Reports of attrition rates in the treatment of multiple myeloma vary widely, indicating that despite all innovations in multiple myeloma treatment, many patients do not reach their full treatment potential. In this retrospective study, the attrition rate in the Austrian Myeloma Registry (AMR) was analysed. A total of 571 patients diagnosed between January 2009 and August 2021 were included. The result of attrition in the AMR is very encouraging compared to previous data, with 22% +/− 5% per line of treatment (LoT). Attrition is higher in the elderly and lower in patients with optimal frontline treatment, including stem cell transplantation and maintenance. The importance of achieving an optimal response is highlighted, not only in terms of attrition, but also in terms of the achievable treatment-free intervals. These promising results support the putative key role of liberal universal drug access and reimbursement.Multiple myeloma (MM) is characterized by serial relapses, necessitating the application of sequential lines of therapy (LoT). Reports on attrition rates (ARs) vary widely. The present study analysed ARs from the Austrian Myeloma Registry. Attrition was defined as being either deceased, progressive without having received another LoT, or lack of follow-up for ≥5 years. A total of 571 patients diagnosed between January 2009 and August 2021 were included . Some 507 patients received at least one LoT. Of the total, 43.6% underwent autologous stem cell transplantation ) with primarily VRd  given as induction (26.5%), followed by lenalidomide maintenance in 55.7% of cases. Transplant-ineligible (NTE) patients were predominantly treated with Vd , receiving maintenance in 27.1%. A total of 37.5% received a second LoT. ARs across one to five LoTs were 16.7–27%. Frontline induction/ SCT followed by maintenance reduced ARs associated with age and achievement of deep remission in the frontline. Deep remission prolongs follow-up and time-to-next-treatment (TTNT), while high-risk-cyctogenetics negatively affected these outcomes. Our results demonstrate considerably lower ARs for MM patients within the AMR data versus other healthcare systems. Young age and the achievement of significant remissions after optimal frontline therapy resulted in particularly low ARs. These promising results support a key role for the ease of drug access and reimbursement policies in governing long-term MM patient outcomes. Multiple myeloma (MM) is a chronically relapsing hematological malignancy of plasma cells associated with numerous clinical complications such as anemia, renal dysfunction, and the development of bone lesions. Treatment options have improved considerably in recent years with the increasing use of novel agents and anti-myeloma antibodies ,2. NeverIn 2016, Yong et al. described an increase in clinical complications and symptom burden with each LoT, showing that only a minority of patients received more than five LoTs . A recenThe increasing improvement of treatment options and possibilities in advanced LoTs underlines the need to encourage patients to stay on therapy and fully exploit available treatment options and sequencing strategies. Patient comorbidities, age, as well as the presence of high-risk cytogenetic profiles and advanced MM stages , have been proposed to play a particular role for high ARs . In contTo identify the causes of high ARs in order to challenge the impediments to successful treatment outcomes in the real-world-setting is of the highest interest. The present study aims to assess ARs in the Austrian health care system using the AMR database.Patients documented via the AMR were included in this retrospective analysis if they received any myeloma treatment between January 2009 and August 2021. Patients with plasma cell leukaemia or smouldering myeloma are assessed in the AMR, while patients with AL amyloidoses are not included. Exclusion factors included lack of sufficient clinical data such as age, sex, and initial date of diagnosis. The starting point (JAN 2009) was chosen to exclude patients treated by outdated chemotherapy protocols alone, while the endpoint of the recruitment period was chosen to guarantee a minimal follow up. Due to decreasing patient numbers in advanced LoT cohorts, LoTs are recorded up to the fifth LoT. Subsequent LoTs are noted as >5 LoT without further discrimination. The analyses distinguished between patients eligible for autologous stem cell transplantation (TE) and patients not eligible for autologous transplantation (NTE). Patients who received a transplant were documented as TE. Therapeutics were stratified into proteasome inhibitors, immunomodulators, antibodies, and chemotherapy. The type of therapy is divided into doublet, triplet and quadruplet regimens. Duration of treatment (DoT) was defined as the duration between day 1 and the last day of respective LoT. Induction therapy, stem cell transplantation and maintenance therapy were considered as one LoT according to the International Myeloma Working Group (IMWG) recommendations. Time to next treatment (TTNT) was the period between the end date of the previous LoT and the start date of the subsequent LoT. Follow-up is reported and analysed as a relative indicator of the duration of the disease. The application of maintenance until toxicity/progression with lenalidomide was documented. Patient characteristics such as the Revised International Staging System (R-ISS), comorbidities according to the Charlson Comorbidity Index, and cytogenetic risk features were documented. For cardiovascular disease, myocardial/heart failure and peripheral/cerebrovascular disease were grouped together. Comorbidity graduation is documented for renal failure, liver disease and diabetes. High-risk cytogenetic features included hypodiploid karyotypes, translocation t, translocation t, deletion del(17p) and amplification 1q of copy numbers. Initial performance status in line with WHO criteria was assessed at the beginning and end of treatments, while ECOG scores were assessed at the first diagnosis. For each LoT, remission status, best response and the attrition rate were recorded. Deceased patients and patients “lost to follow-up” were included in the attrition cohort. Patients without any documentation ≥ 5 years in the registry were identified as “lost to follow-up”. Patients with progressive disease without any additional LoT were likewise added to the AR cohort. Categorical data was given with numbers (n and percentages (%) and continuous data with means and standard deviations (SDs), as well as median and interquartile range (IQRs). Statistical testing was performed using cross-tabulations and a chi-square test to assess associations between categorical variables while an analysis of variance (ANOVA) was performed for continuous data. Time-to-event data was presented using the Kaplan Meier approach together with log-rank testing. p < 0.05 considered statistically significant.Finally, logistic regression analyses were used for multivariate analysis to further investigate the influence of univariate significant variables and clinically important confounders on ARs and response. Odds ratios (OR) and 95% confidence intervals (95%CI) were reported for the main outcomes, with 571 patients could be identified from the AMR database in line with the defined inclusion and exclusion criteria. For a consort diagram see The median (IQRs) age (years) of patients was 72 (63–80) years, with a median follow-up time (months) of 50.8 (24–96.8) months. A total of 57.1% of the patients were male (n = 326) and 42.9% were female (n = 245). Patient characteristics  are depicted in p < 0.001). Autologous SCT was performed in 77.5% of patients ≤ 65 years. Induction therapy followed by autologous stem cell transplantation (SCT) was performed in 43.2% of patients . The median age was 64 (56–70) years. Patients undergoing autologous SCT were significantly younger than patients not eligible for autologous SCT (78 (71–82) years; The predominant induction regimens in the autologous SCT cohort were VRd  and VTd . Triplet regimens were commonly used . Maintenance therapy with Lenalidomide was prescribed to most patients . p < 0.001). Two LoTs were performed in 38.4% (n = 84) of patients, while 16.0% fulfilled the attrition criteria (n = 35)After one LoT, 68.9% of patients (n = 151) demonstrated a very good partial response (VGPR) or better response, while only 5.5% were primary progressive . Autologous SCT improved the chance of achieving VGPR or better by 3.7-fold . No difference was observed in the proportion of cytogenetic high risk patients or patients with R-ISS-III status between the TE and NTE cohorts  was 78 (71–82) years. Bortezomib/Dexamethasone  and VRd  represented the predominant induction regimens among these patients. Most patients received triplet regimens . Continuous therapy was used in 27.1% of patients (n = 78). VGPR or better was observed in 31.9% of patients (n = 92); PR, minimal response (MR) or stable disease (SD) in 29.5% (n = 85) of patients; and PD in 12.8% (n = 37) of patients. No remission status was documented in 74 patients. (25.7%). VGPR or better was achieved more frequently in patients receiving triplet or a clinical trial regimen ( cohorts . Some 37p = 0.002). Patients receiving maintenance therapy had a longer DoT of 15.9 (10.1–32.3) months vs. 5.3 (2.9–7.5) months. No relevant differences were observed in patient characteristics such as high-risk cytogenetics, treatment patterns or response.The median time of treatment (DoT) was 6.6 (3.8–6.7) months, with a significant difference between TE and NTE patients (7.9 (5.3–17) months vs. 5.5 (2.8–11.9) months; p = 0.009). Moreover, a significant difference was also observed in patients achieving a VGPR or better versus those who did not (10.7 (3.7–27.5) months vs 1.4 (0.4–4.7) months; p < 0.001). Maintenance therapy had no relevant effect on TTNT. The median time to next treatment/treatment-free period (TTNT) was 3.8 (1.1–20.5) months with a significant difference in TE vs. NTE patients (5.3 (2.8–23.5) months vs. 2 (0.5–11.5) months; p = 0.016, 6.8 months, 1.5–23.6 months), independent of containing Anthracyclines , Cyclophosphamide  or Melphalan  (p = 0.271). The effect of chemotherapy loses significance without TE patient contrast; only 2% of patients (n = 7) completed the treatment with a quadruplet regimen, so any analysis of this new approach must wait. In patients with high-risk cytogenetics, TTNT was shorter than in patients without this feature . No difference in outcome was observed in patients with a high comorbidity index, age > 70 years and R-ISS III status. Chemotherapy but no other types of treatment significantly prolonged TTNT (p = 0.006).Patients treated with recently approved therapeutics naturally have a shorter the median follow-up as, for example, antibody treatment with 21.8 (15–27.5) months (p < 0.001) while “older treatments” such as chemotherapy had the opposite effect . This can be explained by the time in which these treatments were mainly used. The use of triplet treatments positively influenced follow-up . Patients with high cytogenetic risk tend to have significantly shorter follow-up , as did patients with a high comorbidity index ≥ 4 points  and an ECOG score > 2 points . An R-ISS status of llI also provided negative effects on follow up .Median follow-up was likewise positively influenced by the application of an SCT , suggesting that fewer fragile patients are able to take up further LoTs.A total