Data on drug-coated balloon (DCB) treatment in the context of diabetes mellitus (DM) and multivessel coronary artery disease (CAD) are limited. We aimed to investigate the clinical impact of DCB-based revascularization on percutaneous coronary intervention (PCI) in patients with DM and multivessel CAD.A total of 254 patients with multivessel disease (104 patients with DM) successfully treated with DCB alone or combined with drug-eluting stent (DES) were retrospectively enrolled (DCB-based group) and compared with 254 propensity-matched patients treated with second-generation DES from the PTRG-DES registry  (DES-only group). Major adverse cardiovascular events (MACE) comprised cardiac death, myocardial infarction, stroke, stent or target lesion thrombosis, target vessel revascularization, and major bleeding at 2 years.The DCB-based group was associated with a reduced risk of MACE in patients with DM  at the 2-year follow-up. In patients with DM, the risk of cardiac death was lower in the DCB-based group than the DES-only group, but not in those without DM. In both patients with or without DM, the burdens of DES and small DES (less than 2.5 mm) used were lower in the DCB-based group than in the DES-only group.In multivessel CAD, the clinical benefit of a DCB-based revascularization strategy appears to be more evident in patients with DM than in those without DM after 2 years of follow-up.  Patients with diabetes mellitus (DM) undergoing percutaneous coronary intervention (PCI) have worse clinical outcomes, such as increased risk of in-stent restenosis (ISR), stent thrombosis, myocardial infarction, and death, compared with that of patients without DM –3. FurthDrug-coated balloon (DCB) treatment leaves nothing of lesions behind, and it reduces the risk of stent-associated maladaptive biologic responses causative of restenosis and thrombosis, and allows for favorable natural vascular healing , 7. In phttps://www.clinicaltrials.gov) (unique identifier: NCT04734028). This consortium combined nine prospective registries from 32 Korean academic centers, contributing data from 13,160 patients who were treated with DES between July 2003 and August 2018 [A total of 254 patients with successful PCI for multivessel CAD including patients with DM who received DCB alone or in combination with DES were retrospectively enrolled between 2012 and 2020 from three teaching hospitals in South Korea  with experienced physicians providing treatment for patients with multivessel CAD using DCB . Eligible patients were those who had lesions with ≥ 50% narrowing and who the investigator considered to require PCI for two or more major epicardial coronary lesions. Patients with DM were defined as patients with a history of DM under medication or fasting plasma glucose ≥ 126 mg/dL. All patients were diagnosed with Type 2 DM in this study. Patients were excluded from the analysis if they had previously undergone CABG; presented with cardiogenic shock, thrombolysis before PCI, single-vessel disease, or suboptimal or failed PCI for target lesions; or were lost to follow-up. Additionally, patients’ vessels were required to be sufficiently large to accommodate DES implantation. The results of the hybrid approach in these patients were compared with those of 254 propensity-matched patients from the PTRG-DES consortium, who were treated with DES-only , as a carrier for the drug. After DCB use, the final assessment was performed at least 5 min after administering a bolus of an intracoronary vasodilator, to prevent any remaining acute vessel closure. In cases of high thrombus burden, a bailout glycoprotein IIb/IIIa receptor inhibitor strategy was used. The duration of the prescribed dual antiplatelet therapy was at the discretion of the attending physician.For patients with multivessel disease, the PCI target lesions were first determined, then balloon angioplasty was performed to determine whether DCB treatment would be possible. The DCB-based treatment group received interventions performed according to international and Asia-Pacific consensus recommendations for DCB treatment , 11. SpeAll 508 patients underwent a clinical follow-up following the index procedure via telephone interviews and outpatient clinic visits. The study endpoint was cumulative major adverse cardiac events (MACE) at 2 years, a composite of cardiac death, myocardial infarction (MI), stroke, probable or definite stent or target lesion thrombosis, target vessel revascularization (TVR), and major bleeding. Cardiac death was defined as any death that was not clearly of extracardiac origin, including MI, according to previously published guidelines . AdditioClinical characteristics are reported as percentages for categorical variables and as means with standard deviations for continuous variables. Comparisons between groups were made using either Pearson’s chi-squared test or Fisher’s exact test for categorical variables, and Student’s t-test for continuous variables, as appropriate. In comparing clinical outcomes between the groups, the cumulative incidences of MACE and other outcomes were estimated using the Kaplan–Meier method, and the curves were compared using the log-rank test. To reduce the effect of potential confounding factors, we used propensity score matching to adjust for differences in baseline characteristics. The propensity score was estimated using logistic regression by considering demographic and clinical variables . Without setting the caliper size , patients were 1:1 matched using the nearest-neighbor method with respect to the calculated score. All p-values were two-sided, and a value of < 0.05 was considered statistically significant. R version 4.1.2  was used for all statistical analyses in this study.Among a total of 508 patients with multivessel disease, 219 patients (43.1%) composed the DM group, and 289 patients (56.9%) composed the non-DM group. DCB-based treatment was performed in 47.5% (n = 104) of the DM group and 51.9% (n = 150) of the non-DM group. The baseline clinical and procedural characteristics of the patients are described by DM group and treatment strategy in Table Table The main findings of this study were as follows: (1) in patients with DM, DCB-based treatment significantly reduced the risk of MACE and cardiac death compared with DES-only treatment for de novo multivessel CAD at 2-year follow-up; (2) in patients with non-DM, the clinical outcomes were similar with both DCB-based treatment and DES-only treatment for multivessel CAD. Therefore, a DCB-based revascularization strategy may be an acceptable approach for patients with DM and multivessel CAD.DM accelerates atherosclerosis in multiple vascular beds and is associated with a significantly higher risk of CAD, and its prevalence is still growing globally . It has The current guidelines suggest that for patients with DM and multivessel CAD, revascularization with CABG might be the preferred approach , 19. In The advantages of DCB treatment include homogeneous drug delivery to the vessel wall, immediate drug release without the use of a polymer, and the freedom of leaving no foreign object behind in the vessel. The DCB treatment, involving no-metallic stent struts or polymer, may reduce intimal hyperplasia and vessel inflammation, preserving vessel anatomy and flow compared with DES. Furthermore, although the exact mechanism of late lumen increase is not well understood, DCB treatment of de novo coronary lesions after predilation was known to lead to late lumen enlargement. Therefore, considering the nature of DM in CAD, a DCB-based strategy  may be a good alternative to a DES-only strategy in treating multivessel CAD in patients with DM.In the present study, we showed that in patient with DM who had multivessel CAD, DCB-based treatment was significantly associated with a lower risk of MACE and cardiac death than DES-only treatment; however, this association was not seen in patients without DM. Although it showed no statistical significance, we demonstrated that the need for TVR in the DM group was numerically lower with DCB-based treatment compared with DES-only treatment . Our study results are consistent with those of previous studies on the long-term clinical impact regarding TVR of DCB versus DES treatment in patients with DM having de novo coronary lesions . A previPatients with DM have a relatively smaller vessel caliber, with longer and more diffuse de novo lesions, compared to patients who do not have DM . This maThere are some limitations in this study. First, this study has the innate limitations of its observational nature and the use of registry data. Laboratory test results such as glycosylated hemoglobin A1c (HbA1c) during the follow-up period, which may be associated with DM management status and could impact the outcome, but we could not provide these data. In addition, leaving the choice of treatment strategy to the discretion of the physician inevitably introduces the limitation of selection bias. We addressed this issue by applying extensive sensitivity analyses in which measured or unmeasured confounders were adjusted to minimize the bias from different baseline characteristics. Second, each patient enrolled in this study was treated at an expert center in DCB-only treatment for de novo CAD. Thus, these results may not be reproducible without an adequate learning curve. Third, differences between the enrollment periods of the two groups might have led to differences in results related to technological changes. However, although the PTRG-DES registry was established in 2003, the patients whose data were used in the propensity match analysis had received second-generation DES. Therefore, differences between groups related to device development and PCI technique improvement are not expected to be significant. Further prospective randomized non-inferiority or superiority clinical trials with larger numbers of patients are needed to evaluate long-term outcomes after DCB-based treatment in patients with DM and multivessel CAD.In multivessel CAD, a DCB-based treatment approach  was associated with a reduced risk of MACE in patients with DM, but not in patients without DM. The role of DCB in this setting should be assessed in prospective randomized controlled trials.


