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References | PMC10258964 |
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Background | CYTOTOXICITY, HER2+ BREAST CANCER | The phase II neo-adjuvant clinical trial ICORG10-05 (NCT01485926) compared chemotherapy in combination with trastuzumab, lapatinib or both in patients with HER2+ breast cancer. We studied circulating immune cells looking for alterations in phenotype, genotype and cytotoxic capacity (direct and antibody-dependent cell-mediated cytotoxicity (ADCC)) in the context of treatment response. | PMC10491671 |
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Methods | Peripheral blood mononuclear cells (PBMCs) were isolated from pre- ( | PMC10491671 |
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Results | ER+ tumours | RESIDUAL DISEASE | Treatment attenuated the cytotoxicity/ADCC of PBMCs. CD3+/CD4+/CD8+ T cells increased following therapy, while CD56+ NK cells/CD14+ monocytes/CD19+ B cells decreased with significant post-treatment immune cell changes confined to patients with residual disease. Pembrolizumab-augmented ex vivo PBMC ADCC activity was associated with residual disease, but not pathological complete response. Pembrolizumab-responsive PBMCs were associated with lower baseline TIL levels and ER+ tumours. | PMC10491671 |
Conclusions | PBMCs display altered phenotype and function following completion of neo-adjuvant treatment. Anti-PD-1-responsive PBMCs in ex vivo ADCC assays may be a biomarker of treatment response. | PMC10491671 |
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Subject terms | PMC10491671 |
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Introduction | breast cancers, breast cancer | CYTOTOXICITY, HER2+ BREAST CANCER, BREAST CANCER | Human epidermal growth factor receptor 2-positive (HER2+) breast cancer accounts for approximately 20% of all breast cancers [Trastuzumab was the first HER2-targeted therapy approved for the treatment of HER2+ breast cancer. Trastuzumab targets a juxtamembrane epitope in subdomain IV of the extracellular portion of the HER2 protein. Once bound, trastuzumab suppresses HER2 intracellular signalling, and trastuzumab’s human IgG1 isotype framework was deliberately designed to elicit antibody-dependent cell-mediated cytotoxicity (ADCC) [Lapatinib is a small molecule tyrosine kinase inhibitor (TKI) that targets EGFR and HER2 and it is approved for use in combination with capecitabine as second-line therapy for metastatic HER2+ breast cancer that is refractory to trastuzumab, or to treat advanced hormone receptor positive HER2+ breast cancer in combination with letrozole [Other clinical trials investigating the combination of trastuzumab and lapatinib in the neo-adjuvant setting have been conducted: CHER-LOB [ | PMC10491671 |
Materials and methods | PMC10491671 |
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Cell lines and reagents | Cell lines were cultured at 37 °C/5% CO | PMC10491671 |
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Patient population and sampling time points | HER2+ BREAST CANCER | HER2+ breast cancer patients ( | PMC10491671 |
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Sample processing | BLOOD | Blood was collected in EDTA blood tubes (BD Vacutainer #367525) and processed within 4 h of blood draw. PBMCs were isolated using Ficoll-Paque (ThermoFisher 11778538)-based density centrifugation. PBMCs were frozen in vials at 1 × 10 | PMC10491671 |
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Immune cytotoxicity assays | ALDRICH, CYTOTOXICITY | The immune cytotoxicity assays described are based on flow cytometry-based protocols utilised previously, involving carboxyfluorescein succinimidyl ester (CFSE) (Sigma Aldrich 21888) staining of target cells and the use of aminoactinomycin-D (7AAD) (Sigma Aldrich 9400) as the membrane permeable dead cell dye [ | PMC10491671 |
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Fc gamma receptor genotyping | Patient samples ( | PMC10491671 |
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Peripheral blood immunophenotyping | Healthy volunteer and patient PBMCs were isolated, thawed and counted as previously described. PBMC samples were brought to a concentration of 3 × 10 | PMC10491671 |
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Tumour infiltrating lymphocytes | tumour | TUMOUR, INFILTRATING | Pre-treatment and on-treatment (20 days post cycle 1) stromal, tumour and overall (stromal + tumour) infiltrating lymphocyte counts for ICORG 10-05 were determined previously [ | PMC10491671 |
Ethical approval | ONCOLOGY | The ICORG 10-05 clinical trial was conducted in accordance with the Declaration of Helsinki. The trial was approved by the relevant ethics committees/institutional review boards and health authorities at all participating sites in Ireland under the direction of the study sponsor, the All-Ireland Clinical Oncology Research Group (ICORG). Informed, written consent was obtained from all participants. Healthy volunteer samples were collected under DCU Ethics Committee approval with informed, written consent obtained from all volunteers. | PMC10491671 |
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Statistical analysis | A pooled Student’s | PMC10491671 |
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Results | PMC10491671 |
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Patient characteristics | Eighty-eight patients were enrolled on the TCHL study, comprising of TCH ( | PMC10491671 |
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Breakdown of available samples. | Consort diagram ( | PMC10491671 |
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Downregulation of peripheral cytotoxic immune response following completion of neo-adjuvant chemotherapy | CYTOTOXICITY | The cytotoxicity levels of PBMCs pre- and post-treatment were examined in all available samples. An optimal pooled | PMC10491671 |
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Changes to immune cell subsets following neo-adjuvant therapy | CD45+ PBMCs from patients that have completed neo-adjuvant chemotherapy regimens displayed alterations in immunophenotype. The immunophenotype data, and all following data presented in Figs. | PMC10491671 |
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Pre- and post-treatment immune cell subsets. | Proportion of CD45+ cells staining positive for | PMC10491671 |
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Direct cytotoxicity and ADCC by pCR status. | CYTOTOXICITY | Paired pre- and post-treatment direct cytotoxicity levels of patient PBMCs against | PMC10491671 |
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Immune cell subsets by pCR status. | tumour | CYTOTOXICITY, TUMOUR | Proportion of CD45+ cells which stained positive for The proportion of CD3+ T cells significantly increased following neo-adjuvant treatment (Fig. Diametric changes were observed in other circulating immune cell populations. There was a significant decrease in the proportion of CD56+ NK cells (Fig. Peripheral T cells are not expected to elicit cytotoxic effects in the assays used in this study as they have not been antigen-primed or otherwise activated. However, NK cells and monocytes are mediators of direct cytotoxicity against tumour cells and ADCC [Expression of the immune checkpoint PD-1 was investigated in CD45+ PBMCs to assess potential immunosuppressive phenotypes. CD45+PD-1+ immune cell levels were unchanged between pre- and post-treatment samples (Fig. | PMC10491671 |
Post-treatment cytotoxic immune response did not vary based on pCR and no pCR | CYTOTOXICITY | To ascertain if variances in in vitro PBMC cytotoxicity due to neo-adjuvant treatment could be associated with patient response to therapy, the in vitro cytotoxicity data was broken down by pCR. There was no change in direct cytotoxicity against the K562 or SKBR3 cell lines based on clinical response between pre- and post-treatment samples (Fig. | PMC10491671 |
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Post-treatment changes in immune cell subsets are driven by the No pCR cohort | Immunophenotype data was also assessed in the context of pCR. There were no significant differences in the immune cell populations pre- and post-treatment within the pCR cohort (Fig. | PMC10491671 |