of 191 patients received a second LoT; 60.7% (n = 116) were male with a median age of 72 (63–79) years. An ECOG level of >2 points at baseline was recorded in 18.8% (n = 36) of patients. Only two patients had a comorbidity status ≥ 4 points (1.1%). In 24.6% (n = 47) a high risk cytogenetic profile was documented, while an R-ISS status of III was found in 16.8% of patients (n = 32). A second LoT was applied in significantly fewer patients with a low-performance status > 2 points and an ECOG score > 2 points at baseline  than NTE patients (74 (65–81) years. Most patients received triplet regimens as induction therapy , VRd  and Rd  in particular. Continuous treatment was prescribed in 75% of patients (n = 24) more commonly than in NTE patients . More patients with R-ISS III status were encountered in the SCT Cohort than in NTE patients . In this cohort, VGPR or better could be achieved in 56.3% of patients (n = 18), with three patients being progressive on treatment (9.4%). The attrition criteria were met by 18.8% of patients (n = 6), with four patients deceased and two patients lost to follow up. TE patients (16.8% n = 32) were obviously significantly younger (63 (59–71.3) years, p = 0.015). NTE patients (n = 159) were commonly treated with doublet regimens  including Rd  and Vd . Some 32.7% of patients received continuous treatment (n = 52). VGPR or better was reported in 27% of patients (n = 43) within this group, and PD in 19.5% of patients (n = 31). The response again differed significantly between TE and NTE patients (p < 0.001). The AR in the second LoT was 19.5% (n = 31), with 9.4% (n = 15) being lost to follow-up and 9.4% (n = 15) having died. Continuous treatment significantly prolonged DoT, with 22.6 (12.5–44.1) months compared to 7 (2.4–13.1) months (p < 0.001). No significant statistical difference in TTNT was observed with respect to continuous treatment and high risk cytogenetics. TTNT was improved by the achievement of VGPR or better to 4.4 (1.4–16.1) months vs. 0.7 (0.2–2.5) months; p = 0.002). Further high-risk cytogenetics (47 (22.9–75.8) months vs. 74 (43.3–112.8) months for absence of such features; p < 0.001) and R-ISS status of III negatively affected time on follow-up 48.2 (16.1–80.5) months vs. 73.8 (37.8–112.9) months; p = 0.008.Median time on follow-up was higher when continuous treatment was applied, at 82.2 (45.7–121.8) months compared to 65.9 (27.7–93.8) months with fixed duration therapy. A response of VGPR or better was also associated with a prolonged follow-up of 92.1 (54.6–113.3) months. vs. 52.8 (27.5–96-5) months for inferior responses;  were male. Their median age was 72 (64–79.5) years. Only two patients had a comorbidity score of ≥4 points (2.4%), with 23.5% of patients indicating an ECOG score >2 points at baseline (n = 20). 31.8% (n = 27) harboured high risk cytogenetics. An R-ISS of III was recorded in 18.8% (n = 16). The predominant treatment regimens were triplets , immunomodulators  such as Pomalidomide/Dexamethasone , and antibody- containing combinations ; late and/or second autologous SCTs were performed in 11.8% of patients (n = 10). Continuous therapies were applied in 14.1% (n = 12). of patients. A subsequent LoT was administered in 37 patients (43.5%) with an AR of 23.5% (n = 20); 10 patients died, and 10 patients were lost to follow-up.37 patients received a fourth LoT with 75.7% of patients (28n.) being male and having a median age of 72 (62–80) years. In the 4th LoT, 16.2% of patients presented an ECOG score > 2 at baseline (n = 6). No patients with a comorbidity status ≥ 4 points was recorded. High risk cytogenetics were found in 16.2% (n = 6). A total of 18.9% of patients had an R-ISS of III (n = 7). A total of 70.3% patients (n = 26) were treated with triplet regimens, mainly CD38-antibody containing regimens , most commonly in a combination of Pomalidomide/Daratumumab/Dexamethasone . Only 8.1% (n = 3) of patients received a first or further autologous SCT. Some 18 patients (48.6%) received a subsequent LoT. The AR was 27% (n = 10); one patient was lost to follow-up, while all others died.A fifth LoT was received by 18 patients . They had a median age of 79 (66.3–81.3) years. An ECOG score >2 at baseline (n = 3) was documented in 16.7% of patients. Again, no patients with comorbidity status ≥ 4 points were observed. A total of 16.7% of patients were classified as high risk by cytogenetics (n = 3). An R-ISS of III was recorded in 5.6% (n = 1). Doublet and triplet regimens were commonly administered, with eight patients (44.4%) on each regimen. Treatment regimens were found to be more diverse with each subsequent LoT. The most common treatment in the 5th LoT was Dexamethasone/Daratumumab (Dd) in two patients (11.1%). Late or second autologous SCT was performed in three patients (16.6%), and maintenance therapy was initiated in 4 patients (22.2%). More than five LoTs were recorded in another 61.1% (n = 11) of patients. The AR with respect to LoT 5 was 16.7% (n = 3); two patients died, and 1 patient was lost to follow-up.The AR across LoTs 1 to 4 was more or less constant at 22% +/− 5%, worsening only slightly with each subsequent LoT. At the fifth LoT, an AR of 16.7% (n = 3) was an outlier, which can be explained by the small number of patients pts (n = 11). The percentage of patients receiving a subsequent LoT was found to be increasing with each LoT. . p = 0.011), increasing the relative risk of attrition by 86% . The total AR across all five LoTs was 33.5% (n = 172). A total of 58.7% in this cohort were NTE (n = 101) patients. In a multivariate analysis, patients fulfilling AR criteria were significantly older (75 (64.8–82) years. vs. 70 (61–78) years; p < 0.001). Autologous SCT in a frontline regimen decreased the risk of AR by 31.8% . Achieving VGPR or better in the first LOT further improved AR and reduced its risk by 63.5% . The effect is illustrated by the Kaplan-Meier curves in The use of frontline maintenance reduced the risk of AR by 50.5% . Age is a key factor in the decision to undergo autologous SCT and hence proved to be a confounder in multivariate analyses, as displayed in As demonstrated by multivariate analyses, the achievement of VGPR or better was a confounder for maintenance. The use of maintenance therapy positively influenced response by increasing the chance of achieving VGPR or better by 3.6-fold  had a statistically significant impact on attrition. The DoT decreased continuously with a median of 6.7 (3.8–13.6) months in LoT-1 to 4 (0.5–7.3) months in LoT-5. The exception was the second LoT. This can be explained by the higher amount of patients still on treatment in the first LoT with a consecutively lower transition to the second LoT. TTNT decreased similarly with 3.8 (0.9–16.2) months in the first LoT compared to 1.5 (0.3–3.4 m) months in the fourth LoT. The course of DoT and TTNT is presented in The depth of remission decreased with each subsequent LoT, as PD was present in 9.7% of patients after the first LoT compared to 50% of patients in LoT-5. VGPR or better was achieved in 47.9% of patients in the first LoT, but only 7.1% of patients in the fifth LoT .The influence on attrition across lines of therapy (LoT) 1–5 is given with an odds ratio (OR) in the upper table. The influence of sex, age over 70 years, performance status over 2 points at baseline, Revised International Staging of III (R-ISS), and high-risk cytogenetics is illustrated. The effect of stem cell transplantation (SCT) and maintenance therapy becomes less important with increasing age and the achievement of deep response (≥VGPR) in the first LoT. A possible interpretation is provided in the subsequent second table indicating factors that influence the achievement of VGPR or better in the first LOT. The categorization is performed as in the previous table. Statistically significant data are marked with *.This retrospective real world-study using the AMR dataset highlights a promising AR of 16.7–27% for LoTs 1–5 compared to previously published data on the basis of a median follow up of 49.7m in both transplant-eligible and non-transplant-eligible patients ,4. Our rThe use of SCT shows a favorable impact on attrition with a decrease in risk to 31.8%. This is consistent with previous data . WhetherMaintenance is standard of care  after SCp < 0.001). The use of triplet regimens improved the chance of achieving VGPR or better, while doublet regimens did not have the desired effect. This effect is consistent with earlier data such as the SWOG trial S0777 [Depth of remission was mainly dependent on the use of maintenance and SCT  is encouraging given the data from the US with the predominant use of Vd  and oldeThe use of ani-CD38 antibodies increased with each LoT. This is consistent with its recent approval as a first line treatment . A similDepth of remission naturally had an impact on TTNT, in line with Dimopoulos . DoT andp < 0.001). However, the effect of chemotherapy loses significance without TE patients, so the use of chemotherapy-containing regimens in induction before transplantation cannot be separated from the ASCT effect.The discrepant results in the fifth LoT (14 patients) and in TE patients in the third LoT (eight patients) is not seen as representative due to the small number of patients. The reported effect of chemotherapy on TTNT could be explained by the number of patients who completed the first LoT with chemotherapy (89%) compared to other therapies  is consistent with previous data . Less thThe impact of comorbidity and high risk cytogenetics on TTNT and follow-up was as expected. Follow-up decreases from the first to the fifth LoT according to disease duration. The results of follow-up in relation to maintenance, SCT and patient characteristics are interesting, as follow-up is a relative marker for disease duration. However, the results of follow-up are only representative to a limited extent due to the observation period, which is inherently limited for patients who have been diagnosed recently. The importance of evaluating cytogenetics is underscored to predict the probable course of disease. The prospect of further personalising myeloma treatment as discussed by Atrash et al. corroborates our data . The lowA demand-based treatment with easy access to drugs might play a role in this.Overall, our results with regard to factors impacting the attrition rate is in accordance with recent data . NeverthCertain limitations of this study have to be taken into account. The number of patients in the registry is limited; this limitation was accepted in the choice of observation period. The selected observation period primarily presents data on currently relevant therapies. Documentation in clinical practice is not always complete, but incomplete data have been highlighted. Nevertheless, the real word data of clinical practice are of the significant interest for the evaluation of patient management, as not all of our questions will be addressed in randomized controlled and well-designed clinical trials.In summary, in utilizing the AMR database our results demonstrate considerably lower ARs for MM patients within the Austrian versus other health care systems. The low risk of attrition in our analyses was particularly associated with young age, the achievement of deep and long-lasting remissions after frontline induction/ autologous SCT, and continuous treatment. Importantly, our results emphasize a major role for a liberal drug access and reimbursement policy in governing long-term MM patient outcomes.


