M. da Silva Cardoso

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.

Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1

Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314–330 (EP3) covering the central conserved sequence of this domain. Anti‐hepatitis C virus‐positive blood donors were screened for anti‐EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA‐confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti‐EP3‐producing heterohybridoma cell lines: U1/F30 and U1/F31 produced IgM‐κ, whereas U1/F32 and U1/F33 secreted the isotypes IgG1‐λ and IgG1‐κ, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies U1/F32 and U1/F33 have dissociation constants to the peptide of ∼ 10−9 M, binding to recombinant E1 protein expressed in COS‐7 cells was different. Only U1/F33 detected envelope protein of ∼ 24–35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1.

PCR Screening in the Routine of Blood Banking of the German Red Cross Blood Transfusion Service of Baden-Würtemberg

© 1998 S. Karger GmbH, Freiburg Fax (07 61) 4 52 07 14 www.karger.com Received: October 29, 1997 Accepted: March 9, 1998 Dr. Marcia da Silva Cardoso DRK-Blutspendedienst und Abteilung für Transfusionsmedizin, Universität Ulm Helmholtzstraße 10, D-89081 Ulm (Germany) Tel. +49 731 150-119, Fax -175 E-mail marcia.cardoso@medizin.uni-ulm.de This article is also accessible online at: http://BioMedNet.com/karger PCR Screening in the Routine of Blood Banking of the German Red Cross Blood Transfusion Service of Baden-Württemberg M. da Silva Cardoso K. Koerner B. Kubanek

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.

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

Deutsche Gesellschaft fü Transfusionsmedizin und Immunhämatologie

die Anwendung von Blutzellseparatoren mit extrakorporalem Kreislauf der Spender und Patienten. Sie ermöglicht dadurch die direkte Auftrennung von Blut in verschiedene Bestandteile während des Entnahmevorgangs. Mit Hilfe dieser Zellseparationsverfahren können einzelne Blutbestandteilkonzentrate (Thrombozyten, Granulozyten, Lymphozyten, Blutstammzellen) oder gleichzeitig verschiedene Blutkomponenten (Multikomponentenspende mit Plasma, Erythrozyten und Thrombozyten) gewonnen werden. Die übrigen Blutbestandteile werden dabei dem Spender direkt wieder zugeführt. Der extrakorporale Kreislauf und der Einsatz von Zellseparatoren am Menschen stellen eine wesentliche Änderung im Vergleich zur konventionellen Vollblutspende dar und erfordern besondere Sicherheitsund Vorsichtsmaßnahmen, die in diesen Empfehlungen festgelegt sind. Es gibt kontinuierliche (ständige Entnahme, Auftrennung von antikoaguliertem Vollblut und Rückgabe der nicht gesammelten Blutbestandteile) und diskontinuierliche Separationsmethoden (abwechselnde Sammlung und Rückgabe der Blutbestandteile) zur Herstellung von zellulären Blutbestandteilpräparaten für die Transfusion und Transplantation.

Identification of Infection Routes for HCV in Blood Donors from Germany

Background: The objective of this study was to assess the relevant routes of infection for hepatitis C virus in blood donors from Baden-Württemberg (Germany). Participants and Methods: For this purpose an epidemiological questionnaire was elaborated and sent to 528 blood donors. One half of these donors had an anti-HCV-positive status confirmed by supplemental test (case group); the other half comprised blood donors who were seronegative for all infectious markers (control group). 72% of the enrolled donors (n = 380) sent back the answered questionnaire for evaluation. Results: From this investigation we could identify three major routes of HCV infection: a previous history of blood transfusion (p < 0.05), drug abuse (p < 0.05) and, in male participants, tatooing (p < 0.05). Seropositivity to HCV was significantly related to episodes of hepatitis in adult age (p < 0.05). Conclusion: Additional approaches to the usual preprinted donor’s questionnaire are needed, particularly to inquire about drug abuse and tatooing, since the established procedure seems to be quite inefficient.

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.

Release of mycoplasmavirus L1 upon transfection ofAcholeplasma laidlawii with homologous and heterologous viral DNA

SummaryThis communication reports on the release of Mycoplasmavirus L 1 after infection ofAcholeplasma laidlawii with purified L 3 virus. Release also occurred after transfection with certain restriction fragments from MV-L3 and MV-L1 genomes. Since circular molecules are efficiently taken up in polyethylene glycol-mediated transfection, inducing fragments were applied cloned inE. coli plasmids. Release was also observed after electroporation of cells incubated with MV-L 1 replicative intermediate DNA and linear MV-L 3 DNA isolated from virus particles, respectively. Released MV-L 1 viruses were identified after virus plaque formation on indicator lawns according to plaque morphology and hybridization with labeled viral DNA probes as well as by DNA restriction analysis. Uninfected and untransfected cells from six laboratory strains ofA. laidlawii (including a MV-L 1 resistant one) were examined for the presence of MV-L 1 DNA. They all bear MV-L 1 DNA integrated in their genomes.