The respondents declaring both sweet and salty food products as tempting were characterized by a lower SREBQ score than those declaring only sweet, only salty, or declaring no tempting products . The major factors associated with SREBQ categories were gender, body mass index change, and tempting food products. A higher share of respondents characterized by a high self-regulation of eating behaviors was observed for males than for females ; for respondents losing weight during the COVID-19 pandemic than for respondents gaining weight ; as well as for respondents declaring no tempting products than those declaring both sweet and salty food products . It was concluded that the self-regulation of eating behaviors in adolescents is closely associated with food products perceived as tempting by them, as well as with gender. During the COVID-19 pandemic, the low self-regulation of eating behaviors was a significant determinant of the body mass gain. Taking this into account, female adolescents characterized by a low self-regulation of eating behaviors especially should be subjected to a dedicated intervention program to prevent overweight and obesity.The self-regulation of eating behaviors , has been an important determinant for adopting a healthy lifestyle in the period of the COVID-19 pandemic in adults, but there have been no such studies conducted in the population of adolescents in this period. The aim of the presented study is to analyze self-regulation of eating behaviors in the population-based sample of Polish adolescents within the Polish Adolescents’ COVID-19 Experience (PLACE-19) Study. The Self-Regulation of Eating Behavior Questionnaire (SREBQ) was used to assess self-regulation of eating behaviors in the studied population of 1126 Polish adolescents , aged 15–20, recruited based on a random quota sampling within a national sample. Based on the screening questions of the SREBQ, 145 individuals were excluded from the analysis. The participants of the study were categorized based on their gender, body mass index, body mass index change during the COVID-19 pandemic, and food products declared as tempting. The major factors associated with SREBQ score were body mass index change and tempting food products. The respondents losing weight during the COVID-19 pandemic were characterized by a higher SREBQ score than those maintaining stable body mass or gaining weight (3.4 vs. 3.2 vs. 3.2;  Self-regulation of eating behaviors, also called self-control of eating behaviors, is defined as suppression of the behavioral short-term impulse to consume food products in the interest of pursuing long-term weight goals . The genA higher level of self-regulation of eating behaviors leads to a healthier diet being followed, and as a result, it may prevent excessive weight gain . In geneThe level of self-regulation of eating behaviors results from numerous determinants, including cognitive restraint, moderation, mindfulness, disinhibition, delayed gratification, emotions and moods, self-efficacy, social support, environment, and physical activity . The devFor adolescents, development of self-regulation of eating behaviors is especially important to be prepared to withstand the obesogenic food environment . Taking The outbreak of Coronavirus Disease 2019 (COVID-19), as well as related social isolations and lockdowns, caused significant changes in eating habits in a population of adolescents . At the However, there have been no studies of self-regulation of eating behaviors in the population of adolescents during the COVID-19 pandemic conducted so far. Taking this into account, the aim of the presented study is to analyze self-regulation of eating behaviors in the population-based sample of Polish adolescents within the Polish Adolescents’ COVID-19 Experience (PLACE-19) Study.The presented study was conducted on the basis of the approval of the Ethics Committee of the Central Clinical Hospital of the Ministry of Interior and Administration in Warsaw (No. 2/2021). It was conducted in agreement with the guidelines of the Declaration of Helsinki. Each participant of the study and their parents/legal guardians provided their informed consent for participation.The presented analysis was conducted by the Institute of Human Nutrition Sciences, Warsaw University of Life Sciences (WULS-SGGW) within the PLACE-19 Study. The PLACE-19 study was conducted in the population-based sample of Polish adolescents, recruited from the Polish secondary schools, aged 15–20 . The PLACE-19 Study assessed various behaviors during the COVID-19 pandemic: within the first phase of the study, hygienic and personal protective behaviors ,19,20; wThe presented phase of the PLACE-19 Study was conducted from 21 January 2021 to 17 February 2021, as presented in the previous study .The random quota sampling of secondary schools was conducted within voivodeships and counties—from each voivodeship (16 voivodeships in Poland) 5 random counties were sampled , and from each county 5 random secondary schools were sampled . The principal of each sampled secondary school was informed about inviting the school to participate in the study, and if they agreed, the study was conducted; the principal invited students, received access to an electronic version of the questionnaire, and provided it to each participant of the study.–Students of the sampled secondary schools;–Aged 15–20 years;–Informed consent to participate in the study provided by students and their parents/legal guardians.The following inclusion criteria were formulated:–Participation in the previous phases of the PLACE-19 Study;–Missing/unreliable data provided in the completed form.The following exclusion criteria were formulated:The total number of 1126 secondary school students  participated in the study, while they represented 19 secondary schools in all the regions of Poland.Within the studied population, on the basis of the screening questions of the Self-Regulation of Eating Behaviour Questionnaire (SREBQ), applied within the study, 145 respondents were excluded from the further analysis of self-regulation of eating behaviors , resulting in the final sample of 981 secondary school students .When the PLACE-19 Study was conducted, education in all secondary schools was suspended and remote learning was introduced, as per the decision of the Polish Ministry of Education . TherefoThe self-regulation of eating behaviors was assessed using the Self-Regulation of Eating Behaviour Questionnaire (SREBQ), developed and validated by Kliemann et al.  for the (1)I give up too easily on my eating intentions (reverse statement);(2)I’m good at resisting tempting food;(3)I easily get distracted from my eating intentions (reverse statement);(4)If I am not eating in the way I intend to I make changes;(5)I find it hard to remember what I have eaten throughout the day (reverse statement).The SREBQ is based on 5 questions about self-regulation of eating behaviors–formulated as statements describing specific behaviors declaring how often those behaviors are presented by the respondent. The respondent is asked to specify the frequency of presenting specific behaviors as never, rarely, sometimes, often, or always, which are attributed to 5-point Likert scale from 1 (never) to 5 . In the case of 3 reverse statements, the reverse scale is applied from 1 , to 5 (never). The following self-regulation of eating-behavior statements are formulated within SREBQ:On the basis of the score for each statement, the total score is calculated as the mean score of the 5 statements. Afterwards, the total score may be interpreted while using the following cut-offs: <2.8—low self-regulation of eating behaviors; 2.8–3.6—medium self-regulation of eating behaviors; >3.6—high self-regulation of eating behaviors .The additional element of the SREBQ are screening questions about food products that respondents find tempting, about intentions to not eat too many tempting food products and about intentions to have a healthy diet. Those questions are included in order to assess self-regulation of eating behaviors only for those respondents who either have the intention to have a healthy diet or have the intention to not eat many foods they find tempting .On the basis of the multiple-choice question about tempting food products with the following suggested answers: chocolate, crisps, cakes, ice cream, bread/toast, fizzy drinks, biscuits, sweets, popcorn, pastries, pizza, fried foods, chips, other foods (to be specified), respondents were categorized as those indicating sweet products as tempting, those indicating salty products as tempting, and those indicating both sweet and salty products as tempting. The last possibility was to not indicate any product as tempting (to declare that they do not find any food product tempting) and those respondents formulated the additional subgroup.The additional questions included into the applied questionnaire assessed self-reported body mass and body mass changes during the COVID-19 pandemic. Each respondent was asked about their current body mass and height (February 2021), as well as about their body mass and height before the COVID-19 pandemic. In order to allow secondary-school students easier recognition of the period before the COVID-19 pandemic, respondents were asked about a specific period defined as the time before transition into a remote-education system (March 2020).2), normal weight (18.5–24.9 kg/m2), overweight (25.0–30.0 kg/m2), or obese (>30.0 kg/m2), on the basis of BMI cut-offs by the WHO [On the basis of the provided information about current body mass and height, as well as about body mass and height before the COVID-19 pandemic, for each period the body mass was assessed based on the body mass index (BMI) calculation . Afterwa the WHO . The res the WHO  and usin the WHO . On the  the WHO .The body mass changes during the COVID-19 pandemic were defined based on changes in BMI value (for respondents aged >18 years), or changes in BMI percentile (for respondents aged <18 years), and respondents were categorized as losing weight, maintaining stable weight, or gaining weight during the COVID-19 pandemic (from March 2020 to February 2021).2, comparative fit index (CFI), Tucker–Lewis index (TLI), root-mean-square error of approximation (RMSEA), and standardized root-mean-square residual (SRMR). The cut-offs were applied as follows: p for χ2 > 0.05 [2 = 6.658 (p = 0.155), CFI = 0.994, TLI = 0.985, RMSEA = 0.026, SRMR = 0.025, indicating positive validation of the model for the Polish version of the SREBQ.In order to assess the internal reliability of the Polish version of the SREBQ, the standardized factor loadings within the confirmatory factor analysis (CFA) were analyzed, similarly as in the German study by Schmalbach et al. . The mod2 > 0.05 ; CFI ≥ 02 > 0.05 ; TLI ≥ 02 > 0.05 ; RMSEA ≤2 > 0.05 ; SRMR ≤ 2 > 0.05 . The mod–n = 718) and male respondents (n = 