OBJECTIVE: Hypovitaminosis D is prevalent in epidemic proportions in many developed countries. The aim of this study is to investigate the prevalence of adequate 25-hydroxyvitamin D [25(OH)D] levels in two Mediterranean countries, Greece and Cyprus. METHODS: Data such as 25(OH)D, the month of blood sample collection, and demographic information were blindly collected from 8780 Greek and 2594 Cypriot individuals over 5 years. Comorbidities were also recorded for 839 Greek subjects. Univariate and multivariate analyses were used to examine the relationship between these variables and 25(OH)D levels. RESULTS: In the samples studied, 72.7% of the Greek and 69.3% of the Cypriot population sample had inadequate levels of 25(OH)D. The mean level for the Greek subjects was 25.1 ng/mL and for Cypriots 25.8 ng/mL. For both samples, only month and gender were significantly associated with 25(OH)D levels, and the highest mean levels were recorded in September. For the recorded diseases, the lowest levels were recorded in sickle cell anaemia 13.6 ± 10.2 ng/mL, autoimmune diseases 13.0 ± 8.4 ng/mL, and cancer 22.6 ± 9.5 ng/mL. CONCLUSIONS: The prevalence of vitamin D deficiency is paradoxically high in both Mediterranean countries. Vitamin D plays a pivotal role in bone health and calcium metabolism , but it Vitamin D status and vitamin D3 supplementation affect broad gene expression in humans ,7. VitamAdditionally, vitamin D downregulates the expression of proinflammatory cytokines ,8,11 andThe seasonal periodicity of autoimmune diseases suggests a possible correlation with 25(OH)D levels. It was observed that reduced maternal sun exposure and the resulting hypovitaminosis D in utero might increase the risk of multiple sclerosis development in offspring born in spring . 25(OH)DHypovitaminosis D has reached epidemic proportions around the globe, including Southern Europe . Olmos eIn Greece, 6.6% of a tested sample of Athenian preschool children had vitamin D deficiency with potential adverse developmental outcomes . It has This pattern of findings has raised concerns regarding the prevalence of vitamin D deficiency in Greece and Cyprus. To our knowledge, there are no available up-to-date data for the vitamin D status of Cypriot adults. Therefore, our findings could potentially lead to increased awareness of the necessity for national health guidelines, targeted and focused on these populations needs.In this retrospective cross-sectional study, data from 8780 Greek and 2594 Cypriot individuals were blindly collected for an approximately 5-year period (January 2013–October 2017). Data regarding 25(OH)D levels and other patient data including sex, age, and the month at which the blood sample was drawn were collected from the Nuclear Medicine Department of the tertiary healthcare centre AHEPA University Hospital in Thessaloniki , and a primary healthcare centre in Nicosia, Cyprus “Agios Loukas” , respectively. Comorbidities were documented in the hospital’s online laboratory information system only for a Greek subgroup of patients and were also blindly collected. Every individual who had a vitamin D measurement at the respective laboratories from January 2013 to October 2017 was included in the study. Individuals with more than three missing data values were excluded.The Department of Nuclear Medicine at the University Hospital AHEPA of Thessaloniki used a radioimmunoassay (RIA) method by Immunotech & Beckman–Coulter to measure 25(OH)D serum concentrations. Adamou Lab in Nicosia conducted the measurements for the Cypriot database, and quantitative electrochemiluminescence method (ECLIA) was used for the determination of 25(OH)D concentrations using a Cobas 6000 Diagnostics instrument and reagents . The laboratory assays used remained unchanged during the study period. Additionally, the technical characteristics  of these two laboratory assays were comparable. Following the Endocrine Society guidelines , vitaminggplot2 . There were two main objectives: first, to assess the levels of 25(OH)D in the two population samples, and second, to examine any demographic factors associated with the observed levels. Univariate (crude) linear regression was used to analyse the relationship between age and 25(OH)D levels. The distribution of 25(OH)D levels was explored via histograms. The results were expressed as mean ± standard deviation (SD) for numerical variables. The statistical analysis was performed in R 3.6.1 (R Core Team (2019), Vienna, Austria). The graphs were produced in R with the package t-test was used to investigate the relationship between sex and 25(OH)D levels, and ANOVA to investigate the relationship between month or comorbidities and 25(OH)D levels. The assumptions on the normality of residuals for ANOVA and t-tests were verified via descriptive measures and plots. Pearson correlation analysis was used to investigate the association of age and 25(OH)D levels. Multivariate two-way ANOVA analysis with independent variables, the month of the year in which the blood sample was drawn, and sex were used to investigate their concurrent association with 25(OH)D levels. An unpaired t-test and a linear regression model were used to investigate whether there was a statistically significant difference in 25(OH)D levels between the groups of patients with and without a recorded disease. Linear regression analysis was also performed separately for the disease subgroup of the Greek database. Individuals with any missing values were excluded from the multivariate analysis. The significance level  was set to 5% and confidence intervals to 95%. A result was considered statistically significant when p < 0.05.Unpaired The Greek database included 8780 subjects over a period stretching from January 2013 to October 2017. Approximately 40% of the sample (N = 3504) had Vitamin D deficiency, and 33% (N = 2913) had insufficiency. The mean levels of 25(OH)D for the total Greek database were 25.08 ± 14.4 ng/mL. The sample included both females  and males . The mean 25(OH)D level for females was 25.7 ± 15.0 ng/mL and for males 23.6 ± 13.0 ng/mL a. For 11The database contained subjects from 2 weeks to 96 years old. For 1975 individuals, age was not recorded. The mean age of the subjects for whom age was recorded was 49.3 ± 20.0 years old; 6.6% were children below 12 years old, 2.3% were adolescents between 12 and 17 years old, 81.7% belonged to the 18–74 age group, and 9.4% were elderly of 75 years or above.Amongst all age groups, elders >75 years old had the lowest mean levels 23.6 ± 13.8 ng/mL, while 12–18-years-old adolescents had the highest mean levels 27.5 ± 14.6 ng/mL. Children below 12 years old had mean levels of 26.3 ± 12.4 ng/mL. March was the month with the most drawn samples (N = 955), and August had the least (N = 377). September was the month with the highest-recorded mean levels (30.4 ± 17.4 ng/mL), and February was the one with the lowest (20.6 ± 11.9 ng/mL). The fluctuance of 25(OH)D levels by month in the Greek database is presented in In addition to the above, the Greek database had recorded comorbidities for 839 individuals. The mean 25(OH)D level of the total subgroup of patients with a recorded disease was 24.5 ± 14.2 ng/mL. The most recorded diseases were thalassemia 51.8% (N = 435), multiple sclerosis (MS) 22.4% (N = 188), neurological diseases other than MS 11.08% (N = 93) , and sickle cell anaemia 4.17% (N = 35). The most frequently recorded in the dataset diseases and the mean 25(OH)D levels are presented in The Cypriot database included 2594 subjects between September 2013 and May 2017. There was a significant vitamin D insufficiency in the sample studied, with mean 25(OH)D levels 25.8 ± 10.9 ng/mL. Approximately 31% of the individuals had levels less than 20 ng/mL (deficiency), and 38.3% had insufficient levels. Regarding sex, the sample was composed of 1668 (64.3%) females and 907 (34.9%) males. The sex of 19 patients was not recorded. The mean level for females was 25.2 ± 10.8 ng/mL and for males 26.8 ± 10.9 ng/mL b.The age span of the sample was 2–96 years old. The mean age of the subjects, whose age was recorded, was 39.9 ± 19.4 years old; 9% were children below 12 years old, 11.4% were adolescents between 12 and 17 years old, 75.6% belonged to the 18–74 age group, and 4% were 75 years old or above. For 269 individuals, age was not recorded. September was the month when most samples were collected (N = 301), and August was the one with the least (N = 139). The month with the highest mean 25(OH)D levels was September (mean = 29.7 ± 10.8 ng/mL), and the lowest was January (mean 22.1 ± 8.7 ng/mL). The fluctuance of mean 25(OH)D levels by month for the Cypriot database is presented in t-test analysis showed that sex was associated with 25(OH)D levels . One-way ANOVA revealed that the month of the year was also associated with 25(OH)D levels in both databases p < 0.001. Age was not associated with 25(OH)D levels, as demonstrated by correlation analysis in both samples . The multivariate two-way ANOVA analysis model for the Greek sample showed that month (p < 0.001) and sex (p < 0.001) were significantly associated with 25(OH)D levels. Similar were the findings of the multivariate two-way ANOVA analysis for the Cypriot sample, where the month (p < 0.001) and sex (p = 0.002) were significantly associated with vitamin D.For the total Greek and Cypriot population sample, the unpaired t-test analysis for the combined Greek and Cypriot samples to observe the general trend. The mean level of females (Greek and Cypriots combined) was 25.6 ± 14.2 ng/mL, which was statistically significantly higher than that of males, 24.4 ± 12.5 ng/mL , p < 0.001 and the result was statistically significant. From the above analysis, we obtained contradictory results concerning the association of sex to vitamin D in the studied Greek and Cypriot samples. Females had higher 25(OH)D levels than males in the Greek sample and lower levels than males in the Cypriot sample. Therefore, we performed an unpaired 2 = 0.09), which only included the subgroup of 839 Greek patients with a recorded disease, the month was not significantly associated with 25(OH)D levels , whereas age  and sex  were statistically significantly associated with 25(OH)D levels. In this model, females had lower levels by 5.57 on average compared to the reference value, the males. Age was found to be inversely proportional to 25(OH)D levels for the subgroup of patients with a recorded disease, which was statistically significant. Sickle cell anaemia was the sole disease that was strongly associated with lower vitamin D levels compared to the healthy individuals  in the linear regression model, which used the ‘healthy’ group as a reference level. Amongst the disease group (reference level: MS), sickle cell anaemia was associated with significantly lower 25(OH)D levels compared to MS . Lastly, t-test analysis showed there was not a statistically significant difference in the mean 25(OH)D levels between the total subgroup of patients with a recorded disease and those without a recorded disease.In the linear regression model  in the Greek sample was 33% and in the Cypriot sample 38%, while 40% of the Greeks and 31% of the Cypriots had vitamin D deficiency levels < 20 ng/mL). In the Greek sample, elders had the lowest mean levels at about 24 ng/mL, which is not surprising as cutaneous vitamin D synthesis declines with age 0 ng/mL. .Although levels below 30 ng/mL were prevalent in all age groups and age was not found to be significantly associated with 25(OH)D levels, the findings regarding preschool children were most concerning: 31% of preschool children in Greece and 17.6% of preschool children in Cyprus had levels below 20 ng/mL. In our Greek sample, 10.5% had levels < 10 ng/mL, similar to the findings by Nicolaidou et al., in Athenian preschool children, who found a prevalence of 6.6% [For both the total Greek and Cypriot samples, the highest mean 25(OH)D levels were recorded in the summer and early autumn, agreeing with other studies conducted in the Mediterranean ,19 and GAlthough our study did not investigate cultural behaviour, nutrition, or religious beliefs, these have been suggested as potential explanations for the variation of levels in countries with significant levels of sunshine. The modern lifestyle of office work with less sun exposure, despite the darker skin tones, has been suggested as a possible reason ,18,27. VDiseases characterised by insufficiency or failure of organs involved in vitamin D metabolism, such as liver and kidney dysfunction, have been associated with vitamin D deficiency . TherefoThe low mean levels reported in many Mediterranean and Middle Eastern countries ,29,34,35To our knowledge, this study included the largest Greek population sample investigating the prevalence of hypovitaminosis D to date, and it is the first published study to explore this topic in the adult Cypriot population. Most previous studies in Greece were conducted in adolescents, neonates, and pregnant women ,28,29,38The very large sample, and the use of data collected over different seasons for several years, are some of the strengths of this study. However, as it is a retrospective cross-sectional study, it can only suggest an association between variables and vitamin D levels. Other limitations include the lack of simultaneous measurements and collection of parameters related to bone turnover, e.g., parathyroid hormone, calcium, alkaline phosphatase, and lack of data about habits like sun exposure, diet, and skin phenotype. Therefore, further prospective studies are needed to investigate causality between vitamin D levels and associated risk factors.Our findings suggest that the abundant sunlight exposure in Greece and Cyprus is not sufficient to prevent hypovitaminosis D. This study showed a significant prevalence of vitamin D deficiency and insufficiency in both population samples. Vitamin D deficiency is more pronounced during the winter and early spring months, and it is encountered in both genders and across every age group. Physicians need to be highly suspicious of hypovitaminosis D in patients with certain autoimmune or haematological diseases who might need surveillance. There is still a need for prospective longitudinal studies to investigate the scale and the biological mechanisms associated with vitamin D and other micronutrients’ deficiencies. Furthermore, interventional studies investigating the beneficial effects of vitamin D supplementation are essential. This might have important implications for public health policies and individualised medicine to avoid a possible resurgence of vitamin D-related diseases, such as rickets, especially with the recent lockdown measures taken by the government to combat the current SARS-COVID-19 pandemic.


