Prevotella salivae or Veillonella infantium (< 4.2 and 1.7% respectively), and a low abundance of a pathway associated with LPS biosynthesis (< 0.1%) were highly predictive of developing the need for acute respiratory support (82 and 91.4% respectively). These findings suggest that the composition of the oropharyngeal microbiome in COVID-19 patients may play a role in determining who will suffer from severe disease manifestations.The oropharyngeal microbiome, the collective genomes of the community of microorganisms that colonizes the upper respiratory tract, is thought to influence the clinical course of infection by respiratory viruses, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Infectious Disease 2019 (COVID-19). In this study, we examined the oropharyngeal microbiome of suspected COVID-19 patients presenting to the Emergency Department and an inpatient COVID-19 unit with symptoms of acute COVID-19. Of 115 initially enrolled patients, 50 had positive molecular testing for COVID-19+ and had symptom duration of 14 days or less. These patients were analyzed further as progression of disease could most likely be attributed to acute COVID-19 and less likely a secondary process. Of these, 38 (76%) went on to require some form of supplemental oxygen support. To identify functional patterns associated with respiratory illness requiring respiratory support, we applied an interpretable random forest classification machine learning pipeline to shotgun metagenomic sequencing data and select clinical covariates. When combined with clinical factors, both species and metabolic pathways abundance-based models were found to be highly predictive of the need for respiratory support . To determine biologically meaningful and highly predictive signals in the microbiome, we applied the Stable and Interpretable RUle Set to the output of the models. This analysis revealed that low abundance of two commensal organisms,  COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has sickened an estimated 605 million people and caused 6.5 million deaths, likely an undercount, since the start of the pandemic, of those nearly 95 million cases and in excess of one million deaths in have been in the United States alone . A distiEvidence is emerging that the microbiome can influence clinical disease in COVID-19. Dysbiosis in both the gut and oral microbiomes have been associated with disease symptoms or severity . The oroThe oropharyngeal and nasopharyngeal microbiomes, the collective genomes of microorganisms that colonize the human upper airway, have been hypothesized to influence host immune responses to respiratory viral and bacterial infections . Viral cHere, we hypothesize that the makeup of the oropharyngeal microbiome, including the presence and relative abundance of microbial species as well as metabolic gene content, collected at admission may be predictive of the clinical trajectory of acute respiratory illness in COVID-19, specifically in terms of the need for receiving respiratory support . To test this hypothesis, we investigated the oropharyngeal microbiome of individuals presenting with symptoms suggestive of COVID-19 and positive molecular testing for SARS-CoV-2. We used machine learning-based modeling to examine associations between the microbial residents of the oral cavity and a manifestation of severe COVID-19, specifically, the need for respiratory support. To extract easily interpretable predictive rules from the machine learning model, we then implemented a rule-based classification algorithm. These rules utilize the measured abundance of specific bacterial species and metabolic pathways identified by our machine learning models to give insight into how specific features of the oropharyngeal microbiome impact the clinical course of COVID-19, specifically with respect to why some patients need respiratory support during initial SARS-CoV-2 infection. Our findings further strengthen the links between microbiomes of the upper airways and COVID-19 disease manifestations.via the EMR.Patients presenting with COVID-19 symptoms at the UMass Memorial Medical Center Emergency Department or while admitted to UMass Memorial COVID-19 treatment units were approached for enrollment in the study. Some individuals had known COVID-19 status when approached on inpatient COVID-19 wards, but the majority were approached in the Emergency Department prior to receiving results of molecular tests for SARS-CoV-2. Enrollment and sample collection took place April 2020 through March 2021, before vaccines were widely available, and no subjects had been vaccinated against COVID-19. Enrolled patients were followed prospectively through the Electronic Medical Record (EMR). We collected information on disease outcomes of COVID-19 for their initial visit including need for respiratory support, the results of all laboratory testing, and mortality via nasal cannula or face mask, non-invasive positive pressure ventilation, or intubation. If a patient had a Do Not Intubate (DNI) order but died of COVID-19 symptoms, we considered that patient has having respiratory failure severe enough to require intubation and classified the sample as being from a patient who was intubated. Patients were considered as having in-hospital mortality from COVID-19 if this was listed as a cause of death on hospital death records.Oropharyngeal samples were classified as being collected from a patient with acute COVID-19 (COVID+) if they had a documented positive rtPCR testing for SARS-CoV-2 and self-reported symptoms for 14 days or less. The need for respiratory support was classified as positive if the patient required any intervention to support breathing. This included supplemental oxygen Oropharyngeal samples were collected using OMNIgene•ORAL collection kits . Briefly, the posterior oropharynx was swabbed for 30 s and collected as per manufacturer protocol. Samples were heated at 65–70°C for 1 h  to ensurShotgun metagenomic reads were first trimmed and quality filtered to remove sequencing adapters and host contamination using Trimmomatic  and Bowtn = 115; 74 COVID-19+ and 41 COVID-19–) with the Bonferroni correction for multiple comparisons.To determine the association between microbial species abundance and COVID-19 diagnosis, we performed a non-parametric Wilcoxon Rank Sum test for species with at least 5% prevalence and a minimal average relative abundance of 0.01% across all samples -based pipeline in R. For each subset of data, the pipeline was run six times from six different random seeds, and statistics for the model’s classification performance and variables contribution to class discrimination were calculated for each seed. The first step of the pipeline is a leave-one-out cross-validation split of the data. Leave-one-out cross-validation is a model validation scheme in which a data set of BioRender.com.In our pipeline, the training set resulting from each leave-one-out cross-validation fold was used for the following steps of the pipeline. Feature selection using Boruta  was run p < 0.05), our cohort of COVID-19+ patients had similar characteristics between those requiring respiratory support and those who did not  were treated with supplemental oxygen via facemask, 6 patients were treated with positive pressure ventilation (12%), and 11 (22%) were intubated. There were 2 patients who died of COVID-19 but had Do Not Intubate (DNI) orders; accordingly, they were considered as having respiratory failure severe enough to require intubation. Broad measures of microbiome diversity in our samples  were similar between groups . Resulting sequences were profiled for microbial abundances and metabolic pathways. Samples with very low diversity  were discarded as these contained mostly contaminating human DNA. Reads coming from host contamination versus microbial species was not significantly different between COVID-19+ and COVID-19− cohorts  modeling for analysis. ML is apt for overcoming the mentioned challenges and enables the integration of complex, highly variable microbiome data and clinical variables . RFC ML Using RFC, we sought to define the microbiome’s role in predicting the need for respiratory support in our COVID-19+ cohort. To demonstrate the utility of combining multimodal inputs, we trained our RFC model on (1) patient features alone, (2) microbiome data alone, and (3) microbiome plus clinical data and computed F1-scores from leave-one-out cross-validated data across multiple random seeds . F1-score is the harmonic mean between precision and recall, which accounts for both prediction errors and the specific type of prediction error. We have chosen the F1-score as our main model evaluation metric because the F1-score accounts for imbalanced data and extremes in either recall or precision while maximizing both. Thus, this metric is best suited for cases where both false positives and false negatives are undesirable. A model trained with host specific features, which we define as both sample-level Shannon’s alpha diversity index and clinical covariates, including age, BMI, ethnicity, and selected medical comorbidities available at admission , performP. salivae is the most important predictor needed to accurately classify an individual for respiratory support outcomes , two microbes found at significant levels, is associated with an 82% probability of needing respiratory support. Both rules were found to be the most selected across the different cross-validation folds and were more generalizable across individuals compared to rules that account for clinical factors/demographics including BMI and age . Adding clinical covariates available at admission to the model resulted in only a slightly higher F1-score of 0.821 ± 0.004 . Additiomanno-octulosonate biosynthesis), mycolate biosynthesis, and L-threonine biosynthetic pathways (manno-octulosonate biosynthesis) was highly predictive of the probability of needing respiratory support (p = 91.4%). Similarly, a very low abundance of mycolate biosynthesis also predicted needing respiratory support with a probability of 87.5%. Higher abundances of pathways encoding L-Threonine biosynthesis was associated with the need for respiratory support, although to a lesser degree than low LPS or mycolic acid (p = 60%).The metabolic pathways most important in predicting the need for respiratory support by our modeling were LPS biosynthesis , mycolate biosynthesis, and a lower abundance of genes associated with L-threonine biosynthesis were found to be protective against severe respiratory manifestations of COVID-19 requiring respiratory support. By combining analysis of microbial abundances with metabolic pathways, we can gain deeper insight into microbiome “profiles” which may be predictive of certain clinical outcomes.Here we show that the severity of respiratory illness in acute COVID-19 is associated with certain features of the oropharyngeal microbiome. Specifically, the relative abundances of several Gram-negative and Tropheryma, a potential respiratory pathogen  was ranked 7th among the top 15 significant predictors of the need for respiratory support in acute COVID-19  can be found at: The studies involving human participants were reviewed and approved by University of Massachusetts Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.EB, JH, BM, and AM conceived and led the study. JH, EB, and CT supervised the conduct of the study and data collection. LC, M-CS, SM, CT, and PD managed the clinical data, including quality control. LC and M-CS handled the sample collection and storage. DW managed sample extraction and sequencing and performed metagenomic profiling. AZ and VB performed statistical analysis on the microbiome data and performed all ML modeling. EB, AZ, VB, and JH wrote the manuscript with input from all authors. All authors contributed to the article and approved the submitted version.This work was supported by the Society For Academic Emergency Medicine’s 2020 COVID-19 Research Grant, internal funding to support COVID-19 research early in the pandemic from the Dean of the University of Massachusetts Medical School and the National Institute of Health grant # 1RF1AG067483–01.