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Baseline circulating immune cells from patients with no pCR have augmented cytotoxic response after treatment with pembrolizumab | Examining PD-1 expression on CD56+, CD8+, and CD14+ subsets reveals that PD-1 expression was present but there was no difference between pre- and post-treatment when divided based on treatment response (Supplementary Fig. | PMC10491671 |
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Pembrolizumab-responsive PBMCs in the No pCR cohort. | Trastuzumab ADCC levels with and without pembrolizumab against SKBR3 cells using pre-treatment patient PBMCs from In addition, the pre-treatment PD-1 inhibited ADCC response (Fig. | PMC10491671 |
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TIL levels are lower in patients with in vitro PBMC response to pembrolizumab | Matched pre-treatment TIL data were available for 13/21 pre-treatment PBMC samples utilised in the pembrolizumab ADCC assays [ | PMC10491671 |
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Treatment arm did not affect immune cytotoxicity or PBMC immunophenotype | CYTOTOXICITY | No significant difference was found when post-treatment direct cytotoxicity (K562/SKBR3) or ADCC (SKBR3) were compared to pre-treatment levels within arms (Supplementary Fig. | PMC10491671 |
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Discussion | non-small cell lung cancer, ER+ tumours, melanoma, breast cancer, tumours, TNBC, immune-suppressed tumour | NON-SMALL CELL LUNG CANCER, MELANOMA, BREAST CANCER, TUMOURS, CYTOTOXICITY, HER2+ BREAST CANCER | We report that neo-adjuvant treatment reduced the direct cytotoxic and ADCC capacity of circulating immune cells for all patients on the ICORG 10-05 trial, irrespective of FCGR SNP status. This corresponded with post-treatment increases in circulating CD8+ and CD4+ T cells, and decreased levels of NK cells, monocytes and B cells. When assessed in the context of treatment response, these changes were predominantly associated with patients who did not achieve a pCR. In addition, functionally pembrolizumab-responsive ADCC-capable immune cells were only detected in the pre-therapy blood of patients who did not achieve a pCR, had lower TIL levels and were more likely to have ER+ tumours.This study is the first to genotype, phenotype and functionally assess patient PBMCs in vitro in the context of pCR following 6 rounds of neo-adjuvant anti-HER2 targeted therapy/docetaxel/carboplatin. We found no statistically significant evidence that the HER2-targeted therapy given influenced the in vitro cytotoxicity or immune profile of samples (Supplementary Figs. Previous studies, such as those from Gennari et al. and Varchetta et al., have shown an increase in trastuzumab-mediated ADCC capabilities of patient immune cells in vitro after receiving neo-adjuvant trastuzumab monotherapy [The increase in post-therapy T cell levels indicates a triggering of an adaptive immune response. When assessed by pCR, it was found that the No pCR cohort was making the biggest contribution to the observed post-treatment changes for all immune cell subtypes (Fig. Our results highlight functional assessment of immune cells as an important adjunct to phenotypic analysis, which in this case did not definitively identify PD-1+CD16+ NK cells or monocytes as markers of treatment response in the population as a whole (Fig. The reduced TIL count in tumours from patients with PBMCs that responded to pembrolizumab in vitro is an important indication that the circulating immune cells are associated with an immune-suppressed tumour microenvironment (Fig. It should also be noted that the presence of PD-1-inhibited immune cells could also identify patients who will benefit from the addition of anti-PD-1 immune checkpoint inhibitors in the neo-adjuvant setting. Clinical trials examining checkpoint inhibitors in breast cancer are established, with anti-PD-L1 therapy atezolizumab approved to treat TNBC following the IMpassion130 trial [In summary, we hypothesise that circulating anti-PD-1-sensitive ADCC-capable immune cells identify immune-suppressed tumour phenotypes and could function as a biomarker of response to standard chemotherapy regimens and immune checkpoint inhibitors. Current efforts are focussed on prospective collection of pre-treatment blood samples from early stage HER2+ breast cancer patients and non-small cell lung cancer and melanoma patients scheduled to receive immune checkpoint therapy to provide larger datasets to assess the potential of the functional biomarker assay described in this study. | PMC10491671 |
Supplementary information | The online version contains supplementary material available at 10.1038/s41416-023-02375-y. | PMC10491671 |
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Acknowledgements | Cancer | ONCOLOGY, CANCER | We wish to thank the patients who participated in the ICORG 10-05 clinical trial and the healthy volunteers who provided blood samples. The clinical trial was sponsored by the All-Ireland Clinical Oncology Research Group (ICORG), now known as Cancer Trials Ireland – CTRIAL-IE, which received funding from GlaxoSmith-Kline, the owners of lapatinib at the initiation of the study. Lapatinib is now a commodity of Novartis. | PMC10491671 |
Author contributions | NG, AB, SFM, ST, and DMC conceived/designed experiments and acquired and interpreted data. DK, AJE, MSJM, and AC acquired and interpreted data. BM, JMF, NOD, and JC conceived/designed experiments and interpreted data. JSR, AT, and BTH interpreted data. All authors partook in manuscript drafting/revision, approved the final version, and agreed to be accountable for all aspects of the work. | PMC10491671 |
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Funding | Cancer | CANCER | This translational study was funded by The Caroline Foundation and the Cancer Clinical Research Trust (CHY12210). Open Access funding provided by the IReL Consortium. | PMC10491671 |
Data availability | The datasets generated during and/or analysed during the current study are not publicly available due to the terms of ethics approval/consent for the study and data privacy regulations but are available from the corresponding author on reasonable request. | PMC10491671 |
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Competing interests | CMO, cancer | ONCOLOGY, CANCER | JC has received honoraria from Pfizer, MSD Oncology, Pierre Fabre, and AstraZeneca; has acted in a Consulting or Advisory Role for AstraZeneca, Novartis, MSD, Cepheid and received Speakers' Bureau from Pfizer; has received research funding from Boehringer Ingelheim, Roche, Puma Biotechnology Regeneron, Novartis, MSD Oncology, BMS GmbH, and Co. KG; has received travel, accommodations, and expenses/conference registration from Roche, Daiichi Sankyo, AstraZeneca, MSD, Pfizer, Novartis, and Regeneron; is the Chief Medical Officer (CMO) at OncoMark Ltd and is employed with OncoAssure Ltd, and has associated stock and ownership interests with these entities. MSJM is a co-founder of and shareholder in TORL Biotherapeutics LLC and 1200 Pharma LLC. DMC has received research funding and research materials from Roche/Genentech, WntResearch; research materials from Sanofi; and has received research funding and consultancy fees from Puma Biotechnology, Inc. DMC, NG, NO, and JC report the filing of patent application WO2020011770A1—A method of predicting response to treatment in cancer patients. All other authors have declared no conflicts of interest. | PMC10491671 |
References | PMC10491671 |
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Background | Leprosy, leprosy, infectious disease | LEPROSY, LEPROSY, INFECTIOUS DISEASE | Leprosy is an ancient infectious disease with an annual global incidence of around 200,000 over the past decade. Since 2018, the World Health Organization (WHO) recommends single-dose rifampicin as post-exposure prophylaxis (SDR-PEP) for contacts of leprosy patients. The Post ExpOsure Prophylaxis for Leprosy (PEOPLE) trial evaluated PEP with a double dose of rifampicin in Comoros and Madagascar. Preliminary results of this trial show some reduction in leprosy incidence in intervention villages but a stronger regimen may be beneficial. The objective of the current Bedaquiline Enhanced ExpOsure Prophylaxis for LEprosy trial (BE-PEOPLE) is to explore effectiveness of a combination of bedaquiline and rifampicin as PEP. | PMC10169125 |
