nausea, diarrhea, anosmia and dysgeusia. A dry cough without dyspnea appeared later. A PCR for SARS-CoV-2 performed in his primary care center two days before admission was positive. Vital signs on admission were as follows: blood pressure 130/93 mmHg, heart rate116 bpm, axillary temperature 39 °C, respiratory rate 26 breaths per minute. Respiratory and cardiac auscultations were normal. The chest X-ray showed a condensation in the right upper lobe (RUL) with air bronchogram (Fig. 1 A). Blood tests showed the following results: CRP 306 mg/L, ferritin 1303 ng/mL, procalcitonin 2.81 μg/L, sodium 128 mmol/L, alanine aminotransferase 26.4 U/L, LDH 262 U/L, leukocytes 10,510 × 109/L (85.9% neutrophils), lymphocytes 890 × 109/L, ferritin 2353 ng/ml, and D-dimer 638 ng/mL. The baseline arterial blood gas showed: pH 7.56, pCO2 21.0 mmHg, pO2 69.0 mmHg, and bicarbonate 18.8 mmol/L. The electrocardiogram was normal.Fig. 1 Radiological evolution. (A) At emergency room; (B) 48 h post admission; (C) just before patient's discharge; (D) 2 months after hospitalization. The patient was admitted with a diagnosis of community-acquired bacterial pneumonia and SARS-CoV-2 coinfection. Treatment was started with low-flow oxygen therapy and empirical antibiotic therapy with ceftriaxone and azithromycin. Culture of sputum, pneumococcal urinary antigen test and HIV serology were negative, and the urinary antigen test for Legionella (STANDARD F Legionella Ag FIA, SD BIOSENSOR, Republic of Korea) was positive. At this point, ceftriaxone was withdrawn. The patient was still febrile and presented minimal effort cough 48 h after admission. The chest X-ray showed an increase in RUL condensation and bilateral peripheral patchy infiltrates, which was interpreted as progression to inflammatory pneumonia due to SARS-CoV-2 (Fig. 1B). Treatment with dexamethasone (6 mg/24 h) was added. At this point the fever subsided, the tachypnea decreased and low oxygen flow achieved good saturations. The inflammatory parameters progressively decreased and the RUL condensation improved, while the bilateral peripheral infiltrates persisted (Fig. 1C). The patient was discharged on the ninth day after admission, continuing with oral azithromycin until completing 2 weeks of treatment. Follow up was satisfactory, with resolution of the pulmonary infiltrates (Fig. 1D). Bacterial coinfection in patients with COVID-19 has been less frequently reported than in other viral infections, such as influenza.1, 6, 7 Coinfection of SARS-CoV-2 with L. pneumophila has been reported in 4 cases, two diagnosed by urinary antigen test and two by culture of respiratory secretions.2, 3, 4, 5 Another 7 cases8, 9 of possible coinfection with L. pneumophila have been reported, but this diagnosis was based solely on positive IgM, which does not constitute a diagnostic criterion for legionellosis. Three studies that investigated coinfection in more than 1900 patients admitted for COVID-19 did not identify any case of coinfection with L. pneumophila.6, 7, 10 Our patient was diagnosed of Legionnaires’ disease by a qualitative analysis in a urine sample, using an immunochromatographic assay with a specificity > 95% in detecting the disease caused by L. pneumophila serogroup 1.11 Respiratory and digestive symptoms can coexist in patients with COVID-19, mimicking other diseases such as legionellosis. In SARS-CoV-2 pneumonia, the most common radiological findings are peripheral bilateral infiltrates, while consolidation occurs more rarely. In general, unilobar consolidation suggests bacterial infection and can be found in about 70% of cases of legionellosis. Although pneumonia due to L. pneumophila can progress to bilateral infiltrates, in this case the confirmation of SARS-CoV-2 infection in the previous days and the peripheral distribution of the infiltrates, without signs of airspace consolidation, were highly suggestive of the development of an inflammatory pneumonia due to SARS-CoV-2. The diagnosis of coinfection in these patients is hampered by the lack of specific symptoms. Consequently, coinfection might be underdiagnosed, and broad-spectrum antibiotics overprescribed.10 In Catalonia the reported incidence of Legionnaires’ disease is higher than in other Spanish autonomous communities. Specifically, in our Vallès Occidental area, the incidence rate in 2019 was 9.94 cases/100,000 persons (95%CI 8.11–12.19). In addition, in the two weeks prior to the patient's admission, two other cases of legionellosis were diagnosed in the city where the patient resided, although no common source was identified for the three cases (data obtained from the Health Department, Government of Catalonia). On the other hand, it must be taken into account that, due to his work, the patient entered many homes and commercial premises. These data, together with the season of the year and the clinical picture of a male with a lobar condensation, hyponatremia, diarrhea, and a negative determination of pneumococcal antigen in urine, made it necessary to consider the diagnosis of Legionnaires’ disease. Once the diagnosis was established, treatment with azithromycin was continued, since there is no difference in the observed outcomes for patients with Legionella spp. pneumonia treated with azithromycin vs. levofloxacin.12 In conclusion, this case demonstrates the importance of making an etiological diagnosis of bacterial coinfection in patients with COVID-19 when there are suggestive clinical and/or radiological findings. We consider that in areas with a relatively high incidence of legionellosis, the urinary antigen test for Legionella should be performed in cases of community-acquired pneumonia with epidemiological suspicion, or with criteria for hospital admission and a negative pneumococcal urinary antigen test. With a specific diagnosis the antibiotic treatment can be targeted, thus reducing the threat of bacterial resistance. Funding This research did not receive funding from the public, commercial, or not-for-profit sectors. Conflict of interests The authors declare no conflict of interests. ==== Refs References 1 Lansbury L. Lim B. Baskaran V. Lim W.S. Co-infections in people with COVID-19: a systematic review and meta-analysis J Infect 81 2020 266 275 10.1016/j.jinf.2020.05.046 32473235 2 Adler H. Ball R. Fisher M. Mortimer K. Vardhan M.S. Low rate of bacterial co-infection in patients with COVID-19 Lancet Microbe 1 2020 e62 10.1016/S2666-5247(20)30036-7 32835331 3 Yu N. Li W. Kang Q. Xiong Z. Wang S. Lin X. Clinical features and obstetric and neonatal outcomes of pregnant patients with COVID-19 in Wuhan, China: a retrospective, single-centre, descriptive study Lancet Infect Dis 20 2020 559 564 10.1016/S1473-3099(20)30176-6 32220284 4 Arashiro T. Nakamura S. Asami T. Mikuni H. Fujiwara E. Sakamoto S. SARS-CoV-2 and Legionella co-infection in a person returning from a Nile cruise J Travel Med 27 2020 taaa053 10.1093/jtm/taaa053 32297939 5 Kolenda C. Ranc A.G. Boisset S. Caspat Y. Carricajo A. Souche A. Assessment of respiratory bacterial coinfections among severe acute respiratory syndrome coronavirus 2-positive patients hospitalized in intensive care units using conventional culture and BioFire Film Array Pneumonia Panel plus assay Open Forum Infect Dis 7 2020 ofaa484 10.1093/ofid/ofaa484 33204762 6 Garcia-Vidal C. Sanjuan G. Moreno-García E. Puerta-Alcalde P. Garcia-Pouton M. Chumbita M. Incidence of co-infections and superinfections in hospitalized patients with COVID-19: a retrospective cohort study Clin Microbiol Infect 27 2021 83 88 10.1016/j.cmi.2020.07.041 32745596 7 Rothe K. Feihl S. Schneider J. Wallnöfer S. Wurst M. Lukas M. Rates of bacterial co-infections and antimicrobial use in COVID-19 patients: a retrospective cohort study in light of antibiotic stewardship Eur J Clin Microbiol Infect Dis 40 2021 859 869 10.1007/s10096-020-04063-8 33140176 8 Tang M.L. Li Y.Q. Chen X. Lin H. Jiang Z.C. Gu D.L. Co-infection with common respiratory pathogens and SARS-CoV-2 in patients with COVID-19 pneumonia and laboratory biochemistry findings: a retrospective cross-sectional study of 78 patients from a single center in China Med Sci Monit 27 2021 e929783 10.12659/MSM.929783 33388738 9 Xing Q. Li G. Xing Y. Chen T. Li W. Ni W. Precautions are needed for COVID-19 patients with coinfection of common respiratory pathogens medRxiv 2020 10.1101/2020.02.29.20027698 10 Hughes S. Troise O. Donaldson H. Mughal N. Moore L.S.P. Bacterial and fungal coinfection among hospitalized patients with COVID-19: a retrospective cohort study in a UK secondary-care setting Clin Microbiol Infect 26 2020 1395 1399 10.1016/j.cmi.2020.06.025 32603803 11 Pierre D.M. Baron J. Yu V.L. Stout J.E. Diagnostic testing for Legionnaires’ disease Ann Clin Microbiol Antimicrob 16 2017 59 10.1186/s12941-017-0229-6 28851372 12 Gershengorn H.B. Keene A. Dzierba A.L. Wunsch H. The association of antibiotic treatment regimen and hospital mortality in patients hospitalized with Legionella pneumonia Clin Infect Dis 60 2015 e66 e79 10.1093/cid/civ157 25722195	36464477	PMC9712296	NO-CC CODE	2022-12-02 23:21:38	no	Enferm Infecc Microbiol Clin (Engl Ed). 2022 Dec 1; 40(10):578-579	utf-8	Enferm Infecc Microbiol Clin (Engl Ed)	2,022	10.1016/j.eimce.2021.08.016	oa_other
==== Front Enferm Infecc Microbiol Clin (Engl Ed) Enferm Infecc Microbiol Clin (Engl Ed) Enfermedades Infecciosas Y Microbiologia Clinica (English Ed.) 2529-993X Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. S2529-993X(22)00243-X 10.1016/j.eimce.2021.12.016 Scientific Letter Possible intrauterine transmission of SARS-CoV-2: Ultrasound findings and viral molecular detection in amniotic fluid Posible transmisión intrauterina del SARS-CoV-2: Hallazgos ecográficos y detección molecular viral en líquido amnióticoMaia Maria Carolina Andrade a Tavares Carolina Santos Souza b Santos Cliomar Alves dos c Martins-Filho Paulo Ricardo b⁎ a Division of Gynaecology and Obstetrics, University Hospital/EBSERH, Federal University of Sergipe, Aracaju, Brazil b Investigative Pathology Laboratory, Federal University of Sergipe, Aracaju, Brazil c Health Foundation Parreiras Horta, Central Laboratory of Public Health (LACEN/SE), Sergipe State Health Secretariat, Aracaju, Brazil ⁎ Corresponding author. 1 12 2022 12 2022 1 12 2022 40 10 586587 © 2022 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved. 2022 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcDear Editor, The coronavirus disease-19 (COVID-19) is an infectious disease associated to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel single-stranded RNA-enveloped virus primarily transmitted person-to-person by close contact through respiratory droplets. In addition, studies have shown the effects of SARS-CoV-2 infection during pregnancy including potential vertical transmission of the virus.1, 2, 3 Although the mechanisms of intrauterine transmission are not fully understood,4 it has been found that SARS-CoV-2 infection may lead to placental vascular lesions, breakdown of the placental barrier and fetal intrauterine viremia.5 In this rare report, we present fetal ultrasound (US) findings and RT-PCR results in a possible case of vertical transmission of SARS-CoV-2. We describe the case of a 42-year-old pregnant woman with past medical history of hypertension and gestational diabetes. At 11 and 20 weeks of gestational age, she had flu-like symptoms (runny nose, sore throat, cough, sneezing and headache) and the results of RT-PCR for SARS-CoV-2 were positive at both time points. At 25 weeks of gestation, US findings showed symmetric hyperechogenic signals (“in mirror”) in the right and left fetal hypochondrium suggesting liver calcifications, and placenta exhibiting punctate hyperechogenic foci also compatible with calcifications (Fig. 1 A and B). At 28 weeks of gestation, there was a significant progression in calcifications and changes in placental shape (Fig. 1C). In the 32nd week, the pregnant woman was vaccinated with AstraZeneca/Oxford COVID-19 vaccine.Fig. 1 Ultrasound images at 25 weeks of gestation showing liver (A) and placental (B) calcifications. At 28 weeks of gestation, there was progression of calcifications and changes in placental shape (C). At 36 weeks of pregnancy, the patient presented amniochorial detachment (D). Two days after birth, the child had persistent calcifications in the right and left liver lobes (E). STORCH (syphilis, toxoplasmosis, rubella, cytomegalovirus, and herpes simplex virus) screening test results were negative. At 36 weeks of pregnancy, there was a reduction in fetal movements with amniochorial detachment (Fig. 1D), and the patient was referred for a cesarean delivery. On admission for delivery, the patient had no symptoms suggestive of COVID-19 and the RT-PCR result from nasopharyngeal swab was negative. A full-term female infant weighing 2238 kg was born uneventful with an Apgar score of 9 and 10 in 1 and 5 min, respectively. Intraoperative amniotic fluid was collected for RT-PCR analysis, which showed a positive result for SARS-CoV-2 (cycle threshold value: 30). On the 2nd postpartum day, nasopharyngeal swabs were collected from the mother and newborn and the RT-PCR results were negative. SARS-CoV-2 IgG antibodies were detected in neonatal blood serum two days after birth, but IgA results were negative. Intrahepatic calcifications continued to be found on ultrasound examination after birth (Fig. 1E). No clinical or neurological complications were documented during the first six months of life. Vertical transmission can be defined as the generational transmission of viruses from mothers to their offspring, whether intrauterine, intrapartum or in the early postnatal period. There is limited evidence on the vertical transmission of SARS-CoV-2. In a review of 51 studies with 336 infants, only one amniotic fluid sample was positive for SARS-CoV-2 by RT-PCR.6 It is known that SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) receptor for the entry into the host cells especially in kidneys, heart, respiratory and gastrointestinal tract tissues. In addition, previous studies have also shown the expression of ACE2 in the placenta.7 Despite these findings, the potential mechanisms of maternal-fetal transmission of SARS-CoV-2 remain unclear. A recent study has found a high expression of ACE2 in the placenta during the first and second trimesters of pregnancy, which suggests a potential risk for intrauterine infection, placenta dysfunction, and pregnancy complications from SARS-CoV-2.8 Contrasting results have been observed during the third trimester of pregnancy. In a prospective cohort study performed by Edlow et al.,9 it was suggested a nonoverlapping placental ACE2 and TMPRSS2 (a vital serine protease for the SARS-CoV-2 infectivity) expression as a protective mechanism against vertical transmission of SARS-CoV-2 during the third trimester, despite the decreased transplacental transfer of anti–SARS-CoV-2 antibodies to the fetus. In this report, our patient had two positive results for SARS-CoV-2 infection during the first 20 weeks of pregnancy and placental changes in the following weeks. In addition, hepatomegaly and persistent calcifications were found in the fetus. Although intrahepatic calcifications are common findings in chromosomal disorders and infections,10 there was no diagnosis of aneuploidy or congenital malformation and the results for STORCH were negative. Furthermore, there is evidence of increased expression of ACE2 in liver fibroblasts and hepatocytes from first to second trimester, which suggests that fetal liver is a vulnerable target organ of SARS-COV-2.8 Our findings are suggestive of a possible in utero SARS-CoV-2 transmission, since there is evidence of maternal SARS-CoV-2 infection during the first weeks of pregnancy, placental and fetal changes suggestive of viral infection, and a positive RT-PCR result for the detection of SARS-CoV-2 RNA from a sterile sample (amniotic fluid) during childbirth. Although the child had no clinical or neurological complications after delivery, this case reinforces the possibility of vertical transmission of SARS-CoV-2 and the need for vaccination of pregnant women against COVID-19. Authors’ contributions All authors contributed equally to the manuscript. Funding The authors declare no financial support. Conflict of interest The authors have no competing interests to declare. ==== Refs References 1 Kumar P. Fadila Prasad A. Akhtar A. Chaudhary B.K. Tiwari L.K. Vertical transmission and clinical outcome of the neonates born to SARS-CoV-2-positive mothers: a tertiary care hospital-based observational study BMJ Paediatr Open 5 2021 e001193 Available from: https://bmjpaedsopen.bmj.com/lookup/doi/10.1136/bmjpo-2021-001193 2 Yu N. Li W. Kang Q. Xiong Z. Wang S. Lin X. Clinical features and obstetric and neonatal outcomes of pregnant patients with COVID-19 in Wuhan, China: a retrospective, single-centre, descriptive study Lancet Infect Dis 20 2020 559 564 Available from: https://linkinghub.elsevier.com/retrieve/pii/S1473309920301766 32220284 3 Knight M. Bunch K. Vousden N. Morris E. Simpson N. Gale C. Characteristics and outcomes of pregnant women admitted to hospital with confirmed SARS-CoV-2 infection in UK: national population based cohort study BMJ 2020 m2107 Available from: https://www.bmj.com/lookup/doi/10.1136/bmj.m2107 32513659 4 Vivanti A.J. Vauloup-Fellous C. Prevot S. Zupan V. Suffee C. Do Cao J. Transplacental transmission of SARS-CoV-2 infection Nat Commun 11 2020 3572 Available from: http://www.nature.com/articles/s41467-020-17436-6 32665677 5 Stonoga E.T.S. de Almeida Lanzoni L. Rebutini P.Z. Permegiani de Oliveira A.L. Chiste J.A. Fugaça C.A. Intrauterine transmission of SARS-CoV-2 Emerg Infect Dis 27 2021 638 641 Available from: https://wwwnc.cdc.gov/eid/article/27/2/20-3824_article.htm 33185524 6 Tolu L.B. Ezeh A. Feyissa G.T. Vertical transmission of severe acute respiratory syndrome coronavirus 2: a scoping review PLOS ONE 16 2021 e0250196 Available from: https://dx.plos.org/10.1371/journal.pone.0250196 33886645 7 Pringle K.G. Tadros M.A. Callister R.J. Lumbers E.R. The expression and localization of the human placental prorenin/renin–angiotensin system throughout pregnancy: roles in trophoblast invasion and angiogenesis? Placenta 32 2011 956 962 Available from: https://linkinghub.elsevier.com/retrieve/pii/S0143400411004802 22018415 8 Li M. Chen L. Zhang J. Xiong C. Li X. The SARS-CoV-2 receptor ACE2 expression of maternal–fetal interface and fetal organs by single-cell transcriptome study PLOS ONE 15 2020 e0230295 Available from: https://dx.plos.org/10.1371/journal.pone.0230295 32298273 9 Edlow A.G. Li J.Z. Collier A.Y. Atyeo C. James K.E. Boatin A.A. Assessment of maternal and neonatal SARS-CoV-2 viral load transplacental antibody transfer, and placental pathology in pregnancies during the COVID-19 pandemic JAMA Netw Open 3 2020 e2030455 Available from: https://jamanetwork.com/journals/jamanetworkopen/fullarticle/2774428 33351086 10 Bronshtein M. Blazer S. Prenatal diagnosis of liver calcifications Obstet Gynecol 86 1995 739 743 Available from: http://linkinghub.elsevier.com/retrieve/pii/002978449500278Y 7566840	36464479	PMC9712297	NO-CC CODE	2022-12-02 23:21:38	no	Enferm Infecc Microbiol Clin (Engl Ed). 2022 Dec 1; 40(10):586-587	utf-8	Enferm Infecc Microbiol Clin (Engl Ed)	2,022	10.1016/j.eimce.2021.12.016	oa_other
==== Front Enferm Infecc Microbiol Clin (Engl Ed) Enferm Infecc Microbiol Clin (Engl Ed) Enfermedades Infecciosas Y Microbiologia Clinica (English Ed.) 2529-993X Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. S2529-993X(22)00236-2 10.1016/j.eimce.2021.02.013 Original Article First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant Primera confirmación de importación y transmisión en España de la variante B.1.1.7 del SARS-CoV-2 recientemente identificadaBuenestado-Serrano Sergio ab1 Recio Raúl cd1 Sola Campoy Pedro J. ab Catalán Pilar abm Folgueira M. Dolores cd Villa Jennifer cd Muñoz Gallego Irene cd de la Cueva Víctor Manuel ab Meléndez M. Angeles cd Andrés Zayas Cristina be Losa-García Juan E. f Goyanes M. José f Fraile Torres Arturo gh Von Wermitz Andres gh Fradejas-Villajos Isabel f del Arco Carmen hi Campelo-Gutiérrez Carolina f González Bodi Sara cd López-Wolf Daniel j Iglesias-Franco Higinio k Pérez-Lago Laura ab Arce Arnaez Araceli l Rodriguez Baena Elena l Ordobas Gavin María l Muñoz Patricia abmn Delgado Rafael cd Cardeñoso Laura gh Viedma Esther cd2 García de Viedma Darío abm2⁎ on behalf of the Gregorio Marañón Microbiology-ID COVID 19 Study Group, 12 de Octubre Microbiology COVID 19 Study Group, La Princesa Microbiology-ID COVID 19 Study Group, Hospital Alcorcón Microbiology-ID COVID 19 Group 3 a Servicio de Microbiología Clínica y Enfermedades Infecciosas, Gregorio Marañón General University Hospital, Spain b Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM), Madrid, Spain c Servicio de Microbiología 12 de Octubre General University Hospital, Spain d Instituto de Investigación Sanitaria 12 de Octubre, Madrid, Spain e Genomics Unit, Gregorio Marañón General University Hospital, Madrid, Spain f Servicio de Microbiología, Hospital Universitario Fundación Alcorcón, Alcorcón, Spain g Servicio de Microbiología, Hospital Universitario La Princesa, Spain h Instituto de investigación Sanitaria La Princesa, Madrid, Spain i Emergency Service, Hospital Universitario de la Princesa, Spain j Servicio de Medicina Interna, Hospital Universitario Fundación Alcorcón, Alcorcón, Spain k Centro de Salud Laín Entralgo, Alcorcón, Spain l Subdirección General de Epidemiología, Madrid, Spain m CIBER Enfermedades Respiratorias (CIBERES), Spain n Departamento de Medicina, Universidad Complutense, Madrid, Spain ⁎ Corresponding author. 1 Contributed equally as first authors. 2 Contributed equally as senior authors. 3 The list of members of the Gregorio Marañón Microbiology-ID COVID 19 Study Group, 12 de Octubre Microbiology COVID 19 Study Group, La Princesa Microbiology-ID COVID 19 Study Group, and Hospital Alcorcón Microbiology-ID COVID 19 Group can be consulted in the supplementary material to this article. 1 12 2022 12 2022 1 12 2022 40 10 546549 20 1 2021 9 2 2021 © 2021 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved. 2021 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Introduction A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. Material and methods We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. Results We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. Conclusion The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country. Introducción Recientemente, ha surgido en Reino Unido una nueva variante de SARS-CoV-2, VOC202012/01, que origina el linaje B.1.1.7. Su rápida distribución en Reino Unido ha alertado a otros países a vigilar su presencia. Material y métodos El rastreo inicial de la variante B.1.1.7 se basó en la ausencia de amplificación del gen S en el ensayo TaqPath, causado por la deleción 69/70. Todos los casos candidatos de corresponder a la variante B.1.1.7 con este criterio fueron posteriormente confirmados por secuenciación de genoma completo. Resultados Describimos los primeros 3 casos importados de esta variante, desde Londres hasta Madrid, con la posterior transmisión domiciliaria de uno de estos casos a 3 familiares y, adicionalmente, los 2 primeros casos con la variante sin vínculo epidemiológico con Reino Unido. Uno de los casos requirió hospitalización. En todos los casos el criterio de no amplificación del gen S identificó con precisión la variante B.1.1.7, como demostró posteriormente la presencia de las 17 mutaciones marcadoras de este linaje. Conclusión Las primeras identificaciones de la variante B.1.1.7 de SARS-CoV-2 indican un papel solapante de las introducciones independientes desde Reino Unido, con eventos de transmisión comunitaria, incluso desde los primeros momentos de la presencia de esta variante en nuestro país. Keywords COVID-19 SARS-CoV-2 B.1.1.7 Importation Transmission WGS Palabras clave COVID-19 SARS-CoV-2 B.1.1.7, Importación Transmisión WGS ==== Body pmcA newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the UK, is responsible for 85% of all cases in south east England1 at the time of writing this manuscript. This variant has also been reported in other European countries and in most distant locations, e.g., Japan, Australia, or Singapore. B.1.1.7 has higher transmissibility in comparison to other SARS-CoV-2 lineages.1 It carries an unusually high number of specific mutations, 17, most of which are non-synonymous and eight concentrate in the S gene.2 Among the mutations mapping in the S gene, some are linked to relevant functional roles, e.g., immune response evasion, (69/70 deletion)3 or increased affinity to the ACE2 receptor (N501Y).4, 5 These findings have raised the alarm of having to face a new variant with the potential ability to accelerate the spread of the pandemic, although no association was has found initially between the new variant and greater COVID-19 severity or more frequent hospitalizations6, a recent report finds a realistic possibility (as per PHIA probability yardstick) that B.1.1.7 is associated with an increased risk of death compared to infection with no-B.1.1.7 variants.7 From a diagnostic perspective, the presence of the 69/70 deletion allows a rapid screening of the B.1.1.7 variant, as it impairs the hybridization of the S probe in the TaqPath assay (ThermoFisher, Waltham, USA). It causes a drop-out in the S signal, accompanied by a normal positive signal for the other two genes targeted by the assay.8 Here, we communicate the first eight confirmed SARS-CoV-2 B.1.1.7 variants identified in Spain, all diagnosed in Madrid, with epidemiological support to the UK for six of the cases: three cases who recently travelled from London to Madrid (December 19–20, 2020) and three household members related to one of these cases. The other two cases had no known epidemiological relationship with the UK, neither trips nor contact with people coming from that country. One of the cases required hospitalization and the others were indicated to keep quarantine and isolation measures along with their close contacts. All except the two cases without links with the UK and the hospitalized case were positive for the COVID-19 rapid antigen test (Panbio™ COVID-19 Ag rapid test device, Abbott) and all showed an S gene target failure with the TaqPath RT-PCR COVID-19 kit. The B.1.17 variant was confirmed by identifying its 17 specific mutations by whole genome sequencing (WGS). WGS for Cases 2, 3, 7, and 8 was performed with the Artic_nCov-2019_V3 panel of primers (Integrated DNA Technologies, Inc., Coralville, Iowa, USA) (artic.network/ncov-2019). Libraries were prepared using the Nextera Flex DNA Library Preparation Kit (Illumina lnc, California, USA). WGS for Cases 1, 4–6 followed the MinION procedure (Oxford Nanopore Technologies, Oxford, United Kingdom). Sequences were deposited at GISAID (EPI_ISL_756271, 756272, 756273 and 756274). Imported cases Case 1. A 33-year-old male London resident, who returned to Madrid from the UK on December 19, 2020 started with fever and myalgia on December 20, and the next day with cough and diarrhoea. He consulted his general practitioner and had his first positive RT-PCR for which he was prescribed symptomatic treatment. On December 28, he went to the emergency room due to dyspnoea. Physical examination revealed tachypnea, bilateral crackles, and hypoxemia, and the chest X-ray showed multilobar bilateral infiltrates. Lymphocyte count, D-dimers, and ferritin were normal and C-reactive protein value was 16 mg/L (normal < 5). Patient was treated with oxygen, dexamethasone, and prophylactic heparin with favourable clinical evolution. On December 30, 2020, the patient had a second positive RT-PCR result. By that time, his two household contacts were negative. Case 2. A 30-year-old female without any comorbidity who travelled from London to Madrid on December 20, 2020 and started with fever asthenia, cough, myalgia, and dyspnoea the night of her arrival. She went to the emergency department on December 22 where an antigenic COVID-19 rapid test and RT-PCR were performed, both with positive result; furthermore, unilateral pneumonia was diagnosed. Transmission event Case 3 (imported index Case). A 27-year-old male with a past medical history of protein C deficiency who arrived to Madrid from London on December 19, 2020 and presented two days later to the emergency department with a chief complaint of fever, generalized headache, and mild non-productive cough the night before. The SARS-CoV-2 RT-PCR was positive after testing negative on December 16. Physical examination and vital signs were normal and therefore symptomatic treatment and prophylactic anticoagulation regimen were indicated. Cases 4–6 (household contacts). Three household contacts of the imported index case went to the emergency department on December 23, 2020 due to fever and general malaise and had positive SARS-CoV-2 RT-PCRs. Two female aged 61 and 31 years and a 55-year-old male. None had underlying diseases nor clinical or radiographic signs of pneumonia. Community cases without epidemiological link to UK Case 7. A 36-year-old male without any relevant medical history who attended the emergency department on December 27, 2020 after contact with a friend who tested positive for SARS-CoV-2 on December 23. The patient complained of headache and productive cough that had started 24 h before. He was tested positive result for SARS-CoV-2 RT-PCR. Case 8. A 30-year-old female without any relevant clinical history, who went to the emergency department on December 27, 2020 after close contact with her mother-in-law and partner, who were SARS-CoV-2 positive. The patient presented asthenia, non-productive cough, and diarrhoea, and positive SARS-CoV-2 RT-PCR. Comparative analysis of strains We compared the sequences obtained from the cases identified in Spain; among them and against two UK original B.1.1.7 consensus references (GISAID: EPI_ISL_601443 and 581117; 20 and 21 September 2020) (Fig. 1 ). 3–7 SNPs were observed between our cases and the UK references, consistent with the time since the new variant has been circulating in UK and its import to Spain. Nearly identical sequences (differing in 1 SNP in one of the cases) were obtained for the cases involved in the household transmission. The remaining cases showed 1–4 private SNPs each, consistent with independent importations.Fig. 1 Comparison of the sequences obtained from the cases identified in Spain; among them and against two UK original B.1.1.7 consensus references (GISAID: EPI_ISL_601443 and 581117; 20 and 21 September 2020). Conclusions We report three independent imported cases from the UK into Spain of the new SARS-CoV-2 B.1.1.7 variant and a rapid household transmission linked to one of the cases. We also communicate the two first identifications of the B.1.1.7 variant in cases without any epidemiological suspicion supporting it, which indicates its circulation in the community. Only one case required hospitalization. At the moment of closing the final revision of this manuscript, B.1.1.7 variant constitutes around 15–20% of the cases in our population. This makes meaningless to apply a criterion of epidemiological suspect and makes unaffordable to perform continuous confirmatory sequence analysis of all new candidate cases with a drop out signal in the RT-PCR. Further efforts are currently being made in order to constantly update the magnitude of the presence of the B.1.1.7 variant in our population, based on the S dropout feature. Updatings are either population-based (in those laboratories systematically applying the TaqPath test), or on a random subsampling of positive specimens pretested by another diagnostic RT-PCR. Conflict of interest The authors declare that they have no conflict of interest. Appendix A Supplementary data The following are the supplementary data to this article: Acknowledgements This work was supported by Instituto de Salud Carlos III (Ref COV20/00140: SeqCOVID – Consorcio para la epidemiología genómica de SARS-CoV-2 en España) and by Consejo Superior de Investigaciones Científicas (CSIC) (PTI Salud Global). We are grateful to Dainora Jaloveckas (cienciatraducida.com) for editing and proofreading assistance. Appendix A Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.eimce.2021.02.013 ==== Refs References 1 Volz E. Mishra S. Chand M. Barret J. Johnson R. Geidelberg L. Transmission of SARS-CoV-2 Lineage B.1.1.7 in England: Insights from linking epidemiological and genetic data 2020 MRC Centre for Global Infectious Disease Analysis Report 42 2 Rambaut A. Loman N. Pybus O. Barclay W. Barrett J. Carabelli A. Preliminary genomic characterisation of an emergent SARS-CoV-2 lineage in the UK defined by a novel set of spike mutations ARTIC Netw 2020 3 Kemp S. Harvey W. Datir R. Collier D. Ferreira I. Carabelli A. Recurrent emergence and transmission of a SARS-CoV-2 Spike deletion H69/V70 BioRxiV 2020 4 Chand M. Hopkins S. Dabrera G. Achison C. Barclay W. Ferguson N. Investigation of novel SARS-COV-2 variant: Variant of Concern 202012/01 Public Health England 2020 5 Chen J. Wang R. Wang M. Wei G. Mutations strengthened SARS-CoV-2 infectivity J Mol Biol 432 2020 5212 5226 32710986 6 Horby P. Davies N. Edmunds J. Ferguson N. Medley G. Hayward A. NERVTAG note on B.1.1.7 severity. New and Emerging Respiratory Virus Threats Advisory Group NERVTAG. 2021 7 ECDC Risk related to spread of new SARS-CoV-2 variants of concern in the EU/EEA 2020 8 ECDC Rapid increase of a SARS-CoV-2 variant with multiple spike protein mutations observed in the United Kingdom THREAT ASSESSMENT BRIEF 2020	36464472	PMC9712298	NO-CC CODE	2022-12-02 23:21:38	no	Enferm Infecc Microbiol Clin (Engl Ed). 2022 Dec 1; 40(10):546-549	utf-8	Enferm Infecc Microbiol Clin (Engl Ed)	2,022	10.1016/j.eimce.2021.02.013	oa_other
==== Front Enferm Infecc Microbiol Clin (Engl Ed) Enferm Infecc Microbiol Clin (Engl Ed) Enfermedades Infecciosas Y Microbiologia Clinica (English Ed.) 2529-993X Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. S2529-993X(22)00239-8 10.1016/j.eimce.2021.06.005 Brief Report Acute bronchiolitis during the COVID-19 pandemic Bronquiolitis aguda durante la pandemia de COVID-19Flores-Pérez Patricia a Gerig Nathalia b Cabrera-López Mª Isabel a de Unzueta-Roch José L. a del Rosal Teresa b Calvo Cristina b⁎ COVID-19 Study Group in Children◊ a Pediatric Department, Hospital Universitario Niño Jesús, Madrid, Spain b Pediatric Infectious Diseases Department, Hospital Universitario La Paz, Fundación IdiPaz, Traslational Research Network in Pediatric Infectious Diseases (RITIP), Madrid, Spain ⁎ Corresponding author. ◊ COVID-19 Study Group in Children is provided in Appendix A. 1 12 2022 12 2022 1 12 2022 40 10 572575 7 4 2021 2 6 2021 © 2021 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved. 2021 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Introduction The autumn and winter bronchiolitis epidemics have virtually disappeared in the first year of the COVID-19 pandemic. Our objectives were characterised bronchiolitis during fourth quarter of 2020 and the role played by SARS-CoV-2. Methods Prospective multi-centre study performed in Madrid (Spain) between October and December 2020 including all children admitted with acute bronchiolitis. Clinical data were collected and multiplex PCR for respiratory viruses were performed. Results Thirty-three patients were hospitalised with bronchiolitis during the study period: 28 corresponded to rhinovirus (RV), 4 to SARS-CoV-2, and 1 had both types of infection. SAR-CoV-2 bronchiolitis were comparable to RV bronchiolitis except for a shorter hospital stay. A significant decrease in the admission rate for bronchiolitis was found and no RSV was isolated. Conclusion SARS-CoV-2 infection rarely causes acute bronchiolitis and it is not associated with a severe clinical course. During COVID-19 pandemic period there was a marked decrease in bronchiolitis cases. Introducción La epidemia de bronquiolitis de otoño e invierno prácticamente desapareció durante el primer año de la pandemia de COVID-19. Nuestros objetivos eran caracterizar la bronquiolitis durante el cuarto trimestre de 2020 y determinar el papel desempeñado por el virus SARS-CoV-2. Métodos Estudio multicéntrico prospectivo realizado en Madrid (España) entre los meses de octubre y diciembre de 2020, que incluyó a todos los niños ingresados con bronquiolitis aguda. Se recogieron los datos clínicos y se realizó una PCR múltiple para virus respiratorios. Resultados Se hospitalizó a treinta y tres pacientes con bronquiolitis durante el periodo del estudio: 28 correspondieron a rinovirus, 4 a SARS-CoV-2 y uno presentaba ambos tipos de infección. Las bronquiolitis por SAR-CoV-2 fueron comparables a las bronquiolitis por rinovirus, salvo por una estancia hospitalaria más corta. Se detectó una reducción significativa en la tasa de ingresos por bronquiolitis y no se aisló VSR. Conclusión Es raro que la infección por SARS-CoV-2 cause bronquiolitis aguda y no se asocia a una evolución clínica grave. Durante la pandemia de COVID-19 se produjo un descenso pronunciado de los casos de bronquiolitis. Keywords Bronchiolitis SARS-CoV-2 COVID-19 Sincitial respiratory virus Rhinovirus Pandemic Children Palabras clave Bronquiolitis SARS-CoV-2 COVID-19 Virus sincitial respiratorio Rinovirus Pandemia Niños ==== Body pmcIntroduction Acute viral bronchiolitis is the main cause of hospitalisation in children under 1 year of age. Although all respiratory viruses can cause bronchiolitis, respiratory syncytial virus (RSV) is the primary agent in more than 70% of cases.1 The coronavirus disease 2019 (COVID-19) pandemic has drastically changed the epidemiology of other viral respiratory infections in both children and adults. Worldwide, the autumn and winter RSV epidemics have virtually disappeared, and in some countries in the Southern Hemisphere like South Africa or Australia, the RSV season has moved to spring, marking an unprecedented phenomenon.2 The incidence of symptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children is relatively low, and the disease causes lower symptom severity compared to adults. There is little information about the role of SARS-CoV-2 in bronchiolitis, although experimental studies performed in ferrets have shown lung lesions compatible with bronchiolitis.3 In addition, other human coronaviruses (HCoV) have been detected in up to 5% infants with bronchiolitis, with OC43 and 229E being associated with greater disease severity.4 We aimed to describe the epidemiology of bronchiolitis requiring hospitalisation during the fourth quarter of 2020 and characterise bronchiolitis associated with SARS-CoV-2 infection. Additionally, we compare the number of cases during the same period in 2019. Methods This is a prospective multicentre study carried out in 2 large university hospitals in Madrid (Hospital La Paz and Niño Jesús Children's Hospital) between 1 October and 31 December 2020. Inclusion criteria were infants under 1 year of age admitted with a diagnosis of acute bronchiolitis (first acute episode of wheezing with respiratory dyspnoea and catarrhal prodrome). Local research teams conducted structured interviews, reviewed medical records, and collected supplemental test results during hospitalisation. Within 24 h of admission, all children underwent testing with SARS-CoV-2 polymerase chain reaction (PCR) assay of nasopharyngeal swabs (Vircell; sensitivity 96%, specificity 100%) as well as a respiratory virus PCR panel that included RSV (FTD Respiratory Pathogens 21, Fast Track Diagnostics, Siemens). Patients with a severe underlying disease such as haematological cancer, immunodeficiency, or chronic pulmonary disease (e.g., cystic fibrosis, obliterative bronchiolitis, interstitial disease) and those undergoing chronic immunosuppressive treatment were excluded. Additionally, we reviewed the number of cases and the proportion of RSV bronchiolitis recorded during the same period in 2019. Qualitative data were expressed as absolute and relative frequencies and quantitative data as median and interquartile range (IQR). Categorical variables were compared using the chi-square and Fisher's exact test, and continuous variables were analysed with non-parametric tests as appropriate. A two-tailed p value <0.05 was considered statistically significant. All analyses were performed using the Statistical Package for the Social Sciences, version 21.0 (IBM Corp., CA, USA). The study was approved by the ethics committee of both hospitals and parents signed informed consent. Results Out of 33 children hospitalised with bronchiolitis, 4 had SARS-CoV-2 infection and 28 had rhinovirus (RV) infection. One patient presented coinfection with both viruses and was excluded from the analysis. No cases of RSV were identified. The median age at admission was 99 days (IQR 39–257) and 24 were male. Clinical characteristics of patients with RV and SARS-CoV-2 infection are shown in Table 1 . SARS-CoV-2 bronchiolitis was associated with a shorter hospital stay: median 3 days (IQR 2.5–3) vs 4 days (IQR 2.2–6.7); p = 0.003, likely related to the reduced need for oxygen therapy [median 1 day (IQR 1.0–1.5) vs 4 days (IQR 2.0–6); p = 0.006]. In terms of clinical presentation, the groups had a similar course, although fewer patients with RV bronchiolitis developed fever (50.0% vs 10.7%, p = 0.043) and diarrhoea (25.0% vs 0%, p = 0.007). No patients with SARS-CoV-2 bronchiolitis required intensive care unit (ICU) admission or advanced life-support treatments such as nasogastric tube feeding or non-invasive mechanical ventilation, while 21% of patients in the RV group required such measures.Table 1 Patient characteristics and respiratory pathogens in infants with bronchiolitis. Table 1Variable SARS-CoV-2 infection (n = 4) Rhinovirus infection (n = 28) p Age, d, median (IQR) 133.5 (45.0–248.5) 96.50 (39.25–254.50) 0.381 Sex, n (%) Male 3 (75.00) 21 (75.00) 0.999 Female 1 (25.00) 7 (25.00) Prematurity (gestational age <37 wk), n (%) 2 (50.00) 6 (21.43) 0.217 Confirmed cohabitation with an individual positive for COVID-19, n (%) 3 (75.00) 0 (0) <0.001 Underlying disease (heart disease, Down syndrome, chronic lung disease, immunodeficiency, others), n (%) 0 (0) 7 (25.00) 0.258 Clinical presentation, n (%) Onset of symptoms <24 h 1 (25.00) 5 (17.86) 0.732 Fever 2 (50.00) 3 (10.71) 0.043 Vomiting 0 (0) 7 (25.00) 0.258 Diarrhoea 1 (25.00) 0 (0) 0.007 Refusal to eat 2 (50.00) 9 (32.14) 0.482 Irritability 0 (0) 5 (17.86) 0.358 Wheezing 4 (100.00) 25 (89.29) 0.492 Apnoea 2 (50.00) 5 (17.86) 0.146 Exanthema, conjunctivitis 1 (25.00) 3 (10.71) 0.419 Vital signs on presentaion to the emergency department Tachycardia, n (%) 2 (50.00%) 7 (25.93) 0.322 Tachypnoea, n (%) 2 (50.00) 21 (75.00) 0.298 Baseline oxygen saturation (%); median (IQR) 95 (95–96.5) 94 (92–97) 0.718 Clinical course Length of hospital stay, d, median (IQR) 3.00 (2.5–3.0) 4.00 (2.2–6.7) 0.003 Oxygen therapy, d, median (IQR) 1.00 (1.0–1.5) 4.00 (2.0–6.0) 0.006 Tube feeding, n (%) 0 (0) 8 (28.57) 0.217 Mechanical ventilation, n (%) 0 (0) 12 (46.15) 0.124 Intensive care admission, n (%) 0 (0) 6 (21.43) 0.304 Radiological findings, n (%) Chest X-ray 2 (50.00) 16 (57.14) 0.788 Pneumonia 0 (0) 5 (31.18) 0.358 Pleural effusion 0 (0) 0 (0) NA Treatment, n (%) Oxygen therapy 3 (75.00) 26 (92.86) 0.252 Antibiotic 1 (25.00) 5 (17.86) 0.732 Salbutamol 1 (25.00) 9 (32.14) 0.773 Corticosteroids 0 (0) 5 (17.86) 0.358 Adrenaline 0 (0) 0 (0) NA n: number of cases; p: p-value; d: days; IQR: interquartile range; wk: weeks; h: hours; NA: not available. We found a dramatic decrease in the total number of paediatric admissions between 2019 and 2020 for the same seasonal period (833 vs 435 children) and in the number of admissions due to bronchiolitis (271 vs 33 children), a difference that was statistically significant (32.53% vs 7.59%, p< 0.001). In 2019, most bronchiolitis cases were caused by RSV (241/271 bronchiolitis), while in 2020 there were no cases. Discussion Our results confirm the substantial change in the epidemiology of viral respiratory infections caused by the COVID-19 pandemic. The yearly wave of bronchiolitis admissions was virtually nonexistent, as in other countries.5, 6, 7 The annual RSV epidemic has not taken place yet, and it is unclear whether it will start during the next few months. In Australia, after the reduction of COVID-19 non-pharmacological interventions and the arrival of summer, an unusually delayed and steeper ‘summer bronchiolitis and RSV peak’ has been seen in certain regions, with an older median age and higher total numbers compared to the usual winter peaks.8 We might be pushing the’RSV curve’ forward and may not be able to stop this trend when we arrive at the exponential phase. Due to the small number of patients included, we lack sufficient data to reach precise conclusions about SARS-CoV-2 bronchiolitis and how it differs from other viral infections. In our sample, infection with SARS-CoV-2 was not associated with increased disease severity. SARS-CoV-2 and RV bronchiolitis seem to have a similar clinical course although patients with SARS-CoV-2 bronchiolitis had shorter hospital stay and did not require advanced support treatment or ICU admission. This could be due, at least partially, to none of the COVID-19 patients having underlying diseases and only one being prescribed parenteral antibiotics because of suspected bacterial superinfection. Regarding our case with coinfection due to SARS-CoV-2 and RV, it is known that coinfection is possible but might occur infrequently; not only the elderly or people with systemic diseases are at risk from this coinfection, but healthy children are also,9 which could impact the treatment and prognosis of the disease. The number of bronchiolitis admissions in both centres for the current autumn-winter season has been much lower than predicted based on previous years. More surprisingly, we have not detected any cases of RSV within the population areas served by the 2 hospitals, this despite rapid diagnostic tests performed in the emergency department in all symptomatic cases. Although some authors believe this to be the result of measures instituted to prevent SARS-CoV-2 transmission such as awareness-raising regarding handwashing, mask wearing, and isolation,2 this does not seem to be the only plausible explanation. In our city, nurseries and primary schools have been fully open since the beginning of the academic year (autumn 2020). Although stricter hygiene measures have been implemented and the number of students per class has been limited, the use of masks is not mandatory until age 6 years. Even so, the circulation of respiratory viruses has diminished dramatically, except for SARS-CoV-2. Van Brusselen et al. suggest that this happens because infectious diseases such as bronchiolitis do not become real epidemics when transmission is inhibited by non-pharmaceutical interventions by adults and older children.6 Another hypothesis to explain this phenomenon is competition between respiratory viruses, in this case SARS-CoV-2 and RSV. Some viruses such as RV may interfere with and block infection by other respiratory viruses, either by competition of viral receptors in the mucosa of the respiratory tract or due to the production of interferon by infected respiratory-tract cells. Interactions between co-circulating, taxonomically different respiratory viruses can influence patterns of infection. RV interference may have produced a similar effect during the 2009 H1N1 influenza pandemic in Europe 10and, nowadays, has impaired SARS-CoV-2 replication and spread within the human respiratory epithelium. According to a forthcoming article by Dee et al., RV triggers an IFN response that makes most cells nonpermissive to SARS-CoV-2 infection.11 The susceptibility of SARS-CoV-2 to the IFN response is illustrated by the number of genes present in its genome that are devoted to overcoming the innate immune response.12 If accurate, these findings would support the theory that viral interference may alter the course of an epidemic and we must consider viral interactions in the host as an influence in the observed population dynamics. Our series of cases confirms that SARS-CoV-2 infection may cause acute bronchiolitis as has been previously published in other reports from around the world. Possibly due to various causes including public-health interventions aimed at preventing SARS-CoV-2 transmission and others factors such as the pandemic itself and interference between viruses, we have found an unexpected sharp decrease in admissions from acute respiratory illness and lower frequencies of RSV detection. Conflict of interest The authors declare that they have no conflict of interest. Appendix A COVID-19 Study Group in Children Talía Sainzb, Mª Lourdes Calleja-Geroa, Fernando Baquero-Artigaob, Ana Méndez-Echevarríab, Mª Pilar Gómez-Romerob, Raquel Jiménez- Garcíaa. ==== Refs References 1 Florin T.A. Plint A.C. Zorc J.J. Viral bronchiolitis Lancet 389 2017 211 224 10.1016/S0140-6736(16)30951-5 27549684 2 Britton P.N. Hu N. Saravanos G. COVID-19 public health measures and respiratory syncytial virus Lancet Child Adolesc Health 4 2020 e42 e43 10.1016/S2352-4642(20)30307-2 32956616 3 Kim Y.-I. Kim S.-G. Kim S.-M. Infection and rapid transmission of SARS-CoV-2 in ferrets Cell Host Microbe 27 2020 704 709 10.1016/j.chom.2020.03.023 e2 32259477 4 Calvo C. Alcolea S. Casas I. A 14-year prospective study of human coronavirus infections in hospitalized children: comparison with other respiratory viruses Pediatr Infect Dis J 39 2020 653 657 10.1097/INF.0000000000002760 32453196 5 Friedrich F. Ongaratto R. Scotta M.C. Early impact of social distancing in response to coronavirus disease 2019 on hospitalizations for acute bronchiolitis in infants in Brazil Clin Infect Dis 2020 10.1093/cid/ciaa1458 Online ahead of print 6 Van Brusselen D. De Troeyer K. ter Haar E. Bronchiolitis in COVID-19 times: a nearly absent disease? Eur J Pediatr [Internet] 2021 1 5 10.1007/s00431-021-03968-6 Online ahead of print 7 Pelletier J.H. Rakkar J. Au A.K. Trends in US pediatric hospital admissions in 2020 compared with the decade before the COVID-19 pandemic JAMA Netw Open 4 2021 e2037227 10.1001/jamanetworkopen.2020.37227 33576819 8 Yeoh D.K. Foley D.A. Minney-Smith C.A. Impact of coronavirus disease 2019 public health measures on detections of influenza and respiratory syncytial virus in children during the 2020 Australian Winter Clin Infect Dis 2020 10.1093/cid/ciaa1475 Online ahead of print 9 Chen X. Liao B. Cheng L. The microbial coinfection in COVID-19 Appl Microbiol Biotechnol 104 2020 7777 7785 10.1007/s00253-020-10814-6 32780290 10 Casalegno J.S. Ottmann M. Duchamp M.B. Rhinoviruses delayed the circulation of the pandemic influenza A (H1N1) 2009 virus in France Clin Microbiol Infect 16 2010 326 329 10.1111/j.1469-0691.2010.03167.x 20121829 11 Dee K. Goldfarb D.M. Haney J. Human rhinovirus infection blocks SARS-CoV-2 replication within the respiratory epithelium: implications for COVID-19 epidemiology J Infect Dis 2021 10.1093/infdis/jiab147 Online ahead of print 12 Conti P. How to reduce the likelihood of coronavirus-19 (CoV-19 or SARS-CoV-2) infection and lung inflammation mediated by IL-1 J Biol Regul Homeost Agents 34 2020 333 338 doi:10.23812/Editorial-Conti-2 32228825	36464475	PMC9712299	NO-CC CODE	2022-12-02 23:21:38	no	Enferm Infecc Microbiol Clin (Engl Ed). 2022 Dec 1; 40(10):572-575	utf-8	Enferm Infecc Microbiol Clin (Engl Ed)	2,022	10.1016/j.eimce.2021.06.005	oa_other
==== Front J Nerv Ment Dis J Nerv Ment Dis JNMD The Journal of Nervous and Mental Disease 0022-3018 1539-736X Lippincott Williams & Wilkins 35703234 JNMD_220263 10.1097/NMD.0000000000001558 00005 3 Original Articles Stuck Inside How Social Functioning in Schizophrenia Changed During the COVID-19 Pandemic Minor Kyle S. PhD ∗ Myers Evan J. MS [email protected] ∗ Abel Danielle B. MS [email protected] ∗ Mickens Jessica L. MS [email protected] ∗ Ayala Alexandra BS [email protected] ∗ Warren Kiara K. BS [email protected] † Vohs Jenifer L. PhD [email protected] ‡ ∗ Department of Psychology, Indiana University–Purdue University Indianapolis, Indianapolis, Indiana † Department of Psychology, Louisiana State University, Baton Rouge, Louisiana ‡ Department of Psychiatry, Indiana University School of Medicine, Indianapolis, Indiana. Send reprint requests to Kyle S. Minor, PhD, IUPUI School of Science, 402 N Blackford St, LD 124, Indianapolis, IN 46202. E-mail: [email protected]. 12 2022 14 6 2022 14 6 2022 210 12 915924 Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved. 2022 Wolters Kluwer Health, Inc. All rights reserved. This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections. Abstract Social distancing policies enacted during the COVID-19 pandemic altered our social interactions. People with schizophrenia, who already exhibit social deficits, may have been disproportionally impacted. In this pilot study, we a) compared prepandemic social functioning to functioning during the pandemic in people with schizophrenia (n = 21) who had data at both time points; and b) examined if patterns of decline in schizophrenia differed from healthy controls (n = 21) across a series of repeated-measures analyses of variance. We observed larger declines in social functioning in schizophrenia (η2 = 0.07, medium effect size) during the pandemic compared with the control group. Between-group declines did not extend to other domains, suggesting that declines are specific to social functioning. Our findings signal that treatments focusing on reconnecting people with schizophrenia to their social networks should be prioritized. Future studies should continue tracking social functioning after the pandemic to illustrate patterns of recovery. Key Words Schizophrenia pandemic social interaction psychotic disorders social isolation ==== Body pmcIn response to the COVID-19 pandemic, widespread social distancing policies were enacted across the world. These policies altered how we interacted with friends, family, and acquaintances—eliminating some of these interactions entirely. This sea change in how we interacted with others likely had profound effects on social functioning. People with schizophrenia, who already exhibit deficits in social functioning (Abel et al., 2021; Green et al., 2015), may have been disproportionally impacted. Because social distancing restricted many interactions to online encounters, the smaller social networks and lower rates of Internet access in schizophrenia (Degnan et al., 2018; Young et al., 2020) made connecting with others difficult. When social networks are disrupted in schizophrenia, an array of negative outcomes occur (e.g., increased psychotic symptoms, less focus on mental and physical health; Degnan et al., 2018; see Zhand and Joober, 2021). Thus, it is important to understand how social functioning in schizophrenia changed during the pandemic: was it in step with the declines experienced by healthy adults or did those with schizophrenia have steeper drops in functioning? To answer this question, it is necessary to accurately assess individual responses during the pandemic. This requires baseline measurements of social functioning. Without a baseline, it is difficult to estimate how a person's social functioning differs from their typical course of behavior. To examine if a specific deficit in social functioning exists—rather than a general decline across all areas—it is also important to analyze how other domains were affected during the pandemic. Symptoms, role functioning, and quality of life are good candidates for evaluation; all are negatively impacted in schizophrenia and each has the potential to be affected by the COVID-19 pandemic (Kozloff et al., 2020; Strauss et al., 2022; Zhand and Joober, 2021). To address these issues, we a) recruited people who had participated in studies before the pandemic (i.e., before the first reported statewide case of COVID-19) and b) compared their prepandemic data on functioning and symptoms measures to data collected during the COVID-19 pandemic. To date, few studies have assessed how functioning or symptom domains have changed in schizophrenia during the pandemic. In terms of social activity, two studies found that in-person interactions decreased and communication using digital devices increased in schizophrenia when comparing prepandemic to pandemic activity (Jagesar et al., 2021; Valeri et al., 2021). Mixed results have been observed regarding symptoms (Haddad et al., 2022; Pinkham et al., 2021; Strauss et al., 2022). Pinkham et al. (2021) found that symptoms remained relatively stable while well-being actually increased during the early stages of the pandemic. Strauss et al. (2022) focused on how negative symptoms changed during the pandemic and observed increased severity of both experiential and behavioral symptoms. Although little information exists on changes in role functioning or quality of life, it is likely that increased unemployment rates and social distancing restrictions would be associated with decreases in these constructs as the pandemic continued. There is also evidence that other types of symptoms (e.g., depression), use of maladaptive coping strategies, and loneliness have all increased in schizophrenia as social distancing has remained in place (Rosa-Alcázar et al., 2021; Tso and Park, 2020; Valeri et al., 2021). The COVID-19 pandemic also raises important issues about the intersection between health and social behavior. People with schizophrenia have higher rates of comorbid health conditions (e.g., diabetes, obesity) that place them at greater risk for mortality if COVID-19 infection occurs (see Barcella et al., 2021; Barlati et al., 2021; Kozloff et al., 2020; Maguire and Looi, 2020). A study by Tzur Bitan et al. (2021) found that those with schizophrenia were less likely to be diagnosed with COVID-19; when diagnosed, however, they exhibited more severe cases and were three times more likely to die from infection. Thus, schizophrenia populations may be more likely to receive intensive social distancing and quarantining recommendations than the general public. This may leave two unfavorable options: 1) isolate for a longer period and risk further disconnection from social networks; or 2) break protocol—either by choice or necessity—and face increased risk of developing COVID-19 with severe complications. Observing how those with schizophrenia respond to social distancing policies—and how psychological, health, and socioeconomic variables are related to their responses—is critical for understanding how these options are weighed. Study Objectives and Hypotheses This pilot study had three aims. First, we examined if social functioning declined in schizophrenia during the pandemic and, if so, how these declines compared with those seen in healthy adults. We hypothesized that social functioning would decline in both groups when prepandemic and pandemic scores were compared but that declines would be more severe in schizophrenia. Second, we tested whether symptoms, role functioning, and quality of life changed in schizophrenia during the pandemic compared with controls. Again, we expected to find changes in both groups, with the schizophrenia group exhibiting more significant changes. A goal of this aim was to observe whether changes in those with schizophrenia during the pandemic were specific to social functioning or represented a more global decline across different domains. Third, we identified if psychological (loneliness, resilient coping, optimism toward the future), health (medical conditions linked to worse COVID-19 outcomes), or socioeconomic factors (lack of access to Internet/smartphone) were associated with changes in social functioning during the pandemic. Our expectation was that these constructs would be linked to social declines in both groups. By evaluating these hypotheses, this study has potential to illustrate how social behavior changed in response to the COVID-19 pandemic and to identify constructs that are associated with these changes. This is important for determining the level of vulnerability experienced by those with schizophrenia. It may also allow us to identify treatment targets in schizophrenia that should be prioritized as the pandemic subsides. METHODS Participants Given our focus on testing people with prepandemic data, all participants were recruited from study registries (see Abel et al., 2021; Minor et al., 2022). To be included, those in the schizophrenia group had to a) have a schizophrenia-spectrum disorder diagnosis (e.g., schizophrenia, schizoaffective disorder) listed in their medical record and supported using the Mini International Neuropsychiatric Interview (Sheehan et al., 1998); b) have prepandemic data before March 6, 2020 (i.e., date of first reported COVID-19 case statewide); c) be 18–65 years old; d) exhibit English fluency; e) have no change in outpatient status or medications in the month before testing; f) demonstrate no current substance dependence; and g) have no documented intellectual disability. The healthy control group met similar inclusion criteria with one exception: they could not have a current affective, substance use, or psychotic disorder or have a history of psychosis at baseline or follow-up testing. In total, prepandemic and pandemic data were collected for 42 people (schizophrenia = 21, control = 21). Prepandemic data were collected in person for all participants. Pandemic data were collected primarily using online video programs and/or over the phone (there were also in person procedures for a minority of participants). Figure 1 provides more information on study contacts and rates of participation from those with baseline data. Regarding when pandemic data were collected, the majority (schizophrenia = 11, control = 11) of assessments occurred when businesses in the state were allowed to operate but at ≤50% capacity and only with social distancing guidelines in place (May 20, 2020 to September 22, 2020). Twelve people were assessed (schizophrenia = 5, control = 7) when businesses were allowed to operate at full capacity but before the administration of the first approved vaccine in the state (September 23, 2020 to December 12, 2020). The remaining 8 participants were assessed (schizophrenia = 5, control = 3) when vaccines were available in the state, but those in the participant's age group were ineligible to receive them (December 13, 2020 to February 27, 2021). FIGURE 1 Study participant flowchart. Measures Social Functioning Three instruments were used to capture social functioning: 1) the Global Functioning: Social Scale (GF: Social; Auther et al., 2006); 2) the Interpersonal Relations subscale from the Heinrichs Quality of Life Scale (Heinrichs et al., 1984); and 3) a social activities measure designed specifically for this study. The GF: Social and QLS-IR are clinician-rated measures that have demonstrated good psychometric properties (Cornblatt et al., 2007; Gupta et al., 2000) and have been used previously with schizophrenia samples (Abel and Minor, 2021; Fulford et al., 2020; Minor et al., 2016; Minor et al., 2022). They were administered at both time points (prepandemic, pandemic). The GF: Social assesses functioning in romantic and social relationships as compared with one's peers using a 10-point scale. The QLS-IR uses a 7-point scale and consists of eight items (e.g., active acquaintances, social network sociosexual relations). Increasing scores signify better social functioning on both measures. The measure created for this study was administered at the pandemic time point and assessed social activities across different modalities. Social activities were separated into how interactions occurred (e.g., in person, online using video, texting). Participants estimated how often and the amount of time they spent interacting with others before and during the pandemic. Questions were separated into prepandemic and pandemic categories and administered in an interview format by study personnel (see Table 1 for specific questions). TABLE 1 Social Activity Questions Created for This Study Questions assessing frequency of interactions per week with friends and familya Before March 2020…/since March 2020… How often did you interact with friends or family members online? How often did you interact with friends or family members online using video? How often did you text with friends or family members on the phone? How often did you talk with friends or family members on the phone? How often did you visit a mental health professional? Questions assessing estimated hours of interactions per week with friends and family Before March 2020…/since March 2020… How long (in minutes) would you estimate that you interacted with people outside of your household in person during a typical week? How long (in minutes) would you estimate that you interacted with friends and family online during a typical week? How long (in minutes) would you estimate that you interacted with friends and family online using video during a typical week? How long (in minutes) would you estimate that you texted with friends and family on the phone during a typical week? How long (in minutes) would you estimate that you talked with friends and family on the phone during a typical week? How long (in minutes) would you estimate that you spent visiting a mental health professional during a typical week? aScale: 1 = never, 2 = once or twice per month, 3 = once per week, 4 = more than once per week, 5 = daily, 6 = multiple times per day. Symptoms The Positive and Negative Syndrome Scale (PANSS; Kay et al., 1987) is an interview-rated scale consisting of 30 items across five factors (positive, negative, disorganized, hostility, depression; see Bell et al., 1994; Minor and Lysaker, 2014; Minor et al., 2015a). All items use a 7-point scale, with higher scores signifying greater severity. In past studies, the PANSS has shown good internal consistency (Kay et al., 1987), interrater reliability (Lysaker et al., 2013), and predictive validity (Bell et al., 1992). Here, it was administered at prepandemic and pandemic assessments. Postpandemic PANSS assessments were typically conducted over the phone or by using online video programs. Some symptom variables could not be collected (negative domain: blunted affect, motor retardation, preoccupation; disorganized domain: tension and mannerisms and posturing) when phone-based assessments were the only modality used. In these instances, scores from other items in that symptom category were extrapolated (e.g., “tension” was the mean of other disorganized variables at the pandemic time point). Role Functioning The Global Functioning: Role Scale (GF: Role; Cornblatt et al., 2007) is a clinician-rated scale that was given at prepandemic and pandemic time points. Interviewers selected participants' primary role from three choices (school, work, homemaker). Ratings used a 10-point scale and were based on how well the participant performed in their role compared with their peers. The GF: Role has exhibited strong psychometric properties and been used in past studies with schizophrenia samples (Cornblatt et al., 2007; Minor et al., 2015b). Quality of Life The QLS-IR (Heinrichs et al., 1984) consists of three subscales in addition to the QLS-IR. The Instrumental Role subscale assesses work and school functioning across four items. The Intrapsychic Foundations subscale measures factors such as motivation and sense of purpose using seven items. The Common Objects and Activities has two items that assess one's access to common objects and activities. All items were administered at prepandemic and pandemic assessments and are rated on a 7-point scale, with increasing scores indicating greater quality of life. Psychological, Health, and Socioeconomic Constructs Instruments were also implemented during the pandemic to assess psychological, health, and socioeconomic constructs that may be associated with changes in social functioning. Psychological measures included the UCLA Loneliness Scale (Russell, 2010), the Brief Resilient Coping Scale (Sinclair and Wallston, 2004), and the Revised Life Orientation Test (Scheier et al., 1994). Each has demonstrated good psychometric properties and been used in past studies with schizophrenia samples (Kamenov et al., 2016; Mote et al., 2019; Palmer et al., 2018). For health and socioeconomic variables, questions were designed specifically for this study. Health questions focused on whether participants had been diagnosed with a condition that would place them at higher risk for severe COVID-19 infection if they had been exposed to or developed COVID-19. Socioeconomic questions centered on whether participants had a prolonged period during the pandemic without access to the Internet or a smartphone. Specific questions designed for this study were administered by study personnel in an interview format and are included in Table 2. TABLE 2 Health and Socioeconomic Questions Created for This Study In general, how would you describe your health? (1 = poor, 2 = fair, 3 = good, 4 = very good) (see Subjective Health Rating in Table 3) Do you have at least one health condition listed below? (0 = no, 1 = yes) Cardiovascular condition? (0 = no, 1 = yes) Chronic lung disease? (0 = no, 1 = yes) Obesity? (0 = no, 1 = yes) Diabetes? (0 = no, 1 = yes) Have you experienced any of the following COVID-19 symptoms during the pandemic? (0 = no, 1 = yes) Fever? (0 = no, 1 = yes) Dry cough? (0 = no, 1 = yes) Shortness of breath? (0 = no, 1 = yes) Decreased sense of smell or taste? (0 = no, 1 = yes) Flu-like symptoms? (0 = no, 1 = yes) Have you ever been tested for COVID-19? (0 = no, 1 = yes) Have you ever been diagnosed or had a presumed positive diagnosis of COVID-19? (0 = no, 1 = yes) Are you currently practicing social distancing? (0 = no, 1 = yes) Statistical Analysis Analyses occurred in four parts. First, independent t-tests and chi-square analyses compared demographic and clinical data in schizophrenia and control groups. Second, a series of repeated-measures analyses of variance (ANOVAs) were run with time (prepandemic, pandemic) and group (schizophrenia, control) as independent variables and social functioning as the dependent variable (DV). Partial eta-square values were used to assess the magnitude of time by group interactions. Paired samples t-tests were implemented to capture change from prepandemic to pandemic time points within each group. Third, a series of repeated-measures ANOVAs were also conducted using symptoms, role functioning, and quality of life as DVs. Finally, partial Pearson's correlations were analyzed for each group to determine if psychological, health, or socioeconomic constructs were associated with social functioning during the pandemic while controlling for prepandemic social functioning. For all analyses, an alpha level of 0.05 was used to determine significance. RESULTS Demographic, Clinical, and Health Variables During the Pandemic Regarding demographic data, groups did not significantly differ in age, sex, or race (see Table 3 and Fig. 1). Significant differences in education and marital status were observed, with the schizophrenia group being less likely to progress beyond high school or have been married. Household data indicated no significant group differences in the number of overall people/children in the household, the number of days between prepandemic and pandemic assessments, or the number of days that participants were assessed following the first reported statewide COVID-19 case. TABLE 3 Demographic, Psychological, Health, and Socioeconomic Data Measured During the Pandemic Schizophrenia (n = 21) Control (n = 21) M (SD) M (SD) Test of Significance Age 47.90 (10.30) 50.86 (8.87) t = 1.00 No. people in householda 1.86 (1.35) 2.76 (1.79) t = 1.85b No. children younger than 18 y 0.14 (0.65) 0.52 (0.98) t = 1.48 Days between assessments 567 (427) 537 (405) t = 0.23 Days following initial casec 221 (65) 216 (73) t = 0.24 Loneliness 44.62 (12.19) 34.14 (8.03) t = 3.29d Resilient coping 13.57 (3.40) 16.43 (1.99) t = −3.32d Optimism toward the future 22.24 (7.21) 29.52 (5.13) t = −3.77d Subjective health rating 2.62 (0.92) 2.86 (0.91) t = 0.84 n, % n, % Test of Significance Sex χ2 = 0.76 Male 9, 43% 10, 48% Female 12, 57% 11, 52% Race χ2 = 2.40 African American 14, 67% 9, 43% White 7, 33% 12, 57% Education χ2 = 6.93d ≤High school or GED 8, 38% 1, 5% >High school or GED 13, 62% 20, 95% Marital status χ2 = 3.50e Never married 12, 57% 6, 29% Married (previous or current) 9, 43% 15, 71% ≥1 Health condition 16, 76% 7, 33% χ2 = 7.79d >1 Health condition 8, 38% 3, 14% χ2 = 3.08e Cardiovascular conditions 7, 33% 4, 19% χ2 = 1.11 Chronic lung disease 2, 10% 0, 0% χ2 = 2.10 Obesity 13, 62% 6, 29% χ2 = 4.71e Diabetes 8, 38% 1, 5% χ2 = 6.93d COVID-19 symptoms 2, 10% 9, 43% χ2 = 6.04d Tested for COVID-19 4, 19% 11, 52% χ2 = 5.08e Presumed positive COVID-19 0, 0% 3, 14% χ2 = 3.23e Currently social distancing (%) 19, 90% 20, 95% χ2 = 0.36 Period without Internet or smartphone 6, 29% 2, 10% χ2 = 2.47b aIncludes self. bp < 0.10. cDays assessed after March 6, 2020 (first case in Indiana). dp < 0.01. ep < 0.05. Table 3 also lists psychological, health, and socioeconomic variables. Regarding psychological variables, the schizophrenia group reported greater loneliness, less resilient coping, and less optimism toward the future. Regarding health variables, significant group differences were found for obesity, diabetes, and the presence of one/multiple health conditions that could lead to severe COVID-19 infection. In each instance, the schizophrenia group was more likely to report having a condition. The control group was significantly more likely to report experiencing COVID-19 symptoms, to have been tested for COVID-19, and to have received a positive COVID-19 diagnosis. No significant group differences were found for subjective health ratings, cardiovascular conditions or chronic lung disease, whether participants were currently socially distancing, or if there was a period during the pandemic when participants were without access to the Internet/smartphone. Social Functioning Before and During the Pandemic Repeated-measures ANOVAs revealed significant time by group interactions for the GF: Social and QLS-IR. Main effects were observed for time (GF: Social: F[1,40] = 10.87, p = 0.001; QLS-IR: F[1,40] = 10.38, p = 0.002) and group (GF: Social: F[1,40] = 68.67, p < 0.001; QLS-IR: F[1,40] = 77.14, p < 0.001). Both interview-rated measures showed steeper declines in the schizophrenia group compared with the control group (see Fig. 2). Paired samples t-tests showed significant decreases in social functioning in the schizophrenia group on the GF: Social, t(20) = 3.13, p = 0.003, and QLS-IR, t(20) = 2.86, p = 0.005, during the pandemic. Decreases in the control group on the GF: Social, t(20) = 1.30, p = 0.104, and QLS-IR, t(20) = 1.49, p = 0.076, did not reach the level of significance. FIGURE 2 Social functioning in people with schizophrenia (SZ) and healthy control participants was assessed across prepandemic and pandemic time points. Assessments of social functioning included. A, The Global Functioning Scale: Social (GFS: Social). B, The Heinrich Quality of Life Scale: Interpersonal Relations (QLS: IR). C, Self-reported hours meeting with other people in person. Error bars reflect standard error of the mean variance for each time point. On the study-specific social activity measure, time by group interactions were only observed for time spent interacting with others outside of one's residence. Healthy controls had a sharper decrease in this category compared with those with schizophrenia. Paired samples t-tests indicated that those with schizophrenia had significant changes from prepandemic to pandemic assessments in the frequency they interacted with others online using video (increase), t(20) = −2.65, p = 0.008, the frequency they interacted with mental health professionals (decrease), t(20) = 1.92, p = 0.035, and the number of minutes they interacted with others outside of their residence (decrease), t(20) = 1.82, p = 0.043. Significant changes in controls were observed for the frequency they interacted with others online (increase), t(20) = −2.35, p = 0.015, the frequency (increase), t(20) = −4.51, p = 0.000, and the amount of time they interacted with others online using video (increase), t(20) = 2.68, p = 0.007, the frequency they spoke to others on the phone (increase), t(20) = −3.68, p = 0.001, and the amount of time they interacted with others outside of their residence (decrease), t(20) = 4.80, p > 0.001. Taken together, social functioning declined for both groups (see Table 4). Steeper drops in global social functioning were observed in schizophrenia. Controls, who interacted with others more prepandemic, showed sharper declines in their time spent with others in public during the pandemic. TABLE 4 Pre-Post Differences in Social Functioning Variables Within and Across Groups Schizophrenia (n = 21) Control (n = 21) Pre During Pre During M (SD) M (SD) M (SD) M (SD) F p η2, Size of Effecta Social functioning: GFS: Socialb 6.19 (1.29) 5.24 (1.22) 8.48 (1.25) 8.19 (0.98) 3.15 0.042 0.07, medium Social functioning: QLS-IRb,c 3.39 (1.05) 2.74 (1.11) 5.37 (0.62) 5.17 (0.81) 2.86 0.049 0.07, medium Frequency of interactions per week with friends and familyd Online 2.14 (1.65) 2.29 (1.65) 3.52 (1.97) 4.00 (1.70) 0.94 0.169 0.02, small Online via video 1.90 (1.34) 2.52 (1.66) 1.81 (1.21) 3.00 (1.48) 2.63 0.056 0.06, medium Texting 3.71 (1.98) 4.24 (1.92) 5.14 (1.24) 5.24 (1.22) 1.40 0.122 −0.03, small Phone calls 4.10 (1.61) 4.48 (1.47) 4.45 (1.15) 4.95 (1.23) 0.16 0.347 <0.01, negligible Mental health professional 4.52 (1.36) 4.19 (1.54) 1.05 (0.22) 1.00 (0.00) NA NA Estimated hours of interactions per week with friends and family Time interacting in person 11.43 (18.92) 6.35 (8.18) 31.42 (18.67) 13.33 (16.88) 6.42 0.007 −0.14, large Online 0.72 (1.53) 2.78 (9.82) 6.80 (9.57) 8.50 (10.55) 0.03 0.435 <−0.01, negligible Online via video 0.42 (1.08) 1.85 (4.58) 0.95 (2.10) 2.67 (3.58) 0.03 0.429 <−0.01, negligible Texting 4.77 (12.58) 8.12 (17.50) 8.02 (10.97) 10.23 (13.68) 0.18 0.338 <−0.01, negligible Phone calls 2.90 (3.32) 3.50 (5.98) 5.38 (8.72) 7.23 (7.23) 0.14 0.357 <0.01, negligible Mental health professional 0.65 (0.93) 0.52 (0.83) 0 (0) 0 (0) NA NA aInterpretation of effect sizes made using estimates from Cohen (1988). bGlobal Functioning Scale: Social. cHeinrich Quality of Life Scale: Interpersonal Relations. dScale: 1 = never, 2 = once or twice per month, 3 = once per week, 4 = more than once per week, 5 = daily, 6 = multiple times per day. Symptoms, Role Functioning, and Quality of Life Before and During the Pandemic Repeated-measures ANOVAs did not reveal significant time by group interactions for overall symptoms, any symptom domain, role functioning, or two of three quality of life domains (excluding the QLS-IR; see Table 5). An interaction was observed for Intrapsychic Foundations, with the schizophrenia group exhibiting a sharper decline during the pandemic. TABLE 5 Pre-Post Differences in Clinical Variables Within and Across Groups Schizophrenia (n = 21) Control (n = 21) Pre During Pre During M (SD) M (SD) M (SD) M (SD) F p η2, Size of Effecta Symptoms 58.14 (14.03) 60.86 (16.66) 33.90 (6.15) 34.24 (5.82) 0.80 0.188 0.02, small Positive 14.10 (5.37) 14.48 (7.74) 6.43 (0.87) 6.95 (1.69) 0.02 0.446 <0.01, negligible Negative 14.71 (4.36) 15.52 (6.42) 10.00 (4.00) 8.76 (1.37) 2.20 0.073 −0.05, small Disorganized 12.43 (4.24) 14.38 (5.60) 7.81 (1.66) 8.43 (3.17) 1.52 0.112 −0.04, small Hostility 6.38 (2.77) 6.14 (3.17) 4.81 (1.12) 4.67 (1.15) 0.02 0.439 <0.01, negligible Depression 10.52 (5.48) 10.33 (4.60) 4.86 (1.39) 5.43 (1.96) 0.61 0.220 0.02, small Role functioning 3.74 (2.47) 2.95 (2.70) 8.75 (0.79) 8.20 (1.64) 0.21 0.324 <0.01, negligible Heinrich quality of life Instrumental role 2.00 (1.67) 1.63 (2.00) 5.09 (1.05) 4.64 (1.80) 0.04 0.852 <0.01, negligible Intrapsychic foundations 4.07 (1.22) 3.61 (1.19) 5.52 (0.54) 5.45 (0.59) 2.89 0.048 0.07, medium Common activities/objects 4.39 (1.08) 3.56 (0.98) 5.33 (0.70) 4.81 (0.51) 0.31 0.291 <0.01, negligible aInterpretation of effect sizes made using estimates from Cohen (1988). Paired samples t-tests showed significant declines in role functioning for schizophrenia, t(20) = 1.87, p = 0.039, and control groups, t(20) = 1.76, p = 0.047. Relatively few increases in symptoms were observed during the pandemic: disorganized symptoms in the schizophrenia group was the lone symptom domain that significantly increased, t(20) = −1.97, p = 0.032. For quality of life domains, Intrapsychic Foundations decreased in the schizophrenia group, t(20) = 2.19, p = 0.020, and Common Activities and Objects decreased in schizophrenia, t(20) = 3.79, p = 0.001, and control groups, t(20) = 3.99, p = 0.001. Relationships Between Social Functioning With Psychological, Health, and Socioeconomic Variables Partial correlations between social functioning during the pandemic and psychological, health, and socioeconomic variables were run while controlling for prepandemic social functioning (Table 6). For the schizophrenia group, GF: Social was inversely associated with loneliness and positively associated with optimism toward the future. In the control group, both social functioning measures were inversely related to loneliness and having a period during the pandemic without the Internet or a smartphone. No significant associations were observed for resilient coping or health condition variables in either group. TABLE 6 Partial Correlations Between Psychological Variables and Social Functioning During the Pandemic While Controlling for Social Functioning at Baseline Schizophrenia (n = 21) Control (n = 21) GF: Sociala QLS-IRb GF: Social QLS-IR Loneliness −0.448c −0.142 −0.588d −0.533d Resilient coping 0.219 0.039 0.105 −0.016 Optimism toward the future 0.527d 0.296 0.010 −0.008 ≥1 Health condition (%) −0.153 0.134 0.058 −0.067 >1 Health condition (%) −0.330e −0.162 −0.041 −0.071 Period without Internet or smartphone −0.112 −0.025 −0.590d −0.596d aGlobal Functioning Scale: Social. bHeinrich Quality of Life Scale: Interpersonal Relations. cp < 0.05. dp < 0.01. ep < 0.10. DISCUSSION In this pilot study, we assessed how social functioning changed in schizophrenia during the COVID-19 pandemic and identified psychological, health, and socioeconomic constructs that might help explain lower functioning. Four main findings were observed. First, those with schizophrenia showed larger drops in social functioning than healthy adults during the pandemic. Although both groups exhibited declines, our data suggest that the pandemic had a more deleterious social impact in schizophrenia—where social deficits already existed before the pandemic. Second, in-person social activity decreased for both groups, with healthy adults showing larger declines. Declines were partially offset by a boost in time spent communicating via digital devices, although increases were more likely to be found in healthy adults. Third, role functioning and at least one quality of life domain significantly declined for both groups. Symptoms remained relatively stable across time points; disorganized symptoms in schizophrenia were the only domain that increased. Finally, loneliness was identified as a variable linked to social functioning during the pandemic—while controlling for prepandemic functioning—across groups; optimism toward the future was also inversely related to pandemic social functioning in schizophrenia. The key finding in this study was that the schizophrenia group had a more severe drop in social functioning. The declines found here indicate that those with schizophrenia experienced increased disconnection from their social networks during the pandemic; this is troubling given the importance of social connections to mental health (Bellack et al., 2007; Degnan et al., 2018; Harvey et al., 2007). As many medical and mental health appointments shifted online during the pandemic, there is also evidence that those with schizophrenia had reduced contact with their medical care teams. Dickerson et al. (2022), as well as data here, showed that those with schizophrenia were more likely to miss medical appointments than controls and attended appointments less frequently than before the pandemic. Although preliminary, this raises concerns about access to care and whether a subgroup with schizophrenia had very few social interactions throughout the pandemic. At minimum, it seems that the already limited social support systems available to those with schizophrenia have been reduced significantly compared with before the pandemic. The larger declines experienced in schizophrenia seem to be specific to social functioning. When observed, declines in other domains did not differ according to group status. This has important potential implications for treatment as the pandemic ends. Given the limited resources available in community mental health, findings from this study suggest that interventions focused on improving social connections should be prioritized. This will be challenging because of the widespread need following the pandemic (e.g., see Fares-Otero et al., 2021; Hamada and Fan, 2020). However, if these findings hold true—and replication needs to occur given the limited sample—social functioning in schizophrenia should be a focal point. One point of promise is that past studies have shown that, although social deficits are observed, people with schizophrenia desire social affiliation at similar levels to healthy adults and often list increasing social contacts as a treatment goal (Blanchard et al., 2015; Shumway et al., 2003). This could point to motivation to engage in socially based treatments. Implementing digital or telehealth interventions and providing access to devices may be one way to increase focus more rapidly given the increased implementation of these interventions—and increased acceptance of digital devices—during the pandemic (see Sanchez-Guarnido et al., 2021; Torous and Keshavan, 2020). A potential explanation for the steeper social functioning declines in schizophrenia centers on how social activity occurred during the pandemic. First, the control group reported a greater drop for in-person social activity than those with schizophrenia. To contextualize this finding, however, the control group went from having over 30 hours of weekly in-person activity to just under 15 hours, whereas those with schizophrenia went from just over 11 hours to approximately 6 hours. Thus, the large baseline group differences may signal why the less severe decline in schizophrenia may still hold important implications for global social functioning (i.e., they had less room to decline and still maintain social connections). Second, controls were more likely to report increased use of digital devices during the pandemic. Although those with schizophrenia also exhibited increased use of these devices, their largest increase in reported minutes was with texting. Those in the control group were more likely to report increased frequency of using digital means that more closely approximated in-person interactions. This is important because interactions that more closely approximate in-person conversations may be more substantive; substantive conversations are linked with increased well-being (Mehl et al., 2010) and may foster closer social connections (Rabin, 2010). Given the observed declines in social and role functioning, it was somewhat surprising that symptoms remained relatively stable in schizophrenia. Overall symptoms were similar to prepandemic levels, and four of five symptom domains did not significantly increase. This supports findings from Pinkham et al. (2021), who also reported that symptoms did not change early in the pandemic. We did not replicate findings from Strauss et al. (2022), who observed an increase in negative symptoms during the pandemic. One reason may be that Strauss et al. (2022) implemented a measure designed specifically to assess negative symptoms, whereas we used a broader measure given our focus on a wider range of symptoms. Disorganized symptoms were the lone domain that increased. This increase may be a result of the greater isolation experienced during the pandemic; de Sousa et al. (2015, 2018) have shown that disorganization tends to rise when those with schizophrenia are more isolated. This study had limited success identifying possible correlates for social functioning during the pandemic. Across groups, loneliness showed the most consistent associations with pandemic social functioning, even when controlling for prepandemic functioning. Loneliness has been identified as a major problem for people with serious mental illness during the COVID-19 pandemic, with approximately a third of these individuals reporting high levels of loneliness (Heron et al., 2022). This and findings from our study are not particularly surprising, but they do illustrate how the relationship between loneliness and lower social functioning during the pandemic contributed to one another across schizophrenia and control groups. Optimism toward the future was also associated with pandemic social functioning for one of two measures in schizophrenia. This could point to the importance of hope as a way to maintain social functioning under adverse conditions. The health and socioeconomic variables examined here did not show strong links with social functioning in schizophrenia—despite the higher rates of medical conditions conferring increased COVID-19 risk. The inclusion of participants with prepandemic data and the number of domains tested serve as study strengths. The small sample size is a central limitation. Groups were well-matched, but the low number reduces power to draw inferences. Replication from other studies is needed. A second limitation is that in-person assessments were conducted for prepandemic measures, whereas a mix of online and phone-based assessments was primarily administered during the pandemic. This was necessary due to health and safety protocols. Although other studies have implemented phone-based protocols to deliver psychological assessments (Ainsworth et al., 2013; Eisner et al., 2019), this may have resulted in some unintended differences across time points here. A third limitation is that the activity measure created for this study could only be administered retrospectively, which limits the inferences that can be drawn about how specific types of interactions changed during the pandemic. Potential response bias is a fourth limitation. Although we reached out to all eligible individuals, only approximately 40% per group participated (see Fig. 1 for more information). It could be that those who could not be contacted or declined participation may have responded differently during the pandemic (e.g., contact changed due to no longer being able to pay phone bill, declined participation due to increased symptoms). These limitations should be considered when interpreting findings. CONCLUSIONS In sum, we found that people with schizophrenia demonstrated sharp declines in social functioning during the COVID-19 pandemic. When compared with healthy adults, the steeper declines in schizophrenia seem specific to social functioning—as declines in symptoms, role functioning, and quality of life did not differ based on group status. This signals that reconnecting people with schizophrenia to their social networks should be prioritized. Implementing creative methods of connecting people with social support (e.g., digital or telehealth interventions and programs; see Sanchez-Guarnido et al., 2021; Torous and Keshavan, 2020) is critical for increasing social connections. Regarding correlates of social functioning during the pandemic, loneliness seemed to be the most promising indicator when controlling for prepandemic functioning. Future studies should replicate study findings in larger samples and compare how social functioning responds in healthy adult and schizophrenia groups as the pandemic subsides. DISCLOSURE All authors have read and approved the submitted manuscript. K.S.M. designed the study, performed data analysis, and wrote or cowrote all drafts of the manuscript; E.J.M. performed data analysis, collected study data, created the study database, and cowrote all drafts of the manuscript; D.B.A., J.L.M., A.A., and K.K.W. collected study data and cowrote all drafts of the manuscript; and J.L.V. helped select study instruments and cowrote all drafts of the manuscript. All authors declare that they have no conflicts of interest. All participants signed informed consent documents before engaging in study procedures, and protocols were approved by local institutional review boards. ==== Refs REFERENCES Abel DB Minor KS (2021) Social functioning in schizophrenia: Comparing laboratory-based assessment with real-world measures. J Psychiatr Res. 138 :500–506.33971484 Abel DB Salyers MP Wu W Monette MA Minor KS (2021) Quality versus quantity: Determining real-world social functioning deficits in schizophrenia. 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J Pers Soc Psychol. 67 :1063–1078.7815302 Sheehan DV Lecrubier Y Sheehan KH Amorim P Janavs J Weiller E Hergueta T Baker R Dunbar GC (1998) The Mini-International Neuropsychiatric Interview (M.I.N.I.): The development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10. J Clin Psychiatry. 59 (suppl 20 ):22–33. Shumway M Saunders T Shern D Pines E Downs A Burbine T Beller J (2003) Preferences for schizophrenia treatment outcomes among public policy makers, consumers, families, and providers. Psychiatr Serv. 54 :1124–1128.12883140 Sinclair VG Wallston KA (2004) The development and psychometric evaluation of the Brief Resilient Coping Scale. Assessment. 11 :94–101.14994958 Strauss GP Macdonald KI Ruiz I Raugh IM Bartolomeo LA James SH (2022) The impact of the COVID-19 pandemic on negative symptoms in individuals at clinical high-risk for psychosis and outpatients with chronic schizophrenia. Eur Arch Psychiatry Clin Neurosci. 272 :17–27.33881621 Torous J Keshavan M (2020) COVID-19, mobile health, and serious mental illness. Schizophr Res. 218 :36–37.32327314 Tso IF Park S (2020) Alarming levels of psychiatric symptoms and the role of loneliness during the COVID-19 epidemic: A case study of Hong Kong. Psychiatry Res. 293 :113423.32871487 Tzur Bitan D Krieger I Kridin K Komantscher D Scheinman Y Weinstein O Cohen AD Cicurel AA Feingold D (2021) COVID-19 prevalence and mortality among schizophrenia patients: A large-scale retrospective cohort study. Schizophr Bull. 47 :1211–1217.33604657 Valeri L Wang Z Wang A Eichi HR Liebenthal E Rauch S Schutt R Ongur D Dixon L Baker J Onnela JP (2021) The effect of COVID-19 shelter in place orders on loneliness of schizophrenia and bipolar disorder patients. Biol Psychiatry. 89 :S134–S135. Young AS Cohen AN Niv N Nowlin-Finch N Oberman RS Olmos-Ochoa TT Goldberg RW Whelan F (2020) Mobile phone and smartphone use by people with serious mental illness. Psychiatr Serv. 71 :280–283.31744429 Zhand N Joober R (2021) Implications of the COVID-19 pandemic for patients with schizophrenia spectrum disorders: Narrative review. BRPsychOpen. 7 :e35.	35703234	PMC9712495	NO-CC CODE	2022-12-14 23:29:58	no	J Nerv Ment Dis. 2022 Dec 14; 210(12):915-924	utf-8	J Nerv Ment Dis	2,022	10.1097/NMD.0000000000001558	oa_other
==== Front Phys Life Rev Phys Life Rev Physics of Life Reviews 1571-0645 1873-1457 Elsevier B.V. S1571-0645(22)00049-5 10.1016/j.plrev.2022.07.008 Comment Behavioral and game-theoretic modeling of dengue epidemic: Comment on “Mathematical models for dengue fever epidemiology: A 10-year systematic review” by M. Aguiar et al. Banerjee Malay ⁎ Ghosh Samiran Department of Mathematics & Statistics, IIT Kanpur, Kanpur - 208016, India ⁎ Corresponding author. 17 8 2022 12 2022 17 8 2022 43 2022 21 7 2022 28 7 2022 © 2022 Elsevier B.V. All rights reserved. 2022 Elsevier B.V. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Communicated by M. Frank-Kamenetskii ==== Body pmcDuring the last few decades, we have seen outbreaks of various epidemics e.g., SARS epidemic in 2002−2003 [4], H5N1 influenza in 2005 [12], H1N1 influenza in 2009 [21], Ebola in 2014 [17], dengue outbreaks [13], and currently, COVID-19 since the end of 2019. Mathematical models have helped in understanding the spreading pattern of the epidemic and validating the effectiveness of relevant control measures [9]. People from various countries have been suffering from the dengue epidemic, which is a vector borne disease, for centuries. The first case of dengue was possibly reported in a Chinese medical encyclopedia [16]. Now dengue has become a common epidemic in more than 120 countries [2]. As per the estimates provided in 2013 [6], there are around 390 million cases of dengue infection per year (95% credible interval 284−528 million), of which 96 million people manifest clinically. Regarding the prevalence of dengue virus infection, another study in 2012 [8] estimates that 3.9 billion people are at risk of dengue epidemic every year. The worldwide distribution of dengue infected individuals and death due to dengue is depicted in Fig. 1 . On the other hand, there is no universally acceptable vaccine that can prevent its recurrent outbreaks, and the available vaccines are not affordable for many developing countries [24], [26]. A wide range of mathematical models is proposed and analyzed for dengue epidemic that has played a crucial role in decision making and design of public health policies to fight against the dengue epidemic [28].Fig. 1 (a) A. aegypti and distribution of dengue in 2006; (b) Death caused by dengue fever per million in 2012 (Figure source–[1]). Fig. 1 We strongly believe that the last 10 years review on mathematical modeling of dengue fever infection by M. Aguiar et al. [3], is a very important and timely review article which will help researchers to have clear picture and proceed for further developments regarding dengue epidemic modeling and its effective control. In the said review article, the authors mainly focused on a systemic review of multi-strain modeling frameworks as the secondary infection with heterogeneous serotype is the main risk factor behind the severity of dengue fever. This detailed and systemic review specifically considers three types of multi-strain modeling approaches, namely vector-to-host transmission, host-to-host transmission, and within-host dynamics, with the help of deterministic, stochastic, and spatial models. The said article appropriately highlights several immunological aspects of dengue infection, e.g., temporary cross-immunity, antibody-dependent enhancement, co-infection etc. as well as the effect of these factors on the possibility of recurrent dengue infection to an individual and towards the risk of severity. Unlike to initial mathematical models of dengue epidemic, the multi-strain models can capture the complex oscillatory nature of the sizes of the recurrent outbreaks. The multi-strain model helps to capture the uneven sizes of recurrent outbreaks, as different strains of dengue viruses appear in an irregular manner. Apart from the epidemic aspects of mathematical modeling, the authors have reported that torus bifurcation is responsible for the onset of complex oscillation (namely chaos), which is a dynamic feature of the multi-strain dengue epidemic models [23]. Various optimal strategies regarding vector control and vaccination campaign are reviewed in this article. Nowadays, researchers are using immuno-epidemic models to understand the epidemic progression more accurately [7], [18]. Recent developments of immuno-epidemic models of dengue epidemic are reviewed in this article. Simultaneous infection of a host by multiple pathogens producing similar clinical symptoms is difficult to detect clinically, whereas this co-infection may affect the condition of the patients poorly. Especially, in the current scenario, for a patient with co-infection by dengue and COVID-19, it is challenging to clinically identify the co-infection as some of the symptoms of the two diseases are identical. This scenario is also covered in the review article. Undoubtedly, this review work deserves appreciation from all aspects; still we want to highlight some other modeling approaches that might be worthy of investigation in the days to come. It is a matter of fact that human behavior during the spread of dengue like epidemics can significantly influence the spreading pattern of the epidemic up to a certain extent. During the rainy seasons, the mosquito population grows rapidly in the waterlogged areas, mainly in the developing countries, then people start using mosquito nets at night, spray insecticides in their surroundings, clean the garbage around their habitat etc., which can reduce the rapid growth of the vector. These measures taken by common people, irrespective of the public health policies, have significant impact to reduce the intensity of disease spread. These measures come into action only when the number of infected becomes prominent, and no measures are usually initiated by the competent authorities mainly in the developing countries, which may be due to financial burden. This behavioral change of human beings can influence the dengue epidemic spreading pattern [10], [11], [19], [25]. It is important to mention here that the major behavioral changes due to human activity like, reducing risky behaviors and avoiding the places with high risk of infection, can be captured with the help of non-linear incidence rates and density dependent dispersal of susceptible and infected [14]. The behavioral change of human individuals during the season of dengue epidemic spread can also be modeled with the help of game-theoretic approach by taking into account an individual's action that can maximize their personal payoff. As a result, these will account for the reduction of risk factor of getting infection and finally contribute to the slowing down of epidemic spread at the population level. This approach is known as ‘effect of individual's decision making on global regulation’ [5]. Some preliminary game-theoretic models for dengue are available in literature [15], [22]. Finally, we want to mention that it is really challenging to collect appropriate real data in order to come up with a reliable data-driven model formulation of human behavior based dengue epidemic, however, some initiatives can be taken up to build an efficient mathematical model based upon human behavior. The mathematical modeling of dengue epidemic using neural networks [27] and machine learning techniques [20] also has the potential impact on understanding the spreading pattern of the epidemic and designing effective control measures. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ==== Refs References 1 https://en.wikipedia.org/wiki/Dengue_fever 2 https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue 3 Aguiar M. Anam V. Blyuss K.B. Mathematical models for Dengue fever epidemiology: a 10-year systematic review Phys Life Rev 40 2022 65 92 35219611 4 Anderson R.M. Fraser C. Ghani A.C. Donnelly C.A. Riley S. Ferguson N.M. Epidemiology transmission dynamics and control of SARS: the 2002–2003 epidemic Philos Trans R Soc Lond, B 359 2004 1091 1105 15306395 5 Banerjee M. Demongeot J. Volpert V. Global regulation of individual decision making Math Methods Appl Sci 39 15 2016 4428 4436 6 Bhatt S. Gething P.W. Brady O.J. Messina J.P. Farlow A.W. Moyes C.L. The global distribution and burden of Dengue Nature 496 7446 2013 504 507 23563266 7 Bocharov G. Volpert V. Ludewig B. Meyerhans A. Mathematical immunology of virus infections 2018 Springer 245 8 Brady O.J. Gething P.W. Bhatt S. Messina J.P. Brownstein J.S. Hoen A.G. Refining the global spatial limits of Dengue virus transmission by evidence-based consensus PLoS Negl Trop Dis 6 8 2012 e1760 9 Brauer F. Castillo-Chavez C. Feng Z. Mathematical models in epidemiology, vol. 32 2019 Springer New York 10 Cai L. Guo S. Li X. Ghosh M. Global dynamics of a Dengue epidemic mathematical model Chaos Solitons Fractals 42 4 2009 2297 2304 11 Cai L.M. Li X.Z. Global analysis of a vector-host epidemic model with nonlinear incidences Appl Math Comput 217 7 2010 3531 3541 12 Chen H. Smith G.J.D. Li K.S. Wang J. Fan X.H. Rayner J.M. Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control Proc Natl Acad Sci USA 103 2006 2845 2850 16473931 13 Dick O.B. Martín J.L.S. Montoya R.H. Diego Jd. Zambrano B. Dayan G.H. The history of Dengue outbreaks in the Americas Am J Trop Med Hyg 87 4 2012 584 23042846 14 d'Onofrio A. Banerjee M. Manfredi P. Spatial behavioural responses to the spread of an infectious disease can suppress Turing and Turing–Hopf patterning of the disease Physica A 545 2020 123773 15 Dorsett C. Oh H. Paulemond M.L. Rychtářr J. Optimal repellent usage to combat Dengue fever Bull Math Biol 78 5 2016 916 922 27142427 16 Gubler Duane J. Dengue/Dengue haemorrhagic fever: history and current status Novartis Found Symp 277 2006 3 17319151 17 Frieden T.R. Damon I. Bell B.P. Kenyon T. Nichol S. Ebola 2014—new challenges, new global response and responsibility N Engl J Med 371 2014 1177 1180 25140858 18 Ghosh S. Banerjee M. Volpert V. Immuno-epidemiological model-based prediction of further COVID-19 epidemic outbreaks due to immunity waning Math Model Nat Phenom 17 2022 9 19 Gonzales C.P. Herman K. Murillo D. Sánchez F. Change in host behavior and its impact on the co-evolution of Dengue 2003 20 Hoyos W. Aguilar J. Toro M. Dengue models based on machine learning techniques: a systematic literature review Artif Intell Med 119 2021 102157 21 Jain S. Kamimoto L. Bramley A.M. Schmitz A.M. Benoit S.R. Louie J. Hospitalized patients with 2009 H1N1 influenza in the United States, April–June 2009 N Engl J Med 361 2009 1935 1944 19815859 22 Kabir K.M.A. Tanimoto J. Cost-efficiency analysis of voluntary vaccination against n-Serovar diseases using antibody-dependent enhancement: a game approach J Theor Biol 503 2020 110379 23 Kooi B.W. Aguiar M. Stollenwerk N. Bifurcation analysis of a family of multi-strain epidemiology models J Comput Appl Math 252 2013 148 158 24 Mahoney R. The introduction of new vaccines into developing countries. V: will we lose a decade or more in the introduction of Dengue vaccines to developing countries? Vaccine 32 2014 904 908 24397903 25 Manfredi P. D'Onofrio A. Modeling the interplay between human behavior and the spread of infectious diseases 2013 Springer Science & Business Media 26 Pang E.L. Loh H.S. Towards development of a universal Dengue vaccine–how close are we? Asian Pac J Trop Med 10 3 2017 220 228 28442105 27 Philemon M.D. Ismail Z. Dare J. A review of epidemic forecasting using artificial neural networks Int J Epidemiol Res 6 3 2019 132 143 28 Racloz V. Ramsey R. Tong S. Hu W. Surveillance of Dengue fever virus: a review of epidemiological models and early warning systems PLoS Negl Trop Dis 6 5 2012 e1648	36029602	PMC9712585	NO-CC CODE	2022-12-02 23:21:50	no	Phys Life Rev. 2022 Dec 17; 43:20-22	utf-8	Phys Life Rev	2,022	10.1016/j.plrev.2022.07.008	oa_other
==== Front Genome Res Genome Res genome GENOME Genome Research 1088-9051 1549-5469 Cold Spring Harbor Laboratory Press 9509184 10.1101/gr.277270.122 Corrigenda Corrigendum: Cell cycle arrest explains the observed bulk 3D genomic alterations in response to long-term heat shock in K562 cells Xu Bingxiang Gao Xiaomeng Li Xiaoli Jia Yan Li Feifei Zhang Zhihua 10 2022 10 2022 32 10 1965119651 Published by Cold Spring Harbor Laboratory Press 2022 ==== Body pmc Genome Research 32: 1285–1297 (2022) The authors would like to correct an error in Figure 5A, in which the representative image of cells under the short-term heat shock (SHS) condition was inadvertently duplicated for normal heat shock (NHS). As the panels in Figure 5A only serve as representative images, this error has no impact on any of the analyses or conclusions reported in this article. Figure 5 has been updated in the revised article online. The authors apologize for any confusion this may have caused. doi: 10.1101/gr.277270.122	0	PMC9712624	NO-CC CODE	2022-12-15 23:21:51	no	Genome Res. 2022 Oct; 32(10):19651	utf-8	Genome Res	2,022	10.1101/gr.277270.122	oa_other
==== Front Genome Res Genome Res genome GENOME Genome Research 1088-9051 1549-5469 Cold Spring Harbor Laboratory Press 9509184 10.1101/gr.277322.122 Corrigenda Corrigendum: Diversifying the genomic data science research community The Genomic Data Science Community Network 10 2022 10 2022 32 10 1965219652 Published by Cold Spring Harbor Laboratory Press 2022 ==== Body pmc Genome Research 32: 1231–1241 (2022) The authors would like to provide alternative (alt) text descriptions of the main figures for the visually or print impaired. A pointer to the alt text has been added at the end of the Acknowledgments section in the revised article online and now reads as follows: “Alternative text descriptions of the main figures for the visually or print impaired are available as Additional Supplemental Material.” doi: 10.1101/gr.277322.122	0	PMC9712628	NO-CC CODE	2022-12-15 23:21:51	no	Genome Res. 2022 Oct; 32(10):19652	utf-8	Genome Res	2,022	10.1101/gr.277322.122	oa_other
==== Front J Oral Maxillofac Surg J Oral Maxillofac Surg Journal of Oral and Maxillofacial Surgery 0278-2391 1531-5053 American Association of Oral and Maxillofacial Surgeons S0278-2391(22)00822-9 10.1016/j.joms.2022.08.012 Perspectives Is Prolonged Mask Wearing Associated With Orofacial Pain? Yau Vicky DDS R∗ Liang Hong-Yu MBBS candidate † Sun Chenyu MD, MSc ‡∗ ∗ Resident, Columbia Irving Medical Center, New York City, NY, USA † Medical Student, The Second School of Clincial Medicine, Anhui Medical University, Hefei, China ‡ Attending Physician, AMITA Heath Saint Joseph Hospital Chicago, Chicago, IL, USA ∗ Address correspondence and reprint requests to Dr Sun: AMITA Heath Saint Joseph Hospital Chicago, 2900 N. Lake Shore Drive, Chicago, Illinois, USA 60657 R US/CA OMS Resident. 1 12 2022 12 2022 1 12 2022 80 12 18751877 30 7 2022 14 8 2022 © 2022 American Association of Oral and Maxillofacial Surgeons. 2022 American Association of Oral and Maxillofacial Surgeons Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. ==== Body pmcDuring the pandemic of coronavirus disease 2019 (COVID-19), the Centers for Disease Control and Prevention recommended surgical N95 respirators (also referred as a medical respirator) to be used by healthcare providers for protection from airborne and fluid transmission. With the prolonged use of face masks and surgical respirators, new issues of mask-associated orofacial pain have emerged for oral and maxillofacial surgeons and should be investigated for proper management and prevention. Healthcare workers have reported masks-associated orofacial pain, specifically in areas of temporalis (94.5%), zygomaticus (96.1%), nasalis (96.9%), and auricularis muscle (93.8%) with the use of N95 masks with a mean of 5.9 hours per day.1 The onset of the pain is as immediate as less than 60 minutes after donning, and it usually resolves spontaneously within 30 minutes after doffing of the masks. Such orofacial pain can be secondary to excessive compression caused by straps of masks, the pressure from which irritates superficial sensory nerves, especially trigeminal and occipital1 branches, leading to incidence of orofacial pain, including headache. Orofacial pain associated with mask wearing is often reported by healthcare workers related to masks with designs of ear loop and head strap. The tight seal N95 respirators with head strap compress and create tension of masticatory muscles, including masseter, temporalis, and the pterygoid muscles, while those with ear loop style impose significant pressure on the eminence of concha, leading to the risk of ear deformity and pain radiation to other orofacial muscles. Moreover, another study reported orofacial pain secondary to temporomandibular joint disease (TMD). The authors suggest there is a higher risk of TMD for populations with prolonged use of masks (>8 hours), especially for females.2 In the study, detailed history, including frequent jaw movement for mask adjustment, lifestyle or parafunctional habits, examination, and imaging information were obtained to confirm the presence of muscle or arthrogenous pain. Notably, disk displacement with reduction is reported to be significantly higher in those with frequent jaw movement related to masks.2 The findings can be attributed to the limitation of custom sizes available for mask users with different facial profiles and nasal bridge shapes, whereby repeated jaw motions are needed to adjust the mask position (Fig 1 ). These motions include frequent maximal mandible protrusion and rotation, imposing continuous strain to extracapsular ligaments and eventually associative pain. While TMD and its multifactorial pathoetiology are related to facial morphology, dental occlusion, and psychological and neuromuscular disease, the authors suggest adding mask-related repetitive jaw movements into consideration of the management of TMD as a psychosocial factor. Although a contradictory finding was reported that they found significant decrease in resting temporalis anterior and masseter muscle activity during mask wearing, the observation was only made throughout a period of 4 hours, and only healthy, young females were included in the study,3 making it insufficient to draw a conclusion on correlation between mask wearing and muscle activity.Figure 1 Temporomandibular joint activities during frequent mouth opening and protrusive jaw movement for frequent mask adjustment (created with BioRender.com). It is also important to note that other contributory factors on mask-associated orofacial pain and TMD could be parafunctional activities and physiological changes, including hypoxia and hypoxemia associated with prolonged wearing of masks. Indeed, dental students and dentists who wear N95 respirators during work reported an increase in TMD symptoms and parafunctional movements such as repetitive mouth opening and closing, bruxism, lateral excursions or protrusive movements compared to daily life when without mask.4 Although the association between mask wearing and daytime bruxism is pathophysiologically unclear, underlying reasons can be related to stress with prolonged hours of work with the use of masks, anxiety from discomfort and even claustrophobic reactions from the tight seal of N95 respirators. On the other hand, some dentists reportedly have a significant drop in oxygen saturation 1 hour after wearing N95 respirators, which later increased after another hour but was still lower than the baseline.5 In such cases, changes in breathing pattern and jaw movements, including mouth breathing and bruxism, can collectively contribute to the development of orofacial pain including TMD. Several questions still need to be addressed, as there is limited evidence evaluating the following considerations: (1) assessing facial muscle activity during and/or after prolonged mask wearing, (2) investigating the effects of mask-wearing on orofacial muscles and temporomandibular joints, and (3) exploring the difference between types of masks (N95/KN95/FFP2 respirators vs regular medical masks; masks with ear loop vs masks with head strap/overhead vs tie-on/tie-back surgical masks) regarding their effects on orofacial muscles and temporomandibular joints. Wearing masks has become our daily norm as a result of the COVID-19 pandemic. The N95 respirators used for added protection are distinctive from loose-fitting regular surgical masks due to their tight facial fit, enhanced filtration against 95% or more of 0.3-μm particles, and proven previous success in the prevention of respiratory viral infections including severe acute respiratory syndrome coronavirus 1 and 2 infection. While there are no data on the incidence of orofacial pain and TMD related to mask-wearing prior to the COVID-19 pandemic, with the current prolonged and frequent use of N95 respirators especially, orofacial pain associated with masks should be a topic of discussion considering its clinical relevance. Currently, TMD can be managed conservatively with nonsteroidal anti-inflammatory drugs, hot compressions, soft diet, night guard, and BOTOX injections. It would also be worth considering for manufacturers to design custom masks for people with different mandibular shapes and nasal bridge heights to reduce the need to frequently adjust mask with repeated jaw motions. Yet, there is still much that is unclear regarding mask-related orofacial pain and TMD. There needs to be more studies to address the pathophysiology of mask-related TMD and to develop preventive measures aimed at protecting the 59 million healthcare workers, including oral and maxillofacial surgeons who wear masks for long hours on a daily basis, as well as non-healthcare workers whose jobs require long hours of mask wearing. Conflict of Interest Disclosures: None of the authors have any relevant financial relationship(s) with a commercial interest. ==== Refs References 1 Ong J.J.Y. Bharatendu C. Goh Y. Headaches associated with personal protective equipment – a cross-sectional study among frontline healthcare workers during COVID-19 Headache: The J Head Face Pain 60 5 2020 864 877 10.1111/head.13811 2 Zuhour M. Ismayilzade M. Dadacı M. Ince B. The impact of wearing a face mask during the COVID-19 pandemic on temporomandibular joint: A radiological and questionnaire assessment Indian J Plast Surg 55 01 2022 058 065 10.1055/s-0042-1743131 3 Ginszt M. Zieliński G. Szkutnik J. The effects of wearing a medical mask on the masticatory and neck muscle activity in healthy young women J Clin Med 11 2 2022 303 10.3390/jcm11020303 35053998 4 Akturk E.S. Aydin I. Seker E.D. The Effects of Mask Usage during the COVID-19 Pandemic on Temporomandibular Joint Research Square Platform LLC 2022 10.21203/rs.3.rs-1752353/v1 5 Gaunkar R. Manerkar H. Nagarsekar A. Dhupar V. Khorate M. Assessment of hypoxia and physiological stress evinced by usage of n95 masks among frontline dental healthcare workers in a humid western coastal region of India-A repeated measure observational study Indian J Occup Environ Med 25 4 2021 209 10.4103/ijoem.ijoem_446_20 35197672	36464428	PMC9712832	NO-CC CODE	2022-12-02 23:22:55	no	J Oral Maxillofac Surg. 2022 Dec 1; 80(12):1875-1877	utf-8	J Oral Maxillofac Surg	2,022	10.1016/j.joms.2022.08.012	oa_other
==== Front J Oral Maxillofac Surg J Oral Maxillofac Surg Journal of Oral and Maxillofacial Surgery 0278-2391 1531-5053 Published by Elsevier Inc on behalf of the American Association of Oral and Maxillofacial Surgeons S0278-2391(22)00740-6 10.1016/j.joms.2022.08.006 Letter to the Editor RE: Oral Manifestations of Monkeypox: A Report of Two Cases Mungmunpuntipantip Rujittika PhD Bangkok, Thailand Wiwanitkit Viroj MD Ikeji-Arakeji, Nigeria 1 12 2022 12 2022 1 12 2022 80 12 18741874 © 2022 Published by Elsevier Inc on behalf of the American Association of Oral and Maxillofacial Surgeons. 2022 American Association of Oral and Maxillofacial Surgeons Elsevier has created a Monkeypox Information Center (https://www.elsevier.com/connect/monkeypox-information-center) in response to the declared public health emergency of international concern, with free information in English on the monkeypox virus. The Monkeypox Information Center is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its monkeypox related research that is available on the Monkeypox Information Center - including this research content - immediately available in publicly funded repositories, with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the Monkeypox Information Center remains active. ==== Body pmcDear Editor, We would like to share ideas on the publication “Oral manifestations of monkeypox: A report of two cases”.1 Two unusual instances of monkeypox that presented to the doctor with the first symptom of mouth lesions were documented by Peters et al.1 Currently, monkeypox is becoming a new public health concern. There is little doubt that the recent outbreak's nature means that there are not many data at this time. The distinct clinical appearance of monkeypox must be considered. No fever and an odd skin rash can be atypical clinical symptoms of the sickness.2 Oral presentations sometimes result in mouth ulcers, which can occasionally be accompanied by no fever.3 Recognizing the oral manifestation is crucial. Other unusual clinical symptoms include diarrhea and dysphagia.4 , 5 It is crucial to be aware of these other potential atypical clinical presentations because, in addition to the oral presentation, the maxillofacial surgeon may treat a patient who has another unique clinical monkeypox condition. In light of the current state of the illness outbreak, a global preventative measure must be implemented. Conflict of Interest Disclosures: None of the authors have any relevant financial relationship(s) with a commercial interest. ==== Refs References 1 Peters S.M. Hill N.B. Halepas S. Oral manifestations of monkeypox: A report of 2 cases J Oral Maxillofacial Surg 80 11 2022 1836 1840 2 Wiwanitkit S. Wiwanitkit V. Atypical zoonotic pox: Acute merging illness that can be easily forgotten J Acute Dis 7 2018 88 89 3 Sookaromdee P. Wiwanitkit V. Mouth sores and monkeypox: A consideration J Stomatol Oral Maxillofac Surg 2022 4 Mungmunpuntipantip R. Wiwanitkit V. Dysphagia and Monkeypox: A Consideration Dysphagia 2022 10.1007/s00455-022-10481-x Online ahead of print 5 Mungmunpuntipantip R. Wiwanitkit V. Diarrhea and monkeypox: A consideration Rev Esp Enferm Dig 2022 10.17235/reed.2022.8957/2022 Online ahead of print	36464426	PMC9712877	NO-CC CODE	2022-12-03 23:20:03	no	J Oral Maxillofac Surg. 2022 Dec 1; 80(12):1874	utf-8	J Oral Maxillofac Surg	2,022	10.1016/j.joms.2022.08.006	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01437-X 10.1016/j.apmr.2022.08.847 Research Poster 2194730 Exploring Loneliness in The Older Adults And The Disability Populations 12-Months Post COVID-19 Restrictions Benam Niloufar University of British Columbia Miller William Mortenson W. Ben Tao Gordon Schmidt Julia 1 12 2022 12 2022 1 12 2022 103 12 e154e154 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To identify factors associated with loneliness among the older adults and the disability populations 12-months following COVID-19 physical restrictions. As well as to identify strategies to alleviate the loneliness experienced by these populations. Design The data in this study were taken from a larger study with a longitudinal concurrent mixed-methods design with four data collection points. Data from time point four (12-months post COVID-19 restrictions) were used for our study purposes. Setting This study was completed within the general community of adults living in Canada, British Columbia. The study and data collection were completed through online survey tools. Participants A Survey of a total of 70 British Columbian adults who self-identified as being comfortable writing and speaking in English and belonging within the older adults (>64 years) or the disability populations (self-described and including stroke, spinal cord injury and other disabilities) were included. Interventions N/A. Main Outcome Measures The main outcome measures included scores on the Hospital Anxiety and Depression Scale (HADS), Multidimensional Scale of Perceived Social Support (MSPSS), as well as the UCLA Three-Item Loneliness Scale. These scales measure for anxiety and depression, social support, and loneliness respectively. Data collected through these measures were analyzed using multiple linear regression to investigate the association between the independent variables, anxiety, depression, social support, and the dependent variable, loneliness. Results Our analysis showed a statistically significant positive association between anxiety and loneliness (β = 0.363, p < 0.05), and a statistically significant negative association between social support and loneliness (β = -0.360, p < 0.05). There was no statistically significant association between depression and loneliness (β = 0.142, p > 0.05). Conclusions Anxiety and social support were significant predictors for loneliness in the older adults and disability populations during the COVID-19 pandemic. Facilitating engagement in occupation to reduce anxiety and improve social support may be helpful in reducing loneliness in these populations. Author(s) Disclosures No conflicts of interest. Key Words Loneliness COVID-19 Elderly Disability ==== Body pmc	0	PMC9712906	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e154	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.847	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01437-X 10.1016/j.apmr.2022.08.847 Research Poster 2194730 Exploring Loneliness in The Older Adults And The Disability Populations 12-Months Post COVID-19 Restrictions Benam Niloufar University of British Columbia Miller William Mortenson W. Ben Tao Gordon Schmidt Julia 1 12 2022 12 2022 1 12 2022 103 12 e154e154 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To identify factors associated with loneliness among the older adults and the disability populations 12-months following COVID-19 physical restrictions. As well as to identify strategies to alleviate the loneliness experienced by these populations. Design The data in this study were taken from a larger study with a longitudinal concurrent mixed-methods design with four data collection points. Data from time point four (12-months post COVID-19 restrictions) were used for our study purposes. Setting This study was completed within the general community of adults living in Canada, British Columbia. The study and data collection were completed through online survey tools. Participants A Survey of a total of 70 British Columbian adults who self-identified as being comfortable writing and speaking in English and belonging within the older adults (>64 years) or the disability populations (self-described and including stroke, spinal cord injury and other disabilities) were included. Interventions N/A. Main Outcome Measures The main outcome measures included scores on the Hospital Anxiety and Depression Scale (HADS), Multidimensional Scale of Perceived Social Support (MSPSS), as well as the UCLA Three-Item Loneliness Scale. These scales measure for anxiety and depression, social support, and loneliness respectively. Data collected through these measures were analyzed using multiple linear regression to investigate the association between the independent variables, anxiety, depression, social support, and the dependent variable, loneliness. Results Our analysis showed a statistically significant positive association between anxiety and loneliness (β = 0.363, p < 0.05), and a statistically significant negative association between social support and loneliness (β = -0.360, p < 0.05). There was no statistically significant association between depression and loneliness (β = 0.142, p > 0.05). Conclusions Anxiety and social support were significant predictors for loneliness in the older adults and disability populations during the COVID-19 pandemic. Facilitating engagement in occupation to reduce anxiety and improve social support may be helpful in reducing loneliness in these populations. Author(s) Disclosures No conflicts of interest. Key Words Loneliness COVID-19 Elderly Disability ==== Body pmc	0	PMC9712907	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e54-e55	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.566	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01437-X 10.1016/j.apmr.2022.08.847 Research Poster 2194730 Exploring Loneliness in The Older Adults And The Disability Populations 12-Months Post COVID-19 Restrictions Benam Niloufar University of British Columbia Miller William Mortenson W. Ben Tao Gordon Schmidt Julia 1 12 2022 12 2022 1 12 2022 103 12 e154e154 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To identify factors associated with loneliness among the older adults and the disability populations 12-months following COVID-19 physical restrictions. As well as to identify strategies to alleviate the loneliness experienced by these populations. Design The data in this study were taken from a larger study with a longitudinal concurrent mixed-methods design with four data collection points. Data from time point four (12-months post COVID-19 restrictions) were used for our study purposes. Setting This study was completed within the general community of adults living in Canada, British Columbia. The study and data collection were completed through online survey tools. Participants A Survey of a total of 70 British Columbian adults who self-identified as being comfortable writing and speaking in English and belonging within the older adults (>64 years) or the disability populations (self-described and including stroke, spinal cord injury and other disabilities) were included. Interventions N/A. Main Outcome Measures The main outcome measures included scores on the Hospital Anxiety and Depression Scale (HADS), Multidimensional Scale of Perceived Social Support (MSPSS), as well as the UCLA Three-Item Loneliness Scale. These scales measure for anxiety and depression, social support, and loneliness respectively. Data collected through these measures were analyzed using multiple linear regression to investigate the association between the independent variables, anxiety, depression, social support, and the dependent variable, loneliness. Results Our analysis showed a statistically significant positive association between anxiety and loneliness (β = 0.363, p < 0.05), and a statistically significant negative association between social support and loneliness (β = -0.360, p < 0.05). There was no statistically significant association between depression and loneliness (β = 0.142, p > 0.05). Conclusions Anxiety and social support were significant predictors for loneliness in the older adults and disability populations during the COVID-19 pandemic. Facilitating engagement in occupation to reduce anxiety and improve social support may be helpful in reducing loneliness in these populations. Author(s) Disclosures No conflicts of interest. Key Words Loneliness COVID-19 Elderly Disability ==== Body pmc	0	PMC9712908	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e198	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.021	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01223-0 10.1016/j.apmr.2022.08.633 Research Poster 2184118 Examining Coping and Resilience in People with Acquired Brain Injury During the Pandemic Brisson Amy Ransohoff New York University Steinhardt School of Culture, Education, and Human Development Voelbel Gerald Kim Grace Kim Hayejin Chen Michelle Voelbel Sydney Genova Helen 1 12 2022 12 2022 1 12 2022 103 12 e78e78 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To understand the relationship between use of coping skills and resilience in adults with an acquired brain injury (ABI) during the beginning of the Coronavirus Disease of 2019 (COVID-19) pandemic. Design Survey data was collected via online platform, Research Electronic Data Capture (REDCap). Setting The general community. Participants Participants as part of a convenience sample included self-identified adults with a traumatic brain injury (TBI) or stroke, 18 years or older (n = 99). Exclusion criteria were other neurological diagnoses or serious mental illnesses. Interventions Not applicable. Main Outcome Measures The outcome measures were the following: COVID-19 Experiences subsection of the COVID-19: Impact of the Pandemic and Health Related Quality of Life in Cancer Patients and Survivors Scale (Cov-PHRQoL) and the Brief Resilient Coping Scale. Results There was a significant group difference for race, (p = .04), with the Non-Coping Group having more people of minority status. The Coping Group reported a greater appreciation of family and friends (p= .001), greater appreciation for life (p= .001), more grateful for each day (p= .001), accepting of what they can't change (p= .001), and finding new ways to connect family and friends (p= .001). Compared to the Non-Coping Group, the Coping Group reported significantly greater resiliency and growth from a difficult situation (p = 0.006). Conclusions The COVID-19 pandemic provided a unique opportunity to examine the relationship between coping skills and resilience in people with ABI when faced with a novel stressor. People who utilized coping skills demonstrated positive outcomes in the form of perceived benefits and greater appreciation for who they had in their life. Furthermore, the Coping group was able to reframe difficult situations to find psychological growth. More research must be done to determine the relationship between coping skills and resilience in people with ABI, as well as what aspects of coping skills and resilience are most crucial to producing positive outcomes for people with ABI when faced with novel stressors. Author(s) Disclosures None. Key Words Resilience COVID-19 Brain Injury Stroke ==== Body pmc	0	PMC9712909	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e78	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.633	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01223-0 10.1016/j.apmr.2022.08.633 Research Poster 2184118 Examining Coping and Resilience in People with Acquired Brain Injury During the Pandemic Brisson Amy Ransohoff New York University Steinhardt School of Culture, Education, and Human Development Voelbel Gerald Kim Grace Kim Hayejin Chen Michelle Voelbel Sydney Genova Helen 1 12 2022 12 2022 1 12 2022 103 12 e78e78 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To understand the relationship between use of coping skills and resilience in adults with an acquired brain injury (ABI) during the beginning of the Coronavirus Disease of 2019 (COVID-19) pandemic. Design Survey data was collected via online platform, Research Electronic Data Capture (REDCap). Setting The general community. Participants Participants as part of a convenience sample included self-identified adults with a traumatic brain injury (TBI) or stroke, 18 years or older (n = 99). Exclusion criteria were other neurological diagnoses or serious mental illnesses. Interventions Not applicable. Main Outcome Measures The outcome measures were the following: COVID-19 Experiences subsection of the COVID-19: Impact of the Pandemic and Health Related Quality of Life in Cancer Patients and Survivors Scale (Cov-PHRQoL) and the Brief Resilient Coping Scale. Results There was a significant group difference for race, (p = .04), with the Non-Coping Group having more people of minority status. The Coping Group reported a greater appreciation of family and friends (p= .001), greater appreciation for life (p= .001), more grateful for each day (p= .001), accepting of what they can't change (p= .001), and finding new ways to connect family and friends (p= .001). Compared to the Non-Coping Group, the Coping Group reported significantly greater resiliency and growth from a difficult situation (p = 0.006). Conclusions The COVID-19 pandemic provided a unique opportunity to examine the relationship between coping skills and resilience in people with ABI when faced with a novel stressor. People who utilized coping skills demonstrated positive outcomes in the form of perceived benefits and greater appreciation for who they had in their life. Furthermore, the Coping group was able to reframe difficult situations to find psychological growth. More research must be done to determine the relationship between coping skills and resilience in people with ABI, as well as what aspects of coping skills and resilience are most crucial to producing positive outcomes for people with ABI when faced with novel stressors. Author(s) Disclosures None. Key Words Resilience COVID-19 Brain Injury Stroke ==== Body pmc	0	PMC9712910	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e142	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.813	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01390-9 10.1016/j.apmr.2022.08.800 Research Poster 2184217 Brain MRI and Neuropsychological Findings at Long-Term Follow-Up After COVID-19 Hospitalisation: An Observational Cohort Study Hellgren Lovisa Department of Rehabilitation Medicine, Region Jönköping County, Jönköping, Sweden and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden Thornberg Ulrika Birberg Samuelsson Kersti Levi Richard Anestis 1 12 2022 12 2022 1 12 2022 103 12 e138e138 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To report findings on brain MRI and neurocognitive function, as well as persisting fatigue at long-term follow-up after COVID-19 hospitalisation in patients identified as high risk for affection of the central nervous system. Design Ambidirectional observational cohort study. Setting All patients (n=734) previously hospitalized with a laboratory-confirmed COVID-19 in a total regional population in Sweden during the period March 1st to May 31st 2020. Participants A subgroup (n=185) with persisting symptoms still interfering with daily life at a telephone follow-up 4 months after discharge were invited for a medical and neuropsychological evaluation. Thirty-five of those who were assessed with a neurocognitive test battery at the clinical visit, and presented a clinical picture concerning for COVID-19-related brain pathology, were further investigated by brain MRI. Interventions N/A. Main Outcome Measures Findings on brain MRI, neurocognitive test results and reported fatigue. Results Twenty-five patients (71%) had abnormalities on MRI; multiple white matter lesions were the most common finding. Six patients had had MRI performed in the acute phase during their hospitalisation, and all of these patients had additional white matter lesions at the follow-up MRI. Sixteen patients (46%) demonstrated impaired neurocognitive function, of which 10 (29%) had severe impairment. Twenty-six patients (74%) reported clinically significant fatigue. Patients with abnormalities on MRI had a lower Visuospatial Index (p=0.031) compared with the group with normal MRI findings. Conclusions A majority in this group of patients selected to undergo MRI after a clinical evaluation, showed signs of possible COVID-19 related brain affection detectable by brain MRI and/or neurocognitive test results. Even in a previously fairly healthy group of patients, COVID-19 might have a substantial negative impact on cognition in several domains, persisting several months post discharge. Abnormal findings were not restricted to patients with severe disease. Thus, for clinicians it is important to consider post-covid related changes when facing patients’ reports of neuropsychological deficiency, regardless of severity of disease. Author(s) Disclosures The authors declare no competing interests. Key Words COVID-19 Magnetic Resonance Imaging Cognition Fatigue Rehabilitation Medicine ==== Body pmc	0	PMC9712911	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e138	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.800	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01390-9 10.1016/j.apmr.2022.08.800 Research Poster 2184217 Brain MRI and Neuropsychological Findings at Long-Term Follow-Up After COVID-19 Hospitalisation: An Observational Cohort Study Hellgren Lovisa Department of Rehabilitation Medicine, Region Jönköping County, Jönköping, Sweden and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden Thornberg Ulrika Birberg Samuelsson Kersti Levi Richard Anestis 1 12 2022 12 2022 1 12 2022 103 12 e138e138 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To report findings on brain MRI and neurocognitive function, as well as persisting fatigue at long-term follow-up after COVID-19 hospitalisation in patients identified as high risk for affection of the central nervous system. Design Ambidirectional observational cohort study. Setting All patients (n=734) previously hospitalized with a laboratory-confirmed COVID-19 in a total regional population in Sweden during the period March 1st to May 31st 2020. Participants A subgroup (n=185) with persisting symptoms still interfering with daily life at a telephone follow-up 4 months after discharge were invited for a medical and neuropsychological evaluation. Thirty-five of those who were assessed with a neurocognitive test battery at the clinical visit, and presented a clinical picture concerning for COVID-19-related brain pathology, were further investigated by brain MRI. Interventions N/A. Main Outcome Measures Findings on brain MRI, neurocognitive test results and reported fatigue. Results Twenty-five patients (71%) had abnormalities on MRI; multiple white matter lesions were the most common finding. Six patients had had MRI performed in the acute phase during their hospitalisation, and all of these patients had additional white matter lesions at the follow-up MRI. Sixteen patients (46%) demonstrated impaired neurocognitive function, of which 10 (29%) had severe impairment. Twenty-six patients (74%) reported clinically significant fatigue. Patients with abnormalities on MRI had a lower Visuospatial Index (p=0.031) compared with the group with normal MRI findings. Conclusions A majority in this group of patients selected to undergo MRI after a clinical evaluation, showed signs of possible COVID-19 related brain affection detectable by brain MRI and/or neurocognitive test results. Even in a previously fairly healthy group of patients, COVID-19 might have a substantial negative impact on cognition in several domains, persisting several months post discharge. Abnormal findings were not restricted to patients with severe disease. Thus, for clinicians it is important to consider post-covid related changes when facing patients’ reports of neuropsychological deficiency, regardless of severity of disease. Author(s) Disclosures The authors declare no competing interests. Key Words COVID-19 Magnetic Resonance Imaging Cognition Fatigue Rehabilitation Medicine ==== Body pmc	0	PMC9712912	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e170	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.894	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01390-9 10.1016/j.apmr.2022.08.800 Research Poster 2184217 Brain MRI and Neuropsychological Findings at Long-Term Follow-Up After COVID-19 Hospitalisation: An Observational Cohort Study Hellgren Lovisa Department of Rehabilitation Medicine, Region Jönköping County, Jönköping, Sweden and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden Thornberg Ulrika Birberg Samuelsson Kersti Levi Richard Anestis 1 12 2022 12 2022 1 12 2022 103 12 e138e138 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To report findings on brain MRI and neurocognitive function, as well as persisting fatigue at long-term follow-up after COVID-19 hospitalisation in patients identified as high risk for affection of the central nervous system. Design Ambidirectional observational cohort study. Setting All patients (n=734) previously hospitalized with a laboratory-confirmed COVID-19 in a total regional population in Sweden during the period March 1st to May 31st 2020. Participants A subgroup (n=185) with persisting symptoms still interfering with daily life at a telephone follow-up 4 months after discharge were invited for a medical and neuropsychological evaluation. Thirty-five of those who were assessed with a neurocognitive test battery at the clinical visit, and presented a clinical picture concerning for COVID-19-related brain pathology, were further investigated by brain MRI. Interventions N/A. Main Outcome Measures Findings on brain MRI, neurocognitive test results and reported fatigue. Results Twenty-five patients (71%) had abnormalities on MRI; multiple white matter lesions were the most common finding. Six patients had had MRI performed in the acute phase during their hospitalisation, and all of these patients had additional white matter lesions at the follow-up MRI. Sixteen patients (46%) demonstrated impaired neurocognitive function, of which 10 (29%) had severe impairment. Twenty-six patients (74%) reported clinically significant fatigue. Patients with abnormalities on MRI had a lower Visuospatial Index (p=0.031) compared with the group with normal MRI findings. Conclusions A majority in this group of patients selected to undergo MRI after a clinical evaluation, showed signs of possible COVID-19 related brain affection detectable by brain MRI and/or neurocognitive test results. Even in a previously fairly healthy group of patients, COVID-19 might have a substantial negative impact on cognition in several domains, persisting several months post discharge. Abnormal findings were not restricted to patients with severe disease. Thus, for clinicians it is important to consider post-covid related changes when facing patients’ reports of neuropsychological deficiency, regardless of severity of disease. Author(s) Disclosures The authors declare no competing interests. Key Words COVID-19 Magnetic Resonance Imaging Cognition Fatigue Rehabilitation Medicine ==== Body pmc	0	PMC9712913	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e159	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.861	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01390-9 10.1016/j.apmr.2022.08.800 Research Poster 2184217 Brain MRI and Neuropsychological Findings at Long-Term Follow-Up After COVID-19 Hospitalisation: An Observational Cohort Study Hellgren Lovisa Department of Rehabilitation Medicine, Region Jönköping County, Jönköping, Sweden and Department of Medical and Health Sciences, Linköping University, Linköping, Sweden Thornberg Ulrika Birberg Samuelsson Kersti Levi Richard Anestis 1 12 2022 12 2022 1 12 2022 103 12 e138e138 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To report findings on brain MRI and neurocognitive function, as well as persisting fatigue at long-term follow-up after COVID-19 hospitalisation in patients identified as high risk for affection of the central nervous system. Design Ambidirectional observational cohort study. Setting All patients (n=734) previously hospitalized with a laboratory-confirmed COVID-19 in a total regional population in Sweden during the period March 1st to May 31st 2020. Participants A subgroup (n=185) with persisting symptoms still interfering with daily life at a telephone follow-up 4 months after discharge were invited for a medical and neuropsychological evaluation. Thirty-five of those who were assessed with a neurocognitive test battery at the clinical visit, and presented a clinical picture concerning for COVID-19-related brain pathology, were further investigated by brain MRI. Interventions N/A. Main Outcome Measures Findings on brain MRI, neurocognitive test results and reported fatigue. Results Twenty-five patients (71%) had abnormalities on MRI; multiple white matter lesions were the most common finding. Six patients had had MRI performed in the acute phase during their hospitalisation, and all of these patients had additional white matter lesions at the follow-up MRI. Sixteen patients (46%) demonstrated impaired neurocognitive function, of which 10 (29%) had severe impairment. Twenty-six patients (74%) reported clinically significant fatigue. Patients with abnormalities on MRI had a lower Visuospatial Index (p=0.031) compared with the group with normal MRI findings. Conclusions A majority in this group of patients selected to undergo MRI after a clinical evaluation, showed signs of possible COVID-19 related brain affection detectable by brain MRI and/or neurocognitive test results. Even in a previously fairly healthy group of patients, COVID-19 might have a substantial negative impact on cognition in several domains, persisting several months post discharge. Abnormal findings were not restricted to patients with severe disease. Thus, for clinicians it is important to consider post-covid related changes when facing patients’ reports of neuropsychological deficiency, regardless of severity of disease. Author(s) Disclosures The authors declare no competing interests. Key Words COVID-19 Magnetic Resonance Imaging Cognition Fatigue Rehabilitation Medicine ==== Body pmc	0	PMC9712914	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e206	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.045	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01143-1 10.1016/j.apmr.2022.08.553 Research Paper 2191368 Characterizing Symptoms and Impact of “Long Covid”: A Qualitative Perspective Lerer Lilly University of Chicago Pritzker School of Medicine Cherney Leora Roth Elliot 1 12 2022 12 2022 1 12 2022 103 12 e49e49 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To characterize the nature and impact of physical, cognitive, and mood symptoms associated with Post-Acute Covid-19 Syndrome (PACS). Design Qualitative study utilizing semi-structured interviews adapted from the McGill Illness Narratives Interview (MINI) and the Work Rehabilitation Questionnaire (WORQ). Interviews were coded and analyzed using grounded theory methodology. Setting Outpatient general rehabilitation clinic in Chicago, November 2021-March 2022. Participants Convenience sample of eight adult English-speaking individuals who met WHO Criteria for PACS. Interventions Not applicable. Main Outcome Measures Not applicable. Results Individuals reported a wide range of physical, cognitive and communication, and mood changes associated with PACS. Cognitive changes included slow processing, and difficulties with attention, working memory, short term memory, executive function, abstract reasoning, and cognitive endurance. comprehension, word finding, executive function, working memory, attention, abstract reasoning, cognitive endurance, and short-term memory. Physical changes included profound physical fatigue, insomnia, and daytime tiredness. Mood changes included anxiety, depression, and relapse of previously diagnosed psychiatric conditions. Timeline, duration, severity, and distribution of symptoms varied widely between individuals. Several themes emerged regarding impact of symptoms: (1) difficulty performing instrumental activities of daily living; (2) struggles with verbal communication leading to social isolation, difficulty with collaborative work, and feelings of shame and humiliation; (3) profound impact on self-image and perceptions of the future; (4) profound impact on ability to work, job performance, retirement plans, and relationship with employer; and (5) the utilization of both novel and previously developed coping mechanisms. Finally, individuals reported diverse perspectives on the utility of rehabilitation therapies. These included the perception of rehabilitation therapies as empowering, useful, beneficial, transformative, confidence-inspiring, depressing, confusing, disempowering, useless, or needless. Conclusions In light of the diversity of physical, cognitive, and emotional symptoms associated with PACS, a highly individual approach to rehabilitation planning is needed for individuals with PACS. Further research is needed to understand patient attitudes and perspectives about rehabilitation therapy, which should be utilized to develop patient education efforts in the future. Author(s) Disclosures Supported by KIWANIS Neurologic Research fund. Authors have no disclosures. Key Words Covid-19 Fatigue Cognitive Dysfunction ==== Body pmc	0	PMC9712916	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e49	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.553	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01143-1 10.1016/j.apmr.2022.08.553 Research Paper 2191368 Characterizing Symptoms and Impact of “Long Covid”: A Qualitative Perspective Lerer Lilly University of Chicago Pritzker School of Medicine Cherney Leora Roth Elliot 1 12 2022 12 2022 1 12 2022 103 12 e49e49 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To characterize the nature and impact of physical, cognitive, and mood symptoms associated with Post-Acute Covid-19 Syndrome (PACS). Design Qualitative study utilizing semi-structured interviews adapted from the McGill Illness Narratives Interview (MINI) and the Work Rehabilitation Questionnaire (WORQ). Interviews were coded and analyzed using grounded theory methodology. Setting Outpatient general rehabilitation clinic in Chicago, November 2021-March 2022. Participants Convenience sample of eight adult English-speaking individuals who met WHO Criteria for PACS. Interventions Not applicable. Main Outcome Measures Not applicable. Results Individuals reported a wide range of physical, cognitive and communication, and mood changes associated with PACS. Cognitive changes included slow processing, and difficulties with attention, working memory, short term memory, executive function, abstract reasoning, and cognitive endurance. comprehension, word finding, executive function, working memory, attention, abstract reasoning, cognitive endurance, and short-term memory. Physical changes included profound physical fatigue, insomnia, and daytime tiredness. Mood changes included anxiety, depression, and relapse of previously diagnosed psychiatric conditions. Timeline, duration, severity, and distribution of symptoms varied widely between individuals. Several themes emerged regarding impact of symptoms: (1) difficulty performing instrumental activities of daily living; (2) struggles with verbal communication leading to social isolation, difficulty with collaborative work, and feelings of shame and humiliation; (3) profound impact on self-image and perceptions of the future; (4) profound impact on ability to work, job performance, retirement plans, and relationship with employer; and (5) the utilization of both novel and previously developed coping mechanisms. Finally, individuals reported diverse perspectives on the utility of rehabilitation therapies. These included the perception of rehabilitation therapies as empowering, useful, beneficial, transformative, confidence-inspiring, depressing, confusing, disempowering, useless, or needless. Conclusions In light of the diversity of physical, cognitive, and emotional symptoms associated with PACS, a highly individual approach to rehabilitation planning is needed for individuals with PACS. Further research is needed to understand patient attitudes and perspectives about rehabilitation therapy, which should be utilized to develop patient education efforts in the future. Author(s) Disclosures Supported by KIWANIS Neurologic Research fund. Authors have no disclosures. Key Words Covid-19 Fatigue Cognitive Dysfunction ==== Body pmc	0	PMC9712917	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e106-e107	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.713	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01143-1 10.1016/j.apmr.2022.08.553 Research Paper 2191368 Characterizing Symptoms and Impact of “Long Covid”: A Qualitative Perspective Lerer Lilly University of Chicago Pritzker School of Medicine Cherney Leora Roth Elliot 1 12 2022 12 2022 1 12 2022 103 12 e49e49 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To characterize the nature and impact of physical, cognitive, and mood symptoms associated with Post-Acute Covid-19 Syndrome (PACS). Design Qualitative study utilizing semi-structured interviews adapted from the McGill Illness Narratives Interview (MINI) and the Work Rehabilitation Questionnaire (WORQ). Interviews were coded and analyzed using grounded theory methodology. Setting Outpatient general rehabilitation clinic in Chicago, November 2021-March 2022. Participants Convenience sample of eight adult English-speaking individuals who met WHO Criteria for PACS. Interventions Not applicable. Main Outcome Measures Not applicable. Results Individuals reported a wide range of physical, cognitive and communication, and mood changes associated with PACS. Cognitive changes included slow processing, and difficulties with attention, working memory, short term memory, executive function, abstract reasoning, and cognitive endurance. comprehension, word finding, executive function, working memory, attention, abstract reasoning, cognitive endurance, and short-term memory. Physical changes included profound physical fatigue, insomnia, and daytime tiredness. Mood changes included anxiety, depression, and relapse of previously diagnosed psychiatric conditions. Timeline, duration, severity, and distribution of symptoms varied widely between individuals. Several themes emerged regarding impact of symptoms: (1) difficulty performing instrumental activities of daily living; (2) struggles with verbal communication leading to social isolation, difficulty with collaborative work, and feelings of shame and humiliation; (3) profound impact on self-image and perceptions of the future; (4) profound impact on ability to work, job performance, retirement plans, and relationship with employer; and (5) the utilization of both novel and previously developed coping mechanisms. Finally, individuals reported diverse perspectives on the utility of rehabilitation therapies. These included the perception of rehabilitation therapies as empowering, useful, beneficial, transformative, confidence-inspiring, depressing, confusing, disempowering, useless, or needless. Conclusions In light of the diversity of physical, cognitive, and emotional symptoms associated with PACS, a highly individual approach to rehabilitation planning is needed for individuals with PACS. Further research is needed to understand patient attitudes and perspectives about rehabilitation therapy, which should be utilized to develop patient education efforts in the future. Author(s) Disclosures Supported by KIWANIS Neurologic Research fund. Authors have no disclosures. Key Words Covid-19 Fatigue Cognitive Dysfunction ==== Body pmc	0	PMC9712918	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e82-e83	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.645	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01302-8 10.1016/j.apmr.2022.08.712 Research Poster 2184267 Social Cognitive Predictors of Health Promotion Self-Efficacy among Older Adults during the COVID-19 Pandemic Singh Gurkaran University Of British Columbia Yang Michelle Clayton Cam Harris Devin Pelletier Chelsea Schmidt Julia Zwicker Jill Sakakibara Brodie 1 12 2022 12 2022 1 12 2022 103 12 e106e106 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To examine the relative importance of social cognitive predictors on health promotion self-efficacy among community-living adults, aged >65 years during COVID-19. Design This cross-sectional study was a secondary analysis of baseline data from a larger chronic disease self-management support intervention. Setting This study was conducted in the general community setting. Participants The mean age of participants (n=75) was 72.4 years old (SD=5.8; 44 female). Seventy participants (93%) reported living with long term disability resulting from arthritis (n=33), degenerative disk disease (n=18), stroke (n=6), and other conditions (n=18). Interventions Not applicable. Main Outcome Measures Health promotion self-efficacy was measured using the Self-Rated Abilities for Health Practices Scale [3]. Independent