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Supporting information | PMC9946270 |
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Soluble cytokine concentrations in serum following vaccination. | (A) Heat map of fold changes of all detectable soluble factors in serum following vaccination. (B) Box and whisker plots comparing serum TNF and IL-18 concentrations following the first vaccine dose. The legend denotes color-coding of the seven cohorts. Statistical analyses were performed using nonparametric Friedman and Kruskal-Wallis test with Dunn’s multiple comparison tests.(EPS)Click here for additional data file. | PMC9946270 |
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Sex-based comparisons of final virus nAb titers. | TBEV | (A) Comparisons of male (M) vs female (F) TBEV nAb titers at the final time point in cohort A1 and A2 (left panel), cohort C (middle panel) and combined A and C cohorts (right panel). (B) Comparisons of male (M) vs female (F) JEV nAb titers at the final time point in cohort B1 and B2 (left panel), cohort D (middle panel) and combined B and D cohorts (right panel). (C) Comparisons of male (M) vs female (F) YFV nAb titers at the final time point in cohort E (left panel) and combined A, B, and E cohorts (right panel). The legend denotes color-coding of the seven cohorts. Statistical analyses were performed using nonparametric Friedman and Kruskal-Wallis test with Dunn’s multiple comparison tests. *p < 0.05.(EPS)Click here for additional data file. | PMC9946270 |
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Immunoglobulin expression of circulating plasmablasts. | (A) Median IgG expression of plasmablasts over time following vaccination and comparison of peak expression between cohorts at days (B) 14, (C) 37 and (D) 187. (E) Median IgA expression of plasmablasts over time following vaccination and (F) comparison of lowest expression between cohorts at day14. (G) Median IgG-A- expression of plasmablasts over time following vaccination and (H) comparison of peak expression between cohorts at day14. The legend denotes color-coding of the seven cohorts. All plots are depicted with median values with IQR. Statistical analyses were performed using nonparametric Kruskal-Wallis test with Dunn’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001.(EPS)Click here for additional data file. | PMC9946270 |
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List of adverse events following vaccination. | (PDF)Click here for additional data file. | PMC9946270 |
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Summary of registered adverse events. | P. | RECRUITMENT | (PDF)Click here for additional data file.We thank all study participants for participating in the clinical trial, the Karolinska Trial Alliance staff and research nurses for organizing the recruitment and scheduling of study participants, vaccination, and clinical sampling. Additionally, we thank J. Verner-Carlsson and staff scientists at the Swedish Public Health Agency for support with neutralization assays and BSL-3 instruction. Further thanks to W. Christ for support in the BSL-3 facility, P. Jahnmatz for discussions around the FluoroSpot reader assessment, and D. Wullimann for discussions and advice on the project. | PMC9946270 |
References | PMC9946270 |
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Subject terms | SE, infections | VIRUS, SARS-COV-2 INFECTION, INFECTIONS | Rapid antigen diagnostic (RAD) tests have been developed for the identification of the SARS-CoV-2 infection. However, they require nasopharyngeal or nasal swab, which is invasive, uncomfortable, and aerosolising. The use of saliva test was also proposed but has not yet been validated. Trained dogs may efficiently smell the presence of SARS-CoV-2 in biological samples of infected people, but further validation is needed both in laboratory and in field. The present study aimed to (1) assess and validate the stability over a specific time period of COVID-19 detection in humans’ armpit sweat by trained dogs thanks to a double-blind laboratory test–retest design, and (2) assess this ability when sniffing people directly. Dogs were not trained to discriminate against other infections. For all dogs (n. 3), the laboratory test on 360 samples yielded 93% sensitivity and 99% specificity, an 88% agreement with the Rt-PCR, and a moderate to strong test–retest correlation. When sniffing people directly (n. 97), dogs’ (n. 5) overall sensitivity (89%) and specificity (95%) were significantly above chance level. An almost perfect agreement with RAD results was found (kappa 0.83, SE 0.05, p = 0.001). Therefore, sniffer dogs met appropriate criteria (e.g., repeatability) and WHO's target product profiles for COVID-19 diagnostics and produced very promising results in laboratory and field settings, respectively. These findings support the idea that biodetection dogs could help reduce the spread of the virus in high-risk environments, including airports, schools, and public transport. | PMC9985821 |
Introduction | pneumonia, ’, infection, infected by SARS-CoV-2, RT‐PCR)Based | CORONAVIRUS, INFECTION, PNEUMONIA, SEVERE ACUTE RESPIRATORY SYNDROME | In the past 2 years, great efforts have been made to contain the COVID-19 pandemic worldwide. Due to high contagiousness of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the resultant infection spread extremely fast and widely, provoking a sudden and substantial increase in hospitalisation of patients, due to pneumonia with multiorgan diseaseHowever, implementing conventional rapid diagnostic tests has common potential pitfalls, as sensitivity and specificity may be lower than established laboratory testing (i.e. RT‐PCR)Based on the assumption that, similarly to a variety of other pathologies, including viral infectionsThe results of studies published so far on the accuracy of canine smell in detecting the presence of SARS-CoV-2 in biological samples (e.g., saliva, sweat, urine, trachea-bronchial secretions) from infected people suggest that sniffer dogs might reach percentages of sensitivity and specificity comparable to, or perhaps even higherGiven the previous considerations, once dogs’ ability to detect COVID-19 from biological samples of positive people has undergone laboratory validation, for sniffer dogs’ to be successful in contributing to the sustainable control of the COVID-19 pandemic, they should be brought beyond lab testing and into the field. Scientists suggest that dogs could help control the pandemic because they could screen hundreds of people per hour in crowded places, such as airports or sports stadiumsThe aims of the present study were to (1) assess reliability depending on sensitivity (Se) and specificity (Sp) of purposely trained dogs to indicate an odour associated with COVID-19 in human armpit sweat through a double-blind laboratory test–retest design, and (2) test trained dogs’ screening ability to detect people infected by SARS-CoV-2 by sniffing them directly in public spaces, comparing a scent detection dog test and nasopharyngeal SARS-CoV-2 RAD immunoassays. Community pharmacies and their incoming clients were chosen as settings for the screening in the field (aim 2) because, since Italian Cabinet of Ministers had declared a state of emergency on 31 January 2020 | PMC9985821 |
Results | PMC9985821 |
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Phase 1: Laboratory testing | COVID-19-related VOCs | Data regarding the dogs’ response and the main characteristics of all the samples utilised during both the test and retest phases are reported as supplementary material (Supplementary Table The combined sensitivity and specificity for all three dogs during the test phase was Se = 93% (95% CI = 84–103%) and Sp = 99% (95% CI = 97–101%). The combined sensitivity and specificity for all three dogs during the retest phase was Se = 83% (95% CI = 69–97%) and Sp = 97% (95% CI = 94–99%). Overall and individual Se and Sp are reported in Table Canine ability to detect COVID-19-related VOCs in the laboratory test and retest.Measured and simulated overall positive predictive values (PPVs) and negative predictive values (NPVs) with 95% confidence intervals.Overall, discrepancies between the dogs’ response and the RT-PCR result were observed for 14/360 samples (3.9%), 50% of which were positive. Seventy-one per cent (n = 10) of the discrepancies were found in the retest (Table Main biological characteristics of samples and experimental phase (test/retest) associated to discrepancies between SARS-CoV-2 RT-PCR and the response from the three dogs.As for test–retest reliability overall, an 88% agreement was found between test and retest, which refers to the percentage of trials for which there was no discrepancy between the two phases. Regarding the Spearman’s correlation, one dog (Nala) yielded a Spearman’s r = 1 (100% correct responses). However, the percentage agreement for Nala was 91%. When considering the other two dogs individually, a moderate to strong test–retest reliability was found for Otto (90% agreement, Spearman’s r = 0.7, p = 0.035, 95% CI 0.042–0.917) while the reliability was lower for Helix (83% agreement, Spearman’s r = 0.5, p = 0.082, CI − 0.093 to 0.849).As for GzLM model results, none of the factors included was a significant predictor for the dogs’ correct response (all p > 0,05, data non shown). | PMC9985821 |
