Abstract
BackgroundWith the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination.Methodology/Principal FindingsA large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5′ non-coding region (5′NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%–29.5% of cases, and HCV subtype 1b in 9.5%–8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5′NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5′NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases.Conclusions/SignificanceIn the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5′NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.
Highlights
Over 170 million individuals are infected with hepatitis C virus (HCV) worldwide
It was used to identify the HCV genotype and subtype in 516 treatment-naıve patients included in a multicenter clinical trial assessing different schedules of pegylated IFN-a2a and ribavirin [26]
This is changing with the development of new specific inhibitors of HCV enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV genotype subtype [13,14,15,16,17,18,19,20,21,22]
Summary
Over 170 million individuals are infected with hepatitis C virus (HCV) worldwide. Phylogenetic analyses have shown that HCV strains can be classified into at least 6 major genotypes (numbered 1 to 6), and a large number of subtypes within each genotype [1]. Molecules that have reached clinical development include inhibitors of the nonstructural (NS) 3/4A serine protease and inhibitors of HCV replication that belong to different categories: nucleoside/nucleotide analogue and non-nucleoside inhibitors of the HCV RNA-dependent RNA polymerase (RdRp), NS5A inhibitors and cyclophilin inhibitors [9]. These agents have shown potent antiviral efficacy when used alone, and encouraging results have been recently published showing that HCV clearance can be achieved in approximately 70% of cases when a potent NS3/4A inhibitor is used in combination with pegylated IFN-a and ribavirin [10,11,12]. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination
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