Mycobacterium tuberculosis (MTB) co-infection has become a pressing global public health problem. Although tuberculosis (TB) is both treatable and curable, it has been exacerbated by the HIV/acquired immune deficiency syndrome (AIDS) epidemic. HIV-MTB co-infected patients have a variety of disease-specific, and treatment-related factors that can adversely affect their treatment outcomes. This study was conducted to assess the outcomes of TB treatment and its associated factors among HIV-MTB co-infected patients in Sichuan, Southwest China. A retrospective study was performed on HIV-MTB co-infected patients who were diagnosed and registered in TB designated hospitals in Sichuan from January 1, 2016, to December 31, 2020. Data were collected from patients’ electronic medical records regarding their demographic, clinical, and social support information, and categorical data, such as sex, were reported using numbers and percentages. χ2 and t-tests were conducted to compare groups in relation to different levels of medical institutions. A binary logistic regression model was used to identify the factors associated with unsuccessful TB treatment outcomes. For logistic regression analysis performed using an α of 0.05, odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated for various risk factors. A total of 3677 registered HIV-MTB co-infected patients were enrolled. After adjusting for other variables, male, advanced age, receiving TB treatment at the municipal medical institution, being diagnosed with external pulmonary TB, referral or tracing, being sputum smear positive, not initiating antiretroviral therapy (ART) and not using fixed-dose combinations were the main risk factors for treatment failure of HIV-MTB co-infected patients in Sichuan province. Sex, age, hospital level, patient source, other diagnostic factors , and factors of therapeutic schemes  may serve as risk factors to estimate the likely treatment outcome of HIV-TB co-infection.Human immunodeficiency virus (HIV)- Among HIV-related diseases, tuberculosis (TB) accounts for 26% of AIDS-related deaths, almost (99%) of which occur in developing countries. There were an estimated additional 208,000 TB deaths  among HIV-positive people. Although the deaths of TB patients among PLH account for only 15% of the total TB deaths, in some countries with a high burden of TB, such as South Africa, efforts to control TB are overwhelmed by the rising number of TB cases occurring in parallel with the HIV/AIDS epidemic. The HIV epidemic continues to fuel a TB epidemic.Tuberculosis remains a public health concern among people living with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) (PLH), resulting in increased morbidity and mortality. In an individual host, 2 pathogens, Mycobacterium tuberculosis (M tuberculosis) and HIV, accelerate the deterioration of immunological functions and result in premature death if left untreated.Both TB and HIV can drastically affect the host immune system because they can evade immune surveillance and clearance; however, the underlying mechanisms are not fully understood. As reported in a meta-analysis in China, the overall prevalence of TB infection among PLH was estimated to be 7.2% (range: 4.2–12.3%) and was even higher (22.8%) in AIDS patients. According to the World Health Organization (WHO) policies, China annually provides TB screening for those diagnosed with HIV/AIDS and HIV testing for those diagnosed with TB in regions with high HIV/AIDS epidemics.Even in this circumstance of the introduction of high active antiretroviral therapy, TB infection still places a great burden on PLH in Chinese mainland, a developing country. with the second highest TB caseload among the provinces in China. The HIV testing coverage and HIV-positive case detection rates in Sichuan were estimated to be approximately 76% and 0.9%, respectively, as revealed in the TB report of China 2019 from the Center of Disease Control and Prevention of China. Nonetheless, another investigation in Sichuan showed that HIV testing was not universally available for outpatient TB cases; only 29.5%  of cases had HIV test results. The prevalence of HIV infection among patients with TB is high in locations where the greatest increase in TB cases has occurred. A cross-sectional study conducted in Sichuan, Guangxi, and Henan revealed that among TB patients, 3.3% were HIV-positive.Sichuan Province is characterized by the largest population living with HIV/AIDS in Southwest China. Owing to its remote geographical position and the limitations of local medical services, Sichuan has a high incidence of TB , the mortality of those with HIV-MTB co-infection remains frustrating at 36.5% in southern China according to a recent 5-year-cohort study. Various studies have reported an association between unsuccessful TB treatment and risk factors. However, inconsistent findings from previous studies have made it difficult to assess whether reported factors are applicable to the local environment. In addition, little is known about the factors related to unsuccessful TB treatment outcomes in HIV-MTB co-infected patients who received ART initiation and anti-TB therapy. Consequently, identifying the impact of epidemiological and clinical characteristics on unsuccessful treatment outcomes in patients with HIV-MTB co-infection would provide important insights and a scientific basis for HIV-MTB co-infection prevention, control, diagnosis, and treatment in the local healthcare system.Despite the latest treatment outcome data showing a TB treatment success rate of 84% and antiretroviral therapy (ART) initiation rate of 88% for those with HIV-MTB co-infection in a 2015 cohort in China,This study was conducted in Sichuan, Southwest China. This retrospective study used data from the Chinese National TB Surveillance System (NTSS) database. Following international guidelines, coordinated registration and care systems were initiated between the HIV and TB programs in 2010. In line with WHO policies, care services such as antiretroviral therapy, anti-tubercular treatments, isoniazid preventive therapy, and infection control are now coordinated with the Chinese national HIV and TB programs.Patient data were considered for this study if they were diagnosed with HIV/AIDS and MTB (including pulmonary TB and extra-pulmonary TB) and were reported and registered in NTSS at all designated TB or HIV medical institutions in Sichuan over a period of 5 years (2016–2020).A diagnosis of MTB was made based on the combined evaluation of the clinical, radiological, histopathological, and laboratory features of the patients in accordance with the protocol established by the National Tuberculosis Prevention and Control Program (2008): sample smears/cultures positive or sample smears/cultures negative but met all 3 of the following clinical criteria:Symptoms consistent with TB;chest X-ray suggestive of TB; andpositive anti-TB Rx response.A patient was considered HIV-infected when a screening test by enzyme-linked immunosorbent assay tested positive, HIV strains were isolated from the blood, or both results of the screening test were positive at different times. For TB patients, hospitals provide directly observed therapy (DOT) in both adult and pediatric age groups with free drugs like 2 months intensive phase with rifampicin, isoniazid, pyrazinamide, and ethambutol (2HRZE) and 4 months continuation phase with rifampicin and isoniazid (4RH).Sociodemographic and clinical variables related to the study objectives were obtained from the TB treatment registry. The former included age, sex , ethnicity , occupation , level of medical institution ; the latter included the results of the initial sputum smear test , type of case finding , patient type , HIV antibody test results , site of TB , rifampicin-resistant TB , ART , and fixed-dose combinations . In addition to the standard outcome definitions, we classified the final TB treatment outcome as successful (cure or complete treatment) or unsuccessful .α value of 0.05. Various risk factors were calculated using odds ratios (ORs) at P < .05, and 95% confidence intervals (CIs). The goodness of fit of the model was tested using the Hosmer–Lemeshow test.The abstracted data were entered into Excel 2010 and exported to SPSS, version 17.0 . Chi-square tests and t-tests were conducted to compare groups in relation to different levels of medical institutions. Descriptive statistics of the variables of interest are expressed as absolute and relative frequencies or mean ± standard deviation (SI). The association measures for each variable category were estimated using univariate and multivariate logistic regression models. The outcome variable was whether the patient experienced an unsuccessful outcome after the treatment. A logistic regression analysis was performed using an There were 230,794 cases of TB cases reported and registered in Sichuan between 2016 and 2020, of which 3677 (1.59%) were HIV/AIDS co-infected and were included in the final analysis.Table P = .024). The odds of unsuccessful treatment for patients from city-level hospitals were 2.5 times higher than those from county-level hospitals. Increased age was a statistically significant predictor of unfavorable treatment outcomes among HIV-MTB co-infected patients . In addition, external pulmonary TB patients had a 2.3 times higher chance of developing unfavorable outcomes than pulmonary TB patients did. Relapsed or returned treatment after being lost to follow-up had increased odds of unfavorable outcomes compared to new patients . Patients who visited the clinic due to symptoms  or via referral and tracing mechanisms  had higher odds of unsuccessful outcomes than the others . Moreover, the odds of unsuccessful treatment were 1.6 times higher in smear-positive patients than in smear-negative patients and 1.4 times higher in antiretroviral therapy-free patients than in antiretroviral therapy-treated patients. Patients receiving TB treatment without fixed-dose combinations  had a significantly increased risk of unsuccessful treatment outcomes than those with fixed-dose combinations.The univariate logistic regression model Table  revealedP = .041). For each 1-year increase in age, the odds of unsuccessful treatment increased by 0.9%. Meanwhile, the odds  were higher for patients who sought medical aid at the city-level medical institution than those at the county-level medical institution. Patients who visited the clinic via referral or tracing were 2.2 times more likely to develop an unfavorable outcome than those who visited the clinic through other means, such as contact examination, health examination, and recommendation. External pulmonary TB patients with HIV co-infection were more likely  to develop unsuccessful outcomes than simple pulmonary TB patients co-infected with HIV/AIDS. Sputum smear-positive patients had increased odds of unsuccessful outcomes  compared with sputum smear-negative/non-performed HIV-MTB patients. The odds of unfavorable outcomes were 1.4 times higher in patients who did not receive ART than in those who initiated antiretroviral therapy. Patients without fixed-dose combinations had a statistically significant increase in the chance of developing unsuccessful outcomes compared to those who used fixed-dose combinations .Multivariate analysis of the characteristics associated with unsuccessful treatment is shown in Table  India (80.0%), and Thailand (74.3%).Tuberculosis and HIV co-infection remain among the most serious public health challenges in Southwest China. In this study, we found NTSS an overall TB treatment success rate of 89% for 3677 TB patients living with HIV in 5 years (2016–2020), which is higher than the national treatment success rate of 76.0% in 2018, which is in agreement with studies conducted in Asian countries such as Vietnam (74.0%), Furthermore, males were more at risk for an unsuccessful treatment outcome, which was in agreement with the study by Engelbrecht. The higher rates of unsuccessful treatment outcomes in men may be due to high-risk behaviors, such as alcohol use, smoking, and illicit drug abuse.Based on their self-descriptions, the majority of TB patients living with HIV were male, which is in line with the findings of other studies.,21 Not surprisingly, advanced age is a risk factor for mortality in patients with TB with or without HIV infection.The majority (76.3%) of the HIV-MTB patients were in the age group 33 to 53 years. The rate of