Biomarker of insulin resistance, namely triglyceride-glucose index, is potentially useful in identifying critically ill patients at high risk of hospital death. However, the TyG index might have variations over time during ICU stay. Hence, the purpose of the current research was to verify the associations between the dynamic change of the TyG index during the hospital stay and all-cause mortality.The present retrospective cohort study was conducted using the Medical Information Mart for Intensive Care IV 2.0 (MIMIC-IV) critical care dataset, which included data from 8835 patients with 13,674 TyG measurements. The primary endpoint was 1-year all-cause mortality. Secondary outcomes included in-hospital all-cause mortality, the need for mechanical ventilation during hospitalization, length of stay in the hospital. Cumulative curves were calculated using the Kaplan–Meier method. Propensity score matching was performed to reduce any potential baseline bias. Restricted cubic spline analysis was also employed to assess any potential non-linear associations. Cox proportional hazards analyses were performed to examine the association between the dynamic change of TyG index and mortality.The follow-up period identified a total of 3010 all-cause deaths (35.87%), of which 2477 (29.52%) occurred within the first year. The cumulative incidence of all-cause death increased with a higher quartile of the TyGVR, while there were no differences in the TyG index. Restricted cubic spline analysis revealed a nearly linear association between TyGVR and the risk of in-hospital all-cause mortality  as well as 1-year all-cause mortality . The area under the curve of all-cause mortality by various conventional severity of illness scores significantly improved with the addition of the TyG index and TyGVR. The results were basically consistent in subgroup analysis.Dynamic change of TyG during hospital stay is associated with in-hospital and 1-year all-cause mortality, and may be superior to the effect of baseline TyG index.The online version contains supplementary material available at 10.1186/s12933-023-01874-9. The utilization and costs associated with critical care medicine beds have been consistently increasing in the United States over the past three decades , 2. ThisInsulin resistance (IR) is a prevalent condition in critically ill patients and is considered a marker of systemic inflammatory response and metabolic disorders . PreviouTriglyceride glucose (TyG) index, calculated from fasting triglyceride (TG) and fasting blood glucose (FBG), is a novel marker that has been well-recognized as a simple and reliable surrogate of IR. Recent studies have found that the TyG index is strongly associated with increased all-cause mortality in critically ill patients . HoweverThe data presented in this study was extracted from Medical Information Mart for Intensive Care IV (MIMIC-IV version 2.0), a comprehensive collection of health-related data pertaining to 76,943 ICU stays for 53,150 unique patients (as computed by the authors) who received critical care at the Beth Israel Deaconess Medical Center between 2008 and 2019. It is noteworthy that none of the patients included in the study had contracted COVID-19. In order to preserve patient privacy, all personally identifiable information has been rendered anonymous. Given that all patient records in the MIMIC-IV database were fully de-identified, the requirement for individual patient consent was deemed unnecessary by the institutional review board of the Beth Israel Deaconess Medical Center.Selection criteria, as well as the number of patients, were excluded at each step, were shown in Fig. All baseline characteristics, with the exception of TyG, were extracted within the first 24 h of ICU admission. The study data, which comprised patient demographics, comorbidities, survival outcomes, severity score, laboratory parameters, medication, and other relevant variables, were extracted by author L.C, who completed an online training course from the National Institutes of Health (with certification number 9046642) and obtained permission to access the MIMIC-IV database. The TyG index was determined using the formula ln (fasting triglycerides [mg/dL] × fasting glucose [mg/dL]/2) , 19. TyGTyG baseline was calculated using the first recorded fasting blood glucose and triglycerides after ICU admission. TyG average was defined as the average value of fasting blood glucose and triglycerides recorded on multiple occasions, excluding the first time. All comorbidities were identified based on ICD-9 or ICD-10 codes. Sepsis was defined according to The Third International Consensus Definitions for Sepsis and Septic Shock . Data exThe primary outcome was 1-year all-cause mortality. Secondary outcomes included in-hospital all-cause mortality, the need for mechanical ventilation during hospitalization, length of stay (LOS) in ICU and hospital.For the baseline characteristics, data were presented as the mean with standard deviation (SD) or median with interquartile range (IQR) for continuous variables. The mean of continuous variables was compared using Student’s t-test or the Mann–Whitney U test. Categorical variables were expressed as quantity and frequency and were tested using Pearson chi-square test or Fisher’s exact test. Continuous variables with missing data exceeding 2% were excluded from the analysis to ensure the validity and reliability of the study findings. In order to minimize potential bias between the two study groups, propensity score matching (PSM) without replacement was performed using a caliper width of 0.2 of the pooled standard deviation of the logit of the propensity score. This matching was performed at a 1:1 ratio based on baseline factors to ensure comparability between the groups. An absolute standardized difference (ASD) < 20% for the measured covariate suggests an appropriate balance between groups. The baseline characteristics of the original and matched cohorts were listed separately. To explore the association between TyG index and TyGVR with the primary outcome of interest, the cumulative incidences of 1-year all-cause mortality were estimated using Kaplan-Meier curves and detected by the Log-Rank test. Potential nonlinear for the levels of TyG index and TyGVR with in-hospital and 1-year all-cause mortality measured by restricted cubic spline. Restricted cubic spline was used to assess potential nonlinear relationships between the levels of TyG index and TyGVR with in-hospital and 1-year all-cause mortality, with four knots located at the 5th, 35th, 65th, and 95th percentiles as per Harrell's recommendations , 22. TheAll analyses were performed using STATA MP Version 16.0 , and 2-side P < 0.05 was considered statistically significant.As shown in Table Based on the number of TyG index measurements, the patients were further divided into 2 groups  or those who survived the ICU stay  CKD. This phenomenon seems to contradict previous studies. A prospective study from Zhao et al.  found thThe most strength of the current study is the repeated assessment of TyG index during hospital stay has important implications for critically ill patients. The TyGVR might help stratify critically ill individuals at high risk for all-cause mortality, whether during hospital stay or long-term follow-up. Further research is needed to validate the relationship between the mean changes of TyG index in critically ill patients during hospital stay across various chronic diseases and mortality.As the nature of single-center retrospective study, limited sample size and data bias could be inevitable despite vigorous statistical correction being performed. Additionally, we could provide only the association between TyGVR and mortality rather than causality. Furthermore, although we adjusted for other relevant confounders including obesity, we did not record dietary habits and energy intake that might dramatically affect TG levels. Finally, the data of death is derived from hospital records and state records. The etiology of death, particularly out-of-hospital death, remains unclear. A well-designed prospective study should be conducted to evaluate the causality between TyGVR and mortality in the future.In summary, IR dynamic change, repeatedly assessed by accessible and reliable TyG index, during hospital stay was positively and independently associated with an increased risk for all-cause mortality in individuals with critical illnesses. Moreover, the dynamic change of TyG index may provide more valuable information than the baseline TyG index in identifying patients at high-risk all-cause mortality. Future work should focus on the clinical implications of assessment of TyG index variability across different clinical states.Additional file 1: Table S1. Association of TyG and TyGVR with length of stayAdditional file 2: Figure S1. Area under ROC curve of TyG index and TyGVR combined with various scores for in-hospital mortality and 1-year mortality.


