Direct and indirect effects of Puumala hantavirus on platelet function.

Thrombocytopenia is a cardinal symptom of hantavirus-induced diseases including Puumala virus (PUUV)-induced hemorrhagic fever with renal syndrome (HFRS), which is associated with impaired platelet function, bleeding manifestations and augmented thrombotic risk. However, the underlying mechanisms causing thrombocytopenia and platelet hypo-responsiveness are unknown. Thus, we investigated the direct and indirect impact of PUUV on platelet production, function and degradation. Analysis of PUUV-HFRS patient blood revealed that platelet hypo-responsiveness in PUUV infection was cell-intrinsic and accompanied by reduced platelet-leukocyte aggregates (PLAs) and upregulation of monocyte tissue factor (TF), whereas platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation was comparable to healthy controls. Plasma CXCL4 levels followed platelet count dynamics throughout disease course. PUUV activated both neutrophils and monocytes in vitro, but platelet desialylation, degranulation and GPIIb/IIIa activation as well as PLA formation and endothelial adhesion under flow remained unaltered in the presence of PUUV. Further, MEG-01 megakaryocytes infected with PUUV displayed unaltered polyploidization, expression of surface receptors and platelet production. However, infection of endothelial cells with PUUV significantly increased platelet sequestration. Our data thus demonstrate that although platelet production, activation or degradation are not directly modulated, PUUV indirectly fosters thrombocytopenia by sequestration of platelets to infected endothelium. Upregulation of immunothrombotic processes in PUUV-HFRS may further contribute to platelet dysfunction and consumption. Given the pathophysiologic similarities of hantavirus infections, our findings thus provide important insights into the mechanisms underlying thrombocytopenia and highlight immune-mediated coagulopathy as potential therapeutic target.

Risks of deep vein thrombosis, pulmonary embolism, and bleeding after covid-19: nationwide self-controlled cases series and matched cohort study.

OBJECTIVE: To quantify the risk of deep vein thrombosis, pulmonary embolism, and bleeding after covid-19. DESIGN: Self-controlled case series and matched cohort study. SETTING: National registries in Sweden. PARTICIPANTS: 1 057 174 people who tested positive for SARS-CoV-2 between 1 February 2020 and 25 May 2021 in Sweden, matched on age, sex, and county of residence to 4 076 342 control participants. MAIN OUTCOMES MEASURES: Self-controlled case series and conditional Poisson regression were used to determine the incidence rate ratio and risk ratio with corresponding 95% confidence intervals for a first deep vein thrombosis, pulmonary embolism, or bleeding event. In the self-controlled case series, the incidence rate ratios for first time outcomes after covid-19 were determined using set time intervals and the spline model. The risk ratios for first time and all events were determined during days 1-30 after covid-19 or index date using the matched cohort study, and adjusting for potential confounders (comorbidities, cancer, surgery, long term anticoagulation treatment, previous venous thromboembolism, or previous bleeding event). RESULTS: Compared with the control period, incidence rate ratios were significantly increased 70 days after covid-19 for deep vein thrombosis, 110 days for pulmonary embolism, and 60 days for bleeding. In particular, incidence rate ratios for a first pulmonary embolism were 36.17 (95% confidence interval 31.55 to 41.47) during the first week after covid-19 and 46.40 (40.61 to 53.02) during the second week. Incidence rate ratios during days 1-30 after covid-19 were 5.90 (5.12 to 6.80) for deep vein thrombosis, 31.59 (27.99 to 35.63) for pulmonary embolism, and 2.48 (2.30 to 2.68) for bleeding. Similarly, the risk ratios during days 1-30 after covid-19 were 4.98 (4.96 to 5.01) for deep vein thrombosis, 33.05 (32.8 to 33.3) for pulmonary embolism, and 1.88 (1.71 to 2.07) for bleeding, after adjusting for the effect of potential confounders. The rate ratios were highest in patients with critical covid-19 and highest during the first pandemic wave in Sweden compared with the second and third waves. In the same period, the absolute risk among patients with covid-19 was 0.039% (401 events) for deep vein thrombosis, 0.17% (1761 events) for pulmonary embolism, and 0.101% (1002 events) for bleeding. CONCLUSIONS: The findings of this study suggest that covid-19 is a risk factor for deep vein thrombosis, pulmonary embolism, and bleeding. These results could impact recommendations on diagnostic and prophylactic strategies against venous thromboembolism after covid-19.