263),Gender: female (–n = 32), normal weight (n = 729), overweight (n = 142), and obese respondents (n = 78),Body mass index: underweight (–n = 270), maintaining stable weight (n = 460), and gaining weight respondents (n = 251),Body mass index change during the COVID-19 pandemic: losing weight (–n = 104), salty (n = 71), sweet and salty products (n = 725), as well as declaring no food products as tempting (n = 81).Tempting food products: respondents declaring sweet , with p = 0.0082).The general characteristics of the sample studied within the PLACE-19 Study is presented in p = 0.0003) and tempting food products (p < 0.0001), while neither gender nor body mass index were associated with it. The respondents losing weight during the COVID-19 pandemic were characterized by a higher self-regulation of eating behaviors than those maintaining stable body mass or gaining weight (SREBQ score of 3.4 vs. 3.2 vs. 3.2). At the same time, the respondents declaring both sweet and salty food products as tempting were characterized by a lower self-regulation of eating behaviors than those declaring only sweet, only salty, or declaring no tempting products (SREBQ score of 3.2 vs. 3.4 vs. 3.4 vs. 3.4).The self-regulation of eating behaviors score assessed using the SREBQ within the PLACE-19 Study and in subsamples is presented in p < 0.0001), being good at resisting tempting food (p = 0.0382), and easily getting distracted from the way they intend to eat, there were significant differences between female and male respondents (p < 0.0001). The female respondents declared giving up too easily on their eating intentions, not being good at resisting tempting food, and easily getting distracted from the way they intend to eat  more often than male ones.The self-regulation of eating behaviors assessed using the SREBQ within the PLACE-19 Study and in subsamples stratified by gender is presented in p = 0.0046), easily getting distracted from the way they intend to eat (p = 0.0004), and finding it hard to remember what they have eaten throughout the day, there were significant differences dependent on body mass index (p = 0.0317). Both overweight and obese respondents declared giving up too easily on their eating intentions, easily getting distracted from the way they intend to eat, and finding it hard to remember what they have eaten throughout the day  more often than underweight and normal-weight ones.The self-regulation of eating behaviors assessed using the SREBQ within the PLACE-19 Study in subsamples stratified by body mass index is presented in p < 0.0001), being good at resisting tempting food (p < 0.0001), easily getting distracted from the way they intend to eat (p < 0.0001), and making changes while they are not eating in the way they intend to, there were significant differences between respondents losing and gaining weight (p < 0.0001). The respondents gaining weight declared giving up too easily on their eating intentions, not being good at resisting tempting food, easily getting distracted from the way they intend to eat, and not making changes while they are not eating in the way they intend to  more often than those losing weight or maintaining a stable weight.The self-regulation of eating behaviors assessed using the SREBQ within the PLACE-19 Study in subsamples stratified by body mass index change during the COVID-19 pandemic is presented in p = 0.0209), being good at resisting tempting food (p < 0.0001), easily getting distracted from the way they intend to eat (p < 0.0001), making changes while they are not eating in the way they intend to (p = 0.0001), and finding it hard to remember what they have eaten throughout the day, there were significant differences between respondents declaring no tempting food products and declaring any tempting products (p = 0.0038). The respondents declaring any tempting products declared giving up too easily on their eating intentions, not being good at resisting tempting food, easily getting distracted from the way they intend to eat, not making changes while they are not eating in the way they intend to, and finding it hard to remember what they have eaten throughout the day  more often than those declaring no tempting food products.The self-regulation of eating behaviors assessed using the SREBQ within the PLACE-19 Study in subsamples stratified by tempting food products is presented in p = 0.0142), body mass index change (p = 0.0423), and tempting food products (p < 0.0001), while body mass index was not associated with it. A higher share of respondents characterized by a high self-regulation of eating behaviors was observed for males than for females (27.4% vs. 18.8%); for respondents losing weight during the COVID-19 pandemic than for respondents gaining weight (25.9% vs. 15.5%); as well as for respondents declaring no tempting products than those declaring both sweet and salty food products (38.3% vs. 18.2%).The self-regulation of eating-behavior categories assessed using the SREBQ within the PLACE-19 Study and in subsamples is presented in Within the conducted study, the major factors associated with SREBQ score were body mass index change and tempting food products, while the major factors associated with SREBQ categories were gender, body mass index change, and tempting food products. The lower SREBQ level was observed more often in female respondents, respondents gaining weight during the COVID-19 pandemic, and respondents declaring both sweet and salty food products as tempting than in other subgroups.Gender is an important determinant of food acceptance, food intake, perceived food healthiness, and food avoidance . In adulThe association between self-regulation of eating behaviors and body mass index changes may have been expected, as a body mass gain  is indicated as a potential consequence of the need for immediate pleasure of tempting food products accompanied by consumption driven by emotions, these being elements of a low self-regulation of eating behaviors . In a nuComparable associations between self-regulation of eating behaviors and body mass index changes are indicated for adults. In the study by Ouwehand and Papies  conducteInterestingly, the associations were observed only for body mass index changes during the COVID-19 pandemic, but not for body mass index. It may be explained by the fact that body mass index change during the COVID-19 pandemic may be directly influenced by emotional eating in this period  or beingLast but not least, self-regulation of eating behaviors was in the studied group associated with declaring tempting food products. This association is based on the well-known effect of the strength of temptation on self-control . It resuIt was concluded that the self-regulation of eating behaviors in adolescents is closely associated with food products perceived by them as tempting, as well as with gender. During the COVID-19 pandemic, the low self-regulation of eating behaviors has been a significant determinant of the body mass gain. Taking this into account, female adolescents characterized by a low self-regulation of eating behaviors should especially be subjected to a dedicated intervention program to prevent overweight and obesity.


DNA methylation (DNAm) patterns in peripheral blood have been shown to be associated with aging related health outcomes. We perform an epigenome-wide screening to identify CpGs related to frailty, defined by a frailty index (FI), in a large population-based cohort of older adults from Germany, the ESTHER study. Sixty-five CpGs are identified as frailty related methylation loci. Using LASSO regression, 20 CpGs are selected to derive a DNAm based algorithm for predicting frailty, the epigenetic frailty risk score (eFRS). The eFRS exhibits strong associations with frailty at baseline and after up to five-years of follow-up independently of established frailty risk factors. These associations are confirmed in another independent population-based cohort study, the KORA-Age study, conducted in older adults. In conclusion, we identify 65 CpGs as frailty-related loci, of which 20 CpGs are used to calculate the eFRS with predictive performance for frailty over long-term follow-up. Frailty is associated with an increased risk for negative health outcomes in older populations, and being able to predict frailty could facilitate prevention measures. By performing an epigenome-wide screen, the authors derived a DNA methylation based measure for frailty which can predict both prevalence and longer-term incidence of frailty. It is frequently observed in older adult populations3 and increases the risk of developing negative health outcomes including falls, physical limitations, hospitalization, and mortality5. A growing body of research has reported that this syndrome results from a multidimensional interplay of genetic, biological, psychosocial, and environmental factors8.Frailty is defined as a state of elevated vulnerability to poor resolution of homeostasis11, and they were based on questionnaires, performance measures, routine data, or a combination of any of these. Two principal instruments were widely accepted in clinical practice12, the performance and questionnaire-based approach suggested by Fried et al.4 and the “Frailty index (FI)-approach” suggested by Rockwood et al.10, which can use any kind of data as long as the included items are frailty-related and fulfill specific validated criteria. FI has been shown to be a valid predictor of morbidity and mortality in many different population-based cohorts14.Without a gold standard measurement for frailty, various models were proposed to measure and define frailty in the past years16 and aging-related conditions, such as frailty18. Recently, various DNA methylation (DNAm)-based aging algorithms have been developed23 and have also been shown to be associated with frailty25. However, none of these algorithms was specifically derived for predicting frailty.Epigenetic modifications have been recognized to play a major role in aging21 to derive and validate DNAm signature-based frailty risk score in a large population-based cohort study of older adults. Replication was performed in another independent population-based cohort. Frailty was defined by a frailty index (FI) based on the concept of deficit accumulation26.Here, we followed a previously proposed three-phase procedureP-values > 0.05). The mean age was approximately 62 years, and a slight majority of participants were women. The majority of participants were overweight or obese (approximately 7 out of 10). Approximately half of them had never smoked, and approximately one out of six participants was still smoking at the time of enrollment. In KORA-age, the mean age was about 76 years and half of the participants were females. The education level (27% with ≥12 years of school education), body mass index levels (80% overweight or obese), and the level of alcohol consumption were higher than those in ESTHER.Table Table In the discovery phase, conducted in subset I, 2220 CpGs passed the genome-wide significance threshold (FDR < 0.05). 