predictors of performance including performance accomplishment, vicarious learning, verbal persuasion, and affective states [4] were assessed using the health directed behavior subscale of the Health Education Impact Questionnaire [5], positive social interaction subscale of the Medical Outcomes Survey - Social Support Scale (MOS-SSS) [6], informational support subscale of MOS-SSS, and the Depression Anxiety Stress Scale [7], respectively. Results After controlling for age and sex, linear regression analyses revealed statistically significant associations between health promotion self-efficacy and: (i) performance accomplishment (health-directed behavior; β=2.98, p=0.04); (ii) verbal persuasion (informational support; β=5.30, p=0.01); and (iii) affective state (depressive symptoms; β=-0.84; p< 0.001). Vicarious learning (positive social interaction; β=-1.23; p=0.55) did not significantly predict health promotion self-efficacy. Overall, this model was statistically significant (p< 0.001) and explained 48% of the health promotion self-efficacy variance. Conclusions Verbal communication supports and strategies to address depressive symptoms and facilitate health-directed behaviors may improve health promotion self-efficacy in the context of physical and social distancing (i.e., COVID prevention strategies). A lack of relationship between self-efficacy and vicarious learning highlights the overall impact COVID had on in-person engagement. Author(s) Disclosures We report no real or perceived conflicts of interest. Key Words Self Efficacy Older Adults COVID-19 Pandemic ==== Body pmc	0	PMC9712919	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e106	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.712	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01302-8 10.1016/j.apmr.2022.08.712 Research Poster 2184267 Social Cognitive Predictors of Health Promotion Self-Efficacy among Older Adults during the COVID-19 Pandemic Singh Gurkaran University Of British Columbia Yang Michelle Clayton Cam Harris Devin Pelletier Chelsea Schmidt Julia Zwicker Jill Sakakibara Brodie 1 12 2022 12 2022 1 12 2022 103 12 e106e106 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To examine the relative importance of social cognitive predictors on health promotion self-efficacy among community-living adults, aged >65 years during COVID-19. Design This cross-sectional study was a secondary analysis of baseline data from a larger chronic disease self-management support intervention. Setting This study was conducted in the general community setting. Participants The mean age of participants (n=75) was 72.4 years old (SD=5.8; 44 female). Seventy participants (93%) reported living with long term disability resulting from arthritis (n=33), degenerative disk disease (n=18), stroke (n=6), and other conditions (n=18). Interventions Not applicable. Main Outcome Measures Health promotion self-efficacy was measured using the Self-Rated Abilities for Health Practices Scale [3]. Independent predictors of performance including performance accomplishment, vicarious learning, verbal persuasion, and affective states [4] were assessed using the health directed behavior subscale of the Health Education Impact Questionnaire [5], positive social interaction subscale of the Medical Outcomes Survey - Social Support Scale (MOS-SSS) [6], informational support subscale of MOS-SSS, and the Depression Anxiety Stress Scale [7], respectively. Results After controlling for age and sex, linear regression analyses revealed statistically significant associations between health promotion self-efficacy and: (i) performance accomplishment (health-directed behavior; β=2.98, p=0.04); (ii) verbal persuasion (informational support; β=5.30, p=0.01); and (iii) affective state (depressive symptoms; β=-0.84; p< 0.001). Vicarious learning (positive social interaction; β=-1.23; p=0.55) did not significantly predict health promotion self-efficacy. Overall, this model was statistically significant (p< 0.001) and explained 48% of the health promotion self-efficacy variance. Conclusions Verbal communication supports and strategies to address depressive symptoms and facilitate health-directed behaviors may improve health promotion self-efficacy in the context of physical and social distancing (i.e., COVID prevention strategies). A lack of relationship between self-efficacy and vicarious learning highlights the overall impact COVID had on in-person engagement. Author(s) Disclosures We report no real or perceived conflicts of interest. Key Words Self Efficacy Older Adults COVID-19 Pandemic ==== Body pmc	0	PMC9712920	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e46-e47	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.544	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)00651-7 10.1016/j.apmr.2022.08.061 Systematic & Meta-analytic Review Poster 2184185 Overview of Literature Related to Post COVID-19 Rehabilitation Schertler Sierra Misericordia University Klimas Mikayla Davis Zachary McGee Michael Brogan Laurie 1 12 2022 12 2022 1 12 2022 103 12 e211e211 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objective(s) To examine current literature on the role of physical therapy (PT) in management of post COVID-19 related conditions and common symptomatology in adults, as well as potential guidelines for rehabilitation in the outpatient setting. Data Sources PubMed, CINAHL, Medline, Cochrane. Study Selection Searches were conducted to examine current data related to PT interventions and their effectiveness for treating post COVID-19 conditions. Articles were evaluated for relevance based on the following criteria: articles in English, original peer reviewed articles, adult population (over 18), relevant PT interventions for rehabilitation, post-acute infection of COVID-19. Consensus agreement confirmed approximately 25% of reviewed articles. Data Extraction Articles were analyzed for relevance to implications regarding post COVID-19 and potential PT rehabilitation interventions. Interventions were assessed in feasibility and applicability to an outpatient clinic setting. Independent data extraction followed by consensus discussion was applied. Articles were examined for content regarding the latest updates on disease criteria, manifestations, new classifications, and cohorts emerging as the pandemic progresses as well as management strategies applicable to PT practice. Data Synthesis After article analysis, the findings include a key theme that PT services helped improve overall functional mobility and symptom management in patients after an acute infection of COVID-19. An essential consideration is keeping the interventions specific to the patient and their goals while preventing exacerbations of symptoms that could lead to further setbacks. Conclusions PT has a growing role in the management of post COVID-19 deficits as well as implications related to long COVID sequelae. By choosing the appropriate parameters and having awareness of the varying symptomology amongst patients, physical therapists can improve patients’ functional mobility and post COVID-19 disease management. The focus of future studies should include more specific interventions related to managing conditions and finding the most effective treatment strategies. Author(s) Disclosures No conflicts to disclose. Key Words Post Covid Post-Acute Covid Physical Therapy Physiotherapy ==== Body pmc	0	PMC9712921	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e211	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.061	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)00651-7 10.1016/j.apmr.2022.08.061 Systematic & Meta-analytic Review Poster 2184185 Overview of Literature Related to Post COVID-19 Rehabilitation Schertler Sierra Misericordia University Klimas Mikayla Davis Zachary McGee Michael Brogan Laurie 1 12 2022 12 2022 1 12 2022 103 12 e211e211 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objective(s) To examine current literature on the role of physical therapy (PT) in management of post COVID-19 related conditions and common symptomatology in adults, as well as potential guidelines for rehabilitation in the outpatient setting. Data Sources PubMed, CINAHL, Medline, Cochrane. Study Selection Searches were conducted to examine current data related to PT interventions and their effectiveness for treating post COVID-19 conditions. Articles were evaluated for relevance based on the following criteria: articles in English, original peer reviewed articles, adult population (over 18), relevant PT interventions for rehabilitation, post-acute infection of COVID-19. Consensus agreement confirmed approximately 25% of reviewed articles. Data Extraction Articles were analyzed for relevance to implications regarding post COVID-19 and potential PT rehabilitation interventions. Interventions were assessed in feasibility and applicability to an outpatient clinic setting. Independent data extraction followed by consensus discussion was applied. Articles were examined for content regarding the latest updates on disease criteria, manifestations, new classifications, and cohorts emerging as the pandemic progresses as well as management strategies applicable to PT practice. Data Synthesis After article analysis, the findings include a key theme that PT services helped improve overall functional mobility and symptom management in patients after an acute infection of COVID-19. An essential consideration is keeping the interventions specific to the patient and their goals while preventing exacerbations of symptoms that could lead to further setbacks. Conclusions PT has a growing role in the management of post COVID-19 deficits as well as implications related to long COVID sequelae. By choosing the appropriate parameters and having awareness of the varying symptomology amongst patients, physical therapists can improve patients’ functional mobility and post COVID-19 disease management. The focus of future studies should include more specific interventions related to managing conditions and finding the most effective treatment strategies. Author(s) Disclosures No conflicts to disclose. Key Words Post Covid Post-Acute Covid Physical Therapy Physiotherapy ==== Body pmc	0	PMC9712922	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e88	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.660	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)00651-7 10.1016/j.apmr.2022.08.061 Systematic & Meta-analytic Review Poster 2184185 Overview of Literature Related to Post COVID-19 Rehabilitation Schertler Sierra Misericordia University Klimas Mikayla Davis Zachary McGee Michael Brogan Laurie 1 12 2022 12 2022 1 12 2022 103 12 e211e211 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objective(s) To examine current literature on the role of physical therapy (PT) in management of post COVID-19 related conditions and common symptomatology in adults, as well as potential guidelines for rehabilitation in the outpatient setting. Data Sources PubMed, CINAHL, Medline, Cochrane. Study Selection Searches were conducted to examine current data related to PT interventions and their effectiveness for treating post COVID-19 conditions. Articles were evaluated for relevance based on the following criteria: articles in English, original peer reviewed articles, adult population (over 18), relevant PT interventions for rehabilitation, post-acute infection of COVID-19. Consensus agreement confirmed approximately 25% of reviewed articles. Data Extraction Articles were analyzed for relevance to implications regarding post COVID-19 and potential PT rehabilitation interventions. Interventions were assessed in feasibility and applicability to an outpatient clinic setting. Independent data extraction followed by consensus discussion was applied. Articles were examined for content regarding the latest updates on disease criteria, manifestations, new classifications, and cohorts emerging as the pandemic progresses as well as management strategies applicable to PT practice. Data Synthesis After article analysis, the findings include a key theme that PT services helped improve overall functional mobility and symptom management in patients after an acute infection of COVID-19. An essential consideration is keeping the interventions specific to the patient and their goals while preventing exacerbations of symptoms that could lead to further setbacks. Conclusions PT has a growing role in the management of post COVID-19 deficits as well as implications related to long COVID sequelae. By choosing the appropriate parameters and having awareness of the varying symptomology amongst patients, physical therapists can improve patients’ functional mobility and post COVID-19 disease management. The focus of future studies should include more specific interventions related to managing conditions and finding the most effective treatment strategies. Author(s) Disclosures No conflicts to disclose. Key Words Post Covid Post-Acute Covid Physical Therapy Physiotherapy ==== Body pmc	0	PMC9712923	NO-CC CODE	2022-12-02 23:22:01	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e171-e172	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.897	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01224-2 10.1016/j.apmr.2022.08.634 Research Poster 2194719 Respiratory Function Analysis Of Young Adults Post-Covid-19 Infection And After Immunization: An Observational Prospective Study Freire Ana Paula Central Washington University Amin Shaan Lira Fabio Morano Ana Elisa Ah Pereira Telmo Coelho-E-Silva Manuel-Joao Caseiro Armando Colson Chase Santos Vanessa Silva Bruna 1 12 2022 12 2022 1 12 2022 103 12 e79e79 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To prospectively analyze respiratory function outcomes of young adults post-COVID-19 infection and follow up these individuals after COVID-19 immunization. Design Observational prospective cohort study (partial results from FIT-COVID Study). Participants were recruited after a minimum of 15 and maximum of 180 days of diagnosis by positive PCR test. After a minimum of 6 weeks from the second shot of COVID-19 vaccine participants were once again recruited to evaluations. The Ethical Institutional Review Board approved the study (number: 38701820.0.0000.5402). Setting a Physical Therapy public outpatient clinic in Brazil. Participants We included male and female participants, aged 20–40 years after mild or moderate clinical COVID-19 and infection including slight clinical symptoms, fever, or respiratory symptoms, with previous positive PCR test and that were not admitted to intensive care unit (n=31). An age-matched healthy control group that was negative for COVID-19 was also recruited (n=39). Interventions Dyspnea was measured by the Modified Medical Research Council (MRC). Saccharin transit time test (STT) was performed to evaluate mucociliary transportability. Spirometry was performed using a digital spirometer. Main Outcome Measures Primary outcomes included pulmonary function evaluated by Forced Expiratory Volume in 1 Second (FEV1), Forced Vital Capacity (FVC) FEV1/FVC ratio. Secondary outcomes included STT (in minutes) and MRC score. Results Post-COVID19 group increased FEV1% between baseline and follow up (77.37%±17.6 vs 87.44%±10.28; p=0.003) and presented significant lower values than CG (86.47%±11.433 vs 87.68%±9.29). For STT no differences were observed between groups (p=0.257). Dyspnea scores decreased for Post-COVID19 group between baseline and follow up (0.44±0.61 vs 0.17±0.38; p=0.042) but no differences between groups were observed (p=0.57). Conclusions Pulmonary function was reduced in young adults Post-COVID-19 but improved over follow up analysis. Mucolciliary clearance and dyspnea was not impaired in young adults Post-COVID-19 and did not suffered changes after immunization. Author(s) Disclosures None of the authors have any conflicts of interests. Key Words COVID-19 Pulmonary Function Spirometry ==== Body pmc	0	PMC9712924	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e79	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.634	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01224-2 10.1016/j.apmr.2022.08.634 Research Poster 2194719 Respiratory Function Analysis Of Young Adults Post-Covid-19 Infection And After Immunization: An Observational Prospective Study Freire Ana Paula Central Washington University Amin Shaan Lira Fabio Morano Ana Elisa Ah Pereira Telmo Coelho-E-Silva Manuel-Joao Caseiro Armando Colson Chase Santos Vanessa Silva Bruna 1 12 2022 12 2022 1 12 2022 103 12 e79e79 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To prospectively analyze respiratory function outcomes of young adults post-COVID-19 infection and follow up these individuals after COVID-19 immunization. Design Observational prospective cohort study (partial results from FIT-COVID Study). Participants were recruited after a minimum of 15 and maximum of 180 days of diagnosis by positive PCR test. After a minimum of 6 weeks from the second shot of COVID-19 vaccine participants were once again recruited to evaluations. The Ethical Institutional Review Board approved the study (number: 38701820.0.0000.5402). Setting a Physical Therapy public outpatient clinic in Brazil. Participants We included male and female participants, aged 20–40 years after mild or moderate clinical COVID-19 and infection including slight clinical symptoms, fever, or respiratory symptoms, with previous positive PCR test and that were not admitted to intensive care unit (n=31). An age-matched healthy control group that was negative for COVID-19 was also recruited (n=39). Interventions Dyspnea was measured by the Modified Medical Research Council (MRC). Saccharin transit time test (STT) was performed to evaluate mucociliary transportability. Spirometry was performed using a digital spirometer. Main Outcome Measures Primary outcomes included pulmonary function evaluated by Forced Expiratory Volume in 1 Second (FEV1), Forced Vital Capacity (FVC) FEV1/FVC ratio. Secondary outcomes included STT (in minutes) and MRC score. Results Post-COVID19 group increased FEV1% between baseline and follow up (77.37%±17.6 vs 87.44%±10.28; p=0.003) and presented significant lower values than CG (86.47%±11.433 vs 87.68%±9.29). For STT no differences were observed between groups (p=0.257). Dyspnea scores decreased for Post-COVID19 group between baseline and follow up (0.44±0.61 vs 0.17±0.38; p=0.042) but no differences between groups were observed (p=0.57). Conclusions Pulmonary function was reduced in young adults Post-COVID-19 but improved over follow up analysis. Mucolciliary clearance and dyspnea was not impaired in young adults Post-COVID-19 and did not suffered changes after immunization. Author(s) Disclosures None of the authors have any conflicts of interests. Key Words COVID-19 Pulmonary Function Spirometry ==== Body pmc	0	PMC9712925	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e118	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.746	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01315-6 10.1016/j.apmr.2022.08.725 Research Poster 2184238 Experiences with COVID-19 Pandemic in the Spinal Cord Injury Community Wen Huacong University of Alabama at Birmingham Chen Yuying Botticello Amanda Deutsch Anne 1 12 2022 12 2022 1 12 2022 103 12 e111e111 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives Document the experiences of people with chronic spinal cord injury (SCI) during the COVID-19 pandemic. Design A cross-sectional survey. Setting An academic medical center. Participants A convenience sample of 35 participants from a local SCI Model Systems Center who completed a survey online or by phone between September 2021 and March 2022. Interventions Not applicable. Main Outcome Measures A scale “COVID-19: Impact of the pandemic and HRoL in cancer patients and survivors” obtained from NIH PhenX Toolkit was adapted for SCI population that measures personal COVID-19 exposures and experiences, including COVID-19 specific emotional and physical reactions, health care disruption and concerns, disruption to daily activities and social interactions, financial hardship, perceived benefits, functional social support, and perceived stress management. Results This sample had a mean age of 51.8 years, a mean duration of injury of 22.2 years, and 55.9 % had paraplegia with American Spinal Injury Association Impairment Scale A, B, or C. 17.1% of participants had COVID-19 infection. Forty percent of participants were fully vaccinated which is lower than that of general population in the state (53.5%). More than half of participants were concerned about family members or close friends getting or dying from COVID-19, had feelings of sadness or depression, and experienced disruptions in day to day social interactions with family and/or friends. The majority reported perceived benefits, had functional social supports, and had ability to manage stress. Participants reported varying COVID-19 related impacts, including employment (eg, 8.5% lost job), health care disruption (eg, 37.1% general care disruption), and financial hardship (eg, 34.3% financial difficulties). Conclusions These results provide important markers for developing interventions for SCI population in future crises. Author(s) Disclosures No disclosure. Key Words Spinal cord Injury COVID-19 Health ==== Body pmc	0	PMC9712926	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e111	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.725	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01497-6 10.1016/j.apmr.2022.08.907 Research Poster 2194721 Functional Capacity Of Young Adults After Covid-19 Infection And Immunization: Observational Prospective Study Amin Shaan Central Washington University Lira Fabio S. Morano Ana Elisa Ah Pereira Telmo Coelho-E-Silva Manuel-Joao Caseiro Armando Santos Vanessa Silva Bruna Coalson Chase Freire Ana 1 12 2022 12 2022 1 12 2022 103 12 e175e175 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To prospectively analyze functional capacity of young adults in relation to after COVID-19 infection and follow up these individuals after COVID-19 immunization. Design Observational prospective cohort study (partial results from FIT-COVID Study). After a minimum of 6 weeks from the second shot of COVID-19 vaccine participants were once again recruited to follow up evaluations. Setting Evaluations were performed at a public Physical Therapy outpatient clinic in Brazil. Participants We included male and female participants, aged 20–40 years after mild or moderate clinical COVID-19 with a previous positive PCR test that were not admitted to intensive care unit (COV). Participants were recruited after a minimum of 15 and maximum of 180 days of diagnosis by positive PCR test. An age-matched healthy control group (CT) that was negative for COVID-19 was also recruited. Interventions At baseline and follow up evaluation, functional capacity was assessed using the six-minute walk test (6MWT) and the 30-second stand from chair test (SC). Next, habitual physical activity (HPA) levels were assessed using accelerometry data. Finally, body composition (BC) was assessed using bioelectric impedance. Main Outcome Measures Meters walked during 6MWT, repetitions during SC. Secondarily, sedentary time in minutes, and body composition (lean mass in kilograms). Results Evaluations included Control Group (n=39) and Post-COVID-19 group (n=32). At baseline Post-COVID-19 group presented 626.69±100.68 meters and during the follow-up analysis (626.69±100.685 meters) with no significant difference between moments (p=0.698). No significant differences were observed between groups for 6MWT (p=0.698). In SC, there were no differences between moments (p=0.333) or groups (p=0.26). In the Post-COVID-19 group there was a significant reduction in sedentary time over the follow-up period (4070.36±1524.614 vs 3298.82±786.085; p=0.044). However, no differences were observed between groups (p=0.928). Finally, no differences were observed in body composition between groups or moments (p>0.05). Conclusions Functional capacity in young adults was not affected Post-COVID-19 infection and no changes were observed following immunization. Similar results, were observed for physical activity levels and body composition. Author(s) Disclosures None of the authors have any conflicts of interests. Key Words COVID-19 Physical Function Physical Activity Level Functional Capacity ==== Body pmc	0	PMC9712927	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e175	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.907	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01177-7 10.1016/j.apmr.2022.08.587 Research Paper 2191346 Associations Between Telerehabilitation and Outcomes for Patients with Low Back Pain During the COVID-19 Pandemic Werneke Mark Net Health Systems Deutscher Daniel Hayes Deanna Grigsby David Resnik Linda 1 12 2022 12 2022 1 12 2022 103 12 e62e62 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To examine associations between frequency of telerehabilitation i.e., any few, most or all, and outcomes of functional status, number of visits, and patient satisfaction during COVID-19 and to compare functional status outcomes by telerehabilitation delivery mode i.e., synchronous, asynchronous, mixed, for patients with low back pain. Design Retrospective observational cohort. Setting Outpatient rehabilitation. Participants Sample consisted of 91,117 episodes of care (58% women; mean age = 55 [SD = 18]). Interventions Not applicable. Main Outcome Measures Lumbar Computer Adaptive Test (LCAT) patient-reported outcome measure was administered to assess functional status. LCAT has been shown to be reliable, valid, and responsive. Number of visits during the episode of care were documented at discharge. Data on patient satisfaction with treatment results were collected: “How satisfied were you with overall results of your treatment at this facility?” Results Telerehabilitation was administered in 5013 care episodes (5.5%). Propensity score matching was used to match episodes of care with or without telerehabilitation exposure by the probability of receiving telerehabilitation. Standardized differences were used to compare samples before and after matching. All standardized differences between matched samples were < 0.1. There was no significant difference in functional status points (range = 0–100, with higher representing better functional status) between matched samples, except for episodes that had few (−1.7) and all (+2.0) telerehabilitation frequencies or that involved the asynchronous (−2.6) telerehabilitation mode. These point differences suggest limited clinical importance. Episodes with any telerehabilitation frequency involved significantly fewer visits (0.7 to 1.3) than episodes with no telerehabilitation, except that those with the most telerehabilitation frequencies had nonsignificantly fewer visits (0.6). A smaller proportion of patients with telerehabilitation than of patients with no telerehabilitation (−4.0% to −5.0%, respectively) reported being very satisfied with treatment results, except for those with the ‘all’ telerehabilitation frequency. Conclusions A positive association between telerehabilitation and outcomes was observed, with a trend for better functional status outcomes and fewer visits when all care was delivered through telerehabilitation. Satisfaction tended to be lower with telerehabilitation use. Author(s) Disclosures No conflicts to declare. Key Words Telerehabilitation Outcomes Low Back Pain Outpatient Rehabilitation ==== Body pmc	0	PMC9712928	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e62	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.587	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01387-9 10.1016/j.apmr.2022.08.797 Research Poster 2184251 Challenges in Delivering Vital Services During The COVID-19 Pandemic: Perspectives From Brain Injury Associations Engel Lisa The University of Manitoba / The Institute for Work & Health (Toronto) Salazar Ana Paula Lecours Sophie Swaine Bonnie Schmidt Julia Gignac Monique A.M. Brosda Ashley McDonald Michelle Bottari Carolina 1 12 2022 12 2022 1 12 2022 103 12 e136e137 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To describe community brain injury (BI) associations’ experiences during the COVID-19 pandemic. Design Anonymous online survey study. Setting In January 2022 the project team, including community BI association co-investigators, collected responses from key stakeholders in the 70 eligible BI associations across Canada. Participants Respondents were from 45 associations, including associations in Pacific/Western (n=18; 40%), Central (n=25; 56%), and Atlantic Canada (n=2; 4%). Respondents were primarily paid executive directors (n=35; 78%) representing primarily associations employing 10 or less people (n=33; 77%) but serving 100 or more different clients (n=31; 69%) before the COVID-19 pandemic. Interventions None. Main Outcome Measures The online 26-item survey had quantitative and narrative questions about three main topics: association sustainability, meeting the needs of clients, and addressing public health. Results Thirty-four (76%) associations reported reductions in funding or financial resources during the pandemic which affected provision of programs or services, and only 14 (31%) received sufficient funds to a large or very large extent to cover additional pandemic-related expenses. Yet, twenty-eight (62%) associations reported increased demand for their programs or services, and 42 (93%) innovated their programs or services to meet varied and widespread client needs during the pandemic. Forty-two (93%) associations provided services or information to clients to explain public health guidelines, and forty-one (91%) associations reported clients experienced challenges in understanding and following public health guidelines. Narrative data provided further depth to the quantitative data. Conclusions Community BI associations have a vital role in the long-term rehabilitation and management of BI. However, the COVID-19 pandemic challenged BI associations to remain sustainable and meet the needs of their clients. Researchers and policy makers need to acknowledge and adequately support the indispensable work of associations in the BI rehabilitation continuum, as the loss of community BI associations could have vast ramifications. Author(s) Disclosures This survey study was funded in part by Canadian Institutes of Health Research (CIHR); the funder did not have a role in design, data collection, analysis, or reporting. Key Words Brain Injuries, Stroke, Community Services Community Resources, Community Support Social Support, Pandemics Public Health ==== Body pmc	0	PMC9712929	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e136-e137	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.797	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01387-9 10.1016/j.apmr.2022.08.797 Research Poster 2184251 Challenges in Delivering Vital Services During The COVID-19 Pandemic: Perspectives From Brain Injury Associations Engel Lisa The University of Manitoba / The Institute for Work & Health (Toronto) Salazar Ana Paula Lecours Sophie Swaine Bonnie Schmidt Julia Gignac Monique A.M. Brosda Ashley McDonald Michelle Bottari Carolina 1 12 2022 12 2022 1 12 2022 103 12 e136e137 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To describe community brain injury (BI) associations’ experiences during the COVID-19 pandemic. Design Anonymous online survey study. Setting In January 2022 the project team, including community BI association co-investigators, collected responses from key stakeholders in the 70 eligible BI associations across Canada. Participants Respondents were from 45 associations, including associations in Pacific/Western (n=18; 40%), Central (n=25; 56%), and Atlantic Canada (n=2; 4%). Respondents were primarily paid executive directors (n=35; 78%) representing primarily associations employing 10 or less people (n=33; 77%) but serving 100 or more different clients (n=31; 69%) before the COVID-19 pandemic. Interventions None. Main Outcome Measures The online 26-item survey had quantitative and narrative questions about three main topics: association sustainability, meeting the needs of clients, and addressing public health. Results Thirty-four (76%) associations reported reductions in funding or financial resources during the pandemic which affected provision of programs or services, and only 14 (31%) received sufficient funds to a large or very large extent to cover additional pandemic-related expenses. Yet, twenty-eight (62%) associations reported increased demand for their programs or services, and 42 (93%) innovated their programs or services to meet varied and widespread client needs during the pandemic. Forty-two (93%) associations provided services or information to clients to explain public health guidelines, and forty-one (91%) associations reported clients experienced challenges in understanding and following public health guidelines. Narrative data provided further depth to the quantitative data. Conclusions Community BI associations have a vital role in the long-term rehabilitation and management of BI. However, the COVID-19 pandemic challenged BI associations to remain sustainable and meet the needs of their clients. Researchers and policy makers need to acknowledge and adequately support the indispensable work of associations in the BI rehabilitation continuum, as the loss of community BI associations could have vast ramifications. Author(s) Disclosures This survey study was funded in part by Canadian Institutes of Health Research (CIHR); the funder did not have a role in design, data collection, analysis, or reporting. Key Words Brain Injuries, Stroke, Community Services Community Resources, Community Support Social Support, Pandemics Public Health ==== Body pmc	0	PMC9712930	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e135	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.793	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)00653-0 10.1016/j.apmr.2022.08.063 Systematic & Meta-analytic Review Poster 2184214 Pulmonary Rehabilitation and Breathing Exercise Effectiveness for Post COVID-19 survivors Khan Suheera Northern Illinois University Doctorate Physical Therapy Program McKnight Ryan Bateni Hamid Rehal Aman 1 12 2022 12 2022 1 12 2022 103 12 e211e212 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Objective(s) Residual effects of Covid-19 on respiratory system are often destructive and depilating. Several rehabilitation approach are suggested in literature. This review aims to summarize and compare the available evidence on the current pulmonary rehabilitation therapy following COVID-19. Data Sources A computer search was conducted using PubMed, CINAHL, and ERIC using keywords ‘COVID-19’,‘respiratory therapy,’ OR ‘rehabilitation,’ OR ‘physiotherapy,’ OR ‘physical therapy,’ OR ‘corticosteroid,’ OR ‘respiratory rehabilitation,’ OR ‘traditional Chinese medicine,’ OR ‘aerobic therapy’ AND ‘COVID-19 patients’. A narrative synthesis was used to evaluate the selected literature. The search was limited to the English language, human, and the year 2010 to present. Study Selection Studies were reviewed independently and those with the focus of COVID-19 and medication, traditional Chinese Medication, respiratory rehabilitation, and aerobic exercise interventions were included. Total of 27 articles were included in the final review. Data Extraction An independent extraction of data was performed on demographics, methodology, intervention, and outcomes. Data Synthesis Methods of medication (i.e. corticosteroids, antivirals, and Cardiopulmonary [CP] therapy), traditional Chinese Medicine (i.e. herbal medicine and pediatric massage and acupuncture), respiratory rehabilitation (i.e. incentive spirometer use, pursed-lip breathing, diaphragmatic techniques, and airway clearance techniques) and aerobic exercise were reviewed. Although all approaches showed some level of effectiveness, in medication approach, antivirals and CP therapy seems to be most effective for severe cases of COVID-19. Respiratory rehabilitation appears to best benefit inpatient/acute cases of COVID-19. Aerobic exercise is reported to have a promising positive effects, but a clear outline and parameters are yet to be determined. Conclusions Post COVID-19 pulmonary rehabilitation appears to vary significantly based on the severity of COVID-19 infection/symptoms and individual responses. There is a limited knowledge of the field due to recent occurrence of the disease and lack of information on the new strains of the virus. Author(s) Disclosures There is no conflict of interest associate with this study for any of the authors. Key Words COVID-19 Respiratory Rehabilitation Traditional Chinese Medicine ==== Body pmc	0	PMC9712931	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e211-e212	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.063	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01391-0 10.1016/j.apmr.2022.08.801 Research Poster 2184230 Trajectory of Functional Recovery from 6 to 12 Months in Persons Hospitalized for Severe SARS-CoV-2 Illness Borsi Lydia Spaulding Rehabilitation Hospital, Neurorehabilitation Laboratory Waters Abigail Bodien Yelena Bergin Michael Boudreau Nancy Brown Lorna Chen Roy Corey Kaitlyn O'Brien Anne Vergara-Diaz Gloria Andreu MaryLourdes Lazar Elizabeth Rosand Jonathan Perlis Roy Keysor Julie Zafonte Ross D. Giacino Joseph T. 1 12 2022 12 2022 1 12 2022 103 12 e138e138 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To identify distinct post-acute COVID-19 phenotypes among adults hospitalized for severe SARS-CoV-2 infection and describe multidimensional outcomes and trajectories at 6 and 12 months post-hospitalization. Design Prospective, longitudinal data collection in functional, physical, cognitive, and psychological domains at 3, 6, and 12 months post-hospitalization. Retrospective data collection from the acute care and post-acute care settings. Setting Acute care and post-acute telephone follow-up. Participants English- and Spanish- speaking adults, with decision-making capacity, admitted for inpatient rehabilitation following inpatient rehabilitation for acute COVID-19 related illness (N = 61). Interventions N/A. Main Outcome Measures Physical, cognitive, and psychological symptoms; self-reported employment status and assistance with ADLs. Results Median age 60.8 years; 59% male; 72.1% white; 72.1% non-Hispanic; 26.2% preferred assessment in Spanish. 83% required mechanical ventilation in acute care. Comorbidities were common. We found a high prevalence of persistent symptoms at 6- and 12- months across physical, cognitive, and emotional health outcome domains. Three post-acute phenotypes were identified at 6 months; a "minimally symptomatic" subgroup with minimal symptom endorsement across all domains relative to other subjects (22.95%, n = 14), a “predominantly physical symptoms” subgroup (47.54%, n = 29), and a “globally symptomatic” subgroup (29.51%, n = 18). A similar pattern for phenotypes emerges at 12-months, with 67.21% of subjects falling into the same phenotype at both time points. In the Predominantly Physical Symptom phenotype, 31.0% declined into the Globally Symptomatic Phenotype and 10.3% improved. In the Globally Symptomatic phenotype, 11.1% of participants transitioned to the Minimally Symptomatic phenotype and 16.7% to the Predominantly Physical Symptom phenotype. Compared to premorbid level of employment (50.8%), 24.6% of participants were employed at 12-months. Phenotype at 6-months was a significant predictor of employment at 12-months (B = 2.26, p = .05, OR = 9.6). Conclusions Persons with severe COVID-19 illness experience persistent functional limitations and reduced employment up to 12 months post-hospitalization. Distinct recovery subgroups were found suggesting the need for comprehensive assessment and tailored treatment for recovery. Author(s) Disclosures The authors declare no relevant conflicts of interest. Key Words Post-Acute COVID-19 Recovery Of Function COVID-19/Rehabilitation ==== Body pmc	0	PMC9712932	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e138	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.801	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01515-5 10.1016/j.apmr.2022.08.925 Research Poster 2184258 Feasibility of a Virtual Game-Based Activity Intervention in Breast Cancer Survivors During The COVID-19 Pandemic Wells Stephanie UT MD Anderson Cancer Center Christopherson Ursela Robertson Michael Lyons Elizabeth Martinez Eloisa Silva H. Colleen Golliday Lakesha Swartz Maria 1 12 2022 12 2022 1 12 2022 103 12 e181e181 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives Report the feasibility of a virtual active video game (AVG)-based physical activity (PA) support group intervention for older breast cancer survivors (Pink Warrior 2) during the COVID-19 pandemic. Design A pilot randomized controlled trial. Setting Home-based virtual intervention. Participants Physically inactive breast cancer survivors aged ≥ 55 years. Interventions Pink Warrior 2 was a virtual PA intervention that included 13-weekly AVG sessions, a Fitbit for self-monitoring, and behavioral coaching via videoconference. Participants in the control group participated in a weekly group phone discussion on cancer survivorship topics and wore a Mi band, an electronic activity monitor. Unlike Fitbit's mobile application (app), Mi band's app did not integrate behavioral change techniques (e.g., encouraging steps). Main Outcome Measures Feasibility was assessed by enrollment rate (≥50%), retention rate (≥80%), group attendance rate (≥10 sessions for intervention), percentage of completed assessments (questionnaires and virtual assessments), number of technological issues and adverse events. Results The recruitment rate was 34%. Twenty-seven participants were enrolled, and 20 were randomized into intervention or control groups. The final participant retention rate was 90%. The adherence rate for group attendance was 88% for the intervention group and 82% for the control group. 85% of participants completed all the baseline questionnaires. 96% of virtual assessments were conducted successfully without adverse events. Internet connectivity issues impacted 2 out of 56 (4%) assessments. 14% of assessments were delayed due to Actigraph monitor-related issues or participants or their family members contracting COVID-19. Conclusions Session adherence, retention, virtual assessments, and the number of adverse events met benchmarks for feasibility. Recruitment and completion of paper-based questionnaires were limited by challenges related to COVID-19. Author(s) Disclosures No known conflicts of interest. Key Words Exercise Breast Neoplasms Exergaming Telehealth ==== Body pmc	0	PMC9712933	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e181	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.925	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01282-5 10.1016/j.apmr.2022.08.692 Research Poster 2194714 Characteristics of a Post-Coronavirus-19 Sample Undergoing Rehabilitation: An Exploratory Study Farr Ellen Mayo Clinic College of Medicine and Science Bergquist Thomas 1 12 2022 12 2022 1 12 2022 103 12 e99e100 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives Describe sample presenting to a brain rehabilitation clinic with cognitive impairment after COVID-19 infection. Design Pre- and post-intervention measures from a clinical sample. Setting Outpatient Brain Rehabilitation Clinic. Participants Eight patients undergoing outpatient rehabilitation after COVID-19 infection (Mean age = 47.1, Range: 38-55 years; females = 6, males = 2; 4 hospitalized at time of acute infection, Mean time to treatment 243 days, Range: 84-415 days). Interventions Treatment by an experienced team of interdisciplinary providers with composition and treatment frequency tailored to individual needs. Main Outcome Measures Patient Health Questionnaire (PHQ-9); Participation Index of the Mayo-Portland Adaptability Inventory (M2PI); Satisfaction with Life Scale (SWLS); Neurobehavioral Symptom Inventory – 22 (NSI-22). Results Average number visits = 6.5 (Range: 0-25). Treatment attendance was close to 100%. Mean PHQ-9 on admission 10.625 (Range: 3-18) and at discharge 10.25 (Range: 1-17). Mean M2PI on admission was 13 (Range:8-24, and on discharge was 10.875 (Range: 0-20). Mean SWLS was the same at admission and discharge, 20.625 (Range: Admit: 15-29, Discharge 10-29). NSI-22 mean on admission 37.375 (Range 16-49) and on discharge 31.5 (Range:6-54). Conclusions Most of our sample reported moderate to moderate-severe depression. Most endorsed moderate-severe disability on the M2PI. This changed only slightly over treatment. This sample reported highly variable symptom complaint and life satisfaction. Of 5 patients with worsening scores over treatment, 4 were patients who started treatment well over 200 days after their initial diagnosis (Range: 229-415 days). Our treatment sample reports variable symptomatology and outcomes that may be tied to time to initiating rehabilitation. Author(s) Disclosures None. Key Words Coronavirus Cognition Rehabilitation ==== Body pmc	0	PMC9712934	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e99-e100	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.692	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01195-9 10.1016/j.apmr.2022.08.605 Research Poster 2197257 Relating Acute Care Rehabilitation Use and Discharge Disposition Among Individuals Hospitalized for COVID-19 Malcolm Matt Rocky Mountain University of Health Professions Pennington Ashley Hoffman Amanda Bukhari Rayyan Graham James 1 12 2022 12 2022 1 12 2022 103 12 e69e69 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To examine the extent to which acute care rehabilitation predicts discharge to post-acute care (PAC) in hospitalized COVID-19 patients. To identify social determinants that influence discharge to PAC for hospitalized COVID-19 patients. Design Secondary analysis of de-identified electronic health record (EHR) data obtained from 14 hospitals during January 2020 through April 2021. The dependent variable was discharge disposition (post-acute care or home). Independent variables included age, sex, ethnic/race minority status, presence of significant other, education level, income, insurance type, rural-urban-frontier residence, acute care occupational therapy (OT) and physical therapy (PT), ICU stay. Descriptive statistics and binary logistic regression were employed. Setting Fourteen acute care hospitals that are part of a single health network in Colorado. Participants 5,654 individuals were admitted and diagnosed with COVID-19 during the observation window. To be included in our analyses, cases had to meet the following criteria: survival to discharge, discharged to home or PAC, complete data for variables of interest. After applying these criteria, 979 individuals were excluded resulting in a final sample size of 4,675 individuals. Interventions Receipt of acute care OT or PT. Main Outcome Measures The primary outcome (dependent variable) was discharge disposition, which we categorized as "discharged to Post-Acute Care (PAC)" or "home". Results Age (Odds ratio, OR = 1.03), Medicare (OR = 2.21), receipt of acute care OT (OR = 3.41), receipt of acute care PT (OR = 5.05), and ICU stay (OR = 1.49) significantly predicted discharge to inpatient PAC. Sex, race/ethnicity, significant other status, residence type, education level, and income level were not significant predictors of discharge disposition. Conclusions Individuals who were older, Medicare beneficiaries, received OT or PT, and had an ICU stay were the most likely hospitalized COVID-19 patients to be discharged to inpatient PAC. Contrary to pre-pandemic studies that demonstrate the influence of significant others, residence location (e.g., rural), income, and education level have on discharge disposition, our findings suggest these factors mattered less in the context of COVID-19. Author(s) Disclosures The authors have no conflicts. Key Words Post-Acute Care Rehabilitation COVID-19 ==== Body pmc	0	PMC9712935	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e69	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.605	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01518-0 10.1016/j.apmr.2022.08.928 Research Poster 2194724 Characterizing Patients Referred For Ambulatory Rehabilitation Post COVID-19 Bolt Susan Bryn Mawr Rehabilitation Hospital 1 12 2022 12 2022 1 12 2022 103 12 e182e182 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives to describe baseline characteristics of patients referred for outpatient rehabilitation therapies during the first 7 months of Bryn Mawr Rehabilitation Hospital's Post-COVID Recovery Program. Design Retrospective cohort. Setting Outpatient rehabilitation clinic. Participants All patients referred to the BMRH Post-COVID Recovery Program from May – December 2021, excluding those with neurodegenerative conditions and those already receiving physical, occupational, and/or speech therapy for a pre-existing condition at the time of their COVID-19 diagnosis. Interventions Not applicable. Main Outcome Measures This study focuses on describing baseline characteristics of a relatively new clinical population, ergo there is no main outcome measure. Results Of 116 patients referred to the program, 94 (81.0%) were assessed by at least one therapy discipline. Of patients assessed, 72.3% were assessed by more than one discipline, and 37.2% were evaluated by all three: PT, OT, and Speech. The mean duration from COVID-19 diagnosis to referral was 203.2 days (SD=160.11). The majority of patients, 66.7%, were not hospitalized for COVID-19. Among patients’ chief complaints, the top five were cognitive issues (76.6%), decreased endurance (75.5%), decreased strength (62.8%), pain (56.4%), and balance deficits (50.0%). The most common premorbid conditions were anxiety/depression (37.0%), hypertension (35.3%), lipid disorders (33.6%), current/former smoker (31.0%), and migraines (21.6%). Average body mass index at time of COVID-19 diagnosis was 31.33 (SD=8.89), with 29.15 and 25.88 at the median and first quartile, respectively, indicating that the majority of patients fell within CDC ranges for overweight or obesity. Cognitive complaints were correlated with female assigned sex, lower levels of care received for COVID illness, and preserved strength. Decreased endurance was correlated with higher BMI, lipid disorder, higher level of care, and other physical complaints. Balance deficits were correlated with hypertension and longer duration. Conclusions These results shed light on a relatively new patient population and their rehabilitation needs. As SARS-CoV-2 continues to spread, more and more patients will seek rehabilitation services as part of their recovery. Understanding the characteristics of these patients is key to developing and refining effective treatment protocols. Author(s) Disclosures Nothing to disclose. Key Words COVID-19 Rehabilitation Cognition ==== Body pmc	0	PMC9712936	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e182	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.928	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01518-0 10.1016/j.apmr.2022.08.928 Research Poster 2194724 Characterizing Patients Referred For Ambulatory Rehabilitation Post COVID-19 Bolt Susan Bryn Mawr Rehabilitation Hospital 1 12 2022 12 2022 1 12 2022 103 12 e182e182 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives to describe baseline characteristics of patients referred for outpatient rehabilitation therapies during the first 7 months of Bryn Mawr Rehabilitation Hospital's Post-COVID Recovery Program. Design Retrospective cohort. Setting Outpatient rehabilitation clinic. Participants All patients referred to the BMRH Post-COVID Recovery Program from May – December 2021, excluding those with neurodegenerative conditions and those already receiving physical, occupational, and/or speech therapy for a pre-existing condition at the time of their COVID-19 diagnosis. Interventions Not applicable. Main Outcome Measures This study focuses on describing baseline characteristics of a relatively new clinical population, ergo there is no main outcome measure. Results Of 116 patients referred to the program, 94 (81.0%) were assessed by at least one therapy discipline. Of patients assessed, 72.3% were assessed by more than one discipline, and 37.2% were evaluated by all three: PT, OT, and Speech. The mean duration from COVID-19 diagnosis to referral was 203.2 days (SD=160.11). The majority of patients, 66.7%, were not hospitalized for COVID-19. Among patients’ chief complaints, the top five were cognitive issues (76.6%), decreased endurance (75.5%), decreased strength (62.8%), pain (56.4%), and balance deficits (50.0%). The most common premorbid conditions were anxiety/depression (37.0%), hypertension (35.3%), lipid disorders (33.6%), current/former smoker (31.0%), and migraines (21.6%). Average body mass index at time of COVID-19 diagnosis was 31.33 (SD=8.89), with 29.15 and 25.88 at the median and first quartile, respectively, indicating that the majority of patients fell within CDC ranges for overweight or obesity. Cognitive complaints were correlated with female assigned sex, lower levels of care received for COVID illness, and preserved strength. Decreased endurance was correlated with higher BMI, lipid disorder, higher level of care, and other physical complaints. Balance deficits were correlated with hypertension and longer duration. Conclusions These results shed light on a relatively new patient population and their rehabilitation needs. As SARS-CoV-2 continues to spread, more and more patients will seek rehabilitation services as part of their recovery. Understanding the characteristics of these patients is key to developing and refining effective treatment protocols. Author(s) Disclosures Nothing to disclose. Key Words COVID-19 Rehabilitation Cognition ==== Body pmc	0	PMC9712937	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e88-e89	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.662	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01265-5 10.1016/j.apmr.2022.08.675 Research Poster 2184192 Inequitable Care Delivery for COVID-19 Positive People of Color and People With Disabilities Lee Danbi University of Washington Sabin Janice Mohammed Selina Kett Paula Frogner Bianca 1 12 2022 12 2022 1 12 2022 103 12 e93e93 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To explore provider observations of inequitable care delivery towards COVID-19 positive patients who are Black, Indigenous, and People of Color (BIPOC) and/or have disabilities and to identify ways health providers may be contributing to and compounding inequitable care. Design Qualitative research design using thematic analysis of data collected via individual semi-structured interviews between April and November 2021. Setting Hospitalized and ambulatory care. Participants A total of 19 pandemic frontline health care providers including nine physicians, five nurses, three rehabilitation professionals, and two nurse practitioners from Washington, Florida, Illinois, and New York were recruited through convenience and snowball sampling. The majority of participants were female (n=12) and White (n=11). Interventions Not applicable. Main Outcome Measures A semi-structured interview guide included questions regarding equity in treatments among COVID-19 patients who are BIPOC or have disabilities and perceptions around stigma related to COVID-19. Results Discriminatory treatment included decreased care (i.e., less explanation due to communication barriers, less interaction with health care providers, no access to telehealth follow-ups), delayed care, and fewer options for care. Health care providers’ bias and stigma (racism, ableism, and ageism), patient mistrust and provider-patient disconnect, lack of resources (e.g., staff, interpreter services, funding, or collaboration across health systems), fear of transmission, and burnout were mentioned as drivers for discriminatory treatment. COVID-19 related health system policies such as visitor restrictions and telehealth follow-ups also inadvertently resulted in discriminatory practices towards BIPOC patients and patients with disabilities. Conclusions BIPOC patients and patients with disabilities may experience lower quality of healthcare related to COVID-19 due to factors such as providers’ biases, fear, burnout, and lack of needed resources in their work settings. In addition, COVID-19-related restrictions and policies compounded existing inequitable care for these populations. Author(s) Disclosures Authors do not have conflicts to disclose. Key Words Health Equity COVID-19 BIPOC People with Disabilities ==== Body pmc	0	PMC9712938	NO-CC CODE	2022-12-02 23:22:02	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e93	utf-8	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.675	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01265-5 10.1016/j.apmr.2022.08.675 Research Poster 2184192 Inequitable Care Delivery for COVID-19 Positive People of Color and People With Disabilities Lee Danbi University of Washington Sabin Janice Mohammed Selina Kett Paula Frogner Bianca 1 12 2022 12 2022 1 12 2022 103 12 e93e93 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To explore provider observations of inequitable care delivery towards COVID-19 positive patients who are Black, Indigenous, and People of Color (BIPOC) and/or have disabilities and to identify ways health providers may be contributing to and compounding inequitable care. Design Qualitative research design using thematic analysis of data collected via individual semi-structured interviews between April and November 2021. Setting Hospitalized and ambulatory care. Participants A total of 19 pandemic frontline health care providers including nine physicians, five nurses, three rehabilitation professionals, and two nurse practitioners from Washington, Florida, Illinois, and New York were recruited through convenience and snowball sampling. The majority of participants were female (n=12) and White (n=11). Interventions Not applicable. Main Outcome Measures A semi-structured interview guide included questions regarding equity in treatments among COVID-19 patients who are BIPOC or have disabilities and perceptions around stigma related to COVID-19. Results Discriminatory treatment included decreased care (i.e., less explanation due to communication barriers, less interaction with health care providers, no access to telehealth follow-ups), delayed care, and fewer options for care. Health care providers’ bias and stigma (racism, ableism, and ageism), patient mistrust and provider-patient disconnect, lack of resources (e.g., staff, interpreter services, funding, or collaboration across health systems), fear of transmission, and burnout were mentioned as drivers for discriminatory treatment. COVID-19 related health system policies such as visitor restrictions and telehealth follow-ups also inadvertently resulted in discriminatory practices towards BIPOC patients and patients with disabilities. Conclusions BIPOC patients and patients with disabilities may experience lower quality of healthcare related to COVID-19 due to factors such as providers’ biases, fear, burnout, and lack of needed resources in their work settings. In addition, COVID-19-related restrictions and policies compounded existing inequitable care for these populations. Author(s) Disclosures Authors do not have conflicts to disclose. Key Words Health Equity COVID-19 BIPOC People with Disabilities ==== Body pmc	0	PMC9712939	NO-CC CODE	2022-12-03 23:19:58	no	Arch Phys Med Rehabil. 2022 Dec 1; 103(12):e100	latin-1	Arch Phys Med Rehabil	2,022	10.1016/j.apmr.2022.08.693	oa_other
==== Front Arch Phys Med Rehabil Arch Phys Med Rehabil Archives of Physical Medicine and Rehabilitation 0003-9993 1532-821X Published by Elsevier Inc. S0003-9993(22)01265-5 10.1016/j.apmr.2022.08.675 Research Poster 2184192 Inequitable Care Delivery for COVID-19 Positive People of Color and People With Disabilities Lee Danbi University of Washington Sabin Janice Mohammed Selina Kett Paula Frogner Bianca 1 12 2022 12 2022 1 12 2022 103 12 e93e93 Copyright © 2022 Published by Elsevier Inc. 2022 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. Research Objectives To explore provider observations of inequitable care delivery towards COVID-19 positive patients who are Black, Indigenous, and People of Color (BIPOC) and/or have disabilities and to identify ways health providers may be contributing to and compounding inequitable care. Design Qualitative research design using thematic analysis of data collected via individual semi-structured interviews between April and November 2021. Setting Hospitalized and ambulatory care. Participants A total of 19 pandemic frontline health care providers including nine physicians, five nurses, three rehabilitation professionals, and two nurse practitioners from Washington, Florida, Illinois, and New York were recruited through convenience and snowball sampling. The majority of participants were female (n=12) and White (n=11). Interventions Not applicable. Main Outcome Measures A semi-structured interview guide included questions regarding equity in treatments among COVID-19 patients who are BIPOC or have disabilities and perceptions around stigma related to COVID-19. Results Discriminatory treatment included decreased care (i.e., less explanation due to communication barriers, less interaction with health care providers, no access to telehealth follow-ups), delayed care, and fewer options for care. Health care providers’ bias and stigma (racism, ableism, and ageism), patient mistrust and provider-patient disconnect, lack of resources (e.g., staff, interpreter services, funding, or collaboration across health systems), fear of transmission, and burnout were mentioned as drivers for discriminatory treatment. COVID-19 related health system policies such as visitor restrictions and telehealth follow-ups also inadvertently resulted in discriminatory practices towards BIPOC patients and patients with disabilities. Conclusions BIPOC patients and patients with disabilities may experience lower quality of healthcare related to COVID-19 due to factors such as providers’ biases, fear, burnout, and lack of needed resources in their work settings. In addition, COVID-19-related restrictions and policies compounded existing inequitable care for these populations. Author(s) Disclosures Authors do not have conflicts to disclose. Key Words Health Equity COVID-19 BIPOC People with Disabilities ==== Body pmc	0	PMC9713072	NO-CC CODE	2022-12-02 23:22:06	no	DNP. 2022 Dec 1; 23(6):8	latin-1	null	null	null	oa_other
==== Front Neurocrit Care Neurocrit Care Neurocritical Care 1541-6933 1556-0961 Springer US New York 36450974 1643 10.1007/s12028-022-01643-8 Viewpoint Use of α-Defensins to Help Diagnose Nosocomial Ventriculitis Doub James B. [email protected] grid.411024.2 0000 0001 2175 4264 Division of Clinical Care and Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD USA 30 11 2022 14 12 8 2022 8 11 2022 © Springer Science+Business Media, LLC, part of Springer Nature and Neurocritical Care Society 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Ventriculitis is a severe complication of indwelling neurosurgical devices that is associated with significant morbidity and mortality. The incidence rate of ventriculitis is approximately 10% with external ventricular drains. Obstinately, patients with these indwelling neurosurgical devices are prone to have traditional cerebral spinal fluid parameters that lack sensitivity and specificity in diagnosing nosocomial ventriculitis. In addition, diagnosis can be arduous given that indolent pathogens are commonly implicated. Therefore, diagnosis is difficult but paramount to thwart the morbidity and mortality associated with this infectious condition as well as to reduce the prolonged use of broad-spectrum antibiotics. As we extrapolate from prosthetic joint infections, for which diagnosis can also be challenging, we learn that the use of α-defensins as a diagnostic biomarker for nosocomial ventriculitis may hold promise. Herein, the viewpoint of using α-defensins as a diagnostic biomarker for nosocomial ventriculitis is discussed. Keywords Ventriculitis α-defensins Central nervous system infections Prosthetic joint infections External ventricular drain ==== Body pmcVentriculitis is a severe central nervous system (CNS) condition that involves inflammation of the ependymal lining of the cerebral ventricles [1]. Common symptoms include fever, altered mental status, severe headaches, and sepsis [2]. A wide range of pathogens have been implicated, but with nosocomial ventriculitis, many indolent pathogens are implicated, thus not allowing for these pathogens to be readily cultured [2]. Treatment entails rapid identification and aggressive antimicrobial treatment to prevent long-term sequalae and reduce mortality [1, 2]. Nosocomial ventriculitis is usually associated with neurosurgical devices (external ventricular drains [EVDs] and shunts) but can also occur after neurosurgical procedures and trauma [1]. EVDs are habitually used in Intensive care units to monitor and control increased intracranial pressure in the acute setting [2]. Complications from EVDs include hemorrhage, hygromas, and infections [3]. Infections are usually secondary to microbes colonizing the device at the time of insertion, to introduction with subsequent manipulations, or to retrograde translocation of microbes [2–4]. The incidence of nosocomial ventriculitis varies widely in the reported literature, with EVD-associated ventriculitis being reported as high as 22%, but the current rate is approximately 10% [2]. Incidence is also strongly correlated with the duration of EVD residence, with longer durations being associated with increased risk of ventriculitis [2–4]. The diagnosis of CNS infections is commonly conducted with evaluation of cerebral spinal fluid (CSF) for derangements of protein, glucose, cell count, gram stain, and culture. However, in nosocomial ventriculitis, patients can have abnormal CSF parameters from underlying CNS pathologies, complicating diagnosis [5, 6]. Furthermore, pleocytosis can occur secondary to CNS hemorrhages and from chemical meningitis [2, 6]. Obstinately, patients with hemorrhages and noninfectious ventriculitis often have similar symptoms, such as fever, headache, and altered mental status [2, 6]. Consequently, symptoms and traditional diagnostic CSF testing parameters (cell count, protein level, and glucose level) have significantly less specificity and sensitivity in nosocomial ventriculitis compared to community-acquired meningitis [7]. This has led to the evaluation of several additional parameters to aid in diagnosis, which include CSF lactate level, CSF microbial polymerase chain reaction testing, and procalcitonin level, but these parameters have conflicting data supporting effectiveness [2]. Therefore, novel diagnostics are needed to help clinicians diagnose nosocomial ventriculitis to thereby reduce morbidity and mortality and thwart the use of and the complications associated with broad-spectrum antibiotics. One such biomarker that holds theoretical promise in diagnosing ventriculitis is α-defensins. Human α-defensins are small cationic peptides with 29–33 amino acids that are sequestered in neutrophil granules [8]. These peptides are innate antimicrobial molecules with broad antimicrobial activity to gram-positive and gram-negative bacteria, fungi, and enveloped viruses [8]. They are released by activated neutrophils when they are exposed to microbes to disrupt microbial membrane integrity and function [9]. Low levels of α-defensins can be present in the serum of healthy patients, but levels increase significantly in infectious conditions, causing sepsis, with high levels localized to areas where infections are present [10, 11]. Consequently, α-defensins can be a helpful diagnostic tool whereby increased levels of α-defensins in specific locations are suggestive of an underlying infectious process. This biomarker has been proven effective in periprosthetic joint infections (PJIs) in which diagnosis is important because prosthetic removal is standard care for chronic infections but is associated with significant morbidity, mortality, and financial ramifications [12]. Moreover, in PJI, classic serological and synovial fluid parameters lack sensitivity and specificity, and infections are often secondary to indolent pathogens [13, 14]. The effectiveness of α-defensins in aiding in diagnosing PJI has been proven in several clinical trials, with a meta-analysis showing pooled sensitivity and specificity of 100% and 96%, respectively [13, 14]. Therefore, the use of α-defensins has been incorporated into guidelines for prosthetic joint infection diagnosis [15] and is a commercially available diagnostic test (CD laboratories, Towson, Maryland). Although there are robust data proving the effectiveness of this biomarker in diagnosing PJIs, there are scant data on its use and/or concentrations in the CSF with CNS infections, and no study has evaluated this biomarker in nosocomial ventriculitis. However, three studies do support the use in CNS infections. In one study, α-defensins had higher CSF concentrations in patients with bacterial meningitis than in patients with aseptic conditions and healthy volunteers [16]. In a follow up study, the concentration of α-defensins in the CSF of pediatric patients with meningitis was shown to be significantly elevated compared with those without bacterial meningitis [17]. This study also proved that higher CSF concentrations of α-defensins had greater specificity and sensitivity than lower concentrations [17]. Lastly, in patients with West Nile neuroinvasive disease, there were higher concentrations of α-defensins present compared with patients who did not have neuroinvasive disease [18]. These studies reinforce that neutrophils in the CNS can secrete high concentrations of α-defensins in the CSF when CNS infections are present. Overall, α-defensins hold theoretical promise in aiding in diagnosing ventriculitis. Furthermore, there are several similarities that make diagnosing PJI and ventriculitis difficult but for which α-defensins have been proven to not be affected, thereby aiding diagnosis. In PJI, α-defensins have been demonstrated to be elevated in joints, even despite the use of prolonged systemic antibiotics [13–15]. This is advantageous because conventional CSF parameters and levels of neutrophils are often altered in nosocomial ventriculitis when broad-spectrum antibiotics are used. Likewise, high levels of red blood cells can be present in the joints of patients with PJI, but this does not significantly alter the effectiveness of this biomarker, which is important because in nosocomial ventriculitis, the presence of blood in the CSF can be a common occurrence with some CNS conditions [13, 14]. Additionally, even though α-defensins have very high sensitivities and specificities for diagnosing PJI, they are correlated with conventional parameters obtained from the synovial fluid as part of standard care testing [15]. Therefore, in nosocomial ventriculitis, α-defensins can be paired with conventional CSF diagnostic parameters that are obtained as part of standard care testing. To conduct this research, small proof-of-concept trials will first need to be run, comparing α-defensin concentrations in culture-proven nosocomial ventriculitis with concentrations from patients who do not have nosocomial ventriculitis. To reduce confounding, this would need to be piloted in patients who have EVD in situ and proven or disproven ventriculitis. This would create a foundation to support or refute the use of this biomarker, and if supported, larger studies could then be conducted to establish sensitivity and specificity of this biomarker in ventriculitis. These early studies would first need to use immunoassays, as previously discussed by others [18, 19]. Although the morbidity and mortality associated with nosocomial ventriculitis rivals that of PJIs, the research is unlikely to be chaperoned by large companies given that the financial ramifications are not as significant as those seen in PJI [20]. Therefore, early studies will need to be piloted at a single center or small collaboration of centers and then tested more broadly before being developed into a diagnostic platform. This may be a hindrance because funding would need to be obtained, but the demand for improvements in diagnostic testing and the clinical implications associated with nosocomial ventriculitis support the need to evaluate this novel diagnostic. Without novel diagnostics, patients will continue to either be treated unnecessarily with prolonged antibiotics or have truncated antibiotic courses, thereby placing them at risk for significant morbidity and mortality. Using this biomarker in ventriculitis does have some limitations that will need to be realized. For one, α-defensins are small cationic molecules that could potentially reach the CSF, especially in conditions such as subarachnoid hemorrhages that may disrupt the blood–brain barrier. Subsequently, establishing a cutoff to reduce the confounding serum levels of α-defensins would be needed [17, 18, 21]. As seen in PJI, which has no barrier to protect the joint from systemic α-defensin concentrations, a cutoff of 5.2 mg/L has been established. To maximize the specificity and sensitivity of this diagnostic test, a similar cutoff may be needed but would need to be validated in early ventriculitis studies [20]. Recent literature has shown that interleukin-6 levels are elevated in subarachnoid hemorrhages, as has been shown after joint arthroplasties [22, 23]. Interleukin-6 has been shown to increase the release of α-defensins in patients with COVID-19, but the impact of increased interleukin-6 levels on α-defensins levels in conditions such as subarachnoid hemorrhages is unknown [24]. Consequently, even though surgical trauma induces increased interleukin-6 production, this cytokine will likely need to be evaluated in tandem in early studies to ensure this does not confound the levels of α-defensins. Lastly, the cost of α-defensins in diagnosing PJI is expensive, costing more than 500 dollars per test, but the cost is also associated with additional tests (microbial culture, synovial fluid C-reactive protein, and other synovial fluid tests). The point-of-care lateral flow α-defensin test kit is much cheaper and even less expensive, costing well under 100 dollars per sample. As are serum α-defensin enzyme-linked immunosorbent assays, which could be tailored toward CSF samples to evaluate this biomarker as a diagnostic for ventriculitis. In conclusion, nosocomial ventriculitis can be arduous to diagnosis in part from the limited sensitivity and specificity of traditional CSF parameters and the indolent nature of the causative pathogens. However, the incidence and clinical ramifications of this severe infectious condition should drive research in evaluating α-defensins as a diagnostic biomarker, especially given that this biomarker has been used in a similar fashion with PJIs. As outlined here, α-defensins may hold promise in diagnosing nosocomial ventriculitis, thereby improving morbidity and mortality and reducing the unnecessary use of broad-spectrum antibiotics. Source of Support NonE. Conflicts of interest The author has no competing interests. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Hasbun R Healthcare-associated ventriculitis: current and emerging diagnostic and treatment strategies Expert Rev Anti Infect Ther 2021 19 8 993 999 10.1080/14787210.2021.1866544 33334204 2. 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==== Front Environ Sci Pollut Res Int Environ Sci Pollut Res Int Environmental Science and Pollution Research International 0944-1344 1614-7499 Springer Berlin Heidelberg Berlin/Heidelberg 36449244 24368 10.1007/s11356-022-24368-9 Research Article How does digital technology empower urban green development efficiency in the Beijing-Tianjin-Hebei region—mechanism analysis and spatial effects Yang Yangyang [email protected] 1 Gu Runde [email protected] 1 Ma Shengbin [email protected] 2 Chen Weike [email protected] 1 1 grid.265025.6 0000 0000 9736 3676 School of Management, Tianjin University of Technology, Tianjin, 300384 China 2 grid.30055.33 0000 0000 9247 7930 Department of Construction Management, Dalian University of Technology, Dalian, 116024 China Responsible Editor: Philippe Garrigues 30 11 2022 118 12 9 2022 17 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Under the strategy of “Digital China” and “Sustainable Development,” the synergistic development of digital economy and green economy has become a crucial topic. Based on the panel data of 13 cities in the Beijing-Tianjin-Hebei (BTH) region from 2011 to 2019, this study investigates the direct effect, intrinsic mechanism, and spatial spillover effect of digital technology development (DTD) on urban green development efficiency (GDE). The empirical results show that (1) DTD significantly improves urban GDE in the BTH region, and it passes the endogeneity test, (2) DTD can enhance urban GDE by improving the environmental regulation intensity and technological innovation level in the BTH region; however, the industrial structure optimization weakens the promotion effect of DTD on urban GDE in the BTH region, which shows a “masking effect,” (3) the kernel density estimation method and ArcGIS technology reveal the existence of “digital divide” and GDE differences among cities in the BTH region. Moreover, the spatial distribution pattern of DTD gradually forms “H–H” and “L-L” clusters in the BTH region, and (4) DTD also increases the GDE of neighboring cities through spatial spillover effects in the BTH region, and it passes the robustness test of replacing the spatial weight matrix. This study is important for the BTH region to simultaneously solve economic development and environmental problems in the context of digitalization. Keywords The Beijing-Tianjin-Hebei region Digital technology Green development efficiency Mediating mechanism analysis Spatial spillover effect http://dx.doi.org/10.13039/501100012456 National Social Science Fund of China 18BJY009 17BGL210 Gu Runde Chen Weike http://dx.doi.org/10.13039/501100019062 Tianjin Research Innovation Project for Postgraduate Students 2021YJSB246 Gu Runde ==== Body pmcIntroduction Currently, global warming and resource constraints are becoming increasingly severe. The contradiction between economic development, environmental protection, and resource consumption is prominent in China. Improving green and sustainable development performance is an effective way to resolve the above contradictions, and it is a crucial strategy to achieve high-quality development in China (Zhu et al. 2019; Dong et al. 2021). The Beijing-Tianjin-Hebei (BTH) region accounts for nearly 10% of China’s total economy, and it is a pivotal region for China to explore green and sustainable development (Li et al. 2020). However, the BTH region has prominent problems such as high proportion of heavy industry, resource and environment overload, and serious air pollution. Meanwhile, the BTH region has a disparity in environmental quality, and cities with a high proportion of heavy industry are relatively polluted. Since the release of the Beijing-Tianjin-Hebei Synergistic Development Plan Outline in 2015, deepening the synergistic development of economy, resources, and environment has become the consensus. Liu and Dong (2021) indicated that green development is a growth mode that considers economic growth, environmental protection, and resource conservation. In the context of tighter resource and environmental constraints, the synergistic improvement at urban the green development level has become an essential topic to solve the contradiction between BTH’s environment and economic development (Li et al. 2020). Green development efficiency (GDE) is an effective indicator to measure the synergistic relationship between economy, resources, and environment. It can effectively evaluate the performance of urban green development (Rashidi and Saen 2015). Therefore, this study aims to investigate the new drivers and temporal and spatial evolution characteristics of urban GDE in the BTH region. This can provide new empirical evidence and references for the BTH region to enhance urban GDE and formulate regional synergistic green development policies. With the “Digital China” strategy, the synergistic development of digitalization and greening has become an inherent demand for high-quality economic and social development. The ecological modernization theory shows that digital information technology can mitigate environmental problems (Huber 2000; Popkova et al. 2022) and lays the foundation for exploring the relationship between digital technology development (DTD) and GDE. In practice, the global pandemic of COVID-19 promoted the rapid development of emerging digital technology such as cloud computing, blockchain technology, and artificial intelligence. With the energy crises and environmental problems, the role of digitalization in environmental governance has attracted widespread attention (Wang et al. 2022a), but no general consensus has been reached. The Digital China Development Report (2020) shows that during the 13th Five-Year Plan period, the value of China’s digital economy core industries reached 7.8% of GDP. In 2020, internet users grew to 989 million, and the internet penetration rate increased to 70.4%. Therefore, as the frontier of China’s digital development, what are the spatial and temporal evolution characteristics of DTD in the BTH region? Can DTD improve urban GDE? If this logic is valid, what are the potential mechanisms of DTD affecting urban GDE? Does DTD have a spatial spillover effect on urban GDE? Clarifying the above issues can provide a robust empirical basis and decision reference for the BTH region and even China to use digital technology to improve urban green development. In summary, based on the panel data of 13 cities above the prefecture level in the BTH region from 2011 to 2019, this study conducts the following extended research around the potential mechanism and spatial effect of DTD on urban GDE. (1) combining the connotations of DTD and GDE, this study improves the evaluation system of DTD and urban GDE based on the reference of existing studies; then, this study uses the principal component analysis method and the super-SBM model to measure each urban DTD level and GDE, respectively, (2) after testing the direct impact of DTD on urban GDE with a fixed-effects model, this study explores the potential mechanism of DTD in urban GDE through mediator variables such as environmental regulation, technological innovation level, and industrial structure optimization, (3) this study analyzes the spatial differences and evolutionary characteristics of DTD and GDE among cities; further, the spatial Durbin model is used to identify the spatial spillover effect of DTD on urban GDE, and (4) this study performs robustness tests by replacing the spatial weight matrix, and endogeneity tests using the two-stage least-squares method after setting instrumental variables. Eventually, based on the empirical results, this study proposes corresponding suggestions and expects to provide a reference for the green development of the BTH region and even China from a digital technology revolution perspective. The potential contributions of this study are as follows. (1) From the perspective of digitally empowered green development, this study innovatively examines the intrinsic relationship between DTD and urban GDE from both theoretical and empirical aspects. It broadens the research perspective of GDE and achieves to analyze the comprehensive effect of digital technology on economy, resources, and environment under the same framework. (2) This study explores the theoretical basis and driving mechanism of DTD on urban GDE from three aspects: environmental regulation effect, technological innovation effect, and industrial structure effect. This improves the effect path of DTD on urban GDE that has not been covered by previous studies. (3) Different from the previous static causality studies, this study uses the kernel density estimation method and ArcGIS technology to effectively reveal the dynamic temporal and spatial evolution characteristics and differences of DTD and urban GDE in the BTH region. (4) This study adds the concept of spatial location and examines the spatial correlation and spatial spillover effects of DTD on urban GDE, which deepens the study on DTD and urban GDE from a spatial perspective. The rest of the structure is as follows. The next section is literature review and mechanism analysis to formulate the hypothesis. The third section is research design including data description, variable selection, and model construction. The fourth section is empirical tests including benchmark and mediated regression results analysis. The fifth section is temporal and spatial evolutionary characteristics and spatial spillover effect analysis. The sixth section is robustness tests. The last section is the conclusions, recommendations, and research prospect. Literature review and theoretical mechanism Literature review Green development efficiency The World Bank emphasizes that green development refers to the coordinated development of economic, social, and environmental resources by strengthening ecological protection under the constraints of ecological capacity and resource-carrying pressure (Meng and Qu 2021). As an important indicator to measure urban economic development activities, GDE can measure the performance of green development by measuring the urban input–output efficiency. Previous literature has studied the connotation (Liu et al. 2022a), measurement, influencing factors, and enhancement pathways of GDE. Regarding the measurement approach, some studies chose to construct an evaluation system to investigate the level of green development in different regions (Sun et al. 2018; Fang et al. 2020; Yang et al. 2022a). Another mainstream approach includes nonparametric data envelopment analysis (DEA) and parametric stochastic frontier analysis (SFA) (Tao et al. 2017; Zhao et al. 2020; Yuan et al. 2022). Regarding influencing factors, previous studies found that environmental regulation (Li and Wu 2017; Fan et al. 2022), fiscal decentralization (Song et al. 2018), factor distortions (Lyu et al. 2022; Lin and Chen 2018), technological innovation (Yang et al. 2022b), and financial development (Liu et al. 2019; Yang and Ni 2022) all impact GDE in different degrees. Furthermore, a few scholars studied the spatiotemporal evolution characteristics and spatial differences of GDE (Zhou et al. 2020; Liu et al. 2022a). Digital technology development Since Solow proposed the “Information Technology Productivity Paradox,” academics have continuously debated on the utility of information technology (Wu et al. 2021), which lays a foundation for the research on digital technology. Popkova et al. (2022) indicated that digital technology includes artificial intelligence, cloud computing, information and communication technology (ICT), and other technologies. Existing studies have examined the welfare effects of digital technology from enterprise innovation performance (Usai et al. 2021), real economy development (Tian and Li 2022), green total factor energy efficiency (Gao et al. 2022), and other aspects. However, regarding the environmental effects of DTD, the few available studies have not yet reached consistent conclusions. Among them, some studies considered that DTD is beneficial to improve the environment. Liu et al. (2022b) showed that DTD not only reduces local carbon emissions but also promotes carbon reduction in neighboring cities. Wang et al. (2022b) noted that digital technology effectively contributes to energy sustainability, which is helpful to reduce SO2 pollution. Some studies also argued that digital development requires more energy consumption and releases large amounts of CO2, which can increase the environmental burden. Lee and Brahmasrene (2014) studied the Association of Southeast Asian Nations and noted that ICT positively affects carbon emissions. Lange et al. (2020) proposed that digitization did not decouple economic growth from energy consumption, and that the energy-increasing effect of digitization was greater than the energy-decreasing effect. Furthermore, Asongu et al. (2018) found that ICT did not significantly affect carbon emissions with data from sub-Saharan Africa. In summary, previous studies mainly investigated the unilateral effect of DTD on economic growth, energy consumption, or environmental pollution. The following research needs to be expanded and supplemented. (1) The effect of DTD on urban GDE lacks systematic investigation. Furthermore, the indirect mechanism of how DTD affects urban GDE lacks in-depth analysis. (2) The quantitative assessment of digitalization is still weak, and single indicators such as internet penetration rate and number of websites need to be expanded (Asongu et al. 2018; Yu 2022). (3) Most studies are based on provincial and municipal samples in China and lack studies focusing on regions, which cannot reflect the imbalances and differences among cities within the region. (4) With the wide application of digital technology, DTD and GDE among cities have become more closely connected in space. However, the existing research has fewer effective explanations for their spatial–temporal evolution characteristics and spatial spillover effect. Therefore, due to the special strategic position of the BTH region in green development and digital development, this study explores the direct effect and mechanism path of DTD on urban GDE in the BTH region. Further, this study further identifies the spatial and temporal evolutionary characteristics and spatial spillover effects of DTD and urban GDE. Theoretical mechanism and research hypothesis Direct effect With modern information networks as an important carrier, digital technology is the key production factor in the digital economy era. Wu et al. (2021) indicated that the Internet created by digital technology can reduce the cost of information collection and improve the management efficiency of enterprises. In terms of system optimization, Thompson et al. (2014) revealed that digital technology can optimize production operation processes, decrease unnecessary losses, reduce negative production externalities, and thus achieve the ultimate goal of environmental protection. Digital technology provides advanced technical support for the intelligent and green transformation of enterprises, which can optimize the efficiency of resource allocation, thus reducing resource input and energy consumption (Litvinenko 2020). Li et al. (2021) proposed that the application of digital technology can diagnose and address inefficient links in the energy production, transportation, and consumption systems, thereby improving energy efficiency. The application of digital technology in socioeconomic activities such as living services, transportation operations, and industrial manufacturing can directly or indirectly reduce resource consumption and environmental pollution, which contributes to improving urban GDE (Jiang et al. 2021). For example, digital transportation systems, paperless transmissions, bike-sharing, and car-sharing can reduce unnecessary logistics and commute (Chen and Yan 2020), thus reducing energy consumption and environmental pollution. Therefore, the following hypothesis is proposed: hypothesis 1: digital technology development can positively affect urban GDE in the BTH region. Indirect effects Environmental regulation driving path Sareen and Haarstad (2021) deemed that environmental regulation plays a crucial role in promoting urban green development. The impact of environmental regulation on urban GDE is mainly realized through the combination of both constraint effect and innovation compensation effect (Fan et al. 2022). From the perspective of the constraint effect, digital technology has broadened the way for social agents to participate in environmental regulation and has driven a stronger implementation of environmental regulation policies and tools. It could form greater constraints on enterprises’ production and operation behaviors (Wang et al. 2021). To satisfy the requirements of environmental regulations, enterprises need to increase relevant investments for energy saving and pollution reduction, which reduces the productivity and desired output level of enterprises (Zhang et al. 2018). Thus, environmental regulation negatively affects urban GDE. However, stronger environmental regulations force enterprises to increase technological innovation. Enterprises gradually form green production styles, which can improve production efficiency and operating profitability (Zhao et al. 2018). The compensation effect of innovation promoted by increased profits offsets and exceeds the constraint effect caused by increased costs. It eventually makes enterprises improve their desired output and ecological quality level (Lee et al. 2022). Thus, environmental regulation positively affects urban GDE. Furthermore, the government improves the efficiency of environmental regulation through digital government platforms, which is helpful to improve urban GDE (Janowski 2016). In summary, the following hypothesis is proposed: hypothesis 2: digital technology development enhances urban GDE in the BTH region by increasing the intensity of environmental regulations. Technological innovation drive path The endogenous growth theory believes that technological innovation is the main factor for the improvement of green economy efficiency. Currently, digital transformation and sustainable development effectively stimulate the market demand for green technological innovation and force enterprises to innovate production technology (Llopis-Albert et al. 2021). Proeger and Runst (2020) indicated that digital technology promotes resource sharing and information flow, and is beneficial for enterprises to acquire and integrate innovation resources. According to Schumpeter’s innovation theory, the recombination of production factors is the process of achieving innovation. As a key production factor in the digital era, data resources can break through the limitations of traditional production factors and innovate production technologies and organizational models through resource reorganization (Michaels et al. 2014). Meanwhile, digital technology helps enterprises identify the direction, potential, and path of innovation. It can reduce the cost and risk of innovation and then facilitate green technological innovation. According to the new information technology paradigm, digital technology can improve the way enterprises utilize resources by accelerating technological innovation (Popkova et al. 2022). Enterprises apply innovative energy-saving and environmental protection technologies, cleaner production technologies, and pollution monitoring technologies to production systems, which can improve production efficiency and reduce negative externalities of production and environmental pollution (Wu et al. 2021). As a result, urban economic growth can be decoupled from resource consumption and environmental pollution, and urban GDE will increase. In summary, the following hypothesis is proposed: hypothesis 3: digital technology development can enhance urban GDE in the BTH region by improving the level of technological innovation. Industry structure drive path Previous studies showed that digital technologies such as the Internet of Things can help traditional industries reorganize factor resources, optimize production processes, and realize transformation from extensive to intensive and high efficiency (Cardona et al. 2013). The application of digital technologies such as 5G, big data, and cloud computing will also give birth to high-tech industries with low energy consumption and high added value, which will optimize the industrial structure (Yu 2022). Zhu et al. (2019) indicated that industrial structure optimization is an effective measure to realize green development. The “structural dividend” brought by industrial structure optimization benefits economic growth and improves environmental quality, thus enhancing urban GDE. Szalavetz (2019) revealed that digital technology promotes the transformation of industrial structure from labor-intensive to technology-intensive. From the perspective of production inputs, industrial structure optimization can improve GDE by reducing energy and resource consumption (Zhang et al. 2022a). From the perspective of output, the process of industrial structure transformation from low-technology to high-technology will inevitably improve environmental performance and green productivity, thereby increasing urban GDE (Gu et al. 2022). Furthermore, digital technology can break through the time and space limitations of industrial development, which is conducive to realizing nonspatial industrial agglomeration. Using the externalities of agglomeration, it can accelerate the elimination of low-quality or backward production capacity, which is conducive to driving the development of green transformation (Ding et al. 2022). In summary, the following hypothesis is proposed: hypothesis 4: digital technology development can enhance urban GDE in the BTH region by promoting industrial structure optimization. Spatial spillover effect OMRI (2020) indicated that digital technology can reduce inter-industry differences and spatial boundaries, thus, promoting the rapid flow of resources and technology in a wider region. Similarly, Paunov and Rollo (2016) confirmed that digital technology can enhance knowledge and technology spillover effects, promote cross-regional resource reorganization, and optimize factor allocation and production layout in adjacent regions. It has a positive synergistic effect on the urban GDE of neighboring regions. Yu (2022) showed that the Internet generates positive spatial spillover effects, which significantly promoted the industrial green productivity of local and neighboring regions. However, Zhang et al. (2022b) found that the digital economy failed to exert a spatial spillover effect on promoting the carbon performance of neighboring cities. Meanwhile, it should not be overlooked that the gap between developed regions and backward regions in terms of DTD level is relatively large. The “digital divide” has intensified the siphoning of talents and resources by developed cities (Wang et al. 2022a). The “competition effect” between cities has led to negative externalities in space. The divergence of digital technology further widens the gap in urban GDE. In summary, the impact of DTD on urban GDE may also have a spatial spillover effect, and the total spillover effect depends on the magnitude of the two effects. Therefore, the following hypothesis is proposed: hypothesis 5: digital technology development has a spatial spillover effect on GDE in neighboring cities of the BTH region. In the comprehensive analysis above, a mechanism diagram of DTD in urban GDE is shown in Fig. 1.Fig. 1 The mechanism diagram of DTD in urban GDE in the BTH region Variable definition and model construction Variable definition Explanatory variable: digital technology development (DTD) index Considering the availability of city-level data, this study measures the urban DTD level in four aspects: digital infrastructure, digital factor inputs, digital technology-related outputs, and digital transaction development (Table 1), with reference to Liu et al. (2022b) and Gao et al. (2022), wherein digital transaction development is measured using the Digital Inclusive Finance Index of Peking University. Since Luo et al. (2022) indicated that the principal component analysis method can assign objective weights based on the characteristics of data and indicators, it can avoid the problems of the high correlation of indicators and subjectivity of weight structure. Referring to Liu et al. (2022b), this study uses the principal component analysis method to calculate the variables in Table 1 according to the relevant process to obtain the comprehensive index of DTD.Table 1 Digital technology development level evaluation index system Target layer Guideline layer Indicator layer Measurement variables Digital Technology Development Index Digital factor input Digital industry workforce size The number of employees in the information transmission, computer services, and software industry accounted for the proportion of employees in urban units Digital technology output Computer service Total telecom services per capita Digital infrastructure Internet penetration rate Internet users per 100 people Mobile phone penetration rate Number of mobile phone subscribers per 100 people Digital trading development Digital financial inclusion Digital inclusive finance index Explained variable Green development efficiency (GDE) The increase of GDE relies on the improvement of urban economic development and environmental quality. This is related to the demographic, social, and resource factors involved in the quality of urban development. Therefore, with reference to Zhao et al. (2020), Luo et al. (2022) and Liu et al. (2022a), this study expands the variables from “inputs-desired outputs-undesired outputs” to construct the urban GDE evaluation index system (Table 2). The system includes economic, natural, and social dimensions. Compared with Dong et al. (2021), Lyu et al. (2022), Yang and Ni (2022), and Ding et al. (2022), the input aspects add land input and policy inputs. The desired outputs referred to in Yang et al. (2022a) additionally consider environmental benefits and social benefits. The undesired outputs add domestic waste emission indicate negative environmental outputs.Table 2 Urban green development efficiency evaluation index system Indicators Category Indicator composition Instructions Inputs Cost inputs Capital input Urban fixed asset investment (billion yuan) Labor input Number of employees in urban units at year-end (million people) Land input Urban built-up area (km2) Energy input Total energy consumption (million t of coal) Policy input Technological and environmental protection expenditures in city budgets (billion yuan) Outputs Undesired outputs Exhaust emission Industrial sulfur dioxide emissions (million t) Wastewater discharge Industrial wastewater discharge (million t) Smoke and dust emission Industrial fume and dust emissions (million t) Garbage consumption Domestic garbage (million t) Desired outputs Economic benefit Gross regional product (million yuan) Environmental benefit Greening coverage area (km2) Social benefit Per capita disposable income of urban residents (million yuan) For indicator measurement, Yuan et al. (2022) indicated that the super-SBM model that considers undesired outputs can solve the slackness problem caused by the traditional data envelopment analysis model. It can also solve the problem that the SBM model cannot rank multiple efficiency effective units. Thus, referring to Zhu et al. (2019), this study uses Tone’s (2004) modified super-SBM model to measure urban GDE with the help of the MAXDEA software. Higher GDE values indicate better urban development benefits and vice versa. Mediating variables Environmental regulation (ER) The “Porter hypothesis” argued that ER can stimulate firms to invest in environmental technology transformation and obtain “innovation compensation.” The “cost constraint theory” argued that ER can increase enterprises’ production costs, thus dragging down economic efficiency (Luo et al. 2022). Referring to Song and Han (2022) and Zheng et al. (2023), this study selects industrial SO2 emissions, industrial smoke (dust) emissions, and industrial wastewater emissions to form the ER index system. Then, the entropy method is used to calculate the comprehensive ER index. Technological innovation level (TIL) The increase in TIL is conducive to improving enterprises’ technical efficiency of production and management level of the environment, which may promote the improvement of urban GDE. Referring to Zheng et al. (2023), this study uses the ratio of science and technology expenditure to local fiscal expenditure to measure TIL. Industrial structure optimization (ISO) Industrial structure has a decisive role in the input–output efficiency of resource factors. Therefore, ISO has a significant role in regulating resource consumption and pollution emission (Song et al. 2021). Following Zhu et al. (2019), this study uses the ratio of the added value of the tertiary industry to the added value of the secondary industry to measure ISO. Control variables To more accurately analyze the correlation between DTD and urban GDE, this study controls for the following variables. ① Population density (POP) is measured by the number of people per unit of land area in the city (Yuan et al. 2022). ② Government intervention degree (GID) is measured by the proportion of total fiscal expenditure in GDP (Luo et al. 2022). ③ Capital labor rate (CLR) is expressed by the ratio of the urban annual fixed asset investment to employment (Liu et al. 2022b). ④ Environmental preference (EP) is described by the urban green coverage (Yuan et al. 2022). ⑤ Infrastructure level (INF) is expressed by the urban road area per capita at year end (Gao et al. 2022). All the variables are logarithmically processed before empirical estimation to eliminate heteroskedasticity in the model. Model construction Fixed-effects model To reasonably examine the effect of DTD on urban GDE, this study selects the panel model type by correlation test. According to the Hausman test, the value of the statistic is 35.78, which passes the 1% significance test. The result supports to adopt the fixed effect model. Therefore, model (1) is constructed to verify the direct impact of DTD on GDE:1 lnGDEit=α0+α1lnDTDit+α2lnPOPit+α3lnGIDit+α4lnCLRit+α5lnEPit+α6lnINFit+ui+vt+εit where GDEit is the GDE in period t of city i and DTDit is the DTD level in period t of city i. ui is the unobserved individual fixed effect, vt is the control time fixed effect, and εit is the random disturbance term. Mediating effect model Models (2) and (3) are used to test the role of environmental regulation, technological innovation level, and industrial structure optimization in the path of DTD affecting urban GDE:2 lnESIit=β0+β1lnDTDit+β2lnPOPit+β3lnGIDit+β4lnCLRit+β5lnEPit+β6lnINFit+ui+vt+εit 3 lnGDEit=φ0+φ1lnDTDit+φ2lnESIit+φ3lnPOPit+φ4lnGIDit+φ5lnCLRit+φ6lnEPit+φ7lnINFit+ui+vt+εit where ESIit represents the mediating variables, which are ER, TIL, and ISO in period t in city i. β1 × φ2 is the mediating effect indicating DTD’s indirect impact on urban GDE through ER, TIL, and ISO. Spatial Durbin model (SDM) Since urban DTD has spatial network characteristics such as mobility and replicability, it may have spatial spillover effects. The green development level of a local city will impact neighboring cities, and there may also be a correlation in space. To explore the spatial spillover effect of DTD on urban GDE in the BTH region, the SDM is constructed by adding a spatial interaction term in model (4):4 lnGDEit=λ0+ρWlnGDEit+λ1lnDTDit+φ1WlnDTDit+λ2Xit+φ2WXit+ui+vt+εit where ρ is the spatial autocorrelation coefficient. W is the spatial weight matrix. Xit is the control variables. φ1 and φ2 are the coefficients of independent and control variables’ spatial interaction terms, respectively. Considering the high economic closeness of cities in the BTH region, W used the economic geographic weight matrix to measure the spatial correlation of DTD between cities. Formula (5) is as follows:5 Wi,j=GDPi×GDPjdi,j2,i≠j0,i=i where GDPi and GDPj represent the GDP of the city i and j, respectively. dij represents the geographic distance between city i and city j. We considered that Lesage and Pace (2009) indicated that the regression coefficient of DTD in the SDM model failed to reflect its marginal effect on urban GDE. Therefore, referring to the process of Ding et al. (2022) and Liu et al. (2022b), this study decomposes the direct, indirect, and total effects of DTD on urban GDE using the SDM partial differential method, which will investigate the spatial spillover effects in more detail. Data description and descriptive statistics This study uses 13 cities in the BTH region from 2011 to 2019 as the research samples. The panel data of each city are obtained from the China Statistical Yearbook, China Urban Statistical Yearbook, China Environmental Statistical Yearbook, and statistical bulletins from 2011 to 2019. Furthermore, the missing data are supplemented by the interpolation method. The results of descriptive statistics for each variable are shown in Table 3.Table 3 Descriptive statistics of variables Variables Explanations Average St.d Min Max GDE Green development efficiency 0.752 0.127 0.514 1.039 DTD Digital technology development 0.103 0.0601 0.0328 0.376 ER Environmental regulation 0.109 0.127 6.30e–05 0.815 TIL Technological innovation level 11,526 25,191 165 131,716 ISO Industrial structure optimization 1.205 0.883 0.516 5.169 POP Population density 3339 2115 690 8461 GID Government intervention degree 0.182 0.0567 0.0812 0.394 CLR Capital labor rate 41.49 18.85 8.531 92.13 EP Environmental preferences 42.63 7.242 29.28 92.87 INF Infrastructure level 16.79 3.593 5.260 27.95 Empirical analyses Benchmark regression results The regression results of model (1) are shown in Table 4. The regression results show adjusted R2 > 0.5 in all cases, indicating that the model has a high fit. The coefficient values of lnDTD in columns (1) and (2) are 0.242 and 0.316, and both are significant at the 1% level regardless of whether control variables are included. It indicates that DTD significantly enhances the urban GDE in the BTH region. Specifically, for every 1% increase in DTD, urban GDE will improve by 0.316%, which confirms hypothesis 1. The possible explanation is that the wide application of digital technology within various fields in the BTH region made the production process efficient and low carbon, forming a green development model with low energy consumption and high output. Furthermore, the construction of a “smart city” through digital technology improved the quality of regional environmental governance, ultimately realizing the improvement of urban GDE in the BTH region.Table 4 The regression results of direct effect and explanatory variable on mediating variables Variables (1) lnGDE (2) lnGDE (3) lnER (4) lnTIL (5) lnISO lnDTD 0.242* (11.38) 0.316* (13.34) − 1.986* (− 6.50) 2.706* (16.84) 0.648*** (15.07) lnPOP − 0.024* (-1.59) 0.249 (1.27) 0.599 (5.79) 0.099* (3.58) lnGID − 0.224* (− 7.09) 0.216 (0.53) − 1.079* (− 5.04) 0.420* (7.35) lnCLR − 0.026 (− 1.08) 0.831* (2.72) − 0.811* (-5.04) − 0.153* (-3.55) lnEP 0.202*** (− 2.93) 1.663* (1.88) − 1.332* (− 2.86) 0.456* (3.66) lnINF 0.017 (0.32) 1.889* (2.80) − 0.342 (− 0.96) − 0.333* (− 3.50) Constant 0.282* (5.42) 1.061* (3.38) − 24.015* (− 5.93) 16.758* (7.87) 1.310 (2.30) Year Yes Yes Yes Yes Yes City Yes Yes Yes Yes Yes Adjust R2 0.530 0.688 0.601 0.821 0.892 * p < 0.01, ** p < 0.05, and * p < 0.1 For the control variables, the coefficient of lnPOP and lnGID are − 0.024 and − 0.224, which are significant at the 10 and 1% levels, respectively. They indicate that population size and government intervention suppress urban GDE. The reason may be that the BTH region failed to convert population size into human capital benefits during the development process. The “negative effects” caused by overpopulation such as air pollution and resource and environmental constraints are detrimental to urban green growth. Moreover, the stronger the government intervention in the BTH region, the more local governments tend to adopt administrative environmental regulations for pollution reduction. It creates resource distortion and is not conducive to improving urban GDE. The coefficient of lnEP is 0.202 at the 1% significance level, indicating that environmental preferences contribute to improving urban GDE. Cities with stronger environmental preferences in the BTH region usually focus on ecological and environmental management. They actively guide enterprises to make a green and high-quality transformation, thus obtaining higher urban GDE. Transmission mechanism analysis Referring to the mediation effect test procedure of Ding et al. (2022), Lee et al. (2022), and Luo et al. (2022), this part uses a stepwise test method to validate models (2) and (3). To further ensure the robustness of the mediating test results, this study uses the Bootstrap test to strengthen the verification of the mediating effect. The regression results of the explanatory variable on mediating variables are shown in Table 4. The coefficient of lnDTD in column (3) is − 1.986, with significance at the 1% level. Since ER is measured by three waste emissions, its smaller value indicates the higher intensity of ER, which is a negative indicator. Therefore, it indicates that DTD significantly improves the intensity of ER in the BTH region. The coefficient of lnDTD in columns (4) and (5) is 2.706 and 0.648, and both are significant at the 1% level. It indicates that DTD has obviously contributed to the TIL and ISO in the BTH region. The possible explanations are as follows. First, since the collaborative development of the BTH region, cities have focused on environmental governance and leveraged digital technology to build a “smart city,” which improved the intensity of ER. Second, the BTH region has abundant digital resources and excellent infrastructure conditions for using digital technology to improve urban TIL. Furthermore, the BTH region has achieved deep integration with the industrial sector using digital technologies, thus promoting the ISO of traditional industries. The regression results of the explanatory variable on the explained variable after adding the mediating variables are shown in Table 5. Overall, the inclusion of mediating variables does not affect the promotion effect of DTD on urban GDE. The coefficient of lnER in column (1) is − 0.020, which is significant at the 1% level. However, ER is an inverse indicator. It means that ER significantly increases urban GDE, which confirms hypothesis 3. The above study shows that the “technology spillover effect” of environmental regulation offsets and exceeds the “cost constraint effect” in the BTH region, which is consistent with Porter’s hypothesis. In reality, the ER in the BTH region improved the environmental requirements for industrial development. In summary, ER effectively reduced the emission of pollutants and improved the urban GDE in the BTH region.Table 5 Regression results of DTD on urban GDE through ER, TIL, and ISO Variables (1) lnGDE (2) lnGDE (3) lnGDE lnDTD 0.277* (10.22) 0.162* (3.90) 0.344* (8.27) lnER − 0.020* (− 2.76) lnTIL 0.057* (4.36) lnISO − 0.043 (− 0.81) lnPOP − 0.019 (− 1.30) − 0.058* (- − .62) − 0.020 (− 1.25) lnGID − 0.219* (− 7.15) − 0.162* (− 5.00) − 0.206* (− 5.33) lnCLR − 0.009 (− 0.38) 0.021 (0.84) − 0.032 (− 1.28) lnEP − 0.169** (− 2.48) − 0.126* (− 1.90) − 0.182** (− 2.50) lnINF 0.054 (1.03) 0.036 (0.74) − 0.029 (− 0.58) Constant 0.584* (1.67) 1.064* (2.92) 1.116* (3.46) Year Yes Yes Yes City Yes Yes Yes Adjust R2 0.709 0.735 0.690 * p < 0.01, p < 0.05, and * p < 0.1 In column (2), the coefficient of lnTIL is 0.057, which is significant at the 1% level, indicating that the level of technological innovation promotes the urban GDE of the BTH region. It indicates that the DTD in the BTH region increases urban GDE by enhancing the TIL, so hypothesis 4 is confirmed. Driven by digital technology, the technological advances in the BTH region help improve the efficiency of environmental management. It changes the previous development style of high energy consumption, high pollution, and low technology, and then promotes high-quality green development in the BTH region. In column (3), the coefficient of lnISO is − 0.043, which is significant at the 5% level, indicating that ISO inhibits urban GDE in the BTH region. Specifically, β1 × φ2 = 0.648 × (− 0.043) = − 0.028; however, φ1 = 0.344, and the two values have different signs. The total effect of DTD on urban GDE (α1: 0.316) is smaller than the direct effect (φ1: 0.344). Therefore, the indirect effect of ISO shows a “masking effect.” Next, this part further uses the Bootstrap test to verify the “masking effect” of ISO. The results show that the indirect effect of DTD on urban GDE through ISO is − 0.028, with a 95% confidence interval [LLCI = − 0.186, ULCI = − 0.051], excluding 0. Therefore, the “masking effect” of ISO is further confirmed, and hypothesis 5 is not supported. The reason may be that due to the different development orientations of the three cities in the BTH region, there are obvious gradient differences and imbalances in their industrial structure, which makes ISO negatively affect the urban GDE in the BTH region. Especially, Hebei faced a tough period of industrial restructuring and environmental governance due to its rough industrial structure, and its economic growth rate slowed down. Similarly, through the Bootstrap test, the indirect effects of ER and TIL are 0.039 and 0.154, which are significant at the 5 and 1% levels, respectively. Their 95% confidence intervals are LLCI = 0.007 and ULCI = 0.072 and LLCI = 0.082 and ULCI = 0.226; neither of which excludes 0. Thus, the mediating effects of ER and TIL are further confirmed. Spatiotemporal evolutionary characteristics and spatial spillover effects Spatiotemporal evolutionary characteristics As a nonparametric estimation method, kernel density estimation can describe the distribution conditions of research objects and is specifically described in Zheng et al. (2023). Then, this part uses Matlab16 to plot the kernel density figure (Fig. 2a) to explore the evolution trend of urban DTD. From Fig. 2a, the wave crest of the DTD index for each year is roughly around 0.1 during 2011–2019. Meanwhile, the DTD index shows a trailing trend, indicating the presence of cities with higher DTD levels in the BTH region. Combined with the time trend figure (Fig. 2b), the DTD level of the BTH region shows an overall upward trend, but Beijing, Tianjin, and Shijiazhuang are further away from other cities. This is due to their strengths in economic development and technological innovation, which are favorable to enhancing higher levels of urban DTD.Fig. 2 a Kernel density figure. b Time trend figure Then, this part draws the spatial distribution figure (Fig. 3) by using the ArcGIS 10.7 software to visualize the geographical distribution of urban DTD in the BTH region. Overall, the urban DTD faces the problem of unbalanced development. It reflects in the fact that Beijing’s DTD level is always the highest, and that the DTD level of neighboring cities such as Langfang increases rapidly. Furthermore, Tianjin and Shijiazhuang also consistently maintain high DTD levels. However, southeastern cities such as Handan, Xingtai, Hengshui, and Cangzhou are long-lagging behind in DTD levels. Therefore, it is still an urgent task to narrow the urban DTD gap in the BTH region to avoid the expansion of the “digital divide.”Fig. 3 Spatial distribution of urban DTD levels Similarly, from the kernel density figure (Fig. 4a), the height of the wave crest gradually decreases, and the width subsequently increases, indicating that the differences in urban GDE are becoming more significant. From the time trend figure (Fig. 4b), the urban GDE in the BTH region overall showed a fluctuating upward trend, and the mean value of urban GDE increased from 0.694 in 2011 to 0.831 in 2019. The urban GDE in the BTH region can be roughly divided into the “oscillation period” (2011–2014) and the “rising period” (2015–2019). The early rough growth model of the BTH region caused the growth of urban GDE to fluctuate. With the improvement of regional environmental awareness and sustainable development requirements, cities relied on green transformation and technological innovation to drive the GDE to steadily increase.Fig. 4 a Kernel density figure. b Time trend figure From the spatial distribution figure (Fig. 5), the urban GDE shows a “ladder” characteristic, with apparent high and low partitions. In 2011, only Beijing and Tianjin are highly efficient cities, and most Hebei cities are low efficiency. By 2019, the urban GDE of Tangshan and Langfang had significantly improved, while Xingtai and Handan had dropped to inefficient levels. The growth rate of urban GDE in the northeast part of the BTH region is faster than that in the southwest region. It shows a trend of spreading from the center to the surrounding areas. Furthermore, the “agglomeration” differentiation of cities is obvious. It has formed high-efficiency areas centered in Beijing, Tianjin, and Langfang, and inefficient areas mainly in Handan, Xingtai, and Hengshui.Fig. 5 Spatial distribution of urban GDE Spatial correlation test Before exploring the spatial effect of DTD on urban GDE, it is necessary to test whether DTD is spatially correlated. This study uses global Moran’s I to test whether the DTD in the BTH region has spatial autocorrelation on a global scale. It reflects the overall characteristics of the spatial correlation of urban DTD. The expression is as follows:6 Moran′sI=n∑i=1n∑j=1nωijyi-y¯yj-y¯∑i=1n∑j=1nωij∑i=1ny-y¯2 Here, n is the total number of spatial units. yi is the DTD level of spatial unit i. ωij is the spatial weight matrix coefficient. Moran’s I ∈ [− 1,1], which measures the overall correlation of spatial unit elements. Moran’s I > 0 means positive correlation, Moran’s I < 0 means negative correlation, and Moran’s I = 0 means no correlation. To more visually display the relative geographic distribution of urban DTD, this study plots the global Moran’s I scatter figure (Fig. 6). The global Moran’s I value of lnDTD is 0.614 and passes the 1% significance test, which indicates that urban DTD has a significant positive correlation in space. Except for Beijing and Tianjin, the distribution of DTD in most cities is concentrated in the first and third quadrants, showing obvious H–H and L-L clustering. Furthermore, the DTD levels of 13 cities in the BTH region have jumped to different degrees. Tianjin shifted from the fourth quadrant to the first quadrant, and the other cities mostly shifted from the third quadrant to the first quadrant. It also shows a positive correlation, indicating that the DTD in the BTH region jumped from generally lower levels to higher levels during 2011–2019. In summary, urban DTD has a significant positive spillover effect, and it is necessary to use a spatial regression model for empirical analysis.Fig. 6 Moran’s I scatter plot for urban DTD Spatial spillover effect This study conducted LR and LM tests to select the appropriate spatial econometric model (Table 6). The LM test results show that LM-lag, robust LM-lag, LM-error, and robust LM-error are significant at the 1% level. It indicates that compared with the spatial auto-regressive model (SAR), the spatial error model (SEM) is more appropriate. The LR test results show that SDM cannot be degraded to the SEM or SAR model. Combined with the Hausman test, the results show that using the fixed-effects model is better than using the random-effects model. Therefore, this study uses SDM with fixed effects to test the spatial spillover effect.Table 6 Spatial econometric model tests Inspection content Test method Statistical values P-value SEM and SAR model test LM-error 17.049 0.000 Robust LM-error 11.571 0.001 LM-lag 9.357 0.002 Robust LM-lag 19.971 0.000 Simplified test of SDM LR-SDM-SAR 65.26 0.000 LR-SDM-SER 58.10 0.000 Similar to Fan et al. (2022), this study decomposes the effect of DTD on urban GDE into direct, indirect, and total effects (Table 7). The coefficient of lnDTD in column (1) is 0.120 and significant at the 5% level, indicating that DTD improves the local urban GDE in the BTH region. The spatial lag term of lnDTD in column (2) is 0.687 and significant at the 1% level, indicating that the local DTD has a positive spillover effect on the GDE of neighboring cities in the BTH region. The coefficients of lnDTD in columns (3), (4), and (5) are significantly positive at the 5 and 1% levels, which indicates that DTD positively affects the GDE in both local city and neighboring cities in the BTH region. Specifically, the coefficient of lnDTD in column (4) is larger than that in column (3), which confirms that urban DTD has a strong spatial spillover effect on the BTH region. The possible explanation is that the increased level of local DTD improves green production technology through the technology spillover effect. Thus, it positively affects the local urban GDE in the BTH region. Meanwhile, neighboring cities can imitate or absorb high-level technology and production experience at a lower cost, and local DTD has a “diffusion effect” on neighboring cities. Therefore, local DTD can promote the improvement of GDE in neighboring cities of the BTH region.Table 7 Spatial Durbin model regression results Variables (1) lnGDE (2) Spatial lag term lnGDE (3) Direct effect lnGDE (4) Indirect effect lnGDE (5) Total effect lnGDE lnDTD 0.120 (2.31) 0.687* (6.80) 0.107 (2.15) 0.644* (5.39) 0.751* (7.66) lnPOP 0.006 (0.39) 0.199* (3.84) 0.002 (0.09) 0.179* (3.75) 0.181* (3.23) lnGID − 0.179* (− 6.95) − 0.118 (− 1.14) − 0.175* (− 6.37) − 0.113 (− 1.12) − 0.288* (− 2.84) lnCLR − 0.076* (− 2.60) − 0.052 (− 0.75) − 0.073 (− 2.29) − 0.039 (− 0.59) − 0.111 (− 1.48) lnEP 0.221* (3.31) 0.647* (3.66) 0.222* (3.07) 0.614* (3.29) 0.836* (3.65) lnINF 0.071* (1.77) 0.276** (2.35) 0.067* (1.65) 0.256 (2.04) 0.327 (2.26) Spatial rho − 0.087* (− 0.53) Variance sigma2-e 0.004* (7.80) Adjust R2 0.593 * p < 0.01, ** p < 0.05, and * p < 0.1 For the control variables, the spatial lag term of lnPOP in column (2) is significantly positive at the 1% level, indicating that POP can raise the GDE of neighboring cities in the BTH region. Fang et al. (2020) showed that over-concentration of population inhibits urban GDE. Local cities share a large amount of population migration from neighboring cities, which will increase the GDE of neighboring cities. The coefficients of lnGID and lnLCR are significantly negative at the 1% level in column (1) and not significant in column (2). It indicates that GID and LCR suppress local urban GDE and that the suppression effect is regional with no spatial spillover in the BTH region. The possible reason is that with the increase of GID, the BTH governments tend to use stricter administrative environmental regulations to constrain social production and development. Due to resource misallocation and green transition, the productivity of enterprises fluctuates in the short term, which is not conducive to improving urban GDE. The relatively small correlation coefficients show that little green benefits are obtained in the short term from fixed asset investments in the BTH region. The coefficients of lnEP and lnINF are significantly positive at the 1 and 10% levels in column (1), and the spatial lag term in column (2) is significantly positive at the 1 and 5% levels. It indicates that EP and INF raise the GDE of both local city and neighboring cities in the BTH region. With stronger environmental preferences in the BTH region, cities have cooperated in environmental management and developed a green economy that has reaped effective benefits. Furthermore, the increase in infrastructure level has achieved regional connectivity, thus promoting regional green synergistic development in the BTH region. Robustness tests Replacement space weight matrix This part uses the inverse geographic distance matrix to verify the spatial spillover effect of DTD on urban GDE. The regression results of the SDM are shown in Table 8. Except for the significant reduction of EP and INF, the signs of regression coefficients of other variables are consistent with the above studies. Only the size and significance of the values are relatively improved, which shows that DTD still has a significant spillover effect on urban GDE in the BTH region. This confirms the robustness of the above empirical results.Table 8 Robustness test: SDM regression results Variables (1) lnGDE (2) Spatial lag term lnGDE (3) Direct effect lnGDE (4) Indirect effect lnGDE (5) Total effect lnGDE lnDTD 0.451* (12.94) 1.580* (6.37) 0.395* (9.28) 0.977* (3.79) 1.372* (4.81) lnPOP 0.039* (2.70) 0.341*** (4.44) 0.024* (1.83) 0.224* (3.68) 0.248* (3.62) lnGID − 0.367* (− 11.90) − 1.793* (− 8.21) − 0.297* (− 7.98) − 1.161* (− 4.57) − 1.458*** (− 5.21) lnCLR − 0.051* (− 1.90) − 0.600* (− 4.12) − 0.025 (− 0.94) − 0.409* (− 3.44) − 0.433* (− 3.30) lnEP 0.110 (1.97) 0.443 (1.16) 0.102* (1.80) 0.301 (1.02) 0.403 (1.29) lnINF 0.076 (1.63) 1.042* (3.83) 0.034 (0.69) 0.736* (2.92) 0.770*** (2.71) Spatial rho − 0.488* (− 1.92) Variance sigma2-e 0.004* (7.95) Adjust R2 0.551 * p < 0.01, ** p < 0.05, and * p < 0.1 Endogeneity test Considering the possible reverse causality between urban GDE and DTD, this study uses an instrumental variable method to alleviate the endogeneity problem. Referring to Bartik (2009), this study constructs an instrumental variable “Bartik instrument,” that is, the interaction term (lnDTDi,t−1 × ΔlnDTDt,t−1) of the lagged one-period term (lnDTDi,t−1) and the first-order difference term (ΔlnDTDt,t−1). The reasons are as follows: first, the lag period of the DTD index is significantly correlated with urban GDE, so there is no weak instrumental variable; second, the interference term of the current period cannot affect the result of the lag period of the DTD index, which satisfies the exogenous constraint. Based on this instrumental variable, the two-stage least squares method (IV-2SLS) is used for estimation. The model is as follows:7 lnDTDit=β0+β1lnIVit+β2lnPOPit+β3lnGIDit+β4lnCLRit+β5lnEPit+β6lnINFit+ui+vt+εit 8 lnGDEit=β′0+β′1lnDTDit+β′2lnPOPit+β′3lnGIDit+β′4lnCLRit+β′5lnEPit+β′6lnINFit+ui+vt+εit As shown in Table 9, the coefficients of instrumental variables in the first-stage regression results are significantly positive. The unidentifiable test (Kleibergen–Paap rk LM test) is significant at the 1% level. For the weak instrumental variable test, the Kleibergen–Paap Wald rk F statistic is higher than 16.38 (the critical value at the 10% level of stock Yogo weak identification test). The above test proves the selection of the instrumental variable is reasonable. After considering the endogeneity, the coefficient of lnDTD is still positive, and the regression results in the second stage pass the significance test at the 1% level. It also shows that DTD can improve urban the GDE in the BTH region, which confirms the robustness of the previous regression results.Table 9 Robustness test: IV-2SLS regression results Variables Phase 1 lnDTD Phase 2 lnGDE lnDTDi,t−1ΔlnDTDt,t−1 0.405 (3.05) lnDTD 0.453* (4.98) Controls Yes Yes Year Yes Yes City Yes Yes Constant − 2.549 (− 2.13) 1.492* (3.41) Adjust R2 0.667 N 104 104 Kleibergen–Paap rk LM statistic 7.561 [0.006] Kleibergen–Paap Wald rk F statistic 19.93 {16.38} () is the standard error value, [] is the p value, and {} is the critical value at the 10% level of the stock Yogo weak identification test. * p < 0.01 and p < 0.05 Conclusions and recommendations In the critical period of China’s economic digitalization and green development, how to use digital technology to promote urban green development has become the focus of practice and academia. In this context, based on the panel data of 13 cities in the BTH region from 2011 to 2019, this study examines the effects and mechanisms of DTD on urban GDE with multi-dimensional by using the fixed effect model, mediation effect model, and spatial Durbin model. This study provides a strong empirical basis for Chinese regions to exert the driving effect of digital tools on urban green development. The following interesting and noteworthy findings can be identified from the empirical analysis.Unlike the conclusion of Asongu et al. (2018), the results confirm that DTD is conducive to improving urban GDE in the BTH region. The mediating effect test shows that DTD increases urban GDE by improving environmental regulation intensity and technological innovation level. Interestingly, the industrial structure optimization shows a “masking effect.” This is different from Zhu et al. (2019), which concludes that industrial structure adjustment can improve GDE. The DTD is generally on an upward trend in the BTH region, but there is a “digital divide” between cities. The urban GDE shows a fluctuating upward trend, which can be divided into “shock period” and “rising period.” The geographical distribution shows a “ladder” characteristic, with obvious high-low partitions. The DTD shows a positive spatial autocorrelation in the BTH region, exhibiting a spatial distribution pattern of “H–H” and “L-L” clustering. The DTD has a positive spatial spillover effect on urban GDE in the BTH region. It indicates that DTD promotes the GDE of neighboring cities. Around the above conclusions, this study proposes the following recommendations.The BTH region should increase the construction of digital infrastructures such as 5G and artificial intelligence. Meanwhile, the BTH region should fully utilize digital technology to empower the green transformation for key emission and carbon reduction fields in life services, transportation, and industrial manufacturing. By using digital technology to optimize production processes and resource allocation efficiency, it is possible to increase productivity while reducing environmental pollution. In this way, the urban GDE can be improved. This can provide a Chinese paradigm for the new pattern of global green development. ① In terms of environmental regulation, the BTH region can use digital technology to innovate intelligent environmental regulation manners. It can help the government to propose targeted environmental management strategies. Moreover, the BTH region needs to improve the digital government platform and environmental management information system. This will facilitate the government to formulate dynamic urban green development policies according to social needs. ② In terms of technological innovation, the BTH region should use digital technology to promote green technological innovation. Through clean technology innovation, enterprises can develop green and low-carbon production methods. Furthermore, the BTH region can build a collaborative innovation platform to acquire, integrate, and share innovation resources. It can help enterprises innovate production technology through resource restructuring, thus increasing productivity. ③ In terms of industrial structure optimization, the BTH region should vigorously develop high-tech industries with low energy consumption and high added value spawned by digital technology. Meanwhile, the BTH region needs to deepen the intelligent use of digital technology in manufacturing to improve the energy use efficiency and environmental management level of traditional industries. This will help the traditional industry develop a green manufacturing mode and boost the green development of the city. Due to the differences in digital resources and green development among cities, the BTH region should properly plan the progress and patterns of DTD and green development among cities. Beijing and Tianjin should exert the radiation-driving effect of DTD and increase the spillover effect of their knowledge and technology to neighboring cities. Meanwhile, the BTH government should formulate favorable tax concessions and fiscal expenditure policies to support the DTD and green development in relatively backward cities. This will promote the synergistic development of digitalization and greening in the BTH region. Nevertheless, there is still the following space to be filled. First, the multi-layer measurement system of DTD under the Chinese background still needs to be refined and supplemented. Second, this study mainly controls the variables at the city level and lacks discussion on the control variables of financial development and foreign trade. Furthermore, this study only briefly analyzes the spatial evolution of DTD and GDE in the BTH region. On this basis, subsequent research can focus on analyzing their spatial logical relationship and other impact mechanisms. Author contribution Yangyang Yang: conceptualization, methodology, formal analysis, data acquisition, visualization, validation, and writing (original draft preparation and reviewing and editing). Runde Gu: formal analysis, data acquisition, validation, writing (reviewing and editing), and funding acquisition. Shengbin Ma: data Acquisition, visualization, and writing (original draft preparation). Weike Chen: conceptualization, methodology, formal analysis, writing (reviewing and editing), supervision, and funding acquisition. Funding This research is supported by the National Social Science Foundation of China (No. 18BJY009), National Social Science Foundation of China (No. 17BGL210), and Tianjin Research Innovation Project for Postgraduate Students (No. 2021YJSB246). Data availability Available upon request by contacting the author. Declarations Conflict of interest The authors declare no competing interests. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References Asongu SA Roux SL Biekpe N Enhancing ICT for environmental sustainability in sub-Saharan Africa Technol Forecast Soc 2018 127 209 216 10.1016/j.techfore.2017.09.022 Bartik T (2009) How do the effects of local growth on employment rates vary with initial labor market conditions? 