Methods | leprosy, tuberculosis | LEPROSY, TUBERCULOSIS | BE-PEOPLE is a cluster-randomized trial in which 44 clusters in Comoros will be randomized to two study arms. Door-to-door screening will be conducted annually during four years, leprosy patients identified will be offered standard of care treatment. Based on study arm, contacts aged five years and above and living within a 100-meter radius of an index case will either receive bedaquiline (400-800 mg) and rifampicin (150-600 mg) or only rifampicin (150–600 mg). Contacts aged two to four years will receive rifampicin only. Household contacts randomized to the bedaquiline plus rifampicin arm will receive a second dose four weeks later. Incidence rate ratios of leprosy comparing contacts who received either of the PEP regimens will be the primary outcome. We will monitor resistance to rifampicin and/or bedaquiline through molecular surveillance in all incident tuberculosis and leprosy patients nationwide. At the end of the study, we will assess anti- | PMC10169125 |
Discussion | leprosy | LEPROSY | The COLEP trial on PEP in Bangladesh documented a reduction of 57% in incidence of leprosy among contacts treated with SDR-PEP after two years, which led to the WHO recommendation of SDR-PEP. Preliminary results of the PEOPLE trial show a lesser reduction in incidence. The BE-PEOPLE trial will explore whether reinforcing SDR-PEP with bedaquiline increases effectiveness and more rapidly reduces the incidence of leprosy, compared to SDR-PEP alone. | PMC10169125 |
Trial registration | NCT05597280. Protocol version 5.0 on 28 October 2022. | PMC10169125 |
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Keywords | PMC10169125 |
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Background | Leprosy, leprosy, infection, chronic infectious disease | LEPROSY, MAY, LEPROSY, INFECTION, CHRONIC INFECTIOUS DISEASE | BE-PEOPLE is a randomized controlled trial assessing the effectiveness of bedaquiline and rifampicin as post-exposure prophylaxis (PEP) for leprosy in Comoros. Leprosy is an ancient chronic infectious disease caused by In 2000, the World Health Organization (WHO) declared leprosy eliminated as public health problem based on a prevalence below 1 per 10,000 worldwide [The BE-PEOPLE trial described in this manuscript is a sequel of the PEOPLE trial conducted between 2019 and 2023 in Comoros and Madagascar. In PEOPLE, three modalities of SDR-PEP were compared to a control arm (arm 1), in which annual door-to-door case finding was conducted yet no PEP provided. Randomization was at the village level. The dosage of SDR-PEP used in the PEOPLE trial was double the WHO-recommended dose, i.e. 20 mg/kg. In arm 2 villages only household contacts received PEP. In arm 3 villages blanket PEP coverage was provided to anyone living within a 100-meter of an index case or to the entire village if more than 50% of the village population were included in this perimeter. In arm 4 villages, screening was accompanied by a serosurvey based on anti-PGL-I IgM (the first serosurvey with UCP-LFA on such a large scale in the field), an antibody marker of infection with BE-PEOPLE was preceded by a phase 2 study to confirm the safety of BE-PEP. For this purpose, a leprosy endemic village that had been part of arm 1 of the PEOPLE trial was selected. In May 2022, the entire population of approximately 900 was screened for leprosy. After the screening, 300 eligible participants divided over three age brackets were randomized to either BE-PEP (rifampicin 600 mg + bedaquiline 800 mg for adults) or SDR-PEP (rifampicin 600 mg for adults) in an age de-escalating design. All participants had an ECG recorded pre-treatment and one day after treatment, and venous blood samples were collected pre-treatment and 14 days after treatment to be tested for liver functions (Aspartate aminotransferase AST/Alanine aminotransferase ALT). No major safety concerns emerged and the independent data safety and management board gave the green light for proceeding to phase 3, which is the subject of the current manuscript. | PMC10169125 |
Methods/design | PMC10169125 |
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Objectives and hypothesis | leprosy | ADVERSE EVENT, SECONDARY, LEPROSY | The primary objective of BE-PEOPLE will be to compare effectiveness of BE-PEP to that of SDR-PEP at individual level. As a secondary objective, we will compare overall leprosy incidence between villages randomized to BE-PEP and villages randomized to SDR-PEP. Adverse events will be closely monitored and quantified per study arm.Other objectives are to quantitatively assess anti-PGL-I IgM seropositivity in the population as a proxy for the population burden of | PMC10169125 |
Study design | TB, Leprosy, leprosy, Mohéli | ADVERSE EVENTS, LEPROSY, LEPROSY | The BE-PEOPLE trial is a cluster randomized trial in which 44 clusters from the islands of Anjouan and Mohéli (Comoros) will be randomized to two study arms. These include 34 out of 48 villages that were also part of the PEOPLE trial as well as nine new villages of which one has been divided into two. For randomization, clusters will be listed in order of decreasing baseline leprosy prevalence by island and by former PEOPLE trial study arm. They will then be randomized to arm 1 (BE-PEP), intervention, or arm 2 (SDR-PEP), comparator arm, of BE-PEOPLE. Participants residing in villages arm 1 who are between two and five years of age and with a weight of 10–20 kg are not eligible for BE-PEP but will be offered SDR-PEP instead.A first round of PEP will be provided in 2023, within one month following the screening in each village. This will be considered the start of follow-up. Screening will take place on an annual basis until the fourth and final round in 2026, which will be the end of follow-up. Leprosy incidence will be measured at the individual level among those that received either SDR-PEP or BE-PEP, excluding those below five years of age or weighing less than 20 kg because they are not eligible for BE-PEP. We will also compare incidence at village level between arm 1 and arm 2 villages, irrespective of whether or not an individual received PEP and of the kind of PEP received.Each individual provided PEP will be revisited the day after PEP intake to document any adverse events.Throughout the study skin biopsies will be collected of all consenting leprosy patients and slit skin smears of multibacillary patients, as well as sputum samples of all consenting TB patients. In addition nasal and tongue swabs, tongue scraping, and face mask samples will be collected from both TB and leprosy patients. Those that are PCR-positive for Cost data will be collected alongside the trial.Immediately following the final survey round in 2026, a serosurvey quantitatively assessing anti-PGL-I IgM antibodies will be conducted in BE-PEOPLE villages that were previously (since 2019) part of study arm 4 of the PEOPLE trial. | PMC10169125 |
Setting | TB, Mohéli, deformities, Leprosy, Tuberculosis, leprosy | TUBERCULOSIS (TB), LEPROSY, LEPROSY, TUBERCULOSIS | The Union of Comoros is an archipelago in the Indian Ocean, north of Madagascar, and includes the islands Grand Comore (with the capital Moroni), Anjouan, and Mohéli. Since 2011, around 400 new leprosy cases are notified annually. The vast majority of these cases come from Anjouan and Mohéli with an estimated population of 450,000, equivalent to 888 new leprosy cases/ 1 million population per year.The National Tuberculosis and Leprosy program strictly implements the strategies recommended by WHO, ensuring early diagnosis. Less than 3% of new leprosy cases have visible deformities and treatment completion rates are above 85% for both multibacillary (MB) and paucibacillary (PB) cases [In contrast with leprosy, tuberculosis (TB) in Comoros is mainly found on the main island, Grande Comore, contributing around 150 new TB cases notified annually [ | PMC10169125 |