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Phase 2: in field work | In total, 97 volunteers (54% women) underwent both sniffer dog and RAD test (results are reported in Tables Summary of results from volunteers who tested positive on the rapid antigen test (RAD) and were sniffed by the sniffer dogs in the field work, grouped based on being sniffed by a single dog (one) or multiple dogs (multiple).n. correct choices/total positive = 82% (13/16)n. incorrect choices/total positive = 18% (3/16)n. correct choices/total positive = 100% (1/1)n. incorrect choices/total positive = 0% (0/1)n. correct choices/total positive = 100% (10/10)n. incorrect choices/total positive = 0% (0/10)n. correct choices/total positive = 100% (1/1)n. incorrect choices/total positive = 0% (0/1)Names of sniffer dogs with wrong choices are displayed in bold.Summary of results from volunteers who tested negative on the rapid antigen test (RAD) and were sniffed by the sniffer dogs in the field work, grouped based on being sniffed by a single dog (one) or multiple dogs (multiple).n. correct choices/total negative = 100% (2/2)n. incorrect choices/total negative = 0% (0/2)n. correct choices/total negative = 95% (20/21)n. incorrect choices/total negative = 5% (1/21)n. correct choices/total negative = 100% (7/7)n. incorrect choices/total negative = 0% (0/7)n. correct choices/total negative = 100% (1/1)n. incorrect choices/total negative = 0% (0/1)Name of sniffer dogs with wrong choices are in bold.The rapid antigen test gave a positive result in 39 volunteers (40%) (Table Of the 58 (60%) volunteers tested negative on rapid antigen test (Table Regarding the overall Se and Sp of sniffer dogs working in field, the Se was 89% (95% CI 78–95%) and the Sp was 95% (95% CI 88–98%). Thus, the 95% CI did not overlap by 50%, being far from randomness.Overall, there was an almost perfect concordance between the dogs’ detection and RAD results (kappa 0.84, se 0.047, p = 0.001). | PMC9985821 |
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Discussion | In the present study, firstly, three family dogs were purposely trained to detect COVID-19 related VOCs in human armpit sweat. | PMC9985821 |
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Double-blinded test–retest reliability assessment in laboratory | RECRUITMENT | The prime contribution of the current paper lies in answering the empirical question of whether trained sniffer dogs’ ability is relatively stable over time in a laboratory setting. The current results support such possibility, at least in two of the three dogs involved. The dogs’ ability was analysed through test–retest reliability, in order to verify whether their performance achieved the same results when repeated after a short period of time. According to the results, Otto and Nala had high Se and Sp, which remained high, or even increased in the case of Nala, between the test and the retest. By contrast, Helix had the highest test’s Se and Sp (100%) but was markedly less successful during the retest. It could be useful to report thatDuring the laboratory test, the three trained dogs showed an overall sensitivity rate of 93% (90–100%), and an overall specificity rate of 99%, which ranged from 98 to 100%. These values fall within the upper end of published ranges. In fact, in studies conducted recently on olfactory detection of COVID-19 in sweat samples by sniffer dogsThe high sensitivity and specificity and low inter-dog difference observed in the present study likely originate from two conditions: (1) the “ubiquitous” recruitment system and the rigorous sample collection, and (2) the consistent high-quality training performed.In our study, positive and negative samples were collected from both hospitals and outside hospitals, symptomatic and asymptomatic volunteers, vaccinated and non-vaccinated, so that the actual target population could be accurately covered and potential misleading of dogs due to hospital-associated odours avoided. In addition, as recommendedFollowing the example of Kantele et al. | PMC9985821 |
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In field training procedure | lung cancer, human lung cancer | LUNG CANCER | As for phase 2, the present study is the first evaluating trained sniffer dogs as point-of-care test for SARS-CoV-2 in one of the types of public facility most prone to overload, the community pharmacies. Since late 2020, in Italy, the demand for COVID-19 testing has quickly transformed the pharmacies in points-of-care. Approximately 70% of the 20.000 pharmacies across Italy were offering swabs at the time of our study, performing hundreds of swabs per day, especially amid variant surges. Their involvement indeed caused a 80% increase in the country administration of swabsThere was a difference between the duration of training of dogs in laboratory settings and the time needed to train the other five dogs in field. This was shorter compared to the duration of laboratory training for Nala and Otto, which can be explained by a different individual attitude of the dogs and by the fact that working in a laboratory setting, on samples contained in stainless steel supports, requires a longer time for learning than working in conditions that are way more natural to the dogs, namely outdoors, in public places to which they are already used, in a task that consists in sniffing out people. For Helix, the duration of lab training was shorter compared to Nala and Otto, which can be explained by taking into account that, as we explained in the methods section, she was already engaged in bio-detection work with human lung cancer. That work was carried out in the same lab, with the same scientists: therefore, her training did not require time to get used to the environment, but only to the new smell and to the sample supports, that in this study were aluminium tracks positioned on the ground, while in the lung cancer study they were 60-cm high aluminium stands.The Sp reached by our five dogs in the field (94%) was disappointingly lower than the 97% threshold set by the WHO for validation of antigen tests. Although such value could still be acceptable for screening | PMC9985821 |
Conclusions | ’ personalities | In conclusion, the sniffer dogs involved in the current study demonstrated the foundations of a good screening test: cause minimal discomfort to the human volunteers, be inexpensive to perform, easy to administer, reliable and valid in discriminating diseased and non-diseased humans. Therefore, they may be potentially useful for mass detection and contact tracing for COVID-19, being implemented in various public settings, such as schools or airports, where conventional testing is not feasible or easily accessible. Moreover, the potential usefulness of dogs could be extended to other potential health screening applications. Although, after 4 years, COVID-19 continues circulating, driven by the wildly transmissible new variants, these findings suggest that dogs could be trained and deployed as a first response to new threats or future pandemics.Sniffer dogs performed better than RAD under controlled settings but were similar to RAD in terms of sensitivity in field conditions. However, compared to RAD, sniffing dogs hold some relevant advantages: for example, they do not impose unpleasant procedures, as the nasopharyngeal/nasal swabs, thar are commonly perceived to be uncomfortable, or even painful; they provide timely identification and isolation of the largest number of patients in real-life contexts, thus improving access to communities and simplifying logistics; and do not require operators to come into direct contact with potentially infectious materials. Further efforts seeking standardisation and validation of the processes are needed to better understand the potential and limitation of using sniffing dogs for the detection of COVID-19 and verify them in appropriate populations. It is worth noticing that the consistency in error rates for positive early-stage COVID-19 between the laboratory test and in-field test was potentially the source of the increased error rates in in-field test, in addition to the change from the sweat sample to the entire body odours including clothes, cosmetics, and so on. In our study, training with diverse positive and negative volunteers, we aimed to improve the correct rates in COVID-19 detection by the sniffer dogs. Nevertheless, further research should include more high quality exploration of the influence on the odour signature (or the perception of it) by variables such as training sample number and type, sampling method, dogs’ personalities | PMC9985821 |
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Methods | ONCOLOGY | The study was approved both by the University of Milan Ethical Committee (CE_26_21 and CE 84/21) and the Institutional Review Board of the European Institute of Oncology (R90/14-IEO102), in accordance with the relevant guidelines and regulations, and individual written informed consent was obtained from all the study participants after appropriate information concerning the study was provided. Eligible participants were people of either gender, older than 7 years; in the case of minors, written informed consent was provided by a legal tutor. Participation to provide a sweat sample or to be sniffed by trained dogs was volunteer. Anti-COVID-19 vaccination was not an exclusion criterium. Whenever possible, the type, number of dose and date of vaccine administration were also recorded after the information was provided on a voluntary basis.) | PMC9985821 |
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Phase 1: Laboratory training and testing | The first phase of the study consisted of training dogs to discriminate in the laboratory between sweat samples from patients with COVID-19 and sweat samples from healthy controls. | PMC9985821 |
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Participants and sample sourcing | INFECTIOUS DISEASES, THORACIC, ONCOLOGY | The samples were collected from: the III Infectious Diseases Unit, L. Sacco Hospital, ASST Fatebenefratelli-Sacco, Milan, Italy; the Division of Thoracic Surgery of the European Institute of Oncology (IEO), Milan, Italy; COVID-19 screening stations across the North of Italy.Participants were divided into two groups as shown in Table Groups and sample size of training and test–retest phases.
