How are states’ firearm laws associated with firearm-related deaths in nearby states?In this pooled cross-sectional analysis involving firearm laws and firearm-related deaths from 2000 to 2019 in the 48 contiguous states, a permit requirement for purchasing all firearms had an interstate association with decreased total firearm-related deaths and homicide, whereas the prohibition of firearm possession for individuals who have committed a violent misdemeanor had an interstate association with decreased firearm suicide.These findings suggest that synergic legislative action to implement firearm laws in proximate states may help prevent firearm-related deaths. This cross-sectional study analyzes the interstate association of firearm-related deaths with firearm laws in contiguous states. Firearms are easily transported over state borders; hence permissive firearm laws in one state may have an interstate association with firearm-related deaths in nearby states.To examine whether certain firearm laws have an interstate association with firearm-related deaths in nearby states.This cross-sectional observational study used data on state firearm-related deaths in the 48 contiguous states of the US between January 1, 2000, and December 31, 2019. A spatial autoregressive model with fixed effects for state and year was used to evaluate within-state, interstate, and overall associations between firearm laws and firearm-related deaths. Analyses were performed during January 2022.The following 9 types of laws were evaluated: universal background checks for all firearms purchase, background checks for handgun sales at gun shows, license requirement to purchase all firearms, state dealer license requirement for handgun sales, requirement of retaining records of handgun sales, ban on purchasing a handgun on behalf of another, prohibition of firearm possession by persons who committed violent misdemeanors, required relinquishment of firearms for persons becoming prohibited from possessing them, and discretion in granting a concealed carry permit.State-level total firearm-related death rates, suicide rates, and homicide rates.In sum, the study period included 662 883 firearm-related deaths of all intents. License requirement for firearm purchase had a within-state association , interstate association , and overall association  per 100 000 population decrease in total firearm-related deaths. This law also had within-state association , interstate association , and overall association  per 100 000 population decrease in firearm-related homicide.The findings of this pooled cross-sectional analysis suggest that certain firearm laws in one state were associated with other states’ firearm-related deaths. Synergic legislative action in adjacent states, federal firearm legislation, and measures that reduce migration of firearms across state borders should be part of the overarching strategy to prevent firearm-related deaths. On June 23, 2021, the Biden-Harris administration announced its strategy to combat gun violence using funding from the Rescue Act,2 a critical component of which addresses the flow of firearms used to commit crimes by launching multijurisdictional firearms trafficking strike forces to stop illegal gun trafficking across state lines.Gun violence continues to be a major public health problem in the US. The years 2020 and 2021 have seen extraordinarily high numbers of gun-related fatalities.5 Although actual firearm migration is difficult to measure, there is a growing body of research on interstate differences in gun regulations and within-state firearm-related violence.9 A recent study by Liu et al9 used data on all states from 2000 to 2017 and found that weaker firearm laws in neighboring states were associated with higher within-state firearm-related deaths and, further, that failing to account for weaker laws in neighboring states led to underestimation of the impact of the state’s own laws on within-state firearm-related deaths.Guns are easily transportable, and there are relatively few barriers to gun migration and gun trafficking from states with weak gun regulations to states with strong gun regulations. For example, crime gun tracing data suggest that, in high-regulation states, crime guns are more likely to have been purchased out of state.9 in 2 critical ways. First, instead of considering the laws in neighboring states only, we used a spatial analysis approach that considers the geographical distance between all contiguous states in the US . Second, instead of aggregating across firearm laws, this study used a more granular approach toward categorizing firearm laws to better inform policy makers on which laws were most strongly associated with firearm-related deaths.The present study extends the work of Liu et alSTROBE) reporting guideline.All states of the US were included in this cross-sectional study except Alaska and Hawaii because they are noncontiguous with other US states. The District of Columbia was excluded because it has no relevant entries on state laws in the State Firearm Law Database. The final analysis included the remaining 48 states. The study period was from January 1, 2000, to December 31, 2019. This study was deemed exempt from human participant review by the institutional review board of the University of Alabama at Birmingham. The study followed the Strengthening the Reporting of Observational Studies in Epidemiology  of the Centers for Disease Control and PreventionThe rates of total firearm-related deaths of each state and year were calculated as the main outcome variable. The rates of firearm-related homicide and suicide were also calculated as secondary outcomes.11 developed by Siegel et al.12 This database tracks the presence of 134 laws in 14 categories across all 50 states from 1991 to 2020. Categories include buyer regulations, dealer regulations, background checks, prohibition of gun purchase and possession, domestic violence–related gun laws, “stand your ground” laws, concealed carry permitting laws, assault weapons regulations, gun trafficking laws, and restrictions on places where guns may be carried. Each of the 134 laws are coded based on their year of implementation as being either present (1) or absent (0) for each state during each year. The deciles of the total number of all firearm laws were used as an index to capture the strictness of states’ own firearm regulations.Information on state firearm laws was obtained from the State Firearm Law Database17: (1) requiring universal background checks at the point of purchase for all firearms (background check laws); (2) requiring background checks for handgun sale at gun shows (gun show laws); (3) requiring a license or permit to purchase all firearms (permit laws); (4) requiring a state dealer license for sale of handguns ; (5) requiring all private sellers and licensed dealers to keep and retain records of handgun sales (record-keeping laws); (6) prohibiting any person from purchasing a handgun on behalf of another person (straw purchase laws); (7) prohibiting firearm possession for people who have committed a violent misdemeanor (violence prohibition laws); (8) requiring people to relinquish their firearms after they become prohibited from possessing them (relinquishment laws); and (9) providing authorities with discretion in deciding whether to grant a concealed carry permit, or the law bans all concealed weapons (may-issue laws).For this study, the following categories of laws were identified based on previous literature as having potential association with interstate movement of firearms and firearm-related mortality18 These covariates included population size, proportion 65 years or older, race and ethnicity , unemployment rate, poverty rate, and proportion of population 25 years or older without a high school diploma, which were obtained from the US Census Bureau for 2000 to 2019.19 Property crime rates were obtained from the Federal Bureau of Investigation’s summary reporting system via Crime Data Explorer20 as a measure of the propensity for crimes in the state. The per capita number of licensed gun dealers, obtained from the US Bureau of Alcohol, Tobacco, Firearms and Explosives,21 and the percentage of the state’s population holding a hunting license, obtained from the National Survey of Fishing, Hunting, and Wildlife-Associated Recreation,22 were included as indicators of household gun ownership.24 As a measure of the general state sentiment toward firearm regulation, the vote share differences between the Republican and Democratic presidential candidates in each presidential election year within the study period25 were included, and extrapolated for years between presidential elections.We controlled for additional state-level and potentially time-varying covariates that may be associated with the number of violent deaths based on previous literature.26:27 that compares what would happen in the  scenario in which all those 48 contiguous states have that particular law with the  scenario in which no state has that law. Furthermore, this total effect can be decomposed into within-state (direct effect) and interstate (indirect effect or spillover effect) associations.27 Details are shown in eMethods and eFigure 1 in the To investigate the association between states’ firearm laws and other states’ firearm-related deaths (interstate association), we built a spatial autoregressive linear model , accounting for geographically correlated dependent and independent variablesP = 0. Then each law of interest was added to the basic model one at a time, and we selected laws that resulted both in a decreased Akaike information criterion and P < .10 for the likelihood ratio test between the model including this law and the basic model for the final model. Then we built a pairwise correlation matrix for the selected laws to check for the potential collinearity, and highly collinear variables were removed from the final model. The final model was also used for analyzing the secondary outcomes.To account for the distance decay of the association between firearm laws and outcome, the inverse distance squared weight matrix was used for the primary analysis, because the contiguity matrix may underestimate the association. First, a basic model without any of the 9 laws of interest was built  with 28 fixed effects, (2) using a contiguity matrix instead of an inverse distance squared matrix, and (3) adding a 1-year lagged total firearm-related death term to build a dynamic spatial Durbin model and address potential temporal autocorrelation of firearm-related deaths within state.We performed the following sensitivity analyses: (1) using state random effects in addition to year and census divisionP < .05. Stata, version 16.1 (StataCorp LLC) was used for all analyses. We built spatial Durbin models using the xsmle package developed by Belotti et al,29 which had been used for spatial analysis in the previous literature.31For all models, the effect sizes (the number of deaths per 100 000 population) with 95% CIs of the within-state, interstate, and overall associations between firearm laws and firearm-related deaths were reported. Standard errors were clustered within each state. Statistical significance was set at 2-sided Baseline variables are summarized in The effect sizes of within-state, interstate, and overall associations between each law and total firearm-related deaths rate from models where those laws were included individually are shown in eTable 1 in the P = .06). The effect sizes of the associations reflect the differences of the outcome in the hypothetical scenario when all 48 contiguous states have that law vs none of those states have that law. eFigures 2 and 3 in the The following 5 laws were included in the final model: (1) permit, (2) gun show, (3) violence prohibition, (4) relinquishment, and (5) may-issue , all of which had 50% or more observations missing, were removed from the analysis of firearm homicide (additional details are provided in the eMethods in the In this cross-sectional study, we used data from 2000 to 2019 to investigate the association between firearm laws and firearm-related deaths both within state and interstate. Our findings suggest that having a permit requirement for purchasing all firearms was associated with a decrease of any firearm-related death and firearm-related homicide within state, interstate, and overall. The prohibition of firearm possession for individuals who have committed a violent misdemeanor had within-state and overall associations with decreased total firearm-related death rates. This law also had within-state, interstate, and overall associations with decreased firearm-related suicide rates.34 States with strict laws also host fewer firearm-manufacturing establishments than states with