unsuccessful treatment was significantly associated with advanced age. Several studies have reported that advanced age is associated with unsuccessful treatment. and a study done in northern Ethiopia. HIV-MTB co-infected patients who visit county-level medical institutions mostly seek medical advice after they have high-risk behaviors that may lead to HIV infection so that they can know their HIV infection status in a timely manner and be diagnosed early. They can then receive antiviral and anti-TB treatments. In contrast, patients who choose to seek treatment at city-level medical institutions might be because their immune system has already been seriously harmed, and opportunistic infections or AIDS-related diseases are detected when they seek medical treatment, thus affecting the treatment effect of patients with dual infections. This area needs to be studied in the future to determine which risk factors exist in different locations that may put people at a higher risk of unfavorable outcomes in TB treatment.In this study, the medical institution where an HIV-MTB co-infection patient sought medical aid was found to be associated with unsuccessful outcomes. The odds of an unsuccessful TB treatment outcome were 2.367 times higher for patients seeking medical assistance at city-level hospitals than at county-level institutes. This is similar to an earlier study conducted in Zambia and Jacobson.We also found that identification of patients through referral or tracing was a predictor of unsuccessful treatment outcomes. This is partly because people who were identified with TB through contact or health examination, recommendations, and other methods are more likely to be compliant with care and to pay attention to their own health status than those identified through referral or tracing; therefore, they are more likely to have successful treatment outcomes. Our study sheds light on a neglected issue that requires attention, namely, that identifying patients earlier through voluntary counseling and examining programs is essential to help reduce TB mortality in co-infected patients. This result is consistent with that reported by Teasdale This is consistent with the current study, which found that external pulmonary TB-HIV infection is a 1.98-fold risk factor for unsuccessful outcomes. Another study showed that external pulmonary TB is more common in the HIV-associated TB population, becomes increasingly prevalent with progressive immunodeficiency, and contributes to unfavorable treatment outcomes.Khan et al showed that external pulmonary TB-HIV is a risk factor for poor TB outcomes. and eastern Ethiopia. In contrast, a study conducted by Mizan Tepi demonstrated that HIV-MTB co-infected patients with smear-positive pulmonary TB had a higher chance of successful TB treatment outcomes.The chance of unsuccessful outcomes was higher among HIV-MTB co-infected patients with smear-positive sputum samples. Similar findings have been reported in other studies conducted in South IndiaHalf (58.14%) of the patients did not receive ART at the time of initiation of TB treatment. An example of the difficulties faced by this group is that in some Brazilian cities, patients can receive high active ART but not food vouchers or transportation subsidies. The absence of these 2 services makes successful treatment virtually impossible.Not using fixed-dose combinations was significantly associated with unsuccessful treatment outcomes in patients with HIV co-infection. One of the factors for unsuccessful treatment outcomes might be the load of TB drugs and HIV-drug interactions. HIV-MTB co-infected patients use more drugs and are more likely to experience more drug side effects than patients taking HIV ART or anti-TB drugs only. Other studies have also reported that drug interactions have more side effects on the treatment outcomes of HIV-MTB co-infection.Surprisingly, rifampicin-resistant HIV-MTB, HIV antibody test results, and relapse or return due to loss to follow-up were not significantly associated with unsuccessful treatment outcomes in multivariate hazard ratio analysis of treatment outcomes. Similarly, a study conducted in Nigeria found no association between new and relapsed patients.A major strength of this study is that the large sample size allowed us to identify the independent risk factors for unsuccessful treatment outcomes. In addition, the study was carried out under routine conditions, meaning that the data were representative of the real situation; thus, the findings are generalizable to most of the populous provinces in China, which account for a large proportion of pulmonary TB patients with HIV co-infection in the country.Apart from these significant findings, this study also has several limitations that are characteristic of retrospective studies. All data were dependent on paper registers kept in the hospital and health clinics, which means that it was neither feasible to correct any obvious errors in the data nor could missing data be captured. This excluded other important variables that could have influenced the outcome of TB treatment in HIV-MTB co-infected patients. For example, cluster of differentiation 4 T cell count, viral load, education level, smoking, and other co-infections. Although we attempted to locate missing information from the HIV/AIDS surveillance system this was not always possible because there was not yet a publicly available list of patients to match the TB surveillance system. Therefore, this missing information may have biased risk estimates. Bias is always a concern in secondary data sources. The NTSS database has served as a reliable source for several other population-based studies in China.In this study, the overall success rate of TB treatment was almost in line with the WHO target of 90%. Age, sex, facility level, type of case finding, anatomic site of TB result of sputum examination, ART initiation, and fixed-dose combinations use were predictors of unsuccessful TB treatment outcomes in HIV-MTB co-infected patients in Sichuan. These findings have broad policy implications. Providing better social support to patients could be key to improving treatment outcomes. Therefore, we would like to propose the following recommendations:(1) HIV-MTB co-infected patients who did not receive ART or who did not use fixed-dose combinations due to side effects should be encouraged individually for counseling in the directly observed therapy (DOT) clinic on the side effects of anti-TB/ anti-virus drugs and the risk associated with disrupting TB treatment or not initiating antiretroviral therapy.(2) There is a pressing need to strengthen cooperation between TB and HIV control programs as well as communication between patients and health care workers. Specific initiatives could include incorporating HIV/AIDS counseling and screening for HIV among TB patients into current TB control program assessments, which could achieve better TB treatment outcomes in southwest China.The authors would like to thank Dr Zhang and Dr Kang for linguistic assistance.Ni Yang designed the study, performed statistical analyses and data interpretation, and wrote the main manuscript. Jing Li and Yin Zhong contributed to the data acquisition. Chuang Chen contributed to manuscript revisions. Jinge He supervised this study. All the authors have reviewed the manuscript.Data curation: Yin Zhong, Jing Li.Supervision: Chuang Chen.Validation: Jinge He.Writing – original draft: Ni Yang.Writing – review & editing: Ni Yang.


BackgroundThe widespread societal effects of the COVID-19 pandemic connote public health and epidemiological changes for orthopedic injuries. The epidemiology of upper extremity injuries and the effects of the pandemic on these nationwide trends is poorly defined.MethodsThis cross-sectional, descriptive epidemiological study compares epidemiological trends among upper extremity (UE) orthopedic injuries presenting to emergency departments (EDs) prior to and during the COVID-19 pandemic. Upper extremity fracture and dislocation data was sourced from the National Electronic Injury Surveillance System (NEISS) database in years prior to (2015-2019) and during the pandemic (2020-2021). Data on incidence, patient demographics, injury patterns, mechanisms of injury, incident locale, and patient disposition were collected and compared between years.ResultsThe pre-COVID-19 incidence rate (IR) of UE fractures at 2.03 per 1,000 persons (n=3038930 from 2015-2019) decreased to 1.84 per 1,000 in 2020 (n=474805) and 1.82 per 1,000 in 2021 (n=471793). Dislocation rates were largely unchanged at 0.34 per 1,000 people (n=476740) prior to the pandemic and with incidence rates of 0.33 per 1,000 (n=85582) and 0.34 per 1,000 (n=89386) in 2020 and 2021, respectively. Female patients over 65 had the highest injury IR at 4.85 per 1,000 (n=976948). Finger fractures  overtook hand fractures  as more common during COVID-19 in males, while wrist  fractures remained most common in females. Injuries from individual sports, such as skateboarding and bicycling, increased during the pandemic, while injuries from team sports decreased. Hospital admission and observation increased in 2020, while discharge and transfer rates decreased. Admission, observation, and discharge rates moved closer to pre-pandemic levels in 2021.ConclusionsThe COVID-19 pandemic was associated with epidemiological and activity changes regarding UE fractures and dislocations presenting to EDs. The present study demonstrates notable decreases in rates of upper extremity fractures and dislocations, increases in rates of injuries related to outdoor and individual sports such as skateboarding with corresponding decreases in rates of injuries related to organized sports such as basketball, increases in the rates of injuries occurring in homes and in association with pet supplies, and decreases in rates of injuries occurring in schools and places of recreation observed during the pandemic. Additionally, trends observed among patient disposition specific to the pandemic, such as increasing rates of patient admission, observation, and against medical advice (AMA) departure with decreasing rates of discharge and transfer, offer insight into the burden of upper extremity injuries on the healthcare system during this critical time. While upper extremity orthopedic injuries remained common through the pandemic, the early pandemic was associated with higher rates of hospital admission that normalized closer to pre-pandemic levels by 2021, which may herald a shift and return to pre-pandemic trends. Future research will determine the long-term downstream effects of COVID-19 on activity-related orthopedic injuries and bone health. The novel coronavirus COVID-19) emerged in China at the end of 2019, rapidly spreading worldwide to reach pandemic status by early 2020 9 emerged,4, and tDespite such massive paradigm shifts in society, there is limited literature discussing orthopedic epidemiology on a national level and the burden of orthopedic injuries on the emergency health system, particularly regarding longer-term trends seen when entering the second year of the pandemic. Prior to COVID-19, the incidence of upper extremity fractures was estimated at 0.28 - 0.72 per 1,000 individuals, while the incidence of upper extremity dislocations was estimated at under 0.27 per 1,000 individuals ,7. An ItMany of the studies discussing the orthopedic implications of COVID-19 are specific to a single institution or region. We aim to identify the national implications of the COVID-19 pandemic on upper extremity orthopedic injuries and to better quantify the burden of these injuries on the emergency health system. Using the National Electronic Injury Surveillance System (NEISS), we will explore the effect of the COVID-19 pandemic on upper extremity fractures and dislocations with the hypothesis that emergency department (ED) visits for these injuries decreased during pandemic years and describe changes in injury presentation, trends in patient disposition, and potential implications for future practice.This study offers descriptive epidemiological insight into the impact of COVID-19 on orthopedic upper extremity injuries presenting to emergency departments (EDs) in the US. Data originates from the National Electronic Injury Surveillance System (NEISS) database, which was established by the US Consumer Product Safety Commission (CSPC) in 1978 to aid governmental agencies in monitoring