Hepatitis C Virus Genotyping Based on 5′ Noncoding Sequence Analysis (Trugene)

Abstract

Hepatitis C virus (HCV) genotyping of samples from 184 patients with chronic HCV infection by the Trugene 5NC genotyping kit, based on sequence analysis of the 5 noncoding region (5 NCR), and the InnoLiPA assay was evaluated. In addition to these methods, the 184 samples were also analyzed by sequencing of part of the NS5B of the HCV genome after in-house PCR amplification, as a means of validating results obtained with the 5 NCR. The distribution of the genotypes typed by NS5B sequence analysis was as follows: 1a, 41 samples; 1b, 58 samples; 1d, 1 sample; 2a, 5 samples; 2b, 2 samples; 2c, 7 samples; 3a, 46 samples; 4a, 7 samples; 4c, 1 samples; 4e, 9 samples; 5a, 6 samples; 6a, 1 sample. The Trugene and InnoLiPA assays gave concordant results within HCV types in 100% of cases. The ability to discriminate at the subtype level was 76 and 74% for the Trugene and the InnoLiPA assays, respectively.Hepatitis C virus (HCV) is considered the major cause of posttransfusion non-A, non-B hepatitis. The viral genome, a positive-sense single-stranded RNA of about 9,400 nucleotides (5), is characterized by a high genetic heterogeneity like other RNA viruses. HCV isolates show four levels of genetic variability: types, subtypes, isolates, and quasispecies (3). An HCV genotype is therefore used with the histological results from liver biopsy and viral load for counseling individual patients about the risk-benefit ratio of therapy (17,21,22). HCV genotypes are distributed differently depending on geography and the etiology of infection (15,25). For the purpose of nomenclature, it has been proposed that HCV be classified into types, corresponding to the main branches in the phylogenetic tree, and subtypes, corresponding to the more related sequences within the major groups (23,24). HCV genotypes can be established by methods based on PCR typing and/or serological typing (2, 4, 16). The high degree of conservation in the 5Ј noncoding regions (5Ј NCR) has made it the target of choice for reverse transcriptase PCR-based detection assays. Moreover, several PCR typing methods, such as reverse dot blot (26), restriction fragment length polymorphism (10), cleavase fragment length polymorphism, dideoxy fingerprinting, heteroduplex mobility analysis (28), and hybridization to genotype-specific probes (18), exploit sequence-based differences and/or differences in secondary structure of the 5Ј NCR for HCV genotyping (9). Nucleotide sequence analysis is the reference method for identifying different genotypes of HCV (9, 13). However, because this method is expensive and timeconsuming and requires special equipment for sequencing, it has been restricted to the research setting and considered impractical for large clinical studies. A standardized sequencing assay has recently been developed for routine determination of HCV genotypes. In order to determine whether direct sequencing could be a routine tool for the determination of hepatitis C virus genotype, we assessed this first commercial type of HCV genotyping method (Trugene 5ЈNC HCV genot...

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

“…Also, in accordance with previous studies 6,8,12,14,17,18,21 , discrepant results at the subtype level, mainly subtypes 1a and 1b, were found here (two samples identified as 1a by NS5B sequencing were subtyped as 1b by 5' NC sequencing). Although both regions provide similar results at the genotype level, which is adequate for clinical practice, especially in the management of antiviral therapy, the NS5B sequencing appears to be more useful for epidemiological investigations.…”

“…As observed by other authors 6,8,12,18 , no difference at the genotype level was found for the 42 samples that could be successfully amplified and sequenced in the the 5' NC and NS5B regions. Also, in accordance with previous studies 6,8,12,14,17,18,21 , discrepant results at the subtype level, mainly subtypes 1a and 1b, were found here (two samples identified as 1a by NS5B sequencing were subtyped as 1b by 5' NC sequencing).…”

Geographic distribution of hepatitis C virus genotypes in Brazil

“…Sequencing the HCV 5'UTR has proved effective in discriminating the major genotypes, despite some mistyping in the annotation of subtypes (34,35). Sequencing other viral regions is indicated for more accurate subtyping and molecular epidemiological studies (36), but this would require re-amplification of all samples after RNA detection with different pairs of primers.…”

Detection of HCV by PCR in serum and PBMC of patients with hepatitis C after treatment

“…Primers KY78 and KY80 were used in the reaction, as previously described, to amplify a sequence of 244 nucleotides within the conserved 5'UTR of the HCV genome. Amplified DNA was detected using target-specific oligonucleotide probes that permitted independent identification of HCV amplicons and internalcontrol amplicons [11,12].…”