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- Widell, Anders, et al.
(author)
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Genotyping of hepatitis C virus isolates by a modified polymerase chain reaction assay using type specific primers: epidemiological applications
- 1994
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In: Journal of Medical Virology. - : Wiley. - 1096-9071 .- 0146-6615. ; 44:3, s. 272-279
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Journal article (peer-reviewed)abstract
- A polymerase chain reaction (PCR)-based assay using primers against the hepatitis C core gene has been described [Okamoto et al. (1992a): Journal of General Virology 73:673-679]. Within the two major HCV genotypes 1 and 2, the Okamoto system identifies two subtypes each (1a, 1b and 2a, 2b, respectively). Typing is achieved by a primary PCR with consensus primers followed by a nested PCR with type specific primers. The original assay was modified by addition of a parallel second PCR identifying the recently described major genotype 3. The assay also identifies in duplicate subtype 1b (type II by Okamoto), suggested to respond poorly to interferon. Reaction conditions were reviewed and melting temperatures of all typing primers equalised to increase strigency. The modified system functioned well and typing results were supported by partial core sequencing. The following distribution of genotypes was found in 53 hepatitis C virus (HCV) infected Swedish blood donors: genotype 1a (57%), 3 (19%), 1b (13%), and 2b (11%). In six recipients of HCV infected blood identified in a retrospective study, the recipient HCV genotype was identical to donor HCV genotype. Furthermore, in HCV positive couples identical genotype was observed when only one partner had an external risk factor; whereas genotypes were often diverse if both sex partners had parenteral risk factors. Finally, a cluster of hepatitis C cases in a haemodialysis unit was evaluated retrospectively. Eight patients had genotype 1b, two had mixed 1a and 1b, and one had type 1a. The modified HCV genotyping assay was of value in examining different epidemiological situations and can be expanded presumably to include future genotypes.
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| 2. |
- Widell, Anders, et al.
(author)
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Hepatitis C virus RNA in blood donor sera detected by the polymerase chain reaction: comparison with supplementary hepatitis C antibody assays
- 1991
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In: Journal of Medical Virology. - : Wiley. - 1096-9071 .- 0146-6615. ; 35:4, s. 253-258
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Journal article (peer-reviewed)abstract
- The low specificity of screening ELISAs for antibodies to hepatitis C virus in blood donors has called for confirmatory tests. Two types of supplementary antibody assays are available, recombinant immunoblot assays (RIBA-1 and RIBA-2) and an antibody consumption test referred to as a neutralization assay. Amplification of viral nucleic acid by the polymerase chain reaction (PCR) provides an antibody independent mode of detecting viral infection. We applied reverse transcription-double PCR to detect an HCV 5'-noncoding viral RNA sequence in serum specimens and compared PCR findings with confirmatory antibody tests. This study includes sera from 37 blood donors found positive by the Ortho anti-HCV (C100-3) ELISA out of 14,591 donations. Of the 37 positive sera, 8 were positive by RIBA-1 and 1 further by RIBA-2. Seven of the RIBA positive sera contained HCV RNA by PCR. Among the 8 indeterminate and the 21 negative donor sera by RIBA-I, no PCR positive serum was found. The 37 anti-HCV positive donor sera identified by Ortho ELISA were also tested by Abbott anti-HCV (C100-3) ELISA whereby 22 were positive. Of these 22 sera plus 1 further with ELISA OD just below cutoff, 8 were positive by the neutralization assay, (Abbott Laboratories, North Chicago, IL, USA) and 6 of these, including the borderline serum, were PCR positive. One of the two neutralizable but PCR negative sera was RIBA positive and the other was indeterminate. However, one ELIS. A (Abbott Laboratories) positive (OD 1.99) serum was not neutralizable but nevertheless contained HCV RNA by PCR. Thus in blood donor sera, anti-HCV (C100-3) reactivity confirmed by RIBA or neutralization correlated well with the presence of HCV RNA.
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