Cases, N. 146 patients COVID-19 positive, who had tested positive by RT-PCR for SARS-CoV-2 within 24 h prior collection, regardless of COVID-19 symptoms.Controls, N. 571 patients COVID-19 negative, who had tested negative by RT-PCR for SARS-CoV-2 within 24 h prior collection.The number of samples was calculated based on the number of training and test–retest sessions required according to the training technique we developed, so that dogs could be presented with several samples while ensuring a case: control ratio of 1:5. Samples collected were used multiple times only during training. | PMC9985821 |
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Sample collection and handling | Armpit sweat samples were collected following the same standard protocol for all volunteers and using the same media across all locations. The volunteers were asked to hold an inert polymer tube (3.6 cm length, 0.8 cm thickness) commonly used for adsorbing VOCs for explosive, drug, or criminology detection (Getxent, Neuchâtel, Switzerland) simultaneously under each armpit for 20 min. Each individual polymer tube was immediately placed in a sealed envelope, bearing the subject's ID. The samples were then shipped while refrigerated (+ 4 to + 8 °C) to the laboratory of the dog training/testing centre, in separate packaging for cases and controls, and kept stored at a temperature of 4 °C. The choice to work with sweat samples was made, in accordance with what reported by Grandjean et al. | PMC9985821 |
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Animals | Dogs were provided by local dog owners and screened for inclusion in the study. The initial screening phase included the assessment of the effectiveness of food as a reinforcer for each dog and the dog's suitability for work in a laboratory environment in the presence of non-familiar people. Out of the 6 dogs that met the inclusion criteria, 3 dogs underwent the entire training session, while the others were exempted, due to either health or management problems, or low motivation for working in the scent line-up. One 8 years old Belgian Malinois female dog (Nala), an 8-year-old female mixed-breed dog (Helix), and a 5-year Dachshund male dog (Otto) underwent a training period (two weekly training sessions) to discriminate between the sweat of people with COVID-19 and the healthy controls. The training lasted 5 months for Helix and 7 months for Nala and Otto. All dogs had previous experience with bio-detection work (Helix: human lung cancerThe dogs were not used for other tasks during the study. All dogs lived at home with their owners, they were handled by professional dog trainers or behaviour scientists during the training and testing sessions, and were cared for by their owners between sessions. | PMC9985821 |
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Sniffing room and experimental equipment | Dog training and testing was conducted at the laboratory of Animal PhysioEthology of the Department of Veterinary Medicine and Animal Sciences, University of Milan, Lodi, Italy. Two tracks of three samples, especially designed for this study, and constructed of aluminium, were positioned in a single straight line, spaced 40 cm apart, on the floor, as in Fig. ( | PMC9985821 |
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Training procedure | sweats | The training aimed to teach the dogs to discriminate Case and Control sweats and indicate the cases. The training procedure was carried out by Medical Detection Dogs Italy’s bio-detection trainers and was based on operant conditioning, with a food reward provided for correct behaviour as described previouslyHelix ( | PMC9985821 |
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Double-blinded test–retest reliability assessment | The test and the re-test aimed to measure the accuracy of the training. Each dog was naïve to the samples they used during both the test and retest phases, meaning that they had never been presented before with one of those samples. However, more than one dog could be presented with the same sample from a given volunteer and dogs could sniff during the test or the re-test samples that another dog had used in training. For this reason, during these phases the three dogs were presented with a total of n. 360 unfamiliar samples (n. 300 negatives and n. 60 positives), who were collected from a total of n. 282 participants (n. 233 RT-PCR negative, n. 43 RT-PCR positive). During testing and re-testing, only the trainer was inside the room, and she remained hidden by the wooden screen (Fig. At the beginning of the test, before the dog and the trainer entered the room, one positive and five negative samples were selected and placed in the six holders by an experimenter, who left the laboratory soon after. The location of each sample was pre-determined by this experimenter in a pseudo-random order (designed using The test took place on 2 days, 1 week apart, on which every dog performed 1 session of 5 trials. For each trial, a new set of samples of volunteers was used. However, some of the samples might have been used during training by another dog. At the end of each trial, the dog was rewarded verbally regardless of whether the marking was correct or not.To assess the repeatability (or test–retest reliability) | PMC9985821 |
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Phase 2: field study | PMC9985821 |
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Animals | Five dogs underwent the training process for the field study. One of these was Nala, the Belgian Malinois who was also involved in the laboratory testing phase. The other dogs were Chaos (Golden retriever, male, 4.5 years old), Hope (Border collie, female, 6 years old), Iris (Golden retriever, female, 3 years old) and Nim (mixed breed, female, 12 years old). Only Nala and Nim had previous experience of scent work (phyto aromas and | PMC9985821 |