65 CpGs located at 47 genes across 21 chromosomes were successfully replicated in the validation phase in subset II and were deemed as frailty-related methylation loci. When more comprehensively adjusted for further potential confounders using model 2, 53 CpGs were associated with FI, of which 43 CpGs were inversely associated with FI, with a decrease in FI (95% CI) per one standard deviation (SD) increase in methylation ranging from 0.92% units (0.06–1.77) to 3.07% units (1.05–5.08) , cardiovascular disease , diabetes mellitus , and Alzheimer’s Disease .We performed a literature search in PubMed to obtain information on the genes that contain the 65 frailty-related CpGs  in the PPI network were seen  with the loci cg02867102, cg07312601, and cg11700584, respectively.Supplementary Data rSp, 0.443 and 0.267, respectively), but the correlation of FI with age increased with increasing length of follow-up (up to rSp = 0.397 at 11-year follow-up). Correlation coefficients between eFRS at baseline and FI at baseline and the various follow-up times were all in the range of 0.2–0.3, and correlation coefficients between the FI at various points of time were all in the range of 0.6–0.9. Essentially very similar correlations between eFRS and FI at baseline and the various follow-ups were seen with Pearson’s correlation coefficients (rSp similarly ranged from 0.2 to 0.3). Approximately 6%, 9%, 9%, 9%, and 5% variation of frailty of at baseline, 2-, 5-, 8-, and 11-year follow-up can be explained by eFRS. Furthermore, we observed a significant correlation of eFRS with AccAgeGrim . Replicated analyses in KORA-age showed consistent patterns and similar correlations. eFRS showed a slightly stronger correlation with age (rSp = 0.427) than FI at baseline (rSp = 0.411). Correlation coefficients between eFRS and FI at baseline and the two follow-ups similarly ranged between 0.2 and 0.3. About 6%, 7%, and 7% variation of frailty at baseline, 4-, and 8-year follow-up can be explained by the eFRS. All correlations in ESTHER and KORA-age were highly statistically significant (P < 0.001).Figure Supplementary Table Supplementary Fig. Associations of eFRS with being frail at baseline and various follow-up times in the two validation subsets are shown in Fig. We further conducted sensitivity analyses with models that adjusted for smoking status using the Maas 13-CpGs model rather than self-reported smoking status in ESTHER. The positive associations were highly consistent between both types of models 25. However, the single CpG is not among the CpGs identified in our study. The differences in identified CpGs within corresponding gene clusters between the two studies might be due to the different measurements for frailty. Bellizzi et al. 17 defined frailty status using cluster analysis and reported that global DNA methylation was lower in frail individuals than in the non-frail participants. This is in line with our study, which observed that 43 out of 53 CpGs were inversely associated with FI in an adjusted model. Another study by Collerton et al. 18 using the Fried frailty definition in a cohort with 85 years old participants found that the genome-wide methylation was not associated with frailty status. Recently, several DNAm-based algorithms, i.e. Hannum’s blood-specific clock19 and GrimAge23, have been derived to estimate ‘epigenetic age’ and were suggested to closely correlate with frailty30.To our knowledge, few studies have specifically assessed the relationship between DNAm patterns and frailtyGAPDH) contains two of the frailty-related CpGs (cg00252813 and cg02519286) and has been found to be likely related to the pathogeneses of amyotrophic lateral sclerosis and Huntington’s disease32, various forms of cancers33, and neurodegenerative disorders33. Three CpGs  are located at Solute Carrier Family 1 Member 5 (SLC1A5), a high-affinity l-glutamine transporter that was highly expressed in several cancer types36. The remaining CpGs and correspondent genes, such as SARS, SCRN1, PIK3CD, RUNX1, NCAPH, HDAC4, and VAC14, were also observed to be associated with multiple diseases. The roles of the identified CpGs in the development and/or progression of diseases may explain the association of eFRS with frailty.The identified frailty-related CpGs highlight several genes or genetic regions and might promote further investigation of the biological mechanism of frailty. Glycolytic glyceraldehyde-3-phosphate dehydrogenase . Moreover, cg02867102 and cg11700584 were significant signals in previous EWASs on smoking and aging45, which points to the major role of smoking in adverse health outcomes in old age including frailty.Several accurate composite algorithms of chronological age or lifespan have been built based on DNAm, including the Mortality Risk Score (MRscore) by Zhang et al.46. Third, in the discovery phase, only the CpGs included in both the 450K and EPIC array were included in the EWAS. Potential frailty-related CpGs exclusively covered by the EPIC array might have been missed.The current study has several strengths including the large-scale random samples from the general population, the long-term follow-up with repeated measurements of frailty at multiple points of time during follow-up, and replication in a completely independent cohort. However, it is also necessary to consider the limitations of the present study when interpreting the results. First, the deficits for the measurement of FI were self-reported which might have led to potential reporting bias. However, in the ESTHER cohort, self-reported diseases which account for a large share of the self-reported deficits were found to be in high agreement with medical records in careful validation steps carried out at each follow-up. Second, the recruitment of the participants was conducted during a voluntary health check-up. Therefore, the participants of the ESTHER cohort might not be a fully representative sample of the general population. Nevertheless, the prevalence of risk factors and chronic diseases has been found to be comparable to those observed in the corresponding age range in a representative health survey from Germany which took place at the same time as ESTHER baseline recruitment didIn conclusion, in this EWAS on frailty conducted in a large population-based cohort, we identified 65 frailty-related CpGs and derived an epigenetic algorithm of frailty based on 20 CpGs. The DNAm-based eFRS was demonstrated to be strongly associated with long-term frailty and validated both internally in an independent subset and in an external population-based cohort. Further studies should investigate the associations of eFRS with additional health outcomes and its potential use for earlier detection of frailty risk and designing, monitoring, and evaluating prevention measures.47. Briefly, men and women aged 50–75 undergoing a general health check-up in Saarland, a small federal state in southwestern Germany, from 2000 to 2002 were eligible for participation. At the time of recruitment, these general health exams were routinely offered every 2 years to people aged 35 years and older by their general practitioners (GPs). Overall, 9940 adults aged 50–75 years were recruited by their GPs and were followed by participant and GP questionnaires after 2, 5, 8, and 11 years During baseline enrollment and each follow-up, standardized questionnaires for participants and their GPs were used to collect extensive basic data on sociodemographic characteristics, risk factors, lifestyle factors, and medical history. Moreover, whole blood samples were collected at baseline from which DNA was extracted. The ESTHER cohort was found to be representative of the older German population with respect to major sociodemographic, lifestyle, and medical characteristics46.The epigenome-wide association study (EWAS), including derivation and internal validation, is based on the ESTHER study, an ongoing prospective, population-based cohort study of older adults conducted in the federal state of Saarland, Germany. Details of the study design and population have been reported previously48. Subset III has a nested case-control design for mortality-related methylation signatures and 548 participants were randomly selected as the subcohort irrespective of death status21. Subset I was used as a discovery panel in the epigenome-wide screening for CpG sites related to frailty at baseline. Subset II was utilized as the first internal validation panel to further select CpG sites to construct a methylation-based epigenetic frailty risk score (eFRS). The associations of eFRS with FI at baseline and during 11 years of follow-up were further validated in subset III.Three subsets were randomly selected in three different rounds of methylation analysis from the ESTHER cohort for epigenome-wide DNA methylation data measurements. Subsets I and II included 998 and 741 randomly selected subjects for whom DNAm measurements were performed in August 2018 and July 2019 for various projects49. In 2008 and 2009, 4123 participants aged ≥65 years from four population representative surveys conducted between 1984 and 2001 were enrolled and completed the baseline assessments. Then, an age- and sex-stratified random sub-sample (n = 1079) additionally completed medical examinations and was followed up in 2012 and 2016. Methylation data were available for 1010 participants from this random sub-samples . The baseline, 4- and 8-year follow-up data of these 1010 participants were used to validate the associations of eFRS with FI in an independent cohort.Replication in an independent cohort was performed in the KORA-Age study, a population-based cohort study conducted in the region of Augsburg, Southern Germany, whose study population and design have been described in detail previouslyThe ESTHER study was approved by the ethics committees of the medical faculty of Heidelberg University and of the medical board of the state of Saarland. The KORA-Age study was approved by the Ethics Committee of the Bavarian Medical Association (EK No. 08064). All ESTHER and KORA-Age participants provided written informed consent.48. Genome-wide DNAm profiling was conducted by the Genomics and Proteomics Core Facility of the German Cancer Research Center according to the manufacturer’s protocol. In data pre-processing, signals of probes with detection P-value > 0.01, missing values >10%, and probes targeting the X and Y chromosomes were excluded. Only the CpGs that are covered by both the 450K array and EPIC array were included in the EWAS. In the KORA-Age study, data quality control and pre-processing were conducted following the CPACOR pipeline50. Probes with detection P-value > 0.01 and missing values >5% were removed. Quantile normalization was then performed following a coherent