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Irradiation induces distinct cellular responses such as apoptosis, necroptosis, iron-dependent cell death (a feature of ferroptosis), senescence, and mitotic catastrophe. Several of these outcomes are immunostimulatory and may represent a potential for immunogenic type of cell death (ICD) induced by radiotherapy triggering abscopal effects. The purpose of this study is to determine whether intra-tumoral ICD markers can serve as biomarkers for the prediction of patient’s outcomes defined as the metastasis status and survival over a 5-year period. Thirty-eight patients with locally advanced cervical cancer, treated with neoadjuvant chemoradiotherapy using cisplatin were included in this study. Pre-treatment tumor biopsy and post-treatment hysterectomy samples were stained for cell death markers and danger associated molecular patterns (DAMPs): cleaved caspase-3 (apoptosis), phosphorylated mixed lineage kinase domain like pseudokinase , glutathione peroxidase 4  and 4-hydroxy-2-noneal , high mobility group box 1 (HMGB1) and calreticulin. Although these markers could not predict the patient’s outcome in terms of relapse or survival, many significantly correlated with immune cell infiltration. For instance, inducing ferroptosis post-treatment seems to negatively impact immune cell recruitment. Measuring ICD markers could reflect the impact of treatment on the tumor microenvironment with regard to immune cell recruitment and infiltration.Cell death readouts during neoadjuvant chemoradiation in cervical cancer According to the World Health Organization (WHO), cervical cancer (CC) is the fourth most prevalent cancer in women worldwide. Standard of care for patients with locally advanced CC (LACC) usually consists in a chemoradiation regimen, with a dose of about 45-50 Gy using external beam radiotherapy on tumor, affected and elective lymph nodes, followed by a boost of sequential brachytherapy aiming for an on-tumor cumulative dose up to 90 Gy , 2. GhenSince the discovery of X-rays by Röntgen in 1895, radiotherapy (XRT) was shown to improve clinical outcomes in many types of cancer . AlthougThe cell death modality is decisive in the activation of the immune response. One of the strategies to increase the immune response against tumor cells is through the induction of immunogenic cell death (ICD) . ICD stiHmgb1 gene expression is upregulated in most cervical cancers  and high mobility group protein B1 (HMGB1), released from dying cells act as adjuvants and signal the state of danger to the organism , 15, 31. cancers . It has  cancers .A previous study  from ourPatient selection was previously described . All patPre-treatment biopsies and post-treatment hysterectomies of 38 patients were obtained from the department of pathology of Ghent University Hospital . Each block contained formalin-fixed, paraffin-embedded tissue.2O2 in methanol for 10 minutes. Following 3 additional washes with PBS, tissues were incubated with a blocking buffer (PBS containing 5% goat serum and 1% BSA) for 30 min. The blocking buffer was removed and sections were incubated overnight at 4°C with primary antibodies: calreticulin 1:1000, HMGB1 1:5000, pMLKL 1:1000, GPX4 1:1000, 4-HNE 1:1500 and cleaved caspase-3 1:100 in PBS containing 1% BSA. Slides were incubated with appropriate secondary antibodies  and specific signals were enhanced by use of the ABC-kit . DAB (Vector) was used to detect the signal. The sections were counterstained with Hematoxylin, rinsed and dehydrated. Slides were mounted with a Xylene-based mounting medium. Images were acquired on a Zeiss Axio Scan.Z1 and ZEN 2 software (Zeiss). Images from the whole tissues were quantified with QuPath software (version 0.1.2) with a script enabling the identification of positively stained cells within the tissue. A percentage of positive cells per tissue was obtained with this quantification. The quantitative data of 38 patients were analyzed (1 patient diagnosed with an immunologic disorder was excluded) for correlations with tumor-infiltrating immune cells (TILs) and for changes between pre- and post-treatment samples. Staining of the same patient material (consecutive slicing) for TILs has been performed simultaneously in another project . A citrate buffer, pH6 DAKO, S2031) was used for the following antibodies: calreticulin , HMGB1 , pMLKL , GPX4  and cleaved caspase-3 . An EDTA buffer, pH 9 (Vector H-3301) was used for 4-HNE . Slides were washed 3 times for 5 min with PBS. Peroxidase was blocked with 3% H1 was usevia positron emission tomography with 2-deoxy-2-[fluorine-18] fluoro-D-glucose integrated with computed tomography (18-FDG PET/CT). Cause-specific survival (CSS) was used to analyze the prognostic value of the different markers therefore excluding four patients from the analysis who died due to intercurrent diseases. CSS was defined as the time between the end of therapy  and either the date of death from CC or the date of last follow-up and was analyzed over a 5-year period.Metastasis status was assessed at diagnosis and was followed Two-sided test of correlation was done on the five markers quantified in this study  and the TILs scores previously determined , and the patients’ outcomes (relapse and CSS) was done in R, making use of a generalized linear model of the binomial family. For CSS, a generalized mixed effect model was used. The following mean models were used:With µ the modeled mean. Both models contain all the main terms (fixed effects). In addition, the model on CSS corrected for the number of months between the last biopsy and last contact with the patient or death. Associations were corrected for multiple testing by controlling the false discovery rate at 5% making use of the Benjamini-Hochberg principle.Baseline characteristics from these 38 patients were described previously , B cells and CD163+ cells (possibly M2 macrophages). Patients with high levels of calreticulin have lower PD-L1 expression on immune cells . In both biopsy and hysterectomy samples, we observed a trend towards higher levels of calreticulin and HMGB1 in patients who died or relapsed (patients with CSS = 1 and Relapse = 1)  which are recognized by the immune system as non-self. Adjuvanticity is modulated by DAMPs such as calreticulin and HMGB1 which act as adjuvants to boost the anti-cancer immune response , a key regulator of the antioxidant system and leading to radioresistance . The oxidalities . We havedalities .Additionally, we found that some cell death markers and DAMPs were significantly correlated with distinct TILs in the biopsy as well as the hysterectomy samples. In the pre-treatment samples, we found that high levels of immune cells in the tumor site were not associated with apoptosis nor necroptosis. Thus, in the biopsy samples, before treatment, it seems that a strong immune cell infiltration is installed in the absence of cell death such as apoptosis and necroptosis. One hypothesis could be that this is the result of an efficient killing, phagocytosis of dying tumor cells, or as mentioned above, tumor cell death is no longer detected. The presence of DAMPs was correlated with a low PD-L1 expression on immune cells and lower levels of activated macrophages. Since PD-L1 downregulates the immune response in the context of cancers, a low expression might be beneficial for the patient. Furthermore, the low level of PD-L1 observed on immune cells could also be due to a lower amount of immune cells in the post-treatment samples.When we assessed the changes in levels found in hysterectomy samples as compared with the biopsy samples, we found that increased post-treatment levels of cleaved caspase-3 were associated with a decrease in the PD-L1 expression on immune cells post-treatment. This result Underlines a positive role of treatment-induced apoptosis in these patients since the decrease in PD-L1 levels might lead to a better anti-tumoral immune response. On the other hand, an increase in lipid peroxidation was associated with an increase in PD-L1, suggesting that ferroptosis would not have a positive impact in the patients.In the patients with an incomplete response to the treatment, increased levels of 4-HNE, which could indicate lipid peroxidation induced by chemoradiation correlated with decreased levels of TILs (CD20 and FoxP3). Also, treatment-induced increased in 4-HNE correlated with an increase in PD-L1 on immune cells. We recently reported that ferroptosis impedes antigen presentation. After this sentence, we could add “we recently reported that cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity”. Citation: Wiernicki B, Maschalidi S, Pinney J, Adjemian S, Vanden Berghe T, Ravichandran K, Vandenabeele P. Cancer cells dying from ferroptosis impede dendritic cell-mediated anti-tumor immunity. Nature Communications. In press. In line with this, high GPX4 expression was also shown to be correlated with better survival and induction of an immune response . MoreoveTo date, up to 30% of patients of LACC patients treated with CRT will suffer a recurrence with a subsequent short life expectancy and only few treatment options. Currently, all LACC patients are treated equally, both in the primary and recurrent setting. Biomarkers will allow us to evolve towards a personalized and patient-individualized setting with potentially radiation dose-(de)escalation in responders/non-responders and addition of chemotherapy, immunotherapy (anti-PD(L)1) and targeted therapy in those patients predicted to recur. Identification of biomarker data that can help to enrich the population that is most likely to benefit, would be highly beneficial and avoid treatment-related toxicity  in those patients that will not benefit. Gaining insight in the mechanisms of treatment response and correlation with tumor immune status may enable research towards new synergistic treatment combinations for the optimal priming of the immune system or even new treatment pathways.Altogether, our results suggest that features of ferroptotic cell death seem to be induced following chemoradiation and that ferroptosis  post-treatment seems to negatively impact immune cell recruitment. Although not induced in the majority of patients, apoptosis and necroptosis also occurred upon chemoradiation, and several patients presented more than one cell death modality.Our study presents several limitations. The sample size is small, with 38 patients included. A sample size calculation using Gpower  showed that in function of the significance level, 42 up to 99 patients are required to detect an association between CSS and pMLKL with a power of 0.800. Next to age and FIGO stage, other factors might also influence the data , and it would have been interesting to analyze their correlation with survival, relapse and biomarkers. The markers used in this study also have cell-death independent functions, and thus might not reflect accurately the levels of intra-tumor death. Despite these shortcomings, we believe that this study is a first attempt to show the association between cell cell death markers and immune cells in cervical cancer. Our findings should be confirmed by a study with a larger experimental setup.As the treatment of cancer becomes more and more personalized, monitoring the interplay between tumor cells and the immune microenvironment is becoming increasingly important and provide clues about how each tumor respond to the treatment. Systematic studies exploring treatment regimen where tumor tissues are retrieved prior and after treatment could contribute to this knowledge and might help in selecting more appropriate treatments.