product-related injuries across the country. Data collection for the NEISS is sourced from approximately 100 EDs that represent a probability sample of the over 5000 EDs across the country, which allows results from the NEISS database to be extrapolated to calculate national estimates of injury rates. On an ED level, individual patient data are collected and deidentified by a designated NEISS hospital coordinator through clinical records and follow-up telephone communication. NEISS data for the previous year are released in April of the following year. Database queryThe NEISS database was queried for all primary fracture and dislocation injuries occurring in patients 18 years or older between 2015 and 2021 in the following anatomic regions: shoulder, upper arm, elbow, lower arm, wrist, hand, and finger. Pre-COVID-19 years were defined as 2015-2019, while COVID-19 years were defined as 2020 and 2021. Data CollectionEstimated national incidences were calculated based on patients presenting in the NEISS system, the NEISS weighted estimates, and existing census data and population projections. Patient demographics include patient age and sex, with patient age categorized as 18-44, 45-64, and over 65 years old. Of note, the NEISS database only included non-binary codes for patient sex as early as January 1st, 2021. Injury patterns were classified as either fractures or dislocations and categorized according to the corresponding body part. The mechanism of injury was collected based on the primary product linked to the injury. Mechanisms of injury were classified as “sports” and “non-sports” related categories for further analysis. The incident locale was defined as the location where the injury took place and included home, farm/ranch, street/highway, other public property, manufactured (mobile) home, industrial place, school, and place of recreation or sports. Patient disposition was coded as treated and released; treated and transferred; treated and admitted; held for observation; left either against medical advice (AMA) or without being seen; or fatality, which includes death prior to ED arrival, death in the ED, and death after admission.Statistical analysisRaw and weighted descriptive statistics were calculated for included patients. Incidence rates (IRs) per thousand persons at risk and corresponding 95% confidence intervals (CIs) were also calculated based on US census data population and by demographic-based distribution numbers. All descriptive statistics are reported based on weighted population estimates as well as based on raw NEISS data, with relevant population sample sizes and raw NEISS data sample sizes reported for each. Incidence rate ratios (IRRs) were used to compare pre-pandemic and during-pandemic injury rates as well as injury rates between specific years. IRRs are considered statistically significant if both upper and lower limits of the 95% CI are either greater than or less than one. Student t-tests were used to compare injury rates over consecutive years with significance defined as an alpha of 0.05. IncidenceIn the years prior to COVID-19, an estimated 2.42 per 1000 people presented to EDs with upper extremity fractures or dislocations . Injury rates decreased to 2.17 and 2.16 per 1000 people in 2020 and 2021, respectively   presented to EDs with upper extremity fractures while 0.38 per 1000 people  presented with upper extremity dislocations prior to 2020. During COVID-19, these incidences decreased 9.9% and 10.5% . There were no significant differences observed in rates of injury between the years 2015 and 2019 (t(8)=0.819, p=.436). Between 2020 to 2021, the incidence of fractures presenting to EDs decreased  while the incidence of dislocations presenting to EDs increased . DemographicsIncidence rates varied significantly between male and female patients in both pre-COVID-19 years (t(8)=2.696, p=.027) and COVID-19 years (t(2)=8.320, p=.014). Proportionally more female patients sustained upper extremity fractures  while more male patients sustained dislocations   followed by males ages 18-44  and age groups with the highest incidence of fractures (over 65 years), the incidence rate of dislocations among the highest age group (patients ages 18-44) decreased more than the incidence rate of fractures in patients over 65 . Injury patternsPrior to COVID-19, fractures in male patients most commonly involved the hand , finger , or shoulder  . Hand fractures in males were observed at 0.710 (CI=0.704-0.715), the rate of the prior incidence during COVID-19, while finger fractures were not correlated with a significant change in IRR . Both prior to and during COVID-19, fractures in female patients most frequently involved the wrist , upper arm , or lower arm . Wrist fractures in females were observed at 0.955 (CI=0.949-0.960) of the prior incidence during COVID-19. Shoulder dislocations were the most common dislocation across all patient sexes . Mechanism of injury Table Floors and flooring materials were the most common non-sports-related product associated with orthopaedic upper extremity injuries, most frequently causing upper arm fractures both prior to and during COVID-19 . Bicycles were the most common sports-related product associated with injuries and most commonly were associated with shoulder fractures . Compared to years prior to COVID-19, the incidence of bicycle-related injuries increased in 2020 but decreased in 2021 . Of the top ten products, incidence rates of injuries related to pet supplies and skateboarding increased . Incidence rate ratios of injuries related to pet supplies increased more in 2021 than in 2020 , while rates of injuries related to skateboarding increased more in 2020 than in 2021 . Of all sports-related injuries, rates of basketball injuries decreased the most .Incident localeIncidence of upper extremity fractures and dislocations occurring in the home was 1.12 per 1000  prior to COVID-19, with a decreased incidence observed in 2020  and more markedly decreased incidence observed in 2021   while discharge and AMA departure rates increased . Transfer rates did not change significantly between 2020 and 2021 .The overall number of upper extremity fractures and dislocations presenting to US EDs decreased substantially in both 2020 and 2021, directly in relation to the rise of the COVID-19 pandemic. Fracture rates were highest among female patients over 65, and dislocation rates were highest among male patients aged 18-44. Finger fractures overtook hand fractures as the most common fracture pattern in male patients, while wrist fractures remained the most common in female patients. The incidence of injuries related to skateboarding increased, with the most drastic decrease observed among basketball injuries. There was an increase in injuries related to pet supplies. Injuries occurring in homes decreased more in 2021 than in 2020, while injuries in schools decreased more in 2020 than in 2021. Additionally, the pandemic engendered increased admission, observation, and AMA departure rates, as well as decreased discharge and transfer rates. Ultimately, the COVID-19 pandemic appears to have altered the epidemiology and ED course of upper extremity orthopedic injuries; however, the transition to more normalized injury patterns in 2021 may herald a shift to pre-pandemic injury rates and patterns as society adapts to COVID-19. Early pandemic-related literature documents global decreases in patient volume across the trauma-care spectrum. Considerable reductions in ED trauma volumes were noted internationally, with rates of patients presenting to US EDs decreasing as much as 42% in general and 22% for musculoskeletal complaints -11. SpecThe majority of non-sports-related products implicated in upper extremity fractures and dislocations are related to structural components of buildings and homes, such as floors, stairs, walls, beds, and doors. With high rates of wrist and upper arm fractures in female patients over 65, a substantial portion of injuries from structural components such as floors and stairs may represent fragility fractures or fractures secondary to accidental falls. The decreasing rates of upper extremity injuries from these fixtures during the pandemic may reflect a greater propensity for injury in non-home buildings that are less familiar to patients than a home environment, which many were confined to, particularly the elderly. As previously discussed, the effects of decreased activity on bone health and the risk for fragility fractures remain to be seen. Interestingly, injuries related to pet supplies and equipment increased substantially during the pandemic. While there is debate surrounding whether rates of pet ownership increased during the COVID-19 pandemic , individChanges in injury trends during COVID-19 offer valuable insight into the evolution of athletic activities and sports during the pandemic. With many efforts to promote social distancing, including the suspension of organized sports, it is no surprise that sports-related mechanisms of injury exhibited sizeable decreases during the pandemic . ReflectThe start of the COVID-19 pandemic certainly impacted patient disposition following presentation to the ED. The number of patients admitted in 2020 is substantially higher than in prior years, although admission rates decreased closer to pre-pandemic levels by 2021. When interpreted in the context of decreased rates of fractures and dislocations in 2020, increased admission rates may be explained by patients who tested positive for COVID-19 upon presentation or were admitted for requisite COVID testing prior to operative management. Alternatively, the same factors that Lim et al.  attributAs with any database-driven study, limitations to our data are primarily the result of limitations of the NEISS database. Rates of fractures and dislocations in our studies are limited to injuries that presented to US EDs; subsequently, these rates may underestimate patients who did not receive medical attention for fractures to non-weight-bearing upper extremities or patients that presented to orthopedic or urgent care clinics either virtually or in-person. The societal effects of COVID-19 in the US occurred at different rates nationwide, so trends in the NEISS database may reflect pre-COVID-19 years in 2020 for different geographical regions. Additionally, data parameters were limited by what is reported in the NEISS database, as well as the potential for human error, given how NEISS data are collected by individuals at geographically and institutionally diverse hospitals across the US. Population estimates also introduce an additional challenge during the COVID-19 pandemic. Although NEISS data is intended to calculate population estimates based on census data, census projections were used for the 2020 and 2021 population data and, unfortunately, do not account for deaths due to COVID-19. Furthermore, the NEISS population weights may not be as generalizable to the nation as in typical years since COVID-19 influenced behaviors differently in the region for a variety of societal reasons as well as local case levels.The COVID-19 pandemic was associated with epidemiological and activity changes regarding upper extremity fractures and dislocations presenting to EDs. The present study demonstrates notable decreases in rates of upper extremity fractures and dislocations, increases in rates of injuries related to outdoor and individual sports such as skateboarding with corresponding decreases in rates of injuries related to organized sports such as basketball, increases in the rates of injuries occurring in homes and in association with pet supplies, and decreases in rates of injuries occurring in schools and places of recreation observed during the pandemic. Additionally, trends observed among patient disposition specific to the pandemic, such as increasing rates of patient admission, observation, and AMA departure with decreasing rates of discharge and transfer, offer insight into the burden of upper extremity injuries on the healthcare system during this critical time. While upper extremity orthopedic injuries remained common through the pandemic, the early pandemic was associated with higher rates of hospital admission that normalized closer to pre-pandemic levels by 2021, which may herald a shift and return to pre-pandemic trends. Future research will determine the long-term downstream effects of COVID-19 on activity-related orthopedic injuries and bone health.