relatively permissive laws.35 However, firearms are easily transported across state lines, and crime guns in states with restrictive firearm laws often originate in states with more permissive rules,5 with stronger trafficking flows detected when such states are near each other.36 Hence, a small but growing literature9 has started exploring whether permissive firearm laws in one state have spillover effects on measures of firearm violence in other states. More recently, Morrison et al36 used county-level firearm-related homicide data to evaluate the spillover effects of different categories of firearm laws. They found that the benefits of within-state firearm laws in reducing firearm-related homicide were not independent of firearm laws in nearby states, which is supported by our study findings.Firearm laws vary substantially across states in the US. A rich literature has established associations between stricter state firearm laws and reduced firearm-related violence within those states.17 are also protective for neighboring states, suggesting that more restrictions on the eligibility of obtaining a gun also discourage out-of-state persons from buying firearms from those states. Further, the laws prohibiting firearm possession for people who have committed a violent misdemeanor were associated with decreased firearm-related suicide rates within state, interstate, and overall and with decreased total firearm-related death rates both within state and overall. Prior studies have found that safe storage laws37 and alcohol regulation38 were associated with within-state declines in firearm suicides. At the same time, other studies40 have established correlations between committing acts of violence and inflicting self-harm,40 and our findings imply that regulations preventing firearm possession among those who commit violence in turn also reduce extreme self-harm in both within-state and nearby state populations. We speculate that spillover protective effects occur because the presence of these laws disincentivizes gun traffickers from acquiring guns in these states to send to neighboring states and disincentivizes individuals in neighboring states from crossing state borders and purchasing a weapon.Our study adds to this literature by evaluating specific firearm laws individually rather than as an aggregated index. Particularly, our findings suggest that permit-to-purchase laws, which research finds to be protective against within-state firearm-related deaths,In models that included laws individually with other state covariates, may-issue and record-keeping laws also showed associations with total firearm-related deaths. However, collinearity issues (eTable 3 in the We acknowledge several limitations to this study. First, our analytical strategy used state fixed effects to minimize the effect of time-invariant state-level confounders and hence relied on within-state variation in firearm laws to estimate effect sizes; however, many states had minimal variations in firearm laws during the study period, and states implementing laws such as background check and record keeping often implemented them at similar times, leading to collinearity issues. These factors may have limited the ability of our model to detect statistical effects for certain laws. Second, the presence of laws per se may not indicate the diligence with which laws are enforced in different states. Third, results may be sensitive to alternate approaches to selecting or grouping firearm laws of primary interest. Fourth, the effect sizes can be affected by the matrix accounting for the geographic correlation. Moreover, the weight matrix used in this study solely focused on the distance. Combination of distance with other factors, such as population, firearm manufacturing facilities, and number of interstates and other highways, might be more accurate to describe the attractiveness of firearms for a state and the ease of firearm movement between states. Fifth, because the analysis of firearm-related homicide did not use data from all states, caution should be used when generalizing the findings to the whole country. Last, as in the case of all ecological studies, caution should be exercised when drawing causal inferences from the results.In this pooled cross-sectional analysis of firearm laws and firearm-related deaths from 2000 to 2019, we found that permit-to-purchase laws were associated with decreased firearm-related death rates both within state and interstate. The presence of interstate association between firearm laws and firearm-related deaths suggests that policy initiatives to reduce gun trafficking—such as those adopted by the Biden-Harris administration—are an important component of eliminating firearm violence. It also underlines the importance of synergic legislative action to implement laws such as permit requirements in proximate states as an effective approach to reduce firearm-related deaths.


We aimed to assess the diversity and practices of existing studies on several assays and algorithms for serial measurements of high-sensitivity cardiac troponin (hs-cTn) for risk stratification and the diagnosis of myocardial infarction (MI) and 30-day outcomes in patients suspected of having non-ST-segment elevation MI (NSTEMI).We searched multiple databases including MEDLINE, EMBASE, Science Citation Index, the Cochrane Database of Systematic Reviews and the CENTRAL databases for studies published between January 2006 and November 2021. Studies that assessed the diagnostic accuracy of serial hs-cTn testing in patients suspected of having NSTEMI in the emergency department (ED) were eligible. Data were analysed using the scoping review method.We included 86 publications, mainly from research centres in Europe, North America and Australasia. Two hs-cTn assays, manufactured by Abbott (43/86) and Roche (53/86), dominated the evaluations. The studies most commonly measured the concentrations of hs-cTn at two time points, at presentation and a few hours thereafter, to assess the two-strata or three-strata algorithm for diagnosing or ruling out MI. Although data from 83 studies (97%) were prospectively collected, 0%–90% of the eligible patients were excluded from the analysis due to missing blood samples or the lack of a final diagnosis in 53 studies (62%) that reported relevant data. Only 19 studies (22%) reported on head-to-head comparisons of alternative assays.Evidence on the accuracy of serial hs-cTn testing was largely derived from selected research institutions and relied on two specific assays. The proportions of the eligible patients excluded from the study raise concerns about directly applying the study findings to clinical practice in frontline EDs.CRD42018106379. This is the first scoping review that explored the diversity of study methodologies adopted in studies on testing with serial high-sensitivity cardiac troponin measurements in patients with suspected non-ST elevation myocardial infarction in the emergency department.The review method included a comprehensive literature search, duplicate assessment of eligibility, double extraction of data and descriptive synthesis through tables and graphs.The assessed specifications included the regions and sources of studies, targeted participants and their study flow, troponin assays, sampling algorithms and direct comparisons thereof, and definitions of outcomes.The quantitative results about the accuracy and clinical outcomes were not assessed because this was beyond the scope of the review.Acute coronary syndrome (ACS) is considered a major cause of death worldwide.3 4For the clinical management of NSTEMI, cardiac troponin (cTn) has been used as the mainstay of clinical diagnosis since 2000.3 4However, the comparative effectiveness of management strategies based on serial hs-cTn measurements has not been fully elucidated, because several alternative assays are clinically available and existing reports are from studies with different designs and inconsistent testing algorithms. Thus, this study aimed to explore the diversity of the methodologies used in primary studies on serial hs-cTn measurements in patients suspected of having ACS in the emergency department (ED). We constructed an evidence map of existing studies on serial hs-cTn testing for diagnosing NSTEMI and predicting 30-day clinical outcomes. We critically appraised the currently available evidence and highlighted the issues that need to be addressed in future research.https://bmjopen.bmj.com/content/9/3/e026012.long.14This study is a focused analysis performed in conjunction with a registered systematic review project (PROSPERO registration number CRD42018106379). The protocol for the original systematic review is available at We searched Ovid MEDLINE, EMBASE, Science Citation Index and Cochrane Database of Systematic Reviews for studies published between 1 January 2006 and 17 November 2021 with no restrictions of language or publication status. The search terms included “chest pain”, “acute coronary syndrome,” “myocardial infarction,” “cardiac troponin”, “emergency room” and their synonyms.10.1136/bmjopen-2022-066429.supp1Supplementary dataWe included prospective and retrospective studies that evaluated patients aged ≥18 years who were suspected of having NSTEMI in an ED and had two or more serial troponin measurements using an hs-cTn assay. Eligible were studies that reported the diagnostic accuracy of AMI and/or 30-day clinical outcomes. We only included single-gate design studies, that is, studies that consisted of a single group of subjects based on a single eligibility criteria.The following data were extracted: (1) publication and study characteristics: authors, journal name, publication year, enrolment years, number of eligible and included patients, study design, the name of the study cohort(s), geographical region(s), participant age and use of ECG to exclude patients; (2) test characteristics: assays, the timing of blood sampling, cut-off values, algorithms adopted  and (3) reference standard characteristics: specific diagnostic criteria of MI, such as those defined in clinical guidelines and/or versions of the universal definition of MI, and the assessors of the final diagnoses.Our target population was a group of patients suspected of having NSTEMI who presented at the ED. We recorded the numbers of patients suspected of having NSTEMI who presented at the ED, patients who completed one or more study-specific serial testing algorithm(s), and patients who were assessed for test accuracy and/or 30-day clinical outcomes. A testing algorithm was specified based on the number and timing of hs-cTn measurements. The results for the algorithm were typically reported as a single value measured at presentation, together with an absolute or a relative difference between specific measurement time points , which was categorised as delta or percent change in the hs-cTn concentrations. We classified the studies that involved two hs-cTn measurements into three groups, namely, 0 and 1 hour, 0 and 2 hours, and 0 and 3 hours, based on the blood sampling timing (in hours) of the first and second samples. Other algorithms involving three or more blood samples were grouped into a separate category, labelled as ‘others.’ Assays were classified by specific manufacturers, that is, Abbott , Roche , Siemens  and Beckman . Assays by other manufacturers were categorised as ‘miscellaneous.’To assess the evidence, we used the study with the largest sample to avoid double-counting when multiple studies reported  overlapping patient populations. For the study locations, we assumed that each specific research institution involved in the study assessed patients residing within its geographical region only. Comparative studies were defined as studies that adopted a paired design to assess multiple assays on the same study participants and directly compared the diagnostic accuracy for AMI or 30-day clinical outcomes. This review did not standardise the definition of AMI or the 30-day clinical outcomes and adopted the study-reported outcome definitions as specifically reported.We considered each publication to be the unit of analysis and performed descriptive analyses by using percentages or medians and ranges. We combined data as a weighted average only if the pertinent data were available for specific subgroups. The assessed design specifications included the regions and sources of studies, characteristics of targeted participants and their study flow, specific troponin assays used, sampling algorithms with their operational characteristics, direct comparisons of two or more algorithms and/or assays, and definitions of the index MI and 30-day outcomes.https://www.mapcustomizer.com/).The volume of clinical evidence was assessed with graphs and tables using Stata V.17.0 (Stata). The graphical presentation of the study locations was constructed using Google Maps  and Mapcustomizer (available at We did not involve patients or the public in the preparation of this scoping review.Eighty-three studies (97%) were based on prospectively collected blood samples, and the median sample size was 1391 (range 160–6403) . The mosOf the 86 cohorts that reported accuracy and/or 30-day outcome data using a specific assay and a sampling algorithm, only 42 (49%) were considered unique, which included 78 606 non-overlapping patients . Of the Nineteen studies compared two or more assays using a paired design . The mos17 51Typically, the studies reported only the number of enrolled patients , and the complete data on all clinically relevant patients  were missing. Fifty-two studies (60%) reported quantitative data on patients who failed to complete a study-specific serial testing algorithm(s) or patients whose diagnosis or 30-day clinical outcomes could not be established . VariousThe mean or median participant age ranged from 53 to 73 years, and most studies involved patients in their 40s–70s . Only on52Forty-seven studies (55%) assessed the three-strata algorithms. These studies stratified patients into ‘rule-out (low-risk),’ ‘observational zone (intermediate-risk)’ and ‘rule-in (high-risk)’ groups according to two sets of diagnostic criteria based on the concentrations of hs-cTn at baseline, 1–3 hours, and/or the difference between the hs-cTn concentrations of the two samples. Other studies conventionally categorised the patients into two strata ’ and ‘rule-in (high-risk)’ groups) based on a single set of criteria. The majority of data were based on assays manufactured by either Abbott or Roche, and the 0 and 1-hour algorithm was the most frequently reported .Eighty-four studies (98%) adopted the universal definition of MI 34–37 45 57 62 65 75 94 95To the best of our knowledge, this is the first scoping review to comprehensively assess the reports on testing with serial hs-cTn measurements in patients with suspected NSTEMI in the ED. We summarised how studies measured serial hs-cTn and assessed its diagnostic accuracy for AMI and the 30-day clinical outcomes. Our results showed that most existing data were based on the Abbott cTnI or Roche cTnT assays, and the timing of the blood measurements and the diagnostic algorithms varied. The number of studies assessing these two assays using the 0 and 1-hour, 0 and 2 hours, and 0 and 3 hours protocols has been continuously increasing since 2011 when guidelinesWe comprehensively explored the existing evidence, focusing on how the studies were designed, analysed and reported and, the implications for clinical practice of the identified limitations and concerns. Previously reported systematic reviews focused on a two-strata rule-out strategy using unique serial measurements of hs-cTnT assay onlyThis scoping review performed a focused analysis on the reported study methodologies and did not address the primary objectives of the originally planned systematic review. Therefore, several limitations need to be discussed. First, we did not assess the quantitative results on accuracy and other clinical outcomes because this was beyond the scope of the present scoping review. Second, we focused on studies that assessed only test accuracy and 30-day outcomes. Therefore, data on studies that evaluated long-term outcomes have not been addressed. Third, since we excluded conference abstracts, this review may have missed newer relevant publications. Fourth, the recently developed clinical prediction rules (eg, History, ECG, Age, Risk factors and Troponin Score,34 36 37 45 57 62 65et al proposed a high-accurate risk-assessment tool for NSTEMI using a serial hs-cTn measurements based on an international consortium of prospectively registered data from 15 cohorts. Of these, nine cohorts were also assessed in this scoping review, at least some portions of which should have contributed to the dataset deriving the risk-assessment tool. Given the methodological concerns raised in this review, depending on how the study methodologies in the rest of the data have been addressed and/or improved, uncertainties still exist in its individualised application in real-life clinical practice. First, ACS and MI are common in elderly populations as well as patients with CKD,Recently, a pooled study conducted by Neumann Data on diagnostic test accuracy and short-term outcomes by serial hs-cTn measurements were largely derived from particular research institutions in Europe, North America or Australasia and based mainly on two specific assays. The exclusion of variable proportions of eligible patients, which was inevitable even in well-conducted prospective studies, raised concerns regarding the studies’ generalisability and direct applications in real-world ED clinical practice.


Due to the widespread Omicron variant of SARS-CoV-2 in Thailand, the effectiveness of COVID-19 vaccines has become a major issue. The primary objective of this study is to examine the real-world effectiveness of COVID-19 vaccines based on secondary data acquired under normal circumstances in a real-world setting, to protect against treatment with invasive ventilation of pneumonia during January to April 2022, a period when Omicron was predominant. We conducted a nationwide test-negative case–control study. The case and control were matched with a ratio of 1:4 in terms of age, date of specimen collection, and hospital collection specimen and the odds ratio was calculated using conditional logistic regression. Overall, there was neither a distinction between mix-and-match regimens and homologous mRNA regimens against severe symptoms, nor was there a decline of the protective effect over the study period. The third and fourth dose boosters with ChAdOx1 nCoV-19 or mRNA vaccines provided high levels of protection against severe outcomes, approximately 87% to 100%, whereas two doses provided a moderate degree (70%). Thus, this study concludes that current national vaccine strategies provide favourable protective benefits against the Omicron variant. All Thais should receive at least two doses, while high-risk or vulnerable groups should be administered at least three doses. As of 5 May 2022, there have been more than 500 million confirmed cases of coronavirus disease 2019 (COVID-19) and over 6 million deaths worldwide since the pandemic began ,4,5.Thailand began vaccinating its population on 28 February 2022 . Since tThailand is currently experiencing the country’s fifth wave of the COVID-19 pandemic, with the highly contagious Omicron variant accounting for 70% of all strains in January, and 100% in April 2022 . Since 1As COVID-19 is going to be an endemic disease, not only in Thailand but also worldwide, this study pays attention to the severe outcomes of the disease rather than overall number of infections. The aim of this study is to determine the real-world effectiveness of COVID-19 vaccine regimens against treatment with invasive ventilation in Thailand.An individually matched test-negative case–control study was conducted with a case–control ratio of 1:4. The study population was Thai individuals tested for SARS-CoV-2 between 1 January and 30 April 2022 by healthcare professionals. The indications for testing included those at high-risk of being in contact with a COVID-19 case and active case finding in communities by outbreak investigators.A COVID-19 case was classified as an individual who had a positive test for SARS-CoV-2 by reverse transcriptase polymerase chain reaction (RT-PCR) or professional-use antigen test kit (ATK) at the laboratory certified by National Institute of Health. Additionally, the case had no prior history of SARS-CoV-2 detection in the preceding three months. A control was an individual who had not been detected with SARS-CoV-2 by RT-PCR or ATK. Case and control subjects were matched with ratio of 1:4 with respect to age . The other two matched variables included date of laboratory collection  and site of specimen collection.We analyzed vaccine effectiveness using secondary data, which were retrieved from four main national health databases: Co-Lab , Co-Ward , COVID-19 Death , and MOPH-IC . We used the national identification numbers of each individual as a unique identifier to link the same person across databases.We used the Co-Lab database to access demographic data of individuals, test results, indications of testing, and laboratory collection sites. The Co-Lab database retrieved laboratory test results from public and private hospitals, provincial public health offices, and regional offices of disease prevention and control.For assessing the severity of COVID-19 cases, we used information from the Co-Ward database and the COVID-19 death database. The Co-Ward database collects data of COVID-19 cases severity admitted in both public and private health facilities. The database stores information about the severity of COVID-19 cases , medications, bed occupancy, and types of oxygen support. Severe cases were defined as cases with pneumonia-like symptoms and with oxygen saturation of less than 95% in room air .For vaccination status, the MOPH-IC database contains information on all individuals administered with COVID-19 vaccines in Thailand. It is a mandatory health data system; therefore, the likelihood of missing records is limited. The MOPH-IC database collects information about national identification number, risk category, vaccination date, and vaccine manufacturers.We started with descriptive statistics to examine the overview of the data. Categorical data were presented descriptively using frequency counts and proportions while continuous variables were presented with mean and standard deviation (SD).For the analysis on vaccine effectiveness (VE), we used conditional logistic regression where the primary outcome was severe COVID-19, characterized as requiring invasive oxygen support. Controls were participants who did not meet the severe definition. The secondary outcome was any infection, defined as individuals who were detected with SARS-CoV-2, by RT-PCR or ATK whereas controls were those showing negative test results. In terms of death, the case was a participant who died of COVID-19 while controls composed of non-SARS-CoV-2 infected individuals and non-dead COVID-19 cases.Vaccination status served as the independent variable. Participants were regarded as a vaccine recipient if they were administered the vaccine at least fourteen days prior to the sample collection. We excluded participants who had received the last dose of vaccine within fourteen days before the sample collection. Unvaccinated individuals were defined as those who had not been vaccinated prior to laboratory collection.An odds ratio (OR) was calculated with a 95-percent confidence interval and vaccine effectiveness was defined as one minus the OR multiplied by one hundred. Matching was used to account for potential confounders, namely, age, address and residential area of the hospitals, and time of infection (months of SARS-CoV-2 test).We assessed the vaccine effectiveness by the number of vaccines administered stratified by age group . The time interval between vaccination date and sample collection was also categorized and stratified to explore the vaccine effectiveness over the six-month course. Additionally, we evaluated the effectiveness of two, three, and four doses of COVID-19 vaccine by each regimen.The study was approved by the institutional review board of Thai Department of Disease Control, Ministry of Public Health .We obtained a total of 3,059,616 records including: 1015 cases of COVID-19 pneumonia requiring invasive ventilation (0.16%) from 652,854 cases with SARS-CoV-2 detection and 2,406,762 controls or non-SARS-CoV-2 detection, during 1 January to 30 April 2022. As shown in This study is a large study using nationwide surveillance data. For the real-world effectiveness of overall vaccine combinations, we found that as the dose of vaccines increased, the severity of COVID-19 assessed by oxygen therapy decreased. There is no distinction between mix-and-match regimens and homologous mRNA regimens against severe symptoms and deaths. The study also emphasizes the importance of the third dose and fourth dose boosters with ChAdOx1 nCoV-19 or mRNA vaccines as they can provide substantial protection against pneumonia requiring invasive ventilation and fatalities in adults and the elderly; however, three doses of the COVID-19 vaccine provided minimal protection against COVID-19 infection. As this study was conducted using data obtained during January to April 2022, (which is when the Omicron variant (BA.1 and BA.2 lineage) became a dominant strain in Thailand from more than 80% coverage in January to 100% in