Nasopharyngeal antigen Rapid Diagnostic Tests (RDTs), saliva RT-PCR and nasopharyngeal (NP) RT-PCR have shown different performance characteristics to detect patients infected by SARS-CoV-2, according to the viral load (VL)—and thus transmissibility.In October 2020, we conducted a prospective trial involving patients presenting at testing centres with symptoms of COVID-19. We compared detection rates and performance of RDT, saliva PCR and nasopharyngeal (NP) PCR, according to VL and symptoms duration.6 copies/ml, detection rates were 30.3% (27.3–33.3), 31.4% (28.4–34.5), 31.5% (28.5–34.6), and 31.6% (28.6–34.7%) respectively.Out of 949 patients enrolled, 928 patients had all three tests performed. Detection rates were 35.2% (95%CI 32.2–38.4%) by RDT, 39.8% (36.6–43.0%) by saliva PCR, 40.1% (36.9–43.3%) by NP PCR, and 41.5% (38.3–44.7%) by any test. For those with viral loads (VL) ≥105 and 96.5% (93.6–98.3%) for those with VL≥106. Sensitivity of STANDARD-Q®, Panbio™ and COVID-VIRO® Ag tests were 92.9% (86.4–96.9%), 86.1% (78.6–91.7%) and 84.1% (76.9–89.7%), respectively. For those with VL≥106, sensitivity was 96.6% (90.5–99.3%), 97.8% (92.1–99.7%) and 95.3% (89.4–98.5%) respectively. No patient with VL<104 was detected by RDT.Sensitivity of RDT compared to NP PCR was 87.4% (83.6–90.6%) for all positive patients, 94.5% (91.5–96.7%) for those with VL≥10Specificity of RDT was 100% (99.3–100%) compared to any PCR. RDT sensitivity was similar <4 days  and ≥4 days  after symptoms onset (p = 0.6). Sensitivity of saliva and NP PCR were 95.7% (93.1–97.5%) and 96.5% (94.1–98.1%), respectively, compared to the other PCR.RDT results allow rapid identification of COVID cases with immediate isolation of most contagious individuals. RDT can thus be a game changer both in ambulatory care and community testing aimed at stopping transmission chains, and even more so in resource-constrained settings thanks to its very low price. When PCR is performed, saliva could replace NP swabbing.NCT04613310 (03/11/2020).ClinicalTrial.gov Identifier:  The difference was not significant between NP PCR and saliva PCR test status (p = 0.71), but was so between RDT and NP PCR or saliva PCR (p<0.001). Specificity of RDT was 100% (99.3–100%), which means that there was no false positive RDT. In two instances, RDT was positive, and NP PCR was negative, but the saliva PCR was positive with viral loads of 4.0x107; IQR 2.7x106-1.0x108) than in those with RDT negative  (p<0.001). Among patients with an RDT positive, those with a colour band of high intensity had also significantly higher viral loads by NP PCR  than in those with a colour band of low intensity  (p<0.001)  were significantly higher in patients with RDT positive (median 1.9x10p<0.001) .The sensitivity of RDT according to symptoms duration varied between 80% and 90%. It was lowest the day of symptoms onset  and highest on day 4  . There w7; IQR 1.3x106-8.4x107) in patients with major symptoms than in those with minor symptoms only  (p = 0.007), but the number of positive patients with minor symptoms was very small (8).Viral loads (by NP PCR) were significantly higher  were significantly lower than those with positive NP PCR  (p<0.001). Among 285 patients with VL≥106 by NP PCR, 10 (3.5%) were negative by RDT. Among 87 patients with VL<106 by NP PCR, 50 (57.5%) were positive by RDT  than those with low volume  (p = 0.051)  were not significantly higher in patients with high volume of saliva (median 1.3x10= 0.051) .The two PCR were equivalent also in patients with ageusia or sore throat and no other symptom outside anosmia (p = 0.7).6 copies/ml. The sensitivity of RDT compared to NP PCR was above 85%, and above 95% for patients with a high VL, and was similar between the three RDT brands, with an advantage for STANDARD Q®. The sensitivity varied slightly according to symptoms duration but remained above 80%, even after 7 days despite a progressive drop in VLs. Actually, a lower sensitivity after the acute phase of disease might be an advantage to prevent unnecessary isolation of patients who are, for most of them, no more contagious, despite a positive PCR result (known to sometimes persist for a long time) [The results of the present study show that the detection rate of positive COVID-19 cases by RDT was high, especially for those with a VL of ≥10ng time) ,28. Our The detection rate of SARS-CoV-2 by PCR performed on a saliva sample was equivalent to that of RT-PCR performed on NP swabs. The sensitivity of PCR of one type of sampling compared to the other were similar and above 95%. The two positive saliva PCR but negative NP PCR patients who were still detected by RDT illustrates that some but rare false negative results of tests based on NP swabs are likely due to sampling procedure.6 copies/ml means that these rapid tests are likely to identify reliably individuals that are contagious, which would, if largely deployed, reduce transmission more substantially than what would be expected from its imperfect overall sensitivity. Furthermore, the short turn-around time to return the result to the patient allows more rapid isolation of cases and efficient contact tracing, which should also contribute to more efficient pandemic control.The sensitivity of RDT of more than 95% in patients with VL≥10TM (86%) and COVID-VIRO® (84%), but all met the threshold of 80% sensitivity and 97% specificity of the WHO recommendations for use [There was a slight variability in performance between the three different RDTs with STANDARD Q® having a higher sensitivity (93%) than those of Panbio for use . The sen for use –15. Our In our study, the specificity of all three tests was 100%, which is impressive considering the potential for inter-observer variation in RDT test line reading. This observation implies that the assessed RDTs brands are easy to read, and that faint lines can still be easily detected. This excellent specificity, which was also shown in the other studies on high quality RDT, allows to state that there is no need to confirm a positive RDT test result by an additional PCR test.One of the strengths of our study also lies in the fact that the study population represented that of routine COVID-19 diagnostic centres, namely symptomatic outpatients with fever or cough or anosmia/ageusia or symptomatic close contacts. The study was performed at the end-user level in real-life conditions, which is in agreement with WHO recommendations for evaluating the performance of new diagnostic tests . Real-liHaving a detection rate of saliva PCR equivalent to that of NP PCR is in line with a previous study done on 70 patients COVID-19 positive that showed excellent concordance between the two sampling methods . Other sThe median SARS-CoV-2 VL in saliva was approximately two log lower than that in the NP swab. With such a difference, an overall lower sensitivity of saliva PCR when compared to NP PCR would have been expected. This was not the case in the present evaluation because it is essentially the peak and not the extremes of the VL distribution curve that is shifted towards a lower value for saliva.In terms of procedures, patients were able to easily perform the saliva sampling on themselves after getting a precise explanation by the health professional. Some were not able though to drool saliva in the tube, but this did not affect the sensitivity, the VL being not significantly lower in this group.et al [The FDA has granted emergency use authorization to various saliva-based assays for the SARS-CoV-2. Our pragmatic approach, using the same transport medium tube as for NP swabs, can be applied in any testing facility. The similar sensitivity and specificity achieved by sampling the saliva instead of the nasopharynx validates the sampling method and procedure, at least in this outpatient population with relatively high viral load. The results of the present study, together with that of Wyllie et al  and otheet al ,32, provIf RDT would be used in settings with a lower SARS-CoV-2 prevalence, such as 10%, a negative test would have a negative predictive value (NPV) of 98.6%. Such an NPV is acceptable if the patients do not belong to high-risk populations . If the prevalence would be only 1%, the NPV would be 99.9%. Considering a specificity of 100%, other NPV, whenever for RDT or PCR, can be simply calculated using the following formula: (1-P)/1-SP .Regarding the positive predictive value (PPV), even taking the lowest specificity confidence interval (99.3%), it would be 93% at 10% prevalence, which is high enough. At a lower prevalence, the PPV would drop, and the solution would be to restrict testing to patients with a high enough pre-test probability rather than confirming each positive case by PCR.5 and 106 suggests that RDTs can be safely used for screening schools, university students or contacts of SARS-CoV-2 positive patients [The present study was conducted in a well-defined outpatient population with usual testing criteria for COVID-19 and presenting within 7 days after symptom onset for most of them. Our results might not apply in a setting where patients would have lower viral loads and/or attend after one week of symptoms, keeping in mind that these outpatients would be much less likely to transmit . We did patients .The good performance of the best available RDTs allows point of care management of patients at primary care level, as well as community testing aimed at stopping transmission chains. RDT results allow immediate isolation of the vast majority of contagious individuals, without confining unnecessarily those who are not. The almost perfect concordance between saliva PCR and NP PCR results, the ease of administration and the safety of the procedure could trigger change in sampling method using saliva as reference standard, at least in outpatients who have higher viral loads than inpatients. RDT complies with the ASSURED  criteria, which makes them very useful in primary care practices, and, thanks to its very low price, even more so in resource-constrained settings . The varS1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(XLSX)Click here for additional data file.