P = 0.001). Both FA-guided and ICGA-guided half-dose PDT were effective in the treatment of non-resolving CSCR, with favorable functional and anatomical outcome. In FA group, PDT with smaller spot sizes and fewer numbers of spots were applied.To compare the functional and anatomical outcome of fluorescein angiography (FA) versus indocyanine green angiography (ICGA) guided photodynamic therapy (PDT) in the treatment of non-resolving central serous chorioretinopathy (CSCR). In this prospective interventional case series, all patients with non-resolving CSCR, defined as persistent SRF involving subfoveal area for at least three months, were nonrandomly assigned to receive either FA or ICGA-guided half dose PDT. Baseline and 4 months post-treatment data including best corrected visual acuity (BCVA), the status of foveal subretinal fluid, subfoveal choroidal thickness, choroidal vascularity index, pigment epithelial detachment area, treatment and PDT spot numbers were collected. Thirty-six eyes were included; 24 received ICGA-guided and 12 received FA-guided PDT. Overall, improvement in BCVA and choroidal parameters were observed in all patients. There was no significant difference in baseline parameters as well as follow-up measurements between groups. However, the mean total energy dose and spot number in the IGCA-guided PDT were significantly higher than the FA-guided PDT group ( Neurosensory retinal detachment is the predominant morphological feature of this disorder, which mainly involves the macular region. Decreased visual acuity, vision distortion, relative scotoma, dyschromatopsia, micropsia, and reduced contrast sensitivity are among the early symptoms of this disorder2. Therefore, the current trend is more toward treatment rather than observation. Although several medical and laser treatments have been evaluated, photodynamic therapy (PDT) has been found to be the most effective method of treatment for acute and chronic CSCR7.Most cases resolve spontaneously, however, chronic or non-resolving CSCR may progress to the atrophy of the retinal pigment epithelium (RPE) and retina with eventual vision loss8. Recognition of the areas of hyperpermeability of choroidal vessels, even in subclinical phase of CSCR, is best done using Indocyanine Green Angiography (ICGA)7. Many specialists apply PDT spots on the pathologic regions illustrated in ICGA, however, this dye is expensive and may not be available in all centers.During PDT, a light-sensitive pigment called Verteporfin is employed to reduce the permeability of the choroidal arteries and therefore resolution of the subretinal fluid (SRF)9.Fluorescein angiography (FA) can show areas of the leakage through RPE defect and is a less expensive and more accessible image modality. Koytak and colleagues applied PDT on pathologic areas shown on FA and obtained good results in terms of vision improvement and resolution of SRF11. In the current study, we aimed to compare changes in parameters such as BCVA, choroidal thickness, the status of SRF, choroidal vascularity index (CVI), pigment epithelial detachment (PED) area, number of applied laser spots and total PDT laser energy, between FA and ICGA-guided half-dose PDT in patients with non-resolving CSCR.Few studies have compared FA based PDT versus ICGA based PDT for treatment of non-resolving CSCR, and the results regarding resolution of subretinal fluid, improvement in best corrected visual acuity (BCVA), central macular thickness and subfoveal choroidal thickness (SFCT) are contradictoryIn this prospective interventional case series, all patients with non-resolving CSCR who were referred to Farabi Eye Hospital from December 2020 to April 2021 were enrolled. Non-resolving CSCR was defined as persistent subretinal fluid involving foveal area for at least three months based on enhanced depth imaging optical coherence tomography (EDI-OCT) images.History of steroid usage (either systemic or ocular), Cushing's syndrome, diabetic retinopathy, high myopia (6 D and above), previous PDT or focal laser photocoagulation, treatment with anti-VEGF agents, choroidal polypoid vasculopathy, congenital macular or retinal disease, pregnancy, and posterior uveitis were noted as exclusion criteria. Written informed consent letter was obtained from all participants. The ethics committee of Tehran University of Medical Sciences approved this study (Code: IR.TUMS.FARABIH.REC.1399.025). The study adhered to the tenets of the Helsinki Declaration.Demographic data was collected and a comprehensive ocular examination and dye-based angiography  was performed for all patients. Additionally, patients underwent EDI-OCT  and OCT angiography (OCTA)  at baseline. Patients were non-randomly allocated to receive either FA guided or ICGA guided PDT. As our allocation was not random, there was a concern for unequal distribution of FA leakage patterns among the two groups. Therefore, from both groups, we selected eyes having both FA and ICG available, and compared the patterns of FA leakage between them. These FA leakage patterns were classified into discrete, multiple discrete, and diffuse leakage. We also evaluated areas of abnormal leakage in both FA and ICGA images. We determined whether these areas are compatible with each other, in both treated and asymptomatic fellow eye.2) over eight minutes and were treated with laser two minutes later. The standard dose of 50 J/cm2 laser was applied for 83 s. For those who underwent ICGA, an interval of at least three days from ICGA, was considered for PDT application.In FA group, areas of active leakage and in ICGA group, areas of choroidal vascular hyperpermeability in middle and late phases were considered for laser application. Meticulous attention was paid to exclude areas of window defect and staining in FA group, from laser treatment. All patients received intravenous infusion of Verteporfin , SFCT, CVI, PED area, total PDT laser energy dose, area and number of spots applied.http://imagej.nih.gov/Fiji/). Both EDI-OCT and OCTA images were exported into FIJI software. Imaging measurements were performed by two investigators who were blind to the treatment label . The measurement of retina and choroid was performed manually in horizontal B-scan EDI-OCT images centered at fovea; thickness of the choroid was considered as the distance between the outer border of the RPE and the choroid–sclera border, and the thickness of retina was considered as the distance between internal limiting membrane and RPE  area. Images with poor quality or from eyes with media opacity precluding acceptable image acquisition, were excluded from the study. Segmentation errors were manually corrected by a blind expert investigator (A.M). Image analysis was conducted by FIJI  and reported by descriptive statistics such as mean, standard deviation for quantitative values, and number and percentage for qualitative ones. The normality of the quantitative data was assessed using the Kolmogorov-Smirnov test. Inter- and intra-group analyses of normally-distributed data at baseline and four months after PDT were performed by independent t-test and paired t-test, respectively. Non-normally distributed data at baseline and four months after were compared by Mann-Whitney U test and Wilcoxon test. The analysis of covariance (ANCOVA) was used to adjust for baseline measures and to provide an unbiased estimate of the mean group difference of the 4 months results in two groups. Also, in this study, qualitative variables were compared using Fisher's exact test and Chi-square. A p-value less than 0.05 was determined as statistically significant. Statistical analysis was performed by a statistician blinded to group label (KF).P value: 0.023, and 0.016; respectively). Hence, the total PDT energy was higher in ICGA-guided group .In the present study, we included 36 eyes; 24 eyes received ICGA-guided PDT and 12 eyes received FA-guided PDT. Twenty-eight eyes (77.8%) of males and 8 eyes (22.2%) of females were assessed. The mean age of patients was 44 ± 8.8 years. Table To assess the distribution of various patterns of FA leakage between our two study groups, we selected 28 eyes for whom both FA and ICG were available. Evaluating FA, 17 (47.2%) eyes had one discrete leaking area, 15 (41.7%) eyes had multiple discrete leaking areas, and 4 (11.1%) eyes showed diffuse leakage. Patterns of leakage in FA were equally distributed among FA-guided and ICGA-guided groups . Leakage areas in FA were compatible with ICG in 19 of 28 (68%) treated eyes. While six eyes showed a staining spot in FA of their asymptomatic fellow eyes, 21 of 28 (75%) eyes with ICGA had hyper-fluorescence in the asymptomatic fellow eyes. Hence, disparity between FA and ICG was observed in 9 of 28 (32%) treated eyes, whereas this was remarkable in 20 of 28 (72%) fellow eyes.Table p value: 0.827). Complete resolution of FSRF occurred similarly in both groups; 10 (83.3%) eyes in FA- and 17 (70.8%) in ICGA-guided groups, respectively . Any amount of fluid reduction was also similar between the two groups .With treatment, 34 (94.4%) eyes showed a reduction of FSRF; twenty-seven (75%) eyes had complete resolution of fluid. Two eyes, one in FA group and one in ICG group, with persistent FSRF, received another PDT treatament session; one eye had complete resolution of fluid and the other one showed a decrease in FSRF. Vision improvement was not statistically different among eyes with or without complete fluid resolution  eyes become completely PED free, one from FA and 4 from ICGA group, and this was similar between the two groups . None of the eyes experienced recurrence or exacerbation at the final visit.We found a significant improvement in functional and anatomic parameters in both FA-guided and ICGA-guided PDT in our patients with non-resolving CSCR. However, the FA group achieved this with significantly lower laser energy required and smaller areas to be covered during PDT. We observed similar rates of BCVA improvement and fluid resolution in both groups. Additionally, we found that the reduction of CVI and PED area is the comparable between the two groups.FA is a less expensive imaging modality than ICGA and is widely available in retina clinics. Based on our finding, FA can be employed for guiding PDT in patients with non-resolving CSCR, with smaller spot size and fewer number of laser spots compared to ICGA.14. Several modifications involving time and fluence have been made to compensate for complications. However, occurrence of irreversible damage still remains a concern. Studies reported the possibility of choriocapillaris hypoperfusion and resultant decreased vision with PDT15. Bearing this in mind, the chances of complications would increase with the application of higher laser energy by including more extensive areas in PDT. According to Yannuzi et al.7 PDT area with spot sizes larger than 6000 µm, did not achieve visual improvement.PDT is an effective treatment modality to tackle complications of non-resolving CSCR. However, it may cause ischemic damage to choriocapillaris with consequent RPE atrophy resulting in unfavorable visual outcome. The most effective and safe PDT protocol for CSCR treatment is still an ongoing challenge for ophthalmologistsDue to intrinsic difference between ICGA and FA, subclinical cases of CSCR that have not developed RPE damage with subsequent