April) our findings conclude that the current national vaccine strategies provide favourable protective benefits against the Omicron variant .Previous evaluations of three-dose vaccine effectiveness against SARS-CoV-2 infection in Thailand, when the Delta variant assumed the dominant share, reported the effectiveness level against SARS-CoV-2 infection at around 80–90% for three doses ,17,18. TWhile three doses of the COVID-19 vaccine demonstrated little protection against SARS-CoV-2 infections with the Omicron variant, four- and five- dose regimens showed moderately high degrees of protection (over 70%), which is consistent with Chariyalertsak’s study . HoweverAlthough the protective effect of three doses of the COVID-19 vaccine against any infection of SARS-CoV-2 in this study was low, the vaccine effectiveness of at least a two-dose regimen against severe pneumonia and deaths was obviously high. Our result is similar to a previous report from the UK which showed that two and three doses of the COVID-19 vaccine obviously provided protective benefits against hospitalization requiring oxygen support or intensive care, and the duration of effectiveness can be prolonged to more than 175 days during the Omicron epidemic . Fourth In our study, the third dose and fourth dose of COVID-19 vaccine offer a high level of protection against severe outcome in the elderly. The finding is consistent with studies by Grewal et al. and Arregocés-Castillo et al. that demonstrated a high vaccine effectiveness against hospitalization and fatalities ,25. The In terms of severe outcomes, the vaccine effectiveness for those receiving two-dose mixing regimens was comparable to those acquitting homologous mRNA vaccines. All three-dose regimens provided a very high protective effect (80% to nearly 100%). The results are compatible with a previous study by Wing Ying Au et al. which found that heterologous regimens exhibited comparable effectiveness to homologous mRNA regimens . HoweverOur findings suggested that the current regimens of three doses of COVID-19 vaccines, homologous and heterologous, showed a high degree of protection against severe outcomes. This result supports the current national vaccine strategy of Thailand that ratifies the use of heterologous regimens. The booster dose of COVID-19 should be considered and administered in addition.The study contains a few limitations. As the study used routine secondary data collected from various sources, the collected variables were limited, and the quality of data varied. This made the analysis prone to residual confounding and information bias. Information on some potential confounders such as underlying disease and detailed risk behavior were not available due to data inaccessibility. However, we attempted to limit the extent of confounding by including age in the modelling process. Age can be considered to be a proxy of underlying disease. Despite the matching on age to reduce the confounding effect of underlying diseases, we also limited the participants to those with the testing indications of contact with COVID-19 cases or being at risk of contracting the disease, which could be considered as a proxy of risk behavior. Due to a limited sample, the effectiveness of some vaccination regimens and doses could not be evaluated. Especially the time interval stratification results, we could not estimate effectiveness by a shorter interval, less than a three-month interval. The cases in the study included people who had a SARS-CoV-2 detection by either RT-PCR or ATK test. A person with ATK negative results might be actually a case  due to ineffective sample collection, ability of the test and inaccurate reporting, resulting in the misclassification of the outcome. This error from the sample collection is limited as samples were collected by professional health workers; however, further misclassification from other causes could not be avoided. For infection outcomes, misclassification of the outcome could not be evaded. This might be due to the test performance itself (self-ATK testing) and that the current reporting system in Thailand does not require all individuals with positive self-test ATK to report to the authorities. This meant the results of the self-test ATK were not mandatorily reported or linked with the Co-Lab surveillance. Then, some cases might be misclassified as control. Therefore, the effectiveness of COVID-19 infection from the study should be interpreted with caution. For severity outcomes, pneumonic cases needing invasive ventilation support were less likely to face misclassification of outcomes as the protocol of most hospitals in Thailand requires a patient with a severe condition to undertake RT-PCR before being admitted as an inpatient. For this reason, we decided to regard a severe condition as the primary outcome of this study. Note that non-differential misclassifications with the direction from non-severe to severe might still exist due to a lack of updated data on clinical severity after admission. In addition, as the study was conducted during the widespread of COVID-19 and could capture only those who tested positive for COVID-19 at hospitals, some samples may already have the protective immunity from infection prior to receiving the COVID-19 test at hospitals. This situation may diminish the estimated effectiveness of the COVID-19 vaccine. The waning of vaccine effectiveness could not be excluded as most of the samples were tested within five months after vaccine administration, especially the fourth and fifth doses which had only around two months of time interval between sample collection date and vaccination date. The results of the overall COVID-19 effectiveness, not stratified by vaccine regimen, have limited generalization, and should be interpreted with caution because it was based on a mix of the COVID-19 regimens that may differ to other countries. However, the effectiveness of some specific regimen combinations demonstrated in this study could be useful for other countries’ consideration. Data on individual-level variants was not available for all cases. We thus could not estimate the effectiveness of variant-specific outcomes. Instead, we relied on Thailand’s national variant coverage summary data to reaffirm that, at the time of the analysis, the Omicron variant was the most common circulating strain in the country. Lastly, as the study was conducted using a test-negative case–control study design which faced several limitations by itself, including the inability to estimate the actual infection rate of infection over time from the perspective of vaccination status. Thus, a cohort study or other study designs should be further conducted to identify the rate of infection and compare with the result of this study.Despite our results that two and three doses of COVID-19 vaccination did not provide enough protection against any infection, the vaccines provided favourable effectiveness against severe pneumonia. The protective effect was even more notable among the three-dose vaccinees. No variation in vaccine effectiveness was detected across different age groups, and there was no remarkable decline in effectiveness against severe outcomes, at least within 180 days. The combination of vaccine regimens yielded satisfactory protection against serious illness. The Thai government should recommend its population receives the booster shot (third dose and onward). Monitoring the effectiveness of various vaccine regimens while accounting for the advent of any emerging SARS-CoV-2 variants and the waning time of vaccine protectiveness using cohort study or other study designs is recommended for sub-category analyses, particularly for the regimens with small numbers.


In practical scenarios,viologen-derivatives face anaccelerateddegradation in the unavoidable presence of traces of oxygen in large-scaleredox flow batteries. Herein, we confirm the primary degradation mechanismand propose a straightforward, cheap, and fast method to evaluatethe stability of viologen-derivatives toward this degradation. Consideringthat the cleavage of the N-substituent is the main proposed pathwayfor viologen degradation, a new viologen-derivative, bearing an alkylsulfonatechain with a secondary carbon center joined to the N atom, is synthesizedto illustrate how molecular engineering can be used to improve stability. So far, many organic molecules such as anthraquinone-derivatives,2 phenazine-derivatives,3 or fluorenone-derivatives4 have beenshown to deliver high performance in alkaline media. At neutral pH,viologen-derivatives are the most commonly used active species foranolytes.1 Their performances in termsof energy density and cycle stability have been improved in recentyears by modification of the basic structures.6 The1,1′-bis[3-sulfonatopropyl]-4,4′-bipyridinium (BSPr-Vi)is one of the most commonly used viologen-derivatives in the literature,achieving high cycle stability, which is a key parameter for stationaryenergy storage, at laboratory scale.7 Oneof the main differences for viologen-derivatives at laboratory scaleis the extremely protected atmosphere for the anolyte, as the bestcyclability performance is achieved using an Ar-filled glovebox.9Generationof energy from renewablesources such as wind and solar radiation has become a key priorityfor our society. Unfortunately, the unpredictable intermittency ofthese sources requires energy storage to match energy production anddemand. Among the various energy storage systems, redox flow batteriesare called to play a key role in the transition toward a sustainableand environmentally friendly energy system. Featured by its independentscalability of energy and power, the redox flow battery is a promisingalternative for stationary energy storage. The all-vanadium flow batteryis the most mature redox flow battery technology.10 This capacity fading is not related to degradationof active species but faradaic imbalance. For research purposes, thisissue is overcome by carrying out the measurements inside an Ar-filledglovebox in the complete absence of oxygen.9 Alternatively,faradaic imbalance can be addressed for academic purposes by oversizingthe positive reservoir so that a large excess of electroactive speciesis added to the positive compartment.11 While these approaches are very useful to study theintrinsic properties at the research level, complete absence of oxygenfor long periods (>15 years) at large scale (>MWh) will be verychallenging,if not impossible, to be achieved. Fortunately, several strategieshave been recently proposed to amend this issue through various faradaicrebalance approaches.14 These strategies have been successfully implementedin all-vanadium flow batteries as well as aqueous organic flow batteriesin alkaline media at laboratory scale. However, they have not beenimplemented in AORFBs in neutral pH. Since these scalable strategiesare not based on complete exclusion of air, one needs to assess thestability of the state-of-the-art viologen-derivatives in a redoxflow battery having an oversized positive compartment so that theintrinsic stability of the viologen-derivatives under “standardexclusion of air” needs to be determined. standard air exclusion, and an excess of potassium ferrocyanidewas added to the positive compartment. In contrast to these results,reports in the literature that were carried out inside an Ar-filledglovebox show higher capacity retention values.9 This indicatesthat the presence of small amounts of oxygen remaining in the anolytefor our experiment triggers an accelerated degradation of this state-of-the-artviologen-derivative. To confirm this, the negative compartment wasbriefly exposed to air after being operated for 40 cycles under Aroverpressure  giving rise to the C–Nbond cleavage, which was initially suggested by Bird and Kuhn16 as well as Rieger and Edwards17 and recently revisited by Aziz’s group,18 gained our attention. It should be noted thatthe reduction of oxygen generates hydroxide anions, increasing thepH value of the electrolyte (O2 + 2 H2O + 4e– → 4 OH–).The presence of oxygen dissolved in the electrolyte needsto beavoided, especially for the negative comportment. The irreversiblereduction of oxygen, either through electrochemical reaction in theelectrodes or spontaneous charge transfer with reduced active speciesin the anolyte, leads to the irreversible consumption of charges duringthe charge process. As a result, the anolyte does not reach a fullstate of charge at the end of the charge process, limiting the dischargecapacity and shorting the energy storage capacity. Importantly, thepresence of oxygen leads to faradaic imbalance between the anolyteand catholyte, since the fully charged catholyte cannot be fully discharged,resulting in a progressive loss in charge capacity upon