Correlation of CD38 expression with the progression of hemorrhagic fever with renal syndrome.

To assess the relationship between the expression of CD38 and the progression of hemorrhagic fever with renal syndrome (HFRS), we determined the levels of CD38 during different phases of HFRS and evaluated the relationship between changes in CD38 expression and the progression of HFRS. The expression of CD38 in 68 patients with HFRS was analyzed by flow cytometry, and this method was also used to determine the levels of CD4+T, CD8+T, and B lymphocytes and NK cells. Furthermore, creatinine (Cr), uric acid (UA), and urea in serum at each stage of HFRS were measured using commercial kits. The basic clinical reference values for leukocytes, platelets (PLT), and red blood cells were determined by conventional methods. The colloidal gold method was used to measure HFRS antibody levels in the patients. A significant change in CD38 expression was observed from the fever phase to the recovery phase in patients with HFRS. Moreover, the expression of CD38 was proportionally correlated with the levels of Cr, UA, and urea in serum. In contrast, there was an inverse correlation between CD38 and PLT. Interestingly, an increase in CD38 expression correlated with an increase in CD8+T lymphocytes, B cells, and NK cells, but with a decrease in CD4+T lymphocytes. The expression of CD38 is associated with the progression of HFRS, suggesting that it may be a potent indicator of the stages of this disorder.

Clinical course and long-term outcome of hantavirus-associated nephropathia epidemica, Germany.

Human infection with Puumala virus (PUUV), the most common hantavirus in Central Europe, causes nephropathia epidemica (NE), a disease characterized by acute kidney injury and thrombocytopenia. To determine the clinical phenotype of hantavirus-infected patients and their long-term outcome and humoral immunity to PUUV, we conducted a cross-sectional prospective survey of 456 patients in Germany with clinically and serologically confirmed hantavirus-associated NE during 2001-2012. Prominent clinical findings during acute NE were fever and back/limb pain, and 88% of the patients had acute kidney injury. At follow-up (7-35 mo), all patients had detectable hantavirus-specific IgG; 8.5% had persistent IgM; 25% had hematuria; 23% had hypertension (new diagnosis for 67%); and 7% had proteinuria. NE-associated hypertension and proteinuria do not appear to have long-term consequences, but NE-associated hematuria may. All patients in this study had hantavirus-specific IgG up to years after the infection.

Urine soluble urokinase-type plasminogen activator receptor levels correlate with proteinuria in Puumala hantavirus infection.

OBJECTIVES: Urokinase-type plasminogen activator receptor (uPAR) is upregulated during inflammation and known to bind to ß3 -integrins, receptors used by pathogenic hantaviruses to enter endothelial cells. It has been proposed that soluble uPAR (suPAR) is a circulating factor that causes focal segmental glomerulosclerosis and proteinuria by activating ß3 -integrin in kidney podocytes. Proteinuria is also a characteristic feature of hantavirus infections. The aim of this study was to evaluate the relation between urine suPAR levels and disease severity in acute Puumala hantavirus (PUUV) infection. DESIGN: A single-centre, prospective cohort study. SUBJECTS AND METHODS: Urinary suPAR levels were measured twice during the acute phase and once during convalescence in 36 patients with serologically confirmed PUUV infection. Fractional excretion of suPAR (FE suPAR) and of albumin (FE alb) was calculated. RESULTS: The FE suPAR was significantly elevated during the acute phase of PUUV infection compared to the convalescent phase (median 3.2%, range 0.8-52.0%, vs. median 1.9%, range 1.0-5.8%, P = 0.005). Maximum FE suPAR was correlated markedly with maximum FE alb (r = 0.812, P < 0.001) and with several other variables that reflect disease severity. There was a positive correlation with the length of hospitalization (r = 0.455, P = 0.009) and maximum plasma creatinine level (r = 0.780, P < 0.001) and an inverse correlation with minimum urinary output (r = -0.411, P = 0.030). There was no correlation between FE suPAR and plasma suPAR (r = 0.180, P = 0.324). CONCLUSION: Urinary suPAR is markedly increased during acute PUUV infection and is correlated with proteinuria. High urine suPAR level may reflect local production of suPAR in the kidney during the acute infection.