Participants | cough, liver or kidney disease, leprosy, allergy | LEPROSY, ALLERGY | Participants will be enrolled from 44 clusters: 34 in Anjouan and 10 in Mohéli. We will screen for leprosy including all residents and all ages, whenever leprosy is diagnosed treatment will be provided according to national guidelines. At baseline, all permanent residents aged two years or above, living within 100 m of an index case diagnosed during the period of 2018–2023, will be eligible for PEP. If this entails more than 50% of the population, the entire village population will be eligible for PEP. In arm 1 the BE-PEP regimen will be offered, except for participants not eligible because of age (below five years) and/or weight (below 20 kg) criteria. Such individuals may still receive SDR-PEP if otherwise eligible. In both arms, the exclusion criteria include cough of more than two weeks’ duration (presumptive pulmonary TB), signs of extrapulmonary TB, self-reported pregnancy or breastfeeding, antecedents of liver or kidney disease (clinically or documented by lab tests), allergy to rifampicin, and treatment with rifampicin in the last two years. For arm 1 there will be some additional criteria which include use of medications in the three weeks preceding PEP that are not included in the safe list for bedaquiline. | PMC10169125 |
Randomization | The 44 study clusters will be grouped into 10 categories, by island (Anjouan and Mohéli) and in relation to the PEOPLE trial (former arm 1,2,3, or 4, or new). Within each group, they will be ordered by decreasing baseline prevalence and pairs will be constituted of successive clusters. Within each pair, one will be randomized to arm 1, and the other to arm 2. An independent sponsor biostatistician will prepare the randomization schedule using SAS 9.4 (SAS Institute, Cary NC). | PMC10169125 |
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Outcome measures | TB, leprosy | ADVERSE EVENTS, LEPROSY, SECONDARY | The primary outcome will be the leprosy incidence rate ratio between contacts who received SDR-PEP and contacts who received BE-PEP, excluding those in arm 1 who received SDR-PEP because of not being eligible for BE-PEP due to age and/or weight restrictions. In a secondary analysis, we will calculate the incidence rate ratios at the village level between arms 1 and 2, including all participants irrespective of whether or not they received PEP.Other outcome measures will be the frequency of adverse events by PEP regimen and costing of both study arms, and serosurvey in 2026 quantitatively assessing anti-PLG-I IgM antibodies in a subset of villages. We will also report proportions of leprosy or TB patients with resistance to either of the study drugs and assess any trends if present. A cost-effectiveness of BE-PEP compared to SDR-PEP will be done if BE-PEP is shown to be more effective. | PMC10169125 |
Intervention implementation and data collection | TB, scabies, Mohéli, vomiting, leprosy, loss of sensation, eczema, Vomiting | SCABIES, ADVERSE EVENTS, EVENT, MYCOSES, LEPROSY, SKIN DISEASES, ECZEMA | Before the study begins, village elders will be informed about study objectives and procedures, followed by community sensitization. Annual door-to-door screening for leprosy will be conducted from 2023 to 2026, by teams that consist of experienced health services staff and community volunteers. Data to be collected include geographic coordinates of all households visited as well as individual data, in particular demographic data and for those present during the visit, health, and medical data, results of examinations for leprosy and/or TB, eligibility for PEP, acceptance/refusal of PEP, and occurrence of adverse events. Data entry will be performed directly in the field, making use of an Android app in REDCap (Research Electronic Data Capture). REDCap is widely used in research and is compliant with standards and applicable regulations of good clinical practice (GCP). Data can be entered in REDCap offline, to be uploaded to a secure server whenever internet connection is available. We will also make use of a paper form, one form per household, to record name, age, and gender of each individual enrolled. These forms have a pre-printed unique barcode for each individual recorded as well as a unique household ID. They will be entered in an MS Access database, from which new forms with household ID, names, ages, and barcodes can be printed each time a new survey round starts. The field staff will use these forms to find back households previously visited. When entering data in the REDCap forms, they will use these barcodes and household IDs, no other personal identifiers will be recorded.TB patients diagnosed nationwide will be asked to sign an informed consent and will have sputum, nasal and tongue swabs, tongue scraping, and face mask sampling for genotyping of The diagnosis of leprosy will be following WHO guidelines, based on three cardinal signs: patch with loss of sensation, enlarged peripheral nerves, and/or slit-skin smear (SSS) positive for acid-fast bacilli. All leprosy cases diagnosed will be confirmed by experienced national leprosy control program health staff. All new leprosy cases detected will be treated according to the national guidelines. Other skin diseases such as mycoses, scabies, or eczema will also be treated free of charge. Subject to informed consent, all incident leprosy patients in Anjouan and Mohéli will be enrolled in a sub-study in which slit skin smears, nasal and tongue swabs, tongue scraping, facial mask sampling, and skin biopsies from non-facial lesions will be sampled to be tested with quantitative polymerase chain reaction (qPCR). If sufficient DNA is available, we will conduct further molecular tests for During the first round of annual door-to-door screening for leprosy, residents will be informed about objectives and study procedures, and requested to provide written informed consent. They will then be individually assessed for PEP eligibility. Upon completion of screening in a village, a PEP eligibility zone will be determined based on presence of leprosy index cases within 100 m. If more than 50% of the population lives within a 100-meter radius of an index case, the entire village will be eligible. Then, provision of PEP will be organized according to the study arms in another visit. All new leprosy cases diagnosed in between annual screening rounds will also be taken into account in the study. Eligible contacts of these patients will receive PEP during the next round of PEP administration.All participants who received PEP will be re-visited the next day for assessing vomiting and (serious) adverse events ([S]AE). Vomiting will be recorded but the dose will not be repeated. In case of SAE, the health worker will record the event and report it within 24 h to the sponsor, refer the patient for appropriate care, and follow up until resolution. Household contacts provided BE-PEP will be offered a second dose four weeks later, as preliminary data from the PEOPLE trial documented that their residual risk is still up to three times higher compared to the rest of the population. SDR-PEP will be offered only once. In addition, there will be passive reporting of adverse events over the period of up to 30 days post-PEP administration, afterwards only adverse events that are linked to the PEP intake will be documented. Pregnancy is an exclusion criterion but in case an existing pregnancy at the time of PEP administration only becomes apparent afterwards, the participant will be followed up until the time of delivery. | PMC10169125 |
Post-exposure prophylaxis | PMC10169125 |
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Single dose rifampicin | leprosy | LEPROSY | A single dose of rifampicin at 10 mg/kg is the current standard for PEP in leprosy for contacts aged two years and above, as per the 2018 WHO guidelines [ | PMC10169125 |
Rationale for using serology | Although detection of | PMC10169125 |
