Genetic detection of Dobrava-Belgrade hantavirus in the edible dormouse (Glis glis) in central Serbia.

Hantaviruses are endemic in the Balkans, particularly in Serbia, where sporadic cases and/or outbreaks of hantaviral human disease have been reported repeatedly, and evidenced serologically. Here, we present genetic detection of Dobrava-Belgrade virus (DOBV) hantaviral sequences in wild rodents trapped in central Serbia. All the animals were pre-screened serologically by indirect immunofluorescence (IF) test and only those with a positive finding of hantaviral antigens were further tested by polymerase chain reaction. Of the total of 104 trapped animals, 20 were found to be IF positive and of those three were positive for hantaviral RNA: one Microtus arvalis for Tula virus, and one each of Apodemus agrarius and Glis glis for DOBV. Phylogenetic analysis of the obtained sequences implies putative DOBV spillover infection of A. agrarius and G. glis from Apodemus flavicollis. However, future investigations should help to identify the most common natural host and geographical distribution of DOBV in its reservoir hosts in Serbia.

The effect of parathyroidectomy on insulin sensitivity in patients with primary hyperparathyroidism - an never ending story?

Previous studies demonstrated insulin resistance and increased prevalence of impaired glucose tolerance and type 2 diabetes mellitus in patients with primary hyperparathyroidism (PHPT). The effect of curative parathyroidectomy on insulin sensitivity was associated with conflicting results depending on which method for measuring the insulin sensitivity has been used. There was no improvement using HOMA and QUICKI while minimal model demonstrated significant improvement in insulin sensitivity. The aim of our study was to evaluate the insulin sensitivity before and after parathyroidectomy in patients with PHPT using a euglycemic clamp. 44 patients with PHPT and 11 age and body mass index matched healthy controls participated in study protocol. Before surgery M values and HOMA IR suggest insulin resistance in patients with PHPT. There was no difference in M index (3.74±1.89 vs. 4.62±2.27, p>0.05), HOMA IR (2.94±1.39 vs. 3.29±0.81, p>0.05), AUC glucose (863.0±261.3 vs. 842.3±165.5, p>0.05), AUC insulin (7068.7±4159.0 vs. 7229.6±2581.7, p>0.05), ISI (4.73±2.77 vs. 4.25±2.94, p>0.05) and AIR (47.89±32.05 vs. 38.96±21.20, p>0.05) between patients with PHPT and HC. There was significant improvement in insulin sensitivity after parathyroidectomy but both preoperative and postoperative M values were not significantly different in comparison to HC. There were no significant changes in HOMA IR, AUC glucose, AUC insulin, ISI and AIR before and after therapy. In conclusion, we observed significant improvement in insulin sensitivity after parathyroidectomy in patients with PHPT. There was no difference in parameters of insulin secretion before and after parathyroidectomy in patients with PHPT.

Epidemic hemorrhagic fever with renal syndrome in Yugoslavia, 1986.

An epidemic of hemorrhagic fever with renal syndrome (HFRS) occurred in Yugoslavia May-November 1986; all Republics and Provinces were involved. Serum samples were received from 260 of 276 persons with symptoms clinically compatible with a diagnosis of HFRS. Presumptive infection with a hantavirus was determined serologically for 161 of these. Many patients with serious clinical pictures, including severe renal insufficiency and shock, were hospitalized; 11 died. Indirect fluorescent antibody tests with antigens of 4 hantaviruses (Hantaan, Fojnica, Puumala, and the Vranica strain of Puumala virus) showed that greater than 1 serotype was circulating during this epidemic. Hantavirus antigens were detected in the lungs of 86 of 302 (28.5%) wild-caught small mammals.

Hemorrhagic fever with renal syndrome in Yugoslavia: antigenic characterization of hantaviruses isolated from Apodemus flavicollis and Clethrionomys glareolus.

