Hantaviruses are globally distributed viruses that cause haemorrhagic fever with renal syndrome (HFRS) in Europe, a disease characterised by acute kidney failure and, in some cases, significant bleeding complications. The mechanisms underlying clotting abnormalities in HFRS remain poorly understood. This study aims to investigate the pathological mechanisms of clotting dysfunction in hospitalised HFRS patients, assess the impact of different hantavirus types on disease severity, and evaluate the accuracy of a severity scoring system developed in China for predicting mortality in European patients. Hospitalised patients with laboratory-confirmed HFRS will be prospectively recruited from University Medical Centre Ljubljana, Slovenia. Blood samples will be analysed for routine laboratory markers, thromboelastography (TEG) will assess real-time clotting function, and transcriptomic analysis will identify hantavirus strains and gene expression patterns linked to disease severity. Patients will be stratified into haemorrhagic and non-haemorrhagic groups, with statistical analyses comparing clinical and laboratory parameters to identify predictors of bleeding risk. Findings from this study may contribute to improved risk stratification and potential therapeutic targets for HFRS.
Hantaviruses are globally distributed viruses that are transmitted to humans through the inhalation of viral particles found in the urine and faeces of infected rodents. In Europe, hantaviruses cause haemorrhagic fever with renal syndrome (HFRS), a disease characterised by acute kidney failure and, in some cases, significant bleeding complications. Despite advances in understanding HFRS, the mechanisms underlying these bleeding abnormalities remain poorly understood. Several hypotheses suggest that hantaviruses may interfere with the function of blood clotting factors, but definitive evidence is lacking. Slovenia, a country in southern Europe, has one of the highest incidences of HFRS relative to its population, with recent years seeing large outbreaks. Discussions with leading hantavirus experts in Slovenia have highlighted the urgent need for further research, particularly to understand why some patients with HFRS develop severe bleeding complications. Currently, no licensed treatments exist for HFRS, raising concerns about future outbreaks, which are expected to become more frequent due to climate change. By investigating the mechanisms leading to clotting dysfunction, this study aims to identify potential targets for future therapeutic interventions. This study will explore the pathological mechanisms driving clotting abnormalities and bleeding in hospitalised patients with HFRS. Additionally, it will assess whether different hantavirus types influence disease severity and haemorrhagic complications and evaluate the applicability of a severity scoring system-originally developed for HFRS cases in China-in predicting outcomes among Slovenian patients. Patients with laboratory-confirmed HFRS will be prospectively recruited from University Medical Centre Ljubljana, Slovenia. Blood samples will be collected at multiple time points during their illness and tested for standard laboratory markers, including clotting factors, blood cell counts, kidney function, and liver function. Additionally, thromboelastography (TEG) will be performed to assess real-time clotting dynamics, providing a more detailed evaluation of clotting abnormalities. An additional blood sample will undergo transcriptomic analysis using nanopore sequencing. This will identify the specific hantavirus strain in each patient and analyse gene expression patterns associated with disease severity and bleeding risk. Patients will also be assigned a severity score based on their clinical presentation, laboratory results, and symptoms, using a scoring system developed in China to predict HFRS-related mortality. This study will evaluate the reliability of this scoring system in a European cohort. To better understand the factors contributing to bleeding in HFRS, patients will be categorised into two groups: those with haemorrhagic manifestations and those without. Statistical analyses will compare laboratory and clinical findings between these groups, aiming to identify key predictors of bleeding complications. The findings from this research may help inform future therapeutic strategies for HFRS.
Study Type
OBSERVATIONAL
Enrollment
62
Two blood samples will be collected at admission for thromboelastography using the TEG 6s platform (Haemonetics®). One sample will be collected in a citrated blood tube for global haemostasis assessment, and one sample will be collected in a heparinised tube for platelet function analysis.
Two blood samples will be collected 3 - 7 days after initial thromboelastography for follow-up analysis using the TEG 6s platform (Haemonetics®). One sample will be collected in a citrated blood tube for global haemostasis assessment, and one sample will be collected in a heparinised tube for platelet function analysis.
One blood sample will be collected at admission for transcriptomic analysis. Blood sample will be collected into a PAXgene® RNA tube and analysed using nanopore sequencing to characterise the viral and host transcriptome.
Routine clinical/demographic/epidemiological data will be collected at admission and throughout hospitalisation. This will relate to clinical presentation (day of illness at presentation, presenting symptoms); demographics and epidemiology (age, gender, site of infection); clinical course during hospitalisation (maximum level of care, dialysis use, blood product use, survival outcome).