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Rationale bedaquiline and rifampicin combination | Bedaquiline, the first new drug to be developed against The bioavailability of bedaquiline increases with food (a 2-fold increase in AUC, see label). The exposure of rifampicin, when administered with food, decreases slightly but this is not considered clinically relevant. Therefore, we will offer a snack when PEP is administered. | PMC10169125 |
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Data analysis | TB, leprosy | LEPROSY | To assess the effectiveness of PEP at the individual level, we will fit a Poisson model adjusted for follow-up time as an offset term with the villages nested in islands as a random effect and type of PEP (BE-PEP or SDR-PEP) as an explanatory variable, with SDR-PEP as reference category. We will exclude participants below the age or weight limits for BE-PEP, even if they have received SDR-PEP. For each individual, follow-up time will start the day he or she was last examined before the first dose of PEP was administered and will end either at the time of their last survey visit or at the time the individual was diagnosed with leprosy. All incident leprosy cases diagnosed after the first door-to-door screening in 2023 until the final survey in 2026 will be considered. Participants lost to follow-up after their first intake of PEP will be excluded.Using a similar Poisson model, we will calculate the incidence rate ratio of leprosy at the village level between arms 1 and 2, including all participants, irrespective of whether they received PEP and the regimen received. Follow-up time will start on the median date of first PEP administration in each village and end on the median date of the final survey round. For incident leprosy cases follow-up time will end on the date of diagnosis. Although villages will be randomized, we will explore baseline prevalence of leprosy as a potential confounder.In the villages included in arm 4 of the PEOPLE trial, we will conduct an anti-PGL I survey in 2026 to compare the seroprevalence rates with those of 2019 in different age groups at the village and island level.We will calculate costs per person screened for leprosy and the cost per person provided either SDR-PEP or BE-PEP. If BE-PEP is found to be more effective, then the cost-effectiveness analysis will be performed with the average cost per case of leprosy averted per arm and island. Also, incremental costs per person treated will be calculated using SDR-PEP as a baseline.Finally, the prevalence of bedaquiline and/or rifampicin-resistant strains will be calculated per island. We will include in the numerator all leprosy or TB patients with resistance to rifampicin and/or bedaquiline, and all leprosy or TB patients tested in the denominator. Any apparent annual trends will be tested with chi-square for trend to assess statistical significance. | PMC10169125 |
Sample size | For sample size calculations we used the methodology described by Hayes and Bennet for cluster randomized trials [To assess the effect of BE-PEP at the village level, the entire population examined will be considered in the analysis. If incidence at the village level is 1.1 per 1,000 per year, as observed in the PEOPLE trial, and a 50% reduction is achieved with BE-PEP, the available sample size would provide a power of approximately 90% over a three-year follow-up period. | PMC10169125 |
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Ethics | The study will be carried out according to the principles stated in the Declaration of Helsinki, Good Clinical Practice (GCP), General Data Protection Regulation (GDPR), and all applicable regulations and according to established international scientific standards. A yearly update on the status of the study will be provided as required.In Comoros, the BE-PEOPLE trial was approved by ‘Comité National d’Ethique pour les Sciences de la Vie et de la Santé’ (CNESS) (Réf.N°23/03/CNESS/PR) as well as by the ‘Direction Générale de la Santé’ (Réf.N°23/33/MSSPSPG/DGS). Approval was also received from the Institutional Review Board (IRB) of the Institute of Tropical Medicine (ITM). In addition, the study has been approved by the Ethics Committee (EC) of the University of Antwerp Hospital in Antwerp.The study protocol has been included in the Clinicaltrials.gov public registry (on 28 October 2022, | PMC10169125 |
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Acknowledgements | Leprosy, Tuberculosis | LEPROSY, TUBERCULOSIS | We thank the National Tuberculosis and Leprosy Program of the Union of the Comoros Damien Foundation, and the Institute of Tropical Medicine of Antwerp Belgium for their support. | PMC10169125 |
Author Contribution | RS | AY, SNS, BAT, AP, NA, SHG, SMB, AT, AB, AM, ZS, MA, SG, RS, PC, NOG, CH, AG, BCDJ, and EH designed the study. Drafting the manuscript was performed by AY, NOG, EH, and BCDJ. All authors revised the manuscript. | PMC10169125 |
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Funding | This research will be funded by Janssen Pharmaceutica NV. The funders had no role in development, implementation, analysis of results, or preparation of the manuscript. | PMC10169125 |
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Data Availability | The data supporting the findings of this publication will be retained at the Institute of Tropical Medicine, Antwerp, and will not be made openly accessible due to ethical and privacy concerns. Data can however be made available after approval of a motivated and written request to the Institute of Tropical Medicine at [email protected]. | PMC10169125 |
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Declarations | PMC10169125 |
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Ethics approval and consent to participate | The BE-PEOPLE trial was approved by ‘Comité National d’Ethique pour les Sciences de la Vie et de la Santé’ (CNESS) (Réf.N°23/03/CNESS/PR) as well as by the as well as by the ‘Direction Générale de la Santé’ (Réf.N°23/33/MSSPSPG/DGS) in the Union of Comoros. Approval was also received from the Institutional Review Board (IRB) of the Institute of Tropical Medicine (ITM). In addition, the study has been approved by the Ethics Committee (EC) of the University of Antwerp Hospital in Antwerp. Written individual informed consent will be obtained from all participants included in the study. As the legal age of consent in Comoros is 18 years old, for participants between 2 and 18 years old informed consent of the parents or guardian will be sought. Consent information will be read to illiterate participants and written informed consent will be obtained from their respective legal authorized representative. Prior to the start, this study has been included in the Clinicaltrials.gov public registry (NCT05597280, on 28 October 2022, | PMC10169125 |
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Consent for publication | Not applicable. | PMC10169125 |
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Competing interests | The authors have no competing interests to declare. | PMC10169125 |
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List of Abbreviations | Tuberculosis | LEPROSY, TUBERCULOSIS | Adverse EventPhenolic glycolipid-IAdverse ReactionBacille de Calmette GuérinDamien FoundationInstitutional Review BoardInstitute of Tropical MedicineMulti bacillaryPaucibacillaryNational Tuberculosis and Leprosy Control ProgramPost ExpOsure Prophylaxis for LEprosy in the Comoros and MadagascarPost Exposure ProphylaxisQuantitative Polymerase Chain ReactionResearch Electronic Data CaptureSerious Adverse EventSingle Double Dose Rifampicin Post-Exposure ProphylaxisSingle Dose RifampicinSlit-skin smears | PMC10169125 |
References | PMC10169125 |