cycling.pressure 1B. WhileSection S1). For instance, aCoulombic efficiency in the first cycle of 98%, which is a very goodvalue without a glovebox, results in a pH change from 4 to 12 dueto the logarithmic scale of pH values. Likewise, four cycles at 99.5%due to a very small temporary leakage of air would have the same impactin the pH value. This means that any electroactive species that aredesigned to be implemented in neutral pH at large scale should withstandmild alkaline media, since the unavoidable entry of oxygen will changepH values to mild alkaline values. Even if the pH value is correctedby, e.g., intentionally evolving oxygen in the positive compartment,the electroactive molecule will be temporarily exposed to mild alkalinevalues at some point of its expected long cycle life. While a pH buffercan temporally maintain the pH value, it will require the additionof chemicals in the electrolyte, which will be eventually depleted.Thus, a pH probe was immersedin the negative reservoirto monitorthe evolution of the pH value during the first few cycles. The results1C clearlN-alkylated 4,4′-bipyridine derivative, 3-propane-1-sulfonate(MSPr), should be a degradation product derived from the nucleophilicattack of hydroxide anions, this compound was independently synthesizedand characterized. NMR analysis of the anolyte after 3 days of cyclingunder Ar overpressure revealed that there is a correlation betweenthe capacity fading and the presence of MSPr in the NMR spectra bis(butane-1-sulfonate)(BS3Bu-Vi), as displayed in 19 The detailed synthetic proceduresare described in Section S3. Gratifyingly,cyclic voltammetry  of the new viologen (BS3Bu-Vi) presents similar electrochemicalbehavior to that of the state-of-the-art viologen (BSPr-Vi).Fortunately, one major advantage of organic electroactive speciesis the tunability of their properties through molecular engineering.This feature could be potentially used to improve the stability ofviologen-derivatives against mild alkaline values for enhancing thepractical uses of this family of molecules. First, the degradationmechanism was further investigated. Since the mono- spectra 2, which 20 For a fair comparison, bothbattery cells were evaluated for 3 days (72 h), and their capacityfading (% h–1) was calculated as the total capacityloss after 3 days normalized by 72 h (–1 was obtained for theBSPr-Vi and the new viologen (BS3Bu-Vi), respectively, confirmingthe higher stability of the latter and the ability to engineer viologen-derivativesfor improving their stability . It should be noted that Coulombic efficiencies in bothcases were rather high considering that glovebox was not used: ca.98% in the first cycle and >99.9% after a few cycles. As mentionedbefore, the 2% Coulombic inefficiency in the first cycle would translatein a change in pH from 4 to 12. Indeed, the pH value of the electrolytewas measured after cycling for 3 days, and values above 10 were obtained.It should be noted that crossover of viologen through the membranedid not contribute significantly to the capacity fading as revealedby the postmortem cyclic voltammetries of the catholytes after cycling. NMR postmortemanalysis of the anolyte after cycling revealed the presence of mono-N-alkylated 4,4′-bipyridine (MS3Bu), confirming thedealkylation as the main degradation mechanism for the new viologenas well .Since the degradation mechanism requires theconsumption of onehydroxide anion per molecule of viologen-derivative (equation in  by 72 h 1A and 3BIn conclusion, the generation of hydroxide anionsfrom the reductionof oxygen present in the anolyte of viologen-based redox flow batteriesis confirmed to accelerate their degradation via dealkylation throughnucleophilic attack, leading to a decrease in capacity retention.Thus, the new generation of viologen-derivatives should withstandmild alkaline media for being deployed at large-scale under realisticconditions, as the entry of small amounts of oxygen in large flowingdevices is very challenging to prevent. Herein, it was shown thatmonitoring of the pH starting from mild alkaline media is a fast,cheap, and direct method to evaluate the chemical stability againstdealkylation by nucleophilic attack. In addition, a new viologen-derivativewas proposed to demonstrate that stability can be increased throughmolecular engineering. The introduction of a methyl group in the alkylchain of the viologen at the α-position with respect to theN atoms resulted in higher stability, which is attributed to a greaterdifficulty for the nucleophilic attack of the hydroxide. Because thisone major challenge for practical uses of viologen-derivatives wasidentified, a simple method for evaluation was proposed, and a newviologen-derivative was synthesized for probing the suitability ofmolecular engineering to address this issue, which clearly illustratesthat molecular engineering will play a key role in progressing towardpractical deployment of viologens.


The United States has experienced high levels of excess mortality during the COVID-19 pandemic and also has high prevalence of overweight and obesity, which increases the risk of severe infection and death from the virus. This study uses multiple cause of death data to estimate excess premature cardiovascular disease mortality in the USA in 2020 for which overweight and obesity was a risk factor.The contribution of overweight and obesity to premature (35–74 years) cardiovascular disease mortality was measured as cardiovascular disease reported on the death certificate with one or more of diabetes, chronic kidney disease, obesity, lipidemias or hypertensive heart disease (DKOLH-CVD). Excess mortality was calculated as the difference between actual and expected age-standardised death rates. Expected deaths were estimated using negative binomial regressions of monthly deaths during 2010–19.Excess DKOLH-CVD mortality in March-December 2020 was 29%  for males and 30% (28–32%) for females, much higher than for all causes , females 16% (14–17%)). Excess mortality was higher where two or more DKOLH conditions , females 41% (37–44%)) or obesity , females 47% (43–51%)) were reported. One-half of excess DKOLH-CVD mortality was reported as due to COVID-19, lower than the four-fifths of excess all-cause deaths. For home deaths, just over 10% of excess mortality for each cause classification was reported as due to COVID-19.Excess premature cardiovascular disease mortality in the USA for which overweight and obesity was a risk factor was considerably higher than for all causes, exacerbating adverse pre-pandemic trends. The contribution of COVID-19 to excess mortality appears significantly under-reported for home deaths. The COVID-19 pandemic has had a profound impact on mortality in the United States. Estimates of excess mortality—the difference between the actual and expected number of deaths—range from 930,000–1.13 million deaths or 15–18%) higher than expected for 2020 and 2021 . Excess –18% highThere has been considerable epidemiological interest in the contribution of overweight and obesity to excess mortality during the COVID-19 pandemic , 8. SeveHowever, it is challenging to accurately measure the contribution of overweight and obesity to mortality. Medical certifiers have the option of reporting overweight or obesity on a medical certificate of cause of death (or death certificate), but this is under-reported and so does not reflect its true contribution to mortality . The conDKOLH-CVD) [An alternative is to utilise multiple cause of death data, which comprise all conditions reported on the death certificate. A previous study in the USA and Australia used multiple cause of death data to estimate the contribution of overweight and obesity to cardiovascular disease mortality using deaths where at least one of diabetes, hypertension, chronic kidney disease, lipidemias (high cholesterol) and obesity were reported on the death certificate together with at least one cardiovascular disease (referred to as the acronym OLH-CVD) . These DOLH-CVD) , 22–25. OLH-CVD) .Multiple cause of death data, which is available for all registered deaths in the USA, can reveal much about the contribution of overweight and obesity to mortality during the COVID-19 pandemic. In particular, it allows for measurement of the contribution of overweight and obesity to mortality using unit record data, and so provides more granular evidence of differentials in the population compared with the population attributable fraction method. This study hence uses multiple cause of death data to estimate excess premature cardiovascular disease mortality (at 35–74 years) for which overweight and obesity was a risk factor, using DKOLH-CVD and measures of more severe overweight/obesity, in the US during the COVID-19 pandemic in 2020 and to assess its contribution to all-cause excess mortality. Excess mortality, and the percentage reported as due to COVID-19, is analysed by sex, age, month of death and place of death, to understand the demographic and temporal variation in these indicators and to compare between hospitals and other places of death  where diagnosis of COVID-19 is more difficult. These analyses include comparison with excess deaths from other causes.The study uses unit record data from the Mortality Multiple Cause Files from the National Center for Health Statistics from 2010 to 2020 . Unit reDKOLH2-CVD: The same as DKOLH-CVD, except that two or more of diabetes, chronic kidney diseases, obesity, lipidemias or hypertensive heart disease are reported on the death certificate. This is expected to represent people with higher BMI compared with DKOLH-CVD.Obesity: Deaths where a cardiovascular disease (excluding cardiac arrest) and just obesity are mentioned on the death certificate. This is expected to represent even higher BMI than DKOLH-CVD and DKOLH2-CVD because it is a direct measure of obesity of the deceased individual rather than an indirect estimate of both overweight and obesity.DKOLH-CVD: Deaths where a cardiovascular disease (ICD-10 codes I00-I99) is mentioned on the death certificate (excluding cardiac arrest (I46) which is a more immediate cause of death where all other reported causes can be unrelated to cardiovascular diseases) along with any of the following: diabetes (E10-E14), chronic kidney diseases (N18), obesity (E65-E66), lipidemias (E78) or hypertensive heart disease (I10-I13).Other CVD (i.e. excluding DKOLH-CVD): Deaths where a cardiovascular disease is mentioned on the death certificate, excluding DKOLH-CVD deaths.Non-CVD: All deaths excluding those where a cardiovascular disease is mentioned on the death certificate.This study used entity axis data that show the International Classification of Diseases 10th Revision (ICD-10) codes on the death certificate based on how they appeared on the death certificate, consistent with previous application of the DKOLH-CVD measure , 27. TheCOVID-19 was reported in the data as ICD-10 code U07.1. Other variables analysed were age at death, sex of deceased, date of death and place of death , home, nursing home/hospice, other). However, no geographic data (e.g. county or state of residence) were available in the dataset and so no spatial differences could be identified, which other studies have found for excess mortality and also longer-term diabetes and chronic kidney disease mortality in the USA –31. The Excess mortality was measured as the actual age-standardised death rate minus the expected age-standardised death rate (absolute) and the actual age-standardised death rate divided by the expected age-standardised death rate minus 1 (relative). The expected age-standardised death rate for each sex was estimated using a negative binomial regression of monthly deaths during 2010–19, with covariates of year of death, a third-degree polynomial of month of death, and five-year age groups (35–39 to 70–74 years), offset by the natural log of population. The negative binomial regression results were used to predict deaths by age and month in 2020, from which age-standardised death rates by month in 2020 and annualised age-standardised death rates for March–December 2020 were calculated . Regressions were conducted for each combination of sex, cause of death and place of death to calculate expected deaths for each. Uncertainty intervals of the expected age-standardised death rates were calculated based on 1000 simulations of the predicted age-standardised death rate from the negative binomial models while uncertainty intervals of actual age-standardised death rates were calculated assuming a poisson distribution of deaths. Excess mortality with uncertainty was then calculated by conducting 1000 simulations incorporating the uncertainty of both the actual and expected age-standardised death rates.The proportion of excess mortality due to COVID-19 was