Hantavirus antigens were detected in lung tissues of 8/113 Apodemus flavicollis and 2/17 Clethrionomys glareolus captured in 1984 in Fojnica, a region of Yugoslavia endemic for hemorrhagic fever with renal syndrome; hantavirus antigens were not detected in lung tissues from 126 other mammals collected in Fojnica. Three hantaviruses, 2 from A. flavicollis and 1 from C. glareolus, were isolated directly in Vero E6 cells and were partially characterized. The isolates from A. flavicollis, designated Fojnica virus, were antigenically similar but not identical to Hantaan virus strain 76-118, whereas the isolate from C. glareolus was antigenically indistinguishable from Puumala virus, strain Hällnäs B1. These data are consistent with previous studies that indicate the existence of at least 2 hantavirus serotypes in Yugoslavia.

Different pathohistological presentations of acute renal involvement in Hantaan virus infection: report of two cases.

We present two patients with Hantaan virus infection, admitted to the Department of Nephrology, Skopje, at the same time, with the same clinical presentation (chills, fever, abdominal pain, hemorrhages, nausea, headache, proteinuria, hematuria, oliguria, acute renal failure) but with different pathohistological findings and different disease courses. In the first case diffuse proliferative glomerulonephritis was found, with a complete recovery of renal function after a month, with a mild proteinuria and erythruria during the second and the third month. In the second case, glomeruli were normal in general, with slight mesangial proliferation found in two out of twenty, but interstitial edema, lymphocyte infiltrations and tubular changes were noted. Complete recovery was not noted after 3 months of follow-up. The patient is now without hemodialysis treatment, with polyuria, in the stable phase of chronic renal failure which is not improving.

Evidence for Hantavirus disease in Slovenia, Yugoslavia.

In Slovenia, North-Western part of Yugoslavia, 17 clinically documented Hantavirus disease cases (HVD) were serologically confirmed so far. Previously HVD was reported in the Southern part of Yugoslavia. By the indirect fluorescent antibody test (IFA), the prevalence of IgG class antibodies against different Hantaviral antigens was demonstrated in human sera collected in Slovenia. Three different reactivity patterns were observed. Majority of the IFA-positive human sera were confirmed by the immunoblot method. The distribution of Hantaviral infections was examined in small mammals captured in two natural foci of HVD, where clinical documented cases were reported. Hantaviral antibodies and antigens were demonstrated in C. glareolus, A. flavicollis, A. sylvaticus, and M. musculus.

[Clinical and epidemiologic characteristics of hemorrhagic fever with renal syndrome]. / Klinicke i epidemioloske znacajke hemoragijske groznice s bubreznim sindromom (HGBS).

Clinical and epidemiological data on 16 patients with HFRS admitted to the University Hospital of Infectious Diseases in Zagreb during the past 10 years (1977-1986) are reported. In 13 of them, the diagnosis was confirmed serologically by indirect fluorescent method. All but two were men between 20 and 45 years of age (80% of patients). The disease appeared sporadically only. According to the domicile, way of living and working all the patients but three mentioned the contact with rodents in the fields or in other places (mill, storehouse). Three patients live in Zagreb and there was no possibility to get infected out of the town. The disease had moderate course, more rarely severe course when shock and acute renal failure developed. General symptoms with fever and algias dominated (febrile stage). Some of the patients had ophthalmic disturbances. Patients with a pronounced gastroenteritis developed shock the most frequently. In some of the patients back and abdominal pains followed by obstipation appeared. HFRS was incriminated disease because of febrile stage followed by the acute renal failure, oliguria, azotemia, polyuria and low urine osmolity. One female patient with active rheumatoid arthritis died after two-week staying in hospital due to irreversible shock followed by cardiopulmonary and renal failure.

[Hemorrhagic fever with renal syndrome--etiology]. / Hemoragicna groznica sa bubreznim sindromom (HGBS)--etiologija.

HFRS was confirmed serologically in two patients, who were infected in the natural focus near Zupanja. Using the bigger number of virus antigens in indirect immunofluorescent antibody test, afterwards was proved the agent could be classified into the serotype 3 of Hantaviruses, and antigenically is identical with virus NE (strain Hällnäs), respectively Yugoslav isolate "Vranica". Serotype differentiation of the agent in patient who were infected in the area of Igman, near Sarajevo, has not been performed by the same test. There was assumed a possibility for circulation of "new", up to now non-identified, serovariant of Hantaviruses in this natural focus.