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Subject terms | gastroesophageal junction adenocarcinoma | REGRESSION, LIVER METASTASES, LIVER METASTASES, GASTROESOPHAGEAL JUNCTION ADENOCARCINOMA | The liver is one of the most ordinary metastatic sites of gastroesophageal junction adenocarcinoma and significantly affects its prognosis. Therefore, this study tried to construct a nomogram that can be applied to predict the likelihood of liver metastases from gastroesophageal junction adenocarcinoma. 3001 eligible patients diagnosed with gastroesophageal junction adenocarcinoma between 2010 and 2015 in the Surveillance, Epidemiology, and End Results (SEER) database were involved in the analysis. Patients were randomly divided into a training cohort and an internal validation cohort using R software, with an allocation ratio of 7:3. According to the consequences of univariate and multivariate logistic regression, we constructed a nomogram for predicting the risk of liver metastases. The discrimination and calibration ability of the nomogram was appraised by the C-index, ROC curve, calibration plots, and decision curve analysis (DCA). We also used Kaplan–Meier survival curves to compare differences in overall survival in patients with gastroesophageal junction adenocarcinoma with and without liver metastases. Liver metastases developed in 281 of 3001 eligible patients. The overall survival of patients with gastroesophageal junction adenocarcinoma with liver metastases before and after propensity score matching (PSM) was obviously lower than that of patients without liver metastases. Six risk factors were finally recognized by multivariate logistic regression, and a nomogram was constructed. The C-index was 0.816 in the training cohort and 0.771 in the validation cohort, demonstrating the good predictive capacity of the nomogram. The ROC curve, calibration curve, and decision curve analysis further demonstrated the good performance of the predictive model. The nomogram can accurately predict the likelihood of liver metastases in gastroesophageal junction adenocarcinoma patients. | PMC10329020 |
Introduction | gastroesophageal junction adenocarcinoma | GASTROESOPHAGEAL JUNCTION ADENOCARCINOMA | The incidence of gastroesophageal junction adenocarcinoma (GEJA) has increased markedly in Western countries over the past few decades | PMC10329020 |
Methods | PMC10329020 |
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Patients | LIVER METASTASES, GASTROESOPHAGEAL JUNCTION ADENOCARCINOMA | We screened 3001 GEJA patients newly diagnosed between 2010 and 2015 from the SEER database who met our inclusion criteria, of which 281 developed liver metastases. The exclusion process is shown in Fig. Patient screening flowchart. This figure contains how we screened 3001 Siewert type II gastroesophageal junction adenocarcinoma patients from the SEER database. | PMC10329020 |
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Statistical analysis and optimal cutoffs | tumor | TUMOR | We used x-tile v3.6.1 (Yale University) software to determine optimal cutoff values for tumor size and age | PMC10329020 |
Results | PMC10329020 |
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Characteristics of GEJA patients | tumor | TUMOR, LIVER METASTASES, BRAIN METASTASES, LUNG METASTASES, BONE METASTASES | We included 3001 patients with GEJA diagnosed between 2010 and 2015 in this retrospective study, with 9.3% (n = 281) had liver metastases, 3.5% (n = 106) had lung metastases, 2.5% (n = 77) had bone metastases, 0.4% (n = 14) had brain metastases. Table Characteristics of all 3001 patients before and after PSM.We used the x-tile v3.6.1 (Yale University) to determine the optimal cutoffs for tumor size and age. PSM:propensity score matching. | PMC10329020 |
Survival analysis of liver metastases from gastroesophageal junction adenocarcinoma | gastroesophageal junction adenocarcinoma | LIVER METASTASES, GASTROESOPHAGEAL JUNCTION ADENOCARCINOMA | Using R software v4.3.0, we performed a 1:3 propensity score matching of patients with gastroesophageal junction adenocarcinoma based on the presence or absence of liver metastases, and finally, 221 patients who had liver metastases were matched with 471 patients without liver metastases. The median follow-up for the pre-and post-PSM cohorts was 22 months (interquartile range: 10–44 months) and 11 months (interquartile range: 5–22 months), respectively. As shown in Table Kaplan–Meier curves of the pre- and post-PSM cohort. Kalpan–Meier curves of ( | PMC10329020 |
The diagnostic likelihood of liver metastases in GEJA patients | tumor | REGRESSION, TUMOR, LIVER METASTASES | We randomly divided the patients into a training cohort and an internal validation cohort with an allocation of 7:3 ratio by R software v4.3.0. More information about the training and validation cohorts is shown in Table Patient characteristics of the training and validation cohorts.We used the x-tile v3.6.1 (Yale University) to determine the optimal cutoffs for tumor size and age.Univariate and multivariate logistic regression for analyzing associated factors for developing liver metastases.HR: hazard ratio; CI: confidence interval. | PMC10329020 |
Construction and validation of a predicted nomogram | gastroesophageal junction adenocarcinoma, adenocarcinoma of the gastroesophageal junction | LIVER METASTASES, GASTROESOPHAGEAL JUNCTION ADENOCARCINOMA, REGRESSION, LIVER METASTASIS, ADENOCARCINOMA OF THE GASTROESOPHAGEAL JUNCTION | Based on the risk factors for liver metastases identified by multivariate logistic regression, we created a nomogram to predict the risk of liver metastases in GEJA patients. (Fig. Nomogram to predict the risk of liver metastases in gastroesophageal junction adenocarcinoma patients. From this nomogram, we can determine the risk of liver metastasis of gastroesophageal junction adenocarcinoma.ROC curves of the nomogram. ROC curves of the nomogram in the training cohort (calibration curves of the nomogram. The calibration curves of the nomogram for gastroesophageal junction adenocarcinoma patients in the training cohort (Evaluation of decision analysis (DCA) curves for nomination charts decision analysis curves (DCA) for patients with adenocarcinoma of the gastroesophageal junction in the training cohort ( | PMC10329020 |
Discussion | tumor, T stage, colorectal cancer, gastroesophageal junction malignancies, liver metastases, Malignant tumors, gastric cancer | BRAIN METASTASIS, TUMOR, COLORECTAL CANCER, LIVER METASTASES, LUNG METASTASIS, MALIGNANT TUMOR, LUNG METASTASES, GASTROESOPHAGEAL JUNCTION ADENOCARCINOMA, LYMPH NODE METASTASIS, REGRESSION, BONE METASTASES, GASTRIC CANCER | The survival of patients with metastatic Siewert type II GEJA is influenced by multiple factors, such as pathological type, age, metastatic pattern, degree of differentiation, and treatmentIn this retrospective study, a nomogram that can predict the risk of liver metastases resulting from GEJA was constructed. And its accuracy was verified by the ROC curve, C index, and calibration curve. Through univariate and multivariate logistic regression, we finally identified age, tumor size, N stage, T stage, bone metastases, and lung metastases as factors affecting liver metastases in gastroesophageal junction adenocarcinoma.We found that the likelihood of liver metastases in GEJA patients decreased with increasing age. A previous study found that colorectal cancer incidence increases with age, but metastatic spread decreases with ageWe found that tumor size was also an important factor affecting the happening of liver metastases in GEJA patients, and the larger the tumor size, the higher the risk of liver metastases. It has been previously reported that the risk of lymph node metastasis increases with increasing tumor size in patients with Siewert type II T1-T3 GEJAAs shown in Fig. T staging of gastroesophageal junction malignancies is based on the degree of invasion. However, to our surprise, we found that the higher the T stage, the lower the risk of liver metastases in GEJA patients. We also found that the risk of liver metastases in gastric cancer decreased with increasing T stage. Nevertheless, more evidence is needed to confirm the relationship between the T stage and liver metastases in GEJA patients.Malignant tumors can metastasize to other organs in the body through blood spread. Therefore, it is not surprising that there is a correlation between bone metastases and lung metastases, and liver metastases. However, we found no correlation between brain and liver metastases in our study (Therefore, age, tumor size, T stage, N stage, lung metastasis, and brain metastasis all affect the occurrence of liver metastases in GEJA patients. The nomogram we constructed can accurately predict the likelihood of liver metastases in GEJA patients and better guide clinical practice. | PMC10329020 |