Telehealth has become widely used as a novel way to provide outpatient care during the COVID-19 pandemic, but data about telehealth use in primary care remain limited. Studies in other specialties raise concerns that telehealth may be widening existing health care disparities, requiring further scrutiny of trends in telehealth use.Our study aims to further characterize sociodemographic differences in primary care via telehealth compared to in-person office visits before and during the COVID-19 pandemic and determine if these disparities changed throughout 2020.We conducted a retrospective cohort study in a large US academic center with 46 primary care practices from April-December 2019 to April-December 2020. Data were subdivided into calendar quarters and compared to determine evolving disparities throughout the year. We queried and compared billed outpatient encounters in General Internal Medicine and Family Medicine via binary logic mixed effects regression model and estimated odds ratios (ORs) with 95% CIs. We used sex, race, and ethnicity of the patient attending each encounter as fixed effects. We analyzed socioeconomic status of patients in the institution’s primary county based on the patient’s residence zip code.P=.006). Encounters with the following patients were less likely to be via telehealth compared to in-person office visits: patients who self-identified as Asian  and Nepali , patients insured by Medicare , and patients living in zip code areas with high utilization rate of supplemental nutrition assistance . Many of these disparities persisted throughout the year. Although there was no statistically significant difference in telehealth use for patients insured by Medicaid throughout the whole year, subanalysis of quarter 4 found encounters with patients insured by Medicaid were less likely to be via telehealth .A total of 81,822 encounters in the pre–COVID-19 time frame and 47,994 encounters in the intra–COVID-19 time frame were analyzed; in the intra–COVID-19 time frame, a total of 5322 (11.1%) of encounters were telehealth encounters. Patients living in zip code areas with high utilization rate of supplemental nutrition assistance were less likely to use primary care in the intra–COVID-19 time frame (OR 0.94, 95% CI 0.90-0.98; Telehealth was not used equally by all patients within primary care throughout the first year of the COVID-19 pandemic, specifically by patients who self-identified as Asian and Nepali, insured by Medicare, and living in zip code areas with low socioeconomic status. As the COVID-19 pandemic and telehealth infrastructure change, it is critical we continue to reassess the use of telehealth. Institutions should continue to monitor disparities in telehealth access and advocate for policy changes that may improve equity. The impact of the SARS-CoV-2 strain of coronavirus  pandemic was felt throughout the world in 2020 and will influence the health care landscape for years to come. The COVID-19 pandemic has highlighted existing health care disparities; even when comorbidities are controlled for, Black patients have 2.7 times increased odds of hospitalization from COVID-19 compared to non-Hispanic White patients . PrimaryTelehealth is defined by the Centers for Medicare and Medicaid Services as “exchange of medical information from one site to another through electronic communication .” At theDespite the swift increase in telehealth use, studies during the COVID-19 pandemic have demonstrated that Black  and HispOur study aimed to further characterize telehealth disparities in adult primary care and to assess how the increase in telehealth use impacted existing health care disparities in primary care, specifically examining differences by race or ethnicity, insurance status, and geographic location in who is using in-person office visits versus telehealth intra–COVID-19. We wanted to determine if any existing disparities were significant only at the beginning of the COVID-19 pandemic related to inequity with initial use or if these disparities continued despite increased clinician and institutional comfort with telehealth use.We acquired retrospective data from an informational database for the electronic health record of The Ohio State University Wexner Medical Center, which allows researchers to access deidentified clinical data. All billed outpatient encounters in the Division of General Internal Medicine and Department of Family and Community Medicine were examined in 2 time periods: pre–COVID-19 (April-December 2019) and intra–COVID-19 (April-December 2020). These ranges were picked to have comparative time points for the pre- and intra–COVID-19 time frames during the initial peak of the pandemic. Our study aimed to determine how health care disparities may have changed throughout the first year of the COVID-19 pandemic, and data were subdivided into calendar quarters .Included variables were type of encounter, age, self-identified race, ethnicity, zip code, insurance type, and visit date. Exclusion criteria were ages outside the range of 18-99 years and encounters in departments other than Family Medicine and General Internal Medicine to focus on primary care usage only. Insurance types were separated into Medicaid, Medicare, private, marketplace (including exchange and marketplace policies), worker’s compensation, self-pay, and uninsured.Our analysis included zip codes for Franklin County, the primary catchment area for patients at The Ohio State University. Franklin County has a population of 1.3 million, of which around 13.5% live below the federal poverty line; 66.8% of the population is White and 23.8% is Black. There are multiple refugee populations located in Franklin County, particularly Nepali  and Somali  .Data were deidentified prior to being given to the research team. The deidentified data request was approved by the Honest Broker Committee (HBOC Study ID #1273), which oversees all clinical data sets within The Ohio State University. Given that the data were deidentified and there were no interventions, the study was designated exempt from institutional review board approval.To determine how demographics changed between the pre–COVID-19 and intra–COVID-19 time frames and how they were associated with encounter type  during the intra–COVID-19 time frame, we applied a binary logistic mixed effects regression model and estimated odds ratios (ORs) and 95% CIs. Time period and encounter type were used as the outcome variables for the respective analyses. Sex, race, and ethnicity of the patient attending each encounter were used as fixed effects. The model was fixed to account for within-office correlation. Because a patient could have multiple encounters, we attempted to fit a mixed effects model in which a random effect for patient was used to account for correlation of encounters by the same patient. However, the mixed effects model indicated no detectable within-patient correlation, so the random effects were dropped from the model.Zip code data and insurance provider were analyzed via separate models. US Census Bureau data for the 48 zip codes within Franklin County was used  to deterA total of 81,822 encounters in the pre–COVID-19 time frame and 47,994 encounters in the intra–COVID-19 time frame were analyzed . The samP=.006). The proportions of encounters with patients covered by private insurance, Medicare, and Medicaid were similar between pre- and intra–COVID-19 time frames.There was a substantial decrease in overall primary care encounters during the intra–COVID-19 time frame compared to pre–COVID-19 time frame: between April-December 2019 and April-December 2020, the volume of overall primary care encounters decreased by over 40% . AlthougP=.006).Encounters with patients living in Franklin County data were then separately analyzed (Table S1 in The intra–COVID-19 data (April-December 2020) were then analyzed separately . A totalP<.001) and Nepali . In quarterly subanalyses  were less likely to be via telehealth (P=.03).Compared to private insurance, overall encounters in 2020 with patients covered by Medicare  throughout 2020 were less likely to be via telehealth compared to in-person office visits  or if telehealth visits were done by video or telephone, which provides important context. We did not assess patient’s primary language, which may be an additional barrier to telehealth . Our stuNationally, telehealth accounted for 14% of commercially insured ambulatory encounters in 2020 , and the


Clear evidence of an increased risk for SARS-CoV-2 infection among smokers has not been established. We aimed to investigate associations between cigarette smoking or use of snus (snuff) and other nicotine-containing products and a positive SARS-CoV-2 test, taking test behavior into account.n = 78,860) and antibody measurements (n = 5581). We used logistic regression models stratified by gender and adjusted for age, education, region, number of household members, and work situation.Current tobacco use and testing behavior during the pandemic were recorded by adult participants from the Norwegian Mother, Father and Child Cohort Study and The Norwegian Influenza Pregnancy Cohort. SARS-CoV-2 infection status was obtained from The Norwegian Surveillance System for Communicable Diseases (MSIS) in May 2021 (Snus use was more common among men (26%) than women (9%) and more prevalent than cigarette smoking. We found no clear associations between cigarette smoking or snus and a COVID-19 diagnosis among men. Associations among women were conflicting, indicating that cigarette smoke was negatively associated with a diagnosis , while no association was found for snus use . Compared with non-users of tobacco, both cigarette smokers and snus users had increased odds of being tested for SARS-CoV-2.Cigarette smoking, but not snus use, was negatively associated with SARS-CoV-2 infection in women. The lack of an association between snus use and SARS-CoV-2 infection in this population with prevalent snus use does not support the hypothesis of a protective effect of nicotine.The online version contains supplementary material available at 10.1186/s12889-023-15822-5. By December 2022, WHO have reported more than 640 million