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In field training procedure | The trainers and the method (operant conditioning with positive reinforcement and a food reward provided for correct behaviour) employed were the same as in laboratory testing except they did not use the apparatuses to hold the samples. Training for the field screening was carried out from September 2021 to mid-January 2022, and passed through two steps, as described below, during which the dogs learnt to identify COVID-19 patients by sniffing the volunteers directly. Step one training sessions were conducted at various drive-thru COVID-19 testing points after volunteers had received their negative antigen test result. This was necessary so that, adding a positive scent or not, could reliably allow for presenting the dogs with positive and negative scents on humans. Round-shaped metallic stainless-steel boxes with 6 holes (height: 1.2 cm; diameter: 3.6 cm), typically employed in nose-work, were used during the training. The boxes contained either a positive or negative sweat-stained cylinder. The boxes used for positive samples were not mixed with the boxes used for negative samples. In each training session, the dogs had to enter a room where a maximum of 5 volunteers lined up having a box hidden in their clothing, preferring areas where people are expected to sweat the most (e.g., sleeves, shoes, etc.). The location of the person holding the positive sample was always randomised in the line-up. The volunteers could either be sitting or standing in a queue, thus simulating a typical situation in a testing point, including hospitals and pharmacies, and dogs were required to indicate that they recognised the scent of COVID-19 by stopping in front of the person holding the box with the target odour (positive scent). As in the training phase, the scent was obtained from patients with a diagnosis confirmed by RT-PCR. Overall, the dogs were trained using 131 positive samples. None of the samples presented to Nala (who had participated in phase 1) came from volunteers who had participated in the laboratory testing phase. This training step lasted 2 months for each dog.In step 2 of the field study, 192 incoming clients at community pharmacies volunteered for the study. In each trial, the dogs could sniff any part of the body of one volunteer and they were allowed to touch with their noses the body of the volunteer. This training step lasted 2.5 months for each dog.The trials in step 1 were unblinded (e.g., the handlers knew the position and number of the sniffers containing the positive cylinders) to observe the spontaneous behavioural cues offered by each dog when alerting on the target odour (positive scent). In step 2, the trials were single-blinded, as the handlers were unaware of who had received a positive diagnosis. By contrast, the volunteers and the director of training, who was present during both phases, were always unblinded, and the director informed the dog-trainer dyad of every correct alert. The results of this training step are reported in Table Training of sniffer dogs for field work on volunteers. | PMC9985821 |
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In field screening procedure | MAY | In mid-January 2022, in-field screening begun on human volunteers in North Italy’s pharmacy COVID-19 testing queues, in order to screen the general population. Sessions were carried out once a week, between February 2022 and May 2022. During each daily session, each dog was asked to investigate a maximum of 10 people, depending on their motivation. The director of training was always present as an external controller, ensuring that the predetermined protocol was followed. For each session, the tests were triple-blinded with the diagnosis being unknown to everyone involved (handler, director of training, and participants). As in the in field study by Vesga et al. | PMC9985821 |
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Statistical analysis | Statistical analysis was performed using IBM SPSS Statistics for Windows, version 27.0 (Armonk, NY: IBM Corp).As for phase 1 (laboratory testing), the sensitivity (Se) and specificity (Sp) of a dog's indication of samples compared with the true diagnosis confirmed by RT-PCR were calculated. The sensitivity refers to the conditional probability of the dog indicating COVID-19 when the condition was present, and specificity refers to the conditional probability of the dog ignoring a sample from a healthy donor. Both sensitivity and specificity were expressed as proportions. Point estimates were calculated with 95% confidence intervals. The probability of a perfect test trial (finding the right sample and ignoring the controls) by chance was 1/6 (17%). Similarly to a previous studyPercentage of agreementAs for phase 2 (field work), Se and Sp were calculated compared with the RAD result. In each trial, the probability of success or failure was 50%. Thus, we calculated 95% CI of the Se and Sp and considered statistically different from a random choice those that did not overlap 50%, which is the randomness region.Cohen’s Kappa was used to measure the agreement of the two methods (sniffer dog and RAD) to screen people. Results were interpreted as follows: values ≤ 0 as indicating no agreement, 0.01–0.20 as none to slight, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as substantial, and 0.81–1.00 as almost perfect agreementFor all the analyses, a two-sided p < 0.05 was considered statistically significant. | PMC9985821 |
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Ethical statement | We confirm that the procedures comply with national and EU legislation. Research was performed in accordance with the Declaration of Helsinki. The study was approved by the Animal Welfare Committee (OPBA) of the University of Milan (OPBA_06_2021). Before participating in the olfactory detection test, each dog owner gave informed written consent for using their dogs’ test results in research. Reporting of results follows the recommendations of the ARRIVE guidelines. Informed consent was given by each subject for publication of identifying images in an online-access publication. | PMC9985821 |
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Supplementary Information | The online version contains supplementary material available at 10.1038/s41598-023-30897-1. | PMC9985821 |
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Acknowledgements | La Spina | We are very grateful to Sara La Spina and Elisa Marconato and all the handler-dog dyads for their invaluable contribution to dog training that made the project successful. | PMC9985821 |
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Author contributions | Conceptualization, all authors; methodology, A.L.S., P.P.; validation, M.A., P.P. and F.P.; formal analysis, F.P.; investigation, M.A., A.L.S., P.P. and F.P.; data curation, M.A.; writing—original draft preparation, F.P.; writing—review and editing, all authors; supervision, M.A.; project administration, M.A. | PMC9985821 |
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Data availability | The data that support the findings of this study are available from the project supervisor M.A. upon reasonable request. | PMC9985821 |
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Competing interests | The authors declare no competing interests. | PMC9985821 |
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References | PMC9985821 |
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Background | comorbidity | Computer Assisted Design and Computer Assisted Manufacturing (CAD/CAM) have revolutionized oncologic surgery of the head and neck. A multitude of benefits of this technique has been described, but there are only few reports of donor site comorbidity following CAD/CAM surgery.
