title: 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.
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 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.
authors.affiliation: Microbiology Department, Hospital Universitari Germans Trias i Pujol, Ctra de Canyet, s/n. 08916 Badalona, Spain.
authors.forename: Elisa
authors.initials: E
authors.lastname: Martró
authors.forename: Victoria
authors.initials: V
authors.lastname: González
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Buckton
authors.forename: Verónica
authors.initials: V
authors.lastname: Saludes
authors.forename: Gema
authors.initials: G
authors.lastname: Fernández
authors.forename: Lurdes
authors.initials: L
authors.lastname: Matas
authors.forename: Ramón
authors.initials: R
authors.lastname: Planas
authors.forename: Vicenç
authors.initials: V
authors.lastname: Ausina
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics*; D006526:Hepatitis C / Q000175:diagnosis / Q000821:virology*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D044967:Serum / Q000821:virology; D013223:Statistics as Topic; D014759:Viral Envelope Proteins / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral; D014759:Viral Envelope Proteins; C088325:gene 5b protein, hepatitis virus