Acknowledgements | The authors thank the participants and their families in this study. | PMC10329020 |
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Author contributions | M.Z., W.Y., BH., C.C., D.Z., and Y.Y. made substantial contributions to conception and design; M.Z., W.Y., C.C., D.Z., and Y.Y. made substantial contributions to the acquisition, analysis, and interpretation of data. All authors made substantial contributions to the drafting of the manuscript and revising of the manuscript and the final approval of the manuscript to be published. All authors agree to be accountable for all aspects of the work and ensure its accuracy and its integrity. | PMC10329020 |
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Funding | Supported by Natural Science Foundation of Gansu Province (21JR1RA118) and Gansu Provincial Youth Science and Technology Fund (21JR1RA107, 18JR3RA305). | PMC10329020 |
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Data availability | The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. It can also be downloaded directly from the SEER database. ( | PMC10329020 |
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Competing interests | The authors declare no competing interests. | PMC10329020 |
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References | PMC10329020 |
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Subject terms | acute kidney injury | SECONDARY INFECTION | SARS-CoV-2 and its different variants caused a “wave and wave” pandemic pattern. During the first wave we demonstrated that standardized Brazilian green propolis extract (EPP-AF®) reduces length of hospital stay in adult patients with COVID-19. Afterwards, we decided to evaluate the impact of EPP-AF in hospitalized patients during the third wave of the pandemic. BeeCovid2 was a randomized, double-blind, placebo-controlled clinical trial in hospitalized COVID-19 adult patients. Patients were allocated to receive an oral dose of 900 mg/day of EPP-AF® or placebo for 10 days. The primary outcome was length of hospital stay. Secondary outcomes included safety, secondary infection rate, duration of oxygen therapy dependency, acute kidney injury and need for intensive care. Patients were followed up for 28 days after admission. We enrolled 188 patients; 98 were assigned to the propolis group and 90 to the placebo group. The post-intervention length of hospital stay was of 6.5 ± 6.0 days in the propolis group versus 7.7 ± 7.1 days in the control group (95% CI − 0.74 [− 1.94 to 0.42]; | PMC10611696 |
Introduction | non-vaccinated | CORONAVIRUS, DISEASE, CORONAVIRUS DISEASE 2019, SEVERE ACUTE RESPIRATORY SYNDROME | Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant concern regarding its global impact on healthcare settingsAfter the viral replication phase, there is an enormous immunological and inflammatory challenge since both innate and adaptive immunity may be disorderly activated by SARS-CoV-2 infectionPropolis is a natural resin produced by bees from various parts of plants. It has several properties, such as antioxidant, anti-inflammatory and immunomodulatory activitiesA recent open-label, randomized clinical trial in adults hospitalized due to COVID-19 has demonstrated the safety and efficacy of Brazilian green propolis (EPP-AF) to decrease length of hospital stay among non-vaccinated patients with moderate to severe symptoms of the disease (including those patients in mechanical ventilation and intensive care therapy) during the first wave of the pandemicAs the pandemic progressed and in light of experimental evidence and properties of propolis, additional clinical trials to gather more evidence are needed. We designed this randomized, double-blind clinical trial to assess safety and impact of EPP-AF on this population during the third wave of the pandemic. | PMC10611696 |
Methods | PMC10611696 |
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Trial design and oversight | BeeCovid2 was a randomized, double-blind, placebo-controlled clinical trial to evaluate safety and efficacy of EPP-AF in hospitalized patients with COVID-19 who were not in mechanical ventilation. The study was conducted at Hospital São Rafael, a tertiary 600-bed hospital in Salvador, Bahia, northeastern Brazil, from April to August 2021. The protocol was approved by the local Ethics Committee (registration number 43265321.9.0000.0048) on February 25, 2021, and the trial was registered on March 16, 2021, on ClinicalTrials.gov (NCT04800224). More details can be found in the previously published study protocolThe study was conducted in accordance with the principles of the Declaration of Helsinki and the guidelines of Good Clinical Practice of the International Harmonization Conference. All participating patients and/or their legal representatives were duly informed about objectives and risks of participation before signing the informed consent form. The informed consent form is electronically signed, stored on the Research Electronic Data Capture (REDCap) platform, and automatically made available to participants via download. The electronic informed consent form was evaluated and approved by the local Ethics Committee.All authors guarantee data integrity and fidelity to the study protocol. This study was an initiative of the principal investigator and correspondent. An independent commission from D’Or Institute for Education and Research, an institutional platform to support research, monitored the research documents, ensuring greater information security and fidelity. Any novel information arising from the study were promptly reported to the Ethics Committee during the study. | PMC10611696 |
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Participants | fungal infection, cancer, heart failure, human immunodeficiency virus, sepsis, hypersensitivity | SARS-COV-2 INFECTION, HUMAN IMMUNODEFICIENCY VIRUS, FUNGAL INFECTION, LIVER FAILURE, CANCER, HEART, HEART FAILURE, END-STAGE KIDNEY DISEASE, SEPTIC SHOCK, SEPSIS, HYPERSENSITIVITY | It was considered eligible to study participation patients aged 18–80 years hospitalized with SARS-CoV-2 infection, diagnosis confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) and symptoms starting within 14 days of the randomization date.Exclusion criteria included patients undergoing mechanical ventilation at randomization, pregnancy or lactation, known hypersensitivity to propolis, propolis use within the prior 30 days, active cancer, human immunodeficiency virus positivity, solid organ or bone marrow transplant, use of immunosuppressive drugs, bacterial or fungal infection at randomization, sepsis or septic shock, inability to administer medication orally or via a nasoenteral tube, known liver failure, advanced heart failure (New York Heart Association [NYHA] class III or IV) or end-stage kidney disease. | PMC10611696 |