Introduction: Dyslipidemia refers to the presence of abnormalities in lipid parameters. It has become a global issue with a high risk of cardiovascular diseases (CVDs). The aim of the investigation was to find out the pattern and prevalence of dyslipidemia among patients with the acute coronary syndrome (ACS).Methodology: A cross-sectional study design was used. Data were collected using convenient sampling from 101 patients presenting with ACS, admitted at the critical care unit (CCU) / Rasheeda Begum Cardiac Centre (RBCC) of Shalamar Hospital, during a 12-month period from January 2020 to December 2021. Dyslipidemia is diagnosed by testing the lipid profile when there are one or more abnormal readings of the lipid profile.Results: Nearly 43 (42.6%) had ST-segment elevation myocardial infarction (STEMI), 27 (26.7%) had non-ST segment elevation myocardial infarction (NSTEMI), and 31 (30.7%) were categorized as unstable angina (USA). Overall dyslipidemia was present in 84 (83.2%) patients. The prevalence of dyslipidemia was 55 (65%) in male patients and 29 (34.5%) in female patients. Dyslipidemia was present in 39 (90.7%) patients with STEMI, 25 (80.6%) in the USA, and 20 (74.1%) with NSTEMI.Conclusion: The prevalence of dyslipidemia was quite high among ACS patients. The proportion of obese patients was also high in our study. However, dyslipidemia was more frequent in overweight patients. Acute coronary syndrome (ACS) is defined as a range of clinical conditions characterized by a sudden reduction of blood flow to the heart. This may lead to permanent death of heart tissue and myocardial infarction. The term ACS is used in patients in which there is suspicion or confirmation of ischemia of heart muscles or infarction. This can be classified into non-ST-elevation myocardial infarction (NSTEMI), ST-elevation MI (STEMI), and unstable angina . Dyslipidemia is an abnormal amount of lipids  in the blood. It has become a global issue with a high risk of cardiovascular diseases (CVDs) . The assIncreased total cholesterol (TC), LDL cholesterol, and non-elevated high-density lipoprotein (HDL) cholesterol were seen to correlate directly with mortality. In the Asian-Pacific region, one main reason was coronary heart disease (CHD). The major purpose of strategies to avoid CVDs is to reduce LDL with lipid-lowering therapies (LLTs). This might be due to the scarcity of strong evidence . ReducedGaziano et al. showed for the first time that the triglyceride (TG)/HDL ratio was a solid forecaster of myocardial infarction. Furthermore, TG/HDL ratio is closely associated with cardiovascular risk factors and may predict CAD development and cardiovascular mortality. TG/HDL ratio also reflects the balance between atherogenic and protective lipoproteins . TypicalDyslipidemia is a modifiable risk factor for most common CVDs; TG/HDL is unidentified until spotted during the initial presentation with ACS . This stA cross-sectional study design was used. Data were collected using convenient sampling from 101 patients presenting with ACS admitted to the critical care unit (CCU) / Rasheeda Begum Cardiac Centre (RBCC), Shalamar Hospital, Lahore during a 12-month period from January 2020 to December 2021. History, clinical examination, and electrocardiography (ECG) were done and serum troponin was measured with fasting lipid profile during index admission or during the previous 30 days prior to admission. The exclusion criteria were stable ischemic heart disease (IHD), valvular heart disease, and cardiogenic shock/left ventricular failure (LVF). All patients with rales on lung auscultation decreased breath sounds on lung auscultation, Q waves, LV hypertrophy (LVH), and a widened QRS complex in ECG was classified as LVF in this study. The minimum sample size was calculated as 92 by using a 95% confidence coefficient, and 39% as the prevalence of dyslipidemia in IHD patients [The study protocol was approved by the Shalamar Medical and Dental College Institutional Review Board (SMDC-IRB/AI/67/2020). Consent was taken from the patients before the collection of data. Dyslipidemia was diagnosed by testing the lipid parameters having one or more of the following disorders: blood cholesterol > 5.2 mmol/L (200 mg/dL), triglyceride > 1.7 mmol/L (150 mg/dL), LDL cholesterol > 2.58 mmol/L (100 mg/dL), very low-density lipoprotein (VLDL) normal range from 2 to 30 mg/dL, and HDL cholesterol < 1.03 mmol/L (40 mg/dL). The frequency and percentage of categorical variables were reported. The mean and standard deviation of the numeric variable were calculated. Data analysis was done using IBM SPSS 26.0 .The mean age of the patients who presented with ACS to the Medical ICU was 56.72 + 11.85 SD (in years). Out of the total patients, 68 (67.3%) were males and the remaining 33 (32.7%) were females. About 43 (42.6%) had ST-segment elevation myocardial infarction (STEMI), 27 (26.7%) have non-ST elevation myocardial infarction (NSTEMI), and 31 (30.7%) were categorized as unstable angina (USA). A coronary angiogram of 77 (76.2%) patients was done. The mean + SD of abdominal girth was 102.48 + 17.68 SD (cm). The mean + SD (units) of the lipid profile of the patients is given in Table Overall dyslipidemia was present in 84 83.2%) patients. The prevalence of dyslipidemia was seen in 55 (65%) male patients and 29 (34.5%) female patients. The distribution of dyslipidemia according to the lipid profile is given in Table % patientIn order to compare the TG/HDL ratio among patients with normal and abnormal lipid profiles, an independent sample t-test was used. TG/HDL was found statistically significant for patients with normal and abnormal TC, TG, HDL, and VLDL. TG/HDL ratio was significantly high among patients with TC greater or equal to 200 mg/dL. However, the difference between TG/HDL ratio among patients with less than 100 mg/dL and greater or equal to 100 mg/dL was statistically insignificant. TG/HDL ratio was higher among patients with normal VLDL. We observed that dyslipidemia was common among 72.3% of the patients with low HDL, 53.5% of the patients with high TC, and 50.5% of the patients with high LDL. Dyslipidemia was present in 39 (90.7%) patients with STEMI, 25 (80.6%) in the USA, and 20 (74.1%) with NSTEMI. Body mass index (BMI) less than 18.5 was taken as under-weight, 19-24.99 was normal, 25-29.99 was over-weight and greater and equal to 30 was used as obese. About 39 (38.6%) of the patients were obese, 35 (34.7%) were overweight, 26 (25.7%) were normal, and 01 (1.0%) were underweight. Out of the total patients who were obese, dyslipidemia was present in 29 (34.5%). Dyslipidemia was more frequent in overweight patients. Dyslipidemia was present in about 30 (35.7%) of the overweight participants, and 24 (28.6%) of the normal participants.The current study investigated the patterns of dyslipidemia among patients presenting with ACS. We observed that dyslipidemia was present in 83.2% of cases in our study which was quite similar to another study conducted on diabetic patients in Pakistan. A recent study conducted in Pakistan among type 2 diabetic patients observed that 83.5% of the patients had dyslipidemia . A recenAnother study conducted in Pakistan showed the prevalence of dyslipidemia among stroke patients. It showed the prevalence of dyslipidemia as 39.4% which was relatively low compared to our study . A studyThe prevalence of dyslipidemia in our study was 65.5% among male patients and 34.5% among female patients is almost double in male patients compared to female patients. The prevalence of dyslipidemia was 68.4% among men as compared to 43.7% in females in another study from Pakistan . DyslipiThe prevalence of STEMI was higher in patients with dyslipidemia followed by unstable angina and NSTEMI. Among those patients who presented with STEMI, 39 (90.7%) were found to have dyslipidemia, 25 (80.6%) had dyslipidemia with unstable angina, and 20 (74.1%) had dyslipidemia with` NSTEMI. We observed that dyslipidemia was more common among patients with STEMI followed by unstable angina. The least common ACS was NSTEMI with dyslipidemia. A recent study done to observe the patterns and prevalence of dyslipidemia among ACS patients showed that dyslipidemia was highly prevalent among patients with unstable angina at 64.3% followed by NSTEMI at 48.1% and STEMI at 45.3% . The finIn our study, the lipid abnormalities were a low level of HDL and a higher level of TC and LDL. A recent study reported that among the patients with dyslipidemia, 70.0% of the patients had high LDL, and 34.0% of patients with non-elevated HDL . AnotherSome of the limitations of our study were cross-sectional/descriptive studies conducted in a single center with convenient sampling. Hence drawing a convenience sample and that too from the already diseased people presenting in the hospital may have its limitations. Moreover, there is no time dimension involved in cross-sectional studies and therefore no time interval between “exposure” and “outcome” .The prevalence of dyslipidemia was quite high among ACS patients. It was relatively more prevalent in patients with STEMI and overweight patients. Preventive strategies are required for obese, overweight, and STEMI patients. Dyslipidemia is an important risk factor in heart attacks and early detection can reduce the burden of morbidity, especially among high-risk cases. Moreover, associated risk factors such as obesity, overweight, and other general lipid abnormalities in ACS patients should be considered for the prevention of a rising trend of CVD and stroke among the Pakistani population.


