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 (5NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5NC reverse hybridization (method 2; InnoLiPA HCV II) and 5NC sequencing (method 3; Trugene HCV 5NC). 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.Hepatitis C virus (HCV) is the most important cause of chronic liver disease and is the leading indication for liver transplantation (2). It is estimated that HCV infects 3% (170 million people) of the world's population (26). HCV possesses a positive-sense single-stranded RNA genome of approximately 9.5 kb, which is flanked by noncoding (NC) regions. The HCV genome encodes at least 11 proteins, which include both structural and nonstructural (NS) proteins (5, 7).HCV is known to have a high rate of genetic heterogeneity. This has allowed HCV strains to be classified into a number of genetically distinct groups, known as genotypes, subtypes, isolates, and quasispecies (5). Genome sequence heterogeneity arises due to poor fidelity of the viral polymerase during replication. Sequence variability is not evenly distributed throughout the genome. The lowest sequence variability is found in the 5ЈNC region, which is the target of choice for many molecular diagnostics assays, including genotyping tests. Nevertheless, nucleotide sequencing coupled with phylogenetic analysis of more-variable genomic regions has been recommended for HCV genotyping in consensus proposals (27). As patients infected with different genotypes respond differently to antiviral drug therapy, identification of the infecting genotype ha...

Hepatitis C Virus Genotyping Using an Oligonucleotide Microarray Based on the NS5B Sequence

“…In this work, use of probes complementary to subtype-specific sequences of the NS5B region enabled us to identify more than 20 HCV subtypes in the specimens tested. Other methods, such as realtime PCR, can identify a limited number of subtypes and genotypes (1a, 1b, 2a, 2b, 2c, 3, 4, 5, and 6) (23,44). Only the clip sequencing method can, in theory, discriminate as many subtypes as can our procedure (35).…”

“…Several methods has been proposed for HCV genotyping (50), including commercially available techniques based on real-time PCR: the HCV genotyping analyte-specific reagent (ASR) assay (Abbott Molecular Inc., Des Plaines, IL) (23), semiautomated sequencing (the TruGene HCV 5ЈNC genotyping kit; Bayer HealthCare, Berkeley, CA) (10), and automated reverse hybridization (the Inno-LiPA HCV II assay; Innogenetics, Ghent, Belgium) (46,49). Most HCV genotyping methods are based on analysis of the 5Ј noncoding (NC) region of the HCV genome because the 5Ј NC region is regularly amplified for HCV molecular diagnosis and quantification of the viral load.…”

A single-step multiplex quantitative real time polymerase chain reaction assay for hepatitis C virus genotypes

“…NS5b was used due to the presence of specific variations in its sequence for different genotypes. [28][29][30] The reporter dyes for Taqman chemistry based assays were selected according to the available emission filters. Previous studies have reported sensitive real time assays for quantitative detection of HCV when using FAM, HEX, etc., as reporter dyes.…”

Occult Hepatitis C Virus Infection: Where are We Now?

“…For example, 20-30% of subtype 1a are not correctly identified (Chen and Weck, 2002). Real-time PCR methods and DNA biochip methods are now available (Gryadunov et al, 2010, Mao et al, 2010, Martro et al, 2008, Nakatani et al, 2010, Park et al, 2009, Verbeeck et al, 2008. Methods based on analysis of the NS5B region are better for discriminating between HCV subtypes (Table 2) (Gryadunov et al, 2010.…”