Urinary genome detection and tracking of Hantaan virus from hemorrhagic fever with renal syndrome patients using multiplex PCR-based next-generation sequencing.

BACKGROUND: Hantavirus infection occurs through the inhalation of aerosolized excreta, including urine, feces, and saliva of infected rodents. The presence of Hantaan virus (HTNV) RNA or infectious particles in urine specimens of patient with hemorrhagic fever with renal syndrome (HFRS) remains to be investigated. METHODOLOGY/PRINCIPAL FINDINGS: We collected four urine and serum specimens of Republic of Korea Army (ROKA) patients with HFRS. We performed multiplex PCR-based next-generation sequencing (NGS) to obtain the genome sequences of clinical HTNV in urine specimens containing ultra-low amounts of viral genomes. The epidemiological and phylogenetic analyses of HTNV demonstrated geographically homogenous clustering with those in Apodemus agrarius captured in highly endemic areas, indicating that phylogeographic tracing of HTNV genomes reveals the potential infection sites of patients with HFRS. Genetic exchange analyses showed a genetic configuration compatible with HTNV L segment exchange in nature. CONCLUSION/SIGNIFICANCE: Our results suggest that whole or partial genome sequences of HTNV from the urine enabled to track the putative infection sites of patients with HFRS by phylogeographically linking to the zoonotic HTNV from the reservoir host captured at endemic regions. This report raises awareness among physicians for the presence of HTNV in the urine of patients with HFRS.

Cystatin C, a novel urinary biomarker for sensitive detection of acute kidney injury during haemorrhagic fever with renal syndrome.

To explore the value of cystatin C for evaluating acute kidney injury (AKI) in haemorrhagic fever with renal syndrome (HFRS), the concentrations of cystatin C in serum and urine samples from HFRS patients were determined. The serum and urinary cystatin C concentrations significantly increased in HFRS patients compared with normal controls (p < 0.001). In the acute phase of HFRS, urinary cystatin C increased to higher levels than serum creatinine, especially in severe or critical cases in the oliguric stage. Furthermore, higher levels of urinary cystatin C in the acute phase positively correlated with increased severity of the subsequent kidney injury. In conclusion, urinary cystatin C is a more sensitive clinical marker for AKI in HFRS, which may enable us to initiate treatment measures as early as possible.

Glomerular filtration barrier dysfunction in a self-limiting, RNA virus-induced glomerulopathy resembles findings in idiopathic nephrotic syndromes.

Podocyte injury has recently been described as unifying feature in idiopathic nephrotic syndromes (INS). Puumala hantavirus (PUUV) infection represents a unique RNA virus-induced renal disease with significant proteinuria. The underlying pathomechanism is unclear. We hypothesized that PUUV infection results in podocyte injury, similar to findings in INS. We therefore analyzed standard markers of glomerular proteinuria (e.g. immunoglobulin G [IgG]), urinary nephrin excretion (podocyte injury) and serum levels of the soluble urokinase plasminogen activator receptor (suPAR), a proposed pathomechanically involved molecule in INS, in PUUV-infected patients. Hantavirus patients showed significantly increased urinary nephrin, IgG and serum suPAR concentrations compared to healthy controls. Nephrin and IgG levels were significantly higher in patients with severe proteinuria than with mild proteinuria, and nephrin correlated strongly with biomarkers of glomerular proteinuria over time. Congruently, electron microcopy analyses showed a focal podocyte foot process effacement. suPAR correlated significantly with urinary nephrin, IgG and albumin levels, suggesting suPAR as a pathophysiological mediator in podocyte dysfunction. In contrast to INS, proteinuria recovered autonomously in hantavirus patients. This study reveals podocyte injury as main cause of proteinuria in hantavirus patients. A better understanding of the regenerative nature of hantavirus-induced glomerulopathy may generate new therapeutic approaches for INS.

Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection.

Rapid point-of-care testing is a megatrend in infectious disease diagnosis. We have introduced a homogeneous immunoassay concept, which is based on the simultaneous binding of antigen and protein L to a given immunoglobulin molecule. The complex formation is detected utilizing time-resolved Förster resonance energy transfer between antigen-attached donor and acceptor-labeled protein L, hence the name LFRET. Here, we demonstrate that urine can be used as a sample matrix in LFRET-based serodiagnostics. We studied urine samples collected during the hospitalization and recovery of patients with acute Puumala orthohantavirus (PUUV) infection. We compared PUUV antibody-specific LFRET signals in urine to those in plasma, and found excellent correlation in the test outcomes The LFRET test from urine was positive in 40/40 patients with acute PUUV infection. PUUV causes a mild form of hemorrhagic fever with renal syndrome, characterized by acute kidney injury and proteinuria. Immunofluorescence and western blotting demonstrated PUUV-IgG and -IgA in urine, however, the presence of intact immunoglobulins did not fully explain the LFRET signals. We purified free light chains (FLCs) from both urine and serum of healthy volunteers and patients with acute PUUV infection, and verified the presence of antigen-specific FLCs. Antigen-specific FLCs provide a new means for non-invasive antibody detection and disease diagnosis.

[Long-term outcomes and formation of chronic disease of the kidneys in patients with a history of hemorrhagic fever with renal syndrome].

AIM: To evaluate renal function, persistence of renal dysfunction and probability of chronic renal pathology in convalescents of hemorrhagic fever with renal syndrome (HFRS). MATERIAL AND METHODS: A total of 370 HFRS convalescents were examined with estimation of renal functional reserve, albuminuria, uric acid clearance, activity of urine N-acetil-beta-D-hexosaminidase in the urine, 18-h deprevation test, duplex scanning of renal vessels. Correlation between prevalence of chronic renal failure in Udmurtia and HFRS incidence was analysed. RESULTS: Glomerular and tubular dysfunctions in HFRS convalescents (intraglomerular hypertension, albuminuria, regress of a concentration ability of the kidneys, impairment of tubular transport) are characterized by persistence in the presence of renal hypoperfusion and hypoexcretory hyperuricemia. 13% convalescents developed chronic disease of the kidneys (CDK) which clinically presented as chronic tubulointerstitial nephritis. HFRS may contribute to formation of population of patients with chronic renal failure in the territory of active natural foci. A significant positive correlation was registered between mean annual levels of HFRS morbidity and prevalence of chronic renal failure in different regions of Udmurtia. According to clinical data, chronic renal failure develops in patients who earlier have suffered from renal disease. CONCLUSION: Persistance of renal dysfunctions in HFRS convalescents and possible onset of chronic disease of the kidneys necessitate active follow-up of the disease convalescents.

Urinary Clusterin Is Upregulated in Nephropathia Epidemica.

Kidney insufficiency is a hallmark of nephropathia epidemica (NE). Little is known about the mechanisms of the NE kidney pathology, with current knowledge mainly based on findings in postmortem tissue. We have analyzed kidney damage biomarkers in urine collected from early- and late-phase NE using Bio-Plex kidney toxicity panels 1 and 2. To determine the disease specificity, kidney damage biomarkers were also analyzed in urine samples from patients diagnosed with gout, type 2 diabetes, systemic lupus erythematosus, and chronic kidney insufficiency. Analysis of 12 biomarkers suggests damage to the kidney proximal tubule at the onset of NE. Also, upregulation of biomarkers of inflammation and leukocyte chemotaxis were detected in NE urine. Furthermore, increased clusterin levels were found in early- and late-phase NE urine. Comparative analysis revealed that clusterin is a biomarker, upregulated in NE urine.

Severe hantavirus disease in children.