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ghent University Hospital (B670201732304). The patients/participants provided their written informed consent to participate in this study.SA, PV, and KV designed and supervised the study. KV supervised sample collection and clinical annotation. TO, KL, and SA performed immunohistochemistry staining. TO and SA made the figures. MV and CT performed data analysis. TO, SA, and PV wrote the manuscript. HD, SA, and LL contributed to critical data interpretation. All of the authors have read and provided comments on the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a young-investigators-proof-of-concept grant from the CRIG obtained by KV. TO held a doctoral fellowship from FWO (Flanders Research Organization) (1S72616N). KV held a mandate for clinical and translational research funded by the Foundation against Cancer (Stichting tegen Kanker). SA held a post-doctoral fellowship from FWO. CDT holds an FWO grant (12S9418N). LL is funded by an Emmanuel van der Schueren scholarship of KOTK. PV is senior full professor at Ghent University and senior PI at the VIB-UGent Center for Inflammation Research (IRC). Research in the Vandenabeele group is supported by EOS MODEL-IDI (30826052), EOS- CD-INFLADIS (40007512), FWO research grants , Methusalem (BOF16/MET_V/007), iBOF20/IBF/039 ATLANTIS, Foundation against Cancer , CRIG and GIGG consortia, and VIB.Author MV was employed by Gnomixx.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Frailty is an age-associated state of increased vulnerability to stressors that strongly predicts poor health outcomes. Epidemiological evidence on frailty is limited during the COVID-19 pandemic, and whether frailty is associated with the risk of infection is unknown.We derived a robust Frailty Index (FI) to measure the prevalence of frailty and its risk factors in community-dwelling older adults in Southern Switzerland (Ticino), and we explored the association between frailty and serologically confirmed SARS-CoV-2 infection.In September 2020, we recruited a random sample of community-dwelling older adults (65 +) in the Corona Immunitas Ticino prospective cohort study (CIT) and assessed a variety of lifestyle and health characteristics. We selected 30 health-related variables, computed the Rockwood FI, and applied standard thresholds for robust (FI < 0.1), pre-frail (0.1 ≤ FI < 0.21), and frail (FI ≥ 0.21).p < 0.001) per one-year increase in age. Out of 481 participants with a valid serological test, 11.2% were seropositive to either anti-SARS-CoV-2 IgA or IgG. The frailty status and seropositivity were not statistically associated (p = 0.236).Complete data for the FI was available for 660 older adults. The FI score ranged between zero (no frailty) and 0.59. The prevalence of frailty and pre-frailty were 10.3% and 48.2% respectively. The log-transformed FI score increased by age similarly in males and females, on average by 2.8% (Advanced age increases the risk of frailty. The risk of COVID-19 infection in older adults may not differ by frailty status.The online version contains supplementary material available at 10.1186/s12877-023-03730-7. The world’s population aged 65 years or older is expected to more than double from 0.7 billion in 2019 to 1.5 billion in 2050 [Frailty is a geriatric condition characterized by increased vulnerability to endogenous and exogenous stressors, which is strongly associated with dependency and increased mortality , 4. HowePrevious studies showed that frailty might contribute to dysregulations of the immune system including low grade chronic inflammation, altered general and specialized immune response , which mWe derived a robust FI combining a wide array of measures from an existing population-based study on the impact of the COVID-19 pandemic. We aimed to 1) calculate the prevalence of frailty in community-dwelling older adults in Southern Switzerland (Ticino); 2) explore the risk factors of frailty; and 3) explore the association between frailty and serologically confirmed COVID-19 infections.Corona Immunitas Ticino (CIT) is a population-based prospective seroprevalence study that started in July 2020, after the first COVID-19 epidemic wave and lockdown in Southern Switzerland. The main aim of CIT was to assess the spread and impact of the COVID-19 pandemic. CIT is part of Corona Immunitas (CI), a nationwide research program led by the Swiss School of Public Health (SSPH +) . DetaileCIT was conducted in Southern Switzerland—Canton Ticino, which borders Lombardy, Italy, the epicentre of the epidemic in Europe. We recruited participants using age- and sex- stratified random sampling based on regional registries of the Federal Office of Statistics. For this study on frailty, we considered 874 older adults aged 65 years and older, who agreed to participate in September and November 2020, provided informed consent and responded to the baseline questionnaire. Of these, we excluded 114 participants due to missing values for the computation of the FI (described below). This reduced the analytical sample size (n1) for the FI construction to 660. The included and excluded participants had similar age distributions. For the further assessment of the relationship between frailty and seroprevalence, we analysed data of the subsample of older adults who took part in the serosurvey and who had a valid serological test (n2 = 481).The Research Electronic Data Capture (REDCap) , 19, a sWe asked participants to complete a baseline questionnaire following registration with the study. One week after the completion of the baseline assessment, we administered monthly and weekly questionnaires for repeated measures. We enquired about socio-demographic characteristics, physical and psychological health status, social relationships, and lifestyles of participants and their household environments.To measure seropositivity to SARS-CoV-2, we invited participants to two rounds of blood testing. Professional nurses collected peripheral venous blood samples at a chosen healthcare facility or in their homes. The samples were analysed with the Luminex binding assay SenASTrIS  to detect SARS-CoV-2 antibodies. Previous validations in population-based samples showed high sensitivity and specificity .For the current paper, we used data from the baseline questionnaire, the third monthly questionnaire and the laboratory results from the serological test performed between November 2020 and January 2021 (before the introduction of vaccines).We derived the Rockwood FI using the method described by Searle et al. . We inclFor the analysis of potential risk and protective factors of frailty, we used sociodemographic and lifestyle measures collected at baseline including age ; sex , income satisfaction , education  and smoking .To explore whether there is any relationship between frailty and serologically confirmed SARS-CoV-2 infection, we included in our models the abovementioned sociodemographic factors, multimorbidity and frailty status . The multimorbidity was operationalized as a binary variable (yes/no). Participants who reported at least two chronic conditions were considered having multimorbidity: cardiovascular diseases , chronic respiratory conditions , allergy , immune disorders, hypertension, diabetes mellitus, cancer or other conditions.We used complete case analysis in our study because those participants with missing values on the items used to construct the FI were not significantly different from those with no missing values across socio-demographic characteristics. We first describe the socio-demographic characteristics of the sample. Means and standard deviation (SD) were used for normally distributed data, median and interquartile range (IQR) for skewed data, and Spearman correlations and Wilcoxon rank sum tests to compare groups.We used Poisson regression to estimate the prevalence of frailty by sex and age group. We used log-transformed linear regressions to explore the association between age and FI. In addition, we used univariate and multivariable multinomial logistic regressions to assess associations between age group, sex, income satisfaction, education level and smoking status and frailty status.p value less than 0.05 was considered statistically significant. We performed all analyses using Stata Version 17.0 [Potential risk factors for SARS-CoV-2 seropositivity including age group, sex, smoking status, multimorbidity and frailty status, were tested separately using logistic regressions. A ion 17.0 .We conducted two sets of sensitivity analysis. Firstly, we used two-sample Kolmogorov–Smirnov test and the Mann–Whitney U test to compare the distribution of FI and the mean score of FI between the analytical sample who participated in the serological blood sampling and the sample who did not. We assumed that frail older people might be less likely to participate in the serological blood sampling which may contribute to dilute the results of the potential relationship between frailty and COVID-19 infection.Secondly, we explored whether there is any potential relationship between sub-components of frailty and COVID-19 seropositivity using logistic regression. The seven subcomponents of the FI and the sum of all frailty components were included individually to the model as independent variables. The chronic diseases component’s score ranged from zero to eight, the one about basic activities of daily livings ranged from zero to five, instrumental activities of daily livings from zero to eight, and psychological symptoms and signs ranged from zero to six. The other three components  all ranged from zero to one. The overall sum of the frailty items ranged from zero to 30.Out of 874 respondents aged 65 years and over, complete data for the FI calculation was available for 660 participants (75.4%). The mean age of the study sample