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Evaluation of a New Assay in Comparison with Reverse Hybridization and Sequencing Methods for Hepatitis C Virus Genotyping Targeting Both 5' Noncoding and Nonstructural 5b Genomic Regions

Elisa Martró,^1,2, Victoria González,^1,2, Andrew J. Buckton,³ Verónica Saludes,^1,2 Gema Fernández,¹ Lurdes Matas,^1,2 Ramón Planas,^4,5 and Vicenç Ausina^1,6*

Microbiology Department, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain,¹ Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain,² Virus Reference Department, Health Protection Agency, London, United Kingdom,³ Liver Unit, Hospital Universitari Germans Trias i Pujol, Badalona, Spain,⁴ Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain,⁵ Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), Bunyola, Mallorca, Spain⁶

Received 14 August 2007/ Returned for modification 2 October 2007/ Accepted 25 October 2007

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 nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5' noncoding region (5'NC) (method 1; HCV genotyping analyte-specific reagent 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 were 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 results 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.

Published ahead of print on 7 November 2007.

Equal contributors.