cases and six million deaths globally . NoThe aim of this study was to investigate associations between cigarette smoking or use of other nicotine-containing products (including snus) and a positive SARS-CoV-2 test during March 2020 to May 2021, as measured by i) a COVID-19 diagnosis obtained by linkage to The Norwegian Surveillance System for Communicable Diseases (MSIS) and ii) presence of SARS-CoV-2 antibodies in serum. A secondary aim was to examine associations between tobacco use and having been tested for SARS-CoV-2 in the same period, to explore if the risk of getting a registered diagnosis could be explained by testing behavior. We examine these associations cross-sectionally among participants from two large pre-existing cohorts, at a time point covering the pre- and early vaccination periods of the pandemic in Norway.The current study was based on The Norwegian Mother, Father and Child Cohort Study (MoBa)  and The Sub-studies were initiated in both cohorts when the COVID-19 pandemic hit Norway. Since March 2020, participants have been invited to answer electronic questionnaires every 14 days with questions regarding illness, testing for COVID-19, and more. The response rates to the questionnaires distributed between March 2020 and May 2021 have been 50–80% in both cohorts.For the current study, 146 516 eligible participants included all adult cohort members who were invited to answer a questionnaire in January 2021 about tobacco use. An inclusion criterion for the main analysis (Sample 1) was available national registry data on SARS-CoV-2 infection status, which was obtained for MoBa only.n = 642), Fig. For analyses using seropositivity as outcome (Sample 2), the inclusion criterion was available data from analysis of SARS-CoV-2 antibodies in serum, which was performed in a subsample from both cohorts. A total of 6917 eligible participants from both cohorts residing in the Oslo area were recruited for antibody analysis. Of these, 6223 (90%) had available information about tobacco use. We excluded MoBa participants who according to SYSVAK data had been vaccinated prior to antibody assessment (th 2020 to May 25th 2021 or ii) the presence of antibodies for SARS-CoV-2, collected April 28th 2020 to April 29th 2021. Seropositivity was obtained as a conclusion made by the laboratory at Oslo University Hospital, based on concentrations of antibodies to SARS-CoV-2 the receptor binding domain of the spike protein (RBD) and nucleocapsid protein, measured as previously described [Our primary outcome was having acquired a SARS-CoV-2 infection, measured as i) a positive test for SARS-CoV-2 based on PCR obtained from The Norwegian Surveillance System for Communicable Diseases (MSIS), registered in the period March 6escribed , 14.th 2021. Participants answering “no” and “don’t know” were grouped together.A secondary outcome was whether participants had been tested for SARS-CoV-2 (both negative and positive results). We defined being tested as participants who answered “yes” at least once to the following question: “Have you been tested for coronavirus infection the last 14 days?” from 30 questionnaires administered every 14 days between March 31th 2020 and May 11Current cigarette smoking status was obtained from two questionnaires, one was administered in June 2020 and another one in January 2021 , and those who were tested for other reasons .From the existing cohort databases, we included variables on the participants’ age  and gender (defined by cohort member role as mother or father). County of residence was obtained through linkage to The Norwegian Population Registry and grouped into six geographical regions . As proxies of number of social contacts, we included information on use of home office, job loss, or sick leave , as well as number of people in the household. As proxy of socioeconomic status, we included educational level  reported by participants in August 2021. For those with missing current information (19%), we used most recent reported education level recorded in the existing cohort database . We also included information on body mass index  and underlying chronic illness . From each round of 30 questionnaires administered biweekly between March 31th 2020 and May 11n = 78,860), were stratified by gender to account for the dependency within families. Adjusted models included the following covariates as fixed effects: Age, education level, number of household members, and work situation. We have based our analyses on a causal framework (of available variables in our data) illustrated by a directed acyclic graph (DAG) in Supplementary Figure SWe estimated associations between tobacco use and SARS-CoV-2 infection using binomial regression models with logit link function and robust standard errors. All associations were reported as odds ratio (OR) with 95% confidence intervals (CI). Analyses using the large study sample, Sample 1 (2), to examine a possible effect modification. We also performed the analysis while excluding those who tested positive for SARS-CoV-2 before June (first assessment of smoking status) to account for exposure misclassification of those who may have stopped smoking after getting infected, and while excluding those who reported smoking in January, but not in June (possible exposure misclassification of “new” smokers). None of the two latter approaches changed our findings (data not shown). Lastly, we excluded vaccinated subjects and performed the analysis in a subset who were not yet vaccinated in May 2021. Statistical analyses were performed in R [mice [lmtest [sandwich [.As sensitivity analyses, we analyzed associations between tobacco use and SARS-CoV-2 infection a) in a subset who reported to have been tested at least once during the study period, to account for testing behavior; and b) stratified by BMI , and region (6.9%), followed by number of cohabitants 2.7%) and education level (1.0%). We performed multiple imputation with chained equations and 20 imputations.7% and e%, and reCurrent snus use was more common among men (26%) than women (9%), while current cigarette smoking was less common than snus both among men 5%) and women (7%) Table . 1.3% re% and wom2, and not among women with BMI < 25 kg/m2 . Both male and female snus users had higher odds for being tested for COVID-19  for women  use and having had a positive test for SARS-CoV-2 between March 2020 to May 2021. During the study period, the SARS-CoV-2 infection rate in Norway was still low, and only 2% in our study sample had a positive SARS-Cov-2 test. Also, most subjects were still unvaccinated, which enabled investigation of these associations without potential effect modification by vaccination hesitancy and/or effect. We found no associations between nicotine use, with either cigarette smoking or snus use, and a COVID-19 diagnosis among men. Similarly, snus use was not associated with a COVID-19 diagnosis in women. In contrast, we found negative associations between cigarette smoking and a COVID-19 diagnosis among women. Female smokers were more likely to be tested compared to non-smoking women, which could contribute a higher occurrence of positive tests. However, both cigarette smokers and snus users were more likely to take a COVID-19 test than non-users of tobacco, thus the difference in associations with SARS-CoV-2 between these groups were not explained by testing behavior alone.Hypotheses for both protective and detrimental effects of smoking on COVID-19 have been proposed in the literature. Early epidemiological studies reported an underrepresentation of smokers among patients hospitalized with COVID-19 , 21. AlsIn two recent studies based on Finnish and Swedish general population samples, we did not find conclusive evidence of an association between current smoking and COVID-19 diagnosis , 26, wheCigarette smokers and snus users in our study differed in characteristics and behavior, and potential bias in analyses of the two exposures may also differ, which may contribute to the inconsistency in our findings. For instance, snus users were more likely to have higher education and cigarette smokers lower education, suggesting that these groups may differ in many unmeasured aspects associated with educational level. Also, snus users may have higher occupational risk of acquiring an infection, as snus use may be more acceptable than cigarette smoking in settings where people meet, especially indoor. We found that female snus users had increased odds of having been tested for SARS-CoV-2 than non-tobacco users. This could theoretically hide a decreased risk of infection among snus users in our analysis if the prevalence of COVID-19 in the control group (non-tobacco users) was underestimated. However, when restricting our analyses to only those who had been tested, we still found no association between snus use and a COVID-19 diagnosis.There are several limitations of this study. We did not have recent information about former tobacco use. Use of snus and other nicotine-containing products was reported during the second wave of increased infection rates in Norway, and for some participants after acquiring an infection. This may have resulted in exposure misclassification of those who changed their snus use, for instance after infection. Despite the large sample size, the SARS-CoV-2 infection rates in Norway during the pre-vaccination period were low, which reduced the statistical power and increased the uncertainty in our analyses. Due to this limitation, we did not investigate how different virus variants, season, or other time-varying factors during the study period may have affected the associations. While positive tests were obtained from a registry based on PCR tests, we used self-reported data on testing behavior. Some participants may have reported other types of testing (i.e. rapid antigen test) that was not performed through the health services. Another draw-back with self-reported data is that those who experienced symptoms of COVID-19 may be more likely to contribute data in the ongoing surveys than those who were asymptomatic. Yet, the proportion of participants diagnosed with COVID-19 in this study reflects the (low) infection rates