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Methods | comorbidity | REGRESSION | This study investigated comorbidity of the hip following deep circumflex iliac artery (DCIA) graft raising using CAD/CAM techniques. A cross-sectional examination was performed to determine range of motion, muscle strength and nerve disturbances. Furthermore, correlations between graft volume and skin incision length with postoperative donor site morbidity were assessed using Spearman's rank correlation, linear regression and analysis of variance (ANOVA). | PMC9830896 |
Results | Fifteen patients with a mean graft volume of 21.2 ± 5.7 cm | PMC9830896 |
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Keywords | Open Access funding enabled and organized by Projekt DEAL. | PMC9830896 |
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Background | death, pain, gait disturbances, OSCC, neurosensory deficits | ADVERSE EFFECTS, DISEASES, ORAL SQUAMOUS CELL CARCINOMA | Oral squamous cell carcinoma (OSCC) is the sixth most common cause of death among all cancer-related diseases with an increasing incidence, [In surgical treatment of OSCC, tumor-free resection is aspired and therefore radical surgery is performed [After the introduction of microsurgical techniques, different donor-sites were described for harvesting of osteocutaneous free flaps [Due to the widespread use of computed tomography (CT) and computer-assisted image processing, the options for preoperative planning have improved significantly in recent decades [Besides considerations focusing on the oncological as well as the functional and aesthetic outcome of the stomatognathic system, comorbidities of the donor-site have to be taken into account. Current literature report controversial results regarding donor-site morbidity after harvesting of DCIA graft and is mostly based on retrospective reviews of medical records. Chronic pain, gait disturbances and neurosensory deficits are only a few of the known adverse effects described in the context of DCIA graft reconstructions [The first objective of this study is to investigate the extent of donor site morbidity in a study group receiving CAD/CAM driven DCIA graft reconstruction of the mandible to allow comparison with existing publications reporting donor site morbidity in conventional cases. The second aim of this study is to assess if there is a correlation of the graft volume or the incision length with postoperative donor-site morbidity following CAD/CAM driven DCIA graft harvesting. For this purpose, thorough chart review, evaluation of imaging data and postoperative physical examination was performed. | PMC9830896 |
Materials and methods | This study was conceived in accordance with the Declaration of Helsinki and has been approved by the ethics committee of the Albert-Ludwigs-University Freiburg, Germany (573/19). All participants gave written informed consent for study implementation. The structuring of the manuscript was performed regarding to the STROBE guidelines [ | PMC9830896 |
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Study group | OSCC | This cross-sectional study investigated donor-site morbidity of patients who received a CAD/CAM driven DCIA graft reconstruction of the mandible following resection of OSCC in our single tertiary center (Clinic of Oral and Maxillofacial Surgery, Medical Center— University of Freiburg, Germany). Patients who underwent CAD/CAM driven DCIA graft reconstruction of the mandible were identified by thorough review of all electronic patient charts of our clinic and were invited for physical follow-up examination. At the time of study conception, CAD/CAM was already introduced as standard operating procedure for bony reconstruction after ablation of OSCC at our clinic. Conventional operations were only performed in exceptional cases and could therefore not be investigated as a control group. | PMC9830896 |
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Inclusion and exclusion criteria | OSCC | DISEASES | Patients included in this study were at least 18 years old, signed a written declaration of consent, could attend an appointment for physical examination and were able to cooperate during physical examination. The period between surgical treatment and physical examination had to be at least twelve months.Exclusion criteria were as follows: patients under 18 years of age, patients who did not agree with study implementation and patients with previous surgeries in the pelvic area. Reconstructions of the mandible due to diseases other than OSCC were excluded to rule out a confounding influence caused by different diagnoses. Secondary reconstructions, or reconstructions which were performed without the use of CAD/CAM techniques were excluded likewise. | PMC9830896 |
Preoperative planning procedure | CREST | Preoperative planning was performed in collaboration with a technician of the virtual planning vendor (KLS Martin Group, Tuttlingen, Germany) during a web meeting using high-resolution CT scans of the head, neck and pelvis (slice thickness ≤ 1 mm). The underlying CT data was segmented and displayed for virtual 3D-visualization. Considering the patient-specific anatomy, the resection margins, the section and size of the iliac crest used for graft harvesting and, if necessary, osteotomies within the graft were determined (Fig. Preoperative planning of best possible mandibular reconstruction ( | PMC9830896 |
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Surgical procedure and inpatient care | Surgery was performed under general anesthesia using a two-team approach to reduce surgery time. Team time-out was carried out before surgery. Graft harvesting was always performed by the same team. | PMC9830896 |
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Raising of the microvascular DCIA graft | ischemia | ISCHEMIA, ANTERIOR, CREST | After standardized surgical site skin preparation and application of local anesthesia, a premarked skin incision was made. The abdominal fascia was exposed and incised one fingerbreadth above the inguinal ligament. The deep circumflex iliac artery was located and vessels to the iliac artery were traced. Transection of the externus, internus and transversus abdominis muscle was performed. Subsequently, the externus abdominis muscle, the gluteus medius muscle and the tensor fascia latae muscle were detached from the external side of the iliac crest using a raspatorium (Fig. Exposure of the iliac crest after careful preparation of the overlying soft tissue layers. (1) Anterior superior iliac spine. (2) Iliac crest. (3) Branch of the deep circumflex iliac artery. (4) Pelvic boneInsertion and fixation of the patient individual cutting and drilling jig. The correctly adapted cutting guide allows realizing the preoperative plan and acquiring the best possible configuration of the graftIliac crest after osteotomy and withdrawal from the pelvis. The vessels are not cut until resection is completed to guarantee shortest possible ischemia timeIliac crest after completion of internal osteotomies and fixation of the miter-cut parts along the patient individual osteosynthesis plate. The pedicle is marked with a red vessel-loop | PMC9830896 |
Inpatient care | Postoperative pain, swelling | CAVITY | Patients were transferred to intensive care unit (ICU) for airway monitoring after surgical treatment. The endotracheal tube was usually removed within the first 2 days after surgery if the swelling of the oral cavity allowed spontaneous breathing. As soon as the patient was stabilized hemodynamically and endotracheal tube was removed, transfer to surgical ward was conducted. In the first week after surgery, nutrition was managed using a nasogastric tube. If the patient was not able to eat soft or liquid food for longer than 1 week, percutaneous endoscopic gastrostomy was performed. During stay in surgical ward, speech and language therapists as well as physical therapists performed daily therapy to enable fast rehabilitation. Postoperative pain was managed using non-steroidal anti-inflammatory drugs and transdermal fentanyl patches (usually 12 µg/h every third day). | PMC9830896 |
Data acquisition | After review of all documented surgeries performed at the Clinic of Oral and Maxillofacial Surgery, Medical Center— University of Freiburg, Germany in a period between June 2017 and June 2020, patients who underwent CAD/CAM driven DCIA graft reconstruction of the mandible were identified. All collected data was saved in a spreadsheet (Microsoft Excel | PMC9830896 |
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Review of patient charts and imaging data | blood loss, tumor | BLOOD LOSS, TUMOR | By reviewing all electronic patient charts, information regarding epidemiologic data (sex, age at the time of surgery), hospital stay (length of stay at ICU and surgical ward), surgical therapy (operating time, ischemic time, blood loss and weight of resected tumor sample) and postoperative outcome parameters was collected. Postoperative imaging data was used to determine the volume of the raised DCIA graft. Using the open source software 3D slicer the CT-scan was segmented and the volume of the inserted graft was determined by volumetric measurement of the graft [ | PMC9830896 |
Interviews | postoperative pain | DIABETES MELLITUS, AIDS, AUTOIMMUNE DISEASES | Before physical examination a structured interview was conducted by one investigator (TM) using a predefined questionnaire. The questionnaire included questions regarding general health conditions (nicotine or alcohol consumption, autoimmune diseases, diabetes mellitus) and postoperative features of daily life (postoperative pain, intake of analgesics, limitation in daily or physical activities and use of walking aids). | PMC9830896 |
Physical examination | Physical examination was performed by one investigator (TM) on both legs in order to directly compare the operated leg with the unaffected leg. (1) The range of motion (ROM) of the hip joint was measured using a goniometer and given as angular degrees for the following directions of movement: flexion/extension, abduction/adduction, and interior rotation/external rotation. (2) The muscle strength during hip flexion was determined using the muscle function test according to Janda [ | PMC9830896 |
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Statistical analysis | The collected data was transferred into the Statistical Package for the Social Sciences | PMC9830896 |
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Results | PMC9830896 |