was 73 years, 57% were female, and 77% completed at least higher secondary education. Nearly 40% were either current or past smokers Table . Overallp = 0.005) but was similar for men and women at approximately 10%.The distribution of frailty was right skewed Fig. . The FI p < 0.05). Those who did not complete compulsory education had a median FI score of 0.136 (IQR = 0.054), and people who received university education had a lower median score  0.099 (IQR = 0.080). We found no difference in FI scores by sex (p = 0.275) or between smokers and non-smokers (p = 0.612). In Fig. p < 0.001) per one-year increase in age.Participants with higher income satisfaction had a higher median FI score compared to those who reported lower income . Median FI scores were also inversely associated with higher education level (p < 0.05), and higher income satisfaction was related to lower risk of frailty (p < 0.05) but not with pre-frailty  had developed antibodies against SARS-CoV-2. The proportion of seropositivity increased with frailty status. In the frail group, around 17% had positive serological results compared to 11% in the pre-frail group and 10% in the robust group. The frequencies and proportions of serological results of anti-SARS-CoV-2 antibodies stratified by frailty status are presented in Table The associations between potential risk factors for COVID-19 infection, including age, sex, smoking status, multimorbidity, self-reported health status, and frailty status were not statistically significant. Odds ratios (ORs) for these null associations with 95% robust CIs are presented in Supplementary Table p = 0.202), we could not reject the null hypothesis that the distributions of FI were the same in those who participated in serological blood sampling and those did not. Moreover, Mann–Whitney U test showed that the mean of FI was not statistically significantly different between these two groups . In the second set of sensitivity analysis, neither of the seven sub-components of FI nor the overall sum score of frailty item were associated with COVID-19 infection , and almost half of the study participants were identified as pre-frail. Advanced age and lower income satisfaction were both positively associated with frailty. Neither frailty nor its sub-components was associated with serologically confirmed COVID-19 infection.Our results on the occurrence of pre-frailty at around 48% are consistent with recent figures from a large meta-analysis that included studies conducted with people aged 50 years and over in 62 countries that found a pre-frailty prevalence of 49% . ConversSimilar to other studies, the distribution of frailty was right skewed and upper limit was less than 0.7 , 26, 28.Evidence suggests that older age, low income and multimorbidity may increase the risk of COVID-19 infection, while current smoking may lower it –33. EvidOur study has several limitations. Firstly, issues of directionality cannot be addressed in our analysis due to the cross-sectional design, which is, however, robust for the descriptive findings, including estimation of the prevalence of frailty. Moreover, as not all measures were repeated over time, we were unable to explore changes in frailty status through the pandemic waves. We know that frailty is a dynamic health condition , and it Our study provides novel prevalence estimates of frailty based on the operationalization of the FI during the COVID-19 pandemic in Southern Switzerland. Epidemiological evidence on frailty is patchy or outdated in both Western and non-Western countries, due to inherent difficulties in recruiting and assessing frailty in representative samples of older adults.The FI approach used in this study has several advantages over the traditional Fried frailty phenotype approach . For insIn conclusion, the prevalence of frailty and pre-frailty was high among older adults in Southern Switzerland after the first lockdown and during the COVID-19 pandemic. Our study is exemplar of turning a dreadful public health emergency into an opportunity to collect and use epidemiological data to fill evidence gaps. Prospective data are warranted to monitor changes in frailty occurrence, to study its impact on individuals and society, and to gauge mid- and long-term consequences of the COVID-19 pandemic on frail and pre-frail older adults. However, our findings highlight prevention opportunities of public health significance because of the steady expansion of the older segment of the population in the region and globally.Additional file 1: Supplementary Table 1. Thirty health deficits and their scoring used to derive the Frailty Index. Supplementary Table 2. Univariate logistic regression model for potential risk factors of SARS-CoV-2 infection. Supplementary Table 3. Univariate logistic regression model to assess the association between sub-components of Frailty Index and SARS-CoV-2 infection—a sensitivity analysis.


Myasthenia gravis is a neuromuscular junction disorder characterised by fluctuating muscle weakness, improved by using anti-cholinesterase drugs. In addition to the autoimmune aetiology, various factors such as infections, surgery, and drugs are known to precipitate the condition. We report a case of a 15-year-old boy with D-penicillamine-induced myasthenia gravis who presented with facial diplegia, dysphagia, and drooling of saliva, 6 years after the initiation of treatment for Wilson's disease. Therefore, clinicians should be more vigilant while prescribing patients with chelating drugs like D-penicillamine with regular monitoring of the new symptoms and keeping a very low threshold for the suspicion of myasthenia gravis. As he had severe dysphagia, he was being fed with a nasogastric tube that was inserted at a local hospital. He had first presented to us 6 years ago with complaints of generalized weakness, spastic limbs, bilateral hands tremor, and drowsiness over a month's duration for which he was evaluated and was diagnosed to have Wilson's disease based on the presence of Kayser-Fleischer (KF) ring, low ceruloplasmin, increased serum copper, increased 24-hour urinary copper, and hyperintensity in abnormal bilateral basal ganglia in T2-weighted (T2WI) magnetic resonance imaging (MRI) .There were no other significant past medical illnesses or family history. For the past 6 years, he has been treated with D-penicillamine 250 mg twice daily, zinc 20 mg three times a day, and pyridoxine 40 mg once daily.On examination at this visit, the KF ring was absent. He had bilateral facial weakness , generalHis complete blood count, bood sugar, liver function test, renal function test, thyroid function test, creatine phosphokinase (CPK) and electrolytes  were normal. The RNS of the facial nerve (recording from orbicularis oculi muscle) showed a >10% decremental response suggesting postsynaptic neuromuscular junction disorder .Serum AChR-Ab was positive 14.3 nmol/l . Serum anti MuSK antibody report was negative 0.13U/ml . Computed Tomography (CT) scan of the chest was done to evaluate for the thymoma was normal.The diagnosis of drug-induced myasthenia gravis was considered and D-penicillamine was stopped. He was initially treated with pyridostigmine 60 mg twice a day and then later increased to four times a day.On follow-up at two months, he reported having significant improvement. He could close both his eyes and had substantial improvement in his facial weakness . DespiteTo the best of our knowledge, this is the first case report of D-penicillamine-induced myasthenia gravis in a patient treated for Wilson's disease from Nepal.5 However, D-penicillamine is known to cause several autoimmune diseases, including MG.6 Approximately 1-7% of patients treated with D-penicillamine are reported to develop MG.6 The exact mechanism is unknown; however, D-penicillamine has been hypothesized to cause direct modification of major histocompatibility complex (MHC) molecules, as well as peptides on the antigen-presenting cell surface, causing autoimmunity against acetylcholine receptors.7D-penicillamine is a pyridoxine antagonist and a potent chelator proven to be very effective in the treatment of the conditions like Wilson's disease, rheumatic arthritis, cystinuria, and lead poisoning.8 Additionally, our patient had severe dysphagia which was unusual and led us to think that the clinical presentation could be of advanced or inadequately treated Wilson's disease itself. However, KF-ring was absent and the repeated MRI brain was interestingly normal with the resolution of the previous basal ganglia lesion. Therefore, alternative diagnoses of drug-induced MG were considered and evaluated.Drug-induced MG predominantly presents with ptosis and diplopia as seen in our patient.8 Our patient developed myasthenic symptoms after 6 years of treatment for Wilson's disease. Our patient tested positive for AChR-Ab. The test is found to be positive in 80-90% of the generalised MG induced by D-penicillamine.8 The antibody titer is uniquely associated with the disease severity, with higher titer manifesting with more severe disease. As our patient had a high AchR-Ab titer, this could explain the severe dysphagia present in our patient. Anti-MuSK-Ab was negative in our case, although it has been reported to be positive in drug-induced myasthenia. However, recent literature emphasises that double seropositivity is rare in drug-induced MG.9 RNS study (using 10 stimulation trains and 2 Hz frequency) of the seventh cranial nerve and the response recorded from orbicularis oculi muscle revealed significant decremental response (>10% decrement) in our case. The specificity of RNS has been reported to be very high (about 95%) in both ocular and generalised MG, while the sensitivity is lower .10 Although we could not perform SFEMG and did not perform an Edrophonium test to evaluate the neuromuscular junction (NMJ) disorder in our case, the detailed neurological examination, RNS, and antibody tests we performed are adequate to diagnose MG in routine clinical practice.11MG symptoms are known to usually manifest one month to 8 years after the initiation of D-penicillamine.6 It has been assumed that drug-related adverse events that are caused by interference with the NMJ last only for a short period and depend on the half-life of the drug and patient-related factors such as gender, age, renal function, etc. influencing its elimination.12 Our patient showed remarkable improvement within 2 months. This rapid improvement is likely due to the pyridostigmine, younger age, and good renal function. We decided to treat him with pyridostigmine because of his severe symptoms and have planned to stop in the next follow-up.Fortunately, withdrawal of the offending drug leads to recovery of myasthenic symptoms. Myasthenic symptoms are reported to subside gradually over 6-10 months after discontinuation of the offending drug in 70% of cases.We suggest that clinicians should be more vigilant while prescribing patients with D-penicillamine with continuous monitoring of new symptoms and maintaining a very low threshold for the suspicion of myasthenia gravis. Although discontinuation of offending drugs recovers the symptom, pyridostigmine may help accelerate recovery in such cases.