in Norway . The preA main strength of our study is that we could examine both registry-based diagnosis, seroprevalence results and testing behavior as outcomes, with two commonly used nicotine products (cigarettes and snus) as exposures. We were also able to compare testing behavior between users and non-users of nicotine products. Another main strength is the large sample size and high participation rate. Both genders and participants from all parts of the country are well represented, and there is a wide age span. Using pre-existing cohorts as opposed to panels recruited during the pandemic may be advantageous because they are less subject to selection bias.In conclusion, our study showed inconsistent associations between nicotine use and being infected with SARS-CoV-2 in Norway during the pre- and early vaccination periods. Cigarette smoking was negatively associated with acquiring a SARS-CoV-2 infection among women only, while no associations were found among men or women between snus use and a COVID-19 diagnosis. Testing behavior in nicotine users (smokers and snus users) differed from non-users, but were similar for snus users and smokers. The absence of an association between snus use and a SARS-CoV-2 infection in a population with prevalent snus use does not support the hypothesis of a protective effect of nicotine.Additional file 1.Supplementary Table S1. Frequency of cigarette smoking in MoBa , comparing answers at the two timepoints. Supplementary Table S2. Results from multiple imputation with chained equations of missing values in covariates. Total sample size is n=78,860. Supplementary Table S3. Complete case analysis of associations between tobacco use and a COVID-19 or testing behavior. Unadjusted and adjusted analyses were performed on the same dataset, excluding cases with missing observations in covariates. Supplementary Table S4. Study population characteristics and incident cases of COVID-19 between March 2020 and May 2021, among exclusive cigarette smokers, exclusive snus users and non-users of tobacco. Supplementary Table S5. Study population characteristics in the two cohorts with available SARS-CoV-2 antibody data. Supplementary Table S6. Associations between tobacco use and SARS-CoV-2 seropositivity. Supplementary Table S7. Tobacco use and MSIS diagnosis, in gender and BMI strata, complete case analysis. Supplementary Table S8. Tobacco use and MSIS diagnosis in a subset who had been tested at least once during the study period, complete case analysis. Supplementary Table S9. Tobacco use and MSIS diagnosis in a subset who were unvaccinated by May 2021, complete case analysis. Supplementary Table S10. Associations between tobacco use and having been tested for SARS-CoV-2, stratified by gender and having been tested because of symptoms or not. Figure S1. Suggested causal framework for the association between current smoking/tobacco use and risk of being infected with SARS-CoV-2. Minimal adjustment set of confounders for estimation of total effect includes sex, age, SES, county, number of household members, and work situation (home office/lost job). Chronic illness was considered as a potential mediator on a causal pathway between former/current tobacco use and infection status and not included in adjusted models


Extracorporeal cytokine adsorption aims to reduce cytokine levels in critically ill patients. However, little convincing data exist to support its widespread use. This retrospective study compared interleukin-6 (IL-6) levels in patients treated with or without cytokine adsorber (CytoSorb®). Intensive care patients between Jan 2017 and Dec 2021 who had at least two IL-6 measurements were included. They were divided into an adsorber group and a standard of care group. We screened 3865 patients and included 52 patients in the adsorber group and 94 patients in the standard of care group. Matching was performed and the groups were compared regarding IL-6, lactate, CRP, procalcitonin, vasopressor requirement, and mortality rate. After matching, there were 21 patients in each group. Patients had similar age, ECMO and renal replacement therapy use, baseline noradrenaline requirement, serum lactate, pH, CRP, and IL-6 levels. There were no significant differences in the time course of IL-6, lactate, CRP, procalcitonin and noradrenaline requirement between groups. Two-day and ICU mortality and Kaplan-Meier estimated survival were also comparable. In this matched case-control study no difference in IL-6, inflammatory parameters, noradrenaline requirement or mortality was observed between patients treated with adsorber or standard of care. Cytokine storm was historically poorly defined, with a recent update2. Despite the fact that high levels of cytokines are associated with mortality5, it is not clear whether or in which patients and/or in which clinical setting extracorporeal lowering of serum cytokine levels can improve survival6. Despite very promising conceptual idea, technologically impressive design , apparent lack of serious adverse effects and relatively widespread use in some centers, the amount of data from controlled studies, showing efficiency of IL-6 removal and clinical improvement is very scarce. Nevertheless, a number of clinicians believe  that high cytokine levels ) are an indication for extracorporeal cytokine removal. A step forward was made recently with the proposal of a “dynamic scoring system (DSS)” to support the prescription of adsorption therapy8, not without a strong critique from peer-experts9. Altogether, the indications and/or patient selection are not yet clear.Extracorporeal cytokine adsorption therapy remains controversial. The underlying premise for the use of extracorporeal cytokine adsorption is to reduce elevated levels of cytokines during a “cytokine storm” and the theoretical concepts of this therapy are very nicely laid out in Honore et al.10. However, at baseline concentration of only 500–1000 pg/mL and without concomitant production, as is the case in vitro, it is difficult to extrapolate these results to the clinical setting. There are many case reports and uncontrolled case series, which cannot avoid bias, because the natural course of most cytokines is a rapid decrease in the absence of a trigger12. Furthermore, there is a well-known higher long-term mortality after severe sepsis, possibly due to immunosuppression13 and cytokine adsorption could additionally enhance these immunosuppressive effects. Research on this latter aspect is only in a development phase14. A number of randomized control trials25 and two recent meta analyses27 failed to establish a benefit for treated patients. There are few reports of potential adverse effects. Given the high number of procedures performed to date, it is unlikely that serious short-term adverse effects would be overlooked. However, the high variability of published data might easily hide more subtle problems. The removal of other non-toxic medium-to-high molecular weight molecules including antibiotics, non-antibiotic drugs and nutrients can be a problem but is rarely reported31.In vitro data on cytokine reduction are excellent: the removal of cytokines is large, e.g. 99.6% for IL-6In this study, we aimed to compare the time-course of IL-6 in patients treated with or without hemoadsorption and to compare clinical adsorber efficiency in terms of hemodynamic stability and mortality with standard treatment. We hypothesized that there is no significant difference in serum IL-6 levels and clinical course  between the groups.This was a single-center, retrospective, matched, case-control study. Patients treated in the medical intensive care unit (ICU) of University Medical Center Ljubljana, Slovenia, were screened for inclusion. Effectiveness of cytokine adsorption therapy in terms of IL-6 removal and noradrenaline requirement was compared to standard of care (SOC). Baseline parameters that were different between preliminary groups were used as confounders for matching. Matching with genetic matching method was used to simulate randomization, and matching success was assessed in with the use of standardized mean differences (SMD). Patient data  were collected from hospital electronic health records. The laboratory results were determined using standard chemistry test and IL-6 measurements were made using the electrochemiluminescence assay . We calculated baseline SAPS II score, gathered data regarding renal replacement therapy (RRT), the use of extracorporeal membrane oxygenation (ECMO), mean arterial pressure (MAP) and norepinephrine/noradrenaline requirement at “time zero” (T0) and 4, 8, and 12 hours afterwards. When RRT was used at the same time as the adsorber, it was always connected serially, with CytoSorb cartridges placed in pre-filter position. CytoSorb was coupled with continuous veno-venous hemodialysis (CVVHD) with the ST100 filter in patients with severe hemodynamic instability or requirement for sustained fluid removal. CytoSorb was coupled with intermittent hemodialysis (IHD) with a synthetic high-flux dialyzer in patients with severe hyperkalemia, severe metabolic acidosis or hyperammonemia. Two-day and ICU mortality was recorded. The study complied with the Declaration of Helsinki (as revised in Tokyo 2013) and the National medical ethics committee approved the study (No. 0120-533/2019/5). Due to the retrospective nature of the study, the need for informed consent was waived.Patients treated between 01.01.2017 and 31.12.2021 in the medical ICU were included if they met the following criteria: (a) age > 18 years, (b) at least two IL-6 measurements, (c) if the patient was not treated with adsorption, there should be at least one IL-6 measurement after the maximum IL-6 value, (d) the patient was not treated with tocilizumab or an adsorber other than CytoSorb, (e) sufficient data for per-protocol analysis . Patients, transferred out of the ICU were censored for survival. Patients were divided in two groups based on treatment received: the adsorber group (patients treated with adsorber (CytoSorb) for at least 3 hours) and the SOC group.The primary outcome was the reduction in serum IL-6 levels and secondary outcomes were reduction of