calculated, for each cause classification (i.e. DKOLH-CVD etc.), as the age-standardised COVID-19 mortality rate (COVID-19 as the underlying cause of death) divided by the excess age-standardised death rate, with the remainder of excess mortality classified as excess mortality due to non-COVID causes. In some instances, the COVID-19 death rate was higher than the excess death rate and hence mortality due to non-COVID causes was negative (i.e. lower than expected). All analyses were conducted using Stata 16.0 . Code usExcess premature all-cause mortality in the USA in March–December 2020 was 19%  higher than expected for males and 16% (14–17%) higher for females Table .Table 1ACOVID-19 comprised 77% of male and 81% of female excess premature mortality for all causes during March–December 2020 , and declined gradually to 70–74 years (just over 25%) Fig. .Fig. 2ExExcess DKOLH-CVD mortality was higher in hospitals , females 37% (34–41%)) than in the home , females 30% (27–33%)) Fig. . These tThe impact of the COVID-19 pandemic in the USA was particularly profound for cardiovascular disease mortality of people aged 35–74 years for which overweight and obesity was a risk factor. Excess DKOLH-CVD premature mortality was 30% in March-December 2020, much higher than that for other causes, while it was even higher for DKOLH2-CVD and obesity . This supports previous research showing higher risk of COVID-19 mortality for overweight and obesity . The relOnly about half of excess premature cardiovascular disease mortality for which overweight and obesity was a risk factor could be attributed to reported COVID-19, compared with about four-fifths for other causes. The major contribution to this difference was that the reported COVID-19 death rate was about 80% of excess DKOLH-CVD mortality in hospitals compared with more than 100% for all causes, while the respective figures for home deaths were just over 10%. The figures for hospital deaths are likely to be far more accurate than for home deaths because of much better testing and diagnosis of COVID-19. Another reason for the lower proportion of excess home deaths being due to COVID-19 could be that over-burdened hospitals were unable to accept some non-COVID patients and many people avoided hospitals due to concerns of being infected, and hence they died at home. This may also be reflected by non-COVID deaths also making a net negative contribution to excess hospital mortality for all causes and some specific cause classifications. However, the absolute net negative contribution of non-COVID deaths to excess mortality from all causes in hospitals was only a very small proportion of the absolute positive contribution of non-COVID deaths to excess home deaths (based on data in Table A limitation of this study is that it uses a group of conditions (DKOLH-CVD) to measure cardiovascular disease mortality for which overweight and obesity was a risk factor, without having direct evidence of the BMI of the deceased individuals. However, in the absence of other evidence, the identification of conditions in multiple cause of death data for which overweight and obesity are major risk factors can be used to estimate its contribution to premature cardiovascular disease mortality. The Global Burden of Disease Study estimates that 81% of all deaths at 35–74 years in the US attributed to overweight and obesity have an underlying cause of either diabetes, chronic kidney disease, or cardiovascular diseases (which includes hypertensive heart disease) . FurtherA major advantage of this study’s approach is that by using data of all registered deaths it allows for granular analyses by conditions, age, sex, month and place of death which provide insights into the differentials in excess mortality by cause group and various characteristics. This method overcomes the use of indirect population attributable fractions, which would be challenging to measure for this study because of not having reliable and accessible BMI data by place of death for 2020. Another advantage of multiple cause data is that it includes all deaths, unlike other analyses which have focused on hospital deaths only . FinallyThe extent of excess premature cardiovascular disease mortality in the USA in 2020 for which overweight and obesity was a risk factor was immense by any historical comparison, and was likely similar in 2021 based on other estimates of overall excess mortality . AdverseSupplementary material


Background and objectiveThe majority of glaucoma patients are asymptomatic and are usually diagnosed at an advanced stage of the disease. This study aimed to assess the outcomes of glaucoma screening among known first-degree relatives of primary glaucoma patients.Materials and methodsThis study involved primary angle-closure glaucoma (PACG), primary open-angle glaucoma (POAG), and juvenile open-angle glaucoma (JOAG) patients who attended the glaucoma clinic at the Hospital Universiti Sains Malaysia between January 2014 and December 2015. First-degree relatives of the patients underwent a preliminary eye-screening evaluation, including visual acuity (Snellen chart), intraocular pressure (IOP) measurement (air-puff tonometry), and non-mydriatic fundus photography. Patients with visual acuity worse than 6/12, IOP measuring more than 21 mmHg or a difference of more than 3 mmHg between the eyes, and a vertical cup-disc ratio (VCDR) of 0.7 or higher were given a comprehensive eye examination.ResultsSeventy indexed glaucoma patients were recognized, and 368 first-degree relatives were identified. Forty-five relatives underwent the preliminary screening. Of these, 29 showed normal findings (62%), one had corneal pathology (2%), and 16 (36%) underwent a complete eye examination after failing the initial screening. Among the indexed JOAG group, five relatives (11%) were diagnosed as having JOAG; two were treated medically, while the remaining three required surgical intervention.ConclusionOpportunistic glaucoma screening of high-risk groups, especially JOAG is a feasible and cost-effective way to detect early glaucoma and prevent irreversible blindness. However, improvement in our healthcare system, including the involvement of multicentre clinics in other states in screening initiatives, is required to promote and facilitate the response to screening opportunities. Glaucoma is the leading cause of irreversible blindness worldwide and the second most common cause of blindness after cataracts . GloballThe Thessaloniki Eye Study conducted in Greece reported a high prevalence of undiagnosed glaucoma in the studied population 50%) . This re0% . ThisEffective screening can reduce morbidity and improve the quality of life among glaucoma patients ,8. PopulThis cross-sectional study was exempted from ethical review by the Human Research Ethics Committee of Universiti Sains Malaysia as all the information obtained was anonymized. The study was carried out at the ophthalmology clinic of the Hospital Universiti Sains Malaysia between January 2014 and December 2015.First-degree family members of known glaucoma patients with primary angle-closure glaucoma (PACG), POAG, and juvenile open-angle glaucoma (JOAG) were included in the study. Family members of probands with secondary open-angle glaucoma or pseudoexfoliation glaucoma were excluded from the study. Family members with incomplete data during screening were also excluded.Three hundred and sixty-eight first-degree relatives of 70 indexed glaucoma probands were identified and divided into three groups: PACG, POAG, and JOAG. The pedigree of each patient was charted, and the contact details of first-degree relatives were obtained with permission from the probands. Only 151 of the 368 first-degree relatives identified were residents of Kelantan. The first-degree relatives were contacted and invited to undergo a screening test. However, only 45 relatives attended the screening. The examinations performed by paramedics during the eye screening included visual acuity (Snellen chart), IOP measurement with air-puff tonometry, and ONH photo using non-mydriatic funduscopy. The cut-off points for referral for comprehensive examination were vision worse than 6/12, IOP more than 21 mmHg or a difference greater than 3 mmHg between the eyes, and a vertical cup-disc ratio (VCDR) of 0.7 or higher.Individuals who met these criteria were classified as suspicious of having glaucoma and underwent a detailed examination to obtain a definitive diagnosis. Relatives who were diagnosed with glaucoma were managed with anti-glaucoma medications and surgical treatment. Other ocular abnormalities were referred to a general eye clinic for further evaluation. Information about glaucoma was also provided during the screening.All data were entered and statistical analyses were performed using IBM SPSS Statistics version 21 .While a total of 151 (41%) relatives resided in the Kelantan state, only 45 of these relatives attended the glaucoma screening: 20 (44%) relatives with a family history of POAG, 23 (51%) with a family history of JOAG, and two (4%) with a family history of PACG. The majority of the probands were Malays (95%). Figure Of the 45 first-degree relatives who were screened, 16 (36%) were classified as suspicious for glaucoma, while 28 (62%) had normal findings, and one (2%) had a cornea problem. The screening outcomes are provided in Table Among the indexed JOAG group, five relatives (11%) were diagnosed with JOAG; two of them were treated with topical eye drops alone, and three required a combination of topical eye drops and surgical intervention, as shown in Table In this study, we assessed the prevalence of glaucoma among the first-degree relatives of PACG, POAG, and JOAG patients. We screened the first-degree relatives of primary glaucoma patients to explore the feasibility of identifying individuals at risk of glaucoma based on their family’s medical history.Several previous studies have shown that primary glaucoma is more likely to affect persons with a family history of the disease, and a positive family history has been assumed to be associated with a significant risk of glaucoma ,10. One The glaucoma-screening tools used in our study were selected based on their feasibility in our local setting. The instruments were both user-friendly for our paramedics and non-invasive, which helped to eliminate anxiety among the individuals who were screened. We measured IOP using air-puff tonometry, as it is relatively quick and does not require any contact, thus reducing anxiety in the person being screened. In addition, the non-mydriatic fundus camera is a desirable screening tool for glaucoma, as it is widely available at primary healthcare clinics in the country and thus contributes to the accessibility of glaucoma-screening programs. Other tools that can be used to assess risk factors for the development and progression of glaucoma include optical coherence tomography (OCT) for structural assessment, the Humphrey VF analyzer to assess VF defects, and the ultrasonic pachymeter to assess the central cornea thickness .This study was conducted in Kelantan state, one of Malaysia’s most economically underdeveloped areas, located in the north-eastern region of Peninsular Malaysia. According to the Department of Statistics in Malaysia, the gross domestic product (GDP) per capita in Kelantan was Malaysian ringgit (RM) 12,075 in 2015, while the national per capita income was RM 37,104. Thus, it is not surprising that the majority of first-degree relatives were living and working outside the Kelantan state. This explains the poor attendance rate for this screening program. Another possible cause for the low attendance rate for the screening program in this study was the lack of awareness about glaucoma among relatives. Several previous studies have highlighted that a family history of glaucoma and level of education were determinants of glaucoma awareness . This taThe study was limited by the fact that it was performed at a single center with a significantly low rate of participation by the targeted subjects. Effective glaucoma screening among relatives of JOAG patients, in particular, could be enhanced by setting up user-friendly screening centers across the country at the primary healthcare level and by raising glaucoma awareness among relatives.In our pilot study, glaucoma screening among first-degree relatives showed tangible benefits, especially among the JOAG group. However, it was inconclusive for the POAG and PACG groups. Opportunistic screening of high-risk groups and collaboration between multiple centers across the country would improve the feasibility of early glaucoma detection. More efforts should be undertaken to educate the community about glaucoma, which may in turn improve participation in glaucoma-screening programs.