[Serologic diagnosis of Crimean hemorrhagic fever in Kosovo and Metohia]. / Seroloska dijagnostika Krimske hemoragijske groznice na Kosovu i Metohiji.

In the period June-November 1995, 292 sera from 159 patients and 80 healthy persons from Kosovo and Metohia were tested for the presence of antibodies against the causative agents of Crimean hemorrhagic fever (CHF) and hemorrhagic fever with renal syndrome (HFRS). Diffusion precipitation test in agar gel and indirect immunofluorescence assay were used. Specific antibodies against CHF virus were found in 6.9% and against HFRS virus in 15.1 patient's sera. Antibodies against CHF virus were proved in sera of patients on the fifth day from the disease onset at the earliest. No wanted antibodies were found in healthy persons' sera. The results of sera testing on antibodies against CHF virus were analyzed in detail.

[Belgrade and Hantaan hantaviruses--the causative agents of haemorrhagic fever with renal syndrome in children in Serbia]. / Hantavirusi Beograd i Hantan--uzrocnici hemoragijske groznice s bubreznim sindromom kod dece u Srbiji.

During an outbreak of haemorrhagic fever with renal syndrome (HFRS) in 1989, five children (3 girls, 2 boys, aged 6.8-16 years) with severe clinical form of the disease, were treated at the Institute of Mother and Child Health of Serbia; four of them were followed-up 22-28 months thereafter. The main clinical features were: fever, headache, myalgia, abdominal and back pains, and vomiting in all, and haemorrhagic syndrome in three; renal syndrome with severe acute renal failure in all five patients. All the patients recovered. Serological confirmation by an indirect immunofluorescence assay, enzyme immunoassay for IgM antibodies, and by plaque reduction neutralization test revealed that the infection was caused by Belgrade virus in three, and by Hantaan virus in two patients. It was not possible to differentiate these two serogroups on the basis of clinical features. This finding gave further evidence of circulation of different hantaviruses causing severe HFRS in Serbia.

Belgrade and Hantaan hantaviruses--the causative agents of severe haemorrhagic fever with renal syndrome in children in Serbia.

During an outbreak of haemorrhagic fever with renal syndrome (HFRS) in 1989, five children (3 girls, 2 boys, aged 6.8-16 years) with a severe form of the disease were treated; four of these were followed for 22-28 months. The main clinical features in all five patients were: fever, headache, myalgia, abdominal and back pain and vomiting; haemorrhagic syndrome was present in four children. Renal syndrome presented with severe acute renal failure in all five patients. All patients recovered. Serological confirmation by an indirect immunofluorescence assay, by enzyme immunoassay for IgM antibodies and by plaque reduction neutralization test showed infection by Belgrade virus in three and by Hantaan virus in two patients. It was not possible to differentiate these two serogroups on the basis of clinical features. This study provides further information on the circulation of different hantaviruses causing severe HFRS in Serbia.

Clinical characteristics of haemorrhagic fever with renal syndrome in children.

From January 1988 to September 1989, seven patients (4 girls and 3 boys, aged 3-12 years) with haemorrhagic fever with renal syndrome (HFRS) were hospitalised at the University Children's Hospital in Belgrade. In four patients the disease appeared as a family outbreak, the others were sporadic cases. In six patients the clinical presentation was suggestive of HFRS, as they had fever with headache, myalgia, sore throat and gastrointestinal illness followed by renal abnormalities. However, severe haemorrhagic syndrome with petechia, haematoma, haematemesis and melaena was present in one patient only. Renal disease presented as nephritic syndrome and/or acute renal failure. Five patients recovered after 2-3 weeks without sequellae, one patient had decreased renal function 17 months after the start of the disease and the remaining patient died. In six patients the diagnosis of HFRS was confirmed serologically by a significant rise in antibody titres against hantaviruses, while in the patient with the fetal and fulminant course of the disease, the diagnosis was established on the basis of epidemiological and autopsy findings. We suggest that children living in endemic areas who develop an ill-defined, febrile and gastrointestinal disease with renal dysfunction should be evaluated for HFRS.