approximated values of serum IL-6, reduction of noradrenaline requirement, 2-day and ICU mortality. In the adsorber group, we defined “time zero” (T0) as the time of IL-6 measurement closest to the start of adsorption therapy (within – 10 to + 4 hours), while in the SOC group, T0 was defined as the peak IL-6 value. Other available IL-6 measurements were assigned to the nearest time point: 4, 8, 12, 16, and 20 hours after T0. Due to short IL-6 half-life, we chose a time interval of 4 hours to compare the elimination/reduction of laboratory values.33 generally support this simplification. We estimated missing data points using exponential curve extrapolation based on available IL-6 measurements. Each pair of available data points was used to calculate in-between missing data points. The first and last two points were also used to calculate marginal points.IL-6 has a short half-life in the absence of persistent stimuli. However, frequent measurements are neither feasible nor economical in everyday clinical practice and, in addition, measurements rarely follow precise time intervals. Therefore, to increase the number of data points in an extended analysis, we extrapolated missing values of IL-6 in the following manner: we used a simplistic modeling of IL-6 elimination as first-order kinetics; case-reports34.Baseline parameters were analyzed with Shapiro test for normality, and all were non-normally distributed. Between group differences were compared using Wilcoxon rank-sum test for continuous data and Chi-squared test for categoric data. Genetic matching with a 1:1 ratio without replacement was used to create two matched cohorts. Confounders for matching were selected according to baseline differences. The process was iteratively repeated until a reasonable number of matched pairs with acceptable residual standardized mean differences between groups was achieved. Because matching algorithm requires all data to be non-missing, missing values for selected confounders  were replaced with our population median value. The IL-6 levels and noradrenaline requirement were compared between groups using the Wilcoxon test for each selected time point. We considered and reported statistical significance at p < 0.05; we did not use multiple comparison correction. Mortality was assessed as a categorical parameter using Chi-squared test. Furthermore, Kaplan-Meier survival curves were compared with Log-rank test. Data were processed and analyzed using R and the following packages: tidyverse (processing), ggplot (plotting), MatchIt, matching, and cobalt (matching), survival, and survminer Komisija Republike Slovenije za medicinsko etiko, No. 0120-533/2019/5) approved the study and waived the need for the informed consent due to the retrospective nature of the study.The study complied with the Declaration of Helsinki . The Slovenian national ethics board  and absolute doses of noradrenalin were low. In the following 12 hours, there were no significant differences within groups at predefined time points Table  and alsoTwo-day and ICU mortality did not differ between groups Table . Kaplan-In this study, we retrospectively analyzed the time course of IL-6 in patients treated with CytoSorb adsorber or SOC and found that in the matched groups the time course was comparable. There were also no differences in lactate, noradrenaline requirement, inflammatory markers or mortality.2. We chose IL-6, which is the most commonly used and has also been implemented into our local clinical practice. Measurement of other cytokines, such as TNF-alpha or IL-1, could be more suitable for sepsis-associated hyperinflammation. There is an association between high IL-6 levels and mortality4 as well as the need for organ-supportive therapies5. We also found that IL-6 and lactate were significantly lower in survivors at 12 hours , cytokines levels spontaneously decrease very rapidly37 and that lower serum IL-6 levels at 48-72 hours are associated with successful antibiotic therapy35. Therefore, in the absence of positive randomized studies there is no definite answer as to whether extracorporeal elimination of cytokines truly contributes to improved outcome of patients with cytokine storm6.Elevated levels of IL-6 in conjunction with appropriate clinical settings are usually interpreted as a ‘cytokine storm’. However, it is unclear which cytokines are the best markers of sepsis associated cytokine stormrs Table , while W38. However, due to slightly different patient selection, the results are not directly comparable. Our matched cohorts had similar age, sex, BMI, 2-day mortality, SAPS II score, CRP and proportion of ECMO patients; additionally, patients in both cohorts were treated with RRT. The fact that all patients in matched cohorts were treated with RRT removes any potential bias due to additional treatment effect of RRT itself. A considerable number of patients were treated with ECMO, similar to Scharf’s study38. In their study, patients were also more ill at baseline: noradrenaline requirement was higher , and IL-6 was much higher in Scharf's group 38. Although comparable between groups, the noradrenaline requirement and serum lactate levels indicate that our patients  were not at the high-end of the spectrum of hemodynamic instability. As reported previously, we tend to use Cytosorb relatively early, but only after a stabilization period, allowing for some patients to improve with SOC only39. It is noteworthy that a considerable proportion of our patients had an additional vasopressor, which is often used in ICU as a norepinephrine-sparing strategy. Taken altogether, although patients in Scharf’s group had more severe disease, the conclusions are similar. There was no survival advantage, no difference in the reduction of IL-6 and lactate and no difference in hemodynamic stabilization also in our cohort of less severely ill patients treated with the Cytosorb adsorber. Therefore, we cannot confirm that selecting patients according to severity of illness would be appropriate for the use of CytoSorb.Our study was conducted as a replication of the study by Scharf et al.37 and the reduction of IL-6 in our study was observed in both our groups (adsorber and SOC), the question arises as to the appropriateness of measuring serum IL-6 as a marker of clinically relevant improvement in patients with septic shock treated with hemoadsorption. To address this question, we additionally evaluated clinical efficacy of hemoadsorption in terms of hemodynamic stability, inflammatory markers and mortality. Early reports suggested lower vasopressor requirement40 with the use of CytoSorb. A randomized study reported vasopressor requirement reduction in patients treated with adsorber for 24 hours, but not in the control group17. A matched-controls study by Rugg et al.41 showed similar results with lower vasopressor requirements, but the groups differed significantly (p = 0.014) in terms of starting noradrenaline dose despite the matching process. In contrast, our study showed no benefit in terms of vasopressor reduction, as was also observed by other authors43. In addition, serum lactate and inflammatory markers  also did not differ substantially.Because cytokine levels decrease spontaneously very rapidly in the absence of a triggering signal (DAMP/PAMP)39 and in contrast to the propensity-score-weighted retrospective study, in which CytoSorb was associated with a decreased observed versus expected 28-day all-cause mortality compared with continuous renal replacement therapy alone44. On the other hand, our results are analogous to Scharf et al.38, where matched groups had similar 2-day and in-hospital mortality.Finally, mortality was also not different between our unmatched and matched groups. This is in contrast to our retrospective study, where lower observed versus predicted mortality in patients treated with CytoSorb was reported41, compared with SOC alone8. A recent report of successful treatment of septic shock patients with adjuvant therapeutic plasma exchange (TPE)45 also supports the need to »act fast«: in a small bicenter randomized trial, the use of TPE in the first 24 hours led to significant (p < 0.0001) noradrenaline reduction 6 hours after randomization. Additional studies should be performed to determine the characteristics of patients who might benefit from CytoSorb therapy and when is the optimal time to start adsorption therapy. Secondly, because of the lack of stringent criteria for adsorbent use, selection bias is always introduced in non-randomized studies. The selection of data introduced bias because only patients with a documented reduction in IL-6 levels were included in the control group. Furthermore, retrospective data should always be analyzed and interpreted with the greatest caution, but matching is one of the best methods for reducing group imbalances and provides a way to extract a hidden ‘quasi-randomized’ subpopulation from a larger observed population. Several matching methods were used in a comparable way to asses adsorber effectiveness 44. We selected genetic matching, which is a computational method combining benefits of Mahalanobis matching and propensity-score matching. Since the data were collected retrospectively our analysis suffered from a limited number of IL-6 measurements. As to strengthen our results, we used a model for IL-6 kinetics approximation.In our two matched groups, we found that CytoSorb hemoadsorption did not seem to provide a survival benefit compared with SOC. However, our study has several limitations. First, we did not report the time interval between ICU admission and the start of adsorber and SOC treatment. Some clinical reports describe rapid hemodynamic stabilization and better survival with early use of CytoSorb as an adjunctive therapy to SOCIn conclusion, time course analysis of IL-6 in patients treated with CytoSorb or SOC was comparable in the matched groups and no differences in lactate, noradrenaline requirement, inflammatory markers or mortality between matched groups were confirmed. Additional prospective studies should therefore be designed to elucidate the role, timing and duration of adsorbent treatment in septic patients.Supplementary Tables.