approach as described by Lehne et al.50. In addition, leukocyte composition was estimated in ESTHER and KORA-Age using the algorithms of Houseman et al.51 for adjustment.DNAm profiles of subsets I and II from ESTHER, and subset IV from KORA-Age were assessed with the Infinium Methylation EPIC BeadChip kit , and DNAm profiles of subset III from ESTHER were determined with the earlier introduced Infinium Human Methylation450K BeadChip Assay . Details of the methylation analysis in the ESTHER study have been reported previously52, frailty in ESTHER and KORA-Age was assessed using a frailty index (FI), which is defined as the proportion of presented deficits of a predefined list of all deficits. As previously described, 31 and 33 deficits were selected for the assessment of FI in ESTHER48 and KORA-Age53, respectively. The lists of deficits used to define the FI in ESTHER and KORA-Age are presented in Supplementary Tables 54, participants were deemed frail if their FI was ≥0.250, pre-frail if their FI was >0.100 and <0.250, and non-frail if their FI was ≤0.100.Following a standard procedure55, CpGs that reached genome-wide significance [false discovery rate (FDR) < 0.05] were validated in subset II. Similar to the discovery phase, linear mixed regression models were conducted with baseline FI as the dependent variable and additionally adjusted for age and sex. Again, CpGs with FDRs <0.05 were selected and were deemed as frailty-related loci. Then, we applied LASSO regression with a regularization parameter chosen by ten-fold cross-validation following the ‘one standard error’ rule to select candidates among identified CpGs and construct eFRS.The DNAm-based eFRS was developed using a three-phase process. A flowchart of the three-phase procedure is presented in Fig. http://emea.support.illumina.com/array/array_kits/infinium-methylationepic-beadchip-kit/downloads.html#), which is based on the University of California Santa Cruz (UCSC) and RefGene. To analyze the underlying roles of these genes, we used the Metascape online tool (https://metascape.org)56 to perform Gene Ontology (GO) analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein-protein interaction (PPI) network. Kappa scores were used as the similarity metric when performing hierarchical clustering on the enriched terms and sub-trees with a similarity of >0.3 were considered a cluster. In order to link genetic variants to variations of CpGs included in eFRS, methylation quantitative trait loci (mQTL) analysis was applied for the preliminary association analysis of single nucleotide polymorphism (SNP) sites with CpG sites. SNP-DNAm site pairs with a maximum distance of 1 Mb were tested. The analysis of mQTL was performed using the online tool mQTLdb (http://www.mqtldb.org)57.Frailty-related CpGs were annotated to genes with the information provided in the Illumina manifest file . In further analyses, smoking status  and alcohol consumption (grams per day) were additionally included as fixed effects (model 2). By categorizing baseline and various follow-up frailty statuses into two groups , the associations were also estimated using a logistic regression model adjusting for all variables in model 2. In addition to logistic regression models including eFRS as a continuous variable, we also categorized eFRS according to quartiles and run logistic regression models including eFRS as a categorical variable. FDR was also applied for multiple comparisons among results at baseline and various follow-ups and P  <  0.05 was considered statistical significance after multiple testing.Linear mixed regression and LASSO regression were performed to identify frailty-related CpGs and the eFRS in subsets I and II as aforementioned. The correlations of chronological age, eFRS, and FI at baseline and various follow-ups were assessed using Spearman correlation coefficients in subset III and subset IV. We also evaluated the correlation of eFRS and DNAm-based algorithms of aging in subset III, the AccAgeGrim (GrimAge age acceleration)58 rather than self-reported smoking status. Next, we assessed the association of AccAgeGrim with FI at baseline and various follow-ups.To assess potential additional variance from passive smoking, we conducted sensitivity analyses with additional logistic regression models that controlled for smoking status using a DNAm-based proxy (the Maas 13-CpGs model)Furthermore, we conducted subgroup analyses for the associations of the eFRS with frailty in which only non-frail participants at baseline were included for the outcome being pre-frail or frail, and only non-frail or pre-frail participants at baseline were included for the outcome being frail using model 2 for adjustment as described above.We also systematically screened PubMed for previously reported CpGs associated with frailty and assessed the associations of these CpGs with frailty by linear-mixed regression models using model 2 for adjustment as described above.59. All the other statistical analyses in the ESTHER study were carried out in SAS 9.4  and the analyses in the KORA-age study were conducted in R (version 4.0.1).The LASSO regression analyses were conducted using R programming  package ‘glmnet (version 4.1-4)’Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


This study investigated the associations between health status before SARS-CoV-2 infection and persistent symptoms after acute infection. Data were collected from participants older than 18 years and more than 28 days after acute SARS-CoV-2 infection using an online survey. Sociodemographic data, comorbidities, and daily medication before infection, as well as acute and persistent symptoms were analysed. Among the 1050 participants , 538 (51%) reported being healthy and 762 (73%) reported not taking any daily medication prior to infection. Positive laboratory testing was reported by 965 (92%) participants; asymptomatic infection was reported by 30 (3%); and 999 (95%) stayed at home during their acute infection. Reduced physical capacity (40%), fatigue (39%), cognitive difficulties (30–34%), altered sense of smell (24%), headache (20%), tachycardia (20%), unstable mood (19%), hair loss (17%), and insomnia (17%) were the most often reported symptoms. Those taking daily medication before infection reported increased frequency of both acute and persistent symptoms, except for decreased frequency of persistent altered smell and taste. The presence of persistent symptoms was predicted by taking daily medication before infection and by the total number of acute symptoms. Comorbidities before infection did not predict persistent symptoms. Therefore, the role of medication needs further investigation in both acute SARS-CoV-2 infection and post-COVID-19 condition.  Since December of 2019, the COVID-19 pandemic has affected several hundred million people all over the world, resulting in millions of deaths and overloaded healthcare systems in many countries. The World Health Organization (WHO) has also alerted healthcare providers regarding post-COVID-19 condition and estimates that approximately 10% of all infected people may suffer from it [It is estimated that the incidence of persistent symptoms in non-hospitalized patients is between 22% and 38% ,5, whileThe predictors of post-COVID-19 condition have been studied in hospitalized patients, reporting that comorbidities might predict persistent symptoms in both inpatients and outpatients , while oThe aim of this study was therefore to describe the persistent symptoms after SARS-CoV-2 infection in mainly non-hospitalized persons and any association with comorbidities or medications prior to the infection in the Lithuanian population during the pandemic’s first waves in 2020–2021. The hypothesis was that comorbidities and daily medication prior to infection are associated with symptomatology during acute infection as well as during the period after infection.ClinicalTrials.gov ID: NCT05000229 (accessed on 11 August 2021). Materials concerning ethical permission, consent information given to participants, and the study protocol (slightly updated after the 1st survey) are available through a link to the questionnaire at the university’s website . Questions used in the survey were designed to capture main sociodemographic data; data related to acute SARS-CoV-2 infection, including laboratory findings, treatment during acute infection, comorbidities, and daily use of medication prior to infection; persistent symptoms after the infection; and attitudes related to the need for rehabilitation. The questions were exploratory, and no validated questionnaire was used in the survey. Informed consent was obtained from all participants. Participants were invited to participate in the study by anonymously answering an internet-based questionnaire created with Google Drive. The questionnaire was distributed in the Lithuanian language through Lithuanian websites, including private/public Facebook groups, city/town/district hospitals, and in media outlets . The onlThe following inclusion criteria were required for participating: (1) age 18 years or older, (2) known SARS-CoV-2 infection with or without specific diagnostic tests or laboratory findings , and (3) at least 28 days after acute infection when participating in the survey. The following exclusion criteria were applied: hospitalized patients still receiving treatment or rehabilitation after SARS-CoV-2 infection, unstable or untreated comorbidities, or ongoing stabilization of comorbidities. None of the participants indicated the presence of exclusion criteria in the questionnaire.Regarding comorbidities, the participants were asked to indicate the presence of any chronic disease prior to SARS-CoV-2 infection. Furthermore, the comorbidities were selected from preselected choices for 21 disorders along with an optional choice to write in other diseases. Two of the authors (DV and IBL) analysed and grouped the comorbidities because participants could indicate comorbidities either as preselected choices and/or describe them by name as an optional choice. Analysis of all answers resulted in 49 comorbidities, and all reported comorbidities were included in the statistical analysis.Regarding acute and persistent symptoms, there were 18 preselected acute symptoms and 62 preselected persistent symptoms as well as the possibility to leave additional symptoms or comments. Medication was asked about as the presence or absence of “daily” medication before SARS-CoV-2 infection. Medication for acute SARS-CoV-2 infection was also asked about to understand if there was a difference between the studied groups during the acute course. p < 0.05. Data were downloaded into Microsoft Excel and further analysed using SPSS version 27. Nominal data were compared using two-tailed Chi-square tests and are presented as numbers/percentages. Symptoms were also re-calculated as the total number of acute and persistent symptoms. Spearman