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==== Front Cell Death Differ Cell Death Differ Cell Death and Differentiation 1350-9047 1476-5403 Nature Publishing Group UK London 36450825 1095 10.1038/s41418-022-01095-9 Article PML at mitochondria-associated membranes governs a trimeric complex with NLRP3 and P2X7R that modulates the tumor immune microenvironment http://orcid.org/0000-0003-4530-617X Missiroli Sonia 1 http://orcid.org/0000-0002-8617-2705 Perrone Mariasole 1 Gafà Roberta 2 Nicoli Francesco 3 http://orcid.org/0000-0002-5752-3318 Bonora Massimo 1 Morciano Giampaolo 1 Boncompagni Caterina 1 Marchi Saverio 4 http://orcid.org/0000-0002-2725-5551 Lebiedzinska-Arciszewska Magdalena 5 http://orcid.org/0000-0001-5869-6301 Vezzani Bianca 1 Lanza Giovanni 2 Kricek Franz 6 Borghi Alessandro 7 Fiorica Francesco 8 http://orcid.org/0000-0002-6702-9735 Ito Keisuke 9 Wieckowski Mariusz R. 5 http://orcid.org/0000-0003-3566-1362 Di Virgilio Francesco 1 http://orcid.org/0000-0002-0344-4841 Abelli Luigi 10 http://orcid.org/0000-0001-7108-6508 Pinton Paolo 1 http://orcid.org/0000-0002-2494-7405 Giorgi Carlotta [email protected] 1 1 grid.8484.0 0000 0004 1757 2064 Department of Medical Sciences, Section of Experimental Medicine and Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy 2 grid.8484.0 0000 0004 1757 2064 Department of Translational Medicine, University of Ferrara, Ferrara, Italy 3 grid.8484.0 0000 0004 1757 2064 Department of Chemical, Pharmaceutical and Agricultural Sciences, University of Ferrara, Ferrara, Italy 4 grid.7010.6 0000 0001 1017 3210 Department of Clinical and Molecular Sciences, Marche Polytechnic University, Ancona, Italy 5 grid.419305.a 0000 0001 1943 2944 Laboratory of Mitochondrial Biology and Metabolism, Nencki Institute of Experimental Biology, Warsaw, Poland 6 NBS-C Bioscience & Consulting GmbH, Vienna, Austria 7 grid.416315.4 Department of Medical Sciences, Section of Dermatology and Infectious Diseases, University Hospital of Ferrara, Ferrara, Italy 8 Department of Radiation Oncology and Nuclear Medicine, AULSS 9 Scaligera, Verona, Italy 9 grid.251993.5 0000000121791997 Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Departments of Cell Biology and Medicine, Albert Einstein College of Medicine, Bronx, NY USA 10 grid.8484.0 0000 0004 1757 2064 Department of Life Sciences and Biotechnology, Section of Biology and Evolution, University of Ferrara, Ferrara, Italy 30 11 2022 113 21 6 2022 3 11 2022 11 11 2022 © The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Uncontrolled inflammatory response arising from the tumor microenvironment (TME) significantly contributes to cancer progression, prompting an investigation and careful evaluation of counter-regulatory mechanisms. We identified a trimeric complex at the mitochondria-associated membranes (MAMs), in which the purinergic P2X7 receptor - NLRP3 inflammasome liaison is fine-tuned by the tumor suppressor PML. PML downregulation drives an exacerbated immune response due to a loss of P2X7R-NLRP3 restraint that boosts tumor growth. PML mislocalization from MAMs elicits an uncontrolled NLRP3 activation, and consequent cytokines blast fueling cancer and worsening the tumor prognosis in different human cancers. New mechanistic insights are provided for the PML-P2X7R-NLRP3 axis to govern the TME in human carcinogenesis, fostering new targeted therapeutic approaches. Subject terms Cancer microenvironment Cell biology Inflammasome https://doi.org/10.13039/501100003196 Ministero della Salute (Ministry of Health, Italy) GR-2019-12369646 Missiroli Sonia https://doi.org/10.13039/501100005010 Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research) ID: 26665 IG 22883 IG-23670 IG-23670 Perrone Mariasole Di Virgilio Francesco Pinton Paolo Giorgi Carlotta National Science Centre, Poland (UMO-2018/29/B/NZ1/00589https://doi.org/10.13039/100010663 EC \| EU Framework Programme for Research and Innovation H2020 \| H2020 Priority Excellent Science \| H2020 European Research Council (H2020 Excellent Science - European Research Council) 853057-InflaPML Giorgi Carlotta ==== Body pmcIntroduction Promyelocytic Leukemia Protein (PML), a nuclear moonlighting protein with an established role in genetic control, also acts at the endoplasmic reticulum (ER)-mitochondria interface, namely, at mitochondria-associated membranes (MAMs) [1]. PML behaves as a tumor suppressor, regulating cell fate via Ca2+ transfer to mitochondria and thereby regulating cellular metabolism and death [2, 3]. Recently, MAMs were identified as platforms for inflammatory signaling that are regulated by the multiprotein complex NOD-like receptor family, pyrin domain containing-3 protein (NLRP3) inflammasome [4]. At rest, NLRP3 resides at the ER, whereas in response to danger signals it redistributes to MAMs together with its adapter ASC (apoptosis-associated speck-like protein), driving inflammasome assembly and maturation thus leading to IL-1β and IL-18 secretion [4, 5]. Inflammasome activation leads to defense against several pathogens, but lack of shutdown may be attributed to many diseases, including neurodegenerative, metabolic, and autoimmune diseases as well as cancer [6]. The negative feedback dampening NLRP3 inflammasome activation is poorly understood but essential [7]. The purinergic P2X7 receptor (P2X7R) is one of the most potent activators of the NLRP3 inflammasome. Well known to be a ubiquitous damage-associated molecular pattern (DAMP) receptor and to trigger the release of substantial amounts of IL-1β and IL-18 in response to extracellular ATP [8, 9], P2X7R was recently shown to interact with NLRP3 directly [10]. Here, we put forward the hypothesis of a new PML function in macrophages as a modulator of the P2X7R-NLRP3 axis at MAMs for a new understanding of the rational design of novel anti-inflammatory therapies. Results The PML/NLRP3/P2X7R complex at MAMs Membranes at the ER-mitochondria interface (mitochondria-associated membranes; MAMs) have been reported as a platform for activating the NLRP3 inflammasome [4]. We previously reported that PML localizes at the ER and mitochondria contact sites, forming PML MAMs-bodies (PML-MaBs) and acting as a scaffold protein to regulate mitochondrial metabolism and cell death [2, 3]. Therefore, we hypothesized that PML at MAMs plays a role in controlling NLRP3 activation to trigger inflammation. Co-immunoprecipitation (Co-IP) and proximity ligation assays (PLA) revealed close proximity of PML and NLRP3 in macrophages ER (Fig. 1A, B, and Supplementary Fig. S1A).Fig. 1 PML at MAMs orchestrates the interaction between P2X7R and NLRP3. A Endoplasmic reticulum (ER) fraction obtained from the liver of wild-type (WT) mice was used for co-immunoprecipitation (Co-IP) of endogenous PML with NLRP3. Using PML as a bait, the levels of NLRP3 were detected, showing the interaction between NLRP3 and PML. B Proximity ligation assay (PLA) showing the interaction of NLRP3 with PML (red dots) in the ER fraction of WT resident peritoneal macrophages (pRMØs) left untreated or treated with LPS and nigericin. PDI (green) was used as an ER marker (scale bars, 20 μm). In the bottom panel, the zoomed regions display interaction sites at the ER (scale bar, 2 μm). In the upper right panel, the columns show the quantification of the PLA signal between NLRP3 and PML. Bars: mean ± SEM. (n = 3 independent experiments) C Confocal immunofluorescence staining images of NLRP3 (red) and calnexin (used as an ER marker, green) in WT and Pml−/− pRMØs untreated or treated with LPS and nigericin (scale bars, 5 μm). Merged images of the two immunostainings are shown. The right panel shows the proportion of clustered NLRP3 on the total NLRP3 signal. Bars: median. p < 0.05. D Left panels show immunoblots of subcellular fractions isolated from WT and Pml−/− BMDMs. Where indicated, cells were exposed to LPS and nigericin treatment. The following markers were used: IP3R3 for the ER, VDAC for mitochondria (Mp, mito pure), Sigma 1-R for MAMs, and GAPDH for Cyt (cytosol). H: homogenate. Right panels show the quantification of the P2X7R and NLRP3 proteins in the ER and MAM fractions normalized to the amounts of IP3R3 and Sigma 1-R. The middle lines of each bar represent the mean values (n = 3 independent experiments). E ER fractions obtained from the livers of WT and Pml−/− mice were used for the Co-IP of endogenous NLRP3 with P2X7R. Using NLRP3 as bait, the presence of P2X7R was detected, showing that the interaction between NLRP3 and P2X7R depends on whether PML is lacking or after inflammasome activation. The lower panel shows the fold change of the NLRP3-P2X7R interaction; the middle lines of each bar represent the mean values (n = 3 independent experiments). F PLA shows the interaction between P2X7R and NLRP3 (red dots) in the ER of WT and Pml−/− pRMØs, untreated or treated with LPS and nigericin. PDI (green) was used as an ER marker. Scale bars, 20 μm. Higher magnifications show interaction sites at the ER (scale bars, 2 μm). In the right bar graphs, quantification of the number of PLA red dots per cell revealed an increased P2X7R-NLRP3 interaction in Pml−/− pRMØs. Data are shown as the mean ± SEM. Two independent experiments were carried out in triplicate. p < 0.05, ***p < 0.0001. G Co-IP of endogenous PML and P2X7R in ER fraction obtained from WT mouse liver (as in a). Using PML as bait, the expression of P2X7R was detected, showing the interaction between P2X7R and PML. H Representative PLA (red dots) images of WT pRMØs untreated or treated with LPS and nigericin. PDI (green) was used as an ER marker. Scale bars, 20 μm. Higher magnifications show interaction sites at the ER (scale bars, 2 μm). The column graph on the right shows a quantitative analysis of the PLA signal between PML and P2X7R. Bars: mean ± SEM. Two independent experiments were carried out in triplicate. We then investigated the ER localization of NLRP3 in wild-type (WT) and PML-depleted (Pml−/−) cells under both the resting condition and after treatment with the inflammasome inducers lipopolysaccharide (LPS) and nigericin. Co-immunostaining of NLRP3 and calnexin, an ER marker, revealed enhanced accumulation of NLRP3 at the ER in stimulated resident peritoneal macrophages (pRMØs) from Pml−/− mice (Fig. 1C). The purinoceptor P2X7R is highly expressed within cells of the innate immune system, including macrophages, and its activation is a powerful signal to activate the NLRP3 inflammasome [11]. We thus examined whether P2X7R, in addition to its canonical plasma membrane expression, also localizes to the ER/MAM compartments, where NLRP3 resides and accumulates upon its activation, to mediate NLRP3 complex assembly and maturation. We performed subcellular fractionation analyses of WT and Pml−/− bone marrow-derived macrophages (BMDMs) (Fig. 1D) at rest and after inflammasome stimulation with LPS and nigericin, focusing on the mitochondria, ER, and MAM fractions. As expected, NLRP3 was detected in both the ER and MAM compartments, redistributing more to MAMs in Pml−/− cells than in WT cells after inflammasome activation (Fig. 1D). Consistently, we revealed a previously unknown localization of P2X7R at the ER/MAMs, wherein it accumulated considerably more in the absence of PML than in WT conditions (Fig. 1D). Furthermore, Co-IP and PLA revealed a stronger interaction between NLRP3 and P2X7R at the ER/MAMs in the absence of PML (Fig. 1E, F and Supplementary Fig. S1B). Given the predominant role of PML as a scaffold protein, we examined its potential to interact with and function in complex with NLRP3 and P2X7R at the ER/MAMs. PML, NLRP3, and P2X7R co-immunoprecipitated at the ER (Fig. 1A, E, G and Supplementary Fig. S1C), suggesting that they may form a trimer. Such findings were further confirmed by PLA, which showed a direct interaction between endogenous PML and endogenous P2X7R in ER macrophages (Fig. 1H), similar to what was observed for PML and NLRP3 (Fig. 1B). The capability of NLRP3, P2X7R, and PML to form a trimolecular binding complex was assessed by qualitative surface plasmon resonance (SPR) measurements. In sequential injection experiments, we assessed the simultaneous binding of PML and P2X7R as analytes of NLRP3, immobilized on an optical sensor chip surface by GST tag capturing. The resulting sensorgrams (Supplementary Fig. S1D) showed that binding signals on the NLRP3 ligand were generated for both analytes, indicating a trimolecular interaction. The experiment shown in Supplementary Fig. S1E revealed that the two analytes PML and P2X7R were also able to interact with each other in the absence of NLRP3 (Supplementary Fig. S1E). Altogether, these data suggest that PML, NLRP3, and P2X7R work as triumvirates at MAMs, depicting a multiprotein complex in which PML acts as a negative regulator of NLRP3 function. In PML absence, NLRP3 and P2X7R lose their restraints and tighten together (Supplementary Fig. S1F). PML tones down macrophage NLRP3 arousal NLRP3 inflammasome activation and IL-1β release are thereby primarily triggered by macrophages, which are crucial for generating the inflammatory milieu that can sustain tumor growth [12]. We investigated whether the interaction among PML, P2X7R, and NLRP3 has functional consequences for NLRP3 activation and IL-1β production. pRMØs and BMDMs isolated from WT and Pml−/− mice and PML-silenced human peripheral blood mononuclear cells (PBMCs) (Supplementary Fig. S2A) were first primed with LPS and then stimulated with either the ATP analog benzoyl-ATP (Bz-ATP) or nigericin according to the established protocol to trigger NLRP3 activation and IL-1β release [13]. Lack of PML resulted in increased IL-1β release in response to both stimuli compared to that observed in the WT conditions (Fig. 2A–C). Consistently, the levels of caspase-1 p10 (an autoprocessed fragment of caspase-1) and cleaved IL-1β were higher in the media from activated Pml−/− pRMØs than in that from WT pRMØs (Fig. 2D and Supplementary Fig. S2B). Transduction of an ErPML chimera, which redistributed to only the MAM sites [2], into Pml−/− pRMØs ameliorated the IL-1β release to the levels measured in WT pRMØs, while the nuclear-targeted PML chimera was ineffective [2] (Supplementary Fig. S2C, D). Therefore, the localization of PML at MAMs indicates its relevance to repressing the activation of the NLRP3 inflammasome.Fig. 2 PML deficiency in the host favors increased IL-1β secretion in response to inflammasome activation, which in turn promotes tumor growth. pRMØs (A) and BMDMs (B) from WT (red columns) and Pml−/− mice (black columns) were left untreated or stimulated with LPS and Bz-ATP or LPS and nigericin. The IL-1β levels in the supernatants were determined by ELISA. Error bars indicate the SEM of 3 independent experiments carried out in triplicate. p < 0.05, p < 0.01 C ELISA of mature IL-1β in the supernatants of siRNA-transfected PBMCs upon stimulation with LPS together with Bz-ATP. PBMCs were transfected with scramble or PML siRNA. Error bars indicate the SEM of 3 independent experiments carried out in triplicate. p < 0.01. D The culture media and total cell lysates of pRMØs from WT and Pml−/− mice were collected before and after activation with LPS plus ATP or LPS and nigericin and were resolved by SDS–PAGE. The results shown are representative of 3 independent experiments. ELISA of IL-1β from peritoneal exudate (E) and blood (F) samples from WT (red columns) and Pml−/− mice (black columns) that were untreated or treated with LPS plus nigericin. Bars: mean ± SEM of 3 independent experiments carried out in triplicate. p < 0.01, p < 0.001. G ELISA of IL-1β in Pml−/− pRMØs stimulated with LPS and Bz-ATP after transfection with scramble, siRNA NLRP3 and siRNA NLRC4. Bars: mean ± SEM of 3 independent experiments. p < 0.01. H WT (red) and Pml−/− (black) C57BL/6 mice (7 per group) were injected subcutaneously with B16-F10 melanoma cells (1 × 106). The left graph shows tumor growth for 2 weeks after the injection. The middle graphs show the quantified tumor volumes and weights at 14 days post-injection. Error bars indicate SEM. p < 0.01, *p < 0.0001. The right panel shows representative tumors excised at the same time point (scale bar: 1 cm). I Immunohistochemistry (IHC) revealed much higher IL-1β immunoreactivity (IR) in the peritumoral areas of melanoma-bearing Pml−/− mice than in that of WT mice. TM: tumor mass. Nuclei were counterstained with Harris’ hematoxylin (HH). Scale bars: 50 μm. J Pml−/− C57BL/6 mice were injected subcutaneously with B16-F10 melanoma cells (1 × 106). An experimental group (green; n = 6) was injected IP with IL-1β antiserum (50 µg twice weekly; clone B122; BioXCell), while a blank group (black; n = 6) was injected IP with equal amounts of isotope control antibodies. The left graph shows tumor growth for 14 days after the injection. The middle graphs show the quantified tumor volumes and weights at 14 days post-injection. Error bars indicate SEM. p < 0.05, ***p < 0.001. The right panel shows representative tumors excised at the same time point (scale bar: 1 cm). K IHC revealed an almost complete lack of IL-1β-IR in the peritumoral areas of melanoma-bearing Pml−/− mice treated in vivo with IL-1β antiserum compared with those of the blank group. TM tumor mass. Nuclei were counterstained with HH. Scale bars: 50 μm. To validate these in vitro results, we directly treated WT and Pml−/− mice with LPS in combination with nigericin by intraperitoneal (IP) injection and measured the level of IL-1β 4 h later. The release of IL-1β into the peritoneal exudate, blood, and liver as well as the caspase-1 activity, were strongly increased in the Pml−/− mice compared with the WT mice (Fig. 2E, F and Supplementary Fig. S2E, F). Pharmacological inhibition of NLRP3 activation by MCC950 (a known NLRP3 inhibitor) [14] or AZ10606120 dihydrochloride (a known P2X7R inhibitor) [15] counteracted the IL-1β increase in Pml−/− mice both in vitro and in vivo (Supplementary Fig. S2G–J). To further determine whether PML is capable of controlling the activation of NLRC4 (CARD12, IPAF), another inflammasome complex known to drive IL-1β release [16], we genetically interfered with NLRC4 and NLRP3 in Pml−/− pRMØs. Unlike silencing NLRP3, NLRC4 silencing did not attenuate IL-1β secretion in response to stimulation, indicating that PML is involved in specifically suppressing NLRP3-dependent inflammation (Fig. 2G and Supplementary Fig. S2K). Accordingly, the NLRP3-stimulated secretion of IL-18 was also upregulated in the absence of PML (Supplementary Fig. S2L), whereas this effect was not observed for other pro-inflammatory cytokines independent of NLRP3 stimulation, such as IL-6 (Supplementary Fig. S2M) and TNFα (data not shown). Altogether, these data suggest that PML is a major driver of NLRP3 and P2X7R localization at MAMs, limiting their capacity to activate the inflammasome. PML inactivation boosts tumor growth It is well known that sustained activation of the NLRP3 inflammasome promotes tumorigenesis at several stages [6, 17]. Hence, high levels of IL-1β have been implicated in carcinogenesis and tumor progression [18]. We investigated whether a Pml−/− host, which was skewed toward NLRP3-dependent inflammation, would provide a more permissive environment for tumor proliferation than a WT host. We compared the growth of B16-F10 melanoma and LL/2 Lewis lung carcinoma syngeneic cell lines, which express endogenous PML (Supplementary Fig. S3A). These cells were injected subcutaneously into either WT or Pml−/− mice. Tumor growth was greatly accelerated in Pml−/− mice (Fig. 2H and Supplementary Fig. S3B, C) and associated with the hypersecretion of IL-1β in peritumoral tissues (Fig. 2I and Supplementary Fig. S3D) compared with that observed in WT mice. Cancer cell proliferation is stimulated by inflammation [19]. To assess whether the accelerated tumor progression in the Pml−/− host was dependent on enhanced IL-1β secretion and thus on increased NLRP3 activation, we used an anti-IL-1β serum to deplete the cytokine in the TME. IL-1β immunoblockade dampened tumor growth in the Pml−/− hosts (Fig. 2J, K) without significantly affecting tumor development in WT hosts (Supplementary Fig. S3E). Thus, a fertile environment for accelerated tumor growth in PML-null hosts is generated by the increased release of IL-1β into the TME. Accordingly, knocking down the IL-1β receptor (IL-1R1) in B16-F10 melanoma cells by CRISPR/CAS9 (Supplementary Fig. S3F, G) reduced both B16-F10 melanoma cell proliferation and the tumor size in the Pml−/− hosts (Supplementary Fig. S3H), highlighting the fundamental tumor-promoting role of IL-1β. PML deficiency drives M2 polarization in the TME Macrophages that infiltrate tumor tissues, also known as tumor-associated macrophages (TAMs), are the major component of the highly complex TME, in which cancer cells coexist with immune and nonimmune cells [20]. TAMs preferentially express an M2-like phenotype and promote tumor growth [21]. We investigated whether PML-dependent activation of the NLRP3 inflammasome contributes to the polarization of macrophage phenotypes within the TME. Flow cytometry revealed no significant differences in the percentages of both Ly6C+ and Ly6C- monocytes between WT and Pml−/− mice under resting conditions (Supplementary Fig. S4A), indicating no variation in the number of these circulating cells. We then investigated whether the different amount of IL-1β released into the TME by WT and Pml−/− mice was dependent on the M1/M2 polarization of TAMs. Immunohistochemical and FACS analyses (Supplementary Fig. S4B for gating strategy) revealed in Pml−/− mice a significant shift in TAMs toward the M2 phenotype compared with WT mice, with the loss of M1-associated expression markers (Fig. 3A–C).Fig. 3 Misdirected TAMs accelerate tumor progression in Pml−/− hosts. A Representative tumor sections excised from WT (upper panels) and Pml−/− mice (lower panels), stained with H/E or immunostained for F4/80, iNOS, CD206 (mannose receptor; manR) and Ly6g; nuclei were counterstained with HH. Note the high number of free cells in the peritumoral areas and their larger occurrence in Pml−/− mice. TM: tumor mass. Scale bars: 50 µm. B The peritumoral tissue of Pml−/− mice (black columns) is distinctly enriched with F4/80-, mannose receptor- and Ly6g-immunoreactive cells and almost devoid of iNOS-immunoreactive cells compared with that of WT mice, depicting an immunosuppressive environment. The data are expressed as the mean ± SEM. Significantly different from WT mice p < 0.05, p < 0.01, p < 0.0001. C Absolute number of TAMs (CD3- CD45R- F4/80+ CD11b+)/tumor mass from WT and Pml−/− mice (n = 7 per group). TAM polarization was analyzed by measuring CD86 and MHC-II expression based on the mean fluorescence intensity (MFI). Left panels: the dots indicate the results of individual mice, and the lines indicate the mean values. p < 0.01. Right panels: representative histogram plots from 2 mice/group of CD86 and MHC-II expression in macrophages. D Analysis of tumor growth after bone marrow replacement. WT mice were lethally irradiated to induce myeloablation and then transplanted with bone marrow from WT (PML-WT > PML-WT) or Pml−/− (PML-KO > PML-WT) animals. Bone marrow reconstitution was monitored for 16 weeks (Supplementary Fig. S4 and Methods section), then chimeric mice were subcutaneously injected with 1 × 106 B16-F10 melanoma cells. Tumor growth was monitored at the indicated time points. Numerical results are expressed as the mean ± SEM (n = 4 per group). E Co-culture with activated Pml−/− macrophages showed a larger tumor cell growth-promoting effect compared to that exerted by WT macrophages. B16-F10 cells were co-cultured with pRMØs from WT and Pml−/− mice and treated daily with LPS and Bz-ATP. After one week, the proliferation of B16-F10 cells was analyzed by crystal violet staining (595 nm absorbance). Experiments were performed twice, and the error bars indicate SEM. **p < 0.0001. In most cancer types, TAMs are recruited from bone marrow progenitors [22, 23]. To verify the essential non-cell-autonomous role of Pml−/− immune cells in providing a tumor-supportive microenvironment, we generated bone marrow chimeras (Supplementary Fig. S4C–F). We injected subcutaneously B16-F10 melanoma syngeneic cells into WT mice transplanted with either WT or Pml−/− bone marrow-derived mononuclear cells (BMMNCs) (Fig. 3D). Tumor growth was significantly faster and greater in mice transplanted with Pml−/− cells than in those transplanted with WT BMMNCs (Fig. 3D), highlighting the role of PML-deficient hematopoietic cells in supporting increased tumor growth. Next, to assess the direct involvement of TME macrophages in supporting cancer cell proliferation along the PML/P2X7R/NLRP3 axis, we established a co-culture model. pRMØs from WT and Pml−/− mice were cultured in removable inserts on the top of the plate, activated by LPS and Bz-ATP as inflammasome activators, and incubated with B16-F10 melanoma cancer cells seeded on the bottom of the plate; the cells were separated by a porous membrane that allowed the free exchange of soluble factors and cytokines (Supplementary Fig. S5A). Co-culture with activated Pml−/− macrophages exerted a larger tumor cell growth-promoting effect compared to that of WT macrophages (Fig. 3E). Direct stimulation of cancer cells with LPS had no effect on their growth (Supplementary Fig. S5B). Pretreatment with anti-IL-1β serum before macrophage activation or IL-1R1 knockdown considerably reduced the growth of cancer cells (Supplementary Fig. S5C, D). Accordingly, the H460 human lung cancer cell line in contact with activated PML-silenced PBMCs showed an increased growth rate compared with that of control PBMCs (Supplementary Fig. S5E). Altogether, these results suggest that PML loss in macrophages favors tumor establishment and progression by enhancing IL-1β secretion from M2-polarized TAMs. The PML/NLRP3 axis in human cancers After elucidating the PML-dependent NLRP3 activation pathway, we next explored its clinical relevance in human cancers. Two retrospective cohorts of patients diagnosed with cutaneous melanoma or non-small-cell lung cancer (NSCLC) (see Table S1) were selected. Within these cohorts, we distinguished two groups of patients based on 5-year follow-up data: patients with long survival and NO survival times. Immunohistochemical analysis on melanoma and NSCLC tissue samples (Fig. 4A and Supplementary Fig. S6A) revealed that, compared with the long-survival group, the short-survival patients had more CD68- and CD206-immunoreactive TAMs and greater IL-1β immunoreactivity in the peritumoral stroma (Fig. 4B, C and Supplementary Fig. S6B, C), and these parameters were highly correlated. However, the number of PML-immunoreactive cells did not differ significantly between the two observation groups (data not shown). Indeed, other parameters were carefully evaluated and proven to be very informative, such as (i) the positive linear correlation between inflammatory signs (e.g. number of free cells recruited to the TME) and the IL-1β level in the peritumoral area observed in individual tumor specimens and (ii) the inverse correlation between nuclear and extranuclear PML localization and the release of IL-1β into the TME (Fig. 4A–D and Supplementary Fig. S6A–D).Fig. 4 Inverse correlation between PML expression and IL-1β release in patients’ TME. A Histological sections of human melanoma tissue samples representative also of the peritumoral tissue and belonging to long-survival (up to 5 years) and short-survival (less than 5 years) patients were immunostained for CD68, CD206, IL-1β and PML (see labels). Representative areas are shown at higher magnification to detail the protein expression in macrophages (CD206; arrows) and the differential subcellular localization of PML (arrows). IL-1β levels were closely correlated with the occurrence of CD68+ TAMs, the differential subcellular localization of PML, and poor prognosis. B Patient survival was negatively correlated with TAM M2 polarization and IL-1β expression. C Il-1β expression levels in human melanoma tissue samples and 5-year survival rates. D The nuclear localization of PML was predictive of poor prognosis for melanoma patients. The long-survival group achieved a complete response (CR) without any recurrences, whereas all patients in the short-survival group died within 5 years after diagnosis (Fig. 4C and Supplementary Fig. S6C). IL-1β levels were closely correlated with the numbers of CD68+ or CD206+ TAMs (Fig. 4B and Supplementary Fig. S6B) and might indeed be predictive of poor prognosis after the onset of melanoma or NSCLC (Fig. 4C and Supplementary Fig. S6C). The most important discovery was that PML exhibited differential subcellular localization inside the TAMs in long- and short-survival patients’ tissue samples, with mostly cytoplasmic localization being observed in the former group and nuclear localization being observed in the latter (Fig. 4A, D and Supplementary Fig. S6A, D). Altogether, the data from human samples likely confirm those obtained from mouse models and indicate that a shift in the subcellular localization of PML from an extranuclear region to the TAMs nuclei drives their M2 polarization and promotes tumor development boosted by excessive inflammation. P2X7R/NLRP3 targeting counteracts PML-loss driven tumorigenesis To determine whether targeting NLRP3 could prevent the generation of pro-inflammatory, and tumor-promoting stimuli derived from Pml−/− macrophages, we used three different strategies in Pml−/− mice: (i) NLRP3 blockade by the selective inhibitor MCC950; (ii) genetic deletion of NLRP3; and (iii) genetic deletion of P2X7R, the most powerful NLRP3 activator (Supplementary Fig. S7A, B). Consistent with a reduction in the PML/NLRP3/IL-1β axis, we observed lower amounts of IL-1β in pRMØ, BMDM, peritoneal exudate, and blood samples collected from Pml−/−/P2x7r−/− and Pml−/−/Nlrp3−/− double-knockout (DKO) mice than in those collected from Pml−/− mice (Fig. 5A–D).Fig. 5 Genetic ablation of the P2X7R/NLRP3 axis reduces IL-1β levels and cancer development. pRMØs (A) and BMDMs (B) from Pml−/−, Pml−/−/P2x7r−/− and Pml−/−/Nlrp3−/− mice were left unstimulated or stimulated with LPS plus Bz-ATP. The IL-1β levels in the supernatants were determined by ELISA. Bars: mean ± SEM of 3 independent experiments carried out in triplicate. p < 0.0001. ELISA of IL-1β levels in peritoneal exudate (C) and blood (D) samples from Pml−/−, Pml−/−/P2x7r−/− and Pml−/−/Nlrp3−/− mice after treatment with LPS and nigericin. Bars: mean ± SEM of 3 independent experiments carried out in triplicate. p < 0.001. *p < 0.0001. E Pml−/− (black line) and Pml−/−/Nlrp3−/− (purple line) C57BL/6 mice (6 per group) were injected subcutaneously with B16-F10 melanoma cells (1 × 106). Left panel, the graph shows the tumor kinetics at the indicated time points. The middle panels show the quantified tumor volumes and tumor weights at 14 days post-injection. Error bars indicate SEM. p < 0.01, ***p < 0.0001. The right panel shows representative tumors excised at the same time point (scale bars: 1 cm). F. Pml−/− (black line) and Pml−/−/P2x7r−/− (gray line) C57BL/6 mice (6 per group) were injected subcutaneously with B16-F10 melanoma cells (1 × 106). Tumor growth was analyzed as described in e. Error bars indicate SEM. p < 0.05, p < 0.01, p < 0.001. Scale bar: 1 cm. G Representative tumor sections from specimens of different mouse groups: Pml−/− (upper panels), Pml−/−/Nlrp3−/− (middle panels) and Pml−/−/P2x7r−/− (lower panels). The sections were stained with H/E or immunostained for IL-1β, F4/80, iNOS, mannose receptor (manR) and Ly6g. Nuclei were counterstained with HH. TM: tumor mass. Bars: 50 µm. It is worth noting the massive recruitment of free cells and IL-1β immunoreactivity in the peritumoral areas of Pml−/− mice, which was otherwise hardly detectable in double-knockout mice. H Quantification of the cell density (N/mm2) in peritumoral tissues revealed significantly higher numbers of F4/80-, mannose receptor-, and Ly6g-immunoreactive cells in Pml−/− mice than in Pml−/−/P2x7r−/−and Pml−/−/Nlrp3−/− mice, while no significant differences were found in the numbers of iNOS-immunoreactive cells among the groups. Error bars indicate SEM p < 0.05, *p < 0.001, **p < 0.0001. Moreover, genetic or pharmacological inhibition of P2X7R or NLRP3 drastically reduced B16-F10-induced melanoma growth driven by PML deficiency (Fig. 5E, F and Supplementary Fig. S7C). Interestingly, a significant reduction in the levels of secreted IL-1β, a reduced number of tumor – infiltrating macrophages, and a decreased ratio of M2-like macrophages in peritumoral tissues were observed after NLRP3 inhibition compared with those in Pml−/− mice (Fig. 5G, H). These results strongly indicate a crucial role of NLRP3 in M2 polarization driven by PML loss, which leads to enhanced tumorigenesis. Discussion PML is a tumor suppressor that helps to control gene expression by associating with nuclear bodies (NBs). While PML exerts its main effects in the nucleus, we showed that it is also expressed at MAMs, the region in which the ER and mitochondria are juxtaposed [2]. In this cellular compartment, PML determines the fate of the cell, controlling cell metabolism or death, by modulating Ca2+ transfer to mitochondria [2, 3]. In particular, we proposed that PML forms NBs-like structures (PML-MaBs) at the ER-mitochondria contact sites, where it orchestrates various functions by interacting with several proteins and generating multiprotein complexes. Interestingly, MAMs have been shown to function as a platform for inflammatory signaling regulated by the most widely characterized inflammasome, NLRP3 [4]. Two previous studies revealed a possible liaison between PML and NLRP3 [24, 25]. The earlier study demonstrated that PML could promote NLRP3 activation [24], while the latter proposed PML to be a novel regulator of ASC nuclear localization, thereby limiting NLRP3 activation [25]. To date, the mechanisms that control the formation and activation of NLRP3 at MAMs are unknown, as is the role of PML in this cell compartment. It is worth elucidating this PML-mediated inflammatory response due to obvious clinical implications. In this study, we provide evidence that PML at MAMs represses NLRP3 activation in the context of a molecular triumvirate that includes the purinergic P2X7R. In the ER/MAM compartments, PML has been reported to directly interact with both NRLP3 and P2X7R. These three proteins form a multiprotein complex in which PML plays a fundamental role in counteracting NLRP3 activation mediated by P2X7R. The loss of PML promotes a NLRP3-related cytokine storm in response to stress conditions due to the increased redistribution and interaction of NLRP3 and P2X7R at MAMs. This mechanism is consistent with the hypothesis that PML is involved in retaining ASC, the adapter protein required for proper NLRP3 assembly, and the prevention of inflammasome hyperactivation, in the nucleus. In fact, PML deficiency may lead to higher redistribution of ASC in the cytoplasm, wherein it initiates inflammasome assembly at ER-related compartments together with NLRP3 [25]. We thus revealed that PML loss might boost tumorigenesis via the NLRP3-mediated fueling of the inflammatory response. Indeed, PML deficiency in the TME favors tumor nesting and development by increasing IL-1β secretion. This in turn promotes tumor growth, exerting a direct effect on cancer cells. Tumor growth was strongly reduced by inhibiting host NLRP3, acting on P2X7R, a major inflammasome activator, or directly impeding NLRP3 assembly. Macrophages are the main immune cell type that specifically populates the TME and are highly prevalent in inflammation-sustained tumors. We found that PML-deficient macrophages under stress conditions were skewed toward the M2 phenotype and exacerbated NLRP3-mediated inflammation, exerting a promotional effect on tumor growth via the hypersecretion of IL-1β. Recent findings have indeed revealed the powerful pro-inflammatory potential of M2 macrophages in pancreatic and liver cancer [26, 27], although earlier data primarily associated them with anti-inflammatory action (in contrast to M1). Consistent with our observation from syngeneic mouse models, we found an inverse correlation between PML and IL-1β amounts in the peritumoral areas of patient specimens, and a higher rating of PML inside the nucleus in those with a bad prognosis. Further studies are needed to elucidate the mechanisms that modulate PML localization and expression in the TME. The recent identification of a new PML ubiquitination and degradation pathway [28] supports our findings that host PML loss may drive an immune response that worsens rather than ameliorates cancer, thereby enhancing IL-1β release and opening a new route for PML protein stabilization. In summary, we identified a novel function of PML in undermining the P2X7R/NLRP3 liaison in ER/MAMs. This notion highlights the importance of these structures, first described by our research group and named PML-MaBs. We demonstrated how PML-deficient macrophages would drive tumor progression (in association with poor prognosis) by hyperactivating the IL-1β-driven inflammatory response. Our study thus elucidated pleiotropic tumor-promoting mechanisms due to PML loss, and expanded to outside the tumor, in the TME (Supplementary Fig. S7D). In addition, we suggest that the nuclear localization of PML should be evaluated (with simple, rather inexpensive, and routine analyses) in larger cohorts to determine whether it is predictive of poor prognosis in patients with melanomas, NSCLC, and possibly other solid tumors. Finally, our findings may have implications for severe inflammatory syndromes including pathogen-related ones (as those related to COVID-19) and tissue specific ones (as neuroinflammation) for the following reasons. (i) Several viruses have been shown to disrupt PML-NBs [29]; the same could also happen to PML-MaBs, with consequent mislocalization of PML from the ER/MAM region, which would lead to enhanced IL-1β release; (ii) the induction of IL-1β by herpes simplex virus (HSV-1) was markedly enhanced in PML-deficient BMDMs [25]; this fueling of IL-1β release could presumably also be induced by other pathogens, as SARS-Cov-2, in PML-deficient macrophages; (iii) in brain pathologies NLRP3 dysregulation promotes the neuroinflammatory process exacerbating neuronal death [30]; in this context, NLRP3 driven inflammation might be supported by PML-deficient microglia. The following key questions remain: (i) Can PML-MaBs disorders determine inflammation-related diseases severity (COVID-19 and neurodegenerative disorders)? (ii) Can PML localization at MAMs play a fundamental role in limiting inflammasome activation in response to pathogens (such as SARS-Cov-2)? We believe that these questions are worthy of experimentation. The models we propose might indeed pave the way for new studies on the mechanisms underlying severe inflammatory diseases. Methods Cell culture and transfection B16-F10 melanoma cells (kindly provided by Pier Paolo Di Fiore’s lab at IFOM) were grown in RPMI medium supplemented with nonessential amino acids (Sigma-Aldrich), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (P/S). The LL/2 mouse Lewis lung carcinoma cell line (from Sigma) and H460 cells (kindly provided by the Pagano laboratory [31]) were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS, 1% glutamine, and 1% P/S. Bone marrow-derived macrophages (BMDMs) from WT, Pml−/−, Pml−/−/P2rx7−/− and Pml−/−/Nlrp3−/− mice were isolated as described [32] and differentiated for 7 days in Iscove′s modified Dulbecco′s medium (IMDM) supplemented with 15% FBS (Gibco), 1% glutamine, 1% P/S and 40 ng/ml M-CSF. Resident peritoneal macrophages (pRMØs) from WT, Pml−/−, Pml−/−/P2rx7−/− and Pml−/−/Nlrp3−/− mice were isolated as described [33] and cultured in IMDM supplemented with 15% FBS (Gibco), 1% glutamine and 1% P/S. pRMØs from Pml−/− mice were transfected with siRNAs targeting NLRP3 and NLRC4 using Lipofectamine RNAiMAX (Invitrogen) or with the erPML or nuPML chimeric plasmid using jetPEI-Macrophage (Polyplus transfection). Human PBMCs were isolated as described [34], cultured in RPMI medium supplemented with nonessential amino acids (Sigma-Aldrich), 10% FBS and 1% P/S, and transfected with a siRNA targeting PML using Lipofectamine RNAiMAX (Invitrogen). All cells were grown in a 5% CO2 incubator at 37 °C and tested with a Mycoplasma PCR Detection Kit from Sigma (MP0035-1KT). Reagents ELISA kits for mouse IL-1β, human IL-1β, and mouse IL-6 were purchased from R&D Systems, for mouse IL-18 was purchased from MBL. The anti-IL-1β (clone B122) was purchased from BioXCell. The PML, NLRP3 and NLRC4 siRNAs were purchased from Thermo Fisher Scientific. AZ10606120 dihydrochloride was purchased from Tocris; lipopolysaccharide (LPS) from Escherichia coli 055:B5 (L2880), 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-trisphosphate triethylammonium salt 95% (Bz-ATP) (B6396), ATP (A3377) and nigericin sodium salt (N7143) were purchased from Sigma, and MCC950 (HY-12815A) was purchased from Invivogen. Caspase-1 assay kit was from Abcam. Immunoblotting Total cell lysates from BMDM, pRMØs, PBMCs, B16-F10, and LL/2 cells were lysed in a buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, and 0.2% SDS supplemented with protease and phosphatase inhibitor cocktails. Protein extracts were quantified by the Lowry method, and 10 μg of proteins were loaded and separated on a NuPAGE Novex 4–12% Bis-Tris Gel (Life Technologies) and transferred onto a nitrocellulose membrane. Primary antibodies were raised against human PML (Abcam, ab72137), mouse PML (Millipore, MAB3738), mouse PML (Abcam, #67761), the IL-1 receptor (I/IL-1R1, Abcam, ab107270), Sigma-1R (Sigma-Aldrich, HPA018002), IP3R3 (BD Biosciences, 610312), GAPDH (Cell Signaling, #2118), VDAC (Abcam, ab15895), β-tubulin (Cell Signaling, #55685), NLRP3 (Adipogen, AG-20B-0014-C100), P2X7 (Alomone, APR-004), P2X7 (Sigma-Aldrich, P8232), Caspase-1 (Novus Biological, #14F468), IL-1β (Cell Signaling Technology, #12242), and β-Actin (Sigma-Aldrich, #A2668). HRP-conjugated antibodies (#31430; #31460. WB, 1:5,000 from Thermo Fisher Scientific) were used, followed by detection using ImageQuant LAS 4000 (GE Healthcare) (see Supplemental Material). The western blots shown in the figures are representative of at least 3 independent experiments. Inflammasome stimulation BMDMs were seeded at a density of 5 × 105 cells per well in 24-well plates, while pRMØs were seeded at 3 × 105 and 1 × 106 cells per well in 24- and 6-well plates, respectively. Human PBMCs were seeded at a density of 5 × 105 cells per well in 24-well plates. The next day, the overnight (O/N) medium was replaced, and cells were stimulated with LPS (1 μg/mL) in fresh medium for 2 h and then stimulated with one of the following inflammasome activators: 5 mM ATP (1 h), 100 μM Bz-ATP (30 min) or 10 μM nigericin (1 h). Supernatants were assayed by ELISA according to the manufacturer’s instructions (R&D Systems or MBL). The data shown in the figures are representative of at least 3 different independent experiments. Analysis of cleaved Caspase-1 and cleaved IL-1β levels in conditioned cell culture media A total of 1 × 106 pRMØs from WT and Pml−/− mice were cultured in complete IMDM. The next day, the O/N medium was replaced, and the cells were stimulated with LPS (1 μg/mL) for 3 h and then with the inflammasome activator 5 mM ATP (1 h) or 10 μM nigericin (1 h). The conditioned cell culture media were then collected and concentrated using Pierce Protein Concentrators PES 10 K MWCO (Thermo Fisher) and centrifuged (3000 g for 15 min). Aliquots of media (10 μl) were analyzed by SDS–PAGE and immunoblotting (Ponceau S staining was used as a loading control). The levels of cleaved Caspase-1 and cleaved IL-1β were detected with anti-Caspase-1 (Novus Biological, #14F468) and anti-IL-1β (Cell Signaling Technology, #12242) antibodies, respectively. Gene knockout by CRISPR/Cas9 The procedure was performed in accordance with a previously published protocol [35]. To prevent off-target effects, RNA guides (gRNAs) were purchased from Integrated DNA Technologies IDT. The 20-nucleotide RNA guide sequence was transfected into B16-F10 cells with Streptococcus pyogenes Cas9 (IDT) using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher). After two days of incubation, single cells were isolated by serial dilution and expanded for 5 weeks. Gene KO was assessed by WB and confirmed by RT–PCR. Co-culture assay pRMØs from WT and Pml−/− C57BL/6 mice were seeded in 6-well plates until 80% confluence and co-cultured in an insert chamber placed on top of the B16-F10 cells. IL-1R1 KO B16-F10 cells generated using the CRISPR/Cas9 technique were co-cultured with pRMØs from Pml−/− mice. The bottom of the insert chamber had 0.4-μm pores, allowing cytokines and growth factors produced by pRMØs to reach the lower chamber containing B16-F10 cells or IL-1R1 KO B16-F10 clones. The cells were cultured for up to 1 week, and pRMØs were treated daily with LPS (1 μg\ml) plus Bz-ATP (100 μM) in the absence or presence of anti-IL-1β (50 pg/ml). Equal amounts of PBS or the isotype control antibody were used as controls. H460 cells were co-cultured with human PBMCs transfected with the scramble or PML siRNA and treated daily with LPS and Bz-ATP. After one week, the inserts were removed, and the tumor cells (B16-F10, IL-1R1 KO B16-F10 clones and H460 cells) were stained with crystal violet. After lysis with 10% acetic acid, the absorbance was read at 595 nm. Subcellular fractionation WT and Pml−/− C57BL/6 mice were treated with LPS (250 μg/Kg IP for 1 h) and nigericin (2.5 mg/kg IP for 1 h), and their livers were surgically removed after anesthetic overdose. BMDM from WT, Pml−/− were obtained and isolated as described above and were treated with LPS (1 μg/mL) for 2 h and then stimulated with 10 μM nigericin (1 h). Fractionation was performed as described previously [36, 37]. Briefly, BMDM (109) were harvested, washed by centrifugation at 500 × g for 5 min with PBS, and resuspended in homogenization buffer (225 mM mannitol, 75 mM sucrose, 30 mM Tris-HCl pH 7.4, 0.1 mM EGTA and PMSF) and gently disrupted by Dounce homogenization. Livers were fragmented and homogenized in homogenization buffer (225 mM mannitol, 75 mM sucrose, 30 mM Tris-HCl pH 7.4, 0.5 mM EGTA and 0.5% BSA). The homogenate was centrifuged twice at 600 × g for 5 min, then the supernatant was centrifuged at 10,000 × g for 10 min to pellet crude mitochondria. The resultant supernatant was centrifuged at 20,000 × g for 30 min at 4 °C. Further centrifugation of the obtained supernatant at 100,000 × g for 90 min (70-Ti rotor, Beckman, Milan, Italy) at 4 °C results in the isolation of cytosolic fraction (supernatant) and ER (pellet). The crude mitochondrial fraction was resuspended in isolation buffer (250 mM mannitol, 5 mM Hepes (pH 7.4) and 0.5 mM EGTA), subjected to Percoll gradient centrifugation (Percoll medium: 225-mM mannitol, 25-mM HEPES (pH 7.4), 1-mM EGTA and 30% Percoll (vol/vol) at 95,000 × g for 30 min (SW40 rotor). A dense band containing purified mitochondria was recovered approximately at the bottom of the ultracentrifuge tube, washed by centrifugation at 6300 × g for 10 min, and resuspended in isolation buffer. The MAMs fraction was removed from the Percoll gradient, diluted in isolation buffer, and centrifuged at 100,000 × g for 90 min (70-Ti rotor); the pellet was the MAMs fraction. IP3R3, Sigma 1-R, GAPDH, and VDAC were used as markers of the ER, MAMs, cytosol, and pure mitochondria, respectively. In vivo LPS challenge WT, Pml−/−, Pml−/−/P2rx7−/− and Pml−/−/Nlrp3−/− C57BL/6 mice were treated with LPS (250 μg/kg IP for 4 h) and nigericin (2.5 mg/kg IP for 1 h), and their blood and peritoneal exudates were collected after anesthetic overdose. Pml−/− mice were pretreated with MCC950 (20 mg/kg, IP), AZ10606120 (5 mg/kg, IP) or vehicle (PBS, IP) 30 min before treatment with LPS (250 μg/Kg IP for 4 h) and nigericin (2.5 mg/kg IP for 1 h). Thereafter, their blood and peritoneal exudates were collected after anesthetic overdose. The peritoneal exudate samples were concentrated using Pierce Protein Concentrators PES 10 K MWCO (Thermo Fisher), and the supernatant was centrifuged (3000 × g for 15 min). The concentrated medium (10 μl) was separated by SDS/PAGE and transferred onto a nitrocellulose membrane for standard WB. For the caspase-1 assay, WT and Pml−/− C57BL/6 mice were treated with LPS (250 μg/kg IP for 1 h) and nigericin (2.5 mg/kg IP for 1 h). Their livers were removed after an anesthetic overdose and homogenized with a Potter pestle in lysis buffer (300 mM sucrose, 1 mM K2HPO4, 5.5 mM D-glucose, 20 mM HEPES, 1 mM phenylmethylsulfonylfluoride, and 0.5% IGEPAL). Tissue extracts were then centrifuged (12,000 × g at 4 °C for 15 min). Protein extracts (200 μg) were assayed for caspase-1. Mouse plasma was collected after blood centrifugation (1000 × g, 10 min at 4 °C), and the IL-1β levels were determined by ELISA according to the manufacturer’s instructions. Tumor generation and in vivo drug administration Procedures involving animals and their care were in conformity with institutional guidelines, and the Animal Ethics Committee approved all experimental protocols (authorizations n. 481/2017-PR and CBCC2.N.BH4 approved by the Italian Ministry of Health). To generate DKO C57BL/6 mice, the Pml−/− mice were crossed either with the P2rx7−/− strain (a gift from F. Di Virgilio) or the Nlrp3−/− strain. All mice were housed in a temperature-controlled environment on a 12 h light/dark cycle and received food and water ad libitum. A total of 1 × 106 B16-F10 melanoma cells, IL-1R1 KO B16-F10 clones or LL/2 cells were injected subcutaneously into different groups of 6- to 8-week-old C57BL/6 female mice. Tumor growth was monitored daily, and tumor volumes were measured with calipers according to the following equation: Volume = π/6 × (a × b2), where a is the major diameter and b is the minor diameter. The mice were randomly divided into the treatment and respective control groups (5–6 mice per group). For NLRP3 inflammasome inhibition, mice were treated with MCC950 (20 mg/kg IP) or AZ10606120 (5 mg/kg intratumor) three times a week. For the cytokine neutralization experiments, mice were treated with anti-IL-1β serum (BioXCell, clone B122) (50 µg IP, twice weekly). Controls were treated IP with an equal amount of isotype control antibodies or an equal volume of PBS. After an anesthetic overdose, tumors were surgically removed from all mice that reached the experimental endpoint, after which they were weighed and trimmed for immunohistochemistry or FACS analyses. Histology and immunohistochemistry Tumor masses, including peritumoral stroma, were collected at 14 days postinoculation, fixed in Bouin’s liquid for 7 h at 4 °C, dehydrated in a cold-graded ethanol series and embedded in paraffin. For each specimen, at least ten sets (50 µm away) of five serial sections (6 µm thick) were stained with Harris’ hematoxylin and eosin (H/E) for general histology or processed for immunohistochemistry, which was performed using ABC-peroxidase. Adjacent sections were incubated for 1 h at room temperature with antibodies against F4/80 (CI: A3-1) (Santa Cruz Biotech; sc-59171), IL-1β (H-155) (Santa Cruz, sc-7884), iNOS (Abcam, ab15323), mannose receptor antibody (Abcam, ab64693) and Ly6g [RB6-8C5] (Abcam, ab25377). The antibodies were diluted 1:10 to 1:50 in 0.01 M PBS (pH 7.3) containing 0.1% sodium azide. A hybridoma irrelevant supernatant or normal rabbit serum was substituted for the primary antibody in the negative controls. The antibodies were tested on sections after or without antigen retrieval steps (40 min at 95 °C in 0.01 M, pH 6.0 sodium citrate buffer containing 0.05% Tween 20, followed by cooling at RT and rinsing with PBS Tween 20). Thereafter, the sections were incubated for 60 min with biotinylated goat anti-rat IgG (mouse adsorbed) or biotinylated goat anti-rabbit IgG sera (Vector Labs., Burlingame, USA) and diluted 1:200 or 1:1000, respectively, with PBS containing 0.1% sodium azide and 1% BSA. Some sections (not treated with any primary antibodies) were incubated with biotinylated horse anti-mouse IgG serum (Vector) diluted 1:1000 to label B cells. Thereafter, the sections were incubated for 60 min with avidin-biotinylated peroxidase complex (ABC, Vectastain Elite, Vector). Following rinsing and staining (diaminobenzidine), the sections were slightly counterstained with Harris’ hematoxylin, rinsed, dehydrated, mounted and examined under bright-field illumination. Immunoreactive cells (nucleated only) in 1 mm2 areas of peritumoral stroma were counted by an observer unaware of the treatments using a computer-assisted image analysis system, which included a Nikon Eclipse E600 microscope equipped with a DS-5M digital color video camera. Images were captured through a PC interface using the software package Lucia G 4.81 (Laboratory Imaging Ltd., Praha, Hostivar). The number of immunoreactive cells in each group was then calculated by averaging the cell numbers from all specimens. The mean and SEM were calculated for each parameter. Two-way ANOVA was used to assess the treatment effects, and one-way analysis of variance and Tukey’s multiple comparison post hoc test were used to determine differences among groups. P < 0.05 served as the level for statistical significance. Immunohistochemistry in human tissue specimens A total of 39 patients with lung carcinoma (18 patients) or melanoma (21 patients) who underwent surgical resection at the University Hospital of Ferrara were included in the study. Their clinical features are reported in Table S1; for all of the cases, histopathological parameter analysis was performed by pathologists of the Pathology Unit of the University Hospital of Ferrara. The study was approved by the Local Ethics Committee (CE: 1016/2020/Oss/UniFe). The informed consent was collected from patients whenever it was possible. CD68 immunodetection was performed on formalin-fixed paraffin-embedded tissue sections (4 µm thick) using the antibody CD68 (PG-M1, Diagnostic Biosystems) and the Multimeric Detection Kit (Universal DAB Detection Kit Ultraview, Roche Tissue Diagnostics (CH)) were assessed on a Benchmark XT automated immunostainer (Roche T. D.). For the immunohistochemical evaluation of PML, IL-1β and CD206, 4 µm-thick tissue sections were immunostained with anti-PML (Abcam ab72137, 1:250 dilution), anti-IL-1β (Αbcam ab9722, 1:500 dilution) and anti-CD206 (Abcam ab64693, 1:1000 dilution). After deparaffinization, immunohistochemistry was performed according to [38]. The immunoreactivities of CD68-, PML-, IL-1β−, and CD206 in the peritumoral stroma were evaluated by two independent clinical pathologists (blinded to the patient outcomes) and scored for occurrence/absence. Immunofluorescence assay pRMØs from WT and pml−/− mice were seeded on 24-mm glass coverslips; the next day, the O/N medium was replaced, and cells were stimulated with LPS (1 µg/ml for 2 h) plus nigericin (10 µM for 1 h). Then, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min and washed three times with PBS. The cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS and blocked with PBS containing 2% BSA and 0.1% Triton X-100 for 1 h. The cells were incubated O/N at 4 °C with the following antibodies: calnexin (Santa Cruz Biotechnology, sc11397) and NLRP3 (Adipogen, AG-20B-0014-C100). Appropriate isotype-matched AlexaFluor-conjugated secondary antibodies (diluted 1:1000) were then used (Life Technologies A11008 488 goat anti-rabbit and A-11005 goat anti-mouse 594). The coverslips were mounted with ProLong Gold Antifade reagent (Life Technologies), and immunofluorescence analysis was performed with a confocal laser scanner microscope (Olympus FV3000) equipped with a 63x oil objective. The NLRP3 speckle/NLRP3 total ratio was quantified. Co-immunoprecipitation Co-immunoprecipitation was carried out using protein Gibco CTS Dynabeads (Thermo Fisher) in accordance with the manufacturer’s instructions. Protein extractions from the ER fractions were carried out by adding 50 mM NaCl and 1% NP-40 to the homogenization buffer. All the buffers were supplemented with a proteases inhibitor mixture and Phos‐STOP Phosphatase Inhibitor Cocktail (Roche Applied Science). The same amount of extracted proteins for each condition was incubated O/N with the specific primary antibody PML (Millipore, no. MAB3738), P2X7 (Alomone, APR-004), or NLRP3 (AG-20B-0014-C100, Adipogen). The immunocomplexes were captured with the appropriate dynabeads (Thermo Fisher). Beads were pelleted and washed three times. The bait was eluted in Laemmli sample buffer and denatured for 5 min at 100 °C. Samples were proceeded by SDS–PAGE and analyzed by standard (WB) blotting technique. RT-PCR Total RNA was extracted with TRIzol reagent (Life Technologies) and then purified with the Pure Link RNA Mini Kit (Invitrogen) in accordance with the manufacturer’s instructions. The RNA was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Milan, Italy). One microgram of the total RNA extract was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystem, Carlsbad, CA, USA). qRT PCR was performed on a Step One Real-Time PCR System (Applied Biosystems). Two microliters of cDNA were used as a template. Amplification was performed using predesigned TaqMan probes (Applied Biosystems) for NLRP3, NLRC4 and GAPDH as housekeeping genes. For the screening of CRISPR/Cas9 KO clones, RT–PCR was performed with Two-Step Real-Time PCR on the Rotor Gene Q system (Qiagen). Two microliters of cDNA were used as a template. Amplification of IL-1R1 and GAPDH, a housekeeping gene, was performed using predesigned primers (IDTs). PCR DNA was prepared from tail biopsies and used in 50 μl reaction mixtures that included the following specific primers for each selective gene being amplified: (I) 5′-TGCCCATCTTCTGAACACC, 5′-CTTCCTCTTACTGTTTCCTCCC, and 5′-GCAAGGCGATTAAGTTGGG for P2X7R; (II) 5′-GTCCAGGACATACGTCTGGA, 5′TGAGGTCCACATCTTCAAGG and 5′TTGTAGTTGCCGTCGTCCTT for NLRP3; and (III) 5′-TTGGACTTGCGCGTACTGTC, 5′-CGACCACCAAGCGAAACA, and 5′-TTTCAGTTTCTGCGCTGCC for PML. WT genes corresponded to specific amplicons (568 bp for P2x7r; 327 bp for Nlrp3; 400 bp for Pml), unlike the respective KO gene amplicons (393 bp for P2x7r−/−; 589 bp for Nlrp3−/−; 700 bp for Pml−/−). The reactions were analyzed on a 2% agarose gel containing Midori green. Flow cytometry Tumors were cut into 1–2 mm3 pieces and digested with DNAase (100 μg/ml, Sigma) and collagenase A (20 units/ml, Roche) in RPMI at 37 °C for 1 h. TAMs were blocked with an anti-mouse FcR antibody (CD16/CD32, BD) for 5 min at 4 °C; subsequently, the cells were surface stained in the dark for 15 min at room temperature with directly conjugated monoclonal antibodies (mAbs) and analyzed after the exclusion of nonviable cells using the amine-reactive viability dye Aqua (Life Technologies) as previously described (1, 2). The following mAbs were used to identify macrophages (see Extended Data Fig. 4b for the gating strategy) and analyze their polarization: APC-conjugated anti-CD3 (clone: 145-2C11, eBioscience), APC-conjugated anti-CD45R (clone: RA3-6B2, eBioscience), PE/Cy7-conjugated anti-CD11b (clone: M1/70, Biolegend), APC/Cy7-conjugated anti-F4/80 (clone: BM8, Biolegend), PE-conjugated anti-CD86 (clone: PO3, Biolegend), and Pacific Blue-conjugated anti-MHC-II (clone: M5/114.15.2, Biolegend). To determine the percentage of circulating monocytes, samples of whole blood were immunostained using the following mAbs: PE-conjugated anti-45 (clone: 30F11, Miltenyi), APC-conjugated anti-CD11c (clone: N418, Biolegend), PE/Cy7-conjugated anti-CD11b (clone: M1/70, Biolegend), PerCP-vio700-conjugated anti-Ly-6C (clone REA796, Miltenyi), and VioBlue-conjugated anti-Ly-6G (clone: REA526, Miltenyi). Samples were acquired on a BD FACSCantoII (BD Biosciences) flow cytometer. Data were analyzed using FlowJo version 10 (Tree Star Inc.). Bone marrow replacement experiment B6. SJL-Ptprca Pepcb/BoyJ (Jackson Laboratory) recipients received 950 cGy of total body irradiation, followed by the intravenous injection of 106 bone marrow mononuclear cells derived from WT or pml−/− C57BL/6J donor mice. Reconstitution of donor cells and repopulation of donor myeloid and lymphoid cells were monitored by the staining of peripheral blood cells with antibodies against CD45.1, CD45.2, CD3 (T cell), B220 (B cell), CD11b and Gr-1 (myeloid) every 4 weeks starting at 4 weeks after transplantation. At 16 weeks post transplantation, the engraftments were considered stable, and the mice were subjected to tumor engraftment. Experiments were authorized by the Institutional Review Board for Human Research at Albert Einstein College of Medicine. Surface plasmon resonance (SPR) experiments Recombinant P2X7 protein (GST tag; Cat. No. ABIN1313816; Fc tag; Cat. No. ABIN16964308), NLRP3 protein (AA 1-1036; GST tag; Cat. No. ABIN1312675) and PML protein (Transcript Variant 9, Myc-DYKDDDDK tag; Cat. No. ABIN2729208) were purchased from Antibodies Online, Germany. SPR measurements were performed on a Biacore 3000 instrument (GE Healthcare/Cytiva). GST-tagged NLRP3 protein was captured at low surface density on a polyclonal goat anti-GST antibody, covalently coupled to the surface of a Biacore CM5 optical sensor chip, using and following the protocol of the Biacore GST Capture Kit (Order Code: BR-1002-23). Amine activated flow cell 1 (FC1) of the sensor chip was used as a reference to allow the generation of background-subtracted binding sensorgrams. After GST-tagged NLRP3 or P2RX7 ligand capturing, remaining free anti-GST binding sites were saturated with excess recombinant GST provided with the Biacore GST Capture Kit to allow specific sequential binding of other GST-tagged analytes to the captured ligand protein. Proteins PML and P2X7R were passed as analytes in sequential injections over the captured NLRP3 ligand at single concentrations of 167 nM at a flow rate 30 μl/minutes in HBS-EP buffer (0.1 M HEPES, 1.5 M NaCl, 0.03 M EDTA and 0.5% v/v Surfactant P20). PML was also passed over captured P2X7R in a separate experiment. After the experiments bound captured ligand and analyte proteins were removed by anti-GST surface regeneration with 10 mM HCl. Proximity ligation assay pRMØs from WT and pml−/− mice were seeded on 16-well microarray slides (Thermo Fisher) at a density of 105 cells. Twenty-four hours after seeding, the cells were primed with LPS (1 µg/ml for 2 h) and then with nigericin (10 µM for 1 h). The cells were then fixed with 4% PFA in PBS for 10 min and washed three times with PBS. The cells were permeabilized for 10 min with 0.1% Triton X-100 in PBS and blocked with PBS containing 5% BSA and 0.1% Triton X-100 for 1 h. Then, the cells were hybridized (O/N at 4 °C in a humid chamber) with the following conjugated primary antibodies (PLA probes) overnight: one MINUS (P2X7R) and one PLUS (NLRP3) for the P2X7R-NLRP3 interaction, one MINUS (PML) and one PLUS (NLRP3) for the PML-NLRP3 interaction, and one MINUS (P2X7R) and one PLUS (PML) for the P2X7R-PML interaction. The next day, the PLA was conducted in accordance with the manufacturer’s instructions. After the PLA assay, the cells were fixed with 100% methanol for 20 min at −20 °C and then permeabilized for 10 min with 0.1% Triton X-100 in PBS. The cells were blocked with PBS containing 5% milk and 0.1% Triton X-100 for 1 h and then incubated O/N at 4 °C with an antibody against PDI (Abcam, ab3672). An appropriate, isotype-matched AlexaFluor-conjugated secondary antibody (Life Technologies, A11008, 488 goat anti-rabbit, diluted 1:1000) was then applied, and the PLA signal was detected by an Olympus Xcellence widefield system and deconvolved using Fiji. After 3D digital deconvolution, the PLA signal was quantified as dots within each PDI-positive cell or DAPI-positive cell. Statistics analyses The data were analyzed by Prism 6 (GraphPad) or Microsoft Excel (Microsoft Co.). Unless otherwise specified, the data are representative of at least three biologically independent experiments. Two-group datasets were analyzed by Student’s t test for unpaired data. For multiple comparison (≥3 experimental groups) analysis, one-way ANOVA followed by Tukey’s or Dunnett’s test was used where appropriate. A P value <0.05 was considered significant. Supplementary information Supplemental material Original western blots checklist Supplementary information The online version contains supplementary material available at 10.1038/s41418-022-01095-9. Acknowledgements The authors thank C. Bosi, F. Poletti, A.C. Sarti, and S. Falzoni for their contribution to this study. PP is grateful to C. degli Scrovegni for her continuous support. Author contributions CG conceived the study. SM performed most of the experiments and prepared the figures. MP and SMa performed the proximity ligation assay studies. MP generated the CRISPR knockout clones. CG directed and coordinated the study, oversaw all the experiments, and wrote the manuscript. PP supervised the study and oversaw the experiments. F.D.V. collaborated in experiments related to P2X7R. GM, MRW, and MLA performed the co-immunoprecipitation experiments. RG, GL and LA performed the immunohistochemical analysis. LA helped in writing the manuscript. SM and MP provided advice for the in vivo experiments. FN performed the flow cytometry experiments. MB and KI performed the bone marrow replacement experiment. CB and BV helped with some experiments. SM performed the Western blot, ELISA, and subcellular fractionation studies. FK performed the SPR studies. FF, AB, and RG helped in the patient’s cohorts selection. All authors read and approved the final manuscript. Funding The Signal Transduction Laboratory is supported by the Italian Association for Cancer Research (IG-23670 to PP, IG-19803 to CG and IG 22883 to FDV), A-ROSE, Progetti di Rilevante Interesse Nazionale (PRIN2017E5L5P3 to PP and PRIN20177E9EPY to CG), the Italian Ministry of Health (GR-2019-12369646 to SM), the European Research Council (853057-InflaPML to CG) and local funds from the University of Ferrara to CG and PP. MRW was supported by the National Science Centre, Poland (UMO-2018/29/B/NZ1/00589). MLA was funded by a Polish National Science Centre grant (UMO-2015/17/D/NZ1/00030). MP is supported by an AIRC research fellowship (ID: 26665). Data availability The data analyzed during this study are included in this article and the supplemental data files. Additional supporting data are available from the corresponding authors upon reasonable request. Competing interests The authors declare no competing interests. Ethics approval Procedures involving animals and their care were in conformity with institutional guidelines, and the Animal Ethics Committee approved all experimental protocols (authorization nos. 481/2017-PR and CBCC2.N.BH4 approved by the Italian Ministry of Health). The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the University Hospital of Ferrara (CE-AVEC: 1016/2020/Oss/UniFe). Edited by P Salomoni Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Pinton P Giorgi C Pandolfi PP The role of PML in the control of apoptotic cell fate: a new key player at ER-mitochondria sites Cell Death Differ 2011 18 1450 6 10.1038/cdd.2011.31 21475307 2. 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==== Front Med Sci Educ Med Sci Educ Medical Science Educator 2156-8650 Springer US New York 1692 10.1007/s40670-022-01692-w Keynote Lecture Bouncing Forward: Mental Wealth for All http://orcid.org/0000-0003-2433-6572 Bishop Jo [email protected] grid.1033.1 0000 0004 0405 3820 Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Australia 1 12 2022 16 12 11 2022 © The Author(s) under exclusive licence to International Association of Medical Science Educators 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcWhat is wellbeing and why is it important? The WHO definition reads “Health is a state of complete physical, mental, and social wellbeing and not merely the absence of disease or infirmity. Mental health is a state of well-being in which an individual realizes his or her own abilities, can cope with the normal stresses of life, can work productively and is able to make a contribution to his or her community” [1]. There is currently a significant and increasing amount of interest worldwide in how we can best support our learners and faculty, including health professional educators, to maintain and nurture their wellbeing, in challenging circumstances. Does your institution currently assess wellbeing, through an annual survey, focus groups or involvement in strategic discussions? There is also a focus on strengthening an individual’s ability to overcome adversity, manage stress, and thrive in their personal and professional life and in embedding wellbeing into academic and clinical organisational curricula, plans, policies, and practices. We should be putting people at the heart of what we do! “An individual’s well‐being impacts on their capacity to learn and perform competently. Learner well‐being and supporting their acquisition of skills in self‐care for lifelong practice is therefore core business for educators” [2]. Maslow's theory describing “hierarchy of needs” (see figure) is often portrayed in the shape of a pyramid with the largest, most fundamental needs at the bottom and the need for self-actualization and transcendence at the top [3, 4]. An individuals’ most basic needs must be met before they become motivated to achieve the higher-level needs. The theory states that people are motivated to achieve certain needs and that some of their needs take priority over others. One of our most basic needs is physical survival,this could be in the form of good sleep hygiene, nutrition, all of which can impact our behaviour if not kept in check. Each level needs to be “completed” before we can reach the next level. We work with cultural and interpersonal diversity daily, and we should recognise and address unconscious biases. These biases may include small behavioural cues that signal whether others are valued, whether others are being included or spoken to. Consider the signals you’re sending, your body language, your interaction style, casual comments, and feedback that you deliver. Now look around and consider the environmental signals within your institution, what photos are on your walls, and lastly consider your external signals that may present on your website as images, marketing materials, and which faculty or students are chosen to represent the organisation. Does it suggest all are welcome at this institution? This can really impact the levels of “safety, belonging and esteem” of learners (and faculty). When considering learners life cycle in parallel to this pyramid, not only are there colliding shapes (!) but there are key life changes and transitions that impact their ability to flourish and self-actualise. A key example are the first few days of university, and therefore the role of a dean of student within faculty is to work with the university to ensure there is a balance in the information shared, enough to support but not overwhelm. Welcome sessions can be led by student societies or supported by student leadership. Key information should be given priority, e.g. academic integrity workshops and the respectful community sessions to encourage student transition and to improve social interaction. In addition, pre-arrival information should be co-designed with student leadership and near peers. Within the educational context when we consider the learner life cycle, from student recruitment and admission to their learning and then assessment. This then cycles to results and awards/graduation and finally as alumni. During their life cycle, successful progression of learners requires them to be connected, equipped, motivated, and settled. Why would learners seek support?Difficulty in transitioning to university or new country Difficulty sustaining academic workload and multiple competing tasks Social isolation, social anxiety, loneliness, and victimisation Poor cultural assimilation or English ability Poor resilience or maladaptive coping mechanisms Financial debt or hardship Personal life events A medical condition or disability A psychological predisposition, condition, or disability Emotional instability following a physical or psychological trauma Substance abuse or addiction Why do learners not seek support?Lack of time Lack of confidentiality Fear of negative impact on career Fear of documentation on record Stigma of mental health care Not knowing who to go to Difficulty accessing services Fear of unwanted intervention Fear of legal consequences “Using services will mean I’m weak” Cost My problems are not important “Nobody will understand” How would educators recognise learners in distress?Marked change in academic performance or attendance Persistently late to class or in turning in assessments Highly disorganised Withdrawn from social situations and communication with others Persistent low mood, low motivation, or loss of interest Marked change in hygiene and general appearance Acute stress, anxiety, or panic attacks Unpredictable or irrational thoughts, moods, attitudes, or behaviours Being under the influence of drugs or alcohol Disorientation, delusional, dissociated thoughts, or behaviours Extreme distorted thinking or excessive unwarranted worrying Persistent disruptive or volatile physical or verbal behaviour Alarming material presented in a student’s written work The stigma of mental health is prevalent in healthcare students and practitioners. This may result in learners being reluctant to identify as suffering from a mental health condition and from seeking the required support and management. Learners, health science educators, and practitioners may be wary of accessing support due to number of reasons—including those listed above. To create safe environments for learning and personal growth, we must ensure.Barriers to seeking help are removed. There is access to role model, e.g. near peer or faculty. Confidentiality maintained following disclosure. Fears around progression/career concerns are considered and discussed. Counselling for all is encouraged. Transparency around the benefits of self-care, including the care of others and ensuring safety of patients. Help seeking pathways are accessible and visible. When considering putting strategies into practice to make a difference the institute should monitor policies to promote wellbeing, this can be achieved by finding an existing framework and completing a gaps analysis. At Bond we created a “Promoting Health and Wellbeing working group” with champions from across the university that included student leaders, professional/administrative staff, faculty, and members of the medical services and human resources. We recognised that many initiatives are fairly siloed across faculties; it was realised that it would be helpful to consider how some of these initiatives can be rolled out across the university (e.g. mentoring programmes/counselling offerings) to the benefit of all. We proposed a review of various faculty-specific initiatives with a view to considering resourcing/embedding of support initiatives across the University? Our suggested priorities are listed below including finalising a Mental Health Strategy complete Bond University Wellbeing Research Project.Review and report on the wellbeing and mental health needs of allUsing “A framework for promoting student mental wellbeing in universities” (Baik and Larcombe [5]) Plus consider a whole of university Wellbeing Research Project Reduce the stigma of mental healthPart of the Wellbeing Research Project Work of the Promoting Wellbeing working group (and individual portfolios) Improve education and access to information and promotion of wellbeing within teaching and learning initiatives within the classroom using established frameworkUsing “A framework for promoting student mental wellbeing in universities. https://unistudentwellbeing.edu.au” as indicated by the 5 domains (see image) Increase the volume and visibility of information, resources, and support available in extracurricular endeavours by promoting internal and external servicesContinue to work with the student society, medical services, and our insurance provider to consider appropriate resources Promotion and review of the webpage or create a shared site for all Consider a variety of support methods for allListen to the community (research project and the work achieved in other faculties)—sharing is caring Highlight support needs at key points in the semesterReview transition points—is there a student journey or life cycle that could be considered Improve communication around support strategiesFaculty/student training—mental health first aid Co-design wellbeing events with staff and studentsFunding and support regular events—key dates Establish and promote wellbeing champions Training and support for all A fellow member of the Bond University Collaboration for Research in Understanding Stigma in Healthcare [6] research group and I began the Bond University Mental Health & Wellbeing Project (Bannatyne [7]). Preliminary findings have indicated that during 2020 when reflecting on the impact of COVID-19 on mental health and wellbeing, over half the sample (54.4%) reported a decline in their mental health and wellbeing. 10% of the student population completed the survey with awareness of various support services at Bond University was mixed. Approximately, one-quarter had accessed psychological services. Barriers to accessing University Psychological Services complemented those described earlier, e.g. confidentiality. Notably, over two-thirds of the sample (66.6%) believed Bond University should be actively trying to reduce mental health stigma within the university community. Ideas suggested included sharing lived experience stories via social media, more visible promotion of mental health statistics and support services, a culture of accountability for student leadership, embracing and supporting mental health week and other wellbeing activities, mental health literacy included as a component of core subjects, peer support programmes, and compulsory mental health first aid training for all educators (Bannatyne [7]). Institutions should provide as minimum services such as student counselling service, medical clinic, accessibility & inclusion advisors, security, academic skills centre, first nations support centre, LGBTIQ + services & support, career development centre, and student business centre. Wherever possible consider the role of student leaders, peers who can state and validate “I’ve been there, I get it!”. Engage learners as stakeholders by having learner representatives as a voice for their peers and promote partnerships in health promotion activities and address misconceptions (bust the myths) and open discussion during distressing events. Consider who are your national learner society representatives, does the learner society have a wellbeing officer? Now you? As an educator, who would you turn to, where can you go when need support, who supports the supporters? When questioned, faculty stated they sought support from.Manager/supervisor Executive dean Programme director Faculty-peer support services GP Psychiatrist Mental health professional Clergy member Employee assist programme Internet sources: discussion forums, social media The role descriptors of any educator have had to adapt have and the true extent of their academic workload, and daily pressures should not be underestimated. Institutions are responsible for staff wellbeing as it can affect learner wellbeing and, in the healthcare, setting it can impact patient care. Apart from accreditation and legal requirements to look after health and safety of staff, there are cogent reasons why medical schools should actively promote teacher and staff wellbeing. Who supports the supporters? At Bond, we have supported colleagues by establishing mental health first aid training. This skills based, early intervention training programmes “mobilise and empowers communities by equipping people with the knowledge and confidence to recognise, connect and respond to someone experiencing a mental health problem or mental health crisis. Anyone can have a conversation that may save a life. Everyone should know how” [8] In April 2020, the Student Support Network was established for faculty across the 23 medical schools in Australia and New Zealand. It was realised as a key initiative as we traversed the unprecedented times of the pandemic to ensure those who supported students were also cared for. The once weekly meetings now occur monthly and allow sharing of best practice and resources. Members highlight that they value this community or practice and are empowered to review difficult or complex situations involving students with advice provided by experienced colleagues in confidential manner [9] Mentors Across Borders is a dynamic and diverse international community of Leaders in Health Professions Education formed in 2020, united by a shared passion for mentoring and nurturing fellow educators as well as scholarship. This community aims to promote collegial global conversations about important educational topics in a psychologically safe space to interact, share success stories and challenges, and potentially enhance professional identity (BEI [10]). We have recognised that what learners want and what faculty need is relevant, timely information, in the moment. As educators, you should draw on the expertise of the educational community through membership of organisations such as Australian and New Zealand Association for Health Professional Educators (ANZAHPE), an International Educational Association for Health Professional Educators (AMEE), and the International Association for Medical Science Educators (IAMSE). The publication on redefining scholarship for health profession education [11] emphasises the below resources to support educators:Published research Engagement with a faculty learning community Community of practice Engage with colleagues locally Professional development workshops and opportunities External conference Online webinars No need for face to face, formation of special interest groups, etc. Community can include quality assurance, benchmarking, and peer review Evaluation and student satisfaction Faculty development courses have also been established including an Essential Skills in Medical Education (ESME) course focussing on wellbeing (WESME) (AMEE [12]). In 2019, colleagues published the medical student wellbeing—a consensus statement from Australia and New Zealand (Kemp et al. [13]) that summarised that we should.Design curricula that promote peer support and progressive levels of challenge to students. Employ strategies to promote positive outcomes from stress and to help others in need. Design assessment tasks to foster wellbeing as well as learning. Provide mental health promotion and suicide prevention initiatives. Provide physical health promotion initiatives. Ensure safe and health-promoting cultures for learning in on-campus and clinical settings. Train staff on student wellbeing and how to manage wellbeing concerns. The editors of the understanding medical education textbook recognised the need for a learner wellbeing chapter with recommendations for learner support system and approaches that educators could use (Bishop et al. [2]). The chapter complimented the consensus statement and also suggested the following:To care for patients, health professionals must care for themselves, and they need to learn about and develop this capability. Programmes and interventions can be categorised as general support to all learners, preventive support in anticipation of challenges, and additional support for learners in need. Educators should consider their roles as individual teachers, programme designers, and in creating supportive and safe learning environments that will help learners negotiate future challenges in the clinical workplace. Educators should consider professional boundaries in their actions to promote well‐being and welfare. Whilst many educators may draw upon their experiences as clinicians, learners are not their patients. Future Directions: Building the Future Together The recent publication by Medical Deans Australia and New Zealand Inc. (Medical Deans) on Inclusive medical education is designed to assist medical schools in their approach to and discussions with prospective and current students with a disability and to identify and consider the adjustments or supports that may be needed for them to commence or continue in a medical programme [14] In addition, the National Framework, Every doctor every setting; Improving the wellbeing of doctors and medical students is a key enabler of quality patient care and healthier communities. The framework is based on available evidence and advice from doctors, doctors-in-training, medical students, mental health and suicide prevention experts, and other key stakeholders. The overall consideration is that all jurisdictions, settings, services, and stakeholders must be involved to ensure immediate, sustained, and coordinated action (Everymind [15]). There are predictable times of distress for all of us as we navigate life, and therefore, preventative measures and sharing of a toolbox of skills and resources whilst understanding the “currency of mental wealth” are essential. There are five types of wellbeing described: emotional, physical, social, workplace, and societal wellbeing. Many of us will have a preference—for me, physical activity, weather walking, cycling, or strength training allows me to perform at my best. Emotionally, I am aware of my own requirements and needs to have time out and reconnect. I have a supportive social community with family and friends, in my workplace I have a sense of purpose and drive and thrive during times of challenge. Societal can be difficult when faced with dilemmas outside of our control. Resilience is often defined as bouncing back from adversity; in reality, no one ever returns to how they were before the event. There is often a required time to reflect and to heal, but more than often there is personal growth; the timelines can vary person to person. Using the narrative, “bouncing forward” gives permission to allow the individual to grow and gain strength from these challenging times. I have gained strength in sharing my own vulnerability and hope this will help others. Brené Brown debunks some myths about vulnerability, the most popular being that vulnerability is a sign of weakness. “When we think of times that we have felt vulnerable or emotionally exposed, we are actually recalling times of great courage; Vulnerability is the birthplace of love, belonging, joy courage, empathy, and creativity” (Brown [16]). Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 15 KB) Author Contribution Not applicable. Availability of Data and Material Not applicable. Declarations Ethics Approval and Consent to Participate Not applicable. Consent for Publication Not applicable. Conflict of Interest The author declares no competing interests. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. WHO. Health and well-being. 2022. Retrieved from World Health Organization: https://www.who.int/data/gho/data/major-themes/health-and-well-being. 2. Bishop J, Horton G, Hu W, Vogan C. Learner support and wellbeing. In Swanwick T, Forrest K, O’Brien B, editors. Understanding medical education: evidence, theory and practice. 3rd Edition. Wiley-Blackwell: 2019. p485. 3. Maslow AH A theory of human motivation Psychol Rev 1943 50 4 370 396 10.1037/h0054346 4. Maslow AH Motivation and personality 1954 New York Harper and Row 5. Baik C, Larcombe W. Enhancing student wellbeing. 2016. Retrieved from http://unistudentwellbeing.edu.au/. 6. CRUSH. Collaboration for Research in Understanding Stigma in Healthcare (CRUSH). Retrieved from Bond University. 2022. https://bond.edu.au/researchers/research-strengths/faculty-research-profiles/health-sciences-medicine/crush. 7. Bannatyne A, Bishop J, Tomyn A. Bond University Mental Health & Wellbeing Project. Gold Coast: in progress. 2021. 8. MHFA. Mental Health First Aid Australia. 2022. Retrieved from https://mhfa.com.au/. 9. Bishop J. Sharing is caring. ANZAHPE Festival; Partnerships. 2022. https://eventstudio.eventsair.com/anzahpe-2022/. 10. BEI BH. Mentors across borders. 2022. Retrieved from Brigham Health BEI: https://bei.brighamandwomens.org/mentors-across-borders. 11. Cleland JA, Jamieson S, Kusurkar RA, Ramani S, Wilkinson TJ, van Schalkwyk S. Redefining scholarship for health professions education: AMEE Guide No. 142. Med Teach. 2021;43(7):824–838. 10.1080/0142159X.2021.1900555. Epub 2021 Apr 7. PMID: 33826870. 12. AMEE. Retrieved from An International Association for Health professions Education. 2022. https://amee.org/. 13. Kemp S Hu W Bishop J Medical student wellbeing – a consensus statement from Australia and New Zealand BMC Med Educ 2019 19 69 10.1186/s12909-019-1505-2 30832630 14. Medical Deans Australia and New Zealand Inclusive medical education – guidance on applicants and students with a disability 2021 Sydney Australia 15. Everymind. Every doctor, every setting: a national framework to guide coordinated action of the mental health of doctors and medical students. 2019. Accessed 15 July 2022. 16. Brown B. DARING GREATLY: How the courage to be vulnerable transforms the way we live, love, parent and lead. London, England: Portfolio Penguin. 2013.	36471878	PMC9713081	NO-CC CODE	2022-12-02 23:22:06	no	Med Sci Educ. 2022 Dec 1;:1-6	utf-8	Med Sci Educ	2,022	10.1007/s40670-022-01692-w	oa_other
==== Front Med Sci Educ Med Sci Educ Medical Science Educator 2156-8650 Springer US New York 1692 10.1007/s40670-022-01692-w Keynote Lecture Bouncing Forward: Mental Wealth for All http://orcid.org/0000-0003-2433-6572 Bishop Jo [email protected] grid.1033.1 0000 0004 0405 3820 Faculty of Health Sciences and Medicine, Bond University, Gold Coast, Australia 1 12 2022 16 12 11 2022 © The Author(s) under exclusive licence to International Association of Medical Science Educators 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcWhat is wellbeing and why is it important? The WHO definition reads “Health is a state of complete physical, mental, and social wellbeing and not merely the absence of disease or infirmity. Mental health is a state of well-being in which an individual realizes his or her own abilities, can cope with the normal stresses of life, can work productively and is able to make a contribution to his or her community” [1]. There is currently a significant and increasing amount of interest worldwide in how we can best support our learners and faculty, including health professional educators, to maintain and nurture their wellbeing, in challenging circumstances. Does your institution currently assess wellbeing, through an annual survey, focus groups or involvement in strategic discussions? There is also a focus on strengthening an individual’s ability to overcome adversity, manage stress, and thrive in their personal and professional life and in embedding wellbeing into academic and clinical organisational curricula, plans, policies, and practices. We should be putting people at the heart of what we do! “An individual’s well‐being impacts on their capacity to learn and perform competently. Learner well‐being and supporting their acquisition of skills in self‐care for lifelong practice is therefore core business for educators” [2]. Maslow's theory describing “hierarchy of needs” (see figure) is often portrayed in the shape of a pyramid with the largest, most fundamental needs at the bottom and the need for self-actualization and transcendence at the top [3, 4]. An individuals’ most basic needs must be met before they become motivated to achieve the higher-level needs. The theory states that people are motivated to achieve certain needs and that some of their needs take priority over others. One of our most basic needs is physical survival,this could be in the form of good sleep hygiene, nutrition, all of which can impact our behaviour if not kept in check. Each level needs to be “completed” before we can reach the next level. We work with cultural and interpersonal diversity daily, and we should recognise and address unconscious biases. These biases may include small behavioural cues that signal whether others are valued, whether others are being included or spoken to. Consider the signals you’re sending, your body language, your interaction style, casual comments, and feedback that you deliver. Now look around and consider the environmental signals within your institution, what photos are on your walls, and lastly consider your external signals that may present on your website as images, marketing materials, and which faculty or students are chosen to represent the organisation. Does it suggest all are welcome at this institution? This can really impact the levels of “safety, belonging and esteem” of learners (and faculty). When considering learners life cycle in parallel to this pyramid, not only are there colliding shapes (!) but there are key life changes and transitions that impact their ability to flourish and self-actualise. A key example are the first few days of university, and therefore the role of a dean of student within faculty is to work with the university to ensure there is a balance in the information shared, enough to support but not overwhelm. Welcome sessions can be led by student societies or supported by student leadership. Key information should be given priority, e.g. academic integrity workshops and the respectful community sessions to encourage student transition and to improve social interaction. In addition, pre-arrival information should be co-designed with student leadership and near peers. Within the educational context when we consider the learner life cycle, from student recruitment and admission to their learning and then assessment. This then cycles to results and awards/graduation and finally as alumni. During their life cycle, successful progression of learners requires them to be connected, equipped, motivated, and settled. Why would learners seek support?Difficulty in transitioning to university or new country Difficulty sustaining academic workload and multiple competing tasks Social isolation, social anxiety, loneliness, and victimisation Poor cultural assimilation or English ability Poor resilience or maladaptive coping mechanisms Financial debt or hardship Personal life events A medical condition or disability A psychological predisposition, condition, or disability Emotional instability following a physical or psychological trauma Substance abuse or addiction Why do learners not seek support?Lack of time Lack of confidentiality Fear of negative impact on career Fear of documentation on record Stigma of mental health care Not knowing who to go to Difficulty accessing services Fear of unwanted intervention Fear of legal consequences “Using services will mean I’m weak” Cost My problems are not important “Nobody will understand” How would educators recognise learners in distress?Marked change in academic performance or attendance Persistently late to class or in turning in assessments Highly disorganised Withdrawn from social situations and communication with others Persistent low mood, low motivation, or loss of interest Marked change in hygiene and general appearance Acute stress, anxiety, or panic attacks Unpredictable or irrational thoughts, moods, attitudes, or behaviours Being under the influence of drugs or alcohol Disorientation, delusional, dissociated thoughts, or behaviours Extreme distorted thinking or excessive unwarranted worrying Persistent disruptive or volatile physical or verbal behaviour Alarming material presented in a student’s written work The stigma of mental health is prevalent in healthcare students and practitioners. This may result in learners being reluctant to identify as suffering from a mental health condition and from seeking the required support and management. Learners, health science educators, and practitioners may be wary of accessing support due to number of reasons—including those listed above. To create safe environments for learning and personal growth, we must ensure.Barriers to seeking help are removed. There is access to role model, e.g. near peer or faculty. Confidentiality maintained following disclosure. Fears around progression/career concerns are considered and discussed. Counselling for all is encouraged. Transparency around the benefits of self-care, including the care of others and ensuring safety of patients. Help seeking pathways are accessible and visible. When considering putting strategies into practice to make a difference the institute should monitor policies to promote wellbeing, this can be achieved by finding an existing framework and completing a gaps analysis. At Bond we created a “Promoting Health and Wellbeing working group” with champions from across the university that included student leaders, professional/administrative staff, faculty, and members of the medical services and human resources. We recognised that many initiatives are fairly siloed across faculties; it was realised that it would be helpful to consider how some of these initiatives can be rolled out across the university (e.g. mentoring programmes/counselling offerings) to the benefit of all. We proposed a review of various faculty-specific initiatives with a view to considering resourcing/embedding of support initiatives across the University? Our suggested priorities are listed below including finalising a Mental Health Strategy complete Bond University Wellbeing Research Project.Review and report on the wellbeing and mental health needs of allUsing “A framework for promoting student mental wellbeing in universities” (Baik and Larcombe [5]) Plus consider a whole of university Wellbeing Research Project Reduce the stigma of mental healthPart of the Wellbeing Research Project Work of the Promoting Wellbeing working group (and individual portfolios) Improve education and access to information and promotion of wellbeing within teaching and learning initiatives within the classroom using established frameworkUsing “A framework for promoting student mental wellbeing in universities. https://unistudentwellbeing.edu.au” as indicated by the 5 domains (see image) Increase the volume and visibility of information, resources, and support available in extracurricular endeavours by promoting internal and external servicesContinue to work with the student society, medical services, and our insurance provider to consider appropriate resources Promotion and review of the webpage or create a shared site for all Consider a variety of support methods for allListen to the community (research project and the work achieved in other faculties)—sharing is caring Highlight support needs at key points in the semesterReview transition points—is there a student journey or life cycle that could be considered Improve communication around support strategiesFaculty/student training—mental health first aid Co-design wellbeing events with staff and studentsFunding and support regular events—key dates Establish and promote wellbeing champions Training and support for all A fellow member of the Bond University Collaboration for Research in Understanding Stigma in Healthcare [6] research group and I began the Bond University Mental Health & Wellbeing Project (Bannatyne [7]). Preliminary findings have indicated that during 2020 when reflecting on the impact of COVID-19 on mental health and wellbeing, over half the sample (54.4%) reported a decline in their mental health and wellbeing. 10% of the student population completed the survey with awareness of various support services at Bond University was mixed. Approximately, one-quarter had accessed psychological services. Barriers to accessing University Psychological Services complemented those described earlier, e.g. confidentiality. Notably, over two-thirds of the sample (66.6%) believed Bond University should be actively trying to reduce mental health stigma within the university community. Ideas suggested included sharing lived experience stories via social media, more visible promotion of mental health statistics and support services, a culture of accountability for student leadership, embracing and supporting mental health week and other wellbeing activities, mental health literacy included as a component of core subjects, peer support programmes, and compulsory mental health first aid training for all educators (Bannatyne [7]). Institutions should provide as minimum services such as student counselling service, medical clinic, accessibility & inclusion advisors, security, academic skills centre, first nations support centre, LGBTIQ + services & support, career development centre, and student business centre. Wherever possible consider the role of student leaders, peers who can state and validate “I’ve been there, I get it!”. Engage learners as stakeholders by having learner representatives as a voice for their peers and promote partnerships in health promotion activities and address misconceptions (bust the myths) and open discussion during distressing events. Consider who are your national learner society representatives, does the learner society have a wellbeing officer? Now you? As an educator, who would you turn to, where can you go when need support, who supports the supporters? When questioned, faculty stated they sought support from.Manager/supervisor Executive dean Programme director Faculty-peer support services GP Psychiatrist Mental health professional Clergy member Employee assist programme Internet sources: discussion forums, social media The role descriptors of any educator have had to adapt have and the true extent of their academic workload, and daily pressures should not be underestimated. Institutions are responsible for staff wellbeing as it can affect learner wellbeing and, in the healthcare, setting it can impact patient care. Apart from accreditation and legal requirements to look after health and safety of staff, there are cogent reasons why medical schools should actively promote teacher and staff wellbeing. Who supports the supporters? At Bond, we have supported colleagues by establishing mental health first aid training. This skills based, early intervention training programmes “mobilise and empowers communities by equipping people with the knowledge and confidence to recognise, connect and respond to someone experiencing a mental health problem or mental health crisis. Anyone can have a conversation that may save a life. Everyone should know how” [8] In April 2020, the Student Support Network was established for faculty across the 23 medical schools in Australia and New Zealand. It was realised as a key initiative as we traversed the unprecedented times of the pandemic to ensure those who supported students were also cared for. The once weekly meetings now occur monthly and allow sharing of best practice and resources. Members highlight that they value this community or practice and are empowered to review difficult or complex situations involving students with advice provided by experienced colleagues in confidential manner [9] Mentors Across Borders is a dynamic and diverse international community of Leaders in Health Professions Education formed in 2020, united by a shared passion for mentoring and nurturing fellow educators as well as scholarship. This community aims to promote collegial global conversations about important educational topics in a psychologically safe space to interact, share success stories and challenges, and potentially enhance professional identity (BEI [10]). We have recognised that what learners want and what faculty need is relevant, timely information, in the moment. As educators, you should draw on the expertise of the educational community through membership of organisations such as Australian and New Zealand Association for Health Professional Educators (ANZAHPE), an International Educational Association for Health Professional Educators (AMEE), and the International Association for Medical Science Educators (IAMSE). The publication on redefining scholarship for health profession education [11] emphasises the below resources to support educators:Published research Engagement with a faculty learning community Community of practice Engage with colleagues locally Professional development workshops and opportunities External conference Online webinars No need for face to face, formation of special interest groups, etc. Community can include quality assurance, benchmarking, and peer review Evaluation and student satisfaction Faculty development courses have also been established including an Essential Skills in Medical Education (ESME) course focussing on wellbeing (WESME) (AMEE [12]). In 2019, colleagues published the medical student wellbeing—a consensus statement from Australia and New Zealand (Kemp et al. [13]) that summarised that we should.Design curricula that promote peer support and progressive levels of challenge to students. Employ strategies to promote positive outcomes from stress and to help others in need. Design assessment tasks to foster wellbeing as well as learning. Provide mental health promotion and suicide prevention initiatives. Provide physical health promotion initiatives. Ensure safe and health-promoting cultures for learning in on-campus and clinical settings. Train staff on student wellbeing and how to manage wellbeing concerns. The editors of the understanding medical education textbook recognised the need for a learner wellbeing chapter with recommendations for learner support system and approaches that educators could use (Bishop et al. [2]). The chapter complimented the consensus statement and also suggested the following:To care for patients, health professionals must care for themselves, and they need to learn about and develop this capability. Programmes and interventions can be categorised as general support to all learners, preventive support in anticipation of challenges, and additional support for learners in need. Educators should consider their roles as individual teachers, programme designers, and in creating supportive and safe learning environments that will help learners negotiate future challenges in the clinical workplace. Educators should consider professional boundaries in their actions to promote well‐being and welfare. Whilst many educators may draw upon their experiences as clinicians, learners are not their patients. Future Directions: Building the Future Together The recent publication by Medical Deans Australia and New Zealand Inc. (Medical Deans) on Inclusive medical education is designed to assist medical schools in their approach to and discussions with prospective and current students with a disability and to identify and consider the adjustments or supports that may be needed for them to commence or continue in a medical programme [14] In addition, the National Framework, Every doctor every setting; Improving the wellbeing of doctors and medical students is a key enabler of quality patient care and healthier communities. The framework is based on available evidence and advice from doctors, doctors-in-training, medical students, mental health and suicide prevention experts, and other key stakeholders. The overall consideration is that all jurisdictions, settings, services, and stakeholders must be involved to ensure immediate, sustained, and coordinated action (Everymind [15]). There are predictable times of distress for all of us as we navigate life, and therefore, preventative measures and sharing of a toolbox of skills and resources whilst understanding the “currency of mental wealth” are essential. There are five types of wellbeing described: emotional, physical, social, workplace, and societal wellbeing. Many of us will have a preference—for me, physical activity, weather walking, cycling, or strength training allows me to perform at my best. Emotionally, I am aware of my own requirements and needs to have time out and reconnect. I have a supportive social community with family and friends, in my workplace I have a sense of purpose and drive and thrive during times of challenge. Societal can be difficult when faced with dilemmas outside of our control. Resilience is often defined as bouncing back from adversity; in reality, no one ever returns to how they were before the event. There is often a required time to reflect and to heal, but more than often there is personal growth; the timelines can vary person to person. Using the narrative, “bouncing forward” gives permission to allow the individual to grow and gain strength from these challenging times. I have gained strength in sharing my own vulnerability and hope this will help others. Brené Brown debunks some myths about vulnerability, the most popular being that vulnerability is a sign of weakness. “When we think of times that we have felt vulnerable or emotionally exposed, we are actually recalling times of great courage; Vulnerability is the birthplace of love, belonging, joy courage, empathy, and creativity” (Brown [16]). Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 15 KB) Author Contribution Not applicable. Availability of Data and Material Not applicable. Declarations Ethics Approval and Consent to Participate Not applicable. Consent for Publication Not applicable. Conflict of Interest The author declares no competing interests. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. WHO. Health and well-being. 2022. Retrieved from World Health Organization: https://www.who.int/data/gho/data/major-themes/health-and-well-being. 2. Bishop J, Horton G, Hu W, Vogan C. Learner support and wellbeing. In Swanwick T, Forrest K, O’Brien B, editors. Understanding medical education: evidence, theory and practice. 3rd Edition. Wiley-Blackwell: 2019. p485. 3. Maslow AH A theory of human motivation Psychol Rev 1943 50 4 370 396 10.1037/h0054346 4. Maslow AH Motivation and personality 1954 New York Harper and Row 5. Baik C, Larcombe W. Enhancing student wellbeing. 2016. Retrieved from http://unistudentwellbeing.edu.au/. 6. CRUSH. Collaboration for Research in Understanding Stigma in Healthcare (CRUSH). Retrieved from Bond University. 2022. https://bond.edu.au/researchers/research-strengths/faculty-research-profiles/health-sciences-medicine/crush. 7. Bannatyne A, Bishop J, Tomyn A. Bond University Mental Health & Wellbeing Project. Gold Coast: in progress. 2021. 8. MHFA. Mental Health First Aid Australia. 2022. Retrieved from https://mhfa.com.au/. 9. Bishop J. Sharing is caring. ANZAHPE Festival; Partnerships. 2022. https://eventstudio.eventsair.com/anzahpe-2022/. 10. BEI BH. Mentors across borders. 2022. Retrieved from Brigham Health BEI: https://bei.brighamandwomens.org/mentors-across-borders. 11. Cleland JA, Jamieson S, Kusurkar RA, Ramani S, Wilkinson TJ, van Schalkwyk S. Redefining scholarship for health professions education: AMEE Guide No. 142. Med Teach. 2021;43(7):824–838. 10.1080/0142159X.2021.1900555. Epub 2021 Apr 7. PMID: 33826870. 12. AMEE. Retrieved from An International Association for Health professions Education. 2022. https://amee.org/. 13. Kemp S Hu W Bishop J Medical student wellbeing – a consensus statement from Australia and New Zealand BMC Med Educ 2019 19 69 10.1186/s12909-019-1505-2 30832630 14. Medical Deans Australia and New Zealand Inclusive medical education – guidance on applicants and students with a disability 2021 Sydney Australia 15. Everymind. Every doctor, every setting: a national framework to guide coordinated action of the mental health of doctors and medical students. 2019. Accessed 15 July 2022. 16. Brown B. DARING GREATLY: How the courage to be vulnerable transforms the way we live, love, parent and lead. London, England: Portfolio Penguin. 2013.	0	PMC9713082	NO-CC CODE	2022-12-02 23:22:06	no	Haut in Form. 2022 Dec 1; 15(4):4-7	latin-1	null	null	null	oa_other
==== Front Soft comput Soft comput Soft Computing 1432-7643 1433-7479 Springer Berlin Heidelberg Berlin/Heidelberg 7713 10.1007/s00500-022-07713-5 Retraction Note Retraction Note: The prediction of the lifetime of the new coronavirus in the USA using mathematical models http://orcid.org/0000-0002-6122-3342 Selvakumar K. [email protected] [email protected] 12 Lokesh S. 3 1 grid.252262.3 0000 0001 0613 6919 Department of Science and Humanities, Anna University, Chennai, India 2 University College of Engineering, Nagercoil, Tamil Nadu 629004 India 3 Department of Computer Science and Engineering, Hindustan Institute of Technology, Othakalmandapam, Coimbatore, Tamil Nadu 641032 India 30 11 2022 11 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. ==== Body pmcRetraction Note to: Soft Computing (2021) 25:10575–10594 https://doi.org/10.1007/s00500-021-05643-2 The Editor-in-Chief and the Publisher have retracted this article. The article was submitted to be part of a guest-edited issue. An investigation by the Publisher found a number of articles, including this one, with a number of concerns, including but not limited to compromised editorial handling and peer review process, inappropriate or irrelevant references or not being in scope of the journal or guest-edited issue. Based on the investigation's findings, the Editor-in-Chief therefore no longer has confidence in the results and conclusions of this article. S Lokesh disagrees with this retraction. K. Selvakumar has not explicitly stated whether they agree or disagree with this retraction. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	36471808	PMC9713083	NO-CC CODE	2022-12-02 23:22:06	no	Soft comput. 2022 Nov 30;:1	utf-8	Soft comput	2,022	10.1007/s00500-022-07713-5	oa_other
==== Front J Gen Intern Med J Gen Intern Med Journal of General Internal Medicine 0884-8734 1525-1497 Springer International Publishing Cham 36451016 7801 10.1007/s11606-022-07801-0 Original Research Influence of Cost-Related Considerations on Clinical Trial Participation: Results from the 2020 Health Information National Trends Survey (HINTS) http://orcid.org/0000-0001-5328-7517 Williams Courtney P. DrPH [email protected] Geiger Ann M. MPH PhD Norton Wynne E. PhD de Moor Janet S. PhD MPH Everson Nicole Senft PhD grid.48336.3a 0000 0004 1936 8075 Division of Cancer Control and Population Sciences, National Cancer Institute, Rockville, MD USA 30 11 2022 17 19 4 2022 8 9 2022 © The Author(s), under exclusive licence to Society of General Internal Medicine 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Background People experiencing financial burden are underrepresented in clinical trials. Objective Describe the prevalence of cost-related considerations influential to trial participation and their associations with person-level characteristics. Design This cross-sectional study used and assessed how three cost-related considerations would influence the decision to participate in a hypothetical clinical trial. Participants A total of 3682 US adult respondents to the Health Information National Trends Survey Main Measures Survey-weighted multivariable logistic regression estimated associations between respondent characteristics and odds of reporting cost-related considerations as very influential to participation. Key Results Among 3682 respondents, median age was 48 (IQR 33–61). Most were non-Hispanic White (60%), living comfortably or getting by on their income (74%), with ≥ 1 medical condition (61%). Over half (55%) of respondents reported at least one cost-related consideration as very influential to trial participation, including if usual care was not covered by insurance (reported by 42%), payment for participation (24%), or support for participation (24%). Respondents who were younger (18–34 vs. ≥ 75, adjusted odds ratio [aOR] 4.3, 95% CI 2.3–8.1), more educated (high school vs. <high school, aOR 2.1, 95% CI 1.1–4.1), or with lower perceived income (having difficulty vs. living comfortably, aOR 2.1, 95% CI 1.1–3.8) had higher odds of reporting any cost-related consideration as very influential to trial participation. Non-Hispanic Black vs. non-Hispanic White respondents had 29% lower odds (95% CI 0.5–0.9) of reporting any cost-related consideration as very influential to trial participation. Conclusions Cost-related considerations would influence many individuals’ decisions to participate in a clinical trial, though prevalence of these concerns differed by respondent characteristics. Reducing financial barriers to trial participation may promote equitable trial access and greater trial enrollment diversity. Supplementary Information The online version contains supplementary material available at 10.1007/s11606-022-07801-0. KEY WORDS clinical trials financial hardship cost of care health care access ==== Body pmcBACKGROUND Clinical trials provide the evidence-base for new medical, surgical, and behavioral therapies. Demographically and clinically diverse participants, including those of minoritized racial and ethnic groups, those of sexual and gender groups, or those with comorbid conditions, are necessary to ensure clinical trial results are generalizable at the population level and do not widen existing health disparities.1 Individuals who more often experience financial burden are another population group systematically underrepresented in clinical trials, potentially due to their associated costs.2–4 Underrepresentation of economically vulnerable individuals in clinical trials may exacerbate existing disparate outcomes due to associations with health care access issues.1 Recent policy efforts have sought to diversify clinical trial participation by improving coverage of routine clinical care costs associated with participation.5 However, trial participants are still faced with non-routine care costs, such as those for treating trial-related symptoms or side effects, transportation, lodging, meals, child or elder care, and productivity losses.6 Little is known about how these cost-related considerations influence trial participation among US adults. Understanding cost-related influences on trial participation is key to informing policy and trial design efforts aimed at promoting more diverse and representative clinical trial enrollment. Therefore, this study describes the prevalence of cost-related considerations influential to clinical trial participation using a large sample of US adults. Additionally, we explore associations between these cost-related considerations and person-level characteristics related to clinical trial participation in previous studies, including race or ethnicity, household income, geographic residence, education, and insurance status.3,7 METHODS Study Design and Sample This cross-sectional study used data from the Health Information National Trends Survey (HINTS) 5 Cycle 4, collected February through June 2020 via mailed questionnaires. HINTS is a publicly available, cross-sectional, nationally representative survey of civilian, non-institutionalized US adults that assesses knowledge of, attitudes toward, and use of health-related information.8, 9 Respondents’ return of the completed survey indicated consent to participate. HINTS data are deidentified and thus exempt from review by the US National Institutes of Health Office of Human Subjects Research Protections. Outcome: Cost-Related Considerations for Hypothetical Participation in Clinical Trials After reading a plain language definition of clinical trials, with a drug trial and behavioral intervention provided as examples, respondents were asked to imagine being invited to participate in a clinical trial for a health issue (Supplemental Figure 1). Respondents then rated the extent to which (1) receiving payment for participation (“I would get paid to participate”), (2) support for participation (”I would get support to participate such as transportation, childcare, or paid time off from work”), and (3) if usual care was not covered by insurance (“If the standard care was not covered by my insurance”) would influence their decision to participate. Responses were captured on a 4-point scale (a lot, somewhat, a little, not at all) and then dichotomized for analysis using top-box scoring as “a lot” versus the combined scores of “somewhat,” “a little,” and “not at all” to differentiate the most influential cost-related considerations on decision to participate in a clinical trial.10–12 Covariables Demographic variables included age, sex, race/ethnicity, education, objective household income, subjective feelings about present income, marital status, rural/urban residence,13 census region, employment status, and health insurance status. Medical histories included number of diagnosed medical conditions, self-reported health, and annual health care visits. Statistical Analysis All analyses were weighted to estimate nationally representative estimates using complex survey methodology with jackknife replicate weights for accurate standard errors.8 Respondent demographic and clinical characteristics were described for three groups: (1) overall sample, (2) respondents who reported at least one cost-related consideration as very influential to trial participation, and (3) respondents who reported no cost-related considerations as very influential. Survey-weighted medians, interquartile ranges, frequencies, and 95% confidence intervals (CI) were calculated as appropriate. Adjusted odds ratios (aOR) and 95% CIs from an exploratory, survey-weighted logistic regression model estimated associations between respondent characteristics and odds of reporting a cost-related consideration as very influential to trial participation. Covariable multicollinearity was examined using the variance inflation factor (range=1.0–3.6). Sensitivity analyses estimated associations between respondent characteristics and cost-related considerations dichotomized using bottom-box scoring (a lot/somewhat/a little vs. not at all), and individual cost-related consideration as separate outcomes. All analyses were performed using SAS 9.4 (SAS Institute, Cary, NC). RESULTS A total of 15,347 HINTS 5 Cycle 4 surveys were mailed, 3865 were returned and eligible for analysis (37% weighted response rate), and 3682 with complete information on cost-related considerations for trial participation were included in our sample. The median age of respondents was 48 (IQR 33–61), most were non-Hispanic White (60%), employed (58%), living comfortably or getting by on their present income (74%), and had ≥ 1 medical condition (61%; Table 1). Over half (55%, 95% CI 51–59%) of respondents reported at least one cost-related consideration as very influential to trial participation (vs. somewhat/a little/not at all). Of cost-related considerations, respondents most often reported usual care uncovered by insurance as very influential to participation (42%, 95% CI 38–45%), followed by receiving payment for participation (24%, 95% CI 21–28%), and receiving support for participation (24%, 95% CI 21–26%; Fig. 1). Table 1 Descriptive Statistics and Multivariable Exploratory Analysis of Reporting at Least One Cost-Related Consideration as Influential to Hypothetical Clinical Trial Participation by Respondent Demographic, Clinical, and Health Behavior–Related Characteristics (N=3682) Total, N=3682 Reported cost-related consideration, n=1897 Did not report cost-related consideration, n=1785 Adjusted odds of reporting at least one cost-related consideration vs. none, N=3682 n (weighted %) n (weighted %) n (weighted %) aOR (95% CI) Age (weighted median, IQR) 48 (33–61) 45 (31–57) 52 (37–65) 18–34 479 (26.0) 308 (64.1) 171 (35.9) 4.28 (2.26–8.14) 35–49 688 (25.2) 423 (62.4) 265 (37.6) 4.00 (2.16–7.41) 50–64 1110 (27.2) 566 (50.9) 544 (49.1) 2.18 (1.24–3.84) 65–74 820 (11.3) 379 (44.8) 441 (55.2) 1.51 (1.03–2.21) ≥ 75 487 (8.0) 181 (33.5) 306 (66.5) 1 [Reference] Sex Male 1507 (48.0) 737 (54.0) 770 (46.0) 0.84 (0.63–1.12) Female 2102 (50.3) 1132 (56.4) 970 (43.6) 1 [Reference] Race and ethnicity Non-Hispanic White 2087 (59.7) 1142 (58.2) 945 (41.8) 1 [Reference] Non-Hispanic Black 456 (10.3) 237 (53.1) 219 (46.9) 0.71 (0.52–0.97) Hispanic 515 (14.1) 246 (50.3) 269 (49.7) 0.62 (0.41–0.95) Other race/multiracial 317 (9.3) 155 (52.1) 162 (47.9) 0.67 (0.43–1.03) Education Less than high school 244 (7.6) 80 (33.6) 164 (66.4) 1 [Reference] High school degree 655 (21.4) 304 (53.1) 351 (46.9) 2.12 (1.10–4.06) Some college 1046 (38.6) 550 (57.6) 496 (42.4) 2.26 (1.19–4.29) College graduate or higher 1627 (30.0) 917 (58.8) 710 (41.2) 2.17 (1.08–4.34) Annual household income $0–$19,999 582 (13.7) 278 (50.0) 304 (50.0) 1 [Reference] $20,000–$34,999 428 (10.4) 218 (56.5) 210 (43.5) 1.25 (0.67–2.31) $35,000–$49,999 444 (11.6) 209 (55.3) 235 (44.7) 1.26 (0.76–2.12) $50,000–$74,999 575 (16.9) 305 (55.0) 270 (45.0) 1.13 (0.62–2.08) $75,000–$99,999 397 (11.6) 216 (56.0) 181 (44.0) 1.05 (0.55–2.02) ≥ 100,000 905 (28.1) 525 (58.0) 380 (42.0) 1.16 (0.58–2.32) Feelings about present income Living comfortably on present income 1393 (34.6) 696 (50.2) 697 (49.8) 1 [Reference] Getting by on present income 1379 (39.5) 715 (56.6) 664 (43.4) 1.38 (1.00–1.89) Finding it difficult on present income 507 (15.1) 282 (59.5) 225 (40.5) 1.70 (1.14–2.52) Finding it very difficult on present income 211 (6.2) 127 (63.2) 84 (36.8) 2.08 (1.14–3.82) Marital status Married/living as married 1921 (53.5) 998 (54.0) 923 (46.0) 1 [Reference] Divorced/widowed/separated 1025 (13.5) 490 (49.0) 535 (51.0) 0.93 (0.72–1.22) Single, never married 623 (30.3) 364 (59.9) 259 (40.1) 1.00 (0.72–1.39) Residence Rural 292 (8.2) 142 (51.2) 150 (48.8) 0.84 (0.54–1.33) Urban 3390 (91.8) 1755 (55.4) 1635 (44.6) 1 [Reference] Region Northeast 547 (17.5) 274 (54.0) 273 (46.0) 1 [Reference] Midwest 620 (20.9) 337 (56.1) 283 (43.9) 1.06 (0.65–1.72) South 1644 (38.0) 847 (54.8) 797 (45.2) 1.10 (0.79–1.52) West 871 (23.6) 439 (55.4) 432 (44.6) 1.15 (0.79–1.68) Employment status Employed 1832 (58.0) 1049 (60.3) 783 (39.7) 1 [Reference] Retired 1135 (18.1) 501 (41.8) 634 (58.2) 0.83 (0.56–1.23) Unemployed/disabled 322 (9.8) 173 (56.4) 149 (43.6) 0.99 (0.63–1.57) Other 288 (11.7) 127 (48.9) 161 (51.1) 0.66 (0.37–1.18) Health insurance status Private/employer sponsored 1517 (48.2) 867 (58.5) 650 (41.5) 1 [Reference] Medicare 1124 (18.8) 502 (45.2) 622 (54.8) 1.37 (0.88–2.12) Medicaid 329 (11.3) 179 (56.4) 150 (43.6) 0.95 (0.57–1.59) Dual eligible 225 (3.9) 98 (39.8) 127 (60.2) 0.87 (0.41–1.81) Other* 254 (7.7) 138 (64.1) 116 (35.9) 1.53 (1.03–2.27) Uninsured 192 (9.0) 94 (54.9) 98 (45.1) 0.93 (0.44–1.98) Number of medical conditions 0 1121 (38.4) 595 (56.9) 526 (43.1) 1 [Reference] 1 1141 (30.8) 589 (55.0) 552 (45.0) 0.96 (0.69–1.34) 2 796 (18.9) 413 (57.0) 383 (43.1) 1.16 (0.74–1.80) ≥ 3 592 (11.5) 291 (47.3) 301 (52.7) 0.93 (0.56–1.55) Self-rated health Excellent/very good 1741 (50.0) 945 (57.8) 796 (42.2) 1 [Reference] Good 1340 (35.8) 655 (52.1) 685 (47.9) 0.88 (0.67–1.16) Fair/poor 585 (13.9) 291 (53.5) 294 (46.5) 1.01 (0.64–1.62) Saw provider in last year Yes 3185 (83.0) 1687 (56.4) 1498 (43.6) 1.52 (1.10–2.08) No 471 (16.4) 203 (48.9) 268 (51.1) 1 [Reference] aOR, adjusted odds ratio; CI, confidence interval *Includes coverage by the VA, TRICARE/other military health care, Indian Health Service, or a response of “any other type of health insurance or health coverage plan” Figure 1 Respondent reporting of the extent to which cost-related considerations would influence hypothetical participation in clinical trials (N=3682). In descriptive comparisons, respondents more often reporting at least one cost-related consideration as very influential to trial participation were younger (64% aged 18–34 years vs. 34% aged ≥ 75 years), more educated (59% college graduate or higher vs. 34% < high school education), employed compared to retired (60% vs. 42%), enrolled in private or employer-sponsored health insurance compared to dually enrolled in Medicare and Medicaid (59% vs. 40%), or had lower perceived income (63% finding it difficult vs. 50% living comfortably on their present income; Table 1). Associations between reporting cost-related considerations as very influential to trial participation and respondent characteristics were seen in multivariable model results (Table 1). Compared to those aged 75 and older, younger respondents had higher odds of reporting any cost-related considerations as very influential to clinical trial participation (aged 18–34 aOR 4.28, 95% CI 2.26–8.14; aged 35–49 aOR 4.00, 95% CI 2.16–7.41; aged 50–64 aOR 2.18, 95% CI 1.24–3.84; aged 65–74 aOR 1.51, 95% CI 1.03–2.21). Non-Hispanic Black respondents had 29% lower odds (95% CI 0.52–0.97) and Hispanic respondents 38% lower odds (95% CI 0.41–0.95) of reporting cost-related considerations as very influential to trial participation when compared to non-Hispanic White respondents. Respondents with a high school education or more had two-times higher odds of reporting cost-related considerations as very influential to trial participation compared to those with less than a high school education (high school degree aOR 2.12, 95% CI 1.10–4.06; some college aOR 2.26, 95% CI 1.19–4.29; college graduate or higher aOR 2.17, 95% CI 1.08–4.34). Subjective income also showed associations with hypothetical trial participation, with respondents getting by on their present income having 38% higher odds (95% CI 1.00–1.89), respondents finding it difficult on their present income having 70% higher odds (95% CI 1.14–2.52), and respondents finding it very difficult on their present income having 108% higher odds (95% CI 1.14–3.82) of reporting cost-related considerations as very influential to trial participation compared to those living comfortably on their present income. Respondents who saw their health care provider in the previous year also had 52% higher odds of reporting a cost-related consideration as very influential compared to those who did not (95% CI 1.10–2.08). Sensitivity analyses showed similar associations and directionality to the multivariable model results (Supplemental Table 1). DISCUSSION Over half of respondents to this nationwide survey reported at least one cost-related consideration as very influential in their decision to participate in a hypothetical clinical trial. Respondents most often reported usual care not covered by insurance would influence participation, followed by receipt of payment or support for participation. Furthermore, cost-related considerations influential to trial participation differed by person-level characteristics. Respondents who were younger, non-Hispanic White, more educated, not living comfortably on their present income, or who saw their health care provider in the previous year had higher odds of reporting at least one cost-related consideration as influential to participation in a hypothetical clinical trial. Our results are unique, since data on cost-related considerations associated with clinical trial participation are rarely captured in disease agnostic patient samples, yet are needed for government and payer reimbursement policies to address all disease settings. Our results also point to the importance of including socioeconomically diverse patients for clinical representativeness, as well as the potential to increase trial enrollment for individuals who are financially sensitive by addressing perceived and actual cost-related barriers for a wide range of patients. Financial barriers to clinical trial participation are prevalent, with 55% of our survey respondents reporting a cost-related consideration as influential to trial participation. This suggests future work should test strategies focused on reducing financial barriers to participation. Reimbursement for direct and indirect costs (e.g., travel, parking, lodging, caregiving expenses) is one potential strategy that could aid in reducing the financial burden experienced by participants and allow for more generalizable trial samples. Reimbursement for trial-related travel and lodging costs was shown to increase cancer clinical trial enrollment for younger, lower income patients experiencing financial burden.14 It is important to note the difference between reimbursement for actual trial-related expenses and undue influence of financial incentives or coercion, which is cited as an ethical concern associated with payment or support for clinical trial participation. From the patient perspective, current clinical trial participants believed monetary reimbursement would not coerce individuals to participate if they found the trial protocol unacceptable at enrollment, and would instead act as beneficial compensation reflecting the time, inconvenience, and risks related to participation.15 Researchers have also postulated that Institutional Review Boards should consider whether reimbursements for research participation are high enough to protect against patient exploitation or overburden.16 This stance is emphasized by the US Food and Drug Administration (FDA), which does not consider payment for research participation as a benefit for research participation, but rather a “just and fair” practice.17 The American Society of Clinical Oncology echoes this viewpoint and includes removal of “impediments to ethically appropriate financial compensation for trial-related out-of-pocket costs” and eliminating the perception of patient financial support as undue influence as one of its four policy recommendations to decrease financial barriers to cancer clinical trial participation.4 These stances were recently reflected at the state levels in enacting legislation which provides patient expense reimbursement for cancer clinical trial participation. However, as Largent and Lynch argue, these laws should be expanded to all states and to include all areas of clinical research to ensure equitable trial access and increased trial enrollment for individuals with and without financial burden considering participation.18 Our results showed increasing odds of reporting a cost-related consideration influential to trial enrollment as age decreased. Younger respondents may face more financial barriers related to employment, including productivity loss or lost wages due to time off work to receive trial-related care, when compared to older, potentially retired respondents. Our descriptive results support this hypothesis, since respondents who were employed more often reported a cost-related consideration as very influential to trial participation compared to retired adults. In a study of patients with myeloproliferative neoplasms, 21% reported having missed more workdays while on trial compared to if they had not participated, and 12% reported they would not have participated if they had known about the associated financial consequences.19 However, few studies have explored how concerns about potential employment disruptions, including lost wages, lost productivity, and job loss, influence clinical trial participation. Likewise, patient-level data collected during clinical trials typically do not capture adverse employment outcomes due to trial participation or trial therapy-related side effects, complications, or adverse events. The FDA has recently recommended collecting employment-related outcomes for individuals enrolled in cancer clinical trials.20 Collecting employment-related outcomes during trial screening, enrollment, and monitoring could aid health care organizations and clinicians in understanding how to decrease employment-related barriers to trial participation. Furthermore, scheduling care around a patient’s work schedule, proactively managing work-limiting side effects, receiving reimbursement for lost wages, or receiving paid time off work could increase younger adult participation in clinical trials. Our descriptive results revealed respondents enrolled in private or employer-sponsored health insurance more often reported cost-related considerations as very influential to trial enrollment when compared to respondents enrolled in Medicare, Medicaid, dually enrolled, or even those uninsured. Though this association was not seen in multivariable models, it is still important since considering insurance enrollment only may mask differences in actual insurance coverage. Clinical trials are commonly offered to patients with chronic and costly health conditions, and trial participation increases interactions with the health care system. Thus, patients with private, high deductible plans could face substantial out-of-pocket trial-related expenses before insurance covers care costs. Though insurers cover costs related to routine care received during the trial, research costs not covered by the trial sponsor, such as the trial treatment itself, trial-related labs, imaging, or procedures, or out-of-network trial-related care is at the expense of the patient.21, 22 For example, individuals interested in a cancer clinical trial often receive trial treatment at a National Cancer Institute (NCI)–designated cancer center, yet only 41% of Affordable Care Act Marketplace plans include an NCI-designated cancer center in-network.23 It is therefore important to ensure potential trial enrollees do not incur high out-of-pocket costs associated with trial participation due to their health insurance coverage status. In our study, respondents not living comfortably on their present income had 38–108% higher odds of reporting a cost-related consideration influencing trial participation compared to those living comfortably on their income. Interestingly, no associations were found between measures of objective annual household income and report of cost-related considerations in our study. This contrasts with other studies, where individuals earning <$50,000 of annual household income had 32% lower odds of cancer clinical trial participation compared to those earning higher incomes.2 Our results may point to the importance of differentiating between cost and affordability when assessing financial barriers to trial participation. Potential participants may have similar incomes, but unequal participation affordability due to the number of people being supported on their household income, household expenses, or differences in trial participation costs such as transportation or childcare. Thus, clinicians should consider collecting both subjective and objective measures of patient-reported income data as part of their research since both measures may be important to clinical trial participation. Non-Hispanic Black or Hispanic respondents had lower odds of reporting a cost-related consideration as very influential to trial participation compared to non-Hispanic White respondents in our study. Few data exist on the association between race and ethnicity and cost-specific barriers to clinical trial participation. One study found that patients who are African American or Hispanic discuss trial treatment costs more frequently than non-Hispanic White patients. However, it is unclear how these discussions affect participation.24 We can hypothesize that other factors related to trial participation, such as trust in the health care system or their provider,25 may be more influential in the decision to participate compared to cost. Our results also showed associations between higher education levels or more frequent interactions with the health care system and reporting cost-related considerations as very important to trial participation. These results may be secondary to a patient’s level of activation, defined as the knowledge, skills, and confidence to manage one’s health.26, 27 We hypothesize that respondents with higher levels of activation may have higher levels of education and more frequently utilize the health care system, and thus have more awareness of the costs related to trial participation. Likelihood of trial participation increased for patients with cancer involved in a demonstration project sponsored by the Education Network to Advance Cancer Clinical Trials, which utilized patient navigators to aid in increasing patient activation and trial-related education.6 However, it is unknown if and how trial cost discussions were incorporated into the activation and education strategies. More research is needed to understand associations between education, health care use, and cost-related considerations for clinical trial participation to tailor interventions which drive increases in trial participation. The results of our study should be considered within the context of several limitations. Selection or response bias may exist due to HINTS survey methods and response rates.28 However, the complex survey methodology used when analyzing HINTS data adjusts for non-responders based on the measured characteristics of HINTS sample members known to be related to response propensity, which minimizes non-response bias. Model misspecification may exist based upon omitted variable bias due to the use of secondary survey data, overfitting due to including an irrelevant covariable, or potential covariable measurement error. Questions surrounding cost-related considerations for clinical trial participation were based on hypothetical trial participation, which may differ from respondent considerations when making actual trial participation decisions. Because this survey was fielded in Spring 2020, results may also be influenced by increased awareness of trials or financial issues faced by American public due to the COVID-19 pandemic. However, proportions of respondents reporting cost-related considerations to trial participation were similar for surveys returned both before and after March 11, 2020, the date the pandemic was declared. CONCLUSION Cost-related considerations were influential in the hypothetical decision to participate in a clinical trial, with 55% of survey respondents reporting at least one consideration. These results suggest that financial barriers to trial participation are one of many important factors to consider in assessing participation equity and can inform clinical trial decision-makers at multiple levels. Policy makers and payers should consider creating trial cost policies that are easily accessible and straightforwardly conveyed to individual enrollees. Trial sponsors should consider reimbursement for non-medical costs related to trial participation. Health care systems and clinicians who offer trials should be aware of trial-related costs, and clearly communicate these potential costs to eligible and interested patients. Finally, trial decision-makers should work to better quantify trial-related costs, including out-of-pocket medical and non-medical costs, employment outcomes, and how costs may differ for various patient sociodemographic groups, to better understand the financial impact of trial participation. Because our results suggest cost-related considerations influencing clinical trial participation may differ in their appeal to particular patients, more empirical evidence is needed to test cost-offsetting mechanisms, which could aid in increasing trial participation, and thus improve socioeconomic equity in access to trials and increase socioeconomic generalizability from clinical trials to a broader range of participants. Supplementary Information ESM 1 (DOCX 405 kb) Data Availability The Health Informatics National Trends data are publicly available from the National Cancer Institute, https://hints.cancer.gov/. Declarations Conflict of Interest The authors declare that they do not have a conflict of interest. Disclaimers The observations and conclusions expressed in this article are those of the authors and this material should not be interpreted as representing the official viewpoint of the US Department of Health and Human Services, the National Institutes of Health, or the National Cancer Institute. Prior presentations: This study was presented as part of a symposium at the Society of Behavioral Medicine’s 43rd Annual Meeting & Scientific Sessions, April 6–9, 2022. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Diversity & Inclusion in Clinical Trials. National Institute on Minority Health and Health Disparities, National Institutes of Health. https://www.nimhd.nih.gov/resources/understanding-health-disparities/diversity-and-inclusion-in-clinical-trials.html. Accessed July 2022. 2. Unger JM, Hershman DL, Albain KS, Moinpour CM, Petersen JA, Burg K, et al. Patient income level and cancer clinical trial participation. 2013;31(5):536. 3. Chino F, Zafar SY. Financial toxicity and equitable access to clinical trials. 2019(39):11-8. 10.1200/edbk_100019. 4. Winkfield KM, Phillips JK, Joffe S, Halpern MT, Wollins DS, Moy B. Addressing financial barriers to patient participation in clinical trials: ASCO policy statement. J Clin Oncol. 2018;36(33):3331-9. 5. Clinical Treatment Act, Stat. 913 (December 22, 2020, 2019). https://www.congress.gov/bill/116th-congress/house-bill/913/all-info. 6. Nipp RD, Hong K, Paskett ED. Overcoming barriers to clinical trial enrollment. 2019;39:105-14. 7. Williams CP, Senft Everson N, Shelburne N, Norton WE. 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More than Tuskegee: understanding mistrust about research participation. J Health Care Poor Underserved 2010;21(3):879. 26. Greene J, Hibbard JH. Why does patient activation matter? An examination of the relationships between patient activation and health-related outcomes. J Gen Intern Med. 2012;27(5):520-6. 10.1007/s11606-011-1931-2. 27. Hamel LM, Penner LA, Albrecht TL, Heath E, Gwede CK, Eggly S. Barriers to clinical trial enrollment in racial and ethnic minority patients with cancer. Cancer Control : Journal of the Moffitt Cancer Center. 2016;23(4):327-37. 10.1177/107327481602300404. 28. Maitland A, Lin A, Cantor D, Jones M, Moser RP, Hesse BW, et al. A nonresponse bias analysis of the Health Information National Trends Survey (HINTS). J Health Commun. 2017;22(7):545-53. 10.1080/10810730.2017.1324539.	36451016	PMC9713084	NO-CC CODE	2022-12-02 23:22:07	no	J Gen Intern Med. 2022 Nov 30;:1-7	utf-8	J Gen Intern Med	2,022	10.1007/s11606-022-07801-0	oa_other
==== Front Cybern Syst Anal Cybern Syst Anal Cybernetics and Systems Analysis 1060-0396 1573-8337 Springer US New York 506 10.1007/s10559-022-00506-6 Article Model for Regulating the Reproduction Process in the Economy Iefymenko T. I. [email protected] Dunaev B. B. [email protected] Lyubich A. A. [email protected] State Educational and Research Institution “Academy of Financial Management”, Kyiv, Ukraine 1 12 2022 113 19 11 2021 © Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The authors analyze the regulation of the reproduction process in the real and financial sectors of the economy by variations in the amount of cash in circulation and through the investment policy of the state, as well as the consequences of the collapse of the market value of securities. The production sphere is shown to unite the production system (production), banking system, market system, domestic households, and the rest of the world. It is shown that the regulators of the economic reproduction are the banking system that provides credits to production and the necessary amount and velocity of money in the real sector, the financial sector that is selling securities for cash and investing in production, and the state that pursues an investment policy for the expanded reproduction of production capital. The reproduction process of the economy of Ukraine is modeled for 2022–2024 by the statistical data of 2020 and 2021. Keywords reproduction economy regulation market equilibrium labor capital money interest rate exchange rate crisis inflation targeting ==== Body pmcTranslated from Kibernetyka ta Systemnyi Analiz, No. 5, September–October, 2022, pp. 64–78. ==== Refs References 1. Robinson J Robinson J The rate of interest The Generalization of the General Theory and Other Essays 1952 MacMillan London 67 142 2. Borzenko OO Structural Deformations in the World Financial Markets after the 1990s [in Ukrainian] 2019 Vinnytsia DonNU imeni Vasylya Stusa 3. Stiglitz JE Freefall: America, Free Markets, and the Sinking of the World Economy 2010 W. W Norton 4. B. Dunaev, “Regulation of inflation in macroeconomics,” Nauk. Pr. NDFI, No. 4, 80–94 (2020). 10.33763/npndfi2020.04.080. 5. Iefymenko TI Dunaev BB Lyubich AA Inflation targeting model in macroeconomics Cybern. Syst. Analysis 2021 57 6 968 977 10.1007/s10559-021-00422-1 6. O. M. Didenko, “Regulation of banking activity in the context of reconciliation of government and business interests,” Doctoral Thesis in Economics, SumDU, Sumy (2016). 7. B. B. Dunaev, Well-Being: Labor, Capital, and Money. Fundamentals of Reproduction Theory [in Russian], Interdruk, Kyiv (2013). 8. Dunaev BB Kirilenko LV Deflationary regulation of market equilibrium Cybern. Syst, Analysis 2018 54 2 258 270 10.1007/s10559-018-0027-y 9. Dunaev BB Banking regulation of macroeconomic processes Cybern. Syst, Analysis 2021 57 1 108 123 10.1007/s10559-021-00334-0 10. Sachs JD Larraín FB Macroeconomics in the Global Economy 1993 Prentice Hall 11. Dunaev BB Non-inflationary consumer demand Cybern. Syst. Analysis 2016 52 4 588 599 10.1007/s10559-016-9861-y 12. Dunaev BB Lyubich AA A model of economy operation under currency market rate Cybern. Syst. Analysis 2020 56 1 126 138 10.1007/s10559-020-00228-7 13. Dunaev BB Optimization of production income tax rate Cybern. Syst. Analysis 2019 55 3 430 440 10.1007/s10559-019-00150-7 14. Dunaev BB Dynamics of economic cycles Cybern. Syst. Analysis 2017 53 2 293 307 10.1007/s10559-017-9929-3 15. J. Bullard, “President’s message: Quantitative easing — uncharted waters for monetary policy.” URL: https://www.stlouisfed.org/publications/regional-economist/january-2010/quantitative-easinguncharted-waters%2D%2Dfor-monetary-policy. 16. M. Draghi and V. Constâncio, “Introductory statement to the press conference (with Q&A),” January 19 (2017). URL: http://www.ecb.europa.eu/press/pressconf/2017/html/is170119.en.html#qa. 17. M. Draghi and L. de Guindos, “Introductory statement to the press conference,” September 12 (2019). URL: https://www.ecb.europa.eu/press/pressconf/2019/html/ecb.is190912~658eb51d68.en.html. 18. “Investment strategy 2020–2021.” URL: https://www.finambank.ru/files/u/dw/file/presentations/analyst/strategies/investment_strategy_2021.pdf. 19. “Inflation in the US has reached a 40-year high.” URL: https://finclub.net/ua/news/richna-inflyatsiya-u-ssha-priskorilasya-do-6-8protsen.html. 20. Gorbachuk VM Macroeconomic Methods [in Ukrainian] 1999 Kyiv Alterpress 21. Gorbachuk VM Macroeconomic Methods: Theories and Application [in Ukrainian] 2000 Kyiv Kyi 22. “Monetary-credit and financial statistics.” URL: http://www.bank.gov.ua. 23. World Economic Outlook Database. October 2021 IFM. URL: https://www.imf.org/external/pubs/ft/weo/2020/02/weodata/index.aspx. 24. “Economic statistics /2019/2020, National accounts.” URL: http://ukrstat.gov.ua/operativ/menu/menu_u/nac_r.htm.	36471721	PMC9713085	NO-CC CODE	2022-12-08 23:16:07	no	Cybern Syst Anal. 2022 Dec 1; 58(5):727-739	utf-8	Cybern Syst Anal	2,022	10.1007/s10559-022-00506-6	oa_other
==== Front Int J Technol Des Educ Int J Technol Des Educ International Journal of Technology and Design Education 0957-7572 1573-1804 Springer Netherlands Dordrecht 9798 10.1007/s10798-022-09798-3 Article Exploring the impact of self-regulated learning intervention on students' strategy use and performance in a design studio course http://orcid.org/0000-0003-4351-6800 Ateş Akdeniz Aysun [email protected] grid.10516.33 0000 0001 2174 543X Istanbul Technical University, Istanbul, Turkey 1 12 2022 135 25 11 2022 © The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. A growing number of studies indicate that self-regulated learning plays a significant role in students’ academic achievement. However, research studies on design studio education in self-regulated learning are under-researched globally, including in Turkey. In this study, I developed a self-regulated learning intervention for industrial design studio education and examined its impact on students’ self-regulated learning strategies and design performance. Twenty six third-year industrial design students were tracked in a mixed-method research study conducted during a design studio course. Following the study, quantitative and qualitative data were collected from the students using self-report questionnaires and interviews. The jury grades of the students in the experimental group were compared with the grades of the students in the control group. Integrated data analysis indicated that activities for promoting self-regulated learning strategies such as goal-setting, self-monitoring, self-evaluation, self-efficacy, and seeking help and information in design studios can assist design students to improve their strategy use and design performance. Keywords Self-regulated learning Industrial design Design studio education Design learning Design performance ==== Body pmcIntroduction Self-regulation consists of cyclical thoughts, feelings, and attitudes that individuals develop to help them reach their goals (Zimmerman, 2000). Since 1980, A growing body of literature has investigated self-regulated learning (SRL). Schunk (2014, as cited in Sakız, 2014) categorizes SRL studies into three eras—the developmental era, the intervention era, and the processing era—during which cyclical and dynamic SRL processes have been investigated. While digitalization link education and psychology to assessment, interventions, and the use of technology (Bembenutty et al., 2013), the rapid changes in learning environments require more studies. Multiple SRL models have been proposed based on various theoretical perspectives. Although their focus is different, all the models refer to self-regulation as a phenomenon that includes certain processes (e.g., preparation, realization, and post-evaluation) and occur in different dimensions (cognitive, metacognitive, motivational, and behavioral, among others (Sakız & Yetkin-Özdemir, 2014). Within the various theories and models, Bandura’s research findings and Zimmerman’s implementation of the social cognitive approach are the most frequently cited in the literature (Oz, 2019). According to Schunk and Usher (2013, as cited in Sakız, 2014), Zimmerman’s SRL model is the best example of a model in which both process and component-oriented classifications co-exist in one approach. Panadero (2017) compares different models and asserts that Zimmerman’s model has been more frequently cited because it includes a greater number of specific subprocesses that provide a comprehensive vision. Therefore, Zimmerman’s social cognitive model may prove to be more helpful when researching creative tasks or processes in different learning environments (Rubenstein et al., 2018). Students who self-regulate their learning process are active in their learning. Using metacognitive, behavioral, and motivational strategies, they proceed through three cyclical phases (Zimmerman, 2002; Zimmerman & Pons, 1986). Zimmerman (2000) expands on the notion of the student’s feedback loop which is a central feature of academic learning and includes the phases of forethought (before the study), performance (during the study), and self-reflection (after the study) (Zimmerman, 1989; Zimmerman & Cleary, 2009). Each phase covers a set of procedures that a person may employ when seeking to learn, improve, or perform a skill. Each phase is expected to impact (or feed) the next phase (Rubenstein et al., 2018). The model has multiple sub-processes with significant in-between correlations (Zimmerman, 2008). The theoretical framework Bandura proposed for self-regulation has a process-oriented approach (Wirth & Leutner, 2008), which focuses on the actions and events that enable self-regulation and examines what the person did or should do during these actions (Ader, 2014). The interaction between personal, behavioral, and environmental processes is consistently intertwined; at any one time, one or more of these elements may exert a greater effect than the others (Thomas, 2013). A different learning environment can cause a learner's SRL skills to alter, therefore learners should be expressly encouraged to develop SRL abilities. As a learning environment with quality process-oriented and collaborative facilities, instructors at a design studio need to consider the relationship between students and the context. A social-cognitive perspective provides a design studio learning environment with an appropriate foundation for learners’ varying internal and external factors. In this study, I did not attempt to provide an analysis of all SRL theories or models and the advantages of each. Rather, I employed Zimmerman's (2000) model to show how design learning in the studio might be included in the SRL framework. Design Studio and Self-Regulation University students need to be independent learners with the capacity to plan, monitor, and evaluate their work and control their motivations and emotions (Vosniadou, 2020). Some professions demand these skills using different approaches, creating characteristic forms of teaching and learning. Shulman (2005) defines these unique preparations for the professions as “signature pedagogies” and describes their characteristics as having three levels: (a) a surface structure of teaching and learning activities, (b) a deep structure of assumptions about transferring a particular piece of knowledge, and (c) an implicit structure of a moral dimension, including professional beliefs, values, and attitudes. These three aspects affect the ability to think and act like a professional (Shulman, 2005), which is the main goal of higher education. Professionalism also includes dealing with the uncertainties that signature pedagogies contain. Every professional must provide skills to manage and balance the intrinsic tension of having to make judgments under uncertain situations (Shulman, 2005). Design studio education is one of these signature pedagogies with its distinct pedagogical method (Shreeve, 2015; Shulman, 2005; Zairul, 2018) that includes learner-centered activities, knowledge construction through interaction, and the studio as a social environment (Yorgancioglu, 2020). Design as a discipline involves a highly organized mental process related to manipulating and blending many kinds of information into ideas and generating their realization (Lawson, 1980). It usually starts with a project brief which includes the conditions, needs, or restrictions about the subject. This text is then transformed into an experiential or tangible output. While this transformation requires a high-level cognitive process including several stages of information analysis and synthesis (Lawson, 2005), learning to design to deal with complex problems requires relatively more advanced knowledge levels (Ertmer & Newby, 2013). In the design pedagogy literature, the interactional relation in design studio mostly unfolds over one-to-one conversations between student and instructor (e.g., Goldschmidt et al., 2010; McDonald & Michela, 2019; Oh et al., 2013; Peterson, 1979; Uluoǧlu, 2000). This is one of the features of design studio that sets it apart from lectured class format. The term studio in the design education refers to both the physical space where students and instructors meet and the pedagogical approach applied within it (Crowther, 2013). Two main stimuli help students to sustain their design learning in studio: the individual way of students approaching their design and the pedagogical way of instructors feeding back to students’ design (Kowaltowski et al., 2010). The individual approach of a student is developed progressively through repeated design exercises, largely based on trial–error and feedback (Casakin & Goldschmidt, 1999). Design students are expected to develop solutions and bring them to the studio showing weekly progress to discuss with the instructor or sometimes with peers and guests (Goldschmidt et al., 2010). It requires students to study and develop creative ideas by themselves and this necessitates a profound cognitive process. In these individual self-study times, they are expected to criticize their own work and make progress, which also involves behavioral and motivatinal factors. Hence, self-regulation is one of the important progressive tools in the design learning process. The pedagogical approach is the second stimuli that influence developing skills and self-perception (Yorgancioglu & Tunalı, 2020). The tacit form of design knowledge and the different ways in which designers choose to acquire this knowledge make it impossible for design instructors to develop a specific teaching strategy (Ozdemir, 2013). Interaction with the instructors as experienced professionals through unstructured talks about the design challenges are key components of this instructional approach (Kowaltowski et al., 2006). Instructors provide feedback on the students’ work, and students need to respond to that in each class and juries in which students present their projects. Feedbacks help students to move forward while developing their projects and iteratively provide them with a constructive learning process (Tovey, 2015). Nevertheless, conversation between the student and the instructor centers around the student’s work (Schön, 1985). Thus, students are the main actors and are fully responsible for constructing self-knowledge. They need to have a good self- understanding and self-evaluation about the various stages of their designing process (Almendra & Christiaans H., 2011). This critical and self-constructive process may create challenges for novice students who struggle to engage with the design learning context. Additional capacity and motivation are required by them to deal with the unpredictable and serendipitous studio environment (Crowter, 2013) and the ambiguous pedagogy (Austerlitz et al., 2008). Even though studio instructors encourage students to reflect on their creative process to self-regulate their learning (Greene et al., 2019), neither instructors nor students in design studios are fully aware of SRL strategies. Leading a novice to attempt design learning environment entails a more social and cognitive approach to sustain students in going beyond their capability and self-understanding. There is a fundamental lack of educational learning theories in design education since design learning has not been extensively studied from a cognitive perspective (Oxman, 1999). More research is needed for a comprehensive understanding of design learning with its personal, behavioral, and environmental factors. When we consider the intense cognitive and social processes in design studio, the social-cognitive learning theorem provides an appropriate foundation for studying design learning. There are a limited number of studies on SRL in design studio education and they generally focus on components of self-regulation such as metacognition or motivation (see Hargrove, 2007, 2011; Kavousi, 2017; Kavousi et al., 2019, 2020; Oluwatayo et al., 2015) or a single aspect of SRL such as peer review (Zairul, 2018). The exception is Powers (2006, 2016) who researched SRL in the design studio. He proposed an SRL methodology for design education incorporating the process of design learning. However, the lack of an intervention study to evaluate the assumptions limit this study. It should be said that even the limited number of studies contain valuable insights on SRL in design studios. Industrial design (ID) studios have their domain-specific dynamics and therefore a deeper understanding of the SRL approach is needed. In this study, I address this need for a deeper understanding by studying the effects of SRL strategies (e.g., the intervention) on design students within an experimental design framework. Developing SRL Strategies Existing literature has illustrated that support for SRL noticeably nourishes strategy development in learning environments such as STEM, history, and language, among others, (see Azevedo & Cromley, 2004; Masui & de Corte, 2005; van den Boom et al., 2007; Zimmerman & Kitsantas, 1997). These studies focus on applying an SRL model, designing a new model, or developing and validating self-regulation scales. Jivet et al.’s (2018) study on thirteen different SRL tools indicates that SRL can be supported through tools that provide awareness for learners and trigger reflection on the learning process. Nevertheless, more evaluations are needed to understand the characteristics to be considered in designing new tools, since few if any guides on this topic are available (Pérez-Álvarez et al., 2016). According to the studies on successful interventions, the common elements required are as follows: discipline-oriented content (Hattie et al., 1996; Perels et al., 2009); implementation by the class teacher (Ader, 2014); scaffolding and faded support for students (Greene, 2018); direct and explicit promotion of, and information on, SRL (Kistner et al., 2010). To sum up, for success in SRL development, discipline-focused interventions should be implemented by classroom teachers who provide faded support along with explicit information on SRL. Accordingly, the intervention in this study was designed with attention to the recommendations defined above. Research Methodology Research Design This study is a follow-up intervention application of an earlier exploratory study investigating the differences in the SRL strategies used by design students (see X). The main purpose of this study was to determine how SRL interventions in a design studio affect students’ use of SRL strategies and design performance. I used a quasi-experimental research design framework and mixed-method approach in which both qualitative and quantitative data were collected. In experimental design research, the independent variable or variables are manipulated to determine its/their effect on the dependent variable/s (Teddlie & Tashakkori, 2009). In this study, I used a predetermined intact group of design students studying at a university. All the students underwent the regular studio process, except for the students in the experimental group who practiced SRL interventions. Since participation was voluntary, the research design in this study was quasi-experimental (Campbell & Stanley, 1963). Participants and Setting Thirty seven ID students from the same third-year studio class participated in this study. All students were asked, via email, if they wanted to join this working group. They were also told they would be provided with study information, and that their participation would remain confidential. Eleven students volunteered for the intervention. They were on average 20.9 years old (SD = 1.2), enrolled in the fifth semester, and native Turkish speakers. Therefore, the intervention was offered in Turkish. The remaining 26 students were assigned to the control group. Since SRL focuses on individual learning development, the intervention sessions were carried out during the individual project development phase. The mid-term jury was included during the sessions since it was used as a self-evaluation tool. Therefore, the intervention was conducted from the 5th to the 9th week of the 14-week term. Due to the Covid-19 pandemic, all educational activities had to be shifted to digital platforms, referred to as emergency remote learning because the courses were not built for online delivery originally (Winters, 2021). Teachers had little time to prepare and pupils had to quickly adapt to online sessions. Similarly, the emergency remote studio was a digital version of the physical studio. In the studio in this study, instructors and students interacted virtually using video conferencing platforms. Students presented their work on a computer or paper, received criticism from professors and peers, and were expected to study during the four-hour or longer online session. The instructor's absence gave students the impression that time and space were flexible in the virtual design studio. In this unfamiliar terrain, students could easily stop paying attention. Since self-regulated learners need less of a teaching presence (Pool et al., 2017), supporting design students with SRL strategies can help improve their use of time in the virtual studio. Due to these practical changes, the intervention study was developed and executed online. The content of the study was developed based on the content and examples from pre-exiting online and regular classroom interventions and customized to the emergency remote studio environment. The Content of the Intervention The content of the intervention in the emergency remote ID studio was based on prior studies on design studios and Zimmerman’s (2000) SRL process model. It consisted of four sessions. Three sessions were developed based on the SRL model’s three phases, and the fourth session served as a reminder of the process loop. Table 1 shows the content and process of the intervention study.Table 1 The content and process of the intervention study Process Content Ethical permissions SSRL questionnaire Administering the scale on self-regulation in learning (SSRL), a self-report questionnaire to all 3rd-grade ID students Research Jury Announcement E-mailing invitation posters and initial information about the process The working group Arranging working groups and sending out the announcement for the first session 1st session Goal Setting and Planning in the Studio (Part of the Forethought Phase of SRL): Students introduce themselves Introduction to learning How does learning take place in the studio? Thinking and writing about the project process in the studio What are the SRL and SRL loops? When and how is SRL used? What is the effect of motivation? How to implement SRL in the studio Task Analysis: Re-reading the brief of the project Discussing the learning outcomes and evaluation criteria Goal setting and planning practice: Questionnaire 1 2nd Session Self-observation in the Studio (Part of the Performance Phase of SRL): Discussion of the answers in questionnaire 1 Discussion of individual differences in learning Reflection on the individual learning process: How do I learn? Metacognitive Monitoring: Self-observation My belief in myself in this project (avg. 3.2/5) In which subject do I feel successful, strong, or lucky? At what point do I feel unsuccessful, powerless, unlucky? Developing the weakest point: Questionnaire 2 Midterm Jury 3rd session Self-evaluation in the Studio (Part of the Reflection Phase of SRL): Watching self-presentation and commentary records of the mid-term jury Answering the questions: – How do I feel after the jury? – Commenting on presentation and comments – The strongest part of my project – The weakest part of my project Self-evaluation of the mid-term jury: Questionnaire 3-a Re-planning after mid-term jury: Questionnaire 3-b One-to-one process tracking: My design process 4th session The Loop of SRL Practice thinking on the project process: – What did the studio want from me? – What was my purpose? – Where am I in my project, what am I doing? – What subjects am I bad at, and what can I do better? – What will I do now? Activities on Miro application: – Collective activity: What do you need to be successful in the project? Individual activity: Process follow-up of My design process, the 5-day & 5-week plan Design students’ reliance on studio instructors and other external factors hinder their progress (Ates-Akdeniz & Turan, 2022). The first session of the intervention was intended to be a break point when students became aware of their dependence on others. Students were encouraged to ask questions about their studio experience and to set goals. During the intervention, students were given explicit self-regulation strategies and learning tools and informed about the SRL phases. The project brief was reread and the learning outcomes and evaluation criteria were discussed. Students were then asked to describe their design process and prioritize their goals and plans via an online questionnaire. In a design studio, motivation is another factor that is constantly being tested. Unfair public criticism could cause students to lose confidence in their design abilities (Powers, 2006). Low-achieving design students in particular prize studio success as positive critique and are vulnerable to harsh criticism (Ates-Akdeniz & Turan, 2022), to which they may respond by losing interest in their studies. Low-achieving design students also tend not to seek help from others to avoid feeling demoralized. The second session of the intervention targeted these flaws. In the intervention study, students discussed their goals and plans in the first session. This discussion was aimed at developing peer interaction, which can improve self-efficacy as students share comparable issues and learn from one another. Another direct SRL topic was individual differences. Students were encouraged to continue using their learning styles to develop self-confidence. Later, during the metacognitive monitoring activity, students’ strengths and weaknesses, as per the studio’s assessment criteria, were discussed. Finally, students were asked to fill out an online questionnaire answering questions such as “How to get an A in the design studio”? Nilson (2013) proposed this question as an SRL activity for underperformers to help them learn why and where they failed and how they could improve their learning methods. Making a connection between success and effort is defined as an instructional approach that promotes SRL (Paris & Paris, 2001; Sungur & Gungoren, 2009). Therefore, this question was meant to motivate design students to think about success criteria and compare their performance against them. Microteaching is a pre-teacher education technique that uses a video recording of a scaled-down lesson conducted by a pre-teacher to use later as an evaluation tool. Pre-service teachers examine their video-recorded microlessons with colleagues and instructors to assess, reflect on and develop their teaching skills (Ostrosky et al., 2013). This process, which includes playing and replaying the recording, is intended to improve pre-service teachers’ learning experience by providing detailed feedback (Brent et al., 1996), identifying strengths and weaknesses, and highlighting mistakes (Marulcu, 2014). The third session's content was created using this video recording method to encourage self-criticism among design students. Students were asked to watch their recorded mid-term jury presentation on Zoom and fill out a series of self-evaluation questionnaires. Criticism and feedback, based on the evaluation criteria of the mid-term jury and from peers were analyzed by the researcher. Each student’s prior presentation performance was also discussed during this feedback session. The SRL method emphasizes individuality. Students should be aware that each learning activity is their own and should be identified, tried, evaluated, changed, and improved. The fourth session of the intervention was designed to help students comprehend the SRL loop and show them how to use it in practice. They were prompted to think about the project using Miro, an online collaborative whiteboard. Using Miro, students created individual boards with their ideas on success criteria and discussed them on a general board. Afterward, they planned and discussed their five–day-week goals with the researcher individually. Thus, students were instructed to create new goals and plans based on their earlier SRL loop reflections. Data Collection Assessing and developing SRL skills has not been sufficiently explored. Although researchers have shown that self-report measures like questionnaires and interviews are reliable and useful in monitoring SRL strategies (Roth et al., 2016), utilizing a single measure has been criticized since it limits the ability to assess multiple learning strategies (Perry, 2002). Employing multiple methods to investigate SRL helps researchers build theory inductively (Butler, 2002). This intervention study sought two research questions: (1) What is the impact of SRL intervention on students’ awareness and use of SRL strategies? (2) What is the impact of SRL intervention on students design performance in the studio. To answer these questions both quantitative and qualitative methodologies were employed. Figure 1 depicts the research design.Fig. 1 The research design To evaluate the influence of the intervention study on students' awareness and usage of SRL methods, a mixed-method design was used to collect both quantitative and qualitative data. To measure quantitative data, the “Scale on Self-Regulation in Learning” (SSRL), a self-report questionnaire developed by Erdogan (2012) was used before and after the intervention study. The experimental group's pre- and post-test scores were compared to evaluate how the intervention affected students' use of SRL strategies. After the intervention, students in the experimental group were interviewed to assess their engagement with and awareness of SRL methods. The interviews were guided by a semi-structured interview protocol. The questions were designed to urge participants to elaborate on the intervention's efficacy in regulating their learning. To understand the intervention's impact on students' design performance, the experimental and control groups' jury grades were tracked and compared. Designing is a process that transforms the designer; this transformation is a dimension of learning (Findeli, 2001). Therefore, each student was graded for both the design process and the design outcome. Students studied individually during the concept development and design development phases including the mid-term and final juries. To measure the effects of the intervention on students’ design learning process, students’ grades on the concept development process, design development process, participation, and also their midterm and final jury, and term grades were factored into comparison tests. Quantitative Analysis and Results SSRL Self-Report Questionnaire ThE SSRL pre and post-test scores were compared statistically. Pre-test Cronbach’s alpha coefficients were 0.88 for the whole scale, 0.87 for self-regulated learning skills, and 0.88 for motivation. Post-test Cronbach’s alpha coefficients were 0.85 for the whole scale, 0.82 for self-regulated learning skills, and 0.86 for motivation. Table 2 shows the descriptive statistics of all variables. The pre-test total score was 224.55 (SS = 20.7) and the post-test total score was 246.45 (SS = 15.9). The pre-test SRL mean was 151.64 ± 15.8 while the post-test SRL mean was 176.73 ± 13.4. The motivation mean was 72.91 ± 9.87 for the pre-test and 78.73 ± 9.79 for the post-test.Table 2 Descriptive statistics of pre-and post-test scale results SSRL dimensions Tests N Number of items Mean Std. deviation Minimum Maximum SSRL total Pre-test 11 67 224.55 20.729 205 258 Post-test 11 67 246.45 15.977 222 274 SRL total Pre-test 11 45 151.64 15.870 126 175 Post-test 11 45 167.73 13.417 150 188 Motivation total Pre-test 11 22 72.91 9.874 56 92 Post-test 11 22 78.73 9.799 68 96 Before study Pre-test 11 13 44.64 5.085 36 51 Post-test 11 13 49.00 4.796 41 55 During Study Pre-test 11 19 60.09 7.752 50 72 Post-test 11 19 68.18 7.360 58 78 After study Pre-test 11 13 47.64 6.217 35 56 Post-test 11 13 51.36 7.159 33 61 Arrangement of study time Pre-test 11 4 12.55 2.162 9 16 Post-test 11 4 12.91 2.119 9 15 Planning Pre-test 11 5 15.91 2.212 12 19 Post-test 11 5 18.09 3.419 13 22 Environmental structuring Pre-test 11 4 16.18 2.601 13 20 Post-test 11 4 18.00 1.789 16 20 Organizing and transforming Pre-test 11 5 17.09 4.847 11 24 Post-test 11 5 19.00 4.000 14 24 Seeking appropriate information Pre-test 11 3 9.18 1.991 6 13 Post-test 11 3 11.45 1.635 9 14 Seeking easily accessible information Pre-test 11 2 3.82 1.722 2 7 Post-test 11 2 4.00 1.897 2 7 Rehearsing and memorizing Pre-test 11 4 12.55 2.162 8 16 Post-test 11 4 13.55 2.544 10 18 Self-monitoring Pre-test 11 2 5.91 3.015 2 9 Post-test 11 2 8.09 1.640 5 10 Seeking peer. teacher or adult assistance Pre-test 11 3 11.55 2.252 6 13 Post-test 11 3 12.09 1.973 8 15 Self-evaluation Pre-test 11 6 24.36 3.295 20 30 Post-test 11 6 25.55 4.591 16 30 Self-consequences after success Pre-test 11 4 11.00 4.313 4 17 Post-test 11 4 13.45 2.945 8 17 Self-Consequences After failure Pre-test 11 3 10.55 3.475 5 15 Post-test 11 3 11.55 2.115 9 15 Self-efficacy Pre-test 11 5 18.27 3.524 14 23 Post-test 11 5 21.09 1.758 18 23 Goal orientations Pre-test 11 3 10.27 2.102 7 14 Post-test 11 3 10.18 3.430 4 15 Task value Pre-test 11 5 19.73 4.077 14 25 Post-test 11 5 21.27 3.663 14 25 Attributions for failure Pre-test 11 4 10.82 3.281 7 17 Post-test 11 4 10.73 3.690 8 18 Anxiety Pre-test 11 5 13.82 3.341 9 20 Post-test 11 5 15.45 3.908 11 22 The Wilcoxon signed-rank non-parametric test was performed to assess for a statistically significant difference between pre-and post-test scale scores since each group’s sample size was less than thirty (N = 11, N = 26). As presented in Table 3, the results revealed that total SSRL scale scores were found to be statistically significantly higher in post-tests (Mdn = 248) than in pre-tests (Mdn = 218), T = 0.00, z = –2.940, p < 0.003. In addition, the SRL total scores of the students were found to be statistically significantly higher for the post-test scale (Mdn = 166) than for the pre-test scale (Mdn = 149), T = 0.00, z = –2.937, p < 0.003. Furthermore, motivation total scores of the students were found to be statistically significantly higher for the post-test scale (Mdn = 67) than the pre-test scale (Mdn = 59), T = 1, z = –2.501, p < 0.012. There was also a statistically significant difference in pre-and post-test scores for various sub-dimensions such as planning, seeking appropriate information, self-monitoring, self-evaluation, self-consequences after success, and self-efficacy.Table 3 Wilcoxon test results of pre-and post-tests’ scale scores Negative ranks Positive ranks Test statistics SSRL dimensions n Mean rank Sum of ranks n Mean rank Sum of ranks Ties Z Asymp. Sig. (2-tailed) SSRL total 11 6.00 66.00 0 0.00 0.00 0 –2.940b 0.003* SRL total 11 6.00 66.00 0 0.00 0.00 0 –2.937b 0.003* Motivation total 8 6.50 52.00 2 1.50 3.00 1 –2.501b 0.012* Before study 9 5.83 52.50 1 2.50 2.50 1 –2.555b 0.011* During study 11 6.00 66.00 0 0.00 0.00 0 –2.947b 0.003* After study 9 5.72 51.50 1 3.50 3.50 1 –2.469b 0.014* Arrangement of study time 3 3.33 10.00 2 2.50 5.00 6 –.677b 0.498 Planning 7 5.71 40.00 2 2.50 5.00 2 –2.101b 0.036* Environmental structuring 7 4.00 28.00 0 0.00 0.00 4 –2.388b 0.317 Organizing and transforming 6 6.25 37.50 3 2.50 7.50 2 –1.799b 0.072 Seeking appropriate information 9 5.00 45.00 0 0.00 0.00 2 –2.701b 0.007* Seeking easily accessible information 2 1.50 3.00 0 0.00 0.00 9 –1.414b 0.157 Rehearsing and memorizing 6 7.75 46.50 5 3.90 19.50 0 –1.209b 0.227 Self-monitoring 8 4.50 36.00 0 0.00 0.00 3 –2.539b 0.011* Seeking peer. Teacher or adult assistance 5 4.60 23.00 2 2.50 5.00 4 –1.561b 0.119 Self-evaluation 5 3.90 19.50 3 5.50 16.50 3 –.213b 0.031* Self-consequences after success 6 5.50 33.00 2 1.50 3.00 3 –2.108b 0.035* Self-consequences after failure 5 4.20 21.00 2 3.50 7.00 4 –1.190b 0.234 Self-efficacy 7 4.00 28.00 0 0.00 0.00 4 –2.375b 0.018* Goal orientations 4 5.25 21.00 5 4.80 24.00 2 –.178c 0.858 Task value 6 5.17 31.00 2 2.50 5.00 3 –1.845b 0.065 Attributions for failure 4 5.25 21.00 5 4.80 24.00 2 –.181c 0.856 Anxiety 8 4.88 39.00 1 6.00 6.00 2 –1.970b 0.049 Bold values indicate the significance of p < .05 bBased on positive ranks; cBased on negative ranks Grade Comparison The Mann–Whitney U test was used to test whether there were significant grade differences between groups. The intervention was completed after the mid-term jury and design development process. The test results revealed that the final jury grades (mean rank = 24.05, U = 87.5, z = –1.488, p < 0.035), participation grades (mean rank = 23.41, U = 94.5, z = –1.651, p < 0.042), and term grades (mean rank = 22.18, U = 108.0, z = –1.166, p < 0.024) of the students in the experimental group were statistically higher than that of the students in the control group (see Table 4). This could signify that the intervention had a positive impact on students' design process. These findings support my supposition that the SRL intervention would help students to improve their academic achievement, including in the design studio. However, it remains unclear to which degree the increase in the grades can be attributed to the SRL intervention. Therefore, more data analysis will be required to determine the assumptions underlying this study and the validity of the results.Table 4 Mann–Whitney U test results for between groups Grades Groups N Mean rank Sum of ranks Mann–Whitney U Wilcoxon W Z Asymp. sig. (2-tailed) Concept dev process Experimental 11 23.41 257.5 94.5 445.500 –1.613 0.107 Control 26 17.13 445.5 Mid-term jury Experimental 11 17.91 197.00 131.000 197.000 –0.402 0.688 Control 26 19.46 506.00 Design dev process Experimental 11 19.73 217.00 135.000 486.000 –0.266 0.790 Control 26 18.69 486.00 Final jury Experimenal 11 24.05 264.50 87.500 438.500 –1.488 0.035* Control 26 16.87 438.50 Participation Experimental 11 23.41 257.50 94.500 445.500 –1.651 0.042* Control 26 17.13 445.50 Term grade Experimental 11 22.18 244.00 108.000 459.000 –1.166 0.024* Control 26 17.65 459.00 Bold values indicate the significance of p < .05 Another Wilcoxon signed-rank test was run to see if the experimental group's grades changed significantly before and after the intervention study. The mid-term and final jury grades were utilized because they were graded similarly and their timing was suitable for pre and post-comparison. As shown in Table 5, the difference in the mid-term (Mdn = 60.0) and final jury grades (Mdn = 72.6) of the students in the experimental (intervention) group are statistically significant T = 0.00, z = –1.689, p < 0.04. This means that participant students’ final jury grades were an improvement on their mid-term grades.Table 5 Wilcoxon signed-rank test results within the experimental group Juries Negative ranks Positive ranks Test statistics n Mean rank Sum of ranks n Mean rank Sum of ranks Ties Z Asymp. sig. (2-tailed) Pre-tests (Midterm Jury) – Post-tests (Final Jury) 3 4.67 14.00 8 6.50 52.00 0 –1.689 0.04* Bold value indicate the significance of p < .05 Qualitative Analysis and Results The answers given by the 11 interviewees were analyzed using the content analysis method, which classifies data without any theoretical assumptions (Elo & Kyngäs, 2008; Schilling, 2006). The analysis process was conducted in three phases—preparation, organizing, and reporting (Elo & Kyngäs, 2008). In the preparation phase, the questions were converted into headings and the codes were derived from the students' answers via open coding. General views on the intervention, activities, and students’ self-comments were the main categories. During the organizing phase, the coding scheme, shown in Table 6, was developed. In the reporting phase, the frequency of the use of these categories in students’ interviews and direct quotes were included. The MAXQDA’20 qualitative data analysis program was used for all three phases.Table 6 The coding scheme of the content analysis 1.0 General views of students on the SRL intervention 1.1 A useful activity 1.2 Time to analyze yourself 1.3 Good timing in the studio process 1.4 "I was not alone" 1.5 Comfortable environment 1.6 Earlier need in university life 2.0 General views of students on the activities practiced in the SRL intervention 2.1 Setting and achieving goals 2.2 Project analysis/rereading the brief 2.3 Project analysis/replaying mid-term records 2.4 Most useful activity: miro 3.0 Students' comments on themselves after the SRL intervention 3.1 Strategy development 3.2 Increase in self-confidence 3.3 Influenced by peers 3.4 Continuity 3.5 Learning experience In qualitative research, reliability is defined as the consistency of data code groups derived by multiple researchers (Creswell, 2014). Therefore, the researcher collaborated with another scholar on the content analysis. Because several codes were incompatible, they were then grouped with other categories or eliminated and the reliability of coders to create themes was agreed upon by each coder. Inter-coder reliability is defined as the ratio of the sum of agreements to all agreements and disagreements amongst coders (Miles & Huberman, 1994). The inter-coder reliability of the researcher’s and the expert’s themes and codes was 83%. To obtain a clear picture of the participants' opinions, direct statements from them were required. This section includes key findings on the students' engagement in the intervention study. The content analysis yielded three main themes: views on the intervention, views on the activities, and students' self-comments after the intervention. The letters and numbers in parentheses (P1, P2, etc.) indicate students whose statements were directly quoted in Tables 7, 8 and 9.Table 7 General opinions about the intervention Categories ƒ Student expressions A useful activity 11 “I don't see it from the point of view of the grade. It was useful, I started to think about something about myself, about what I want, about the future. For example, I am planning to spend more time on coding.” (P1) “It was very useful for me, I felt that I improved myself and my goals.” (P3) “The practices we did make me realize things I knew but did not pay much attention to.” (P8) Time to analyze yourself 9 I became aware of myself, that is, I feed off what they (other students in the group) said and what we did together.” (P4) “After the study, I looked back at myself to see how I could work and express myself better.” (P10) “I found the opportunity to analyse myself better.” (P7) Good timing in the studio process 9 “I think the timing was very good. After the midterm, everyone was devastated, some of the friends who expected high grades got low grades. This study helped us to recover ourselves after the midterm.” (P2) “It was very good timing. Since it was on Tuesday mornings, it was really motivating me before the studio hours. Even if I had only one hour until the studio started, it made me want to do something [study for the project]. So, I was sitting and doing something for my project, saying ‘What do I have to lose?’