Testicular germ cell tumour is a rare and highly curable malignancy with excellent survival rates. Guideline and institutional protocols differ in the modality, number, and timing for follow-up investigations and can lead to suboptimal care of survivors. More prospective trials are needed to assess how patients can be followed to ensure high adherence, define the role of miR-371a-3p in follow-up, and develop protocols for follow-up after curative treatment in the metastatic setting. The aim of this review is to describe the proportion of testicular germ cell tumours (tGCTs) with recurrence, and the timing and anatomical sites of relapse across different disease stages and after different treatment options. We summarise published follow-up protocols and discuss current and future developments to personalise follow-up for patients with tGCT.A systematic literature search was conducted and current guidelines and selected institutional follow-up protocols were reviewed.Of 302 publications, we screened 68 full texts and included 29 studies; 22 of these were retrospective and seven were prospective in nature, contributing data for 20 570 patients. The number of patients included per study ranged from 119 to 2483. We compared the guideline follow-up protocols of the European Society for Medical Oncology, European Association of Urology, National Comprehensive Cancer Network, and American Urological Association, as well as institutional follow-up protocols. The protocols differed in terms of the number, time points, and type of follow-up investigations.Future research should assess how tGCT can be followed to ensure high adherence, define the role of miR-371a-3p microRNA during follow-up, and develop follow-up protocols after curative treatment in the metastatic setting.In this review of follow-up protocols for men with testis cancer, we observed different recommendations and discuss future research areas to improve follow-up for these patients. Fortunately, approximately 95% of patients with tGCT overall and 80% with metastatic disease can be cured Metastatic relapse in men with localised disease is classified according to the International Germ Cell Cancer Collaborative Group (IGCCCG), and men in the good prognosis group require only three cycles instead of the four cycles of cisplatin-based combination chemotherapy required for men in the intermediate or poor prognosis group. An important aim of follow-up investigations is therefore to detect disease recurrence at an early stage, while the disease is classified in the IGCCCG good prognosis group, in which case less treatment is required for cure and the prognosis is better. The aim of this review was to summarise published follow-up protocols and discuss current and future developments to personalise the oncological follow-up for patients with tGCT.2We performed a literature search on March 28, 2022 to summarise follow-up protocols published in the literature  and in c3We identified 302 publications that met the initial search criteria and proceeded with a title and abstract screening review of 68 full texts . In tota3.13.1.1For clinical stage I seminoma, both surveillance and adjuvant chemotherapy are valid options after orchiectomy, as overall survival for these patients is excellent with both treatments and does not significantly differ between them The aforementioned site-specific and modality-specific recurrence patterns have resulted in recommended follow-up that includes abdominal CT scans every 6 mo in the first 2–3 yr and every 12 mo in years 4 and 5 . Those rThere is a rationale for personalising follow-up protocols on the basis of risk factors for recurrence Because isolated pelvic metastases are rare, some authors have suggested omission of a pelvic scan, which would decrease the cumulative radiation exposure 3.1.2For stage I nonseminoma, both surveillance and adjuvant therapy  are valid options after orchiectomy; approximately 80% of patients on surveillance are cured and will not need further treatment +, 89% in LVI−) within the first 2 yr. After the second year (late and very late relapses) 6% of all recurrences are detected and after the third year it is only 1% Recurrences are detected in 80% of stage I nonseminoma cases within the first year and 90%  and the Toronto group (five chest CTs) during follow-up is remarkable As with stage I seminoma, isolated pelvic metastases with stage I nonseminoma are rare, and some authors have thus suggested omitting a pelvic scan, which would decrease cumulative radiation exposure 3.1.318F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)-CT at 6–8 wk after the end of systemic treatment 18F-FDG PET-CT in this setting, optimal management of masses positive on 18F-FDG PET-CT remains disputable and may well only lead to closer follow-up schedules rather than immediate initiation of salvage treatment, as high-level evidence is lacking For metastatic seminoma, chemotherapy and radiotherapy are treatment options. After first-line chemotherapy, 30% of patients with metastatic seminoma experience a complete clinical response , while 60% achieve a favourable response  After first-line chemotherapy for advanced seminoma, <20% of patients experience relapse at a median of 9 mo. According to the IGCCCG-Update Consortium from 2021, 89% of patients relapse in good prognosis and 79% in intermediate prognosis group Therefore, current follow-up protocols recommend abdominal CT scans every 6–12 mo during the first 5 yr, together with STM measurement . Follow-The NCCN guidelines also provide distinct follow-up recommendations for clinical stage IIA and nonbulky stage IIB versus bulky stages IIB, IIC, and III metastatic seminoma. For stage IIA and nonbulky stage IIB, STM measurement is optional, whereas for advanced stages of the disease, STM measurements are recommended ever 2–3 mo in the first 2 yr and every 6 mo thereafter. Chest X-rays are also recommended every 2–3 mo in years 1–2 and then annually until year 5. For stages IIA and IIB, chest X-rays are only performed at 6-mo intervals for the first 2 yr. In this stage, only four CT scans are performed in the first 3 yr (three CT scans in the first 2 yr), whereas for advanced stages of the disease, seven CT scans are recommended up to year 4 (five CT scans in the first 2 yr).3.1.4For stage II nonseminoma negative for STMs, both RPLND and chemotherapy are valid options. After primary RPLND without additive chemotherapy, 12–40% of patients experience relapse, whereas only 0–4% of patients experience relapse after RPLND and additive chemotherapy . The only guideline specifying a follow-up protocol after primary RPLND is the NCCN: after primary RPLND without additive chemotherapy, several measurements of serum tumours markers are recommended in the first 5 yr . Similar3.1.5Among men with nonseminomatous tGCT and a complete response to first-line chemotherapy, approximately 20% experience relapse at a median of 3 mo. Recurrence occurs within the first 2 yr in nearly all patients; recurrence is observed in the retroperitoneum (33%) and pelvis (25%), where it can be detected via abdominopelvic CT, and in the lungs (33%). STM measurement can detect recurrence in 75% of patients . In 25% of relapse cases, STMs are negative 3.1.6More recent publications have defines relapses in the first 2 yr, after 2–5 yr, and after 5 yr as early relapse (ER), late relapse (LR), and very late relapse (VLR), respectively To detect LRs, the NCCN advises annual STM measurement after 5 yr up to year 10. SWENOTECA recommends STM measurement and MRI of the retroperitoneum after 7 and 10 yr for patients with intermediate or poor prognosis, residual tumours, teratoma in testis without RPLND, or teratoma in residual tumour resections.3.2We investigated what proportion of tGCT cases recur and described the timing and anatomical sites of relapse across different disease stages and after different treatment options. Those key statistical figures are the basis for follow-up protocols, and only a limited number of prospective trials comparing different follow-up protocols are available. In addition, we summarised published follow-up protocols and discuss current and future developments to personalise follow-up for patients with tGCT. In summary, we believe that future research to improve tGCT follow-up should focus on (1) determining how patients with tGCT can be monitored to ensure high adherence to follow-up protocols, (2) defining the role of miR-371a-3p as a new and potentially more accurate biomarker during follow-up, and (3) developing new follow-up protocols for the metastatic setting.18F-FDG PET-CT should only be used in highly selected cases, such as men with pure seminomatous postchemotherapy residual disease. In general, most of the follow-up protocols published have proven to be safe, meaning that for most men any recurrence can be detected while they are still in the IGCCCG good prognosis group. This suggests that all the above-mentioned protocols seem to be oncologically safe as long as there is adherence to close follow-up.To decrease cumulative radiation exposure in this young patient population mainly presenting with localised disease, the number of follow-up scans recommended has decreased over time Low adherence to published protocols represents a major challenge. Nowadays, patients with tGCT are geographically very mobile; this could explain the low adherence to follow-up protocols, possibly leading to a high proportion of relapses detected at an advanced cancer stage requiring intense treatment, which can lead to treatment-related toxicity To personalise follow-up protocols, a correct histopathological diagnosis of the presence or absence of rete testis or LVI is important. Such a personalised approach might be limited by the considerable inter-reader discrepancy of 20–30% for these variables, as suggested in the literature Studies are needed to indicate how measurement of circulating miR-371a-3p levels could be used to detect relapse Findings for two large cohorts  have not yet been reported, and may confirm the accuracy of circulating miR-371a-3p measurements during follow-up and decrease the number of regular cross-sectional imaging investigations during surveillance. These changes would in turn reduce health care costs and radiation exposure. In particular, men with pure seminomatous tGCT might only require cross-sectional imaging when circulating miR-371a-3p levels increase. At present, regular imaging cannot be entirely omitted for men with nonseminomatous GCT, as recurrences with pure teratomatous GCT are not detected by miR-371a-3p measurement; however, future follow-up protocols will recommend a lower number of cross-sectional scans and therefore reduce costs and exposure to ionising radiation.Because most GCT relapses occur within 2 yr, 2-yr disease-free survival was historically considered to be equivalent to cure, and later relapse was described as a distinct clinical entity. For men with localised disease at initial presentation and without adjuvant treatment, ER, LR, and VLR are observed in 15%, 5%, and 1% of seminoma cases, and 30%, 2%, and 1% of nonseminoma cases, respectively By contrast, after first-line chemotherapy, men with LR/VLR more commonly present with both elevated STMs and retroperitoneal disease in comparison to men with ER 4We summarised published follow-up protocols and discuss current and future developments to personalise the oncological follow-up for patients with tGCT. Functional follow-up, including screening for late toxicities, represents another important aspect of follow-up in this patient population.Author contributions: Christian Daniel Fankhauser had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Study concept and design: Fankhauser, Kaufmann, Antonelli.Acquisition of data: Kaufmann, Antonelli.Analysis and interpretation of data: Fankhauser, Kaufmann.Drafting of the manuscript: Fankhauser, Kaufmann.Critical revision of the manuscript for important intellectual content: Fankhauser, Kaufmann, Albers, Cary, Gillessen Sommer, Heidenreich, Oing, Oldenburg, Pierorazio, Stephenson.Statistical analysis: Fankhauser, Kaufmann.Obtaining funding: None.Administrative, technical, or material support: Fankhauser.Supervision: Fankhauser.Other: None.Financial disclosures: Christian Daniel Fankhauser certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript , are the following: None.Funding/Support and role of the sponsor: None.


