S. Epple

Prevalence of HCV-RNA-positive blood donors and correlation to ELISA and RIBA status

SummaryIn Germany, transmission of hepatitis C virus by blood transfusion is prevented by screening the donations for anti-HCV and ALT. The specificity of the anti-HCV screening in low seroprevalence populations has been questioned. In order to evaluate this screening policy we wanted to estimate the prevalence of viremic and potentially infectious donors by the HCV-RNA polymerase chain reaction (PCR) in our donor population of southern Germany. Donors (n=301) were divided into four subgroups according to anti-HCV status and ALT levels. HCV sequences were detected by nested PCR, using primers for the most conserved region of the viral genome. The recombinant immunoblot assay (RIBA-4) was applied to the same samples. PCR detected 4.2% HCV-RNA carriers in the subgroup anti-HCV−/ALT−; 3% in the subgroup anti-HCV−/ALT+; 19.4% in the subgroup anti-HCV+/ALT−; and 59.4% in the subgroup anti-HCV+/ALT+. It was concluded that, on the one hand, the lack of specificity of the anti-HCV ELISA gives rise to many false-positive results; on the other hand, a minority of infected donations will not be detected by the screening procedure. ALT in conjunction with anti-HCV improves the quality of screening for potentially infectious donors.

The serology of hepatitis C virus (HCV) infection: antibody crossreaction in the hypervariable region 1

SummaryWe determined the NS1/E2 N-terminal sequence including the hypervariable region 1 (HVR1) from five individuals chronically infected with HCV: two from the Czech Republic and three from Germany. From each sequence, six 12-mer overlapping peptides were synthesized and used in a peptide scan to evaluate seroreactivity of each of those patients, as well as three anti-HCV positive blood donors to the different isolates. We could show the general presence of antibodies to multiple HVR1 specific sequences reflecting the existence of multiple variants in infected persons. Finally, we observed the persistance of HCV infections in all individuals despite an active humoral response directed against the virus.

Isotype-specific immune response to a single hepatitis C virus core epitope defined by a human monoclonal antibody: diagnostic value and correlation to PCR

SummaryIn this study we tested the seroreactivity of 223 selected anti-HCV-reactive blood donors to the human B-cell epitope N-VYLLPR-C (C34–39) of the hepatitis C virus core antigen. The epitope was recently identified and characterized by the human monoclonal IgG antibody Ul/F10 [23] and is located within the amino acid residues 34–39 of the aminoterminal core region. The blood donor sera were selected from anti-HCV ELISA (Ortho, 2nd generation)-reactive samples. Sixty-seven of these sera were further reactive in RIBA (Ortho, 2nd generation). According to their RIBA pattern, these samples were divided into four groups. Samples in the first group (n=18) reacted to all four recombinant HCV antigens. The samples of the second (n=9) and third group (n=8) reacted to c223/c33c and c22-3/cl00-3, respectively. Sera from group 4 (n=32) showed a RIBA indeterminate pattern with reactivity only to c22-3. All 223 samples were analyzed for anti-C34–39 antibodies by ELISA, and the 67 RIBA-reactive samples were additionally tested for the presence of HCV RNA by RT/PCR. In groups 1 and 2, over 80% of the samples showed anti-C34–39 reactivity which was restricted to the IgG1 isotype. In contrast, in groups 3 and 4, antibodies to the epitope C34–39 were detected in less than 10% of the samples. Interestingly, the anti-C34–39 response correlates with the presence of HCV RNA; 95.5% of the samples had coincident results in all subgroups. None of the RIBA-negative sera showed a specific seroreaction to the C34–39 peptide.

Safety of Blood Products Derived from Plasma Pools: The Positive Impact of Anti‐HCV Screening on the Quality of Such Products

Anti‐HCV screening of volunteer blood donors was introduced by the Red Cross Blood Service of Baden‐Württemberg (over 400,000 donations/year) in June 1990. At that time, donors were tested with a first‐generation screening test, which was able to detect antibodies to a single recombinant HCV antigen: c100‐3. Test performance was neither very sensitive nor specific [ 1 ]. The anti‐HCV prevalence observed then was 0.53%. The second generation of screening tests relied on the detection of antibodies to three HCV recombinant antigens c100‐3, c22‐3 and c33c showing improved sensitivity and specificity [2]. It was implemented in Baden‐Württemberg in November of 1991. Anti‐HCV prevalence reached 0.47% after the implementation of this test. Finally, since June 1994 all blood donations are screened with the third generation of antibody tests, which includes the recombinant antigen NS5 and some biochemical modifications on the c33c antigen. The present anti‐HCV prevalence in this population is 0.23%.