correlation was used to investigate the associations between the persistent symptoms and the following parameters of interest: chronic disease at the time of acute infection, taking daily medication prior to acute infection, and acute symptoms during infection. Spearman correlation analysis was also performed between taking any daily medication and reporting any comorbidity prior to acute infection. The predictors of persistent symptoms (dependent variable) were analysed with a binary logistic univariate regression model regarding the following covariates: the presence/absence of chronic disease and medication prior to SARS-CoV-2 infection and the total number of acute symptoms. Gender and age were included as control variables. Statistical significance was considered as The final study cohort included 1050 participants. The mean age was 41 years , and 95% of the participants were younger than 60 years. Among the total cohort, 927 (88%) were women and 123 (12%) were men . A majorp = 0.023, Chi-square 2-tailed test). A weak but statistically significant correlation was found between taking daily medication prior to acute infection and having a chronic disease . Among the 1050 participants, 538 (51%) reported not having any chronic disease and 762 (73%) reported not taking any daily medications prior to their SARS-CoV-2 infection. Among those reporting comorbidities (n = 512), only 31% (n = 157) were taking daily medication prior to the SARS-CoV-2 infection, which was higher compared to healthy participants taking medication   revealed 49 comorbidities . The totRegarding acute infection, 21 (2%) participants reported becoming “officially” healthy from acute infection within 10 days, 625 (60%) within 2 weeks, 305 (29%) within 4 weeks, and 99 (9%) longer than 4 weeks. This means that a primary health care doctor terminated isolation and granted the participant´s return to work and/or social activities. Therefore, most participants (91%) officially recovered from their acute symptoms within 4 weeks. The numbers of participants who did not receive any treatment, those who consumed prescribed medication, and those who treated themselves (not being prescribed any specific medication) during the acute SARS-CoV-2 period were approximately the same at 277 (27%), 403 (38%), and 370 (35%), respectively. s = 0.02, p < 0.61, N = 1050).Regarding the development of symptoms during the acute infection, half of the participants (51%) reported variation in their symptoms, while the remaining participants reported that their symptoms continued to be the same (26%) or that they developed new symptoms (18%). The total number of acute symptoms ranged from 0 to 18 with a median of 6 symptoms. The frequency of acute symptoms is presented in s = 0.1, p < 0.001, N = 1050). A weak but statistically significant correlation was also found between acute and persistent symptoms .Persistent symptoms were reported by 812 (77%) participants. The total number of persisting symptoms ranged from 0 to 48 with a median of 4 symptoms. The predictors of persistent symptoms were analysed with a binary logistic regression model using the presence/absence of persistent symptoms as the dependent variable and the presence of daily medication prior to SARS-CoV-2 infection, the total number of comorbidities, and the total number of acute symptoms as the covariates. Gender and age were included as control variables. In the present study we identified 49 disorders prior to SARS-CoV-2 infection and 62 persistent symptoms after 4 weeks post-infection. However, not all participants with chronic disorders reported taking daily medication prior to the infection. This was further confirmed by a weak correlation between the presence of comorbidities and taking daily medication before SARS-CoV-2 infection. We report here that those participants who took daily medication prior to their SARS-CoV-2 infection more often reported both acute and persistent symptoms, which is a key finding of this study. Among the persistent symptoms; fatigue; pain; and symptoms related to the nervous system, including the autonomic nervous system; as well as gastrointestinal/metabolic disturbances were reported more often. The fact that not all 62 symptoms were more often reported by participants taking daily medication prior to the infection might explain a weak but significant correlation coefficient between these parameters. The study cohort involved persons with milder forms of SARS-CoV-2 infection who mainly stayed at home during the acute infection instead of going to the hospital and who mainly were well-educated working women. The majority of participants were working at the time of the survey, indicating that despite persistent symptoms there was a restoration of working capacity. This confirms that a relatively healthy population was involved in the study. Similar findings regarding age, gender, education, and employment were obtained in a Swedish study recruiting persons with persistent post-COVID-19 condition for an online rehabilitation programme . The lack of correlation between comorbidities and daily medication before infection might be explained by a relatively healthy population in the present cohort and broadly accepted non-pharmacological treatments in Lithuania, such as alternative medicine and physical rehabilitation medicine. On the other hand, some participants reporting to be healthy prior to SARS-CoV-2 infection indicated pharmacological treatment, which might include temporary over-the-counter medication for general or milder conditions such as pain, allergic reactions, etc. Participants in the present study were recruited by social media (Facebook), including three websites dedicated to medical staff. Hypothetically, this might explain the particular findings of a weak correlation between medication and comorbidities in this cohort. per se for remaining symptomatology, even if such medication is essential for the management of both severe acute and post-COVID-19 conditions. Careful follow-up of prescribed medication and restoration of impaired functions by rehabilitation instead of overflow medication might be important in preventing persistent symptoms and/or breakthrough infections after vaccination. In clinical practice, comorbidities are often considered as risk factors for further symptoms or new disorders. For SARS-CoV-2 infection, cardiovascular and endocrine comorbidities were reported to be associated with increased severity of infection in hospitalized patients . In thisOur results are in line with previous reports on post-infection symptoms after SARS-CoV-2 infection such as fatigue, cognitive symptoms ,19,20,21Age, BMI, and female gender have been shown to be predictors for post-COVID-19 condition using an online survey . In prevThe following limitations should be considered: 1) our online survey with limited spread in social media mostly recruited middle-aged women and did not include other populations, i.e., people who do not use the internet or do not pay attention to or participate in online surveys, which limits generalizability; (2) the anonymity of our survey did not allow contacting the participants in case of uncertainties regarding the inclusion criteria or missing data; (3) our first collection of data did not include a vaccination question (mass vaccination in Lithuania started in May 2021), which hypothetically could be an important confounder; (4) more data regarding comorbidities, the use of medication are required, for example, ongoing, prescribed, and non-prescribed medication as well as information regarding time frame for taking or not taking any medication; and (5) we used a retrospective design based on self-reported data. Moreover, the present study was initiated and performed prior to the publication of the WHO´s definition of post-COVID-19 condition and its report on persistent symptoms after SARS-CoV-2 infection [ our onliThe strengths of the present study are (1) the breadth of our questionnaire in regards to health status and medication before infection; (2) the inclusion of mostly middle-aged participants (40–49 years old), who statistically represent the age group most often infected with SARS-CoV-2 in Lithuania ; and (3)The results of the present study indicate that daily medication before SARS-CoV-2 infection might be important for both acute and persistent symptomatology. This should be further studied in both hospitalized and non-hospitalized populations as well as for different drug groups, including non-prescribed drugs. Our findings also point out the importance of non-pharmacological strategies in the management of both comorbidities and persistent symptoms, for example, by endorsing exercise, psychotherapy, and rehabilitation interventions.


The continuous variability of SARS-CoV-2 and the rapid waning of specific antibodies threatens the efficacy of COVID-19 vaccines. We aimed to evaluate antibody kinetics one year after SARS-CoV-2 vaccination with an mRNA vaccine in healthcare workers (HCW), with or without a booster. A marked decline in anti-Spike(S)/Receptor Binding Domain (RBD) antibody levels was registered during the first eight months post-vaccination, followed by a transitory increase after the booster. At three months post-booster an increased antibody level was maintained only in HCW vaccinated after a prior infection, who also developed a higher and long-lasting level of anti-S IgA antibodies. Still, IgG anti-nucleocapsid (NCP) fades five months post-SARS-CoV-2 infection. Despite the decline in antibodies one-year post-vaccination, 68.2% of HCW preserved the neutralization capacity against the ancestral variant, with a decrease of only 17.08% in the neutralizing capacity against the Omicron variant. Nevertheless, breakthrough infections were present in 6.65% of all participants, without any correlation with the previous level of anti-S/RBD IgG. Protection against the ancestral and Omicron variants is maintained at least three months after a booster in HCW, possibly reflecting a continuous antigenic stimulation in the professional setting. The newly emerged Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused an unprecedented global pandemic with more than 513 million confirmed cases and 6.2 million deaths by 1 May 2022. In Romania, as of 1 May 2022, there are more than 2.8 million confirmed cases and 65,000 deaths ), while it decreases slowly, but significantly, at 12 months after the second dose in subjects without a booster dose (median 88.92% [95% CI: 85.48–90.97]). Neutralizing capacity against the Omicron variant is significantly lower compared to the ancestral variant (77.07% [95% CI: 25–82.65]) in subjects without booster doses, but still significant in HCW with booster doses (82.75% [95% CI: 76.17–87.04%]). There is no difference in terms of neutralizing capacity for individuals with breakthrough infections compared with those who remained uninfected , slow decrease (60–89%), and moderate decrease (30–59%). None of the participants had a rapid decrease in neutralizing capacity (below 30%); instead, for the majority (68.2%), the neutralization capacity against the ancestral variant was preserved over time, 27.6% had a slow decrease, and only 4.13% had a moderate decrease in neutralizing capacity A.At the same time, the vast majority of participants (87.39%) experienced a rapid decrease in anti-S/RBD IgG levels, 10.03% experienced a moderate decrease, and only 2.58% had a slow waning pattern. Interestingly, participants who tend to maintain anti-S/RBD IgG overtime had lower initial levels of antibody 1 month after the initial vaccination regimen B.In this study we report the dynamics of the anti-SARS-CoV-2 antibody response during a one year period following vaccination with the BNT162b2 vaccine,  in HCW from Romania.We confirm previous reports  of a marAt 3 months post-booster dose we report a marked decline in the anti-S/RBD IgG levels in individuals without SARS-COV-2 infection prior to vaccination, compared to those previously infected. These results are in agreement with other studies that suggest a limited time effect of the booster dose on the antibody level . HoweverA plethora of studies demonstrate that both natural infection and vaccination induce strong, yet short lived, protection against reinfection. Some studies suggest that immunity after infection may offer even better protection against reinfections compared to vaccination with two doses of an mRNA vaccine, but data are not fully consistent . In a naA study by Levin et al. about waning immunity after vaccination concluded that antibody levels substantially decrease 6 months after vaccination with two doses, especially in elderly men and immunocompromised persons; unexpectedly, it is maintained at high levels in obese persons .Little is known about the risk factors that identify people prone to infections. Some reports indicate high rates of reinfection in the elderly, immunocompromised patients, those with severe comorbidities, or subjects at risk of exposure  ,26,27. IIt is important to mention that our study was conducted during a period of high prevalence of the Delta variant, and only shortly after the emergence of the Omicron variant. In the past few months, real-world studies illustrate that vaccine efficacy against Omicron symptomatic infections is close to non-existent/very low, while protection against hospitalization for severe disease is maintained, although at lower levels compared to that against the previously dominant Delta variant ,30,31. LThe continuous antigenic drift of the Omicron VOC, with periodic displacement of circulating sub-lineages by others that are more immune evasive and as such, more prone to spreading in a highly immunized population, will raise important public health problems, with periodic waves of infections and potential severe consequences in highly vulnerable populations. Recent studies suggest that in the presence of BA.2, BA.4, and BA.5 Omicron subvariants, protection against severe disease is decreased in recipients of two vaccine doses and in previously infected, but unvaccinated persons.Moreover, unvaccinated individuals who acquire an infection with one of the initial sub-lineages of the Omicron variant (BA.1) develop low levels of cross-neutralizing antibodies and may be susceptible to reinfections with another sub-variant  . AntigenThis study has a series of limitations. Study results may not be representative of the general population, as participants were mostly healthy, without age- or disease-related immunosuppression, and were more frequently exposed to natural infections in healthcare facilities. There is a certain age bias, as more than 47% of individuals enrolled in the study were aged 35–50 years, while the most vulnerable age group, persons >65 years, who might have a less robust immune response due to immunosenescence, are under-represented (2.4% of the study subjects). Yet, the enrolled population is representative of the age structure of the healthcare work force in Romania, and the dynamics of the immune response are relevant for further specific recommendations in the national vaccination strategy. The study stopped during the initial phase of the Omicron wave in Romania, in which most infections were registered in children and young adults; it is probable that many more reinfections with the highly transmissible and antigenically unrelated Omicron variant will also be recorded during the next few months in healthcare workers. It was not possible to monitor the dynamics of the humoral immune response at all points for all enrolled participants, as there were several missing samples for at least one of the collection points; nevertheless, data were available for a sufficient number of participants to ensure statistical significance as defined by the study methodology.In conclusion, the present study shows that one year after vaccination there is a consistent and constant decrease in the level of anti-S/RBD antibody, even in persons who receive a booster dose 8 months after completion of the initial regimen. Nevertheless, people with a prior SARS-CoV-2 infection develop significantly higher levels of anti-S IgG antibodies and long lasting IgA anti-S antibodies after a booster dose compared to naive subjects. A significant neutralizing capacity against the ancestral and Omicron variant is maintained at least 3 months after a booster dose, and the number of breakthrough infections in HCW is limited, possibly reflecting a continuous antigenic stimulation following frequent exposures in their professional setting.Establishment of an antibody level cut-off value as a correlate for protection remains elusive. There are no statistically significant differences between the anti-S/RBD IgG levels in subjects with and without breakthrough infections, suggesting that frequent boosting might not be the most appropriate action in immunocompetent persons. Differences in cellular or innate immunity, or even individual genetic differences, are probably more important to the evolution of an infection post-vaccination. Longitudinal studies of memory B cells’ response will inform future vaccination strategies, especially in the presence of a highly immune-evasive viral variant with a constant evolution via antigenic drift.


Management of acute respiratory distress syndrome (ARDS) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still a challenge for the staff on intensive-care units (ICU's) around the world. Many of these patients are treated with invasive ventilation. Sometimes, the occurrence of pneumothorax and/or pneumomediastinum can complicate the course of the disease because initiation of invasive ventilation might be fatal in those patients. Venovenous extracorporal membrane oxygenation (vv-ECMO) is increasingly used to prevent patients with severe ARDS from hypoxia. However, clear recommendations for or against the initiation of vv-ECMO in awake patients are currently lacking. We present the case of a 42-year-old patient with COVID-19-associated severe ARDS, pneumothorax, and pneumomediastinum. To preserve sufficient oxygenation and to avoid invasive ventilation, we implanted a vv-ECMO while the patient was awake. The patient recovered and was discharged home 41 days after transfer to our hospital. We therefore suggest that awake implantation of vv-ECMO might be useful in a subgroup of patients with severe ARDS caused by SARS-CoV-2. However, further evidence is needed to verify our hypothesis. The proportion of patients receiving invasive mechanical ventilation (IMV) ranges from 29.1% [IMV died . This miIf the occurrence of pneumomediastinum and/or pneumothorax contributes to high mortality is currently unclear, data are limited to case reports or case series, respectively: a literature review found 35 case reports of spontaneous pneumomediastinum in COVID-19 patients with a mortality rate of 28.5% (10 of 35) . HoweverTo prevent patients from hypoxia in severe acute respiratory distress syndrome (ARDS), venovenous extracorporeal membrane oxygenation (vv-ECMO) is increasingly used . A multiHereinafter, we describe the successful treatment of a COVID-19 patient with severe ARDS, spontaneous pneumomediastinum, and pneumothorax using awake implantation of vv-ECMO.2) was transferred from another hospital to our intensive care unit (ICU). He was first tested positive for COVID-19 eight days before he was admitted to our unit. Besides a fructose intolerance, his medical history was empty. At admission to the first hospital, he presented himself with dyspnea and intermittent episodes of fever (temperature maximum 38.2°C). The patient was treated with dexamethasone 6 mg i.v., and intermittent noninvasive ventilation (NIV) was started due to ARDS and dyspnea. During the stay, the patient developed a spontaneous right side pneumothorax and a massive pneumomediastinum. A thoracic drainage had been placed in the right hemithorax, and the patient was transferred to our unit seven days after admission. A chest CT after transfer indicated bilateral ground glass opacities with consolidations and pneumomediastinum  found that barotrauma might occur more frequently in patients with ARDS due to COVID-19 than in patients with ARDS from other origins [The mechanism of occurrence of pneumomediastinum or pneumothorax, respectively, in COVID-19 patients is unclear: on the one hand, barotrauma leading to rupture of alveoles due to positive pressure ventilation might be responsible for mediastinal emphysema: a systematic review including 13 studies n = 181 patientsOn the other hand, lung frailty due to COVID-19 itself may also be a contributing factor: a retrospective study including 323 ARDS-patients receiving IMV concluded that pneumomediastinum was sevenfold increased in ARDS due to COVID-19 compared to ARDS from other origins; interestingly, no difference in PEEP, plateau pressure, peak pressure, compliance, and tidal volume/ideal body weight ratio between COVID-19 patients with and without pneumomediastinum was observed . The autEvidence of application of vv-ECMO in awake patients is currently shaky . A retroBesides vv-ECMO, another technique for extracorporal gas exchange has emerged in the past several years: the pumpless interventional lung assist (iLA). While the vv-ECMO drains venous blood through a gas exchange membrane before returning it to the venous system using a pump, the iLA-system works with a gas exchange membrane being integrated within an extracorporal arteriovenous bypass using the arteriovenous pressure gradient . As noneAll in all, we hypothesize that the use of vv-ECMO to avoid IMV in patients with pneumothorax and/or pneumomediastinum might be considerable and applicable in a subgroup of patients  with severe ARDS due to COVID-19. Further evidence is needed to confirm or discard our hypothesis.