.” (P10) "I am not alone" 8 “When everyone talked about themselves, I realized that we are all alike.” (P11) “I noticed people in the same situation as me. I felt that I was not alone.” (P4) “It was nice to have the environment of those who experienced the same situation. I wasn't alone. It felt good to know that there are people who can tell and understand what I went through.” (P9) Comfortable environment 7 “At first, I wasn't sure if I would attend, but when you said that there are motivational techniques and that I could learn them, the event caught my attention. Also, since we could share our thoughts, it was like a therapy session.” (P3) “The working environment was a more comfortable and freer environment, unlike our studios. That's why I was able to express myself.” (P7) Earlier need in university life 7 “I wish this study was in the previous year. The second year of my university was a complete mess. The worst period of my life. My most hated period. If only it was then, it would have been a less bad semester for me.” (P2) “It might be even better for freshmen. After making a few mistakes, they can reflect on themselves and say ‘Where have I made the mistake?’. The sooner the better.” (P9) Table 8 Students’ views on the activities practiced in the intervention Categories ƒ Student expressions Setting and achieving goals 11 “I wanted to learn software-related coding. I watched tutorials on Udemy. Because the project was bad anyway, I planned to focus on my other goals. I thought I could improve myself in the field I want.” (P1) “I know that my product (design) is not super great in my project, but I researched everything I put in my project. I decided on this approach after the goal planning activity where you made us set goals. I planned as my goal that I will think in detail about every decision and move I make. I'm going to put something in my project, how can I put it, what is this in real life, and so on. I felt so good knowing that if you break a dryer, I can fix it again. I've been working on it a lot. But I know I wouldn't have done this without the intervention study.” (P2) “I was away from the rhino [computer-aided 3d drawing software] at the beginning. Then I set to study on rhino as a goal for my project, and when I did it, I liked it, I remembered, I continued, I even tried different renders, etc. In that sense, I felt that I improved myself a little more, rather than designing for a company or a studio. I started to trust my project more. I realized that I could speak more freely. I was able to present more comfortably in this term.” (P4) “I set a goal to find out how the heating and turning functions work together. This gave me the determination to solve. In my previous projects, I did not set my own goal as much as this one.” (P6) Most useful activity: miro 9 “The most useful part is the one with the chart in Miro. I used it to phase my project process in steps. I have used it on my personal plans, too. I’m still using it.” (P3) “The applications in the Miro definitely worked a lot. The parts we commented on together at the same time were very useful. I realized that the difficulties I was challenged with were not special to me.” (P4) “Especially the post-it works in Miro stuck in my mind. Sometimes I was thinking about how much I had done. After the final jury, I thought how many of my goals in the post-its I have completed.” (P7) Project analysis/replaying midterm records 8 “It was the most helpful activity. It was good to understand what I should concentrate on. Listening to the comments again was good feedback for me.” (P6) “We had never done this before. This was the first time. When the midterm jury finished, we forgot everything about it. We celebrated that we somehow progressed, another big assignment is finished, as if we passed the exam. I didn't care about the critics I got. But in this study, it was very useful to watch it again. Later, I watched it 2–3 times more.” (P7) Project analysis/rereading the brief 6 “Before, I never reread the briefs. Now I am reading the briefs.” (P11) “I read it at first, but I did not read it again. Actually, it was helpful to understand the expectations of the course.” (P5) “It was extremely useful for me. I never read the brief. I don't even look at what I do first. I always look ahead. I like to watch movies a lot, and I knew that in my second watch I perceive something different before then. I did not realize that I did not apply this approach outside of the cinema. I didn't apply it to the brief reading. I realized that in the second read of the brief I can perceive something else. Now, I’m trying to interpret the brief differently in every read.” (P10) Table 9 Students' Comments on Themselves After the Intervention Categories ƒ Student expressions Strategy development 9 “I started to work with watercolor. I'm planning to start to work with my iPad now. I was studying by writing the same notes over and over for exams, I never understand by reading. But I understood that it was too much work. I've tried to get over this, and finally this semester I achieved a first in my 15 years of education life. I managed to change my way of studying. It was a starting point for me.” (P2) “I focused on my process in the project, and I think it was not a bad decision. I tried to have something to show every week because I think the process was given more importance. Most of the percentage of evaluation included the process, the rate was almost the same as a jury.” (P3) “I realized that I like to take notes and write with nice pens. I realized that when I write with beautiful pens, I am more encouraged to do it. That's why I'm trying to work with paper now.” (P5) “I never thought before that [intervention] what I should do for the better. But what we did in the study affected me, [showing] that instead of trying to learn something without liking it, I will try to make it interesting.” (P7) Increase in self-confidence 8 “During this period, I realized that I know what to do. This study gave me the first step to try.” (P11) “I became aware of myself. I can dare and trust my project. This study showed me that I need to be confident in myself. To be honest, I used to study to get good grades. This year, I approached [my work] boldly. I persisted on what I was doing. In the past, when I thought I couldn't do it and that people were doing better than me, I always pulled away. But now I start to think that I do things better than some other people.” (P4) “I think if I can continue what we tried during the study, I will increase my self-confidence even more. Because normally, I would pull away from myself with the smallest negativity. But this semester, I felt self-confident, I think that I did what I really wanted to do in my project.” (P6) “My project was the best project of the entire studio. I’m satisfied with everything I did. What I received (grade) was not satisfying, but I stand behind my product. I generally don't feel comfortable with my projects, I always find something to criticize. But I feel very satisfied with this project.” (P10) Learning orientation 8 “Before, when the brief arrived, we tried to do whatever is requested. Our aim was always to satisfy the teacher to get good grades. When they commented on our works in different ways, we got lost. The reason for that was being focused on what the teachers wanted to get high grades. This year, after our sessions, I felt this emptiness about myself, I realized how nice it is to do projects for myself, not for them. I also found what I want to do in my graduation project. It was definitely the studio I enjoyed the most in my life.” (P2) “In fact, I got a better grade than my previous projects, but I had aimed for a much better grade this term, but I couldn’t [get that]. But I am happy with my process because I had a very productive process for myself. I think that what I learned is more than my grade.” (P8) “I ask myself whether I did something I felt good about or not. I liked the last product I designed for my project very much. Maybe I didn't get a good response as a grade, but it made me make some decisions about myself.” (P6) Influenced by the sample/peer 6 “In one of our sessions, you [the researcher of this thesis] gave an example about your experience with finding your drawing style. If you look at my process, the styles of my drawing changed completely after that week. Even though it took more time, including watercolor while using markers for drawing helped me to start studying beforehand. From now on, I want to develop it [further].” (P9) “From the moment you explained that this [your learning method and strategies] can be learned, the event caught my attention.” (P3) “You told us about your experience of finding your drawing style in a session. After that, I criticized myself to see how I could work and express myself better. I realized that I stopped taking notes which were one of my favorite things, colored pencils, writing with images, etc. After that speech, I bought a new notebook for the project and spent the semester with it.” (P4) Continuity 5 Then I used our page on Miro again. Now this semester, I will prepare a page for myself. With the help of this study [the intervention], I learned how to do it [planning to study]. I know how to study much better now.” (P2) “Watching midterm jury records were very useful. I watched it 2–3 more times after that. I will record and watch or listen to my presentations again from now on.” (P7) The Views on the Intervention Students were asked about their general opinion on the intervention study. As shown in Table 7, their answers were grouped under six main categories. The study appears to have benefited the participants. Students’ consensus was that the project's intensity overrode their wishes and feelings. They could go forward by focusing on their goals when they had time to analyze themselves. Eight of them called the study “self-analyzing time,” and said that the feeling of “I’m not alone” made them feel more comfortable while self-evaluating. This shared experience provided a friendly and sympathetic environment where students could express themselves and pursue self-directed goals. Seven students compared the study to the studio and said they could express themselves more freely. It was observed that the students need communication at this level during studio hours, too. Nine students' responses indicate that the timing of the intervention was helpful since it took place before the studio starts, a period that is typically stressful, uncertain, and unmotivated in the studio process. Seven students also indicated that an intervention study would be helpful in the first or second year of university because of the new learning environment and the intensity of the uncertain processes in the studio. Students' Views on the Activities Students were asked about their opinions on the intervention activities in detail. As shown in Table 8, their responses were classified into four main categories. In the interviews, all students praised the goal planning activity and stated that they achieved their project goals. It was observed that this activity improved students’ self-efficacy by directing attention from grades to learning. Students also worked in groups and individually on Miro to identify and plan for challenges in their projects. Nine students noted that in the group activity, everyone had similar difficulties and this gave them comfort during self-evaluation. They said that having their goals on post-its helped them remember and track their progress. Also, students said the Miro activity was the most beneficial in the study. The reasons for this may include Miro’s easy interface process and students’ familiarity with it (e.g., they could use it interactively and open private pages to review their work). Students' interpretations of the project analysis activities indicated that all students had not re-read the project brief after the first studio day. Re-reading the brief revealed new meanings to them. The students were initially embarrassed and refused to watch their mid-term jury recordings since they did not like watching themselves. They agreed to participate after being encouraged to focus on content rather than visuals. They watched in separate Zoom rooms and were pleased to recognize their areas of weaknesses (e.g., could not convey what they wanted to say, saying things that the jury would misunderstand, omitting important project details, etc.). Students' Comments on Themselves After the intervention, students were asked about their self-assessment. As shown in Table 9, the students' perceptions of their advancement were classified into five main categories. Students were encouraged to devise strategies that would help them achieve their study goals. As evidenced by their remarks, students found new methods to express themselves and created process-oriented strategies. They were more focused on themselves than on their grades. Learning about their abilities and desires strengthened their self-efficacy. Eight of them utilized self-confidence and self-discovery terms frequently. These outcomes were attributed to a learning-focused development strategy. During the intervention sessions, the researcher shared their own experiences of identifying a learning strategy, such as discovering their sketching style. Six students reported that they were motivated to take action to identify their strategies. In addition, five students said they would employ some of the activities in the next semester. This was an important indicator of SRL skills since task perseverance is an outcome of self-efficacy and self-regulation (Schraw, 2006; Zimmerman et al., 1992). The Integrated Analysis To evaluate the effect of the intervention on the use of the SRL strategy, two types of data were collected and analyzed together for a deeper understanding of the intervention. A joint comparison matrix was used to determine the agreement levels between the data sets (Creswell & Plano Clark, 2018; O’Cathain et al., 2010). Table 10 presents the coherence of the findings. Improvements in some SRL strategies were prominent in both data sets—planning, seeking appropriate information, self-monitoring, self-evaluation, and self-efficacy significantly increased, statistically, post-intervention. Qualitative data provided explicit descriptions of these strategies. The findings also included some contradictions. Qualitative findings revealed modest improvements in seeking assistance, goal orientation, and task value although no statistically significant increases were revealed in the quantitative analysis of the data. This enabled to interpret unanticipated quantitative results qualitatively. Table 10 Joint comparison matrix SRL strategies and motivational factors Quantitative (SSRL pre-post test results) Qualitative (content analysis of interview) Agreement, partial agreement, dissonance/expansion, no match Arrangement of study time – No match Planning + + + + Agreement Environmental structuring – No match Organizing and transforming – No match Seeking appropriate information + + + + Agreement Seeking easily accessible information – No match Rehearsing and memorizing – No match Self-monitoring + + + + Agreement Seeking peer, teacher, or adult assistance – + Dissonance/expansion Self-evaluation + + + + Agreement Self-consequences after success + + No match Self-consequences after failure – No match Self-efficacy + + + + Agreement Goal orientations – + Dissonance/expansion Task value – + Dissonance/expansion Attributions for failure – No match Anxiety – No match + + : exact information related to a finding + : supporting/related information related to a finding –: contrasting information related to a finding No symbol: no information Discussion In this experimental study, I evaluated a self-regulation intervention in an ID studio. The aim was to understand the effect of self-regulated intervention in studio on design students’ (1) use of SRL strategies and (2) design performance. Concerning the first aim, convergent mixed methods were employed. The integrated analysis indicated that design students who engaged with the intervention demonstrated increases in metacognitive strategies (e.g., goal planning, self-monitoring, and self-evaluation); motivation level (e.g., self-efficacy), and behavioral strategies (e.g., seeking information and help). Concerning the second aim, students’ jury grades were tracked and compared within and across groups. The comparison analysis revealed that design students who participated in the intervention received higher jury grades than the students who did not participate. The following paragraphs discuss the changes in strategies and design performance. Change In Metacognitive Strategies Metacognitive strategies help students to monitor themselves. These strategies teach students to plan goals, activate prior knowledge, and prepare the materials according to their learning style (Pintrich, 1999). In collaborative learning environments (e.g., design studio), metacognition has a central role in supporting individual and group regulations (Järvelä et al., 2021). Goal setting is one of the fundamental metacognitive features of self-regulation (Boekaerts & Niemivirta, 2000). During the intervention, students were encouraged to define and plan for their personal project goals. This approach was intended to help students reflect on their learning processes and strategies. The integrated analysis demonstrated that goal-setting activities supported the development of SRL skills. Participating students managed to set future process and learning-oriented goals. Two participants expressed their willingness to set goals for other courses and also their social life. Another student proudly mentioned the accomplishment of her goals. Yet another student stated that he still followed his off-project goals (e.g., doing sport regularly) that he set during the intervention. These findings support the notion that planning goals and monitoring them throughout learning can increase learning efficiency (van den Hurk, 2006). Design students are exposed to emotional triggers because they require constant feedback and approval to continue developing ideas, which is a sensitive process (Goldschmidt et al., 2010). Some students can be overly invested in the instructor's negative or favorable comments, which hinders the learning process (Kavousi et al., 2020). These students need a reference to assess and interpret criticism with their ideas and aims. Goals are like safety jackets for students. They can help reduce external dependency and syncretize comments with their own goals. When actively participating in SRL activities, design students were more likely to use effective learning strategies, set goals, customize goals as per their preferences, track their progress, and evaluate themselves using the tools offered. Also, metacognitive strategies scores increased as per the quantitative analysis. Such improvements in both data sets show that students’ use of metacognitive strategies changed positively after the intervention. The integrated findings indicated an increase in the use of self-monitoring strategies. Students reported using new notebooks and/or pens to take notes on their design process. They also watched themselves (i.e., self-watching)) on their jury presentation recordings, an example of a self-monitoring method. Students stated that they would use video or voice recording in future studios. Thus, learning self-monitoring skills is beneficial for students (Chang, 2007). While self-monitoring is required for self-regulation, it is not sufficient (Schunk, 1995), because self-monitoring is influenced by both individual processes and behavioral factors (Zimmerman, 1989). Self-monitoring strategies like self-trial and self-recording require systematic and frequent tracking (Bandura, 1986). Design involves long and complicated cognitive processes during which ideas and/or comments can be forgotten. Therefore, self-monitoring demands this type of tracking method. Self-monitoring strategies would help design students track their progress and reflect on their ideas and comments, thus providing the necessary materials to self-evaluate. Schunk (1995) suggests that explicit self-monitoring of skill acquisition through self-evaluation of capabilities improves student achievements. For a productive learning environment in a design studio, these two self-regulation skills—self-monitoring and self-evaluation—should be linked to student goals. During the intervention, a micro-teaching method from the education literature was employed to create an environment for self-evaluation. Students watched their mid-term jury recordings and evaluated their projects and presentations using the jury requirements. They reported hypothetical concerns before watching themselves and stated that self-watching helped them identify their weaknesses and mistakes and ponder necessary improvements. Thus, students were able to plan their goals more effectively because they were aware of their future needs. Some students talked about their plans to develop their knowledge base, which they thought inadequate (e.g., sketching, computer-base drawing, design history). Students were able to concentrate on the process since the mid-term jury was not the final assessment. They could embrace their weaknesses and strengths and become more learning-oriented through discussion on individual differences. These findings support the notion that observing self-performance enables one to monitor SRL behaviors and self-learning processes in a real-world and dynamic social setting (Kohen & Kramarski, 2012). A critical instructional perspective creates an environment of rich externalizations and interaction for learner experience. In this environment, students can develop their value-driven design expertise and design identity (Gray et al., 2020)). Both the design process and design education should include the designer’s self-processes. In the design studio, instructors' criticism is the key evaluation method, while students' remarks are rarely heard. According to Chien et al. (2021), self-evaluation improves ID students’ self-efficacy. Our study revealed that self-criticisms enabled by micro-teaching help students understand their values and develop their learning identity. Finally, self-evaluation behavior increases students’ self-efficacy, indicating that motivation is an essential component of the SRL cycle (Zimmerman, 2000). Change in Motivational Level During the intervention, participants were encouraged to compare their performance with the success criteria of the design studio. This increased students' self-efficacy and persistence (Zimmerman, 2000), which guarded against the adverse effects of harsh criticism. Self-belief and its challenges in the design studio are discussed by Ochsner (2000) from a psychoanalytic perspective:Many students are motivated to apply to architecture school by an idealism about the environment and a wish to contribute to human betterment. Some are also clearly motivated by the kind of experience they will find in a design studio—they are seeking a place where they can draw on ways of being and thinking that they sense are possible, but they have not found widely understood or recognized. They may not be able to articulate this consciously, but many are seeking to recover aspects of the transitional space of creative play lost since childhood. To do this requires a suspension of disbelief and an acceptance of the process before the results can be assured. For students, this can be exhilarating, but the uncertainties and ambiguities can also be frightening (p.203). As this quote shows, design students need to develop their self-belief to capture and use their creative side. However, this can be frustrating due to the complexity of design studios. Design students who lack self-efficacy may underperform even if they are skilled designers (Powers, 2006). The role of design instructors should also include developing students’ self-efficacy. Self-efficacy beliefs are shaped by earlier self-experiences but can also be influenced by the experience of others, verbal convincing, and the learner’s physiological reactions (Bandura, 1986; Bong & Skaalvik, 2003; Pajares & Valiante, 2002, as cited in DiFrancesca et al., 2016). During the intervention, sharing personal experiences prompted students to relate to their own experiences. The qualitative analysis revealed that design students were more likely to share positive or negative experiences with peers when the environment was supportive because it helped them to feel less lonely and increased their self-efficacy. The quantitative analysis also revealed an increase in students’ self-efficacy scores. Both data sets provide evidence of the strengthening of students’ motivation levels. Teacher training studies (Dermitzaki & Kriekouki, 2017; Liu, 2016), found that experience sharing could be a potential SRL training method for students. Powers (2006) states that low-achieving design students are less likely to utilize goals to connect with peers, as a result of which they interact less with their peers as they deliberate on their studio performance. To better interact with peers, students should be encouraged to share positive or negative experiences. By reflecting on similar stories, they may overcome their frustrations as they struggle to improve their skills. Change in Behavioral Strategies Seeking information is a process of selecting the most suitable information sources (Uçak, 1997). Due to the rapid evolution of technology, the ability to quickly and efficiently access information has become a requirement of the modern era (Oz, 2019). The self-regulated learner is expected to gather and organize appropriate information. During the intervention, students were encouraged to think about relevant information sources for their personal project goals. Searching for information to inspire rather than imitate was emphasized. Both qualitative and quantitative findings showed that when students advanced in learning goals, they sought out more learning-oriented information. Students were willing to thoroughly research their topics and align their findings with their goals and project requirements. In a design studio, students begin by developing ideas and conducting ongoing research. This can be difficult for underachieving students with limited and/or negative experiences. These students encounter two challenges while collecting information to address ill-defined design problems. Specifically, they have trouble defining the problem and knowing where to begin (Rittel & Webber, 1984). According to Powers (2006), low achievers prefer the trial-and-error method using first online information they reach since they have less ownership of their project and focus on grade rather than learning. Guiding low-performing students to understand the importance of goals and develop their self-efficacy helps them to engage with external resources. Chen (2016) discovered that while senior design students use external resources such as objects (e.g., internet, books, magazines, products), strategies (e.g., brainstorming, discussion, observation, interview, survey, practice, computer-aid), and their local amenities (e.g., library, workshop, processing factory, department store) to address learning problems, junior design students rely more on human resource (e.g., instructors, peers, technicians, experts, family, friend). A scaffolding approach in which, instructors can direct students to use information as a tool, not a goal, would be useful for students of all levels. Seeking help is the ability to use peers, teachers, or other adults as resources to deal with learning uncertainties and difficulties (Newman, 2008). Unlike other self-regulated strategies, help-seeking requires social interaction. During the intervention, the main topics included being open to criticism, sharing opinions, integrating opinions with individual preferences, and connecting every project detail to learning goals. According to students' statements, they were conscious of their need for assistance and preferred to consult senior peers. This finding is per Karabenick and Knapp (1988) who suggest that students may be ashamed to ask questions because of emotional pressure in the studio and prefer to hear feedback from peers outside the studio. Both reasons indicated a need for more focused and flexible communication in the studio. The “mystery-mastery” syndrome which was defined as the inaccessibility of the instructors in the studio by Schön (1987) may cause low achievers to feel out of place and prevent them from asking for help. Notably, in a design studio where collaboration is expected, underachieving students displace themselves by refusing to communicate. Sungur and Yerdelen (2011) attributed underperformers’ poor usage of help-seeking strategies to the exam-driven educational system in Turkey. Due to national prescriptive tests for gaining admission to good high schools and universities, students’ competitive behavior begins in middle school (Sungur & Yerdelen, 2011). Accordingly, when students encounter a studio that combines individual and collective procedures, they need to adapt their learning practices. Schön (1985) states that the design studio’s purpose is built on teaching the language of design and designing. However, due to its complex instructional structure, this new language is not easily explained or transferred to novices. If poor performers cannot receive adequate explanations from studio instructors, they turn to their peers for assistance. Help-seeking can be stigmatizing due to its implications of inadequacy, resulting in personal consequences like feeling obligated to compensate the helper (Karabenick & Gonida, 2018). Therefore, underperformers are reluctant to ask for further assistance. This impediment to help-seeking should be removed by clear communication on studio education. During the intervention, students expressed their feelings of loneliness while working in the studio. Although scale scores did not support this outcome, the qualitative findings indicated that students felt less alone after talking about their struggles with their peers and were able to express their thoughts about their processes more easily. This conclusion is consistent with Bilgin and Akkapulu’s (2007) findings that peer attachment reduces loneliness and promotes social self-efficacy in adolescents. Sherer et al. (1982) define social self-efficacy as one dimension of self-efficacy and confidence in self-social skills. The design studio requires high social skills students who can present their work with panache to instructors, guests, and peers. Dunbar et al. (2018) found that collaborative learning environments promote social self-efficacy, which in turn promotes academic performance. Peer interaction in the design studio contributes to a collaborative learning structure (Wang, 2010) so that students become proactive, self-regulating learners (Crolla et al., 2019). Change in Design Performance Before and after the intervention, the grades of all students were tracked and then compared vis-a-vis their groups (i.e., the grades of the students in the experimental group were compared with the grades of the students in the control group). The comparison analysis for each evaluation criterion of the jury indicated that the students in the experimental group had higher grades than the students in the control group following the intervention even though there was no statistical difference between the groups before the intervention. Participants' grades improved following the intervention, indicating a positive relationship between SRL strategies and design achievement. Our results support the positive correlation between SRL and academic achievement (Araz & Sungur, 2007; DiFrancesca et al., 2016; Erdogan & Senemoglu, 2016; Loeffler et al., 2019; Zimmerman, 1990). Rubenstein et al. (2018) highlight the challenges of evaluating creative processes given their psychological and social patterns and intentional actions. Nevertheless, they suggest SRL measurement methods to assess students’ creative processes. Studies in art and design education have also shown a strong relationship between SRL techniques and creative performance (Greene et al., 2019; Hargrove, 2007, 2013; Sawyer, 2017). However, it should be noted that my study did not focus solely on the creativity of students’ final projects. Instead, I centered on academic design performance, which included creative performance as a criterion. Higher education generally assigns higher-order abilities to creativity, and evaluation typically mirrors that in practice with an emphasis on the outcome and the craft skills of implementation (Cowdroy & de Graaff, 2005). Academic design performance is assessed differently from creative (i.e. professional) design performance. Buchanan (1998) characterizes design education as a distinct field that complements, but does not duplicate, professional practice. Thus, the emphasis of design studio evaluation should not be only on the creative output, but also on the learning process that underpins it. Design instructors evaluate not only the creativity of the final project but also the idea’s continuity, timing, and development throughout the design learning process. This study on learning in the design studio provides a vital starting point for discussion and further research on design performance with SRL strategies. Limitations The intervention study was participated by 11 junior ID undergraduates on a voluntary basis within a studio course at Istanbul Bilgi University that had its own instructional goals and processes. Even though this study verifies previous findings, brings light to new ones and signifies relationships from the data, we acknowledged that there were limitations. The size of the sample group presented limitation regarding external validity in terms of generalizability to other subjects, settings, or time. On the other hand, no unanticipated challenge emerged during the study due to the size of the sample. Mixed method research design was utilized and data was collected through both quantitative and qualitative methods to minimize the limitations. More than collecting one type of data provided to make more accurate interpretations as pointed out by Creswell (2014). Besides, the inter-coder reliability for qualitative findings was 83%, which also indicates a good level of consistency of data code groups. This study attempted to establish a foundation for understanding the SRL process in terms of the ID studio rather than drawing generalizations and making meta-analysis for design education. The sample held information relevant for the aim of the study, sample specificity was acceptable and was appropriate for the analysis strategy. Finally, the results drawn out were supporting the inital hypothesis. The findings are promising with regard to the establised theories, yet a larger representative sample size is needed to generalize across design sub-disciplines, settings, and education levels. Conclusion In this study, I aimed to better understand the design learning process and provide insights for redesigning the studio learning experience through the application of SRL strategies. The social-cognitive view of SRL allowed the researcher to develop and apply a domain-specific introductory-level SRL intervention in the design studio, which opened new communication channels for design students. The findings illustrate that activities for promoting SRL in design studios can assist design students to improve their design learning experience and performance. Both theoretical and practical implications can be drawn from the findings. This section summarizes some pedagogical insights for design education derived from this study. Florishing Personal Goals High dependency on external factors has a detrimental effect on design process. Personal goals provide students with a reference point to interpret the design feedbacks. Encouraging and orienting personal goals address the internal characteristics of the design students (e.g., their judgment, perception, appreciation, empathy, courage, imagination), which is also suggested for design studio education by Buchanan et al. (2013). Metacognitive activities such as goal planing provide proper content for students’ individualistic features to discuss with them. The aims of the project and the goals of the students should be differentiated and then syncretized together through the conversations in the studio. By doing this, students can comprehend the importance of their own goal plans and orient them towards learning. Personal taste and attitudes of the students can flourish through such an approach which also helps to increase their self-efficacy and decreases the high external dependency. Watching Self-Recordings Due to its complex longitudinal process, the most frequent problem of designing is forgetting or misunderstanding the ideas and/or comments on one’s design. Design learners need methods and tools to trace their designing process. Design instructors should encourage students to record their own process via either digital (video, audio recording) or physical (note-taking, drawing) tools. Since the student’s emotional experience is higher while presenting or explaining their work, this recording activity should include especially critic sessions and jury presentations. For underperforming students, note-taking can be frustrating since they do not count on their abilities enough. Therefore, especially these students should be presented with easy recording methods such as audio or video records. However, recording is not a satisfactory learning strategy without reviewing. Specific time allocations should be included in the studio process for evaluating these records. These sessions can be conducted within student groups or individually. However in both, the instructor should be included in the process to lead the students in self-evaluation. Having a metacognitive view through self-evaluation encourages low performer design students to regulate their learning strategies, develop self-belief, and explore their ways of designing which is the main purpose of studio education. Questioning the Studio In self-regulation, outcomes of the learning activities feed the other strategic learning steps. While metacognitive strategies such as planning goals, monitoring, and evaluating help to increase the self-efficacy and motivation of the learners, they also give students a point of view to regulate their behavioral strategies. Help-seeking as a behavioral strategy is the only strategy that requires social interaction in SRL. Design students may have problems reaching the studio instructors for asking for help when they are insecure about their process. They are mostly afraid of being criticized and consequently demoralized or feel too embarrassed to ask questions. The educational system that they were previously part of was based on a competitive approach, so they need to adapt to the features of the collaborative learning environment. In some cases, reflective conversations with the instructors are not enough to make students understand the learning process. They need more explicit and flexible communication in the studio. Questioning the studio at a meta-level can be helpful to develop more sincere dialogues between instructors and students, which also helps to create a more collaborative environment. In these meta-studio activities, students should be encouraged to compare the criteria of success and their performance in the design studio explicitly, so that they can define their weaknesses and strengths. This activity also allows students to question the decision-makers of the studio and decrease their high dependency on them. Critical thinking requires questioning minds so that all kinds of information can be synthesized. The fewer the number of external factors that students depend on, the more agency and awareness they can develop. Sharing Experiences In the design studio, knowledge is constructed mostly through reflective conversations between the instructor and the student. These conversations vary according to the works and activities of the students. In the case of no development in the project or no activity during the week, the effectiveness of the critic session decreases. Instructors may not look for the reasons for these situations, and they might simply assume that students are not investing enough time in their studies. Instead, instructors should pay attention to the motivational factors which have a big effect on the learning process. Even though students try to apply learning strategies, motivational deficiency can cause them to underperform. Experience sharing is a motivational strategy that can be promoted through group tasks or activities in the studio process. Design instructors can share their experiences of learning and designing to encourage students to share their difficulties and problems with their peers as well. This creates another level of communication for these students and increases social self-efficacy by empowering them via making them feel less alone in this journey. Higher self-efficacy helps the students to trust their abilities and regulate their learning strategies accordingly. The design domain in SRL studies is under-researched globally and has not been studied in Turkey. To the best of our knowledge, this study is known to be the first to examine SRL strategies quantitatively and qualitatively through SRL intervention in an industrial design studio. This study fills a gap within the existing body of design pedagogy and instruction in industrial design relative to self-regulated learning. It highlights the importance of students’ self-awareness, learning strategy preferences, and motivational aspects in the studio education process. Design studios will not fulfill their potential to foster SRL skills through the signature pedagogy unless individual student differences are paid attention to. Studio education needs improvement to encourage students to develop their learning skills. The implementation of SRL strategies in design learning environments can help to improve the design performance, especially, of less accomplished students. More empirical studies are required to verify and enhance design education. Our study will hopefully serve as a base and starting point for discussion and further research. Acknowledgements The author gratefully acknowledges the support provided by Istanbul Bilgi University and would like to thank the students of Industrial Design department for their contribution to this research. Funding This research did not receive any specific grant from any organization in the public, commercial or not-for-profit sectors. Declarations Conflict of interest The author has no competing interests to declare that are relevant to the content of this article. Ethical approcal This study was based on author’s PhD dissertation titled “Developing student performance in industrial design studios through self-regulated learning strategies”, Istanbul Technical University, Graduate School. The author immensely grateful to Prof. Dr. Gülname Turan, the thesis advisor, for her valuable critics, guidance and encouragement. The study was conducted in accordance with the ethical principles for research involving human subjects and ethical approval was provided by the University Ethical Committee. Informed consent All participants provided informed consent. 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==== Front Diabetologie Die Diabetologie 2731-7447 2731-7455 Springer Medizin Heidelberg 977 10.1007/s11428-022-00977-3 Leitthema Telehealth als Therapieoption in der juvenilen Adipositasprävention Telehealth as a therapeutic approach in the management of childhood obesityReschke Felix [email protected] 12 Kapitzke Kerstin 12 Weiskorn Jantje 12 Galuschka Laura 12 Meister Daniela 12 Sadeghian Evelin 12 Guntermann Cathrin 12 von Stülpnagel Kisa 23 Weiner Chantal 12 Danne Thomas 1 1 Diabetologie, Endokrinologie, Gastroenterologie und Klinische Forschung, Kinder- und Jugendkrankenhaus AUF DER BULT, Janusz-Korczak-Allee 12, 30173 Hannover, Deutschland 2 KiCK – Kindergewicht intensiv Coaching im Krankenhaus, Kinder- und Jugendkrankenhaus AUF DER BULT, Janusz-Korczak-Allee 12, 30173 Hannover, Deutschland 3 grid.9463.8 0000 0001 0197 8922 Institut für Sportwissenschaft, Universität Hildesheim, Universitätsplatz 1, 31141 Hildesheim, Deutschland 30 11 2022 17 25 10 2022 © The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Übergewicht und Adipositas im Kindes- und Jugendalter gehen nicht nur mit ausgeprägten medizinischen und psychologischen Begleit- und Folgeerkrankungen einher, sondern stellen auch aus ökonomischer Sicht eine der größten Herausforderungen für das Gesundheitssystem dar. Die Beschränkungen im Rahmen der weltweiten COVID-19-Pandemie (COVID-19: „coronavirus disease 2019“) führten zu einer weiteren Aggravierung der vorbestehend hohen Prävalenz an juvenilem Übergewicht. Da pharmakologische Behandlungsmöglichkeiten im Kindes‑/Jugendalter im Allgemeinen nicht zugelassen sind und daher keine Option darstellen, spielt die Lebensstilveränderung aus therapeutischer Sicht eine gesonderte Rolle. Dabei haben multimodale Beschulungskonzepte mit Beratungen durch Mitarbeitende verschiedener Fachbereiche (beispielsweise Psychologie, Ernährung, Sport, Medizin) aktuell die besten Erfolgsaussichten. Durch den gezielten Transfer dieser Schulungsprogramme in telemedizinische Konzepte könnten deren Wirkungsgrad nachhaltig verbessert und dabei Ressourcen sowohl auf therapeutischer Seite als auch der der Betroffenen geschont werden. Overweight and obesity in childhood and adolescence are not only associated with severe medical and psychological complications and sequelae, but also represent one of the greatest challenges for the healthcare system from an economic point of view. The restrictions imposed by the global COVID-19 (coronavirus disease 2019) pandemic have further aggravated the already high prevalence of juvenile obesity. Since pharmacological treatment options are generally not approved in childhood/adolescence and therefore they are not an option, lifestyle modification has a separate role from a therapeutic perspective. Multimodal training concepts from various disciplines (e.g., psychology, nutrition, sports, medicine) currently show the best prospects of success. The targeted transfer of these training programs into telemedical concepts could sustainably improve their effectiveness and reduce resource requirements on both the therapeutic and patient side. Schlüsselwörter Übergewicht Schulung, multidisziplinäre Programme Prävention Gruppentherapie Gesundheitsschulungen, videobasiert Keywords Overweight Teaching, multicomponent programs Primary prevention Group therapy Health education, video-based ==== Body pmcDie Zahl an Kindern und Jugendlichen mit Adipositas zeigt seit Jahren einen ansteigenden Trend, welcher durch die Lockdownbestimmungen im Rahmen der COVID-19-Pandemie (COVID-19: „coronavirus disease 2019“) noch verstärkt wurde. Juveniles Übergewicht geht bereits in dieser Altersperiode häufig mit Insulinresistenz, Dysglykämien, Fettstoffwechselstörungen, arterieller Hypertension und Transaminasenerhöhung sowie weiteren Vorboten des metabolischen Syndroms einher. Therapieoptionen stellen Schulungsprogramme dar. Der Einsatz von Telehealth könnte helfen, diesem Problem überregional Sorge zu tragen. Juveniles Übergewicht und Adipositas Juveniles Übergewicht und Adipositas stellen aktuell und zukünftig eine der größten Herausforderungen für das Gesundheitssystem dar. In Deutschland ist seit geraumer Zeit ein ansteigender Trend an Übergewicht bei Jugendlichen im Alter von 14–17 Jahren auf 16,2 % bei Mädchen bzw. 18,5 % bei Jungen zu beobachten [1]. Dabei sind Kinder und Jugendliche mit niedrigem sozioökonomischem Status etwa 4‑mal häufiger betroffen. Die zurückliegenden Restriktionsmaßnahmen im Rahmen der COVID-19-Pandemie in Deutschland haben diesen Trend noch verstärkt: Ergebnisse der Schuleingangsuntersuchungen in der Region Hannover [2] zeigten eine signifikante Zunahme von Übergewicht und Adipositas von 8,9 % im Jahr 2017 auf 14,4 % im Jahr 2022 (p < 0,001). Störungen im Zuckerstoffwechsel liegen bereits bei ca. 12 % der Jugendlichen mit Adipositas vor. Die jährliche Inzidenz von juvenilem Typ-2-Diabetes (T2D) zeigt ebenso einen steigenden Trend und liegt aktuell mit ca. 170 Neuerkrankungen zwar noch hinter dem internationalen Spitzenreiter USA, allerdings muss von einer hohen Dunkelziffer ausgegangen werden. Ende 2020 nahm die Prävalenz von T2D in Nordrhein-Westfalen mehr als doppelt so schnell zu wie die von Typ-1-Diabetes [3]. Kinder und Jugendliche mit Adipositas leiden überwiegend an einer alimentären Adipositas Auch wenn es seltene Fälle monogenetischer Adipositasformen gibt, leidet der überwiegende Anteil der Kinder und Jugendlichen mit Übergewicht an einer alimentären Adipositas, bei welcher eine Disbalance zwischen Kalorienaufnahme und -verbrennung besteht. Die nachgewiesenermaßen häufigste adipogene Verhaltensweise ist der übermäßige Konsum gesüßter Getränke sowie nährstoffarmer, fettreicher Lebensmittel in Kombination mit geringer körperlicher Aktivität, einem hohen Maß an sitzenden Tätigkeiten sowie einer verkürzten Schlafdauer [4]. Ernährungsinhalte und -verhalten, körperliche Aktivität, Bildschirmzeit und Schlafverhalten werden durch eine Vielzahl von Faktoren und Wechselwirkungen beeinflusst, zu denen neben (epi-)genetischen insbesondere auch Parameter wie zwischenmenschliche Beziehungen und das familiäre Ernährungs- und Aktivitätsverhalten gehören [5]. In Studien wurde ein enormer Einfluss der Bildschirmzeit auf die Entstehung und Weiterentwicklung der juvenilen Adipositas festgestellt. So ist erwiesen, dass die Medienzeit von Kindern kongruent ist zum Konsum von Snacks, gesüßten Getränken, Fast Food und gleichzeitig diametral zur Einnahme von Obst und Gemüse [6]. Zudem wirkt sich die Medienzeit negativ auf Schlafverhalten, -dauer und -muster von Kindern aus, was ebenfalls mit einer gesteigerten Rate an Adipositas im Kindes- und Jugendalter einhergeht [7]. Kinder und Jugendliche mit Adipositas leiden bereits in dieser Altersperiode mitunter massiv an Begleiterkrankungen, die psychosozialer, respiratorischer, metabolischer, endokrinologischer, orthopädischer, neurologischer und psychiatrischer Natur sein können (Abb. 1). Zudem wurde nachgewiesen, dass sie im Vergleich zu schlanken Altersgenoss:innen fast doppelt so viele und auch deutlich größere Fettzellen haben. Dabei kommt es durch eine Hyperaktivität fettzellassoziierter Makrophagen zu einer chronischen Entzündungsreaktion im Fettgewebe von Kindern und Teenagern mit Übergewicht, und gleichzeitig wird die Sekretion von Leptin und Adiponektin, Hormone, die das Hungergefühl und den Stoffwechsel beeinflussen, krankhaft verändert [8]. Das dadurch dysfunktional veränderte Fettgewebe trägt massiv zur Entstehung von Folgeerscheinungen der Adipositas bereits im Kindesalter, wie der Insulinresistenz, bei, weswegen eine frühzeitige Prävention einen hohen gesundheitlichen Nutzen hat. Ohne Intervention besteht bei über 85 % der Kinder und Jugendlichen mit Adipositas diese auch im Erwachsenenalter fort. Hier potenzieren sich v. a. die metabolischen und kardiovaskulären Folgeerkrankungen. Es besteht eine eindeutige Assoziation zwischen juvenilem Übergewicht und der Entstehung von T2D, einer Steatosis hepatis und kardiovaskulären, aber auch onkologischen Folgeerkrankungen [9]. Therapieoptionen für juveniles Übergewicht Medikamentöse Therapie Im Gegensatz zu Erwachsenen gibt es für Kinder nur wenige zur Behandlung der Adipositas zugelassene Medikamente. Eine Ursache ist sicherlich, dass es schwierig ist, die Sicherheitsprofile zentral wirkender Medikamente im Kontext von Wachstum und Entwicklung nachzuweisen. Im Juni 2021 wurde mit Liraglutid (Saxenda®) das erste Medikament zur Behandlung der juvenilen Adipositas zugelassen. Es ist verordnungsfähig für Jugendliche ab 12 Jahren mit einem Ausgangs-BMI-Wert (BMI: Body-Mass-Index), der nach internationalen Cut-off-Punkten (IOTF [International Obesity Task Force] für Adipositas) mindestens dem eines Erwachsenen mit einem BMI von ≥ 30 kg/m2 entspricht, sowie einem Körpergewicht von über 60 kg. Die europäische Arzneimittelbehörde (EMA [European Medicines Agency]) schreibt ergänzend eine Kombination mit einer Lebensstilveränderung durch gesündere Ernährung und erhöhte körperliche Aktivität vor. Die Therapie mit Liraglutid soll neu bewertet oder gar abgesetzt werden, wenn die mit 3,0 mg/Tag oder der maximal verträglichen Dosis Behandelten nach 12 Wochen ihren BMI oder BMI-Z-Score nicht um mindestens 4 Prozentpunkte verringern konnten. Ein weiteres Manko stellt die fehlende Kostenübernahme von Liraglutid für den pädiatrischen Sektor durch die GKV (gesetzliche Krankenversicherung) dar. Die Therapie muss von den Betroffenen bzw. deren Eltern selbst getragen werden. Andere zugelassene pharmakologische Therapieoptionen bestehen für den Kinder- und Jugendbereich nicht. Präsenzschulungen Für weitere Therapien der juvenilen Adipositas gibt es im Allgemeinen wenig Evidenz. Unter den verschiedenen Optionen, die hierfür in Frage kommen, gibt die familienzentrierte ganzheitliche ambulante Adipositasschulung am meisten Hoffnung [10]. In Studien wurde darüber hinaus ein Vorteil für multimodale Therapiekurse mit Beschulung aus verschiedenen Fachbereichen (beispielsweise Ernährungsberatung, medizinisch-kognitiv edukative Therapie, Sporttherapie, psychosoziale Beratung usw.) gegenüber monomodalen Schulungsmodellen nachgewiesen [11]. Diese Aussagen werden durch die aktuelle Leitlinie der Endocrine Society zur Therapie des juvenilen Übergewichts gestützt, in der empfohlen wird, dass sich die therapeutischen Ansätze zur Behandlung der pädiatrischen Adipositas auf die Beratung über körperliche Aktivität und die Änderung des Lebensstils beschränken sollten [12]. Diese Kurse scheinen am effektivsten zu sein, wenn die Schulungsinhalte in einem Kombinationskonzept aus Individual- und Gruppentherapie multiprofessionell vermittelt werden (sog. individualisierte Gruppentherapie – s. Abb. 2).Viele dieser Kurse sind durch ein hohes Maß an Präsenzveranstaltungen geprägt. Dies bedeutet, dass Kinder und Jugendliche, die beispielsweise in eher ländlichen Regionen wohnen, auf Erwachsene angewiesen sind, die sie zum Kurs begleiten, oder auf die Nutzung öffentlicher Verkehrsmittel, was einem hohen zeitlichen Aufwand entspricht, der leider häufig nicht auf sich genommen wird. Folglich erreichen viele dieser Schulungsprogramme Familien nur in einem engen regionalen Umfeld des Veranstaltungsortes. Einen weiteren komplizierenden Faktor stellen eine fehlende Compliance und hohe Abbruchquoten bei diesen Kursen dar. Eventuell könnte die Ergänzung von Programmen zur Änderung des Lebensstils durch pharmakologische Behandlungen zukünftig eine wirksame therapeutische Strategie in der Pädiatrie darstellen. Einsatz von Telehealthkomponenten Wie erwähnt sind Lebensstilinterventionen zwar als die erfolgreichste Form der Adipositastherapie im Kindes‑/Jugendalter anzusehen, stellen aber aufgrund der häufigen Vorstellungstermine eine enorme Herausforderung an die damit verbundenen Ressourcen mit Blick auf den Mangel an Unterrichtsräumen oder die begrenzte Zeit vieler Fachkräfte einerseits und dem hohen Zeitaufwand für die Teilnahme an den Sitzungen auf Seiten der Teilnehmenden andererseits dar. Dieser Faktor wird durch eine nicht flächendeckend vergleichbar gute Finanzierungsstruktur in den verschiedenen Zentren oder Situationen mit Kontaktbeschränkungen, wie in Zeiten einer globalen Viruspandemie, noch potenziert. Dadurch sind die Reichweite, die Nachhaltigkeit und der Effekt von Lebensstilinterventionen für Familien von Kindern und Jugendlichen mit Übergewicht oder Adipositas eindeutig begrenzt. Telehealth kann eine praktikable und ressourcenschonende Option für die Behandlung von Adipositas im Kindesalter sein Telehealth als Begriff einer multiprofessionellen Bereitstellung gesundheitsbezogener Dienstleistungen via Telekommunikation stellt einen vielversprechenden Weg zur Behandlung mittels Lebensstilinterventionen dar und kann vielen der oben genannten limitierenden Faktoren begegnen. Es wurde nachgewiesen, dass sie eine praktikable Option für die Behandlung von Adipositas im Kindesalter sein kann [13]. In einer 2017 veröffentlichten Studie wurden monatliche Telefonkontakte (Dauer 15 min) mit einer in adipositasrelevanten Themen geschulten Pflegefachkraft und 2‑mal/Jahr erfolgende persönliche Arztvorstellungen in einem Adipositaszentrum (Dauer 45 min) über einen Zeitraum von etwa 28 Monaten verglichen. Bezogen auf den BMI-SDS-Wert (SDS: „standard deviation score“ [Standardabweichung]) konnte kein Unterschied zwischen den beiden Gruppen festgestellt werden, obwohl die Teilnehmenden (Alter 5–15 Jahre) beider Gruppen nach der Intervention einen reduzierten BMI-SDS aufwiesen (Telehealth: ∆ BMI-SDS = −0,16 vs. Arztvorstellung: ∆ BMI-SDS = −0,12; [14]). Pbert et al. verglichen in der 2016 veröffentlichten FITLINE-Studie (n = 37, 8–12 Jahre) den Effekt einer telefonbasierten Beratung (30 min Dauer) in einem 6‑wöchentlichen Intervall über einen Zeitraum von 3 Monaten gegenüber einer Kontrollgruppe, die keinerlei Beratung erhielt. Sie stellten für die telefonisch beratene Gruppe einen signifikanten Abfall des BMI-SDS um −0,1 fest. In der Kontrollgruppe blieb dieser unverändert [15]. In einer anderen Studie mit Crossoverdesign wurde der Effekt einer videobasierten Beratung von Jugendlichen mit Adipositas (n = 40, 10–17 Jahre) durch Hausärzt:innen im 3‑monatlichen Intervall mit dem zusätzlichen Einsatz von Videokonferenzen mit Adipositasspezialist:innen verglichen. Nach 6 Monaten erfolgte der Crossover bei einem Gesamtbeobachtungszeitraum von 12 Monaten. Nach den ersten 6 Monaten wies der BMI-SDS in der Telehealthgruppe einen signifikanten Abfall gegenüber dem Startzeitpunkt auf (∆ BMI-SDS 6 Monate: −0,11), der nach 12 Monaten und Crossover allerdings nahezu wieder verschwunden war (∆ BMI-SDS 12 Monate: −0,04). In der Vergleichsgruppe wurde nach 6 Monaten lediglich eine Verminderung des BMI-SDS um −0,06 festgestellt, was sich allerdings nach 12 Monaten und Crossover in die Telehealthgruppe auf eine Reduktion von −0,011 veränderte [16]. Diese Ergebnisse zeigen, dass bereits ein niedrigschwelliger Einsatz von Telehealthmaßnahmen einen günstigen Effekt auf die Gewichtsentwicklung haben kann. Interessanterweise gaben die Patient:innen und deren Eltern in den genannten Studie zu sehr großen Teilen an, dass sie von der monodisziplinären Telehealthkonsultation nachhaltig profitiert hatten, und zeigten sich überwiegend zufrieden (Tab. 1).Autorengruppe Bewertung Bohlin et al. 2017 [14] 70 % der Eltern bewerteten das Programm als gut, 76 % als hilfreich 63 % der Eltern bewerteten die Telehealthmaßnahmen als gut in den Alltag integrierbar Pbert et al. 2016 [15] In qualitativen Interviews berichteten die Eltern über einen großen Vorteil der flexibleren Beratungsmöglichkeiten via Telehealth im Vergleich zu Präsenzterminen. Die Teilnehmenden berichteten zu 97,6 %, von den Programminhalten gelernt zu haben Fleischman et al. 2016 [16] 15/26 (58,7 %) berichteten, dass sie an einem konventionellen Programm mit Beratungsterminen in Präsenz nicht teilgenommen hätten Pecoraro et al. 2021 [17] 74,6 % der Teilnehmenden und Eltern stimmten einer Teilnahme an einem Telehealthprogramm zu Reschke et al. 2022 [18] Eltern bewerteten die Durchführung und den Erfolg des Programms mit 9,4 (7,0–10,0), Kinder und Jugendliche mit 9,2 (6,0–10,0) auf einer 10-Punkte-Skala mit 10 entsprechend höchster Akzeptanz und Erfolg Telehealth für juvenile Adipositas im Pandemielockdown Im Rahmen der weltweiten COVID-19-Pandemie konnten viele Zentren den Erfolg von videobasierten und digitalen Gesundheitskursen nachweisen. Pecoraro et al. [17] berichteten über den Effekt eines videobasierten multidisziplinären Adipositaskurses über 3 Monate im Zeitraum des ersten Lockdowns in Italien (März 2020 bis Mai 2020). An diesem Kurs nahmen insgesamt 117 Kinder und Jugendliche (5–18 Jahre) teil. Sie wurden alle 3 Wochen für 30 min von Ernährungsberater:innen, Psycholog:innen und Sporttherapeut:innen individuell beraten. Darüber hinaus wurden im 2‑wöchentlichen Rhythmus sog. Snackmeetings veranstaltet, bei denen 4–5 Jugendliche zu einem videobasierten Austausch ohne professionelle Unterstützung zusammenkamen, um sich über ihr Ernährungsverhalten und den Umgang mit Appetit auszutauschen. Zu diesen Meetings war es den Teilnehmenden erlaubt, zu essen, um eine gemeinsames Ernährungserlebnis zuzulassen, von welchem man sich einen positiven Effekt auf das Ernährungsverhalten erhoffte. Zusätzlich wurden Tutorials (web- und videobasiert) zur Verfügung gestellt, welche die Jugendlichen im Alltag unterstützen sollten, ihr Gewicht zu kontrollieren. Teilnehmende des Kurses konnten über den Zeitraum des ersten Lockdowns ihr Gewicht halten (BMI-SDS = 0,0) und die freie Fettmasse (gemessen via Bioimpedanzanalyse) sogar um 0,9 % verbessern. Die Autor:innen werteten dies als Erfolg, da in der Vergleichskohorte an deren Zentrum, die nicht an der Telehealthkomponente partizipiert hatte (n = 7), der BMI-SDS um 0,1 im gleichen Zeitraum signifikant anstieg [17]. Die multiprofessionellen Adipositasschulungsprogramme werden von den Kassen oft nicht oder nur teilweise finanziert Aus Hannover wurde der Effekt der Umstellung eines Präsenzschulungsprogramms für Kinder und Jugendliche mit Adipositas (Kindergewicht intensiv Coaching im Krankenhaus [KiCK]) auf ein rein videobasiertes Schulungsmodell im multidisziplinären Setting unter Beibehaltung der Präsenzpflicht berichtet (Video-KiCK). Dabei wurden wöchentliche videobasierte Einheiten zum Thema Sport (90 min/Woche) und einem zusätzlichen Thema aus den Bereichen Ernährung, Psychologie oder Medizin (45–90 min) durchgeführt. Die Sportkurse erfolgten in Gruppen von 12 bezüglich des Alters angepassten Kindern/Jugendlichen, die anderen Beschulungen teilweise in Gruppen oder in individuellen Familiengesprächen. Das Programm wurde und wird über einen Zeitraum von 12 Monaten durchgeführt und ist von der Arbeitsgemeinschaft Adipositas im Kindes‑/Jugendalter zertifiziert (www.adipositas-gesellschaft.de). In einer Auswertung für Programmteilnehmende von März 2020 bis März 2021 wurde ein deutlicher Abfall des BMI-SDS (∆ BMI −0,18) und des HOMA-Indexes (∆ −1,4, HOMA: „homeostasis model assessment“) festgestellt. Die körperliche Leistungsfähigkeit, gemessen im 6‑min-Gehtest, stieg nachweislich (∆ 97 m), und das körperliche Wohlbefinden zeigte einen signifikant positiven Trend (WHO-5-Index: ∆2,5 [WHO: World Health Organization]). Die Autor:innen verglichen die Ergebnisse mit den Daten ihres Programms mit Präsenzterminen vor der COVID-19-Pandemie und fanden vergleichbare Ergebnisse für diese Kontrollgruppe. Die vorhandenen und aufgeführten Daten und Studien zeigen ein sehr positives Bild für den Einsatz von Telehealthmaßnahmen zur Therapie der juvenilen Adipositas. Leider besteht aufgrund sehr unterschiedlicher Studiendesgins, häufig kurzer Beobachtungszeiträume und unterschiedlicher Schulungsinhalte noch wenig Evidenz. Ein begrenzender Faktor für multiprofessionelle Adipositasschulungsprogramme ist die häufig fehlende, aufwendige oder nur teilweise vorhandene Finanzierung durch die gesetzlichen Krankenkassen. Zukünftige Studien werden den positiven Effekt der bisherigen Ergebnisse noch bestätigen müssen. Wünschenswert wären interagierende, überregionale Arbeitsgruppen zur Förderung der Evidenz der durchgeführten digitalen Schulungskonzepte. Kinder und Jugendliche könnten aufgrund ihres vergleichsweise hohen technischem Verständnisses und ihrer Screenaffinität eine Gruppe sein, die besonders von digitalisierten und Telehealthkomponenten profitiert. Gleichzeitig jedoch stellt die Bildschirmzeit einen bedeutenden Risikofaktor für Adipositas von Kindern und Jugendlichen dar. Diesem Faktor müssen zukünftige Therapiekonzepte Rechnung tragen. Es könnte sein, dass Kombinationsmodelle aus Präsenzterminen und Telehealth für die Versorgung von Kindern und Jugendlichen mit Adipositas eine Lösung darstellen, die bei günstigem Effekt auf die Gewichtsentwicklung und Begleiterkrankungen dennoch ressourcenschonend ist und im Rahmen derer Patientenzufriedenheit und -compliance nachhaltig positiv beeinflusst werden. Einsatz digitaler Apps im Kontext der Adipositasprävention Digitale Gesundheitsanwendungen (DiGA) erleben international derzeit einen großen Zulauf. In der Behandlung von Übergewicht und Adipositas sind sie auch in Deutschland gefragt. Aktuell stehen für Erwachsene mit Adipositas mit zanadio® (PZN: 16898701) und Oviva Direkt® (PZN: 17850527) 2 verschreibungsfähige DiGA zur Adipositastherapie zur Verfügung, die ebenfalls mit einem multimodalen Ansatz konzipiert sind. Für zanadio® wurde unlängst in einer kontrollierten Studie (150 Teilnehmende [91 % Frauen] – 77 in der Behandlungs- und 73 in der Kontrollgruppe) eine Gewichtsabnahme von 7,7 % (95 %-KI = −9,63;−5,82 [KI: Konfidenzintervall]) festgestellt [19]. Insgesamt konnten rund 57 % der Teilnehmer:innen der Interventionsgruppe ihr Gewicht um mindestens 5 % reduzieren, 31 % der Interventionsgruppe nahmen sogar 10 % oder mehr ab. Für die Kontrollgruppe blieb das Gewicht unverändert. Leider stehen in Deutschland für den pädiatrischen Bereich bisher keine entsprechenden verordnungsfähigen und geprüften oder zertifizierten Anwendungen zur Verfügung. Bestehende Apps, wie beispielsweise yuBuddy® (Dr. Schupfner, Hamburg, Deutschland – www.yubuddy.com), eine Smartphone-App, die sich an Kinder zwischen 8 und 12 Jahren richtet und v. a. die Motivation zu mehr Bewegung fördern soll, stimmen zwar hoffnungsvoll, müssen allerdings hinsichtlich ihres Nutzens noch bewertet werden. In europäischen Nicht-EU-Ländern (EU: Europäische Union) gibt es bereits entsprechende DiGA (Schweiz: Pathmate®; Großbritannien: NoObesity®), mittels welchen versucht wird, sich auf digitaler Ebene diesem Problem zu stellen und dabei auch die Grenzen der Anwendung auf Benutzerseite aufzuzeigen [20, 21]. Zusammenfassung und Ausblick Juveniles Übergewicht und Adipositas stellen mit ihren vielen Komorbiditäten im Kindes‑/Jugendalter und bei Folgeerkrankten eine der größten Herausforderungen an unser Gesundheitssystem dar. Multiprofessionelle ganzheitliche und familienzentrierte Schulungsprogramme über einen Zeitraum von 6–12 Monaten sind die aktuell hoffnungsvollsten Therapieformen. Leider gibt es derzeit weder eine flächendeckende Versorgung von Betroffenen noch eine einheitliche Kostenübernahmestruktur. Telecaremaßnahmen können evtl. helfen, auch Familien in ländlichen Gebieten kostengünstig zu versorgen. Seitens des Gesetzgebers ist die Entwicklung eines Disease-Management-Programms (DMP) Adipositas vorgesehen [22]. Allerdings ist bisher unklar, ob neben dem geplanten DMP für Erwachsene auch eines für Kinder und Jugendliche konzipiert wird und wann dies umgesetzt werden kann. Eine Einbindung telemedizinischer Komponenten in die Versorgung von pädiatrischen Patienten mit Übergewicht und Adipositas ist wünschenswert, allerdings ist die Evidenz über deren Nutzen aktuell noch zu gering. Daher sollten auch Projekte und Studien durchgeführt werden, die helfen, den Effekt dieser Maßnahmen noch besser bewerten zu können. Fazit für die Praxis Pädiatrisches Übergewicht und Adipositas zeigen seit Jahren einen ansteigenden Trend, welcher durch die Einschränkungen im Rahmen der Coronapandemie verstärkt wurde. Adipositas geht bereits im Kindes‑/Jugendalter mit signifikanten Veränderungen des Fett- und Zuckerstoffwechsels sowie der Leberwerte einher und ist Vorbote eines Typ-2-Diabetes und metabolischen Syndroms. Für das Kindes‑/Jugendalter stehen kaum pharmakologische Therapieoptionen zur Verfügung. Den größten Erfolg zeigen multimodale Schulungskonzepte aus den Bereichen Ernährung, Sport, Psychologie und Medizin. Strukturierte Telehealthmaßnahmen können helfen, die Reichweite, die Nachhaltigkeit und den Effekt der Schulungskonzepte zu erhöhen und stehen auch in Zeiten von Kontaktbeschränkungen zur Verfügung. Eine finanzielle Kostendeckung seitens der Krankenkassen besteht weder für konventionelle Schulungsprogramme noch für Telehealthkomponenten, wodurch die Versorgung von Kindern und Jugendlichen mit Adipositas enorm bedroht ist. Einhaltung ethischer Richtlinien Interessenkonflikt F. Reschke, K. Kapitzke, J. Weiskorn, L. Galuschka, D. Meister, E. Sadeghian, C. Guntermann, K. von Stülpnagel, C. Weiner und T. Danne geben an, dass kein Interessenkonflikt besteht. Alle beschriebenen Untersuchungen am Menschen wurden mit Zustimmung der zuständigen Ethik-Kommission, im Einklang mit nationalem Recht sowie gemäß der Deklaration von Helsinki von 1975 (in der aktuellen, überarbeiteten Fassung) durchgeführt. Von allen beteiligten Patienten liegt eine Einverständniserklärung vor. QR-Code scannen & Beitrag online lesen ==== Refs Literatur 1. Danne T, Holder M, Kapellen T Diabetes bei Kindern und Jugendlichen. In: Deutscher Gesundheitsbericht Diabetes 2022 Herausgeber Deutsche Diabetes Gesellschaft. 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Reschke F Galuschka L Landsberg S Weiner C Guntermann C Sadeghian E Lange K Danne T Successful telehealth transformation of a pediatric outpatient obesity teaching program due to the COVID-19 pandemic—the „Video KiCK“ program J Pediatr Endocrinol Metab 2022 35 6 803 812 10.1515/jpem-2022-0104 35575788 19. Roth L Ordnung M Forkmann K Mehl N Horstmann A Die Evaluation von zanadio – einer digitalen Gesundheitsanwendung für Erwachsene mit Adipositas Adipositas 2022 16 03 176 10.1055/s-0042-1755692 20. Meinert E Rahman E Potter A Lawrence W Van Velthoven M Acceptability and usability of the mobile digital health app noobesity for families and health care professionals: protocol for a feasibility study JMIR Res Protoc 2020 9 7 e18068 10.2196/18068 32706703 21. De-Jongh González O Tugault-Lafleur CN Buckler EJ Hamilton J Ho J Buchholz A Morrison KM Ball GD Mâsse LC The aim2be mhealth intervention for children with overweight or obesity and their parents: person-centered analyses to uncover digital phenotypes J Med Internet Res 2022 24 6 e35285 10.2196/35285 35731547 22. Joisten F Gellhaus I Kauth T Leipold G Wabitsch M Weihrauch-Blüher S Wiegand S Doetsch J Fischbach T Die Versorgungslage von Kindern und Jugendlichen mit Adipositas – ist ein Disease Management Programm (DMP) eine Lösung ? Adipositas 2022 16 149 158 10.1055/a-1912-0686	0	PMC9713089	NO-CC CODE	2022-12-02 23:22:07	no	Diabetologie. 2022 Nov 30;:1-7	utf-8	null	null	null	oa_other
==== Front Pediatr Surg Int Pediatr Surg Int Pediatric Surgery International 0179-0358 1437-9813 Springer Berlin Heidelberg Berlin/Heidelberg 36449111 5308 10.1007/s00383-022-05308-7 Original Article High intestinal vascular permeability in a murine model for Hirschsprung’s disease: implications for postoperative Hirschsprung-associated enterocolitis Suda Kazuto [email protected] 1 Yamada Shunsuke 1 Miyahara Katsumi 1 Fujiwara Naho 1 Kosaka Seitaro 1 Abe Kumpei 1 Seo Shogo 1 Nakamura Shinji 2 Lane Geoffrey J. 1 Yamataka Atsuyuki 1 1 grid.258269.2 0000 0004 1762 2738 Department of Pediatric General and Urogenital Surgery, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421 Japan 2 grid.258269.2 0000 0004 1762 2738 Division of Biomedical Imaging Research, and Division of Ultrastructural Research, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421 Japan 30 11 2022 2023 39 1 1514 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose Intestinal vascular permeability (VP) in a murine model for Hirschsprung’s disease (HD) and postoperative Hirschsprung-associated enterocolitis (HAEC) were investigated. Methods Intestinal VP was determined using a Miles assay using 1% Evans blue injected into a superficial temporal vein of newborn endothelin receptor-B KO HD model (KO) and syngeneic wild-type (WT) mice (n = 5, respectively). Extravasated Evans blue in normoganglionic ileum (Ng-I), normoganglionic proximal colon (Ng-PC) and aganglionic distal colon (Ag-DC) was quantified by absorbance at 620 nm. Quantitative polymerase chain reaction (qPCR) for Vascular Endothelial Growth Factor A (VEGF-A), VEGF-B, CDH5, SELE and CD31, and immunofluorescence for CD31 were performed. Results VP was significantly higher in Ng-I, Ng-PC, and Ag-DC from KO than WT (p < 0.01, p < 0.05, and p < 0.05, respectively). qPCR demonstrated upregulated VEGF-A in Ng-I and Ag-DC, VEGF-B in Ng-I, and SELE in Ng-I and Ng-PC (p < 0.05, p < 0.05, p < 0.05, p < 0.01 and p < 0.05, respectively), and downregulated CDH5 in Ng-I and Ng-PC from KO (p < 0.05, respectively). Expression of CD31 mRNA in Ng-I and Ag-DC from KO was significantly higher on qPCR (p < 0.05) but differences on immunofluorescence were not significant. Conclusions VP may be etiologic for postoperative HAEC throughout the intestinal tract even after excision of aganglionic bowel. Keywords Hirschsprung’s disease Hirschsprung associated enterocolitis Endothelin receptor-B Vascular permeability Vascular integrity Vascular endothelial growth factor Juntendo University Project Grant2021-27 2021-27 2021-27 2021-27 2022-33 2022-33 2022-33 2022-33 Suda Kazuto Yamada Shunsuke Abe Kumpei Nakamura Shinji Japan Society for the Promotion of Science “KAKENHI” grant18K08601 Miyahara Katsumi President's Grant for Young Researchers Juntendo University2021-07 Kosaka Seitaro issue-copyright-statement© Springer-Verlag GmbH Germany, part of Springer Nature 2023 ==== Body pmcIntroduction Hirschsprung’s disease (HD) is characterized by intestinal dysmotility due to an absence of ganglionic cells differentiated from enteric neural crest cells (ENCC). Resection of aganglionic bowel and pulling-down normoganglionic bowel to the anus are performed to normalize bowel function [1]. Hirschsprung associated enterocolitis (HAEC) causes morbidity and can be serious enough to cause death in HD patients both before and after definitive pull-through surgery [2]. While its etiopathogenesis remains unknown, hypoperistalsis due to a lack of ganglionic cells has been conventionally regarded as responsible for the development of HAEC. However, its incidence can range from 15–50 to 2–33% for pre and postoperative HAEC, respectively [3], suggesting a more generalized cause involving the entire intestinal tract rather than a nerve cell distribution anomaly causing localized aganglionic bowel [4]. In addition to ganglion and nerve cell anomalies that are pathognomonic for HD, altered vascular density has also been reported in the gastrointestinal tract (GIT) of HD patients [5]. For example, increased submucous microvascular density has been observed in aganglionic colon from HD patients compared with ganglionic colon [5] and vascular networks comprised of a variety of fluid and blood-conducting vessels with the overall goal of perfusing all metabolic tissues of the intestines are disrupted with significantly decreased blood vessel density in the distal colon of rearranged during transfection (RET) knockout mice, an animal model for HD compared with controls [5]. These findings may just be the result of aberrant neovascularization and their specific clinical importance is unclear but their association with HD is relevant. Vascular endothelial growth factor A (VEGF-A) mediates angiogenesis and is also involved in the etiopathology of several diseases such as inflammatory bowel, chronic inflammatory, and autoimmune diseases [6]. Tissue vascular permeability (VP) can be enhanced by VEGF-A activation and this mechanism is closely linked to serious inflammatory conditions such as inflammatory bowel disease due to disrupted endothelial barrier function [6–9]. In addition, abnormal vascularity caused by irregular VEGFA-induced angiogenesis has increased permeability and susceptibility to invasive cancer cells [10, 11]. Despite these known actions, there are no reports of increased VP in the GIT of HD patients. The status of abnormally permeable vessels and aberrant angiogenesis-mediated stimuli observed in the GIT of Endothelin receptor-B null mice (KO), a representative murine model for HD presenting with distal colorectal aganglionosis [12] was investigated as a possible cause for HAEC. Although HAEC can also arise preoperatively, bowel dysmotility associated with HD affects its clinical presentation. Postoperative HAEC arises in the absence of aganglionic bowel, allowing the etiology of HAEC to be investigated more specifically. As a result, this study focuses on postoperative HAEC because it is more distinct clinically than preoperative HAEC, although the morbidity involved is essentially the same. Material and methods Animal model KO mice were originally obtained from Jackson Laboratory (Bar Harbor, USA). Homozygous KO mice were raised at Juntendo University School of Medicine by crossing pairs of heterozygous littermates. Genotypes were determined by polymerase chain reaction (PCR). Newborn homozygous KO mice and syngeneic wild-type (WT) mice obtained within 24 h of birth were used to minimize the effect of growth related inflammation that might occur. Approval for this study was obtained from the Animal Care and Use Committee (registration number: 1570, permission number: 2022280) at Juntendo University School of Medicine. GIT specimens studied were normoganglionic ileum (Ng-I), normoganglionic proximal colon (Ng-PC), and aganglionic distal colon (Ag-DC) from KO mice, and ileum (I), proximal colon (PC), and distal colon (DC) from WT mice. The miles assay VP was determined using the Miles assay [13]. Briefly, 30 μL of 1% Evans blue dye solution dissolved in phosphate-buffered saline (PBS) was injected into a superficial temporal vein just anterior to the ear bud of neonatal KO and WT mice using a 31-gauge needle under stereomicroscopic control (n = 5, respectively). Mice were perfused with 200 μL of PBS through the left ventricle 2 h after injection. GIT specimens (Ng-I, Ng-PC, Ag-DC from KO and I, PC and DC from WT as well) were harvested from mice sacrificed with carbon dioxide, cleaned, and immediately placed in a centrifuge tube containing 1.5 mL of formamide. After incubation for 24 h at 60 °C, the formamide extract was collected, and colorimetric measurements performed with a Nano Drop 1000 spectrophotometer (ND-1000; Thermo Scientific, Yokohama, Japan) at 620 nm. The Evans blue content of extracts was quantified with a standard curve. RNA isolation from gut, complementary DNA (cDNA) synthesis and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from GIT specimens taken from subject mice [14]. Real-time PCR (RT-PCR) was performed for VEGF-A, VEGF-B, endothelial integrity-associated genes such as SELE and CDH5, and CD31, a marker for endothelial cells, using the 7500 Fast RT-PCR system (Applied Biosystems, Forster City, Calif), according to the manufacturer’s specifications. All results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to compensate for differences in the amount of cDNA. Sequences of primers were as follows: Forward 5′ GACATCAAGAAGGTGGTGAAGCAG 3′ and Reverse 5′ ATACCAGGAAATGAGCTTGACAAA 3′ for GAPDH; Forward 5′ CACTGCTTTGGGAGCCTTC 3′ and Reverse 5′—GGGGCAGCGATTCATTTTTCT—3′ for CDH5; Forward 5′ ATGCCTCGCGCTTTCTCTC 3′ and Reverse 5′—GTAGTCCCGCTGACAGTATGC—3′ for SELE; Forward 5′ AGGCAGACTATTCAGCGGAC 3′ and Reverse 5′—CCAACCTCCTCAAACCGTTG—3′ for VEGFA; Forward 5′ GCCAGACAGGGTTGCCATAC 3′ and Reverse 5′—GGAGTGGGATGGATGATGTCAG—3′ for VEGFB; Forward 5′ AACAGAAACCCGTGGAGATG 3′ and Reverse 5′—GTCTCTGTGGCTCTCGTTCC—3′ for CD31. Immunofluorescence, confocal microscopy, and image quantification GIT specimens were fixed in 4% paraformaldehyde, embedded in OCT Mounting Compound (VWR International, Leuven, Belgium), frozen at − 80 °C, sectioned transversely at a thickness of 5 μm and mounted on Superfrost Plus slides (VWR International). Preparations were washed three times in PBS + 0.5% Triton X-100 (PBST). Primary antibody to CD31 (Rabbit, Abcam, 1:1200) was diluted in blocking solution (PBST + 5% BSA) and slides were incubated with primary antibody at 4 °C overnight. After several washes with PBS, a secondary antibody was added in the blocking solution for 1 h at room temperature at the following dilution: anti-rabbit Alexa Flour 488 (Invitrogen, UK) 1:200. Finally, slides were washed with distilled water and mounted with coverslips using Vectashield containing DAPI (Vector Laboratories, USA). Images were acquired on a confocal laser-scanning microscope (LSM780, Zeiss, Germany). Immunostaining was repeated on at least 3 tissue sections per tissue block. Only representative immunostainings are presented in this report. CD31 fluorescence was quantified by calculating corrected total cell fluorescence (CTCF) using the formula: CTCF = integrated density-(area of selected cell x mean fluorescence of background readings) using image J software. Statistical analysis Differences between two groups were tested using the unpaired t-test. All statistical tests were two-sided and all data were expressed as mean ± standard deviations. A p-value of 0.05 or less was considered statistically significant. Results VP was higher in the GIT of neonatal KO mice Extravasated Evans blue was observed macroscopically in harvested GIT specimens from KO mice using the Miles assay (Fig. 1a). Quantifying the amount of extravasated Evans blue by absorbance at 620 nm demonstrated significant differences for Ng-PC and Ag-DC and particularly Ng-I, indicating increased VP in KO mice (Fig. 1b). Interestingly, the results for extravasated Evans blue were similar for PC and DC from WT mice, and also Ng-PC and Ag-DC from KO mice, suggesting that these findings are independent of whether bowel was normoganglionic or aganglionic.Fig. 1 Assessment of vascular permeability by the Miles assay in the GIT of neonatal mice. A Typical views of harvested intestines. Extravasated Evans blue is more readily observed macroscopically KO mice compared with WT mice. Arrow heads indicate the cecum. B Summary of intestinal vascular permeability by the Miles assay in neonatal mice. Quantitatively increased absorbance of extravasated Evans blue at 620 nm in KO mice for Ng-I, Ng-PC, and Ag-DC (*p < 0.01, and p < 0.05) Expression of permeability associated gene was altered in KO mice Relative mRNA expression was assessed by qPCR for CDH5, a representative marker for vessel integrity that can be downregulated in permeable endothelial cells [15], and SELE, an endothelial adhesion molecule, that can be activated in highly permeable endothelial cells [16]. A significant decrease in CDH5 mRNA (Fig. 2a) and increase in SELE mRNA (Fig. 2b) was observed in Ng-I and Ng-PC from KO mice compared with WT mice, which was compatible with increased VP especially in Ng-I from KO mice. In contrast, mRNA expression of CDH5 and SELE were similar between Ng-PC and Ag-DC from KO mice, and in PC and DC from WT mice (Fig. 2c, d), indicating that these altered gene expressions also appear to be independent of whether bowel was normoganglionic or aganglionic.Fig. 2 Expression of permeability associated gene assessed by qPCR. A, B Significantly decreased CDH5 (A) and increased SELE (B) in Ng-I and Ng-PC from KO mice is shown (*p < 0.01, and p < 0.05). C, D No significant differences in expression of CDH5 and SELE between segments of intestine from WT mice (C) and KO mice (D) Expression of angiogenesis promoting gene was activated in the intestine of KO mice qPCR for VEGF-A/B to test whether increased VP was associated with aberrant expression of major angiogenesis-mediating factors in the GIT of KO mice. qPCR showed significantly increased mRNA expression of VEGF-A in Ng-I and Ag-DC (Fig. 3a) and VEGF-B in Ng-I (Fig. 3b) in KO mice. Expression of both substances in KO mice was relatively higher than in WT mice, but differences were not statistically significant compared with other GIT specimens. The mRNA expression of VEGF-A and VEGF-B were similar for Ng-PC and Ag-DC from KO mice, and in PC and DC from WT mice (Fig. 3c, d), indicating that these data were independent of whether bowel was normoganglionic or aganglionic.Fig. 3 Expression of angiogenesis promoting gene assessed by qPCR. A, B Significantly increased VEGFA in Ng-I and Ag-DC (A) and VEGFB in Ng-I (B) in KO mice is shown (p < 0.05). C, D No significant differences in the expression of VEGF-A and VEGF-B between segments of the intestine from WT mice (C) and KO mice (D) Distribution of endothelial cells in the GIT did not differ between KO and WT mice The extent of angiogenesis induced by activated VEGF-A/B signals was assessed using expression of CD31 in KO mice detected by qPCR and tissue immunofluorescence. As expected, CD31 mRNA was higher in Ng-I and Ag-DC of KO mice than WT mice (Fig. 4a). However, the distribution of CD31 + cells was similar or slightly increased in entire regions of KO mice on tissue immunofluorescence (Fig. 4b). Despite the relative increase observed in KO mice, differences were not statistically significant when quantified by CTCF calculation (Fig. 4c).Fig. 4 Assessment of CD31 expression in the gastrointestinal tract of neonatal mice. A Significantly increased CD31 mRNA in Ng-I and Ag-DC from KO mice is shown by qPCR (p < 0.05). B Immunofluorescence shows the distribution of CD31 + endothelial cells (green). Fluorescent images presented with DAPI staining. Scale bar: 50 μm. C Quantitative analysis by image-J for fluorescence level of CD31 in intestine. There were no significant differences between KO mice and WT mice Discussion Our data demonstrated increased intestinal VP associated with activated VEGF-A/B in KO mice. The mechanism for VEGF-mediated VP has been broadly accepted as an exacerbating factor in a variety of diseases such as Covid-19 induced pneumonia and inflammatory bowel disease [6, 17–19]. These findings may indicate a potential etiopathologic role in the development of HAEC. Of note is that the amount of extravasated Evans blue and expression of SELE, CDH5, and VEGF-A/B in were not affected by whether bowel had normal or abnormal ganglion distribution in KO mice; in other words irrespective of whether bowel was ganglionic or aganglionic, extravasation was different based on the type of mouse. This indicates that the mechanism by which HAEC may be induced in association with increased VP will be activated even after resection of aganglionic bowel which suggests that increased VP with activated VEGFA/B is caused by the genetic loss of endothelin receptor-B in KO mice rather than direct association with neural abnormality due to dysfunctional ENCC in this mouse model. The primary role of endothelin receptor-B signaling is to inhibit the differentiation of ENCC and to maintain them in a proliferative state [20]. Endothelin receptor-B is also expressed in the endothelial cells of several tissues and plays an important role in the control of vascular tone by causing constriction [21]. Carpenter et al., reported that loss of endothelin receptor-B predisposed pulmonary endothelial cells to high permeability in rats, via increased VEGF expression induced by secondarily elevated endothelin-1 peptide [22], a similar situation to the findings of this study. In another study using cultured rat cardiac microvascular endothelial cells in vitro, endothelial junction integrity determined by transendothelial electrical resistance was not changed by an endothelin receptor-B blocker [23]. The effect of endothelin receptor-B on endothelial permeability through inactivation might be diverse and depend on various factors such as signal alteration, environment (in vitro or in vivo), or type of tissue. Higher CD31 mRNA and relatively increased distribution of CD31 protein signal was observed in the GIT of KO mice but differences were not statistically significant. Although there was induction by upregulated VEGF-A/B, it was ineffective for excessive angiogenesis. Nevertheless, upregulated VEGF-A/B in KO mice was sufficient to promote GIT VP [24]. Carpenter et al. also reported that overexpressed endothelin-1 in Endothelin receptor-B KO could still act via endothelin receptor-A to stimulate VEGF mRNA and protein which supported the findings of the present study [22]. In contrast, Schrenk et al. reported their observation of degraded density of intestinal vessels with disorganized structure in RET KO HD model mice [5], interesting because the resultant aberrant vascular formation was opposite in a different murine model for HD. Thus, there may be other mechanisms for causing HAEC involving the induction of distinct genetic features. Increased SELE mRNA and decreased CDH5 mRNA shown by qPCR in association with enhanced VP seen in KO mice was consistent with published reports [7, 15]. Especially in oncology, expression of endothelin receptor-B in human hepatocellular and lung carcinoma was positively correlated with CDH5 expression [25, 26] and in the lungs of smokers who underwent lung resection for lung cancer or transplantation for advanced chronic obstructive pulmonary disease [27], endothelin receptor-B was significantly downregulated whereas there was a twofold increase in the expression of SELE. Collectively, endothelin receptor-B appears to be an adequate mediator maintaining vascular integrity by regulating these substances. Conclusions In the present study, experimental murine model evidence was instrumental for demonstrating increased GIT VP with altered expression of VEGF irrespective of whether the bowel was normoganglionic or aganglionic or whether the bowel had been resected or not. This observation is reported for the first time and may contribute to causing HAEC. Of note, some 5% of HD patients have a documented alteration of the endothelin receptor-B gene [28]. Further research on the biological effect of this mechanism and its implication for causing HAEC and its severity is planned and an enterocolitis model evaluating endothelial barrier function targeting etiopathogenesis for potential clinical application is being developed. Acknowledgements We thank the Laboratory of Morphology and Image Analysis and the Laboratory of Molecular and Biochemical Research, Research Support Center, Juntendo University Graduate School of Medicine for technical assistance. This study was supported by a Japan Society for the Promotion of Science “KAKENHI” Grant (18K08601), Juntendo University Project Grant (No. 2021-27 and No. 2022-33), and President's Grant for Young Researchers Juntendo University (No. 2021-07). We thank Mirei Takahashi, Aki Ishizaki, and Maika Ozawa for assistance with experiments. Author contributions KS, SY, and KM designed the study. NF, SS, SN, and AY provided the conceptual advice. KS, SY, KM, SK, KA, and SN performed experiments. KS, SY, KM, and SN analyzed the data. KS, KM, GL, and AY wrote the manuscript. Data availability statement Data are available on reasonable request from the authors. Declarations Conflict of interest The authors declare no conflict of interest. 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==== Front Curr Obes Rep Curr Obes Rep Current Obesity Reports 2162-4968 Springer US New York 36451065 487 10.1007/s13679-022-00487-9 Psychological Issues (LJ Heinberg and K Goodpaster, Section Editors); An Updated Review of Night Eating Syndrome: An Under-Represented Eating Disorder http://orcid.org/0000-0002-2341-0534 Lavery Megan E. [email protected] 1 Frum-Vassallo Deirdra [email protected] 2 1 Christiana Care Bariatric Surgery Service, 501 West 14th Street, Wilmington Hospital, Gateway Building, 2nd Floor, Wilmington, DE 19801 USA 2 grid.413840.a 0000 0004 0420 1678 Northport VA Medical Center, 79 Middleville Rd, Northport, NY 11768 USA 1 12 2022 110 15 9 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose of Review Night eating syndrome (NES) is an eating disorder that has historically been under-studied. The current review aims to summarize the most up-to-date research on NES to support better awareness. Recent Findings Since NES was recently included as a formal diagnosis, research on the prevalence of NES is ever evolving. Current studies underscore the high comorbidity between NES and other eating disorders, with additional complexities for patient with comorbid eating disorders. Recent findings also support the association between NES and sleep correlates, a relationship that has remained during the COVID-19 pandemic. Emerging research confirms correlates of distress in NES across cultures. There remain mixed findings between NES and BMI. There is also debate around whether age is a risk factor. Bariatric surgery research has focused on the re-emergence of NES post-operatively. Summary Our understanding of the correlates of NES is increasing. However, research on the treatment for NES remains particularly under-studied and requires further attention. Keywords Night eating syndrome Nocturnal eating Delayed food consumption Evening hyperphagia insomnia Eating disorder ==== Body pmcIntroduction The current article aims to synthesize the growing body of research and clinical knowledge focused on night eating syndrome (NES), with particular emphasis placed on the most recent literature. Although the syndrome was first described in 1955, NES has remained an under-studied disorder [1]. Our current understanding of its complexities is ever evolving. with many correlates of NES not fully understood. It is our hope that an updated review will help providers to identify and treat this less recognized and understood disorder. Overview NES is generally classified within the eating disorder domain. It was recently included in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-V) for the first time under “other specified feeding or eating disorders [2].” NES is conceptualized as a circadian delay in food intake, evidenced by evening hyperphagia (at least 25% of daily food intake ingested after the evening meal) and/or nocturnal awakenings with food ingestion at least two times per week. According to the DSM-V criteria, patients with NES must also experience at least three of five associated features: (a) a lack of desire to eat in the morning and/or breakfast skipped four or more mornings per week; (b) a strong urge to eat between dinner and bedtime and/or during the night; (c) sleep-onset and/or sleep maintenance insomnia at least four nights per week; (d) a belief that one must eat in order to sleep; and (e) depressed or worsening mood in the evening. This constellation of symptoms must be accompanied by marked distress or impairment over a period of 3 months or more [2, 3]. For a NES diagnosis, it is important that a patient has a level of awareness with recall of nocturnal ingestions the following day. Although most patients with NES are easily able to recollect eating episodes, a distinct subset endorse decreased consciousness and seemingly automatic behavior [3]. However, even with this diminished awareness, when prompted, patients with NES should be able to remember some detail, with insight often increasing with treatment [3–5]. According to Lundgren and Pona (2017), “How do you know you have consumed food at night?” can be a key question in assessing awareness [6]. Ultimately, a minimal level of awareness is necessary for a NES diagnosis. For patients without awareness, sleep-related eating disorder (SRED) may be considered (SRED will be reviewed in an upcoming section). Due to varying criterion utilized across studies, the prevalence of NES is difficult to ascertain. Generally, the estimates of NES within the general population ranges from 1 to 2%. However, the incidence of the disorder has evidenced significant variation within clinical samples. For example, de Zwaan et al. [7] found that across studies, the prevalence of NES in pre-operative bariatric patients ranged from 6 to 64% [7]. In attempt to elucidate the prevalence of NES using only the DSM-5 diagnostic criteria, Kaur et al. [8••] reviewed the most up-to-date empirical literature, concluding that the overall prevalence of NES ranged from 2.8 to 8.2% across eating disorder, obesity, and bariatric surgery populations. Further, Kaur et al. noted that when the cut-off score on the Night Eating Questionnaire (NEQ) (an assessment tool detailed in the assessment section) was decreased from 30 to 25, the prevalence of NES within these clinical populations increased (6.9 to 15.2%) [8••]. These findings underscore the increased frequency of NES across clinical populations, suggesting some populations may be more vulnerable to developing NES. There is consensus throughout the literature that many sociodemographic factors do not differ significantly between patients with NES and controls, including gender [9, 10•], education level [11, 12], cigarette consumption, and income [13]. The association between age and NES is less clear. While several empirical investigations suggest that age is not a risk factor [9, 10•], recent research indicates that there are higher rates of NES in older individuals [12, 14]. These results are striking given evidence that NES typically first appears in young adulthood [15]. It is possible that Latzer et al. [12] findings capture amplified distress as NES symptoms sustain throughout life. In this way, age may increase the likelihood of disturbance or impairment associated with NES. Assessment Tools Several self-report tools have been developed and validated to assess for NES. The NEQ is the most frequently used scale clinically and within research. It is a 14-item questionnaire that uses a 5-point Likert scale. Items assess for symptoms consistent with a diagnosis of NES, such as morning hunger and control over nighttime eating. A total score of 25 or higher is suggestive of NES. There is a positive predictive value (PPV) of 40.7%; meaning, there is a 40.7% chance that a patient who has a positive score has NES. A score of 30 or more is an even stronger indicator of NES, with a PPV of 72.7%. The NEQ is thus able to capture many patients with NES, particularly as scores increase. The questionnaire has also demonstrated good psychometric properties, including internal consistency (Cronbach’s alpha = 0.70), fixed factor structure, and convergent validity [16]. It is limited by a lack of items measuring perceived distress and functional impairment. Two items were added to assess these factors but are not included in the final score [17••]. Overall, the NEQ is a good option to utilize when assessing patient’s risk and to track treatment progress. The NEQ and its scoring are available in the public domain. The Night Eating Symptom Scale (NESS) was developed based on the NEQ. On the NESS, each item requires a specific number to assess the frequency of awakening and nocturnal ingestions. The questions are focused on symptoms from the previous week to allow for tracking changes. This scale can be particularly useful in monitoring symptoms across time [15]. The NESS is not available in public domain. In addition, the Night Eating Diagnostic Questionnaire (NEDQ) is another paper-and-pencil option. The NEDQ asks “yes” and “no” questions designed to measure the presence of symptoms. This scale aims to classify patients into categories (non-NES verses NES). When NES is present, the questionnaire further delineates “mild,” “moderate,” and full” NES classifications [15, 17••]. The NEDQ is particularly valuable in regard to quantifying the severity of a patient’s symptoms. The scale is also available in the public domain. In addition to paper-and-pencil questionnaires, the Night Eating Syndrome History and Inventory (NESHI) is another tool used to evaluate NES symptoms. It was developed as a semi-structured interview to assess a patient’s typical 24-h food intake. The interview includes items from the NEQ while also gathering information about the frequency of nocturnal ingestions, sleep routine, mood, stress, weight and diet history, medical history, and past treatment [15, 17••]. Although one notable downside of this tool is its length, it can provide rich clinical information. It is also important to note the NESHI is unpublished and not available in the public domain. In addition to formal assessment tools, having a patient keep a food record can aid in a provider’s understanding of the clinical picture, elucidating triggers and behavioral patterns. Figure 1 provides an example of a typical food log for a patient with NES.Fig. 1 An example of a typical food log for a patient with NES NES and Other Eating Disorders When assessing for NES, it is important to consider other eating- and sleep-related disorders that can present with over-lapping symptoms. A large body of literature has specifically explored the relationship between NES and binge eating disorder (BED). While some findings have highlighted marked differences [15, 18], other research has underscored a strong association between the two disorders. In fact, between 18 and 50% of patients with BED have been found to meet criteria for NES [18–20]. Evidence also suggests that there is considerable overlap in genetic factors impacting these two disorders. For instance, one study found that the genetic association between NES and BED was 0.60 in a sample of 11,604 adult twins [21]. These findings suggest that genetic markers may increase a patients’ vulnerability to both disorders. Clinically, this underscores the importance of assessing for BED if a patient meets criteria for NES and vice versa. Given the rates of comorbidity between NES and BED, recent research has examined correlates of a NES-BED diagnosis. Emerging data indicates that patients with comorbid NES-BED experience significantly higher rates of binge eating days, as well as increased energy and fat consumption when compared to patients with BED-only [12]. In addition, patients with NES-BED may experience significantly more distress than patients with NES only. When comparing these two subpopulations, patients with comorbidity have been found to have greater state anxiety, hunger, shape/weight concerns, disinhibition, and appearance dissatisfaction [18, 20, 22]. Building upon this research, a 2020 study found that female patients with NES-BED endorsed significantly higher rates of childhood physical abuse when compared to patients with BED and BN only [14]. Taken together, these findings highlight the complicated relationship between NES and BED. It appears likely that patients who meet criteria for both disorders represent a higher risk subpopulation, which may necessitate additional intervention. Given these complexities, it is important that clinicians accurately capture a patient’s disordered eating symptoms. When assessing for NES verses BED, there are key areas to help distinguish between the disorders. More specifically, the size of the nocturnal ingestion can be a central feature separating NES and BED. While nocturnal ingestions are frequently equivalent to the size of a snack or small meal, binge eating episodes require an objectively large amount of food. Studies also highlight how the disorders can be differentiated by the level of weight/shape concern and motivation to eat, with night eating often precipitated by a desire to fall asleep verses a craving or emotional distress [23, 24]. In addition, loss of control can be an important symptom to access. While LOC is needed for a BED diagnosis, only some patients with NES experience LOC. Thus, LOC is not needed for an NES diagnosis. Table 1 further illuminates these clinical differences. Ultimately, carefully assessing the amount of food intake and factors around eating can help elucidate a diagnosis.Table 1 Comparison of night eating syndrome and binge eating disorder Criterion Night eating syndrome Binge eating disorder Amount of food consumed Often the size of a small meal or snack For a core criterion needs to be objectively large Degree of control Can range from complete control to minimal (with or without loss of control) Minimal to none; loss of control is needed Associated mood Evening dysphoria Increased depressive symptoms but not associated with time of day Associated sleep concerns Sleep-onset or maintenance insomnia common Not associated with diagnosis Motivation/precipitants Belief that one needs to eat to fall to sleep or back to sleep Variable; can include urges and emotional eating components Level of weight/shape concern Typically lower level of concern; may be associated if patient experiences weight gain Often connected with weight and body image concerns Although BES has been the most studied eating disorder in relation to NES, researchers have increasingly explored other eating disorders [25, 26•]. Tu et al. exemplifies this research. Tu and colleagues studied characteristics of NES in patients seeking eating disorder treatment. Similar to previous findings, the researchers found a high prevalence of NES in patients with BN and BED (34.9 and 51.7%, respectively). When comparing NES-only patients to BN-only patients, comparable levels of disordered eating, sleep disturbance, depression, and functional impairment were found. The investigators postulated that the strong urge to eat at night may be akin to the experience of loss of control with BN. Outside of greater eating and weight concerns, the NES-BN group evidenced no significant differences across eating pathology, psychopathology, and functional impairment domains when assessed alongside the BN-only group. These findings underscore similar psychopathology and correlates of distress for patients with NES, BN, and dual diagnoses, contrasting previous research suggesting higher levels of eating disturbance in patients with BN. Given that Tu and colleagues’ study examined patients seeking treatment, it is possible that the NES group was in more distress than the average NES patient. Ultimately, as our understanding continues to grow, it is critical that we elucidate how comorbid eating disorder diagnoses impact symptomatology, severity, and clinical outcomes. NES and Sleep Correlates Akin to its relationship with eating disorders, NES shares important over-lapping features with several sleep disorders. In particular, sleep-related eating disorder (SRED) is a parasomnia that serves as an important differential. The most critical diagnostic criteria separating NES from SRED are the nature of the nocturnal ingestions. Specifically, while NES is characterized by awareness, SRED is composed of involuntary eating episodes during sleep with impaired recall. Bruzas and Allison (2022) further note that is not uncommon for patients with SRED to consume unusual food or inedible objects [27]. Thus, there are key features that help distinguish between NES and SRED (see Table 2 for further information).Table 2 Comparison of night eating syndrome and sleep-related eating disorder Criterion Night eating syndrome Sleep-related eating disorder Volition Can be planned episodes but requires a minimal level of intent Involuntary without volition Awareness Some level of awareness around episode Complete lack of awareness Content Enjoyable or otherwise edible foods Can include odd combinations or inedible substances Motivation Triggered by hunger, a compulsion to eat or belief that eating is necessary to sleep Not associated with hunger or thirst Categorization Eating disorder; delay in circadian food intake Parasomnia In addition to SRED, an increasing body of literature has examined the relationship between NES and sleep problems. In fact, within their review of the literature, Kaur et al. [8••] contend that being a nocturnal eating disorder, NES is inherently associated with worse sleep-related outcomes [8••]. Research demonstrates that patients with NES experience higher levels of insomnia [9] and poorer sleep quality [10•, 28]. Expanding on this research, when comparing patients with NES with evening hyperphagia to patients with nocturnal ingestions, Loddo et al. found differences in sleep features across NES subgroups. The researchers observed higher levels of total duration of eating episodes, eating latency following wakening, and sleep latency following eating episodes in the evening hyperphagia group [29]. These findings contrast previous research suggesting that sleep duration, rapid eye movement, and sleep efficiency are not affected in NES [28]. It may be that sleep disturbance is heightened in patients with evening hyperphagia. Additional research is needed to continue to fully understand sleep concerns across NES subgroups. Several recent investigations have explored the association between NES and sleep during the COVID-19 pandemic. More generally, there is research demonstrating increased sleep disturbance during the COVID-19 pandemic, with incidence of insomnia noted to have increased 23% [30]. As a result, there has been concern about the potential for greater vulnerability for NES. Much of the recent research investigating sleep and NES during the pandemic has specifically examined sleep quality [31–33]. Akdevelioglu et al. exemplifies this research. The investigators found that the positive association between poor sleep quality and higher NES scores remained during the COVID-19 pandemic [31]. In addition, Kwan et al. [32] results suggest that poor sleep quality and higher BMI were risk factors for NES during the COVID-19 pandemic [32]. Given this growing body of literature, treatment targeting sleep symptoms may help to reduce risk for NES during times of chronic stress, such as the pandemic. It is important to note that the generalizability of these conclusions is limited by research conducted with university student samples. NES and Psychological Distress A large body of research has established a relationship between NES and psychiatric correlates, including increased rates of anxiety, depression, and substance abuse [34]. Research suggests stress and psychiatric symptoms often precede the onset of NES and are associated with symptom maintenance [35]. One recent study examining NES in veterans also demonstrated a significant relationship between NES and posttraumatic stress disorder [9]. Additional research has underscored the increased risk of depression for patients with subclinical and clinically significant NES [9, 12, 26•, 34, 36, 37]. Given this body of research, it is not surprising that many patients with NES present with heightened distress and comorbid psychiatric concerns. In an attempt to further elucidate the relationship between NES and depression, Riccobono et al. investigated the relationship between NES, depression, and chronotypes within a non-clinical sample [38]. Chronotype is a term used to capture behavioral and biological rhythms in relation to the timing of activity and sleep. While the morning type experiences heightened alertness and energy in the morning, the evening type evidences peak performance and attentiveness later in the day. Riccobono et al. observed a significant relationship between the evening dimension, Beck Depression Inventory (BDI) scores, and NES. These findings suggest that patients with NES may not only experience a circadian delay in food intake, but a delay in overall functioning. Further, it appears that the evening chronotype is a risk factor for depression and NES. The association between these variables may in part help to explain the effectiveness of phototherapy or bright light therapy (BLT) for NES (see “Treatment” section). To date, most of the studies examining psychological correlates of NES have been conducted in Western societies. Given the impact of sociocultural factors in eating disorders, several researchers have hypothesized that NES may present differently across cultures. For this reason, limited research has begun to explore the relationship between psychiatric correlates and NES within eastern populations [39, 40]. For example, using the Chinese-NEQ, He et al. [40] found that 2.8% of participants met criteria for NES. NES was significantly correlated with psychological distress, including depression, anxiety, and overall stress. He et al.’s findings support emerging research suggesting that the association between NES and psychiatric concerns remains across cultures. More empirical attention is needed to clarify if other clinical associations are evidenced across cultures. Body Mass Index and NES The relationship between NES and body mass index (BMI) has been the subject of long-standing interest. Although NES is associated with higher BMI in some studies [14, 32, 41], other research has found no significant relationship [9,10•,36, 42-44]. In a 2012 review, researchers postulated that these conflicting findings might be the result of NES being conceptualized differently across studies [45]. Following an updated review of the empirical literature, Bruzas and Allison concluded that the research remains split [46••]. Given this discrepancy over time, it is likely that moderating variables affect the relationship between NES and BMI. Researchers have suggested the role of several factors, including activity level, dietary restriction, psychopathology, insomnia severity, and genes [45]. There is currently some evidence to suggest that emotional eating and age may also help to explain the variability. Specifically, while a significant relationship was found between NES and weight in individuals with high emotional eating and those between ages 31 to 60, the association did not remain in individuals evidencing low emotional eating or younger/older individuals [47, 48]. Likely, there are other unmeasured or unknown variables that impact this relationship. Bariatric Patients and NES Up to 65% of patients undergoing bariatric surgery demonstrate symptoms of NES. Bariatric surgery has shown potential in reducing NES [17••]. However, treatment effects have not been found to have longitudinal value. Despite initial reduction in NES severity after metabolic and bariatric surgery, Nasirzadeh et al. [49] found that symptoms increased significantly 1 to 3 years post-surgery [49]. These results suggest that initial remission of eating pathology is often temporary and does not guarantee long-term resolution. A similar pattern has been found with depression. Studies suggest that depression often improves after surgery but recurs in later years [50]. Ultimately, NES symptoms may re-emerge as the physiological effects of the surgery become less acute. Life stressors may also contribute to the return of symptoms. Given the temporary remission of NES, recent literature has sought to understand the trajectory and impact of NES symptoms post-operatively. Pinto et al. [51] exemplify this research. The investigators sought to assess the effects of metabolic and bariatric surgery on night eating and depressive symptoms. Data suggested that reductions in depression and night eating were seen predominantly in patients with pre-operative depression [51]. These findings support hypotheses around the important role of low mood in NES. The results are striking given the improvements in depression often observed initially after surgery. Many bariatric programs require patients who meet criteria for NES to obtain treatment prior to surgery, with the rationale that untreated disordered eating could negatively impact outcomes. However, there is limited evidence that pre-surgical symptoms of NES affect weight loss following surgery [24, 49]. In fact, research suggests that although binge eating at year one is associated with lower total weight loss at 2 years post-surgery, night eating is not predictive of weight outcomes [49]. Clinically, even if NES is not related to poorer weight loss, one must question the clinical and psychological impact of the re-emergence of NES symptoms after surgery. Supporting this concern, additional research has examined the impact of disordered eating post-operatively. Specifically, Ivezaj et al. [10•] followed 131 patients who sought treatment for eating and weight concerns 6 months following the sleeve gastrectomy. Fifteen percent of the sample met criteria for NES, while another 21.4% were night eating regularly, not meeting full criteria. The researchers concluded that the co-occurrence of loss of control (LOC) eating and night eating following surgery may represent a more severe subgroup with elevated psychopathology, poorer sleep, and lower percent weight loss [10•]. These findings build upon previous research suggesting that LOC in bariatric surgery patients is associated with increased psychological burden, including greater depressive symptoms and poorer mental health-related quality of life [52]. Researchers have postulated that LOC eating could be a potential phenotype marker for NES in bariatric patients. Taken together, these findings may have practical implications when evaluating and following patients for bariatric surgery. Treatment The treatment of NES includes pharmacological and behavioral interventions. Treatments targeting the regulation of circadian rhythms, mood, stress, and faulty cognitions have been considered. Pinto et al. [53] conducted a critical review of treatment for NES and concluded that serotonergic agents and psychological interventions, particularly cognitive behavioral therapy (CBT), have shown effectiveness [52]. However, Muscatello et al. contends that there is still a paucity of research in this area, with treatment for NES remaining an emerging field [17••]. Pharmacological Treatment Studies examining pharmacological treatments largely consider drugs affecting the serotonergic system. Serotonin has a role in eating, sleep, and mood. It is therefore thought to be connected to NES. Decreased serotonin levels have been hypothesized to lead to the alteration of circadian rhythms and increase risk of evening hyperphagia [53]. Night eaters have also been found to have higher levels of serotonin transporter in the temporal lobe, which contributes to changes in circadian rhythms and appetite [54]. Antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), have been the focus of much research for NES treatment [55]. Muscatello’s literature review reports that sertraline has been found to reduce nocturnal ingestions and evening hyperphagia, improving mood, quality of life, and weight [17••]. Paroxetine and fluvoxamine have further demonstrated positive reductions in night eating episodes. Escitalopram has also been studied, but results have been contradictory, with some studies showing no effect [56] and others evidencing significant improvement of NES symptoms [57]. In addition to SSRIs, researchers have examined the off-label use of several medications. For example, topiramate, an anticonvulsant and nerve pain medication, has been studied for NES. There have been sparse reports of its benefit with short-term use, although results have underscored how side effects outweigh benefits from a short-term perspective. In 2015, Macdonald et al. conducted a case study with long-term use of low dose topiramate, with sustained results of remittance of NES symptoms and weight loss over a period of 5 years with no adverse side effects [58]. Melatonin agonists have also been studied to a lesser degree. Matsui et al. [59] conducted the most robust study to date with 49 patients included in the analysis. The investigators found that 42.9% of their participants evidenced a positive response. The researchers also found that the use of ramelteon allowed for significant reductions in benzodiazepine and Z-drugs use [59]. Taken together, these studies suggest increasing support for medications outside of SSRIs. Non-Pharmacological Treatment CBT is a well-validated treatment for depression. It has also received increasing empirical support in the treatment of a variety of behavioral health problems, including insomnia, eating disorders, and weight loss. CBT for NES integrates behavioral interventions with cognitive techniques aimed at increasing awareness during eating episodes and addressing faulty cognitions. The treatment also employs thought records, Socratic questioning, stimulus control, and relapse prevention techniques [60]. CBT has evidenced effectiveness in reducing NES symptoms, including evening caloric intake, awakenings, and nocturnal ingestions. A pilot study of a CBT protocol with tailored psychoeducation, nutrition, and sleep hygiene rules was found to be comparable to a randomized controlled trail of sertraline [17••]. Unfortunately, limited data is available on the stability of these improvement over time. Additional studies examining the use of CBT for NES with long-term follow-up are greatly needed. Research has also explored the use of relaxation techniques. Progressive muscle relaxation (PMR) has been of particular interest. This technique teaches participants to slowly tense and relax muscle groups. Although no known recent studies in the past 5 years have examined this topic, Vander Wal et al. demonstrated that PMR practice results in a reduction of depression and perceived stress. Techniques such as PMR likely help to mitigate the known relationships between stress, insomnia, and NES symptoms [61]. In addition, phototherapy or BLT has been explored as a treatment for NES. Phototherapy involves exposure to certain wavelengths of light by using a light box. Phototherapy has been applied for the treatment of mood disorders and sleep disorders. It’s effects on melatonin and post-synaptic serotonin prompted studies for NES. Though randomized controlled trails are needed, preliminary evidence supports the use of phototherapy for the treatment for NES [59]. Future Directions Despite NES being described in the 1950s, NES research is still emerging with several areas with conflicting or limited results. Our understanding of NES and the generalizability of previous findings would benefit from continuing to conduct studies across cultures. Additional research elucidating the relationship between NES and BMI is also needed. Further, it would be helpful to clarify how to assess and treat higher risk subsets of patients with NES, such as those with NES-BED and post-operative bariatric surgery patients. Perhaps most importantly, the literature analyzing treatments for NES has been particularly limited and is in dire need of more research. To more effectively treat patients, it is imperative that we continue to explore the long-term outcomes of CBT and other treatments. Conclusion It is our hope that the current review sparks clinical curiosity and needed additional research. The most recent literature underscores the complex relationships between NES and other eating and sleep disorders. Additional research is needed to fully appreciate psychiatric, sleep, and physical correlates across NES subgroups and different cultures more broadly. It is likely that subsets of the NES population, such as those with NES-BED, experience greater distress and impairment. It is important that we continue to understand differences within the NES population. Ultimately, the lack of vigorous empirical research regarding the treatment of NES remains perhaps one of the most critical holes in the research, thus limiting our clinical understanding. Declarations Conflict of Interest The authors declare no competing interestss. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors. 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==== Front Pediatr Surg Int Pediatr Surg Int Pediatric Surgery International 0179-0358 1437-9813 Springer Berlin Heidelberg Berlin/Heidelberg 36449114 5291 10.1007/s00383-022-05291-z Editorial The 35th International Symposium on Pediatric Surgical Research Puri Prem [email protected] grid.7886.1 0000 0001 0768 2743 Beacon Hospital, University College Dublin, Dublin, Ireland 30 11 2022 2023 39 1 16© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. issue-copyright-statement© Springer-Verlag GmbH Germany, part of Springer Nature 2023 ==== Body pmcDue to the global COVID-19 pandemic and resulting travel restrictions, the 33rd and 34th ISPSR were held on line. We were delighted that after a lapse of 2 years we again had the opportunity to meet face to face to attend the 35th ISPSR held at the Osaka International Conference Centre, Osaka, Japan, from 21 to 23 October, 2022. The first day of the Meeting was devoted to 15 Keynote lectures on recent advances in pediatric surgery by world experts. On the other 2 days, 102 papers on clinical and basic science research were presented. As always, the highlight of the symposium was the Prize Session. There were ten excellent papers presented at this session. The Prem Puri Basic Science Research Prize was awarded to Dr. Naho Fujiwara from Tokyo, Japan, for her presentation on “In Vitro Transplantation of Enteric Neural Crest Cells From Sox10-Venus Mouse Embryonic Stem Cells in the Gut of the Endothelin Receptor B Null Mouse Model”. The Michael Hollwarth Prize for the Best Clinical Research Paper was awarded to Dr Kazuto Suda, also from Tokyo, Japan, for his presentation on “Successful Engraftment of Bladder Organoids in De-Epitheliallized Mouse Colon”. This year we received 60 manuscripts for consideration for publication in the Pediatric Surgery International. This is the highest number of manuscripts ever submitted from any of the ISPSR Meetings. The Publication Committee selected 24 manuscripts for publication. The ISPSR 2022 in Osaka was a highly successful meeting, both scientifically and socially. The International Board of Pediatric Surgical Research thanks Professor Takashi Doi, Dr. Hiroki Nakamura and the entire team from the Department of Pediatric Surgery, Kansai Medical University for organising the most memorable ISPSR meeting. Prem Puri On behalf of the Scientific Committee of the International Board of Pediatric Surgical Research. Data availability Author declares that the data supporting the findings of this study are available within the article. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.	0	PMC9713093	NO-CC CODE	2022-12-02 23:22:07	no	Pediatr Surg Int. 2023 Nov 30; 39(1):16	utf-8	Pediatr Surg Int	2,022	10.1007/s00383-022-05291-z	oa_other
==== Front Mark Lett Mark Lett Marketing Letters 0923-0645 1573-059X Springer US New York 9657 10.1007/s11002-022-09657-0 Article How uncertainty affects information search among consumers: a curvilinear perspective http://orcid.org/0000-0003-0101-0393 He Sharlene [email protected] 1 Rucker Derek D. [email protected] 2 1 grid.410319.e 0000 0004 1936 8630 John Molson School of Business, Concordia University, Montreal, QC Canada 2 grid.16753.36 0000 0001 2299 3507 Kellogg School of Management, Northwestern University, Evanston, IL USA 1 12 2022 114 7 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Abstract Uncertainty is an inherent part of consumers’ environment. A large literature in marketing and related disciplines has found a positive relationship between uncertainty and information search: as consumers’ uncertainty about a brand, product, or service increases, so does their inclination to seek out and engage with information. In contrast to this conventional view, the present research proposes and demonstrates a curvilinear (inverted-U) relationship between uncertainty and information search. Conceptually, we put forth theoretical insight for this relationship: uncertainty increases both accuracy and efficiency considerations, presenting an inherent tradeoff. This tradeoff is perceived to be more favorable at moderate levels of uncertainty relative to low and high levels. Empirically, we observe an inverted-U relationship between uncertainty and information search across three experiments and find evidence consistent with our theorizing. This research suggests that the conventional view is incomplete and points to the importance of exploring uncertainty at multiple levels. Supplementary Information The online version contains supplementary material available at 10.1007/s11002-022-09657-0. Keywords Uncertainty Information search Accuracy Efficiency Curvilinear relationship http://dx.doi.org/10.13039/100007059 Northwestern University http://dx.doi.org/10.13039/501100002914 Concordia University ==== Body pmcIntroduction Uncertainty is an inherent part of consumers’ environment. Consumers experience uncertainty about many things, including products and services, topical issues, and other people. One adaptive function of uncertainty is to provide a signal that one should search for more information. For example, uncertainty about a product may encourage consumers to obtain more information via online reviews, websites, or friends. A substantial literature has documented that as consumers’ uncertainty about a stimulus increases, so does their tendency to search for and process information pertaining to that stimulus, i.e., a positive relationship (e.g., Grant & Tybout, 2008; Heslin et al., 1972; Lanzetta & Driscoll, 1968; Maheswaran & Chaiken, 1991). Indeed, this relationship has been found in the domains of marketing (e.g., Murray, 1991; Sun et al., 2012), economics (e.g., Kohn & Shavell, 1974; Mele & Sangiorgi 2010), communications (e.g., Bradac, 2001; Kuhlthau, 1993; Wilson, 1999), and psychology (Pelham & Wachsmuth, 1995; Weary & Jacobson, 1997). We suggest the conventional view—uncertainty exerts a positive effect on consumers’ information search—reflects a useful but incomplete representation of how consumers respond to uncertainty. We advance the hypothesis that the relationship between uncertainty and information search can be curvilinear—specifically, an inverted-U form. Conceptual development Conventional view: uncertainty increases information seeking The experience of uncertainty has been defined as the subjective assessment of a gap in one’s information or understanding of a stimulus (Clore & Parrott, 1994; Kagan, 1972; Milliken, 1987). The reduction of uncertainty is viewed as a critical driver of human behavior (Berlyne, 1960; Inglis, 2000; Kagan, 1972). Since uncertainty arises from a perceived gap in information or understanding, acquiring information is a natural means to reduce uncertainty. For example, in past research, consumers who were more uncertain about whether they could predict another person’s behavior sought more information about that person (Weary & Jacobson, 1997). Similarly, consumers who were more uncertain about a disease were more inclined to seek information (Rosen & Knäuper, 2009). Indeed, the idea that uncertainty increases consumers’ desire to seek out or engage with information has been used to explain diverse phenomena from how people evaluate new products (Grant & Tybout, 2008) to how emotions affect information processing (Tiedens & Linton, 2001). Based on these and other findings (e.g., Desender et al., 2018; Heslin et al., 1972; Maheswaran & Chaiken, 1991; Pelham & Wachsmuth, 1995; Sun et al., 2012; Tormala et al., 2012), one might conclude that uncertainty governs consumers’ information search in a simple fashion: consumers are more prone to engage in search as uncertainty increases. This positive relationship can be explained by the idea that consumers’ uncertainty provides an internal signal that additional information will be useful (Chaiken et al., 1989; Weary & Jacobson, 1997). Unexplained findings The idea of a positive relationship is parsimonious, intuitive, and consistent with the vast majority of prior findings in the literature. Yet, such a conclusion overlooks a handful of findings that suggest uncertainty can reduce information search—i.e., a negative relationship. For example, Urbany et al. (1989) found that consumers with greater uncertainty about product offerings were less inclined to seek information. Elsewhere, for decision problems high in uncertainty, further uncertainty appeared to negatively correlate with information search (Driscoll & Lanzetta, 1964). While intriguing, prior research has not put forth an integrative solution for these findings against the larger literature documenting a positive relationship between uncertainty and information search. Existing research is also marked by a critical empirical limitation. Namely, experimental manipulations of uncertainty have usually focused on two levels of uncertainty—lower versus higher (e.g., Grant & Tybout, 2008; Heslin et al., 1972; Maheswaran & Chaiken, 1991; Rosen & Knäuper, 2009; Sun et al., 2012; Tormala et al., 2012; Weary & Jacobson, 1997). With two levels of uncertainty, it was only possible to test for a linear effect of uncertainty on participants’ desire for information (i.e., positive, negative, or null). Theorizing and findings of the conventional positive relationship may thus be predicated on this empirical limitation. Of interest, some prior research has found evidence of an inverted-U relationship between a related construct—prior knowledge—and the extent of information search or processing (Bettman & Park, 1980; Johnson & Russo, 1984; Moorthy et al., 1997; but see Brucks, 1985; Punj & Staelin, 1983). While this finding provides initial reason to believe that uncertainty may exert an inverted-U effect on search, the present research is distinct in two important respects. First, uncertainty is distinct from knowledge (Alba & Hutchinson, 1987; Rheingold, 1985). A consumer with low knowledge may nevertheless feel highly certain (Dunning, 2011; Fischhoff et al., 1977). Second, and equally important, we put forth a novel theoretical account. The intuition for the curvilinear effect of knowledge is that knowledge increases both the ability to process more information (increasing search) and the ability to search in a more effective manner (decreasing search). In contrast to the ability-centric explanation for knowledge, our theoretical account for the effect of uncertainty involves judgment goals. We present this theorizing next. Conceptual framework We propose that the relationship between uncertainty and information search may often be curvilinear across the uncertainty continuum. Our proposition rests on the assumption that consumers strive to strike a balance between two common judgment goals—accuracy and efficiency (e.g., Bettman, Luce, and Payne 1998; Forster et al., 2003; Payne et al., 1988). An accuracy goal refers to a desire to reach a judgment or decision that is valid and correct. An efficiency goal refers to a desire to be economical with one’s time and energy in making the judgment or decision. Consumers often seek to allocate their time and energy in a manner that offers the greatest value (Fiske & Taylor, 1991; Higgins & Bargh, 1987; Sherman et al., 1989)—that is, to balance acceptable accuracy with reasonable efficiency. We propose that uncertainty itself impacts consumers’ perceptions of accuracy versus efficiency considerations in information search. Because uncertainty arises from a perceived gap in information or understanding, higher uncertainty about a stimulus implies to the consumer that more information is needed to achieve accuracy in their judgment about the stimulus. At the same time, seeking and processing this additional information would require further time and effort, thus hindering efficiency. In other words, rising uncertainty increases both the accuracy and efficiency considerations of information search, presenting an inherent tradeoff—accuracy considerations create a driving force for search while efficiency considerations create a deterring force (e.g., Bettman, 1979; Schmidt & Spreng, 1996). The tradeoff between these competing forces shapes and helps to explain the relationship between uncertainty and information search. We hypothesize that as uncertainty rises from lower to moderate levels, the driving force predominates. At lower levels of uncertainty, the tradeoff favors accuracy because additional information is perceived to substantially increase accuracy while efficiency concerns remain tolerable. Thus, initially, a rise in uncertainty causes information search to increase. However, as uncertainty rises further from moderate to higher levels, the deterring force predominates. At higher levels of uncertainty, the tradeoff favors efficiency because additional gains in accuracy are perceived to come at the cost of substantial decrements in efficiency. Thus, a further rise in uncertainty causes information search to decrease. This theorizing predicts that search peaks at moderate levels of uncertainty. Put simply, moderate uncertainty signals a favorable balance between accuracy and efficiency, increasing the value of search. If our hypotheses are true, then the relationship between uncertainty and information search would take on an inverted-U form. Or, put differently, the conventional positive relationship between uncertainty and information search would emerge from low to moderate uncertainty, but turn negative at higher levels of uncertainty, ultimately producing a curvilinear relationship across the uncertainty continuum. Empirical overview We report three experiments which investigate the inverted-U relationship in different domains of judgment. Study 1 provides an initial demonstration of this relationship in a product purchase context. Study 2 assesses natural variation in consumers’ uncertainty towards mask-wearing during the COVID-19 pandemic and examines their information search behavior. Lastly, study 3 examines the proposed mechanism by testing whether a perceived accuracy-efficiency tradeoff mediates the inverted-U relationship. Sample sizes were determined prior to data collection and were based on a consideration of study design, collection method, and participant availability. Data from each study were analyzed only once data collection was complete. Any additional measures and analyses are reported in the Supplemental Appendix. Data are available at https://osf.io/xpbj2/?view_only=003050cdf25247f4a993ab3b9ca5db98. Study 1 Prior research, which usually operationalized uncertainty at only two levels, could not test a curvilinear relationship. As such, study 1 manipulated uncertainty at three levels to test whether an inverted-U relationship would emerge. Method One hundred fifty participants on Amazon Mechanical Turk completed this study (97 females, Mage = 35.35). Participants were randomly assigned to one of three between-participant conditions: low, moderate, or high uncertainty. We manipulated participants’ sense of uncertainty about a product or service based on a procedure adapted from past research (Cheatham & Tormala, 2017). This task involved participants writing about a potential gift (a product or service) about which they felt “very certain” (low uncertainty), “somewhat, but not totally, certain” (moderate uncertainty), or “very uncertain” (high uncertainty) (see Supplemental Appendix for the complete verbatim instructions). Next, participants read a scenario in which they were deciding among gift options. Participants were further informed that they had some time, but not a lot of time, to purchase a gift. Thus, the scenario was designed to encourage participants to allocate their resources strategically, as they often would in everyday life. Participants then completed two measures assessing their intention to search for information about the product or service they had described in the writing task: the extent to which they would want to find out more information about the option they described (1 = not at all; 7 = very much so), and how likely they were to seek more information about the option (1 = not likely at all; 7 = very likely), r = 0.86. The expectation was that participants’ uncertainty regarding the product or service they described should affect the extent to which they were willing to search for information about this option. Next, as a manipulation check, four items assessed participants’ uncertainty about the product or service they described (1 = very uncertain, 7 = very certain; see Supplemental Appendix for items), α = 0.88. Lastly, two items assessed participants’ evaluation of the product or service (1 = very negative; 7 = very positive; 1 = very unfavorable, 7 = very favorable), r = 0.76. Results and discussion We analyzed the data using one-way ANOVA. Manipulation check The manipulation had a significant overall effect, F(2, 147) = 76.68, p < 0.0001, η2 = 0.51. With lower values representing higher uncertainty, participants were more uncertain in the moderate uncertainty condition (M = 4.81, SD = 1.18) compared to the low uncertainty condition (M = 6.22, SD = 0.75), t(101) = 6.60, p < 0.001, d = 1.43, and more uncertain in the high uncertainty condition (M = 3.54, SD = 1.28) than in the moderate uncertainty condition, t(95) = 5.78, p < 0.001, d = 1.04. Information search A trend analysis testing for an inverted-U effect was significant, F(1, 147) = 22.44, p < 0.0001. Individual contrasts revealed that intentions to search increased from low (M = 3.99, SD = 1.92) to moderate levels of uncertainty (M = 5.87, SD = 1.16; t(101) = 5.90, p < 0.001, d = 1.18), then declined from moderate to high uncertainty (M = 5.10, SD = 1.65; t(95) = 2.36, p = 0.02, d = 0.55) (see Fig. 1). For robustness, we also ran an ANCOVA in which participants’ evaluation of the product or service was included as a covariate. The effect of this covariate was nonsignificant, F(1, 146) = 0.04, p = 0.844, ηp2 = 0, and did not affect the significance of the trend analysis or the individual contrasts.Fig. 1 The curvilinear effect of uncertainty on information search (study 1). Note: The error bars represent the standard error of each mean Study 1 offered evidence that uncertainty can have a curvilinear effect on information search. While the established positive effect was observed from low to moderate levels of uncertainty, the relationship reversed and became negative at a higher level of uncertainty. Study 2 Study 2 measured natural variation in consumers’ uncertainty towards wearing a mask during the COVID-19 pandemic and examined its effect on the search for information about mask-wearing. Method One hundred forty-one undergraduate participants at a large Canadian university completed this study as part of online lab sessions in June 2020 (83 females, Mage = 22.58). Participants responded to three items assessing their uncertainty about wearing a mask, specifically how uncertain they were about (1) the effect of wearing a mask for COVID-19 prevention, (2) public health recommendations for wearing a mask during the COVID-19 pandemic, and (3) whether they were going to wear a mask during the COVID-19 pandemic (1 = not uncertain at all; 7 = very uncertain), α = 0.92. Next, participants were allowed to select up to six pieces of information to read about different aspects of mask-wearing, including the science of masks, the impact of masks, the appropriate use of masks, and when to wear a mask. The number of items selected served as the dependent variable. Participants then viewed their chosen information items on the next screens. Finally, participants completed measures of their attitude towards mask-wearing and behavioral intentions to wear a mask. Results Twenty-five participants failed the attention check for the online lab session (see Supplemental Appendix), and their responses were excluded from the analysis. To test for an inverted-U relationship between uncertainty and information seeking, we analyzed the data using the two-line test recommended by Simonsohn (2018). This method estimates two regression lines, one for lower values of uncertainty and one for higher values of uncertainty, and tests whether the slopes of the two lines are opposite in sign. An inverted-U relationship would be indicated by a positive slope for the first line (lower values of uncertainty positively predict information seeking) and a negative slope for the second line (higher values of uncertainty negatively predict information seeking). We found a positive effect of uncertainty for the first line (b = 0.81, z = 2.31, p = 0.021) and a negative effect for the second line (b = − 0.38, z = 2.32, p = 0.021). Thus, an inverted-U relationship emerged (see Fig. 2).Fig. 2 Uncertainty about mask-wearing had a curvilinear effect on information search (study 2) Increased information seeking had a positive effect on attitudes and behavioral intentions to wear a mask. These results are reported in the Supplemental Appendix. Study 2 offered additional evidence for a curvilinear relationship between uncertainty and information search. Study 3 Our theoretical account proposes that, as uncertainty increases, more information search should be perceived to help enhance accuracy but also to hinder efficiency. The tradeoff between these two competing considerations should be viewed as relatively optimal at a moderate level of uncertainty, where acquiring information offers a satisfactory gain in accuracy for an acceptable amount of efficiency. To test this mechanism, this experiment presented participants with three alternatives that varied in uncertainty (low, moderate, and high). For each alternative (i.e., each level of uncertainty), we measured participants’ perceptions of the tradeoff between accuracy and efficiency and tested whether this perceived tradeoff mediated the curvilinear effect of uncertainty on information search. Method This pre-registered experiment manipulated uncertainty within participants (preregistration available at https://aspredicted.org/bb6q3.pdf). All participants were presented with three alternatives, each associated with a different level of uncertainty (low, moderate, and high). One hundred twenty participants completed this study online through the Prolific panel (75 females, mean age = 32.67). Participants read a scenario involving buying a vacuum cleaner. They were shown a set of three alternatives, each accompanied by a photo, the average consumer rating, and some text. The average rating was the same for all three alternatives (4 stars), thus holding evaluative information constant. The degree of uncertainty associated with each alternative was varied based on the amount and source (first- or second-hand) of participants’ existing information about the alternative (Fazio & Zanna, 1981). Specifically, for the low uncertainty alternative, participants were told that it was made by a brand whose vacuum cleaners they had used in the past. For the moderate uncertainty alternative, participants were told that they had not used the brand before but their friend owned this vacuum cleaner. Lastly, for the high uncertainty alternative, participants were told that they had not used the brand before and their friends could not tell them much about it either. This manipulation was pretested and found to vary uncertainty as expected1 (see Supplementary Appendix for stimuli and pretest results). To assess information search, participants rated how likely they would be to learn more about each alternative (1 = very unlikely, 7 = very likely). Next, to assess participants’ tradeoff between efficiency and accuracy, they rated the extent to which it would be worthwhile to spend time getting a more accurate impression of each alternative (1 = not at all, 7 = very much so). Lastly, as a manipulation check, participants rated how certain they were about how the product would perform (1 = very uncertain, 7 = very certain). Results and discussion The data were analyzed using repeated measures ANOVA. Manipulation check The uncertainty manipulation had a significant overall effect, F(2, 238) = 279.64, p < 0.0001, ηp2 = 0.70. With lower values indicating higher uncertainty, participants were more uncertain about the moderate uncertainty alternative (M = 4.92, SD = 1.06) than the low uncertainty alternative (M = 6.11, SD = 0.86), t(119) = 8.68, p < 0.001, d = 1.24. They were also more uncertain about the high uncertainty (M = 2.88, SD = 1.47) relative to the moderate uncertainty alternative, t(119) = 14.71, p < 0.001, d = 1.59. Information search A trend analysis to test for an inverted-U effect was significant, F(1, 238) = 34.54, p < 0.0001. Individual contrasts revealed that participants were more likely to engage in information search for the alternative they were moderately uncertain about (M = 5.46, SD = 1.28) than the one they had low uncertainty about (M = 4.88, SD = 1.96), t(119) = 2.66, p < 0.001, d = 0.35. By contrast, they were less likely to seek information for the alternative they were highly uncertain about (M = 3.81, SD = 1.83) compared to the one they were moderately uncertain about (M = 5.46), t(119) = 7.52, p < 0.001, d = 1.05. Mediation analysis Participants’ tradeoff perceptions were more favorable for the moderate-uncertainty alternative (M = 5.49, SD = 1.35) than for the low-uncertainty (M = 4.51, SD = 2.05; t(119) = 4.67, p < 0.001, d = .57) and high-uncertainty alternatives (M = 4.31, SD = 1.68; t(119) = 5.58, p < 0.001, d = .77). We performed a multilevel mediation analysis with the tradeoff measure as a mediator of the inverted-U effect. Uncertainty (coded as a quadratic contrast term) positively predicted tradeoff perceptions, and tradeoff perceptions positively predicted search (see Fig. 3). The indirect effect was significant (bootstrapped: b = 0.24, z = 6.03, p < .001, C.I.[0.16, 0.32]). The inverted-U effect of uncertainty on search decreased in magnitude when the mediator was included (β = 0.13, z = 2.54, p = .011) compared to when the mediator was not included (β = 0.37, z = 5.66, p < .001). Conditional on the assumptions of our mediation model, our statistical test showed that tradeoff perceptions can account for a significant portion of variance.Fig. 3 Perceptions of an accuracy-efficiency tradeoff mediated the inverted-U effect of uncertainty on information search (study 3) General discussion Substantial prior research has found that uncertainty increases information search. The present work builds on this foundation by offering an alternative perspective. While a positive relationship is observed when examining low to moderate levels of uncertainty, the relationship can become negative between moderate to high levels of uncertainty, thus producing a curvilinear relationship across the uncertainty continuum. We suggest the relationship between uncertainty and search is shaped by a tradeoff between accuracy and efficiency, which varies across different levels of uncertainty. This research can offer insight about why consumers may not seek information in some situations involving uncertainty. For example, marketers may want consumers to seek information about new products, and policy makers may often want consumers to be well-informed in domains such as health (e.g., a new vaccine) or personal finance (e.g., investing for retirement). The present work suggests consumers may search less if they are highly uncertain about the product or issue at hand. Rather, an “optimal” level of uncertainty—determined based on the relative importance of accuracy and efficiency considerations in a given situation—may be most effective in motivating search. This research also highlights the importance of operationalizing uncertainty at multiple levels. If researchers only operationalize uncertainty at two levels, its effects may depend on whether “low uncertainty” in the study is actually low or moderate and whether “high uncertainty” is actually moderate or high. Indeed, the present findings suggest that much of prior research may have unknowingly operationalized low and moderate certainty, respectively. This reveals the importance of examining multiple levels of uncertainty to understand its effects more fully. This work contributes to a broader literature on the antecedents of consumer information search, which include factors such as involvement, need for justification, and product characteristics, among others (Beatty & Smith, 1987; Moore & Lehmann, 1980; Punj & Staelin, 1983; Schmidt & Spreng, 1996; Srinivasan & Ratchford, 1991). In fact, some of these factors may shape the relationship between uncertainty and information search by affecting the perceived accuracy-efficiency tradeoff. For example, high involvement may increase the importance of accuracy in the tradeoff, which may lead the inflection point of the curvilinear effect to occur at a higher level of uncertainty. We also note the proposed accuracy-efficiency tradeoff mechanism has theoretical roots in the benefit–cost perspective of search (e.g., Bettman, 1979). A novel insight of the present research is this tradeoff varies across different levels of uncertainty, which helps to explain why uncertainty has a curvilinear effect on search. Although this work focused on efficiency concerns as a driver of the downturn in search at high uncertainty, we recognize other factors might drive this downturn. For example, high uncertainty may be associated with stronger negative emotions (Bar-Anan et al., 2009), which could discourage search in some situations. A fruitful avenue for future research is to explore additional mechanisms that may underlie the relationship between uncertainty and search. In sum, while a great deal of research has found that uncertainty increases information search, the present research suggests that the effect of uncertainty on search is more complex than previously understood. We hope this research challenges scholars to engage in greater exploration of the important link between uncertainty and information search. Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 170 KB) Funding This work was supported by funding from the Kellogg School of Management and the John Molson School of Business. Declarations This research was conducted in accordance with the APA’s guidelines on the ethical treatment of human subjects. The data in this manuscript have not been published elsewhere, and the manuscript is not under consideration for publication elsewhere. All authors have seen and approved the manuscript. 1 A potential limitation of this manipulation, as pointed out by an anonymous reviewer, is that it contains social information. As a result, while clearly varying uncertainty, it is possible that the manipulation may have also varied additional factors. 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==== Front J Nephrol J Nephrol Journal of Nephrology 1121-8428 1724-6059 Springer International Publishing Cham 36450998 1523 10.1007/s40620-022-01523-6 From Distant Places COVID-19 disease in peritoneal dialysis patients: a single centre experience from India Lakshmi P. Aiswharya Christopher Latha Margarate Hemalatha N. Fahima S. Sunnesh A. Mathini S. Kumar N. Prasanna Rao G. Vishwaeswar Amarendra M. Raja Naveen K. Bhatt G. Gayathri Srilakshmi G. Manuel Maria Bethasida Alekhya B. Virali G. Yagnapriya C. Sindhu M. Pravallika K. Ram R. [email protected] Kumar V. Siva Vengamma B. grid.416288.1 0000 0004 1767 3463 Sri Padmavathi Medical College Hospital, Sri Venkateswara Institute of Medical Sciences University, Tirupati, India 30 11 2022 14 1 8 2022 29 10 2022 © The Author(s) under exclusive licence to Italian Society of Nephrology 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Background This study presents our data on mortality in end stage renal disease (ESRD) patients on peritoneal dialysis (PD) who developed COVID-19. Materials and methods Sri Padmavathi Medical College Hospital, Sri Venkateswara Institute of Medical Sciences University, was designated the State COVID Hospital in March 2020. In a retrospective observational study, we collected the data of ESRD patients on PD and identified the risk factors for mortality. Results Prior to the pandemic, 136 patients with ESRD were on peritoneal dialysis at our Institute. Among them, 27 (19.8%) eventually developed COVID-19, and 14 of them (51.8%) died. Serum albumin levels were lower and D-dimer levels were significantly higher in deceased patients than in survivors. Discussion The mortality rate in ESRD patients on PD with COVID-19 at our institution was higher than in other published studies. Supplementary Information The online version contains supplementary material available at 10.1007/s40620-022-01523-6. Keywords End stage renal disease Peritoneal dialysis SARS-CoV-2 COVID-19 Oxygen saturation Non-invasive ventilation Serum albumin D-dimer Mortality ==== Body pmcIntroduction On 28 March, 2020, Sri Padmavathi Medical College Hospital, Sri Venkateswara Institute of Medical Sciences University, was designated the State COVID Hospital for the state of Andhra Pradesh, India, the population of which is approximately fifty million people. The aim of this article is to report our experience with end-stage renal disease (ESRD) patients on peritoneal dialysis (PD) with COVID-19. Materials and methods We collected data concerning ESRD patients on peritoneal dialysis consecutively presenting to our hospital between March 2020 and December 2021 in a register. Based on the information in this register, data were retrieved from the patients' files and a retrospective observational study was carried out. We included demographic, clinical, and laboratory data, as well as treatment schedules of peritoneal dialysis and COVID-19. For the purpose of this study, we used the following definitions; (1) non-invasive ventilation at admission (NIVa) for patients requiring non-invasive ventilation at admission, (2) non-invasive ventilation during hospitalization (NIVh) for patients requiring non-invasive ventilation at any time during hospitalization, (3) oxygen-dependent for patients requiring oxygen via non-rebreather mask or simple mask. None of the patients in the latter group required NIV during hospitalization. At admission, we initiated patients on remdisivir 200 mg on day 1, followed by 100 mg/day from day 2 to day 5, which was extended up to day 10 as per the clinical condition, dexamethasone 0.1 mg/kg/day i.v., and low molecular weight heparin till discharge. All peritoneal dialysis inpatients were treated with automated peritoneal dialysis (APD). Patients who were in home isolation were managed with an APD machine that had the advantage of remote patient monitoring (RPM). Statistics The data were entered into a Microsoft Excel spreadsheet. The frequency and percentage were calculated for qualitative variables. The mean and standard deviations were calculated for quantitative variables. An independent sample t test was used to test the significant difference between the two means. A Chi-square test was used to test the significant difference between proportions. An odds ratio with 95% confidence intervals was calculated. The IBM SPSS 26th version was used. A P value of less than 0.05 was considered statistically significant. We obtained clearance from the institutional ethics committee, number 1249. Results With regard to all COVID-19 patients, we admitted and managed 15,719 COVID-19 patients from March 28, 2020 to December 31, 2021.The mortality rate was 18.3% (2878 deaths out of 15,719 patients). Concerning demographics and admission, prior to the pandemic, 137 patients with ESRD were on peritoneal dialysis at our Institute, of whom 27 (19.7%) eventually developed COVID-19. From March 2020 to March 2021, we treated 10 ESRD patients on peritoneal dialysis with COVID-19, while from March 1, 2021, to December 31, 2021, we admitted 17 ESRD patients on peritoneal dialysis with COVID-19. Seventeen (62.9%) patients were admitted to hospital as inpatients, while home isolation was adopted in 10 (37.0%) subjects. Patients ranged in age from were 17–80 years, and 17 were males (62.9%). Aetiology of end stage renal disease varied. Diabetes mellitus was the cause of ESRD in eight (29.6%) of the 27 patients, hypertension in 11 (40.7%), and other aetiologies were present in the remaining eight (29.6%). Hypertension was identified as the cause of ESRD in 11 (40.7%) patients. However, 17 (62.9%) patients had hypertension at the time of admission or during their hospital stay. The mean systolic and diastolic blood pressures at admission were 141.8 and 85.5 mmHg. Of 17 patients who were admitted to the institution, NIVa was needed by three (17.6%) patients, eight (47.0%) patients required oxygen, while the remaining six (35.2%) patients did not require oxygen during their hospital stay. However, nine out of 17 (52.9%) patients required a NIVh. This group included patients who were switched from oxygen to NIV or who were admitted without requiring oxygen. Peritoneal dialysis was performed on 17 inpatients using APD. Patients were given combinations of 5.0 L solutions of 1.5% and 2.5% dextrose exchanges over 15 h/day. APD machine management and patient connections were carried out by peritoneal dialysis nurses. The mean ultrafiltration rate in inpatients was 996 ± 227 mL/day and the mean urine output was 258 ± 151 mL/day. The 10 patients who were in home isolation were managed with an APD machine that had the option of RPM. Patients connected and disconnected themselves from the APD machines at home. RPM was done by the peritoneal dialysis nurse at our Institute. The mean ultrafiltration rate in patients at home was 818 ± 203 mL/day and the mean urine output was 388 ± 111 mL/day. The demographic, clinical, and laboratory values are provided in Supplementary Table 1. Comparison between clinical and laboratory markers (Table 1) identified serum albumin and D-dimer levels as being significantly higher in deceased patients than in survivors on peritoneal dialysis with COVID-19. We did not observe any adverse effects of remdesivir.Table 1 Comparison between clinical and laboratory markers between deceased s and surviving hospitalised COVID-19 patients Variable Non-survivors (n = 11) Mean (SD) Survivors (n = 6) Mean (SD) P value Duration of staya 15.7 (16.6) 16.6 (11.3) 0.305 Age 50.7 (18.5) 40.8 (14.6) 0.277 SPO2 at admission 76.6 (37.9) 76.5 (37.6) 0.994 Haemoglobinb 8.9 (2.4) 9.2 (2.7) 0.856 Total leucocyte countb 9054.5 (5617.7) 8280.0 (4975.6) 0.796 Neutrophilsb 80.9 (11.5) 76.0 (11.4) 0.440 Lymphocytesb 11.5 (6.1) 19.4 (10.7) 0.078 ESRb 82.1 (32.9) 51.6 (43.2) 0.141 Platelet countb 1.8 (0.8) 1.9 (0.7) 0.881 Blood ureab 124.5 (35.3) 107.8 (45.9) 0.435 Serum creatinineb 9.8 (3.3) 8.2 (2.1) 0.357 Serum potassiumb 4.5 (0.9) 4.3 (1.2) 0.680 Serum Sodiumb 136.0 (7.1) 137.2 (2.3) 0.722 Serum bilirubinb 0.6 (0.3) 0.5 (0.1) 0.419 SGOTb 36.0 (33.9) 58.0 (52.9) 0.328 SGPTb 33.4 (45.7) 30.4 (7.7) 0.889 ALPb 88.9 (41.2) 116.0 (58.5) 0.302 Serum total proteina 4.5 (2.4) 6.1 (0.4) 0.200 Serum albuminb 2.2 (1.1) 3.2 (0.4) 0.030 CRPb 44.3 (66.6) 97.8 (89.9) 0.201 Procalcitoninb 0.2 (0.4) 08 (1.4) 0.137 Serum ferritinb 314.4 (495.8) 211.0 (307.9) 0.677 D-dimerb 0 (0) 0.2 (0.3) 0.024 Serum IL-6b 0 (0) 42.0 (93.9) 0.143 LDHb 129.9 (258.6) 380.0 (510.6) 0.287 SPO2 oxygen saturation of blood, AST aspartate aminotransferase, SGOT serum glutamic-oxaloacetic transaminase, ALT alanine transaminase, SGPT serum glutamic-pyruvic transaminase, IL-6 interleukin-6, LDH lactate dehydrogenase, ESR erythrocyte sedimentation rate aNumber of non-survivors = 10 bNumber of survivors = 5 Fourteen out of 27 (51.8%) ESRD patients on peritoneal dialysis with COVID-19 died. There were 11 (64.7%) deaths among the 17 inpatients, and three (30%) deaths among the 10 patients in home isolation. Two, four and five deaths occurred in NIVa, oxygen at admission and in non-oxygen-dependent patients, respectively. There were no significant differences in mortality rates among these three groups. Supplementary table 2 shows the characteristics of ESRD patients on peritoneal dialysis between March 2020 and December 2021 according to infection status. Mortality rates of PD patients who were not affected by COVID-19 were significantly lower than in those affected by COVID-19. Discussion With the understanding that the outcomes of the general population and of patients admitted to hospital are not comparable, we state that at our Institute, the number of deaths reported was 2878 (18.3%) of the 15,719 COVID-19 admitted patients. The reported mortality worldwide, in India and in the state of Andhra Pradesh was 1.9% [1], 1.38% [2], and 0.64% [3] respectively. The mortality rate in ESRD patients on maintenance haemodialysis (MHD) with COVID-19 at our Institute was 34.1% (203 deaths out of 595 patients). Fourteen out of 27 (51.8%) ESRD patients on peritoneal dialysis died. We observed that serum albumin and D-dimer levels were significantly higher in patients who eventually died than in survivors. Ten of our peritoneal dialysis patients (37.0%) were in home isolation after initial consultation at the Institute. Important investigations, including chest radiograph, were carried out during the initial consultation. These patients were taught to recognise hypoxia by means of pulse oximeter and were also trained to take a 6-min walk test. They were already well trained in the technique of PD. In addition, these patients were under RPM. We had three deaths in this home isolation group, all of whom were transferred to the hospital before they died. We must confess that the Institute failed in its careful monitoring of these patients. This could partly explain the higher mortality rate of the peritoneal dialysis patients than haemodialysis patients at our institute and also as compared to other publications. The earliest report of COVID-19 in ESRD patients on peritoneal dialysis came from none other than Wuhan [1, 4]. Supplementary Table 3 contains data from published studies [2–9]. This study has some limitations: first of all, we could not compare COVID-19 patients on peritoneal dialysis to those on MHD. We also could not compare COVID-19 patients on dialysis to those not on dialysis. Moreover, ours is a retrospective, single centre study with a small sample size. Finally, we were not able to obtain the vaccination status of patients, and its modulating effect remains unknown. Our work underlines the fragility of peritoneal dialysis patients and the need to protect them from severe forms of SARS-CoV-2 infection since they are affected by a higher mortality rates related to COVID-19. Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 17 kb) Funding None. Declarations Conflict of interest The authors have declared that no conflict of interest exists. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study. Compliance with ethical standards Yes. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. https://covid19.who.int/?mapFilter=cases 2. https://www.mohfw.gov.in/ 3. http://hmfw.ap.gov.in/covid_dashboard.aspx/ 4. Jiang HJ Tang H Xiong F Chen WL Tian JB Sun J Dong JW Wang XH Jin XF Ding YQ Xu L Miao XP Zhang C COVID-19 in peritoneal dialysis patients Clin J Am SocNephrol 2020 16 1 121 123 10.2215/CJN.07200520 5. Sachdeva M Uppal NN Hirsch JS Ng JH Malieckal D Fishbane S Jhaveri KD Northwell COVID-19 Research Consortium and the Northwell Nephrology COVID-19 Research Consortium COVID-19 in hospitalized patients on chronic peritoneal dialysis: a case series Am J Nephrol 2020 51 8 669 674 10.1159/000510259 32731215 6. Kazancıoğlu R Öztürk Ş Turgutalp K COVID-19 infection in peritoneal dialysis patients: a comparative outcome study with patients on hemodialysis and patients without kidney disease Turk J Nephrol 2022 31 1 33 42 10.5152/turkjnephrol.2021.21045 7. Ronco C Manani SM Giuliani A Tantillo I Reis T Brown EA Remote patient management of peritoneal dialysis during COVID-19 pandemic Perit Dial Int 2020 40 4 363 367 10.1177/0896860820927697 32597314 8. Quintaliani G Reboldi G Di Napoli A Nordio M Limido A Aucella F Messa P Brunori G Italian Society of Nephrology COVID-19 Research Group Exposure to novel coronavirus in patients on renal replacement therapy during the exponential phase of COVID-19 pandemic: survey of the Italian Society of Nephrology J Nephrol 2020 33 4 725 736 10.1007/s40620-020-00794-1 32621109 9. Ouni A Geudri Y Sahtout W Haj Brahim M Mrabet S Ben Aycha N Fradi A Boukadida R Sabri F Zallema D Azzebi A Achour A POS-703 COVID-19 in patients on peritoneal dialysis: a monocentric experience KidneyInt Rep 2022 7 S1 S436	36450998	PMC9713098	NO-CC CODE	2022-12-02 23:22:07	no	J Nephrol. 2022 Nov 30;:1-4	utf-8	J Nephrol	2,022	10.1007/s40620-022-01523-6	oa_other