Association between pharmaceutical modulation of oestrogen in postmenopausal women in Sweden and death due to COVID-19: a cohort study.

OBJECTIVE: Determine whether augmentation of oestrogen in postmenopausal women decreases the risk of death following COVID-19. DESIGN: Nationwide registry-based study in Sweden based on registries from the Swedish Public Health Agency (all individuals who tested positive for SARS-CoV-2); Statistics Sweden (socioeconomical variables) and the National Board of Health and Welfare (causes of death). PARTICIPANTS: Postmenopausal women between 50 and 80 years of age with verified COVID-19. INTERVENTIONS: Pharmaceutical modulation of oestrogen as defined by (1) women with previously diagnosed breast cancer and receiving endocrine therapy (decreased systemic oestrogen levels); (2) women receiving hormone replacement therapy (increased systemic oestrogen levels) and (3) a control group not fulfilling requirements for group 1 or 2 (postmenopausal oestrogen levels). Adjustments were made for potential confounders such as age, annual disposable income (richest group as the reference category), highest level of education (primary, secondary and tertiary (reference)) and the weighted Charlson Comorbidity Index (wCCI). PRIMARY OUTCOME MEASURE: Death following COVID-19. RESULTS: From a nationwide cohort consisting of 49 853 women diagnosed with COVID-19 between 4 February and 14 September 2020 in Sweden, 16 693 were between 50 and 80 years of age. We included 14 685 women in the study with 11 923 (81%) in the control group, 227 (2%) women in group 1 and 2535 (17%) women in group 2. The unadjusted ORs for death following COVID-19 were 2.35 (95% CI 1.51 to 3.65) for group 1 and 0.45 (0.34 to 0.6) for group 2. Only the adjusted OR for death remained significant for group 2 with OR 0.47 (0.34 to 0.63). Absolute risk of death was 4.6% for the control group vs 10.1% and 2.1%, for the decreased and increased oestrogen groups, respectively. The risk of death due to COVID-19 was significantly associated with: age, OR 1.15 (1.14 to 1.17); annual income, poorest 2.79 (1.96 to 3.97), poor 2.43 (91.71 to 3.46) and middle 1.64 (1.11 to 2.41); and education (primary 1.4 (1.07 to 1.81)) and wCCI 1.13 (1.1 to 1.16). CONCLUSIONS: Oestrogen supplementation in postmenopausal women is associated with a decreased risk of dying from COVID-19 in this nationwide cohort study. These findings are limited by the retrospective and non-randomised design. Further randomised intervention trials are warranted.

Inequitable impact of infection: social gradients in severe COVID-19 outcomes among all confirmed SARS-CoV-2 cases during the first pandemic wave in Sweden.