There is a need for short and safe all-oral treatment of rifampicin-resistant tuberculosis. We compared outcomes up to 24 months after treatment initiation for patients with rifampicin-resistant tuberculosis in South Africa treated with a short, all-oral bedaquiline-containing regimen (bedaquiline group), or a short, injectable-containing regimen (injectable group).Patients with rifampicin-resistant tuberculosis, aged 18 years or older, eligible for a short regimen starting treatment between Jan 1 and Dec 31, 2017, with a bedaquiline-containing or WHO recommended injectable-containing treatment regimen of 9–12 months, registered in the drug-resistant tuberculosis database (EDRWeb), and with known age, sex, HIV status, and national identification number were eligible for study inclusion; patients receiving linezolid, carbapenems, terizidone or cycloserine, delamanid, or para-aminosalicylic acid were excluded. Bedaquiline was given at a dose of 400 mg once daily for two weeks followed by 200 mg three times a week for 22 weeks. To compare regimens, patients were exactly matched on HIV and ART status, previous tuberculosis treatment history, and baseline acid-fast bacilli smear and culture result, while propensity score matched on age, sex, province of treatment, and isoniazid-susceptibility status. We did binomial linear regression to estimate adjusted risk differences (aRD) and 95% CIs for 24-month outcomes, which included: treatment success  versus all other outcomes, survival versus death, disease free survival versus survival with treatment failure or recurrence, and loss to follow-up versus all other outcomes.vs 57%); loss to follow-up was 4% (1–8) lower in the bedaquiline group (6% vs 12%); and disease-free survival was 2% (0–5) higher in the bedaquiline group (99% vs 97%). The bedaquiline group had 8% (4–11) lower risk of mortality during treatment (17·0% vs 22·4%), but there was no difference in mortality post-treatment.Overall, 1387 (14%) of 10152 patients with rifampicin-resistant tuberculosis treated during 2017 met inclusion criteria; 688 in the bedaquiline group and 699 in the injectable group. Four patients (1%) had treatment failure or recurrence, 44 (6%) were lost to follow-up, and 162 (24%) died in the bedaquiline group, compared with 17 (2%), 87 (12%), and 199 (28%), respectively, in the injectable group. In adjusted analyses, treatment success was 14% (95% CI 8–20) higher in the bedaquiline group than in the injectable group (70% Patients in the bedaquiline group experienced significantly higher rates of treatment success at 24 months. This finding supports the use of short bedaquiline-containing regimens in eligible patients.WHO Global TB Programme.For the French translation of the abstract see Supplementary Materials section. Mycobacterium tuberculosis strains resistant to rifampicin—the most effective tuberculosis drug—reduce the probability of treatment success. South Africa, like many other African countries, has a high burden of HIV, tuberculosis, and rifampicin-resistant tuberculosis.Tuberculosis is a public health emergency, resulting in around 1·5 million deaths annually.Evidence before this studyIn 2017, 57% of patients worldwide treated for rifampicin-resistant tuberculosis had a successful outcome (cure or complete treatment). Early discontinuation of therapy based on patient's decision (loss to follow-up) was a common reason for poor outcomes; loss to follow-up is associated with 18–24-month treatment duration and medication toxicity. In 2013, WHO first recommended use of a standard shorter regimen based on observational studies in Bangladesh. In 2017, this regimen was introduced in South Africa. However, high levels of ototoxicity were noted shortly after introduction. Based on South Africa's successful experience replacing these injectables with the new oral drug bedaquiline in longer rifampicin-resistant tuberculosis regimens, South Africa began also replacing the injectables used in short regimens with bedaquiline under programmatic conditions, and slowly expanded its implementation. We searched PubMed on Nov 15, 2021, using the terms ((((tuberculosis) AND ((rifampin-resistant) OR (rifampicin-resistant))) AND ) AND (bedaquiline)) AND (short) without language restrictions. We identified only one study evaluating a bedaquiline-containing all-oral short regimen for rifampicin-resistant tuberculosis, although the regimen differed from the one we evaluated. Despite being a non-comparative study and including only three health facilities in South Africa, the study found a high treatment success rate with the bedaquiline-containing regimen.Added value of this studyIn this study used to inform WHO rifampicin-resistant tuberculosis treatment guidelines, we report on the outcomes from the introduction of the bedaquiline-containing short regimen in South Africa. We showed that all-oral bedaquiline-containing short regimens were associated with an absolute improvement in rifampicin-resistant tuberculosis treatment success of 14% when compared with injectable-containing short regimens. Patients receiving the bedaquiline-containing regimen had risks of mortality during treatment that were 8% lower than patients receiving injectable-containing regimen. Disease recurrence after treatment success was rare and post-treatment mortality was similar between groups. These results were robust in several subgroup analyses.Implications of all the available evidenceResults from our large programmatic cohort in a high HIV-burden country provide evidence that bedaquiline used in a shorter rifampicin-resistant tuberculosis treatment regimen is associated with higher rates of treatment success 24 months after treatment initiation and associated with lower risk of mortality during treatment than an injectable-containing regimen. These results led to the 2020 WHO recommendation for the use of bedaquiline in shorter rifampicin-resistant tuberculosis regimens under programmatic conditions. Despite a short duration of treatment, disease recurrence, as measured by re-initiation of rifampicin-resistant tuberculosis treatment, was rare. Although injectable-containing short regimens had substantially lower loss to follow-up than longer regimens in previous studies, we found bedaquiline-containing short regimens might be associated with even further reductions. Clinicians and policy makers in countries that are still using injectable-based regimens might use our analysis to re-evaluate their practices.Mycobacterium tuberculosis strains that are resistant to rifampicin, an injectable, and a fluoroquinolone.South Africa implemented the bedaquiline clinical access programme (BCAP)A shorter, injectable-containing, WHO-recommended rifampicin-resistant tuberculosis regimen was introduced in South Africa in 2017.The objective of this study was to compare 24-month outcomes between patients initiated on an injectable-containing or bedaquiline-containing short regimen for rifampicin-resistant tuberculosis in South Africa in 2017.plus and GenoType MTBDRsl . Stable patients in fair to good clinical condition initiate rifampicin-resistant tuberculosis treatment as outpatients, whereas ill or unstable patients in poorer condition are hospitalised and initiate rifampicin-resistant tuberculosis treatment as inpatients.Management of rifampicin-resistant tuberculosis in South Africa is decentralised and handled at the subdistrict level across 658 facilities. All individuals with presumptive tuberculosis are tested for tuberculosis and rifampicin-resistance using the Xpert MTB/RIF assay . South Africa implemented the Xpert MTB/RIF Ultra in October 2017 in a phased approach. Guidelines state all rifampicin-resistant patients should be tested for isoniazid, rifampicin, fluoroquinolone, and second-line injectable drug resistance using GenoType MTBDRRecords of all patients initiating treatment for rifampicin-resistant tuberculosis with or without isoniazid resistance from Jan 1 to Dec 31, 2017 using a short treatment regimen with an intended duration of less than 12 months registered in the national drug-resistant tuberculosis database EDRWeb were eligible for inclusion. In South Africa, to be eligible for either short regimen, patients had to be newly diagnosed with rifampicin-resistant tuberculosis, without history of previous treatment with second-line tuberculosis drugs, and without confirmed or suspected resistance to fluoroquinolones, second-line injectables, or both. Additional inclusion criteria for the analysis were: treatment duration of 13 months or less and receipt of the injectable-containing or bedaquiline-containing regimen. Exclusion criteria were use of cycloserine or terizidone, para-aminosalicylic acid, delamanid, carbapenems, or linezolid, and patients with missing information on the regimen they received. Additionally, we excluded patients who were aged younger than 18 years old, or had unknown HIV status, sex, or age, or their national ID number was not recorded in EDRWeb. Patients who initially received an injectable and later bedaquiline (or vice-versa) were also excluded.This analysis was approved by Human Research Ethics Committee, Medical of University of Witwatersrand . The requirement to obtain informed consent for individual patients was waived since this was a retrospective record review. No patients were contacted directly, nor underwent any study specific procedures.The short, WHO-recommended, injectable-containing regimen consisted of 9–12 months of moxifloxacin  or levofloxacin , clofazimine , ethambutol , and pyrazinamide , supplemented with high-dose isoniazid , ethionamide or prothionamide , and an injectable , delivered intramuscularly, for the first 4–6 months.End-of-treatment outcomes were obtained from EDRWeb. End-of-treatment outcomes were recorded by the treating clinician following South Africa guidelines. To obtain information on death up to 24 months after initiation of treatment, the national ID numbers recorded in EDRWeb were matched with national death registry data.Tuberculosis smear microscopy and culture was conducted routinely on sputum samples collected from all patients at treatment start, and monthly while on treatment thereafter. Laboratory tests, including electrolytes, renal function, liver function, thyroid function test, and full blood count, were done on the same schedule. High-frequency-enabled audiometers were used to monitor auditory function in patients receiving injectable rifampicin-resistant tuberculosis drugs. As per WHO recommendationsEnd-of treatment outcomes included cure, complete treatment, treatment failure, loss to follow-up during treatment, and death during treatment.Recurrence of tuberculosis among people whose end-of-treatment outcome was recorded as complete treatment or cure was ascertained by probabilistic matching using name, date of birth, and national ID number in EDRWeb to determine if the same person was reinitiated on rifampicin-resistant tuberculosis therapy within 24 months of initiating treatment.In this study, we defined treatment success as an end-of-treatment outcome of cure or complete treatment without evidence of death or recurrence up to 24 months after treatment initiation; failure or recurrence was defined as survival at least 24 months after treatment initiation and an end-of-treatment outcome of failure  or an end-of-treatment outcome of success with evidence of recurrence during follow-up after treatment completion; loss to follow-up was defined as an end of treatment outcome