Sublingual tablet buprenorphine (BUP-SL) and oral liquid methadone (MET) are the daily, standard-of-care (SOC) opioid agonist treatment medications for opioid use disorder (OUD). A sizable proportion of the OUD treatment population is not exposed to sufficient treatment to attain the desired clinical benefit. Two promising therapeutic technologies address this deficit: long-acting injectable buprenorphine and personalised psychosocial interventions (PSI). This study will determine (A) the effectiveness and cost-effectiveness — monthly injectable, extended-release (BUP-XR) in a head-to-head comparison with BUP-SL and MET, and (B) the effectiveness of BUP-XR with adjunctive PSI versus BUP-SL and MET with PSI. Safety, retention, craving, substance use, quality-adjusted life years, social functioning, and subjective recovery from OUD will be also evaluated.This is a pragmatic, multi-centre, open-label, parallel-group, superiority RCT, with a qualitative (mixed-methods) evaluation. The study population is adults. The setting is five National Health Service community treatment centres in England and Scotland. At each centre, participants will be randomly allocated (1:1) to BUP-XR or SOC. At the London study co-ordinating centre, there will also be allocation of participants to BUP-XR with PSI or SOC with PSI. With 24 weeks of study treatment, the primary outcome is days of abstinence from non-medical opioids during study weeks 2–24 combined with up to 12 urine drug screen tests for opioids. For 90% power , 304 participants are needed for the BUP-XR versus SOC comparison. With the same planning parameters, 300 participants are needed for the BUP-XR and PSI versus SOC and PSI comparison. Statistical and health economic analysis plans will be published before data-lock on the Open Science Framework. Findings will be reported in accordance with the Consolidated Standards of Reporting Trials and Consolidated Health Economic Evaluation Reporting Standards.This pragmatic randomised controlled trial is the first evaluation of injectable BUP-XR versus the SOC medications BUP-SL and MET, with personalised PSI. If there is evidence for the superiority of BUP-XR over SOC medication, study findings will have substantial implications for OUD clinical practice and treatment policy in the UK and elsewhere.EU Clinical Trials register 2018-004460-63.The online version contains supplementary material available at 10.1186/s13063-022-06595-0. Opioid use disorder OUD; DSM-5 ) is a de  is a decrack). Patients with dual OUD-CUD find it harder to engage and derive clinical benefit from treatment  and CEQ-F[C])OUD and CUD DSM5 status measured by SCID-5-RVClinician rating of severity, complexity, and recovery strengths by ADAPTClinician rating of global impression (CGI-I anchored on baseline CGI-S)Difficulties in Emotion Regulation — Short Form (DERS-SF)Patient report of depression symptoms (QIDS-SR)Patient report of work and social adjustment functioning (WSAS)Patient evaluation of OUD recovery (SURE)Patient report of OUD improvement (PRO-I anchored on baseline PRO-S)Cognitive function (MoCA)Alcohol use Among participants enrolled in longer-term BUP-XR treatment, the following measures will be administered: heroin, cocaine, and illicit/non-medical benzodiazepine use in past 90 days ; OUD and CUD remission status (SCID-5-RV); somatic symptoms (PHQ-15), emotion regulation (DERS-SF), depression and anxiety symptoms (PHQ-4) and quality of life (OSTQOL)EXPO has the following secondary outcome measures:Informed by the DELTA2 guideline , sample n = 152) versus group 2  comparison, and 300 participants for the group 3  versus group 4  comparison. The Statistical Analysis Plan (SAP) will present a sensitivity check on this power calculation on the assumption of a greater group response.To obtain 90% power, with alpha at 5% and with 15% inflation for attrition, a target total of 304 participants will be needed for the group 1  before data-lock. The senior statistician will be blinded. The junior statistician will be unblinded so that reports can be prepared. The research and clinical team will also be unblinded. Findings will be reported following the Consolidated Standards of Reporting Trials  for a 2-year period before randomisation and follow-upAfter completing and reporting the analyses of the primary and secondary analyses, there is a planned longer-term exploratory analysis of outcomes at 3 and 6 years following randomisation using linked UK registry data. Subject to patient consent and approval from the Office for Health Improvement and Disparities , the Ministry of Justice, and NHS Digital, EXPO participant data will be linked to the following:This analysis of extracts from NDTMS, HES, NHS Digital, and the PNC will be implemented subject to resources and requiring protocol amendment and analysis plans.An independently chaired Trial Steering Committee (TSC) and Data Monitoring Committee (DMC) will oversee this Clinical Trial of an Investigational Medicinal Product, and its integrity, recruitment procedures, research measures and their completion, and the data analysis. These committees will include members with addiction service delivery, commissioning, service management, and patient and public involvement (PPI) expertise. The Trial Management Group will be responsible for the day-to-day running of the study and members will attend meetings of the oversight committees. After approving the protocol, the TSC and DMC will meet approximately two to four times each year. The King’s Health Partner’s Clinical Trials Office (KHP-CTO) will monitor EXPO centres every 14–18 weeks (but can be increased/decreased). While there are no trial stopping rules, the study may be prematurely discontinued by the sponsor, or for reasons reported by the chair of the DMC to the chair of the TSC.Adverse event — will be any untoward medical occurrence in a subject to whom a medicinal product has been administered including occurrences which are not necessarily caused by or related to that productAdverse reaction — will be any untoward and unintended response in a participant to an investigational medicinal product which is related to any dose administered to that participantThe Reference Safety Information for all information pertaining BUP-XR will be the Investigator’s Brochure (IB). The Summary of Product Characteristics (SmPC) will be the reference document for SOC medication. During the study, adverse events will be defined as follows:3.Serious adverse event (SAE) — a serious adverse reaction or suspected unexpected serious adverse reaction — will be any adverse event, adverse reaction, or unexpected adverse reaction, respectively, that results in death, is life-threatening, required hospitalisation or prolongation of existing hospitalisation, results in persistent or significant disability or incapacity, and consists of a congenital anomaly or birth defect4.Important medical events (IME) —will not be immediately life-threatening or result in death or hospitalisation but may jeopardise the patient or may require intervention and will be considered serious. Although not an SAE, any unplanned pregnancy will be reported as an IME.Unexpected adverse reaction — will be an adverse reaction the nature and severity of which is not consistent with the information about the medicinal product in question set out in the SmPC or the IBAll clinical investigators in the study will be provided with full details of possible adverse medical events that may result from study medication. Clinicians will report, and the PI will assess, each adverse event for seriousness, causality , and intensity . All serious adverse events will be promptly reported to the study sponsor no later than 24 h after the research team becoming aware of the event. The CI (or a doctor nominated by the CI) will review every event within one working day of the SAE form being received and determine whether the event was expected or unexpected. The CI may upgrade the causality of an event without PI agreement.All patient data including audio recordings will be assigned a unique numeric identifier and stored on a password-protected computerAll study data will be stored in line with the Medicines for Human Use  Amended Regulations 2006All study data will be archived in line with the Medicines for Human Use  Amended Regulations 2006 (and as defined by the sponsor’s archiving policy and procedure)The CI will have overall responsibility for the trial dataset, supported by the oversight committees, and the co-clinical lead investigator (CCLI) will act as custodian for the trial data under the General Data Protection Regulations. Only the CI, CCLI, KCTU, EXPO statisticians, and EXPO heath economists will have access to the final trial dataset. Participant data (as defined by the Data Protection Act 2018) will not be disclosed to the funder or the sponsor — except where this is required to satisfy safety monitoring — and confidentiality of participant information will be assured through the following adherence:Findings from the study will be communicated as reports to peer-reviewed scientific journals, medical conferences, and other scientific meetings, and in target ways appropriate for service user, public, and professional audiences.The opioid agonist/partial agonist medications BUP-SL and MET are first-line, evidence-based SOC, but not all patients are able to adhere and derive benefit. EXPO is the first randomised controlled trial of BUP-XR versus SOC and the first study to contrast BUP-XR and SOC with personalised PSI.The study has several strengths. Firstly, it has patient and public engagement; is well-powered and pragmatic, with a protocol open to as many members of the target population as possible in well-established specialist NHS services in areas with a high prevalence of OUD; and implemented under routine clinical conditions. This will increase the likelihood that the findings from the study generalise to NHS services in England and Scotland. If the study secures evidence for the relative effectiveness of BUP-XR over SOC, this will have substantial implications for policy and clinical practice in the UK and elsewhere.Secondly, the primary outcome is a well-defined, clinically meaningful and combines patient report and biochemical measure. The collection of outcome measures is timed to coincide with routine clinical follow-up as part of efforts to minimise loss to follow-up. Thirdly, EXPO builds on work to develop and study idiographic, personalised PSI as evaluated in the successful ARC study . If therLimitations of the study include the relatively short 24-week endpoint. This horizon is commonly used in the field, but further exploratory research (as planned in EXPO) will be needed to determine longer-term outcomes on opioid use. Other research questions — for example the deployment of BUP-XR as a taper for medication discontinuation — will need new protocols.An integrated approach to assessment, stratified treatment, and continuing care is now gaining momentum in behavioural