Abstract
BackgroundThe technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5’UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results.Methods100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5’UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank.ResultsAll the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5’UTR and Core. Twenty-three samples were sequenced for two regions: 5′ UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5’UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%.ConclusionsIn this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.
Highlights
The technique most frequently used to genotype hepatitis C virus (HCV) is quantitative RT-PCR
For the determination of the type and G1 subtype, the kappa coefficient was 0.94 and 0.82 respectively, which means that the strength of agreement between VERSANT VERSANT HCV Genotype 2.0 Line Probe Assay (LiPA) assay and 5′ untranslated region (5’UTR) region sequencing was almost in perfect agreement
Of the 9 samples identified as G6 by the VERSANT LiPA assay, 3 were identified as G6 by 5’UTR sequencing, 1 as G1b and 4 were undetermined (Table 3)
Summary
The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. HCV genotyping is part of the evaluation of newly diagnosed patients and has always been important in guiding treatment [4]. In the last EASL recommendations for the treatment of hepatitis C [5], identification of HCV genotype remains an essential consideration. Since the development of pan-genotype direct acting agents (DAA), correct identification of the genotype is important to guide the choice of the DAA combination and the duration of the treatment. Starace et al showed that 14.9% of DAA failure are related to a genotyping error [6] and Di Maio et al report that 6/197 (3%) of DAAfailing patients and in particular 4/7 non responders to a DAA INF-free regimen were impacted on a wrong genotype assignment [7]
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