BACKGROUND: Very recently, a novel European hantavirus, Sochi virus, has been discovered which causes severe courses of hantavirus disease with a case fatality rate of about 15 percent. OBJECTIVES: We aimed to study to which extent and with which clinical severity children were affected by Sochi virus infection. STUDY DESIGN: Sochi virus infection of patients was confirmed by molecular, serological, and epizoonotic studies. Clinical and laboratory parameters were analyzed for the age group of up to 15 years (n = 6) in comparison to all older patients (n = 56). RESULTS: 9.7 percent of patients with hantavirus disease studied (6/62) were up to 15 years old. The children showed moderate to severe clinical courses similarly to the situation in adults. CONCLUSIONS: While children are in general considered to be less affected by hantavirus infections than adults, in case of highly pathogenic hantaviruses, such as Sochi virus, frequency of clinical cases as well as their clinical course are comparable between children and adults. Therefore, hantavirus disease, particularly in regions endemic to highly pathogenic hantaviruses, should be considered in cases of unclear fever and kidney/pulmonary failure in children.

Relationship between circulating vascular endothelial growth factor and its soluble receptor in patients with hemorrhagic fever with renal syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is characterized by endothelial dysfunction with capillary leakage without obvious cytopathology in the capillary endothelium. The aim of the study was to analyze the kinetics of vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-2) in HFRS patients infected with Dobrava (DOBV) or Puumala virus (PUUV). VEGF and sVEGFR-2 levels were measured in daily plasma and urine samples of 73 patients with HFRS (58 with PUUV, 15 with DOBV) and evaluated in relation to clinical and laboratory variables. In comparison with the healthy controls, initial samples (obtained in the first week of illness) from patients with HFRS had higher plasma and urine VEGF levels, whereas sVEGFR-2 levels were lower in plasma but higher in urine. VEGF levels did not differ in relation to hantavirus species, viral load, or the severity of HFRS. The comparison of VEGF dynamics in plasma and urine showed the pronounced secretion of VEGF in urine. Significant correlations were found between daily VEGF/sVEGFR-2 levels and platelet counts, as well as with diuresis: the correlations were positive for plasma VEGF/sVEGFR-2 levels and negative for urine levels. In addition, patients with hemorrhagic manifestations had very high plasma and urine VEGF, together with high urine sVEGFR-2. Measuring the local secretion of sVEGFR-2 in urine might be a useful biomarker for identifying HFRS patients who will progress to severe disease.

Haematuria is a marker for the severity of acute kidney injury but does not associate with thrombocytopenia in acute Puumala hantavirus infection.

BACKGROUND: Puumala hantavirus (PUUV) causes haemorrhagic fever with renal syndrome characterized by thrombocytopenia, capillary leakage and acute kidney injury (AKI) with proteinuria and haematuria. Although the typical histologic lesion is acute tubulointerstitial nephritis, the amount of glomerular proteinuria predicts the severity of upcoming AKI. Here, we studied the associations of haematuria and proteinuria with the severity of emerging AKI, thrombocytopenia and markers of coagulation and fibrinolysis in PUUV infection. METHODS: We examined 205 consecutive patients treated for serologically confirmed acute PUUV infection at Tampere University Hospital during 1997-2014. The patients were divided into three groups according to the combined positive result in urine haemoglobin and albumin dipstick tests: 0-2 + (n = 58), 3-4 + (n = 100) and 5-6 + (n = 47). RESULTS: The medians of maximum creatinine concentrations in the three groups were: 0-2 + 100 µmol/L (range 52-1499), 3-4 + 204 µmol/L (range 65-1071) and 5-6 + 361 µmol/l (range 51-1285) (p < .001). The number of blood platelets (p = .069), and the levels of fibrinogen, prothrombin fragments F1 + 2 and d-dimer (p = .602, p = .113, p = .289, respectively) were not significantly different between the groups. When the amount of haematuria in the dipstick test was examined separately, no association with thrombocytopenia was detected (p = .307 between groups 0, 1+ and 2-3+). CONCLUSIONS: Combined positive result of haematuria and proteinuria in the dipstick test at hospital admission predicted the severity of upcoming AKI in acute PUUV infection. As haematuria was not associated with the severity of thrombocytopenia, it did not indicate increased bleeding tendency, but was rather a marker of acute kidney injury.