leakage into SRF, may not show pathology in FA. In accordance with this fact that FA cannot penetrante to depict RPE/choroidal pathologies, we detected a higher rate of choroidal pathology using ICGA compared to FA in the asymptomatic fellow eyes. However, when the eye becomes symptomatic with FSRF, the disparity between FA and ICGA pathologic areas becomes less apparent (68% percent in asymptomatic fellow eyes versus 28% percent in treated eyes). The impact of this finding on the indication of treatment for asymptomatic eyes is still an ongoing debate. Although our short term results failed to reveal any benefit in treating the areas that show solely choroidal hyperpermeability and congestion.16. However, they did not have a control group of ICGA-guided treatment. Koytak et al., in a subsequent study, compared FA-guided versus ICGA-guided PDT in eyes with chronic CSCR, and showed vision improvement and reduction in retinal and choroidal thickness in both groups9. However, they possibly had a selection bias by recruiting the eyes with discrete leakage area in FA-guided group, and the more diffuse ones in ICGA-guided group. In our study, the baseline characteristics between the two groups, including the pattern of fluorescein leakage were not different among the two groups. Recently, Hayashida et al. compared FA-guided and ICGA-guided half-time PDT for the treatment of chronic CSCR and concluded that ICGA group give a more favorable anatomical outcome while the visual function was similar between their two groups. Their dissimilar result may be due to different treatment protocol. Recent studies assessed alternative imaging modalities for guiding focal laser photocoagulation for the treatment of CSCR. Maltsev et al. used OCT to find the area of RPE defect on which focal laser was applied. They showed both anatomical and functional success with this technique18.The first attempt to incorporate FA in guiding PDT for eyes with chronic CSCR dates back to the investigation in 2020 by Koytak and colleagues, who reported vision improvement and reduction in retinal thickness at one year following FA-guided half dose PDT20. Agrawal and colleagues observed a lower CVI in eyes with resolved CSCR compared to those with acute CSCR19. CVI accounts for a more reliable measurement for choroidal parameters as this ratio is least affected by diurnal variations of choroidal structure. This index has been shown to be plausible for monitoring disease activity19. In this regard, successful treatment of CSCR with half-dose PDT, results in vision improvement as well reduction of CVI and choroidal thickness21. Interestingly, undertreatment with half-dose-half-fluence PDT results in SFCT reduction, in the absence of vision improvement or CVI reduction21. In our study, vision improvement along with CVI reduction in both FA-guided and ICG-guided treated groups, indicates a sufficient initial treatment response.To the best of our knowledge, this is the first study to report the comparison of alterations in CVI and PED area in FA-guided versus ICG-guided PDT in eyes with CSCR. Based on our finding, CVI and PED area reduced significantly following treatment. Based on our intergroup analysis, the decrease in CVI and PED area, was similar between the two groups. According to previous studies, eyes with acute and chronic CSCR have higher CVI compared to normal population23. There is a spatial relationship between the location of PED and the area of choroidal vascular abnormalities in eyes with CSCR24. Therefore, it is reasonable that following treatment of the abnormal choroid, a reduction in the PED area ensues. Our results demonstrated that 5 of 17 (29.4%) eyes with PED achieved a complete PED resolution, and 12 (70.6%) had a decreased PED area following treatment, which was similar between our two groups. This is consistent with previous studies that demonstrated PED improvement following PDT26.The presence of PED is a common feature seen in eyes with CSCR, more prevalent in chronic forms of the diseaseOur study has some limitations. First of all, it is a non-randomized study, that may impose a bias to the results. Additionally unequal sample size between groups might be another source of bias. We applied the following approaches to compensate for these possible sources of bias. The patients had similar baseline characteristics in each group. We ensured that FA leakage patterns are not different among FA-guided and ICGA-guided groups. Additionally, the investigators who performed image and statistical analysis were blind to treatment label. A relatively small sample size and short follow-up time is another limitation of our study.In conclusion, FA and ICGA-guided half-dose PDT showed similar functional and anatomical improvement in patients with non-resolving CSCR at 4 months following laser. FA guide is associated with applying less laser energy compared to ICGA guide and FA is more accessible and less expensive. The decision to surrogate FA with ICGA as a guide for PDT, still awaits further prospective validation with randomized controlled studies with longer follow up time.


Deubiquitinases (DUBs) have been the subject of intense scrutiny in recent years. Many of their diverse enzymatic mechanisms are well characterized in vitro; however, our understanding of these enzymes at the cellular level lags due to the lack of quality tool reagents. DUBs play a role in seemingly every biological process and are central to many human pathologies, thus rendering them very desirable and challenging therapeutic targets. This review aims to provide researchers entering the field of ubiquitination with knowledge of the pharmacological modulators and tool molecules available to study DUBs. A focus is placed on small molecule inhibitors, ubiquitin variants (UbVs), and activity-based probes (ABPs). Leveraging these tools to uncover DUB biology at the cellular level is of particular importance and may lead to significant breakthroughs. Despite significant drug discovery efforts, only approximately 15 chemical probe-quality small molecule inhibitors have been reported, hitting just 6 of about 100 DUB targets. UbV technology is a promising approach to rapidly expand the library of known DUB inhibitors and may be used as a combinatorial platform for structure-guided drug design. Over 40 years ago, the seminal works of Avram Hershko, Aaron Ciechanover, and Irwin Rose  engendered the ubiquitin (Ub) field as we know it today. A first-hand account of these early years, for which the 2004 Nobel Prize in Chemistry was awarded, has been delineated by Varshavsky . It is nThe complexity of molecular outcomes as a result of ubiquitination presents a ubiquitin code ,4. SinceUbiquitination is reversed by the activity of deubiquitinase enzymes (DUBs) . About 1DUBs are essential for maintaining Ub homeostasis by processing Ub precursors   and uncoThe factors dictating DUB activity and substrate specificity include the structure of the catalytic domain, the presence of additional domains for substrate recruitment or Ub binding ,25,26, aexo-cleavage), from within a chain (endo-cleavage), or all at once (en bloc-cleavage) . . 103]. USince inception, UbVs have been developed for almost every level of the UPS. Although binders for the human E1 Ub-activating enzymes have not yet been reported, UbVs targeting Ub-conjugating components at the E2 ,110,111 SP.3 was >250-fold more specific for STAMBP than STAMBPL1, despite a 56% sequence homology. This was determined to be a result of key hotspot mutations in the Ub C-terminal tail and the preservation of wildtype Arg-42 [The site to which a UbV binds dictates its function as an inhibitor, activator, or allosteric modulator. This has been nicely demonstrated for the UbVs targeting the HECT family of E3 ligases . To datee Arg-42 .T9F/T66K with a specificity index of 33 in favor of UCHL1 [Q40V/T9F/V70F) with a specificity for UCHL3 over UCHL1 [Alternatives to the method used by the Sidhu group have been used to produce potent UbVs. Early efforts to optimize an inhibitor for USP7 relied on in silico methods to guide phage display library design . A UbV fof UCHL1 . In a foer UCHL1 , but itsFirst, when considering the vastness of the UPS, only a fraction of the possible UbV targets have been successfully hit. Screening the billions of UbVs in existing libraries will undoubtedly lead to discovering new binders. New randomization strategies for the library design can also help identify novel binders with previously undescribed functions. Further variation of the β1–β2 loop might be key ,129. An Second, a focus should be placed on developing UbVs that bind alternative sites. The intra- and intermolecular modulation of DUB activity is well-characterized ,24. USP7Third, poly-UbVs for DUBs have not yet been appreciably explored. The USP15-targeting di-UbVs 15.1/D and 15.D/1 illustrate how a multisite approach can increase both the affinity and specificity . Strand-UbVs should be treated in a similar manner to chemical probes . HolistiAs described above, target validation for UbVs in live cells or cell lysates is relatively straightforward. Cellular delivery is usually mediated by the transient transfection of a plasmid encoding the UbV of interest or by the generation of inducible stable lines . Alternatively, purified UbVs conjugated to a reactive group can be added to cell lysates to facilitate activity-based protein profiling . However, these methods do not permit in vivo delivery in a drug-like manner. As UbVs are cell impermeable, delivery methods are required to gain access to their intracellular targets and to assess their therapeutic potential in live cells.Methods for delivering biopharmaceuticals have been reviewed previously ,134,135.Activity-based probes (ABPs) have long been used as tools to study the mechanism of metabolic and signaling enzymes, including DUBs. When applied to whole proteomes, ABPs can resolve changes to the enzyme activity independent of the expression levels. They have been used to discover new DUBs and determine the structures of Ub-bound DUBs for compound/inhibitor screening and for Ub substrate profiling. Many excellent reviews have summarized their design, optimization, and application ,143,144.These adaptable molecules incorporate three functional elements: (1) a recognition element that endows the target specificity, (2) a reactive moiety (or warhead) that covalently reacts with a target catalytic residue, and (3) a reporter tag (or handle) to facilitate the detection and/or enrichment of the ABP-labeled target enzyme A. The moendo-cleavage was preferred for K11 and K63 but not K48-linked tri-Ub ABPs that contained a non-cleavable distal linkage and a native, cleavable proximal isopeptide linkage. Interestingly, K48-linked tri-Ub is exclusively processed by the exo-cleavage mode. The ABP that resolved this was a tri-Ub containing a distal Michael acceptor warhead and a native, cleavable-proximal isopeptide bond. These data suggest that USP9X poly-Ub processivity is linkage-dependent and more rapidly deconjugates both K11 and K63 rather than K48-linked chains [A mono-Ub C-terminal electrophile that binds the S1 site is the simplest DUB ABP. To study linkage specificity, poly-Ub ABPs that occupy multiple Ub-binding sites have been developed ,147. Thed chains .Using Ub as the recognition