Comparison of Hepatitis C Virus NS5b and 5′ Noncoding Gene Sequencing Methods in a Multicenter Study

Abstract

A national evaluation study was performed in 11 specialized laboratories with the objective of assessing their capacities to genotype hepatitis C virus (HCV) and define the applicability of a given genotyping method. The panel consisted of 14 samples positive for HCV RNA of different genotypes (including 3 samples with two different artificially mixed genotypes) and 1 HCV-negative sample. Seventeen sets of data were gathered from the 11 participating laboratories. The sensitivities ranged from 64.3 to 100% and from 42.7 to 85.7% for the methods that used sequencing of the NS5b region and the 5 noncoding (5 NC) region, respectively. When the data for the artificially mixed samples were excluded, NS5b genotyping gave correct results for 80% of the samples, 1.7% of the samples were misclassified, and 18.3% of the samples had false-negative results. By 5 NC-region genotyping methods, 58.3% of the results were correct, 29.7% were incomplete, 8.3% were misclassifications, 1.2% were false positive, and 2.4% were false negative. Only two procedures based on NS5b sequencing correctly identified one of the three samples with mixtures of genotypes; the other methods identified the genotype corresponding to the strain with the highest viral load in the sample. Our results suggest that HCV 5 NC-region genotyping methods give sufficient information for clinical purposes, in which the determination of the subtype is not essential, and that NS5b genotyping methods are more reliable for subtype determination, which is required in epidemiological studies.

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

“…However, the genotyping methods employed in those studies, which were based on 5' noncoding (5'NC) region analyses, did not permit the correct identification of HCV subtypes (10)(11)(12)(13)(14)(15)(16)(17). To our knowledge, the present study is the first to analyze Brazilian HCV samples using two genotyping methods: line probe assay (LiPA, based on the 5'NC region) and the nucleotide sequencing analysis of the nonstructural 5B (NS5B) region, which has been used as a reference method for accurate genotyping (10)(11)(12)(13)(14)(15)(16)(17). Thus, our objective was to determine which HCV genotypes and subtypes occur in hemodialysis patients in Central Brazil and also to compare the genotyping efficiency of LiPA and direct sequencing of the NS5B region.…”

“…On the other hand, this region does not contain sufficient information for the recognition of all different types and subtypes. Thus, in the present study we chose the NS5B region for sequence analysis since this region appears to be much more accurate for identifying variations in the nucleotide sequences (10)(11)(12)(13)(14)(15)(16)(17).…”

Hepatitis C: genotyping

“…The HCV viral genotype can be determined in a clinical sample in different forms, and the regions of HCV genome considered appropriate include the core, E1, NS4 and NS5 regions, as well as the 5'UTR, as mentioned in innumerable studies [4,5].…”

“…The most direct method, considered the gold standard, is the sequencing of the HCV genome in a certain region, sufficiently divergent for different genotypes and subtypes to be distinguished [5].…”

“…Therefore, it depends on samples that present viral particles for analysis. These techniques are: sequencing, hybridization with genotype-specific probes, and real-time polymerase chain reaction (PCR) [5,7,9].…”