[Hemorrhagic fever with renal syndrome in children]. / Hemoragijska groznica s bubreznim sindromom kod dece.

Seven patients, 4 girls and 3 boys, aged 3 to 12 years /X = 7.14/ affected by haemorrhagic fever with renal syndrome /HFRS/., were hospitalized at the University Children's Hospital in Belgrade during the last two years /January 1988-January 1990/. The diagnosis was established on the basis of clinical features, epidemiological data and autopsy findings in one patient while in the others the diagnosis of HFRS was confirmed serologically by indirect immunoflorescence tests on Vero E 6 cells. A significant increase in antibody titre against Hantaan virus was found in all serologically tested patients. Three of them had also significant increase of antibody titre against Soeul and one against Puumale virus. In four patients the disease appeared as family outbreak at the end of January 1988 while the others were sporadical cases. All patients but one mentioned contact with rodents at home or in fields. The predominant slynical symptom were: sudden onset of febrile condition with headache, generalized malaise, myalgia, abdominal pain, vomiting, diarrhoea, oliguria and oedema. All patients had haematuria and only one had other severe haemorrhagic manifestations. Four patients were hypertensive. Two patients had renal insufficiency, but only one required haemodialysis. Five patients recovered after 2 to 8 weeks without sequellae, one patient was still /7 months after the beginning of the disease/ in mild renal insufficiency and one patient died. Autopsy findings showed tubular necrosis in the kidney, myocarditis, massive pneumonia with hydrothorax and jejunal haemorrhagia.

[Acute renal insufficiency in hemorrhagic fever with renal syndrome]. / Akutna bubrezna insuficijencija kod hemoragijske groznice sa renalnim sindromom.

In the Clinic for Nephrology of Military Medical Academy, 116 patients with hemorrhagic fever with renal syndrome (HFRS) have been treated in the period from January 1989 to December 1995. The patients were hospitalized with advanced acute renal failure (ARF). The largest number of our patients got ill from HFRS from the middle of March till the end of May and during August when the other frequency elevation was noticed. Most frequently the disease has the course that is followed up by phases. Positive finding of antibodies to HFRS virus was noticed in all 116 patients (Hantaan 45%, Belgrade 35%, Puumala 20%). According to clinical symptoms, signs and syndromes that follow up HFRS, the most of our patients were with more severe disease type. The ARF treatment with dialysis was necessary in 45% our patients. The most severe clinical forms were caused by Belgrade strain. Very favorable effect of "an early dialysis" to the disease course and outcome was observed. By this approach in HFRS therapy, the patient's mortality was decreased to 0.86%. The development of chronic renal failure as HFRS sequela is possible, but not so frequent.

[Hemorrhagic fever with renal syndrome in Yugoslavia 1951-1988]. / Hemoragicna groznica sa bubreznim sindromom u SFRJ u periodu od 1951. do 1988.

Data on hemorrhagic fever with renal syndrome (HFRS) incidence in SFRY 1950-1988 were analysed. Information sources were published papers and official reports of Federal and Republic Institutions for Public Health. Indirect immunofluorescence technique was used in testing 1.842 organs of small wild mammals to the presence of HFRS viral antigen. This antigen was found in the lungs of 11 species. Average incidence of the carrier state was 10.4% Registered were 613 cases within the period 1951-1988. Morbidity rate was 0.05-10.6:1.00.000 for years. Disease have been registered during the whole year with maximum incidence rate in summer months. Men most capable of working were the most frequently affected: farmers, wood workers, soldiers. Average lethality was 5.2%.

[Epidemiologic characteristics of hemorrhagic fever with renal syndrome in a military population]. / Epidemioloske karakteristike hemoragicne groznice sa bubreznim sindromom u vojnickom kolektivu.

In the period 1952-1990 there have been recorded 84 patients with hemorrhagic fever with renal syndrome (HFRS): 81 soldiers and 3 officers of the Y.P.A. The largest number of cases was recorded in three epidemics, 61 or 72.6%. In 94% of cases the infection occurred during camping of units. The disease appeared in all months, but 57.2% of cases occurred in June and July. The mean lethality was 2.4%, in epidemics 1.6% and as sporadic cases 4.3%. In an army unit staying for six months in HFRS focus, 9.8% of soldiers were infected by the causative agent of this infection and only in one case the clinical picture of HFRS was manifested. Serologic tests (IIF and ELISA) confirmed the diagnosis of HFRS. Virus strains of Hantaan, Puumala and Seoul groups were used as antigens.