element affords DUB-targeting ABP broad selectivity. To afford a greater target specificity, UbV ABPs have been utilized in the past ,123,124.Instead of a full-length Ub, smaller recognition elements such as Ub-derived peptides and small molecule inhibitors have also been used ,149,150.The choice of reactive group (cysteine-reactive electrophile) must be carefully considered, as different chemistries will influence the reactivity and selectivity. This was demonstrated recently when comparing the reactivity of a UCHL1-targeting ABP bearing either a propargyl amide (PA) or vinyl methyl ester (VME) warhead . FurtherCell-based assays that successfully address a particular biological question depend on a reliable method with an unadorned readout. For DUBs, a major goal is to establish a more complete understanding of the up- and downstream effectors, interacting partners, substrates, and context-dependent specific activity. To mitigate artifacts is critical and requires highly selective tools and unintrusive methods. In this final section, we discuss genetic and pharmacological approaches to studying DUBs at the cellular level and how high-quality modulators are incorporated to bolster the results.Omics-level analyses of PPIs give a broad view of the molecular networks. Laundry lists of high-confidence interactors serve as great starting points for characterizing novel interactions and their associated biological significance. Classical proteomics workflows rely on AP-MS to identify co-purifiers of an endogenous or overexpressed protein of interest. Using AP-MS, the BioPlex project expanded the known human interactome ,155. The*). A systematic study of DUB interactomes by BioID would be an indispensable asset to the community. Using BioID, USP35 isoforms 1 and 2 were shown to be anti- and proapoptotic, respectively. As baits, isoform 1 was found to associate with cytosolic proteins, whereas isoform 2, containing a transmembrane domain, was associated with ER membrane proteins [Inherent to the AP-MS methods are biases against transient interactions, the isolation of only soluble proteins, and the non-physiological association of once-segregated  proteins upon cell lysis and dilution. To circumvent this, proximity-dependent biotinylation coupled to MS (PDB-MS or BioID) was developed . While tproteins . The disproteins .DUB interactors can be divided into three classes. (1) Constitutive binding partners, often as part of larger macromolecular complexes, e.g., USP22/SAGA-DUBm  and CSN5USP16 is embryonic-lethal in mice [The genetic interference of target genes using RNAi or clustered regularly interspaced short palindromic repeat (CRISPR) technology has become routine in regular laboratories. The challenge of genetic interference is that the encoded proteins are knocked down or removed from cells in their entirety. Since most DUBs have domains other than the catalytic domain that could play essential roles in target localization, trafficking, substrate recruitment, PPIs, or scaffolding, knockdown or knockout of the protein can often have unexpected outcomes that differ from the inhibition of the enzymatic activity. The drawback can be partially salvaged by using CRISPR base editing or mutagenesis techniques ,174. Thi in mice ; therefo in mice  with appThere are still very limited examples of introducing inactivated mutations in DUBs using CRISPR editing technology. It is prudent to compare the results from genetic interference with those from pharmacological interference, which targets the enzymatic activity of DUBs specifically. Done in parallel, these efforts would validate the functional roles of the DUBs under study. In a recent example, the Dirks and Angers groups used genome-wide CRISPR screening and identified USP8 as one of the vulnerable genes from patient-derived glioblastoma stem cells and then validated USP8 to be an actionable therapeutic target using the lentiviral delivery of a UbV inhibitor .DUB research remains a field ripe with opportunity. Both small molecule inhibitors and biological probes, such as ABPs and UbVs, have proven to be powerful tools in studying the biology of the DUB family of enzymes. While small molecules are the gold standard therapeutic scaffold, UbVs are critically important to leverage to expand the available DUB modulators. The demonstration of the druggability of DUBs, especially that of USP7, which has a canonical USP fold lacking the insertions commonly seen in many USP family members, and the recent publications of several high-quality inhibitors for other USPs, shall attract renewed interest in drug discovery for this challenging but important family of proteins. So far, there have been very few chemical probe-quality inhibitors. We observed approximately 15, hitting just 6 of the >100 DUB targets. This constitutes only three of the seven families. Many new structures of DUBs have been reported in recent years, and artificial intelligence-driven, structure-guided small molecule drug discovery would be a particularly interesting application.Given the naturally large Ub-binding pocket of DUBs, the UbV technology is especially suited for finding potent DUB inhibitors. This technology is suitable for generating inhibitors rapidly and cost effectively. These DNA-encoded inhibitors can be delivered using a lentiviral system or plasmid-mediated transfection to study the cellular function of the cognate DUBs. However, the direct delivery of cell impermeable, purified UbVs needs to be addressed further. The recent successful nanoparticle-encapsulated mRNA vaccine for COVID-19 is another potential UbV delivery technique that has not been tried and would be worth exploring.Ubiquitination is an extremely complicated PTM compared to others, such as phosphorylation, methylation, and acetylation. A near-complete set of high-quality tool reagents targeting each individual DUB, combined with genetic intervention and proteomics, would be a dream team for studying the fascinating biology of DUBs at the cellular level.


The differential risk profiles associated with prefrailty may be attributable to underlying intrinsic capacity (IC).We examine (i) effect of a multi-domain physical exercise and nutrition intervention on pre-frailty reversal in community-dwelling older adults at 1-year, and (ii) whether IC contributes to pre-frailty reversal.Prefrail participants in this non-randomized study were invited to attend a 4-month exercise and nutritional intervention following a frailty screen in the community. Prefrailty was operationalized as (i) FRAIL score 1–2 or (ii) 0 positive response on FRAIL but with weak grip strength or slow gait speed based on the Asian Working Group for Sarcopenia cut-offs. Participants who fulfilled operational criteria for prefrailty but declined enrolment in the intervention programme served as the control group. All participants completed baseline IC assessment: locomotion , vitality , sensory (self-reported hearing and vision), cognition , psychological . Reversal of prefrailty was defined as achieving a FRAIL score of 0, with unimpaired grip strength and gait speed at 1-year follow-up.p = 0.206). Reduced prevalence of depression was observed among participants in the intervention group at 1-year relative to baseline . In multiple logistic regression, intervention had no effect on prefrailty reversal, while higher composite IC exhibited reduced likelihood of remaining prefrail at 1-year .Of 81 participants , 52 participants (64.2%) were enrolled in the multi-domain intervention, and 29 participants (35.8%) who declined intervention constituted the control group. There was no difference in age, gender and baseline composite IC between groups. Reversal to robustness at 1-year was similar between intervention and control groups (30.8% vs. 44.8% respectively, Focusing only on the locomotion and vitality domains through a combined exercise and nutritional intervention may not adequately address component domain losses to optimize prefrailty reversal. Future studies should examine whether an IC-guided approach to target identified domain declines may be more effective in preventing frailty progression. Healthy aging has been presented by the World Health Organization (WHO) as the process of developing and maintaining the functional ability to enable well-being in older age . IntrinsSince its inception, cumulative evidence has supported declining IC as being representative of the diminished homeostatic reserves underpinning the excess vulnerability typical of frailty –7. AccorPrefrailty has been recognized as a prodromal state along the frailty continuum. While the consensus established that prefrailty is reversible, the process is likely non-linear with dynamic transitions and trajectories between robust, prefrail and frail states . FurtherWe aim to examine (i) effect of a multi-domain physical exercise and nutrition intervention on pre-frailty reversal in community-dwelling older adults at 1-year, and (ii) whether IC contributes to pre-frailty reversal.For this non-randomized controlled trial of a multi-domain physical exercise and nutritional intervention, potential prefrail participants were identified through our ongoing community frailty screening “Individual Physical Proficiency Test for Seniors (IPPT-S),” which had been previously described . In brieAll participants completed a multi-domain geriatric screen and physical fitness assessment administered by trained study team members at baseline and 1-year follow-up. The multi-domain geriatric screen included risk factors for frailty—mood , cognition , and nutrition  –21. FuncThe physical fitness test battery was modified from the Senior Fitness Test , and par2). Low muscle mass was defined using Asian Working Group for Sarcopenia 2019 (AWGS2019) cut-off values of <7.0 kg/m2 for men and <5.7 kg/m2 for women , with appendicular skeletal mass index (SMI) calculated as the sum of fat-free lean mass of all 4 limbs divided by height-squared  once-weekly group-based exercise classes lasting 1 h each session  with individually prescribed home exercises for maintenance between sessions and (ii) group-based nutritional education (6 sessions). Group size was maintained at 8–10 participants to ensure that each participant received adequate attention. While the intensity of exercise was not measured, the target was to achieve at least moderate intensity as tolerated by the seniors. The exercises focused on strength, balance and endurance training, with a warm-up and cool-down routine. TheraBands and step boards were used for resistance and balance training, respectively. The exercise sessions were designed for progressive intensity  based on the group's progress, while catering for individual variability, with group sessions conducted under the supervision of a physiotherapist or an exercise physiologist. Each session commenced with 5 min of dynamic warm-up e.g., slow marching with small to big arm circles, followed by 45 min of exercises focusing on: (a) balance, coordination and speed e.g., heel to toe walks; (b) strength e.g., rising from a chair and Theraband exercises for lower and upper limbs, respectively; and (c) endurance e.g., fast walking. All sessions ended with 5 min of static cool down e.g., stretching muscles of the thigh and arms with slow breathing. Individually prescribed structured home exercise