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Study group characteristics | After an initial review of all documented surgeries during the mentioned period, 38 patients were eligible for study implementation. Six patients were not available, two patients could not travel to our clinic, 13 patients refused to participate in the study, and two patients died before their examination date. Eventually fifteen patients (five women and ten men) with a mean age of 65.5 ± 10.3 years (minimum: 53; maximum: 84 years) could be included in this study. | PMC9830896 |
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Review of patient charts and imaging data | Blood loss | BLOOD LOSS | Operating time ranged from 5 to 16 h, with a mean of 11.8 ± 3.0 h. Ischemic time was between 60 and 160 min with a mean of 93.5 min. Blood loss was between 300 and 2500 ml with a mean of 1021 ml ± 582 ml. Patients stayed at the ICU and the surgical ward for 5.7 ± 3.5 days and 25.8 ± 15.0 days respectively. The volume of the raised DCIA graft ranged between 12.1 cm | PMC9830896 |
Interviews | SD, low pain | AIDS, DIABETES MELLITUS, AUTOIMMUNE DISEASES | Five patients confirmed regular alcohol consumption, seven patients reported of regular smoking. One patient suffered from diabetes mellitus, two patients of autoimmune diseases. Patients reported of physical limitations in postoperative daily life activities for 12.3 ± 11.9 weeks in mean. Athletic activities were impossible for 6–15 weeks after surgery (38.4 ± 40.0 weeks in mean). Walking aids were used for 2–115 weeks postoperatively (37.1 ± 44.8 weeks in mean). Analgesics were required for a mean of 10.1 ± 6.5 weeks. At the time of examination, patients mostly reported no or low pain sensation of the hip (visual analog scale mean 0.3 ± 0.8). Table Anamnestic features regarding general health conditions reported by the patients during interview (SD = standard deviation)Characteristics of the study group regarding therapy and postoperative rehabilitation identified by interview, chart review and evaluation of postoperative CT-scans (SD = standard deviation)Length of skin incisionin mmGraftat volumein cm | PMC9830896 |
Physical examination | neurosensory deficits, hypesthesia | Range of motion (ROM) and muscle strength of the hip joint, as well as neurosensory deficits of the unaffected and operated side were examined and are presented in Tables Differences between the surgical and unaffected side regarding ROM of the hip joint given in degree of angle (SD = standard deviation)Comparison of the muscle strength of the surgical and unaffected side during flexion of the hip joint given by the total number and proportion of patients (in brackets) with no differences between both sides, weaknesses at the surgical side and weaknesses at the unaffected side (SD = standard deviation)Comparison of the sensibilitiy of cutaneous peripheral nerves of the surgical and unaffected side given by the total number and proportion of patients (in brackets) with no differences between both sides, hypesthesia at the surgical side and anesthesia at the surgical side (SD = standard deviation) | PMC9830896 |
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Statistical analyses | PMC9830896 |
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Parameters acquired by interview | substance abuse, pain | AIDS | Graft volume significantly correlated with the duration of the use of walking aids (R = 0.57; p = 0.033) and impairment in daily life activities (R = 0.65; p = 0.012). There was no statistical correlation of sex, substance abuse, medical preconditions, age, incision length or transplant volume with duration of hospital stay, pain sensation or intake of analgesics.
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Range of motion | Comparing the ROM of the hip joint of the operated leg with the unaffected leg, the Wilcoxon signed-rank test showed significant lower values in flexion (− 10.0° ± 14.9°; Spearman’s rank correlation coefficients for patient specific factors age (in years), follow-up interval (in weeks), length of skin incision (in cm), graft diameter (in cm), graft volume (in cmSignificance level of the correlation is given by the according | PMC9830896 |
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Muscle strength | weakness of the operated leg. | In the investigation of muscle strength during flexion of the hip joint according to Janda, the operated leg revealed to be less powerful in all directions of movement, especially during flexion. Women (5/5) were afflicted more frequently by a weakness of the operated leg than men (6/10; p < 0.05). Moreover, alcohol consumption (p < 0.05) and smoking (p < 0.01) correlated significantly with a detectable weakness of the operated leg. No statistical correlations could be found between age, graft volume, length of skin incision and muscle strength (Table Evaluation of the differences of muscle strength during flexion of the hip joint between the operated and unaffected side in dependence of patient specific factors age (in years), follow-up interval (in weeks), length of skin incision (in cm), graft diameter (in cm and, graft volume (in cmSignificance level of the correlation is given by the according | PMC9830896 |
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Sensitivity of the peripheral cutaneous nerves | loss of sensation | Testing the sensitivity of the peripheral cutaneous nerves, reduction in sensation (n = 8), as well as loss of sensation (n = 3) of the lateral cutaneus femoris nerve was noticeable (Table Evaluation of the differences of nerve disturbances at the operated side in dependence of patient specific factors age (in years), follow-up interval (in weeks), length of skin incision (in cm), graft diameter (in cm) and graft volume (in cmSignificance level of the correlation is given by the according | PMC9830896 |
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Discussion | avascular iliac crest grafts, postoperative impairment of the leg, neurosensory deficits, hypesthesia, abduction, peripheral cutaneous nerves | AIDS, CREST | In this study the extent of donor site morbidity in patients receiving CAD/CAM driven reconstruction of the mandible using DCIA grafts was documented. A correlation between the volume of the raised DCIA graft and postoperative use of walking aids and impairment in daily life activities could be found. Moreover, the incision length correlated with the occurrence of neurosensory deficits of the iliohypogastric nerve. The severity of detected neurosensory deficits correlated with the length of the skin incision: anesthesia of peripheral cutaneous nerves was associated with long skin incisions (280 ± 30.0 mm), whereas patients with hypesthesia and normal neurosensory function had shorter skin incisions (245 ± 10.1 mm and 216 ± 27.7 mm respectively). These results confirm that surgery should be performed as minimally invasive as possible to reduce postoperative donor-site morbidity.Liu et al. found a slightly smaller incision lengths of 186 mm in mean in a study group of CAD/CAM treated patients, [ROM of the hip joint of both legs was investigated during physical examination to quantify the impairment induced by surgery. Flexion and internal rotation were decreased significantly (-10.0° ± 14.9° and − 5.0° ± 5.7 respectively) in comparison to the unaffected leg, while other directions of motion were hardly affected after graft harvesting (Table ANOVA was used to investigate further relations of ROM with patient-specific factors (Table Investigation of muscle strength equally showed that the performed surgery using CAD/CAM impairs functionality on the operated leg. Especially flexion (60% of patients), abduction (40% of patients) and internal rotation (27% of patients) were weaker in comparison to the opposite leg (Table In the last 2 years, three systematic reviews were published summarizing the findings regarding donor-site morbidity after head and neck reconstruction of the last 20 years [Katz et al. performed a retrospective chart review of DCIA graft patients and could acquire information about the graft volume in 65% of the investigated cases. A mean graft volume of 8.4 ± 6.9 cmGhassemi et al. performed a prospective interview with a mixed study group receiving vascular and avascular iliac crest grafts without using CAD/CAM techniques [As CAD/CAM techniques have only been part of the clinical routine for approximately ten years, just one other study (Liu et al.) reports of donor-site morbidity in a study population, in which CAD/CAM was used consistently for DCIA graft harvesting [Investigations regarding neurosensory deficits of the pelvis after conventional iliac crest harvesting have been conducted previously [The localizations of nerve alterations most frequently stated in literature (iliohypogastric nerve and lateral cutaneous nerve of thigh) are consistent with the findings of this study [In general, the small sample size of this study (n = 15) obliges cautious interpretation of the presented results. Anatomical or functional peculiarities of our specific study group may affect statistical analysis, which makes generalizability of our results questionable. Pre- and postoperative CT- or MR-data of the donor sites should be used in future studies to enable a more objective evaluation of the anatomical and functional changes. In fact, it would be useful to compare the results with a control group consisting of conventionally operated patients. Since the advantages of CAD/CAM technology and the associated virtual planning are undeniable, it was considered unethical to conduct a case–control study. Instead, a comparison with existing literature reports on conventionally treated patients was conducted. Nevertheless, advanced investigations with larger study groups may be necessary to reveal the true effect of CAD/CAM driven DCIA graft harvesting on postoperative impairment of the leg. | PMC9830896 |
Acknowledgements | Not applicable. | PMC9830896 |
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Author contributions | RS | LSB contributed to conception and design, to acquisition, drafted and critically revised the manuscript and gave final approval. The author agrees to be accountable for all aspects of work ensuring integrity and accuracy. PJV contributed to conception and design, contributed to interpretation, critically revised manuscript and gave final approval. The author agrees to be accountable for all aspects of work ensuring integrity and accuracy. TM contributed to conception and design, to acquisition and gave final approval. The author agrees to be accountable for all aspects of work ensuring integrity and accuracy. JK, ME, RMR, MCM, RS, PP critically revised the manuscript and gave final approval. The author agrees to be accountable for all aspects of work ensuring integrity and accuracy. JVW contributed to scientific writing, critically revised the manuscript and gave final approval. The author agrees to be accountable for all aspects of work ensuring integrity and accuracy. All authors read and approved the final manuscript.
