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Background The current SARS-CoV-2 pandemic has highlighted a need for easy and safe blood sampling in combination with accurate serological methodology. Venipuncture for testing is usually performed by trained staff at healthcare centres. Long travel distances to healthcare centres in rural regions may introduce a bias of testing towards relatively large communities with closer access. Rural regions are therefore often not represented in population-based data. Aim The aim of this retrospective cohort study was to develop and implement a strategy for at-home testing in a rural region of Sweden during spring 2021, and to evaluate its role to provide equal health care for its inhabitants. Methods We developed a sensitive method to measure antibodies to the S-protein of SARS-CoV-2 and optimised this assay for clinical use together with a strategy of at-home capillary blood sampling. Results We demonstrated that our ELISA gave comparable results after analysis of capillary blood or serum from SARS-CoV-2-experienced individuals. We demonstrated stability of the assay under conditions that reflected temperature and humidity during winter or summer. By assessment of capillary blood samples from 4,122 individuals, we could show both feasibility of the strategy and that implementation shifted the geographical spread of testing in favour of rural areas. Conclusion Implementation of at-home sampling enabled citizens living in remote rural areas access to centralised and sensitive laboratory antibody tests. The strategy for testing used here could therefore enable disease control authorities to get rapid access to information concerning immunity to infectious diseases, even across vast geographical distance.

BACKGROUND: Orthohantaviruses are rodent-borne emerging viruses that cause haemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in America. Transmission between humans have been reported and the case-fatality rate ranges from 0.4% to 40% depending on virus strain. There is no specific and efficient treatment for patients with severe HFRS. Here, we characterised a fatal case of HFRS and sequenced the causing Puumala orthohantavirus (PUUV). METHODS: PUUV RNA and virus specific neutralising antibodies were quantified in plasma samples from the fatal case and other patients with non-fatal PUUV infection. To investigate if the causing PUUV strain was different from previously known strains, Sanger sequencing was performed directly from the patient's plasma. Biopsies obtained from autopsy were stained for immunohistochemistry. RESULTS: The patient had approximately tenfold lower levels of PUUV neutralising antibodies and twice higher viral load than was normally seen for patients with less severe PUUV infection. We could demonstrate unique mutations in the S and M segments of the virus that could have had an impact on the severity of infection. Due to the severe course of infection, the patient was treated with the bradykinin receptor inhibitor icatibant to reduce bradykinin-mediated vessel permeability and maintain vascular circulation. CONCLUSIONS: Our data suggest that bradykinin receptor inhibitor may not be highly efficient to treat patients that are at an advanced stage of HFRS. Low neutralising antibodies and high viral load at admission to the hospital were associated with the fatal outcome and may be useful for future predictions of disease outcome.

International audience; Objective : Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction.Methods : We performed a comprehensive characterisation of longitudinal antiviral B-cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells.Results : We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27−IgD− B cells and CD27−/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection.Conclusion : Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus-infected individuals have significantly elevated levels of extracellular ATP in circulation.

The current SARS-CoV-2 pandemic has highlighted a need for easy and safe blood sampling in combination with accurate serological methodology. Venipuncture is usually performed by trained staff at health care centers. Long travel distances may introduce a bias of testing towards relatively large communities with close access to health care centers. Rural regions may thus be overlooked. Here, we demonstrate a sensitive method to measure antibodies to the S-protein of SARS-CoV-2. We adapted and optimized this assay for clinical use together with capillary blood sampling to meet the geographical challenges of serosurveillance. Finally, we tested remote at-home capillary blood sampling together with centralized assessment of S-specific IgG in a rural region of northern Scandinavia that encompasses 55,185 sq kilometers. We conclude that serological assessment from capillary blood sampling gives comparable results as analysis of venous blood. Importantly, at-home sampling enabled citizens living in remote rural areas access to centralized and sensitive laboratory antibody tests.

Objective Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction. Methods We performed a comprehensive characterisation of longitudinal antiviral B‐cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells. Results We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27−IgD− B cells and CD27−/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection. Conclusion Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus‐infected individuals have significantly elevated levels of extracellular ATP in circulation., We show a comprehensive study to correlate two pathologies associated with haemorrhagic fever with renal syndrome (HFRS), thrombocytopenia and kidney dysfunction, with changes in the circulating B‐cell compartment (cells and antibodies). We show that thrombocytopenia is associated with mobilization of both activated and resting plasmablasts and plasma cells in circulation. However, the level of kidney dysfunction associated with longitudinal development of neutralizing antibodies in patients and associated with the reduction of surface CD27 expression on both plasmablasts and B cells in circulation. The low CD27 expression might be explained by elevated levels of ATP that could contribute to shedding of CD27 from B cells by a MMP‐8‐dependent mechanism.

Human hantavirus infections can cause hemorrhagic fever with renal syndrome (HFRS), major signs of the disease being thrombocytopenia and transient kidney dysfunction. By a comprehensive and longitudinal study of circulating B cells, we demonstrate that these two pathologies associate with distinct effects on the humoral immune system during HFRS. Low thrombocyte counts strongly associated with an abnormal frequency of plasmablasts in circulation, whereas kidney dysfunction was indicative of an accumulation of CD27−B cells and plasmablasts. Finally, we provide evidence that high levels of extracellular ATP in circulation during HFRS correlates with shedding of surface CD27 on B cells via a metallomatrix proteinase-8-mediated mechanism. Since extracellular ATP is known to regulate kidney function, our study reveals a link between kidney dysfunction and the generation of CD27−IgD−B cells, and a potential molecular target for treatment of the symptomatic phase of HFRS.

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, &alpha, 1-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.

Background. Endothelial activation and dysfunction play a central role in the pathogenesis of sepsis and viral hemorrhagic fevers. Hantaviral disease is a viral hemorrhagic fever and is characterized by capillary dysfunction, although the underlying mechanisms for hantaviral disease are not fully elucidated. Methods. The temporal course of endothelial activation and repair were analyzed during Puumala hantavirus infection and associated with disease outcome and a marker for hypoxia, insulin-like growth factor binding protein 1 (IGFBP-1). The following endothelial activation markers were studied: endothelial glycocalyx degradation (syndecan-1) and leukocyte adhesion molecules (soluble vascular cellular adhesion molecule 1, intercellular adhesion molecule 1, and endothelial selectin). Cytokines associated with vascular repair were also analyzed (vascular endothelial growth factor, erythropoietin, angiopoietin, and stromal cell-derived factor 1). Results. Most of the markers we studied were highest during the earliest phase of hantaviral disease and associated with clinical and laboratory surrogate markers for disease outcome. In particular, the marker for glycocalyx degradation, syndecan-1, was significantly associated with levels of thrombocytes, albumin, IGFBP-1, decreased blood pressure, and disease severity. Conclusions. Hantaviral disease outcome was associated with endothelial dysfunction. Consequently, the endothelium warrants further investigation when designing future medical interventions.