Characteristics of Puumala and Dobrava infections in Croatia.

In this study, two different hantaviruses, Puumala virus (PUUV) and Dobrava virus (DOBV), were demonstrated for the first time to coexist and cause hemorrhagic fever with renal syndrome (HFRS) in Croatia. Phylogenetic analysis showed some differences among the nucleotide sequences of PUUV originating from Dinara mountain, which was more closely related to Austrian PUUV than other Croatian PUUV from Mala Kapela mountain. More consistency was found among the Croatian DOBV. HFRS was verified in 85 of 201 suspected cases recorded in 1995 during the largest HFRS outbreak in Croatia. Most of these cases were soldiers. With the exception of the coastal region and islands, all of Croatia was found to be an area endemic for HFRS. A statistically significantly higher proportion of DOBV-infected patients had acute renal failure, visual disturbance, severe thrombocytopenia, and elevated levels of nonsegmented leukocytes, creatine, and total bilirubin. The prevalence of gastrointestinal and electrocardiography disorders also was greater in DOBV-infected patients. Interestingly, significantly more PUUV-infected patients had elevated systolic blood pressure on admission to the hospital. Further prospective studies are necessary to shed more light on differences in HFRS severity associated with PUU and DOB viruses.

Detection of hepatitis C virus RNA in alpha interferon derived from in vitro culture of leukocytes of human origin.

In order to assure the virological safety of blood products, in addition to serological testing of individual donations and virus inactivation steps undertaken during manufacture, routine PCR testing for HCV RNA of starting materials (plasma, cells), intermediates or final product is necessary. The aim of this study was to determine the rate of HCV RNA positive batches of human native leukocyte interferon during large-scale production. Our findings indicate the presence of HCV RNA in 6.1% batches despite acidification of intermediates in order to inactivate Sendai virus.

Incidence of hepatitis C virus RNA in anti-HCV negative plasma pools in Croatia.

The risks of transmitting viral infection by blood and products derived from plasma have long been known and still remain an area of concern. Blood banks and transfusion centres are faced with the imminent introduction of nucleic acid amplification testing (NAT) of plasma pools as used by the plasma industry. In this paper, we show a part of our results of a validation study of an in-house method for routine polymerase chain reaction (PCR) screening for hepatitis C virus (HCV) RNA in plasma pools and the results of testing 2,718 anti-HCV negative plasma pools for the presence of HCV RNA. The European Committee for Proprietary Medical Products (CPMP) recommended that from 1 July 1999, only batches derived from plasma pools tested and found non-reactive for HCV RNA, using validated test methods of suitable sensitivity and specificity, should be batch released by authorities. The quality and efficiency of NAT detection of HCV RNA is among others influenced by the efficacy of RNA isolation, the primer selection and the use of control samples. Using modern molecular biology techniques (sensitive and specific in-house amplification methods for detection of HCV RNA and automated sequencing), we analysed samples of plasma pools from different Croatian transfusion centres. By detection of HCV RNA in an NIBSC working reagent (genotype 3) and a Pelispy HCV RNA run control (genotype 1) we determined a high reproducibility and sensitivity (below 100 International Units (IU)/ml) for our in-house method. By direct sequencing PCR cDNAs we proved the specificity of the test system and the possibility of determining the HCV genotype when the method was used for PCR screening of HCV RNA in single donations. Of 2,718 anti-HCV negative plasma pools we have found that 2.1$ were HCV RNA positive. Results of our investigation confirm the necessity of testing HCV RNA in plasma pools to further increase the safety of human plasma-derived drugs.

Introduction of the gene amplification technique to decrease the risk of hepatitis C virus transmission by plasma products.

The viral safety of plasma-derived products with respect to hepatitis C virus (HCV) is assured by selection of donors, screening of individual donations for antibodies to HCV and the incorporation of effective viral inactivation-removal steps into manufacturing processes. As antibody screening of single donations is not sufficient to completely eliminate HCV RNA positive plasmas from plasma pools, testing for HCV RNA by gene amplification techniques may be necessary to identify positive donations. Using modern molecular biology techniques, we developed a specific, sensitive and reproducible method for routine PCR screening for HCV RNA in plasma pools.

Role of peripheral blood mononuclear cell (PBMC) phenotype changes in the pathogenesis of haemorrhagic fever with renal syndrome (HFRS).

Hantaviruses cause an important human illness, HFRS. Blood samples from 22 HFRS-positive, six seronegative patients and 15 healthy controls were examined in 1995, during the largest HFRS epidemic in Croatia. Results of double- and triple-colour immunofluorescence analysis showed an increased percentage of cytotoxic T cells (CD3+CD8+) in seropositive patients compared with seronegatives and healthy controls. The majority of seropositive HFRS patients expressed activation and memory antigens on T and B lymphocytes. The percentage of CD23+ and CD21+ B lymphocytes was lower in seropositive patients. HFRS patients had elevated levels of sCD23 and five had elevated total IgE. The increased expression of both early and late T cell activation antigens, e.g. CD25, CD71 and HLA-DR, memory cells and sCD23 positively correlated with biochemical parameters (AST, ALT, urea, alpha2-globulin) during the acute phase of HFRS. The phenotypic changes observed, especially early and late T cell activation markers, as well as memory cells, could be useful parameters in the evaluation of HFRS course, and prognostic factors of HFRS severity. Additional attention should be paid to liver involvement in the pathogenesis of HFRS.