Performance comparison of the versant HCV genotype 2.0 assay (LiPA) and the abbott realtime HCV genotype II assay for detecting hepatitis C virus genotype 6

Abstract

The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5' untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1.

FIG 1

Phylogenetic trees of the 222-bp NS5B sequences of 60 CCgenos HCV variants (A) and 355-bp core sequences of 3 variants (B). The bootstrap percentages are shown at the node of each main branch. The bar at the lower left indicates the genetic divergence. The reference sequences are labeled with black circles.

FIG 2

Alignment of the 5′ UTR sequences from samples with atypical LiPA 2.0 line patterns. The regions that reacted with the LiPA probes on lines 7, 17, 18, and 21 (30) are boxed and labeled at the bottom right outer corner of the box. Nucleotides that are identical to those of the consensus sequence are indicated by dots; gaps introduced in the sequences to preserve alignment are indicated by horizontal lines. At the left are the subtypes and CCgenos sample identifications (IDs) or accession numbers of the standard sequences.

FIG 3

Alignment of the 5′ UTR sequences from the samples with inconsistent or indeterminate results using Realtime II version 1.00 and version 2.00 assays. The possible probe hybridization regions of the Realtime II assay are boxed. Box a, Three representative genotype 6a sequences correctly determined to be genotype 6 using the Realtime II; box b, 19 sequences misclassified as a mixture of genotypes 6 and 1 using the Realtime II version 1.00 assay but correctly determined using version 2.00 assay; box c, 4 genotype 6a sequences with indeterminate results using the Realtime II assay; box d, 5 genotype 6n sequences with indeterminate results using the Realtime II assay; box e, 1 genotype 6e sequence misclassified as genotype 1 using the Realtime II assay.