Research on adolescent substance use is of utmost importance. Using local toxicological data, both prevalence and pattern of substance use (SU) and substance-related death (SRD) can be assessed to design effective prevention programs. A retrospective study of toxicology investigations of all adolescents referred to the medico-legal section of the Toxicology Unit of the University Hospital of Lille, France, for a 2-year period from 2017 to 2018. In the total sample of 1961 cases, adolescents accounted for 3.3% of the cases (n = 65). Among the adolescents, 16.9% were aged 10–14 years and 83.1% were aged >14–19 years. About 69.2% were males. Less than 70% of all presented adolescents used substances. More than two-thirds (74%) of positive detections were male. Illicit substances (43%) were the most detected substance followed by alcohol (20%) and prescription substances (20%). Tetrahydrocannabinol (THC) was extremely common as it was found in 29% of all adolescents. Cocaine and amphetamines were detected in 13.8% of total tested adolescents. Polysubstance use was common between alcohol and THC and among males. About one-third of deaths were due to substance use. About 54% of SRD was associated with polysubstance detection. It is recommended that illicit substances, ethanol, and prescription substances are targeted for testing among adolescents in order to provide appropriate prevention. Substance use (SU) represents a preventive medicine dilemma in any society. The users may consume different types of substances that have been legal or accepted for public use , shared for conditions other than medical use without prescription (prescription substances), or prohibited by law for public use (illicit substance) . SU is cAccording to the definition by the World Health Organization, an adolescent is an individual who is aged between 10–19 years . AdolescResearch based on a postmortem database introduces a complementary and different perspective on SU. It includes those persons who do not usually participate in self-reporting studies, and the results depend on actual toxicological analysis; the results of the measurements could temporally connect the toxic substance effect and the fatality . DeceaseA systematic retrospective review of adolescent cases , who were referred to the medico-legal section of the Toxicological Unit of the University Hospital of Lille (North of France), was carried out over 2 years (2017 to 2018) . Autopsyad-hoc dedicated methods using liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) or diode array detector (LC-DAD), and headspace-gas chromatography with flame ionization detection . The therapeutic, toxic, and fatal concentrations were determined according to the published reference tables [Systematic toxicological investigations that were performed in the collected cases included general unknown screenings using liquid chromatography with the high-resolution mass spectrometry method (LC-HRMS) previously reported ,10,11. Me tables . The cause of death variable has 2 categories, including SRDs and non-substance-related deaths. The age variable was recorded into two categories, 10–14 years of age (defined as early adolescence) and >14–19 years of age (late adolescence). The data analysis is mainly descriptive in nature. Statistics consisted of frequencies and crosstab analysis.In the total sample of 1961 cases referred to the medico-legal section of the Toxicology Unit of the University Hospital of Lille, France, during 2017–2018, adolescents constituted 3.3% of the cases (n = 65) . Among tAmong the adolescents in the present study, 69.2% (n = 45) were males and 30.8% (n = 20) were females. In 2017, 68% (n = 17) were males and 32% (n = 8) were females. In 2018, 70% of the cases (n = 28) were males and 30% (n = 12) were females. There were 40 deceased cases whose biological samples were investigated during 2017–2018 . Meanwhile, there were 25 living cases that were investigated during 2017–2018 . In this study, SRDs accounted for 32.5% (n = 13) and 67.5% (n = 27) for non-substance-related deaths. Toxicological data was compiled using all available data from each individual and considered positive if the threshold for positivity was achieved . The mosNo substance was detected in the 10–14 years old group (n = 11). This age group consisted of five males and six females. Two out of 11 were living and the other (n = 9) were deceased. The exclusive cause of death among early adolescents was not related to any substance. The group aged >14–19 years old (n = 54) consisted of 40 males and 14 females. Twenty-three were living and the other (n = 31) were deceased. The commonest detected substance among late adolescents was THC , followed by alcohol . Among males (n = 45), 40 were in the >14–19 years old group. Out of the total number of males in the present study, there were 29 deceased (64.4%). The most common substance detection was THC in 33.3% (n = 15), followed by alcohol in 20% (n = 9) and benzodiazepine-related substances (BZ) in 11% (n = 5) . The SRDAmong females (n = 20), 14 were in the group aged >14–19 years. Out of the total number of females in the present study, there were 11 deceased (55%). The most common substance detection was both THC and alcohol in 20% (n = 4) for each substance followed by both amphetamine and cocaine in 10% (n = 2) for each substance . The SRDIn this study, there were 13 cases  positive for alcohol. All of the cases were in the >14–19 years age group (seven deceased and six living). The majority (9/13 = 69%) were males. The most common cause of death in alcohol-positive cases was substance-related (4/7 57%). Ethanol was quantified in all cases, the cases ranged from 60–2850 mg/L. Ethanol was included in the cause of death for two cases.In the present study, six cases were positive for opioids . The detected substances under this class were morphine, methadone, and codeine. Five cases were males (three died and two living cases) and the last one was a deceased female. All positive opioid cases were in the >14–19 years age group. The sample belonging to living persons (n = 2) were presented to the lab due to the police request after either an accident or violence. The blood analysis of one of them detected morphine (42.3 µg/L), THC (0.8 µg/L), and THC-COOH (20.8 µg/L). For the other living case, blood analysis revealed methadone (2.1 µg/L). The concentration of morphine in the blood of deceased cases in the present study ranged from 5.4 to 73.9 µg/L, with an average of 40.5µg/L (n = 4). The toxicological analysis detected other substances  in three positive opioids cases. SRDs were 50% (n = 2/4) among positive opioid adolescents.Six cases were positive for BZ . Four cases were males (three deceased and one living) and two cases were females (one deceased and one living), and all cases were in the >14–19 years age group. Among positive BZ cases (n = 4), the toxicological analysis detected other substances . SRDs were 50% (n = 2/4) among deceased positive BZ adolescents.N-desmethyltramadol, O-desmethyltramadol) were 194, 153, and 5.8 µg/L respectively. The cause of death was not substance-related.Tramadol was detected in only one case—a 19-year-old deceased male. The concentrations of TR and their metabolites  were positive for one prescription substance, whereas 38.5% (n = 5/13) were positive for poly-prescription use. Of the positive adolescents for prescription substances, 38.5% (5/13) were also positive for illicit substances . In the present study, 43% (n = 28) were positive for illicit substances (15 out of them were deceased). Only 14% (n = 4/28) of positive illicit substances were positive for poly-illicit (two or three) SU such as THC, MDMA, amphetamine, and/or cocaine. All cases with illicit substance positives occurred in the >14–19 year age group. Males  were more likely to be positive for illicit substances than females except for MDMA . SRDs constituted 80% (12/15) of deceased cases with positive illicit substances. Four cases (6.2%) were positive for amphetamine-related substances. All cases were in the age group of >14 to 19 years old (two deceased and two living cases). The positive amphetamine findings occurred equally in both sexes. Amphetamine (2.8 µg/L) was detected in a 17-year-old living male with polysubstance detection . The blood concentrations of 3,4-Methylenedioxymethamphetamine (MDMA) were 117 and 1890 µg/L and the blood concentrations of MDA were 8 and 31 µg/L. In one of these cases, both MDMA and 3,4-Methylenedioxyamphetamine (MDA) were detected in urine . SRDs made up 100% (n = 2/2) of deceased cases with positive MDMA.Cocaine and/or its metabolites were detected in five cases (7.7%). All cases were in the age group of >14 to 19 years old (four deceased and one living case). The positive cocaine findings occurred in three males and two females. In the present study, the blood concentration of cocaine was 3.4 µg/L (n = 1). For benzoylecgonine (BE), the blood concentrations ranged from 3.4–204 µg/L with a mean = 59.4 µg/L. The blood concentrations of ecgonine methyl ester ranged from 1.3 up to 4.1 µg/L with a mean of 1.8 µg/L. SRDs accounted for 75% (3/4) of deceased cases with positive cocaine substances.Cannabinoid was the most frequently detected illicit substance , which occurred exclusively in the >14–19 years age group (nine deceased and ten living cases). Males comprised 78.9% (15/19) of positive THC cases. The THC blood concentration ranged from 0.7 to 40.5 µg/L, with an average of 6.7 µg/L. The average THC-COOH blood concentration was 10 µg/L, with a range from 0.9 to 47 µg/L. When THC and/or metabolites were detected, the most common cause of death was substance-related .In this study, there were 16 cases (six living and 10 deceased) positive for multiple substance detection. Out of 16 cases, 12 cases were with polysubstance detection , and the last four cases were with pure poly-illicit substance detections. The most common polysubstance detection was found between alcohol and THC 43.8% (n = 7) of this group. This pattern of intake was presented exclusively among the >14–19 years (n = 16). The males dominated by a margin of 5:2 in comparison with female cases. SRDs constituted 70% (n = 7/10) of deceased adolescents with multiple substance detection.The death due to substance overdose among adolescents is diagnosed as SRD which is different from positive substance detection . The SRDSU is a prevalent problem in adolescence ,13. The This is a retrospective study of toxicology investigations of (deceased and living) adolescents (10–19 years old) referred to the medico-legal section of the Toxicological Unit of the University Hospital of Lille (North of France) for 2 years from 2017 to 2018. The present study displayed that more than two-thirds (69.2%) of presented adolescents used substances. A previous French study (2006) reported that the prevalence of drug users among a young population of students (n = 3561) from Toulouse University was based on a self-administered questionnaire is 44% . The varThe positive toxicological results in the present study were found exclusively in a group of late adolescence (>14–19 years old). This is in agreement with the World Drug Report (2018), which revealed that SU is typically initiated in late adolescence and peaks in young adulthood . More than two-thirds (74%) of positive detections in the present study were in males. In polysubstance use, males also dominated by a margin of 5:2 in comparison to females. The SRDs occurred more likely among males than females. This is in the same direction as previous studies that confirmed the dominance of males either in SU and its consequences .Illicit substances (43%) were the most commonly detected substance among tested adolescents followed by alcohol 20%) in the present study. THC was extremely common among adolescents as it was found in 29% of all tested cases. Regardless of smoking, a previous French study is in agreement with the present study as it reported that the most consumed products by adolescents are alcohol and cannabis . Another% in the The prevalence of prescription substances in the present study was 20%. The most common detected prescription substances were opioids (9%) and BZ (9%). According to an earlier French study, 1.9% of people took psychotropic medications, mostly BZ, and <1% used opiates . AnotherThe most common polysubstance detection 46.6%) was found between alcohol and THC. Of the positive adolescents for prescription substances, more than three-fourths were also positive for illicit substances or other prescription substances. Polysubstance use is a common pattern among both recreational and regular users. However, polysubstance use among adolescents is linked to an increased risk of developing long-term consequences and engaging in risk-taking through binge drinking or stimulant use such as “ecstasy” .6% was f. In EuroIn the present study, about one-third of deaths (32.5%) were due to SU. About 54% of SRDs in the current study were associated with polysubstance detection. These results are in agreement with previous reports that revealed the extreme outcome of SU is death. Globally, deaths due to SU increased from 2000 to 2015 by 60%. The top two ranks for recorded deaths among different age groups including adolescents were Europe followed by the Americas .Updated revision regarding commonly used substances and identifying the danger directed at adolescent health is needed to provide appropriate prevention. SU becomes a major problem as the adolescent moves into adulthood. If problems persist, polysubstance use and addiction may occur and increase the complexity of the problem. For this reason, increased supervision and effective prevention programs against SU for adolescents are justified, both for surveillance and reduction of harm.