Procedures | Trial randomization was stratified based on clinical parameters related to the need for supplemental oxygen as follows: no use of oxygen, catheter ≤ 5 L/min, nasal catheter > 5 L/min or non-rebreather mask, continuous positive airway pressure (CPAP), or high-flow nasal cannula. Randomization was carried out from permuted blocks listed on the REDCap platform. The permuted block (four patients per block) randomization sequence, including stratification, was prepared by a statistician not involved in the trial. To minimize bias, design and allocation concealment were performed by a trained professional who is not connected to the study.Eligible patients were randomly assigned at a 1:1 ratio to receive Propomax® capsules produced with dehydrated standardized Brazilian green propolis extract, EPP-AF®, for 10 days at 900 mg/day (three 100-mg capsules, three times per day) or placebo (three capsules, three times per day). Hard gelatin capsules with magnesium stearate (1%), silicon dioxide (0.1%), and microcrystalline cellulose qsp (320 mg) were prepared as placebo formulations. Placebo packages had the same labelling of the active product. They were also opaque and had a security system to prevent improper opening. All capsules had the same organoleptic characteristics, making propolis and placebo capsules indistinguishable. The study was double-blinded, so neither the patients, the healthcare providers nor the principal investigator knew to which group the patients were allocated.Both groups received standard-of-care treatment that could include supplemental oxygen, noninvasive or invasive ventilation, corticosteroids, antibiotics and/or antivirals, vasopressor support, renal replacement therapy, intra-aortic balloon pump (IABP), and extracorporeal membrane oxygenation (ECMO), as needed. Supportive treatment was freely selected at the sole discretion of assisting clinical teams in accordance with individual needs of patients, without any kind of interference from researchers.The propolis extract used in this study was prepared from a single batch to guarantee uniformity. High performance liquid chromatography (HPLC) was performed in the propolis batch before the manufacturing of capsules. The standardized Brazilian green propolis extract, which is composed mainly of green propolis produced in southeastern Brazil and processed with specific extraction and drying processes, was selected for this study due to its batch-to-batch reproducibilityPatients were evaluated daily during hospitalization, from days 1 to 28. Patients who were discharged in less than 10 days have completed treatment at home and were followed up by telephone as a means to obtain relevant safety data on the study medication. | PMC10611696 |
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Outcomes | AKI, initiation of renal replacement, acute kidney injury | SECONDARY, SECONDARY INFECTION, ACUTE KIDNEY INJURY, KIDNEY DISEASE | The primary endpoint was the time to clinical improvement, defined as the difference in days in length of hospital stay between groups.The secondary outcomes included percentage of participants who needed mechanical ventilation (MV), secondary infection (defined as cultures of blood, urine or tracheal aspirate positive for fungi or bacteria), incidence of acute kidney injury, as well as need for renal replacement therapy, vasoactive drugs, IABP, or ECMO. We have also assessed the mortality rate by 28 days.Acute kidney injury (AKI) was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) as stage 1 (increase in serum creatinine of 0.3 mg/dl in 48 h or increase in baseline serum creatinine by 1.5 to 1.9 times in 7 days), stage 2 (increase in serum creatinine by 2.9 times in 7 days), or stage 3 (threefold or more increase in serum creatinine in 7 days or initiation of renal replacement therapy). | PMC10611696 |
Safety | death, Cancer | ADVERSE EVENT, ADVERSE EVENT, CANCER | Patients were monitored to ensure safety, which is the major premise of the entire study. Adverse events (AE) that may compromise safety or were regarded as serious or severe (pregnancy, life-threatening illness, new hospitalization, or death) were reported to the local research ethics committee (within 24 h). Adverse events were classified according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. | PMC10611696 |
Statistical analysis | death | REGRESSION | The mean length of hospital stay in the control group of BeeCovid study was 12.6 days with a standard deviation of 6.5 daysThe main analysis of the study was conducted under the intention-to-treat principle. The modified intention-to-treat population consisted of all patients who were randomized and received either EPP-AF or placebo.The primary outcome of the study was defined as length of hospital stay from randomization to 28 days if the patient remained hospitalized after that period. Additionally, if the patient did not survive hospitalization, length of hospital stay was considered as equal to 28 days, even when death occurred less than 28 days after randomization. Data from patients who could not be reached to complete the 28-day follow-up were censored at hospital discharge. The between-group comparison was evaluated by generalized additive models assuming beta-binomial distribution, with adjustments by stratification variable (baseline type of oxygenation) and had 95% confidence intervals.Secondary outcomes, such as the need for dialysis and mortality, were evaluated by logistic regression models and presented relative to chance with respective 95% confidence intervals. Continuous outcomes were described by means and standard deviations and compared by generalized regression models with distributions that best fit the data. More details are provided in the study protocol | PMC10611696 |
Results | PMC10611696 |
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Discussion | critically ill, chronic kidney disease, fever, coronavirus infection, pain, infection, hemodialysisAcute kidney injury | CRITICALLY ILL, CORONAVIRUS INFECTION, DISEASES, INFECTION, SECONDARY INFECTION | In this randomized, double-blind, placebo-controlled study, the use of standardized Brazilian green propolis extract associated with standard-of-care treatment reduced the length of hospital stay by one day compared to a placebo group among hospitalized adult patients with mild to moderate COVID-19. This trend was more noticeable among non-vaccinated patients despite no statistical significance. On the other hand, the use of propolis was associated with three times fewer secondary infections.A recent analysis in a tertiary hospital has demonstrated that each day of hospitalization costs around US$ 900The first randomized study to use EPP-AF in moderate to severe COVID-19 non-vaccinated patients showed a significant decrease in length of hospital staySARS-CoV-2 infection is more associated with secondary infections when compared to non-infected patientsBeeCovid2 has demonstrated that the use of standardized Brazilian green propolis extract in the dose used is safe, even among critically ill patients, without a single case of hospitalization or readmission to the hospital due to EPP-AF side effects. The safety analysis was performed throughout the entire study period. Previous randomized studies that used EPP-AF had already shown the safety profile of this medication, even in patients with chronic kidney disease undergoing hemodialysisAcute kidney injury may occur in a significant number of COVID-19 patientsOur trial has several limitations. Due to the lack of supplies during the pandemic, glomerular filtration was assessed using creatinine levels calculated by dry chemistry method, which suffers interference by dipyrone (metamizole), drug widely administered to fever and pain controlExperimental data show the potential actions of propolis against viral targets such as TMPRSS2, ACE2 receptor and PAK1, which justifies the experimental use of EPP-AF and results in new perspectives to treat diseases that lead to immuno-inflammatory dysregulationWe therefore conclude that coronavirus infection has a negative impact on people worldwide. This immunological challenge opens a window of opportunity to use propolis, a natural immunomodulator. This is important because propolis may be considered and tested to treat other diseases that cause immunological dysregulations. The present study provides clinical evidence that can be explored by other researchers. In this study, the EPP-AF dose tested was safe to use in adult patients with mild to moderate Covid-19. The use of the medication was not significantly associated with decreases in length of hospital stay. More studies are needed. | PMC10611696 |
Supplementary Information | The online version contains supplementary material available at 10.1038/s41598-023-43764-w. | PMC10611696 |
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Acknowledgements | The authors thank the patients for their valuable contributions. The authors also thank Apis Flora Indl. Coml. Ltda. for providing propolis and placebo, the support of the Maria Emília Pedreira Freire de Carvalho Foundation, and the staff of D’Or Institute for Research and Education (IDOR) and Hospital São Rafael for their assistance. | PMC10611696 |
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