Data on routine laboratory parameters will be collected throughout hospitalisation. These will relate to laboratory clotting parameters (platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen, D-dimer); liver function tests (aspartate aminotransferase, alanine aminotransferase); laboratory haematology parameters (haemoglobin, white cell count, blood film); laboratory biochemistry parameters (urea, creatinine); viral load.
A severity score will be assigned to each patient based on clinical and laboratory data at admission according to a pre-defined scoring system.
Change in Reaction Time (R) on thromboelastography from admission to follow-up (3-7 days), assessing clot initiation and its association with haemostatic dysfunction in HFRS.
Reaction time (R): The amount of time between the start of the test and the beginning of coagulation. Measured in minutes (min). Measured according to standardised assay on the TEG 6s haemostasis analyser.
Time frame: Baseline and 3-7 days later
Change in K-Time (K) on thromboelastography from admission to follow-up (3-7 days), evaluating clot kinetics and fibrin polymerisation in relation to haemostatic abnormalities in HFRS.
K-time (K): The speed of formation of the clot from Reaction Time (R) to a specific clot strength. Measured in minutes (min). Measured according to standardised assay on the TEG 6s haemostasis analyser.
Time frame: Baseline and 3-7 days later
Change in Alpha Angle (α-Angle) on thromboelastography from admission to follow-up (3-7 days), reflecting fibrin build-up and clot formation rate in patients with HFRS.
Alpha Angle (α-Angle): The speed of clot strengthening. Measured in degrees (°). Measured according to standardised assay on the TEG 6s haemostasis analyser.
Time frame: Baseline and 3-7 days later
Change in Maximum Amplitude (MA) on thromboelastography from admission to follow-up (3-7 days), assessing overall clot strength and platelet contribution to clot stability in HFRS.
Maximum Amplitude (MA): The ultimate strength of the clot. Measured in millimetres (mm). Measured according to standardised assay on the TEG 6s haemostasis analyser.
Time frame: Baseline and 3-7 days later
Change in Lysis 30 (LY30) on thromboelastography from admission to follow-up (3-7 days), measuring fibrinolysis and clot breakdown in relation to bleeding risk in HFRS.
Lysis 30 (LY30): Percent lysis 30 minutes after Maximum Amplitude (MA) is finalised. The LY30 measurement is based on the reduction of the tracing area that occurs between the time that MA is measured until 30 minutes after the MA is finalised. Measured as a percentage (%). Measured according to standardised assay on the TEG 6s haemostasis analyser.
Time frame: Baseline and 3-7 days later
Change in Percentage Inhibition (% Inhibition) on thromboelastography from admission to follow-up (3-7 days), evaluating the effect of antithrombotic pathways on clot formation in HFRS.
Percentage Inhibition (% Inhibition): Indicates the reduction in platelet contribution to overall clot strength. Measured as a percentage (%). Measured according to standardised assay on the TEG 6s haemostasis analyser.
Time frame: Baseline and 3-7 days later
Change in Percentage Aggregation (% Aggregation) on thromboelastography from admission to follow-up (3-7 days), assessing platelet function and its role in haemostatic dysfunction in HFRS.
Percentage Aggregation (% Aggregation): Indicates the percent of platelets not inhibited, determined by comparing the uninhibited platelet contribution to the baseline platelet contribution. Measured as a percentage (%). Measured according to standardised assay on the TEG 6s haemostasis analyser.
Time frame: Baseline and 3-7 days later
Change in platelet count over the course of illness, assessing thrombocytopenia and its role in haemostatic dysfunction in HFRS.
Platelet count measures the number of platelets in the blood, which are essential for clot formation. Measured as ×10⁹/L. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in prothrombin time (PT) over the course of illness, evaluating coagulation factor activity and clotting dysfunction in HFRS.
Prothrombin time (PT) measures the time taken for blood to clot via the extrinsic pathway. Measured in seconds (s). Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in activated partial thromboplastin time (APTT) over the course of illness, assessing abnormalities in the intrinsic clotting pathway in HFRS.
Activated partial thromboplastin time (APTT) assesses the intrinsic clotting pathway and measures time to clot formation. Measured in seconds (s). Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in fibrinogen levels over the course of illness, investigating fibrinogen consumption and clot formation abnormalities in HFRS.
Fibrinogen is part of clotting assessment and is a key protein involved in clot formation, converted into fibrin to stabilise clots. Measured in g/L. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in D-dimer levels over the course of illness, evaluating fibrinolysis and its association with haemorrhagic complications in HFRS
D-dimer is a marker of fibrin breakdown, used to assess fibrinolysis and clotting activity. Measured in ng/mL. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
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Change in aspartate aminotransferase (AST) over the course of illness, assessing liver involvement and potential contributions to haemostatic dysfunction in HFRS.
