Evaluation of a new assay for hepatitis C virus (HCV) genotyping targeting both 5'NC and NS5b genomic regions, in comparison with reverse hybridization and sequencing methods

Microbiology Department, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain; Centro de Investigación Biomédica en red de Epidemiología y Salud Pública (CIBERESP), Spain; Virus Reference Department, Health Protection Agency, London, United Kingdom; Liver Unit, Hospital Universitari Germans Trias i Pujol, Badalona, Spain; Centro de Investigación Biomédica en red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Spain

^* To whom correspondence should be addressed. Email: vausina{at}ns.hugtip.scs.es.

	Abstract

We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the NS5b subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5'non-coding region (5'NC) (method 1: HCV Genotyping ASR assay). This method was compared with 5'NC reverse hybridization (method 2: InnoLiPA HCV II) and 5'NC sequencing (method 3: Trugene HCV 5'NC). Two hundred-ninety five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment was used to resolve discrepant results. Suspected multiple genotype infections were confirmed by PCR-cloning and Pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct), and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45% and 92% by methods 1, 2 and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2, and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more time-consuming assays.