==== Front J Relig Health J Relig Health Journal of Religion and Health 0022-4197 1573-6571 Springer US New York 36449251 1708 10.1007/s10943-022-01708-0 Original Paper Unpacking the Relationship Between Prayer and Anxiety: A Consideration of Prayer Types and Expectations in the United States Upenieks Laura [email protected] grid.252890.4 0000 0001 2111 2894 Department of Sociology, Baylor University, One Bear Place, Waco, TX 76798 USA 30 11 2022 122 23 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Prayer, considered by some to be the essence of religion, has been a universal behavior throughout human history. Scholars have increasingly recognized that there are different types of prayer and various prayer purposes, but little work has been done to examine their mental health consequences beyond an examination of prayer frequencies. In this study, we draw on nationally representative data from Wave 6 of the Baylor Religion Survey (2021) to examine whether four subtypes of prayer are associated with anxiety: prayer efficacy (the belief that prayer can solve personal and world problems); devotional prayer (praise of God and prayer for the well-being of others); prayers for support (e.g., better health, financial aid); and prayer expectancies (whether God answers prayers). Results suggest that prayer efficacy, prayers for support, and one form of devotional prayer (asking God for forgiveness) all correlate with higher anxiety, while another form of devotional prayer (praise of God) and prayer expectancies are associated with lower anxiety in the American population. We note the importance of capturing multidimensional phenomenon that comprise religious prayer life within the extensive religion and health literature. Keywords Prayer Anxiety Prayer types United States ==== Body pmcIntroduction Prayer, considered by some to be the essence of religion, has been a universal behavior throughout human history (Levin & Taylor, 1997; Woodhead, 2016). At its core, prayer can be defined as “communication addressed to God” (Stark, 2017, p. 45), where an individual can externalize their concerns, intentionally relate to others, and attempt to see reality through the eyes of God or a divine being (Woodhead, 2016, p. 224–225). William James (1902) offered a broad definition of prayer, suggesting that prayer was “every kind of inward communion or conversation with the power recognized as divine” (p. 464). In this sense, prayer is an experience through which believers interact with the divine. Although there is considerable variability in prayer experiences, some practitioners have reported that they can literally hear God “talking back” (Luhrmann, 2012). Over the course of the last several decades, researchers have made considerable strides in the study of prayer. As this literature began to develop, it became quickly evident that prayer is a vast, multidimensional phenomenon (see Froese & Uecker, 2022). Early investigators, for instance, identified different types of prayer, such as ritual, conversational, petitionary, and meditative prayer (Poloma & Gallup, 1991). Alongside research on the types and forms of prayer, theories about how prayer may affect well-being also began to emerge in the literature as well (Baesler et al., 2011; Breslin & Lewis, 2008). Results from existing work on prayer and mental health have been somewhat inconsistent. Indeed, some prior cross-sectional studies point to the beneficial associations of prayer with mental health (Ai et al., 1998; Ellison & Taylor, 1996; Levin, 1996), while others find that prayer may be linked to worse mental health outcomes (Bartkowski et al., 2017; Hank & Schaan, 2008). Quantitative studies of prayer and well-being tend to measure prayer in terms of simple frequencies that fail to represent the variegated experience of this phenomenon. If the effects of prayer are conditional on the type of prayer or perceptions of the divine object of prayer, the limited measurements of previous work may obscure our understanding of the complex relationship between prayer and mental health. In a recent review article, Froese and Jones (2021) suggest that prayer may include at least four dimensions: (1) prayer quantity, including issues such as prayer frequency and consistency, (2) prayer style, or the behavioral rules and norms that underlie prayer, (3) prayer purpose, or what one wishes to accomplish via their prayers, and (4) prayer targets, the supernatural entities that are prayed to. In this study, we center in on the latter two dimensions of prayer through the use nationally representative data from Wave 6 of the Baylor Religion Survey (2021), which provides an unprecedented opportunity to move beyond simple prayer frequencies. Specifically, we look at four subtypes of prayer, including prayer efficacy (the belief that prayer can solve personal and world problems), devotional prayer (praise of God and prayer for the well-being of others), prayers for support (e.g., better health, financial aid), and prayer expectancies (whether God answers prayers). As we outline below, each dimension of prayer may be connected to anxiety in unique ways, in both positive and negative directions. Our reasons for centering on prayer and anxiety are twofold. First, anxiety-related issues are common in the United States, with approximately one in five Americans suffering from anxiety disorders (Horwitz, 2013). Second and more substantively, researchers of religion/spirituality have focused heavily on certain outcomes (e.g., depression and life satisfaction), but far fewer studies have concentrated on other important emotional states, such as anxiety. Thus, after laying out the reasons why prayer typically shows an inconsistent relationship with mental health, we devote significant space to understanding how prayer type, as a feature of this broader religious activity, may correlate with anxiety, a vastly understudied outcome, in the American populace. Literature Review Theoretical Models of Prayer and Well-Being Dating back several decades ago, McCullough (1995) suggested that physiological and psychological spiritual pathways could explain why prayer may be associated, for better or worse, with mental health. McCullough (1995) posited that prayer may activate health-promotive psychological mechanisms such as structure, meaning and hope. In other words, prayer may be useful insofar as it changes the ways in which individuals appraise stressful events. Finney and Malony (1985) suggested that contemplative prayer involves hypnotic suggestion, and a condition of a lower level of arousal through inducing a state of relaxation. The act of prayer has been found to lead directly to a lower heart rate, reduced muscle tension, and slower breathing rate (McCullough, 1995). Clinical studies have also suggested that prayer may also contribute to feelings of tranquility by altering brain chemistry and structure (Newberg & Waldman, 2009). Levin (2020, p. 105) summarizes research which finds a wide swathe of other physiological and neurological outcomes of prayer, including decreased sympathetic nervous system activity, and increased parasympathetic activity (e.g., the opposite of the fight-or-flight response). Taken together, these studies suggest that prayer may put us into a calm or relaxed state by prompting changes in brain chemistry and structure (Levin, 2020). In addition to these physiological mechanisms, the psychological belief that God provides answers could provide rest, inspiration, and a sense of intimacy that can help to ease the burden of stress and provide a sense of purpose in life (McCullough, 1995). As Levin (2020, p. 61) notes, prayer can promote a transcendent experience which “typically evokes a perception that human reality extends beyond the physical body and its psychosocial boundaries” (Levin, 2020, p. 61). According to Levin (1996), hope, love, contentment, empowerment, and other positive emotions might flow from prayer. Prayer may thus foster psychological benefits by creating a positive sense of meaning, hope, and empathy (Levin, 1996; McCullough, 1995). Prayer can also provide a framework for thinking about life and processing the outcomes of life (Ellison, 1995) and cross-sectional research has shown that it may be used as a coping tool that can aid believers in confronting a certain stressor or in overcoming negative emotions (Bade & Cook, 2008; Masters & Spielmans, 2007; Rainville, 2018; Sharp et al., 2016). Conti and colleagues (2003) even suggest that prayer is analogous to a form of psychotherapy in helping people to redefine stressors in less threatening ways through sharing them with a divine power and helping them derive meaning in the face of hardship (see also Ellison & Taylor, 1996). As mentioned at the outset of this study, prayer does not have an unequivocally positive relationship with mental health. From a physiological standpoint, some purposes of prayer have been found to be damaging to the brain if they are focused on elements of fear or anger (Newberg & Waldman, 2009). A unique challenge when studying prayer is that “individuals confronting high levels of stress and distress pray more often” (Bradshaw et al., 2008, p. 654). While prayer as a form of religious coping may help reduce the effects of stress, findings from both cross-sectional (Ellison et al., 2014) and longitudinal studies (Bradshaw & Kent, 2018) suggest that praying to a God who is perceived to be distant and cold undermines the sense of security and significance, and could actually increase psychological distress and anxiety. Perhaps due to this dual process of prayer fostering both positive and negative physiological and psychological processes, at least one cross-sectional study found that prayer frequency is unrelated to anxiety (Ellison, Hill, & Burdette, 2009). Ellison and colleagues (2009) suggest incorporating varieties of prayer types in order to understand which ones are associated with the greatest reductions in anxiety. Therefore, the relationship between prayer and mental health may depend on the extensive variation in prayer experiences, which we outline below. As Brown (1994) argues, what individuals pray about is much more important than how often they do it. Put another way, why someone is praying could matter more than the quantity of prayer. Prayer Purpose & Intentions The purpose of prayer accesses why practitioners say they enter into prayer and seeks to capture their explicit motivations for engaging in this religious activity. According to Froese and Jones (2021), prayer purpose/intention represents the “rational” aspect of prayer, gauging how practitioners rationalize the activity of prayer to themselves. One of the basic stated purposes of prayer is that supplication to and communication with God should produce positive individual and social outcomes. For the purposes of our study, we term this prayer efficacy. Hunter (1990) demonstrated that a majority of Americans felt that prayer before government sessions, sporting events, and school activities leads to greater success. It is possible that using prayer as a vehicle to solve both individual and world problems might be associated with lower anxiety. Equipped with the hope of a positive end result, prayers for assistance to problems at the personal or global level might be helpful in reducing anxiety, at least temporarily. Beyond asking for aid in solving problem, other types of prayer are perhaps more idiosyncratic. Prayers of petition are ones in which the believer asks God to grant them a blessing in this world to fill some specific need, while prayers of intercession ask God to grant a blessing to another person beyond the individual (Janssen et al., 1990). We divide this larger category of petitionary prayer into Devotional Petitions (e.g., asking God for forgiveness, praising God, or praying for the well-being of others) and Prayers of Support (e.g., asking God for guidance in decision-making, support for relationships, better health, and financial help). As a class of prayers, petitionary prayers introduce the idea of a transaction in one’s relationship with God and raise the question of whether God will respond to prayer requests. Asking God for something concrete—be it His forgiveness, good health, or economic aid, for instance, could help to ease some of the anxiety associated with those problems. Sharp (2012, p. 257) notes that having “prayed over” a decision helps to “prevent possible negative characterization,” because the act of prayer helps to give moral legitimacy to whatever decision is rendered. However, the content of petitionary prayers also maps directly onto the type and severity of individual life problems that are being experienced. For example, petitionary prayer is most common among disadvantaged members of American society, including African Americans and those with lower education and lower income (e.g., Baker, 2008; Krause & Chatters, 2005; Spilka et al., 2003). Better health is one of the most commonly made requests through petitionary prayer, as nearly three-quarters of Americans pray for their general health (Brown, 2012). Therefore, to the extent that good health, forgiveness, financial prosperity, support with intimate relationships, and guidance in decision-making are the primary purposes of prayer, this may be indicative of a person experiencing unusually high amounts of stress, perhaps correlating with higher anxiety. One prior study by Winkeljohn-Black and colleagues (2015) found neither a positive nor negative relationship between petitionary prayer and mental health. These authors suggest that this lack of relationship between mental health and petitionary prayer can be explained by the prayer’s lack of meaningful disclosure to God, a topic which we explore in the next section. Prayer as Interaction with God An additional component of prayer could be to offer praise or otherwise deepen a relationship with God or a divine being. Most Americans see themselves as having a two-way, relationally engaged bond with God (Hall & Fujikawa, 2013), a relationship where God speaks to them through prayer (Luhrmann, 2012), is actively involved and intervened in their lives (Froese & Bader, 2010), and collaborates with them to solve problems (Krause, 2005). Stark (2017) argued that one of the primary purposes of prayer is to strengthen the relationship between the believer and the divine. Recent scholarship by Luhrmann et al. (2010) maintained that one’s perception of God’s character is cultivated over time through prayer. The experience of prayer may also determine how God is perceived, which affects the feelings that may be experienced during prayer and the eventual affective outcomes. For instance, the link between a higher frequency of prayer and greater distress exists among people who perceive God as remote and do not view God as loving (Bradshaw & Kent, 2018; Bradshaw et al., 2008). In addition, perceptions of an angry God may inspire feelings of anxiety, while beliefs in a caring God may promote optimism and a sense of agency (Liu & Froese, 2020), both of which might benefit mental health. We focus on prayers of adoration toward God, which involve praise of and interaction with God, as a form of devotional prayer. Prayers of adoration have the psychological benefit of lifting individuals out of themselves and diminish egocentricity (Watts, 2001). Watts (2001) claims that it is psychologically freeing for individuals to open their hearts to God in an act of unwavering appreciation. This form of prayer may help individuals to calm both their mind or body and can help the process of stress recovery. Meisenhelder and Chandler (2001) suggested that this form of prayer is beneficial to mental health because this praise may strengthen one’s relationship with God, fostering a deeper sense of love and appreciation for the divine being. Offering prayers of praise to God as a form of devotion may also increase confidence that the person is praying in accordance with the will of God (Henning, 1981), rather than petitioning for outcomes to happen according to their own timeline. Altogether, prayer with the aim of showing devotion to God could decrease anxiety by reminding individuals that there is a God who cares about our worship. Prayer-Based Expectancies As a final dimension considered in the current study, prayer is often undertaken with an expectation of desired outcomes from God. Many believers hold expectations about whether their prayers are answered, how quickly prayers are answered, and how prayers are ultimately answered by God (e.g., Krause et al., 2000). Existing research focused on prayer expectancies reveal that if people get what they expect, they may experience an enhanced sense of well-being, but if expected outcomes fail to arise, people may feel psychological distress (Olson et al., 1996). Though research on prayer-based expectancies is somewhat limited, older people who endorse trust-based expectancies (that God knows how to best answer prayers and does so in His own time) had higher self-esteem and life satisfaction (Krause, 2004; Krause & Hayward, 2013). Such trust-based prayer-expectancies ae unlikely to be invalidated because individuals are willing to wait for a response from God and may accept responses that differ from their initial request. According to these studies, what one expects from prayer may bear close connection with affective outcomes that may stem from prayer, including anxiety. Some scholars have also hinted at the downsides of unfulfilled prayer expectancies. For example, Epperly (1995) argued that prayer may pose threats to well-being if answers to prayers are viewed solely as a reflection of God’s arbitrary will. If prayers being answered according to human requests are a precondition for viewing God as loving, this may create a dangerous scenario in which a person may only love and trust God if they get the desired responses and feel they are important to God. This claim has been corroborated in prior research, as a study by Exline and colleagues (2021) found that if participants saw God as more responsive to their prayers, they reported less doubt about God’s existence, felt less distant from God, and perceived God as less cruel. The corollary, however, is that if individuals believed God was not responsive to their prayers, their relationship with God tended to be more negative. In her work, Luhrmann (2012) is careful to remind researchers that not all prayer should seek a response back from God. Indeed, when the prime goal of prayer is for God to respond in direct and concrete ways specified by humans, too much emphasis is placed on “outer effects” (Giordan, 2016). Therefore, we might expect, to the extent that individuals expect to receive answers from God to their prayers, this may actually foster higher anxiety, especially if the responses do not align with the recipient’s intentions. Methods We use data from the 2021 Baylor Religion Survey, which was administered by Gallup. Data collection lasted from January 27 to March 21, 2021, during which time 11,000 surveys were sent out to 50 US states and the District of Columbia using an address-based sampling technique. Data were collected with either a mailed or an online questionnaire, which were available in both English and Spanish. The result was a random sample of 1248 US adults. The response rate for the survey was 11.3%. This survey was selected since it was collected recently, is nationally representative, and offers unique measures targeting several aspects of prayer along with items for constructing an index of anxiety. To be eligible for inclusion in our sample and respond to the Baylor Religion Survey Prayer Module questions, respondents had to report praying outside of religious services. Of the 1206 respondents who gave valid responses to the prayer frequency question, 289 reported never praying, yielding a final analytic sample of 917. Removing all cases with missing data using listwise deletion, we are left with a final sample of 823 respondents for analysis. Dependent Variable: Anxiety Three items were used to assess the respondents’ anxiety level. Respondents were asked, “In the past week, how often have you had the following feelings?” Subsequent statements included “I worried a lot about little things,” “I felt tense and anxious,” and “I felt restless.” Again, the answer categories were (1) “never”, (2) “hardly ever”, (3) “some of the time”, and (4) “most of the time”. Responses to these three questions were summed, with a higher score corresponds with a higher anxiety level. This anxiety scale has an alpha reliability coefficient of 0.83. Key Independent Variables: A Variety of Prayer Dimensions Several variables assessing prayer type were used in the current. The first two variables assess the extent to which a person agrees with the following statements about prayer efficacy: (1) “I pray because prayer is the best way to solve world problems,” and (2) “I pray because prayer is the best way to solve personal problems.” Responses to both of these items were scored where 1 = “Strongly disagree” (reference category), 2 = “Disagree,” 3 = “Agree,” and 4 = “Strongly agree.” The next three variable target devotional/intercessory prayer. The first item asks respondents their agreement with the following statement: “I pray to ask God for forgiveness,” coded from 1 = “Strongly disagree” to 4 = “Strongly agree.” The second two items ask respondents how often they use prayer to “praise God” and “pray for the well-being of others,” with both items coded in the following way: 1 = “Never,” 2 = “Rarely,” 3 = “Some of the time,” 4 = “Much of the time,” and 5 = “All of the time.” Our next set of four prayer variables indicate how often a respondent prays for support. These variables are based on the frequency (from 1 = “Never” to 5 = “All of the time”) that a respondent, through prayer, asks for “guidance in decision-making,” “support with relationships,” “better health,” and “financial help.” Finally, our last measure of prayer captures prayer-based expectancies from God by assessing agreement/disagreement with the statement “I pray because God answers my prayers.” This measure is coded from 1 = “Strongly disagree” to 4 = “Strongly agree.” Covariates To ensure that any association between prayer type and mental health was not confounded by other variables that could also predict anxiety, we controlled for several demographic variables, including gender (female = 1), age (years), race and ethnicity (non-Hispanic white, non-Hispanic black, Hispanic, and Other race), marital status (married or in a cohabiting partnership = 1), and education (college degree = 1). Respondents also reported on their total household income last year (before taxes) from 1 = $10,000 or less, 2 = $10,001–$20,000, 3 = $20,001–$35,000, 4 = $35,001–$50,000, 5 = $50,001–$100,000, 6 = $100,001–$150,000, and 7 = $150,001 or more. We also included adjustment for the region of the country that the respondent lived in (Northeast, South, Midwest, and South). Analyses also adjusted for two additional religious covariates in all statistical models. First, religious attendance was assessed by the question, “How often do you attend religious services at a place of worship?” This was coded into a four-category variable, where 1 = Never attends, 2 = Attends yearly, 3 = Attends monthly, and 4 = Attends weekly or more. Analyses also adjust for religious tradition following the RELTRAD scheme proposed by Steensland et al. (2000), which categorizes individuals into Evangelical Protestants (reference group), Mainline Protestants, Black Protestants, Catholic, Jewish, Other religion, and the non-affiliated. Plan of Analysis We estimate a series of ordinary least squares (OLS) regression models to assess the associations of prayer type and anxiety. All regression models use weighted data to enhance representativeness of parameter estimates. After presenting sample descriptive statistics (Table 1) and descriptive statistics for all prayer variables (Table 2), we present results for each category of prayer type (Tables 3, 4, 5, and 6). In the interest of space, we do not show results for the covariates in each table (but results are available upon request).Table 1 Unweighted Descriptive Statistics, 2021 Baylor Religion Survey (Wave 6) (N = 823) Mean/% SD Minimum Maximum Dependent variables Anxiety 7.22 2.35 3 12 Control variables Gender (female = 1) 49.85 Race White, non-Hispanic 65.77 Black 11.46 Hispanic 15.61 Other race 7.15 College educated 53.82 Marital status (Married/Domestic Partnership = 1) 53.59 Age 54.89 17.19 18 98 Household income $10,000 or less 5.47 $10,001–$20,000 8.86 $20,000–$35,000 12.76 $35,001–$50,000 15.00 $50,000–$100,000 26.76 $100,000–$150,000 15.66 $150,000 or more 15.49 Religious attendance Never attends 34.78 Attends yearly 31.18 Attends monthly 8.10 Attends weekly or more 25.94 Religious affiliation Mainline/Black Protestant 21.03 Evangelical Protestant 24.76 Catholic 24.84 Jewish/Other 10.77 Non-affiliated 18.33 Region of the United States Northeast 16.25 South 38.15 Midwest 20.92 West 24.68 Standard deviations are omitted for categorical variables Table 2 Descriptive Statistics for Prayer Variables (% shown) Prayer efficacy Devotional/Intercessory prayer Prayers for support Prayer expectancies (1) Solve world problems (2) Solve personal problems (3) Ask god for forgiveness (4) Praise god (5) Prayer for well-being of others (6) Guidance in decision-making (7) Support with relation-ships (8) Better health (9) Financial help (10) God answers prayers Agreement/Disagreement Strongly Disagree 9.72 6.77 6.07 7.20 Disagree 23.65 18.39 12.47 16.24 Agree 39.63 42.47 43.71 44.30 Strongly agree 27.00 32.37 37.74 32.26 Frequency of prayer type Never 7.97 4.69 5.20 10.95 8.70 25.67 Rarely 8.29 5.74 6.26 14.77 14.85 27.90 Some of the time 18.07 11.30 25.24 26.99 33.40 23.75 Much of the time 20.51 34.18 28.63 22.85 18.66 10.86 All of the time 45.16 44.09 34.68 24.44 24.39 11.82 Table 3 Unstandardized coefficients for Anxiety, Prayer Efficacy, 2021 Baylor Religion Survey (Wave 6), N = 823 (1) Solve world problems (2) Solve personal problems b (SE) b (SE) Agreement/Disagreement (ref = Strongly Disagree) Disagree 0.48 (0.32) 0.45 (0.36) Agree 0.34 (0.32) 0.85* (0.34) Strongly agree 0.39 (0.35) 0.74* (0.37) Results based on linear OLS regressions. Standard errors in parentheses. Models are adjusted for age, race (white = 1) gender (female = 1), education (college degree = 1), marital status (married = 1), age, income category, religious attendance, region of the United States, and religious affiliation (“Evangelical Protestants” are contrasted with Catholic, Mainline/Black Protestants, Other, and Non-Affiliated) p < 0.05, p < 0.01, *p < 0.001 (two-tailed tests) Table 4 Unstandardized Coefficients for Anxiety, Devotional/Intercessory Prayer, 2021 Baylor Religion Survey, (Wave 6), N = 823 (3) Ask god for forgiveness (4) Praise god (5) Prayer for well-being of others b (SE) b (SE) b (SE) Agreement/Disagreement (ref = Strongly Disagree) Disagree 0.54 (0.40) Agree 0.88 (0.37) Strongly agree 0.84* (0.40) Frequency of Prayer (ref = Never) Rarely 0.33 (0.40) −0.42 (0.79) Some of the time 0.30 (0.36) −0.53 (0.67) Much of the time −0.23* (0.11) −0.75 (0.66) All of the time −0.20* (0.10) −0.79 (0.66) Results based on linear OLS regressions. Standard errors in parentheses. Models are adjusted for age, race (white = 1) gender (female = 1), education (college degree = 1), marital status (the married = 1), age, income category, religious attendance, region of the United States, and religious affiliation (“Evangelical Protestants” are contrasted with Catholic, Mainline Protestants, Other, and Non-Affiliated) p < 0.05, p < 0.01, *p < 0.001 (two-tailed tests) Table 5 Unstandardized coefficients for anxiety, prayers for support, 2021 Baylor Religion Survey (Wave 6), N = 823 (6) Guidance in decision-making (7) Support with relation-ships (8) Better health (9) Financial help b (SE) b (SE) b (SE) b (SE) Frequency of Prayer (ref = Never) Rarely 0.09 (0.49) −0.28 (0.32) 0.32 (0.34) −0.03 (0.23) Some of the time −0.12 (0.39) −0.11 (0.29) 0.35 (0.31) −0.04 (0.24) Much of the time −0.35 (0.40) −0.22 (0.30) 0.28 (0.34) 0.19 (0.31) All of the time −0.38 (0.40) −0.03 (0.31) 0.45 (0.22) 0.36* (0.16) Results based on linear OLS regressions. Standard errors in parentheses. Models are adjusted for age, race (white = 1) gender (female = 1), education (college degree = 1), marital status (the married = 1), age, income category, religious attendance, region of the United States, and religious affiliation (“Evangelical Protestants” are contrasted with Catholic, Mainline Protestants, Other, and Non-Affiliated) p < 0.05, p < 0.01, *p < 0.001 (two-tailed tests) Table 6 Unstandardized coefficients for anxiety, prayer expectancies, 2021 Baylor Religion Survey (Wave 6), N = 823 (10) God answers prayers b (SE) Agreement/Disagreement (ref = Strongly Disagree) Disagree 0.71 (0.36) Agree −0.88 (0.34) Strongly agree −0.80 (0.37) Results based on linear OLS regressions. Standard errors in parentheses. Models are adjusted for age, race (white = 1) gender (female = 1), education (college degree = 1), marital status (the married = 1), age, income category, religious attendance, region of the United States, and religious affiliation (“Evangelical Protestants” are contrasted with Catholic, Mainline Protestants, Other, and Non-Affiliated) p < 0.05, p < 0.01, **p < 0.001 (two-tailed tests) Results Table 1 shows descriptive statistics for all non-prayer related study variable. Of note, the BRS Wave 6 sample had average anxiety scores of 7.22 (SD = 2.35) and ranged from 3 to 12. We would also note that over half of our sample had a college degree, and about one-quarter of our sample reported attending religious services weekly. More germane to our central research questions, we also show in Table 2 the percentage of BRS-6 respondents who expressed agreement/disagreement with each of the prayer prompts, as well as the frequencies of specific prayer types. As can be seen there with our two measures of prayer efficacy, more than two-thirds of the sample (66.63) expressed at least some agreement that prayer is the best way to solve world problems. A higher percentage of respondents (74.84%) expressed at least some agreement that prayer is the best way to solve personal problems. For devotional/intercessory prayer, more than 80% of the sample reported that they pray to ask for God’s forgiveness. Almost two-thirds of the sample reported praising God either “much of the time” or “all of the time” in prayer, and over three-quarters of the sample reported that they regularly (much or all of the time) used prayer to pray for the well-being of others. Across the four types of support that respondents could report praying for, respondents were most likely to regularly ask for guidance in decision-making (63.31%), followed by support with relationships (47.29%), then better health (43.05%), and finally, they were least likely to regularly ask for financial help (22.68%). As for our last category of prayer examined, prayer-based expectancies, over three quarters of the sample (76.56%) reported at least some agreement that God answers their prayers. With these descriptive statistics laid out, we now move to our regression analysis, where these various types of prayer are regressed on anxiety and a host of demographic and religious control variable. Multivariable Regression Results Tables 3, 4, 5 and 6 show results for each category of prayer variables outlined above. Beginning with measures of prayer efficacy in Table 3, we see that agreement with the statement “I pray because prayer is the best way to solve world problems” was not significantly associated with anxiety. However, stronger agreement that prayer is the best way to solve personal problems was significantly associated with higher anxiety, with those agreeing (b = 0.85, p < 0.05) and strongly agreeing (b = 0.74, p < 0.05) with that statement more likely to report higher anxiety than those disagreeing with that statement. Moving to the results for devotional/intercessory prayer shown in Table 4, we see that stronger agreement that prayer is used as a means to ask forgiveness with God is also associated with higher anxiety (agree: b = 0.88, p < 0.05, strongly agree: b = 0.84, p < 0.05). However, a higher frequency of prayer that is used to praise God was found to be significantly associated with lower anxiety, for respondents reporting praising God much of the time (b = −0.23, p < 0.05) and all of the time (b = −0.20, p < 0.05) relative to those who never praised God in prayer. Finally, in the last model of Table 4, we see that no frequency of prayer for the well−being of others significantly correlated with the anxiety of the respondents. Table 5 shows results for the four different types of prayer support we examined in this study. Guidance in decision-making was observed to have a null association with anxiety, as was asking for support with relationships through prayer. Respondents who more frequently prayed for better health (“all of the time”) (b = 0.45, p < 0.05) and financial help (b = 0.36, p < 0.05) reported higher anxiety relative to those who never asked for these types of support via prayer. Post-hoc tests also showed that those who frequently prayed for better health and financial help also reported higher anxiety than BRS respondents who prayed for support “some of the time,” but this highest frequency group did not have anxiety scores that differed from those who asked for these two types of support “much of the time.” Finally, Table 6 shows results from a model that shows how prayer-based expectancies, that God answers prayers, is associated with anxiety. As we see there, stronger agreement with the statement, “I pray because God answers my prayers” is associated with lower anxiety for those who agree (b = −0.88, p < 0.05) and strongly agree with this statement (b = −0.80, p < 0.05). In the discussion section that follows, we unpack these findings across these four categories of prayer types and purposes. Supplemental Analyses On almost every dimension of religiosity, women tend to be more religious than men (Schnabel, 2015, 2018), and report a higher frequency of prayer than men (Maselko & Kubzansky, 2006). It has also been suggested that women report higher levels of intimacy with God (Kent & Pieper, 2019) which is an important component of prayer. Previous work by Kent (2020) women that prayer was either non-significant or associated with lower depressive symptoms for women but was associated with higher depressive symptoms for men. We therefore examined in ancillary analyses whether the results we observed in our main analyses had gender contingencies. To do this, we examined if each of the ten dimensions of prayer (grouped into four types of prayer) significantly interacted with gender (male = 0, female = 1) in their associations with anxiety. In no case was a significant interaction term detected. Therefore, on the basis of these supplemental analyses, we did not find evidence that our main findings differed by gender. We return in the discussion section to recommend further analyses on potential status-based differences in the relationship between prayer and anxiety. Discussion Prayer occupies a core position in religious life, a sentiment which earlier social theorists like William James (1902) clearly recognized this when he said, “Prayer is religion in act; that is, prayer is real religion” (p. 464). James (1902) argued that prayer feels like a true conversation or transaction between the person who prays and the divine being that is being prayed to. Despite this central importance of prayer, the sociology and psychology of religion appears somewhat stunted in its study of prayer, particularly in considering measures beyond the frequency of private prayer measured commonly in survey research. Within the religion and health literature, the results from studies on prayer and mental health have been mixed, with some studies identifying a beneficial association of prayer with mental health (Ai et al., 1998; Ellison & Taylor, 1996; Levin, 1996, 2020), and others showing prayer to be linked to unfavorable mental health outcomes, such as lower anxiety (Bartkowski et al., 2017; Hank & Schaan, 2008). We suspect that what may underlie these discrepant set of findings could be the measures and aspects of prayer that were examined. Indeed, not all types and forms of prayer may elevate a person’s mental well-being, and it is possible that some may even be deleterious. To provide a broad scope of the implications of prayer for mental health, the current study drew from a nationally representative sample of Americans and examined ten different types of prayer types/purposes, which we broke down into four sub-categories: prayer efficacy, devotional/intercessory prayer, prayers of support, and prayer expectancies. As our results show, these various types of prayer held both positive and negative associations with anxiety for reasons we expound on below. A series of key findings emerged from our study looking at the various types of prayer and their association with anxiety. First, with regard to prayer efficacy (what people believe prayer can accomplish), we found that stronger agreement with prayer as the solution to personal problems was associated with higher anxiety. A similar finding was not observed for believing that prayer is the best solution to problems of the world. It is possible that world problems may seem too far removed from the perceived effects of personal prayer, and hence bear no association with mental health problems. However, people who report that prayer represents the best solution to personal problems may do so because they are facing a high volume of earthly problems with no other solution. Because we relied on cross-sectional data, we cannot disentangle whether current hardships may select people into different types of prayer, but to the extent that prayer represents a coping response to stress, seeing prayer as a solution to one’s personal problems appears to promote higher anxiety. This finding also dovetails with our results pertaining to prayer and support. Indeed, people who more often sought support with their health and finances through prayer also reported higher levels of anxiety. Turning to prayer when faced with these difficulties could be an enactment of religious coping or could be a frustrating endeavor if there is no improvement in one’s health or financial situations, leading people to question whether their prayers to God are in vein (e.g., DeAngelis et al., 2019). Taken together, more frequent prayer in the midst of personal problems appears to associate with higher anxiety relative to less frequent prayer, perhaps amplifying the angst felt by the experience of life stressors. Several of our findings related to prayer can also be interpreted in light of a person’s relationship with God. Many religious believers in the United States engage in prayer to deepen their relationship with God (Stark, 2017), where God is perceived to speak to them through prayer (Luhrmann, 2012). Prayers of devotion and adoration toward God involving prayer were associated with lower anxiety in our sample. Prayers of adoration have the benefit of shifting the focus from the individual to a more transcendent entity, which could help individuals place their personal problems in a different perspective. Prayers of adoration have been found in previous research with small samples to have a negative relationship with anxiety (Maltby et al., 1999) and a positive association with overall well-being (Maltby et al., 1999), and we show this to be the case here using a larger, nationally representative sample. Some of our other findings surrounding the prayer target of God also hint that the eventual affective outcomes of prayer may be dependent on how God is approached in prayer. For instance, believers who more often sought forgiveness from God through prayer reported higher anxiety. It is possible that the need to seek forgiveness from God might indicate transgressions on the part of the individual that they believe can be damaging to their overall relationship with God, causing anxiety as to how God will respond. Prior work has found that for those with a distant relationship from God, personal well-being is maximized if forgiveness is sought less (Kent et al., 2018), and our results support this finding that seeking out forgiveness may be associated with higher anxiety, perhaps as a response is awaited. It is also worthy of mention that we found that petitionary prayer, that is, prayer for the well-being of others, was not associated with anxiety in either a positive or negative direction. One prior study has found that petitionary prayer is associated with more mental health problems, while another study found no association (Maltby et al., 1999; Winklejohn Black et al., 2015). Because the targets of intercessory prayers are outside of the individual’s self, we may not expect to see strong associations with anxiety, which is experienced as an individual outcome. This does not preclude the possibility, however, that praying for others may be associated for other outcomes that may underlie well-being, such as optimism or empathy. Our final result was that stronger agreement that God answers one’s prayers is associated with lower anxiety. Prayer can sometimes be undertaken with the explicit desire to receive a response from God. Some research suggests that fail to receive an answer from God may lead to heightened distress (Olson et al., 1996), while other studies show that trusting God to answer prayers in His own way and according to His timeline are associated with higher well-being (Krause, 2004; Krause & Hayward, 2013). We show that the belief that God answers prayers is associated with lower anxiety. While our measure of prayer expectations lacks a deeper consideration of whether the person views the answers they receive as coming in a timely fashion and reflecting their inner desires, the mere acknowledgement that God answers prayers was observed to be efficacious. We interpret this finding in light of previous research that has considered the intersection of prayer with one’s attachment to God. The belief that God answers prayers may be one component of what scholars have termed attachment to God. Granqvist and Kirkpatrick (2016) theorize that individuals with higher secure attachment to God, that God is emotionally responsive to them and a secure base from which to view the world, will feel a great deal of emotional safety and assurance when God is their specific prayer target and report better mental health outcomes (Bradshaw & Kent, 2018; Ellison et al., 2014). Prior work has found that seeing God as unresponsive to one’s prayers is associated with viewing God as distant and cruel (Exline et al., 2021) and could thus undermine mental well-being. If God is viewed as responsive to prayers, it follows that people should feel less anxiety because they have a trusted confidant to turn to. Unlike the relationship between other commonly studied religious variables, such as religious attendance and health, our findings from a wide range of prayer measures show that the relationship between prayer and mental health is less straightforward. As a whole, our study shows that not all forms of prayer are created equal, and that an even more complex picture emerges when we move beyond the limits of examining only prayer frequency to encompass various types of prayer. Painting with broad strokes, prayer appears to be associated with better mental health if it facilitates and reinforces a positive relationship with a perceived divine other (God) through praise or belief that one’s prayers are answered (e.g., Whittington & Scher, 2010). It is likely that reinforcing a close connection with a perceived personal God through frequent prayer can bolster some aspects of the self, perhaps by increasing self-esteem or the sense of mattering (e.g., Ellison, 1993; Schieman et al., 2010). On the other hand, prayer tends to be associated with worse mental health if it arises out of a troubled relationship with God, or if prayer is used as a resource of coping during periods of high stress. Limitations Despite the numerous dimensions of prayer experience that were considered in the current study, it is not without limitations. First and foremost, we were limited to the use of cross-sectional data, which does not allow us to extract conclusions regarding causality. As was implied with several of the prayers of support dimensions, prayer is often a response to negative life events or significant hardship. The differences in prayer style chosen by the respondents may be a result of the social contexts people find themselves in, such that devotional/intercessory prayer is sought out by those facing hardship, while prayers of adoration may be more likely to be prayed by those experiencing fewer struggles in their lives. It is therefore also possible that a person who feels more anxious is more likely to pray to God. Ultimately, longitudinal studies will be needed to sort out the temporal direction of stressors, prayers, and mental distress. And as Levin (2020, p. 86) warns, scientific methods “cannot possibly prove or disprove actions of a presumably supernatural being that may exist in part outside of the physical universe.” Thus, any relationship between prayer and well-being are only within the purview of social association. Second, we also acknowledge that the majority of our sample were Christian respondents from the United States. The results of this study should therefore not be generalized to people of other religious affiliations. It should also be in the interest of future research to obtain more diverse samples to determine whether our findings are robust across other ethnic and religious groups, especially given the importance of private prayer to religious life in countries where formal religious attendance may be quite low. Third, our data were collected roughly 1 year into the COVID-19 pandemic in the early months of 2021, which could have also factored into our results. For example, Bentzen (2021) noted a 50% increase in Google searches for topics related to prayer at the start of the pandemic in 2020. If Americans were in fact praying more than previous time periods because of increased hardships and out of greater anxiety about what the future holds, this could have affected our results, particularly with prayers offered for better health and financial aid as well as to solve personal problems. Our results should therefore be replicated with more recent data as the pandemic wanes. Additionally, we acknowledge a lower response rate (11.3%) in the BRS-6. Since the survey was fielded during the COVID-19 pandemic, many areas experienced significant postal delays related to the pandemic. This likely had an impact on response rates, and Gallup did see a significant decline in response rates on other mail surveys fielded during the pandemic. Researchers who consider replicating the current study in its examination of multiple types of prayer may also wish to consider several moderating variables that could be of importance. For instance, studies looking at the demographic correlates of prayer frequency show that women pray more often than men (Schnabel, 2015) and that Black individuals tend to pray more than White individuals (Baker, 2008; Levin & Taylor, 1997). Despite these differences in prayer frequency, and while supplemental analyses showed no gender-based contingencies in the association between prayer types and anxiety, a closer investigation of which types of prayer may matter more or less for the mental health of women and Black Americans is worthy of further study, ideally with longitudinal data. Finally, future investigations may also profitably consider whether how a person sees their communication with God (i.e., as one-way or two-way) and the person’s perceived relationship with God as possible moderators between prayer type and mental health as well. Froese and Bader (2007, 2010), for instance, have noted the diversity in images of how people see God, and these diverse images may account for diversity in types or purpose of prayer. Though beyond the scope of the current study, future work could build on the current findings by considering participants’ images of God as an additional modifier, with positive images of God expected to be more strongly associated with better mental health for types of prayer that bore salubrious associations with anxiety, and more negative or punitive images of God expected to exacerbate any ill effects of prayer for mental health. Conclusion Prayer occupies a prominent place in the study of religiosity. At its core, prayer is an aspect of religious life that exemplifies its very personal, individualized dimension that centers on an individual’s interaction with a perceived deity. Prior research on how prayer is connected with mental health, almost exclusively focusing on the frequency of private prayer, has revealed divergent patterns of both salubrious and detrimental associations with mental well-being. Casting a wide net and examining several types of prayer, our findings exemplify finer grained nuances in the relationship between prayer and well-being. Future research is needed to further understand the complex relationship between prayer and mental health, but we hope that these findings launch an array of studies that move beyond the limits of examining prayer frequency to better capture the multidimensional phenomenon that is religious prayer life within the extensive religion and health literature. Funding None. Declarations Conflict of interest The authors declare that they have no conflict of interest. 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==== Front Int J Inf Secur Int J Inf Secur International Journal of Information Security 1615-5262 1615-5270 Springer Berlin Heidelberg Berlin/Heidelberg 635 10.1007/s10207-022-00635-1 Regular Contribution Design and analysis of DDoS mitigating network architecture http://orcid.org/0000-0001-6934-3173 Swati [email protected] 1 Roy Sangita 2 http://orcid.org/0000-0002-6351-9884 Singh Jawar 1 http://orcid.org/0000-0001-8247-9040 Mathew Jimson 3 1 grid.459592.6 0000 0004 1769 7502 Department of Electrical Engineering, Indian Institute of Technology Patna, Patna, Bihar India 2 grid.412436.6 0000 0004 0500 6866 Department of Computer Science and Engineering, Thapar Institute of Engineering and Technology, Patiala, Punjab India 3 grid.459592.6 0000 0004 1769 7502 Department of Computer Science, Indian Institute of Technology Patna, Patna, Bihar India 30 11 2022 113 © The Author(s), under exclusive licence to Springer-Verlag GmbH, DE 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Distributed Denial of Service (DDoS) attacks have emerged as the top security threat with the rise of e-commerce in recent years. Volumetric attacks are the most common DDoS attacks that aim to overwhelm the victim’s bandwidth. The current mitigation methods use reactive filtering techniques that are not magical and straightforward solutions. In this paper, we propose a network architecture based on the capability to address the threat of DDoS attacks. Physically Unclonable Functions (PUFs) have emerged as a promising solution in security. Motivated by the capability approach, we put forward a network architecture where the routers use Transient Effect Ring Oscillator PUF to generate and verify capabilities. This novel hardware-based solution, to address the problem, has reduced the computational overhead of capability generation. Additionally, the destination has complete control over the incoming traffic in the proposed architecture, resulting in uninterrupted communication with the legitimate clients regardless of the attacker traffic. The large-scale simulation on an open-source Network Simulator (NS-3) has shown that the proposed architecture efficiently mitigates DDoS attacks to a large extend. With our proposed architecture, the throughput was hardly affected when attacker traffic was varied from 10 to 80%. Keywords Network security Distributed denial of service Capability PUF ==== Body pmcIntroduction The outbreak of the Covid-19 pandemic in 2019 has accelerated e-commerce growth, with most businesses going digital. The ubiquity and open nature of the Internet has made digital businesses more vulnerable to hackers. The recent incidences and frequent attacks launched by exploiting Internet of Things (IoT) devices’ vulnerabilities, challenging the cyber security of computer networks have established the need for valuable techniques for desktop systems and their feeble brother. The vulnerability and pervasiveness of IoT devices have attracted many hackers, mainly those launching DDoS attacks to slow down a site to gain competitive advantages or distract the security team and launch an exploit in another [1, 2]. DDoS flooding attacks are launched through botnets comprising many computers and IoT devices, exploited by their vulnerabilities. Once an attacking army has been set up, an attacker can invoke a coordinated, large-scale attack against one or more targets. Botnet-based DDoS attacks on the application layer limit resources, curtail revenue, and yield customer dissatisfaction, among others [3, 4]. The most crucial mechanisms supporting our civilization are those that distribute and regulate energy, like smart grids and industrial ecosystems. In the past, Supervisory Control And Data Acquisition (SCADA) networks, which are used for controlling industrial processes remotely, were isolated from other corporate networks to reduce exposure to insecure networks like the Internet. Gradually, to satisfy the real-time demand, Edge computing (EC) has evolved, improving the quality of service (QoS) in these networks. It offers the benefit of a decreased transmission latency since the compute and storage activities are moved from the distant cloud to the edge clouds. Additionally, EC may handle data from neighboring devices without much data exchange, avoiding network congestion [5]. Despite these benefits, the Internet increased attack opportunities, and all SCADA components now have a higher percentage of zero-day vulnerabilities. EC has significantly intensified security issues since a single-edge server lacks computing and storage capacities making it highly vulnerable to DDoS attacks [6]. Their services must be continuously offered for society’s social and economic well-being. Any vulnerability or failure might have a significant cascade effect on how well other essential infrastructures function. Researchers have developed a substantial number of DDoS mitigation techniques in recent years. Conventional countermeasures for the issue include firewalls, blackhole routing, intrusion detection systems, cloud-based mitigation and fog computing. However, most are based on a prior understanding of attack characteristics. As the victims are always in a passive state, attackers will ultimately discover and exploit system flaws given enough time and reconnaissance efforts. Moreover, the outsourcing approaches as cloud computing are inefficient in detecting and blocking the attack near the attacking sources if the attack is initiated within the local network [7]. The literature argues that to mitigate DDoS attacks effectively, network-wide cooperated work is required and that every party, besides the victim under the DDoS attack, should contribute to the DDoS mitigation. Network-based mechanisms have been known to achieve better DDoS defense efficiency and efficacy. These mechanisms deploy the mitigation functionalities inside networks, mainly routers, and could respond to the attack more accurately. IoT devices, the weakest link in the security chain due to a lack of computational abilities, are an easy target to penetrate a vast and secure network. Sporadic attention to IoT botnet attacks is a concern that cannot be overlooked. The overall number of cyber attacks in 2020 is more than twice as high as in 2019. According to Neustar Inc., an American internet analytics firm, the highest attack size seen this year is also the largest they have ever neutralized. The attack size was found to be 1.17 Terabits per second (Tbps), among the largest ever seen on the Internet. According to them, the most prolonged single attack they neutralized lasted five days and 18 h [8]. India is also not an exception to it. Covid-19 pandemic spurred a significant transition to online life, resulting in unprecedented levels of threat actors. In India, according to Netscout’s Atlas Security Engineering and Response Team (ASERT), advanced threat analytics and response platform, there were around 5.4 million DDoS attacks in 2021 [9]. These reports emphasize the need for more study in this area of cyber security. At present, the potential DDoS victims are dependent either on Cloud Security Service Providers (CSSP) or vendors selling on-premise hardware equipment designed for scrubbing attack traffic. The research community has proposed various approaches to deal with the detection and mitigation of DDoS attacks. These DDoS attacks through IoT botnets need to be checked by lightweight techniques without compromising security. DDoS mitigation techniques can be broadly classified as filtering-based approaches and capability-based approaches [10]. The first approach detects malicious traffic and then filters it at the network layer to protect the victim’s resources [11]. On the other hand, only authorized traffic can reach the host in the latter approach [12, 13]. A capability-based security model is one of the existing models for secure systems. In this model, an unforgeable token is issued for providing access rights. Researchers and companies have incorporated this approach in developing several secure systems for decades. Tahoe Least-Authority File Store (Tahoe-LAFS), a secure open-source distributed file system, is designed on the “Principle of Least Authority” (POLA) using capability for access control [14]. KeyKOS, a pure capability-based operating system, was designed to meet commercial computer service performance and security goals [15]. Fuchsia, Google’s open-source operating system, prioritizes security and performance and supports embedded systems to smartphones, tablets, and computers. It has introduced security mechanisms for a security-oriented design, including capability routing [16]. Our proposed network architecture is inspired by the capability-based approach to deal with DDoS attacks. There has been a venerable tradition of authenticating or identifying objects, systems, and people using their intrinsic random physical features. Similar to the fingerprint identification of humans that dates back to the nineteenth century, PUFs emerged as the prominent solution for increased security demands in the system. The multiple-input, multiple-output, large entropy unclonable physical systems are an excellent choice for generating and storing secret keys [17]. We have used these digital fingerprints of the devices in our model to enhance security and reduce computational load. The motivation of the research is to propose a network architecture that is:Scalable Works against even sophisticated DDoS attack Computationally less expensive Should be able to deliver victim-desired traffic (destination-driven traffic control). Related work Many strategies have been proposed and tested for defending the DDoS attack over the Internet. These strategies are broadly classified based on their deployment location and the point in time that defense takes place. The mechanism is generally deployed at the application level and network or transport level, which can be further categorized as source-based, destination-based, network-based, and hybrid. Moreover, on the basis of time of defense, it is further classified as before the attack, during the attack, and after the attack. Deployment of DDoS defense mechanism at the network/transport level is always a better choice than at the application level, as responding at this level, most of the resources have already been exhausted. Hybrid defense at the network/transport level is the best mechanism to mitigate DDoS attacks as all the nodes collaborate to deal with the attack. Some of the state-of-the-art defense mechanisms are enumerated in this section. Yaar et al. [18] proposed a Path Identification for tracking the path traversed by the packet. A fingerprint is embedded in each packet by the routers to enable the victim to track each packet’s path and discard the packets by the attacker despite the source IP address spoofing. The drawback of this mechanism is that the limitation on the identification field’s size may result in the same path information for different paths, leading to increased false-positive rates. Yaar et al. [12] extended their work to deal with this issue with a capability-based defense mechanism for DoS, Stateless Internet Flow Filter (SIFF), in which every router adds precapability information based on the IP address and timestamp to the IP packet. Based on the precapability provided by the router, server generates a capability and sends it back to the sender. The legitimate sender sends the capability and precapability to the receiver every time, and the server and the routers in the path validate the info to check if the client is a legitimate sender. However, this approach is vulnerable to the DDoS attack during capability setup, and also, there is no packet flow control for the legitimate users having acquired capabilities. Later Yang et al. [13] proposed another method based on SIFF, named Traffic Validation Architecture (TVA). They implemented a capability-based defense mechanism in the TCP/IP layer, addressing earlier proposed systems’ limitations. Nevertheless, the capability and precapability in the authenticated packet could be compromised and used to launch an attack. In this model, the receiver is supposed to distinguish the attack traffic from legitimate traffic. The main challenges to be addressed in the capability-based technique are high processing overheads, secure setup of capability channel, and efficiently choosing the unknown sources’ capabilities. In this study, Liu et al. [19] propose the design and assessment of NetFence, an architecture that sets the network as the first line of defense against DoS attacks. They demonstrated NetFence as an effective and scalable DoS solution using a Linux implementation, ns-2 simulations, and theoretical analysis: it decreases the amount of state retained by a congested router from per-host to at most per-node (Autonomous System). MiddlePolice, the first easily deployable and proactive DDoS prevention system was claimed and presented by Liu et al. [20] in this study. They carefully designed MiddlePolice such that it does not require any modifications to the Internet core or the client network stack, allowing it to be deployed immediately in the present Internet architecture. MiddlePolice can also impose destination-driven traffic management, ensuring that victim-desired traffic is delivered regardless of attacker techniques. They created a MiddlePolice prototype and demonstrated its viability through rigorous testing on the Internet, a hardware testbed, and a large-scale simulation on NS-3. SIFF, TVA and NetFence use cryptographic algorithms, which leads to the need for secret key management. Table 1 contains the property comparison of other research proposals.Table 1 Property comparison Proposals Upgrade requirements Router support Other requirement TVA[13] Source and destination Cryptography, O(N) states Header update Netfence [19] Source and destination Cryptography O(N) states Header update, Passport Middle Police[20] Only destination None mbox Our approach Source and destination PUF Header update Contribution and plan of this paper Our main contribution in the paper are as follows:This paper proposes a network architecture for DDoS attack mitigation that harness the physical properties of routers. Our approach addresses the limitations of capability-based mechanisms, like high processing overheads and efficient algorithms to secure the capability setup channel. The architecture design is developed considering three main aspects, viz. computation, deployability, and efficiency. Primarily, we attempted to lower the computational cost significantly without compromising with the security. The computations needed to process capabilities where significantly lowered by replacing the cryptographic functions by computationally inexpensive PUFs and XOR operations. Secondly, to deploy the proposed system in the established Internet architecture, we have to upgrade the hosts. We will need to place processing boxes at points of congestion to process the packets in transition. Lastly, the maximum extent up to which the proposed system can check the DDoS attack. There is always a trade-off between performance based on cost and efficiency. We have given more emphasis on making the system more efficient performance-wise rather than considering cost-benefits. We propose an architectural level solution based on hardware, which can handle the capability-based defense mechanism in a faster and more secure way for limiting the DoS and DDoS attacks. Since this method alters the IP packet structure, we propose a solution for its implementation for the existing IoT devices. We formally prove the security of the proposed system. We have simulated the proposed system on an open-source simulator NS-3 to analyze its performance for volumetric attacks. A detailed discussion on our work is done in the remainder of this paper. Section 2 discusses fundamental ideas in DDoS attacks and PUF to establish the groundwork. In Sect. 3, system and adversary models considered in formulating the problem are discussed, and Sect. 4 presents the design of the proposed architecture. The security analysis of the proposed model is done in Sect. 5. Performance evaluation of our approach using NS-3 simulator is discussed in Sect. 6. Section 7 comprises the modifications required for the IoT, and finally, we summarize our work in Sect. 8. Preliminaries This section will go over some basic terms that will be used over the paper and PUF security, which will be used to build our suggested system in the next section. Definition 1 Trust boundaries: It is defined as the farthest group of Internet Service Providers (ISPs) with which a web server has an economic relationship. A trust barrier exists everywhere we allow user input in any form. Here, we consider each Autonomous System (AS) as a trust and fate-sharing unit as a trade-off for scalability. We believe that network-managed routers are significantly less likely to be hacked than end systems. As a result, rather than entirely relying on the end systems, we prefer to include routers for the generation of precapability. Definition 2 PUF: These are the physical realization of a random function that is difficult to clone and shows incalculable challenge-response behavior that is ideally difficult to model but can be readily and reliably assessed in other ways. The basic properties of PUFs’s challenge-response defining its behavior used to provide cryptographic security to our proposed method [21, 22]. Evaluable: For given τ and c, it is easy to evaluate r=τ(c), where τ:C→R:τ(c)=r,c∈C,r∈R. Unique: τ(c) contains some information about the identity of the physical unit embedding τ. Reproducible: y = τ(c) is reproducible up to a small error. Unclonable: For a given τ, it is hard to construct a procedure γ, such that ∀c∈C,γ(c)≈τ(c). Unpredictable: It is hard to calculate ru=τ(cu), with only a set Q= (ci,ri) such that ri=τ(ci). One way function: It is hard to evaluate c, for a given r and τ without any physical access to the PUF. Tamper-proof: Changing the physical entity that embeds τ changes it into τ′. However, because the PUF response is unexpected and unclonable, it is assumed that the PUF response cannot be predicted or calculated without physical access to the PUF. It is obvious that the hash function can be replaced with a perfect PUF. Definition 3 Path id: Path identifiers are 32-bit tags issued by edge routers at trust boundaries that serve as an approximate source locator. These are packet markings included in each packet, as route fingerprint, allowing a victim to identify packets transiting the same Internet pathways on a per-packet basis, independent of source IP address spoofing. IP tracing is essential for detecting attack sources and putting in place Internet security measures. System and adversary models Our approach exploits the capacity to ensure that packets are sent smoothly between valid users and destinations, even during packet flooding. We consider all the routers and hosts under the trust boundaries runs our protocol to achieve this. The routers are accompanied by packet processing boxes containing PUFs. These boxes generate precapability for the incoming request packets and verify regular and renewal packets. In this section, we will describe the system and adversary models. System model First, we will have to understand the system requirements to formulate the problem. Currently, most of the victims rely on CSSPs to mitigate DDoS. They provide reactive filters that do not assure complete success as the mitigation takes place after the detection of the attack. Only reactive filters are insufficient to prevent such attacks, but we must ensure that the network discards the unwanted packets before reaching the bottleneck link. Such an approach is effective in dealing with most adversaries’ strategies. The main properties of our proposal are enumerated below. Unforgeable capabilities: The main issue of proactive detection of attacks can be solved using capabilities. These packets with capabilities help the router identify and discard unwanted packets before reaching a congested link. Nevertheless, these capabilities need to satisfy some requirements. Firstly, they must be unforgeable and non-transferable. So that stealing or sharing of capabilities can be checked. Secondly, the routers should verify these capabilities independently without trusting other sources. Finally, the overhead of capability generation and verification should be minimum. The source of a packet can be identified using its IP address or mac address. However, IP addresses can be easily spoofed, and we can identify the mac address only when connected to the same network. So, we have used a path id which is discussed in Sect. 4. To ensure non-transferability, we have included the source and destination IP addresses together with path ids in the precapability generation. We have also used a 16-bit timestamp to bound it with a specific time. It only allows the communication for a specific duration after that capability expires and packets are demoted to a lower priority level. We use PUFs and inexpensive XOR operations to minimize the computational overhead of precapability generation and verification. As the router knows all the inputs needed to verify the capability and we are not using cryptographic functions, there is no need for any secret to be shared or changed. PUFs provide secure and low-cost authentication. The destination authorizes the request packet with precapabilities by sending an ordered list of capabilities. Tackling the unwanted traffic: In our proposed model, even the unwanted packet gets authorized at least once. In a condition when any malicious sender gets authorized access, they may flood the network for the authorized period. Such situations are tackled by rate-limiting the capabilities. When the destination issues capabilities, they authorize the traffic to transfer N bytes for the next T seconds. Any source trying to flood the network will be able to transfer the utmost allotted bytes in its authorized period and, such sources will be demoted and blocklisted by the destination. IP traceback: The challenge of identifying the attack source is also addressed in this study. While identifying the device from which the attack was launched remains a difficult task, we have narrowed the problem of identifying the source of the offending packets whose addresses may be faked. Several approaches based on probabilistic packet marking (PPM) [23–25], deterministic packet marking (DPM) [18, 26–28], deterministic edge router marking (DERM) [29] and distributed star coloring (DSC) [30, 31] have been proposed. We have used a PPM approach to deal with source identification. Each router at the ingress of a trust boundary, e.g., AS edge, tags the request with a 32 bit value derived from its incoming interface that is likely to be unique across the trust boundary. Routers that are not at trust boundaries do not tag requests that have already been marked by the upstream. The tags serve as a network path’s identification. Victim-driven traffic control: The destination is provided with the control of network utilization to mitigate DDoS attacks. A client has to agree upon the policies defined by the destination to communicate further. The fine-grained capabilities with rate-limited and timeout capabilities ensure uninterrupted communication between legitimate users and the destination. Capability-based systems, like CRAFT [32] that enforces per-flow fairness, NetFence enforcing per-sender fairness and SIBRA [33] enforcing per-steady-bandwidth fairness, works with a scheduling policy. We have implemented an idea as in TVA [13], where fair queuing is based on the victim’s authorization. Regular, renewal, request and demoted packets have different priority levels. Regular packets enjoy the highest priority, followed by renewal and request. Adversary models and assumptions Adversary Model: An attacker aims to exhaust the end-system and network resources with excessive traffic. Our proposed model focuses on mitigating such network layer flooding attacks along with application vulnerabilities. We assume that the attacker owns large botnets and can launch strategic attacks. Regarding the capacity of an attacker aiming to attack, some essential assumptions were considered. Assumption 1 The attacker is outside the trust boundaries and cannot corrupt the routers. It is the basis of secure protocol to make sense. Assumption 2 The routers are well connected to the packet processing boxes. These processing boxes are not compromised. Even the secure protocols running on infected devices cannot assure protection. Such integrity of the hardware device is required. Assumption 3 The PUF generates the same response for the same input as a challenge. All the computations of the PUF are done in a trusted zone of the device. Proposed network architecture Design overview This section deals with the key components of the proposed design. The scheme aims at dealing with the flood of packets by generating capabilities, which are short-term authorization provided by the receiver/sink to the legitimate senders. The processing overheads required for generating capability/precapability are reduced without compromising the security and efficiency of the algorithm by including PUFs in the architecture design. PUFs have emerged as the most promising solution to ensure low-cost, secure authentication, access control, and traceability [34]. These PUFs will be synthesized and incorporated into the packet processing boxes of the legacy routers. We recommend TERO PUF, which has optimized power consumption, area efficiency, and reliability, in keeping with the design consideration [35]. According to the definition of PUF, it is said to be secure if the response for two different challenges has at least d1 variation, and any challenge for two different PUFs should produce distinct responses with at least d2 variation where d1 and d2 are the error tolerance threshold for PUF. Mathematically,1 Pr[HD(PUF1(C1),PUF1(C2))>d1]=1-ε,Pr[HD(PUF1(C1),PUF2(C1))>d2]=1-ε,whereεisnegligiblesmallvalue,HDisthehammingdistance,andrandomchallengesC1,C2ϵ{0,1}k Packets with capability Considering that all the routers and hosts in the network run our protocol, the main aim is to drop the unwanted packets before reaching a congested link to prevent a DDoS attack. An early stage detection is only possible when each packet carries information that can be verified to confirm its legitimacy. The packets must be identified and dropped in an early stage. Each packet must carry information that the routers can verify to confirm its legitimacy. Such a token is known as capability. The destination issues these unforgeable capabilities, and their validity is only for a specified period. Figure 1 shows the simple flow of packet for obtaining capability.Fig. 1 Client sending request packet to obtain capability from the server The IP packets are categorized as request, renewal, demoted, and regular packets in the capability-based system. The packets from unknown sources without any capability/precapability are considered the request packet. Each router adds the precapability to such packets, marking its path to the server, and finally, the server computes the capability based on the precapability information. The packets already having the capability issued by the server are known as regular packets. These packets have higher priority than others as the server has already authorized them for further communication for a particular period. The router checks the capability to ensure legitimate traffic. After a specified period for regular packets expiry, the client can renew the capability using its list of capabilities. Such packets are renewal packets, and their list of capabilities is verified to generate a new set. If it fails the capability check, the priority level of such packets is lowered, and they fall in the category of demoted packets. The structure of regular and request capability packets is shown in Fig. 2. Here, the common header carries the information about the type of packet, i.e., regular, request, renewal, or demoted. The request packet carries a request header along with the common header consisting of blank capabilities and path ids.Fig. 2 Types of capability packet Queue management Request sent to the destination as a part of TCP SYN packet to obtain capabilities. The regular packets are further checked by the routers and validated for further communication. The initial request channel must, however, avoid providing a path for DoS attacks, either by flooding a destination or denying requests from authorized senders. In the proposed architecture, flooding of destination was handled by rate-limiting the requests to 5% of the total bandwidth of each link. Moreover, the possible solutions to prevent legitimate requests from the overwhelming attacker traffic with their drawbacks are tabulated in Table 2. In order to localize the impact of an attack, we have used the path identifiers to locate the upstream party, and a fair-queue analogous to TVA [13].Table 2 Possible solutions to prevent attack traffic Methods Drawbacks Per-source fair queuing Spoofing Ingress filtering Unprotected ingress allows remote spoofing Public key infrastructure Deployment hurdle IP traceback The intermediate border routers at the AS edge generate path ids as shown in Fig. 3. These unique tokens are generated by XORing the source IP address with the router IP address. Thus transversing the multiple hops, every path id gets XORed to the IP address of the edge router, and the subsequent path id is generated. The path id generation is given in Algorithm 1. Since XOR is its own inverse, IP tracing the packet using the path ids generated is affordable and straightforward.Fig. 3 Path id generation and path reconstruction The 32-bit marking information fits into the IP header as seen in Fig. 4. The IP identification field, flag bits, and fragment offset field are all overloaded by the path id. According to research, the internet fragments fewer than 0.25 percent of packets [36]. Just like all other PPM methods, we have employed the IP identification field, which is used for IP fragmentation. We have also used the reserved flag bit for packet labeling, as proposed in [37]. Because the fragment offset field loses its relevance when the IP identification field is used for something other than IP fragmentation. However, repeating the fragment offset field is not recommended because the IP datagram with a nonzero value in the fragment offset field is treated as an IP fragment by the destination. The destination host may mix up designated packets with IP fragments. To avoid this kind of perplexity, we propose to use the unused Type of Service (TOS) bits. The 8-bit ToS employs three bits for IP Precedence and four bits for ToS, with the final bit unused. Although specified, the 4-bit ToS field has never been utilized. The last two bits of TOS are used as the Don’t Fragment (DF) flag and More Fragment (MF) flag. While marking a packet, the router sets the DF to be 1 and MF to 0. Thus, the marked packets from legitimate users can be easily identified and will not be fragmented downstream from the marking router. The number of false positives in the upstream router map is reduced using this method.Fig. 4 Encoding path id into IP header Design description We have mainly focused on three points in the proposed protocol: firstly, a secure and lightweight algorithm for the generation of precapability for the request packets by the routers; secondly, the routers’ regular packet handling and finally packet handling by the server and the client. The tokens are verified at each level, and the details of the blocklisted sources are available with the destination. Precapability generation by routers The packets are classified as regular or request based on the availability of capability. Each packet carries a capability header which is a shim layer above IP. The regular packets have capability headers piggybacking the capability information. On the other hand, the request packet carries a blank capability to initiate communication with the server. The router identifies such packets and generates the precapability using the defined algorithm. The algorithm for generating precapability is shown in Fig. 5. We have used both the logical address and path id to generate the precapability for the request packets. The capability/precapability should be secure and bandwidth-efficient, but there is always a trade-off between these two. Small precapability makes it vulnerable to brute force attack, and large precapability will make it bandwidth inefficient. A 64-bit capability ensures security without consuming a large bandwidth. The IP address of the sender and path id generated by the router generates the first 32-bits of precapability. They are XORed to generate a challenge for the PUF, whose the response is again XORed with the 32-bit timestamp generating the first 32 bits of precapability. The next 32-bits of precapability are generated similarly using the IP address of the destination, path id and PUF response of timestamp. The two outputs are finally concatenated to form a 64-bit precapability.Fig. 5 Block diagram for generation of precapability by the routers Packet handling by server The server receives both the request packets as well as the regular packets. For request packets, it has to decide whether to authorize or reject the request. Initially, every request packet is issued capability and timeout. Every capability is valid for a period of the next T seconds and has the authority to send up to N bytes. However, if a client misbehaves by flooding unexpected packets, it is blacklisted and loses its capability. For any received packet, the server first checks its capability, and if it is a request packet with an ordered list of precapability, it generates capability by hashing precapability along with N and T, as shown in Fig. 6.Fig. 6 Capability generation by server Regular packet handling by routers The request packets accepted by the server are issued capabilities. The routers also check such regular packets for balancing the flow of authorized traffics. Every capability generated is only valid for a certain period. If the regular packets fail the check by the router or the capability expires, the packets are demoted to the low priority traffic and have to get their capability reissued by the destination. Figure 7 shows the flow diagram of the regular packet handling algorithm by the routers.Fig. 7 Verification of capability by the routers Performance evaluation In this section, the performance of our proposed model is evaluated on the basis of computational complexity, communication overhead and storage constraints. Computational complexity The primary operations for the generation of precapability in the proposed model are XoR and PUF operations. In order to evaluate the performance of the model, we have expressed time in terms of the required main operations. The notations used are tabulated in Tables 3 and 4 shows the time required for generation of precapability in the proposed model and TVA.Table 3 Notations Symbol Definition NH Time for hashing NPUF Time for PUF response NXoR Time for XoR Table 4 Computational complexity comparison between TVA and proposed model Model Precapability Capability TVA NH NH Our approach 3NPUF + 4NXoR NH Hashing takes longer time and more processing power than PUF operations and XoR operation. Hence, replacing the hash function with these inexpensive functions lowers the computational complexity our proposed approach than TVA. Storage constraints The proposed model does not impose any constraints on the routers for storing sensitive data, as they do not have to store any secret key. Moreover, the header packet only stores the path id and capabilities or precapabilities used as token for communication. On the other hand, TVA uses keyed hash function, which needs a secret key to be shared and stored. Security analysis Forgery or Tempering: The malicious end system may attempt to forge capability or manipulate the authorized access time (T) or bandwidth (N). However, capabilities cannot be spoofed if the PUFs are secure. The use of N and T in the generation of capability prevents any scope of unnoticed tampering with them. The design of PUFs is such that its output depends on the physical microstructure of the system, and it is unpredictable [38]. The PUFs are one-way functions that cannot be replicated. Finally, these functions are hard to predict but easy to construct, making them a perfect choice for lightweight devices [39]. Stealing or sharing of capability In our proposed model, the capabilities are bound with the source and destination IP addresses and the routers to reach the destination, so another sender cannot use the capability. In the case of eavesdrop, the attacker may spoof the IP address and speak for any senders. However, such a situation is also handled in our proposed model. The capabilities are rate-limited and time-bounded. Capabilities need to be renewed after a specific period, and if within authorized time sender misbehaves, they are blocklisted and demoted by the destination. Consequently, compromised senders will not be further able to send the packets. This will only hamper the compromised sender’s traffic, but other legitimate users’ communication will not get affected. Masquerading attack An eavesdrop can launch such an attack by masquerading a receiver, say a compromised router, to send attack traffic. Here, only the queue at the particular router will be affected. As the precapability of downstream routers cannot be forged, their performance will not get affected. Flooding with request Packets The malicious system may attempt to flood the routers with request packets to launch the attack. However, in our proposed system, the priority of different packets is set. The regular packets with capability have the highest priority and will be given preference. Then comes the renewal/demoted packets, and the least priority is given to the request packet. The request packets are rate-limited. It can use a maximum of 5% of the bandwidth. So flooding these packets will not affect the legacy packets. However, the new legitimate request packets may face some delay. Attacker request packet gets approved by Victim The malicious client may get access by the destination for the first request packet received. On receiving the capability and the attacker may try to flood the network. However, it can only send a limited number of bytes in an approved period, and during this time, if the sender misbehaves by exceeding its rate limit, the packets are demoted and blocklisted. Network layer attacks In the network layer, all the network routing takes place using the set protocols such as internet protocol (IP), IPSec, internet control message protocol (ICMP), etc. DDoS attacks in this layer target a computer’s network software instead of a specific port. Smurf, ICMP flood or ping of death and ping flood are some of the most common reported ddos attacks. In the proposed model, rate-limiting the request packets helps check the ping flood and smurf attacks to a great extent. Here, the destinations are allowed to control the bandwidth allocation to each sender, resulting in relatively bandwidth distribution between the colluder and the destination. A Flood of packets around the bottleneck link can cause temporary congestion due to reduced available bandwidth. Nevertheless, each legitimate user will get a lesser share of the bandwidth but will not be starved. All the transfers get complete with a few re-transmissions to finish.Fig. 8 Simulated Topology Fig. 9 Rate of (request, renewal and regular) packets delivered Simulation This section will discuss the design parameters, simulation methodology, and its limitations. We have simulated the proposed design on network simulator NS-3.33 to check the efficiency in mitigating DDoS attacks. In real-world DDoS attacks, millions of bots flood the targeted victim. However, we cannot simulate at such a level with our limited resources. So to simulate the attack scenario, we have fixed the number of nodes and scaled-down the link capacity. We have used the dumbbell topology, which consists of 10 ASes each containing 100 clients, 1 transit AS containing the bottleneck routers, and another AS containing the victim/sink, as shown in Fig. 8. The 10 ASes are connected to the destination AS through the transit AS, which consists of the bottleneck routers. Each of the 10 ASes consists of some malicious clients and a few legitimate clients. The client data rate is 1 Mbps, and the attacker Data Rate is set to 30 Mbps. Bottleneck bandwidth is set to be 1 Gbps, and non-bottleneck bandwidth is 10 Gbps. Link delay is 5–10 ms. Request packets are rate limited to 5% of the link capacity. To flood uplink request capacity by sending requests, the attacker needs to send at the rate of 50 Mbps. We checked the performance by varying the number of attackers. The results were obtained for 10%, 20%, 40% and 80% of attackers. Result and discussion The topology and parameters used for the simulation are discussed in Sect. 6. Now, as per the set conditions, the attack was launched, and the number of attackers and the rate of packets were varied to analyze the performance of the proposed network architecture. The processing time of a request packet without attack was found to be 156 ns and, under attack, 984 ns. The processing overhead of the regular packets with capability was found to be 15 ns. Figure 9 shows the throughput of different types of packets. The variation of outgoing traffic as the incoming traffic is varied is clearly plotted in the figure. The throughput of request packets was found to be the lowest under attack as it reached its bandwidth limit. However, the legitimate users having regular packets with capability did not suffer any loss due to increased traffic. The renewal packets did notice the slight loss of packets.Fig. 10 Cumulative throughput of packets with varying attack traffic With the increased attack, the legitimate request packets also get lost. The user has to keep on trying a number of times to get success. The packet loss rate can be obtained by equation 2, where Ba is the attack bandwidth and Bl is the bottleneck bandwidth.2 p=Ba-BlBa We suppose that unprivileged traffic is clogging up the network’s last ith hops and that there is a εi chance that it will be dropped at any one of those routers. Because inbound packets only often face congestion in routers during flooding attacks, we neglect the possibility that the server’s outgoing packets will be dropped. P(connect after 1 try)= (1-εi)i is the probability that a client and server would be able to establish a privileged channel after one try (by exchanging two packets), as drop probabilities at routers are independent Bernoulli trials. Let N be the connection attempts required by the client for successful connection. Then, the probability of being connected after N tries is as follows:3 P=1-(1-P(connectafter 1try))N=1-(1-(1-εi)i)N⇒N=log(1-P(connect))log(1-(1-εi)i) The logarithmic dependence of N on the connection probability implies that even for more significant εi, a new client can get the capability issued after a reasonable waiting time. Once the packet gets capability, it does not share the same traffic with the attacker. As a result, the file transfer rate is not affected by increased attack traffic. The same phenomenon is shown in Fig. 10. There is no delay time for queuing with 10% and 20% attack traffic, and file transfer is smooth and faster. Nevertheless, when the attack traffic reaches 80%, there is delay and some loss in getting request packet re-transmission, which is evident from the obtained results.Fig. 11 Performance comparison of the network with and without proposed architecture Figure 11 shows the comparison of throughput ratio with and without the proposed architecture when the attack traffic is varied from 10% to 80%. The performance of the network improved significantly with the proposed architecture. The throughput dropped to almost zero with an attack traffic of 60% in the network without the proposed architecture. Modification for IoT Since the above-proposed system changes the existing IP packet structure, IoT clients cannot handle the packet. In such cases, the IoT users need to process the packets at their end. We propose introducing a middleware between the IoT clients and the router to handle the capability packet’s overhead. The idea flow graph is as shown in Fig. 12. The IoT clients send the IP packets encapsulated in an Ethernet frame to the edge device. These IP packets will get processed here and converted into capability packets without modifying their payload. Then, these packets are classified as request or regular packet as per the validity, and it is encapsulated in ethernet frame and forwarded to the internet after Network Address Translation (NAT) handling. The edge device will first process the incoming reply packets from the server. It will store the capability if the timestamp is not expired in a hash table and convert the capability packet to an IP packet. These packets are encapsulated in an ethernet frame and sent to the IoT clients.Fig. 12 Proposed Modification for IoT users Conclusion The proposed capability-based approach for mitigating DDoS attacks addresses three challenges. Firstly, eliminate the need to share or store any secret key for capability generation, which adds to the vulnerability. Secondly, by using PUF, we can make the whole precapability generation process faster and more reliable. Finally, it is destination-driven, as the destination sets the rate of incoming data from legitimate users having capabilities. It is evident from the results discussed in Sect. 6.1 that our motive for limiting the DDoS attack is fulfilled. Most of the data transmitted from legitimate users were received with minimum time lags. The deployability of the approach at a commercial scale remains a matter of concern. In addition to this, the network configuration’s static and homogeneity help the attackers find the vulnerabilities and exploit them. Hence, to deal with such a scenario, it would be interesting if we could reverse the situation dynamically by changing the configuration of the cyber-system. Availability of data and materials Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. Declarations Conflict of interest The authors declare that they have no conflict of interest or competing interests. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent No informed consent is to be reported. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Meneghello F Calore M Zucchetto D Polese M Zanella A IoT: internet of threats? A survey of practical security vulnerabilities in real IoT devices IEEE Internet Things J. 2019 6 5 8182 8201 10.1109/JIOT.2019.2935189 2. Bhuyan MH Bhattacharyya DK Kalita JK An empirical evaluation of information metrics for low-rate and high-rate DDoS attack detection Pattern Recognit. Lett. 2015 51 1 7 10.1016/j.patrec.2014.07.019 3. 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==== Front DNP Die Neurologie & Psychiatrie 2731-8168 2731-8176 Springer Medizin Heidelberg 5546 10.1007/s15202-022-5546-8 Zertifizierte Fortbildung Erworbene Myopathien und ihre neuen Therapien Muskuläre Symptome frühzeitig erkennen und sicher behandeln Gutschmidt Kristina [email protected] 1520254793001 Schoser Benedikt 1520254793002 1520254793001 grid.5252.0 0000 0004 1936 973X Friedrich-Baur-Institut, Neurologische Klinik, Ludwig-Maximilians-Universität München, Ziemssenstr. 1a, 80336 München, Germany 1520254793002 grid.411095.8 0000 0004 0477 2585 Klinikum der Universität München, Friedrich Baur-Institut / Neurologische Klinik und Poliklinik, Ziemssenstraße 1a, 80336 München, Germany 1 12 2022 2022 23 6 5867 © Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. issue-copyright-statement© Springer Medizin Verlag GmbH, ein Teil von Springer Nature 2022 ==== Body pmcEine erhöhte Kreatinkinase, Muskelschmerzen und -schwäche sind häufige Beschwerden myopathisch Erkrankter, nicht selten manifestieren sich erworbene Myopathien durch Rhabdomyolysen. Zeitnah zu diagnostizieren und zu therapieren, kann die Prognose entscheidend verbessern. Aufgrund der Breite und Komplexität des Themas erhebt der Beitrag keinen Anspruch auf Vollständigkeit. Die wichtigsten Entitäten der erworbenen Muskelerkrankungen gliedern sich in Myositiden, endokrine und metabolisch-toxische Myopathien. Eine ausführliche Diagnostik beinhaltet neben der Erfassung eines klinischen Verteilungsmusters der Symptomatik, die Messung der Kreatinkinase (CK) und weiterer spezifischer Laborparameter wie die Antikörpertestung, die elektromyografische Untersuchung sowie eine molekulargenetische Diagnostik und gegebenenfalls eine Muskelbiopsie. Unabhängig von der Genese einer Muskelerkrankung empfiehlt sich in den meisten Fällen ein interdisziplinäres Behandlungskonzept. Das bedeutet, dass neben der kausalen Therapie eine symptomatische Begleittherapie, bestehend aus Physio- und Ergotherapie, Logopädie/Schlucktherapie, gegebenenfalls eine psychosomatische Behandlung unter anderem zur Krankheitsbewältigung sowie eine differenzierte Schmerztherapie erforderlich sein können. Auch eine Hilfsmittelversorgung und eine psychosoziale Beratung sollten frühzeitig eingeplant werden. Myositis-Syndrome Bei den Myositiden handelt es sich um eine Gruppe heterogener entzündlicher Erkrankungen der Skelettmuskulatur, wobei folgende Krankheitsbilder unterschieden werden: zum einem Autoimmunerkrankungen mit Dermatomyositis (DM, juvenile DM - jDM), immunologische Myositiden innerhalb autoimmuner Systemerkrankungen (MIRS, overlap myositis - OM), das Anti-Synthetase-Syndrom (ASyS), die Polymyositis (PM), die immunvermittelte nekrotisierende Myopathie (IMNM) und die Einschlusskörpermyositis (inclusion body myositis, IBM), zum anderen infektiöse Myositiden. Schlüsselsymptom aller Formen ist das subakute bis chronische Auftreten von Muskelschwäche und -schmerzen bei erhaltener Sensibilität und Muskeldehnungsreflexen. Als relevanteste extramuskuläre Symptomatik ist die Lungenbeteiligung bei ASyS zu vermerken, die einen erheblichen Einfluss auf Verlauf und Mortalität der Erkrankung haben können. In schwereren Fällen kann eine Dysarthrie oder eine Dysphagie das klinische Bild ergänzen [3]. Therapie in der Akutphase Kausale Behandlungen der idiopathischen Myositiden sind bislang nicht etabliert, sodass bei allen Entitäten, ausgenommen der IBM, immunsupprimierende oder immunmodulierende Medikamente als symptomatische Therapieansätze zum Einsatz kommen. In der Akutphase mit sehr ausgeprägter Symptomatik oder schwerer Organbeteiligung kann eine Stoßtherapie mit intravenösen Glukokortikoiden (250-1.000 mg Methylprednisolon täglich über 3-5 Tage) in Erwägung gezogen werden. Alternativ können bei IBM intravenöse Immunglobuline (IVIG) mit einer Dosierung von 2 g/kg Körpergewicht (KG) (z. B. 0,4 g/kg KG/Tag über 5 Tage) eingesetzt werden [2, 4]. Therapie bei mäßiger Symptomatik Die Leitlinien der Deutschen Gesellschaft für Neurologie (DGN) aus dem Jahr 2022 empfehlen, mäßig symptomatische DM, PM, OM, ASyS und IMNM initial mit oralen Glukokortikosteroiden (1 mg/kg KG) zu therapieren, bis eine klinische Besserung eintritt [2, 5]. Dies trifft in der Regel nach spätestens zwölf Wochen zu. Die Steroidtherapie bietet sich auch als Fortsetzung der intravenösen Behandlung an. Stabilisiert sich die Erkrankung hierunter oder kommt es gar zu einer Remission der Myositis, sollte spätestens nach sechs Monaten allmählich eine wöchentliche Reduktion der Dosierung um 5-10 mg oder eine alternierende Gabe erfolgen. Ziel ist es dabei, die Erkrankung weitestgehend zu supprimieren und gleichzeitig Glukokortikoid-vermittelte Nebenwirkungen wie eine Osteoporose möglichst zu vermeiden. Die Erhaltungsdosis sollte daher so niedrig wie möglich sein. Ist eine längerfristige Glukokortikoidgabe indiziert, sollte eine Osteoporose-Prophylaxe mit beispielsweise Kalzium und Vitamin D3 erfolgen. Zusätzlich kann bei langfristiger Steroidgabe eine erneute Muskelschwäche als Ausdruck einer möglichen Steroidmyopathie auftreten. Eine probatorische Dosisreduktion der Steroide unter klinischer Überwachung kann hier Aufschluss bringen. Zu empfehlende Präparate sind Prednisolon, Prednison und Methylprednisolon, während bei halogenierten Kortikoiden ein erhöhtes Risiko für die Entwicklung einer Steroidmyopathie besteht. Immunsuppressive Behandlung Als Langzeittherapie mit Rückfallprophylaxe, bei unzureichendem Ansprechen der Patientin oder des Patienten auf Kortikosteroide oder/und ausgeprägten Nebenwirkungen (z. B. entgleister Diabetes mellitus) sowie unter Umständen auch initial bei schweren und rasch progredienten Verlaufsformen sowie insbesondere der Beteiligung der Atemmuskulatur sollte ergänzend eine immunsuppressive Therapie mit Azathioprin (1-3 mg/kg KG/Tag) für einen primär angedachten Zeitraum von ein bis drei Jahren als Kombinationstherapie erfolgen [2]. Dabei ist zu berücksichtigen, dass die Wirkung des steroidsparenden Immunsuppressivums erst nach drei bis sechs Monaten einsetzt. Außerdem ist die kombinierte Gabe von Azathioprin und Allopurinol kontraindiziert. Als Alternative kann eine Behandlung mit IVIG (1-2 g/kg KG, verteilt über 1-5 Tage) versucht werden, mit Wiederholung in Abhängigkeit von der klinischen Symptomatik alle vier bis acht Wochen. Im Jahr 2013 wurde die Off-label-Behandlung mit IVIG als Add-on-Therapie bei therapierefraktärer PM und DM beschlossen. Bei schwerer Symptomausprägung oder in therapieresistenten Fällen kann über die orale Steroidgabe und/oder IVIG hinaus eines der folgenden Immunsuppressiva eingesetzt werden: Methotrexat peroral/subkutan (initial 15 mg/Woche, Zieldosis 10-25 mg/Woche), Mycophenolat mofetil peroral (2 × 1-1,5 g/d, circa 20 mg/kg KG), Ciclosporin A peroral (2,5-5 mg/kg KG/d) oder Tacrolimus peroral (1-2 × 1-3,5 mg/d; circa 0,075 mg/kg KG). Bei juveniler DM mit ausgeprägter Hautsymptomatik wird Methotrexat, bei normaler Nierenfunktion Azathioprin bevorzugt. Bei Methotrexat-Gabe ist eine zeitversetzte Substitution mit Folsäure dosisangepasst obligat; als mögliche Nebenwirkung ist hier eine Pneumonitis zu berücksichtigen, die unter Umständen nur schwer von einer interstitiellen Lungenbeteiligung zu unterscheiden ist, die innerhalb des Myositissyndroms auftreten kann. Aufgrund der variablen Resorption und der dosisabhängigen Nephrotoxizität sollte bei Ciclosporin A im Vergleich zu den anderen Immunsuppressiva verstärkt eine regelmäßige Kontrolle des Serumspiegels und eine Überprüfung der Nierenwerte erfolgen. Zeigt sich die Erkrankung unter den genannten Medikamenten weiterhin therapieresistent, besteht die Möglichkeit, zu eskalieren - mit unter anderem Cyclophosphamid oder Rituximab - beziehungsweise zusätzlich IVIG zu verabreichen. Vor allem bei schwerer Lungenbeteiligung kann eine Kombination aus Mycophenolat mofetil und Tacrolimus in Erwägung gezogen werden [2]. Um die Wirksamkeit der eingesetzten immunsupprimierenden Substanzen sicher zu beurteilen, sollte auf eine ausreichende Dosierung und Dauer geachtet werden (Tab. 1, Abb. 1). Dermatomyositis (DM), immunvermittelte nekrotisierende Myopathie (IMNM), Polymyositis (PM), Anti-Synthetase-Syndrom (ASyS), Overlap-Myositis (OM) Einschlusskörpermyositis (IBM) Initialtherapie GCS 1 mg/kg KG bei sehr ausgeprägter Symptomatik oder schwerer Organbeteiligung ggf. als Stoßtherapie mit 250-1.000 mg/d über 3-5 Tage bei schweren Verlaufsformen: GCS + Immunsuppressivum (AZA 1-3 mg/kg KG/d; MTX Zieldosis 10-25 mg/Woche) IMNM: bei Nachweis von Anti-SRP-AK RTX als Erstlinientherapie Sechsmonatiger Therapieversuch mit circa vierwöchentlichen IVIG-Infusionen (initial 2 g/kg KG, danach 1-2 g/kg KG) vor allem bei Dysphagie ggf. Fortführung bei positivem Therapieeffekt Langzeittherapie — niedrig dosierte GCS-Therapie — in Kombination mit Immunsuppressiva (MTX oder AZA) für einen Zeitraum von 1-3 Jahren oder länger Bei zunehmender Organmanifestation oder fehlendem Ansprechen: MMF, CsA, TAC Eskalationstherapie: RTX, CYC (früher Beginn bei ASyS mit interstitieller Lungenerkrankung) Einsatz von immunmodulatorischen oder immunsuppressiven Strategien nicht empfohlen Intravenöse Immunglobuline (IVIG) Therapierefraktäre Fälle: Versuch mit IVIG 1-2 g/kg KG Über 1-5 Tage verteilt, Wiederholung alle 4-6 Wochen in Abhängigkeit von der klinischen Symptomatik Als Add-on-Therapie im Off-label-Verfahren verordnungsfähig für DM und PM in Deutschland; Einzelpräparate sind zur Behandlung der aktiven Dermatomyositis zugelassen. Bei Dysphagie: experimentelle Therapieverfahren, z. B. lokale Botulinumtoxin-Injektionen, Ballondilatation oder Myotomie (Einzelfallentscheidung) Physiotherapie Abkürzungen: GCS: Glukokortikosteroide, AZA: Azathioprin, MTX: Methotrexat, Anti-SRP-AK: Anti-Signal-Recognition-particle-Antikörper, MMF: Mycophenolat-mofetil, CsA: Ciclosporin A, TAC: Tacrolimus, RTX: Rituximab, CYC: Cyclophosphamid, IVIG: intravenöse Immunglobuline Sonderfall: Einschlusskörpermyositis Die IBM ist überwiegend therapierefraktär. Bei einer fraglichen Wirksamkeit von Glukokortikosteroiden wird ein Therapieversuch mit ihnen für bis zu sechs Monaten als gerechtfertigt eingestuft. Insbesondere bei neu aufgetretener Schluckstörung sollte ein individueller Versuch mit niedrig dosierter Steroidgabe bis 10 mg täglich über etwa drei Monate erfolgen [6]. Der Einsatz von immunmodulatorischen oder immunsuppressiven Substanzen wird dagegen nicht befürwortet, da alle bisherigen überwiegend offene und keine Studien erfolgslos blieben. Da ein individuelles therapeutisches Ansprechen auf IVIG beziehungsweise eine vorübergehende Stabilisierung des Krankheitsverlaufs durch mehrere kleine nicht randomisierte, wenige randomisierte und Placebokontrollierte Studien, Expertenmeinungen und Fallberichte belegt ist, wird es als sinnvoll erachtet, eine Behandlung mit IVIG über sechs Monate zu versuchen: initial am ehesten mit 2 g/kg KG, danach mit 1 g/kg KG (evt. auch 2 g/kg KG, verteilt über 2-5 Tage bei intravenöser Gabe alle 4-6 Wochen oder mehrere subkutaner Infusionen über die Woche verteilt) (Abb. 1). Im Anschluss soll die klinische Symptomatik beurteilt und damit über eine Fortführung der Therapie entschieden werden. Wird sie fortgesetzt, ist eine regelmäßige Reevaluation indiziert. Besonders zu berücksichtigen sind hierbei die Schluckfunktion und das Gehvermögen. Ein Monitoring kann etwa anhand des 6-Minuten-Gehtests, des 10-Meter-Gehtests oder der gemessenen Zeit für ein Time-up-and-go erfolgen. Neue Medikamente mit diversen Therapieansätzen für die IBM wurden in Studien und individuellen Heilversuchen untersucht, einen klaren Durchbruch gab es bislang nicht. Studien zu Rapamycin/Sirolimus befinden sich derzeit in Planung oder sind noch nicht abgeschlossen. Bisherige Ergebnisse aus den abgeschlossenen Phase-II-Studien zu Rapamycin/Sirolimus, die mit einer Verbesserung multipler funktionell relevanter sekundärer Endpunkte nach zwölf Monaten einhergingen, sowie zu Alemtuzumab, das eine Verlangsamung der Krankheitsprogression zeigte, erwecken einen vielversprechenden Eindruck [2, 7]. Größere Studien zu Bimagrumab und Arimoclomol blieben dagegen ohne Erfolg [8]. Supportive Maßnahmen Trotz einer nur niedrigen Evizdenz wird für Myositis-Syndrome, allen voran DM und PM, eine regelmäßige Physiotherapie befürwortet. Alternativ beziehungsweise ergänzend kann ein individuelles submaximales Heimtraining eine Stabilisierung der klinischen Symptomatik bewirken. Dabei steht ein mäßiges Ausdauer- (60 %) und Krafttraining (40 %) im Vordergrund, das vor allem konzentrische Übungen wie Schwimmen, Fahrradergometer, Step-Training, Gehen oder Laufen beinhalten sollte. Ein forciertes Gewichtheben ist beim Krafttraining kontraindiziert. Stattdessen sollte mit kleinen Gewichten und einer hohen Wiederholungszahl trainiert werden. Yoga und Pilates bieten gute Ergänzungen. Die Einnahme von Kreatinmonohydrat als Nahrungsergänzungsmittel erbrachte in einer Studie mit Myositis-Patientinnen und Patienten zwar einen signifikanten Kraftzuwachs, jedoch sind die Ergebnisse aufgrund der fehlenden Kontrollgruppe und Verblindung nur eingeschränkt zu bewerten [9]. Die Wirksamkeit einer Therapie orientiert sich vor allem an der klinischen Symptomatik, unter anderem an der Ausprägung der Muskelkraft, die anhand der Medical-Research-Council(MRC)-Skala gemessen wird. Ein Monitoring des CK-Wertes ist nicht ausreichend. Für IBM-Patientinnen und Patienten spielt aufgrund möglicher progressiver Hand- und Greiffunktionseinschränkungen mit finalem Verlust der Greif- und Streckfähigkeit der Hand und Finger sowie des Schreibens speziell die Ergotherapie eine wichtige Rolle. Steht eine Schluckstörung im Vordergrund, ist neben der medikamentösen Therapie auch ein Schluckassessment (regelhaft über Fiberendoskopie) mit logopädischer Behandlung indiziert. Sehr selten kann eine Dilatation des Ösophagus oder können Botulinumtoxininjektionen in den Musculus cricopharyngeus notwendig sein. Oft muss passierte Kost erwogen werden, gegebenenfalls auch eine perkutane endoskopische Gastrostomie(PEG)-Anlage. Darüber hinaus schaffen regelmäßige Rehabilitationsmaßnahmen die Möglichkeit, ein individuelles multidisziplinäres Therapiekonzept zu erstellen, zu intensivieren und zu optimieren. Früh muss auf die langfristigen Konsequenzen der Diagnose, auf das mögliche körperliche Handicap, hingewiesen werden. Hier ist ein Einstieg in ein "Advance Care Planning" sehr hilfreich. Auf die entsprechenden Angebote der Deutschen Gesellschaft für Muskelkranke e. V., des Myositis-Netzes und des IBM-Patientenregisters sei explizit verwiesen [10, 11, 12]. Therapieassoziierte Myositiden Gesondert zu nennen ist die Gruppe der therapieassoziierten Myositiden, die heutzutage am ehesten auf den Einsatz von Immuncheckpoint-Inhibitoren zurückzuführen sind. Laut Studien mit geringer Fallzahl scheint auch hier der Einsatz von Glukokortikosteroiden, IVIG und Plasmapherese erfolgreich zu sein [13]. Schwangerschaft unter Myositiden Eine Myositis stellt keine Kontraindikation für eine Schwangerschaft dar, wobei das maternale und fetale Komplikationsrisiko zunimmt, je schlechter die Erkrankung kontrolliert ist. Eine besondere Bedeutung nimmt die geeignete Therapeutikaauswahl ein. Gemäß den EULAR-Empfehlungen (European League Against Rheumatism) gilt der Einsatz von Hydrochloroquin, Sulfasalazin, Azathioprin, Ciclosporin A, Tacrolimus und Colchicin als sicher - sowohl präkonzeptionell als auch während der Schwangerschaft. Bei aktiver Erkrankung wird schwangeren Frauen die Gabe von Methylprednisolon oder IVIG empfohlen [2]. Impfempfehlungen unter Immunsuppression Grundsätzlich gilt für Impfungen von Patientinnen und Patienten mit Myositis, dass Lebendimpfstoffe unter einer immunsuppressiven Therapie kontraindiziert sind. Davon abgewichen werden kann, wenn keine immunsupprimierenden Substanzen eingenommen werden oder die Impfung bis zu vier Wochen vor Beginn der Behandlung gegeben wird [14]. Myositis-Betroffene sollten die Standardimpfungen gemäß Robert-Koch-Institut (RKI) erhalten, zusätzlich den Totimpfstoff gegen Herpes zoster (im Abstand von zwei bis maximal sechs Monaten) sowie die Impfung gegen die saisonale Influenza und gegen Pneumokokken als sequenzielle Impfung. Bezüglich einer Impfung gegen COVID-19 gelten die regulären Empfehlungen für Personen mit einer chronisch-entzündlichen Erkrankung beziehungsweise einer Immunsuppression [2, 15]. Da die Antikörperbildung unter Rituximab stark vermindert ist, sollten notwendige Impfungen entweder mindestens vier Wochen vor einer immunsuppressiven Therapie abgeschlossen sein, oder fünf Monate nach Beendigung der Rituximab-Gabe durchgeführt werden. Infektiöse Myositiden In seltenen Fällen können Myositiden auch durch infektiöse Erreger bedingt sein. Darunter fallen Bakterien (vor allem Streptokokken), Viren (Influenza A/B, Coxsackie-Viren, Parainfluenza-, Echo-, RSV-, EBV-, Adeno- und Arboviren, HIV), Pilze (insbesondere Candida) oder Parasiten (etwa Trichinella spiralis, Echinokokken, Toxoplasma gondii, Taenia solium oder Toxocara canis). Die Betroffenen sind meist immunsupprimiert oder weisen offene Muskelverletzungen auf. Sie beklagen oft lokale Beschwerden, wobei generalisierte Myalgien am häufigsten auftreten. Gelegentlich kann es zu einer kardialen Beteiligung mit vitaler Bedrohung kommen. Therapeutisch steht die Behandlung der Grunderkrankung im Vordergrund, also eine adäquate Antibiose, eine antivirale Therapie (derzeit nur für HIV und Herpesviren verfügbar) oder antiparasitäre Maßnahmen, wobei bei vorherigem Auslandsaufenthalt eine mögliche regionale Medikamentenresistenz beachtet werden sollte. Gegebenenfalls muss zusätzlich eine chirurgische Sanierung des Infektionsherdes erfolgen [16]. Endokrine Myopathien Hormone beeinflussen in komplexen Vorgängen den Stoffwechsel der Muskulatur, weshalb ein Ungleichgewicht speziell im Hormonhaushalt der Schilddrüse, der Nebenschilddrüse, der Nebenniere sowie der Hypophyse zu Beeinträchtigungen des Energiestoffwechsels und damit sekundär zu Muskelfunktionsstörungen führen kann [17]. Im Vordergrund der Therapie steht der Ausgleich des hormonellen Ungleichgewichts und dementsprechend die Behebung der zugrundeliegenden endokrinologischen Störung. Bei erfolgreicher Behandlung ist die muskuläre Symptomatik in der Regel reversibel. Maßgeblich ist hierfür ein rascher Beginn der therapeutischen Maßnahmen, da die Fähigkeit zur Rehabilitation durch die Dauer der Grunderkrankung und damit durch das Ausmaß der Muskelschädigung beeinflusst wird. Myopathien bei Nebennierenrindeninsuffizienz Klinische Landmarken einer Myopathie bei Nebenniereninsuffizienz sind allgemeine Müdigkeit, Belastungsinsuffizienz und eine generalisierte Muskelschwäche. Gelegentlich werden auch Muskelkrampi, Myalgien und Muskelkontrakturen genannt. Im Vordergrund stehen jedoch die typischen Symptome bei Morbus Addison. Neben der Behandlung der Grunderkrankung kann eine Substitution von Mineralo- oder Glukokortikoiden sinnvoll sein. Myopathien bei Schilddrüsendysfunktion Da Schilddrüsenfunktionsstörungen häufig sind, haben die daraus resultierenden muskulären Funktionsstörungen eine hohe Relevanz in der medizinischen Praxis. Allgemein wird zwischen einer hyperthyreoten (thyreotoxischen) und einer hypothyreoten Stoffwechsellage unterschieden. Innerhalb einer thyreotoxischen Myopathie tritt bei bis zu 80 % der Patientinnen und Patienten eine Muskelschwäche auf, wohingegen sich bei einer hypothyreotisch ausgelösten Myopathie eine Muskeltonuserhöhung abzeichnet, die von pseudomyotonen Zeichen wie verlangsamter Kontraktion und Erschlaffung begleitet wird. Mit einer niedrigen Inzidenz von 0,1-0,2 % können Menschen während einer thyreotoxischen Krise in seltenen Fällen eine periodische Paralyse (TTP) entwickeln [18]. Diese sind als rezidivierende Episoden von Paralysen und Hypokaliämie definiert. Hierbei ist zu beachten, dass die Lähmungsepisoden mitunter Monate bis Jahre vor Diagnosestellung einer Hyperthyreose auftreten können. Wie bei Myopathien innerhalb einer Nebennierenrindeninsuffizienz besteht die Therapie aus der Behandlung der endokrinologischen Grunderkrankung mit thyreostatischer Medikation, Radioiodtherapie oder operativer Intervention [19]. Der Einsatz von Glukokortikoiden kann bei thyreotoxischer Lage die Umwandlung von T4 in T3 blockieren und somit günstig auf den Krankheitsverlauf einwirken (Tab. 2). Zur Unterstützung können sowohl bei Hyperthyreose als auch bei TTP nicht selektive Betablocker eingesetzt werden [20]. Patientinnen und Patienten mit TTP profitieren unter Umständen ebenfalls von kaliumsparenden Diuretika beziehungsweise von einer direkten Kaliumzufuhr. Da bei intravenöser Gabe die Gefahr einer Verteilungsstörung mit Rebound-Hyperkaliämie besteht, sollte der Elektrolytausgleich oral erfolgen [20]. Trotz adäquater Substitution können bis zu 33 % der Betroffenen mit Hypothyreose im Gegensatz zu Menschen mit Hyperthyreose nach einem Jahr noch Residualsymptome beklagen [18].Myopathie bei Pathogenese Therapie Anmerkung Schilddrüsen- dysfunktion Hyperthyreote (thyreotoxische) Stoffwechsellage Thyreostatische Medikation, Radioiodtherapie, operative Intervention Thyreotoxische Lage: ggf. Blockade der Umwandlung von T4 in T3 durch Einsatz von Glukokortikoiden Hypothyreote Stoffwechsellage Substitution der Schilddrüsenhormone Nebenschilddrüsen-dysfunktion Primärer Hyperparathyreoidismus Operative Intervention (u. a. Entfernung eines Nebenschilddrüsenadenoms) Hyperkalzämische Krise: NaCl-Infusion, Bisphosphonate, Furosemid, Steroide Sekundärer Hyperparathyreoidismus Behandlung der Grunderkrankung (renal, intestinal), phosphatarme Diät, Vitamin-D-Substitution Primärer und sekundärer Hypoparathyreoidismus Gabe von Kalzium- und Vitamin-D-Präparaten Steroidmyopathie Endogen: gesteigerte Freisetzung von ACTH oder Nebennierenrindenhormonen Behandlung der Grunderkrankung, Absetzen oder Dosisreduktion der Steroide Derzeit Gegenstand der Forschung: IGF-1, Glutamin, Kreatin-Supplementation Exogen: therapeutischer Einsatz von Glukokortikoiden Hypophysen- dysfunktion Über sekundäre Schilddrüsen- und Nebennierenrindeninsuffizienz Behandlung der Grunderkrankung NaCl: Natriumchlorid, ACTH: Adrenocorticotropes Hormon, IGF: Insulin-like growth factor Myopathien bei Nebenschilddrüsendysfunktion Ein Hyperparathyreoidismus kann mit einer proximalen Muskelschwäche und einer muskulären Belastungsintoleranz sowie gelegentlich auch positiven Pyramidenbahnzeichen einhergehen. Zu beachten ist eine mögliche Beteiligung der Atem- und Herzmuskulatur. Die Therapie besteht aus der Normalisierung des Parathormons und demzufolge auch der Kalziumwerte. Das kann im Falle eines primären Hyperparathyreoidismus die operative Entfernung eines Nebenschilddrüsenadenoms bedeuten sowie aller Nebenschilddrüsen bei Hyperplasie oder die Entfernung aller Epithelkörperchen beim schweren neonatalen Hyperparathyreoidismus (NSHPT); in Einzelfällen kann bis zur definitiven Therapie die Verwendung eines Kalziummimetikums in Erwägung gezogen werden. In der hyperkalzämischen Krise stehen die NaCl-Infusion sowie die Gabe von Bisphosphonaten (Pamidronat/Neridronat) sowie von Furosemid, Steroiden und eine kalziumfreie Kost im Mittelpunkt [21]. Liegt ein sekundärer Hyperparathyreoidismus vor, sollte neben einer ursächlichen Behandlung (renal, intestinal) eine phosphatarme Diät verschrieben werden und eine Substitution mit Vitamin D erfolgen (Tab. 2) [4]. Der primäre und sekundäre Hypoparathyreoidismus gehen mit einer erhöhten neuromuskulären Erregbarkeit einher, die sich mit Tetanie und episodischen Parästhesien präsentiert. Therapeutisch ist die Gabe von Kalzium- und Vitamin-D-Präparaten hilfreich (Tab. 2) [22]. Myopathien bei Hypophysendysfunktion Im Rahmen eines Hypophysenadenoms, das eine Akromegalie verursacht, kann es bei etwa 50 % der Erkrankten zu einer langsam progredienten proximalen Muskelschwäche kommen, mit zum Teil Myalgien und einem beidhändigen Karpaltunnelsyndrom. Werden die erhöhten Werte der Wachstumshormone normalisiert, bilden sich diese allerdings wieder zurück. Eine Hypophyseninsuffizienz kann über sekundär ausgelöste Schilddrüsen- und Nebennierenrindeninsuffizienzen zur endokrinen Myopathie führen. Den wichtigsten Stellenwert besitzt die Behandlung der zugrundeliegenden Erkrankung. Steroidmyopathie Die Steroidmyopathie ist die häufigste endokrine Myopathie und kann zum Einen endogen durch eine gesteigerte Freisetzung von Adrenokortikotropes Hormon (ACTH) oder endogenen Nebennierenrindenhormonen, zum anderen exogen/iatrogen durch den therapeutischen Einsatz von Glukokortikoiden mit einer Myopathie-Inzidenz von 20-60 % bei einer Äquivalenzdosis von > 10 mg/kg KG täglich für mindestens drei Monate ausgelöst werden [23, 24]. Insbesondere fluorierte Kortikosteroide wie Dexamethason und Triamcinolon sind bei oraler Einnahme mit einem erhöhten Risiko für eine Myopathie vergesellschaftet als die nicht fluorierten Steroide Prednisolon und Hydrocortison. Klinisch betroffen ist vor allem die proximale Muskulatur der unteren Extremitäten, gelegentlich berichten die Patientinnen und Patienten über Myalgien und Krampi. Eine endogene Steroidmyopathie wird durch die Therapie der Grunderkrankung behandelt. Essenziell für die exogen verursachte Myopathie ist daher das Absetzen beziehungsweise die Dosisreduktion der Glukokortikoide. Grundsätzlich sollte bei therapeutischem Einsatz von Kortikosteroiden eine möglichst geringe Dosis verwendet werden; weiterhin sollte frühzeitig über den Beginn einer immunsuppressiven Behandlung diskutiert werden, um Glukokortikoide "einzusparen". Bei zeitnaher Diagnosestellung, entsprechenden therapeutischen Maßnahmen und physiotherapeutischem Training sind die Symptome in der Regel reversibel. Folgende Substanzen sind derzeit Teil der wissenschaftlichen Forschung und ihre Wirksamkeit ist noch eindeutig zu belegen : Insulin-like growth factor 1 (IGF-1), Glutamin, Kreatin-Supplementation (Tab. 2) [25]. Exogen-toxische Myopathien Unter diesem Begriff werden jene Muskelerkrankungen zusammengefasst, die durch die Anwendung von Arzneimitteln oder durch die Zuführung von Noxen wie Alkohol und anderen Suchtmitteln beziehungsweise durch direkte physikalische Einflüsse, wie eine intramuskuläre Injektion, zu einer Schädigung der Muskulatur führen können [17]. Häufig ist jedoch der Ernährungszustand entscheidend, sodass eine vorhandene Mangelernährung zeitgleich ausgeglichen werden muss. Die häufigste Form einer exogen-toxischen Myopathie ist nach wie vor durch Alkohol induziert und kann sich bei Karenz, Muskeltraining und Verbesserung der Ernährungssituation rückläufig verhalten - im Gegensatz zu einer ethyltoxisch bedingten Polyneuropathie. Darüber hinaus sollten aus medikamentös-toxischer Sicht zwei Medikamente aus einer Vielzahl an Arzneimitteln, die eine Myopathie verursachen können, hervorgehoben werden: Steroide (siehe oben) und Statine. In beiden Fällen ist eine Dosisreduktion anzustreben. Sofern die muskuläre Symptomatik auch mit niedrig dosierten Statinen unbeherrschbar bleibt, sind gegebenenfalls ein Absetzen der Lipidsenker sowie eine Lipidapherese indiziert. Mehrere Studien konnten ein erhöhtes Risiko für das Auftreten einer Rhabdomyolyse unter Statin-Medikation nachweisen, vor allem in Kombination mit Fibraten [26]. Behandlung einer Rhabdomyolyse Die Rhabdomyolyse (RML) gilt grundsätzlich als die schwerwiegendste Form einer toxischen Myopathie. Nicht selten manifestiert sich auch eine hereditäre Myopathie erstmals durch das wiederholte Auftreten einer RML. In der Regel wird bereits bei akuten CK-Werten > 1.000 IU/l oder Werten über das Fünffache des oberen Grenzwertes und gleichzeitigem Ausschluss myokardialer, renaler oder zerebraler Ereignisse von einer milden RML gesprochen [26]. Klinisch präsentieren sich Patientinnen und Patienten je nach Schweregrad mit mindestens einem der folgenden Symptome: Muskelschmerzen, Muskelschwäche, Schwellung der Muskulatur sowie eventuell dunkel gefärbtem Urin. Begleitend können systemische Symptome wie Fieber, allgemeines Unwohlsein, Übelkeit und Erbrechen auftreten. Therapeutisch stehen drei Aspekte im Vordergrund [27, 28]: 1. die kausale Behandlung der auslösenden Faktoren, 2. die frühe forcierte Flüssigkeitssubstitution mit am ehesten NaCl-Lösung 0,9 % zum Erhalt der Nierenfunktion und zur Vermeidung eines sekundären akuten Nierenversagens (ANV) und 3. die frühzeitige Berücksichtigung und Prävention potenziell einhergehender lebensgefährlicher Komplikationen wie Herzrhythmusstörungen durch Elektrolytverschiebungen [29]. Die Empfehlungen zur Laufgeschwindigkeit der Kochsalzlösung variieren zwischen 0,5 l/h [30] und 1,5 l/h [31]. Darüber hinaus wird die Verwendung von isotonen Elektrolytlösungen oder einer 5 %-Glukose-Lösung diskutiert, während der positive Effekt einer ergänzenden Verabreichung von Natriumbicarbonat (bspw. 50 mmol alle 2-3 l) umstritten bleibt [32]. Ebenso weist der Einsatz von Mannitol zur Abwendung eines ANV nur eine geringe klinische Evidenz auf, kann jedoch in Einzelfällen versuchsweise gegeben werden [33, 34]. Fazit für die Praxis Eine frühzeitige Diagnostik erworbener Myopathien kann den klinischen Verlauf prognostisch positiv beeinflussen. Die richtige Diagnose ermöglicht einerseits eine adäquate Behandlung der Grunderkrankung, die oftmals im endokrinen Bereich zu suchen ist, andererseits auch den gezielten Einsatz spezifischer Medikamente für die kausale Therapie. So ist teilweise eine vollständige Remission möglich. Vor allem im Rahmen der Myositiden steht eine große Bandbreite an Therapeutika zur Verfügung - von Glukokortikosteroiden bis hin zu immunmodulatorischen und immunsupprimierenden Medikamenten. Weil ihre Behandlung je nach myositischer Unterform teilweise stark variieren kann, ist eine differenzialdiagnostische Beurteilung unter anderem auch mithilfe der entsprechenden Antikörperdiagnostik wichtig. So gelingt eine genaue Zuordnung zu den jeweiligen Entitäten, und damit auch eine bestmögliche Versorgung der Patientin und des Patienten. Trotz bestehender und bewährter Basistherapien unterliegen erworbene Myopathien ebenfalls einem fortschrittlichen, wissenschaftlichen Interesse. Daher sollten insbesondere bei therapierefraktären Verläufen auch innovative Ansätze verfolgt werden. Konstanter Bestandteil jeglichen therapeutischen Konzepts bei neuromuskulären Erkrankungen bleiben jedoch physiotherapeutische Einheiten (Abb. 2). Ergänzende Informationen sind sowohl in den neuromuskulären Zentren sowie auf den Webseiten der Deutschen Gesellschaft für Muskelkranke e. V. erhältlich (www.dgm.org). Darüber hinaus können sowohl Ärztinnen und Ärzte als auch Patientinnen und Patienten Informationen über Diagnostik, Therapie, Beratung oder wissenschaftliche Fortschritte in den entsprechenden Registern sammeln, wie im IBM-Patientenregister unter www.ibm-register.de oder unter www.myositis-netz.de/patienten-diagnosegruppe-myositis. Dr. med. Kristina Gutschmidt Friedrich-Baur-Institut, Neurologische Klinik, Interdisziplinäres Zentrum für Neuromuskuläre Erkrankungen Klinikum der Universität München Ludwig-Maximilians-Universität München Ziemssenstr. 1a, 80336 München [email protected] Prof. Dr. med. Benedikt Schoser Friedrich-Baur-Institut, Neurologische Klinik, Interdisziplinäres Zentrum für Neuromuskuläre Erkrankungen Klinikum der Universität München Ludwig-Maximilians-Universität München Ziemssenstr. 1a, 80336 München [email protected] CME-Fragebogen Erworbene Myopathien und ihre neuen Therapien Teilnehmen und Punkte sammeln können Sieals e.Med-Abonnentin von SpringerMedizin.de als registrierter Abonnent*in dieser Fachzeitschrift zeitlich begrenzt unter Verwendung der abgedruckten FIN. Dieser CME-Kurs ist auf SpringerMedizin.de/CME zwölf Monate verfügbar. Sie finden ihn, wenn Sie die FIN oder den Titel in das Suchfeld eingeben. Alternativ können Sie auch mit der Option "Kurse nach Zeitschriften" zum Ziel navigieren oder den QR-Code links scannen. Dieser CME-Kurs wurde von der Bayerischen Landesärztekammer mit zwei Punkten in der Kategorie I (tutoriell unterstützte Online- Maßnahme) zur zertifizierten Fortbildung freigegeben und ist damit auch für andere Ärztekammern anerkennungsfähig. Für eine erfolgreiche Teilnahme müssen 70 % der Fragen richtig beantwortet werden. Pro Frage ist jeweils nur eine Antwortmöglichkeit zutreffend. Bitte beachten Sie, dass Fragen wie auch Antwortoptionen online abweichend vom Heft in zufälliger Reihenfolge ausgespielt werden. Bei inhaltlichen Fragen erhalten Sie beim Kurs auf SpringerMedizin.de/CME tutorielle Unterstützung. Bei technischen Problemen erreichen Sie unseren Kundenservice kostenfrei unter der Nummer 0800 7780777 oder per Mail unter [email protected]. Welches Medikament setzen Sie bei einem 45-jährigen Menschen in der Akutphase einer Myositis ein? Ciclosporin A Rituximab Azathioprin Methylprednisolon Etanercept Welche Nebenwirkung von Glukokortikoiden ist bei mittel- und langfristiger Gabe zu beachten? Elektrolytverschiebungen Osteoporose Fieber Sensibilitätsstörung Inkontinenz Nach welchem Zeitraum etwa setzt die Wirkung von Azathioprin ein? nach drei bis sechs Monaten sofort nach ein bis zwei Wochen nach drei bis fünf Jahren nach ein bis zwei Jahren Welche der Myositiden gilt am häufigsten als therapierefraktär? Dermatomyositis Myositis im Rahmen immunologischer Systemerkrankungen Einschlusskörpermyositis immunvermittelte nekrotisierende Myopathie infektiöse/erregerbedingte Myositis Welche Impfung ist unter einer immunsuppressiven Therapie kontraindiziert? Hepatitis B Tetanus Keuchhusten Diphterie Masern Welches Medikament würden Sie bei einem 57-jährigen Menschen mit thyreotoxischer Myopathie einsetzen, um die Umwandlung von T4 in T3 zu blockieren? L-Thyroxin Glukokortikoide Betablocker Carbimazol Iod Welches Medikament/Nahrungsergänzungsmittel würden Sie bei Vorliegen eines sekundären Hypoparathyreoidismus verordnen? Kalium Magnesium Glukokortikoide Adrenalin Vitamin D Wobei handelt es sich definitionsgemäß neben einer endogenen auch um eine exogene Myopathie? Myopathie bei Schilddrüsenüberfunktion Myopathie bei Schilddrüsenunterfunktion Myopathie bei Nebenschilddrüsendysfunktion Steroidmyopathie Myopathie bei Hypophysendysfunktion Was wäre bei einer Patientin oder einem Patienten, die oder der über akut aufgetretene Muskelschmerzen, Muskelschwäche, Schwellung der Muskulatur und dunkel gefärbten Urin klagt, Ihr primäres therapeutisches Vorgehen, nach der kausalen Behandlung der auslösenden Faktoren? forcierte Flüssigkeitssubstitution mit NaCl 0,9 % Gabe intravenöser Immunglobuline (IVIG) Glukokortikoid-Stoßtherapie Abwarten Antibiose Der Einsatz welches Medikamentes kann zur Abwendung eines akuten Nierenversagens beitragen? Intravenöse Immunglobuline Vitamin D Mannitol Propafenon Modafinil Interessenkonflikt Die Autorin und der Autor erklären, dass sie sich bei der Erstellung des Beitrages von keinen wirtschaftlichen Interessen leiten ließen. Sie legen folgende potenzielle Interessenskonflikte offen: K. Gutschmidt: keine, B. Schoser: keine. Der Verlag erklärt, dass die inhaltliche Qualität des Beitrags in zwei unabhängigen Gutachten geprüft wurde. Werbung in dieser Zeitschriftenausgabe hat keinen Bezug zur CME-Fortbildung. Der Verlag garantiert, dass die CME-Fortbildung sowie die CME-Fragen frei sind von werblichen Aussagen und keinerlei Produktempfehlungen enthalten. Dies gilt insbesondere für Präparate, die zur Therapie des dargestellten Krankheitsbildes geeignet sind. ==== Refs Literatur 1. Stenzel W, Goebel HH, Aronica E. Review: immune-mediated necrotizing myopathies--a heterogeneous group of diseases with specific myopathological features. Neuropathol Appl Neurobiol. 2012;38(7):632-46 2. Wiendl H. SJea. Deutsche Gesellschaft für Neurologie (Hrsg.), S2k-Leitlinien für Diagnostik und Therapie in der Neurologie - Myositissyndrome. In: Neurologie KLdDGf, editor. Deutsche Gesellschaft für Neurologie 2022 3. Day JA, Limaye V. Immune-mediated necrotising myopathy: A critical review of current concepts. Semin Arthritis Rheum. 2019;49(3):420-9 4. Stephan Z. Muskelerkrankungen: Georg Thieme Verlag KG; 2014 5. Carstens PO, Schmidt J. Diagnosis, pathogenesis and treatment of myositis: recent advances. Clin Exp Immunol. 2014;175(3):349-58 6. Schoser B. Inflammatory myopathies. Z Rheumatol. 2009;68(8):665-75; quiz 76-7 7. Dalakas MC, Rakocevic G, Schmidt J, Salajegheh M, McElroy B, Harris-Love MO et al. Effect of Alemtuzumab (CAMPATH 1-H) in patients with inclusion-body myositis. Brain. 2009;132(Pt 6):1536-44 8. Hanna MG et al. Safety and efficacy of intravenous bimagrumab in inclusion body myositis (RESILIENT): a randomised, double-blind, placebo-controlled phase 2b trial. Lancet Neurol. 2019;18(9):834-44 9. Tarnopolsky M, Martin J. Creatine monohydrate increases strength in patients with neuromuscular disease. Neurology. 1999;52(4):854-7 10. www.ibm-registry.org/de/ 11. www.dgm.org/ 12. www.myositis-netz.de/ 13. Solimando AG et al. Immune Checkpoint Inhibitor-Related Myositis: From Biology to Bedside. Int J Mol Sci. 2020;21(9):3054 14. www.rki.de/SharedDocs/FAQ/Impfen/AllgFr_Kontraindi/FAQ01.html 15. www.dgrh.de/Start/Wissenschaft/Forschung/COVID-19/Impfung-gegen-SARS-CoV2.html 16. DGM-Handbuch Myositis - Ein Patientenratgeber. In: e.V. DDGfM, editor. 2020 17. Katzberg HD, Kassardjian CD. Toxic and Endocrine Myopathies. Continuum (Minneap Minn). 2016;22(6, Muscle and Neuromuscular Junction Disorders):1815-28 18. Wenninger S, Schoser B. Congenital and endogenous endocrine myopathy. Z Rheumatol. 2011;70(9):760-2, 4-6 19. Sindoni A, Rodolico C, Pappalardo MA et al. Hypothyroid myopathy: A peculiar clinical presentation of thyroid failure. Review of the literature. Rev Endocr Metab Disord. 2016;17(4):499-519 20. Hahn S, Rudorff KH, Saller B, Mann K. Thyreotoxic hypokalemic paralysis; presentation of symptoms in three case reports. Internist (Berl). 2001;42(5):748-55 21. S1-Leitlinie - Primärer Hyperparathyreoidismus. In: e.V. DGfKu-dD, editor. 2016 22. S1-Leitlinie - Hypoparathyreoidismus. In: e.V DGfKu-dD, editor. 2016 23. Batchelor TT, Taylor LP, Thaler HT, Posner JB, DeAngelis LM. Steroid myopathy in cancer patients. Neurology. 1997;48(5):1234-8 24. Haran M, Schattner A, Kozak N, Mate A, Berrebi A, Shvidel L. Acute steroid myopathy: a highly overlooked entity. QJM. 2018;111(5):307-11 25. Pereira RM, Freire de Carvalho J. Glucocorticoid-induced myopathy. Joint Bone Spine. 2011;78(1):41-4 26. Vinci P, Panizon E, Tosoni LM et al. Statin-associated Myopathy: emphasis on mechanisms and targeted therapy. Int J Mol Sci. 2021;22(21):11687 27. Stahl K, Rastelli E, Schoser B. A systematic review on the definition of rhabdomyolysis. J Neurol. 2020;267(4):877-82 28. Chavez LO, Leon M, Einav S, Varon J. Beyond muscle destruction: a systematic review of rhabdomyolysis for clinical practice. Crit Care. 2016;20(1):135 29. Sauret JM, Marinides G, Wang GK. Rhabdomyolysis. Am Fam Physician. 2002;65(5):907-12 30. Zimmerman JL, Shen MC. Rhabdomyolysis. Chest. 2013;144(3):1058-65 31. Zutt R, van der Kooi AJ, Linthorst GE, Wanders RJ, de Visser M. Rhabdomyolysis: review of the literature. Neuromuscul Disord. 2014; 24(8):651-9 32. Bosch X, Poch E, Grau JM. Rhabdomyolysis and acute kidney injury. N Engl J Med. 2009;361(1):62-72 33. Better OS, Rubinstein I. Management of shock and acute renal failure in casualties suffering from the crush syndrome. Ren Fail. 1997;19(5): 647-53 34. Gunal AI et al. Early and vigorous fluid resuscitation prevents acute renal failure in the crush victims of catastrophic earthquakes. J Am Soc Nephrol. 2004;15(7):1862-7	0	PMC9713102	NO-CC CODE	2022-12-02 23:22:07	no	DNP. 2022 Dec 1; 23(6):58-67	utf-8	null	null	null	oa_other
==== Front Datenschutz Datensich Datenschutz und Datensicherheit - DuD 1614-0702 1862-2607 Springer Fachmedien Wiesbaden Wiesbaden 1695 10.1007/s11623-022-1695-3 Schwerpunkt KI-Biases im Gesundheitswesen – Teil 1 (Terminologie und Typologie) Wolff Daniel LL.M. (Yale) [email protected] Juniorprofessor für Öffentliches Recht an der Universität Augsburg grid.7307.3 0000 0001 2108 9006 Universität Augsburg, Augsburg, Deutschland 30 11 2022 2022 46 12 733738 © Springer Fachmedien Wiesbaden GmbH 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Der Einsatz von künstlicher Intelligenz im Gesundheitswesen soll dort zu mehr Gleichheit führen, bewirkt bisweilen aber genau das Gegenteil. Dieser Aufsatz behandelt das damit angesprochene Problem von KI-Biases. Während der vorliegende erste Teil eine Bias-Typologie herausarbeitet, widmet sich der in in Heft 01/2023 erscheinende zweite Teil der unionsrechtlichen Adressierung der Bias-Problematik. issue-copyright-statement© Der/die Autor(en), exklusiv lizenziert an Springer Fachmedien Wiesbaden GmbH, ein Teil von Springer Nature 2022 ==== Body pmc	0	PMC9713104	NO-CC CODE	2022-12-02 23:22:07	no	Datenschutz Datensich. 2022 Nov 30; 46(12):733-738	utf-8	null	null	null	oa_other