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Funding | Open Access funding enabled and organized by Projekt DEAL. | PMC9830896 |
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Availability of data and materials | The data presented in this study is available on reasonable request from the corresponding author. The data are not publicly available due to privacy reasons. | PMC9830896 |
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Declarations | PMC9830896 |
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Ethics approval and consent to participate | All experimental protocols were approved by the Ethics Committee of the Albert-Ludwigs-University of Freiburg, Germany (Protocol #573/19). All methods were carried out in accordance with relevant guidelines and regulations. | PMC9830896 |
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Consent for publication | Written informed consent was obtained from all subjects and/or their legal guardian(s). | PMC9830896 |
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Competing interests | There are no competing interest to declare. | PMC9830896 |
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References | PMC9830896 |
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1. Introduction | RS | Resonance Raman spectroscopy (RRS) has been used as a reference method for measuring skin carotenoid levels (SCL), which indicate vegetable and fruit intake. However, RRS is not an easy-to-use method in SCL measurement due to its complicated implementation. In this study, a commercial spectrophotometer based on reflection spectroscopy (RS), which is relatively simple and inexpensive, was evaluated to confirm usability compared with RRS in measuring SCL. To investigate the agreement between RS and RRS, eighty participants were randomly assigned to a high-carotenoid diet group (21 mg/day of total carotenoids) or a control-carotenoid diet group (14 mg/day of total carotenoids) during a 6-week whole-diet intervention period and a 4-week tracking period. Strong correlations between the RS and RRS methods were observed at baseline (r = 0.944) and the entire period (r = 0.930). The rate of SCL increase was similar during the diet intervention; however, the initiation of the SCL decrease in RS was slower than in RRS during the tracking period. To confirm the agreement of RS and RRS from various perspectives, new visualization tools and indices were additionally applied and confirmed the similar response patterns of the two methods. The results indicate that the proposed RS method could be an alternative to RRS in SCL measurements.Carotenoids are important indicators of vegetable and fruit (V & F) intake because they cannot be produced in the human body and must be obtained via external intake [Therefore, noninvasive methods of skin carotenoid level (SCL) measurement have been proposed to estimate V & F intake [Another noninvasive method, a relatively simple and implantable method known as reflectance spectroscopy (RS), has been employed to measure SCL. In the 1960s, predicting carotenoids in human skin using RS with wavelengths near 480 nm was suggested [In this study, we proposed the measurement of SCL by using a commercial spectrophotometer based on RS. To obtain a carotenoid-specific signal, wavelengths of the range related to carotenoids are selected by referring to the study by Ermakov and Gellermann [To verify a commercial RS-based spectrophotometer, a carotenoid diet intervention was performed in this study. Additional approaches were considered for evaluating RS as an alternative to RRS. The degree of agreement between two different measurement approaches has been conventionally evaluated as correlations or Bland–Altman plots [ | PMC10490775 |
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2. Materials and Methods | PMC10490775 |
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2.1. Diet Intervention Study Design | DISEASES | The study was designed as a randomized, parallel-group, whole-diet controlled trial involving a 6-week dietary intervention and a 4-week tracking period (Korean adults aged over 18 without underlying diseases were recruited at SNU, and screening tests (at week 0) were conducted five weeks before the start of the intervention (at week 1). SCL, body mass index (BMI), and waist circumference were measured to analyze their initial status in week 0 and week 1. The participants were informed that they could withdraw from the study at any time if they were willing to stop participating, developed symptoms related to COVID-19, or came into contact with an infected individual. After screening based on eligible criteria (During the 6-week intervention period, the HG was provided with a high-carotenoid diet (including average total carotenoids contents: 21.0 mg/2000 kcal per day), while the CG was provided with a control diet (including average total carotenoids contents: 13.6 mg/2000 kcal per day). The experimental diet amount was provided in proportion to the design content according to the calories closest to the estimated energy requirements of each participant. In compliance with the Helsinki Declaration guidelines, the level of carotenoids in all diets was designed not to exceed the upper limit of vitamin A intake in consideration of the conversion of carotenoids to vitamin A, and the lowest level was above the estimated average requirement of Dietary Reference Intakes for Koreans (KDRI) [After the 6-week diet intervention period, the participants were instructed to maintain their typical lifestyle habits and diet to detect changes during the 4-week tracking period. Restricting the carotenoid intake during the follow-up period could negatively affect health in research ethics; hence, rather than limiting the carotenoid intake during the follow-up period, they were instructed to return to a routine diet to ensure that it could return to the baseline level after a controlled diet. The details of the eligibility criteria and experimental diets are described in the | PMC10490775 |
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2.2. RRS-Based Measurement | To evaluate SCL, a peak reflecting various types of carotenoids was used. As a reference, RRS has been widely used in previous studies because the size of the peak measured at the thumb was highly correlated with the SCL. To minimize the effect on other components of the skin, the right thumb, which has a low melanin concentration and a high carotenoid concentration due to its thick stratum corneum, was selected as the measurement site. The details of the peak size calculation using Raman spectra are described in a previous study [At each visit, the participants were instructed to conduct three trials by placing their right thumb in contact with the device’s glass window for measurement. For each trial, measurement with an exposure time of 400 ms was accumulated for 50 times. The average of the measured values was used for the analyses. | PMC10490775 |
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2.3. RS-Based Measurement | For the RS-based measurement, a commercial spectrophotometer (CM−700d, Konica Minolta, Inc., Tokyo, Japan) composed of an integrating sphere was used (At each visit, participants were instructed to measure 10 trials that consisted of three repetitions of strong pressure, three repetitions of weak pressure, and four repetitions of arbitrary pressure using their right thumb. For each trial, fewer than 5 s were taken from the measurement click to the data acquisition. Because the pressure between the thumb and glass on the measurement site was negatively correlated with the blood volume, high pressure enables blood to diverge from the measurement site to minimize the noise due to the hemoglobin. However, the quantitative control of pressure is not useful because the relationship between physical pressure and the blood clearance may vary among individuals. Accordingly, HbI was monitored instead of pressure to distinguish between trials of strong and weak pressure. The vicinity of minimum and maximum HbI values indicate the conditions of strong and weak pressure, respectively. The maximum and minimum HbI values measured at each participant’s finger are clear. This study’s protocol was specifically designed to obtain data across a wide range of hemoglobin levels. On behalf of the HbI provided in the software, the absorbance at 570 nm (=The wavelength range of the carotenoid spectrum is 400–510 nm, but in previous studies [The peak size of Equation (1) is negatively correlated with HbI ( | PMC10490775 |
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2.4. Statistical Analysis and Evaluation | Correlation coefficients and To confirm the change in SCL, a two-tailed paired | PMC10490775 |