folders comprising pictorials and written explanations were provided at the end of each session, to encourage participants to maintain regular physical exercise between the group classes. Compliance with home exercise was tracked using a weekly diary, and participants were instructed to achieve a target of performing the prescribed home exercises at least 3 days each week. The nutritional intervention was delivered with the aim of facilitating healthy eating habits to achieve adequate protein, energy, calcium and Vitamin D through regular food and beverages that are more specific to the Asian palate. There were 6 sessions over the 4-month intervention period with 2 sessions per month in the first and second months and 1 session per month in the third and fourth months. Each session lasted 1.5 h and was delivered by a trained nutritionist, incorporating a combined modality of teaching methods that included didactics, food-based games and grocery-shopping trips with sponsored vouchers to provide guidance on choosing quality foods within budget.Participants who fulfilled operational criteria for prefrailty but declined enrolment in the intervention programme following IPPT-S screening served as the controls. As part of the IPPT-S screening, these participants (as well as those in intervention group) would have received an individual counseling session with a member of the study team, providing feedback on the results of their screening, along with advice on physical activity and nutrition for frailty prevention reinforced in a personal frailty booklet. They were also invited to attend 4 group-based education classes on frailty that were conducted within the senior activity centers after each screening cycle.Reversal of prefrailty was defined as achieving a FRAIL score of 0, with unimpaired grip strength and gait speed at 1-year follow-up. Hospitalization and falls during the 1-year period were captured based on self-report.t-tests or Wilcoxon rank-rum tests were used for univariate analyses comparing intervention and control groups in baseline characteristics and outcomes. Within-group changes in functional performance and prevalence of frailty risk factors between baseline and 1-year follow-up was examined using Wilcoxon signed-rank or paired sample t-tests and Mc Nemar's tests. Multiple logistic regression was performed to examine the independent effect of intervention and IC on prefrailty reversal, adjusted for age, gender and any significant univariate variables. Two separate models were compared—the first model included individual IC domains and the second model included composite IC score. Statistical analysis was performed using STATA SE 15.0 . All statistical tests were two-tailed, with p-value ≤ 0.05 considered statistically significant.Descriptive data are presented as means (±SD) or median  for quantitative variables and as absolute and relative frequencies for categorical variables. Chi-squared and p = 0.064).Among 209 participants with complete IC data and meeting operational criteria for prefrailty, only 81 were available for 1-year follow-up owing to restrictions imposed by the COVID-19 pandemic. Fifty two participants (64.2%) were enrolled in the multi-domain exercise and nutritional intervention programme, and 29 participants (35.8%) who declined intervention constituted the control group. There was no difference in age and composite IC score between participants who attended 1-year review compared with those who were excluded, although male participants were more likely to have been excluded albeit not statistically significant . Weekly exercise target was achieved only for 5 (interquartile range: 1–9) weeks over the intervention period.p = 0.047), and less likely to be in active employment . Impaired performance on the CMMSE was significantly more prevalent in the intervention group, contributing to significant decline in the cognition domain compared with control . The intervention group also exhibited greater decline in the sensory domain . Locomotion, psychological and vitality domains were similar between intervention and control groups, with both groups having similar composite IC scores . Individual frailty criteria by FRAIL items were similar between intervention and control groups, although the intervention group had significantly more participants with slow gait speed at follow-up. There was no difference between intervention and control groups in 1-year prevalence of cognitive impairment, depression and malnutrition (p = 0.022). Functional performance, life space mobility and physical activity level were similar between intervention and control groups at follow-up, and the measures were stable relative to baseline in both groups. While BMI was similar between groups at follow-up, control group participants registered a significant gain in BMI at 12-month relative to baseline (paired sample t-test p = 0.002). Both intervention and control groups exhibited significant improvement in grip strength at follow-up, with weak grip observed in 48.3% of intervention and 51.9% of control group participants at follow-up, relative to 71.2 and 79.3%, respectively at baseline . 1-year incidence of hospitalization was similar between groups, with a trend for lower falls incidence in the control group (p = 0.065).Sixteen (30.8%) participants in the intervention group no longer fulfilled operational criteria for prefrailty at 1-year, and reversal to robustness was also observed in 13 (44.8%) of control participants (utrition . Howeverp = 0.034). Prefrail participants who were both FRAIL score positive and exhibited slow gait and/or weak grip strength at baseline were most likely to remain prefrail compared with those who were asymptomatic on FRAIL but enrolled due to slow gait speed and/or weak grip strength .In Model 1 including age, gender, active employment status, baseline prefrailty enrolment criteria, and the cognition and sensory domains in isolation, receipt of multidomain exercise and nutrition intervention was not associated with prefrailty reversal. Older participants were significantly more likely to remain prefrail . Higher composite IC at baseline was associated with reduced likelihood of remaining prefrail . Again, intervention had no effect on prefrailty reversal , independent of intervention exposure  and National Innovation Challenge on Active and Confident Ageing (MOH/NIC/HAIG04/2017). The grants funded the research staff, assessment equipment and on-site conduct of the trial, and the researchers were independent from funders.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


These are exciting exploratory times for structural/valvular heart interventions. The growth of medical knowledge is expanding, and novel therapeutic options are constantly emerging. Mitral regurgitation (MR) is the most prevalent valvular heart disease ,2,3,4. MIn the most recent guidelines , percutap = 0.042), the one-year mortality rate was similar between the groups . There was also the improvement of NYHA functional class and sPAP reduction in both. This indicates that PMR could be a viable option for the treatment of patients with severe MR and ADHF [In this Special Issue, we have investigated some of these matters. Medvedovsky et al. explored the impact of percutaneous MR repair (PMR) during acute decompensated heart failure (ADHF). In the study, the researchers compared high-risk patients who underwent PMR during hospitalization due to ADHF with elective patients. From a cohort of 237 patients, 46 patients (19.4%) presented with severe MR of either functional or degenerative etiology who underwent emergent PMR during index hospitalization. These patients were at a higher risk for surgery compared with elective patients. While the thirty-day mortality rate was higher in ADHF patients as compared to the elective group , improved (n = 40), and decreased (n = 41). For a median follow-up time of 2 years, following adjustment to confounders, decreased postprocedural left ventricular ejection fraction (LVEF) was associated with an increased risk of death , whereas increased postprocedural LVEF was associated with a reduced risk of death  compared to the unchanged LVEF group [A second paper on patients with MR looked at the impact of post-PMR change in left ventricular function on survival. The authors, led by Hagnäs et al., studied outcomes of 399 patients who underwent percutaneous edge-to-edge mitral valve repair for secondary MR and divided the cohort into three groups: unchanged . Residual TR was ≤2 in 79% of patients (paired p < 0.001) [Currently, the predominant percutaneous TR therapy is edge-to-edge repair using the MitraClip/TriClip Transcatheter Tricuspid Valve Repair Systems  and the PASCAL transcatheter valve repair system . Medical centers throughout the world are reporting their initial edge-to-edge repair experience for TR. In our Special Issue, Cepas-Guillen et al. report their initial experience with edge-to-edge repair using the MitraClip and TriClip systems, assessing the efficacy and safety in the first consecutive 28 patients with severe TR. Only one patient experienced a procedural complication . At three-month follow-up, 83% of patients were in NYHA I or II  .p = 0.36). Mortality at one year was 8.0%. It thus seems that the tricuspid ViV procedure is feasible and safe, and certainly a less invasive treatment option (versus redo-open heart surgery) for patients with tricuspid valve degeneration.In addition, the valve-in-valve (ViV) technique is becoming a viable percutaneous alternative for the treatment of bioprosthetic structural valve deterioration (SVD) in the tricuspid position. Our Special Issue includes two articles discussing this clinical matter. In a case report and review by Montenegro da Costa et al., they describe the implantation of a 32 mm MyVal transcatheter aortic valve device  in a 58-year-old female with a history of bioprosthetic valve implanted at the tricuspid position 27 years prior to the index event. The valve had deteriorated, resulting in severe tricuspid stenosis and mild TR. Treatment using the ViV technique was successfully performed, resulting in a reduction in the mean gradient to 3.3 mmHg . From ou®, Tricento®, and TrilliumTM systems). The author suggests that procedures with a CAVI-specific device are relatively simple and predictable, requiring fewer anatomical pre-requisites and interventional skills than other percutaneous tricuspid devices. However, the currently ongoing exploratory trials as well as future pivotal randomized studies versus optimal medical treatment and other therapeutic solutions will shed more light on the forecast of CAVI.Finally, Abdul-Jawad Altisent et al. describes the bicaval valve implantation (CAVI) technique in patients with severe TR . The proIn summary, the field of percutaneous treatment of atrio-ventricular valves is gaining momentum as more evidence becomes available regarding the optimal non-invasive assessment of patients with MR and TR prior to the procedure, the best timing and selection of patients who could benefit from intervention, as well as their prognosis. New percutaneous technologies, and the experience gained from the clinical studies assessing them, will likely lead to improved techniques and a better understanding of the broad aspects of these complex valvular heart disease patients.