Human papilloma virus is an etiological risk factor for a subset of head and neck squamous cell carcinomas. HPV has been proven to be a powerful prognostic biomarker for oropharyngeal cancer, but its role in the larynx has not been explored in depth. The developmental mechanisms of laryngeal carcinomas are quite complex and controlled by various factors. Smoking and alcohol are most important risk factors. Recent studies indicate that HPV infection also plays an important role in larynx carcinomas. HPV related laryngeal carcinomas especially occur at the supraglottic region of larynx.We aimed to determine the frequency of HPV/protein16 positivity in patients with laryngeal carcinoma and association of HPV and/or p16 positivity with variables such as age, sex, smoking habits, tumor localization, lymph node metastasis, recurrence and survival in advanced stage laryngeal carcinoma in our study.This retrospective study included 90 patients with advanced laryngeal carcinoma. The Control group was 10 normal larynx mucosa specimens. The presence of HPV was investigated polyclonally by polymerase chain reaction, and protein16 with immunohistochemical method. In HPV positive cases, the presence of HPV types 16, 18 were evaluated by polymerase chain reaction. Demographic features of patients were noted. Patient survival and association with HPV/protein16 was determined.p =  0.011). 55.6% of protein16 positive cases was located in the supraglottic region, 33.3% was glottic and 11.1% was transglottic. Although life expectancy over 5 years were numerically higher in HPV and protein16 positive cases, this was not found to be statistically significant. There was no statistically significant relationship between HPV positivity and mean age, differentiation, smoking and alcohol use, tumor progression, lymph node metastasis, localization, recurrence, cause of mortality and treatment methods in our study. The mean follow-up period of our patients was 6.7 years.Polyclonal HPV positivity was detected in 11 (12.2%) of 90 cases. Out of these 11 cases, HPV 16 was positive in 6, HPV 18 in 4, and both HPV 16 and 18 were positive in 1. In 18 (20%) of the cases, p16 was positive. Six of the cases (6.6%) had both HPV and protein16 positivity. In cases where protein16 alone or HPV and protein16 were co-positive, alcohol use was less and the tumor was found more likely to be localized in the supraglottic area. These ratios were statistically significant. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant (The close relationship between HPV and oropharyngeal squamous cell carcinoma could not be shown in larynx malignancy in many studies, including our study. Our findings support a limited role of HPV in laryngeal carcinogenesis. Protein16 is not a reliable surrogate for HPV status in laryngeal cancers and is not a predictor of laryngeal cancer survival. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant. There is a need for more populated clinical trials, where neoplastic proliferation is better demonstrated and the accuracy of the results obtained is supported by different techniques. Laryngeal cancers comprise more than 3 % of all malignant tumors in the body, and is the sixth most common cancer type around the world.Ninety patients diagnosed with advanced stage epidermoid (squamous cell) carcinoma of the larynx in Cukurova University School of Medicine Department of Otolaryngology between January 2006 ‒ December 2011, who were treatment-naive before, have been included in the study. This study has been approved by the local ethics committee of Cukurova University . Cases with early stage laryngeal cancer, cases that have been diagnosed and treated before, and cases that cannot be followed up regularly have been excluded from the study.Paraffin-biopsy tissues fixed in formaldehyde were used with immuno-histochemical staining method for p16 and 10 normal larynx tissues were used for control group. Tissues detected in 10 % formaldehyde were blocked after tissue follow-up process and Hematoxylin-Eosin (HE) stained preparations were prepared in 5 μm serial cross-sections. They were examined by light microscope. Histological cross-sections were transferred on polylysine slides specific for immuno-histochemical staining. For PCR, 5–10 sheets of 5µ were sampled. For cases taken from the study group, p16  was applied to the cross-sections prepared from paraffin blocks with Streptavidin-Biotin complex immuno-peroxidase method. Normal larynx mucosa was used for all antibodies as positive control. Prepared samples have been assessed in light microscope.Firstly, polyclonal HPV, and then HPV 16 and 18 studies were performed on the cross-sections of cases prepared from paraffin blocks. General HPV 16 and 18 types were studied with PCR method.t-test has been used in the comparison of numeric measurements between groups. The McNemar test has been used for examining concordance between HPV and P16. Statistically significant level was assumed as 0.05 in all tests.IBM SPSS Statistics Version 20.0 package program has been used in the statistical analysis of cases. Categorical measurements have been summarized in number and percentage, and numeric measurements as mean and standard deviation. Chi-square test statistics have been used in the comparison of categorical measurements between groups. Independent Eighty-four (93.3 %) of the cases were male and 6 (6.7 %) were female. The average age was 59.9 + 10.8 (26–83). 46 cases were well-differentiated (51.1 %), 28 (31.1 %) were moderately-differentiated and 16 (17.8 %) were poorly differentiated. 44 tumors (48.9 %) had glottic, 24 (26.7 %) had supraglottic, 22 (24.4 %) had transglottic location. 60 cases (66.7 %) with T3 have been followed-up, and 30 cases (33.3 %) with T4 have been followed-up. Lymph node (LN) metastasis was determined in 29 (32.2 %) patients. While all patients included in the study had a history of smoking, 52 cases (57.8 %) were determined to have regular alcohol use. 27 cases (30 %) were determined to have recurrence, while no recurrence was determined in 63 (70 %) cases. Nine cases (10 %) underwent surgery only as primary treatment, while 40 (44.4 %) underwent post-operative adjuvant radiation, 28 (31.1 %) underwent post-operative adjuvant chemoradiotherapy and 13 (14.4 %) underwent only chemoradiotherapy without surgery. The average follow-up period was determined to be 6.7 years in our patients. Fifty-three (58.9 %) cases had a survival above 5 years, and 37 (41.1 %) had 5 year survival and below. Twenty one of 37 patients (56.8 %) died due to laryngeal cancer, and 16 (43.2 %) died due to others reasons such as cerebrovascular attack, coronary artery disesase and pneumonia.Eleven cases (12.2 %) were determined to have polyclonal HPV positivity with PCR method. HPV 16 was positive in 6 of these 11 cases (54.5 %), HPV 18 was positive in 4 (36.4 %) and HPV 16 and 18 were positive in 1 case (9.1 %). p16, which was determined with immuno-histochemical staining method, was positive in 18 cases (20 %). 6 cases (6.6 %) had both HPV and p16 positivity. The relation between HPV and p16 test results was determined to have non-compliance.p =  0.011). 55.6 % of p16 positive cases was supraglottic, 33.3 % was glottic and 11.1 % was transglottic. In cases with any one of or both of HPV and p16 positivity, location of tumor in the supraglottic area was determined to be increased. The correlation between any one of or both HPV and p16 positivity, and supraglottic area location was determined to be statistically significant (p =  0.008). 52.2 % of these cases were supraglottic, 34.8 % was glottic and 13 % was transglottic.In our study, supraglottic localization of tumor was determined to be increased in p16 positive cases. The correlation between p16 positivity and supraglottic area location was determined to be statistically significant (p =  0.004). 72.2 % of p16-positive cases did not use alcohol and 27.8 % used alcohol.Alcohol use was determined to be lower in p16 positive cases. The correlation between p16 positivity and alcohol use was determined to be statistically significant (p =  0.009). 64.7 % of HPV and p16 positive cases did not use alcohol, while 33.3 % did.Alcohol use was also determined to be lower in HPV and p16 positive cases. The correlation between HPV and p16 positivity and alcohol use has been determined to be statistically significant  2739 LSCC cases were reported to have HPV positivity.While HPV positive cases in the oropharynx area showed significantly better prognosis compared to HPV negative ones in most of the studies, this was not proven in Non-Oropharyngeal Head and Neck Cancers (NOSCC).Morshad et al. have performed one of the largest studies in HPV associated laryngeal cancers. In this study, no significant difference was determined with regard to survival between HPV positive and negative patients in their follow-up in year 3 and 5 after diagnosis.p16 positivity rate in laryngeal cancers has been reported at rates varying from 1 % to 58 % in literature. In the study performed by Xu et al., 51 of 674 LSCC cases (7.57 %) were shown to have p16 positivity, while Chung et al. were determined to have p16 positivity in 11 of 140 LSCC cases (7.8 %) and also Kanyılmaz et al. have reported p16 positivity in 58 (44 %) of 131 LSCC cases.There are studies showing cigarette and alcohol use is lower in p16 positive cases.There are various studies in the literature about p16 positivity and tumor location. Xu et al. reported that p16 positivity was significantly higher in supraglottic cases in their study.While some authors associated p16 presence in LSCC with good prognosis, p16 negativity was associated with poor prognosis and recurrence.In the study performed on the presence of recurrence by Kalfert et al., 8 of 58 LSCC cases (13.8 %) experienced recurrence 3 years after initial diagnosis. It was stated that all recurring cases had p16 negativity.While a relation between p16 positivity in OSCC, and low T stage and Lymph Node (LN) metastasis was shown, this relation could not be shown in LSCC cases.p16 positivity is an important indicator in the diagnosis of HPV infection. In more advanced and sensitive methods, different results on HPV-p16 correlation can be shown. In a study, HPV was determined to be positive in 33 of 674 LSCC cases, while p16 was positive in 51. All of HPV positive cases were also determined to be p16-positive, and p16 sensitivity was stated as 100 % and p16 specificity was stated as 65 %.While it was shown that HPV and p16 positivity together provided better prognosis in OSCC, no significant result could be obtained in LSCC causes which may be due to the low number of HPV and p16 positive cases.Today, the accepted opinion for oncogenesis is that there should be a reciprocal interaction between different molecular factors for the formation of tumor. These factors are protooncogens, tumor suppressor genes and some viral oncogens that regulate cellular proliferation, apoptosis and differentiation. However, due to various factors such as cell type, cell environment and genetic background, it is hard to estimate the process of progress once carcinogenesis activity starts.The close relationship between HPV and OSCC could not be shown on larynx in many studies including our study. Our findings support that p16 is not a reliable surrogate for HPV status in laryngeal cancers and is not a predictor of laryngeal cancer survival. Supraglottic localization of tumor was determined to be increased in p16 positive cases. The correlation between p16 positivity and supraglottic area location was determined to be statistically significant. Larger studies on HPV and p16 positivity in laryngeal cancer will be more useful for treatment, follow-up and prognosis of patients.Cukurova University Scientific Research Project Fund.The authors declare no conflicts of interest.