Urinary excretion of interleukin-6 correlates with proteinuria in acute Puumala hantavirus-induced nephritis.

BACKGROUND: Nephropathia epidemica (NE) is a mild type of hemorrhagic fever with renal syndrome caused by Puumala Hantavirus. Cytokines are thought to have an important role in the pathogenesis of NE. The aim of this study is to evaluate whether cytokines contribute to renal involvement in NE. METHODS: Overnight urinary excretion of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), IL-6, tumor necrosis factor-alpha (TNF-alpha), albumin, immunoglobulin G (IgG), and alpha1-microglobulin and quantitative 24-hour urinary protein excretion were measured for 3 consecutive days from 70 hospitalized patients with acute NE (49 men, 21 women; age, 15 to 70 years; median age, 39 years). Plasma levels of the respective cytokines also were measured. Urinary collections were repeated after 1 year. The control group for blood samples included 400 healthy blood donors. RESULTS: Maximum median urinary IL-6 excretion in the acute phase of NE was increased compared with values detected after 1 year (49.5 versus 0.7 pg/min; P < 0.001). Correspondingly, maximum median plasma IL-6 concentration in patients was increased compared with controls (14.6 versus 1.2 pg/mL; P < 0.001). Urinary IL-6 excretion correlated with urinary albumin, IgG, and protein excretion (r = 0.79; P < 0.001; r = 0.76; P < 0.001; and r = 0.65; P < 0.001, respectively), but not plasma IL-6 levels (r = 0.18; P = 0.148). CONCLUSION: Plasma IL-6 concentrations and urinary IL-6 excretion were markedly increased in patients with acute NE, but there was no correlation between plasma and urinary IL-6 levels. The high urinary IL-6 levels might reflect local production of this proinflammatory cytokine in the kidneys during acute infection.

Nephropathia epidemica (hemorrhagic fever with renal syndrome) in children: clinical characteristics.

The clinical characteristics of serologically verified nephropathia epidemica, the Scandinavian form of hemorrhagic fever with renal syndrome, were studied in Swedish children who were < 15 years of age. In 1990 to 1992, 14 cases were prospectively followed. A retrospective survey during 1984 to 1990 disclosed another 18 cases. Among the 32 cases (20 boys, 12 girls, 3 to 15 years of age; median age, 11 years), the most common symptoms were fever (100%), headache (100%), abdominal pain (93%), vomiting (91%) and back pain (76%). Laboratory findings included elevated serum creatinine concentration (19 of 28) and thrombocytopenia (7 of 22). Urinalysis showed proteinuria (31 of 31 patients) and hematuria (24 of 30). Six children had mild hemorrhagic manifestations (epistaxis, metrorrhagia, and petechiae). No severe complications occurred. The clinical symptoms of children with nephropathia epidemica seem to be similar to those found among adult nephropathia epidemica cases.

Isolation of a strain of a Hantaan virus from a fatal case of hemorrhagic fever with renal syndrome in Slovenia.

Hemorrhagic fever with renal syndrome (HFRS) has been serologically confirmed in Slovenia during the last seven years. There is evidence that three hantaviruses (Hantaan, Puumala, and a newly described form termed Dobrava) circulate simultaneously in this area. Recently, a hantavirus was isolated from the urine and brain tissue of a fatal case of HFRS. Positive immunofluorescent reactions with reference human sera and monoclonal antibodies were first recognized after the second cell culture passage. Extensive cross-reactivity between our isolate and prototype Hantaan virus, strain 76-118, and Hantaan-like isolates from the former Yugoslavia, Fojnica and Plitvice, was revealed by enzyme-linked immunosorbent assay with specific rat antisera. The reaction pattern of the isolate was similar to the prototype Hantaan virus by indirect immunofluorescent assay with a panel of monoclonal antibodies. Furthermore, the specificity of the isolates was confirmed by analysis of polymerase chain reaction products of this virus with five restriction endonucleases. This appears to be the first isolation of a strain of prototype Hantaan virus from a fatal case of HFRS in Europe.