BACKGROUND: The backdrop of the ubiquitous social inequalities has increasingly come into foreground in research on the COVID-19 pandemic, but the lack of high-quality population-based studies limits our understanding of the inequitable outcomes of the disease. The present study seeks to estimate social gradients in COVID-19 hospitalisations, intensive care admissions and death by education, income and country of birth, while taking into account disparities in comorbidities. METHODS: We used a register-based retrospective open cohort design enrolling all 74 659 confirmed SARS-CoV-2-positive cases aged >25 years in Sweden during the first wave of the pandemic (until 14 September 2020). Information was retrieved from multiple registers and linked by the unique Swedish personal identity number concerning COVID-19 case identification; COVID-19 hospitalisations, intensive care admissions and death; comorbidities as measured by the Charlson Comorbidity Index; and sociodemographic information. Social gradients were estimated by the Relative Index of Inequality (RII) using Cox regression. RESULTS: Adjusted analyses showed significant social gradients in COVID-19 hospitalisation, intensive care admission, across education, income and country of birth, which were unaffected by adjustment for comorbidities. Education and country of birth gradients were stronger for hospitalisation and intensive care admissions but small to non-existent for death. In contrast, income gradients were consistent across all three COVID-19 outcomes. CONCLUSION: Social gradients in severe COVID-19 outcomes are widespread in Sweden, but appear to be unrelated to pre-existing health disparities. Inequitable outcomes of SARS-CoV-2 infection may therefore be at least partially avoidable and could rely on equitable management of confirmed COVID-19 cases.

Avoiding bias in self-controlled case series studies of coronavirus disease 2019.

Many studies, including self-controlled case series (SCCS) studies, are being undertaken to quantify the risks of complications following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19). One such SCCS study, based on all COVID-19 cases arising in Sweden over an 8-month period, has shown that SARS-CoV-2 infection increases the risks of AMI and ischemic stroke. Some features of SARS-CoV-2 infection and COVID-19, present in this study and likely in others, complicate the analysis and may introduce bias. In the present paper we describe these features, and explore the biases they may generate. Motivated by data-based simulations, we propose methods to reduce or remove these biases.

Risk of acute myocardial infarction and ischaemic stroke following COVID-19 in Sweden: a self-controlled case series and matched cohort study.

BACKGROUND: COVID-19 is a complex disease targeting many organs. Previous studies highlight COVID-19 as a probable risk factor for acute cardiovascular complications. We aimed to quantify the risk of acute myocardial infarction and ischaemic stroke associated with COVID-19 by analysing all COVID-19 cases in Sweden. METHODS: This self-controlled case series (SCCS) and matched cohort study was done in Sweden. The personal identification numbers of all patients with COVID-19 in Sweden from Feb 1 to Sept 14, 2020, were identified and cross-linked with national inpatient, outpatient, cancer, and cause of death registers. The controls were matched on age, sex, and county of residence in Sweden. International Classification of Diseases codes for acute myocardial infarction or ischaemic stroke were identified in causes of hospital admission for all patients with COVID-19 in the SCCS and all patients with COVID-19 and the matched control individuals in the matched cohort study. The SCCS method was used to calculate the incidence rate ratio (IRR) for first acute myocardial infarction or ischaemic stroke following COVID-19 compared with a control period. The matched cohort study was used to determine the increased risk that COVID-19 confers compared with the background population of increased acute myocardial infarction or ischaemic stroke in the first 2 weeks following COVID-19. FINDINGS: 86 742 patients with COVID-19 were included in the SCCS study, and 348 481 matched control individuals were also included in the matched cohort study. When day of exposure was excluded from the risk period in the SCCS, the IRR for acute myocardial infarction was 2·89 (95% CI 1·51-5·55) for the first week, 2·53 (1·29-4·94) for the second week, and 1·60 (0·84-3·04) in weeks 3 and 4 following COVID-19. When day of exposure was included in the risk period, IRR was 8·44 (5·45-13·08) for the first week, 2·56 (1·31-5·01) for the second week, and 1·62 (0·85-3·09) for weeks 3 and 4 following COVID-19. The corresponding IRRs for ischaemic stroke when day of exposure was excluded from the risk period were 2·97 (1·71-5·15) in the first week, 2·80 (1·60-4·88) in the second week, and 2·10 (1·33-3·32) in weeks 3 and 4 following COVID-19; when day of exposure was included in the risk period, the IRRs were 6·18 (4·06-9·42) for the first week, 2·85 (1·64-4·97) for the second week, and 2·14 (1·36-3·38) for weeks 3 and 4 following COVID-19. In the matched cohort analysis excluding day 0, the odds ratio (OR) for acute myocardial infarction was 3·41 (1·58-7·36) and for stroke was 3·63 (1·69-7·80) in the 2 weeks following COVID-19. When day 0 was included in the matched cohort study, the OR for acute myocardial infarction was 6·61 (3·56-12·20) and for ischaemic stroke was 6·74 (3·71-12·20) in the 2 weeks following COVID-19. INTERPRETATION: Our findings suggest that COVID-19 is a risk factor for acute myocardial infarction and ischaemic stroke. This indicates that acute myocardial infarction and ischaemic stroke represent a part of the clinical picture of COVID-19, and highlights the need for vaccination against COVID-19. FUNDING: Central ALF-funding and Base Unit ALF-Funding, Region Västerbotten, Sweden; Strategic funding during 2020 from the Department of Clinical Microbiology, Umeå University, Sweden; Stroke Research in Northern Sweden; The Laboratory for Molecular Infection Medicine Sweden.