The growth kinetics of German and other European WNV and USUV strains were determined and compared. Subsequently, simultaneous co-infections were performed with selected WNV and USUV strains. The results show that the growth of USUV was suppressed by WNV in all cell lines. This effect was independent of the virus lineage but depended on the set WNV titre. The replication of WNV also decreased in co-infection scenarios on vertebrate cells. Overall, co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts. These interactions can strongly affect the epidemiology of USUV and WNV in areas where they co-circulate.The mosquito-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) pose a significant threat to the health of humans and animals. Both viruses co-circulate in numerous European countries including Germany. Due to their overlapping host and vector ranges, there is a high risk of co-infections. However, it is largely unknown if WNV and USUV interact and how this might influence their epidemiology. Therefore, The online version contains supplementary material available at 10.1186/s12985-023-02206-9. Flavivirus, family Flaviviridae) are two closely related zoonotic mosquito-borne viruses. Both circulate in an enzootic cycle between mosquitoes as biological vectors and birds as primary vertebrate hosts, but can also be transmitted to other mammalian species [West Nile virus (WNV) and Usutu virus  [WNV lineage 1 strains have been circulating in Europe for several decades . In 2004 merula) –16. Curr merula) , 14.The distribution areas of WNV and USUV are increasingly overlapping in central European countries such as in Germany  as well In-vitro studies are a fundamental first step in investigating viral co-infections and their effects on virus replication. A few previously conducted in-vitro co-infection studies examined combinations of WNV with other flaviviruses [iviruses , 22, butiviruses . In thisiviruses . HoweverAnser anser f. domestica)) for both WNV and USUV [Cx. tarsalis)) [For the mono- and co-infection kinetics of WNV and USUV, the well-established mammalian (Vero B4) and mosquito (C6/36) cell lines were used )  were utiCells were seeded on 6-well cell culture plates  24 h prior to infection at the set concentrations  of the virus stock  . Viral RNA was then extracted using the NucleoMag Vet Kit  and BioSprint96  according to the manufacturer´s instructions. The RNA extracts were either examined with the specific USUV RT-qPCR  or the s50; derived from the relative and absolute standard curves) or titration analyses (TCID50) were log transformed and checked for homogeneity of variance across groups by the Levene Test. Following a generalized linear model (RT-qPCR versus titration comparisons) or a multifactorial analysis of variance (ANOVA)  with gamma distribution was applied [Data visualization, analysis and statistics were conducted in R /R Studio (Version 1.4.1103) , 31. Dat applied . Pairwis applied . ResultsGrowth kinetics (mono-infections) were performed with all USUV and WNV strains on Vero B4 and C6/36 Fig. . FurtherAll tested WNV lineages replicated faster to higher viral titres in mammalian (Vero B4) and mosquito cell lines (C6/36) than the two USUV strains Fig. . As showThe agreement of the titres determined by titration and RT-qPCR  the WNV and USUV viral titres were lower compared to those of the single infection Fig. . InteresWhen comparing the co-infections of WNV lineage 2 from Germany 2018) or WNV linage 1 from Italy (2009) with both USUV lineages, respectively, results were similar although less prominent (Fig. 8 or WNV With the introduction of WNV into Germany, there is a need to understand the role that co-infections with WNV and USUV might play in the enzootic transmission cycle. The focus of this study was therefore to investigate the viral replication as well as potential interactions in co-infections of German and other European WNV and USUV strains.Aedes albopictus for WNV and USUV [Culex tarsalis for WNV [in-vitro experiments [In-vitro attenuations of the virus isolates to certain cell lines must also not be disregarded.All WNV and USUV isolates demonstrated a rapid viral growth followed by a steady decline of the titres due to a strong CPE in both vertebrate cell lines. Maximum titres were higher in the avian GN-R cells than in the mammalian Vero B4 cells, similar to results from previous studies . This isand USUV , 39 and  for WNV . Overalleriments , 41. Deceriments , 43. In-in-vivo observations in geese where two different WNV strains, whose isolates were also used in the current study, caused comparable pathology [in-vitro study reported differences in viral replication of USUV strains in cell culture as well as virulence in mice [When comparing the viral growth kinetics within one virus species, USUV Europe 3 demonstrated slower viral growth than USUV Africa 3 on CT cells but not on C6/36 cells. A possible explanation might be the presence of a functional RNA interference pathway, which is the main antiviral pathway in mosquitoes , and proathology , 26. In  in mice . Overall in mice , 47 and  in mice  as well  in mice .in-vitro co-infection studies with flaviviruses mostly reported a competition between both viruses, resulting in the inhibition of at least one virus [Other ne virus , 48, 49.ne virus , resultine virus . This isThe viral interference appeared not to be strain or lineage dependent as similar results were found for combinations with other virus isolates. This was not surprising as almost all strains had similar viral kinetics. It must be noted that, even though marked differences in virulence were not observed between the virus strains used in this study, it is not uncommon for WNV to show variances in its efficiency to replicate and become neuroinvasive, as shown for Australian strains in cells and an established mouse model . Interesin-vivo findings in birds and mosquitoes. Birds that were co-infected with both viruses had higher viral loads of WNV than USUV [Cx. pipiens biotype pipiens that were simultaneously infected with WNV [in-vivo could not be confirmed for every mosquito species [Overall, WNV appears to have an advantage over USUV, possibly due to the observed different replication kinetics in host and vector cells. This is in accordance to han USUV , and USUwith WNV , 52. TakBelow is the link to the electronic supplementary material.Supplementary material: additional file 1: Fig.


The proportion of ewes conceiving triplets is increasing on farms across Australia as sheep producers adopt more fecund genetics and ewe management practices to increase lamb output per ewe mated. The aims of this research were to consult producers to quantify rates of ewe and lamb mortality in this cohort and identify priorities for future research to reduce these losses. Surveys of producers with experience in managing triplet-bearing ewes indicated the average mortality of triplet-bearing ewes was 6.4%, and the survival of triplet-born lambs was 59%. There was significant variation in the actual targets adopted by different producers for ewe condition score at lambing, mob size during lambing and feed-on-offer at lambing, and no differences in the average rate of mortality of triplet-bearing ewes or lamb survival between producers that prioritised adoption of certain management practices. The highest priorities for further research identified by producers from surveys, workshops and a webinar were ewe condition score, mob size, feed-on-offer at lambing and mineral supplementation. p < 0.05). The key strategies adopted to reduce the mortality of triplet-bearing ewes and their lambs included management of condition score, feed-on-offer, mob size at lambing and use of shelter. There were no differences (p > 0.05) in the average mortality of triplet-bearing ewes or their lambs between producers that prioritised the adoption of certain management practices. However, significant variation existed between producers in their targets at lambing for ewe condition score (2.8 to 3.5), mob size (10 to 150 ewes) and feed-on-offer (800 to 2500 kg dry matter/ha). Overwhelmingly, the highest priorities for further research identified by producers from surveys, workshops and a webinar were ewe condition score, mob size, feed-on-offer at lambing and mineral supplementation. This study informs benchmarks for mortality of triplet-bearing ewes and their lambs under extensive grazing conditions in Australia, and the priorities for future research to reduce these losses.Consultation with sheep producers was used to quantify the mortality of triplet-bearing ewes and their lambs, identify management practices adopted by producers to reduce these losses and prioritise future research needs to improve the survival of triplet-bearing ewes and their lambs. Surveys were completed by 64 producers across Australia who identified and separated triplet-bearing ewes from twin-bearing ewes in 2017 and/or 2018. On average, 5.9% of all ewes mated were identified as carrying triplets (6.6% of non-Merino ewes and 2.9% of Merino ewes). The average mortality of triplet-bearing ewes was 6.4%, and ewe mortality did not differ significantly between ewe breeds. The average survival of triplet-born lambs was 59%, and survival was significantly higher for lambs from non-Merino compared to Merino ewes (60.1 vs. 52.9%,  Increasing lamb production can improve the profitability of sheep enterprises, especially for non-Merino sheep and when sheep meat prices are high ,2. Lamb Triplet lambs are born lighter, are more metabolically challenged, have lower body temperature and impaired behavioural development, and receive less colostrum and milk than their twin counterparts. Combined, these result in higher rates of mortality of triplet-born lambs . Kenyon Management guidelines based on achieving ewe condition score, feed-on-offer (FOO) or mob size targets at lambing have been developed for single- and twin-bearing Merino and non-Merino ewes in Australia ,13,14. Hn = 64) or who always scanned for multiple-bearing ewes only and did not manage triplet-bearing ewes separately from twin-bearing ewes . Contact details for producers to survey were obtained mostly through professional networks. Pregnancy scanning contractors and sheep production consultants also provided contacts of their clients who had confirmed their interest and eligibility for participation in the surveys. The ewe management system for a farm was classified as ‘Separated’ if the triplet-bearing ewes from at least one ewe flock on their farm were identified and separated from twin-bearing ewes in either 2017 or 2018, where ‘flock’ represented all adult Merino or non-Merino ewes on a farm. In other words, over the two years, a farm classified as ‘Separated’ could include both separated and combined flocks. Telephone or in-person surveys were conducted in 2018 for 95 producers who had either pregnancy scanned to identify some triplet-bearing ewes and managed them separately from twin-bearing ewes , South Australia , Tasmania , Victoria  and Western Australia   . The proThe producers that had some experience in separating and differentially managing twin- and triplet-bearing ewes were asked to rank the primary causes of mortality of triplet-bearing ewes and lambs, as well as the key practices they had adopted to improve survival of triplet-bearing ewes