==== Front Curr Psychol Curr Psychol Current Psychology (New Brunswick, N.j.) 1046-1310 1936-4733 Springer US New York 4046 10.1007/s12144-022-04046-2 Article Parental attachment and cyberbullying among college students: the mediating role of loneliness and the moderating role of interdependent self Fang Yong 12 Fan Cuiying [email protected] 1 Cui Jia [email protected] 13 Zhang Xuechen 1 Zhou Ting 14 1 grid.411407.7 0000 0004 1760 2614 School of Psychology, Central China Normal University, Wuhan, 430079 Hubei Province China 2 grid.257143.6 0000 0004 1772 1285 School of Nursing, Hubei University of Chinese Medicine, Wuhan, 430065 Hubei Province China 3 grid.412787.f 0000 0000 9868 173X Department of Student Affairs, Wuhan University of Science and Technology, Wuhan, 430065 Hubei Province China 4 grid.410654.2 0000 0000 8880 6009 School of Education and Sports Science, Yangtze University, Jingzhou, 434023 Hubei Province China 1 12 2022 19 16 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Although considerable research has shown that attachment is a protective factor for cyberbullying, little research has investigated the mechanisms underlying this relationship among college students. This study examined whether loneliness mediates the association between parental attachment and cyberbullying and whether this process is moderated by interdependent self. A sample of 1125 college students (Mage = 19.14, SDage = 1.52 years) in China completed a questionnaire measuring parental attachment, cyberbullying, loneliness, and interdependent self. Loneliness partially mediated the relationship between parental attachment and cyberbullying. Moreover, the mediation effect of loneliness was moderated by interdependent self. A moderated mediation analysis further revealed that interdependent-self moderated the predictive effect of loneliness on cyberbullying. Specifically, the predictive effect of loneliness on cyberbullying was only significant among college students with low interdependent self. The study highlights the complex nature of the association between parental attachment and cyberbullying. These findings provide new perspectives for intervention and prevention of cyberbullying among college students. Keywords Parental attachment Cyberbullying Loneliness Interdependent self Humanities and Social Sciences Research Funds from the Ministry of Education of ChinaProject no. 21JDSZ3067 Fang Yong Opening Research Program Funds of Key Laboratory of Adolescent Cyberpsychology and Behavior (Central China Normal University), Ministry of EducationGrant Number 2019B05 Fan Cuiying ==== Body pmcIntroduction With the rapid development of an internet-based society, electronic communication technology has had a profound effect on people’s lives. According to the 49th China Statistical Report on Internet Development, by December 2021, the number of internet users in China had reached 1.032 billion (China Internet Network Information Center, 2021). However, with this growth come several problems that require urgent attention. One such problem is cyberbullying (Jing et al., 2017). Cyberbullying refers to the behavior of individuals or groups who repeatedly bully vulnerable individuals or groups via digital media for the purpose of causing harm (Tokunaga, 2010). Research has revealed that cyberbullying can be divided into seven categories: flaming, online harassment, cyberstalking, denigration, masquerading, outing, and exclusion (Li, 2007). Cyberbullying is closely related to individual negative emotions, such as depression, anxiety, and loneliness. It also has negative effects on social interaction and behavior, leading to psychosocial maladjustment and behavioral disorders among cyberbullying victims, perpetrators, and even bystanders (Kowalski et al., 2014). Most research on cyberbullying to date has emphasized cyberbullying behavior in middle and high school students (Cassidy et al., 2013), whereas relatively fewer studies have investigated college students. However, previous studies on cyberbullying among college students have found prevalence rates of cyberbullying around 10%–15% (Hemphill et al., 2012; Hinduja & Patchin, 2010; Schenk & Fremouw, 2012). Moreover, the incidence rates may even be higher (Gibb & Devereux, 2014; Selkie et al., 2015). In China, students in junior and senior school bear the pressure of further study compared with college students. Some surveys in China have shown that cyberbullying among college students is an emerging issue warranting attention (Chen et al., 2020; Leung et al., 2018; Wang et al., 2019). In addition, previous studies have analyzed the causes of cyberbullying from the perspective of parental attachment, which is helpful for the development of effective intervention programs (Paez, 2020; Zhu et al., 2021). However, for Chinese college students, there are few studies verified the relationship between parental attachment and cyberbullying, as well as its associated psychological factors (e.g., a specific emotion, one type of self) and mechanisms. The current study aims to fill this gap. Parental attachment and Cyberbullying Family is the primary place for individual socialization and development, and the family system plays a key role in individual psychological and behavioral development (Bronfenbrenner, 1979). Among the many related factors in the family system, the level of attachment between individual and parents is a key factor, and the relationship between parental attachment and problematic behavior has remained an important research topic in clinical psychology (Frits et al., 2000). Parental attachment refers to the emotional connection and bond between an individual and their parents, which affects the quality of interpersonal relationships across the life span (Pallini et al., 2014). Social control theory, also known as “social key theory” or “social bonding theory”, suggests that the attachment component is a primary “social key”; that is, the stronger the emotional attachment between individuals and people around them, especially their parents, the more likely they are to feel that people around them are friendly and trustworthy. Strong social relationships allow them to realize the need for autonomy, further leading to less problem behavior (Hirschi, 1969). However, poor social connections, and especially unhealthy parental attachment, can disrupt individual self-affirmation and have negative effects on interpersonal relations and increase the risk of problematic behavior (Pallini et al., 2014). Several empirical studies have demonstrated a significant negative correlation between parental attachment levels and individual problem behavior (Chen et al., 2015) and that individuals with low parental attachment levels show more bullying behaviors compared with individuals with high levels of parental attachment (Chang et al., 2015). Cyberbullying is an extension of traditional bullying (Smith, 2015). The influence of the individual family system on bullying behavior is thought to extend from the real world to the online world. Studies have shown that family upbringing style (He et al., 2016) and parental psychological control (Fan et al., 2017) predict individual cyberbullying. For adolescents, some studies have emphasized the need to ensure strong, positive parent–child attachment to enhance cyberbullying prevention efforts (e.g., Paez, 2020; Zhu et al., 2021). However, in general, the number of studies on this relationship among young adults is small, especially for Chinese college students (Canestrari et al., 2021; Martínez-Monteagudo et al., 2019). On the basis of the attachment theory, early attachment experience, mainly parents attachment, is the starting point of personality and social development, which has a lasting and extensive influence on the physical, mental and social adaptation of individuals. Chinese college students, whom were influenced by Chinese traditional culture such as Confucianism, should attach more importance to relations with parents, and Zeng (2009) found that Chinese college students’ parents attachment has important effect on somatopsychic adaptation. In summary, the specific mechanism underpinning the relation between parental attachment and cyberbullying in the family system remains unclear, and the effects of parental attachment on cyberbullying merit further discussion. Accordingly, we hypothesize that a high level of parental attachment is associated with less cyberbullying (H1). Loneliness as a mediator Uruk and Demir (2003) found that family relationships, especially the relationship between individuals and their parents, are an important, long-term factor affecting individual loneliness. Loneliness refers to an individual's subjective feelings about the quantity and quality of a person’s social interactions, which is different from the objective social isolation from society in connotation. Such subjective feelings occurs when the social network is less satisfying than she/he would like (Russell et al., 1980). As a complex interplay of emotion and cognition, loneliness involves the frustration and negativity that an individual feels from not being accepted in intimate or social relationships (Heinrich & Gullone, 2006). Wiseman et al. (2006) conducted a study on the loneliness of freshmen and found that students’ loneliness in college was significantly and negatively correlated with early parent–child relationships. Some studies have also found that individual parental attachment level affects loneliness (Zhang et al., 2017). Davis (2001) proposed a cognitive-behavioral model of pathological internet use (PIU), which distinguishes between specific PIU and generalized PIU. Specific PIU refers to the condition in which an individual pathologically uses the internet for a particular purpose, such as online sex or online gambling, whereas generalized PIU describes a more global set of behaviors, where individual deviant behavior is caused by risk factors such as substance dependence, social isolation, or loneliness. Relevant empirical studies have shown that factors such as loneliness are important explanatory variables for poor online relationships and can predict cyberbullying (Brewer & Kerslake, 2015). Thus, we infer that the cognitive-behavioral model of PIU can also explain the specific deviant behavior of cyberbullying. In addition, the general aggression model (GAM) posits that input variables (personal and situational factors) can affect an individual’s internal state (e.g., cognition and emotion), thereby affecting an individual’s judgment and behavior (Anderson & Bushman, 2002). Loneliness is a typical individual internal state, and researchers often investigate loneliness as a mediator variable (Sun et al., 2017). However, previous studies have primarily focused on the mediating effect of loneliness on individual aggression in real environments. The present study examines the mediating role of loneliness in the internet environment. Based on the above discussion, it is reasonable to predict that loneliness may mediate the relation between parental attachment and cyberbullying (H2). Interdependent self as a moderator Lazarus and Folkman (1984) suggested that cognitive appraisals, rather than the event itself, determine what coping style we adopt. According to the transactional theory of stress (Lazarus & Folkman, 1984), people react differently to the same thing and reach different outcomes. However, personality variables play an important role in understanding the situation (Garrido, 2016; Vollrath et al., 1998; Zhou et al., 2017). Davis (2001) suggested that individual deviant behavior on the internet can be affected by personality variables. The self is an important concept for individual personality, and its positive development corresponds with positive personality development. It is also an important protective factor for psychological and behavioral development (Xu et al., 2017). Markus et al. (1991) proposed that there are different types of self from the perspective of the relationship between individuals and others and that interdependence is a critical type. Singelis (1994) further suggested that the interdependent self is defined by social relations. It emphasizes the relation and interdependence between the individual and others and may be necessary toward maintaining mutual relationships with others, where the self exists as part of one’s social relationships. It is thought that individuals with different self-types show differences in thinking patterns and information-processing methods in dealing with interpersonal relationships (Hong & Chang, 2015), which then have an impact on individual motivation and behavior (Ding et al., 2016). In general, individuals who are high in interdependent self value the needs and views of others, which is critical in interpersonal situations (Cross et al., 2002). Therefore, we speculate that individuals who pay little attention to others’ needs and feelings are more likely to become involved in cyberbullying to deal with loneliness, whereas those with the same degree of loneliness and high interdependent self may show less cyberbullying in order to maintain positive interpersonal relationships. Consequently, this study further introduces the moderating variable of “interdependent self.” Specifically, we predict that interdependent-self moderates the relationship between loneliness and cyberbullying, and specifically, the latter half of the mediating role of loneliness (H3). The present study In summary, drawing from classical theories and models, such as social control theory, the cognitive-behavioral model of PIU, and the self from the perspective of the relationship between individuals and others, the purpose of this study is to explore the influence of parental attachment on cyberbullying. Specifically, we aimed to investigate (a) whether parental attachment negatively predicts cyberbullying and (b) whether loneliness mediates the relationship between parental attachment and cyberbullying and (c) whether interdependent-self moderates the indirect relations between parental attachment and cyberbullying (i.e., the latter half of the mediating role of loneliness). Methods Participants Prior to conducting the present study among Chinese college students, we have sought the opinions of the students' parents and obtained the approval of school authorities and the University Academic Committee. Participants who did not have one or both of the parents raising them were not required to fill out the questionnaire. A total of 1336 online questionnaires were collected, and after discarding questionnaires based on item scoring rules, short answer time, and other problematic reasons, 1125 valid questionnaires were obtained. Of these, 364 were from men and 761 were from women, and their mean age was 19.14 years (SD = 1.52). Before starting the survey, participants were informed that they were free to refuse or discontinue participation at any time. Researchers clearly stated and emphasized the fact that any information the participants provided would not be revealed to anyone. Lastly, participants completed four online questionnaires (described below). Measures The parent attachment scale We adopted the Chinese college students’ parental attachment questionnaire (Zeng, 2009), which includes four subscales: father attachment intimacy, mother attachment intimacy, father attachment autonomy, and mother attachment autonomy. There are 30 items in the questionnaire, and the score for each item ranges from 1 to 6. The total score of this questionnaire reflected the level of participant’s parental attachment (Cui et al., 2021). In the present study, the CFA showed an acceptable fit: RMSEA = 0.04, NFI = 0.86, CFI = 0.89, TLI = 0.91. The Cronbach’s alpha for the total score was 0.91. Cyberbullying perpetration subscale To capture cyberbullying, the cyberbullying perpetration subscale in the revised cyberbullying inventory (Chu & Fan, 2017) was adopted. The subscale contains 14 items depicting cyberbullying activities that respondents may have perpetrated over the past 6 months, and each item is graded from 1 to 4 (1 = “never implemented,” 2 = “1 time,” 3 = “2–3 times,” and 4 = “more than 3 times”). The higher the score, the more cyberbullying students are involved in. In this study, Cronbach’s alpha for the total scale was 0.71. Loneliness scale Individual general loneliness was assessed with the University of California, Los Angeles loneliness scale. This scale is used to evaluate the gap between the desire for social interaction and the actual level and is a one-dimensional scale (Russell et al., 1978, 1980). Wang (1995) translated and revised the scale into Chinese, and the revised version has good reliability and validity. The scale used here consists of 20 items, where participants rate each item on a 4-point scale from 1 to 4. A high total score indicates a high level of individual loneliness. In this study, Cronbach’s alpha for the total score was 0.83. Interdependent self-construal scale Singelis (1994) developed the self-construal scale (SCS). Tang (2010) revised the scale and studied on characteristics of Chinese college students' self-construals in the collectivism cultural background. We used the revision interdependent self-construal scale, which is the subscale of SCS and consists of 15 items. This subscale ranges from 1 to 5, where higher scores indicate higher levels of interdependent self-construal. For the present sample, the internal consistency of scale was acceptable (Cronbach’s α = 0.81). Control and assessment of common method biases Considering the common biases associated with different measurement methods, some approaches were taken to control the analyses or to evaluate bias. First, this study followed previous advice (Podsakoff et al., 2003) advocating anonymous procedures and reverse scoring of some items. Second, Harman’s single-factor test was conducted to assess common method bias. The results showed that the eigenvalues of nine factors were greater than 1.0, and the variance explained by the first factor was 25.50%, less than the critical standard of 40%. This suggests that there was no serious common method bias in this study, which can be used to inform subsequent statistical analysis. Statistical analysis All of the statistical analyses were conducted using SPSS 22.0 and SPSS macro PROCESS 2.16. First, we analyzed descriptive statistics and correlations among variables. Second, Model 4 of the PROCESS macro (Hayes, 2013) was used to test Hypotheses 1 and 2. Finally, we used Model 14 to test the moderated mediation for Hypothesis 3. This approach has been widely accepted for testing complex models, including moderated mediation (e.g., Shi et al., 2021). We test the significance of the effects using a bootstrapping approach, which constructs confidence intervals (CIs) derived from many bootstrap resamples (here, n = 5000). If the CI excludes zero, the effect is considered statistically significant. The method has been extensively used to calculate estimates because it can implement statistical tests without needing to satisfy the standard assumption of normality. Results Prevalence and correlational analysis In the current sample (n = 1125), 20.71% of the college students reported that they had bullied someone online at least once in the past 6 months. The correlation analysis in Table 1 shows that there was a significant negative correlation between parental attachment and cyberbullying, as well as with loneliness. In turn, interdependent-self construal was negatively correlated with cyberbullying and loneliness. There was a significant positive correlation between parental attachment and interdependent-self construal, as well as between loneliness and cyberbullying.Table 1 Descriptive statistics and correlations between variables M SD 1 2 3 4 1 Parental attachment 4.13 0.62 1 2 Cyberbullying 1.12 0.39 − 0.22 1 3 Loneliness 2.13 0.47 − 0.49 0.16 1 4 Interdependent self 4.90 0.67 0.32 − 0.18 − 0.30 1 * p < 0. 05, p < 0. 01, * p < 0.001. An identical system is used in subsequent tables Mediation analysis Controlling for demographic variables such as gender and grade, we used the SPSS macro PROCESS (Model 4; Hayes, 2013) to conduct mediation analysis. As shown in Table 2, parental attachment significantly predict cyberbullying, which is the total effect. When parental attachment and loneliness were added to the regression equation as predictors of cyberbullying, parental attachment had a significant negative effect on loneliness, what's more, both parental attachment and loneliness significantly predict cyberbullying. The statistic (β) for indirect effects was − 0.04, and the bootstrap 95% CI did not include 0 (− 0.06, − 0.01). Therefore, loneliness partially mediated the relationship between parental attachment and cyberbullying, and the mediating effect accounted for 16.87% of the total effect (Fig. 1).Table 2 Mediation test Outcome Predictors R2 F-value β t-value Cyberbullying Gender 0.05 18.34* − 0.07 − 1.06 Grade 0.00 0.15 PA − 0.21 − 7.26* Loneliness Gender 0.24 119.04* − 0.08 − 1.38 Grade − 0.02 − 0.89 PA − 0.48 − 18.01* Cyberbullying Gender 0.05 13.95* − 0.06 − 0.97 Grade 0.01 0.20 PA − 0.18 − 6.23* Loneliness 0.07 2.76* Bootstrap sample size = 5000; PA Parental attachment. The same is true for Fig. 1. Fig. 1 The mediation model examining the mediating effect of loneliness Moderated mediation analysis Controlling for demographic variables such as gender and grade, we used the SPSS macro program PROCESS (Model 14; Hayes, 2013) to conduct a moderated mediation analysis. The results are shown in Table 3. After the interdependent-self construal variable was added in the model, the product (interaction term) of loneliness and interdependent self significantly predicted cyberbullying. This indicates that the latter half of the mediating effect between parental attachment and cyberbullying was moderated by interdependent self (Fig. 2).Table 3 Moderated mediation effect test Outcome Predictors R2 F-value β t-value Cyberbullying Gender 0.08 11.29* − 0.08 − 1.27 Grade 0.00 0.09 PA − 0.15 − 5.07* Loneliness 0.07 2.69 IS − 0.10 − 2.31* Loneliness × IS − 0.13 − 3.49*** IS Interdependent-self Fig. 2 Moderated mediation model with the moderated effect of loneliness To clarify the moderating effect, the high and low groups were divided based on 1 SD of interdependent self-construal, and the regression slope of loneliness on cyberbullying was tested in each group (i.e., simple slope test), and the simple effect is shown in Fig. 3. When the level of interdependent self was low (M − 1 SD), loneliness positively predicted cyberbullying (simple slope = 0.25, t = 5.70, P < 0.001). When the level of interdependent self was high (M + 1 SD), loneliness did not have an effect on cyberbullying (simple slope = 0.01, t = 0.29, P > 0.05). Thus, with higher interdependent self-construal, the predictive effect of loneliness on cyberbullying decreased. That is, interdependent self can buffer the influence of loneliness on cyberbullying.Fig. 3 Simple effect analysis of the effect of interdependent self on the second-half mediation path Discussion Based on previous empirical and theoretical evidence, this study constructed a moderated mediation model of cyberbullying, with loneliness as a mediating variable and interdependent self as a moderating variable. We investigated two specific questions: first, whether parental attachment affects cyberbullying among college students and, second, how parental attachment among college students predicts cyberbullying. The results have theoretical and practical implications for furthering our understanding of the relationship between parental attachment and cyberbullying. First, this study investigated the relationship between parental attachment and cyberbullying among college students, and the results showed that parental attachment is negatively correlated with cyberbullying. Specifically, lower levels of parental attachment were associated with greater likelihood of engaging in cyberbullying. This result supports the conclusions of previous similar studies (Paez, 2020; Yusup et al., 2018). Thus, the emotional connection between children and their parents has a direct effect on the likelihood and extent of cyberbullying. This study not only verified the effect of social connection on deviant behavior in the context of social cybernetics (Hirschi, 1969) but also provided empirical support for potential causes of cyberbullying. Individual attachment with parents leads to the formation of stable mental states (Shaver & Mikulincer, 2002) reflecting the “internal working model” of one’s attitude toward others. This in turn affects the way individuals engage in interpersonal interaction with others. Individuals with high levels of parental attachment exhibit less aggressive and hostile sexual behavior as compared with those with low levels of parental attachment (Ooi et al., 2006). However, previous studies have shown externalization problems caused by low parental attachment in non-online domains, such as smoking, alcoholism, substance abuse, and antisocial behavior. As the internet has become an essential part of daily life, this study demonstrated that the influence of parental attachment on individual psychology and behavior extends from the real world to the online world. This finding not only enriches the applicability of attachment theory in developmental psychology in the online domain, but also suggests that creating a warm family atmosphere and improving attachment relationships among parents and other family members has implications for online behavior. Second, we confirm that loneliness can predict cyberbullying behavior (Brewer & Kerslake, 2015), supporting the applicability of the cognitive-behavioral model of PIU in explaining the causes of cyberbullying behavior (Davis, 2001). That is, under the influence of psychopathological risk factors such as loneliness, individuals may abuse others on the internet, socialize in an “eye for an eye” way, isolate others, and so on. Critically, this study shows that loneliness plays a mediating role between parental attachment and cyberbullying, similar to previous findings (Sun et al., 2017). Individuals with poor parental attachment are often high in loneliness (Zhou et al., 2019). In addition, given the accessibility and anonymity afforded by the internet, individuals who feel lonely can easily vent negative emotions online without concern for implications. We have tested the GAM (Anderson & Bushman, 2002) in an online environment, showing that loneliness—a complex cognitive and emotional construct—is an important mediating variable in the relation between parental attachment (environmental input) and cyberbullying (explicit aggressive behavior). Finally, we also found that the mediating effect of loneliness on the relation between parental attachment and cyberbullying was moderated by interdependent self. Specifically, the indirect effect of loneliness was only non-significant in individuals with high interdependent self, consistent with previous findings on self-construction (Cross et al., 2002; Ding et al., 2016). This is likely because individuals with high interdependent self tend to connect closely with others, pay attention to the needs and opinions of people around them, and thus maintain harmonious and beneficial communication (Cross et al., 2000; Xie, 2018). Even if such individuals experience loneliness, this does not impact their interpersonal relationships. According to self-construal theory (Markus & Kitayama, 1991; Singelis, 1994), when the level of interdependent self was not high and in the context of negative emotions such as loneliness, individuals will do not prioritize their relationships with others, even carry out cyberbullying. This study focused on the relationships among parental attachment, cyberbullying, loneliness, and interdependent self and identified a mechanism, parental attachment might explain cyberbullying among college students. Owing to limited conditions, the present study had some limitations that must be addressed. First, we used a cross-sectional design to examine the relationship between parental attachment and cyberbullying, as well as the mediating effect of loneliness and the moderating effect of interdependent self. In the final model on cyberbullying, the explained variance is relatively low. Longitudinal research or intervention experiments can be used to further explore the relationships. Second, the variables were all collected by questionnaire and subjective report. Although we identified no serious common method bias, the results were inevitably affected by social expectations and might not be sufficiently objective. Thus, future studies should collect multiple types of data (e.g., behavioral experiments or electroencephalograph recordings). Despite these limitations, the findings of this study have several significant implications. First, according to social control theory (Hirschi, 1969), attachment plays a fundamental role in crime reduction. Improve parental attachment relationships is always important. Second, the study was conducted during COVID-19, when college students did not return to school. The lower the level of parental attachment, the higher the loneliness and the more cyberbullying behaviors. In particular, during the COVID-19 pandemic, emotional self-efficacy and emotion regulation mediate the relation between cyber victimization and well-being among youths (Schunk et al., 2021). This study suggests that we should prioritize reducing individual loneliness when preventing and providing intervention for cyberbullying. Finally, this study found that the deviant behavior of individuals on the internet is influenced by their own personality, which provides avenues for changing problematic cyberbullying behavior. We should also concentrate on the individual’s understanding of the relationship between the self and others. Conclusion Parental attachment negatively predicted cyberbullying. Loneliness partially mediated the relationship between parental attachment and cyberbullying. The mediating effect of loneliness was moderated by interdependent self. The predictive effect of loneliness on cyberbullying was only significant among college students with low interdependent self. Acknowledgements This study was funded by Humanities and Social Sciences Research Funds from the Ministry of Education of China [Project no. 21JDSZ3067], Opening Research Program Funds of Key Laboratory of Adolescent Cyberpsychology and Behavior (CCNU), Ministry of Education: The Research of Mechanism of Internet Use on adolescents’ mental health [Grant Number 2019B05]. Data availability No datasets have been shared by the author for the following reason: The respondents were assured that raw data would remain confidential and would not be shared. Declarations Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study. 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==== Front J Racial Ethn Health Disparities J Racial Ethn Health Disparities Journal of Racial and Ethnic Health Disparities 2197-3792 2196-8837 Springer International Publishing Cham 36449130 1475 10.1007/s40615-022-01475-4 Article Racism During Pregnancy and Birthing: Experiences from Asian and Pacific Islander, Black, Latina, and Middle Eastern Women http://orcid.org/0000-0003-1185-045X Nguyen Thu T. [email protected] 1 Criss Shaniece 2 Kim Melanie 3 De La Cruz Monica M. 4 Thai Nhung 5 Merchant Junaid S. 1 Hswen Yulin 6 Allen Amani M. 78 Gee Gilbert C. 9 Nguyen Quynh C. 1 1 grid.164295.d 0000 0001 0941 7177 Department of Epidemiology & Biostatistics, University of Maryland School of Public Health, College Park, MD 20742 USA 2 grid.256130.3 0000 0001 0018 360X Department of Health Sciences, Furman University, Greenville, SC 29613 USA 3 grid.40263.33 0000 0004 1936 9094 Department of Anthropology, Brown University, Providence, RI 02912 USA 4 grid.47840.3f 0000 0001 2181 7878 School of Social Welfare, University of California, Berkeley, CA 94720 USA 5 grid.47840.3f 0000 0001 2181 7878 Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720 USA 6 grid.266102.1 0000 0001 2297 6811 Department of Epidemiology and Biostatistics, Bakar Computational Health Sciences Institute, University of California San Francisco, San Francisco, CA USA 7 grid.47840.3f 0000 0001 2181 7878 Division of Epidemiology, University of California, Berkeley, CA 94704 USA 8 grid.47840.3f 0000 0001 2181 7878 Division of Community Health Sciences, University of California, Berkeley, CA 94704 USA 9 grid.19006.3e 0000 0000 9632 6718 Department of Community Health Sciences, University of California, Los Angeles, CA 90095 USA 30 11 2022 111 4 10 2022 18 11 2022 21 11 2022 © W. Montague Cobb-NMA Health Institute 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Background Despite persistent racial disparities in maternal health in the USA, there is limited qualitative research on women’s experiences of discrimination during pregnancy and childbirth that focuses on similarities and differences across multiple racial groups. Methods Eleven focus groups with Asian American and Pacific Islander (AAPI), Black, Latina, and Middle Eastern women (N = 52) in the USA were conducted to discuss the extent to which racism and discrimination impact pregnancy and birthing experiences. Results Participants across groups talked about the role of unequal power dynamics, discrimination, and vulnerability in patient-provider relationships. Black participants noted the influence of prior mistreatment by providers in their healthcare decisions. Latinas expressed fears of differential care because of immigration status. Middle Eastern women stated that the Muslim ban bolstered stereotypes. Vietnamese participants discussed how the effect of racism on mothers’ mental health could impact their children, while Black and Latina participants expressed constant racism-related stress for themselves and their children. Participants recalled better treatment with White partners and suggested a gradient of treatment based on skin complexion. Participants across groups expressed the value of racial diversity in healthcare providers and pregnancy/birthing-related support but warned that racial concordance alone may not prevent racism and emphasized the need to go beyond “band-aid solutions.” Conclusion Women’s discussions of pregnancy and birthing revealed common and distinct experiences that varied by race, skin complexion, language, immigration status, and political context. These findings highlight the importance of qualitative research for informing maternal healthcare practices that reduce racial inequities. Keywords Discrimination Maternity care Pregnancy Birthing Focus groups http://dx.doi.org/10.13039/100006545 National Institute on Minority Health and Health Disparities R00MD012615 R01MD015716 R01MD016037 Nguyen Thu T. Nguyen Quynh C. http://dx.doi.org/10.13039/100000092 U.S. National Library of Medicine R01LM012849 Nguyen Quynh C. ==== Body pmcIntroduction Racial and ethnic disparities in pregnancy-related outcomes are an enduring problem in the USA [1, 2]. The role of racial bias in patient-provider experiences contributes to racial disparities in maternal health but can also serve as levers for change in policies aimed at reducing healthcare inequalities [1, 3–5]. However, there is little qualitative research examining women’s experiences of racism and discrimination in the context of pregnancy and birthing, and even fewer studies present perspectives from multiple racial and ethnic groups. Moreover, most studies were developed prior to the COVID-19 pandemic, which may not reflect contemporary experiences. Our study addresses this gap in the literature by synthesizing experiences of racism during pregnancy and birthing across four different racial/ethnic groups of women from across the USA. The term “obstetric racism” describes the differential treatment of childbearing people based on race or ethnicity [6]. Research on this topic builds on medical racism research, which focuses on the mistreatment of people of color in clinical settings. For instance, a study examining a multi-ethnic sample of 2700 birthing women found that one in six women had experienced mistreatment by healthcare providers, but mistreatment varied by race/ethnicity—greater proportions of Indigenous (38%), Hispanic (25%), and Black (22.5%) women reported experiencing mistreatment compared to White women (14.1%) [7]. Importantly, women of color were twice as likely as White women to report that a healthcare provider had ignored, refused, or delayed treatment [7], which is particularly problematic because the delayed clinical response is associated with maternal mortality [4, 7]. Moreover, although racial disparities in birth outcomes, such as fetal deaths, stillbirths, or maternal deaths during hospitalizations, remained consistent with pre-COVID-19 levels [8, 9], women of color reported increased challenges in access to obstetric and mental healthcare compared to White women [10, 11]. Visitor restriction policies were more strictly enforced for women of color compared to White women, which isolated them from the social support systems that provided a source of advocacy and protection against discriminatory treatment by healthcare workers [12]. These findings highlight the importance of centering on women’s experiences of racism during pregnancy and birthing, with a more nuanced focus on the factors impacting different racial groups. Much of the qualitative work on obstetric racism has focused on Black mothers’ experiences with racism during pregnancy and childbirth. Black women experience racialized pregnancy stigma—stereotypes that devalue Black pregnancies and motherhood—in everyday interactions, healthcare settings, and social services [13]. Stereotypes include the assumption that Black women are low-income, dependent on government resources like welfare, single, and have multiple children, regardless of their socioeconomic or marital status [13–15]. Within institutional settings, Black women report being ignored, having their experiences of pain dismissed, being treated by trainees disproportionately more than physicians, feeling like they received a different recommendation based on race, and feeling judged by social service providers [13, 16, 17]. In their gendered roles as nurturers, Black mothers and mothers-to-be cite the need to protect their children from the stressors of racism [13, 18, 19]. The stress of racism even impacts the decision to have children, with many Black women expressing fear of getting pregnant because of stories of trauma during the delivery they heard from other Black mothers [16]. Although Black mothers’ pregnancy-related health disparities have been the focus of the majority of the research [20, 21], revealing higher rates of anxiety, inflammatory response, and sleep disturbances in Black compared to White mothers [22, 23], research has also demonstrated disparities across minority racial groups in other pregnancy-related complications. For instance, research has demonstrated higher rates of intracerebral hemorrhage during pregnancy and postpartum in Black, Hispanic, and Asian mothers compared to White mothers [24]. Differences in health disparities observed between minority race populations can be attributed to a myriad of historical, political, and societal factors that individuated the experiences of racism across racial and ethnic groups. For example, language barriers in patient-provider interactions contribute to reductions in healthcare delivery for patients who do not speak the majority language, conferring a unique challenge for racial groups with large immigrant populations (e.g., Latina and Asian women in the USA) [10, 25], while policies leading to higher rates of incarceration are more specific to low-SES, Black birth outcomes [26]. In general, there is limited qualitative research on the experiences of racism for AAPI and Middle Eastern women, specifically during pregnancy and delivery. Thus, examining the lived experiences of pregnancy and birthing from women of different racial/ethnic backgrounds addresses a critical gap in order to better inform practices that can reduce maternal health disparities. For the current paper, we adopted the health equity conceptual framework by Kramer and colleagues [27] (Fig. 1), which shifts focus from conventional clinical risk factors to provide a more holistic account of the challenges faced by women of color. This framework was originally used to explain racial and geographic disparities in maternal mortality, but the framework can be applied to reproductive health broadly. Under this conceptual framework, there are two dimensions that situate the birthing person. The first dimension focuses on the life course and health trajectory of the birthing person and the accumulation of exposures over the lifespan. The other dimension represents the multilevel determinants of health, from biomedical conditions and health services environment to social support and community environment. Racism in this model is considered a social determinant of health. We sought to understand the constraints and opportunities of women’s reproductive health environment toward a better understanding of how racism works as a determinant of their reproductive health.Fig. 1 Conceptual framework for reproductive health. Reprinted from Kramer et al. Changing the conversation: applying a health equity framework to maternal mortality reviews. Am J Obstet Gynecol. 2019;221:609.e1-609.e9. with permission from Elsevier Qualitative research can provide an in-depth understanding of the human experience and provide insight into the “how” and “why” of a specific phenomenon and perspective [28]. The objective of the present paper is to explore the influence of racism on pregnancy, birthing, and child-rearing experiences from the perspectives of AAPI, Black, Latina, and Middle Eastern women. We also seek to gain an understanding of policies and programs that could support and promote maternal and child health from the perspectives of our racially and ethnically diverse focus group participants. Methods We recruited a purposive sample of 52 participants for eleven focus groups by posting flyers via social media (Twitter and Facebook) and by contacting student and community organizations (e.g., YMCA, Korean American Community Foundation NY, and Chicana Latina Foundation). Eligibility criteria included women who were at least 18 years old, use social media (Twitter, Facebook, etc.), have had children or are open to having children in the future, and self-identified as Black, Latina, Middle Eastern, Asian, or Pacific Islander and were available to participate in a 90-min focus group via Zoom. All participants received a $50 gift card. We conducted race- and ethnic-specific focus groups. Those interested were invited to complete a brief online survey to collect basic demographic information, including race, region of residence, contact information, and their availability to participate at the scheduled times. Focus Group Guide Development We used a semi-structured focus group guide developed based on a review of the literature and our research aims. The research questions necessitated that we include specific topics such as the extent to which racism and discrimination impact pregnancy and birth experiences, the influence of racism on mental and physical health during pregnancy, and whether there are policies or programs to help protect people from the impact of racism during pregnancy and birthing. Specific questions included the following: “Do you think racism and discrimination impact pregnancy and birth experiences? Why or why not? If so, how?”; “Are there any kinds of policies or programs that you think could help protect people from how racism impacts their pregnancy and birthing experiences?”; “What are some steps you have taken to protect yourself or somebody else from the impact of racism?”. Data Collection and Analysis The 90-min focus group sessions were audio-recorded, later transcribed, translated into English (if conducted in another language), and de-identified. Two study team members attended each focus group to provide support. We utilized rev.com for the transcription services. We had two transcripts that needed translation, which was conducted by study team members—one fluent in Spanish and the other fluent in Vietnamese. Those transcripts maintained the original language along with the English translation in order to be able to compare and clarify meaning when needed. The initial codebook was based on topics from the developed focus group guide. We evaluated the codebook through consensus-building discussions to ensure the accuracy and clarity of code definitions. We used Nvivo, a qualitative data analysis software package, to code the data based on the codebook. The team discussed coding disagreements and came to a consensus for all coding to prepare the data for theme development. The NVivo coding reports were a compilation of the focus groups based on race, with one report for the AAPI, Black, Latina, and Middle Eastern participants. We analyzed the NVivo coding reports through a series of team meetings to establish themes and connections within and across the focus groups for the different racial/ethnic groups. Throughout the process, we sought to maintain data trustworthiness, adhering closely to the transcripts from the participants for interpretations by using the constant comparison approach [19]. This approach involves the continuous interplay of comparing and contrasting textual data points to other data points in the dataset in order to discern conceptual similarities and solidify themes [29, 30]. We also utilized multiple data analysts with different racial/ethnic backgrounds. This study was approved by the University of California, San Francisco Institutional Review Board (18–24,593). Results Focus group participants included 15 Black, 21 Asian and Pacific Islander, 8 Middle Eastern, and 8 Latina women. The mean age was 35 years old. Most participants (75%) had a bachelor’s or graduate degree. The majority of focus group participants had children (75%), and of those with children, most had two children (71%). Of the focus group participants who did not have children, the majority stated that they planned to have children within 5 years (61%). Participants resided in the Western (n = 19), Northeastern (n = 16), and Southern (n = 17) USA. Three themes related to experiences of racism during pregnancy and birthing emerged: (1) vulnerability and voice in pregnancy and birth experiences, (2) mental and physical health, and (3) representation and advocacy for appropriate care. We describe these themes in more depth below and provide some quotes as examples. Additional illustrative quotes are presented in Table 1.Table 1 Themes with illustrative examples Vulnerability and voice in pregnancy and birth experiences “As well as just the subtle inherent racism that was part of every day of just the questions and assumptions that were made around what kind of job I had. Did I have this type of insurance? What did my spouse do? What was his role when he showed up? Questions that really… I don't know why they're pertinent to my medical history anyway but those types of conversations happened and were part of it. And I will fully acknowledge going out to that mental part. And then the inability to check folks because you're vulnerable to them in those moments” (Black FG 1) “I’m sorry. I’m actually the opposite. I will check you. And I feel like I have to let you know, of course in a right way, I have to let you know. I had an incident when my son was born. He was in NICU. And we had a pretty traumatic birth experience. And I remember one of the main doctors on the floor being very rude and condescending to me and he was breaking down things but it was in a condescending manner. …It was just so inappropriate. And I remember checking him as well as going up the ladder so much so that I didn’t see him anymore for the rest of our stay. He was not there at all. And I made sure to let them know I don’t want him around my child. I don’t even want to see him when I come and visit. And they made that happen. They took it seriously” (Black FG 1.) Vulnerability from the stigma related to being Muslim I think something at that large of a scale, for the entire nation to say, ‘Hey, you guys are banned,’ is a free pass for everyone and every group and agency and department, to say, ‘Okay, we’re banning you too in some kind of way. We’re not banning you, verbatim we say you’re banned. We’re going to treat you as such and make you feel as such, because our entire country is giving us a free pass to do that.’ You can feel that everywhere. You can feel that walking into a store. You’re going to for sure feel it in a medical space as well” (Middle Eastern FG 9) Fear of mistreatment in hospital setting “I used to work for a project that was helping Latino women who had a baby or were pregnant… and a lot of those participants were experiencing fear in being in immigrant related situations, to being worried and everything related to their mental health. And one of the worries they had was if they were going to be accepted in hospital, if they were going to have trouble or what’s going to happen. They were uncertain to go to a hospital even” (Latina FG 11) Another participant from the Spanish-speaking focus group revealed her thoughts about entering the healthcare setting: “Will they judge me when I go to the doctor? The receptionist is going to look at me with a bad face. They won’t understand me when I go to get the medicines.’ All that has an effect. Also, something that at least I would feel, that worries me a lot…” (Latina FG 10, translated) “Ma’am, I’ve had two C-sections, I wanted to get in and I wanted to get out. So I wanted to get the care I needed, but didn’t want to labor it, I didn’t want to hang in there…and have my baby in the nursery kind of thing. I wanted to take advantage of the services, but I also wanted to come out of there because I know that there are or there is potentially, just being treated differently because you are a Black female or a brown person” (Black FG 2) Differences in treatment “My daughter’s father is white, and so I noticed difference in treatment by staff, and just in the world, I think, when I had been with him versus when I was not. In spaces where you would totally just be ignored, you realize there's a difference, like,” “Oh. They are acknowledging me now. They are speaking to me now. I am actually here, and they see me, and they’re treating me with a little bit more visibility than otherwise” (Middle Eastern FG 9) “Just for example, in the same hospital where I gave birth to my daughter, I recommended that hospital for other women who were going to have their babies, and she was traumatized, and she was a non-English speaking person. She didn’t have someone that looked like my ex [who is white], and didn’t have anyone to advocate for her, or felt like they could advocate for her, and so she was treated horribly. She left, or stayed, in that space in tears because of staff basically almost verbally abusing her. And I was just so shocked and surprised by that treatment, as compared to mine” (Middle Eastern FG 9) “I would say that every time I go into the hospital with a brown and black person, and see the level of condescension and just being pushed around and bullied and coerced into doing things in the hospital. I would say it impacts me negatively. I feel like I have vicarious trauma and a lot of rage, so watching that happen, and it's compounded by the fact that, because I have light skin and the ability to just code switch, and also channel my inner white lady to step up in these spaces that it highlights the injustice that all it takes is to be able to code switch or look a certain way to be able to navigate the hospital” (Pacific Islander FG 6) Another participant reported the discriminatory treatment based on skin color: “I want to say my prenatal experience being a brown woman and trying to utilize general hospital… I just felt like I wasn’t treated with a lot of respect or value, and so… Just often, I wasn’t receiving the support that I needed” (Pacific Islander 6) Positive health care experiences “I feel very lucky that during the time that I was pregnant with my baby, my doctor was Vietnamese. When I delivered my baby, the entire doctor team was White and were very kind and motivated me through the process. When I said that I am not fluent in English, they went to find me a translator. The way they treated me made me feel loved and respected so I was very thankful and grateful to them” (Vietnamese FG5, translated) Mental and physical health “I think something that keeps coming up to mind is if I want to bring a child into the world that’s going to experience systematic oppression. …I know that these stressors that I’ve experienced the last 5, 10 years are stressors that more than likely will continue and will affect not only my physical well-being and mental well-being, but also my child’s wellbeing… I think it’s really fear-inducing to think about being pregnant or getting pregnant, if I would continue to deal with the same stressors that I have now…I think it would take a major toll on me” (Latina FG 11) COVID-19 pandemic “I have had a lot of friends who have babies during the COVID, so I’ve had seen how they were doing during this COVID, and I think they had a lot of difficulties. For example, they cannot go to the hospital with their husband… And usually, they are all Korean friends, so their parents can come to take care of them after giving birth, but they cannot have that. So I guess at COVID, they had a lot of difficulties carrying the baby and giving birth. And after that, they also had a lot of limited experience when they were having a birth. So some of my friends got vaccinated, but some people who weren't or who didn’t want get vaccinated, they were just staying home for nine months. And then after birth, they still had to stay in home for babies, so I think definitely COVID has impacted pregnancy experiences for women a lot” (Korean FG 4) Representation and advocacy for appropriate care “I just delivered a baby around 8 months now. When I was pregnant, I did see the doctor and the baby was a bit big so many doctors refused to see me, only the Vietnamese doctor agreed to see me. At that moment, I realized that the Vietnamese people care for me and my baby while doctors of other nationalities refused” (Vietnamese FG5, translated) “I think representation matters. …Hospitals and healthcare facilities need to recruit folks, even from high school, like black and brown people, to get into the healthcare industry” (Middle Eastern FG 9) Doulas and pregnancy groups “…a doula that may be able to be right there with you and educate you and share experiences with you. Someone to be right there in the process from beginning to end” (Black FG 1) and “[The formal group] was only for black women who were pregnant, who are delivering around the same time. We all were going through the same thing and we’d be able to meet every week and share our experiences” (Black FG 3) “…in which pregnant mothers can talk about their feelings and situations and receive resources, support…and speak English the way it comes, because you know that you are not the only person who is experiencing that situation” (Latina FG 10, translated) Abortion and financial services “I mean I think one thing that I think of is, who has access to abortion and not just who has access to abortion, but also in some places, there are a lot of women who have that option and maybe don’t actually want to have an abortion, but choose to do so because they don’t have the financial means to support a child…a social policy that I think is really useful is just making sure that people can afford living and having a home and food and knowing that. That is a guaranteed right” (Middle Eastern, FG8) Mental health services and empathy “Programs that help people who can see their mental health compromised, especially for pregnant women. I also think that it is very important to educate the alleged aggressor… Creating empathy, creating classes at school that open that universe to you as a person, how to be empathetic to the reality of others, that you know that there is a world beyond [the US]” (Latina FG 10, translated) Empathy classes for children: “it could be something social where they teach you to have empathy for others and value what is empathy, consideration. It is not only from the face to the outside, but it really increases those values and in a certain way also incorporates the parents in those things, because what today’s children learn is what they see at home” (Latina FG 10, translated) “Respectfully, I'm not referring to y’all, but just in general, we look at band-aid solutions to this really big problem. I think doula programs are great, having staffers or interpreters that are there that can understand culturally, linguistically as well, the patient… I work in a school district, we have a lot of these ABAR trainings, that are called anti-racist, anti-Blackness workshops in order to help promote inclusion and diversity and all the equity and things like that. Even with that, I still also think it’s a band-aid solution, but that can also be helpful for healthcare workers to try to understand and listen, you know what I mean? I think each healthcare institution can have a panel… can listen to the community, and kind of see where they are demographically and understand that these are people, even if they were lower income, even if they might not understand you, they’re also people, they bleed like you, and these are their needs, and this is where they came from." (Middle Eastern, FG 9) Vulnerability and Voice in Pregnancy and Birth Experiences Many of the participants across focus groups reported the role of unequal power dynamics within the patient/provider relationship. As a result, women described feeling vulnerable and feeling that their voice had limited power, particularly in responding to discrimination. One participant shared a very uncomfortable situation while being examined:“The first person who asked me about the [baby’s] name, literally had a wand in my vagina doing the sonogram. [The doctor asks,] ‘What are you thinking about naming your child?’ And when I said, David. “Oh good, a nice normal name.’ …So it catches me off guard sometimes because I wasn’t expecting it to happen or to come up again in these spaces… “I’m not going to check you and say, ‘You shouldn’t have said that’s a nice, normal name.’ Because I don’t know what you’re going to do next...” (Black FG 1). In the Latina focus groups, participants reported that much of their vulnerability stemmed from their immigration status and speaking a different language. Middle Eastern participants expressed increased vulnerability from the stigma related to being Muslim after the Muslim ban, which was viewed as an informal signal that discrimination is permitted. Participants shared their fears about going to the hospital, whether they would be accepted or what treatment they would receive, which influenced their healthcare decisions. One participant discussed how concerns and fears around potential mistreatment in the medical system impacted her decision to have elective C-sections.“Ma’am, I’ve had two C-sections, I wanted to get in and I wanted to get out. So I wanted to get the care I needed, but didn’t want to labor it, I didn’t want to hang in there…and have my baby in the nursery kind of thing…because I know that there are or there is potentially, just being treated differently because you are a Black female or a brown person” (Black FG 2). A Black participant responded directly to discriminatory experiences in the hospital by making it known that she did not want the provider near her or her child after the provider was rude and condescending. However, this kind of direct response was rare. Focus group participants shared how experiences in the health care setting can vary based on race/ethnicity, skin color, and language. One participant observed that she receives better treatment when her ex-partner, who is White, accompanies her to her medical visits. While she had a positive delivery experience, when she recommended the hospital to a non-English speaking woman, she stated the woman “did not have anyone to advocate for her” and was “treated horribly.” Participants in the Pacific Islander focus group reported how treatment varies based on skin complexion, with darker-skinned women receiving worse treatment. A participant who was light-skinned shared that she witnesses poorer treatment: “every time I go into the hospital with a Brown and Black person, and see the level of condescension and just being pushed around and bullied and coerced into doing things in the hospital” (Pacific Islander, FG 6). Mental and Physical Health Participants reported that racism took a toll on their mental and physical well-being before, during, and after their pregnancy. Latina and Black participants expressed how chronic racism-related stress created a constant worry for the well-being of oneself and one’s child. Compounding the existing racism-related stress, many of our participants reported that they experienced pregnancy during hospital lockdowns due to the COVID-19 pandemic, which was marked by periods of isolation away from friends and family members during pregnancy, labor and delivery, and the postpartum period. This isolated them from sources of support and advocacy. Vietnamese participants reported, “Vietnamese people need to have a happy mindset so the baby can be born healthy" (Vietnamese FG 5, translated). Another participant shared how racism could negatively impact the mom and baby through the mindset: “I think there is a small impact if their relatives or themselves experience racism since the mother will not be happy and that will somewhat affect the baby” (Vietnamese FG 5, translated). Despite the racism-related mental health concerns, most participants across focus groups stated they would not change their decision for future children based on the presence of racism, though some participants shared that racism would impact where they raised their children. A South Asian participant said,“The only thing it may trigger in the future is geography, whether it’s high racism against my culture or race is a place I would want to bring up a child. But apart from that, whether I would want to have a child in this world based on racism, my race, is not a factor” (South Asian, FG7). Representation and Advocacy for Appropriate Care Participants across racial and ethnic groups emphasized the importance of culturally and racially concordant care. One Vietnamese participant shared that while many doctors refused to see her because her baby was estimated to be large, she was able to find a Vietnamese doctor who agreed to see her. Black participants reported how a non-Black physician stated that her son had jaundice, while her Black physician did not think the baby had jaundice by responding, ‘No. We’re yellow.’… I wonder how many times he’s had that conversation. But yeah, representation really does matter. It matters that we see ourselves reflected in the services that are provided to us in all walks of life” (Black, FG1). Other Black participants warned that racism could still be present within the medical system, even if they had a Black doctor. One participant said,“Every time I talked to somebody and I was just like, ‘I don’t think you all do this to White women.’ I can’t guarantee, but I feel in my body and my soul that this is something you’re doing and these are Black physicians who I cherish, but they’ve been steeped. I'll use that word in White medical education, just like me…. Even in the midst of having Black providers, there still ways for that racism to creep in there" (Black FG 3). In addition to a discussion about representation, focus group participants saw the value of doulas and pregnancy groups for providing resources, guidance, and social support. A Middle Eastern participant emphasized the need to go beyond doula programs and anti-racist workshops, which she viewed as “band-aid solutions to this really big problem [racism].” Multiple Vietnamese participants said that they were not aware of any pregnancy and birth support or programs. One participant said, “I only know to call 911.” (Vietnamese FG5, translated). Some participants (from the Latina and Middle Eastern focus groups) mentioned the need for increased access to abortion services and financial support services. The need for mental health services for pregnant women was also expressed. Women in the Middle Eastern and Latina focus groups discussed ways to increase empathy. A Latina participant recommended empathy courses for children that would also incorporate participation from parents. A Middle Eastern participant recommended that hospitals have a panel to listen to the community to understand “they bleed like you, and these are their needs, and this is where they came from." Discussion The current study provided nuanced insights about differences and similarities in the types of discrimination experienced during pregnancy and birthing from the perspectives of women from four different racial and ethnic groups. We apply Kramer and colleagues’ [27] conceptual framework to understand our findings by focusing on the social-contextual contributors to racial disparities. One of the two dimensions of the conceptual framework is the life course and health trajectory of the birthing person. Women in our study reported chronic experiences of racism even prior to their pregnancy and discussed the toll of this stress on their and their children’s well-being. The framework examines racism as a fundamental social determinant of health, systematically determining the risk, constraints, and opportunities across the life course. Women in our study discussed their health environment and how their identities interacted with the broader social and cultural context to influence their healthcare interactions and daily life experiences. Spoken language and immigration status were more prominent factors influencing treatment for Latina women compared to Black women. The Muslim ban increased experiences of vulnerability and discrimination for Middle Eastern women in any public space they entered, including the healthcare setting. For some women, the geographic location where they chose to raise their children was impacted by whether they felt the area was welcoming to their cultural or racial identity. In addition to the constraints of their environment, the women also highlighted opportunities or protective factors to support a positive birthing experience. Related to representation and advocacy for appropriate care, participants shared the importance of having healthcare providers that represented their racial/ethnic identity, while others pointed out the importance of addressing systemic racism within the medical system. Our study provides support for Kramer et al.’s [27] conceptual framework in highlighting the role of social determinants of health and the socio-spatial context in shaping the reproductive health of birthing people. Several studies indicated that minoritized women are mistreated at greater rates in the clinical setting [6, 7, 31], from physicians yelling at patients [7] to withholding treatment plans [31]. This included another qualitative study conducted by the study team utilizing Twitter data to conduct a content analysis of tweets related to COVID-19 vaccines and race/ethnicity. That study had a theme of medical racism that included both subtle and overt examples [32]. In the current study, there was also a wide spectrum of perceptions and experiences described by the women that included both subtle and more overt experiences of racism. Our study participants described a nuanced perspective on mistreatment. For instance, as described above, one participant described how her physician remarked about her baby’s name. While this may have appeared subtle, she described being in the vulnerable position of having a vaginal examination. The combination of the physical proximity, the power dynamics of the physician–patient relationship, and the words culminated in a threatening situation for our participant, compounded by her perceived powerlessness. Chronic worry about racial discrimination has been found to partially explain Black-White disparities in preterm birth [33]. Medical mistrust was common among the study participants. Medical mistrust is defined as a lack of confidence in the intentions and work of medical systems and providers, stemming from a long history of racism in the healthcare system as well as personal experiences of discrimination and maltreatment [14, 34, 35]. Research has found medical mistrust negatively influences the utilization of healthcare services [34]. This was evidenced in our study by participants’ experiences in clinical settings, some who emphasized that they just wanted to “get in… and get out.” These findings align with the literature on the experiences of many Black women, whose mistrust of healthcare providers led to suspicion of medical treatment and a reluctance to seek care [14, 36]. A study among Hmong women and men, another ethnic and cultural minority group, found that medical trust generally stemmed from unfamiliarity with Western medicine, a fear of being studied, and, relevant to our study findings, negative experiences with providers [37]. A recent review of literature by Ho et al. [35] found a paucity of research on medical mistrust among Asian Americans, and especially Arab Americans. Our study adds to the research examining how anti-Arab and anti-Muslim climates affect healthcare experiences and medical mistrust. The study findings here suggest that while there are similarities across racial and ethnic groups, there are also unique concerns. Literature on racism within the health care setting emphasizes the centrality of patient-centered care to improve patient experiences [16]. For instance, one study of Black patients and their non-Black physicians at a primary care clinic found that non-Black physicians’ racial biases, as well as Black patients’ history of racial discrimination, were associated with talk times in provider-patient interactions [38]. Other research shows that many Black women feel powerless when interacting with physicians, specifically gynecologists and obstetricians, and that they participate less in decision-making [14]. Participants in a study of Black women in the South reported avoiding health care or no longer asking questions during visits because healthcare providers dismissed or did not believe their health concerns [16]. Experiences of racialized pregnancy stigma cause reduced access to and quality of health care services, social support, and other resources [6, 13]. In contrast, women who had non-judgmental interactions with their providers felt more comfortable sharing relevant health information, such as risk factors important for predicting maternal and infant outcomes [13]. Prior studies have also emphasized that collaborative communication was associated with better medication adherence for patients without racially/ethnically concordant care [39]. Participants in our study emphasized particular aspects of their identities that influenced their perceived quality of health care. For example, Pacific Islander participants spoke about how appearing “lighter-skinned” or having a White or English-speaking advocate seemed to act as protective factors against the mistreatment of mothers of color. Other literature also found that participants perceived that lighter skin or higher education influenced the quality of healthcare they received [40]. Our study also emphasized the difficulty and vulnerability introduced by speaking a different language and the fear of being judged by physicians or other healthcare workers based on appearance. Prior research has found that language barriers often cause miscommunication between care providers and women and may increase the risk of negative obstetric outcomes [41]. Many participants in our study emphasized the importance of racially/ethnically concordant and culturally competent care, including having doctors, interpreters, and advocates like doulas of the same race. One Black participant highlighted how a Black physician was able to identify health issues better based on sharing the same race as the patient. Racial congruence is a key element to building trusting relationships between patients and providers, particularly as physicians of color tend to be more aware of and impacted by structural racism; thus, they are able to understand the struggles of patients and provide a sense of community in the medical environment [40]. However, participants also noted that racial and ethnic concordance does not guarantee anti-racism and should not be the only solution. Prior research noted the disparity between the general population of Blacks, Latinxs, and American Indians or Alaska Natives and the number of physicians of that race/ethnicity [42]. As of 2019, Black individuals have 11 times fewer physicians and 6 times fewer OB/GYN options of the same race compared to their White counterparts [43]. Currently, there is no publicly available database that lists the race/ethnicity of maternal health providers in a patient’s area [43]. Our study helps to highlight the potential value of such a resource. This lack of representation is related to the sense of vulnerability addressed in our study between patient and provider. However, some studies have found factors other than racial concordance to be more predictive of patient outcomes [44]. Factors such as age, education, and patient-centered communication influenced perceptions of similarity to their providers. Perceived personal similarity led to more trust in the physician, satisfaction with care, and stronger intention to adhere to recommendations [44]. Patient-centered behavior was central to the patient’s belief that the physician shared a common understanding of their health conditions [44]. Participants mentioned the systemic nature of racism within health care and highlighted the need to go beyond “band-aid” approaches. In recent years, there have been changes to the Medicaid program, which is the health insurer for 45% of total births in the country [45]. Many states have adopted support for different models of prenatal and delivery care, including support for interdisciplinary teams of health workers, doulas, and midwives and adopting person-centered models of care like group prenatal care and pregnancy medical homes. In addition, some state Medicaid policies have changed reporting requirements to increase data collection and disaggregate these data by race to understand disparities in maternal and birth outcomes for race-specific interventions [46]. These policy changes are consistent with some of the suggestions voiced by the participants. Study Strengths and Limitations Examining the lived experiences of pregnancy and birthing from women of different racial/ethnic backgrounds addresses a critical gap in order to better inform practices that can reduce maternal health disparities. However, our study had some limitations. For some focus groups, we encountered challenges in recruitment. For example, within the AAPI group, there were only two Pacific Islander women; yet these two women were considered leaders in their community and were able to provide an in-depth perspective. In addition, not all US areas were represented, as there were no participants from the Midwest. Our sample was also highly educated, with 75% of participants having a college or graduate degree, compared to 38% of the US population having a college degree, so they may not be representative of US women [47]. Conclusion Pregnancy represents a critical time period in the life of a mother and her baby. Pregnancy and birth complications can have long-lasting effects on maternal and child mortality and morbidity. Despite this, little is known about the experiences of birthing women of color within the healthcare setting. Our study sheds light on some of the commonalities in experiences across racial and ethnic groups, as well as some of the unique experiences faced by women of distinct racial and ethnic groups. The women tell us of experiences within and outside of the healthcare setting that facilitates or inhibits feeling supported and heard during this time. These experiences varied by race, ethnicity, skin complexion, language, immigration status, and historical and current political context. Our study helps to provide a deeper understanding of the complexities of racism across several races/ethnicities and their impact on provider-patient interactions. Understanding the problems and solutions from the perspective of birthing women is a key process in creating an optimal and patient-centered healthcare environment. Author Contribution Thu T. Nguyen: funding acquisition, conceptualization, methodology, data curation, formal analysis, writing—original draft preparation, review, and editing; Shaniece Criss: conceptualization, data curation, formal analysis, writing—original draft preparation, review, and editing; Melanie Kim: writing—original draft preparation, review, and editing; Monica M. De La Cruz: writing—original draft preparation, review, and editing; Nhung Thai: writing—original draft preparation, review, and editing; Junaid S. Merchant: review and editing; Yulin Hswen: review and editing; Amani M. Allen: review and editing; Gilbert C. Gee: review and editing; Quynh C. Nguyen, PhD: conceptualization, review, and editing. Funding Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities [R00MD012615 (TTN), R01MD015716 (TTN), and R01MD016037 (QCN)] and the National Library of Medicine [R01LM012849 (QCN)]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Data Availability Focus group transcripts are housed with the corresponding and senior authors. Code Availability Not applicable. Declarations Ethics Approval This study was approved by the University of California, San Francisco Institutional Review Board (18–24593). Consent to Participate All study participants provided consent to participate in this study. Consent for Publication The authors consent to the publication of this manuscript. Conflict of Interest The authors declare no competing interests. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Artiga S, Pham O, Orgera K, Ranji U. Racial disparities in maternal and infant health: an overview - issue brief [Internet]. KFF. 2020. 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Association between provider-patient racial concordance and the maternal health experience during pregnancy. J Patient Exp. SAGE Publications Inc; 2022;9:23743735221077520. 44. Street RL, O’Malley KJ, Cooper LA, Haidet P. Understanding concordance in patient-physician relationships: personal and ethnic dimensions of shared identity. Ann Fam Med. 2008;6:198–205. 45. Markus AR Andres E West KD Garro N Pellegrini C Medicaid covered births, 2008 through 2010, in the context of the implementation of health reform Womens Health Issues Elsevier 2013 23 e273 e280 10.1016/j.whi.2013.06.006 46. Khanal P, McGinnis T, Zephyrin L. Tracking state policies to improve maternal health outcomes [Internet]. 2020 [cited 2022 Sep 17]. Available from: https://www.commonwealthfund.org/blog/2020/tracking-state-policies-improve-maternal-health-outcomes. 47. Schaeffer K. 10 facts about today’s college graduates [Internet]. Pew Res. Cent. 2022 [cited 2022 Sep 12]. Available from: https://www.pewresearch.org/fact-tank/2022/04/12/10-facts-about-todays-college-graduates/.	36449130	PMC9713108	NO-CC CODE	2022-12-02 23:22:07	no	J Racial Ethn Health Disparities. 2022 Nov 30;:1-11	utf-8	J Racial Ethn Health Disparities	2,022	10.1007/s40615-022-01475-4	oa_other
==== Front Intensive Care Med Intensive Care Med Intensive Care Medicine 0342-4642 1432-1238 Springer Berlin Heidelberg Berlin/Heidelberg 36450929 6936 10.1007/s00134-022-06936-2 Letter Mental health symptoms are not correlated with peripheral inflammatory biomarkers concentrations in COVID-19-ARDS survivors Chean Dara 12 Benarroch Samuel 12 Pochard Frédéric 12 Kentish-Barnes Nancy 12 http://orcid.org/0000-0002-8162-1508 Azoulay Élie [email protected] 12 the Famirea study groupResche-Rigon Matthieu Megarbane Bruno Reuter Danielle Labbé Vincent Cariou Alain Géri Guillaume Meersch Guillaume Kouatchet Achille Guisset Olivier Bruneel Fabrice Reignier Jean Souppart Virginie Barbier François Argaud Laurent Quenot Jean-Pierre Papazian Laurent Guidet Bertrand Thiéry Guillaume Klouche Kada Lesieur Olivier Demoule Alexandre Guitton Christophe Capellier Gilles Mourvillier Bruno Biard Lucie 1 grid.413328.f 0000 0001 2300 6614 Médecine Intensive et Réanimation, APHP, Hôpital Saint Louis, Paris Cité University, 1 Avenue Claude Vellefaux, 75010 Paris, France 2 grid.50550.35 0000 0001 2175 4109 Psychiatry Department, Lariboisière Fernand-Widal University Hospital, Paris, France 30 11 2022 13 22 9 2022 7 11 2022 © Springer-Verlag GmbH Germany, part of Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. http://dx.doi.org/10.13039/501100004792 Ministère de l'Enseignement Supérieur et de la Recherche COVID2020 Azoulay Élie ==== Body pmcDear Editor, Clinically significant and long-lasting symptoms of anxiety, depression, and post-traumatic stress disorder (PTSD) are common in acute respiratory distress syndrome (ARDS) survivors [1]. Post-ARDS psychiatric morbidity is associated with younger age, female sex, prior psychological distress, unemployment, greater alcohol and opioid use, and the number of traumatic memories. In patients survived after ARDS due to coronavirus disease 2019 (COVID-19), high levels of acute stress disorders have been reported [2]. Previous studies assessing mental health symptoms in ARDS survivors did not report any significant difference in the prevalence and the severity of PTSD-related symptoms between COVID-19-ARDS and other ARDS etiologies [3]. In general, studies of PTSD have consistently found increased concentrations of inflammatory cytokines and imbalances in immune cell proportions [4, 5]. Along this line, plasma concentration of C-reactive protein (CRP) has been associated with the development of PTSD-symptoms in war zone-deployed Marines [4], with each tenfold increment in CRP concentration being associated with an odds ratio of PTSD symptoms of 1.51 (95% CI 1.15–1.97). However, in quarantined COVID-19 patients in whom very high rates of symptoms of anxiety and depression were self-reported [5], CRP concentrations and other inflammatory markers did not show statistical difference between patients with and without psychological symptoms. Identifying patients at higher risk of mental health symptoms would offer the opportunity of preventive measures to avoid or alleviate the prevalence and severity of psychological burden We sought to correlate peripheral inflammatory biomarkers to the development of symptoms of anxiety, depression and PTSD in critically ill patients with severe COVID-19 ARDS admitted to 23 intensive care units (ICUs) in France during the first COVID-19 wave (March and April 2020). These patients were reported elsewhere [3]. CRP, procalcitonin (PCT) and ferritin were sampled at ICU admission. During the day-90 telephone interviews, the patients completed the Impact of Event Scale Revised (IES-R) to assess PTSD-related symptoms and the Hospital Anxiety and Depression Scale (HADS) to assess the prevalence of symptoms of anxiety and depression. The Pearson correlation coefficient (R) was used to measure the strength and the direction of the linear relationship between the concentrations of inflammatory biomarker and the IES-R, or the anxiety and the depression subscales of the HADS. Among the 178 ICU patients with COVID-19 ARDS, assessment of mental health symptoms was available in 156 (88%, median age 59 (IQR 50–68), 118 (76%) men and 38 (24%) women). Mechanical ventilation was required in 114 (73%) patients and 42 (27%) required high flow nasal oxygen only. Symptoms of anxiety (HADS-anxiety subscale > 7), depression (HADS-depression subscale > 7), and PTSD (IES-R > 22) were found in 25%, 22% and 22% of the patients, respectively. Symptoms of anxiety and depression were found in 55% and 42% of patients with PTSD, respectively, as compared to 9% and 8% of patients without PTSD, respectively. CRP concentration at admission was 157 mg/l (IQR 86–246), procalcitonin was 0.37 ng/ml (0.16–1.17), and ferritin was 1280 μγ/l (830–2605). Pearson correlation coefficient for the association between CRP and the anxiety subscale of the HADS was 0.03, showing a poor association between the two scores (Fig. 1). Similar figures were found for the association between CRP and the depression subscale of the HADS or the IES-R (R = 0.009 and R = 0.06, respectively). Similarly, PCT concentrations at admission were poorly correlated with the anxiety or depression subscales of the HADS or with the IES-R (R = 0.02, R = 0.1, and R = − 0.04, respectively). Last, ferritin concentrations at admission were also poorly correlated with mental health symptoms (R = 0.05 for the anxiety subscale of the HADS, R = 0.03 for the depression subscale of the HADS, and R = 0.11 for the IES-R).Fig. 1 Relationship between the concentrations of inflammatory biomarker and the scales for PTSD (Impact of event Scale-revised, IES-R), or anxiety and depression (Hospital anxiety and depression Scale, HADS) In conclusion, peripheral inflammatory biomarkers are poorly correlated with further occurrence of symptoms of anxiety, depression or PTSD in survivors of COVID-19 severe ARDS. Until PTSD-specific biomarkers can be identified validated in this setting, early screening of mental henalth symptoms in ARDS survivors should be offered regardless the concentrations of initial peripheral inflammatory biomarkers. Acknowledgements The Famirea study group: Matthieu Resche-Rigon: Clinical Research Unit, APHP, Saint Louis University Hospital, Paris, France; Bruno Megarbane: Medical Intensive Care Unit, APHP, Lariboisière University Hospital, Paris, France; Danielle Reuter: Medical-Surgical Intensive Care Unit, CH Sud Francilien, Corbeil, France; Vincent Labbé: Medical-Surgical Intensive Care Unit, APHP, Tenon University Hospital, Paris, France; Alain Cariou: Medical Intensive Care Unit, Cochin University Hospital, APHP, Centre–Université de Paris, Paris, France; Guillaume Géri: Medical-Surgical Intensive Care Unit, APHP, Ambroise Paré University Hospital, Boulogne, France; Guillaume Van der Meersch: Medical-Surgical Intensive Care Unit, APHP, Avicenne University Hospital, Bobigny, France; Achille Kouatchet: Medical Intensive Care Unit, Angers Teaching Hospital, Angers, France; Olivier Guisset: Medical Intensive Care Unit, Saint-André Hospital, Bordeaux, France; Fabrice Bruneel: Intensive Care Unit, André Mignot Hospital, Le Chesnay, France; Jean Reignier: Medical Intensive Care Unit, University Hospital Centre, Nantes, France.; Virginie Souppart: Famirea Study Group, Medical Intensive Care Unit, APHP, Saint Louis University Hospital, Paris, France; François Barbier: Medical Intensive Care Unit, La Source Hospital, CHR Orléans, Orléans, France; Laurent Argaud: Medical Intensive Care Department, Edouard Herriot Hospital, Hospices Civils de Lyon, Lyon, France; Jean-Pierre Quenot: Medical Intensive Care Department, University Hospital, Dijon, France; Laurent Papazian: Respiratory and Infectious Diseases Intensive Care Unit, APHM Hôpital Nord, Marseille, France; Bertrand Guidet: Medical Intensive Care Unit, APHP, Saint-Antoine University Hospital, Paris, France; Guillaume Thiéry: Medical Intensive Care Unit, Saint-Etienne, University Hospital, Paris, France; Kada Klouche: Department of Intensive Care Medicine, Lapeyronie Hospital, Montpellier, France; Olivier Lesieur: Medical-Surgical Intensive Care Unit, La Rochelle Hospital, La Rochelle, France; Alexandre Demoule: AP-HP, Groupe Hospitalier Universitaire APHP-Sorbonne Université, Pitié-Salpêtrière site, Service de Pneumologie, Médecine Intensive et Réanimation (Département R3S) and Sorbonne Université, INSERM, UMRS1158 Neurophysiologie Respiratoire Expérimentale et Clinique, F-75005 Paris, France; Christophe Guitton: Medical Intensive Care Unit, Le Mans Hospital, Le Mans, France; Gilles Capellier: Medical Intensive Care Unit, Besançon, University Hospital, Besançon, France; Bruno Mourvillier: Medical Intensive Care Unit, Reims University Hospital, Reims, France; Lucie Biard: Clinical Research Unit, APHP, Saint Louis University Hospital, Paris, France. Funding All financial support for the research was provided by the French Ministry of Health (Grant COVID2020). Declarations Conflicts of interest Authors declare no conflict of interest in relation with this manuscript. The members of “The Famirea study group” are processed under acknowledgements. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Bienvenu OJ Psychiatric symptoms after acute respiratory distress syndrome: a 5-year longitudinal study Intensive Care Med 2018 44 38 47 10.1007/s00134-017-5009-4 29279973 2. Mongodi S High prevalence of acute stress disorder and persisting symptoms in ICU survivors after COVID-19 Intensive Care Med 2021 47 616 618 10.1007/s00134-021-06349-7 33730197 3. Azoulay E Association of COVID-19 acute respiratory distress syndrome with symptoms of posttraumatic stress disorder in family members after ICU discharge JAMA 2022 327 1042 1050 10.1001/jama.2022.2017 35179564 4. Eraly SA Assessment of plasma C-reactive protein as a biomarker of posttraumatic stress disorder risk JAMA Psychiat 2014 71 423 431 10.1001/jamapsychiatry.2013.4374 5. Guo Q Immediate psychological distress in quarantined patients with COVID-19 and its association with peripheral inflammation: a mixed-method study Brain Behav Immun 2020 88 17 27 10.1016/j.bbi.2020.05.038 32416290	36450929	PMC9713109	NO-CC CODE	2022-12-02 23:22:07	no	Intensive Care Med. 2022 Nov 30;:1-3	utf-8	Intensive Care Med	2,022	10.1007/s00134-022-06936-2	oa_other
==== Front SN Bus Econ SN Bus Econ Sn Business & Economics 2662-9399 Springer International Publishing Cham 385 10.1007/s43546-022-00385-1 Original Article How Covid-19 changed economic and health-related worries in Italy Stella Gian Paolo [email protected] 1 Filotto Umberto [email protected] 2 Cervellati Enrico Maria [email protected] 3 1 grid.17682.3a 0000 0001 0111 3566 Department of Business Economics and Quantitative Studies, University of Naples “Parthenope”, Via Generale Parisi, 13, 80132 Naples, Italy 2 grid.6530.0 0000 0001 2300 0941 Department of Management and Law, University of Rome “Tor Vergata”, Via Columbia 2, 00133 Rome, Italy 3 grid.7841.a Department of Human Science, University of Rome Link Campus, Via del Casale di S. Pio V, 44, 00165 Rome, Italy 30 11 2022 2022 2 12 19718 5 2022 23 11 2022 © The Author(s), under exclusive licence to Springer Nature Switzerland AG 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. The Covid-19 pandemic has been a huge challenge for governments all over the world, as well as for the World Health Organization (WHO) and the pharmaceutical companies in charge of creating the vaccines against the coronavirus. The success of all the efforts and the measures put in place to contain the spread of the contagion and to immunize people, however, also depends on people social compliance. In this study, we thus investigate how demographic and socio-economic variables affected individuals’ economic and health-related worries due to the Covid-19 pandemic. Using questions created by the WHO, we surveyed about 3000 Italians between May and June 2020. Our results show that individuals’ socio-demographic and socio-economic characteristics are engaged with distinct types of worries due to Covid-19, such as health-related worries, economic-related worries and worries connected to restrictions on movements. Our findings have implications for decision makers and policy makers in showing how important is to consider demographic and socio-economic differences between individuals, to better understand how people are differently affected by different worries and which actions and policies may be more effective in protecting and supporting people especially the most vulnerable ones. Keywords Covid-19 coronavirus pandemic Demographic and socio-economic individual characteristics Economic worries Health-related worries Italy issue-copyright-statement© Springer Nature Switzerland AG 2022 ==== Body pmcIntroduction and literature review The Coronavirus pandemic is one of the greatest challenges of recent decades. Governments struggled to implement measures to contain the virus spreading. Almost everywhere, governments proposed “soft measures” such as disinfection guidelines and behavioral recommendations, but as the situation worsen, they took drastic measures, such as lockdowns. Italy was an exception as the government quickly decided to move from soft to hard containment measures, due to the severity of Covid-19. At the beginning of the pandemic, before it started to hit other European countries, a debate on the existence of an “Italian case” asked why Italy was so deeply impacted by coronavirus compared to other European countries. The Italian government was accused to have acted poorly to contain the virus and it was perceived as creating the conditions for the virus to spread. However, later on, the opposite seemed true, and it was understood that being the first western country severely hit by the Coronavirus, Italy had just been ill-fated, and it was simply unprepared to face this huge challenge. Eventually, it has been internationally recognized that the measures put in place by the Italian government to contain the virus were probably among the most effective ones (Armillei and Filippucci 2020). If on one hand the total lockdown served to save thousands of lives, on the other hand it had a tremendous impact from an economic point of view, in a sort of macabre trade-off between economic and casualties’ numbers. It is important to stress that the effectiveness of government measures depends on citizens’ social compliance and responsibility, that, in turn, depend on how worried people are, both from a health and economic point of view. Müller and Rau (2021) propose a survey study, analyzing the extent to which three key economic preferences (risk, time, trust) are able to predict citizens’ social compliance to government’s policy measures. They investigate how economic preferences and social responsibility shape people’s perception of the dangers associated with coronavirus as well as people behaviors related to avoiding crowds, staying at home, panic buying, and willingness to get tested for Covid-19. They found that higher risk tolerance is associated with a lower propensity to avoid crowds, higher patience increases compliance with government’s measures, while present bias leads to panic buying. Furthermore, they find a positive relationship between social responsibility and compliance to government’s measures. Our main goal is to discern which were the main economic and health worries of people during the pandemic, classifying people depending on demographic and socio-economic variables to verify if these variables are able to explain why different people were impacted differently by the distinct worries connected to the Coronavirus. As shown by Cervellati et al. (2022), while socio-economic characteristics are usually considered as mere control variables, in the new pandemic context, they may represent important explanatory variables as the coronavirus differently affected people with distinct individual characteristics. Based on previous findings in the literature, we consider the following variables: age, gender, civil status, education, work status, income, area of residence, economic satisfaction changes due to the pandemic (described in detail below). With regard to age, it should be considered that younger people are likely to have a better general health condition, as well as to (subjectively) feel more vigorous, compared to older ones, thus they may have been less worried about the health issue. Older people, instead, are on average more likely to health problems compared to younger ones. In addition, this awareness of greater vulnerability was probably intensified by the pandemic’s toll among elders, as the number of casualties due to the Covid-19 were terrible among older Italians in the first months of the pandemic in spring 2020. Thus, older people may have been more worried (or even scared) of getting the virus, compared to younger ones. On the other hand, older people typically have higher accumulated wealth than younger ones, so they may be less worried from a personal economic point of view, which is about not being able to purchase what they need, even in case of shortages that could have driven up prices. In addition, the sudden deprivation of social contact (e.g., dating, hanging out with friends, parties, sports—that is particularly important to young people—due to the lockdown and to the other restrictions on movements, may have been more stressful to them than to older people. Thus, we may expect younger people to be more worried than older ones about restrictions on movements. Gender differences may explain different behaviors and degrees of life and economics satisfaction. During the lockdown, women in general bore the greatest part of household duties and children care, having to combine them together with smart working. In some cases, women eventually resigned from their job to take care of their families. Of course, also some men did so, but to a lesser extent. Thus, gender appears to be an important explanatory variable of the economic worries. With regard to health-related worries, Cervellati et al. (2022) showed that Italian men exhibited less healthy behaviors than women, probably due to trait impulsivity Clay and Parker (2020), which is the tendency to act without adequate forethought. During the pandemic period, especially during the lockdown months, civil status played a major role with respect to changes in life and economic satisfaction and to health-related and economic worries. People happiness is affected by the time people spend with others, thus, during the lockdown, it made a difference if individuals were alone or with their spouse, kids, parents, and etcetera. Adjusting for several socio-demographic and economic variables, Cervellati et al. (2022) find that satisfaction among married people increases as the time spent with the spouse increases. Since the lockdown forced married people to spend time with their spouses, the author’s simulations show that their happiness increased compared to before the lockdown. Instead, their happiness decreased when they were losing work time (for example due to child caring) or when their income decreased. For singles, on the other side, life satisfaction decreased as time spent alone increased: lockdown forced solitude on singles and their happiness decreased in general (they were alone to cook, grocery shopping, etcetera, but also in terms of affect they may feel alone), in particular for whom also suffered income and work time losses. Huebener et al. (2020) investigate the effects of movement restrictions that the German government put in place to contain the spread of the virus on individuals with dependent children. Through a survey administered in May and June 2020, when day care centers and school were closed in Germany, but other government’s measures had been relaxed and new infections were low, the authors’ goal was to analyze the effect of such closures on parental wellbeing. They find that the Covid-19 crisis decreased the wellbeing of individuals with children, especially for those with young children, for women, and for people with lower levels of education. Thus, their study shows that government’s measures to contain crisis such as Covid-19 do have large effects on family wellbeing, with implications both for parents’ work and for child development. With regard to Italy, Cervellati et al. (2022) found that married individuals (single, divorced, or widowed) behaved in a healthier way compared to unmarried ones. They were probably able to eat healthier than unmarried ones that, in addition, probably due to loneliness caused by isolation during the lockdown months and consequent higher levels of stress and worries, tended to drink more alcohol and to exhibit more precautionary buying than married ones. This evidence is in line with previous studies showing how alcohol misuse (Clay and Parker 2020) and unhealthy eating (Rundle et al. 2020) have been more common among unmarried people. At the same time, unmarried people adhered more closely to social distancing practices than married ones. If, on one hand, single, divorced, and widowed people may have felt deeply the loneliness, stress, and worries caused by the pandemic, especially during the lockdown, thus being tempted to meet family members and friends, on the other hand they may have preferred to comply with social distance to protect themselves and others. In addition, it should be kept in mind that every person in a household adds to the size of its network of social contacts, thus to the desire and need for contact with others. Not all couples, however, agreed with regard to how many visitors to allow in their home or who to visit. Unmarried people living alone, instead, being alone, had full control over these decisions, not having to compromise with a spouse. Thus, in respect to restriction on movements worries, we do not have an aprioristic expectation, given the above-mentioned contrasting evidence. Education is usually significant in explaining individual differences, and all other things being equal, increases the levels of life and economic satisfaction. However, in the novel coronavirus environment it is not straightforward to discern what kind of impact education had on health and economic worries. It could be that more educated people did trust the “experts” (e.g., government officials and scientists) more than less educated people, recognizing the role of experts for guidance, or vice versa that less educated people trusted and followed experts more than more educated ones that could have been more skeptical. In this respect, Cervellati et al. (2022) found that more educated people were more resilient in responding to the pandemic, keeping more healthy habits compared to less educated ones. As a possible explanation, the authors claim that it could be that higher educated people may engage in healthier behaviors because they better consider the future consequences of less healthy choices, for them and others, compared to less educated ones. Following their reasoning, we may thus expect that more educated people may be more sensitive to health-related worries, as well as to country economic worries. We do not have a prior, instead, with regard to their personal economic worries, since on one hand more educated people may worried, anticipating also for themselves financial difficulties in the future, on the other hand they are, on average, higher income earner, thus they may be less worried about their personal economic situation. We additionally claim that work status is important in explaining the differences in life and economic satisfaction during the lockdown, because individuals’ personal situation changed dramatically if they were occupied (full vs. part-time) or unemployed. At the same time, as suggested by Cervellati et al. (2022), it is also important to consider the joint effect of demographic and socio-economic variables. In this respect, they found that young people possess, on average, lower level of income and poorer working status compared to older ones, thus we may expect younger respondents to our survey to be more worried than older ones from an economic point of view. Of course, also income is connected to both economic worries and health-related ones since people with higher income may be less worried about incurring in financial difficulties due to the pandemic or not being able to cover health-related expenses. We also consider people area of residence, distinguishing between small towns and big cities (based on the number of inhabitants), to ascertain if the size of the city where respondents live had an effect on changes in life and economic satisfaction and (economic and health-related) worries during the pandemic. The reason of considering the area of residence is that previous studies in the literature on life satisfaction (e.g., Helliwell et al. 2020) show that, in normal situations, life satisfaction in industrialized countries is usually higher in big cities with respect to small towns. However, the novel Covid-19 situation was far from normal, thus we want to understand if and how life satisfaction changed during the pandemic for people living in big cities versus small towns. Armillei and Filippucci (2020) found that in Italy the epidemic had a geographical heterogeneous impact, also within most highly infected zones. They relied on a novel dataset with wide granular information, finding that higher mortality rates across municipalities were associated to people with low education and income levels, as well as with low household dimension. This evidence suggests that peripheral areas were more severely hit by the virus. We also found differences in people worries, depending on if they were living in big cities or small towns. A first possible explanation of this evidence is that since in Italy the first cases of Covid-19 were discovered in small towns, while in big cities the coronavirus seemed initially to spread less—as a matter of fact a lively debate followed trying to understand the reasons of this initial evidence—people living in small towns may have been more worried about the virus. A second potential explanation is that the spread of information differs depending on the city dimension. In small towns, people may be more affected by their small circles of acquaintances or misinterpret contagion data based on “small sample bias”. Still with regard to the Italian case, Cervellati et al. (2022) found that people living in smaller towns also displayed more precautionary buying compared to people living in bigger cities, probably because smaller cities could have lower food supplies during the pandemic, compared to big cities. Therefore, we may expect people living in small towns to be more worried than people living in big cities. Finally, with regard to changes in economic satisfaction—that is if respondents to our survey claimed that their economic situation worsen, stayed constant, or improved during the pandemic—we expect people claiming that their economic situation improved to be less worried than others, especially from an economic point of view, but potentially also with regard to health-related worries, since they may have felt more confident in facing potential health-related expenses. Data and methodology We collected data through questionnaires administered in Italy from the beginning of May 2020 until the end of June 2020. We used the Computer Assisted Web Interview (CAWI) survey methodology. We invited to participate about 3000 randomly selected individuals, and ended up with collecting 2950 questionnaires that, after cleaning them canceling the ones with missing values, reduced to 2872. The questionnaire included socio-economic information and worries related the health and economic domains, as well as to restrictions to movements. With regard to worries, we used the first release of the “Survey Tool and Guidance: Behavioural Insights on COVID-19” issued by the World Health Organizations (WHO). As far as we know, we are among the first ones to use the WHO tool in Italy (see also Cervellati et al. 2022), and we use the first release of it, including questions on worries that the second release does not include. In Table 1, we describe the variables that we use and how we codified them for the analyses that follow.Table 1 Variables’ descriptions and codification Variable Description and codification Age Respondent’s age Gender Female = 0; male = 1 Civil status Single = 1; married = 2; divorced = 3; widowed = 4 Education 0–9 Years = 1; 9–12 years = 2; more than 12 years = 3 Work status Full-time (> 35 h/week) = 1; part-time (< 35 h/week) = 2; housekeeper = 3; student = 4; retired = 5; disabled or unable to work = 6; unemployed = 7 Income < €10,000 = 1; between €10,000 and €20,000 = 2; between €20,000 and €40,000 = 3; between €40,000 and €80,000 = 4; > €80,000 = 5; refuse to answer = 99 Area of residence Small towns (< 20,000 inhabitants) = 1; City (> 20,000 inhabitants) = 2 Economic satisfaction changes Better = 1; worse = 2; unchanged = 3 To analyze the impact of Covid-19 on respondents’ worries, we used 14 specific questions provided in the first release of WHO (2020). To answer, respondents used a 10-point Likert scale ranging from 1 (Don’t worry at all) to 10 (Worry a lot), as shown in Table 2.Table 2 Covid-19-related worries questions Item Worries Range 1 Losing someone I love 1–10 2 Health system being overloaded 1–10 3 My own mental health 1–10 4 My own physical health 1–10 5 My loved ones’ health 1–10 6 Restricted liberty of movement 1–10 7 Loosing vacation opportunities 1–10 8 Small companies running out of business 1–10 9 Economic recession in my country 1–10 10 Restricted access to food supplies 1–10 11 Becoming unemployed 1–10 12 Not being able to pay my bills 1–10 13 Not be able to visit people who depend on me 1–10 14 Having to defend a decision not to participate in a social event which my family or friends expect me to attend 1–10 Results Descriptive results Respondents’ age ranges from 18 to 96 years, with an average of 41.67 years, and a standard deviation (SD) of 14.20. Respondents are almost equally distributed between women (47.9% of the sample) and men. With regard to civil status, 45.6% of respondents were married, 42.3% single, 8.8% divorced and 3.3% widowed. Regarding education, 14.9% of respondents completed less than 9 years of education, 23.3% between 9 and 12 years, and 62.5% more than 12 years. In terms of work situation, 49.7% of respondents worked full time, 16.2% part-time, 4.9% were housekeepers, 16.4% students, 6.3% retired, 0.6% disabled or unable to work, and 6% unemployed. By “housekeeper” we mean a man or a woman staying at home, keeping care of the house, that is, we do not mean a professional hired housekeeper. With regards to income,19.2% preferred not to answer;8.6% had an annual income of less than €10,000; 23.3% between €10,000 and €20,000; 31.7% between €20,000 and €40,000; 13.8% between €40,000 and €80,000; 3.5% had an income of more than €80,000. In terms of area of residence, 71.7% of respondents lived in a city (more than 20,000 inhabitants) whereas 28.3% in a small town (less than 20,000 inhabitants). Regarding income variable, the option “Preferred not to say”, coded 99, was omitted from the descriptive analysis to avoid abnormal fluctuations in the values of the average and, consequently, of the standard deviation. Thus, for this variable the number of observations is lower, that is, 2320. The changes about economic satisfaction related to the effects of Covid-19 shows that 41.7% of respondents declared that their economic satisfactions decreased during the pandemic, 50.9% claimed that their situation remained unchanged and only 7.3% to be better off. Factor analysis We conducted a factor analysis (using SPSS 26 software). The Kaiser–Meyer–Olkin (KMO) index is equal to 0.853, whereas the Bartlett test is significant (p = 0.000). These results reject the null hypothesis that the matrix of correlations between variables is an identity matrix. (Hair et al., 1995). According with the K1 criterion (Kaiser 1960), factor analysis proposes the extraction of four factors, which explain in cumulative terms 70.195% of the variance. According with the standards of Peterson (2000), in Table 3, we show that the explained variance (59.23%) can be considered acceptable.Table 3 Total variance explained Factor Initial eigenvalues Extraction sums of squared loadings Rotation sums of squared loadings Total % Of variance Cumulative % Total % Of Variance Cumulative % Total 1 5.437 38.834 38.834 4.976 35.54 35.54 4.266 2 1.895 13.535 52.369 1.437 10.263 45.803 3.529 3 1.279 9.139 61.509 0.904 6.457 52.26 2.826 4 1.216 8.687 70.195 0.976 6.975 59.235 2.612 Extraction method: maximum likelihood We analyzed the information provided by the worries questions through a maximum likelihood (ML) factor analysis with Promax method. Indeed, Maximum Likelihood algorithm allows reducing the dimension analysis, optimizing the dimensionality of our indicator in order to yield a more robust statistical analysis (Costello and Osborne 2005). The Pattern matrix shows that the four-factor solution is clean. The pattern matrix detects the loading of each item on the factors. Specifically, the matrix highlights whether the correlation between each item and the factor. See Table 4. The matrix has only one cross loading to be managed present in the second factor which presence, according to Krishnan (2011), is not so relevant.Table 4 Pattern matrix Factors Item (1) Health worries (2) Personal economic worries (3) Country economic worries (4) Restrictions on movement worries Losing someone I love 1 0.938 My loved ones’ health 5 0.894 Health system being overloaded 2 0.768 My own physical health 4 0.555 My own mental health 3 0.512 Not be able to visit people who depend on me 13 0.352 Not being able to pay my bills 12 0.991 Becoming unemployed 11 0.873 Restricted access to food supplies 10 0.421 Economic recession in my country 9 0.833 Small companies running out of business 8 0.813 Loosing vacation opportunities 7 0.777 Restricted liberty of movement 6 0.689 Having to defend a decision not to participate in a social event which my family or friends expect me to attend 14 0.316 0.405 Maximum likelihood as the estimation method and a promax as rotation method We named the four factors as follows: (1) Health Worries; (2) Personal Economic Worry; (3) Country Economic Worry; (4) Restrictions on Movements Worry. We thus distinguish between health-related, economic-related worries—further dividing the latter in worries related to the personal situation from the ones related to the overall country economic situation—and worries related to restrictions on movements. We use each one of the above-mentioned four factors as dependent variables in the linear regression models that follow (see Table 6 later on). For the reasons described in detail in “Introduction and literature review”, we use as independent variables the demographic and socio-economic variables presented in Table 1. Correlation matrix Before presenting our regression analyses, in Table 5, we show the correlation between economic and health-related worries and the above-mentioned demographic and socio-economic variables. The correlations are high and statistically significant, suggesting that individual’s demographic and socio-economic characteristics may help explaining different degrees of worries related to the Covid-19.Table 5 Correlations matrix between socio-economic variables and worries related to the Covid-19 No Variable 1 2 3 4 5 6 7 8 9 10 11 12 1 Age 1 2 Gender − 039* 1 3 Civil status 0.504* − 071* 1 4 Education − 143* − 046 − 078* 1 5 Work status − 053* − 066* − 153* − 090* 1 6 Income 0.116* 0.113* 0.119* 0.223* − 0.163* 1 7 Area of residence 0.002 0.040 − 0.037 0.131* − 0.067* 0.161 1 8 Economic satisfaction changes − 0.016 0.075* − 0.028 0.056* − 0.016 0.210 0.03* 1 9 Health worry 0.012 − 0.221*** 0.031* 0.043 0.005 − 0.086 − 0.043 − 160* 1 10 Economic worry − 0.125* − 0.125* − 0.028 − 0.045 0.044 − 0.235 − 0.108* − 0.277* 0.543* 1 11 Country economic worry 0.083* − 0.158* 0.066* 0.091* − 0.056* 0.003 − 0.016 − 0.144* 0.577* 0.394* 1 12 Restriction of movements worry − 0.079* − 0.055* − 0.022 − 0.007 0.011 − 0.006 − 0.024 − 0.048 0.443* 0.598* 0.305* 1 Asterisks indicate the significance of the coefficients at 10% (), 5% () and 1% () Investigating the correlation matrix, it is useful to discuss the linkages among demographic and socio-economic variables and between these variables and economic and health-related worries. Later on, discussing the empirical findings resulting from our regression analysis, we will discuss further the importance of demographic and socio-economic variables in defining economic and health-related worries related to the Covid-19 pandemic. The negative correlation coefficient between education and age (− 0.134) suggests that in our sample older respondents possess a lower level of education. The negative coefficient between work status and age (− 0.053), instead, suggests that older respondents are better employed compared to younger ones, as we could expect. Instead, the positive correlation between income and age (0.116) suggests, in line with our intuition, that older people possess, on average, higher income. The correlation coefficients of age with “Area of residence” (0.002), “Economic satisfaction changes” (− 0.016) and “Health Worry” (0.012) are not statistically significant suggesting no big differences in these dimensions depending on age. Instead, “Economic Worry” correlation with age is negative and statistically significant (− 0.125), suggesting that older respondents worry less than younger ones from an economic point of view, maybe also because, as mentioned above, they appear to be better employed. Interestingly, the correlation coefficient between age and “Country Economic Worry” (0.083), as well as “Restriction of Movements Worry” (− 0.079) suggest that older respondents are more worried compared to younger ones. These two statistically significant positive correlation coefficients seem to suggest that older people are more worried for the economic perspective of the country, in contrast with their personal economic situation. A possible explanation is that while on one hand they may count on a better employment compared to younger people, older respondents appear to me more worried for the perspective of the country as a whole, from an economic point of view. On the other hand, older people may be more worried about restrictions of movement for several reasons, for example with regard to the possibility of not being able to meet their children or grandchildren, their families and friends, more in general. With regard to gender, men seems to possess, on average, a slightly lower level of education with respect to women, given the negative correlation coefficient (− 0.046). The negative coefficient between gender and work status (− 0.066) seems instead to indicate that women are, on average, slightly better employed than men. The correlation is instead positive between gender and income (0.113) suggesting that men, all other things being equal, earn higher levels of income compared to women. The correlation between gender and area of residence is slightly positive (0.040), suggesting that there are, among our respondents, slightly more men than women in bigger cities. The coefficients related to both “Economic satisfaction changes”, “Economic Worry” “Restrictions on Movements worries” are not statistically significant, suggesting no big differences between men and women. Instead, men seem to be more worried than women in terms of “Health worry” (0.031) and “Country economic worry” (0.066). With regard to education, in contrast with our intuition, the negative correlation coefficient (− 0.090) suggests that, among our respondents, the higher the respondents’ level of education, the worse their employment status. Of course, there may be several explanations of this evidence, depending on the field of study of our respondents and on other socio-economic and demographic characteristics. The regression analysis will be useful to shed additional light on this issue. Instead, in line with our intuition, the correlation coefficient between education and income is positive (0.223) indicating higher income levels as the education level increases. Also, in line with our expectations, people living in bigger cities possess a higher level of education (0.131). Big cities usually offer more opportunities both from an educational point of view, but also in terms of job perspectives, attracting more educated people. The positive correlation coefficient between education and “Economic satisfaction changes” (0.056) suggests that the personal economic situation of more educated respondents improved during the observation period. Among worries, the only not statistically significant is the one related to “Restrictions on Movements worries”. Instead, with regard to economic and health-related worries, in line with our intuition, the negative correlation coefficient (− 0.045) suggests that more educated people were less worried about their personal economic situation—probably because, as mentioned above, the higher the level of education, the higher the income—while instead more worried about “Health worry” and “Country Economic Worry”, suggesting that less educated people may have underestimated the negative effects of the pandemic. With regard to the work status, as expected, we find a negative correlation with income (− 0.163)—since the scale of “work status” takes higher values passing from fully employed to unemployed—suggesting that the better the employment, the higher the income. Respondents living in big cities seem to have a better employment (correlation equal to − 0.067). There were no statistically significant differences with respect to changes in economic satisfaction during the period. The only worries to be statistically significant are the economic ones, while the ones related to health and to restrictions to movements seem not to significantly worry the respondents. Interestingly, personal “Economic worry” display a positive correlation, meaning that they are higher among unemployed people, as we might expect. Instead, the coefficient associated with “Country Economic Worry” is negative, suggesting that better employed people were more worried about the country economic perspectives, with respect to respondent with worse employment situation, probably because they were more focused on their personal economic situation. With regard to the income variable, we find it to be positively correlated with living in bigger cities (0.161)—as expected since they typically offer better employment opportunities, but also because the cost of living is usually higher, thus discouraging lower earning people—as well as with increases in economic satisfaction, suggesting that the pandemic hit more the lower earning respondents. It is interesting to note that the coefficients related to “Health worry” and “Economic worry” are both negative (respectively, equal to − 0.086 and − 0.235), suggesting that higher earning people were less worried both from an economic point of view, but also with respect to health. Instead, the coefficients related to both “Country economic worry” and to restrictions to movements are not statistically significant. With respect to changes in economic satisfaction, our respondents claimed that their situation improved more if they lived in bigger cities (0.031), while, as expected, we find a negative correlation with economic-related worry (− 0.108) and health-related worries (− 0.043), since a decrease in economic satisfaction is usually linked to a worsening of the economic situation. Instead, worries related to “Country economic worry” and to restrictions to movements are not statistically significant. As expected, all the correlation coefficients linking changes in economic satisfaction to worries are all negative and statistically significant, suggesting that respondents which situation improved during the pandemic were also less worried on an economic ground, in line with our intuition, but also with respect to health-related worries. Interestingly, all worries are positively correlated with other worries, health-related, economic-related and the ones associated to restrictions of movements. Thus, it seems that the pandemic impacted negatively on all respects and that they are linked to one another. In the regression analyses that follow, we test if the above-mentioned evidence in terms of correlation coefficients are supported from an econometric point of view. Regression results In Table 6, we present the results from four regression analyses using the same demographic and socio-economic explanatory variables, but distinct dependent variables, related to the four above-mentioned worries: health-related, personal economic worries, country economic worries, and worries related to restrictions on movements.Table 6 Regression models with robust errors Model 1 Model 2 Model 3 Model 4 Health worries Personal economic worries Country economic worries Restrictions on movements worries Age 0.004 − 0.010* 0.007* − 0.006* (0.002) (0.002) (0.002) (0.002) Gender − 0.408* − 0.172* − 0.294* − 0.104 (0.039) (0.037) (0.038) (0.036) Civil status − 0.115* 0.0329 − 0.069 0.010 (0.032) (0.028) (0.032) (0.028) Education 0.060 − 0.023 0.120* − 0.032 (0.029) (0.027) (0.029) (0.027) Work status − 0.007 − 0.005 − 0.022 0.006 (0.011) (0.011) (0.011) (0.011) Income − 0.040* − 0.134* 0.014 0.026 (0.022) (0.020) (0.021) (0.020) Area of residence − 0.086 − 0.154* − 0.043 − 0.031 (0.043) (0.041) (0.041) (0.041) Economic satisfaction changes − 0.206* − 0.352* − 0.195* − 0.055* (0.033) (0.029) (0.033) (0.029) σ 0.80 0.137 0.60 0.013 Adjusted R2 0.076 0.134 0.057 0.010 No 2872 2872 2872 2872 p < 0.05, p < 0.01, **p < 0.001 Before commenting in detail the findings of the four regression analyses, we underline that in all models the demographic and socio-economic variables are often statistically significant, highlighting their importance in helping explain different degrees of worries between different people, in line with our previous results in terms of correlations and with previous findings in the literature (see Cervellati et al. 2022). In regression model 1, we use as dependent variable the first factor found through the factor analysis, that we called “Health Worries” because all the worries that constitute the components of this factor relate to worries associated to the health sphere. Except for “Work Status”, all the explanatory variables in this model are statistically significant. In particular, the positive coefficient associated with Age suggest that older respondents were more worried regard their health, with respect to younger ones. This result is in line with our expectations, as during the first phase of the pandemic in Italy, older people were hit more severely by the coronavirus, both in terms of contagions and of casualties. The negative coefficient associated with the dummy variable Gender suggests that women were more worried than men with regard to their health. A possible explanation of this evidence is that men are on average more overconfident than women, thus they may have underestimated the threat of Covid-19. A second explanation is that women have a stronger attitude to self-care compared to men. A third one is that since men, compared to women, on average display more trait impulsivity. (i.e., the tendency to act without proper forethought), they may be less worried from a health-related point of view, thus displaying less healthy attitudes and behaviors (Clay and Parker 2020). The positive coefficient associated with Education seems to suggest that the higher the number of years of education, the higher the worries with regard to health. More educated respondents could have a better appraisal of how serious the situation was and how high were the dangers for their health. Work Status is not statistically significant, suggesting no relevant differences with regard on health worries among (fully or partially) employed or unemployed respondents. As a matter of fact, work status may affect more economic worries than health worries. The negative coefficient associated with Income suggests that the higher the annual personal income, the lower the health worries. Respondents with higher income may count on more money to face medical expenses. The negative coefficient associate with Area of residence suggests that people living in smaller towns are more worried about health with respect to respondents living in bigger cities. Big cities usually have better hospitals, thus reassuring the people living there. The negative coefficient associated with Economic Satisfaction Changes suggests that people that saw their personal situation improved during the pandemic were less worried with regard to their health. In regression model 2, we use as dependent variable the second factor found in the factor analysis, that we called personal “personal economic worries” because all the worries that are the components of this factor relate to worries associated to the respondents’ personal economic sphere. In this second regression, not only work status, but also civil status and education are not statistically significant. In addition, the coefficient of Age is now negative, suggesting that the older the respondent, the lower the economic worries. As mentioned above, on average older people have higher income and better employment position, thus they may be less worried of not being able to pay their bills or having a restricted access to food supplies or even becoming unemployed, with respect to younger ones that on average have lower available income and more precarious jobs. As for health worries, also with regard to economic worries men appear to be less worried. While similar explanation can be used for economic worries as for health worries, in terms of income and occupancy, men have on average higher income and better jobs. As a matter of fact, Income appears to be the variables that mostly affect economic worries, with a coefficient equal to − 0.134, suggesting that respondents with higher income, in line with intuition, are less worried in economic terms, counting on higher available income and better jobs. The coefficients associated with Area of residence is negative as in regression model 1, suggesting that respondents living in bigger cities are less worried not only with respect to the health dimension, but also with regard to the economic sphere. Respondents living in bigger cities may also be less worried in general about becoming unemployed and finding a new job is usually easier in bigger cities. Finally, also with respect to “personal” economic worries, as for health worries, respondents with an improvement in economic satisfaction during the pandemic, record lower levels of economic worries. In our third econometric model, as dependent variable, we use the third factor determined through our factor analysis, that we name “Country Economic Worries”, which components are worries related to a possible economic recession in Italy and to the possibility that small companies could run out of business. The second component, related to small companies, is particularly relevant in Italy because the Italian economic system is dominated by small firms. Some results are in line with our regression model 1, dealing with “personal” economic worries, while other are at the opposite. In particular, the coefficient associated with Age is negative, while it was positive with respect to “personal” economic worries. While older people may be less worried about their personal economic situation, because on average they have higher available income and better jobs, it seems that they are more worried for the country as a whole, with respect to younger respondents. The reason could be that older people are more patriotic, or also that being less worried for their personal situation, they may see the “big picture” of the country or being more worried for their children, grandchildren or for young people (future generations) in general. This evidence may be coupled with the one on Civil Status, which negative coefficients suggests that married respondents are more worried for the economic condition of the country as a whole, probably again because of the worries for their children and future generations. As before, instead, men are less worried than women also with regard to the country economic situation. Even in this case, the above-mentioned explanations applied, together with a probable higher level of confidence that usually characterize more men than women. Interestingly, this is the only econometric model in which Work Status is statistically significant. The Work Status variable goes from full employment to unemployment and the negative coefficient suggest that respondents with poorer job positions worries a country economic downturn more than fully employed people. As in the previous two regression models, also in the third one a higher Economic Satisfaction Changes is associated with lower worries, also in terms of Country Economic Worries. Finally, in our fourth regression model, we analyze the fourth factor found in the factor analysis, which we called “Restrictions on Movements worries” since its constituents all refer to restrictions to movements. Interestingly, older respondents appear to be less worried compared to younger ones with respect with such a dimension. A possible explanation is that, given the fact that on average they were more severely hit by the pandemic, during the lockdown they did not want to move as much as younger people did. On the other hand, being older could also be associated with a higher degree of social responsibility. Even in this case men are less worried than women, as found in the other regression models. Interestingly, civil status, education, work status, area of residence and even income are not statistically significant. Thus, it seems that these variables do not play a role in explaining differences in worries with regard to the freedom of movement. The only other statistically significant variable in the fourth regression model is Economic Satisfaction Changes, stressing even further the importance of such a construct. Conclusions The purpose of this study is to investigate how demographic and socio-economic variables affected individuals’ economic and health-related worries due to the Covid-19 pandemic. To achieve our aim, we conducted a survey in Italy in May and June 2020 on about 3,000 individuals. Italy represents an interesting case because it was the first western country to be severely hit by the pandemic, but also because the Italian government put in place strong measures to contain the virus such as the full lockdown of the country. The effectiveness of these measures also depends on citizens’ responsibility in complying with them, that, in turn, depends on how worried people are, both from a health and economic point of view. This is why it is important to analyze how different demographic and socio-economic characteristics differently affected individuals’ economic and health-related worries. Our main goals are thus on one hand to discern which were the main economic and health-related worries of people during the pandemic, classifying them based on demographic and socio-economic characteristics and, on the other hand, to verify if these variables are able to explain why different people were impacted differently by the distinct worries connected to the Coronavirus. While these characteristics are usually considered as mere control variables, in the new pandemic context, our findings support the idea that they represent important explanatory variables to understand how people with distinct individual characteristics were differently worried about the Covid-19. We consider the following variables: age, gender, civil status, education, work status, income, area of residence, economic satisfaction changes due to the pandemic. Our results show that indeed individuals’ demographic and socio-economic characteristics are related with distinct types of worries due to Covid-19, which are health-related worries versus economic-related worries—further dividing the latter between worries on the personal economic situation versus worries on the whole Country economic situation—and finally on worries connected to restrictions on movements. Based on the empirical evidence, we can draw the following main conclusions. We find women and people that are older, unmarried, more educated, low earning, living in small towns, and for which the economic satisfaction worsen during the pandemic, to be more worried about health-related issues. With respect to personal economic worries, we find that women and people that are younger, low earning, living in small towns, and for which the economic satisfaction worsen during the pandemic, to be more worried. Instead, with regard to the general economic situation of the country, we find that women and people that are older, unmarried, more educated, unemployed, and for which the economic satisfaction worsen during the pandemic, to be more worried. Finally, we find higher health-related worries among older people, women, single, less educated, lower income earners, living in small towns and with a decrease level of economic satisfaction. The importance of considering individual differences regards several realms and has implications for decision makers and policy makers. First, it allows to transform the cold numbers on infected people and casualties into useful information to achieve a better knowledge of the dynamic of the pandemic, considering the distinct reaction of different people. Second, it helps explaining the distinct degree of social compliance to governments’ measures to contain the virus, since they alter perceptions, affect worries and behaviors. Third, it allows governments and other public institutions to understand which actions and policies may be more effective in protecting and supporting people, especially the most vulnerable ones, helping them in several ways, thus reducing their worries, improving their behaviors, with a positive effect on the society as a whole. An increased attention to individual differences may thus help decision makers and policy makers design actions to support people effectively, avoiding a one-size-fits-all approach. Acknowledgements We would like the Editorial Board and the anonymous referee for useful comments and suggestions on a previous version of the paper. Author contributions GP conceived the research idea, methodology and elaborated the results. UF wrote the conclusions and corrected the paper from typos. EC wrote the abstract and introduction of the paper. Funding The authors did not receive support from any organization for the submitted work. Data availability Not applicable. Declarations Conflict of interest The authors have no competing interest to declare that are relevant to the content of this article. Ethical approval Not applicable. ==== Refs References Armillei F, Filippucci F (2020) “The heterogenous impact of Covid-19: evidence from Italian municipalities”, working paper, Aug 3, 2020. Available on line at: https://www.localopportunitieslab.it/wp-content/uploads/2020/08/The-heterogenous-impact-of-Covid19-Evidence-from-Italian-municipalities-Armillei-and-Filippucci.pdf Cervellati EM Stella GP Filotto U Maino A How consumer spending and other consumer behaviours changed during the COVID-19 pandemic: evidence from Italy Glob Financ J 2022 51 2022 100680 10.1016/j.gfj.2021.100680 Clay JM Parker MO Alcohol use and misuse during the COVID-19 pandemic: a potential public health crisis? 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Unpublished manuscript, Early Child Development Mapping (ECMap) Project, Alberta, University of Alberta, Edmonton, Canada Müller S Rau HA Economic preferences and compliance in the social stress test of the Corona crisis J Public Econ 2021 194 104322 1 12 10.1016/j.jpubeco.2020.104322 Peterson RA A meta-analysis of variance accounted for and factor loadings in exploratory factor analysis Mark Lett 2000 11 3 261 275 10.1023/a:1008191211004 Rundle AG Park Y Herbstman JB Kinsey EW Wang YC COVID-19–related school closings and risk of weight gain among children Food Nutr 2020 16 226 233 10.1002/oby.22813 World Health Organization (2020) “Survey tool and guidance: Rapid, simple, flexible behavioural insights on COVID-19.” World Health Organization (WHO) Regional Office for Europe, July 29. https://apps.who.int/iris/bitstream/handle/10665/333549/WHO-EURO-2020-696-40431-54222-eng.pdf?sequence=1&isAllowed=y	36471718	PMC9713110	NO-CC CODE	2022-12-02 23:22:07	no	SN Bus Econ. 2022 Nov 30; 2(12):197	utf-8	SN Bus Econ	2,022	10.1007/s43546-022-00385-1	oa_other