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2.5. Visualization Using a Difference Vector | To visualize the change in SCL for respective participants, difference vectors were calculated. The use of difference vectors in this study is described in | PMC10490775 |
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2.6. Hit Rate | The proportion of participants whose The chance level of the index was 0.5, which implies that the change was random. The probability of the hit rate was calculated to confirm significance, as shown in Equations (4)–(6). The variable | PMC10490775 |
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2.7. Subgroup Classification | To compare the kinetics of the different initial SCL, we divided the participants into four groups on the basis of the SCL obtained using RRS in the screening week. The SCL obtained from the screening test ranged from approximately 50 AU to 250 AU, and the subgroups were divided into 50 AU intervals (Group 1 (G1): Over 200 AU, Group 2 (G2): 200–150 AU, Group 3 (G3): 150–100 AU, Group 4 (G4): Below 100 AU). In the HG, 5, 8, 14, and 8 participants belonged to groups G1, G2, G3, and G4, respectively. In the CG, 2, 10, 12, and 8 participants belonged to groups G1, G2, G3, and G4, respectively. | PMC10490775 |
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2.8. Sample Size | The effect size was considered in previous studies with regard to the effect of the carotenoid intake on skin and blood carotenoid levels [ | PMC10490775 |
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3. Results | PMC10490775 |
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3.1. Participants’ Characteristics | RS | MAY | Out of the total 152 individuals who enrolled in the study between December 2020 and February 2021, eight participants did not attend the screening test, 59 did not meet the inclusion criteria, and five declined to participate. The 80 eligible participants provided written and informed consent. All participants were randomly allocated to the high-carotenoid diet group (HG, n = 40) and control-carotenoid diet group (CG, n = 40) for the 6-week whole-diet intervention and 4-week tracking periods, from February to May 2021. In the analysis, which was based on the average SCL or correlations between the two methods, RS and RRS, we included 67 participants who attended every week. Of these, 32 participants were part of the CG, while the remainder belonged to the HG. The analysis was conducted without any change in the group initially assigned. In the analysis using difference vectors or hit rates, all participants of each week were included ( | PMC10490775 |
3.3. Change Rate between RRS and RS | The strength of the change was calculated as the slope from the beginning to the last point. During the intervention period in the HG, the The results of the paired The changes in subgroups G1 to G4 divided by the criteria before the start of the study were compared. The SCL of the low-initial-level groups (subgroups G3 and G4) increased relatively faster than that of the high-initial-level groups (subgroups G1 and G2). During the intervention period, the During the four weeks of tracking in the HG, | PMC10490775 |
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3.4. Vector Field between RRS and RS | A vector field comprises the difference vectors defined in By employing a differences vector, | PMC10490775 |
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3.6. Hit Rates of RRS and RS | The hit rate, the proportion of participants that show the intended change in the carotenoid level, is presented in During the tracking period, the hit rates of | PMC10490775 |
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4. Discussion | PMC10490775 |
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4.1. Principal Findings | RS | This study entailed a whole-diet randomized controlled trial to evaluate the performance of a commercial RS-based spectrophotometer in measuring SCL as compared with RRS. Similar response patterns in the RRS and RS methods were identified using correlation analysis and the ratio of change during the intervention and tracking periods. Before the intervention, the correlation between When comparing the correlation by referring to RRS, the correlation with commercial devices (r = 0.92 to 0.96) in this study was higher than that of RS devices (r = 0.76 to 0.86) developed for SCL measurement [ | PMC10490775 |
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4.2. The Strengths of This Study | RS | This study reported the characteristics of SCL changes and applied a novel analysis method to dynamic responses at the individual level. The current study revealed three distinctive tendencies for changes in the SCL. First, the change rate of the SCLs measured by both approaches depends on the initial level. In the case of participants with a low initial SCL, the difference between the amount of carotenoids intake before and after the intervention period begins is larger, which may result in a larger increase in Notably, the pattern of differences between RS and RRS in the initial tracking period suggests that Additionally, the new visualization tools and indices were proposed and confirmed for observing SCL changes from an individual rather than an average perspective. In previous studies, the effects of carotenoid-containing diets and supplement intake were confirmed by analyzing a trend of average values [Furthermore, the use of the vector fields was newly proposed to visualize the behavior of | PMC10490775 |
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4.3. Limitations | A limitation of this study is that only participants with skin tones of an intermediate color were included. Therefore, an improved model for a general skin tone should be investigated in future studies. To cover the full range of skin tones such as Fitzpatrick skin types I to VI, the model should consider the effects of melanin, and the features with additional wavelengths should be designed. To resolve this issue, using RRS as a reference should be carefully considered because the approach is affected by chromophores [To quantify the usual carotenoid intake, it is necessary to further examine whether the relationship between the equilibrium SCL and usual intake exists. However, both the | PMC10490775 |
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5. Conclusions | RS | Compared to RRS, RS-based commercial equipment is useful because commercial RS has faster measurement times without calibration every time, is convenient to measure at reasonable prices, and is simple to implement. Therefore, a commercial device applying the proposed method is expected to be used as an alternative to conventional RRS in future applications for SCL measurement. Consequently, the results of this study enable more convenient V & F intake monitoring and will contribute to an improvement of dietary management for personal health. | PMC10490775 |
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6. Patents | SAMSUNG ELECTRONICS Co., Ltd. (Suwon, Republic of Korea) has a patent (Patent No.: US11359965) for RS-based skin carotenoid level measurement. | PMC10490775 |
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Supplementary Materials | The following supporting information can be downloaded at: Click here for additional data file. | PMC10490775 |
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Author Contributions | J.-E.H. set up the experiments, performed the data analysis, and drafted the manuscript as the first author. J.-Y.P. and H.J. administrated the project and reviewed the manuscript. M.H.J., K.E. and H.S.M. participated in the project and conducted the experiments. Y.J.K. designed the study, performed the data analysis, and drafted the manuscript as the corresponding author. All authors have read and agreed to the published version of the manuscript. | PMC10490775 |
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Institutional Review Board Statement | The study was approved by the Institute Review Board of Seoul National University (IRB No. 2010/001-014). | PMC10490775 |
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Informed Consent Statement | Informed consent was obtained from all subjects involved in the study. | PMC10490775 |
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Data Availability Statement | The data that support the findings of this study are available from the first and corresponding authors upon reasonable request. Furthermore, all data are the property of SAMSUNG ELECTRONICS Co., Ltd., and permission is required to export it. | PMC10490775 |
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Conflicts of Interest | The authors declare no conflict of interest. | PMC10490775 |
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