Is maternal COVID-19 vaccination during the second or third trimester of pregnancy associated with reduced risk of COVID-19 within the first 4 months of life in their infants?In this register-based cohort study of all live-born infants in Norway, there was a lower incidence of a positive SARS-CoV-2 test result in infants born to women vaccinated with a messenger RNA vaccine during pregnancy. The risk was lower during the period dominated by the Delta variant than during the Omicron-dominated period.The study results suggest that maternal COVID-19 vaccination during pregnancy could protect against infant SARS-CoV-2 infection in the early months of life. Pregnant women are recommended to receive COVID-19 vaccination to reduce risk of severe COVID-19. Whether vaccination during pregnancy also provides passive protection to infants after birth remains unclear.To determine whether COVID-19 vaccination in pregnancy was associated with reduced risk of COVID-19 in infants up to age 4 months during COVID-19 pandemic periods dominated by Delta and Omicron variants.This nationwide, register-based cohort study included all live-born infants born in Norway between September 1, 2021, and February 28, 2022.Maternal messenger RNA COVID-19 vaccination during second or third trimester compared with no vaccination before or during pregnancy.The risk of a positive polymerase chain reaction test result for SARS-CoV-2 during an infant’s first 4 months of life by maternal vaccination status during pregnancy with either dose 2 or 3 was estimated, as stratified by periods dominated by the Delta variant  or Omicron variant . A Cox proportional hazard regression was used, adjusting for maternal age, parity, education, maternal country of birth, and county of residence.Of 21 643 live-born infants, 9739 (45.0%) were born to women who received a second or third dose of a COVID-19 vaccine during pregnancy. The first 4 months of life incidence rate of a positive test for SARS-CoV-2 was 5.8 per 10 000 follow-up days. Infants of mothers vaccinated during pregnancy had a lower risk of a positive test compared with infants of unvaccinated mothers and lower risk during the Delta variant–dominated period  compared with the Omicron period .The results of this Norwegian population-based cohort study suggested a lower risk of a positive test for SARS-CoV-2 during the first 4 months of life among infants born to mothers who were vaccinated during pregnancy. Maternal COVID-19 vaccination may provide passive protection to young infants, for whom COVID-19 vaccines are currently not available. This cohort study examines the association of COVID-19 vaccination during pregnancy with reduced risk of COVID-19 in Norwegian infants up to age 4 months during COVID-19 pandemic periods dominated by Delta and Omicron variants. Transplacental transfer of maternal vaccine-derived antibodies against pertussis and seasonal influenza has been demonstrated following vaccination during pregnancy, and maternal immunization provides passive protection against infection to infants during the first months after birth.3 It is plausible that COVID-19 vaccination during pregnancy could provide passive protection from COVID-19 to infants during their first months of life.4 Vaccine-derived maternal antibodies have been identified in cord blood after COVID-19 vaccination during pregnancy, and a recent study found maternal vaccination to be associated with a 61% reduced risk of infant hospitalization for COVID-19.7 This study evaluated the association between maternal COVID-19 vaccination during pregnancy and incidence of infant SARS-CoV-2 infection during the first 4 months of life, as well as whether the association differed according to Delta variant and Omicron variant–dominated time periods.11The risk of critical illness because of COVID-19 has been reported to be higher in infants younger than 1 year compared with older children.STROBE) reporting guidelines and was approved by the Regional Committee for Medical and Health Ethics of South/East Norway. This committee waived the need for informed consent from participants in this registry-based study.This study followed the Strengthening the Reporting of Observational Studies in Epidemiology ,14 contains registrations of all COVID-19 vaccinations, including dates of all doses and the type of vaccine product. Vaccine doses reported fewer than 20 days after the previous dose were not included. Women who received a second or third dose of a messenger RNA (mRNA) vaccine after gestational day 83 and up to 14 days before delivery were considered vaccinated. Infants born to women who received their third or fourth vaccine dose between 13 and 7 days before birth were excluded, as they would be censored before birth in the statistical analyses. We excluded infants born to women vaccinated outside Norway while pregnant and those vaccinated exclusively before pregnancy or who only received dose 1 during pregnancy because the maternal antibody level and possible transplacental transfer of antibodies for these scenarios are uncertain.17The Norwegian Immunization Register18 We included the first positive PCR test for SARS-CoV-2 registered at least 1 day after birth and within 122 days after birth (4 months of age). Similarly, we identified women with a positive SARS-CoV-2 test 14 days or more before giving birth. In Norway, PCR tests were free of charge and widely available.During the study period , the date of testing and results of all SARS-CoV-2 polymerase chain reaction (PCR) tests were registered in the Norwegian Surveillance System for Communicable Diseases.From the Medical Birth Registry of Norway, we derived maternal age at conception  and extracted information on parity  and calendar week of birth. The registered gestational age in the birth registry was estimated from routine ultrasonography assessments or last menstrual period if there were no ultrasonography estimates. From the Population Registry of Norway, we obtained information on maternal country of birth  and current county of residence . From Statistics Norway we obtained information on the highest maternal educational level as of 2019 .20We calculated incidence rates of SARS-CoV-2 infection  in infants within 4 months after birth by maternal vaccination status. We split the follow-up time on December 31, 2021, by introducing a variable with 2 categories that corresponded to the time before this date (proxy for the Delta-dominated period) and after this date (proxy for the Omicron-dominated period). Using a Cox proportional hazards model, we estimated hazard ratios (HRs) for infant SARS-CoV-2 infection using calendar time (in days) as the time axis. Infant follow-up began on the date of birth. Observations were censored at age 4 months, death or emigration, date of when a woman received a vaccine dose outside Norway, or on April 4, 2022, whichever came first. For women vaccinated at the end of pregnancy or after pregnancy, infants were censored 14 days after the vaccination date of dose 1 and 7 days after dose 3 or 4. This was done because maternal vaccination within the last 14 days of pregnancy could potentially provide an exclusive protective effect from antibody transfer through breastfeeding or cocooning .We assessed differences in the Delta and Omicron periods by using linear combinations of the coefficients for vaccination status and an interaction term between vaccination status and period. Multivariable analyses adjusted for maternal age at conception, parity, educational level, county of residence, and maternal country of birth.5 Second, in separate analyses, we restricted the sample to (1) term-born infants, (2) infants who had the opportunity to reach 42 completed gestational weeks by the end of the inclusion period to avoid oversampling preterm births, (3) infants born to Scandinavian-born women, (4) infants born to first-time mothers, and (5) infants born to women who only received mRNA vaccines for all doses. Third, to obtain more robust results for the Omicron-dominated period, we used an alternative follow-up period starting on January 15, 2022, as after this date the circulating virus was more certain to be Omicron. Finally, as testing and risk of infection may differ by infant age, we used infant age in days as the time axis, while adjusting for week of birth.First, we excluded infants born to women who had a positive SARS-CoV-2 test more than 14 days before delivery, as women with a history of COVID-19 might be less likely to become vaccinated during pregnancy and could transfer anti–SARS-CoV-2 antibodies across the placenta.21 we explored the risk of infant hospitalization for COVID-19. All hospital admissions were registered with admission and discharge dates, as well as diagnostic codes, using the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10). We identified all infants with a hospitalization with the ICD-10 code U07.1  as the main diagnosis within the first 4 months of life and reported the crude proportions by maternal vaccination status.Using the Norwegian Patient Registry,To assess associations between maternal vaccination, even if not fully vaccinated, and incidence of a positive PCR test for SARS-CoV-2 among infants, we conducted a secondary analysis in which we compared the risk of a positive SARS-CoV-2 test in infants born to unvaccinated women with those with mothers who had received a first dose of an mRNA vaccine during the second or third trimester . Infants were censored 14 days after a woman received the second dose postpartum or during the last 14 days of pregnancy and 14 days after the first dose among the unvaccinated group. We compared the timing of vaccination in pregnancy in those who received only 1 dose in pregnancy with those who were fully vaccinated. Furthermore, we conducted a secondary analysis stratifying on whether the woman received COVID-19 vaccine dose number 2 or 3 during pregnancy.To explore possible differences in the likelihood of being tested in infants among vaccinated and unvaccinated mothers, we assessed the proportions in each group that had at least 1 registered SARS-CoV-2 PCR test (positive or negative) before age 4 months. We calculated crude incidence rates for registration of a PCR test by maternal vaccination status. Again, infants were censored at time of death, emigration, or maternal receipt of a vaccine dose outside Norway. All analyses were conducted using Stata, version 16.0 SE (StataCorp).Of 21 643 newborns included in the study, 9739 (45%) were born to women who received a second or third dose of a COVID-19 mRNA vaccine during the last 2 trimesters of pregnancy . Fewer tA total of 906 infants (4.1%) were registered with a positive PCR test for SARS-CoV-2 during the first 4 months of life. Infants born to vaccinated mothers had a lower incidence of SARS-CoV-2 . During Results were robust in sensitivity analyses, although estimates