Endometrial cancer (EC) incidence has been rising over the past 10 years. Delays in diagnosis reduce survival and necessitate more aggressive treatment. We aimed to develop and validate a simple, noninvasive, and reliable triage test for EC to reduce the number of invasive diagnostic procedures and improve patient survival.We developed a test to screen and triage women with suspected EC using 726 cervical smear samples from women with and without EC, and validated the test in 562 cervicovaginal samples using three different collection methods  and four different settings .Women's cancer risk IDentification – quantitative polymerase chain reaction test for Endometrial Cancer (WID-qEC), a three-marker test that evaluates DNA methylation in gene regions of GYPC and ZSCAN12. In cervical, self-collected, and vaginal swab samples derived from symptomatic patients, it detected EC with sensitivities of 97.2% , 90.1% (83.6 to 94.6), and 100% (63.1 to 100), respectively, and specificities of 75.8% (63.6 to 85.5), 86.7% (79.3 to 92.2), and 89.1% (77.8 to 95.9), respectively. The WID-qEC identified 90.9%  of EC cases in samples predating diagnosis up to 1 year. Test performance was similar across menopausal status, age, stage, grade, ethnicity, and histology.We describe the The WID-qEC is a noninvasive reliable test for triage of women with symptoms suggestive of ECs. Because of the potential for self-collection, it could improve early diagnosis and reduce the reliance for in-person visits. EC patient survival is strongly dependent on stage at diagnosis,3 with delays in diagnosis and treatment resulting in significant adverse impacts on survival.4 The current route of diagnosis for suspected EC is transvaginal ultrasound (TVUS) followed by hysteroscopy and endometrial biopsy.5 During the COVID-19 lockdown, referrals via the 2-week-wait urgent pathway for suspected cancer in England, United Kingdom, decreased by up to 84%. A 3-month delay in EC diagnosis in England alone has been suggested to cause a loss of 6,305 life-years.6 A rapid triage modality that could rule out malignancies without the need for initial specialist referral could improve patient care and reduce time to diagnosis.Endometrial cancer (EC) is among the tumor types with the sharpest rising incidence over the past 10 years.Key ObjectiveWomen's cancer risk IDentification – quantitative polymerase chain reaction test for Endometrial Cancer (WID-qEC), a patient-friendly tool for screening and triaging women with suspected EC.Endometrial cancer (EC) is among the most common cancers in women. Tests for suspected EC are invasive, expensive, and require a specialist setting. Assessing DNA methylation in 1,288 cervicovaginal specimens, we developed and validated the Knowledge GeneratedThe WID-qEC identified 100% of ECs within a cohort of women presenting with postmenopausal bleeding at 89% specificity. Moreover, the test could also be performed on self-collected samples and resulted in higher accuracy than ultrasound and somatic mutation analysis in our setting.RelevanceThe WID-qEC may enable rapid, noninvasive screening and triage for symptomatic women at greatest risk, avoiding invasive investigations in healthy women. Future prospective studies will confirm whether it could be used to replace ultrasound to triage women with suspected EC, and further clarify its utility for screening in general and high-risk populations.7 In Black women, the performance is poor and offers a sensitivity of only 43.7%.8 A positive triage result needs be followed up by histologic diagnosis, such as via hysteroscopic assessment and endometrial sampling. The low specificity of TVUS as an initial triage test, therefore, results in potentially high numbers of unnecessary invasive follow-up procedures.Current triage investigations available for suspected EC suffer from several limitations. Assessment of endometrial thickness using TVUS, the most frequently used initial investigation, is only feasible in postmenopausal women, and a cutoff of ≥ 5 mm has a sensitivity of 96.2% and specificity of 51.5%.9 As summarized in our recent review,10 we13 and others22 have previously assessed DNA-based markers in cervicovaginal samples for EC detection. DNA methylation changes in cancer appear across an entire region rather than single sites, for example, all cytosine nucleotides followed by guanine nucleotides (CpGs) in a given CpG island may be hypermethylated. This results in a substantially increased signal-to-noise ratio compared with single point mutation analysis.23 Sensitivities using molecular methods in cervical samples reached ≈80%,22 but previous studies were affected by various issues, including low sample numbers (most studies included < 40 EC cases), lack of independent validation, low sensitivities, and no standardized test format that could be applied in samples obtained via different collection strategies.Alternatives to ultrasound investigation using molecular testing have been developed. A blood-based test using cell-free DNA sequencing has reported sensitivities of 28% and 16.7% for detecting overall and stage I uterine cancers, respectively.Women's cancer risk IDentification – quantitative polymerase chain reaction test for Endometrial Cancer (WID-qEC) in several different settings. The test is based on quantitative, methylation-specific polymerase chain reaction (PCR) targeting one region in the gene ZSCAN12 and two regions in GYPC. Our data indicate that the WID-qEC may be amenable for use in self-collected samples and could improve triage and earlier diagnosis by reducing the need for in-person consultations and invasive testing caused by low specificity of TVUS. Future prospective studies comparing TVUS and the WID-qEC side-by-side will guide clinical application of this methylation-based test.A simple test to triage women with improved, or at least equivalent, accuracy to current standards  without the need for specialist referral is urgently needed. Ideally, such a test should also be applicable across different sample types, including self-collected samples. Here, we developed and applied the We identified the most informative regions in an epigenome-wide screen in cervicovaginal specimens from 716 women and developed the PCR-based WID-qEC test in 40 individuals (FORECEE Pilot), 30 of whom were present in the epigenome-wide screen .We validated the test in an independent group of 562 volunteers all > 18 years) in three diagnostic and two predictive settings .For all EC cases, histology data following biopsy or hysterectomy were available to confirm diagnosis and deemed as the reference standard. For controls, histology was not always available. In addition to STARD diagrams, the Data Supplement details where confirmation of diagnosis using reference standard was available. Briefly, most controls in the FORECEE study did not undergo biopsy/histology, except for controls with benign gynecologic conditions. In the Barcelona Validation set, all controls with benign gynecologic conditions underwent biopsies while hospital controls (attending for other conditions) did not. In the PMB Cohort, only individuals with abnormal ultrasound underwent biopsies and histologic confirmation, while the rest without ultrasound abnormalities did not. All individuals in the Lynch Cohort, except for one participant who refused, underwent a biopsy and histologic assessment. In the Karolinska Cohort, only EC cases underwent biopsies.24 The epigenome-wide study and details of the methylation-specific PCR-based MethyLight assay11 are described in the Data Supplement.WID-qEC test regions were discovered in an epigenome-wide approach . Cervicovaginal samples from 572 controls and 144 women with EC (FORECEE study) were subjected to the Illumina MethylationEPIC array following a previously established pipeline.PTEN, TP53, PIK3CA, ARID1A, and CTNNB1) that led to the highest sensitivity of identifying cancers  in The Cancer Genome Atlas data set, as previously described.25Details of DNA mutation analysis are described in the Data Supplement. Briefly, DNA mutation analysis was performed for five genes . Details for statistical methods are presented in the Data Supplement. Original data are available in the Data Supplement, and all analysis code is available on github and is archived on Zenodo.v non-White), and cancer characteristics .The workflow for WID-qEC test development and assessment is shown in Figure Validation of the WID-qEC in 137 cervical smear samples  led to a 97% sensitivity at 76% specificity and—assuming a prevalence of 9% in symptomatic women—PPV and NPV of 28% and 100%, respectively  Evalyn brush-collected samples , 90% of ECs were correctly identified at 87% specificity (Table For a subset of EC cases in this set (n = 109), DNA MMR status was available. The WID-qEC performed similarly in detection of MMR-proficient (n = 84) and MMR-deficient (n = 25) ECs (Data Supplement).15 In the Barcelona Validation set, ultrasound and DNA mutation data were available for subsets of women in addition to the reference standard , which enabled an initial comparison of the WID-qEC with different triage modalities.Ultrasound assessment is the most common initial assessment for suspected ECs, and DNA mutations in cervicovaginal samples were recently shown to indicate ECs.The WID-qEC offered similar sensitivity but significantly increased specificity compared with qualitative ultrasound assessment . AUCs for the WID-qEC score and quantitative ultrasound data  were similar Fig A, but thTo assess the test in a real-life setting, we recruited 63 women who presented consecutively because of postmenopausal bleeding at an outpatient clinic (PMB Cohort). Health care professionals in the clinic collected a vaginal swab sample from the posterior fornix. The WID-qEC correctly identified 100% of women who were subsequently diagnosed with EC at 89% specificity . The WID-qEC was negative in all women who did not show any signs of cancer at the time of sample collection. In the single patient with a positive WID-qEC test (Data Supplement), the concurrent biopsy detected an endometrioid stage 1, grade 1 EC. The sensitivity of the WID-qEC was lower in this group compared with other settings . Applying prespecified thresholds across settings and collection modalities, our data indicate that the WID-qEC performs at least equally well, if not better, than other strategies currently in use to screen and triage women with EC, importantly ultrasound investigation.21 Wang et al22 used a sensitive targeted sequencing method evaluating 18 genes of interest in a large case-control study (382 cancer cases and 714 controls): 81% of EC cases were identified in Pap brush samples. However, the specificity of this approach remains uncertain as controls were substantially younger than patients with EC 22 and aging is strongly associated with the accumulation of somatic mutations.27 We used matched controls and observed high performance across study designs and populations. WID-qEC detection of ECs did not seem to vary by histology, grade, stage, age, ethnicity, and menopausal status in the cohorts studied here. However, future large-scale prospective studies will be required to further strengthen these data, in particular for covariates for which sample numbers in the current study were small . Our data indicate that the WID-qEC could be suitable for use with a variety of collection devices. This, in combination with the high sensitivity and specificity of the test particularly in symptomatic settings, could make the WID-qEC test especially valuable in conditions where access to specialists or even any health care professional might be restricted .Previous proof-of-concept studies, albeit using limited sample numbers, demonstrated the feasibility of discriminating between EC and benign conditions using genomic or epigenomic testing on Pap brush samples, tampons, or vaginal self-samples.assumed population prevalence, meaning that changes in the assumed population prevalence could alter the estimated PPV/NPV. We note that although the PPV was sensitive to changes in estimated prevalence, the NPV remained relatively stable (Data Supplement). Nonetheless, diagnostic accuracy including PPV/NPV of the WID-qEC should ultimately be evaluated in large-scale prospective clinical studies in each target population. A limitation in the predictive evaluation using the Karolinska Cohort was that diagnoses beyond the end of 2015 were not known; hence, some controls could potentially have been diagnosed with EC after the last follow-up. Finally, we were unable to analyze 40/291 (13.7%) self-collected samples in the Barcelona Validation set because of insufficient DNA. Low yield is a common problem with self-sampling methods, but could be addressed with clear sampling guidance and optimized extraction protocols.The inherent limitations of case-control studies also apply to our study. We strived to mitigate bias by inclusion of several study designs (including cohorts) to ensure the robustness of the findings. We also included different sample collection methods to enhance generalizability and evaluate the potential of self-collection. Thresholds fixed during test development in the small FORECEE Pilot set detected ECs in all other sample sets and support the generalizability of our findings. A further limitation relates to the calculation of PPVs and NPVs, which were derived from the 8 For example, although the number of non-White women in our settings was low, the performance of the WID-qEC to detect EC was similar in White and non-White women; and (2) women at high risk of developing EC. The WID-qEC exhibited a high NPV in all settings.10Two predefined diagnostic thresholds (high sensitivity or high specificity) were applied depending on the clinical context of each sample set. The appropriate threshold for clinical implementation depends on the setting: in low-risk populations, false positives should be avoided as they lead to unnecessary invasive procedures. Thus, a high-specificity approach is suitable (threshold 2). Conversely, in high-risk and/or symptomatic populations, high sensitivity is desired (threshold 1). Women who might benefit most from the WID-qEC in the near future might be (1) women presenting with abnormal bleeding or other symptoms suggestive of ECs undergoing triage for malignancies, in particular those for whom currently available tests  are less reliable.28 As a relatively low-cost PCR-based test (estimated costs below $200 USD) with the potential for self-collection of samples, thus reducing the need for specialist referral, the WID-qEC offers several benefits compared with current clinical practice.Pelvic ultrasound costs in the United States range from $220 US dollars (USD) to $3,200 USD, with a national average cost of $575 USD.Initial clinical implementation of the WID-qEC test in circumstances where TVUS is inconclusive, not available, or declined by the patient is warranted to triage and prioritize patients with the highest cancer risk for hysteroscopy and endometrial biopsy. Given the potential benefits of earlier diagnosis of ECs for survival and reduced health care costs, future studies could also evaluate the test's potential for screening of asymptomatic women with increased risk, for instance women with obesity or Lynch syndrome, or women in the general population age > 50 years who are participating in routine cervical screening.In conclusion, the WID-qEC could represent a patient-friendly test for the screening and triage of women with symptoms suggestive of EC or those at risk of EC. Because of its suitability for use in self-collected samples, the WID-qEC may be a suitable tool for managing women with abnormal bleeding, particularly when access to specialist care is restricted. Further research will determine the most appropriate preventive screening and early detection settings in which to deploy this test.