Delayed onset of diuresis in a patient with acute renal failure due to hemorrhagic fever with renal syndrome who also developed anterior hypopituitarism.

A 23-year-old man developed acute renal failure (ARF) due to hemorrhagic fever with renal syndrome (HFRS). The patient also developed anterior hypopituitarism as a complication of HFRS. The patient's oliguric phase was very much prolonged for over 10 days before the diuresis began. The urine output during the oliguric phase was near anuric (< 50 ml/day). Interestingly, the patient began to diurese just after the institution of glucocorticoid and thyroid hormone replacement therapy. The plasma atrial natriuretic polypeptide went up to a smaller peak (150.0 pg/ml) at the onset of diuresis compared with 15 other patients (292.4 +/- 190.4 pg/ml) who did not develop anterior hypopituitarism. The delayed onset of diuresis and smaller increase of plasma ANP may have a causal relationship with the patient's hypopituitarism.

[Using urinary diagnostic indices to evaluate acute renal failure in epidemic hemorrhagic fever].

The urinary diagnostic indices were used to evaluate acute renal failure in 118 cases with epidemic hemorrhagic fever (EHF). When the renal failure was mainly caused by acute tubular necrosis, it would occur earlier and persist longer. The authors suggested that the indices are helpful in documenting the severity and identifying the characteristics of acute renal failure in EHF. The mechanisms for developing acute renal failure in EHF was discussed.

[Structural protein of hemorrhagic fever renal syndrome virus in uric fusion cells in hemorrhagic fever renal syndrome].

The efoliative cells in the urine of 21 hemorrhagic fever patients with renal syndrome (HFRS) were examined with Swiss blue staining. Fusion cells were found in the urine of 18 HFRS patients. The quantity of fusion cells was closely related to the severity of patients' condition and renal damage. Specific antigen of HFRS virus (HFRSV) was found immunohistochemically in all fusion cells. Specific monoclonal antibodies (McAb) of membrane protein and nucleocapsid protein from HFRSV were used for analyzing antigen in fusion cells. The positive rate of membrane protein antigen was 72.2%. Nucleocapsid protein was not found in all fusion cells. The above results suggested that the forming of fusion cells in the urine of HFRS patients is related membrane protein of HFRSV.

[The detection of antibodies to the hantaan virus in the urine of patients with hemorrhagic fever with renal syndrome]. / Obnaruzhenie antitel k virusu khantaan v moche bol'nykh gemorragicheskoi likhoradkoi s pochechnym sindromom.

Using a commercial diagnostic preparation for indirect IFA test, 135 urine samples from 50 patients with HFRS were examined at different periods of the disease. Antibodies were demonstrable in all urine specimens from HFRS patients for 13 days. In 14-20 days they could be detected in half of the patients, and no antibodies could be demonstrated since the 21st day on. The results of urine examination from healthy subjects and some patients with other clinical diagnoses were negative same as controls with normal antigen. The dynamics of antibody titres in the patients' urine differed from that in the blood and was considered as "decreasing" similarly as the clinical disease. The antibody excretion in the urine coincided with the period of renal structure damage and stopped when the normal renal function was restored. The data are discussed from the point of view of the pathogenesis and diagnosis. Special attention was paid to the possibility and advantages of early HFRS diagnosis by antibody determinations in the patients' urine.

[Disruption of the balance of amino acids in patients with hemorrhagic fever with kidney syndrome]. / Narushenie balansa aminokislot u bol'nykh gemorragicheskoi likhoradkoi s pochechnym sindromom.

Changes in amino acids in blood, daily urine as well as amino acid clearance were studied in 20 patients with moderate hemorrhagic fever and renal syndrome. The amino acid balance was impaired and the degree of impairment depended on the type of an amino acid and the duration of a disease. Dysaminoacidemia at the peak of the disease is associated with excretion and reverse transport of some amino acids. It is suggested that hyperaminoacidemia of early and late periods of the disease is compensatory.