Characterization of Dobrava virus: a Hantavirus from Slovenia, Yugoslavia.

Small mammals were collected in natural foci of hemorrhagic fever with renal syndrome (HFRS) in Slovenia, Yugoslavia, and a hantavirus was isolated from the lungs of an Apodemus flavicol lis captured in Dobrava village. This new isolate, Dobrava virus, was compared with representative strains of the Hantavirus genus by serological and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. It was found by cross immunofluorescent and enzyme-linked immunosorbent assays that antigenic properties of Dobrava virus were different from those of other hantaviruses. The RNA of this virus was successfully amplified with hantavirus genus reactive primer sets by reverse transcriptase polymerase chain reaction (RT-PCR); however, PCR-RFLP analysis of the amplified product was shown to be unique among those of the known hantaviruses, further indicating that Dobrava virus represents a new hantavirus serotype.

Hemorrhagic fever with renal syndrome in Yugoslavia: detection of hantaviral antigen and antibody in wild rodents and serological diagnosis of human disease.

Lung tissues from 547 rodents and 26 insectivores captured between 1981 and 1984 in central Bosnia (Fojnica) and central Serbia (Cacak), 2 regions known to be endemic for hemorrhagic fever with renal syndrome (HFRS), were examined for hantaviral antigen by the indirect immunofluorescent antibody technique. Antigen was detected in 17/231 Apodemus flavicollis, 3/187 A. sylvaticus, 1/46 A. agrarius, 4/32 Mus musculus, and 3/28 Clethrionomys glareolus. In addition, antibodies against Hantaan and Puumala viruses were found in serum pooled from 2 C. glareolus captured in Fojnica and 6 Pitimys subterraneus caught in Cacak. Sera of 27 HFRS patients from different parts of Yugoslavia were tested against 3 serotypes of hantavirus. Patients from Bosnia and Serbia had highest titers against Hantaan virus, while patients from Croatia had highest titers against Puumula virus, the agent of nephropathia epidemica.

Belgrade virus, a cause of hemorrhagic fever with renal syndrome in the Balkans, is closely related to Dobrava virus of field mice.

Belgrade virus is a recently described hantavirus that causes severe hemorrhagic fever with renal syndrome (HFRS) in people living in various parts of the Balkan Peninsula. Nucleotide sequencing of the G2-encoding region in the medium (M) segment of the viral genome, reverse transcribed and amplified by the polymerase chain reaction, revealed the Belgrade virus to be substantially different from Hantaan virus and other major serotypes of hantavirus but identical to Dobrava virus, a virus isolated from a field mouse (Apodemus flavicollis) in Slovenia. Belgrade virus may be an important cause of HFRS in the Balkan Peninsula, extending north toward the Alps. It poses a special danger to humans who have close contact with field rodents.

Belgrade virus: a new hantavirus causing severe hemorrhagic fever with renal syndrome in Yugoslavia.

Two biologically and genetically distinct hantaviruses were isolated from blood and urine specimens collected from four Yugoslavian patients with clinically severe hemorrhagic fever with renal syndrome (HFRS). Viral isolates from three patients, designated strains Belgrade 1-3, were distinct from Hantaan, Seoul, Puumala, and Prospect Hill viruses as determined by plaque-reduction neutralization tests and restriction analysis of enzymatically amplified M-segment fragments. The fourth isolate, called Kraljevo, was indistinguishable from Hantaan virus. Strains Belgrade 1 and 2, like the Kraljevo strain, caused a fatal meningoencephalitis in newborn mice inoculated with 100 pfu of virus intracerebrally and intraperitoneally. Strain Belgrade 3 was much less neurovirulent, requiring 30,000 pfu of virus to cause fatal disease in mice. These data indicate that two distinct hantaviruses, one of which constitutes a new serotype, cause clinically severe HFRS in Yugoslavia.