and/or triplet-born lambs. The practices described by producers were distilled into six key themes: (i) ewe condition score at lambing; (ii) FOO during lambing; (iii) ewe mob size during lambing; (iv) shelter during lambing; (v) handling pre-lambing, and disturbance before and during lambing; and (vi) supplementary feeding in late pregnancy with grain and/or minerals. Producers then identified which of these six strategies were their first, second and third priorities for reducing the mortality of triplet-bearing ewes and/or improving the survival of their lambs. Where appropriate, producers also provided the targets they adopted for ewe condition score, FOO and mob size at lambing, including how they compared to targets for twin-bearing ewes. In the context of this paper, a ‘mob’ refers to a group of sheep managed together within the same paddock. Finally, the producers were asked to identify key research priorities to improve the survival of triplet-bearing ewes and their lambs. n = 8), Struan, SA (n = 5), Hamilton, VIC (n = 37), Ballarat, VIC (n = 13) and Holbrook, NSW (n = 15). There was also a webinar conducted involving a further 35 producers who could not attend the workshops. The workshops and webinar started by introducing the purpose of the project and a summary of the literature review on the survival of triplet-bearing ewes and their lambs [Five workshops involving 78 producers were conducted across Australia in 2018, including at Katanning, WA . OveralWhere farms identified and separated twin- and triplet-bearing ewes, ewe mortality and lamb survival for each birth type were analysed by the method of Restricted Maximum Likelihood with ewe breed at the flock level fitted as a fixed effect while year, state (nested within year), farm (nested within state) and flock (nested within farm) were fitted as random effects. Pearson correlation was used to measure the association between any two of the various parameters. The influence of the key management practice adopted by survey participants to reduce mortality of triplet-bearing and/or improve survival of their lambs on actual ewe mortality and lamb survival were analysed by the method of Restricted Maximum Likelihood with the main priority fitted as a fixed effect while year, state (nested within year) and farm were fitted as random effects.Producers that completed surveys managed approximately 352,000 ewes, including 153,000 Merino ewes and 199,000 non-Merino ewes. Farm and flock demographic data for survey participants are shown in At the farm level, participants with non-Merino ewes achieved a higher overall reproductive rate, lamb marking rate and lamb survival than those with Merino ewes only or a combination of both ewe breeds . These eAt the flock level, ewes were mated 17 to 22 days earlier but for 9 days longer on farms that always mixed multiple-bearing ewes together than ewes on farms that identified and separated at least some triplet-bearing ewes from twin-bearing ewes in either 2017 or 2018. There were no significant differences in flock reproductive rate, lamb marking rate or lamb survival between farms that always mixed multiple-bearing ewes together versus those who had identified and separated some triplet-bearing ewes from twin-bearing ewes in either 2017 or 2018. For farms that had separated some triplet-bearing ewes from twin-bearing ewes, flocks that were separated had a higher reproductive rate and marking rate, but there was no difference in reported lamb survival. Like above, this comparison needs to be treated with caution because the reproductive rate is underestimated, and lamb survival is overestimated in flocks where multiple-bearing ewes are combined. There were no significant interactions between ewe breed by ewe management system either at a farm level or flock within the farm level for overall reproductive rate, lamb marking rate or lamb survival. The raw means for Merino and non-Merino ewe flocks that identified and separated triplet-bearing ewes were 150.2% and 172.4% for reproductive rate, 112.8% and 137.4% for lamb marking rate and 75.1% and 79.9% for lamb survival.p < 0.05), but at a given reproductive rate, the proportion of twin- and triplet-bearing ewes appeared to be similar for Merino and non-Merino ewes. The proportion of twin-bearing ewes increased to a maximum of around 60–65% at a corresponding reproductive rate of about 180%, and then started to decline as higher-order multiples increased . By contrast, the mortality of twin-bearing ewes was 3.3% and ranged from 0.5% to 17% (10th percentile = 1.2% and 90th percentile = 5.0%), and the mortality of single-bearing ewes was 1.6% and ranged from 0% to 8% (10th percentile = 0.5% and 90th percentile = 3.0%). There were no differences in the average mortality of single-, twin- or triplet-bearing ewes between ewe breeds . Of the p = 0.06) and was significantly higher for twins and triplets compared to their Merino counterparts . By contrast, the average survival of twin-born lambs was 80% and ranged from 59% to 93% (10th percentile = 71.0% and 90th percentile = 88.6%), and the average survival of single-born lambs was 92% and ranged between 70% and 100% (10th percentile = 86.2% and 90th percentile = 96.8%). On average, the survival of lambs from non-Merino ewes tended to be higher for singles , mob size during lambing (22%), FOO during lambing (11%) and mineral supplementation (9%). Similarly, the top four research priorities identified by producers during the workshops and a webinar were FOO during lambing, mob size during lambing, ewe condition score at lambing and mineral supplementation . BetweenConsultation with sheep producers that had previously identified and differentially managed at least some triplet-bearing ewes indicated that reducing the mortality of triplet-bearing ewes was a high priority. The average mortality of triplet-bearing ewes from the benchmarking surveys was reported to be 6.4%, which was double that for twin-bearing ewes and four times that for single-bearing ewes. To our knowledge, this is the first study to quantify the mortality of triplet-bearing ewes on commercial farms across southern Australia. Kleemann et al.  reportedThe average survival of triplet-born lambs from the benchmark surveys was reported to be 59%, which was 22% and 33% lower than their twin- and single-born counterparts. The survival of triplet-born lambs was considerably lower than the 68% survival reported across 29 research studies, mostly based in New Zealand, which could reflect, in part, that the data in our study were collected from commercial farms, whereas most of the data reported in Kenyon et al.  were froOne-third of producers surveyed did not identify triplet-bearing ewes, and approximately two-thirds of these producers indicated the main reason for their decision was an insufficient number of triplet-bearing ewes. On average, each of these producers managed nearly 4000 breeding ewes and had they identified triplet-bearing ewes, their actual reproductive rate was likely to be around 153% rather than 148% based on scanning for multiples only. Therefore, it is likely thatapproximately 100 triplet-bearing ewes were mixed with the twin-bearing ewes in these flocks. As only 3% of all ewe flocks in Australia are scanned for triplet-bearing ewes , furtherA mixed-method approach involving more than 200 sheep producers from across southern Australia was effective at establishing the research needs and priorities of producers to improve the survival of triplet-bearing ewes and their lambs. Our hypothesis was therefore supported. The top four priorities for further research identified by producers were to establish targets for ewe condition score, FOO, mob size at lambing and quantify the impacts of supplementation with minerals, regardless of the ewe breed managed. These top four priorities represented between 73 and 81% of all responses, despite variations in the consultative processes used to identify the research priorities. The key research priorities to improve the survival of triplet-bearing ewes and their lambs identified from producer consultation in this study align with the knowledge gaps identified by Kenyon et al. . PrioritMost producers indicated the primary reason for managing the condition score of triplet-bearing ewes at lambing was to reduce ewe mortality. On average, the target condition score at lambing for triplet-bearing ewes of 3.3 was similar to that for twin-bearing ewes, but the target varied from 2.8 to 3.5 between producers. To our knowledge, there has been no detailed experimental work relating condition score profile during pregnancy and at lambing to risks of ewe mortality on commercial farms. In contrast to best practice management guidelines for twin-bearing Merino and non-Merino ewes, which require increased feeding to achieve a higher condition score at lambing for most farms ,20,21, nProducers considered FOO at lambing important to reduce both ewe mortality and especially to improve lamb survival. Whilst the three-fold range in target FOO levels from 800 to 2500 kg DM/ha would, in part, reflect different production environments and time of lambing in relation to seasonal pasture supply, the producers indicated there was a need to better define the FOO targets for optimal survival of triplet-bearing ewes and their lambs. Studies in New Zealand found that offering around 800 kg DM/ha from mid-pregnancy until birth reduced birthweights of triplet-born lambs compared to higher FOO levels , but othSheep producers reported that reducing mob size at lambing was a key practice they had adopted to improve the survival of triplet-born lambs, yet this remained a priority for further research. The average mob size at lambing from the benchmark surveys was reported to be 52 triplet-bearing ewes, but it was apparent that producers had very different opinions of the optimum mob sizes, which varied from 10–150 triplet-bearing ewes. Bates et al.  recentlyLittle is known regarding whether mineral supplementation of lambing ewes can reduce ewe and lamb mortality. Subclinical deficiencies of calcium and magnesium are common in lambing ewes in Australia due to imbalances in pasture grazed by the ewes . MineralThe sheep producers consulted in this study represented a biased sample, so caution is needed in extrapolating some of the findings across the national sheep flock, especially those relating to levels of reproductive performance. Producers must have utilised pregnancy scanning for litter size to be eligible for inclusion in the benchmarking surveys, and this only represents about 35% of Australian sheep producers . FurtherSurveys completed by 64 producers across Australia who separated triplet-bearing ewes from twin-bearing ewes found that 5.9% of all ewes mated were carrying triplets. The average mortality of triplet-bearing ewes of 6.4% did not differ between ewe breeds, whereas survival of triplet-born lambs was 60.1% for those from non-Merino ewes compared to 52.9% for Merino ewes. The key strategies adopted to reduce the mortality of triplet-bearing ewes and their lambs were the management of condition score at lambing, FOO and mob size during lambing, and use of shelter. Overwhelmingly, the highest priorities for further research identified by producers were ewe condition score, mob size, FOO at lambing and mineral supplementation.