Cystatin C and α-1-Microglobulin Predict Severe Acute Kidney Injury in Patients with Hemorrhagic Fever with Renal Syndrome.

Puumala orthohantavirus causes hemorrhagic fever with renal syndrome (HFRS) characterized by acute kidney injury (AKI), an abrupt decrease in renal function. Creatinine is routinely used to detect and quantify AKI; however, early AKI may not be reflected in increased creatinine levels. Therefore, kidney injury markers that can predict AKI are needed. The potential of the kidney injury markers urea, cystatin C, α1-microglobulin (A1M) and neutrophil gelatinase-associated lipocalin (NGAL) to detect early AKI during HFRS was studied by quantifying the levels of these markers in consecutively obtained plasma (P) and urine samples (U) for 44 HFRS patients. P-cystatin C and U-A1M levels were significantly increased during early HFRS compared to follow-up. In a receiver operating characteristic (ROC) curve analysis, P-cystatin C, U-A1M and P-urea predicted severe AKI with area under the curve 0.72, 0.73 and 0.71, respectively, whereas the traditional kidney injury biomarkers creatinine and U-albumin did not predict AKI. Nearly half of the HFRS patients (41%) fulfilled the criteria for shrunken pore syndrome, which was associated with the level of inflammation as measured by P-CRP. P-cystatin C and U-A1M are more sensitive and earlier markers compared to creatinine in predicting kidney injury during HFRS.

Circulating Extracellular Vesicle Tissue Factor Activity During Orthohantavirus Infection Is Associated With Intravascular Coagulation.

BACKGROUND: Puumala orthohantavirus (PUUV) causes hemorrhagic fever with renal syndrome (HFRS). Patients with HFRS have an activated coagulation system with increased risk of disseminated intravascular coagulation (DIC) and venous thromboembolism (VTE). The aim of the study was to determine whether circulating extracellular vesicle tissue factor (EVTF) activity levels associates with DIC and VTE (grouped as intravascular coagulation) in HFRS patients. METHODS: Longitudinal samples were collected from 88 HFRS patients. Patients were stratified into groups of those with intravascular coagulation (n = 27) and those who did not (n = 61). We measured levels of circulating EVTF activity, fibrinogen, activated partial prothrombin time, D-dimer, tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), and platelets. RESULTS: Plasma EVTF activity was transiently increased during HFRS. Levels of EVTF activity were significantly associated with plasma tPA and PAI-1, suggesting that endothelial cells could be a potential source. Patients with intravascular coagulation had significantly higher peak EVTF activity levels compared with those who did not, even after adjustment for sex and age. The peak EVTF activity value predicting intravascular coagulation was 0.51 ng/L with 63% sensitivity and 61% specificity with area under the curve = 0.63 (95% confidence interval, 0.51-0.76) and P = .046. CONCLUSIONS: Plasma EVTF activity during HFRS is associated with intravascular coagulation.