medicine, where tailoring variables and measurement-based care actions can improve outcomes , 76. Withttps://www.clinicaltrialsregister.eu/ctr-search/trial/2018-004460-63/GB. The protocol and PIS/PCF materials (available from corresponding author) were approved Health Research Authority (IRAS project number: 255522) via the London-Brighton & Sussex Research Ethics Committee (reference: 19/LO/0483) on 14 June 2019. Participant recruitment commenced on 6 August 2019. It is anticipated that the study will complete recruitment in November 2021.Approval for the study was obtained from the UK Medicines and Healthcare Product Regulatory Agency on 4 March 2019. The study was registered on the EU Clinical Trials Register on 4 March 2019 (number: 2018-00460-63); The latest version of the study protocol is version 5.1 (6 October 2021). There have been seven non-substantial and substantial approved amendments to the protocol, as follows:Version 1.2 (13 June 2019). Non-substantial amendment: (a) use of rescue BUP-SL after first BUP-XR injection and ongoing (to be recorded as concomitant medication); (b) a surgical procedure guideline for the removal of BUP-XR; and (c) all SARs among participants allocated to BUP-XR to be classified as unexpected and reported as SUSARS.Version 2.0 (8 January 2020). Substantial amendment: (a) dosing guidance for participants consenting to receive continued BUP-XR after the study treatment endpoint; (b) optional use of cold press before BUP-XR injection; (c) removal of instruction that BUP-XR can be stored at room temperature for up to 7 days prior to administration.Version 2.1 (9 July 2020). Non-substantial amendment: addition of qualitative interview #1 at South London, West Midlands, Newcastle, and Tayside centres.Version 3.0 (22 July 2020). Substantial amendment: addition of BUP-NX as this is an SOC medication at Tayside site.Version 4.0 (30 September 2020). Substantial amendment: (a) addition of BUP-ESP as this is an SOC medications at Tayside site; (b) clarification that participants allocated to SOC arms can be transitioned between BUP-SL and MET following prescribing guidelines in the SmPC and if there are no known allergic, adverse reactions or contraindications.Version 5.0 (1 June 2021). Substantial amendment: (a) addition of OSTQOL; PHQ-4 and PHQ-15 for continued treatment evaluation; (b) option for research measures recorded during clinic visits at weeks 2, 6, 10, 14, 18, and 22 to be done by telephone (precluding UDS collection) in response to government public health restrictions for COVID-19; (c) clarification that participants can continue in the trial if LFT testing is not taken post-randomisation according to CI, PI, and sub-investigator clinical judgement; (d) clarification that participants can continue in study if LFT testing not done post-randomisation according to CI, PI, and SI clinical judgement.Version 5.1 (6 October 2021). Non-substantial amendment: expanded description of cost-effectiveness analysis for the health economic analyses.Additional file 1: Table S1. Standard Protocol Items for Randomised Trials (SPIRIT) 2013. SPIRIT Checklist.Additional file 2.Additional file 3.


What is the risk of thromboembolism and recurrent atrial fibrillation after infection-related atrial fibrillation?In this cohort study including 274 196 patients with pneumonia, of whom 6553 developed atrial fibrillation, the 1-year risk of thromboembolism was 0.8% in patients without atrial fibrillation vs 2.1% in patients with atrial fibrillation. Among patients with new-onset atrial fibrillation, 32.9% had a recurrent hospital contact with atrial fibrillation and 14.0% initiated anticoagulation therapy during 3-year follow-up.These findings suggest that the concept of infection-related atrial fibrillation as a transient condition needs reconsideration given the high risks of recurrence combined with risks of thromboembolism that may warrant anticoagulation therapy. This cohort study investigates the thromboembolic risks associated with atrial fibrillation (AF) in patients with pneumonia, assesses the risk of recurrent AF, and examines the association of initiation of anticoagulation therapy with new-onset AF. New-onset atrial fibrillation (AF) is commonly reported in patients with severe infections. However, the absolute risk of thromboembolic events without anticoagulation remains unknown.To investigate the thromboembolic risks associated with AF in patients with pneumonia, assess the risk of recurrent AF, and examine the association of initiation of anticoagulation therapy with new-onset AF.This population-based cohort study used linked Danish nationwide registries. Participants included patients hospitalized with incident community-acquired pneumonia in Denmark from 1998 to 2018. Statistical analysis was performed from August 15, 2021, to March 12, 2022.New-onset AF.Thromboembolic events, recurrent AF, and all-cause death. Estimated risks were calculated for thromboembolism without anticoagulation therapy, new hospital or outpatient clinic contact with AF, initiation of anticoagulation therapy, and all-cause death at 1 and 3 years of follow-up. Death was treated as a competing risk, and inverse probability of censoring weights was used to account for patient censoring if they initiated anticoagulation therapy conditioned on AF.Among 274 196 patients hospitalized for community-acquired pneumonia, 6553 patients  developed new-onset AF. The 1-year risk of thromboembolism was 0.8%  in patients without AF vs 2.1%  in patients with new-onset AF without anticoagulation; this risk was 1.4%  among patients with AF with intermediate stroke risk and 2.8%  in patients with AF with high stroke risk. Three-year risks were 3.5%  among patients with intermediate stroke risk and 5.3%  among patients with high stroke risk. Among patients with new-onset AF, 32.9%  had a new hospital contact with AF, and 14.0%  initiated anticoagulation therapy during the 3 years after incident AF diagnosis. At 3 years, the all-cause mortality rate was 25.7%  in patients with pneumonia without AF vs 49.8%  in patients with new-onset AF.This cohort study found that new-onset AF after community-acquired pneumonia was associated with an increased risk of thromboembolism, which may warrant anticoagulation therapy. Approximately one-third of patients had a new hospital or outpatient clinic contact for AF during the 3-year follow-up, suggesting that AF triggered by acute infections is not a transient, self-terminating condition that reverses with resolution of the infection. The decision to initiate OAC therapy should be based on the expected net clinical benefit of OAC therapy, which tracks closely with the absolute risk of stroke and bleeding in patients not receiving OAC therapy. Patients at low stroke risk may have little to no or negative net clinical benefit from therapy. Decision analyses have indicated that the threshold at which OAC treatment yields a net clinical benefit is a stroke risk between 1% to 2% per year.16 However, there is a paucity of data on the absolute thromboembolic risk associated with AF triggered by infections to guide the treatment decision among patients with pneumonia and incident AF.A prevailing thought has been that AF triggered by acute infection is a transient and self-terminating condition that reverses with resolution of infection. However, mounting evidence suggest that AF frequently recurs, carrying an increased risk of stroke.This nationwide cohort study in Denmark sought to investigate the risks of arterial thromboembolism in patients with new-onset AF after community-acquired pneumonia without anticoagulation therapy. In secondary analyses, we sought to clarify the concept of postinfection new-onset AF as a transient condition by estimating risks of recurrent hospital or outpatient clinic contact with AF, OAC therapy initiation, and all-cause mortality.STROBE) reporting guideline for cohort studies.17This cohort study included all patients hospitalized with community-acquired pneumonia in Denmark from January 1998 through June 2018. The study was approved by the Danish Data Protection Agency. Registry studies do not require ethical approval or informed consent in Denmark. The data were provided by the Danish Health Data Authority. We followed the Strengthening the Reporting of Observational Studies in Epidemiology  The Civil Registration system containing information about age, sex, and vital status19; and (3) The Danish National Prescription Registry storing information about all claimed prescriptions from Danish people.20The source population included the entire population of Denmark (5.6 million inhabitants). Denmark has a tax-funded universal health care system, with equal access to hospitals and primary care for all residents and partial reimbursement of medication costs. A unique civil registration number, assigned to all residents, is used to track individual health services in nationwide registries. This study was based on linkage between 3 registries: (1) the Danish National Patient Register, which includes information on all discharge diagnoses from Danish hospitals22 We used the National Patient Register to identify all patients aged at least 18 years at incident primary inpatient pneumonia diagnosis. To exclude confounding from underlying conditions and procedures, we restricted the study to patients with community-acquired pneumonia. Accordingly, we excluded all patients with hospital contact (inpatient or ambulatory) within the 30 days before pneumonia admission. To ensure lookback time for prevalent AF diagnoses, we further excluded patients not residing in Denmark for at least 2 years before pneumonia diagnosis at hospital discharge (index date).We focused on pneumonia because it is a well-characterized prevalent infection that has been consistently linked with cardiovascular disease, including AF.Because we focused on new-onset AF following pneumonia, we screened the pneumonia cohort for status of AF according to diagnoses and prescriptions. We excluded patients with prior AF diagnoses and patients with prior experience of any OAC treatment within 180 days before index date. To investigate risk of outcomes in patients not receiving OAC, we further excluded patients who developed a study end point, initiated OAC treatment, or died during the landmark period of 30 days after discharge from pneumonia hospitalization , ischemic stroke (approximately 97%),2DS2-VASc  score24 as a measure of stroke risk and a HAS-BLED  score25 as a measure of bleeding risk , intermediate risk (1-2 risk factors), and high risk (≥3 risk factors) where points for female sex were not considered.We combined covariate information into the CHA26 Specifically, we used the Aalen-Johansen estimator to estimate risk development for all end points over time, assuming death as competing risk.24 Cause-specific Cox regression with adjustment for inclusion period, sex, and age (included as restricted cubic spline) was used to assess the risk of arterial thromboembolism comparing patients