==== Front Cancer Metastasis Rev Cancer Metastasis Rev Cancer Metastasis Reviews 0167-7659 1573-7233 Springer US New York 36451067 10070 10.1007/s10555-022-10070-2 Non-Thematic Review The vulnerable primed cancer stem cells in disguise: demystifying the role of Maspin http://orcid.org/0000-0001-9338-0652 Sheng Shijie [email protected] 123 Bernardo Margarida 13 Dzinic Sijana H. 23 Chen Kang 34 Sakr Wael A. 13 1 grid.254444.7 0000 0001 1456 7807 Department of Pathology, Wayne State University School of Medicine, Room 640.2, Hudson-Webber Building, 4100 John R Street, Detroit, MI 48201 USA 2 grid.254444.7 0000 0001 1456 7807 Department of Oncology, Wayne State University School of Medicine, Room 640.2, Hudson-Webber Building, 4100 John R Street, Detroit, MI 48201 USA 3 grid.477517.7 0000 0004 0396 4462 The Tumor Biology and Microenvironment Program of the Barbara Ann Karmanos Cancer Institute, Detroit, MI 48201 USA 4 grid.254444.7 0000 0001 1456 7807 Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI 48201 USA 1 12 2022 110 12 9 2022 17 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Epithelial-specific Maspin is widely known as a tumor suppressor. However, while the level of maspin expression is inversely correlated with tumor grade and stage, emerging clinical evidence shows a correlation between seemingly better differentiated tumor cells that express Maspin in both the nucleus and the cytoplasm, (n + c)Maspin, with a poor prognosis of many types of cancer. Biological studies demonstrate that Maspin plays an essential role in stem cell differentiation. In light of the recently established characterization of primed stem cells (P-SCs) in development, we propose, for the first time, that cancer stem cells (CSCs) also need to undergo priming (P-CSCs) before their transition to various progeny phenotypes. We envisage major differences in the steady state kinetics between P-SCs and P-CSCs. We further propose that P-CSCs of carcinoma are both marked and regulated by (n + c)Maspin. The concept of P-CSCs helps explain the apparent dichotomous relationships of (n + c)Maspin expression with cancer diagnosis and prognosis, and is supported by the evidence from mechanistic studies. We believe that the potential utility of (n + c)Maspin as a molecular marker of P-CSCs may significantly accelerate the advancement in our understanding of the genesis of tumor phenotypic plasticity in response to changes of tumor microenvironments (TME) or drug treatments. The vulnerabilities of the cellular state of (n + c)Maspin-expressing P-CSCs are also discussed as the rationale for future development of P-CSC-targeted chemotherapeutic and immunotherapeutic strategies. Keywords Tumor progression Phenotypic heterogeneity Histone deacetylase 1 Cellular stress Immunogenicity Salinomycin ==== Body pmcThe role of Maspin as a tumor suppressor challenged Maspin is a clay B member in the serine proteinase inhibitor (serpin) superfamily, also known as Serpin B5 [1]. The maspin expression is epithelial-specific in normal somatic tissues, and is differentially regulated in carcinomas. Since its discovery, Maspin is widely known as a class II tumor suppressor, i.e., the loss of its expression, but not its genetic mutation or deletion, is a gain of function in tumor progression [2]. The evidence in support to this notion includes the inverse correlation between the total levels of maspin protein and mRNA with tumor grade and stage [3]. For example, the level of maspin mRNA in primary prostate carcinoma (PCa) is comparable or slightly higher than that in adjacent benign prostate epithelial tissues, but is significantly down-regulated in invasive and metastatic diseases ([4], Fig. 1A). Accumulated biological evidence further supports a causative relationship between Maspin and tumor suppression, demonstrating that Maspin blocks tumor invasion and metastasis without affecting cell proliferation [3].Fig. 1 Differential expression of Maspin human PCa. (A): Differential expression of maspin mRNA in adjacent normal tissues (green, n = 59), primary tumor (red, n = 66), metastatic tumors (purple, n = 25). The level of expression is presented as a percent of the maximal expression detected in the cohort. Percentile ranking is based on the level of expression within each subgroup of tissue specimens. The plot is based on the data from GEO: GDS2545 / 862_at. (B) Immunohistochemical staining of maspin (brown color) in human tissues. (a): normal prostate tissue. Black arrow: secretory epithelial cells; Red arrow: basal epithelial cells. (b): nMaspin-expressing wow grade primary PCa; (c): (n + c)Maspin-expressing low grade PCa; (d): PCa bone metastasis. Inlet: low magnification However, clinical investigations into the relationship between Maspin and clinical outcome revealed that better or moderately differentiated primary tumors can be distinguished based on the subcellular localization patterns of Maspin. Specifically, as compared to the tumor cells that express maspin in the nucleus (nMaspin), the subpopulation of tumor cells that expresses Maspin in the nucleus plus cytoplasm ((n + c)Maspin) is specifically correlated with malignant progression and/or earlier tumor relapse [5] of ovarian cancer [6], non-small cell lung cancer [7], and PCa [8], among other types of carcinoma. It is worth noting that the majority of the experimental approaches employed established tumor cell lines or immortalized noncancerous epithelial cell lines, wherein the expression or silencing of Maspin is ectopically controlled constitutively. In in vitro culture, these cell lines express Maspin in the (n + c) pattern, irrespective of its subcellular localization in the original tissue specimens. The apparent dichotomous relationships of (n + c)Maspin expression with tumor cell differentiation and cancer prognosis bring into focus the following important questions: is Maspin exclusively a tumor suppressor? If the (n + c)Maspin-expressing tumor cells represent a bulk tumor cell population that is inhibited in invasion and metastasis, how can they lead to poor clinical outcomes? Is it possible that the (n + c)Maspin-expressing tumor cells in natural tumor progression represent an intermediary cellular state that is not aggressive it itself but has the potential to become aggressive? The (n + c)Maspin-expressing cellular state in stem cells differentiation Cancer mortality is predominantly caused by metastasis and drug resistance, both of which are facilitated by tumor cell genetic instability and phenotypic plasticity. To date, one of the most supported hypotheses for tumor heterogeneity and phenotypic plasticity comes from the concept that a small number of naïve CSCs carry driver oncogenic mutations and, when activated, are capable of self-renewal (SR) and multilineage differentiation. To this end, CSCs share with normal stem cells (SCs) many common cellular characteristics and the underling molecular mechanisms. Maspin is not expressed in naïve embryonic stem cells (ESCs) or somatic SCs, but is involved in their differentiation. During human pregnancy, detection of fetus-derived hypermethylated maspin DNA in the plasma predicts a higher risk of preeclampsia [9]. In mouse embryogenesis, the level of maspin mRNA and protein gradually increases from embryonic day (ED) 1 to ED5, and then decreases on ED6 and ED7. Treatment of pregnant mice with anti-Maspin polyclonal antibodies significantly reduced the number of implanted embryos [10]. Mouse ESCs with homozygous deletion of the Maspin gene were viable in vitro. However, after the implantation, they died at the peri-implantation stage, with disorganized endodermal cell mass and no basement membrane layer in the embryo body [11]. The embryonic lethality of Maspin knockout was also shown by a Cre-recombinase-based breeding scheme at the step of oogenesis [12]. Forced tissue-specific transgenic expression of Maspin curtailed mouse mammary gland development, which was accompanied by increased epithelial apoptosis [13]. On the other hand, through an alternative breeding scheme wherein cytoplasmic Maspin could be carried over from oocyte to the zygote, viable Maspin knockout (Maspin−/−) mice were successfully generated. However, these Maspin−/− mice developed a spectrum of epithelial abnormalities including adenoma and tumors of primitive basal epithelial features in the mammary and prostate glands, and poorly differentiated lung adenocarcinoma [12]. According to the theory of CSC in tumor progression, the activation of CSC differentiation should be expected whenever new colonies of a surviving tumor progeny phenotype emerges. The (n + c)Maspin-expressing tumor cells appear in the same time window. In normal somatic tissues, nMaspin-expression is strictly restricted to terminally differentiated epithelial cells (e.g., luminal cells in the mammary gland and secretory epithelial cells in the prostate gland), whereas the (n + c)Maspin-expressing cells reside in the basal layer of these glands [1, 4]. It is important to note that the basal epithelial layer contains both basal or myoepithelial cells of primitive differentiation and multipotent epithelial stem (precursor) cells [14]. Carcinogenesis is commonly marked by the loss of the normal basal epithelial cells. However, the (n + c)Maspin-expressing cells did not disappear with the basal epithelial layer. Instead, they are detected abundantly at the earliest in situ tumors [1, 15] (Fig. 1Ba–c), exhibiting a gene expression profile similar to that of epithelial precursor cells [16]. In breast cancer lymph node metastasis, the re-appearance of the primary-liked tumor cells was associated with the expression of (n + c)Maspin [17], whereas in PCa bone metastasis, (n + c)Maspin-expressing tumor cells of moderate differentiation are detected sporadically among the majority of tumor cells that do not express Maspin (Fig. 1Bd). In radical prostatectomy specimens of PCa patients who received neoadjuvant anti-androgen treatment, (n + c)Maspin-expressing cells were detected among the debris of the bulk androgen-sensitive tumor cells [18]. The (n + c)Maspin-expressing tumor cells as primed CSCs (P-CSCs) In the last couple of decades, significant advancements have been made to demonstrate the capability of CSCs to initiate tumor, and led to the identification of their cell surface molecular markers [19]. However, there is yet an overarching conceptual framework for how to connect the central control of the CSCs in producing heterogeneous bulk tumor cell populations, shifting the differentiation lineages in different tumor microenvironments (TME), and supporting cancer drug resistance and recurrence. To this end, a recent breakthrough in the field of development demonstrates that naïve SCs that are stimulated to exit the symmetrical self-renewal (SR) are primed prior to making the commitment to their destined differentiation [20]. The primed SC (P-SC) step seems preserved across species, and may be triggered by stress signals, including oxidative stress or nutrition deprivation. The gene expression profiles of P-SCs seem distinct from those of naïve SCs, but have no clear lineage directionality [21]. Cells in the P-SC state are considered epigenetically “paused,” yet with a landscape of chromatin modification poised to respond swiftly to differentiation-specific transcription factors [22]. The P-SCs of adult epithelial cells reside among the basal epithelial cells [23] and exhibit a hybrid basal and luminal cell differentiation profile [14]. Other P-SC features include a decrease in membranous integrity of intracellular organelles and an increase in nuclear-cytoplasmic shuttling of proteins. These cells also have a higher level of tolerance of increased endoplasmic reticulum (ER) stress without elevating unfolded protein response (UPR) which may otherwise prematurely prevent the establishment of the upcoming proteome in the destined phenotype [24]. While P-SCs may survive such contemporaneous stress at least in part by the mechanism of autophagy, they may exhibit an elevated level of immunogenicity which is marked by the increased expression of major histocompatibility complex class I (MHC-I) molecules [25]. These characteristics of P-SCs are also the key features of the (n + c)Maspin-expressing tumor cells that have an expression profile overlapping with that of inflammatory [26] basal epithelial cells [16]. In some cancer patients, anti-Maspin antibodies were also detected [27]. We propose that the (n + c)Maspin-expressing tumor cells are the P-CSCs (Fig. 2A).Fig. 2 A hypothetical model. (A): P-CSC as naïve CSC-derived common precursor of progenitor (Bulk) Tumor cells of diverse phenotypes. Maspin differential expression and subcellular localization are represented by brown color of different intensities. P-CSC: Primed cancer stem cells; SR: self-renewal. (B) The structural basis for direct inhibition of HDAC1 by maspin. Maspin reactive center R340 and the D346 in the KDEF motif are critical for HDAC1 inhibition and maspin subcellular localization, respectively The epigenetic pausing in primed stem cells involves transient asynchronous histone modification [22]. Starting from the step of oogenesis and morphogenesis in development, a dominant histone-modifying enzyme is histone deacetylase 1 (HDAC1) that controls the expression of key regulators of cellular stemness, including Nanog which is a driver of stem cell priming [28, 29]. It came as no surprise that HDAC1 is essential and cannot be complemented by other HDACs in early embryogenesis [30]. Interestingly, HDAC1 may be translocated to the cytoplasm in stressed stem cells [31]. Cytoplasmic HDAC1 may reside in the cytosol and ER [32] to deacetylate proteins such as heat shock protein 90 (Hsp90) [33] and glucose regulated protein 78 (GRP78). In turn, deacetylated Hsp90 and GRP78 are biochemically active to protect new proteomic and transcriptomic profiles to capacitate the phenotypic transition. Conversely, acetylation inactivates Hsp90 and GRP78. A hallmark of Hsp90 inactivation is the decrease of its translocation from the cytoplasm to the nucleus, whereas acetylation-mediated inactivation of GRP78 may alter ER integrity and trigger its dissociation from the ER without initiating UPR [34]. In tumor progression, HDAC1, Hsp90 and GRP78 are commonly upregulated. In addition, HDAC1 expressed in the (n + c) pattern [35], increased nuclear Hsp90 [36], GRP78 dissociation from the ER [34], and GRP78 secretion [37] are each associated with a worse cancer prognosis. Maspin is one of the most ancient and highly conserved clay B serpins [38]. Consistent with the X-ray crystallographic analysis [39], Maspin does not inhibit activated serine proteases [40]. Instead, it inhibits the activation of serine protease zymogens such as single-chain tissue-type plasminogen activator and pro-urokinase-type plasminogen activator [41–43], and inhibits serine protease-like enzyme HDAC1 [44]. Maspin is the only endogenous polypeptide enzymatic inhibitor of HDAC1 identified thus far. As shown in Fig. 2B, Maspin depends on amino acid (aa) residue R340 in its reactive center loop (RCL) to interact with the catalytic domain of the target molecule. As a 42 kDa protein, Maspin can pass the nuclear envelope pores through diffusion. It also has an intramolecular KDEL motif (aa 345–348) for contingent ER retention. Among all serpin homologues, maspin is the only one with this KEDL sequence. Conservative substitution of D346 with E in this motif rendered exclusive nuclear localization of both Maspin and HDAC1 [45]. Thus, the synchronized nuclear-cytoplasmic shuttling of HDAC1 and Maspin may be driven by Maspin. The (n + c)Maspin-associated gene expression profile was highly sensitive to changes of TME. When cells were cultured as three-dimensional (3D) organoids, (n + c)Maspin expression resulted in increased expression of antigen processing and histocompatibility genes, as well as inflammatory cytokines. Under several experimental conditions, (n + c)Maspin commonly upregulated the expression of 31 genes and downregulated the expression of 29 genes [46]. These common changes are involved in stem cell differentiation, adhesion and tumor progression. Bioinformatics analysis identified transcription factors (TFs) that directly control Maspin-regulated gene expression [47]. A general upstream analysis revealed that Maspin downregulates key nodes of the TGF-β signaling network, and upregulates key nodes in the networks of inflammation and selective cell death. Glutathione s-transferase (GST), Hsp90 and GRP78 have also been identified as predominant Maspin-associated proteins [45, 48]. The inhibitory effects of Maspin on HDAC1 is enhanced by its molecular interaction with GST, leading to reduction of cellular oxidative stress [48]. Maspin also abrogates the HDAC1-mediated epigenetic silencing of GST-Pi [49]. Interestingly, a mutually exclusive subcellular localization pattern of Maspin and GRP78 was revealed by dual immunofluorescent staining [45]. This result would be expected if Maspin acts to inhibit HDAC1-mediated deacetylation of GRP78, which leads to the exit of acetylated GRP78 from the ER [34]. These data suggest that the Maspin/HDAC1 axis may exert a contemporaneous and concerted control of cell differentiation, stress response, tumor inflammation and selective cell death pathways at the step of stem cell priming. The “Disguise” of (n + c)Maspin-expressing P-CSCs If priming is a common steady state required for stem cell differentiation, its kinetics may vary under different pathophysiological conditions. In somatic tissues, the elusive quiescent multipotent SCs can go through the steps of self-renewal and differentiation with a high level of fidelity to replenish the function of terminally differentiated cells that are injured or lost [50]. This highly hierarchical process may quickly pass through the step of P-SC to reach the predestined differentiated products. A major disadvantage of CSC study is the lack of clearly defined timing and loci of CSC activation and priming since the onset of cancer is driven by genetic mutations which disrupt the normal predestined temporal and spatial program of differentiation. To maximize the survival of the progeny cells, CSCs may be perpetually primed by their mutator genotypes and the evolving TME, rather than by the finite need for normal tissue morphogenesis or damage repair. Consequently, P-CSCs may be substantially accumulated in the tumor tissues, and easily mistaken as one of the final products of CSC differentiation. In this new thought framework, the evidence that (n + c)Maspin-expressing tumor cells are less invasive and metastatic as compared to more aggressive bulk tumor cells that do not express Maspin makes biological sense, since these cells represent an intermediary steady state between naïve CSCs and their established progeny cells. To this end, the past interpretation of the same data as evidence for a tumor suppressive role of Maspin was inaccurate due to the assumption that the (n + c)Maspin-expressing cells represented an established progeny phenotype of CSC differentiation. In any experimental model wherein ectopic (n + c)Maspin expression is driven by a strong constitutive promoter, the surviving clonal cell lines may resemble a P-CSC state. However, since these experimental P-CSCs are prevented from downregulating the level of maspin expression, their commitment to non-epithelial phenotypes, such as mesenchymal-like or neuroendocrine phenotypes may not ensue or reach fruition. On the other hand, while these results underscore the limitation of using ectopic Maspin expression to study the differentiation of CSCs wherein the temporal and spatial dynamics of Maspin expression is expected to play a key role, they serendipitously helped to exaggerate the manifestation of certain aspects of the P-CSC state. For example, as expected of P-CSCs, (n + c)Maspin-expressing tumor cells, but not the bulk tumor cells that do not express Maspin, have the capacity to undergo induced differentiation. In an earlier study, we developed a 2D/Susp/3D (or xenografts in vivo) scheme [51]. In brief, 2D culture for exponential growth can provide attachment anchors to trigger CSCs to exit from the SR state. Naïve CSCs in 2D culture can be enriched by consecutive passaging of mammospheres in suspension culture (Susp). Cells harvested from either 2D culture or Susp culture can be grown as 3D organoids or as xenografts in immune-compromised mice. In our experiments, a PCa cell line that expresses an undetectable level of Maspin was stably transfected with the maspin cDNA or a mock control. We showed that the “CSC’s” of maspin-transfected cells lost the ability to form symmetrical mammospheres in Susp culture. Instead, they grew as asymmetric cell aggregates with an elevated level of autophagy for up to 7 consecutive passages before dying of senescence. The same cell population failed to produce tumors in the limiting-dilution tumor initiation (LDTI) assay. In contrast, the CSCs of the mock-transfected cells were immortal in Susp culture, were tumorigenic in the LDTI assay, and produced invasive progeny tumor cells. When cells harvested directly from the 2D culture were grown as 3D organoids or as xenografts in a model for PCa bone metastasis, maspin-transfected cells gave rise to better differentiated in situ like tumors whereas the mock-transfected control formed poorly differentiated tumor sheets [52]. In addition, we noted that the maspin-transfected cells in 3D culture and xenografts featured Maspin protein predominantly in the nucleus. These experimental data demonstrate that as the (n + c)Maspin-expressing tumor cells are averted from immortality, they acquire a greater aptitude for differentiation. Further, the elevated levels of proteostatic and ER stress in (n + c)Maspin-expressing P-CSCs also help explain the evidence that maspin-transfected cells elicited a dramatic anti-tumor inflammatory response when they were subcutaneously injected into nude mice. Approximately 40% of injections were quickly “absorbed” and never developed tumors [53]. The “gross tumor volumes” of the maspin-transfected tumors resulting from the remainder injections were significantly greater than those of the mock controls. However, these maspin-transfected tumors sustained dramatic tissue lysis and were infiltrated extensively by neutrophils [53, 54], which targeted maspin-transfected tumor cells specifically in vitro. In addition, anti-Maspin immunoglobulin G (IgG) was detected only in mice bearing maspin-transfected tumor nodules. The vulnerabilities of the P-CSCs and future directions To our knowledge, this is the first time that the concept of P-CSC is raised. It does not contradict the current framework for CSCs, but rather highlights a previously unrecognized step between the naïve CSCs and the bulk cancer cells. It is likely that future studies will identify additional molecular features that are specifically associated with P-CSCs in different organ and tissue microenvironments. For example, a large body of evidence demonstrates that carcinoma is a disease resulting from the combination of genetic mutations and aging. The cellular characteristics and gene expression profiles of (n + c)Maspin-expressing P-CSCs, overlap significantly with those of senescing epithelial cells [51, 55]. In fact, replicatively senescent normal human mammary epithelial strains [56] expressed (n + c)Maspin at a significantly higher level than immortalized normal breast epithelial cell lines [1]. A possibility to be tested is that P-SCs derived from aging SCs with insufficient reprogramming capacity may not complete the full differentiation process, thus may remain in the P-SC state which is otherwise called atrophy [57]. We propose that the (n + c)Maspin-expressing P-CSCs in aging tissues may be similarly accumulated like P-SCs and serve as the immediate precursors of bulk tumor cells of divergent phenotypes. It was reported that the expression of (n + c)Maspin, vs. nMaspin, predicts a worse prognosis in laryngeal cancer patients who were 65 years of age or older [58]. We believe that P-CSCs are at the center in connecting CSCs to the genesis of phenotypic plasticity, tumor metastasis, tumor dormancy and tumor recurrence. It has long been recognized that metastasis is an inefficient process. The chances for circulating disseminated tumor cells to establish metastasis at secondary organ sites is estimated to be less than 0.01%. Such a low efficiency demonstrates that the bulk tumor mass may not be responsible for the colony formation at metastatic sites. The probability of metastasis may depend not only on how many CSCs are disseminated but also on how sensitive these CSCs are to the priming signals from different tissue microenvironments. These additional biological variables may explain why some disseminated “tumor cells” remain dormant for a long time and when they exit the dormancy their phenotypes resemble more closely their matching primary tumors [59]. We propose that the combination of cancer genetics and the quantification of (n + c)Maspin-expressing P-CSCs may accurately predict the propensity of tumor metastasis and cancer lethality. Ideally, naïve CSCs, the seeds of all the bulk tumor cells in the continuum of tumor progression and metastasis, should be eliminated in order to cure the disease. This notion may explain why androgen deprivation therapy (ADT) of PCa always leads to cancer recurrence, since ADT may not be cytotoxic to PCa CSCs that do not express androgen receptor [60]. Yet, naïve CSCs may be only a nominal fraction of tumor cell population and can remain quiescent without distinct phenotypic features to aid specific and effective drug targeting. Our new concept of P-CSC suggests that P-CSCs, not naïve CSCs, may be accumulated, thus presenting a widow of opportunity to nip the devil in the bud. To this end, we have shown that (n + c)Maspin-expressing P-CSCs are resistant to a number of chemotherapeutic agents. However, two characteristics of P-CSCs may be particularly promising drug targets. First, the “intracellular stress” that the P-CSCs have to tolerate may be pushing the cells to the limit of viability, which may prove to be an Achilles’ heel. In particular, the decreased membranous integrity of ER and other organelles may perturb the ion gradients, and render the cells more sensitive to drugs like salinomycin (SAL). SAL is a broad-spectrum ionophore-type antibiotic that is generally nontoxic to healthy people and has been approved by the US Food and Drug Administration (FDA) for treating patients infected by the coronavirus disease 2019 (COVID-19). Of note, SAL is cytotoxic to CSCs both in preclinical studies and in clinical trials [61]. In 2020, HSB-1216, a novel formulation of SAL, was approved by FDA as an orphan drug for treating small cell lung cancer. In our study, we found that the (n + c)Maspin-expressing P-CSCs of PCa were resistant to docetaxel and HDAC inhibitor, but were hyper-sensitive to SAL in the Susp culture, 2D culture and 3D culture [51]. It is intriguing to speculate that the effectiveness of SAL may be due to its dual targeting to both CSCs and (n + c)Maspin-expressing P-CSCs. Second, the (n + c)Maspin-expressing P-CSCs may have an elevated level of immunogenicity of MHC class I binding peptides, which may induce cytotoxic T lymphocyte response. Major breakthroughs in cancer immunotherapies have benefited patients with many types of cancer. However, it remains a challenge to make personalized prediction of the therapeutic response. To this end, in non-small cell lung cancer patients treated with immune checkpoint blockade inhibitors (ICIs), the development of a CD8+/IFN-γ+ T lymphocyte population that is also positive with T cell receptor (TCR) for Maspin peptides was specifically associated with partial or complete remission [62]. While ICIs are not effective in treating PCa, the autologous dendritic cell therapy Sipuleucel-T (Sip-T), the FDA-approved immunotherapy for asymptomatic metastatic castration resistant PCa (mCRPC) [63], produced remission in some patients in clinical trials. Of note, although the overall mortality rate of African American men (AAM) with PCa is more than double any other racial groups, the median overall survival of the AAM treated with Sip-T was 20.6 months longer than that of the European American men (EAM) [64]. It has been shown that the effectiveness of Sip-T depends on tumor epitope spreading (i.e., the broadening of the immune recognition to additional tumor antigens) [65], thus may be dictated by the baseline tumor immunogenicity and inflammation. Indeed, the highly heterogenous PCa is commonly associated with an elevated level of inflammation [66]. In particular, low-grade inflammation is more prevalent in the primary tumors of AAM PCa patients, and AAM PCa patients who responded favorably to Sip-T showed a higher baseline level of IFN-γ than EAM patients [64]. It is intriguing to postulate that the tumor cell population that contributed to antigen spreading and Sip-T sensitivity may be the chemo-resistant (n + c)Maspin-expressing P-CSCs. To explore the possibility of blocking P-CSCs by maspin-targeted therapeutic strategies, it is essential to identify the molecular features of maspin involved in the specific inhibition of HDAC1 in the nucleus and the cytoplasm, respectively. Based on the X-ray crystallographic analysis, future structural–functional relationship studies of maspin may be focused on its the RCL. In addition, the G-helix of Maspin is another highly flexible secondary structure and has been implicated in protein phosphorylation and molecular interaction with co-factors [39]. Our revelation about the role of (n + c)Maspin in the steady state of P-CSCs may serve as a reminder that tumor progression involves changes of transcriptomes, proteomes, as well as protein subcellular distribution. Earlier, we have shown that Maspin is also secreted to extracellular space both as a free molecule and a cargo of exosomes [67]. It is important to understand how Maspin secreted in these two different forms may differentially regulate the biological activities of stem cells and their progenies, respectively. To this end, the effect of (n + c)Maspin on HDAC1 may also shift the trafficking routes of HDAC1 substrates such as Hsp90 and GRP78, both of which may also be secreted to extracellular space. The specific contributions of compartmentalized Hsp90 and GRP78 to the steady state of P-CSCs need to be delineated. Acknowledgements This work was supported by the Ruth Sager Memorial Fund. We thank the assistance of Mr. Din Dzinic in literature search and Mr. Charles T. Jiang for his review and editing of this manuscript. Data Availability The authors confirm that the original data and hypothetical models in support to the current review are available within the article. 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==== Front Indian Geotech J Indian Geotechnical Journal 0971-9555 2277-3347 Springer India New Delhi 694 10.1007/s40098-022-00694-0 Original Paper Seismic Response and Vulnerability Evaluation of Jammu Region (Jammu and Kashmir) http://orcid.org/0000-0003-1515-7628 Ansari Abdullah [email protected] 1 http://orcid.org/0000-0002-1040-803X Zahoor Falak 12 http://orcid.org/0000-0003-1412-9545 Rao K. S. 1 Jain A. K. 1 1 grid.417967.a 0000 0004 0558 8755 Department of Civil Engineering, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016 India 2 grid.444723.2 0000 0004 1756 1373 Department of Civil Engineering, National Institute of Technology Srinagar, Srinagar, Jammu and Kashmir 190006 India 30 11 2022 114 25 4 2022 21 11 2022 © The Author(s), under exclusive licence to Indian Geotechnical Society 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. In this study, an attempt is made to generate hazard maps for the Jammu Region (JR) in Jammu and Kashmir in terms of surface peak ground acceleration (PGAsurface), liquefaction zonation, and vulnerability index (Kg). To do this, the seismic response of 200 sites was examined, and amplification factors at 0.01 s, 0.2 s, 1 s, and 10 s were estimated based on site-specific PGAsurface. The calculated factor of safety from field approaches was normalised to produce an integrated liquefaction zonation map. Field data from geophysical testing was also used to generate a vulnerability distribution for the study area. According to the findings, the southern portion of the JR has young sediments and alluvium deposits, resulting in low shear wave velocity (Vs) and very strong amplification. As a result, these sites are kept in high to very high vulnerable zones with Kg values of more than 35. This study provides a database for designers working on the construction, development, and expansion-related projects in J&K for developing any prospective earthquake-induced liquefaction mitigation strategies. Keywords Seismic response Vulnerability Probability of liquefaction Jammu and Kashmir Indo-Gangetic Plains ==== Body pmcIntroduction Jammu and Kashmir (J&K) is located in the north-western part of the Himalayas triggered during 1555, 1828, 1885, 1905, 2005, 2013, and 2019 earthquake events [1–3]. Jammu Region (JR) and Kashmir Valley (KV) are the two major sections of J&K. JR is located around 232 and 112 km from the epicentres of the 2005 and 2019 earthquakes in Kashmir and Mirpur, respectively [4, 5]. JR experienced property damages, slope failures, and liquefaction hazards during these two far-field earthquakes, particularly in the southern regions. This area is a part of the Indo-Gangetic Plains (IGP), where the availability of soft soil and thick sedimentary strata is at its highest. Significant damages at distant sites demonstrate the impact of the dynamic soil characteristics that lead to local site effectiveness. The clearest illustration to support the idea of liquefaction hazards is the damages seen after the 1985 Mexico earthquake, 1999 Izmit earthquake, 2001 Bhuj earthquake, 2004 Niigata earthquake, and the most recent earthquake in Central Italy in 2016 [6–10]. There are site-specific challenges involved in designing the foundation of buildings and infrastructure utilities in an earthquake-prone area [11]. It becomes essential to comprehend the regional geology and lithological variety of underlying strata that are conducive to strong ground motion resulting in building damages and foundation instability [12–15]. In the present study, an attempt has been made to provide the hazard maps in terms of surface peak ground acceleration (PGAsurface), liquefaction zonation, and vulnerability index (Kg) for the JR. Connectivity and access to the site are difficult and challenging in the Himalayan region. Geotechnical consultants for the study region provided extensive geotechnical data from boreholes (Fig. 1). Tromino® ENGY 3G [16] is used to measure the shear wave velocity (Vs) and resonance frequency (f0). For data acquisition, Multichannel Simulation with One Receiver (MSOR) and Microtremor Horizontal to Vertical Spectral Ratio (MHVSR) techniques are utilised. According to the findings, more than half of the JR is classified under moderate to high-risk zones. The proposed zonation will aid in understanding the dynamics of subsurface layers, local geology, and lithological formations in JR. This study provides a database for designers working on the construction, development, and expansion-related projects in J&K for developing any prospective earthquake mitigation strategies.Fig. 1 Study area map of the Jammu Region (JR) showing the locations for boreholes and geophysical testing Geological Formation and Seismotectonic Settings of Jammu Region (JR) The JR is located in the NW Himalayas, especially in the Lesser Himalaya Zone, from a regional geological standpoint [13, 17]. The Main Central Thrust (T1) in the north and the Main Boundary Thrust (T4) in the south define the Lesser Himalayan Zone [11]. The lower Himalaya's geological unit is mostly made up of sedimentary rocks that have been sheared and over-thrust to the south into the Siwalik Molasse (Sub-Himalaya Zone) following the NNE dipping Reasi Thrust (T8). The southern boundary of the JR including the bank of the Tawi River in the Samba and Kathua area presents the Indus-Alluvium plains [18]. North of this region, the Siwaliks are exposed. Near Reasi and Vaishnodevi, Sirban formation is mostly visible [19]. This formation comprises dark grey dolomite and limestone with a back thrust concerning the overlying Subathu Formation. After a considerable time interval, the fine-grained detrital sediments of the Murree Formation were deposited above the Subathu Formation [20]. The Panjal Volcanics and the Upper Palaeozoic to Lower Tertiary Agglomeratic Slate Sequence are unconformably overlain by the Ramban Formation [21]. Slate, phyllite, quartzite, and gneiss are the main rocks that make up this formation. The Panjal Thrust (T10) is the Main Central Thrust in this area and passes through the Ramban and some parts of Reasi. Rocks of the Salkhala Formation constitute a low to high-grade meta-sedimentary suite comprising phyllite, schist, quartzite, and limestone [22, 23]. The local geology of JR is illustrated in Fig. 2.Fig. 2 Geological formations of the JR showing: a Indus-Alluvium plains, b outcrops of the conglomerate c outcrops of dolomite, d Main Central Thrust (MCT) crossing the Sangaldan area, e contact between the Murre Formation and Ramban Formation, f outcrops of Phyllites The JR has been impacted due to the near-field as well as far-field earthquakes in the Himalayan region extending up to Hindukush in Afghanistan [24, 25]. The Jhelum Fault (F3), Attock Fault (F1), Reasi Thrust (T8), Balakot-Bagh Fault (F8), Deosai Fault (F13), Jwalamukhi Thrust (T3), Hanna Fault (F14), Batal Fault (F12), and Mawer Fault (F7) are few of the active seismic source that surrounds this area [26]. The T1 distinguishes the crystalline rocks of the higher Himalayas from the formations of the lower Himalayas [27]. Along the JR's northern boundary, the T4 and T10 run parallel. NNW-SSE and NW–SE trends are shown by the Jhelum Fault (F3) and Balapur Thrust (T7), respectively [28, 29]. The Kishtwar Window (KW) and Jhelum Fault (F3) are the two major local strike-slip faults in the JR. The F8, which is NE dipping in Pakistan, is the primary cause of the 2005 Kashmir earthquake [30]. Active T8 and Udhampur Fault Zone (F9, F10, and F11) pass through the core centre section of the JR in addition to the T4. As a result of the imbrication of the lower Himalayas into a deeper structural level, the Kishtwar Window (KW) developed inside the crystalline upper Himalayas [31]. Recently occurred far-field June 2022 Paktika earthquake (Mw = 5.9) in Afghanistan and the 2019 Mirpur earthquake (Mw = 5.6) in Pakistan are also showing the active dynamics of the tectonic plates in this region. An updated seismotectonic map of JR is presented in Fig. 3.Fig. 3 Seismotectonic map of the Jammu Region (JR). All active sources located within a 350 km radius around Jammu are covered Subsurface Investigations In the present study, geotechnical and geophysical data were collected from local consultants and field investigations, respectively. An extensive geophysical field study is conducted at 200 locations from where SPT (N) data is provided for boreholes. Site locations are marked in the study area map, as presented in Fig. 1. For data acquisition, Tromino® ENGY 3G [16] is used which is provided by the Geotechnical Division of National Institute of Technology Srinagar, Jammu and Kashmir. The shear wave velocity (Vs) based on MSOR is measured for shallow depth [32–36]. The resonance frequency (f0) is recorded based on the concept of ambient noise measurements [37–39]. The liquefaction features such as sand blows and ground rupture were found in Jatah and Simbal villages. The only factor used to classify a site under the NEHRP is the averaged shear wave velocity to a depth of 30 m (Vs30) [40]. In the northern and western regions (Sonder and Gattigali), where there is access to rock outcrops, Vs30 values have reached as high as 1150 m/s (Fig. 4). The central part of the JR showed Vs30 values ranging from 350 to 425 m/s, whereas locations on the southern side have lower average shear wave velocities of 182 to 312 m/s. Beyond the Ravi River, particularly in the central region of Kathua, where it ranges between 380 and 570 m/s, an increase in Vs30 values is seen. Vs up to 30 m depth for various site locations is shown in Fig. 4.Fig. 4 Depth-wise variation of shear wave velocity (Vs) The shear wave velocity (Vs) is high for sites with high SPT (N) values, according to the results of both geotechnical and geophysical studies. As shown below, a correlation between the Vs and the N has been developed for Jammu (Fig. 5). The performance of the proposed empirical correlation is evaluated by comparing measured and calculated shear wave velocity values (Fig. 6).Fig. 5 Relationship between shear wave velocity (Vs) and SPT (N) Fig. 6 Comparision of relationships between shear wave velocity (Vs) and SPT (N) proposed in the present study with previous studies [41–49] Various researchers have proposed the relationship for various regions. Figure 7 shows a comparison of the proposed correlation with the existing correlations in the literature. Depending upon the variation in shear wave velocity and the frequency of an area, the relationship may differ. This relationship is helpful when SPT (N) values or Vs needs to be established for the locations in and around Jammu. The plotted data are dispersed between lines with slopes of 1:0.5 and 0.5:1, with the majority of the values close to slope line 1:1, demonstrating that the proposed correlation provides an acceptable fit for the assembled data for the tested soils.Fig. 7 Measured versus calculated shear wave velocities for all soils Seismic Response Analysis In this study, ten different bedrock motions from the Himalayan region were considered and a one-dimensional seismic response analysis for the equivalent linear case has been done using DEEPSOIL [4, 50–52] at all sites marked in Fig. 1. Actual ground motions of five earthquakes in the region, i.e. Uttarkashi (1991), Chamba (1995), Chamoli (1999), South Hindukush (2010) and Kishtwar (2013), were considered for analysis. Figure 8 depicts the ground motion history for all input motions at bedrock. To prevent the influence of probable seismic amplification effects, the first criterion used in the selection is to include only accelerograms recorded on outcropping rock [53, 54]. Furthermore, real accelerograms were chosen with a tolerance on seismological parameters, magnitude, and epicentral distance that are regarded adequate for J&K. The deaggregation results were used as a basis to establish the magnitude and distance range. Based on these criteria, 10 records were chosen from a pool of 149. Typically, the effect of magnitude on spectral response is greater than the effect of epicentral distance. As a result, shorter magnitude range and broader epicentral distance range records were chosen. The frequency response of the chosen ground motion is illustrated in Fig. 9 by Fourier amplitude spectra.Fig. 8 Input ground motions at bedrock considered from the Himalayan seismic stations Fig. 9 Fourier amplitude spectra for input ground motions at bedrock considered from the Himalayan seismic stations The damping versus shear strain curves and modulus reduction curves are used to represent the soil parameters of each soil layer. In this study, shear modulus reduction curves and damping ratios for clays and sands have been defined using discrete points by Seed and Sun [55], Seed and Idriss [56], and Idriss [57]. Table 1 shows the estimated amplification factors for 0.01 s, 0.2 s, 1 s, and 10 s. In the study area, amplification is higher at rock sites and lower at alluvium sites for short periods. The amplification is higher at alluvium sites and lower at rocky sites for long periods.Table 1 Amplification factor at various periods for sites in Jammu Region (Jammu and Kashmir) Location Amplification Factor 0.01 s 0.2 s 1 s 10 s Gulab Garh 2.83 3.05 1.08 1.06 Chak Malal 2.96 3.02 1.15 1.08 USBRL T2 Tunnel Portal 2 2.06 3.50 1.02 1.02 Chandial Kot 1.97 2.61 1.04 1.02 Vishwabharti Public School, Panjpeer 2.22 2.46 1.08 1.04 Sazar 2.66 2.83 1.15 1.07 Gurukul Public School, Reasi 2.20 3.27 1.05 1.01 Sonder 2.85 2.83 1.15 1.07 Bagani 2.91 2.46 1.20 1.07 Kangral Sangral 2.87 1.78 1.03 1.01 Khari 2.85 1.59 1.03 1.01 Tahra 2.96 1.87 1.04 1.02 Sawjian 2.53 3.33 1.02 1.03 Sonder 2.69 2.89 1.09 1.06 USBRL Tunnel T2 Portal 1 1.73 2.64 1.05 1.00 Mughal Road, Morha 1.07 1.18 1.00 1.00 Jagti 1.07 1.20 1.01 1.00 Mustafa Nagar 1.09 1.21 1.00 1.00 Kot Bhalwal 1.25 1.22 1.01 1.00 Patel Construction Camp, Bhaga 1.25 1.26 1.01 1.00 Gattigali 1.07 1.18 1.00 1.00 Qadar Pur 1.16 1.35 1.01 1.00 Khan Pora 1.17 1.25 1.01 1.00 Degwar Maldayalan 1.29 1.33 1.01 1.00 Govt. Degree College, Bishnah 1.15 1.24 1.01 1.00 Berigalah 1.16 1.32 1.01 1.00 Gundi 1.16 1.28 1.01 1.00 Phagla 1.23 1.34 1.01 1.00 Mahara 1.16 1.27 1.01 1.00 Sandel 1.29 1.68 1.01 1.01 Siksha Niketan School, Jeevan Nagar 1.20 2.31 1.04 1.05 Govt. School, Reasi 1.66 2.47 1.19 1.23 St. Peter's School, Karan Bagh 2.02 2.66 1.39 1.27 Nagrota 2.36 2.48 1.53 1.27 Chenab Bridge Kauri Side 1.67 2.83 1.17 1.23 Paloura 1.77 2.44 1.18 1.25 Govt. College of Engg. & Tech., Chak Bhalwal 2.43 2.60 1.58 1.27 Domi 1.66 2.45 1.12 1.17 Govt. Ploytechnic, Sanjay Nagar 1.88 1.54 1.27 1.26 Baji Pur 1.23 2.44 1.06 1.07 Dhar Shiv Garh 2.22 2.50 1.53 1.26 Eidgah, Banihal 2.05 3.11 1.40 1.27 Batna 1.63 2.84 1.15 1.21 Kalu Chak 1.78 2.33 1.13 1.17 Kotli Mian Fateh 2.15 2.33 1.54 1.26 Indian Institute of Management (IIM) Jammu, Canal Road 1.34 1.38 1.04 1.00 Rani Park, Near J&K Legislative Assembly 1.56 1.78 1.05 1.00 Abdullah Bridge, Gujjar Mandi 2.36 2.47 1.09 1.00 Khan Market 1.88 3.12 1.07 1.00 Sangaldan 1.97 2.44 1.04 1.00 Mehmoodpur 2.02 2.63 1.04 1.00 Anji Khad Bridge 1.33 1.38 1.04 1.00 Devi Kund 1.71 3.32 1.07 1.00 Jammu University 3.06 3.90 1.80 1.02 Janipur 1.69 1.86 1.06 1.00 Khorbani 1.79 2.60 1.06 1.00 Jatah 1.71 3.32 1.07 1.00 Thiloo 2.26 3.57 1.45 1.02 Simbal 1.96 2.35 1.33 1.01 Kotijijan 2.40 2.49 1.11 1.00 Dangah 2.08 2.51 1.43 1.29 Chak Lalushah 1.70 2.66 1.10 1.13 SIDCO Industries 1.88 2.95 1.41 7.83 Sher-e-Kashmir University of Agricultural Sciences & Technology (SKAUST) 1.82 4.21 1.16 1.23 Geeta Nagar, Reasi 1.23 1.36 1.04 1.05 Sidhra 1.35 1.54 1.05 1.06 Govt. Secondary School, Rehari Colony 1.55 2.63 1.10 1.13 Katli 1.32 1.52 1.05 1.06 Khandwal 1.36 1.95 1.07 1.09 St Xavier School, Barnai 1.93 2.21 1.34 1.26 Patniyal 1.70 2.69 1.10 1.13 Stephens School, Sialkot Road 1.36 2.97 1.38 7.82 Raina Colony 1.60 2.85 1.11 1.14 Transport Nagar 1.32 1.55 1.05 1.07 Chak Lalushah 1.99 2.39 1.40 1.28 The JR is separated into three major classes based on the PGAsurface value, which is fixed at 0.3 g, 0.3–0.45 g, and > 0.45 g. The spectral acceleration values are highest in the northern region for rocky sites compared to alluvium sites in the southern region, which is part of the IGP. The PGAsurface in Shareef Bagh, Banihal, and Baflaiz is greater than 0.45 g. Maximum sites in Jammu, Samba, Kathua, and the central part of Udhampur have very low PGAsurface due to younger sediments and alluvium deposits near the Tawi and Ravi rivers, as illustrated in Fig. 10. PGAsurface value of 0.05 g is considered to be sufficient to cause damage to poorly designed buildings. However, PGAsurface is not the only criteria for damage; there are various additional parameters that are essential from a damage standpoint, such as strong motion duration, building natural period and ground motion amplitude. The main causes of the low PGAsurface are the short duration of input bedrock motion, the longer source-to-site distance, and impedance contrast. Travelling a long distance with weak ground motion is likely to inflict less damage. Local site effects, on the other hand, play a significant part in devastation in the case of far-field earthquakes.Fig. 10 Surface peak ground acceleration (PGAsurface) map for Jammu Region (Jammu and Kashmir) Vulnerability Assessment for Jammu Region The standard penetration test (SPT), cone penetration test (CPT), and seismic tests to assess the shear wave velocity of soil deposits are the most often utilised field testing-based procedures for obtaining soil samples for liquefaction hazard study. The most recent technique proposed for SPT (N) value [58] and shear wave velocity [59, 60] was employed in this work to assess the factor of safety against liquefaction in the present study. The empirical correlation proposed by [61] is used to determine the probability of liquefaction (PL) for all sites. Local geology and field investigation results were considered to change the ranges for liquefaction zones. Table 1 highlights the intended range for several zonation classes (Table 2). Table 2 Liquefaction zonation based on probability of liquefaction (PL) Probability of liquefaction (PL) Zonation [61] Present study (Proposed range) PL≥ 0.85 > 0.75 Very High High 0.51–0.75 High 0.35 ≤ PL < 0.65 0.41–0.50 Moderate 0.15 ≤ PL < 0.35 0.21–0.40 Low PL< 0.15 < 0.20 Very Low The factor of safety is derived using the SPT (N) value, and Vs-based approaches were given equal weightage for normalisation in order to estimate the probability of liquefaction (PL). A liquefaction zonation map for JR is presented in Fig. 11. Majority of the sites in the southern part are classified under High or Very High liquefaction zones. At all of these locations, Vs less than 230 m/s is observed. High amplification is seen due to the presence of young sediments. The northern and northern western ends of JR include rock outcrops. Shear wave velocity of more than 600 m/s was measured at all such sites where very low amplification levels were detected.Fig. 11 Liquefaction zones for Jammu Region (Jammu and Kashmir) Nakamura [38] presented a vulnerability index (Kg) value for precisely predicting earthquake damage to surface ground and buildings. The H/V amplitude (A) and resonance frequency (f0) estimated from the MHVSR can be used to evaluate the vulnerability index, as described in Eq. (1).1 Kg=A2/f0 Based on the calculated values of Kg, the JR is divided into five zones of vulnerability. These zones show the severity as very low, low, moderate, high, and very high. The vulnerability map in Fig. 12 is in accordance with the local lithological and geological formations. The highest point on JR is at Sinthan Top, at the maximum altitude of 3784 m. Mountainous topography may be seen in most places in Reasi, Poonch, Ramban, Doda, and Kishtwar. Testing locations in these districts fall under one of two vulnerability categories: low or very low. The IGP, which makes up the southern portion of JR, has exceptionally soft alluvium. Soft soils and thick sedimentary layers in southern locations make them susceptible to potential damage from intense motion caused by either near-field or far-field earthquakes. Sites near the Tawi, Ravi, and Chenab Rivers have extremely high vulnerability.Fig. 12 Vulnerability zones for foundation soil in Jammu Region (Jammu and Kashmir) In the event of moderate to large magnitude earthquakes, low resonance frequency (f0) and high H/V amplitude (A) are the most lethal and catastrophic damage-producing combinations. Outstanding f0 and A values in the regions of Khorbani, Jatah, and Simbal will cause liquefaction-related damages in these areas. Significant building and structural damages were observed due to the Kashmir and Mirpur far-field earthquakes in 2005 and 2019, respectively, in these regions. Site locations of Laal Haveli and Punjaja with f0 < 1 Hz present the vulnerability index ranging between 15 and 35. The majority of the sites in Poonch, Kishtwar, and the eastern part of Doda have extremely low Kg values (< 5). For Kg > 15, the central portion of the JR displays H/V amplitudes between 4.5 and 6.8. Sites in the southwestern part of Jammu, Kathua, Samba, and central Rajouri where Vs30 ranges between 182 and 560 m/s. These sites exhibit very high amplification and are vulnerable to severe structural damages in case of any seismic activities. Conclusions The Jammu Region (JR) of Jammu and Kashmir is located in the NW Himalayas which is one of the most active tectonic regions. This area has witnessed damage during far-field earthquakes that occurred in 2005 and 2019. In this study, ten different bedrock motions from the Himalayan region were considered and a one-dimensional seismic response analysis for the equivalent linear case has been done. For short periods, amplification is higher at sites with rock outcrops in the north and north-west, and lower at alluvium sites in the south. For long periods, the amplification is greater at Simbal and Jatah-like alluvium sites near the banks of the Tawi, Ravi, and Chenab rivers. In Shareef Bagh, Banihal, and Baflaiz, the PGAsurface is more than 0.45 g. The factor of safety (Fs) against liquefaction is calculated using both SPT (N) value and shear wave velocity (Vs)-based field methods and results are superimposed to generate an integrated map of liquefaction zonation in the JR. The majority of sites in southern Jammu, Samba, and Kathua have Vs of less than 230 m/s. The results of local geology and field investigations were used to adjust the ranges for liquefaction zones. The projected liquefaction zones are labelled as very low, low, moderate, high, and very high, with PL of 0.2, 0.2–0.4, 0.41–0.5, 0.51–0.75, and > 0.75, respectively. The testing sites in the southern section have PL values more than 0.5. These locations are vulnerable to damage from either near-field or far-field earthquakes due to their fragile subsurface strata. The vulnerability indexing-based site grouping in JR was defined by means of resonance frequency (f0) and H/V amplitude. The findings concluded that more than half part of the JR is classified as moderate to high-risk zones. These findings can be used to analyse the seismic response of foundation soil and superstructures. Acknowledgements The authors would like to thank the M/S DMB Pvt. Limited, Space Engineers Consortium Pvt. Limited and, Mount Geotechnical Services Pvt. Limited for providing the SPT data including other geotechnical properties for this study. The authors thank for the technical and logistical assistance offered by the National Institute of Technology Srinagar. The authors are also thankful to the Divisional Commissioner Office of Jammu and Kashmir for providing special permission during the COVID-19 pandemic for fieldwork in Jammu and Kashmir. Funding There has been no significant financial support for this work that could have influenced its outcome. Declarations Conflict of interest The authors declare that they have no conflicts of interest associated with this publication. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Enescu B, Mori J, Miyazawa M. (2007) Quantifying early aftershock activity of the 2004 mid‐Niigata Prefecture earthquake (Mw6. 6). J Geophys Res Solid Earth. 112(B4) 2. Rao KS Rathod GW Seismic microzonation of Indian megacities: a case study of NCR Delhi Indian Geotech J 2014 44 2 132 148 10.1007/s40098-013-0084-0 3. 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==== Front Gene Ther Gene Ther Gene Therapy 0969-7128 1476-5462 Nature Publishing Group UK London 36450833 375 10.1038/s41434-022-00375-w Article In vivo genome editing using novel AAV-PHP variants rescues motor function deficits and extends survival in a SOD1-ALS mouse model Chen Yi A. 1 Kankel Mark W. 2 http://orcid.org/0000-0002-0847-7159 Hana Sam 1 Lau Shukkwan Kelly 1 Zavodszky Maria I. 1 http://orcid.org/0000-0002-1733-2971 McKissick Olivia 1 Mastrangelo Nicole 1 Dion Jessica 1 Wang Bin 1 Ferretti Daniel 1 Koske David 1 Lehman Sydney 1 Koszka Kathryn 1 McLaughlin Helen 1 Liu Mei 1 Marshall Eric 1 Fabian Attila J. 1 Cullen Patrick 1 Marsh Galina 1 http://orcid.org/0000-0002-0983-2589 Hamann Stefan 1 Craft Michael 1 Sebalusky Jennifer 1 Arnold H. Moore 1 Driscoll Rachelle 1 http://orcid.org/0000-0003-0080-8950 Sheehy Adam 1 Luo Yi 1 http://orcid.org/0000-0003-4974-6148 Manca Sonia 1 Carlile Thomas 1 Sun Chao 1 http://orcid.org/0000-0002-7342-5750 Sigrist Kirsten 1 McCampbell Alexander 1 Henderson Christopher E. 1 http://orcid.org/0000-0002-5187-0730 Lo Shih-Ching [email protected] 1 1 grid.417832.b 0000 0004 0384 8146 Biogen Inc., Cambridge, MA USA 2 Apple Tree Partners (ATP) Research & Development, Branford, CT USA 1 12 2022 112 15 1 2022 10 10 2022 8 11 2022 © The Author(s), under exclusive licence to Springer Nature Limited 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. CRISPR-based gene editing technology represents a promising approach to deliver therapies for inherited disorders, including amyotrophic lateral sclerosis (ALS). Toxic gain-of-function superoxide dismutase 1 (SOD1) mutations are responsible for ~20% of familial ALS cases. Thus, current clinical strategies to treat SOD1-ALS are designed to lower SOD1 levels. Here, we utilized AAV-PHP.B variants to deliver CRISPR-Cas9 guide RNAs designed to disrupt the human SOD1 (huSOD1) transgene in SOD1G93A mice. A one-time intracerebroventricular injection of AAV.PHP.B-huSOD1-sgRNA into neonatal H11Cas9 SOD1G93A mice caused robust and sustained mutant huSOD1 protein reduction in the cortex and spinal cord, and restored motor function. Neonatal treatment also reduced spinal motor neuron loss, denervation at neuromuscular junction (NMJ) and muscle atrophy, diminished axonal damage and preserved compound muscle action potential throughout the lifespan of treated mice. SOD1G93A treated mice achieved significant disease-free survival, extending lifespan by more than 110 days. Importantly, a one-time intrathecal or intravenous injection of AAV.PHP.eB-huSOD1-sgRNA in adult H11Cas9 SOD1G93A mice, immediately before symptom onset, also extended lifespan by at least 170 days. We observed substantial protection against disease progression, demonstrating the utility of our CRISPR editing preclinical approach for target evaluation. Our approach uncovered key parameters (e.g., AAV capsid, Cas9 expression) that resulted in improved efficacy compared to similar approaches and can also serve to accelerate drug target validation. Subject terms Gene therapy Gene delivery ==== Body pmcIntroduction Amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig’s disease, is a devastating neurodegenerative disorder that results in the selective loss and dysfunction of motor neurons (MNs) in the brain and spinal cord, resulting in progressive muscle weakness and atrophy [1]. Though the precise etiology of ALS remains unknown, approximately 10% of cases present an inherited or familial form of the disease (fALS), while the remaining 90% are deemed sporadic (sALS). Amongst the 20 or more genes associated with ALS, mutations in the superoxide dismutase 1 (SOD1) gene account for approximately 15–20% of genetically defined ALS cases [2]. Although the exact mechanism of SOD1 mutations remains incompletely understood, there is a consensus that a toxic gain-of-function could disrupt several cellular functions thereby collectively contributing to MN degeneration [3–10]. Thus, lowering levels of SOD1 is predicted to be therapeutic. The SOD1G93A transgenic mouse carries a high number of tandem repeats of a human SOD1 (huSOD1) transgene harboring the G93A mutation and recapitulates many aspects of disease, including progressive muscle atrophy, impaired motor functions and death due to paralysis [6]. It was used to support the preclinical development of Tofersen, an antisense oligonucleotide (ASO) [11] targeting huSOD1 mRNA for degradation through a RNase H1-dependent mechanism [12]. Delivery of SOD1-ASO to the cerebral spinal fluid (CSF) significantly reduced huSOD1 protein expression in the brain and spinal cord of SOD1G93A mice, extended survival and slowed the rate of neurodegeneration [12]. The translatability of these preclinical studies was supported by results from the Tofersen clinical trials [13]. In the Phase 3 VALOR study, SOD1 CSF concentrations decreased in patients receiving 100 mg of Tofersen over 28 weeks. Plasma neurofilament light chain (NfL), a potential marker of neuronal degeneration, also decreased. With the 12-month data from the open-label extension of Tofersen, signs of reduced disease progression across multiple secondary and exploratory endpoints (including motor function, respiratory function and quality of life) were also observed, though the primary endpoint as measured by the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale did not reach statistical significance [14]. In recent years, gene therapy has emerged as a promising modality to treat diseases with defined genetic causes (e.g., monogenic liver, retinal and blood cell diseases). Adeno-associated virus (AAV) vectors allow for prolonged expression of engineered genetic information in diverse cell populations in the central nervous system (CNS) [15, 16] and are the basis of most gene therapy approaches for the treatment of neurodegenerative diseases (e.g., Alzheimer’s disease, spinal muscular atrophy, Parkinson’s disease, Huntington’s disease). If successfully developed, an AAV-based gene therapy could provide a one-time treatment for neurodegenerative diseases, such as SOD1 ALS. Several preclinical studies utilized AAV-delivered RNA interference (RNAi) to decrease huSOD1 mRNA levels in transgenic SOD1G93A mice and showed varying degrees of huSOD1 protein reduction and protection against disease progression [17–21]. Although clearly disease-modifying, the survival benefits reported were incomplete (<70 days extension of median survival) and most animals eventually succumbed to ALS-like symptoms, thus attempts to improve efficacy are warranted either through refinement of RNAi methods or through alternative strategies. Alternative approaches to decrease huSOD1 protein levels include employing AAV9 with genome editing methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 systems [22–27]. The CRISPR-Cas9 system generates DNA double-strand breaks (DSBs) at genomic loci specifically targeted by single-guide RNAs (sgRNAs) and, following DNA repair, introduces insertions and/or deletions (indels) that disrupt gene expression [28]. Two studies utilized CRISPR to target the huSOD1 transgene in SOD1G93A mice using the AAV9 serotype and observed modest survival benefits (extension of median survival by 12 days and 25 days, respectively) [29, 30], perhaps due to insufficient distribution and/or expression of CRISPR-Cas9 components in target cell populations. Here, we set out to assess key parameters affecting CRISPR-based gene therapy, with the goal of increasing huSOD1 protein reduction and assessing whether greater huSOD1 knockdown improved the reported therapeutic benefits in preclinical studies. We screened immortalized cells for sgRNAs directed against huSOD1 to identify sequences that markedly reduced human SOD1 protein levels. To deliver our SOD1-targeting sgRNAs to disease-relevant-cells in the SOD1G93A model, we utilized AAV-PHP.B and AAV-PHB.eB variants, which provide broader tropism and/or transduction rates in mice compared to other serotypes [31–34]. For our in vivo model, we crossed the SOD1G93A transgenic mice with the H11Cas9 knock-in mice [35], which are engineered to express constitutively spCas9 from a CAG promoter. This eliminates the need to package Cas9 into viral constructs and allows for broad distribution of Cas9 expression in all disease-relevant cells. We show that a single intracerebroventricular (ICV) injection of AAV-PHP.B-huSOD1-sgRNA into neonatal H11Cas9 SOD1G93A mice extended disease-free survival in the SOD1G93A model by >110 days and resulted in strong disease suppression through robust and sustained reduction of mutant SOD1 protein in the cortex and spinal cord. Furthermore, we observed improved protection (>170 days in survival extension) using the AAV-PHP.eB capsid to delivery SOD1-targeting sgRNA to pre-onset adult SOD1G93A mice via one-time intrathecal (IT) or intravenous (IV) injections. These results support the notion of applying this approach to multiple genes of therapeutic potential for effects in other CNS disease areas and points to key parameters of preclinical work to support the development of CRISPR-based gene therapy. Materials and methods Animals and general procedures H11Cas9 mice [B6J.129-Igs2tm1.1(CAG-cas9) Mmw/J; stock #028239; laboratory of M. Winslow, Stanford University, Stanford, CA] and transgenic SOD1.G93A mice (B6.Cg-Tg(huSOD1G93A)1Gur/J; stock #: 004435) were purchased from the Jackson Laboratory. All mice were backcrossed for at least eight generations to C57BL/6 mice. Homozygous H11Cas9 female mice and heterozygous SOD1-G93A male mice were crossed to generate H11Cas9−/+; SOD1G93A−/+ mice and age-matched H11Cas9−/+; huSOD1.G93A−/− littermates. Mice were housed in a 12/12 h light/dark cycle in a temperature-controlled room (22–24 °C) with access to food pellets and water provided ad libitum. Behavioral testing occurred between 8:00 A.M. and 5:00 P.M. For survival studies, SOD1-G93A mice were observed daily for paralysis once they reached 130 days old. The animals were sacrificed at the humane endpoint, when they were unable to right themselves within 15 s of being placed on their side, showed 20% weight loss of peak body weight or exhibited hunched posture, as directed by a veterinarian. For biochemical and histological studies, mice were euthanized at the indicated age prior to paralysis, and organs were removed and immediately frozen in liquid nitrogen or fixed in 10% neutral buffered formalin (NBF). Experimenters were blind to treatment. All animal use and treatments were approved by the Biogen Institutional Animal Care and Use Committee (IACUC) and followed the National Institute of Health Guide for the Care and Use of Laboratory Animals. Single guide RNA (sgRNA) design, vector design and production The eight SOD1-targeting spacer sequences of ~20 nucleotides [36] were selected based on Benchling (San Francisco, CA, USA) bioinformatic output [37, 38] against the human SOD1 gene (Table S1). Each sgRNA spacer sequence was used in combination with sgRNA scaffolds, structurally optimized for sp.Cas9 binding [39]. The sgRNAs were synthesized and cloned into the expression constructs by PackGene (Worcester, MA, USA). Each AAV construct contains a U6 promoter that drives expression of a sgRNA, and either a CBA promoter that drives expression of a green fluorescence protein (eGFP) fused with a KASH domain or a CAG promoter that drives expression of a mCherry fluorescence protein. All AAV vectors, including AAV-PHP.B and AAV-PHP.eB, were purchased from PackGene (Worcester, MA, USA). Neonatal intracerebroventricular (ICV) injection, intrathecal injection (IT) and intravenous injection (IV) of AAV Postnatal day 0 (P0) pups were anesthetized by hypothermia for 2–4 minutes until movement ceased. Cryo-anesthetized pups were injected with 4 μl of AAV buffered with PBS containing 0.25% of FastGreen dye into the lateral ventricle(s) (2E11 vg per mouse). Injection sites were located halfway between lambda and bregma, 1 mm lateral to the superior sagittal sinus, to a depth of 2 mm. Injections were performed with a 33-gauge, 10 µL, 45o bevel Hamilton syringe (Hamilton Company, Reno, NV, USA) inserted perpendicular to the surface of the skull. Injection efficiency was monitored by the spread of the dye throughout the lateral and the third ventricles. Mice were euthanized for tissue collection at 20 weeks of age for histological analysis, and at 5, 10 or 20 weeks of age for biochemical and genomic analysis. Animal injection, group allocation and takedown were pseudorandomized. For intrathecal injection (IT), 5 weeks of age mice were anesthetized with isoflurane first in the chamber and then continuously anesthetized with isoflurane to maintain deep anesthesia via a nose cone throughout the procedure pad. The fur on the back of the anesthetized mice were shaved from the tail to the caudal thoracic spine and placed on a sterile sheet laid on top of a heating pad. 100 μl of AAV buffered with PBS (1E12 vg per mouse) was injected into the intrathecal space between the L3 an L4 vertebrae with a 31-gauge, 0.5 mL insulin syringe [40]. A subset of injected mice was euthanized for tissue collection at 15 weeks of age for biochemical analysis. For IV injection, 5 weeks of age mice were placed in a restraint that positioned the mouse tail in a lighted, heated groove (Braintree Scientific, Inc). The tail was swabbed with alcohol and then injected intravenously with 100 μl of AAV buffered with PBS (1E12 vg per mouse). The sample size for all mouse experiments was an n > 6/treatment was determined from prior unpublished data. Open field To assess locomotor activity, mice were placed within an open field apparatus and locomotor activity was captured by video tracking software. After, mice were acclimated to the testing room for 30 min they were individually placed in a cylindrical arena (43 cm × 36 cm: d x h). An overhead camera was used to track the animals using Noldus Ethovision (version 13) for 15 min. Mice were then returned to their home cage, and the arena was thoroughly cleaned prior to the next session. Locomotor activity was assessed on a monthly basis, starting at 7 weeks of age and concluding at 43 weeks of age. Rotarod For the Rotarod testing, mice were placed in one of five lanes on a slowly rotating rod (Ugo Basile) 16 cm above a stainless-steel trip box. Mice were acclimated to the testing room for a minimum of 30 min prior to testing. Once the mice were placed on the rotating rod, it gradually accelerated from 5 to 40 RPM (revolutions per minute) over 5 min. When the mice fell onto the trip box, a magnetic switch was activated and the latency to fall was recorded. For each mouse there were three test trials each day with a minimum 5-min interval between test trials. After each trial the mice were returned to their home cage. Observations were recorded at 11, 15, 19, 21, 23, 25, 27, 29, 31, 35, 39, and 43 weeks of age. Inverted grid Mice were placed on a small, inverted metal grid 25 cm above standard shaving bedding and latency to fall was recorded. Mice were acclimated to the test room for a minimum of 30 min prior to testing. The height of the grid allowed a soft landing when the mice fell but was high enough to prevent the mice from voluntarily jumping down. The time to fall was recorded by an experimenter with a stopwatch. The trial ended when the mouse fell or remained on the grid for 240 s. Up to two trials were done each test day with a rest interval of at least 2 min between trials. The second trial was only performed if the mouse did not reach 240 s on the first trial. After each trial and upon test completion, mice were returned to their home cage. Observations were recorded at 25, 27, 31, 35, 39, and 43 weeks of age. Clasping Mice were observed for limb clasping each day just prior to the rotarod assessment. Mice were lifted by the base of the tail and were observed for 10 sec and then placed back into their home cage. A score was assigned based on the following 5-point scale: limbs splayed outward (away from abdomen) = 0, one limb retracted toward abdomen = 1, two limbs retracted toward abdomen = 2, three limbs retracted toward abdomen = 3, four limbs retracted toward abdomen = 4, and all limbs retracted toward abdomen and body forming a ball =5. Observations were recorded at 11, 15, 19, 21, 23, 25, 27, 29, 31, 35, 39, and 43 weeks of age. Compound Muscle Action Potential (CMAP) Recordings were conducted by an experimenter blinded to genotype and treatment. All measurements were performed with animals maintained under isoflurane anesthesia (1.5–2.5%) and with body temperature maintained around 37 °C. Electrophysiological responses were obtained separately from both the left and right hind limbs of each mouse; the left hind limb was always recorded before the right. The period of testing (and time during which the animal was anesthetized) lasted approximately 10 minutes. Responses were recorded at 5, 8 and 10 weeks of age, and at monthly intervals subsequently until age week 46. Disposable monopolar needle electrodes (Teca 25 mm, 28G electrodes, Natus Medical Inc., San Carlos, CA) were used for both stimulation and recording. The sciatic nerve was stimulated with constant-current monophasic square-wave pulses (0.1 ms duration) produced by a WPI (model 365A) stimulator, with timing controlled by a data acquisition interface (National Instruments USB-6343). Current was delivered via a stimulating cathode placed sub-dermally near the sciatic notch to stimulate the sciatic nerve. The recording electrode was placed 1 mm intramuscularly into the belly of the tibialis anterior muscle and the reference electrode was placed at the ankle. For each recording, stimulation was applied every 2 s with incremental current levels (beginning at 1.0 mA and increasing in 0.5 mA steps) until the CMAP amplitude stopped increasing; recordings were performed using a current level 0.5 mA above this level. This supra-maximal stimulus intensity (typically found with a current between 1.5 mA and 3.5 mA) was used to record four CMAP responses which were then averaged for analysis. The minimum and maximal values of the response waveform were measured beginning 0.8 ms after stimulation to exclude the stimulus artifact. The absolute value of these numbers was taken and then summed for the final CMAP value of a response. The final CMAP value for a given animal is the average of the left and right leg peak-to-peak amplitudes. Each animal is considered one data point for statistical purposes. CMAP data was analyzed with two-way ANOVA followed by Sidak’s multiple comparison test, P < 0.05 is considered significant. NMJ staining and counting Tibialis anterior muscles were dissected and fixed in 10% NBF at 4 °C for 8 h, followed by 2% paraformaldehyde [11] buffered in 0.1 M sodium phosphate at 4 °C for 16 h, and then transferred to 20% sucrose in PBS at 4 °C for 24 h for cryoprotection. Samples were shipped to Jackson Laboratory for cryo-embedding in OCT, staining and analysis. Sections (20 μm) were collected by a cryostat and mounted directed onto glass slides. Sections were incubated overnight in primary antibodies containing a cocktail of mouse monoclonal anti-neurofilament 2H3 and anti-SV2 antibodies (Developmental Studies Hybridoma Bank), washed the following day and incubated overnight with Alexa-Fluor-488 conjugated anti-mouse IgG1 and Alexa-Fluor-594 conjugated α-bungarotoxin (BTX, Invitrogen). Sections were mounted to slides in fluorescence mounting media (DAKO, S3023) and imaged on a Leica SP5 laser confocal microscope. Manual counting of NMJ was performed by blinded individuals under a microscope as innervated, partially innervated (i.e., 50% or less anti-SV2 and anti-neurofilament positive signals overlapping with anti-bungarotoxin positive signals) and denervated NMJ. A total of 970 NMJs were counted in SOD1; sgLacZ group, 1531 NMJs were counted in SOD1; sgSOD1#5 group, and 1949 NMJs were counted in WT; sgLacZ group. Spinal motor neuron staining and quantification Lumbar spinal cord samples were fixed in 10% NBF for 48 h, processed on the Leica Peloris tissue processor and embedded into paraffin in a transverse orientation. Samples were bisected prior to embedding, such that every block contained two segments. Sectioning was performed on a Leica rotary microtome at a 5-micron thickness. Sections were collected at 6 levels, >50 µm apart. 6 sections per animals were used for IHC staining for choline acetyltransferase (ChAT), for a total of 12 technical replicates for quantification of motor neuron counts. Immunohistochemistry was performed for ChAT to assess MN numbers. The automated Ventana Discovery Ultra staining platform was used. Briefly, slides were deparaffinized and rehydrated. All reagents were from Roche Ventana, unless stated otherwise. Epitope retrieval was performed in the CC2 buffer (pH 6.0) for 64 min, at 93 °C. Primary antibody (rabbit monoclonal anti-choline acetyltransferase, clone EPR16590, Abcam, Cat. No. ab178850) was applied at the final concentration of 2 µg/mL for 60 min at ambient temperature. Goat anti-rabbit polyclonal antibody conjugated to nitropyrazole (NP) was applied for 12 min, followed by incubation with anti-NP reagent conjugated to alkaline phosphatase for 12 min. Positive staining was visualized with Discovery Red chromogen applied for 16 min. Slides were counterstained with hematoxylin. Slides were digitized on a 3D-Histech Pannoramic-250 whole slide scanner at 200x magnification. Custom made VisioPharm algorithms were used to quantify motor neurons in the ventral horns of the spinal cord. Tibialis muscle staining and analysis Tibialis Anterior muscle samples were fixed in 10% NBF for 7 days, processed on the Leica Peloris tissue processor and embedded into paraffin in a transverse orientation. Samples were bisected prior to embedding, such that every block contained two segments of the muscle. Sectioning was performed on a Leica rotary microtome at a 5-micron thickness. Gomori method for staining reticulin fibers was applied. Briefly, slides were deparaffinized in a series of xylene and graded ethanol solutions, and rehydrated. Slides were incubated with the following solutions (all from Poly Scientific R&D Corp unless stated otherwise): 0.5% aqueous potassium permanganate (Cat. No. S263), 2% aqueous potassium meta-bisulfite (Cat. No. S2005), 2% aqueous ferric ammonium sulfate (Cat. No. S179), Gomori’s ammonical silver nitrate (Cat. No. S114), 10% buffered formalin (Cat. No. S185), 0.2% aqueous gold chloride (Cat. No. S202), 2% aqueous potassium meta-bisulfite (Cat. No. S2005), and 2% aqueous sodium thiosulfate (Cat. No. S280). Slides were thoroughly washed between each incubation step. All subsequent reagents were from Roche Ventana, unless stated otherwise. Epitope retrieval was performed in the CC2 buffer (pH 6.0) for 64 min at 93 °C. To block the endogenous mouse IgG and non-specific background, slides were incubated with Rodent Block M (Biocare Medical, Cat. No. RBM961) for 16 min. Slides were counterstained with hematoxylin. Slides were digitized on a 3D-Histech Pannoramic-250 whole slide scanner at a 200x magnification. Custom made VisioPharm algorithms were used to assess the average diameter of myosin fibers. Blood neurofilament quantification Roughly 30 μl blood was collected by facial vein puncture at the indicated time points. Serum samples were prepared by centrifugation through BD Microtainer SST Clog Activator/Gel tubes (Becton Dickinson) and stored at −80 °C until used. Levels of pNFH in serum were measured by the ELLA microfluidic ELISA platform according to the manufacturer’s instructions (Protein Simple). Human SOD1 ELISA analysis Cortex or spinal cord tissues were homogenized in tissue lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X, 1% Na-deoxycholate, 0.1% SDS, 8 M Urea, 5 mM EDTA, supplemented with 1 mM dithiothreitol, 1 mM phenylmethanesulfonylfluoride fluoride (Sigma, 93482), complete protease inhibitor (Roche, 04693124001), PhosSTOP phosphatase inhibitor (Roche, 4906845001), 10 mM sodium fluoride, and 1 mM sodium orthovanadate (New England Biosciences, P07585)], by using TissueLyser II (QIAGEN). Homogenates were subsequently cleared via centrifugation at 20,000 x g for 30 min. Total protein concentration of the supernatant was measured by using BCA Protein Assay Kit (Pierce). Human SOD1 protein levels were measured from lysates with equal amount of total proteins, using Human Cu/ZnSOD1 Platinum ELISA Kit according to manufacturer’s instructions (BMS222, Invitrogen). Nuclei isolation, human SOD1 indel analysis and copy number analysis For Cohort #1, at 5-, 10-, or 20-weeks post-injection of AAV-U6-sgRNA-CBA-eGFP-KASH, cortices and half spinal cords were dissected, snap frozen and stored at −80 °C. Frozen tissues were thawed and homogenized in 1.5 mL ice-cold homogenization buffer (HBSS, 25 mM HEPES). The homogenate was passed through a 250 µm filter and spun at 600 x g for 5 min at 4 °C. The cell pellet was gently resuspended in 1 mL FBS and subsequently in 9 mL of 33% Percoll solution (GE Healthcare, Chicago, IL, USA) containing HBSS and 16.7 mM HEPES. An additional 1 mL of 10% FBS solution containing HBSS and 22.5 mM HEPES was carefully layered onto the top of cell suspension layer containing 30% Percoll. Density gradient centrifugation was performed at 800 x g for 15 min at 4 °C (1 acceleration and 1 brake). The supernatant was removed, and the nuclei pellet was resuspended and washed in FACS buffer (HBSS, 1% BSA, 2 mM EDTA, 25 mM HEPES, 0.09% sodium azide). Half a million intact EGFP positive nuclei labeled with Vybrant DyeCycle Violet Stain (Thermo Fisher Scientific) were isolated by FACS using MoFlo Astrios EQ (Beckman Coulter, Brea, CA, USA). Genomic DNA was isolated from the sorted nuclei or total nuclei by DNeasy Blood & Tissue Kit (catalog #: 69504) according to manufacturer’s instruction (QIAGEN). For Cohort #4, at the end of study (~45 weeks post-injection of AAV-U6-sgRNA-CBA-eGFP-KASH), cortices and half spinal cords were dissected, snap frozen and stored at −80 °C. Total genomic DNA were isolated from frozen cortices and spinal cords using DNeasy Blood & Tissue Kit (catalog #: 69504) according to manufacturer’s instruction (QIAGEN). For human SOD1 indel analysis, primers (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGagcttgctggaggttcactg-3′; 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGcacaacacccacctgctgta-3′) with Illumina adaptor (underlined) were used to amplify region of human SOD1 surrounding the sgRNA-targeted locus, using KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA, USA). The library was constructed by indexing individual samples with Illumina’s Nextera XT indices (Illumina, San Deigo, CA, USA) with limited PCR cycles. The library was pooled by volume and purified using AMPure XP beads (Beckman Coulter). The final library pool was quantified by KAPA Library Quantification Kit (Kapa Biosystems) and loaded on to Illumina’s MiSeq at 10 pM for 2 × 150 bp cycle run. For human SOD1 transgene copy number analysis, the following oligonucleotides were used for human SOD1: 5′-GGGAAGCTGTTGTCCCAAG - 3′ (primer #1); 5′- CAAGGGGAGGTAAAAGAGAGC - 3′ (primer #2); 5′-CTGCATCTGGTTCTTGCAAAACACCA - 3′ (probe conjugated with FAM). The follow oligonucleotides were used for ApoB as a reference gene with two copies per cell: 5′-CACGTGGGCTCCAGCATT - 3′ (primer #1); 5′- TCACCAGTCATTTCTGCCTTTG - 3′ (primer #2); 5′-CCAATGGTCGGGCACTGCTCAA - 3′ (probe conjugated with HEX). Oligonucleotides were purchased from IDT (Newark, NJ, USA). The ddPCR 2x Super Mix reagent (186–3010, Bio-Rad) was used for the real-time amplification, along with 5–10 ng of genomic DNA, 900 nM of primers and 250 nM of probe. Prior to thermal cycling, the reaction mixtures were further processed into droplets by QX100 Droplet Generator according to manufacturer’s instructions (1863002, Bio-Rad). After initial activation at 95 °C for 10 min, 40 PCR cycles of 96 °C for 30 s and 59 °C for 60 s were performed, followed by 98 °C for 10 min and then held at 4 °C. Amplified products in the droplets were subsequently analyzed on QX100 Droplet Reader (1863003, Bio-Rad). Tissue culture, transfection, AAV transduction and RNA sequencing (RNAseq) COS1 cells were purchased from ATCC (CRL-1650) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS), 1% Penicillin-Streptomycin (Pen-Strep) and 2 mM L-glutamine and incubated at 37 °C and 5% CO2. FUGENE HD (Promega, San Luis Obispo, CA, USA) was used for transfection according to the manufacturer’s instructions. The expression plasmid CMV-HA-human SOD1, CMV-FLAG-Cas9 and U6-sgSOD1-CAG-mCherry were mixed in a 1:4:5 ratio in Opti-MEM media (Thermo Fisher Scientific, Waltham, MA, USA) for transfection. The transfected COS1 cells were harvested 24 h after transfection. The inducible Neuro-2a cells expressing sp.Cas9 were purchased from GeneCopoeia and maintained in DMEM supplemented with 10% HI-FBS, 1% L-glutamine and hygromycin (100 µg/mL final) and incubated at 37 °C and 5% CO2. Neuro-2a cells were treated with doxycycline inducer at a concentration of 0.01, 0.1, or 1 µg/mL to induce expression of sp.Cas9. The expression plasmid CMV-HA-human SOD1 and U6-sgSOD1-CAG-mCherry were mixed in a 1:9 ratio in Opti-MEM media (Thermo Fisher Scientific, Waltham, MA, USA) for transfection by FUGENE HD. The transfected Neuro-2a cells were harvested 24 h after transfection. HeLa cells stably expressing Cas9 (GeneCopoeia SL503) were cultured in DMEM, 10% FBS, 1% Pen-Strep, 1x final GlutaMAX (Thermofisher Scientific 35050061), and Hygromycin (250 µg/mL final) at 37 °C and 5% CO2. After 24 h of seeding into 24-well PDL-coated tissue culture plates (Corning 354470), cells were transduced with AAV-PHP.B-sgLacZ and AAV-PHP.B-sgSOD1#5 at 1000k MOI (multiplicity of infection) for 48 h or 72 h. The cells were then harvested in QIAzol (Qiagen 79306) for RNA extraction using miRNeasy mini kit (Qiagen 217004). For bulk RNAseq, 250 ng of extracted RNA was used to prepare RNAseq libraries using the Kapa mRNA hyperprep kit (Kapa biosystems KK8581), following the manufactures instructions. Fragmentation of mRNA was carried out for 6 min at 94 °C, followed by RT, A-tailing, adaptor ligation, and 12 cycles of PCR, to produce Illumina sequencing libraries. Tagged libraries were pooled and sequenced on an Illumina NovaSeq6000 sequencer with a run parameter of 2 × 51 bp paired end reads. Quality control was performed using Illumina’s BaseSpace run summary tool, which showed a %Q30 of 94.14%. The fastq files were generated from the bcl files using bcl2fastq v2.20 (Illumina). The RNA-seq analysis pipeline consisted of alignment with STAR version 2.5.2a against human genome version GRCh38 and Gencode gene model release 27 [41, 42]. After alignment, quantification of gene expression was carried out with RSEM v1.2.26 [43]. Data quality metrics and plots for visualization were generated with Quickomics [44]. Differential expression analysis was performed with DESeq2 version 1.30.0 [45]. The function apeglm was used for LFC shrinkage to reduce noise and preserve large differences [46]. Cutoff values used for significant differential expression were fold change >2 and adjusted p-value < 0.05. Immunoblotting and antibodies For the detection of protein expression in total cell lysates, cells were lysed in Novex Tris-Glycine SDS sample buffer containing NuPAGE sample reducing agent (Invitrogen). Lysates were electrophoresed through Novex Tris-Glycine gels or NuPAGE Bis-Tris gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis. The blocking of membranes and subsequent antibody incubations were performed using Odyssey blocking buffer (LI-COR Biosciences) according to the manufacturer’s instructions. Primary antibodies against β-Tubulin (926–42211, LI-COR Biosciences), β-actin (926–42210, LI-COR Biosciences), FLAG (F1804, Millipore Sigma, Burlington, MA), mCherry (ab167453, Abcam, Cambridge, United Kingdom), GAPDH (ab8245, Abcam), and human SOD1 (ADI-SOD1–100, Enzo) were purchased from commercial sources. The IRDye 800CW-conjugated and IRDye 680-conjugated secondary antibodies were obtained from LI-COR Biosciences. Immunoblot signals were visualized by the Odyssey CLx infrared imaging system and quantified by ODYSSEY application software (LI-COR Biosciences). Results Neonatal ICV injection of AAV-sgSOD1 effectively reduces huSOD1 protein in the CNS and generates a high indel rate in the huSOD1 gene To identify CRISPR/sgRNAs that disrupt huSOD1 expression, eight huSOD1-targeting sgRNAs (termed sgSOD1s) were assessed for their CRISPR activity in vitro in two different cell lines. COS-1 cells were co-transfected with expression vectors for huSOD1, Streptococcus pyogenes Cas9 nuclease (spCas9) and eight different sgSOD1s. These eight sgRNAs were compared for their ability to facilitate reduction of exogenously expressed huSOD1 protein by immunoblotting analysis (Fig. S1). Additionally, engineered Neuro-2a cells were induced to express spCas9, and were subsequently co-transfected with expression vectors for huSOD1 and sgSOD1s. Two of the huSOD1-targeting sgRNAs, sgSOD1#1 and sgSOD1#5, consistently resulted in marked reduction of exogenous huSOD1 protein levels in the COS-1 and Neuro-2a cell types and were used in subsequent in vivo studies (Fig. S1). CRISPR-Cas can sometimes cause off-target genome editing which could lead to unintended changes in gene expression. To assess for potential off-target effects in an unbiased manner, we performed bulk RNA sequencing to detect differentially expressed genes (DEGs) in human HeLa cell lines stably expressing Cas9 transduced with AAV9-sgSOD1#5 at an MOI of 1000 K for 48 h or 72 h. The average sequencing depth was 100 million reads per sample, with mapping rates of 97% and 85% of the reads mapping to exons. As expected, AAV-sgSOD1#5 caused a pronounced reduction of 69% and 79% in SOD1 expression at 48 h and 72 h. When compared to the control samples transduced with AAV9-sgLacZ, two genes (SERPINE2, MYEOV) were slightly downregulated by AAV-sgSOD1#5 at 48 h post-transduction (Fig. S2). However, downregulation of these genes was not observed at 72 h post-transduction, suggesting that these genes are unlikely to be targeted directly by sgSOD1#5. Consistent with this notion, neither gene shares homology with the spacer sequence of sgSOD1#5 and neither gene was a predicted off-target based on Benchling’s “CRISPR Guide RNA Design” tool [38]. Taken together, these data indicate that sgSOD1#5 does not display any off-target effects in human HeLa cells. After confirming the ability of sgRNAs to reduce huSOD1 protein in vitro, we evaluated whether a huSOD1-targeting CRISPR system delivered by AAV (AAV-CRISPRSOD1) could reduce huSOD1 protein and alleviate disease progression in SOD1G93A mice crossed to H11Cas9 knock-in mice. sgSOD1#1 and sgSOD1#5 were packaged into AAV-PHP.B capsids, selected for their broad CNS transduction rates [31–34]. sgRNA targeting the non-mammalian LacZ gene (termed sgLacZ) was used as the control. In H11Cas9 SOD1G93A mice, AAV-sgRNAs (2E11 viral genome (vg) per animal) were delivered to the CSF via intracerebroventricular (ICV) injection on post-natal day 0 (P0) (Fig. 1a, Cohort #1). We first analyzed the degree of huSOD1 protein reduction by AAV-sgSOD1 using an ELISA assay. At 5 weeks after P0 ICV injection of AAV-PHP.B-sgRNA directed against huSOD1, we observed ~50–60% huSOD1 protein reduction in the cortex and spinal cord of sgSOD1-treated mice – this level of reduction was sustained at later time points (10 and 20 weeks of age, Fig. 1b). As a direct indicator of CRISPR-mediated gene editing efficiency, we then assessed the indel rates in the huSOD1 transgene by amplicon sequencing. In the cortex, we detected indels in ~20% of the huSOD1 transgenes in all nuclei, which included nuclei of transduced and non-transduced cells. Using FACS to enrich for GFP-positive nuclei transduced by AAV-PHP.B, the indel rate in the huSOD1 transgene doubled to ~40% (Fig. 1c). Consistent with stable reduction of huSOD1 protein over time, the indel rates in the cortex remained stable at 5-, 10- and 20-weeks post-injection. In the spinal cord, huSOD1 transgene indels were detected in ~7% of the total nuclei, as compared to an indel rate of ~17% in the AAV-transduced nuclei after enrichment by FACS (Fig. 1c).Fig. 1 Neonatal ICV injection of AAV-sgSOD1 effectively reduces huSOD1 protein in the CNS and generates high indel rates in the huSOD1 gene. a Study design of histological and biochemical analysis on H11Cas9 SOD1G93A mice treated with AAV-sgSOD1 (Cohort #1). b Percentage of remaining huSOD1 protein in cortex (left) and spinal cord (right) of H11Cas9 SOD1G93A mice treated with AAV-sgSOD1 (Cohort #1) as determined by ELISA. c Indel percentage in cortex and spinal cord of H11Cas9 SOD1G93A mice treated with AAV-sgSOD1 (Cohort #1) as determined by amplicon-Seq of transduced nuclei (left) and all nuclei (right). Data are presented as mean with standard deviation (SD) unless otherwise stated. Welch’s t-test with Bonferroni correction is performed unless otherwise stated. p < 0.05, p < 0.01, and *p < 0.001, n.s. not significant. See also Fig. S3. To assess the frequency of large excisions of SOD1 transgenes, we used droplet digital PCR (ddPCR) to directly assess huSOD1 transgene copy number remaining in the transduced nuclei from the cortex and spinal cord of 10-week-old animals. We found no difference in huSOD1 transgene copy numbers between sgSOD1-treated and sgLacZ-treated mice (Fig. S3), indicating that a large excision could not be detected following CRISPR editing in vivo using ddPCR. Thus AAV-sgSOD1 delivered to neonates generates high indel rates in the huSOD1 gene and effectively reduces huSOD1 protein in the CNS in SOD1G93A mice. While we did not measure indel rates at the endogenous mouse SOD1 gene, sgSOD1#1 targets a region in the SOD1 gene that is 100% conserved between human and mouse. Unlike sgSOD1#1, sgSOD1#5 targets a region in the SOD1 gene that is poorly conserved between human and mouse and is unlikely to induce murine SOD1 suppression. As describe above, sgSOD1#5 did not result in any off-target gene expression changes in human HeLa cells. For these reasons, sgSOD1#5 was selected as the lead sgRNA used in all in vivo study cohorts while sgSOD1#1 was used in a subset of in vivo study cohorts. We previously reported broad neuronal distribution and high rates of transduction in the hippocampus (~80%), cortex (~95%) and in the dorsal (~60%) and ventral (~80%) spinal cord upon neonatal ICV injection of AAV-PHP.B targeting NeuN [33]. As our current analysis used the same AAV-PHP.B serotypes as in the NeuN study, we elected not to perform histological experiments to assess cell type distribution and transduction efficiency. We expect similar cellular distribution and transduction efficiencies as those observed in Hana et al. Neonatal ICV injection of AAV-sgSOD1 prevents motor function decline and extends survival in H11Cas9 SOD1G93A mice We next evaluated whether AAV-sgSOD1 treatment could alleviate disease progression in the H11Cas9 SOD1G93A mouse model of ALS. The effect of AAV-sgSOD1 on survival and motor functions in mice was evaluated in two independent study cohorts (Cohort #2 and Cohort #3, Fig. 2a). Again, AAV-sgRNAs (2E11 vg per animal) were delivered to the CSF via ICV injection on P0.Fig. 2 Neonatal ICV injection of AAV-sgSOD1 prevents motor function decline and significantly extends survival in H11Cas9 SOD1G93A mice. a Study design of two independent cohorts (Cohorts #1 and #2) of H11Cas9 SOD1G93A mice treated with AAV-sgSOD1. b, c Kaplan-Meier survival curves of Cohorts #1 and #2. Log-Rank test, P < 0.0001. d Body weight of mice over time (Cohort #1). Behavioral assessments of SOD1G93A mice treated with AAV-sgSOD1: rotarod performance measured as latency to fall over age (e), total distance travelled in open field over time (f), performance in inverted grid test measured as latency to fall at week 14 and 19 (g), and clasping score over age (h). Data are presented as mean with standard deviation (SD) unless otherwise stated. Two-way ANOVA was performed with all groups compared to WT group in d–f. One-way ANOVA Dunnett’s multiple comparisons test was performed with all groups compared to WT group in g (n.s. is not labeled). p < 0.05, p < 0.01, and p < 0.001, and n.s. not significant (p > 0.05). Strikingly, both huSOD1-targeting sgRNAs extended the life span of H11Cas9 huSOD1G93A mice by >110 days: 137 days (~20 weeks) in Cohort #2 and 113 days (~16 weeks) in Cohort #3 (Fig. 2b, c). As further evidence of efficacy, out of a total of 48 treated animals (21 in Cohort #2 and 27 in Cohort #3), only one exhibited paralysis and succumbed to ALS-like symptoms at 193 days (~27 weeks) whereas 47 died or required euthanasia for humane reasons at ~300 days (~43 weeks) without paralysis or any other ALS-like symptoms. Euthanasia was required in this group because of hunched body posture and overall poor body condition, malocclusion or prolapse, or a combination of the two. These symptoms in our treated SOD1G93A mice could result from CNS-independent SOD1G93A toxicity and warrant further investigation [11, 47–49]. Furthermore, over time, sgSOD1#1- and sgSOD1#5-treated animals maintained body weights that were similar to those of wild type mice (Fig. 2d), indicating effective protection against disease progression. We further performed a battery of motor behavioral tests, including rotarod, open field, and inverted grid (Fig. 2e–g). sgLacZ-treated control mice exhibited motor deficits beginning at 14–15 weeks of age which became more severe by week 19. In contrast, the performance of sgSOD1-treated animals was not significantly different from that of wild type mice, even at 43 weeks of age (Fig. 2e–g). We also used the “clasping” reflex phenotype to monitor motor function (Fig. 2h). This reflex assesses motor control whereby wild type animals lifted by the tail extend their hindlimbs outward. In contrast, mice with motor deficits when handled in this manner exhibit abnormal “clasping” characterized by retraction of the hindlimbs toward the abdomen, an effect observed in SOD1G93A mice as well as other ALS models [50, 51]. While sgLacZ-treated H11Cas9 SOD1G93A mice showed abnormal clasping behavior starting at ~21 weeks of age, animals receiving the huSOD1-targeting sgRNAs showed minimal clasping, resembling wild type mice (Fig. 1h). Together, these data demonstrate that CRISPR-mediated in vivo disruption of the huSOD1 transgenes in SOD1G93A mice prevented the development of motor deficits and markedly extended survival. Neonatal ICV injection of AAV-sgSOD1 prevents neuromuscular junction loss, muscle atrophy and neurodegeneration The first sign of disease pathophysiology in SOD1G93A mice is characterized by denervation of the neuromuscular junction (NMJ) in the hindlimbs [52, 53]. Denervation can be detected at the electrophysiological level as a decline over time in compound muscle action potential (CMAP), followed by MN loss [12]. To test whether CRISPR-mediated huSOD1G93A reduction could affect this phenotype, H11Cas9 SOD1G93A mice were injected with AAV-PHP.B-sgRNAs intracerebroventricularly at P0 and evaluated for CMAP (Cohort #2) and innervation at NMJ (Cohort #1) in the tibialis anterior (TA) muscles in the hind limbs (Figs. 2a, 3a). Remarkably, CMAP measurements of mice treated with either sgSOD1#1 or sgSOD1#5 were indistinguishable from their wild type littermates over the course of 46 weeks or until the end stage, whereas CMAP for sgLacZ-treated SOD1G93A animals was reduced by greater than 75% by 22 weeks of age (Fig. 3b). We double-labeled MN axon presynaptic terminals with anti-neurofilament and anti-SV2, and muscle post-synaptic membranes with α-bungarotoxin to evaluate the integrity of NMJ in the TA muscle. Consistent with electrophysiological preservation of CMAP, this analysis revealed that sgSOD1-treated mice-maintained innervation of TA muscles, whereas sgLacZ-treated SOD1G93A animals showed evidence of full denervation of ~70% of NMJ in muscle endplates by 20 weeks of age (Fig. 3c).Fig. 3 Neonatal ICV injection of AAV-sgSOD1 prevents NMJ loss, muscle atrophy and neurodegeneration in H11Cas9 SOD1G93A mice. a Study design of histological and biochemical analysis on H11Cas9 SOD1G93A mice treated with AAV-sgSOD1 (Cohort #1). b CMAP amplitude over disease progression (time). Two-way ANOVA is performed with all groups compared to WT group. c Percentage of fully innervated, denervated and partially innervated NMJs in male mice at 20 weeks of age. d Diameter of fast myosin muscle fiber of tibialis muscles at 20 weeks of age. e Left, representative images of spinal motor neurons stained by ChAT at 20 weeks of age. (scale bar, 50 µm); Right, spinal motor neuron numbers at 20 weeks of age. c–e One-way ANOVA Dunnett’s multiple comparisons test was performed with all groups compared to WT group. f Serum pNFH levels over disease progression (time). Two-way ANOVA was performed, all groups compared to WT group. b–f Data are presented as mean with standard deviation (SD) unless otherwise stated. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001, and n.s. not significant. Data was from collected from Cohort #3 in B-E and from Cohort #1 in F. A myosin classification system based on ATPase reactivity distinguishes skeletal muscle fibers as either fast-twitch or slow-twitch types [54]. In SOD1G93A mice, fast-twitch muscle fibers are more susceptible to atrophy than slow-twitch fiber types [55, 56]. To determine if AAV-sgSOD1 protects against these changes in the TA muscles, we compared sgSOD1- and sgLacZ-treated H11Cas9 SOD1G93A mice for fast-myosin fiber diameter at 20 weeks of age. As expected, sgLacZ-treated SOD1G93A mice showed significant decrease in fast-myosin fiber diameter, indicating atrophy of fast-twitch muscle fibers (Fig. 3d and Fig. S4). The TA muscle of sgSOD1-treated H11Cas9 SOD1G93A mice did not show any of these phenotypes and were indistinguishable from wild type littermates. Death of spinal MNs represents a hallmark of ALS, a phenotype that is recapitulated in the SOD1G93A mice [6]. To determine if CRISPR-mediated huSOD1G93A reduction can protect against MN loss, we counted MNs stained positive for choline acetyltransferase (ChAT) [18] in the spinal cords of sgLacZ- and sgSOD1-treated animals. We observed a 37% decrease in the number of ChAT+ MNs in sgLacZ-treated H11Cas9 SOD1G93A mice at 20 weeks of age (Fig. 3e), whereas no decrease in MN number was detected in sgSOD1-treated animals, (Fig. 3e). Elevated levels of phospho-neurofilament heavy chain (pNFH), one of the main by-products of axonal damage [57], are detected in CSF and serum from patients with neurodegenerative diseases, including ALS [58]. As similar increases are observed in ALS rodent models [59], sgSOD1-treated H11Cas9 SOD1G93A mice (Cohort #2) showed lower levels of serum pNFH compared to controls (Fig. 3f). This reduction occurred in two phases: an initial rise that never exceeded 40% of the levels detected in SOD1 mice treated with control AAV and a subsequent plateau at <20% of the maximal levels observed in SOD1 controls. Therefore, AAV-sgSOD1 protects against neurodegeneration. Despite full correction of the morphological and functional readouts, AAV-sgSOD1 treatment did not completely extinguish the pNFH biomarker signal. This could not be explained by degeneration caused by the AAV vector alone, since wild type mice injected with control AAV did not show significant pNFH increases. It therefore seems likely that a set of neurons not required for normal function were not transduced and/or changes in pNFH are more sensitive indicators of ongoing low rates of MN degeneration than the functional or morphological assays. Collectively, these data demonstrate that sgRNA-guided in vivo editing of toxic huSOD1 prevented NMJ loss and muscle atrophy, and strongly reduced neurodegeneration in the SOD1G93A mouse model. Intrathecal and intravenous injection of AAV.PHP.eB-CRISPRSOD1 in adult mice fully preserves neuromuscular function and extends survival beyond one year Next, we set out to evaluate whether the therapeutic effects of CRISPRSOD1 could be further enhanced by another novel AAV capsid, AAV.PHP.eB. AAV.PHP.eB was derived from AAV-PHP.B via AAV-targeted evolution for more efficient transduction of neurons and contains two different amino acids at positions 587 and 588 [31]. We treated a small cohort of adult H11Cas9 SOD1G93A mice (Cohort #4) with a single intrathecal (IT) or intravenous (IV) injection of AAV.PHP.eB-sgSOD1#5 (or sgLacZ; n = 10 per group; 1E12 vg per animal) at 5 weeks of age, immediately prior to the onset of CMAP decline (Fig. 4a). Strikingly, sgSOD1 extended survival of treated mice by at least 170 days (~24 weeks) independent of delivery method (Fig. 4b). This is a minimum estimate since we euthanized all sgSOD1-treated animals at postnatal day 340 (~49 weeks) due to COVID-19-related emergency shutdown of the animal facility. None of the treated mice showed any abnormal phenotypes at the time of euthanasia. sgSOD1 treatment prevented body weight loss observed in the sgLacZ-treated mice (Fig. 4c) and CMAP decline in the TA muscle, indicating protection against NMJ denervation (Fig. 4d). Notably, there was no difference in the degree of therapeutic benefit between IT and IV delivery methods, consistent with the report that AAV-PHP.eB crosses the blood-brain barrier efficiently in C57BL/6 mice [31]. When huSOD1 protein levels in the CNS were evaluated by ELISA in IT-injected mice at 10 weeks post injection, we observed a ~85% reduction of huSOD1 protein in the cortex and ~80% reduction of huSOD1 protein in the spinal cord of sgSOD1-treated mice (Fig. 4e). The higher degree of huSOD1 protein reduction (80% in Cohort #4 vs 50–60% in Cohort #1) and stronger therapeutic benefits (>170 days survival extension in Cohort #4 vs 113–137 days in Cohort #2 and #3) may reflect superior transduction efficiency in the CNS by AAV.PHP.eB versus AAV.PHP.B [33, 36] and/or the route of administration, highlighting two key considerations to potentially boost the efficacy of gene therapy. Amplicon sequencing performed on genomic DNA of a subset of sgSOD1-treated mice at the end of the study (~49 weeks) showed indel rates of ~39% in the cortex and ~24% in the spinal cord (Fig. S5). Together, these results demonstrated that AAV-CRISPR-mediated in vivo gene editing achieved robust reduction of huSOD1 transgene expression in the SOD1G93A mice, leading to pronounced survival extension.Fig. 4 IT and IV injection of AAV-sgSOD1 in adult H11Cas9 SOD1G93A mice preserves neuromuscular function and significantly extends survival. a Study design of IT and IV injection of AAV-sgSOD1 in adult H11Cas9 SOD1G93A mice (Cohort #4). b Kaplan-Meier survival curves of Cohorts #1 and #2. Log-Rank test, P < 0.0001. c Body weight of mice over age (Cohort #4). t-test was performed at week 21 before any mock-treated mice reach euthanasia endpoint: IT-sgLacZ vs. IT-sgSOD1; IV-sgLacZ vs. IV-sgSOD1. d CMAP amplitude of TA muscle at week 5, 10 and 14. Two-way ANOVA with Dunnett’s multiple comparisons test was performed. e Percentage of remaining huSOD1 protein at week 14 in cortex (left) and spinal cord (right) of H11Cas9 SOD1G93A mice IT injected with AAV-sgSOD1 (Cohort #4) as determined by ELISA. Unpaired t-test. c–e Data are presented as mean with standard deviation (SD). p < 0.05, p < 0.01, and p < 0.001, **p < 0.0001, n.s, not significant. Color of indicates the group on which statistics was performed. Discussion In recent years, AAVs have been extensively used as vehicles for gene transfer to the nervous system to modulate gene expression, via overexpression, knockdown or genome editing. As a result, gene therapy approaches have greatly facilitated the ability to evaluate potential therapeutic candidates in preclinical proof of concept studies for the treatment of neurological diseases, as performed here. Gene therapy approaches that employ CRISPR-associated genome editing systems hold enormous potential to treat several genetic forms of ALS. Here, we report that one-time delivery of SOD1-targeting sgRNAs using AAV-PHP.B and AAV-PHP.eB to the CNS of H11Cas9 SOD1G93A mice significantly extended disease-free survival through robust and sustained reduction of mutant huSOD1 protein in the cortex and spinal cord and resulted in strong disease suppression corroborated by comprehensive morphological and functional analyses (Summarized in Table 1).Table 1 Summary of all phenotypes assessed in Cohort #1–4. Study Summary Study Cohort 1 2 3 4 4 Serotype AAV-PHP.B AAV-PHP.B AAV-PHP.B AAV-PHP.eB AAV-PHP.eB Delivery ICV ICV ICV IT IV sgSOD1 #1/#5 #1/#5 #1/#5 #5 #5 Survival – +137 days (20 weeks) +113 days (16 weeks) >170 days (24 weeks) >170 days (24 weeks) Body weight – Resemble WT resemble WT Resemble WT Resemble WT Motor functions Rotarod – Resemble WT – – – Open field – Resemble WT – – – Inverted grip – – resemble WT – – “clasping” – Resemble WT – – – CMAP (Tibialis anterior) – No decline – No decline No decline Neuromuscular junction Resemble WT – – resemble WT Resemble WT Motor neuron count Resemble WT – – – – Serum pNFH – Lower than controls – – – Fast-myosin muscle diameter Resemble WT – – – – huSOD1 levels Cortex 50–60% reduction – – 85% reduction – Spinal cord 50–60% reduction – – 80% reduction – Indel rate bulk Cortex 20% – – 39% – Spinal cord 7% – – 24% – Indel rate FACS sorted Cortex 40% – – – – Spinal cord 17% – – – – We found a larger degree of suppression than two previously published studies employing AAV-mediated delivery of CRISPR to treat SOD1G93A mice [29, 30]. We observed a median survival extension of >110 days vs 25 days and 12 days, respectively. Gaj and colleagues employed a single AAV9 vector delivery of saCas9 and sgRNA to introduce indels in the huSOD1 transgene. Lim and colleagues used two AAV9 vectors to deliver a split intein-mediated protein trans-splicing system to introduce early stop codons in the huSOD1 transgene. Several factors could contribute to the improved efficacy in our study. First, in our experiments spCas9 was constitutively expressed in target cell populations rather than being packaged into AAV and delivered exogenously. Second, we used newer AAV variants with reportedly broader distribution and/or higher transduction rates in neurons and astrocytes than AAV9 [33, 34, 36]. Consistent with this, we observed much higher indel rates (~7–17%) in the spinal cord of treated animals as compared to earlier studies (<1.2%). Remarkably, the 80% huSOD1 protein reduction achieved in Cohort #4 (vs. 50–60% in Cohort #1) translated to greater survival extension (>170 days vs >137 days), highlighting that phenotypic efficacy correlates with the degree of huSOD1 protein reduction. Lastly, we cannot exclude the possibility that the nuclease activity of spCas9 generates higher editing efficiency than saCas9 or spCas9-fused CBEs. Future studies to directly compare the nuclease activity of these different Cas proteins are warranted. Taken together, our approach provided a higher degree of therapeutic benefit in SOD1G93A mice, highlighting several key considerations to increase genome editing efficiency, including distribution and expression level of Cas9, and transduction efficiency of AAV capsids on target cell populations. Though successful, our approach has some inherent limitations that preclude it from directly translating into clinical development. First, the introduction of the H11Cas9 transgene into SOD1G93A mice is highly artificial and does not translate as a general strategy for patients. In patients, there is a need to package a complete CRISPR/Cas9 system, which might limit the effectiveness of target modulation. Second, our study did not assess the immunological impact of constitutive Cas9 expression in the CNS, another factor highly relevant to gene therapy patients. Patients undergoing gene therapy treatment would need to be monitored constantly for an immunological response and potentially receive immuno-suppressants to compensate for such a response. A third limitation is the preventive nature of our treatment paradigm—animals were treated immediately prior to symptom onset. This paradigm does not parallel the treatment of patients as they would be treated after symptom onset. Despite this specific concern, other studies also administered treatments prior to symptom onset with less impactful effects on survival than in our study [29, 30, 60]. Finally, though the enhanced CNS tropism of AAV-PHP.B and AAV-PHP.eB is useful for evaluating and validating targets in pre-clinical models, it is not yet translatable to development of therapeutics in humans as their strong transduction in CNS appears to be limited to specific mouse strains, such as C57BL/6 and FVB/NCrl [30, 61–64]. The goal of the current study is not to develop therapeutics for clinical trials, but instead to demonstrate the therapeutic potential of CRISPR editing in preclinical models and identify key parameters (e.g., AAV capsid, Cas9 expression) critical to greater efficacy. Like other work using CRISPR to target huSOD1 [29, 30], we observed a discrepancy between indel rate (~10–20%) and huSOD1 protein reduction (~50–60%) in target tissues. Several reasons could account for this discrepancy. First, while huSOD1 transgene is present in all cells in the target tissue, its expression level varies between different cell types (https://www.brainrnaseq.org/). As the AAV-PHP.B variant has strong tropism towards neurons and astrocytes where huSOD1 expression is also high (https://www.brainrnaseq.org/) [32, 34], it is conceivable that cells with high huSOD1 expression such as neurons and astrocytes also have high indel rates. Other cell types (e.g., microglia, endothelial cells) with low huSOD1 expression might have low indel rates. Single-cell or single-nucleus RNA-Seq could be employed in future studies to confirm this possibility. Secondly, larger deletions may also occur in the vicinity of the Cas9 cleavage sites in addition to small insertions and deletions. These deletions may not be detected by amplicon sequencing due the smaller size (~500 bp) of the amplicons. Indeed, a recent publication detected deletions, ranging in size from a few hundred base pairs (bp) to over 4000 bp by long-range PCR, cloning, and Sanger-sequencing in the vicinity of the Cas9 cleavage site [65]. While we have ruled out large deletions that affect huSOD1 transgene copy number by ddPCR, we cannot exclude the possibility AAV-sgSOD1 introduced intermediate-sized deletions (a few hundred to a few thousand bp), which led to huSOD1 protein reduction. Long-range PCR, cloning, and Sanger-sequencing approaches are needed in future studies to carefully examine this possibility. In summary, our approach provided remarkable protection against multiple ALS-related phenotypes in the SOD1G93A mouse model and, importantly, resulted in substantial disease-free survival extension, highlighting the utility of combining CRISPR-based gene editing with robust novel AAV capsids in evaluating potential therapeutic targets in pre-clinical studies. The positive results from our proof-of-concept study in SOD1G93A mice, supports the notion of introducing the spCas9 gene into other genetic animal models of ALS for CRISPR-based gene editing. This would afford an opportunity to assess nearly any gene in the genome for their potential effects in multiple ALS mouse models. Genes found to be protective across multiple ALS models are more likely to identify potential disease-modifying therapeutic targets for sporadic ALS, which represents ~90% of all ALS cases. Such an approach would ultimately accelerate drug discovery while also aiding in elucidating the etiology and biology of ALS. Moreover, as this strategy is agnostic to disease, it can easily be applied more broadly to other CNS disease areas, potentially benefiting those patient communities as well. Supplementary information Supplementary Material Chen et al SOD1 CRISPR-2022 Supplementary information The online version contains supplementary material available at 10.1038/s41434-022-00375-w. Acknowledgements We thank Jianxin Hu, Brigitte Pettmann and Giulio Srubek Tomassy for helpful discussion of this research; Chia-yen Wu (Jackson Laboratory), Martin Lamb, and Linhong Sun for assistance with experiments; and Dr. Monte M. Winslow for providing constitutive H11Cas9 knock-in mice. Author contributions SCL, YAC, AM, and CEH conceived the project and designed the experiments. OM, NM, SL and SKL performed in vitro screening of sgRNAs. DF, DK. KK, YAC, YL, and KS, performed in vivo studies including injections, survival monitoring, body weight measurement, tissue collection, etc. JD and BW performed CMAP recordings. AF, HM, ML, EM, CS, PC and TC performed indel analysis and RNA sequencing. GM, SH, MC, and JS performed histology analysis. HMA, RD, and AS performed behavioral testing. SM and SL performed huSOD1 protein measurement. MIZ performed sequencing analysis. SCL, YAC, MWK., SH, and CEH wrote the paper. Funding This research was provided by Biogen Inc. Data availability The RNA-seq data generated from HeLa cell lines has been deposited in the Gene Expression Omnibus (GEO tracking number GSE213125). Competing interests The authors would like to declare they are/were employees of Biogen Inc. Ethical approval All animal use and treatments were approved by the Biogen Institutional Animal Care and Use Committee (IACUC) and followed the National Institute of Health Guide for the Care and Use of Laboratory Animals. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Brown RH Al-Chalabi A Amyotrophic lateral Sclerosis N Engl J Med. 2017 377 162 72 10.1056/NEJMra1603471 28700839 2. 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==== Front Pharm Res Pharm Res Pharmaceutical Research 0724-8741 1573-904X Springer US New York 36451070 3445 10.1007/s11095-022-03445-1 Original Research Article Lipid Microparticles Show Similar Efficacy With Lipid Nanoparticles in Delivering mRNA and Preventing Cancer Ji Afang 12 Xu Minghao 123 Pan Yunzhi 24 Diao Lu 34 Ma Lin 4 Qian Li 4 Cheng Junping [email protected] 12 http://orcid.org/0000-0002-5185-5872 Liu Mi [email protected] 34 1 grid.410745.3 0000 0004 1765 1045 Suzhou TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou, 215000 Jiangsu China 2 grid.490559.4 The Fifth People’s Hospital of Suzhou, Suzhou, 215000 Jiangsu China 3 Suzhou Ersheng Biopharmaceutical Co., Ltd, Suzhou, 215000 People’s Republic of China 4 grid.263761.7 0000 0001 0198 0694 College of Pharmaceutical Science, Soochow University, Suzhou, 215123 Jiangsu China 30 11 2022 115 3 6 2022 20 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose Messenger RNA (mRNA) has shown great promise for vaccine against both infectious diseases and cancer. However, mRNA is unstable and requires a delivery vehicle for efficient cellular uptake and degradation protection. So far, lipid nanoparticles (LNPs) represent the most advanced delivery platform for mRNA delivery. However, no published studies have compared lipid microparticles (LMPs) with lipid nanoparticles (LNPs) in delivering mRNA systematically, therefore, we compared the impact of particle size on delivery efficacy of mRNA vaccine and subsequent immune responses. Methods Herein, we prepared 3 different size lipid particles, from nano-sized to micro-sized, and they loaded similar amounts of mRNA. These lipid particles were investigated both in vitro and in vivo, followed by evaluating the impact of particle size on inducing cellular and humoral immune responses. Results In this study, all mRNA vaccines showed a robust immune response and lipid microparticles (LMPs) show similar efficacy with lipid nanoparticles (LNPs) in delivering mRNA and preventing cancer. In addition, immune adjuvants, either toll like receptors or active molecules from traditional Chinese medicine, can improve the efficacy of mRNA vaccines. Conclusions Considering the efficiency of delivery and endocytosis, besides lipid nanoparticles with size smaller than 150 nm, lipid microparticles (LMPs) also have the potential to be an alternative and promising delivery system for mRNA vaccines. Keywords lipid microparticles lipid nanoparticles LMP LNP mRNA vaccine particle size ==== Body pmcIntroduction Vaccines are widely considered one of the greatest public health achievements and vaccinations have greatly decreased the burden of infectious diseases worldwide and saved millions of lives each year [1–3]. A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic world-wide and caused millions of deaths. Given that vaccines are the most important public health approach to protect people from COVID‐19, companies and research institutions competitively devoted to develop SARS-CoV-2 vaccines. So far, researchers have developed inactivated vaccines, viral-vector-based vaccine, subunit vaccines and mRNA vaccines etc. [3–5]. Among them, mRNA vaccines showed excellent efficacy and showed potentials in application beyond preventing infection disease. In addition to infectious diseases, mRNA vaccines can also be used to treat other diseases, such as cancer [4–6]. According to recent studies, nucleic acid-based vaccines could provide efficient protection or treatment, by stimulating both humoral immunity and cellular immunity [7]. Compared to DNA vaccines, mRNA vaccines demonstrated significant advantages, in terms of its high potency, capacity for rapid development and potential for low-cost manufacture and safe administration [4, 7, 8]. The technical challenges associated with DNA vaccines are to ensure delivery into the cell nucleus potential risks of integration into the host genome. While mRNA has no potential risks of infection or genomic integration, thanks to mRNA carries genetic information from the DNA to the cytosol, where it is used by the ribosomes as a template for protein synthesis [3, 6, 9, 10]. Furthermore, mRNA can be degraded by normal cellular processes, and the half-life of mRNA can be regulated through the use of various modifications and delivery methods [3, 11, 12]. However, therapeutics based on mRNA has the challenges of the instability of mRNA and the crossing of membrane barrier [3, 9, 12]. Recent technological advances have now largely overcome these issues, these new technologies include mRNA modification and delivery platforms such as protamine complexes, nanoparticles based on lipids or polymers, and hybrid formulations etc [11–16]. Indeed, a good delivery platform should efficiently bind mRNA, protect mRNA from extracellular RNase degradation, uptake by the desired target cell and express the mRNA in target cells [17]. Sa far, lipid nanoparticles (LNPs) is the most promising and developed technology for mRNA delivery [16, 18, 19]. A typical lipid/mRNA particles formulation is consisted of ionizabe lipids or cationic lipids, neutral helper lipids, sterol lipid and a polyethylene glycol (PEG)-lipid [20–23]. It is reported that mRNA vaccines delivered by lipid/mRNA particles can activate both humoral and cellular immune responses potently. Many factors may affect the potency of mRNA vaccine delivered by lipid/mRNA particles, such as particle size and the incorporation of adjuvants [23, 24]. In previous studies and the FDA-approved mRNA vaccines, researcher applied lipid nanoparticles with the size around 70 nm-100 nm. Though the scientists from Moderna analyzed lipid nanoparticles range from 50 to 200 nm and proved that LNPs, with size from 50 to 200 nm, yielded similar robust immune response [20, 25]. However, no studies have compared the impact of particle size, in a broader range, on the efficacy of lipid particles. Given that the size of particles can impact the migration and uptake of particles, particles with size larger than 200 nm may have particular behaviors [1, 26]. So far, the studies of vaccine delivery by microparticles focus on biodegradable materials-based microparticles, such as PLGA microparticles and the payload of microparticles focus on protein or peptide antigens. Recently, a few SiRNA delivery by microparticles have been explored and these limited number studies of SiRNA delivery by microparticles focus on pulmonary administration or lung-targeting [27–30]. No studies have evaluated the delivery efficacy of mRNA vaccines by microparticles, especially lipid microparticles. Studies have reported that microparticles, loaded with antigens, might have the advantages in activating immune responses, such as microparticles degrade more slowly and can be more taken up by antigen presenting cells, besides endocytosis microparticles can also attach to the surface of antigen presenting cells and deliver antigens, microparticles produced enhanced CD4 + T cell activation compared to smaller size nanoparticles etc [1, 26, 30–41]. Therefore, delivery of mRNA vaccines by microparticles might be a promising strategy for mRNA drugs. Herein, we evaluated the impact of particle size, especially larger size, on cellular uptake and immune responses induction of mRNA lipid particles in a broader size range. Especially, we compared the efficacy of lipid microparticles (LMPs) with LNPs in delivering mRNA. To our knowledge, the most commonly used lipid particles for mRNA delivering are about 50 nm-100 nm, hence 70 nm –100 nm was chosen as the small size of lipid nanoparticles (LNPs). Considering other common applied nanoparticle size, we selected 250 nm -350 nm as the size for medium size lipid nanoparticles (LNPs) and selected 1.1 μm – 1.3 μm as the size for large size lipid microparticles (LMPs). In order to specifically investigate the impact of particle size on vaccine potency, all other factors of the delivery system must be matched [25, 42]. In our study, we changed lipid/mRNA nanoparticles/microparticles size independent of lipid composition by different preparation methods. The different size lipid/mRNA particles with or without adjuvants were prepared by using solvent diffusion method, microfluidics and film dispersion method. To investigate the impact of particle size and adjuvants on the efficacy of mRNA vaccine, we immunized mice with mRNA vaccine encodes ovalbumin (OVA) and then challenge mice with subcutaneously inoculation of OVA expressing E.G7 lymphoma cells. Both lipid nanoparticles and lipid microparticles were investigated and both poly(I:C) and hesperetin were tested when work as adjuvants. According to the results, both poly(I:C) and hesperetin can be used as immune adjuvants to enhance the potency of lipid/mRNA complexes, and all size lipid/mRNA nanoparticles/microparticles produced robust immune responses. Materials & Methods Materials DOTAP, DOPE and DMG-PEG2000 were purchased from Avanti Polar Lipids (Alabama, USA); cholesterol was purchased from Sinopharm Chemical Reagent (China); FITC-DSPE-PEG2000 was obtained from AVT (Shanghai, China); Hesperetin was produced in Yuanye (Shanghai, China); eGFP mRNA was obtained from VectorBuilder (Guangzhou, China); OVA mRNA was purchased from TriLink Bio Technologies (California, USA); Quanti-iT RiboGreen RNA reagent and Kit were obtained from Invitrogen (California, USA); Mouse ovalbumin specific IgG ELISA kit was purchased from Shanghai Enzyme-linked Biotechnology (Shanghai, China); OVA323-339, OVA257-264, OVA208-216, OVA27-35 were purchased from Apeptide (Shanghai, China); Poly(I:C) (HMW) VacciGrade™ was obtained from InvivoGen (San Diego, USA); DMEM medium, RPMI 1640 medium, 1% Penicillin/Streptomycin, PBS and FBS were purchased from Procell Life Science&Technology (Wuhan, China). Cell Culture and Animal Studies DC2.4 (American Type Culture Collection, Manassas, VA, USA) was cultured in the Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (P/S) (Procell Life Science&Technology Co., Ltd., China) in 5% CO2 at 37℃. RPMI 1640 medium containing 10% FBS and 1% PS was used to culture E.G7-OVA cells (BeNa Culture Collection, China). For in vivo assays, 6–8 weeks old female C57BL/6 mice were used. Mice were assigned to treatment groups based on cage numbers. All animal work was approved and monitored by the Animal Ethics Committee of Soochow University. All the mice were ordered from the animal facility platform of Soochow University. Lipid/mRNA Nanoparticle/Microparticle Preparation Lipid/mRNA complexes with different particle size were formulated by solvent diffusion method, microfluidics or film dispersion method. The ethanol phase was prepared by solubilizing a mixture of cationic lipid (DOTAP, Avanti Polar Lipids, USA), DOPE (Avanti Polar Lipids), cholesterol (Sinopharm Chemical Reagent Co., Ltd, China) and DMG-PEG2000 (Avanti Polar Lipids) at a molar ratio of 35:16:46.5:2.5 with ethanol; hesperetin (5 mg/mL) was also solved in the ethanol phase in the particles loaded with hesperetin as immune adjuvants. The aqueous phase was prepared in 10 mM citrate buffer (pH 3) with either EGFP mRNA (VectorBuilder, USA) or OVA mRNA (TriLink BioTechnologies, USA), with or without poly(I:C) (1 mg/mL). Small size lipid/mRNA nanoparticles were prepared via solvent diffusion method by mixing the aqueous phase and ethanol phase in volumetric flow ratios 4:1 and heating the mixture to 55℃ for 10 min. Medium size lipid/mRNA nanoparticles were prepared via microfluidics by mixing the aqueous phase and ethanol phase in volumetric flow ratios 3:1. Large lipid/mRNA microparticles were prepared via film dispersion method. Heating the ethanol phase to 40℃ for 30 min to form a lipid film and adding the aqueous phase to dissolve the film. The molar ratio between mRNA and the cationic lipid was 1:20. The prepared lipid complexes were purified by dialyzing against 1 × PBS solution in a 1000 MWCO dialysis membrane (Spectrum Laboratories, Inc, USA) at 4℃ for 2 h. Before the administration of mRNA lipid particles loaded with poly(I:C) as immune adjuvants, Lipid/ mRNA particles were mixes with 0.1 mL poly(I:C) to load poly(I:C) to the exterior part of the lipid particle. The lipid particles loaded with dyes were prepared the same as described above, but replacing corresponding lipids with FITC-conjugated lipids. Lipid/mRNA Nanoparticle/Microparticle Characterization The size and surface charge of lipid particle were assessed with or without mRNA. The size, polydispersity (PDI) and Zeta potential of the lipid/mRNA particle were measured by a Zeta sizer Nano ZS (Malvern Instruments, UK). A Quanti-iT RiboGreen RNA reagent and Kit(Invitrogen Corporation, USA)was used to calculate the mRNA encapsulation efficiency. The samples were diluted to a concentration of approximately 5 μg/mL in 1 × TE buffer solution. 50 μL of the diluted samples were transferred into a 96 well plate and either 50 μL of 1 × TE buffer solution (measuring “free” mRNA) or 50 μL of a 2% Triton-X100 (measuring total mRNA) was added to the certain wells. The plate was incubated at a temperature of 37℃ for 15 min. The RiboGreen RNA reagent was diluted 1:100 in 1 × TE buffer solution, and 100 μL of this solution was add to each well. The fluorescence intensity was measured using the SPARK® multimode microplate reader (Tecan Trading AG, Switzerland) at an excitation wavelength of about 480 nm and an emission wavelength of about 520 nm. The fluorescence values of the reagent blank were subtracted from that of each of the samples. The percentage of free mRNA was determined by dividing the fluorescence intensity of the sample without Triton-X100 by the fluorescence intensity of the sample with Triton-X100. The morphology of Lipid/mRNA complexes with different particle size were investigated by the transmission electron microscope (TEM). The samples were diluted to a concentration of approximately 100 μg/mL in deionized water (total lipid concentration was approximately 3.8 mg/mL). After dropping 10–20 μL diluted samples onto the copper nets, the nets were dried at 50–60℃ for 3 h. Phosphotungstic acid solution(1%) was added to the prepared copper nets, 1–2 min later, using the filter paper to absorb the excess dyeing solution. The nets were washed 3 times and dried, followed by taking the images. The stability analysis of LNPs and LMPs on particle size were conducted under 4℃ and -20℃. The LNPs and LMPs were formulated as originally described and then stored in the refrigerator (4℃), or in the freezer (-20℃) for 4 weeks. Size was measured using a Malvern Zetasizer Nano (Malvern Instruments, UK) every week. Each LNP sample was measured three times. In Vitro Cellular Uptake FITC-Lipid Nanoparticles/Microparticles Either DC2.4 cells and splenocytes from the mice were applied to conduct the uptake study. Splenocytes were isolated from sacrificed mice by using a 70 μm cell strainer (Sorfa Life Science Research Co., Ltd., China). After the isolation, red blood cells were lysed by using lysing buffer (BD Bioscience). After that, splenocytes were seeded into a 24-well plate at a density of 2.0 × 10 [5] cells/well and cell culture was done in 1.5 mL culture medium for 24 h at 37℃. The blank lipid particles covalently modified with FITC were added into the DC2.4 cells or splenocytes, and then the cells were treated with 2 μg of the FITC- lipid particles for 4 h at 37℃ in a CO2 incubator. After the incubation, DC2.4 cells were washed with PBS, collected and measured by using BD FACSAria™ III Cell Sorter Flow Cytometer (BD, USA). In terms of splenocytes, the cells were washed with PBS, followed by staining with Zombie Aqua™ Fixable Viability Kit (BioLegend, USA) and incubating with Fc block for 10 min. Then, the splenocytes were stained with PerCP/Cyanine5.5 anti-mouse CD11c antibody, APC anti-mouse B220 antibody and PE anti-mouse F4/80 antibody in FACS buffer (1% FBS in PBS). After cell staining, the stained cells were washed with FACS buffer and measured by using Flow Cytometer. Study of Transfection Efficacy In Vitro DC2.4 cells were seeded into a 24-well plate at a density of 5 × 104 cells/well and cell culture was done in 1.5 mL Dulbecco’s Modified Eagle Medium (DMEM) medium for 24 h at 37℃ in a CO2 incubator. Prior to transfections, EGFP mRNA encapsulated in lipid particles were added into cells in DMEM medium and incubated for 24 h at 37℃. After incubation, each well was imaged under a microscopy and flow cytometry was performed to measure the percentage of GFP-positive cells. The transfection efficacy of lipid particles in DC, B cells and macrophage were compared by incubating splenocytes with different lipid particles. Splenocytes were prepared by isolating them from sacrificed mice and filtering through a 70 μm cell strainer (Sorfa Life Science Research Co., Ltd., China), followed by lysing red blood cells by using lysing buffer (BD Bioscience). After that, splenocytes were seeded into a 6-well plate at a density of 1.0 × 106 cells/well at 37℃. EGFP-mRNA encapsulated lipid particles were added into cells in DMEM medium (0.5 μg/mL) and incubated for 36 h at 37℃. After the incubation, splenocytes were washed with PBS, followed by staining with Zombie AquaTM Fixable Viability Kit (BioLegend, USA) and incubating with Fc block for 10 min. Then, the splenocytes were stained with CD11c-PE/Cy7, F4/80-PE and B220-Percp/cy5.5 antibody (anti-mouse) in FACS buffer (1% FBS in PBS). After cell staining, the stained cells were washed with FACS buffer and measured by using BD FACSAria™ III Cell Sorter Flow Cytometer (BD, USA). Analysis of Lipid Nanoparticles/Microparticles Uptake by Antigen-Presenting Cells In Vivo Female C57BL/6 mice (6–8 weeks) were housed in groups of 5 mice per individually ventilated cage in an SPF facility. Mice were subcutaneous injected with FITC- lipid nanoparticles/microparticles. After 16 h, spleen and axillary lymph nodes were collected and processed into single-cell suspensions as previously described. Single-cell suspensions were stained with Zombie Aqua™ Fixable Viability Kit according to the manufacturer’s instructions to exclude dead cells from analysis. After incubated with Fc-block to block nonspecific FcR binding, cells were surface stained with PerCP/Cyanine5.5 anti-mouse CD11c antibody, APC anti-mouse B220 antibody, APC/Cyanine7 anti-mouse F4/80 antibody and PE anti-mouse CD8a antibody for 30 min at 4℃. After cell staining, the stained cells were washed with FACS buffer and analyzed by flow cytometer. Analysis of Biodistribution of Lipid Particles The DiR dye (purchased from absin, abs45153692) was dissolved in ethanol to prepare a concentration of 1 mg/ml and the DiR dye was encapsulated into 3 different lipid particles with the above method. Female BALB/c mice aged 6–8 weeks received DiR via subcutaneously injected with lipid particles loaded with DiR dye, respectively. Imaging in vivo was performed at 6 h, 24 h and 48 h after injection. Meanwhile, the heart, liver, spleen, lung, kidney, draining lymph nodes and non-draining lymph nodes were taken out after 48 h to observe the distribution of DiR loaded in 3 different lipid particles. Fluorescence signals (Ex740nm, Em790nm) were measured by IVIS® Spectrum (PerkinElmer, Waltham, USA). Immunization of Mice With Lipid Nanoparticles/Microparticles Lipid nanoparticles/microparticles loading with OVA-mRNA and poly(I:C) (HMW) VacciGrade™ (InvivoGen, USA) or hesperetin (Yuanye, China) were used to immunize the mice. The C57BL/6 mice were randomized in different treatment groups and subcutaneously injected with mRNA vaccines near the inguinal lymph nodes twice at a weekly interval. Each dose contained 10 μg of mRNA, in the administration with immune adjuvants each dose contained hesperetin 80 μg or 62.5 μg poly(I:C). The body weight of mice were recorded every week. Challenge Immunized Mice With Inoculation of Tumor Cells Mice were injected subcutaneously, on the flank of mice, with 3.0 × 105 E.G7-OVA cells 4 weeks after the first injection of mRNA vaccines. Tumor volume and body weight of the mice were measured every 3 days. The tumor size measurement was stopped when tumor size was exceeded 2000 mm3. Analysis of Cellular Immunity Induced by mRNA Vaccines on Mice The antigen-specific T cells in splenocytes were detected to analyze the cellular immunity induced by mRNA vaccines. Mice were sacrificed when the tumor size exceeded 2000 mm3 and splenocytes were collected, followed by processing into single-cell suspensions by using a 70 μm cell strainer and lysing buffer. And then, cells were seeded into a 12-well plate at a density of 2.0 × 105 cells/well and cell resting was done in 2 mL medium for 12 h at 37℃. After that, the isolated splenocytes were stimulated with peptide antigens (OVA323-339, OVA257-264, OVA208-216, OVA27-35, 10 μg/mL each, Apeptide, China) for 1 h at 37℃ in a CO2 incubator. After adding Brefeldin A (BioLegend, USA) for additional 11 h, the cells were washed with PBS and collected. Single-cell suspensions were then stained with Zombie Aqua™ Fixable Viability Kit and incubated with Fc block to block, followed by staining with APC anti-mouse CD3 antibody, Pacific Blue anti-mouse CD4 antibody, PE anti-mouse CD8a antibody and PE/Cy7 anti-mouse IFN-γ antibody prior to flow cytometry analysis. Analysis of Humoral Immune Responses Induced by mRNA Vaccines on Mice The antigen-specific antibody induced by mRNA vaccines was evaluated by Enzyme linked immunosorbent assay (ELISA). Approximately 200 μL of blood was collected 3 weeks after the second injection of mRNA vaccines for the measurement. After 2 h of incubation at 37℃, the collected blood was centrifuged at 1200 g for serum isolation (10 min at 4℃). Mouse ovalbumin specific IgG concentrations in serum were measured by ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd., China). Briefly, 96-well plates were coated with Ovalbumin. Serial dilutions of serum were added and enzyme-conjugate were added. Then covered with an adhesive strip and incubated for 60 min at 37℃. After the incubation, the plate was washed 5 times with wash buffer. Substrate solution was then added to each well and incubated for 15 min. The reaction was stopped with adding stop solution. Finally, the plate was read at 450 nm absorbance using microplate reader. In each group, samples were analyzed in triplicate. Then construct the standard curve and calculate concentrations according to the manufacturer’s instructions. Analysis of Toxicity of Vaccines by Histological Analysis Tumor-bearing mice were sacrificed when the tumor size exceeded 2000 mm3. Heart, liver, spleen, lung and kidney tissues were collected from tumor-bearing mice and healthy mice. These organs were embedded in paraffin and then the tissue sections were stained and analysis with H&E method. Statistical Analysis All data were evaluated and plotted by using GraphPad Prism 8.0. Student's t-test, two-way ANOVA and Log-rank were used to analyze significant differences in data. A P value < 0.05 was considered statistically significance. Results Characterization of Lipid Nanoparticles/Microparticles Lipid particles with different particle sizes were successfully prepared. The structure of lipid/mRNA particles was shown in (Fig. 1a). The size and zeta potential of lipid particles were measured by DLS and DLS analysis showed that the particle size of small lipid/mRNA nanoparticles, formulated by solvent diffusion method, was approximately 90.15 ± 2.92 nm (PDI = 0.21) and the zeta potential was approximately 8.03 ± 1.40 mV (Fig. 1b-d). The particle size of medium lipid/mRNA nanoparticles, formulated by microfluidics, was approximately 300 ± 40 nm (PDI = 0.25), and the zeta potential was approximately 12.30 ± 1.03 mV (Fig. 1b, e and f). The particle size of large lipid/mRNA microparticles, formulated by film dispersion method, was approximately 1150 ± 100 nm (PDI = 0.47), and the zeta potential was approximately 28.60 ± 2.20 mV (Fig. 1b, g and h). The results acquired from transmission electron microscope (TEM) showed similar as DLS analysis (Fig. 1i, j and k). In addition, the encapsulation efficiencies (EEs) of small, medium and larger size formulations were respectively 93.40% (A-LNP), 88.70% (B-LNP) and 87.07% (C-LMP). The stability studies on particle size showed that LMPs have similar stability with LNPs when stored at 4 ∘C or -20 ∘C for 4 weeks (Fig. 1l and m). A slightly increase in z-average diameter was observed when freezing the lipid particles, indicating that freeze–thaw cycles should be avoided.Fig. 1 Characterization of lipid/mRNA particles. (a). Schematic representation of a lipid particle consisting of lipids and mRNA. (b), The summary of particle size and PDI of lipid particles. (c) and (d), The size and zeta potential of small size lipid nanoparticles. (e) and (f), The size and zeta potential of medium size lipid nanoparticles. (g) and (h), The size and zeta potential of large size lipid microparticles. (i), The image of small size (around 90 nm) LNP captured by TEM. (j), The image of small size (around 90 nm) LNP captured by TEM. (k), The image of small size (around 90 nm) LNP captured by TEM. (l), Investigation of stability of lipid particles on particle size under 4℃. m, Investigation of stability of lipid particles on particle size under -20℃. The Impact of Particle Size on Cellular Uptake of Lipid Particles In Vitro In order to explore the cellular uptake properties of particles with different sizes, we utilized FITC-DSPE-PEG2000 instead of DMG-PEG2000 to prepare lipid particles. According to the analysis results, all 3 different lipid particles could be efficiently uptake by DC2.4 when co-incubation with DC2.4 cell lines (Fig. 2a and b). When we analyze the endocytosis of nanoparticles or microparticles by all antigen-presenting cells (APCs), including dendritic cells (DC), B cells and macrophages, we found that both small size lipid nanoparticles (90 nm LNP) and large size microparticles (1.2 μm LMP) showed significant stronger uptake signals than medium size lipid nanoparticles (300 nm LNP) (Fig. 2c). In the study, we used PerCP/Cyanine5.5 anti-mouse CD11c antibody to label DC. APC anti-mouse B220 antibody and APC/Cyanine7 anti-mouse F4/80 antibody were applied to mark B cells and macrophages, respectively. DC is the most important APC in initiating cellular immunity and humoral immunity. In terms of DC uptake, we discovered that large size microparticles showed significant stronger uptake than medium size nanoparticles and the other comparing didn’t show significant differences (Fig. 2d). In terms of B cell uptake, it was witnessed that both small size lipid nanoparticles and large size microparticles showed significant stronger uptake signals than medium size lipid nanoparticles (Fig. 2e). However, all three size particles didn’t show any significant difference in uptake by macrophages (Fig. 2f). These data indicated that small size lipid nanoparticles and large size microparticles can be uptake by total APCs stronger than medium size lipid nanoparticles.Fig. 2 Cellular uptake FITC-lipid complexes with different particle sizes. (a-b). The percentage of NP+ cells in DC2.4 live cells (n = 6). (c). The percentage of NP+ cells in splenocytes (n = 15). (d). The mean fluorescence intensity (MFI) of B220+ cells (B cells) in splenocytes. (e). The MFI of F4/80+ cells (macrophages) in splenocytes. (f). The MFI of CD11c+ cells (DC) in splenocytes. * means with significant difference and P < 0.05; ns means not significant. The Impact of Particle Size on Transfection Efficiency In Vitro To verify whether particle size can impact the efficacy of mRNA delivery, we tested the transfection efficacy of EGFP-mRNA after delivering by different size of lipid particles. The efficacy of mRNA delivery was examined by determining the quantity of transfected cells and the value of mean fluorescent intensity (MFI). As shown in Fig. 3a and b, all three sizes of lipid/mRNA particles could deliver EGFP-mRNA to cells and cause the transfection of cells. However, compared to medium size lipid nanoparticles (300 nm LNP), small size nanoparticles (90 nm LNP) and large size microparticles (1.2 μm LMP) demonstrated significantly higher transfection efficacy of EGFP-mRNA and induced transfections in more cells. These data illustrated that small size nanoparticles (90 nm LNP) and large size microparticles (1.2 μm LMP) perform better in vitro.Fig. 3 Investigation of transfection efficiency of mRNA particles into DC2.4 (n = 6) and investigation of transfection efficiency of mRNA particles into different antigen-presenting cells insplenocytes in vivo (n = 3). (a-b). Expression of eGFP mRNA in DC2.4 cells and the corresponding transfection efficiency at 24 h after co-incubation with different lipid particles. (c), In vivo expression of eGFP mRNA in B cells of splenocytes at 24 h after splenocytes co-incubating with different lipid particles. (d), In vivo expression of eGFP mRNA in DC of splenocytes at 24 h after splenocytes co-incubating with different lipid particles. (e), In vivo expression of eGFP mRNA in macrophages at 24 h after splenocytes co-incubating with different lipid particles. means significant difference and P < 0.05. Splenocytes were applied to analyze the transfection of different lipid particles, tanks to that splenocytes contain B cells, DC and macrophage. The results showed that the mRNA loaded lipid particles were efficiently uptake and expressed these 3 different antigen-presenting cells. As shown in Fig. 3c-e, there was no significant difference between small size (90 nm) and large size (1.2 μm) lipid particles in the expression of mRNA in B cells, macrophage and DC cells. However, both small size (90 nm) and large size (1.2 μm) lipid particles illustrated stronger expression of mRNA than that of medium size (300 nm) lipid particles in B cells, macrophage and DCs. The Impact of Particle Size on Lipid Particle Uptake In Vivo TO verify the results of endocytosis of lipid particles in vitro, we further conduct an in vivo experiment to study the impact of particle size on the uptake of particles APCs. The same as above, we used PerCP/Cyanine5.5 anti-mouse CD11c antibody to label DC and PE anti-mouse CD8a antibody to identify subgroup of DC related with induction of immune responses. APC anti-mouse B220 antibody and APC/Cyanine7 anti-mouse F4/80 antibody were applied to mark B cells and macrophages, respectively. The gating strategy in flow cytometric analysis is shown in Fig. 4a. Herein, besides CD11c+ DC population, CD11c + CD8 + DC population was also investigated to provide more comprehensively information, given that CD11c+CD8+ DCs is the population in DCs that play critical role in activating T cells and inducing the immune responses. The results revealed that no lipid particles produced a statistically different endocytosis efficiency with other size lipid particles in splenocytes in vivo (Fig. 4b-e). While in the investigation of lipid particle uptake by APC in lymph nodes, it was discovered that DC, CD8a+ DC and B cells, were more inclined to take up large lipid microparticles (1.2 μm LMPs) (Fig. 4f-i), though small size lipid nanoparticle (90 nm LNP) also showed significantly higher uptake than medium size lipid nanoparticles (300 nm LNP). According to previous studies have reported that larger particles stay longer at the injection site, we suspect that micron-sized lipid microparticles have a greater chance of being taken up by antigen-presenting cells at the host site [1, 36, 37, 43–46].Fig. 4 The analysis of in vivo delivery efficiency of lipid particles with different sizes (n = 7). (a). The gating strategy in flow cytometry studies. (b-e). The MFI (FITC) of DC, CD8a+ DC, B cells, and macrophage in splenocytes. (f-i). The MFI (FITC) of DC, CD8a+ DC, B cells, and macrophage in LNs. means significance and P < 0.05. Investigation of Biodistribution of Lipid Particles In vivo imaging results (Fig. 5) showed that, micron-sized lipid particles may reside longer in injection site and draining lymph nodes than nano-sized lipid particles. 6 h after injection of lipid particles with three particle sizes, fluorescence signals were observed at the injection site, and the fluorescence signals of the particles with small particle sizes tend to migrate to the draining lymph node. 24 h after injection, the fluorescence signal of draining lymph nodes could be observed obviously, which suggests that the complexes enter the draining lymph nodes from the injection site. 48 h after injection, the fluorescence signal in draining lymph nodes was enhanced in medium size LNP and large size LMP, but decreased in small size LNP. Meanwhile, fluorescent signals in various organs of mice revealed that draining lymph node has the highest concentration of all 3 types of lipid particles. Besides, the micron-sized lipid particles remain in the lymph nodes, while the small size lipid particles have some migration to liver and medium size lipid particles have some distribution to spleen. These data suggest that lipid microparticles migrate to lymph nodes more slowly than small lipid nanoparticles.Fig. 5 Investigation of biodistribution of lipid particles after subcutaneously injected at the right back. Imaging in vivo is conducted at 6 h、24 h and 48 h after the administration of lipid particles; the distribution of lipid particles in major organs is conducted at 24 h and 48 h after sacrificing the mice. The Impact of mRNA Vaccine Size on Preventing Cancer To further investigate the impact of particle size on vaccine potency, we immunized mice with different size mRNA vaccines and then challenged mice with E.G7-OVA cancer cell, a cell line that can induce T lymphoma. Since, the mRNA in lipid particles can encode the antigen ovalbumin (OVA) and E.G7-OVA cancer cell express the antigen OVA, immunize mice with mRNA vaccines would induce OVA-specific immune responses and thus retard the tumor growth. In the study, mice were vaccinated on day -28 and day -21 with OVA mRNA vaccines, and then on day 0 the mice were subcutaneously inoculated with OVA expressing E.G7 lymphoma cells (Fig. 6a). The tumor growth and survival rate of mice were monitored to evaluate the efficacy of different size mRNA vaccines. As tumor growth curves and survival curve shown in Fig. 6b and c, mice vaccinated with different sized lipid particles showed a significant delay of tumor growth and a prolongation of survival, compared with the mice in PBS control group and blank nanoparticle group. However, there was no significant difference, in terms of tumor growth and mouse survival, between mRNA vaccines with different particle size. Besides, it should be noted that immune adjuvants, either poly(I:C) or hesperetin, could improve the efficacy of mRNA vaccines (Fig. 6d and e), though the cationic lipids in mRNA formulation themselves have partial adjuvant function. Poly(I:C) and hesperetin are easy to be encapsulated into mRNA formulation due to they are also negatively charged, which is the same as mRNA.Fig. 6 Analysis of prevention efficacy of mRNA lipid particles with different sizes. (a),Time line of the mice immunization and tumor inoculation. (b-c), Tumor growth curve and survival rate of mice immunized with different size mRNA vaccines (n = 8). (d-e), Tumor growth curve and survival rate of mice immunized with mRNA vaccines encapsulated with different adjuvants (n = 8). (f-g), Measurement of IFN-γ secreting CD8+ and CD4+ T lymphocytes in splenocytes after stimulating by OVA peptides. and * mean compared with PBS control group; # means compared with medium blank nanoparticle loaded with only poly(I:C); $ means compared with medium blank nanoparticle loaded with hesperetin; & means compared with medium nanoparticle without adjuvants. , #, & and $, P < 0.05; * P < 0.01. The data demonstrated that there was no significant difference between mRNA vaccines encapsulated with poly(I:C) group and mRNA vaccines encapsulated with hesperetin. (Fig. 6d and e). Hesperetin is an active substance from traditional Chinese medicine and potentially has the ability to alter immune responses. The study presented here indicated that hesperetin potentially could be applied as immune adjuvants. Furthermore, the cellular immunity and antigen-specific T cells induced by mRNA vaccines were analyzed by detecting the IFN-γ secreting CD8+ or CD4+ T cells upon stimulating by incubating with OVA peptides antigens. IFN-γ secreting is the sign of T cell activation, therefore we can quantify the amounts of activated antigen-specific T cells by mixing antigens with immune cells. The results showed that the percentage of IFN-γ+CD8+ T cells in mice, immunized by all 3 sizes of mRNA vaccines, are higher than that in the PBS control group (Fig. 6f). Meanwhile, the percentage of IFN-γ+ CD4+ T cells in mice, immunized by large microparticles, was significantly higher than that in the PBS control group (Fig. 6g). These results indicated that all 3 size lipid particles can effectively activate antigen-specific cellular immunity. Investigation of Antigen-Specific Humoral Immunity Induced by mRNA Vaccines To evaluate the impact of particle size on inducing antigen-specific humoral immunity, the OVA-specific IgG concentrations in serum of mice were measured after immunizing mice with mRNA vaccines. In this study, all 3 kinds of mRNA vaccines induced robust humoral immune responses and statistically difference was observed between different vaccine groups (Fig. 7a). Meanwhile, comparing with PBS control group and medium blank nanoparticle control group, it was found that medium lipid nanoparticles without adjuvants can also induced stronger humoral immunity, and the addition of adjuvant hesperetin didn’t increase the humoral immune response (Fig. 7b). These data indicated that in terms of inducing humoral immunity, 90 nm lipid nanoparticle (LNP), 300 nm lipid nanoparticle (LNP) and 1.2 μm lipid microparticle (LMP) have no significance. In addition, the lipids included in the particles also worked as adjuvants in facilitating the activation of humoral immunity.Fig. 7 Analysis antigen-specific humoral immunity induced by lipid nanoparticles or microparticles and evaluation of toxicities of mRNA vaccines by H&E staining. (a-b), Analysis antigen-specific humoral immunity induced by lipid nanoparticles or microparticles by measuring the concentrations of OVA-specific IgG in serum of mice. (c), evaluation of toxicities of mRNA vaccines by H&E staining of major organs (Each figure has at least 3 repeats and the figures are representative one from these 3 repeats). and * mean compared with PBS control group; means P < 0.01; * means P < 0.005. Analysis of Toxicity to Major Organs During Immunization To assess the potential side effects of lipid nanoparticles or lipid microparticles, the major organs were analyzed by H&E staining after vaccine immunization. As shown in Fig. 7c, no obvious pathological changes or toxicities were observed in the collected tissue samples, including heart, liver, spleen, lung and kidney. Discussions and Conclusions Thank to mRNA vaccine platform presents various advantages, such as good safety, rapid manufacture, and co-induction of cellular and humoral responses etc., the studies on mRNA therapy are becoming hot spot and entering a very exciting stage [42, 47]. Once injected through subcutaneously or intramuscularly, mRNA vaccines are uptake by APCs and then transported to the draining lymph nodes (dLNs) by migratory APCs [48]. The crucial APCs for the induction of cellular immunity and humoral immunity are DCs, which are highly specialized to take up and process antigens [1, 45, 49–51]. The efficient uptake of antigen or its delivery platform by APCs depends on properties of particles, such as the size of particles [1, 51]. In reported studies and the FDA-approved mRNA vaccines, scientists applied lipid nanoparticles with the size around 70 nm-100 nm. No researches have compared the impact of particle size on the efficacy of lipid particles in a broader range. Therefore, we we compared the efficacy of lipid microparticles (LMPs) with LNPs in delivering mRNA. In the studies, there typical particle sizes were applied, 90 nm, 300 nm and 1.2 μm. Thus, both lipid nanoparticles (LNP) and lipid microparticles (LMPs) were included. 90 nm LNPs and 1.2 μm LMPs showed stronger cellular uptake and transfection than 300 nm LNPs in vitro. APCs can take up mRNA lipid particles at both the injection site and draining lymph nodes. Particle size determines the mechanism of trafficking to the lymph nodes. According to previous studies, nanoparticles traffic to the draining lymph nodes in a size-dependent manner, the smaller size (smaller than 200 nm) particles can infiltrate into lymph node more easily than larger particles. Whereas, lipid microparticles were uptake similarly by the DCs in lymph node with small size lipid nanoparticles (90 nm). We speculate that this is duo to large microparticles (500 nm—2000 nm) mostly uptake by DCs from the injection site and these DCs carried the lipid microparticles into the lymph node [37, 44, 46]. Literature suggests that lipid nanoparticles smaller than 200 nm freely drain to the lymph nodes [1, 43, 45, 46, 51, 52]. Previous studies showed that smaller lipid particles leave the injection site more readily than larger particles, however, smaller lipid complexes in the 30 nm size range drain rapidly from the site of s.c injection resulting in limited potency [53]. Lipid nanoparticles, with size 30 nm -200 nm, most are uptake by APCs in lymph node and with a part of nanoparticles uptake by APCs at injection site [1, 43]. Theoretically, large lipid microparticles can recruit circulating APCs, such as DCs, and uptake by such APCs, followed by being carried into the lymphatic system by APCs which can squeeze through openings between overlapping endothelial cells [1, 52, 54–58]. So far, limited knowledge exists for whether micron-sized lipid microparticles could be used to deliver mRNA and how it impacts vaccine potency. Given the above situations, our studies, comparing lipid microparticles with lipid nanoparticles in delivering mRNA, provided important information in mRNA delivering area for deeper investigations. According to our in vivo immunization study, all 3 sized mRNA vaccines could induce effective immune responses and showed a significant delay of tumor growth in preventing cancer on mouse model. Besides, significant difference in cellular immunogenicity and humoral immunogenicity was observed between these 3 different lipid nanoparticles (LNPs) and lipid microparticles (LMPs). According to previous studies and our studies on nanoparticles and microparticles, we speculate that these results were caused by the combination of different residence time of particles stay in injecting site and ability of particles infiltrating into lymph node [1, 37, 43–46]. In summary, we prepared 3 different size lipid/mRNA particles without changing their lipid composition. It was witnessed that lipid/mRNA particles encapsulating with immune adjuvants (either poly(I:C) or hesperetin) showed a more significant stronger immune responses than lipid particles without adjuvants. Notably, compared to the most commonly used 50–100 nm lipid nanoparticles (LNP), 1–2 μm sized lipid/mRNA microparticles (LMP) showed similar robust immune response and excellent intracellular delivery. 50–100 nm lipid nanoparticles (LNP) have been approved to be applied in clinic to deliver mRNA vaccines. Therefore, this work proved that lipid microparticles can achieve similar mRNA delivery efficacy with small size lipid nanoparticles, and thus provided us an alternative micron-sized platform for mRNA delivery, besides lipid nanoparticles with size smaller than 150 nm. Fundings This research was funded by Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the Health Talent Training Project of Suzhou (GSWS2019069, GSWS2020094) and Suzhou Science and Technology Development Project for People's Livelihood (SS202011). Author Contributions Conceptualization, M.L.; methodology, M.L, A.J, M.X, Y.P, and L.D.; software, A.J. M.X and L.M; validation, M.X., L.M. and L.M.; formal analysis, M.X, A.J.; investigation, A.J, M.X. and Y.P; resources, M.L and J.C.; data curation, A.J., M.X, and M.X; writing—original draft preparation, A.J.; writing—review and editing, M.L, M.X, and L.Q.; supervision, M.L. and J.C; project administration, M.L.; funding acquisition, M.L and J.C. All authors have read and agreed to the published version of the manuscript. Data Availability The data supporting the findings of this study are included in the paper. All other relevant data are available from the corresponding author upon reasonable request. Declarations Animal Study Statement All animal work was approved and monitored by the Animal Ethics Committee of Soochow University. Conflicts of Interest The authors declare no conflict of interests. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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==== Front J Neurooncol J Neurooncol Journal of Neuro-Oncology 0167-594X 1573-7373 Springer US New York 4185 10.1007/s11060-022-04185-3 Research Neurocognitive functioning after Gamma Knife and LINAC stereotactic radiosurgery in patients with brain metastases http://orcid.org/0000-0002-4083-016X Albers Elaine A. C. [email protected] 1 de Ruiter Michiel B. 1 http://orcid.org/0000-0003-0413-6872 van de Poll-Franse Lonneke V. 123 Merckel Laura G. 5 http://orcid.org/0000-0001-9732-2150 Compter Annette 4 http://orcid.org/0000-0003-0153-8059 Schagen Sanne B. 16 1 grid.430814.a 0000 0001 0674 1393 Department of Psychosocial Research and Epidemiology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands 2 Department of Research and Development, Netherlands Comprehensive Cancer Organization, Utrecht, The Netherlands 3 grid.12295.3d 0000 0001 0943 3265 Department of Medical and Clinical Psychology, Centre of Research on Psychological and Somatic Disorders (CoRPS), Tilburg University, Tilburg, The Netherlands 4 grid.430814.a 0000 0001 0674 1393 Department of Neuro-Oncology, Antoni van Leeuwenhoek, Amsterdam, The Netherlands 5 grid.430814.a 0000 0001 0674 1393 Department of Radiotherapy, Antoni van Leeuwenhoek, Amsterdam, The Netherlands 6 grid.7177.6 0000000084992262 Department of Psychology, University of Amsterdam, Amsterdam, The Netherlands 1 12 2022 110 15 9 2022 26 10 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose Brain metastases (BM) themselves and treatment with stereotactic radiosurgery (SRS) can influence neurocognitive functioning. This prospective study aimed to assess neurocognitive decline in patients with BM after SRS. Methods A neuropsychological test battery was assessed yielding ten test outcomes. Neurocognitive decline at 3 and 6 months post SRS was compared to measurement prior to Gamma Knife (GK) or linear accelerator (LINAC) SRS. Reliable change indices with correction for practice effects were calculated to determine the percentage of neurocognitive decline (defined as decline on ≥ 2 test outcomes). Risk factors of neurocognitive decline were analyzed with binary logistic regression. Results Of 194 patients pre-SRS, 40 GK and 29 LINAC patients had data accessible at 6 months. Compared to baseline, 38% of GK patients declined at 3 months, and 23% declined at 6 months. GK patients declined on attention, executive functioning, verbal memory, and fine motor skill. Of LINAC patients, 10% declined at 3 months, and 24% at 6 months. LINAC patients declined on executive functioning, verbal memory, and fine motor skills. Risk factors of neurocognitive decline at 3 months were high age, low education level and type of SRS (GK or LINAC). At 6 months, high age was a risk factor. Karnofsky Performance Scale, BM volume, number of BM, tumor progression and neurocognitive impairment pre-SRS were no risk factors. Conclusion Neurocognitive decline occurs in a considerable proportion of patients with BM treated with GK or LINAC SRS. Overall, high age appears to be a risk factor for neurocognitive decline after SRS. Supplementary Information The online version contains supplementary material available at 10.1007/s11060-022-04185-3. Keywords Brain metastases Neurocognitive functioning Stereotactic radiosurgery Gamma Knife radiosurgery LINAC radiosurgery The Netherlands Cancer Institute ==== Body pmcIntroduction Incidence of patients with brain metastases (BM) is increasing due to an aging population, advanced imaging techniques to detect smaller metastases, and better systemic treatments resulting in improved survival and higher risk of developing BM [1, 2]. Due to increasing life expectancy, maintaining good neurocognitive functioning (NCF) as long as possible is of growing importance. BM can have unfavorable effects on NCF by affecting healthy brain tissue and brain connectivity [3]. Historically, Whole Brain Radiation Therapy (WBRT) has been the main treatment for BM, targeting the entire brain with a low radiation dose [4]. Nowadays, stereotactic radiosurgery (SRS) offers delivery of a precisely localized, high dose of radiation, while sparing healthy tissue in rest of the brain. SRS has better local tumor control and therefore a beneficial effect on NCF, compared to WBRT [5–12]. Nevertheless, SRS might still damage healthy brain tissue in the vicinity of the BM [8, 13]. Both the linear accelerator (LINAC) and the Gamma knife (GK) are used for SRS. For LINAC, 1–2 mm margins are used for planned target volume (PTV) [14]. GK is a dedicated system designed for intracranial radiosurgery and no margins are needed. Furthermore, dose fall-off of GK is steeper than of LINAC and therefore radiation dose to healthy brain tissue is lower. On average, the GK spares normal brain volume receiving ≥ 12 Gray (V12 Gy) by approximately 20% compared to LINAC [15, 16]. This leads in theory to fewer negative effects on NCF, although there are no studies yet that compared this directly. Studies evaluating changes in NCF at the individual patient level concluded that, in LINAC patients with 1–4 BM, NCF was maintained compared to their pre-treatment level up to 6 months after SRS [17]. Up to 9 months, NCF was maintained or improved compared to pre-treatment levels among GK patients with 1–10 BM [17, 18]. Earlier studies used limited neuropsychological tests [19] and a relatively insensitive method to measure neurocognitive change, without taking practice effects into account [17]. Moreover, in a study by Schimmel et al. (2021), patients could ‘compensate’ for neurocognitive decline on one test by cognitively improving on another test, which potentially masks neurocognitive decline [18]. Therefore, this prospective study aimed to assess neurocognitive decline in patients with BM up to 6 months after GK or LINAC SRS, by using a recommended neuropsychological test battery and a sensitive approach to assess neurocognitive change at the individual level that adjusted for practice effects. Furthermore, predictors of neurocognitive decline were investigated. We hypothesized that patients would show neurocognitive decline after SRS, in particular after LINAC. Methods Study population At the Netherlands Cancer Institute (NKI), neuropsychological assessment (NPA) in BM patients is conducted as part of routine clinical care. BM patients who received GK or LINAC SRS, who had baseline (pre-SRS) NPA between September 2016 and March 2020, and who completed follow-up NPA at 3 and 6 months were included. Patients were excluded when: (i) they did not give consent to use data for scientific research, (ii) received resection or (iii) received additional radiotherapy (including ‘staged’ GK). The local Institutional Review Board approved the study protocol (approved on 10-03-2020, IRBd20-089). Procedures Pre-treatment NPA was scheduled in the week preceding SRS. Follow-up NPAs were scheduled at the same day as the diagnostic MRI scan, approximately 3 and 6 months after SRS. At each time point, NPA was conducted by a trained test leader. Treatment Up till 2018, all patients at the NKI were treated with a LINAC Synergy Agility™ (Elekta A.B., Stockholm, Sweden). Patients underwent a computed tomography (CT) scan in a supine position using a mask for fixation [20]. The CT scan was registered with a 1-mm-thick contrast-enhanced T1-weighted MRI-scan, which was used for delineation of gross tumor volume (GTV) of BM. PTV was created by expanding GTV with 2 mm [14]. Dose prescribed to PTV was 18–24 Gy in a single fraction, 21–27 Gy in three fractions, or 25–30 Gy in five fractions. A treatment plan was created to such that a minimum of 95% of PTV received 100% of the prescribed dose [20]. Since February 2018, patients were treated with the Leksell Gamma Knife® Icon™ (Elekta A.B., Stockholm, Sweden). Immobilization was performed using a mask or rigid headframe. Patients received a dose of 18–25 Gy, with 99–100% coverage of the target and no setup margin was used [20]. Measures NCF was assessed with a test battery consisting of six neurocognitive tests, yielding ten outcomes. Three tests were based on recommendations of the International Cognition and Cancer Task Force (ICCTF) [21]: (1) Hopkins Verbal Learning Test-Revised (HVLT-R) immediate recall, delayed recall and recognition [22], (2) Trail Making Test (TMT) part A and B [23] and (3) Letter Fluency (LF) [24]. Additionally, three tests measuring important daily cognitive functions are included: (1) Grooved Pegboard (GP) (non)-dominant hand [25], (2) Digit Span (DS) [26] and (3) Boston Naming Test (BNT) [27] (Suppl. Table 1). Sociodemographic and clinical characteristics were retrieved from patients’ medical health records. Tumor volume of GK patients was derived from Leksell GammaPlan® for each BM separately, from which total tumor volume was calculated. GTV of LINAC patients was derived from Pinnacle SmartEnterprise version 9.10 (Philips Healthcare, Andover, MA, USA) treatment planning system. Tumor progression was defined as any new, contrast-enhancing lesion on follow-up MRI. Statistical analysis Analyses were done for GK and LINAC patients separately. Independent samples t-tests and Pearson’s chi-square tests were carried out to compare characteristics of both groups. Pre-SRS NCF scores were calculated by converting raw individual scores into age and if suitable education and gender corrected standardized Z-scores, using published normative data or using the Dutch database ‘Advanced Neuropsychological Diagnostics Infrastructure (ANDI)—norms’ [28] (Suppl. Table 1). Lower Z-scores mean worse NCF functioning. Patients were classified as impaired by below-average scores of Z < − 1.5 [29] in ≥ 2 out of 10 test outcomes. To assess change in NCF, differences in scores were calculated from pre-SRS to 3 months and pre-SRS to 6 months. We used the Iverson reliable Change Index (RCI), which takes into account test–retest reliability, Standard Deviation (SD) on the first and second measurement and practice effects. Performance on a test was classified as “changed” when it fell outside the 90% confidence interval: improved performance when RCI values were above + 1.645, decline in performance when RCI values were below − 1.645, stable performance when RCI scores did not exceed these values. We derived the total number of tests on which patients declined. Binary logistic regression was used to identify risk factors for neurocognitive decline. Dependent variable consisted of neurocognitive decline, defined with the commonly used threshold of decline in RCI scores on ≥ 2 out of 10 test outcomes [21, 30]. Considered predictive factors were; age, education level, type of SRS (GK or LINAC), pre-SRS Karnofsky Performance Score (KPS)-score, BM volume, number of BM, tumor progression and neurocognitive impairment pre-SRS [31–33]. Contribution of associated factors was taken into account when logistic regression had a P value < 0.05 and was significant at the level of 0.01. Over time, technical improvements made it easier to radiate more BM, which has led to broadened indications for GK [34]. Patients with an increasing number of BM are now treated with the GK and therefore a significant difference in number of irradiated BM between the GK and LINAC group is expected. GK and LINAC patients with ≥ 6 BM were excluded in additional analyses to match patient characteristics for both radiotherapy techniques. All analyses were conducted using IBM SPSS Statistics, version 25.0. Results Patient characteristics Pre-SRS, 194 patients were included (Fig. 1), of which 98 (50%) completed NPA at 3 months and 69 (36%) at 6 months. Reasons for dropout were; being deceased, finding the NPA too burdensome, or a pause of NPA assessment due to SARS-CoV-2. Median overall survival (OS) of 121 GK patients who completed NPA pre-SRS was 11.0 months (SD = 8.0), with a 1-year survival rate of 37.3%. For 73 LINAC patients, median OS was 15.5 (SD = 15.1), with a 1-year survival rate of 38.8%.Fig. 1 Flow-chart of excluded participants. Pre-SRS before treatment with Gamma Knife or Linear accelerator Stereotactic Radiosurgery; FU follow-up. Note A pause of neuropsychological assessment due to SARS-CoV-2 took place between March 2020 and June 2021. Consequently, patients were not able to do a follow-up neuropsychological assessment in this period Characteristics are summarized in Table 1. We present results of 69 patients who completed NPA at 3 and 6 months. Forty patients were treated with GK and 29 patients with LINAC (mean age 59.1 ± 10.8 years, and 57.7 ± 9.6 years respectively). The GK group had a mean number of 4.8 BM, with a mean GTV volume of 4.1 cm3. Four patients (10%) developed new distant BM at 3 months and 3 (7.5%) at 6 months. Median OS was 16 months (SD = 5.7) and 1-year survival rate was 84%.Table 1 Pre-SRS characteristics separately for patients treated with GK and LINAC No. of GK patients N = 40 No. of LINAC patients N = 29 GK versus LINAC patients, P value Age in years, mean ± SD 59.1 (10.8) 57.7 (9.6) 0.650 Gender 0.404 Male 18 (45) 16 (55) Educational levelA 0.445 Low 8 (20) 3 (10) Middle 12 (30) 12 (41) High 20 (50) 14 (48) Karnofsky Performance Scale 1 (2.5) 1 (3.4) 0.817 < 70 39 (98) 28 (97) ≥ 70 – – Primary tumor 0.057 Melanoma 18 (45) 14 (48) Lung 19 (48) 5 (18) Breast 2 (5.0) 4 (14) Kidney 1 (2.5) 2 (6.9) Other – 4 (14) Target locations SRS Frontal 22 (55) 12 (41) 0.324 Parietal 18 (45) 10 (35) 0.444 Occipital 13 (33) 3 (10) 0.037 Temporal 11 (28) 10 (35) 0.534 Cerebellar 16 (40) 3 (10) 0.008 Subcortical 7 (18) 2 (6.9) 0.215 Other 4 (10) 2 (6.9) 0.683 BM tumor load Total volume (cm3), mean ± SDB 4.1 (4.8) 6.2 (10.4) 0.013 Number, mean ± SD 4.8 (5.3) 1.8 (1.4) 0.001 1 12 (30) 20 (69) 2 6 (15) 2 (6.9) 3 6 (15) 5 (17) 4 1 (2.5) – > 4 15 (38) 2 (6.9) BM symptoms at diagnosis 0.426 SymptomaticC 9 (23) 9 (31) Timing of BM diagnosisD 0.029** Synchronous 6 (15) – Metachronous 34 (85) 29 (100) ChemotherapyE 0.079 Yes 4 (10) – Hormone therapyE 0.237 Yes – 1 (3.4) ImmunotherapyE 0.693 Yes 12 (30) 10 (35) Targeted therapyE 0.809 Yes 10 (25) 8 (28) SD standard deviation; LINAC linear accelerator; SRS stereotactic radiosurgery; BM brain metastases; GK Gamma Knife A P value of < 0.05 was considered statistically significant Indicate a statistically significant difference in the proportion of patients treated with GK and LINAC AThe 7 categories to classify the level of education of the Verhage scale were merged into low (Verhage 1–4), middle (Verhage 5), and high (Verhage 6 and 7) educational level BGTV volume CPatients with epilepsy are categorized as symptomatic DSynchronous are brain metastases found < 30 days after diagnosis of primary tumor. Metachronous are brain metastases found ≥ 30 days after diagnosis of primary tumor EAlone or in combination with other systemic therapies. Yes = currently or in the last 3 months received systemic therapy The LINAC group had a mean number of 1.8 BM, with a mean GTV volume of 6.2 cm3. Three patients (10%) developed new distant BM at 3 months and 4 (14%) at 6 months. Median OS was 17 months (SD = 16.1) and 1-year survival rate was 97%. Characteristics of patients who completed NPA at 3 months versus patients who completed NPA at 3 and 6 months are shown in Suppl. Table 2. There were statistically significant differences in primary tumor, BM symptoms at diagnosis, timing of BM diagnosis and systemic therapy between responders and non-responders at 3 months. No differences were reported at 6 months. Neurocognitive functioning pre-SRS Twenty-three percent of GK patients and 17% of LINAC patients showed neurocognitive impairment pre-SRS according to our pre-defined criteria (against an expected 14% in a healthy population [30]). NCF pre-SRS mean z-scores are shown in Table 2 and additional data from complete case analyses at 3 months are presented in Suppl. Table 3. The GK group showed lowest mean scores and most frequent impairments for verbal memory, fine motor skills and language. The LINAC group showed lowest mean scores and most frequent impairments for executive functioning, verbal memory, fine motor skills and language.Table 2 Neurocognitive functioning pre-SRS in patients who completed NPA at 6 months (n = 69) Mean z-score (SD) No. of patients with impaired NCF GK (n = 39–40) LINAC (n = 27–29) GK (n = 39–40) LINAC (n = 27–29) Attention Trail Making Test-A 0.37 − 0.10 – 2 (6.9%) Digit span 0.19 0.59 1 (2.5%) – Executive functioning Trail Making Test-B 0.10 − 0.27 2 (5.0%) 4 (14%) Letter fluency − 0.33 − 0.61 6 (15%) 4 (14%) Verbal memory HVLT-immediate recall − 0.51 − 0.62 8 (20%) 7 (24%) HVLT-delayed recall − 0.70 − 0.70 10 (25%) 8 (28%) HVLT-recognition − 0.42 − 0.84 7 (18%) 4 (14%) Fine motor skills Grooved pegboard dominant − 0.73 − 1.03 13 (33%) 10 (35%) Grooved pegboard non-dominant − 1.18 − 0.69 11 (28%) 7 (24%) Language Boston naming test − 0.44 − 0.44 6 (15%) 4 (14%) SD standard deviation; NCF neurocognitive functioning; GK Gamma Knife; LINAC linear accelerator *Neurocognitive impairment was defined as a z-score ≤ -1.5 Change in neurocognitive functioning on test level GK patients reported highest percentages of decline between pre-SRS and 3 months or pre-SRS and 6 months on tests of attention, executive functioning, verbal memory, and fine motor skills (Fig. 2(2.1)). Percentage of patients with declined NCF decreased between 3 and 6 months on all tests.Fig. 2 Individual neurocognitive changes at test level over 6 months after SRS for patients treated with 2.1 Gamma-Knife (pre-SRS—T6: n = 39–40) and 2.2 LINAC (pre-SRS—T6: n = 27–29). Numbers are expressed in percentages. T3 3 months; T6 6 months; TMT-A Trail Making Test A; DS digit span; TMT-B Trail Making Test B; LF letter fluency; HVLT-IR Hopkins Verbal Learning Test-Immediate Recall; HVLT-DR Hopkins Verbal Learning Test-Delayed Recall; HVLT-Recog Hopkins Verbal Learning Test-Recognition; GP-D Grooved Pegboard-Dominant; GP-ND Grooved Pegboard Non-dominant; BNT Boston Naming Test LINAC patients reported highest percentages of decline largely in the same domains: executive functioning, verbal memory, and fine motor skills (Fig. 2(2.2)). In general, percentage of patients with declined NCF increased between 3 and 6 months. After excluding GK and LINAC patients with ≥ 6 BM, we observed in the GK group that percentage of decline per test dropped for both pre-SRS vs. 3 months and for pre-SRS vs. 6 months comparisons. This drop was most pronounced for tests of fine motor skills. In the LINAC group, results for pre-SRS vs. 3 months and pre-SRS vs. 6 months comparisons remained essentially the same, as only one patient with 6 or more BM was omitted from the analysis (Suppl. Fig. 1). Additional results of percentage of decline from complete case analyses at 3 months are shown in Suppl. Fig. 2. Cumulative change in neurocognitive functioning on patient level In the GK group, 38% declined on ≥ 2 out of 10 tests at 3 months (Fig. 3(3.1)) and 23% declined at 6 months. In the LINAC group, 10% declined on ≥ 2 out of 10 tests at 3 months and 24% at 6 months (Fig. 3(3.2)). For descriptive purposes, improvement of NCF is shown in Suppl. Fig. 3. After excluding GK and LINAC patients with ≥ 6 BM, percentage of decline dropped for both pre-SRS vs. 3 months and pre-SRS vs. 6 months comparisons in the GK group (Suppl. Fig. 4). In the LINAC group, results for pre-SRS vs. 3 months and pre-SRS vs. 6 months comparisons remained essentially the same, as only one patient was omitted from the analysis (Suppl. Fig. 4). Additional results of percentage of decline from complete case analyses at 3 months are shown in Suppl. Fig. 5.Fig. 3 Individual cumulative neurocognitive decline at patient level over 6 months after SRS, for 40 patients treated with Gamma Knife (3.1) and 29 patients treated with the LINAC (3.2) Prognostic factors of NCF decline At 3 months, overall model had an R2 of 0.50, and was considered significant (P < 0.001). High age [Odds Ratio (OR) 1.07; 95% Confidence Interval (95%CI) 1.00–1.13; P = 0.059], low education level (OR 25.03; 95%CI 3.80–164.79; P = 0.001) and type of SRS (OR 0.17; 95%CI 0.03–0.85; P = 0.031) significantly predicted neurocognitive decline (see Table 3). Pre-SRS KPS score, BM volume, number of BM, tumor progression and neurocognitive impairment pre-SRS were no significant predictors.Table 3 Binary logistic regression analyses of predictive factors for neurocognitive decline at 3 and 6 months post-SRS NCF decline at 3 months NCF decline at 6 months Univariate regression Multivariable regression Univariate regression Multivariable regression Model summary Adjusted R2 0.50 0.32 P value < 0.001 0.004 N 66 66 66 66 Age OR 1.061 1.065 1.107 1.088 P value 0.037 0.054 0.003 0.009 95% CI 1.003–1.122 0.999–1.136 1.036–1.184 1.021–1.176 Education level—low OR 18.000 25.031 2.222 P value < 0.001 0.001 0.286 95% CI 3.742–86.594 3.798–164.778 0.512–9.647 Education level—middle OR 2.667 2.059 P value 0.167 0.255 95% CI 0.664–10.714 0.594–7.130 Type of SRS OR 4.952 0.170 0.924 P value 0.020 0.031 0.889 95% CI 1.292–18.983 0.029–0.852 0.306–2.790 Pre-SRS Karnofsky Performance Scale OR 2.889 3.375 P value 0.461 0.399 95% CI 0.172–48.620 0.200–57.042 Total volume BM OR 0.959 1.046 P value 0.394 0.191 95% CI 0.870–1.057 0.978–1.120 Number of BM OR 1.218 1.013 P value 0.013 0.840 95% CI 1.043–1.422 0.895–1.146 Tumor progression OR 0.706 0.604 P value 0.781 0.688 95% CI 0.060–8.261 0.051–7.100 Pre-SRS neurocognitive impairment OR 0.824 0.620 P value 0.718 0.390 95% CI 0.289–2.355 0.208–1.845 Boldface indicates significance at P<0.05 NCF neurocognitive functioning; N number; OR odds ratio; 95% CI 95% confidence Interval At 6 months, overall model had an R2 of 0.32, and was considered significant (P = 0.004). High age (OR 1.09; 95% CI 1.02–1.17; P = 0.009) was a significant predictor for neurocognitive decline. No other predictors were found. Discussion This prospective study aimed to assess neurocognitive decline in patients with BM up to 6 months after GK or LINAC SRS, using a recommended battery of neuropsychological tests and a sensitive approach to assess neurocognitive decline at the individual level that adjusts for practice effects. Both patients treated with GK and LINAC reported decline in NCF. Most common affected functions prior to SRS included executive function, memory, fine motor skills and language. Neurocognitive decline over 3 and 6 months post SRS was observed in the same cognitive functions, and in attention. Higher age was a strong predictor for neurocognitive decline. Furthermore, low education level and type of SRS (GK or LINAC) were predictors of neurocognitive decline at 3 months. We found that 38% of the GK group declined in NCF at 3 months and 23% at 6 months. In the LINAC group, 10% declined at 3 months and 24% at 6 months. Characteristics of the GK and LINAC group significantly differ on number of BM; 4.8 in the GK group and 1.8 in the LINAC group. This could partly be explained by broader indications for GK, allowing treatment of patients with an increasing number of BM. Since February 2018, GK was introduced at the NKI. We were not able to correct for change in treatment indications for SRS, which likely influenced the results presented in this study. After excluding GK and LINAC patients with ≥ 6 BM, differences in decline of NCF between the GK and LINAC group remained. Maybe a detrimental impact on NCF is partially transient for the GK group, and not for the LINAC group. This can potentially be caused by two factors: (1) Higher number of BM in the GK group can have a more diffuse negative impact on brain functioning and lead to a reduced cognitive reserve. When this reserve is implicated (e.g., with the presence of edema), it may have a greater influence on NCF of GK patients than it would have on LINAC patients with fewer BM. We showed that, in general, there is a comparable percentage of patients who report impaired NCF pre-SRS in the GK and LINAC group. A diminished cognitive reserve can become more apparent over time, causing the diffuse impact on the brain to have a stronger influence on NCF over time. This could be an explanation why GK patients have more impaired NCF at three months compared with LINAC patients. (2) Systemic therapy can influence NCF. In particular, 10% of GK patients received chemotherapy, compared to 0% of LINAC patients. Previous research reported that chemotherapy negatively influences NCF [35]. Overall, there is no straightforward explanation for these observations. Our results are not in line with two recent studies that concluded no decline in patients after SRS. We believe, however, that these studies may have underestimated the rate of cognitive decline due to methodological choices. In the first study by van der Meer et al., 55 LINAC patients were followed up to 6 months after treatment [17]. They concluded that half of the patients showed impaired NCF prior to SRS, mainly in the domain of verbal memory. Patients maintained their pre-SRS NCF level. Changes in neurocognitive functioning were evaluated per domain, thus averaging across test outcomes within a domain. In doing so, impaired test scores can be masked by unimpaired test scores. Furthermore, a relatively insensitive method was used to measure neurocognitive change by analyzing NCF without taking into account test–retest (including practice) effects. In a second study by Schimmel et al. [18], 92 patients were followed up to 9 months after GK. They concluded that 15–55% of patients showed impaired NCF pre-SRS, mainly in domains of attention, executive functioning, and fine motor skills. Patients maintained or improved their pre-SRS NCF level. NCF was corrected for test–retest effects, but an interpretation of neurocognitive decline was used that could result in an underestimation of decline for two reasons: (1) The possibility to lift classification of decline on tests by improved test performance on other tests, and (2) Statistical testing for differences between proportions of patients and controls classified as declined, as conducted by the authors, could be viewed as an overcorrection: cognitive changes in patients were already defined based on reliable change intervals in the control sample. We aimed to overcome underestimation of neurocognitive decline in this study by using a sensitive and commonly used method that corrects for practice effects, whereby we did not additionally compare test outcomes with a control sample in order to prevent overcorrection. Also, our criterion for neurocognitive decline without the possibility to compensate with improved tests is in line with recommended criteria stated by for example the ICCTF [21]. Furthermore, neuropsychological tests are administered as part of usual care and therefore providing a representative sample of patients with BM. Nevertheless, this study has limitations to consider. We excluded patients who did not complete ≥ 1 follow-up NPA post-SRS (n = 96). These patients are expected to have worse NCF pre-SRS. Despite the fact that we have shown that neurocognitive impairment pre-SRS is not a predictor for neurocognitive decline, potentially low scores on tests in this excluded group can be predictive for neurocognitive decline. Furthermore, we excluded patients who received volume-staged GK. Consequently, GK patients with large BM volumes are underrepresented in this study, while they presumably will suffer from decline in NCF. Further research should investigate whether differences of GK and LINAC SRS on NCF as suggested in this study are results of different effects of both types of treatments or results of selection bias, due to e.g., exclusion of staged GK patients. This should be investigated among larger groups of patients and longer time spans (> 6 months post-SRS). Furthermore, matching on BM volume can give more insight in differences in patient characteristics between GK and LINAC SRS [32]. In conclusion, patients with BM show decline in NCF over time when they are treated with GK or LINAC SRS. This suggests that SRS has an influence on NCF of the individual patient. If results of this study are confirmed in future studies, it is recommended to routinely assess neurocognitive functioning in patients with BM when they are treated with GK or LINAC SRS in order to determine risk–benefit aspects of SRS. Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 434 kb) Acknowledgements Not applicable. Author contributions MR and SS contributed to the study conception and design, material preparation and data collection. Analyses were performed by EA and MR. Clinical characteristic information was retrieved by EA, AC, and LM. The first draft of the manuscript was written by EA and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Funding The study was funded by the Netherlands Cancer Institute. Data availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Ethical approval This study was approved by the local Institutional Review Board of the Dutch Cancer Institute. Consent to participate Informed consent was obtained from all individual participants included in this study. Consent to publish The authors affirm that all human research participants provided informed consent for publication of their data. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. 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==== Front Pharm Res Pharm Res Pharmaceutical Research 0724-8741 1573-904X Springer US New York 36451069 3447 10.1007/s11095-022-03447-z Perspectives What Do We Know About the Smallpox Virus? A Journey Between Clinic and Therapy https://orcid.org/0000-0002-6528-2171 Zovi Andrea [email protected] 1 http://orcid.org/0000-0001-9298-6783 Ferrara Francesco [email protected] [email protected] 2 Sorrentino Sarah [email protected] 2 Langella Roberto [email protected] 3 Trama Ugo [email protected] 4 Boccellino Mariarosaria [email protected] 5 https://orcid.org/0000-0003-2623-166X Vitiello Antonio [email protected] 6 1 grid.415788.7 0000 0004 1756 9674 Ministry of Health, Viale Giorgio Ribotta 5, 00144 Rome, Italy 2 Pharmaceutical Department, Asl Napoli 3 Sud, Dell’amicizia street 22, 80035, Nola, Naples, Italy 3 Italian Society of Hospital Pharmacy (SIFO), SIFO Secretariat of the Lombardy Region, Via Carlo Farini, 81, 20159 Milan, Italy 4 Health Protection and Coordination of the Campania Regional Health System, Naples, Italy 5 grid.9841.4 0000 0001 2200 8888 Department of Biochemistry, Biophysics and General Pathology, University of Campania “Luigi Vanvitelli”, Naples, Italy 6 grid.415788.7 0000 0004 1756 9674 Clinical pharmacologist, Ministry of Health, Viale Giorgio Ribotta 5, 00144 Rome, Italy 30 11 2022 17 17 10 2022 22 11 2022 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose Modern research is increasingly focusing on the study of new viruses and the re-emergence of past microbes, such as Coronaviruses, particularly Sars-Cov2 that was responsible for the very recent pandemic. Methods and Results This infection manifested itself and still continues to manifest as a severe respiratory syndrome. The main discriminator of whether or not one succeeds in overcoming this infection may depend on a great many factors, but the main one is definitely determined by vaccination, which has minimized hospitalizations and more severe syndromes. Conclusion Recently, a new virus, the monkeypox virus, which was previously confined to Central and West Africa but is now gradually spreading to more than 30 countries including the United States of America, where such an infection is not endemic, is coming forward again. Keywords epidemiology infectious monkeypox smallpox vaccine virology ==== Body pmcThe Smallpox Smallpox viruses are a very large group of DNA viruses that cause infections in both humans and animals. Smallpox originated with the infection of a subspecies of vertebrates namely cows, and their immunity was responsible for the discovery of a vaccine against smallpox in humans, thanks to Edward Jenner, called the "father of immunization" [1]. Smallpox and monkeypox are closely related, and even the symptoms, fever and skin rushes, are strikingly similar. Monkeypox was discovered in 1970 in the Democratic Republic of Congo, but its epidemiology remains "ad horas" unknown. Viruses that usually infect animals and that are thus confined to their own world, very often can evolve and adapt to the environment in a way that allows a "species jump" and thus leading to infection and transmission among humans. This is what happened with Sars-Cov-2 and is what is progressively happening with Monkeypox [2]. This problem is mainly attributable to the promiscuity that very often occurs between the human and animal worlds in underdeveloped countries and, in fact, before the declaration of some cases of monkeypox in non-African countries, such as the United States of America, this infection was endemic only in: Cameroon, Central African Republic, Democratic Republic of Congo, and Nigeria [3]. Smallpox immunization with the vaccine was also effective against monkeypox. However, it is strongly discouraged to carry out such vaccination after 1980 since this infection shares several clinical and pathological factors with other microbial infections, such as Varicella Zooster Virus, cowpox, and many others [4]. The process of total eradication of smallpox worldwide was initiated in 1958 by the World Health Assembly received a report on the catastrophic consequences of smallpox in 63 countries leading then in 1967, under the tutelage of the World Health Organization to a global campaign that led to the eradication of smallpox finally in 1977. On May 8, 1980, the World Health Assembly announced to the entire momdo that smallpox had been eradicated [5, 6]. Monkeypox The current re-emergence of monkeypox cases may be due both to a decline in vaccination and to a gene development of the virus itself that would then be able to implant itself more easily into the human genome. This should be considered, therefore, an important cue both to continue gene sequencing of the virus by keeping it "under control" and to highlight host preferences and possible hot reservoirs that continue to develop [7]. Monkeypox has a lower incidence of severe forms than COVID-19, although there are many concerns about the geographical spread and further resurgence of the infection [8, 9]. Between 2010 and 2019, a few cases were reported in Nigeria after 40 years of silencing infections. Since 2003, however, cases of monkeypox have also been reported in countries where the disease was not endemic. In particular, there are reports that transmission would have occurred as a result of the importation of rodents from Ghana into the United States. Studies show that the probability of animal-to-human transmission is high [10]. The first cases were probably detected in one of the following countries, Nigeria, the United States, Singapore. [11–13], however to date the first epidemic outbreak is still unclear where it occurred. In the United Kingdom, cases of hospital transmission and cases that occurred via the family route have been identified [14]. Epidemiological data show that infected cases can be carried by travelers [15]. In addition, the epidemic potential corresponding to R0 >1 is evidence of transmission between humans [16]. The outbreak of a high proportion of monkeypox cases as of May 2022 with non-directed transmission from endemic countries has raised several issues referring to how this infection could spread, but especially to the fact that the line of transmission may have changed in the meantime. This "mutated" transmission could thus raise serious health issues, which at the dawn of the end of the Coronavirus pandemic, is certainly one of the major issues to be addressed. Even though, compared with COVID-19 pandemic, it is noteworthy that there are already ready-made vaccines as a weapon for MPX, which is a substantial advantage [17, 18]. Biology and Physiopathology Monkeypox virus (MPXV) belongs to the family Poxviridae [19]. Poxviruses are viruses with a linear double-stranded DNA (dsDNA) genome composed of about 200 genes. Generally, for this type of virus. molecular genetics shows that about half of the genes serve for viral replication, while the remaining half are the so-called accessory genes, which are useful for the virus to penetrate cells into the host organism [20]. The Poxviridae family consists of several viruses, all descended from a rodent probably[21], including cowpox virus (CPXV) camel pox virus (CMLV), and several new variants emerging and identified in recent years [22, 23]. Immunity acquired by infection or vaccination against a virus in this family can also protect against infection of other species. Human monkeypox is typically a zoonosis that clinically resembles smallpox but a lower mortality rate and human-to-human transmission and also differs from smallpox epidemiologically [24, 25]. Biological fluids, respiration, and wound exudate are the main inducer of virus transmission. In addition, the virus is highly stable as demonstrated by laboratory studies [26]. Monkeypoxvirus (MPXV), in spite of its name, is widespread in African rodents especially squirrels, which are now considered the virus' maintenance reservoir [27, 28]. Two strains of MPXV are known today: a more virulent one that can lead to 10% mortality from the Congo Basin, while another milder one is found in West Africa [29]. MPXV can still infect additional animals such as dogs and other rodents with relatively low doses [30–36]. "Wild" mice are susceptible to MPXV (CAST/eij strain of mice) and differ from the more resistant classical mice [37–40]. The CAST mouse/MPXV model may have advantages for studying correlations of immunity and vaccine efficacy. Monkeypox is similar to but milder than the now extinct smallpox. Its manifestation consists of three stages:Incubation: can range from 7 to 14 days, but is generally around 13 days. Prodromal phase: includes fever and lymphadenopathy Skin rash. The lymphadenopathy characteristic of the prodromal phase is the essential element that distinguishes monkeypox from smallpox and chickenpox. The rash also deserves a separate characterization: it is in fact distinguished in turn into several phases. An initial macular phase in which the papules appear rosaceous, but flat and not raised. These papules then become denser, vesicular and pustular. They then evolve further to become scabs that will then inevitably fall off. The rash can affect the face, trunk, and extremities, and sometimes the genitals, and all of these areas are involved at the same level, so the manifestation occurs simultaneously in all of the above areas. Extreme care must be taken with papules in the pustular phase, as they contain active virus that by direct transmission can infect another individual [41]. Secondary symptomatology may be of serious concern compared with primary manifestations. This, in fact, can occur with bronchopneumonia, gastroenteritis, sepsis, encephalitis, and keratitis [42] (Table I).Table I Characteristics Stage for Clinical Symptoms Monkeypox Disease Phase Characteristics Stage Duration Enanthem Lesions form on the tongue and in the mouth (Fever and other prodromal symptoms can occur) 1−2 days Macules Macular lesions (Fever and other prodromal symptoms can occur) (Respiratory and rectal symptoms can occur) 1−2 days Papules Lesions progress from macular to papular (Fever and other prodromal symptoms) (Respiratory and rectal symptoms can occur) 1−2 days Vesicles Lesions become vesicular (Fever and other prodromal symptoms) (Respiratory and rectal symptoms can occur) 1−2 days Pustules Lesions then typically become pustular (Fever and other prodromal symptoms) 5−7 days Scabs By the end of the second week, pustules have crusted and scabbed over. Scabs will remain for about a week before beginning to fall off. 7−14 days Although how monkeypox manages to maintain itself in the wild is still unknown, in recent decades the research world has been asking ever more probing questions to prevent this same virus from implanting itself in new hosts outside endemic areas. Certainly one of the main reasons why this monkeypox manages to stay active in the wild is that it uses different hosts. One of the most concerning alerts is the implantation of this virus in ground squirrels in regions of North America, which would then represent one of the most dangerous outbreaks in the coming years. [43]. The rationale for why this virus is of concern lies in the fact that it can lurk in any mammalian cell, and the interval at which it maintains itself in the host is variable. Infectivity may depend on several factors, chief among them the responsiveness of the host's immune system. An interesting study considered 528 case infections of Monkeypox diagnosed between April 27 and June 24, 2022, in 16 countries. The study showed that during infection, 95% of people presented with a rash, 73% with anogenital lesions, and 41% with mucosal lesions. Common systemic features occurring preceding, concurrently with, or after the rash included fever (62%), lethargy (41%), myalgia (31%), and headache (27%) [44]. Replication Antiviral factor K is a protein that inhibits the multiplication of the viral genetic material and, consequently, the multiplication of the virus, thus causing the infection to stop or be attenuated. It is targeted by two viral genes E3L and K3L. It has been found that primate K protein genes have undergone substantial modifications and are susceptible to inhibition of the K3L gene [44, 45]. The main modification that would have originated is that the viral genome has evolved small inhibitors of human antiviral K protein. For these reasons, it is very likely that much more virulent variants could be generated in humans. In fact, the greatest fear is that other types of variants of the same virus family could be generated. Smallpox virus mutates less rapidly than RNA viruses because the DNA genome has a DNA polymerase that has a 3'-5' exonuclease correction activity [46]. The mutation rate of poxviruses could result in up to 2 nucleotide mutations in the genome per year compared to the dozens that can occur in an RNA virus [47–49]. Poxviruses therefore vary much less even than SARS-CoV-2 [50] however, it must be said that the smallpox virus genome is large and flexible and allows for large changes to the structure causing loss or increase of genes altering viral phenotypes very rapidly. Generally however, mutants have repeats in their genome of a viral gene that is often the direct target of the drug therapy being undertaken [51, 52] and because of this the virus in order to resist the effect of the drug increases the production of that affected gene. Vaccine It is curious to note that the practice used until 1800 for immunization in the East against smallpox was that of "variolation," which consisted of subcutaneous implantation of portions of virus extrapolated from scabs of infected patients. This practice made it possible to avoid the more severe and violent forms of smallpox. Later an English physician Jenner reported a viable alternative. He noticed that the lesions on the hands of milkers who contracted cowpox were similar to those produced by variolation. He then verified whether bovine virus inoculation could avoid the pustules caused by variolation. This finding produced an important assessment: the two viruses, i.e., cowpox and smallpox, are very similar and coming from the same family (Orthopoxvirus) provide cross immunity. The efforts therefore led by WHO to eradicate smallpox ended in 1977 when precisely the last case of smallpox was diagnosed in Somalia. Despite this it should be noted that the vaccine used by Jenner in the beginning was different than the one used in the vaccine campaign. Jenner used cowpox virs (CPXV), while the one used in the vaccine campaign was live vaccinia smallpox virus (VACV). The eradication of smallpox was a formidable, but more importantly encouraging achievement that saved millions of lives and also allowed for the discontinuation of vaccination since it was estimated that vaccination protects for an interval of 20 to 30 years [53]. This remained silent until the first decade of the 2000s when following the Twin Towers terrorist attack, fearing that it might be the prelude to a bioterrorist attack, the United States immediately worked to develop both a vaccine and a pool of antiviral drugs. Currently, two antiviral drugs and two smallpox vaccines are on the market and approved by the FDA that could also have positive efficacy in treating monkeypox virus. The first new generation vaccine was ACAM2000, SIMILAR TO Dryvax because it was obtained on a cell culture with a clone of Dryvax. Vaccines such as the latter generate immunity with antibodies and B cells detected even 60 years later and specific immunity against human monkeypox virus as high as 85 percent [54]. ACAM2000 contains replication competent VACV and is administered by skin scarification. ACAM2000 is contraindicated in pregnancy and in cases of established immunodeficiency and appears to have a risk/benefit profile similar to Dryvax for which the most noted side effect is myopericarditis. The second vaccine synthesized but not yet approved for human use is MVA-BN. It is produced with the modified Ankara vaccine strain (MVA). MVA is altered in replication in most mammalian cells, in part due to the loss of two host genes. It is administered with a double dose of subcutaneous injection 4 weeks apart [55]. The safety profile is extremely encouraging, especially the efficacy shown in studies from animal trials tested for smallpox virus and monkeypox virus. In July 2022, the European Medicines Agency's Committee for Medicinal Products also recommended extending IMNAVEX use in the European Union, for immunization of adults against monkeypox [56]. To confirm this efficacy, the EU will collect data resulting from an observational study conducted during the monkeypox epidemic. Antiviral Drugs Tecovirimat and Brincidofovir are the two drugs to date approved by the FDA for monkeypox. Tecovirimat acts on the virus envelope by preventing virion release and blocking the activity of the VP37 protein, encoded by a gene that is present throughout the orthopoxvirus genus family [57]. Blocking the interaction of VP37 inhibits the activation of the Rab9 GTPase and TIP47 cells, thus preventing the replication of fully functional cells, capable of exiting the cell and spreading the virus long distances passing from cell to cell. In contrast, the second drug is the propharmaceutical of the antiviral cidofovir [58]. There is lipid conjugation with cidofovir, which thus allows this drug to be used at lower concentrations, consequently reducing its toxicity and still allowing it to have a targeted action on inhibition of viable DNA replication. Brincidofovir is already on the market for the treatment of cytomegalovirus. Limited studies of drug use in some cases of monkeypox have shown that tecovirimat is more effective than brincidofovir because the latter can develop pharmacoresistance with mutations in F13L (highly conserved viral membrane protein) and E9L (DNA polymerase) [59, 60]. On July 13, 2018, tecovirimat has been authorized for the placing on the American market as the first drug indicated for the treatment of smallpox, to which the indication was later extended to also treat MPX. Afterwards, Tecovirimat has been also authorized for the placing on the European market on 06 January 2022 [61]. In the context of the monkeypox outbreak, EU government has already been facilitating the conduct of large multinational trials in the EU on the use of the antiviral tecovirimat by reviewing the trial protocols and liaising with the Clinical Trial Coordination Group (CTCG) and national regulatory bodies to coordinate and facilitate the approval of clinical trial applications by national competent authorities. Recent evidence has shown that several interesting studies are underway that are identifying new compounds with antiviral activity against the virus responsible for Monkeypox monkeypox. Tyrosine kinase inhibitors of the Src/Abl family are conceivable as potential inhibitors of viral release from the cell [62]. The methylene blue derivative, PAV-866, has shown in vitro antiviral activity against various Orthopoxviruses. including monkeypox virus by blocking virus binding, fusion, and entry [63]. In order to limit as much as possible a new scenario that could harken back to the recent pandemic from which we may be getting out of, the biology of this virus certainly needs to be studied in depth to try to assess any genetic changes while keeping them under control and limiting transmission to humans as much as possible Fig. 1.Figure 1 The antiviral effect of Cidofovir and Brincidofovir is the result of a selective interaction with the viral DNA polymerases. The mechanism of action of tecovorimat is the viral wrapping inhibition. Expert Opinion Since the beginning of the monkeypox epidemic and until October 4, 2022, there have been approximately 20,000 confirmed cases of monkeypox (MPX) in EU/EEA countries. Spain, France, and Germany are the European countries that have reported the largest cases of infection. The Clade II b variant appears to be responsible for the current epidemic by giving clinically less severe symptomatology with less transmissibility and lower mortality. The person's immune response generates the individual's degree of response to the disease. However, it appears that vaccines on the market for smallpox eradication could provide some immunity against MPX as well. The vaccines approved to date against MPX are recommended only for certain high-risk persons, and there is no universal vaccination. Much confusion still exists about the intradermal or subcutaneous route of administration in different countries, so the important thing today is to ensure the right doses to guarantee immunization. However, the intradermal route, with a lower dose than the subcutaneous route has limited data in the literature to date about its efficacy. In addition, not all countries are aligned on the population categories recommended for smallpox vaccination. To date, there is no approved treatment specifically for Monkeypox infections. However, some antivirals developed for use in smallpox patients may prove useful against MPX. These antivirals include: tecovirimat or ST-246 (TPOXX); brincidofovir (Tembexa); and cidofovir (Vistide). Additionally, intravenous vaccinia immune globulin (VIGIV), which is licensed for the treatment of complications from smallpox (vaccinia) vaccination, may be authorized for use to treat monkeypox and other pox viruses during an outbreak. Clinical evidence on efficacy against MPX is needed for antiviral agents; to date there are few results demonstrating antiviral activity against orthopoxvirus. In addition, no data are available on the efficacy of VIGIV in the treatment of monkeypox virus infection. The use of VIGIV has no proven benefit in the treatment of monkeypox, and it is not known whether a person with severe monkeypox infection would benefit from treatment with VIGIV. Cidofovir, brincidofovir in the treatment of monkeypox cases in people have no supporting clinical evidence, only data of antiviral activity on orthopoxvirus. Well-organized clinical trials are needed to generate the right evidence. Conclusion The epidemic caused by monkeypox unlike COVID-19 is not having the same severity and speed of spread, both because of the different biological characteristics of the virus and the ready availability of different vaccines and antiviral agents for smallpox and monkeypox. The rapid initiation of infection control measures and the use of vaccines and antiviral agents are important strategies for controlling the monkeypox epidemic. To limit as much as possible a new scenario that could resemble the recent pandemic from which we may be about to emerge, the biology of this virus certainly needs to be studied in depth to try to assess any genetic changes while keeping them under control and limiting transmission to humans as much as possible. Regardless, the new reality is that human monkeypox is no longer a rare zoonotic disease and needs more public health attention. Code Availability Not applicable. Data Availability Full availability of data and materials. All stated data can be provided on request to the reader. Declaration Ethical approval Not applicable Consent to participate Not applicable Consent to publish The authors consent to the publication of the manuscript Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare that the opinions expressed are of a personal nature and do not in any way commit the responsibility of the Administrations to which they belong. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ==== Refs References 1. Pal M Mengstie F Kandi V Epidemiology, diagnosis, and control of monkeypox disease: a comprehensive review Am J Infect Dis Microbiol. 2017 5 94 99 2. 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==== Front Soc Psychiatry Psychiatr Epidemiol Soc Psychiatry Psychiatr Epidemiol Social Psychiatry and Psychiatric Epidemiology 0933-7954 1433-9285 Springer Berlin Heidelberg Berlin/Heidelberg 2387 10.1007/s00127-022-02387-8 Review Ethnic disparities in the use of restrictive practices in adult mental health inpatient settings: a scoping review http://orcid.org/0000-0003-3620-3523 Pedersen Martin Locht [email protected] 123 http://orcid.org/0000-0001-9075-6108 Gildberg Frederik [email protected] 23 http://orcid.org/0000-0001-9985-9875 Baker John [email protected] 4 http://orcid.org/0000-0003-1238-2344 Damsgaard Janne Brammer [email protected] 1 http://orcid.org/0000-0001-6464-4911 Tingleff Ellen Boldrup [email protected] 235 1 grid.7048.b 0000 0001 1956 2722 Department of Public Health, Aarhus University, Bartholins Allé 2, 8000 Aarhus C, Denmark 2 grid.10825.3e 0000 0001 0728 0170 Forensic Mental Health Research Unit Middelfart (RFM), Department of Regional Health Research, Faculty of Health Science, University of Southern Denmark, Østre Hougvej 70, 5500 Middelfart, Denmark 3 grid.425874.8 0000 0004 0639 1911 Psychiatric Department Middelfart, Mental Health Services in the Region of Southern Denmark, Østre Hougvej 70, 5500 Middelfart, Denmark 4 grid.9909.9 0000 0004 1936 8403 School of Healthcare, University of Leeds, Baines Wing, Woodhouse Lane, Leeds, LS2 9JT UK 5 grid.10825.3e 0000 0001 0728 0170 OPEN, Odense Patient Data Explorative Network, Odense University Hospital/Department of Clinical Research, University of Southern Denmark, J.B. Winsløws Vej 9 A, 5000 Odense C, Denmark 1 12 2022 118 20 6 2022 14 11 2022 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Purpose To identify and summarise extant knowledge about patient ethnicity and the use of various types of restrictive practices in adult mental health inpatient settings. Methods A scoping review methodological framework recommended by the JBI was used. A systematic search was conducted in APA PsycINFO, CINAHL with Full Text, Embase, PubMed and Scopus. Additionally, grey literature searches were conducted in Google, OpenGrey and selected websites, and the reference lists of included studies were explored. Results Altogether, 38 studies were included: 34 were primary studies; 4, reviews. The geographical settings were as follows: Europe (n = 26), Western Pacific (n = 8), Americas (n = 3) and South-East Asia (n = 1). In primary studies, ethnicity was reported according to migrant/national status (n = 16), mixed categories (n = 12), indigenous vs. non-indigenous (n = 5), region of origin (n = 1), sub-categories of indigenous people (n = 1) and religion (n = 1). In reviews, ethnicity was not comparable. The categories of restrictive practices included seclusion, which was widely reported across the studies (n = 20), multiple restrictive practices studied concurrently (n = 17), mechanical restraint (n = 8), rapid tranquillisation (n = 7) and manual restraint (n = 1). Conclusions Ethnic disparities in restrictive practice use in adult mental health inpatient settings has received some scholarly attention. Evidence suggests that certain ethnic minorities were more likely to experience restrictive practices than other groups. However, extant research was characterised by a lack of consensus and continuity. Furthermore, widely different definitions of ethnicity and restrictive practices were used, which hampers researchers’ and clinicians’ understanding of the issue. Further research in this field may improve mental health practice. Supplementary Information The online version contains supplementary material available at 10.1007/s00127-022-02387-8. Keywords Ethnicity Manual restraint Mechanical restraint Psychiatry Rapid tranquillisation Seclusion ==== Body pmcIntroduction Widespread international efforts have been made to improve mental health practice by reducing the use of restrictive practices, such as manual/mechanical restraint, rapid tranquillisation and seclusion [1–4], but, so far, with little success [5, 6]. Of concern, ethnic minorities appear to be subject to more restrictive practices than others [7–10]. If mental health practices are to be improved, an enhanced understanding of the relationship between restrictive practices and ethnicity is of crucial importance. This paper presented a scoping review of international research literature, which details ethnicity and the use of restrictive practices in mental health inpatient settings, to summarise current knowledge. Background The challenges associated with a multicultural society inhabited by people with different ethnic backgrounds have still not been successfully addressed in mental health [11, 12]. In many cases, treatment and care pathways are offered according to ethnic group [11, 13]. Consequently, mental health practice may be considered institutionally racist, meaning that an organisational inability exists to provide the right service to people due to their ethnic background [14]. This inability places ethnic minorities at a disadvantage and may be seen as discriminatory. Racist stereotyping observed in processes, attitudes and behaviour have been reported [14–16]. Institutional racism in mental health further extends beyond the inability to provide appropriate services; it manifests as harm to individuals and worse outcomes relating to mental illness [17]. Cultural competency, such as knowledge of values, beliefs and practices, is thus required in mental health and may improve treatment and care for ethnic groups [18, 19]. Together with implementation of guidelines targeting ethnic disparities and developing responsive practices, this may deliver ethnic equality [11]. Additionally, in recent years, research has highlighted how ethnic disparities and institutional racism still occur in mental health practice [11, 13, 17]. A review of seven quantitative studies showed that compared with those described as White, ethnic minorities, in this case people described as Black, were more likely to be hospitalised by police and less likely to trigger the involvement of a general practitioner at the first episode of psychosis [20]. Furthermore, in a large and more recent review comprising 71 quantitative studies, Barnett et al. [13] showed that ethnic minorities were generally at a greater risk of compulsory detention than were majority populations. Additionally, researchers have identified delay/gaps in access to mental health treatment and care for ethnic minorities; e.g. among first-generation immigrants with psychosis [21–23]. Several studies have also reported inequalities in the length of mental health hospitalisations among various ethnic groups, with ethnic minorities often experiencing prolonged admissions [24, 25]. Finally, mental health staff have been shown to perceive some ethnic minorities as more dangerously disturbed than others [8, 13, 26]. The above examples of ethnic differences in pathways and mental health practice may contribute to the complex interplay of factors influencing ethnic differences in the rates of different types of restrictive practices that occur in mental health inpatient settings [10]. In mental health inpatient settings, restrictive practices remain common and are largely classified into four main types: manual restraint, mechanical restraint, rapid tranquillisation and seclusion [3, 27]. Although, most mental health acts consider their use to be acceptable as a last resort to prevent people from harming themselves and/or others [28, 29], the practices remain a topic of considerable debate [30, 31]. Their use is considered necessary by some mental health professionals to ‘maintain safety for all’ [32]. However, it is traumatising for the people who are subjected to these practices [4, 33]. Furthermore, physical and psychological harm from the use of restrictive practices to both inpatients and staff are well documented [2, 33–37]. Evidence suggests that certain ethnic minorities are more likely to encounter restrictive practices than patients in general, e.g. foreign-born compared with national people [38, 39], indigenous compared with non-indigenous people [9, 40] and people described as Black compared with those described as White [2, 10]. Furthermore, ethnic minorities are more likely to die from restrictive practices [41, 42]. Outcomes for different ethnic groups are therefore an area of interest when implementing programmes to reduce restrictive practices in mental health [10]. Several reviews have identified ethnicity as a risk factor frequently associated with restrictive practices [7, 8]. However, these reviews were limited to acute/intensive mental health inpatient settings and did not focus on ethnicity specifically but on risk factors generally. Therefore, a need exists to create an overview of knowledge concerning restrictive practices and ethnicity across a wide range of mental health inpatient settings. Considering the above, the purpose of this paper was to conduct a scoping review by covering a broad spectrum of international research literature examining reported ethnicity and the use of common types of restrictive practices to establish a foundation for improving mental health practice and identify knowledge gaps. To the best of our knowledge, no studies have previously synthesised these data. Aim The aim of the study was to review extant international research literature to identify and summarise existing knowledge about patient ethnicity and the use of manual restraint, mechanical restraint, rapid tranquillisation and seclusion in adult mental health inpatient settings. Methods A scoping review inspired by the JBI framework [43, 44] was chosen to identify, select and summarise existing knowledge about patient ethnicity and the use of different types of restrictive practices in mental health inpatient settings. The interpretive framing of data to summarise existing knowledge was rooted in the epistemology of pragmatism and the methodological approach described by Blumer [45], stressing the need for careful and disciplined data examination using open-ended categories inductively for concepts such as ‘ethnicity’ in order not to skew interpretations into ethnocentrism. In line with this framework, the following were undertaken: identifying the review question, identifying relevant studies, screening and selecting studies, extracting data and analysing and presenting results. The PCC (Population, Concept, and Context) elements were incorporated to develop a focused review question [43, 44]: What characterises international research literature on patient ethnicity and the use of manual restraint, mechanical restraint, rapid tranquillisation and seclusion in adult mental health inpatient settings? PCC elements were as follows: (a) population: adults (≥ 18 years old) categorised by ethnicity, defined as the ‘social group a person belongs to, and either identifies with or is identified with by others, as a result of a mix of cultural and other factors’ [46]; (b) concept: restrictive practices, defined as manual restraint, mechanical restraint, rapid tranquillisation (also known as chemical restraint) and seclusion [3, 27]; and (c) context: all types of mental health inpatient settings into which a person may be formally admitted, varying in time until discharge depending on treatment and care needs. The Reporting Checklist for Scoping Reviews (PRISMA-ScR) was used for reporting the findings [47, 48]. Search strategy To identify relevant studies, the literature search followed a three-step process: initially, a search in CINAHL with Full Text (EBSCO) and PubMed (NCBI) was conducted to identify relevant keywords and search subject headings [43]. Secondly, these relevant keywords and search subject headings were combined using the Boolean operators AND/OR in a systematic block search strategy, framed by the above review question (PCC elements) and guided by an informatics specialist [43]. The literature search was conducted in CINAHL with Full Text, PubMed, APA PsycINFO (ProQuest), Scopus (Elsevier) and Embase (Elsevier) (between 1 January 2010 and 22 February 2021). This data range was chosen to ensure a contemporary knowledge base in a field in which interest is growing [49, 50]. As an example, the search in CINAHL with Full Text is shown in Table 1, and the full literature search comprising all the selected databases is shown in the supplementary material. The final step of the literature search process was a ‘citation pearl searching’ [51], i.e. an examination of the reference lists of all included studies.Table 1 Search subject headings and keywords combined with Boolean operators (OR/AND) in CINAHL with Full Text Population: descriptors of ethnicity MH “Ethnic Groups + ” OR MH “Immigrants + ” OR Ethnic OR Refugee OR Ethnology OR Migrant OR Transient OR Emigrant OR Immigrant OR Minority OR Race OR Continental population OR Ethnological OR Ethnicity Concept: restrictive practices Seclusion OR Coercion OR Restraint OR Coercive OR Compulsory OR Involuntarily OR Involuntary OR Forced medic* OR Tranquiliz* Context: mental health inpatient settings MH “Forensic psychiatry + ” OR Psychiatry OR Psychiatric OR Secure service OR Secure setting OR Forensic service OR Forensic setting OR Mental health To identify grey literature, the following were hand searched by the authors: Google, OpenGrey and selected websites (i.e. Danish Health Authority (sst.dk), National Institute for Health and Care Excellence (nice.org.uk), Substance Abuse and Mental Health Services Administration (samhsa.gov), Race Equality Foundation (raceequalityfoundation.org.uk) and Mind (mind.org.uk) [52]. These websites are run by health authorities and interest organisations and therefore considered relevant to the review topic. The grey literature search was conducted in accordance with the limitations in the database search. The authors’ international research network were also contacted regarding knowledge of relevant literature. Source of evidence screening and selection The literature searches and selection process are documented in a PRISMA Flow Diagram [48]. As shown in Fig. 1, initially 6823 studies were identified across the databases. Hereafter, the number of hits was reduced by using relevant automation tools to limit the number of hits in the databases, as follows: language, English; publication year, 2010 to present. Following removal of duplicates, 2325 studies were imported into Covidence [53] to ensure a systematic selection process. This process was guided by the following inclusion/exclusion criteria: the inclusion criteria were (a) all types of research literature, including reviews, qualitative, quantitative and mixed method studies; (b) studies in English; (c) studies about use of restrictive practices (concept) among adults with described ethnicity (population) in a mental health inpatient setting (context). Studies were excluded based on the following criteria: (a) those without reported empirical data; (b) thesis; (c) no full text available; (d) non-mental health setting.Fig. 1 PRISMA flow diagram of the study selection process Initially, titles/abstracts were screened, which excluded 2217 studies. Subsequently, 108 studies were sought for retrieval. Among these, 102 studies were assessed by full-text reading, which excluded an additional 80 studies. The first and last author independently completed the screening and full-text reading. In cases of disagreement, the second author was consulted to reach a final decision. A total of 16 additional studies were identified by other methods (Fig. 1). Finally, 38 studies were included in this review. Data extraction Data were extracted using a charting table inspired by the scoping review framework [43]: (a) general information: author(s) and year of publication; (b) methodological information: study design; (c) context information: mental health inpatient setting and country; (d) sample information: number of participants (primary studies) and number of included studies (reviews); (e) demographic information: gender and ethnicity as defined by the papers; (f) type of restrictive practice(s); (g) key findings relevant to the aim of this review. Data extraction was conducted by the first author and reviewed by the last author. Subsequently, the extracted data were discussed between all authors to ensure a common understanding. If a common understanding of data was not achieved, the authors of the studies reporting the data were contacted for clarification. Analysis and presentation of results According to Krippendorff [54], content analysis is a scientific method for data processing in several type of research, including those that use qualitative and quantitative approaches. The analytic process was initiated by a discussion between the first and last author to determine which applicable data extraction from the included studies should be used for further analysis [54]. Data were assessed for applicability based on the above review question [54]. Then, these data were coded and compared for similarities and differences before being sorted into categories [54]. In keeping with the scope of scoping reviews, the results and ethnic groups are presented descriptively, including tables, and some are described in the supplementary material [43]. Data were included as characterised in the studies. To provide a detailed answer to the review question, the characteristics of the included studies are first presented; this is followed by an overview of the use of the four different types of restrictive practice in relation to reported ethnicity. Results Results of the literature search As shown in Fig. 1, 38 studies were included in this review. One additional study met the inclusion criteria [55] but not included, as an updated version was included instead [2]. Description of studies included Table 2 provides general, methodological, contextual and sample information extracted from the studies, whereas Table 3 provides an overview of the reported ethnic groupings in relation to restrictive practices. In the following, these tables are presented focusing on context and study design. Table 2 General, methodological, context and sample information of the included studies Author(s) Year Study design Mental health inpatient settings as described by the papers Country Sample (n.) PS RS Alda Díez et al. 2010 Case–control Psychiatric ward Spain 204 Bak et al. 2014 Cross-sectional Psychiatric hospital units Denmark/Norway NR Bak et al. 2015 Cross-sectional Psychiatric hospital units Denmark/Norway NR Beames and Onwumere 2021 Systematic review Adult acute inpatient or psychiatric intensive care UK 20 Beghi et al. 2013 Systematic review Acute psychiatry wards Italy 49 Bennewith et al. 2010 Cohort Mental health hospitals UK 773 Bilanakis et al. 2010 Cohort Mental health hospitals Greece 282 Bowers et al. 2012 Cross-sectional Acute psychiatric wards and psychiatric intensive care units UK 522 Bowers et al. 2010 Cross-sectional Acute mental health wards UK NR Collazos et al. 2021 Cross-sectional Hospital psychiatry emergency rooms Spain 397 Cullen et al. 2018 Case–control General adult acute wards and psychiatric intensive care unit UK 4002 Currier et al. 2011 Experimental Psychiatric emergency department USA 151 Drown et al. 2018 Survey Mental health inpatient units New Zealand NR Flammer et al. 2013 Cohort Inpatient psychiatric care Germany 3389 Gowda et al. 2018 Cohort Department of Psychiatry India 200 Happell and Koehn 2010 Survey Mental health inpatient units Australia 3244 Hendryx et al. 2010 Cohort Adult state psychiatric hospital USA 1266 Hui et al. 2016 Literature review Forensic psychiatry within secure hospital settings UK 18 Husum et al. 2010 Cross-sectional Acute psychiatric wards Norway 3462 Jury et al. 2019 Cohort Adult mental health inpatient services New Zealand 11,341 Knutzen et al. 2013 Cohort Acute psychiatric wards Norway 371 Knutzen et al. 2014 Cohort Acute psychiatric wards Norway 373 Knutzen et al. 2011 Case–control Acute psychiatric wards Norway 749 Lai et al. 2019 Ecological Mental health inpatient services New Zealand 10,727 Lay et al. 2011 Cohort Psychiatric hospitals Switzerland 9698 McLeod et al. 2017 Cohort Mental health inpatient units New Zealand 7239 Mellow et al. 2017 Systematic review Mental health settings UK 11 Miodownik et al. 2019 Cohort Acute, closed psychiatric ward Israel 176 Norredam et al. 2010 Cohort Nationwide psychiatry Denmark 312,300 Opitz-Welke and Konrad 2012 Cohort Psychiatric department within a prison hospital Germany 107 Sambrano and Cox 2013 Qualitative Acute mental health facility Australia 3 Tarsitani et al. 2013 Case–control Psychiatric intensive care unit Italy 200 Taylor et al. 2012 Cohort Psychiatric inpatients units USA 3758 Thomsen et al. 2017 Cohort Nationwide psychiatry Denmark 112,233 Trauer et al. 2010 Experimental Acute psychiatric inpatient ward Australia 352 Tyrer et al. 2012 Cohort General adult acute psychiatric unit New Zealand 254 van de Sande et al. 2017 Cohort Acute psychiatric admission wards Netherlands 878 Verlinde et al. 2017 Cohort Mental health hospitals Netherlands 3242 NR not reported, PS primary studies (n = participants), RS reviews (n = included studies) Table 3 Description of ethnicities by restrictive practices Restrictive practices (n.) Description of ethnicity References Definition of the restrictive practice Main categories (n.) Manual restraint (n = 1) Review (n = 1) Hui et al. [2]† Yes Mechanical restraint (n = 8) Migrants and native nationals (n = 6) Alda Díez et al. [38] No Bak et al. [57] Yes Bak et al. [58] Yes Flammer et al. [74] Yes Husum et al. [75] Yes Tarsitani et al. [25] Yes Mixed categories (n = 1) Currier et al. [76] Yes Review (n = 1) Hui et al. [2]† Yes Rapid tranquillisation (n = 7) Migrants and native nationals (n = 3) Flammer et al. [74] Yes Lay et al. [39] No Opitz-Welke and Konrad [66] No Mixed categories (n = 1) Verlinde et al. [78] Yes Religion (n = 1) Gowda et al. [77] Yes Review (n = 2) Beames and Onwumere [7] Yes Hui et al. [2]† Yes Seclusion (n = 20) Indigenous and non-indigenous people (n = 5) Drown et al. [60] Yes Happell and Koehn [9] No Lai et al. [79] Yes McLeod et al. [62] Yes Trauer et al. [65]* Yes Indigenous people (n = 1) Sambrano and Cox [67] No Migrants and native nationals (n = 3) Flammer et al. [74] Yes Husum et al. [75] Yes Trauer et al. [65]* Yes Mixed categories (n = 8) Bowers et al. [1] Yes Bowers et al. [59] Yes Cullen et al. [81] Yes Hendryx et al. [69] Yes Jury et al. [40] Yes Tyrer et al. [80] Yes van de Sande et al. [68] No Verlinde et al. [78] Yes Religion (n = 1) Gowda et al. [77] Yes Review (n = 3) Beames and Onwumere [7] Yes Hui et al. [2] Yes Mellow et al. [82] Yes Multiple restrictive practices (n = 17) Geographical categories (n = 1) Thomsen et al. [64]* Yes Migrants and native nationals (n = 10) Bilanakis et al. [83] Yes Collazos et al. [63] No Flammer et al. [74] Yes Knutzen et al. [71] Yes Knutzen et al. [72] Yes Knutzen et al. [73] Yes Lay et al. [39] Yes Norredam et al. [49] Yes (manual and mechanical restraint only) Opitz-Welke and Konrad [66] No Thomsen et al. [64]* Yes Mixed categories (n = 4) Bennewith et al. [61] No Hendryx et al. [69] No Miodownik et al. [84] Yes Taylor et al. [70] Yes (seclusion only) Religion (n = 1) Gowda et al. [77] Yes Review (n = 2) Beames and Onwumere [7] Yes Beghi et al. [8] No Studies dividing ethnicity into more than one category †Study (a review) investigating restrictive practice; however, no findings were reported Context As shown in Table 2, most studies (n = 20) were conducted in mental health inpatient settings in general. More specifically, the remaining studies were conducted in acute/intensive settings (n = 15), emergency settings (n = 2) and forensic settings (n = 1). According to the World Health Organization [56] guidelines, the studies were mainly conducted in Europe (n = 26), followed by the Western Pacific (n = 8), the Americas (n = 3) and South-East Asia (n = 1). Study design Of the 38 studies, 34 were primary studies, including 33 quantitative and 1 qualitative study. The remaining four studies were reviews. In total, the studies contain findings based on 491,893 participants (255,342 females and 227,986 males) in the 34 primary studies and 98 studies comprising the four reviews. However, four primary studies failed to report the number of participants [57–60], whereas nine studies reported incomplete or no gender information (missing data: n = 8565) [1, 39, 57–63]. Reviews were not comparable by gender. Gender information from all studies is reported in the supplementary material. As shown in Table 3, ethnicity was described and divided into groups in a wide range of manners across the studies, underpinning the heterogeneity of the concept. In two studies, e.g. several ethnic groupings were used [64, 65]. Furthermore, in several studies ethnicity was reported in one way in relation to the description of participants but in different ways in the analysis. The study by Alda Díez et al. [38] may serve to exemplify this; most ethnic minority participants were categorised as Latin Americans, followed by sub-Saharans, Maghrebian and Eastern Europeans; however, in the analysis, immigrants as a single group were compared with nationals. A more accurate description of ethnicity information provided in all studies is reported in the supplementary material, whereas the main categories are presented in Table 3. Most of the 34 primary studies (n = 16) divided ethnicity by migrant/national status (e.g. foreign born, immigrants or refugees and nationals), followed by indigenous (e.g. Māori, Pasifika or indigenous status) and non-indigenous (n = 5), region of origin (n = 1), sub-categories of indigenous people (n = 1) and religion (n = 1). The remaining 12 primary studies used mixed categories (e.g. comparing religion/race and origin). Reviews were not comparable by ethnicity. Ethnicity in relation to restrictive practices As shown in Table 3, restrictive practices were defined and used very differently across the studies. In 12 studies, types of restrictive practices were not defined [8, 9, 38, 39, 49, 61, 63, 66–70]. An overview of definitions of restrictive practices used in the remaining studies is provided in the supplementary material. Moreover, seclusion was the most frequently studied restrictive type (n = 20), followed by mechanical restraint (n = 8), rapid tranquillisation (n = 7) and manual restraint (n = 1). In 17 studies, multiple restrictive practices were investigated concurrently (e.g. both mechanical restraint and rapid tranquillisation [71–73]). From these studies, data on individual restrictive practices could not be extracted. Table 4 summarises available relative risk, odds ratio, confidence interval and p value data, and additional key findings to highlight important reported associations between ethnicity and restrictive practices. As only one study (a review) investigated manual restraint with no reported findings [2], this restrictive type is not listed below.Table 4 Available relative risk, odds ratio (OR), confidence interval and p value data, and additional key findings to highlight important reported associations between ethnicity and restrictive practices Restrictive practices Study Country Variable OR 95% CI p value Notes and additional key findings Mechanical restraint Alda Díez et al. [38] Spain Immigrant 2.6 1.9–3.0 NR Immigrants were significantly balanced with national subjects after 3 years in Spain Bak et al. [57] Denmark/Norway Ethnicity NR NR NR No significant difference between countries were reported in relation to ethnicity However, a small difference was observed in the number of mechanical restraints per unit Bak et al. [58] Denmark/Norway Ethnicity NR NR NR No significant difference in ethnicity between countries Currier et al. [76] USA Race NR NR 0.18 Proportional difference between ethnic groups were reported Tarsitani et al. [25] Italy Immigrant 3.67* 1.05–12.7 0.027 Non-significant results between ethnic groups in relation to rates of repeated mechanical restraints and in the overall duration of restraint Flammer et al. [74] Germany German citizenship 0.56 0.33–0.94 < 0.05 0.29 0.17–0.5 < 0.001 Psychotic subgroup results Husum et al. [75] Norway Other than Norwegian 0.39 0.16–0.96 < 0.05 Adjusted for patients' individual psychopathology Rapid tranquillisation Beames and Onwumere [7]† UK Ethnicity NR NR NR Reporting about significant and non-significant results in the literature Flammer et al. [74] Germany German citizenship 1.17 0.56–2.45 NR 0.88 0.31–2.5 NR Psychotic subgroup results Gowda et al. [77] India Religion 0.43 NR NR Lay et al. [39] Switzerland Foreign national 1.14 1.1–1.18 NR 1.23 0.96–1.5 NR Adjusted for other sociodemographic variables However, proportional difference between ethnic groups was reported Opitz-Welke and Konrad [66] Germany German NR NR NR Proportional difference between ethnic groups was reported Verlinde et al. [78] Netherlands Non-western descent NR NR NR Policy change did not affect the use of rapid tranquillisation Seclusion Beames and Onwumere [7]† UK Ethnicity NR NR NR Reporting about significant and non-significant results in the literature Bowers et al. [1] UK Ethnicity NR NR NR Ethnicity was not reported as being associated with the likelihood of seclusion, number of seclusion episodes or when in the hospital stay seclusion occurs Bowers et al. [59] UK Asian NR NR 0.001 Seclusion was not strongly associated with the type of patients. Additional p values available in the paper. However, the associations were relatively weak and non-significant after adjusted analysis Cullen et al. [81] UK Black African/Caribbean 1.13 0.71–1.79 0.609 Adjusted for all demographic/clinical factors and behavioural precursors. ORs for other ethnic groups are available in the paper. However, all were non-significant. Proportional differences between ethnic groups were reported Drown et al. [60] New Zealand Māori NR NR NR Seclusion among Māori slightly increased between 2007 and 2013, whereas among other groups seclusion decreased (no significant difference) However, in 2014 Māori received seclusion proportionally more often than non-Māori Flammer et al. [74] Germany German citizenship 0.68 0.42–1.11 NR 0.51 0.25–1.07 NR Psychotic subgroup results Gowda et al. [77] India Religion NR NR NR No significant results were reported Happell and Koehn [9] Australia Indigenous people NR NR 0.066 Proportional difference between ethnic groups was reported; with significant results in relation to age group Hendryx et al. [69] USA Black/Hispanic/native NR NR NR No significant differences in relation to ethnicity between people who received seclusion and people who did not. Ethnicity was not a significant predictor of seclusion Hui et al. [2]† UK Ethnicity NR NR NR Reporting about proportional (non-significant) difference between ethnic groups Husum et al. [75] Norway Other than Norwegian 1.15 0.7–1.88 NR Adjusted for patients' individual psychopathology Jury et al. [40] New Zealand Pasifika 1.89 1.44–2.47 < 0.001 Additional significant ORs available in the paper in relation to ethnic group Lai et al. [79] New Zealand Māori NR NR < 0.001 Lower seclusion rates association with higher proportion of Māori McLeod et al. [62] New Zealand Māori 1.39* 1.05–1.83 NR 1.33* 0.97–1.81 NR Adjusted for a range of demographic and admission variables Additional RRs available in the paper, including in relation to various adjustments Age was reported as an important contributor to the ethnic disparities in seclusion Mellow et al. [82]† UK Ethnicity NR NR NR Reporting about experiences of being in seclusion from the literature Sambrano and Cox [67] Australia Indigenous status NR NR NR Indigenous people experienced seclusion as discriminatory and degrading Tyrer et al. [80] New Zealand Māori/European NR NR < 0.05 Trauer et al. [65] Australia Australian born/ Indigenous people NR NR NR No significant differences in relation to ethnicity between people who received seclusion and people who did not van de Sande et al. [68] Netherlands Non-western 1.68 1.06–2.67 0.022 0.45 0.24–0.84 0.012 Adjusted for within-patient variation Verlinde et al. [78] Netherlands Non-western descent NR NR NR Use of seclusion was slightly reduced after policy change Multiple restrictive practices Beames and Onwumere [7]† UK Ethnicity/migrant status NR NR NR Reporting of significant and non-significant results in the literature Beghi et al. [8]† Italy Non-autochthonous NR NR NR Reporting of significant and non-significant results in the literature Bennewith et al. [61] UK Black 2.19 1.47–3.27 NR ORs for ethnicity and other ethnic groups available in the paper However, all were non-significant Black 1.09 0.66–1.81 NR Adjusted for age, gender, diagnosis and mental health trust. ORs for ethnicity and other ethnic groups available in the paper However, all were non-significant Bilanakis et al. [83] Greece Other than Greek NR NR 0.470 Proportional (non-significant) association was reported Collazos et al. [63] Spain North African 4.23 1.26–14.17 < 0.05 Adjusted for patient’s geographical origin. ORs for other migrant groups available in the paper However, all were non-significant North African 2.12 0.54–8.32 NR Adjusted for patient’s geographical origin and further demographic and clinical variables. ORs for other migrant groups available in the paper However, all were non-significant Flammer et al. [74] Germany German citizenship 0.75 0.54–1.05 NR Ethnicity was not related to the number of restrictive practices recorded 0.49 0.32–0.77 NR Psychotic subgroup Gowda et al. [77] India Religion NR NR NR No significant results reported Hendryx et al. [69] USA Black NR NR 0.02 No significant differences in relation to ethnicity between people receiving seclusion and people who did not. However, ethnicity was a significant predictor of restrictive practices Knutzen et al. [71] Norway Immigrant NR NR NR Ethnicity was not related with the duration of restrictive practices or the restrictive type received Knutzen et al. [72] Norway Immigrant NR NR 0.552 Ethnicity was not related with the number of episodes Knutzen et al. [73] Norway Immigrant 1.52 1.05–2.17 0.03 Lay et al. [39] Switzerland Foreign national 1.045 0.838–1.302 NR Adjusted for other sociodemographic variables. However, before this adjustment, there are no reported significant associations either Miodownik et al. [84] Israel Ethnicity NR NR NR No association found between ethnicity and frequency or length of restrictive practices Norredam et al. [49] Denmark Migrant status NR NR NR Use of restrictive practices were about twice as high for both refugees and immigrants as for non-migrant Danes Opitz-Welke and Konrad [66] Germany German NR NR NR Proportional difference between ethnic groups was reported Taylor et al. [70] USA Race NR NR 0.115 Ethnicity was not related to the number of restrictive episodes Thomsen et al. [64] Denmark Immigrant 1.64 1.54–1.74 < 0.001 Adjusted for sex, age and calendar period. ORs for other migrant group and geographical categories available in the paper. However, both significant and non-significant 0.99 0.85–1.17 NR Adjusted for sex, age, calendar period and further demographic variables Europe 0.43 0.35–0.53 < 0.001 Adjusted for sex, age and calendar period. ORs for other migrant group and geographical categories available in the paper. However, both significant and non-significant 0.7 0.51–0.97 < 0.05 Adjusted for sex, age, calendar period and further demographic variables NR not reported †Review study Mechanical restraint As shown in Table 4, four studies reported significant associations between ethnicity and mechanical restraint [25, 38, 74, 75]. People with migrant status, in this case immigrants and non-nationals (Europe-based studies), were significantly more likely to receive mechanical restraint in all but one study where the reverse association was reported after using adjusted analysis [75]. Moreover, a significantly lower frequency of mechanical restraint was identified in one study in those with immigrant backgrounds who had resided in a country for a longer period of time [38]. Proportional (non-significant) findings were reported in one study, where people described as non-White were more likely to receive mechanical restraint [76]. No significant differences in ethnicity were reported in the findings of three studies [25, 57, 58]. Rapid tranquillisation Three studies reported significant associations between ethnicity and rapid tranquillisation [7, 39, 77], of which there was no further description in one study [77]. Ethnic minorities, in this case people of foreign citizenship (Swiss-based study) or not further described (review study), were significantly more likely to receive rapid tranquillisation in the two remaining studies [7, 39]. However, these associations became non-significant after using adjusted analysis, although, proportionally, ethnic minorities were more likely to receive rapid tranquillisation. Proportional (non-significant) findings were reported in one other study, where non-German people were more likely to receive rapid tranquillisation than Germans [66]. No significant differences in ethnicity were reported in two other studies [74, 78]. Seclusion Eight studies reported significant associations between ethnicity and seclusion [7, 9, 40, 59, 62, 68, 79, 80]. Ethnic minorities were significantly more likely to receive seclusion in all but one study, where the inverse association was reported [79]. However, after adjusted analysis, the reverse association was reported in one further study [68], whereas associations became non-significant in three studies [7, 59, 62]. In Western Pacific-based studies, ethnic minorities were indigenous (e.g. Māori) or European people [9, 40, 62, 79, 80]. In European-based studies, they were people of non-Western descent or described as non-White [59, 68], while in the remaining (review) study, ethnic minority status was not further described [7]. Moreover, age was identified in two studies as a significant contributor to ethnic disparities between indigenous and non-indigenous people in relation to the use of seclusion [9, 62]. Proportional (non-significant) findings were reported in three studies where ethnic minorities were more likely to receive seclusion [2, 9, 60, 81]. No significant differences in ethnicity were reported in relation to the findings of nine studies [1, 7, 59, 65, 69, 74, 75, 77, 78]. Additionally, seclusion was reported to be experienced as discriminatory and degrading across ethnicities [67, 82]. Multiple restrictive practices investigated concurrently Eight studies reported significant associations between ethnicity and restrictive practices [7, 8, 61, 63, 64, 69, 73, 74]. Ethnic minorities, in this case people described as Black, with migrant status, of non-European descent or from North Africa (European and US-based studies) or not further defined (review studies) were significantly more likely to receive restrictive practices in all studies, of which the results from two studies were based on adjusted analyses [63, 64]. However, in four studies, associations became non-significant after (further) adjusted analysis [7, 61, 63, 64]. Proportional (non-significant) findings were reported in four studies where foreign nationals were more likely to receive restrictive practices [39, 49, 66, 83]. No significant differences in ethnicity were reported in relation to findings in eleven studies [7, 8, 61, 63, 64, 70–72, 74, 77, 84]. Discussion The present review summarised literature on ethnicity and the use of restrictive practices in adult mental health inpatient settings. It showed that from 2010 to the present, a total of 38 studies were published in this field. The studies were characterised by lacking consensus and continuity, and both ethnicity and restrictive practices were reported with widely differing definitions. Thus, this review provides important understanding of variables that should be considered in future more rigorous analysis of the influence of ethnicity on rates of restrictive practices to support efforts at reducing restrictive practices in mental health inpatient settings [10]. In extant literature, ethnicity is reported as one of the risk factors most frequently associated with the use of restrictive practices [7, 8]. It may therefore be considered surprising that in some of the included studies, ethnicity was not associated with the use of restrictive practices. However, the fact that this lack of effect is stronger in studies after using adjusted analysis underpins the complex interplay of factors influencing ethnic differences in the rates of restrictive practices [10]. The findings of this review showed, e.g. that factors such as residence time in a country and also age contributed significantly to ethnic disparities in relation to the use of mechanical restraint and seclusion, respectively. These findings potentially suggest the importance of a focus on intersectionality and the social determinants of mental health [85, 86]. This would facilitate recognition of multiple sources of disadvantage and how this may contribute to the use of restrictive practices towards ethnic minorities [86–88]. Furthermore, in many cases, ethnic minorities remain proportionally more likely to receive restrictive practices than the majority population, although some findings in this regard are reported as non-significant. Several international analyses in the field confirm this increased likelihood of restrictive practices among ethnic minorities [10, 89–91]. Consequently, although the picture is mixed, it is of concern if ethnic minorities do not receive treatment and care in a respectful, safe and non-restrictive environment [35]. Therefore, further initiatives are warranted both in clinical practice to improve the care of ethnic minorities and in relation to research to ensure that potential institutional racism in mental health inpatient settings may be overcome [14]. We propose that these initiatives may be focused on staff-related factors affecting the use of restrictive practices for two reasons: first, since such practices are initiated by staff [31, 32, 92]; second, because research is largely unanimous that use of restrictive practices in mental health settings is associated with staff-related factors [8, 93]. Furthermore, research has highlighted that disparities in care based on ethnicity may be maintained by staff-related factors such as a lack of cultural understanding and culturally appropriate services and by communication issues [8, 18]. Research into these factors is important for mental health care to become more sophisticated and person centred, to learn about and prevent the use of restrictive practices in minority groups and thereby eliminate ethnic inequalities; especially as these inequalities have been further exacerbated by the COVID-19 pandemic [94, 95]. Such research should account for intersectionality and the social determinants of mental health that are known to be important [86–88], and it should explore the possibility that ethnic disparities in use of restrictive practices may also be influenced by other sources of disadvantage, such as income, living situation and trauma [64, 93]. This review also shows that clarity about institutional racism in mental health inpatient settings is further confused by the very diverse classification of ethnic minorities. For instance, the findings suggested that studies dividing ethnicity into migrant/native status are more likely to report an association between ethnicity and the use of restrictive practices than are studies using mixed categories to describe ethnicity. These conflicting results have meant that frequently discussed comparisons and syntheses are not possible. Like others, we therefore suggest greater standardisation in how ethnicity is categorised [2, 86]. Furthermore, we propose the use of several ethnic divisions, which this review has shown were used only sparingly, to help build an overview of the field and to facilitate specific comparisons between different understandings of ethnicity across contexts. We know that ethnic definitions, terms and their use change over time and between countries [86], being sensitive to the diversity of concepts such as ethnicity, may be more important now than at any other point in time. Therefore, this research has relevance not just in different contexts, but also in the future and to the people in the healthcare system whose conditions we are trying to improve. Additionally, as the number of international migrants is increasing [96] and managing their (mental) health needs may be challenging [12, 97, 98], a stronger focus on ethnicity may be desirable in future systematic reviews. Such focus may help advance our knowledge on one widely reported ethnic group (migrants/natives) encountering ethnic disparities in the use of restrictive practices. Only one study reported data on manual restraint. This is of major concern particularly as death from prone manual restraint is an international issue [41, 42]. Furthermore, manual restraint was typically included in the studies in which several restrictive practices were studied concurrently. Thus, the lack of manual restraint research highlights a problem that exists in many mental health research fields characterised by a trend towards bundling up different types of restrictive practices or coercion [32, 99–101], making it difficult to tease out research on specific restrictive practices such as manual restraint. As argued by several researchers, research designs that distinguish between different restrictive practices is urgently required, as both their use and the negative consequences they have for those affected vary [2, 5, 33, 36, 102]. The trend to bundle different types of restrictive practices and coercion may also explain the low number of included studies investigating mechanical restraint (n = 8) and rapid tranquillisation (n = 7). Therefore, to increase knowledge about the association between ethnicity and the use of restrictive practices, we strongly recommend conducting more research on the association between particular restrictive practices and ethnicity. Limitations Although the use of a broad and systematic search strategy must be considered a strength of this review, inclusion of, e.g. ProQuest Dissertations & Theses Global may potentially have identified additional qualitative studies [103], leading to different findings. Secondly, the language limitations may have impacted the number of identified studies as studies relevant to the purpose of this review have undoubtedly also been drafted in non-English languages. Thirdly, in the context of inclusion/exclusion criteria, the studies by Bak et al. [57] and Verlinde et al. [78] should be mentioned, as these only contain minor elements of relevance to this review. Fourthly, it has been argued that the lack of quality assessment is a limitation of scoping reviews [104, 105]. However, quality assessment of studies is beyond the purpose of a scoping review, which should be used to gauge the size and scope of extant research literature in a field [43, 105, 106]. Therefore, it contributes to the validity and reliability of this review that this part of the scoping review framework was adopted. Fifthly, most studies have been conducted in Western countries. Whilst this was not an unexpected finding in this field [2, 7, 13], the geographical variation of studies, with certain regions being underrepresented or absent, suggests that the risk of instructional racism concerning restrictive practices is not addressed in some countries, or that reporting/publication bias may be prominent. Lastly, the studies comprised by our review were conducted in very different settings. Since the goal was to review existing international research literature, this is a strength of the review, although it should be noted that laws and acceptable treatment/care cultures may vary between settings [5, 102, 107, 108]. Conclusion In this scoping review, we identified the contemporary knowledge about ethnicity and use of restrictive practices. This research is characterised by a lack of consensus and continuity, and widely different definitions of ethnicity and restrictive practices are used in the literature. We conclude that seclusion was most frequently studied, followed by multiple concurrent restrictive practices, mechanical restraint, rapid tranquillisation and, finally, less frequently, manual restraint. Additionally, particular ethnic minorities appeared to be more likely than others to experience restrictive practices. Therefore, further research is warranted exploring how people from different ethnic backgrounds are subjected to restrictive practices in routine care. Standardisation of the language of restrictive practices and ethnicity is vital to truly understand this. Supplementary Information Below is the link to the electronic supplementary material.Supplementary file1 (PDF 143 KB) Supplementary file2 (PDF 646 KB) Acknowledgements The authors are grateful to the research colleagues in our network who guided our literature search in further directions towards potentially relevant literature. We also take this opportunity to thank informatics specialist Anne-Marie Fiala Carlsen, Ronni Laulund and Naja Locht Amsinck for providing valuable input to the literature search and strategy and/or to the manuscript. Author contributions All authors contributed to the study design. MLP, FAG and EBT conducted the literature searches. MLP and EBT undertook the screening, selection of studies for inclusion and data extraction. The analysis was primarily undertaken by MLP, and continuously discussed with FAG, JB and EBT. MLP made the first draft and all other authors critically reviewed and commented on the manuscript. The final manuscript version was approved by all authors before submission. Funding No external funding was received. Data availability All data and material generated and analysed in the present review are available in the published paper or in supplementary material. Declarations Conflict of interest The authors declare that they have no conflict of interest. ==== Refs References 1. Bowers L Ross J Nijman H Muir-Cochrane E Noorthoorn E Stewart D The scope for replacing seclusion with time out in acute inpatient psychiatry in England J Adv Nurs 2012 68 4 826 835 10.1111/j.1365-2648.2011.05784.x 21749438 2. 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==== Front Int J Pept Res Ther Int J Pept Res Ther International Journal of Peptide Research and Therapeutics 1573-3149 1573-3904 Springer Netherlands Dordrecht 36471676 10478 10.1007/s10989-022-10478-y Article A Review: The Antiviral Activity of Cyclic Peptides http://orcid.org/0000-0001-9056-5707 Chia Le Yi 1 http://orcid.org/0000-0003-1129-7084 Kumar Palanirajan Vijayaraj [email protected] 1 http://orcid.org/0000-0001-7515-6921 Maki Marwan Abdelmahmoud Abdelkarim 1 http://orcid.org/0000-0002-8257-5363 Ravichandran Guna 2 http://orcid.org/0000-0001-6460-1612 Thilagar Sivasudha 2 1 grid.444472.5 0000 0004 1756 3061 Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, UCSI University, No. 1, Jalan Menara Gading, Taman Connaught, Cheras, 56000 Kuala Lumpur, Malaysia 2 grid.411678.d 0000 0001 0941 7660 Department of Environmental Biotechnology, School of Environmental Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620024 India 1 12 2022 2023 29 1 716 11 2022 © The Author(s), under exclusive licence to Springer Nature B.V. 2022, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. In the design and development of therapeutic agents, macromolecules with restricted structures have stronger competitive edges than linear biological entities since cyclization can overcome the limitations of linear structures. The common issues of linear peptides include susceptibility to degradation of the peptidase enzyme, off-target effects, and necessity of routine dosing, leading to instability and ineffectiveness. The unique conformational constraint of cyclic peptides provides a larger surface area to interact with the target at the same time, improving the membrane permeability and in vivo stability compared to their linear counterparts. Currently, cyclic peptides have been reported to possess various activities, such as antifungal, antiviral and antimicrobial activities. To date, there is emerging interest in cyclic peptide therapeutics, and increasing numbers of clinically approved cyclic peptide drugs are available on the market. In this review, the medical significance of cyclic peptides in the defence against viral infections will be highlighted. Except for chikungunya virus, which lacks specific antiviral treatment, all the viral diseases targeted in this review are those with effective treatments yet with certain limitations to date. Thus, strategies and approaches to optimise the antiviral effect of cyclic peptides will be discussed along with their respective outcomes. Apart from isolated naturally occurring cyclic peptides, chemically synthesized or modified cyclic peptides with antiviral activities targeting coronavirus, herpes simplex viruses, human immunodeficiency virus, Ebola virus, influenza virus, dengue virus, five main hepatitis viruses, termed as type A, B, C, D and E and chikungunya virus will be reviewed herein. Graphical Abstract Keywords Cyclic peptides Antiviral activity Coronavirus Dengue virus Fundamental Research Grant Scheme (FRGS), Ministry of Higher Education (MOHE), Malaysia.FRGS/1/2021/ SKK0/UCSI/02/5 Research excellence and innovation grant Under Centre of Excellence in Research, Value Innovation and Entrepreneurship (CERVIE), UCSI University, Malaysia.REIG-FPS-2020-052 issue-copyright-statement© Springer Nature B.V. 2023 ==== Body pmcIntroduction In drug discovery, macromolecules, such as antibiotics, proteins, peptides and nucleic acids, are currently attracting increasing interest as therapeutic candidates, although small biological entities remain central to the market (Siang Ong et al. 2017). Peptides have relatively straightforward production, low toxicity, high structural diversity, good selectivity and strong binding affinity towards the desired biological targets (Ayoub and Scheidegger 2006; Passioura et al. 2014; Siang Ong et al. 2017). However, the existing barriers of linear peptide stability in the human body, including low oral bioavailability, high risk of proteolytic degradation and high excretion rate, greatly influence their development in therapeutic fields (Ayoub and Scheidegger 2006; Gongora-Benitez et al. 2014; Wang and Craik 2016; Siang Ong et al. 2017). Although polypeptides are used to treat various ailments and diseases, major drawbacks are their in vitro and in vivo stability. Currently, researchers overcome this problem by developing various novel drug delivery systems (Kumar et al. 2018; Kumar et al. 2019; Chellathurai et al. 2021; Tee et al. 2021). One of the smartest approaches that is predicted is converting these polypeptides into cyclic peptides, but their pharmacological properties and stability need to be proven once again after conversion. The findings from the researchers supported that cyclic peptides, polypeptide chains with a closed structure (Joo 2012), are the more favourable alternatives that are free from the majority of these challenges. Cyclic peptides demonstrate their resistance to exopeptidases and even endopeptidases, showing improved in vivo stability (Joo 2012; Siang Ong et al. 2017). With a bioactive conformation and decreased entropic penalty for binding, target selectivity can be improved and ensures stronger binding interactions (Siang Ong et al. 2017). In addition, cyclization of peptides has the advantage of cell penetration under some conditions (Siang Ong et al. 2017). Thus, drug discovery and development with cyclic peptides has recently become a significant direction of study based on their unique structural features. Due to their wide spectrum of biological activities and the formation of unique folded structures, cyclic peptides have been employed in applications ranging from drug design to nanotechnology (Tapeinou et al. 2015). Recently, the generation of peptide nanostructures, such as nanospheres, nanofibers and nanotubes, has been exploited for the purpose of drug delivery, medical imaging tools, and tissue engineering. Ghadiri et al. demonstrated a pioneering design of a novel class of heterochiral cyclic peptides in nanotubular structures, and this transmembrane channel structure was proposed to be a potential delivery agent into living cells in gene therapy (Mandal et al. 2014). In the pharmaceutical industry, great success in the bioengineering of linear peptide cyclization from the N- to C-terminus has specifically contributed to peptide toxin progression (Thapa et al. 2014). Current therapeutic cyclic peptides permitted by the Food and Drug Administration (FDA) consist of natural derivatives or artificial analogues resembling the natural substrate (Table 1) (Zorzi et al. 2017; Al Shaer et al. 2020; Choi and Joo 2020, Buckton et al. 2021, Zhang and Chen 2021). They are able to exhibit various biological activities. Examples of natural cyclic peptides are gramicidin S with antimicrobial activity, vancomycin with antibacterial effect and cyclosporine A with immunosuppressive activity (Ayoub and Scheidegger 2006; Joo 2012; Jin 2020). In discovering and designing cyclic peptide drugs, various sound approaches have been practised to optimise the desired properties for drug delivery. In addition to the well-established biological methods, such as split intein circular ligation of peptides and proteins system (SICLOPPS), phage display, and mRNA display (Siang Ong et al. 2017; Liras and Mcclure 2019), cyclization of peptides can be achieved chemically via techniques such as head-to-side chain, side chain-to-side chain, head-to-tail and side chain-to-tail, which are determined by the functional groups (Fig. 1) (White and Yudin 2011; Vitali and Fasan 2017; Hayes et al. 2021).Table 1 Cyclopeptides approved in the last 10 years (2011–2021) approved by the EMA and/or FDA Name Activity or indication Route of administration First clinical use [177Lu]-DOTATATE Al Shaer et al. (2020) Gastroenteropancreatic neuroendocrine tumors Intravenous 2018 [64Cu]-DOTATATE Al Shaer et al. (2020) Positron emission tomography (PET) imaging Intravenous 2020 [68 Ga]-DOTATATE Buckton et al. (2021) PET imaging Intravenous 2016 [68 Ga]-DOTATOC Al Shaer et al. (2020) PET imaging Intravenous 2019 [68 Ga]-PSMA-11 Al Shaer et al. (2020) Diagnosis of recurrent prostate carcinoma by PET Intravenous 2020 Bremelanotide Al Shaer et al. (2020) Women hypoactive sexual desire disorder Subcutaneous 2019 Dalbavancin Zorzi et al. (2017) Complicated skin and skin structure infections (CSSSIs) Intravenous infusion 2014 Linaclotide Choi and Joo (2020) Irritable bowel syndrome (IBS-C), chronic idiopathic constipation (CIC) Oral 2012 Oritavancin Zorzi et al. (2017) CSSSIs Intravenous infusion 2014 Pasireotide Zorzi et al. 2017) Cushing’s disease, acromegaly, neuroendocrine tumors Subcutaneous, intramuscular 2012 Peginesatide Zorzi et al. (2017) Anemia associated with chronic kidney disease Intravenous, subcutaneous 2012 Plecanatide Choi and Joo (2020) CIC Oral 2017 Setmelanotide Al Shaer et al. (2020) Obesity Subcutaneous 2020 Voclosporin Zhang and Chen (2021) Lupus nephritis Oral 2021 Fig. 1 Schematic illustration of peptide cyclization modes. Created with BioRender.com Current and emerging viral diseases have evolved as public health threats, causing morbidity and death worldwide over the human development timeline (De Clercq and Li 2016; Hoffmann et al. 2020). However, there are still inadequate effective therapeutics for most viruses to date (Hoffmann et al. 2020). In fact, the current treatment of some viral infections faces a dilemma, as the approved antiviral treatments are less than satisfactory and presented with certain limitations (Balasubramanian et al. 2019; Farooq and Ngaini 2021; O’Donnell and Marzi 2021). Hence, to minimize the impact of virus outbreaks, novel antiviral drug discovery is of utmost importance. In conjunction with the fast-evolving biomedical and biotechnology developments, antiviral cyclic peptides can be synthesized not only through the fermentation of natural sources but also via synthetic modifications from linear peptides to achieve the anticipated biological activities. With high feasibility and possibility, substantial attention is given to cyclic peptides with antiviral activity to occupy this void space to combat viral infections (Hoffmann et al. 2020). In this review, antiviral cyclic peptides targeting different viral strains of infectious diseases will be discussed. Natural and Synthetic Cyclic Peptides With the advancement in the biotechnology, the cyclic peptides can be synthesised or modified accordingly in order to obtain the desired biological activities based on rational design despite the fact that the majority of the clinically approved cyclic peptides in the market are the natural product derivatives (Abdalla and McGaw 2018). Naturally occurring cyclopeptides have been extracted from various sources including algae, fungi, plants, mammals, and bacteria, be it on the land or in the ocean (Agarwal and Gabrani 2021). They have a wide range of size composing of the 20 proteinogenic amino acids with one or more disulphide bonds. The naturally synthesized cyclic peptides share common characteristics of well-defined structures and exceptional stability even in unfavourable biological environment (Bockus et al. 2013). In addition, they typically possess the ability on host defence of their producing organisms (Thorstholm and Craik 2012). On the other hands, the natural linear peptides with significant drawback of instability towards proteolysis require structural modifications to maintain their feasibility for the pharmaceutical industry (Katsara et al. 2006; Tapeinou et al. 2015). To date, diverse strategies have been introduced and concentrated on the remodelling of the cyclic peptidomimetics. There are three principal technologies being introduced chronologically, which are direct isolation from host organism, chemical synthesis via solid phase peptide synthesis (SPPS) or chemo-enzymatic synthesis, and biological synthesis employing recombinant expression in plants or bacteria (Thorstholm and Craik 2012). For example, cyclization to increase stability, incorporate unnatural amino acids to improve specificity and introduce assorted structural restrictions. The novel modality forces the ordered secondary arrangement of a peptide, enhancing its metabolic stability and oral bioavailability. The cyclic mimetics are more preferred over the larger proteins in terms of lower immunogenicity while preserving the selectivity and potency (Tapeinou et al. 2015). However, it is difficult to obtain the native fold when the original sequence of the peptide is altered (Thorstholm and Craik 2012). The Antiviral Activities of Synthetic Cyclic Peptides Cyclic Peptides in Coronavirus Therapy Coronaviruses (CoVs) are grouped under the Coronaviridae family (Ali and Alharbi 2020). The outbreak of CoV disease in December 2019 (COVID-19) is a viral disease caused by severe acute respiratory syndrome CoV 2 (SARS-CoV-2) (Ali and Alharbi 2020; Brodin 2021). In December 2021, the WHO reported that SARS-CoV-2 variants of concern (VOCs) variants are Alpha, Beta, Gamma, Delta, and Submicron, whereas the variants of interest (VOIs) includes the Epsilon, Theta, Lambda and Mu (Konings et al. 2021, Parums 2021, WHO 2021). The mutation in SARS-CoV-2 variants mainly enhances viral attachment to the host cell and therefore facilitates its entry, thus having various degrees of spreadability (Davies et al. 2021). Researchers revealed that in the Omicron variant, there are 15 RBD region mutations among the variations concealed in its spike protein. Unlike Mu, Beta, and Gamma variants having E484K substitutions, E484A and Y505H substitutions are reported in the Omicron variant (Woo and Shah 2021; Kannan et al. 2022). From the investigations by Woo and his colleagues, the E484A substitution in the Omicron variant facilitates evasion of the immune response by significantly reducing the binding energy between its RBD and the approved therapeutic monoclonal antibodies, together with other replacements of T478K, Q493K, and Q498R, which strengthen ACE2 binding (Woo and Shah 2021). With greater binding affinity and antibody neutralization ability, the Omicron variant is estimated to have an infectability that is three to 6 times greater than that of the Delta variant, given the same period of time (Callaway and Ledford 2021). SARS-CoV-2 facilitates its entry into human cells through the binding of the spike protein RBD to cell-surface ACE2 receptors in the lung (Ali and Alharbi 2020; Shang et al. 2020; Norman et al. 2021). Thus, to develop new antiviral agents against SARS-CoV-2, targeting the RBD-ACE2 interaction is potentially an effective strategy (Norman et al. 2021). Norman et al. have utilized mRNA display to discover cyclic peptides with high glycoprotein binding affinity ligands towards SARS-CoV-2 spike protein (Norman et al. 2021). Among the nine target cyclic peptides being evaluated, peptide 4 (Fig. 2a) has the highest affinity to RBD with a detected nanomolar dissociation constant, KD with a value of 15 nM and a noticeably low dissociation rate of 1.4 × 10–3 s−1. Peptide 5 (Fig. 2b) recorded a KD value of 76 nM (Norman et al. 2021). However, useful detection of the spike protein was identified, although the tested peptides did not counteract the cells infected with SARS-CoV-2, which might be due to the distal ligand binding of RBD to the ACE2 binding site (Norman et al. 2021) (Fig. 3). The complexation of peptide 4 with the SARS-CoV-2 spike protein provided a unique binding mechanism of cyclic peptides neighbouring the C- and N-end domains. This result suggested that spike protein restructuring is vital for ligand accommodation. In addition, the possibility of hybridization by designing lipid-linked variants of peptide 4 is another interesting direction of study to improve spike RBD affinity and antiviral activity, as the binding site was found to be located next to a newly revealed SARS-CoV-2 RBD fatty acid binding pocket (Toelzer et al. 2020; Norman et al. 2021).Fig. 2 Cyclic peptide structure of a peptide 4 and b peptide 5 (Norman et al. 2021) Fig. 3 Mechanism of SARS-CoV-2 binding to the human ACE2 receptor and entering the host cell. Cyclic peptide is able to restructure the spike protein, influencing the nature of the interaction between the RBD and the binding site of the ACE2 surface receptor. (Norman et al. 2021) Created with BioRender.com In addition, the main protease (Mpro) of SARS-CoV-2 is utilized as a specific intracellular target for the development of new antiviral agents (Zhang et al. 2021). With the number of cleavage sites of the two polyproteins pp1a and pp1ab not less than eleven sites, together with papain-like protease (PLpro), Mpro has an important role in viral replication and viability (V’kovski et al. 2021). Notably, the highly specific proteolytic activity of the Leu–Gln↓Xaa sequence (as ↓ indicates the cleavage site and Xaa could be Asn, Ala or Ser) has not been reported for human proteases (Zhang et al. 2020, Johansen-Leete et al. 2022). Hence, this unique finding leads to the potential search for peptidomimetic-based or peptide inhibitors of Mpro. Despite the currently available peptidomimetic Mpro inhibitors either in the market or under clinical trials (Boras et al. 2021; Owen et al. 2021; Yang et al. 2021), macrocyclic peptides are attractive candidates because of their high binding selectivity and greater proteolytic stability in comparison with their linear counterparts (Vinogradov et al. 2019). Johansen et al. utilized random nonstandard peptide integrated discovery technology coupling mRNA display with flexizyme-mediated genetic code reprogramming and successfully identified cyclic peptides with potential antiviral activity as inhibitors of SARS-CoV-2 Mpro (Johansen-Leete et al. 2022). The importance of peptide cyclization has been proven to impose strain on the peptide, avoiding the adoption of a wholly relaxed configuration (Johansen-Leete et al. 2022). Cyclic Peptides in Dengue Therapy Dengue virus (DENV) is an enveloped vector-borne Flaviviridae family virus with positive single-stranded RNA (Muller et al. 2017). In the viral infection cycle, structural proteins, especially the envelope (E) protein, facilitate viral entry through interactions with host cell receptors (Fig. 4) (Chew et al. 2017). The E protein can be divided into envelope domains (ED) I, II and III (Rodenhuis-Zybert et al. 2010; Alhoot et al. 2013). The important contribution of EDIII in receptor recognition for viral attachment has been reported by several studies; therefore, targeting DENV E structural protein could be considered an effective strategy to inhibit the entry of the virus into the host cell (Hung et al. 2004; Swaminathan and Khanna 2009). Researchers have gained valuable information and a better understanding of the well-determined E structural protein by utilizing advanced techniques such as cryo-electron microscopy, nuclear magnetic resonance spectroscopy and X-ray crystallography (Modis et al. 2004; Volk et al. 2007; Fibriansah et al. 2015). Thus, various strategies have been applied in the development of antiviral drugs against the E protein. Among them, a novel peptide DN59 at < 25 µM with > 99% inhibition rate of DENV-2 was reported by Hrobowski et al. in 2005 (Hrobowski et al. 2005). However, a sequence of certain mechanisms is required for the peptide to otherwise lose its function in inhibiting DENV infection due to possible unnecessary intramolecular interactions. A constrained structure of the peptide can help overcome this issue. In 2013, a study was conducted to screen marketed cyclic peptides through in silico studies (Parikesit and Tambunan 2013). Among all ligands, brain natriuretic peptide (BNP) (7–32) porcine, amylin human and big endothelin (1–38) human are the three ligands able to enter the envelope protein cavity as a whole. From the molecular dynamic simulation, BNP (7–32) porcine is stable without conformational changes at different temperatures. Despite the promising results, further in vitro and in vivo studies are required to evaluate the practicability of cyclic peptides as effective ligands against DENV. In fact, with the property of a highly conserved E stem region among flaviviruses, it is likely for the antiviral cyclic peptide inhibiting one DENV serotype to exhibit the same action on other serotypes and even closely related flaviviruses, including Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBE), West Nile virus (WNV) and yellow fever virus (YFV) (Hrobowski et al. 2005; Schmidt et al. 2010; Chew et al. 2017).Fig. 4 DENV infection cycle. Virus binding and membrane fusion are organized by the envelope protein in a pH-dependent environment. (Chew et al. 2017) Adapted from “ZIKV Infection Cycle”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates Among the nonstructural (NS) components, NS3 consists of adenosine triphosphate (ATP)-dependent helicase and serine protease at its C-terminus and N-terminal domain, respectively (Moreland et al. 2010; Ross 2010). It is responsible for viral processing. NS2B is an NS3 protease cofactor (Ross 2010). NS2B-NS3 protease is a two-component protease with a serine protease catalytic triad in a combination of Ser135, His51, and Asp75 that has a polybasic substrate recognition profile (Noble et al. 2012; Takagi et al. 2017). This lipophobic catalytic site obstructs the design of potent micromolecule inhibitors that mimic DENV NS2B-NS3 protease. With the incorporation of an electrophilic warhead into substrate mimetic inhibitors, the inhibitory activities can be improved but not much impact on the specificity to DENV protease and the cell penetrability to give an antiviral effect (Takagi et al. 2017). Cyclic peptides target the regulation of protein–protein interfaces and other plane binding surfaces, which is difficult for the characteristic “rule of 5” small entities to accomplish (Matsson et al. 2016). Takagi et al. adopted the cyclization strategy by on-resin head-to-tail for high-throughput cyclic peptide synthesis (Takagi et al. 2017). From their findings, cyclic peptide 22 (Fig. 4, Table 2) was the strongest blocker (IC50 = 0.95 µM, where IC50 is the 50% of the maximal inhibitory concentration in the evaluation of the in vitro enzymatic inhibition). Using molecular modelling, the cyclic peptide consisting of a stable, active β-turn-like conformation with DDLL (D-Phe-D-Pro-L-Lys-L-Arg) sequencing at the P4’—P4—P3—P2 position was docked with the protease. Cyclic peptides with appropriate placement of the hydrophobic aromatic residue and an extra Arg revealed anti-DENV efficacy. Peptide 32 (Fig. 5, Table 2) had a low cytotoxic concentration with a remarkable antiviral effect (EC50 = 2 µM, where EC50 is the half maximal effective concentration to induce antiviral effect by 50% compared to an infected control culture without treatment). It is believed that peptide 32 (Fig. 5, Table 2) is the first cyclic peptide showing a hindering effect against NS2B-NS3 protease and possesses an antiviral effect towards DENV. Unlike peptide 22, peptide 33 (Fig. 5, Table 2), with the replacement of an amino methylene group as a substitute of an amide bond at site P1–P1’, was found to be stable against hydrolysis by the protease enzyme while maintaining enzyme inhibition and antiviral activity (IC50 = 2.1 µM, EC50 = 11.4 µM) (Takagi et al. 2017).Table 2 The moieties at different positions of the cyclic peptides. The amino acids are stereoisomers designated based on the optical direction of the plane-polarized light, with D-indicating dextro-rotatory (to the right) and L-indicating levo-rotatory (to the left). (Takagi et al. 2017) Cyclic peptide Sequence P4 P3 P2 P1 P1′ P2′ P3′ P4′ 22 D-Pro L-Lys L-Arg L-Lys L-Ser L-hPhe L-Ser D-Phe 32 D-Arg L-Arg L-Arg L-Lys D-2Nal L-hPhe L-hPhe D-2Nal 33 D-Arg L-Arg L-Arg L-Lys-Ψ[CH2NH]-L-Ala L-hPhe L-1Nal D-Phe Fig. 5 Cyclic peptide modelled by Takagi et al. (Takagi et al. 2017) Other than the extremely hydrophilic and general nonprime side (P), the prime side (P’) of the active site of DENV protease (Saleem et al. 2019) was also reported to be highly effective against DENV protease (Ki = 26 nM) (Lin et al. 2017). Lin’s group studied the interaction between both sides of the active site of DENV protease and cyclic peptides as potential inhibitors. With an optimized amino acid sequence and length in addition to an appropriate cyclization linkage, the findings showed that the tightest cyclic peptide targeting both sides had the potential to reach specificity and lower hydrophilicity as an inhibitor (Ki = 2.9 µM) against wild-type (WT) DENV3 protease (Lin et al. 2017). However, even with the inclusion of a second loop to support the structure, its flexibility is greater than the equivalent loops in aprotinin, potentially leading to the decreased binding affinity and weaker interactions than those in aprotinin, as evidenced by the fact that aprotinin has a comparatively lower Ki value against DENV3 WT protease (Lin et al. 2017). In addition, the enzymes NS3 and NS5 with known activity in DENV duplication are the supreme targets in the manufacturing of antiviral therapeutic agents (Noble et al. 2010; Lim et al. 2015). The Y-shaped slit of NS5 methyltransferase (NS5 Mtase) allows it to connect to two active sites (the RNA-cap binding site and S-adenosyl-l-methionine (SAM) binding site), where two-step methylation takes place from the binding site of SAM to the guanine base of RNA (Lim et al. 2011; Lim et al. 2013; Brecher et al. 2015). Idrus et al. reported the design of cyclic peptide inhibitors targeting both NS5 methyltransferase binding pockets (Idrus et al. 2012). The cyclic pentapeptide inhibitors consisted of terminal cysteines at both ends with a combination of three other amino acids in the middle, along with an S–S bridge to increase the stability of the ligand. The findings reported that the cyclic peptide CTWYC (Fig. 6a) has a more stable structure to inhibit the SAM active site, whereas the CYEFC structure (Fig. 6b) has better strength to bind with the RNA-cap pocket, with binding free energies (∆Gbinding) of − 30.72 and − 22.89 kcal.mol−1, respectively (Idrus et al. 2012). The lower ∆Gbinding indicated more stable binding of ligands to the target protein compared to the standards (Tambunan et al. 2017).Fig. 6 Cyclic peptides a CTWYC and b CYEFC illustrated by Indrus et al. (Idrus et al. 2012) Cyclic Peptides in Human Immunodeficiency Virus Therapy Human immunodeficiency virus (HIV) is a lentivirus that belongs to the Retroviridae family and causes acquired immunodeficiency syndrome (AIDS) (Girard et al. 2011). CD4+ helper T-cells are the desired target where depletion will cause immunocompromised, increasing the risk of opportunistic infections (Fauci 2003; Girard et al. 2011). The interaction of the HIV Gag p6 region (a domain containing a P(T/S)AP tetrapeptide motif) with the domain ubiquitin E2 variant (UEV) of host protein tumour susceptibility gene 101 (TSG101) is vital to complete the vital life cycle (Garrus et al. 2001, Liu et al. 2008; Tavassoli et al. 2008). Tavassoli et al. reported the practice of the reverse two-hybrid system (RTHS) of bacteria to recognize the cyclic peptides that restrict the interaction between Gag and TSG101. It was observed that cyclic peptides with five amino acids consisting of threonine at the first position and tryptophan at the third position can interrupt and inhibit HIV egress (Tavassoli et al. 2008). Reducing virus-like particle (VLP) release has been suggested as a potential HIV infection treatment in practical use. The selected inhibiting molecule has a primary sequence different from the sites of interaction of Gag and TSG101. The HIV Tat-tagged cyclic peptide inhibited VLP production in addition to enhancing protein translocation across mammalian cell membranes (Tavassoli et al. 2008). The inhibitory effect on PTAP-dependent Gag budding is dose-dependent (IC50 = 7 µM) without any cytotoxic effects (Tavassoli et al. 2008). Therefore, cyclic peptides can interfere with TSG101-Gag protein interactions with the addition of translocation-promoting amino acid sequences, but this has no effect on the Gag protein mutation (Tavassoli et al. 2008). Therefore, conclusions can be made on small biological therapeutics that inhibit specific virus-host protein interactions and are believed to have wide-ranging applicability in antiviral therapy, but existing countermeasures may be readily circumvented by viral mutations, resulting in ineffective targeting of the viral budding process (Tavassoli et al. 2008). In viral replication, Tat and Rev are both basic regulatory proteins of HIV-1 (Sherman and Greene 2002), having a similar arginine rich motif (ARM) that plays a role in the mediation of host-cell nuclear import, translocation over biological membranes, and binding to targeted viral RNA sequences (Futaki et al. 2001; Seelamgari et al. 2004; Hariton-Gazal et al. 2005). Hariton-Gazal et al. used karyophilic backbone cyclic (BC) ARM peptides to target the ARM domain to develop a therapeutic agent against HIV-1 and as a delivery carrier to living cells (Hariton-Gazal et al. 2005). The findings revealed that the BC Rev-ARM analog (Rev-13) (Fig. 7) effectively blocks Rev-GFP from binding to the importin β receptor. Moreover, it influences the penetration of bovine serum albumin (BSA). An ELISA-based study revealed that the majority of backbone cyclic peptides (BCPs) from the library possess both cell-penetrating characteristics and binding ability by transporting BSA into intact Colo-205 cells (Hariton-Gazal et al. 2005). In addition, the BC Rev-13 (Fig. 7) analog showed the ability to inhibit the expression of Rev-induced genes in the low micromolar range (2–5 µM) without showing any cytotoxic effect (Hariton-Gazal et al. 2005).Fig. 7 Peptide Rev-13 (Hariton-Gazal et al. 2005) As mentioned previously, the life cycle of HIV-1 is dependent on Rev protein for the regulation of viral mRNA expression by enhancing the production of viral structural protein precursors (Chaloin et al. 2007). Chaloin et al. investigated in vitro the ability of a series of BCP analogues having a conformational-controlled ARM of Rev for HIV-1 replication suppression. Rev-BCPs showed remarkable inhibitory activity against HIV-1 in persistently infected T lymphocytic cells. In addition, the nuclear import process was affected by Rev-BCPs, and there was significant efficiency in blocking the action controlling polyprotein precursor protein Pr55Gag and envelope glycoprotein precursor gp160env production (Chaloin et al. 2007). Remarkably, Rev binding is necessary for the nuclear export of these mRNA-encoded protein precursors (Smagulova et al. 2005). By utilizing an HIV-1 Gag oligonucleotide probe conjugated with Cy-3, in situ hybridization revealed that Rev-BCPs are able to inhibit the intracellular accumulation of unspliced viral RNA (Chaloin et al. 2007). In silico studies revealed that recombinant simulation proposes that upon binding to the Rev-response element, Rev-BCP 14 (Fig. 8) is the most promising analogue with an observed IC50 = 6.2 µM and was able to broaden the distorted viral RNA major groove (Chaloin et al. 2007). In addition to the strong electrostatic interactions present between the positive charges of the peptides and the phosphate backbone, various contacts between RNA and peptide were discovered within the complex, and some are important for the interactions, for example, the hydrogen bonds and the hydrophobic contacts (van der Waals) between the carboxy-amidated peptide end and the RNA backbone phosphate (Chaloin et al. 2007; Young et al. 2011). Altogether, the use of structurally restricted Rev-BCPs is a promising tactic to develop novel cyclic peptide-based therapeutics against HIV-1, with further improvement on the reduction in the total inhibition concentration (Chaloin et al. 2007).Fig. 8 Peptide REV-14 (Hariton-Gazal et al. 2005) To date, various affinity-based methods, such as encoded synthetic peptide libraries, DNA directed synthesis, and phage and ribosomal display, are commonly enriched for the synthetic and in vitro generation of large libraries of peptides from synthetic and natural amino acids (Sandman et al. 2000; Sattely et al. 2008; Young et al. 2011). Young et al. reported a microbial system for cyclic peptide evolution that utilizes a series of lengthened amino acid building blocks (Young et al. 2011). The combination of SICLOPPS with orthogonal aminoacyl-tRNA synthetase∕tRNACUA pairs was used to biosynthesize a library of ribosomal peptides encompassing amino acids with exclusive reactivity and structure (Young et al. 2011). According to cellular viability, the selection was performed in the peptide library, and the HIV protease inhibitor in Escherichia coli (E. coli) was evolved. After selection and isolation, two cyclic peptides containing p-benzoylphenylalanine (pBzF) amino acids were reported to have a moiety of electrophilic aryl ketone and a fairly large hydrophobic surface area (Young et al. 2011). With observed cyclic IC50 = 0.96 µM and linear IC50 of 6.67 µM, the encoded GIXVSL clone, in which X is pBzF, was the most potent HIV protease blocker (Fig. 9). Therefore, the pBzF amino acid residue is believed to covalently form a Schiff base adduct with the protease by binding to the ϵ-amino group of Lys-14, suggesting that in response to selective pressure, the expansion of the genetic code could have evolutionary benefits. Therefore, further studies could combine chemical building blocks with natural evolutionary processes to develop novel biologically active peptides (Young et al. 2011).Fig. 9 Cyclic peptide HIV protease inhibitor G12 (Young et al. 2011) Cyclic Peptides in Ebola Virus Disease Treatment In 1976, Ebola virus (EBOV) was first identified in the Democratic Republic of the Congo (Jacob et al. 2020). It is an enveloped virus with negative-stranded RNA belonging to the family Filoviridae (Geisbert et al. 2003) and has been discovered to be the causative agent of Ebola virus disease (EVD) (Baize et al. 2014). Studies have been conducted and they found out that by blocking the construction of the six-helix bundle structure of the glycoprotein 2 (GP2) ectodomain can restrict the entry of the genetic material of EBOV into the cytoplasm (Miller et al. 2011; Tambunan et al. 2017). In silico modelling was utilized to investigate the interaction between the modified commercial cyclopeptide ligands and the EBOV N-terminal heptad repeat (NHR) GP2 ectodomain. The ligand that interacts with the amino acid residues Arg B580, Glu C578, Phe C572, and Thr B576 was placed in the active site of the target protein and was found to form a stable protein–ligand complex with the best molecular interaction under both 310 and 312 K (Tambunan et al. 2017). The temperatures mentioned above indicate the normal body condition and the infected human body with theEBOV. As evidenced, its root mean square deviation is 3.0–3.5 Å at 310 and 312 K, whereas ΔGbinding is − 53.6622 kcal/mol. Moreover, the pharmacokinetics evaluation revealed that this particular ligand has good pharmacological characteristics, and no toxic effect was detected. (Garabedian et al. 2018). The conjugation of HIV-1 Tat ligand with commercial cyclic peptides has been proven to be effective in exhibiting inhibitory effects against EBOV (Tambunan et al. 2017). Therefore, further in vitro and in vivo investigations of its activity against EBOV could be conducted for its future therapeutic value. As previously mentioned, Tsg101 UEV domains are potential targets for HIV infection treatment. Studies have shown that the same strategy could also be utilized against EBOV. The goal is to protect the emerging virions from infected cells as immature particles. The resultant virus budding process failure can be achieved by interruption of the binding interaction between the PT/SAP motifs and UEV domain of Tsg101 proteins on EBOV Vp40 proteins (Pornillos et al. 2002; Lin et al. 2020). Due to the limited experimental 3D structure information available, a study conducted by Lin et al. utilized a beneficial methodology that involves various techniques, including Gaussian accelerated molecular dynamics (GaMD), normal molecular dynamics (cMD), solvated interaction energy (SIE), two-dimensional (2D), and potential of mean force (PMF) techniques, with the purpose of examining the binding conformations of cyclic peptides with UEV domain proteins (Lin et al. 2020). Their findings described Trp145, Tyr147, and Trp148 as the hot-spot residues of the cyclo-(Ser-Gly-Trp-Ile-Tyr-Trp-Asn-Val) inhibitor, which play a crucial role in the interaction with the UEV domain proteins via hydrogen bond formation and nonbonding interactions (Lin et al. 2020). Since the single point substitution might influence the 3D structures of the peptide and the protein receptor binding affinity (Iwazaki et al. 2005; Garabedian et al. 2018), Lin et al. revealed that the mutation of Tyr 147 reduces UEV domain protein binding abilities. From this study, the developments in computational approaches and calculating power (Wang and Cheng 2021) might escalate the prediction precision of cyclic peptide-protein conformation, which is able to expedite in tackling the blocking blocks towards cyclic peptide inhibitor design (Lin et al. 2020). Promising Progress of Cyclic Peptides in Chikungunya Therapy Chikungunya, an arthropod-borne virus (CHIKV), causes deleterious health effects globally for more than a half-century. This is distinguished as an alphavirus of the Togaviridae family, mainly spreading by mosquito-borne vectors Aedes albopictus and Aedes aegypti (Sourisseau et al. 2007, for Chikungunya 2021, Jagadesh et al. 2021). To date, there is no approved therapeutic medicine or vaccine available for CHIKV (August et al. 2021). Multiapproach research exploring potential anti-CHIKV drugs is in progress (Kumar et al. 2020; Bappy et al. 2021). Peptides are now receiving attention as potential alternatives to many conventional antiviral drugs available on the market. Since several of these peptides have anionic characteristics, they are easily transported into tumor cells and execute antiproliferative functions (Hoskin and Ramamoorthy 2008). Previously, antibacterial peptides have been widely documented for countering antibiotic resistance (Parachin and Franco 2014). In this milieu, researchers have started experimenting with cyclic peptides to combat multidrug resistive pandemic viruses, as they will have a huge impact on clinical research and medical practice. It is natural and synthetic cyclic peptide products (as pure and formulated) that have been designed to treat CHIKV (Boas et al. 2019). Similarly, attempts to discover cyclic peptide vaccines have also been recently initiated because vaccines can prevent and provide permanent protection against CHIKV infection. In addition to exploring active peptides, subsequently, it is also imperative to develop strategies to construct an appropriate design and predict an appropriate target to have a potential outcome. CHIKV uniquely contains approximately 240 copies of immunogenic E2 protein on its surface, which renders an attractive structure for finding suitable targets for detection as well as designing drugs or vaccines (Voss et al. 2010; Weger-Lucarelli et al. 2015). With the help of a library of one-bead-one-compound cyclic peptides, researchers have developed a multivalent maturation strategy to construct peptide-based capture receptors (peptide format: H2N-Pra-Cy(XXXXX)-TG) with satisfactory thermal stability to target unglycosylated E2 proteins of CHIKV. They synthesized macrocyclic peptides such as Pra-Cy(WIYYI), Pra-Cy(FWQIL), Pra-Cy(IYLRY), and Pra-Cy(YWHWS) (Fig. 10). Using high-performance computing algorithms, Pra-Cy(WIYYI) was found to be the best interacting macrocyclic peptide whose appropriate conformation for the effective E2 protein-peptide receptor affinity was identified (Coppock et al. 2019).Fig. 10 Structure of macrocyclic peptides synthesized by Coppock et al. (2019) The analysis displayed the binding of macrocyclic peptides with lysine residues in the hydrophobic surface of domain B of the E2 protein, K107, K200, and K280. Each of the peptides found interacted with more than one residue. WIYYI, YWHWS, and FWQIL peptides established affinity at all three residues, while YWHWS and IYLRY preferred targeted K200 locations with high enthalpy (Coppock et al. 2019). These predictions are in agreement with previous studies that targeted domain B of the E2 protein (Asnet Mary et al. 2013, Lam et al. 2015, Deeba et al. 2017). After multivalent enhancement, WIYYI macrocyclic peptide top performed with up to ninefold better selectivity and displayed affinity in 50% human serum, which was comparatively better than the commercial anti-E2 antibody. The multivalent enhancement of macrocyclic peptides was primarily attributed to their efficient binding with CHIKV E2. These peptides bind at multiple sites to viral surfaces and exert structural analogues and compatible physical features, such as hydrophobicity. This could limit their specificity to target a potential domain, as multivalency improves macrocyclic peptides to interact with several copies of the same target (Coppock et al. 2019). In terms of thermal stability, macrocyclic peptides are efficient over linear peptides since they have characteristic structural rigidity with stringent intramolecular arrangements, unlike their linear counterparts. Indeed, from the study, the best protein-peptide complex, (Pra-Cy(WIYYI)–CHIKV-E2), has also been successfully demonstrated with commercially available polyclonal antibodies, suggesting that cyclic peptides through this strategy can also be employed for detecting viruses other than CHIKV in sensor-based devices used in clinical diagnosis and environmental monitoring (Coppock et al. 2019). On the other hand, researchers have tried and successfully designed antiviral drugs to mitigate multiplication in the host by blocking the NS proteins that interact with enzymes involved in the replication process. However, unlike other viruses, it is challenging to target CHIKV, as it possesses a complex or intricate NS protein design (Wang et al. 2011; Brandler et al. 2013). Although recombinant CHIKV vaccines have been used to invoke an immune response in the host, they still have not been subjected to clinical trials despite protecting against arthritis in experimental animals. In search for alternatives, researchers are attempting antigenic peptide candidates to explore conserved B cell and T cell epitopes of CHIKV structural polyprotein to discover vaccines that would hopefully evoke a defensive immunogenic response (Tahir ul Qamar et al. 2018). Using respective computational tools, B cell epitopes (PPFGAGRPGQFGDI, IPTGAGKPGDSGRP, NYPASHTTLGVQDI, PFHHDPPVIGREKF, TSAPCTITGTMGHF, TPYELTPGATVPFL, PEGYYNWHHGAVQY and WLKERGASLQHTAP) and T cell epitopes (MHC-I alleles: IFDNKGRVVAIVL, PLVPRNAEL, VMHKKEVVL, LSVTLEPTL, TAECKDKNL, GTLKIQVSL & LQISFSTAL and MHC-II alleles: FKRSSKYDL, LLANTTFPC, LKIQVSLQI, FVRTSAPCTI, VGFTDSRKI, VRYKCNCGG, WVMHKKEVV & LVVAVAALILIVVLCVSFSR) with higher antigenicity, flexibility, accessibility of surface, linearity, and hydrophilicity were predicted and docked with the solved 3D structure of the human HLA-B7 allele. The analysis showed VMHKKEVVL, LSVTLEPTL, GTLKIQVSL, and LQISFSTAL as potentially interacting peptides with minimized binding energy (− 20.2086, − 19.6688, − 17.3569, & − 14.5594) (Tahir ul Qamar et al. 2018). The analysis showed that the best-interacted peptide, VMHKKEVVL, formed stable hydrogen bonds with Lys-243 and Glu-232 of chain A and Tyr-26 and Tyr63 of chain B of HLA-B7. In addition, the peptide also extended a water-mediated interaction with Thr-233 of the A-chain and Thr-10 of the B-chain (Tahir ul Qamar et al. 2018). The above peptides showed no toxicity compliance to the FAO/WHO standards for nonallergenicity. Since these peptides were characterized only through in silico models, they may show unstable physiochemical characteristics if tested in vitro or in vivo. There are some physiochemical modification strategies (multi-valent enhancement, analogue formations, hydrophobic ion pairing, macromolecular conjugation, substitution-based delivery) through which peptides can be improvised as potential targets to construct vaccines (Mahajan et al. 2014). Taking into account all these factors, it seems that cyclic peptides can provide a clear hope to successfully develop novel antiviral agents. In the near future, they may possibly emerge as suitable candidates for designing effective vaccines or medicines to combat the deadliest pandemic viruses. Cyclic Peptides in Influenza Virus Therapy The Orthomyxoviridae is a family of segmented RNA viruses, including influenza viruses (IVs), which are enveloped RNA viruses. Generally, the IVs that affect humans are three types: A, B and C. Type B and C viruses have no known subtypes, while Type A has many subtypes based on antigens (Kumar 2017, Suchitra Rao 2019). Polymerase (PB1, PB2, and PA), hemagglutinin (HA)and neuraminidase (NA)-glycoproteins, nucleoprotein (NP) and member protein (-M1 and -M2) are RNA-coded proteins (7 or 8 segments) of the influenza virus genome (Machała and Brydak 2006). These segments consist of one or more open-reading frames, segment-specific untranslated regions (UTRs) and partially complementary 5′ and 3′ termini. The double-helical rod-like structure is formed by the assembly of viral ribonucleoprotein complexes, as the viral RNA-dependent RNA polymerase bound the 5′ and 3′ termini and the oligomeric viral NP bound the rest of the RNA (Fodor and Te Velthuis 2020). Saito et al. developed a class of macrocyclic peptides (iHAs) targeting the viral envelope HA protein of IV. The in vitro examination of the developed iHAs revealed that iHA-24 and iHA-100 (Fig. 11) are capable of inhibiting the replication of IVs. iHA-100 inhibits HA-mediated adsorption and membrane fusion through binding to the stalk domain of HA. This demonstrates the potential development of mid-sized multifunctional cyclic peptides as next-generation molecules against IVs (Saito et al. 2021).Fig. 11 The chemical structures of iHA-24 and iHA-100 synthesized by Saito et al. 2021 (Saito et al. 2021) Horne et al. reported the selection of eight-residue cyclic d, l-peptide from a directed combinatorial library as a potential molecule to develop a low pH environment at endocytic vesicles, which could stop the emergence of virions from the endosome to prevent adenovirus infections without affecting cell viability. The inhibition of the emergence of IV type A from endosomes by the action of these peptides supports the potential utilization of this approach against other pH-dependent viral infections. Therefore, the discovery/design of broad-spectrum antiviral agents could be achieved by the self-assembling cyclic d, l-peptides as a new rational supramolecular approach. From the 20 hits found, peptide 1 (Fig. 12) was synthesized and used in the study because it significantly inhibited Ad5-mediated gene delivery. α-sarcin uptake has previously been found to correlate with enveloped viral entry. Interestingly, peptide 1 significantly reduced the delivery of influenza virus-mediated α-sarcin (Horne et al. 2005).Fig. 12 Schematic illustration of the antiviral action of membranes associating cyclic d, l-a-peptides to the endosomal membranes during virus internalization by offsetting the formation of a low pH environment inside endosomes via the membrane permeating supramolecular assemblies and the primary stages of the adenovirus infection cycle. a Schematic illustration of the primary stages of the adenovirus infection cycle. b The antiviral action of membranes associating cyclic d, l-a-peptides to the endosomal membranes during virus internalization by offsetting the formation of a low pH environment inside endosomes via the membrane permeating supramolecular assemblies. c The structure and sequence of the peptide identified by Horne et al. (Horne et al. 2005). (a and b are created with BioRender.com) Kadam et al. introduced a strategy to design macrocyclic peptides with the ability to inhibit the fusion of group 1 influenza A viruses (Fig. 13). These peptides are capable of binding to the conserved HA stem with a similar approach of broad neutralizing antibodies, which might inhibit the process of generating escape mutants (Kadam et al. 2017).Fig. 13 Amino acid sequences of cyclic peptides designed by Kadam et al. (Kadam et al. 2017) Cyclic Peptides in Hepatitis Virus Therapy The majority of the global burden of hepatitis is caused by hepatitis A virus (HAV), HBV, HCV, HDV, and HEV. HBV and HCV are responsible for the majority of chronic hepatitis morbidity cases and death (Lanini et al. 2019; Wang and Tsai 2021). Human HBV is characterized by an asymmetric replication mechanism and an unusual organization of its genome. It is circular in shape, with 3200 base pairs and four overlapping ORF regions: PreC/C, X, P, and Pré-S1/Pré-S2/S. The three envelope proteins of HBV, nuclear protein, core protein, surface antigen (HBeAg, HBcAg and HBsAg), viral polymerase (P), and X protein (affect viral proliferation and replication), are all encoded by these genes (Wen et al. 2008; Zhu et al. 2008, Souza et al. 2014). In a preferred but not obligated order, this polyprotein is processed into ten mature proteins; p7 separates the structural proteins (the envelope glycoproteins E1 and E2 and the core) from the NS3, NS4A, NS4B, NS5A and NS5B, which are essential for viral RNA replication (Lohmann 2013; Moradpour and Penin 2013; Niepmann 2013; Simmonds 2013; Madan and Bartenschlager 2015). A study utilizing a binding assay in solution revealed that peptides bearing the amino acid sequences C-WPFWGPW-C and C-WSFFSNI-C tightly bind to HBcAg and inhibit L-HBsAg-HBcAg association with KD rel < 25 nM, which is at least 10 orders of magnitude higher than the peptide (LLGRMK) identified from a linear peptide library (Ho et al. 2003). The cyclic peptide CP11 (CGWIYWNV) inhibits HEV viral protein interaction with the host cell, preventing the release of the virus (Anang et al. 2018). In addition, HCV15R (CR-Nal-RV-(D)-P-Cha-HRYRC-CONH2) cyclic peptide inhibits the entry of HCV by targeting cell receptors (Khachatoorian et al. 2015). Through interactions with HCV RNA, human La protein stimulates the translation of HCV. A cyclopeptide (LaR2C-N7-cy) synthesized by Manna et al. mimics the β-turn of the human La-protein and can interact with HCV-RNA. It significantly inhibits the translation of HCV-RNA better than the corresponding linear peptide (Fig. 14). This cyclic peptide was also found to inhibit the replication of HCV by measuring replicon RNA levels using RT-PCR (Manna et al. 2013).Fig. 14 Chemical structure of LaR2C-N7-cy synthesized by Manna et al. (Manna et al. 2013) The Antiviral Activities of Natural Isolated Cyclic Peptides Naturally isolated cyclic peptides possess medical significance as therapeutic agents with a wide range of biological activities comprising antiviral, anticancer, antibacterial, antifungal effects and so on (Abdalla and McGaw 2018). The cyclic peptides from various natural sources are documented (Table 3) with their respective antiviral activities studied in the previous literature.Table 3 Cyclic peptides from various natural sources with respective enzyme inhibition (IC50) and antiviral activities (EC50) Compound name Class Source IC50/EC50 References Animal (leukocytes) Retrocyclin-2 (RC-2, θ-defensin) Cyclic octadecapeptides Non-human ‘Old World’ primates HIV-1 -IC50: 2.33 µg/mL Wang et al. (2004) Cyclosporine Cyclic oligopeptide Tolypocladium inflatum fungi IV -IC50: 1.45 µM Hamamoto et al. (2013); Abdalla and McGaw (2018) Fungi Marine natural product Aspergillipeptide D Cyclic pentapeptides Marine gorgonian-derived fungal strain Aspergillus sp. SCSIO 41, 501 Herpes simplex virus type 1 (HSV-1) -IC50: 9.5 µM Ma et al. (2017) Asperterrestide A Cyclic tetrapeptide Marine-derived fungus Aspergillus terreus SCSG AF0162 IAV/WSN/33 (H1N1) -IC50: 15.0 µM IAV/Hong Kong/8/68 (H3N2) -IC50: 8.1 µM He et al. (2013) Celebeside A Celebeside C Cyclic depsipeptide Indonesian marine sponge Siliquariaspongia mirabilis HIV-1 -IC50: 2.1 μM -IC50: > 62 μM Kang et al. (2015) Didemnin A Cyclic depsipeptides Marine tunicate Trididemnum solidum Coxsackie virus, equine rhinovirus (both RNA), HSV-2 (DNA) -IC50: 1.5 µg/mL HSV-22 -IC50: 3 µg/mL Mata et al. (2017) Homophymine A-E Homophymine A1-E1 Cyclodepsipeptides Sponge Homophymia sp. III B strain of HIV-1 -IC50: 75 nM Kang et al. (2015) Koshikmaide B Koshikmaide F–H Cyclic peptide lactone Theonella sp. HIV-1 -IC50: 2.3 & 5.5 µM Kang et al. (2015) Mirabamide A Mirabamide C Mirabamide D Cyclic depsipeptides Sponge Siliquariaspongia mirabilis HIV -IC50: 40–140 nM -IC50: 140 nM-1.3 µM -IC50: 190 nM-3.9 µM Plaza et al. (2007) Mirabamide E Mirabamide F Mirabamide G Mirabamide H Cyclic depsipeptides Sponge Stelletta clavosa HIV-1 viral strain YU2-V3 -IC50: 121 nM -IC50: 62 nM -IC50: 68 nM -IC50: 41 nM Lu et al. (2011) Mollamides B Cyclic hexapeptide Indonesian tunicate Didemnum molle HIV-1 -EC50: 48.7 µM Donia et al. (2008) Papuamide A Papuamide B Cyclic depsipeptide Sponge Theonella sp. HIV -EC50: 3.6 ng/mL -IC50: 710 nM Ford et al. (1999); Sagar et al. (2010) Sansalvamide A Cyclodepsipeptide Marine fungus Fusarium sp. Topoisomerase of Molluscum Contagiosum Virus (MCV) -IC50: 124 µM Moghadamtousi et al. (2015) Simplicilliumtide J Cyclolipodepsipeptide Deep see-derived fungal strain Simplicillium obclavatum EIODSF 020 HSV-1 -EC50: 14.0 µM Liang et al. (2017) Theopapuamide A Theopapuamide B Cyclic undecapeptide Sponge Theonella swinhoei Siliquariaspongia mirabilis CEM-TART (T-cells that express both HIV-1 tat and rev) -IC50: 0.5 µM -IC50: 0.8 µg/mL Ngo et al. (2012) Verlamelin A Verlamelin B Cyclic hexadepsipeptide Deep see-derived fungal strain Simplicillium obclavatum EIODSF 020 HSV-1 -EC50: 16.7 µM -EC50: 15.6 µM Liang et al. (2017) Plant Circulin A Circulin B Cyclotide Chassalia parvifolia HIV -EC50: 70 nM Wani et al. (2020) Cycloviolacin VY1 Cycloviolacin Y5 Cyclotide Viola yedoensis IAV (H1N1) -IC50: 2.27 µg/mL HIV-EC50: 40 nM Wang et al. (2008, Liu et al. (2014) Kalata B1 Kalata B8 Cyclotide Oldenlandia affinis HIV-1-EC50: 140 Nm -EC50: 2.5 µM Gerlach and Mondal (2012) MCoT-I MCoT-II Cyclotide (Palicourein) Momordica cochinchinensis HIV -EC50: 100 nM Gerlach and Mondal (2012) Secondary metabolites Antimycin A Cyclic lactone Streptomyces kaviengensis Western equine encephalitis viruses (WEEV) -IC50: 3 nM Yi et al. (2020), Lin et al. (2020) Daptomycin Cyclic lipopeptide Streptomyces spp. ZIKV -IC50: 1 µM Jakubiec-Krzesniak et al. (2018) Aspergillipeptide D Aspergillipeptide D (Fig. 15) is a cyclic pentapeptide collected from the broth culture of the sea gorgonian-derived fungal strain Aspergillus sp. SCSIO 41, 501 (Ma et al. 2017; Abdalla and McGaw 2018; Jing and Jin 2020). It appears as a white crystal with the known molecular formula of C40H49N5O8 (Wang et al. 2020) From Ma et al. aspergillipeptide D (Fig. 15) showed a potent antiviral effect on HSV-1 (Ma et al. 2017). With their noncytotoxic concentrations (TC0) against the Vero cell line, aspergillipeptide D showed an IC50 of 9.5 µM. It also exhibited good antiviral effects towards two isolates, HSV-1-106 and HSV-V-153, which were clinically resistant to acyclovir at a concentration of 12.5 µM with approximately 50% inhibition rates (Ma et al. 2017).Fig. 15 Aspergillipeptide D (Ma et al. 2017) Simplicilliumtide Among the cyclic peptides discovered from the marine-derived fungal strain Simplicillium obclavatum EIODSF 020, Liang et al. showed that the novel cyclolipodepsipeptide simplicilliumtide J (Fig. 16a) has the molecular formula of C46H73N7O11 with the presence of a fatty acid chain moiety and lactone linkage, exhibiting an anti-HSV-1 effect with an EC50 value of 14.0 µM (Liang et al. 2017; Jing and Jin 2020). In addition, its noncytotoxic concentrations TC0 and TC50 values against Vero cells were observed to be 25.1 and 204 μM, respectively (Liang et al. 2017, Yi et al. 2020, Lin et al. 2020). The other simplicilliumtide cyclopeptides, for example simplicilliumtide K (Fig. 16b) and L (Fig. 16c), have less remarkable antiviral activity towards HSV-1 (El Maddah et al. 2020). It is believed that the activity reduction is due to the carbonyl substituent at C-13 or C-14 of (S)-5-hydroxy-13-ketotetradecanoic acid (HKTA) or (S)-5-hydroxy-12-ketotetradecanoic acid (HKTA), respectively (Liang et al. 2017; El Maddah et al. 2020).Fig. 16 Structure of Simplicilliumtides (Liang et al. 2017; El Maddah et al. 2020) Verlamelins Verlamelins A (Fig. 17a) and B (Fig. 17b) are known analogs of the cyclic peptide simplicilliumtide J–M. Recently, they were discovered to be isolated from the same crude fungal strain as simplicilliumtide J–M (Liang et al. 2017). Formerly, verlamelin A (Fig. 17a) was isolated from Verticillium lamellicola because of its antifungal activity (El Maddah et al. 2020). Later, together with verlamelin B (Fig. 17b), both were reported from Lecanicillium sp. HF627 in 2014 for the absolute configuration determination (Liang et al. 2017). Liang et al. determined their anti-HSV-1 activity through plaque reduction assays. Their corresponding EC50 values were 16.7 and 15.6 µM under TC0 against the Vero cell line. Verlamelin A (Fig. 17a) displayed TC0 and TC50 values of 57.2 and 137.0 μM, respectively, whereas verlamelin B (Fig. 17b) reported TC0 and TC50 values of 49.4 and 101.1 μM, respectively (Liang et al. 2017).Fig. 17 Structure of Verlamelins (Liang et al. 2017) Asperterrestide A With the investigation of the chemical constituents in the fermentation broth of Aspergillus terreus SCSGAF0162 isolated from gorgonian Echinogorgia aurantiaca tissue from Sanya, Hainan Province, China (Moghadamtousi et al. 2015), a new cyclic tetrapeptide asperterrestide A (Fig. 18) was isolated along with other compounds (He et al. 2013). From He et al. asperterrestide A (Fig. 18) was allocated with a molecular formula of C26H32N4O5. Asperterrestide A (Fig. 18) consists of a structure with an intermittent 3–OH–N–CH3-Phe moiety (He et al. 2013). From their research, the antiviral activity of asperterrestide A (Fig. 18) was tested on two strains of IV, A/WSN/33 (H1N1) and A/Hong Kong/8/68 (H3N2), by cytopathic effect (CPE) assay. These virus strains are the M2-resistant strain and the M2-sensitive strain, respectively. The results showed its inhibitory ability with corresponding IC50 values of 15 and 8.1 µM (He et al. 2013; Moghadamtousi et al. 2015).Fig. 18 Asperterrestide A (He et al. 2013) θ-Defensins and Retrocyclins θ-Defensins are the sole family of mammalian cyclic peptides found by scientists, in which they are isolated from baboons and rhesus macaque leukocytes (Lehrer et al. 2012). With a premature stop codon, the translation apparatus in human θ-defensin genes is unable to form peptides in human leucocytes, although the precursors are needed (Venkataraman et al. 2009). Thus, retrocyclin (RC) was synthesized with the corresponding sequences with those expressed in the human pseudogenes (Lehrer et al. 2012). Activity Against Human Immunodeficiency Virus Gp120 is a viral surface glycoprotein that engages two target receptors, CD4 and CXCR4 or CCR5. Following this initial binding of HIV, viral entry is mediated by six-helix bundle structure formation due to conformational changes in gp41 (Noah et al. 2008; Regula et al. 2013). Neither cytotoxic nor virotoxic effects of RC-1 inhibit the formation of proviral DNA, preventing viral entry (Lehrer et al. 2012). Several studies revealed that RC-1 can defend human target cells against HIV-1 infection and that HIV-1 strains employ either CXCR4 or CCR5 coreceptors; however, there is not much effect against HIV-2 viral strains and related retroviruses infecting blood cells that do not rely on the co-receptors CXCR4 or CCR5, such as simian immunodeficiency virus type 1 (Simmons et al. 2000; Lehrer et al. 2012). With the cyclic backbone and intact disulphide bonds, RC-1 was found to have high binding affinity to CD4, gp120, and a surface glycolipid that is galactosylceramide with KD values of 31 nM, 35.4 nM, and 24.1 nM, respectively (Lehrer et al. 2012; Xiong et al. 2021). From the fusion assays targeting the envelope glycoproteins of HIV-1, RC-1 showed its ability to prevent the viral entry of HIV-1 with neither the involvement of cross-linking membrane proteins nor impeding the interactions of gp120-CD4 (Cole et al. 2006; Lehrer et al. 2012). RC, which is only 18 residues in length, is of interest for its lack of complexity in designing congeners with greater anti-HIV-1 activity (Penberthy et al. 2011). A congener, RC-101, formed from a charge-conservative substitution with lysine on one of the arginines (Owen et al. 2004), was found to have increased anti-HIV-1 potency (low micromolar EC50 activity of 1–5 µg/mL) compared with the parent molecule (Owen et al. 2004; Wang et al. 2004; Penberthy et al. 2011). Compared to RC1, the RC-101 congener has a much greater binding affinity to gp120 that is approximately 25-fold, while it has no significant superiority in binding to galactosylceramide with identical binding, as proven by the observed values of 20–30 nM (Wang et al. 2004). To date, new HIV-1 viral strains have been found to commonly develop resistance to drugs targeting coreceptors gp41, CXCR5, or CCR4 within weeks after exposure. Therefore, the ability to obstinately block HIV-1 by affecting the key amino acid residues for infection is one of the criteria of anti-HIV-1 therapeutic interventions (Pang et al. 2009; Penberthy et al. 2011) To better understand the latent resistance of HIV-1 to RCs, a study revealed that most HIV-1 strains were expected to be vulnerable to inhibition by RC-101 treatment (Fuhrman et al. 2007). Further support by data reporting RC-101 induced minimal HIV-1 resistance of only 5–10-fold. It is significantly different from other HIV entry inhibitors showing 10,000–20,000-fold HIV resistance in similar passaging tests (Trkola et al. 2002; Nameki et al. 2005). Unpredictably, even with HIV-1 mutations, RC-101 is still able to reduce HIV-1 infectivity, although HIV-1 protein binding is prohibited (Cole et al. 2006; Fuhrman et al. 2007), therefore conveying viral fitness significance to undermining RC action. Activity Against IAV Aside from HIV-1, RC has potent antiviral activity on several viruses as well. For example, RC-1 showedan antiviral effect on IAV. In addition to the impairment of HA-mediated viral entry (Leikina et al. 2005), RC-1 causes the induction of viral aggregation, enhancing IAV ingestion by neutrophils (Doss et al. 2009). RC-2, with an additional arginine in structure, acts against IAV by preventing the membrane fusion mediated byHA (Doss et al. 2009; Lehrer et al. 2012). Similar to RC-1, RC-2, as a multivalent lectin, can form an immobilized surface glycoprotein cross-linkage network to block IAV entry (Leikina et al. 2005; Lehrer et al. 2012). Activity Against HSV From the in vitro testing on the effectiveness of antiviral activity against HSV-1- and HSV-2-mediated cervix epithelial cells infection (Yasin et al. 2004), RC-1, RC-2, and rhesus θ-defensin-3 (RTD-3) showed protective ability, but RC-2 stood out without viral preincubation. With a Kd value of 13.3 nM, RC-2 attached to HSV-2 glycoprotein B, but no binding was observed after deglycosylating glycoprotein B (Lehrer et al. 2012). Diverse Peptides that Resemble RCs Tachyplesin/polyphemusins were isolated from horseshoe crab haemocytes. They are peptides comprising 17, 18 amino acids with an alpha-amide group at the C-terminus that build a rigid double-stranded anti-parallel beta-pleated sheet structure in the connection by a beta-turn (Bulet et al. 2004). Although these natural host-defense peptides (HDPs) exhibit modest antiviral activity against HIV-1 X4 tropic viruses, in which viral entry exploits the CXCR4 coreceptor, they show undesirable haemolytic action against human erythrocytes, and more prominent activity is being observed in high salt environments mimicking the typical marine living settings of horseshoe crabs (Bulet et al. 2004; Penberthy et al. 2011). To reduce haemolytic effects, the backbone of synthetic tachyplesins is manipulated via peptide cyclization. By imitating the HIV-1 protein gp41 portions, synthetic tachyplesins (e.g., T22) bind gp120 and effectively minimize X4-tropic HIV-1 infection of T lymphocytes (Penberthy et al. 2011). Cyclotides, a subset of HDPs that are cyclic in nature, were first identified and isolated from plant sources such as Rubiaceae (coffee), Violaceae (violet), and Cucurbitaceae (cucurbit) families (Gruber et al. 2008; de Veer et al. 2019). Structurally, they are small (approximately 30 amino acids) proteins having a cysteine knot motif from three interlocking disulphide bonds, which is significant for protein stability. In addition to being thermally stable and resistant to proteases, cyclotides are capable of being modified with amino acid sequence replacement to optimize their inherent antiviral activity (Penberthy et al. 2011). Cycloviolins In a study by Hallock and colleagues, an emerging class of cyclic peptides consisting of four new anti-HIV macromolecules with 28–31 amino acid residues, known as cycloviolins A–D, were quarantined from the genuine tropical plant Leonia cymosa from South American collections (Hallock et al. 2000). A combination of methods was employed to determine and analyse the amino acid configuration and sequence in the cyclic primary structure of the discovered entities (Hallock et al. 2000). Six cysteines, presented as three internal disulphide bonds, are commonly found in all cycloviolin structures, contributing to chemical stability and possibly reducing proteolysis resistance (Hallock et al. 2000; Northfield et al. 2014). Cycloviolin B is the smallest member among this peptide class, and a proline residue is absent in its structure, but it has a distinctive TSSQ sequence from residues 17–20 (Hallock et al. 2000). Captivatingly, cycloviolins A–D performed a high level of homology in sequence to the identified circulins A and B as well as cyclopsychotride A from the Rubiaceae family but much less matching with the Viola varv peptides isolated from a member of the Violaceae family (Hallock et al. 2000). Apart from their potential chemotaxonomic interest, an anti-HIV-1 study on cycloviolins A–D reported that these peptides possessed similar antiviral activities with observed EC50 values at approximately 130 nM (Hallock et al. 2000). Compared to circulins A and B, no significant deviations of their direct cytotoxicity to the host cells with the reported EC50 values of approximately 560 nM, concluding that the variations in the amino acid sequence variations have no assessable influence on the general activity profile (Hallock et al. 2000). Prospective Most of the viral infections discovered currently still face the challenges of a lack of proper treatment with desired therapeutic outcomes. Worldwide, populations are being exposed to the public health threats of viral pandemics due to the emergence or mutation of some viral strains, or even worse, the development of viral resistance from the available antiviral therapies. Such alarming conditions should be avoided, as viral diseases can spread uncontrollably all over the world in a highly globalised world. For example, the COVID-19 pandemic has already reported 620,301,709 infected people, including 6,540,487 deaths globally by WHO as of 13 October 2022 since the outbreak was discovered in January 2020 (Ciotti et al. 2020; WHO 2020). Although there are antiviral molecules undergoing clinical trials, the development of novel antiviral cyclic peptides with satisfactory clinical outcomes has been the unique direction of effort by research scientists because of their significant advantageous properties when compared to linear peptides and other biological entities. With the current available naturally isolated cyclopeptides, optimization of cyclic peptide analogues has been the drug discovery trend to achieve better potency and pharmacokinetic properties. Modern genetic recombinant technologies are combined with peptide chemistry to develop more diverse cyclic peptide libraries. Rapid screening with display systems and the introduction of cyclization approaches further promotes its evolution in cyclic peptide discovery. The conjugation of cyclic peptides with nanocarriers plays an important role in improving tissue targetability. Cyclic peptide nanoparticles have excellent capability for a broad array of biomedical applications, especially in drug delivery, due to their great tissue permeation and selectivity to the target site with specific receptor interactions (Abdalla and McGaw 2018, Zhang and Chen 2021). In the future, cyclic peptides will be one of the most important highlights in the advancement of therapeutic agents due to their outstanding characteristics. Significantly, the reduction in production cost will encourage growth and fasten the pave of cyclic peptide drug discovery as well as commercialization. Conclusion In conclusion, polypeptides with restricted structures are potential candidates for antiviral drug development to produce favourable therapeutic outcomes with outstanding properties compared to their linear counterparts. The progress of novel drug discovery towards antiviral therapy is receiving increasing attention and is being supported with sophisticated biotechnologies and useful approaches, from the fermentation of natural products to biological methods and then chemically structural modification techniques, to overcome the limitations and to optimise the antiviral activities of cyclic peptides. In designing cyclic peptides as effective treatment agents for viral infections such as CoVs, DENV, and HIV, a well-established understanding of the virus structures, their mechanism of action, and their interactions with the target site of host cells are vital to ensure the effectiveness and targetability of therapeutic drugs. In fact, the increasing mutation on the viral strains has placed obstacles in the path of the drug investigation in addition to the challenges of the existing antiviral cyclic peptides yet to handle. Nonetheless, because of the desired characteristics of cyclic peptides in medical and pharmaceutical areas, such blocking stones never demotivate researchers to investigate the possibilities of cyclic peptides for antiviral treatment. With continuous effort in optimising and utilising the distinctive properties of cyclic peptides, it will be the cross-century advancement of therapeutic agents. Abbreviations ∆Gbinding Gibbs free binding energy 2D Two-dimensional ACE2 Angiotensin converting enzyme 2 ADMET Absorption, distribution, metabolism, excretion and toxicity AIDS Acquired immunodeficiency syndrome ARM Arginine rich motif ATP Adenosine triphosphate BCP Backbone cyclic peptide bnAbs Broad neutralizing antibodies BNP Brain natriuretic peptide BSA Bovine serum albumin CHIKV Chikungunya virus CIC Chronic idiopathic constipation cMD Classical molecular dynamics CoV Coronavirus CPE Cytopathic effect CSSSIs Complicated skin and skin structure infections DENV Dengue virus E. coli Escherichia coli EBOV Ebolavirus EC50 Median effective concentration ED Envelope domain ELISA Enzyme-linked immunosorbent assay EVD Ebola virus disease FAO Food and agriculture organization FDA Food and drug administration GaMD Gaussian accelerated molecular dynamics Gp Glycoprotein HA Hemagglutinin HDPs Host-defence peptides HIV Human immunodeficiency viruses HKTA Hydroxy ketotetradecanoic acid HSV Herpes simples virus HV Hepatitis virus IAV Influenza A virus IBS-C Irritable bowel syndrome IC50 Median inhibitory concentration JEV Japanese encephalitis virus KD Dissociation constant Ki Inhibitory constant Koff Rate of dissociation MCV Molluscum contagiosum virus MHC-I Major histocompatibility complex class I Mpro Main protease mRNA Messenger ribonucleic acid Mtase Methyltransferase NA Neuraminidase NHR N-terminal heptad repeat NP Nucleoprotein NS Nonstructural ORF Open-reading frames pBzP P-benzoylphenylalanine PET Positron emission tomography PLpro Papain-like protease PMF Potential of mean force RBD Receptor binding domain RC Retrocyclin RdRp RNA-dependent RNA polymerase RTD Rhesus Theta defensin RTHS Reverse two-hybrid system RT-PCR Reverse transcription–polymerase chain reaction SAM S-adenosyl-l-methionine SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 SICLOPPS Split intein-catalysed ligation of proteins and peptides system SIE Solvated interaction energy SPPS Solid phase peptide synthesis S–S Disulphide Tat Transactivator of transcription TBE Tick-borne encephalitis virus TC0 Noncytotoxic concentration TNFSF10 TNF ligand superfamily member 10 tRNA Transfer ribonucleic acid TSG Tumour susceptibility gene UAA Unnatural amino acid UEV Ubiquitin E2 variant UTR Untranslated regions VLP Virus-like particle VOC Variant of concern VOI Variant of interest Vp Viral matrix protein vRNP Viral ribonucleoprotein WEEV Western equine encephalitis viruses WHO World health organisation WNV West Nile virus WT Wild type YFV Yellow fever virus ZIKV Zika virus Acknowledgements This Project is supported by Fundamental Research Grant Scheme (FRGS) (Reference code: FRGS/1/2021/SKK0/UCSI/02/5), Ministry of Higher Education (MOHE), and Research excellence and innovation grant (Project Code: REIG-FPS-2020-052) Under Centre of Excellence in Research, Value Innovation and Entrepreneurship (CERVIE), UCSI University, Malaysia. Author Contributions The conceptualization of the review article was by PVK. CLY, MAAM, and GR searched the related articles and prepared the figures. CLY collected the literature data and prepared the tables. CLY, MAAM, and GR wrote the manuscript. MAAM, PVK and ST revised the manuscript. All authors reviewed the final version of the manuscript for submission. Declarations Conflict of interest The authors have no relevant financial or nonfinancial interests to disclose. 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