Determination of hepatitis C virus genotype by direct sequence analysis ofproducts generated with the amplicor HCV test

Consistent with other members of the family Flaviviridae, hepatitis C virus (HCV) demonstrates a high degree of sequence variation throughout the codi ng regions of its genome. However, there is a high degree of sequence conse rvation found within the 5' untranslated region (UTR) of the genome, making this region a target of choice for most nucleic acid amplification-based d etection assays. In this study, the Amplicor HCV test, a commercially avail able assay which detects the 5'UTR was used for the detection of HCV RNA in 669 serum samples obtained from a cohort of liver transplantation patients . Amplification products obtained from the HCV-positive cases were subjecte d to direct sequencing and genotyping based upon seven phylogenetically inf ormative regions within the 5'UTR, Of the 669 specimens, 416 (62.2%) tested positive for the presence of HCV RNA. Of these, 372 (89.4%) specimens were successfully classified into 11 HCV genotypes and subtypes after computer assisted analysis of the sequence data, Forty-four (10.6%) of the HCV RNA-p ositive specimens were not classifiable, the majority corresponding to low- titer specimens as determined by the Chiron Quantiplex HCV RNA 2.0 assay. A dditional comparative studies targeting the NS-5 region of the viral genome generally confirmed the accuracy and sensitivity of the 5'UTR-based classi fications, with the exception of the misclassification of a small number of type 1a cases as type 1b, We conclude that although the high sequence cons ervation within the 5'UTR results in the misclassification of a small numbe r of HCV subtypes, the overall gains of efficiency, the shorter turnaround time, the inclusion of contamination control measures, and the low rate of test failure compared to that of methods based on the NS-5 gene together co nstitute significant advantages over other techniques.