[Hepatitis C virus genotyping: comparison of the Abbott RealTime HCV Genotype II assay and NS5B sequencing].
ABSTRACT Hepatitis C virus genotyping is needed for treatment decision and monitoring. The results of a genotyping assay based on real-time PCR and TaqMan chemistry were compared with the results of NS5B region sequencing.
One hundred and two sera (genotypes 1-6) were tested. Amplification and detection of viral RNA were performed with the Abbott RealTime HCV Genotype II assay targeting 5'non-coded region (5'NC) for the identification of genotypes 1 to 6 and NS5B, for 1a and 1b subtypes detection. Sequencing of 5'NC fragment was used to resolve discrepant results.
No indeterminate results were obtained. Concordance with NS5B sequencing was 93% (95 on 102), 96% at the genotype level (98 on 102) and 93% for genotype 1 subtyping (40 on 43). Discordant genotyping results were a 2f subtype identified as 5, a 6a typed as 1, a 3a identified as a 1-3 co-infection and a 4r identified as a 1-4 co-infection. Discordant subtyping results were 2 1b subtypes only typed as 1 and a 1e identified as 1a.
Abbott RealTime HCV Genotype II assay is a rapid, automated and simple to interpret method for HCV genotyping. It allows the detection of possible mixed infections which might have a negative impact on therapeutic response. However, the discrepant results found in this small series underline the need for assay optimization.
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ABSTRACT: The Versant HCV genotype 2.0 assay (LiPA 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used for analysis of hepatitis C virus (HCV) genotypes. However, their performances for detecting genotype 6 have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using LiPA 2.0. The genotyping results were confirmed by NS5B or core sequence phylogenetic analysis. A total of 57 samples were confirmed as genotype 6 (51 as genotype 6a, five as genotype 6n and one as genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by LiPA 2.0 were confirmed as genotype 3b. The remaining two samples classified as genotype 6 by LiPA 2.0 were confirmed as genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using Realtime II Version 2.00, 47 genotype 6a samples were identified as genotype 6; one 6e sample was misclassified as genotype 1; four 6a and five 6n samples yielded "indeterminate" results. Nine nucleotide profiles in the 5' untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. Realtime II fails to identify some 6a and all non-6a subtypes and misclassifies genotype 6e as genotype 1.Journal of Clinical Microbiology 08/2014; 52(10). DOI:10.1128/JCM.00882-14 · 4.23 Impact Factor
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ABSTRACT: Background: Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct acting antivirals (DAAs) are subtype-dependent. Currently available genotyping methods have limited subtyping accuracy. Methodology: We have evaluated the performance of a deep sequencing based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Results: Phylogenetic analysis of deep sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott real-time HCV genotype II, failed subtype 1 calling in 13 (16%) samples each, and were unable to identify the HCV genotype and/or subtype in more than half of non-genotype 1 samples. Conclusions: Deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections, and may be more helpful to inform treatment strategies with new DAA-containing regimens, across all HCV subtypes.Journal of Clinical Microbiology 11/2014; DOI:10.1128/JCM.02093-14 · 4.23 Impact Factor
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ABSTRACT: The hepatitis C virus (HCV) is a globally prevalent human pathogen that causes persistent liver infections in most infected individuals. HCV is classified into seven phylogenetically distinct genotypes, which have different geographical distributions and levels of genetic diversity. Some of these genotypes are endemic and highly divergent, whereas others disseminate rapidly on an epidemic scale but display lower variability. HCV phylogeny has an important impact on disease epidemiology and clinical practice because the viral genotype may determine the pathogenesis and severity of the resultant chronic liver disease. In addition, there is a clear association between the HCV genotype and its susceptibility to antiviral treatment. Similarly to other RNA viruses, in a single host, HCV exists as a combination of related but genetically different variants. The whole formation is the actual target of selection exerted by a host organism and antiviral therapeutics. The genetic structure of the viral population is largely shaped by mutations that are constantly introduced during an error-prone replication. However, it appears that genetic recombination may also contribute to this process. This heterogeneous collection of variants has a significant ability to evolve towards the fitness optimum. Interestingly, negative selection, which restricts diversity, emerges as an essential force that drives HCV evolution. It is becoming clear that HCV evolves to become stably adapted to the host environment. In this article we review the HCV phylogeny and molecular evolution in the context of host-virus interactions.Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 11/2013; 21. DOI:10.1016/j.meegid.2013.10.021 · 3.26 Impact Factor