Healthcare workers (HCWs) are at higher risk of SARS-CoV-2 infection than the general population. This group is pivotal to healthcare system resilience during the COVID-19, and future, pandemics. We investigated demographic, social, behavioural and occupational risk factors for SARS-CoV-2 infection among HCWs.HCWs enrolled in a large-scale sero-epidemiological study at a UK university teaching hospital were sent questionnaires spanning a 5-month period from March to July 2020. In a retrospective observational cohort study, univariate logistic regression was used to assess factors associated with SARS-CoV-2 infection. A Least Absolute Shrinkage Selection Operator regression model was used to identify variables to include in a multivariate logistic regression model.Among 2258 HCWs, highest ORs associated with SARS-CoV-2 antibody seropositivity on multivariate analysis were having a household member previously testing positive for SARS-CoV-2 antibodies (OR 6.94 (95% CI 4.15 to 11.6); p<0.0001) and being of black ethnicity (6.21 (95% CI 2.69 to 14.3); p<0.0001). Occupational factors associated with a higher risk of seropositivity included working as a physiotherapist (OR 2.78 (95% CI 1.21 to 6.36); p=0.015) and working predominantly in acute medicine (OR 2.72 (95% CI 1.57 to 4.69); p<0.0001) or medical subspecialties (not including infectious diseases) (OR 2.33 (95% CI 1.4 to 3.88); p=0.001). Reporting that adequate personal protective equipment (PPE) was ‘rarely’ available had an OR of 2.83 . Reporting attending a handover where social distancing was not possible had an OR of 1.39 .The emergence of SARS-CoV-2 variants and potential vaccine escape continue to threaten stability of healthcare systems worldwide, and sustained vigilance against HCW infection remains a priority. Enhanced risk assessments should be considered for HCWs of black ethnicity, physiotherapists and those working in acute medicine or medical subspecialties. Workplace risk reduction measures include ongoing access to high-quality PPE and effective social distancing measures. A strength of this study was the use of a large, well-defined cohort of UK healthcare workers (HCWs).The identification of actionable risk factors for mitigation of HCW infection.Representative and transferable conclusions for acute hospital trusts.Limitations include some potential retrospective recall bias of subjective questionnaire responses.The COVID-19 pandemic continues to overwhelm healthcare services globally with substantial morbidity and mortality.HCWs are at a disproportionately high risk of infection from SARS-CoV-2Cambridge University Hospitals NHS Foundation Trust (CUH) is a tertiary referral centre and teaching hospital with 1000 beds and 11 545 staff serving a population of 580 000 people in the East of England. The facility was equipped with 43 intensive care unit (ICU) beds prior to the pandemic, rising to 103 ICU beds at the peak of the pandemic, and an emergency department that receives ~14 000 attendees a month. During the study period (between March and June 2020), CUH treated 525 patients with PCR-confirmed COVID-19. The peak of COVID-19 admissions occurred in late March and early April 2020, with comparatively few COVID-19 admissions from June 2020 to November 2020. The definition of COVID-19 working for the purpose of risk stratification included clinical areas caring for patients with PCR-confirmed SARS-CoV-2 infection and those with patients for whom there is a high clinical suspicion of COVID-19, awaiting the results of SARS-CoV-2 PCR tests.According to the 2011 England and Wales census,A staff screening programme for SARS-CoV-2 serological testing was available from 10 June 2020 to 7 August 2020 and has been described previouslyAs no prior data were available to assess between-group differences on the metrics assessed in this study, a formal sample size calculation was not feasible.A questionnaire covering demographic, occupational and behavioural factors potentially associated with risk of infection was designed with input and pretesting from infectious disease physicians, occupational physicians, virologists, microbiologists and epidemiologists. Formal reliability testing was not performed. Participants previously enrolled in a longitudinal HCW serological study (as described above) were invited by email to complete an online form containing the questionnaire in English, with the option to request the questionnaire in another language. A copy of the questions included in this questionnaire is included in the 10.1136/bmjopen-2022-063159.supp1Supplementary dataSerological testing for antibodies directed against SARS-CoV-2 was performed using the Centaur XP SARS-CoV-2 Total Antibody assay . This method is a fully automated high throughput enzyme-linked chemiluminescent bridging immunoassay which targets the S1RBD antigen of SARS-CoV-2 and can detect all immunoglobulin subclasses . The method was independently validated by Public Health England and has a reported sensitivity and specificity of 98.1% (95% CI 96.6% to 99.1%) and 99.9% (95% CI 99.4% to 100%),18Univariate logistic regression was used to assess each variable in the questionnaire for association with positive SARS-CoV-2 antibodies. Variables with a p value of <0.05 on univariate analysis were included in a Least Absolute Shrinkage and Selection Operator (LASSO) regression analysis with post-estimation extended Bayesian information criterion commands for variable selection to include in a multivariate logistic regression model. The LASSO method of variable selection was used, in preference to the older stepwise selection method, because it has been shown to lead to higher prediction accuracy and variable selection that is less sensitive to small changes in the data.Staff at CUH contributed to study and questionnaire design.A total of 2258 of 5698 (40%) invited HCWs responded to the invitation to complete an online questionnaire. Of the participants who responded to join the study, 19.65% (400 of 2044 responses) were male, the median age was 42 years (IQR 32–53 years) and 27.7% (618 of 2044) reported working in a designated COVID-19 ‘red’ area during the first wave of the pandemic. Notably, 9.8% (n=222 of 2044) of the cohort tested seropositive for SARS-CoV-2 antibodies. The demographics of the study group are shown in 10.1136/bmjopen-2022-063159.supp2Supplementary dataResponses demonstrated to have a significant association (p<0.05) with seropositivity in a univariate analysis are described in Renting a room in a shared house (OR 1.84 (95% CI 1.22 to 2.74); p=0.003) and living with another healthcare worker (OR 1.49 (95% CI 1.10 to 2.02); p=0.009) were further demographic factors associated with a significantly higher risk of infection on univariate analysis.Other than job role, specialty and direct COVID-19 patient care, a number of other occupational factors were associated with higher odds of infection, including working night shifts (OR 1.68 (95% CI 1.26 to 2.25); p<0.001), using the doctors’ mess (OR 1.77 (95% CI 1.17 to 2.69); p=0.007), spending rest or meal time with colleagues ‘most of the time’ (OR 1.99 (95% CI 1.19 to 3.33); p=0.009) and using hospital-supplied scrubs (OR 1.15 (95% CI 1.04 to 1.27); p=0.007). Those reporting having received formal personal protective equipment (PPE) training had a 40% higher risk of infection (OR 1.4 (95% CI 1.05 to 1.85); p=0.02) than those who did not. A higher proportion of those who worked in COVID-19 red areas reported receiving formal PPE training  than those not working in COVID-19 red areas , and formal PPE training no longer remained significant when controlling for ‘red area’ working (OR 1.2 (95% CI 0.88 to 1.63); p=0.20).Those reporting having adequate PPE available ‘some of the time’ (OR 1.93 (95% CI 1.22 to 3.05); p=0.005) or ‘rarely’ (OR 3.60 (95% CI 1.71 to 7.57); p=0.001) were associated with higher odds of infection compared with those who reported adequate PPE being available ‘all of the time’. A higher proportion of those reporting PPE being available ‘some of the time’  worked in COVID-19 red areas compared with those reporting PPE being available ‘all of the time’ . Attending shift handover (a staff meeting prior to shift change) where social distancing was not possible was associated with a higher risk of infection (OR 1.74 (95% CI 1.31 to 2.30); p<0.001). Working predominantly from home between March and June 2020 was associated with a protective effect (OR 0.60 (95% CI 0.39 to 0.91); p=0.016), as was working from home between June and July 2019 (OR 0.58 (95% CI 0.36 to 0.94); p=0.026).Reporting being a smoker was associated with a lower risk of infection (OR 0.37 (95% CI 0.18 to 0.76); p=0.007) among behavioural risk factors. Reporting drinking alcohol was associated with a lower risk of infection (OR 0.74 (95% CI 0.55 to 0.98); p=0.38); however, frequency of drinking alcohol had no effect on risk of infection. Having food or grocery deliveries to home ‘daily’ was associated with a higher risk of infection between March and May 2020 (OR 5.38 (95% CI 1.27 to 22.8); p=0.022) and June and July 2020 (OR 6.1 (95% CI 1.01 to 36.7); p=0.049) compared with those who reported ‘never’ having food or groceries delivered. Exercising outdoors ‘daily’ was associated with a lower risk of infection between March and May 2020 (OR 0.58 (95% CI 0.39 to 0.86); p=0.007) and between June and July 2020 (OR 0.56 (95% CI 0.37 to 0.84); p=0.005) compared with those who reported exercising outdoors less than once per week.The variables selected by the LASSO model are shown in We used a multivariate logistic regression model to include all variables selected by LASSO modelling. In this model, working in a designated COVID-19 area and having a household member with a previous positive SARS-CoV-2 PCR swab were not significantly associated with the participant having a positive antibody test result (p>0.05) and were dropped from the final model. A total of eight variables were included in the final multivariate logistic regression model .In this resulting model, the highest reported adjusted ORs (aORs) associated with participants testing seropositive for SARS-CoV-2 antibodies were having a household member who had previously tested positive for SARS-CoV-2 antibodies (OR 6.94 (95% CI 4.15 to 11.6); p<0.0001) and being of black ethnicity (6.21 (95% CI 2.69 to 14.3); p<0.0001). Occupational factors associated with a higher risk of seropositivity were working as a physiotherapist (aOR 2.78 (95% CI 1.21 to 6.36); p=0.015) and reporting that they predominantly worked in acute medicine (aOR 2.72 (95% CI 1.57 to 4.69); p<0.0001) or medical subspecialties (not including infectious diseases) (aOR 2.33 (95% CI 1.4 to 3.88); p=0.001). Reporting that adequate PPE was ‘rarely’ available was associated with an aOR of 2.83  and reporting attending a handover where social distancing was not possible was associated with an aOR of 1.39 .In this systematic evaluation of demographic, occupational and behavioural risk factors associated with COVID-19 seropositivity among HCWs, we have identified several targetable risk factors for HCW infection from SARS-CoV-2. These may also serve as a framework for targeting HCW risk during future respiratory pathogen pandemics. The ability of healthcare systems to cope with the surge of infections requiring hospitalisation has been challenged in a number of countries including the UK,Both a positive SARS-CoV-2 antibody in a household member and prior symptoms in a household member were significantly associated with seropositivity in a multivariate model. The finding that a positive PCR test in a household member was not associated with seropositivity on multivariate analysis may reflect a proportional relationship between viral load and transmissibility in asymptomatic infections. A study of cycle threshold (Ct) values  in uncomplicated community SARS-CoV-2 demonstrated that self-reported symptoms were an independent predictor of lower Ct value , and that Ct values were significantly higher in those who remained antibody negative.The finding that black ethnicity remained highly significantly associated with seropositivity after controlling for many plausible explanations is concerning. The effect of increased risk of infection in certain ethnicities has been reported elsewhere; the reasons for this are complex and remain poorly understood but may include increased risk of household transmission.34 36The subjective feeling that adequate PPE was rarely available remained highly statistically significant in the final multivariate model. While interesting, this finding requires a careful consideration of context and the subjective nature of the question. The availability and standard of PPE at CUH have been reported as exceeding that recommended by Public Health England for HCWs during the period of the study.40The impact of social distancing on the risk of COVID-19 infection is now well documented.Physiotherapy played a key role in both ICU and acute medical wards with therapeutic positioning, early mobilisation and breathing exercises.These analyses have limitations. By their nature, questions about behavioural factors contain subjective answers and must be interpreted with caution, including the subjective experience of availability of PPE. In addition, the questionnaire was sent to participants 3–7 months following the period encompassed by the questions, which could add imprecision. This delay leaves responses open to recall bias;however, most important factors assessed here  are objective and are unlikely to have changed in the intervening period. Participants were aware of their serostatus at the time of completing the questionnaire, which may also have influenced responses to subjective questions, particularly around the availability of PPE. We have previously shown that porters and domestic staff are at a higher risk of infection47Our work identified a number of targetable risk factors for mitigation of the risk of HCW infection during the ongoing COVID-19 pandemic. Maintaining vigilance and providing adequate social distancing space for shift change handover are likely to reduce the risk of HCW infection. The subjective experience of staff towards PPE should be considered when providing adequate and safe PPE provision and training. In addition, there are a number of non-modifiable risk factors, which nevertheless are feasible for extra mitigation strategies for healthcare professionals working within a health service to reduce the risk of HCW infection.