with new-onset AF vs those without new-onset AF.Baseline characteristics at pneumonia diagnosis were summarized using means and SDs for continuous measures and percentages for categorical measures. We used time-to-event analyses to analyze outcome risks measuring time at risk from the 30-day landmark to outcome of interest, emigration, death, or end of follow-up, whichever came first. We investigated the absolute risk of events, taking into account the competing risk of death, and hereby incorporating the diminishing at-risk population under the expectation of a relatively high risk of death and number of outcomes.27 Risk of OAC therapy initiation during follow-up was estimated using a Fine-Grey regression model with death as competing risk and accounting for baseline and time-varying confounding factors, including age, sex, heart failure, hypertension, stroke, diabetes, vascular disease, use of diuretics, renin-angiotensin system inhibitors, β-blocker, or calcium-channel blocker. Recurrent hospital or outpatient clinic contact with AF during follow-up was also included as a time-varying covariate as it may trigger OAC therapy initiation. To allow for time-varying covariates, the data set was structured in the counting process style with multiple rows of data per patient by splitting the follow-up into 7-day periods and evaluate covariates and outcomes at every interval. The IPCW given by the inverse of the survival function of the Fine-Grey model for the outcome of OAC initiation was subsequently estimated by a weighted Cox proportional hazards model.28 To account for repeated observations, we applied a sandwich estimator of the covariance matrix. To reduce influence of extreme IPCWs, the weights were stabilized by the conditional probability of treatment initiation only, given that the inclusion period accounted for the competing risk of death. The 95% CIs for arterial thromboembolic risk were estimated by bootstrapping with 1000 samples. Because of small sample size and few events, we decided post hoc not to use IPCW methods for patients with low stroke risk, because this may reduce the fit of the censoring model and precision of estimated weights. IPCW methods were not used for assessment of secondary outcomes, because patients were not censored according to OAC therapy use.For analyses of arterial thromboembolism in patients with new-onset AF, we censored patients when they started OAC therapy to estimate risks had these patients not initiated anticoagulation and to avoid potential structural selection bias between groups . We used an inverse probability of censoring weighted (IPCW) analysis to handle informative censoring related to OAC therapy initiation.29 Therefore, we conducted a sensitivity analysis excluding patients with baseline aspirin treatment to ascertain whether this would affect thromboembolic risk.In sensitivity analyses, we examined potential outcomes of changing guidelines for OAC treatment. First, we stratified the baseline hazard function by inclusion period  and included interactions between inclusion period and all covariates when estimating IPCW weights. Next, we examined outcome risks with restriction to the most recent period, 2014 to 2018. Finally, although aspirin does not provide optimal stroke protection in patients with AF, there is evidence of some benefit in terms of lower stroke rates compared with no treatment.P < .05) was used to evaluate significance. Analyses were performed using Stata/MP statistical software version 16 (StataCorp) and R statistical software version 3.1.1 . Statistical analysis was performed from August 15, 2021, to March 12, 2022.Statistical tests represent 2-sided hypotheses and a 5% level  developed new-onset AF ; adjusted hazard ratio [HR], 1.49 ) . When weAt 1 year, the risk of thromboembolism was 0.8%  in patients without AF vs 2.1%  in patients with new-onset AF not receiving anticoagulation. Among patients with new-onset AF, the risk was 1.4%  in patients at intermediate stroke risk and 2.8%  in high-risk patients . At 3-yeAmong patients with new-onset AF, 8.7%  claimed a prescription for OAC therapy during the first year after hospital admission with community-acquired pneumonia, increasing to 14.0%  at 3 years . ConcurrOne-year and 3-year risks of thromboembolic events among patients with new-onset AF changed little when accounting for temporal changes by stratifying the baseline hazard function by inclusion period and including interactions between inclusion period and all covariates in the Fine-Grey regression model  had a new hospital or outpatient clinic contact with AF and 14.0%  initiated OAC during follow-up.Among 293 051 patients with community-acquired pneumonia and no history of AF, 3.8% developed AF after pneumonia hospitalization. Without OAC therapy, new-onset AF was associated with a 2.1% 1-year risk of arterial thromboembolic events (1.4% among patients with intermediate stroke risk and 2.8% among patients at high risk). The 3-year risks were 3.5% and 5.3%, respectively. These estimates exceed the general threshold of 1.7% per year for vitamin K antagonists and 0.9% for the non–vitamin K OACs for obtaining a positive net clinical benefit of stroke prevention12 and in patients with infections without AF.13 Among patients with infection-related AF, pneumonia was associated with the highest risk of thromboembolic events,13 but the association has also has been demonstrated in patients with sepsis.30 The benefit of OAC therapy was similar between matched patients with infection-related AF and non–infection-related AF; for infection-related AF, the adjusted HR for thromboembolism was 0.75  compared with those without OAC therapy, and for non–infection-related AF the HR was 0.70 .10 Most previous studies have only reported the relative risks, which does not inform about baseline risk and may obscure the magnitude of the association. In many situations, the absolute risk gives a better representation of the clinical situation, also from the patient’s point of view.31 Our study reporting absolute risks and focusing on a specific infection in patients without OAC, therefore, supports and strengthens the validity of previous findings.This study expands the evidence suggesting that there is an increased risk of thromboembolism in patients with infection-related AF, and it adds to the notion that new-onset AF should not be regarded as self-limiting and benign. Previous studies have demonstrated similar relative risk of thromboembolism in patients with infection-related AF compared with patients with AF without infection matched by baseline OAC treatment status32 New-onset AF after pneumonia may identify a subset of patients who have a substrate for thromboembolism regardless of the arrhythmia. A key finding of our study was that the AF episode was not a transient, self-limited event, because approximately one-third of patients had a new hospital AF diagnosis during follow-up. This corroborates previous findings33 and suggests that restoration of sinus rhythm with resolution of infection may not protect against AF recurrence. In this respect, infection may merely be a stress test that demonstrates the likelihood for future AF. The substantial observed risk of recurrent AF suggests that individuals with new-onset AF in the context of pneumonia may have underlying predisposition to AF. Indeed, patients with AF in our cohort were older and had a high prevalence of comorbidities, both associated with high risk of AF and thromboembolism.Several mechanisms explain why infections can trigger AF, including systemic inflammation, electrophysiological disturbances, metabolic imbalances, hypoxia, and dysvolemia.34 According to current guidelines, patients with non–infection-related AF receive OAC therapy according to their perceived stroke risk rather than the heart rhythm displayed at a particular time.36 The updated European Society of Cardiology guidelines35 state that the clinical pattern of AF  should not condition the indication to thromboprophylaxis. In the era of non–vitamin K OAC therapy, perhaps it is time for infection-related AF to be treated in the same way as non–infection-related AF, with long-term anticoagulation maintained in patients with manifested clinical stroke risk factors. However, the decision to initiate anticoagulation treatment in the setting of severe infections is complex. High bleeding risk due to systemic activation of the inflammatory response and depletion of coagulation factors and platelets may outweigh the benefits of anticoagulation. Most trials investigating different OAC therapies for stroke prophylaxis excluded patients with AF due to reversible conditions,40 and high-quality evidence regarding the role of OAC in patients with infection-related AF is lacking.From a clinical point of view, our study has 2 major implications. First, our findings suggest that new-onset AF during pneumonia is a marker of future AF. This implies that vigilance for recurrent AF may be warranted and that attention should be given to each episode of infection-related AF, regardless of its duration, to tailor surveillance in patients developing this complication. Second, our findings suggest that new-onset AF occurring with pneumonia is associated with a risk of thromboembolism that reaches a level where OAC therapy is considered beneficial. The question remains whether these patients should initiate lifelong OAC therapy. Previously, a UK study found that patients with resolved AF remained at higher risk of stroke than patients without AF.Our study was strengthened by the completeness of data in a nationwide cohort of patients experiencing incident AF after community-acquired pneumonia. The Danish health care system provides equal access to health care services for all residents regardless of socioeconomic and insurance status. In Denmark, OAC therapy can be purchased only through prescription, and all Danish pharmacies register redeemed prescriptions ensuring complete and accurate registration.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision diagnosis, and we may have missed episodes given the known risk of brief, self-terminated AF episodes that may be undetected or unreported. Therefore, our findings may not generalize to very brief, self-terminating AF episodes. We lacked information on duration and number of AF episodes during admission, and detailed classification of strokes as cardioembolic vs other was not feasible.Our study also has limitations. New-onset AF was defined by a record of an Among patients with community-acquired pneumonia, new-onset AF was associated with increased risks of thromboembolism, which may cross the threshold where OAC therapy is considered beneficial. One-third of patients had a new hospital AF diagnosis during follow-up, indicating that the concept of infection-related AF as a transient condition may need reconsideration. These findings may have rhythm monitoring and treatment implications, and improved communication and monitoring of long-term AF risks is warranted.