of loss to follow-up  without evidence of death; and death was defined as death of any cause up to 24 months after treatment initiation.We retrospectively extracted the data for the patient cohort from EDRWeb on June 30, 2019, and collected 24-month outcomes for all included patients on July 31, 2020.The primary analysis compared 24-month outcomes among patients receiving the injectable-containing regimen (injectable group) with those receiving the bedaquiline-containing regimen (bedaquiline group). We considered multiple outcome comparisons between the regimens: success versus all other outcomes; survival versus death; disease-free survival versus survival with treatment failure or recurrence (excluding patients who died); and loss to follow-up versus all other outcomes.To compare outcomes between treatment groups, we did binomial linear regression. We used a combination of exact and propensity-score based matching to generate similar patient populations and minimise confounding. To assess which variables to exactly match and which variables to propensity-score match for multivariable analysis, we conducted univariable analysis on a-priori selected covariates for the comparison of success versus all other outcomes. This comparison was chosen because it encompasses the most important comparison used in country-level and WHO-level measures of effectiveness of rifampicin-resistant tuberculosis treatment. These variables included age, sex, HIV , previous treatment with first-line drugs, isoniazid resistance, acid fast bacilli (AFB) smear positivity 4 weeks before or 2 weeks after treatment start, and culture positivity 4 weeks before or 2 weeks after treatment start. Using this analysis, we exactly matched patients on HIV , AFB smear , culture , and previous treatment with first-line drugs (yes or no), whereas propensity-score matched using a caliper distance of 0·5 SDIn secondary analysis, we first constructed curves for the cumulative incidence of death from treatment initiation to 24 months between the injectable and bedaquiline group on the matched population from our primary analysis of survival versus death. Next, using the entire population, we performed an analysis of factors associated with death at any time, death during treatment, and death post-treatment. In this analysis, we did not use matching as we were interested in the potential role of different risk factors. We did multivariable binomial linear regression on the total population  and on the population stratified by received treatment; we included the same variables in the multivariable model as we did in the matched analysis. As we noticed the strongest effect on mortality was early during treatment, we conducted a post-hoc sensitivity analysis evaluating factors for early death (death within 3 months of treatment initiation) and late death (death at least 3 months after treatment initiation). Finally, we estimated median time to death from treatment initiation in the bedaquiline and injectable groups and compared them using a Kruskal-Wallis test.All data analysis was performed in R (version 4.0.3), with matching done using the package MatchIt (version 4.1.0).The sponsor of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.At the time of data extraction, 10 152 patients were initiated on rifampicin-resistant tuberculosis treatment in South Africa during 2017. Among these patients, 1516 (15%) had resistance to a fluoroquinolone, second-line injectable, or both, and 588 (6%) had received previous treatment with second-line drugs, making them ineligible for short regimens. Of the remaining 8048 patients, 5279 (66%) were excluded, most commonly for receiving a longer  regimen . 3246 patients had received a short regimen, but 477 were excluded, mainly because the treatment end date was more than 13 months after treatment starting date (n=354). Of the 2769 patients eligible for inclusion, 1382 patients were excluded, most commonly due to receiving both bedaquiline and an injectable drug (n=665). Overall, 1387 patients were included in this analysis, of whom 688 were in the bedaquiline group and 699 were in the injectable group . The disPatients in the bedaquiline group were older , with a The characteristics of patients eligible for inclusion in the study who were ultimately included versus those excluded are shown in Among patients included in the analysis, 661 (96%) of 688 patients in the bedaquiline group and 663 (96%) of 699 patients in the injectable group had a valid culture, smear, or Xpert result in the baseline period. Of patients with a valid result, 602 (91%) of 661 patients in the bedaquiline group and 605 (91%) of 663 patients in the injectable group had at least one of these tests positive.At the end of rifampicin-resistant tuberculosis treatment, 507 (74%) of 688 patients in the bedaquiline group and 421 (60%) of 699 in the injectable group were considered to have treatment success. After successful treatment and up to 24 months from treatment initiation, one (<1%) of 507 patients experienced recurrence in the bedaquiline group compared with four (1%) of 421 patients in the injectable group. Four patients (1%) had treatment failure or recurrence, 44 (6%) were lost to follow-up, and 162 (24%) died in the bedaquiline group, compared with 17 (2%), 87 (12%), and 199 (28%), respectively, in the injectable group.Univariable analysis of different covariates showed that HIV, previous treatment, baseline AFB smear positivity, and baseline culture were most strongly associated with 24-month outcomes  and thusIn multivariable analysis, the matching procedure reduced the standardised mean difference between groups for all variables , retainiA total of 361 deaths occurred within 24 months of treatment initiation. Among the 162 (24%) of 688 patients in the bedaquiline group who died within 24 months of treatment initiation, median time to death was 4·9 months (IQR 1·2–11·0); and among the 199 (28%) of 699 patients who died in the injectable group, the median time to death was 2·5 months . Among the matched population (485 patients in each group), the cumulative proportion of death was significantly higher in the injectable group . In the Sex and isoniazid resistance were not associated with risk of mortality. HIV and ART status, previous tuberculosis treatment, and increasing age were significantly associated with mortality during treatment. The characteristics associated with post-treatment mortality were baseline culture, increasing age, and history of previous tuberculosis treatment .When stratifying the analysis according to treatment received , we founIn sensitivity analysis evaluating death within the first three months of treatment versus after three months, 171 (47%) of 361 deaths (63 in the bedaquiline group and 108 in the injectable group) happened within the first three months of treatment initiation, and 190 (53%) deaths (99 in the bedaquiline group and 91 in the injectable group) happened after three months of treatment. Findings from regression analyses were qualitatively similar to those of on-treatment mortality versus post-treatment mortality .We measured and compared treatment outcomes for patients with rifampicin-resistant tuberculosis treated with a short, bedaquiline-containing regimen and patients treated with a short, injectable-containing regimen. We found that the bedaquiline-containing regimen was associated with higher probability of treatment success 24 months after treatment initiation, lower risk of loss to follow-up, and lower risk of mortality during treatment. Importantly, PLHIV receiving ART did not appear to be at increased risk of mortality compared with HIV negative patients in the bedaquiline group.Poor outcomes in the context of rifampicin-resistant tuberculosis treatment are driven by high death rates and high losses to follow-up.The reduction in mortality associated with bedaquiline has previously been demonstrated in longer rifampicin-resistant tuberculosis treatment regimens.The findings of increased risk of unfavourable rifampicin-resistant tuberculosis treatment outcomes among PLHIV who were not on ART further supports the need to establish HIV infection status and initiate ART. Furthermore, our findings of similar risk of mortality among HIV-negative people and PLHIV on ART in the bedaquiline group support the use of bedaquiline among PLHIV on ART and reinforce the importance of timely initiation of ART regimens among PLHIV.The bedaquiline-containing regimen presented in this study has a significant pill burden; however, its shorter length is likely to make it preferable than a regimen with a smaller daily pill burden lasting 18–20 months.The South African National HIV/TB strategic plan provides a target of 75% treatment success rate for all rifampicin-resistant tuberculosis or multidrug-resistant tuberculosis by 2022.Our study has several limitations. It is based on observational data that cannot determine physician's reasoning to start someone on a particular regimen and might not capture all important patient characteristics, which could cause residual confounding. Different provinces had differential roll out of bedaquiline. We attempted to account for differences between patients treated across provinces by including it as a variable in propensity score matching. The assessment of outcomes post-treatment required a valid national ID to be registered, which might lead to selection bias. Although this analysis included many patients, they were all from South Africa and a majority had HIV-coinfection. Further research is required to ensure our findings are generalisable, although stratified analyses did not identify important differences in outcomes among PLHIV and people without HIV. We did not assess safety of the regimens in this study, however previous analyses have shown bedaquiline is safervs 1% injectables), similar to those observed in a multinational study of the injectable-containing regimen,vs 7·4% injectables). The availability of individual-level data allowed adjustment for potential confounders and helped reduce biases that could emerge from identifiable patient and clinical characteristics. These findings add to the literature regarding effectiveness of shorter rifampicin-resistant tuberculosis treatment regimens.A major strength of our study is the 24-month follow-up of patients initiating short rifampicin-resistant tuberculosis regimens in routine conditions. The results reported in this study led to new WHO recommendations for the use of bedaquiline in shorter rifampicin-resistant tuberculosis treatment regimens.The use of bedaquiline instead of an injectable in a shorter rifampicin-resistant tuberculosis treatment regimen is associated with significant improvement in treatment success 24 months after treatment initiation, and significantly lower mortality and loss to follow-up during treatment. Bedaquiline could replace injectables in short regimens for all patients who are eligible, while efforts continue to further improve the effectiveness, safety, and completion of rifampicin-resistant tuberculosis regimens.Norbert.Ndjeka@health.gov.za or norbertndjeka@gmail.com).The data used for this analysis in the form of deidentified participant data and a data dictionary will be made available after publication. Investigators wishing to access these data will need to have an approved research proposal and complete a data access agreement. All inquiries should be sent to the corresponding author (We declare no competing interests.