were attenuated when restricting to infants born to Scandinavian-born women  compared with those with a second dose   born to unvaccinated mothers and 1206 infants (12.4%) born to vaccinated mothers . CorrespThis cohort study of all live births in Norway between September 1, 2021, and February 28, 2022, found that COVID-19 vaccination during pregnancy was associated with a reduced risk of an infant receiving a positive PCR test for SARS-CoV-2 during the first 4 months of life. This association was present during periods dominated by the Delta and Omicron variants, although it was stronger in the former. Results were robust in sensitivity analyses, although the number of cases during the Delta-dominated period was low in some of the subgroups. The association was somewhat attenuated when restricted to infants born to Scandinavian-born women.3 Because the newborn’s immune system is naive, with limited antibody response during the first months of life, an important protection against infection comes from maternally transferred antibodies.3 Infants are at higher risk of severe COVID-19 compared with older children.1 As to our knowledge no COVID-19 vaccines are licensed for use in children younger than 5 years, an added benefit of maternal vaccination during pregnancy could be a protection of infants against SARS-CoV-2 infection during the first months of life.4It is not unexpected that maternal COVID-19 vaccination during pregnancy could reduce infant risk of COVID-19, as similar protective benefits against infant infection have been documented for pertussis and influenza vaccination during pregnancy in randomized clinical trials and observational studies.11 Infants born to women with only 1 mRNA vaccine dose received during pregnancy also had a lower risk of a positive SARS-CoV-2 test than those born to unvaccinated women, but results were not statistically significant.We observed a lower risk of infection among infants born to women who received their third dose in pregnancy compared with the second, suggesting a stronger level of protection following the booster dose. This aligns with studies showing a waning of vaccine effect after the second dose unless a booster is received.6The strengths of this study include the use of registry data covering the whole Norwegian population and many individuals vaccinated during pregnancy. Mandatory reporting to registries  limited potential selection bias and provided detailed information on clinical and sociodemographic variables. We believe our study results are generalizable to other pregnant populations. This assumption is strengthened by the fact that the findings align with the results from the US study examining maternal COVID-19 vaccination and risk of infant hospitalization for COVID-19.11The limitations of this study included the lack of information on the infant’s test with a SARS-CoV-2 variant. However, there were distinct periods of dominance with the different variants in Norway during the study period, and we believe the defined periods capture risk with the different variants. The differences we observed in estimates for the Delta and Omicron-dominated periods support this, as the vaccines generally have been shown to be less effective against Omicron than Delta.22 there could be a possible added effect of transfer of SARS-CoV-2 antibodies through breastmilk in these children,24 as more than 90% of infants in Norway are breastfed.26 We did not have individual-level information on breastfeeding and were unable to directly address whether this differed by maternal vaccination status.Although we did not include vaccinations during the last 14 days of pregnancy or after pregnancy in the vaccinated group to allow for sufficient time of transplacental transfer of antibodies before birth,27 or other unmeasured differences in characteristics between women who got vaccinated during pregnancy and those who did not.We adjusted for potential confounders, which did not substantially affect the estimates. Still, there may be residual confounding because of healthy vaccinee biasThe distribution of follow-up time in the vaccinated and unvaccinated groups varied across the 2 periods. We used calendar time as the underlying time scale in the analyses to ensure that comparisons were made on the same calendar days. This was important, as maternal vaccination status and risk of SARS-CoV-2 infection varied substantially over the study period. Although we did not have information on the number of household members or positive SARS-CoV-2 tests among them, we adjusted for maternal parity as a proxy.We did not have information on disease symptoms in the infants. Thus, we could not assess the severity of the infections and whether this differed by maternal vaccination status. As the number of infants hospitalized for COVID-19 was low, we could not perform robust analyses to discern whether this differed by maternal vaccination status. We found that infants born to unvaccinated women were more likely to be tested for SARS-CoV-2, and this could be because of higher incidence of COVID-19 or a higher likelihood of symptomatic disease leading to testing. Although we cannot exclude differential test behavior according to maternal vaccination status, we believe it is unlikely. However, women who got vaccinated may have behaved differently , which could have biased the estimates. Still, this is unlikely to account for all of the substantial reduction in risk that we observed.In this nationwide registry-based cohort study, we found that infants born to women who received a second or third COVID-19 vaccine dose during the last 2 trimesters of pregnancy had a lower incidence of SARS-CoV-2 infection within the first 4 months of life compared with infants born to unvaccinated women. The reduction in infant infection risk was greater during the Delta-dominated period compared with the Omicron-dominated period. The findings of this study provide early evidence to suggest that infants benefit from passive protection from SARS-CoV-2 infection following maternal COVID-19 vaccination during pregnancy.


BRCA1 and BRCA2 in breast cancer risk. However, it is known that a single genetic variant is not enough for cancer to develop leading to the theory of multistage carcinogenesis. In many cases, it is a sequence of events, acquired somatic mutations, or simply polygenic components with strong epigenetic effects, such as in the case of brain tumours. The expression of many genes is the product of the complex interplay between several factors, including the organism’s genotype (in most cases Mendelian-inherited), genetic instability, epigenetic factors (non-Mendelian-inherited) as well as the immune response of the host, to name just a few. In recent years the importance of the immune system has been elevated, especially in the light of the immune checkpoint genes discovery and the subsequent development of their inhibitors. As the expression of these genes normally suppresses self-immunoreactivity, their expression by tumour cells prevents the elimination of the tumour by the immune system. These discoveries led to the rapid growth of the field of immuno-oncology that offers new possibilities of long-lasting and effective treatment options. Here we discuss the recent advances in the understanding of the key mechanisms controlling the expression of immune checkpoint genes in tumour cells.Cancer is a disease of the genome, therefore, its development has a clear Mendelian component, demonstrated by well-studied genes such as  BRCA1 and BRCA2 in breast cancer risk , genetic instability level, epigenetic factors (non-Mendelian inheritance) as well as immune cells of the host, to name just a few Fig. . Here weOne of the most incredible powers of the human immune system is its constant ability to recognise cells as being ‘foreign’ or ‘self’, thus, to distinguish perfectly between self-antigens and non-self-antigens. Within this complex process, it is important to mention the immune checkpoint molecules: small proteins present at the cell’s membrane Fig. . They neT cell-dependent immune mechanisms are known to protect an organism from cancer Dustin . The detEven though checkpoint proteins are absolutely necessary for the proper functioning of the immune system, in some situations checkpoint molecules might help the disease to progress. This is the scenario we often observe in cancer—tumour cells at some point of cancer development might escape from immune recognition and attack. One of the well-described mechanisms used by cancer cells to escape from immune system recognition is simply to stop the immune cells (mostly lymphocytes) from launching an attack on them  is another major checkpoint protein that limits T cell activity Fig. . Its twoDespite the overall similarity in their effects on T cells, CTLA-4 and PD-1 operate at distinct stages during immune cell activation, with CTLA-4 required earlier than PD-1  in HLA genes, are usually highly predictive of the risk of developing specific autoimmune syndromes and, thus, may contribute to the development of immune checkpoint inhibitor-induced autoimmunity . The expression of PD-L1 is generally lower in individuals with predicted African ancestry, although no single cis-expression Quantitative Trait Locus (eQTL) correlated with PD-L1 expression  mutations in NSCLC epithelial cells have been shown to induce and enhance PD-L1 expression  are abundant small non-coding RNAs composed of 22–24 nucleotides  in gene promoters results in stable transcriptional silencing of gene expression , in particular, it is placed de novo mostly by DNMT3A and DNMT3B and maintained by DNMT1 Fig. . OverexpThe genome of all organisms is organised into chromatin—a complex of DNA, RNA and associated proteins  by Merck and CheckMate-026 (Opdivo)  by Bristol-Myers used IHC to determine the levels of PD-L1 biomarker. The major difference in the design between these trials was the biomarker threshold level—set more restrictively in KEYNOTE-024 (Keytruda). Clinical trials for Keytruda met their endpoint successfully with 305 enroled participants, while Opdivo was unsuccessful with a cohort of 1325 participants  measurements of PD-L1 are routinely used to stratify patient cohorts for clinical trials. However, setting up a threshold even for a single protein is a challenging problem that can determine the success or failure of a clinical trial. For example, two clinical trials of checkpoint inhibitor drugs for Non-Small Cell Lung Cancer: KEYNOTE-024 (Keytruda)  upstream or downstream in the regulatory pathway. Hence, a panel of multiple biomarkers may produce better prediction power than a single biomarker. Indeed, adding the RNA-seq expression estimates of other biomarkers increases the predictive power of anti-PD-1 therapy efficacy in comparison to using PD-L1 levels alone  benefits patients and further expands our knowledge about cancer.