Aspartate aminotransferase (AST) is an enzyme released from damaged liver cells, used to assess liver injury. Measured in U/L. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in alanine aminotransferase (ALT) over the course of illness, evaluating liver injury and its association with disease severity in HFRS.
Alanine aminotransferase (ALT) is an enzyme released from damaged liver cells, used to assess liver injury. Measured in U/L. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in haemoglobin levels over the course of illness, assessing anaemia and its potential link to bleeding severity in HFRS.
Haemoglobin (Hb) measures the oxygen-carrying protein in red blood cells. Measured in g/L. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in white cell count (WCC) over the course of illness, evaluating immune response and its association with disease severity in HFRS.
White cell count (WCC) reflects the number of white blood cells, indicating immune response or infection. Measured in ×10⁹/L. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in blood film findings over the course of illness, identifying morphological changes in blood cells associated with HFRS severity.
A blood film is a microscopic evaluation of blood cells to assess morphology and detect abnormalities. This is a descriptive report with no numerical value. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in creatinine levels over the course of illness, evaluating renal impairment and disease progression in HFRS.
Creatinine is a marker of renal function, indicating kidney filtration efficiency. Measured in µmol/L. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Change in urea levels over the course of illness, assessing kidney dysfunction and its relationship to HFRS severity.
Urea is a waste product of protein metabolism, used to assess kidney function. Measured in mmol/L. Change over the course of illness, measured in days.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Day of illness at hospital presentation, assessing timing of medical intervention in relation to disease progression.
The number of days of illness experienced prior to presentation to hospital, inclusive of day of presentation.
Time frame: Baseline (at admission)
Frequency of symptoms at presentation and throughout hospitalisation, characterising the clinical course and severity of HFRS.
The occurrence and frequency of key symptoms (such as fever, bleeding, or kidney dysfunction) at the time of hospital presentation and during the hospital stay, used to characterise the clinical course and severity of HFRS.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Maximum level of clinical care required during hospitalisation, assessing disease severity and need for intensive care in HFRS patients.
The highest level of medical care required by the patient during their hospitalisation (e.g., general ward, high-dependency unit, or intensive care unit), used to assess disease severity and the need for intensive care in HFRS patients.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Survival outcome, evaluating overall mortality and predictors of fatal HFRS cases.
Outcome of each illness, recorded as either 'survival' or 'death'. Outcomes will only be observed from the point of illness onset until discharge from hospital.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Frequency of blood product use during hospitalisation, assessing transfusion requirements as a marker of haemorrhagic severity in HFRS.
The number of patients receiving blood products during hospitalisation. A blood product refers to any therapeutic component derived from human blood, including red blood cells, platelets, plasma, and clotting factor concentrates, used for transfusion or treatment of coagulation disorders.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Frequency of acute renal replacement therapy use during hospitalisation, evaluating the need for dialysis as an indicator of severe renal dysfunction in HFRS.
Number of patients requiring acute renal replacement therapy (RRT) during hospitalisation. Acute RRT refers to short-term treatments used to support kidney function in patients with acute kidney injury (AKI), including haemodialysis, haemofiltration, or continuous renal replacement therapy (CRRT), to remove waste products, excess fluids, and toxins from the blood.
Time frame: Baseline (day 1) and daily thereafter through hospitalisation, up to 21 days
Viral RNA genomic analysis and its association with disease severity and haemostatic dysfunction
Nanopore sequencing of hantavirus RNA to identify the specific hantavirus strain present, using blood samples collected at the time of hospital admission. This will help determine if certain hantavirus strains are associated with more severe disease and increased haemostatic dysfunction in patients with HFRS.
Time frame: Baseline (at admission)
Host RNA genomic analysis and its association with disease severity and haemostatic dysfunction
Nanopore sequencing of host RNA to identify differentially expressed genes between cohorts, using blood samples collected at the time of hospital admission. This analysis will help investigate any host genetic factors that may be linked to disease severity and haemostatic dysfunction in patients with HFRS.
Time frame: Baseline (at admission)
Validation of a severity score for predicting risk of death from HFRS in European patients
Each patient will be assigned a severity score upon admission based on a scoring system developed by researchers in China, designed to predict the risk of death from HFRS. Each patient will be assigned a score of 0 - 22 according to the published scoring matrix. Scores of 0 - 10 are categorised as 'Low Risk'; scores of 11 - 14 are categorised as 'Medium Risk'; scores of 15 - 22 are categorised as 'High Risk'. The score will be calculated at baseline and then assessed for its correlation with the disease outcome (survival vs. death) to determine if it is applicable for use in European patients with HFRS.
Time frame: Baseline (at admission)