Identification of the source of infection through HCV genotyping: HCV look-back II.
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Biomedical subjects
Publications and source records attributed to K Koerner.
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Platelet concentrates prepared from buffy coat were pooled and stored for 6 days after removal of leukocytes by filtration. The platelets were stored in plasma or in an additive solution, Plasmalyte-A. In vitro platelet function was better preserved using Plasmalyte-A than plasma with regard to osmotic reversal and aggregation. No significant differences for the release of platelet markers beta-thromboglobulin, platelet factor 4, or lactate dehydrogenase pre- and post-filtration and storage in plasma or Plasmalyte-A was observed. Expression of the surface membrane glycoproteins Ib, Ia/IIa, IIb/IIIa, and IV measured by flow cytometry after binding of monoclonal antibodies did not change during storage. The expression of activation-dependent alpha-granula glycoprotein GMP140, the thrombospondin, and the glycoprotein 53 from the lysosomal granules was not different between platelet pools stored in plasma or in Plasmalyte-A. The in vitro quality of platelets stored as pools is comparable for plasma and the additive solution Plasmalyte-A.
The contribution of hepatitis C virus (HCV) infection to liver disease after bone marrow transplantation (BMT) was retrospectively evaluated in 61 patients treated with BMT. HCV genome, as well as antibodies to HCV, was analyzed in sera collected before and serially after BMT. Six patients had been infected with HCV before BMT and three patients acquired the infection during or shortly after BMT. All patients infected before BMT died within 10 weeks after transplantation. Five of these six patients (83%) died of veno-occlusive disease (VOD), compared with nine of 52 patients (17%) not infected with HCV (P < .005). Risk factors for VOD other than HCV were not more prevalent in these patients compared with uninfected patients. Parallel to the development of VOD, replication of HCV increased, as demonstrated by rising concentrations of viral RNA in serum. HCV infection acquired during or after BMT caused only mild acute hepatitis C, which progressed to chronic hepatitis C in one patient surviving 10 years after BMT. These data suggest that patients with liver disease caused by HCV infection are at high risk of developing lethal VOD after BMT.
In 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 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 c22-3/c33c and c22-3/c100-3, 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.
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.
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In this study we describe the establishment of two hybridoma cell lines secreting human monoclonal antibodies to the 22-kD nucleocapsid protein (core, p22) of the hepatitis C virus (HCV). For this purpose we isolated B lymphocytes from an anti-HCV positive blood donor and infected them with Epstein-Barr (EBV). We obtained several lymphoblastoid cell clones secreting antibodies to the recombinant HCV core protein. The B-cell cultures were oligoclonally expanded and two of them were fused with the (mouse:human) heteromyeloma cell line K6H6/B5. The resulting stable hybridomas produce antibodies of the IgG1/kappa (U1/F10) and the IgM/kappa (Ul/F11) isotype reacting specifically with the recombinant core protein p22. To identify the epitopes recognized by these antibodies we synthesized overlapping peptides (13-mer and 6-mer) from the amino terminus of the core amino acid sequence. Antibody reactivity to these peptides was analyzed in an immunoblot assay. Finally, we were able to define a linear epitope recognized by the Ul/F10 antibody on the nucleocapsid protein. The antibody shows specificity to the sequence N-VYLLPR-C, which corresponds to the amino acids 34-39 of the core sequence.
Three different synthetic media without glucose were studied for platelet storage. The first medium contained acetate and gluconate. The second contained acetate, gluconate and citrate. Finally the third contained phosphate and mannitol. The purpose of the study was to investigate whether there were differences among the various media in terms of preservation of platelet quality. Pools of platelet concentrates were prepared from buffy coats. In vitro function and metabolic parameters were measured during 5 days of storage in these additive solutions as well as in plasma. Platelet aggregation, hypotonic shock response and release of beta-thromboglobulin, platelet factor 4 and lactate dehydrogenase of the cytosol were equivalent in the media containing acetate compared to plasma storage. In vitro platelet functions and pH in these two media were better preserved compared to the medium with phosphate and mannitol. In addition bacteriological studies using platelets suspended in additive solutions or in plasma were carried out. Carryover of 20% of plasma to the synthetic media necessary for successful platelet storage in these additive solutions allows bacteriological growth. As shown, inoculation of 1 colony/ml Staphylococcus epidermidis leads to 10(6)-10(7) organisms/ml after 5 days of storage.