Prevalence of HCV-RNA-Positive Patients in a Dialysis Unit in Germany

Dr. Marcia da Suva Cardoso, German Red Cross Blood Bank and, Department of Transfusion , Medicine, University of Ulm, Helmholzstrasse 10, D-89081 Ulm (Germany) Dear Sir, In order to establish the prevalence of HCV infectious patients in the Dialysis Unit of the Board of Dialysis of Transplantation Ulm, we investigated a group of 195 patients by HCV RT/PCR. For this purpose we used a well-established HCV RT/PCR protocol, which we used in previous studies with blood donors and hepatitis patients [1,2]. This protocol is based on the use of at least two nested primer sets, either two sets for the 5’-noncoding region (5’-NCR) or one set for the 5’-NCR and another for the core region of the HCV genome. As a result, we could detect the presence of HCV-RNA in serum from 30 of these dialysis patients. Serum samples from all 195 patients were also tested for the presence of HCV antibodies with the ELISA second generation test from Ortho Diagnostic Systems. From these individuals 165 were anti-HCV negative and 30 anti-HCV positive. Twenty-one out of the 30 anti-HCV-positive samples could be confirmed positive by the recombinant immuno-blot RIBA second generation (RIBA II). The other 9 samples resulted either indeterminate (1) or negative (8) by RIBA II. In summary, HCV-RNA could be found in 22 of the anti-HCV-positive samples and in 8 of the anti-HCV-negative samples (table 1). From these 22 HCV-RNA-/anti-HCV+, 21 were RIBA II positive and 1 indeterminate due to seroreactivity to the c-22 recombinant antigen. None of the HCV-RNA+/anti-HCVwere RIBA positive, what led us to repeat the PCR assay, finally confirming the previous results. The HCV-RNA prevalence in our unit though is 15%, being not different from the anti-HCV prevalence, although not all the samples are simultaneously HCVRNA+/anti-HCV+. The data presented here suggest that in the hemodialysis population the antibody status of a patient has a very high prognostic value for viremia: we obtained over 95% correlation between HCV and RIBA in the antibody-positive group. This contrasts very much with what has been observed in a low prevalence population, e.g. blood donors [2]. Furthermore, we could observe a direct correlation between HCV-RNA positivity to the duration of dialysis, which has also been

Identification of the Source of Infection through HCV Genotyping: HCV Look‐Back II

In a recent publication we estimated the risk of HCV transmission through blood transfusion in Germany to be less than 1:40,000 [I]. For this purpose we retrospectively investigated all repeat blood donors who have been identified as anti-HVC-positive by RIBAII in 1993 (27 of 378,548 blood donations). Sixteen living recipients of blood/blood components from 10 of these donors could be found and were enrolled in the study. Serum from these individuals were tested by second-generation serological assays (ELlSA and RIBA) and by HCV RT/PCR. In the year 1994 another 5 pairs of donorsirecipients were identified by the same retrospective method and included in this lookback study (look-back 11). In order to investigate whether the infectivity of antiHCV-positive blood donations is directly related to HCVRNA titer in serum, we determined the virus RNA titer in serum of all positive donors who were identified in the retrospective study (look-back 11). Furthermore genotyping of isolates from all HCV-RNA-positive individuals (donors and recipients) was carried out, not only to investigate the infectivity of the different genotypes, but also as a way to certify that blood transfusion had indeed been the source of infection in those patients.

Analysing the Performance of Anti-HCV Supplemental Tests in German Blood Donors

In order to test the performance of the presently available anti-HCV supplemental tests, we selected 241 ELISA 2nd-generation reactive samples, previously tested by RIBA 11, to be further investigated with the RIBA 111 (all three tests from Ortho Diagnostic Systems, Germany). From these, 240 samples could be tested by Matrix (Abbott, Germany) and 236 by Innolia (Innogenetics, Belgium). This panel of samples resulted from the selection of matching samples (age, sex, city of origin) from the three subgroups of antiHCV ELISA reactive donations (ELISA+/ RIBA 11-, n = 75; ELISA+/RIBA 1I+, n = 91; ELISA+/RIBA I I indeterminate, n = 75) which were collected by the Blood Transfusion Service of Baden-Wurttemberg in 1993. In our Blood Transfusion Service, serum from anti-HCV ELISA reactive donations are routinely frozen at -20°C for future investigation. The results produced by the different supplemental tests were interpreted strictly according to the manufacturers recommendations. A validated RT/PCR protocol [ I , 21 was applied to all samples and its results were taken as reference, hence sensitivity and specificity of the above-cited supplemental tests were calculated in relation of them. Analysing the Performance of Anti-HCV Supplemental Tests in German Blood Donors

RIBA Performance in the Routine Diagnostic of HCV

Der DRK-BSD BaWu testet seit 1993 alle anti-HCV-ELISA-positiven Blutspenden (0,4%) mit RIBAII. RIBA-II-positive Spender werden von weiteren Blutspenden ausgeschlossen. Die Rate an RIBA-II-positiven Bl

Analysing the sequence diversity of the 5′ NC-Region of HCV-isolates found in Southern Germany

In this study we compared the nucleotide sequence of the 5′ NC-region of the HCV genome isolated from seven patients and two blood donors from Southern Germany. We could identify two very distinct groups of isolates: the first very similar to the HCV prototype sequence (HCV1) with homology ranging from 99.5% to 98.7%; the second showing 91.7% homology to the HCV1. Group 1 isolates could be found in five patients and the two blood donors. Group 2 isolates could be found in the two other patients. Finally, we could observe neither nucleotide insertion nor deletion in the isolates described here.