Buffycoat free red cell concentrates in a medium composed of sodium chloride, adenine, glucose and mannitol (SAGM) stored for 42 days at +4 degrees C were rejuvenated by a solution which contained pyruvate, inosine, disodiumphosphate and adenine (PIPA). The rheological behaviour of red cells was measured before and after rejuvenation by viscosimetry of red cell suspensions (hematocrit 45%) at various intervals during the storage period. The deformability of the red cells during storage was determined also before and after rejuvenation by measurement of the viscosity of hard packed cells (hematocrit 98%). The results show improvement of the rheological properties by rejuvenation. Moreover it is shown that incubation of the red cells at 37 degrees C for two hours without rejuvenation solution decreases the viscosity.
In 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.
Manipulation checks should be used in psychotherapy trials to confirm that therapists followed the treatment manuals and performed the therapy competently. This article is a review of some strategies that have been used to document treatment integrity; also, their limitations are discussed here. Recommendations for improving these checks are presented. Specific guidelines are offered regarding when and how to assess both therapist adherence to treatment protocols and competence.
In this paper we discuss the value of a new methodology for the production of monoclonal antibodies by recombinant techniques. This approach is especially useful for human monoclonal antibodies due to instability of human antibody-producing cell lines. As an example, we present a recombinant human antibody to the hepatitis C virus (HCV) core protein.
OBJECTIVE: Assessment of the present risk of blood-borne infection associated with homologous blood transfusion in Central Europe with particular emphasis on HIV and HCV infections. DATA SOURCES: The relevant literature in the English and German language and the authors' data. STUDY SELECTION: No special study has been carried out for the present paper. RESULTS: The recognition of the risk for transmitting HIV by transfusion has led to a bundle of measures which have improved the safety of blood supply in Germany. A stricter donor selection as well as screening for HIV 1 and HIV 2 have reduced the risk to transmit HIV to the order of 1 per 1 million units transfused. The transmission of hepatitis B is estimated to be in the order of 1:50,000. The anti-HCV testing, introduced in 1990, has markedly reduced the transmission of HCV to less than 1:5,000 per unit as judged from our own data. A further reduction is expected by an improved HCV screening in the near future. Fatal disease from bacterial contamination is rare, with an estimated risk of one in a million units. Syphilis transmitted by transfusion is virtually not occurring anymore. However, increased efforts should continue to enhance the safety of blood, bearing in mind that a zero risk is not achievable for effective therapies. The estimation of risks is a dynamic, time-dependent value which has to be estimated for a geographically defined population for a given time period. CONCLUSIONS: The risk of transfusion-associated infection has been markedly reduced in recent years. Albeit there is a small but definitive risk, which is often overestimated. The risks have to be defined to estimate the risk/benefit ratio of the homologous as well as the autologous transfusion for the individual patient.
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Autologous platelets of 5 alloimmunized patients with acute leukemia in remission were cryopreserved with 5% dimethylsulfoxide in liquid nitrogen and retransfused in the following therapy-induced thrombocytopenic phase. The mean platelet recovery after freezing, thawing and washing was 85 +/- 6%. The mean corrected 1-hour increment in platelet counts was 11 (4-27) x 10(9)/l, i.e. 61% in comparison with fresh platelet transfusions in other patients. In vivo function of frozen platelets was documented by improvement of the posttransfusion bleeding time. Cryopreserved autologous platelets function hemostatically and can be used even for completely refractory patients and thus permit curative antileukemic therapy.
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In this study, we report the investigation of a hemophiliac cohort, in which the last HIV-1 seroconversion occurred in 1985. We wanted to evaluate whether 5 years later the results of serological screening and polymerase chain reaction (PCR) technology would match. We examined 61 German patients with congenital deficiencies of factors VII, VIII-c, and IX, and 16 sexual partners (15 partners of anti-HIV-1-negative hemophiliacs). Patients' and partners' anti-HIV-1 status was determined by ELISA and Western blot. Further, we applied the PCR to investigate the possible presence of HIV sequences in anti-HIV-1-negative individuals. Four sets of primers were used in four separated reactions to avoid false-negative results due to genetic variation, as well as false-positive results due to DNA carryover. The data by PCR were not different from the data attained by conventional serological methods. The prevalence of serological markers for HBV and HCV was determined.
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