Development and evaluation of an automated hepatitis C virus NS5B sequence-based subtyping assay

Virco BVBA, Mechelen, Belgium.
Clinical Chemistry and Laboratory Medicine (Impact Factor: 2.71). 08/2010; 48(8):1095-102. DOI: 10.1515/CCLM.2010.236
Source: PubMed


Hepatitis C virus (HCV) genotyping and accurate subtype determination is becoming increasingly important to better understand viral evolution, the development of resistance to STAT-C, and possibly even for the treatment and management of chronic HCV-infected patients.
A subtyping assay based on a 329-bp sequence of the NS5B region, together with an automated subtype interpretation tool was developed. Clinical samples of the six major genotypes were used to assess assay performance characteristics.
The NS5B BLAST-based subtyping assay showed clinical sensitivity for amplification of 89% (n=603 random samples). Assessment of analytical sensitivity of amplification for genotypes 1, 2, 3 and 4 revealed a suitable performance for high viral load samples and decreased only with low viral loads. The results were 100% and 99% accurate at the genotype and subtype level, respectively. A concordance of 97% on genotype level and 62% on subtype level was observed by comparison with subtype results from 5' non-coding-based assays with a panel of 276 isolates.
The HCV NS5B subtyping assay has been validated for research use. Based on its performance, it is the method of choice in cases where subtype rather than genotype information is needed.

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    • "However, current HCV NS5B-based genotyping uses only partial short nucleic acid sequences of NS5B. Koletzki et al. [32] used a 329-bp NS5B gene fragment (nucleotides 8280–8610 in HCV sequence) to classify HCV and showed an 89% clinical sensitivity for amplification. Cantaloube et al. [30] used 339-bp nucleotide segments (nucleotides 8370–8708) in the NS5B region to classify HCV and achieved a 98.32% of sensitivity for TABLE 2. The theoretical amplification ability of the potential primers for all NS5B from hepatitis C virus (HCV) database "
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    ABSTRACT: Nucleotide sequencing of the phylogenetically informative region of NS5B remains the gold standard for HCV genotyping. Here we developed a new methodology for sequencing new NS5B regions to increase the accuracy and sensitivity of HCV genotyping and subtyping. The 8 new primers were identified by scanning the full-length NS5B regions from 1127 HCV genomic sequences found in HCV databases. The ability of each pair of primers to amplify HCV subtypes were scored, and the new primers were able to amplify the NS5B region better than the previously used primers, therefore more accurately subtyping HCV strains. Sequencing the DNA amplified by the new primer pairs can specifically and correctly detect the 5 standard HCV subtypes (1a, 2a, 3b, 6a, and 1b). We further examined patient samples and found that the new primers were able to identify HCV subtypes in clinical sample with high sensitivity. This method was able to detect all subtypes of HCV in 567 clinical samples. Importantly, 3 novel HCV subtypes (1b-2a, 1b-2k, and 6d-6k) were identified in the samples, which have not been previous reported in China. In conclusion, sequencing the NS5B region amplified by the new NS5B primers is a more reliable method of HCV genotyping and a more sensitive diagnostic tool then sequencing using the previously described primers, and could identify new HCV subtypes. Our research is useful for clinical diagnosis, guidance of clinical treatment, management of clinical patients, and studies on the epidemiology of HCV. Copyright © 2015. Published by Elsevier Ltd.
    Clinical Microbiology and Infection 06/2015; 21(9). DOI:10.1016/j.cmi.2015.05.034 · 5.77 Impact Factor
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    ABSTRACT: With the development of freeware bioinformatics software as well as the availability of web-based software, it is now possible to use various bioinformatics tools to identify viral subtypes such as hepatitis C virus (HCV). This study aimed to demonstrate the role of bioinformatics tools in identifying HCV subtypes and to compare the accuracy of HCV-1 subtyping by 5'UTR PCR-RFLP analysis and DNA sequencing. From a clinical viewpoint, accurate genotype and subtype identification of HCV are important because this may be used as guide for deciding which therapy is appropriate to use for a particular patient. From 2005 up to 2008, we had a total of 30 HCV genotype 1 (HCV-1) positive samples. HCV-1 subtypes were identified by an in-house PCR-RFLP analysis and through direct nucleic acid sequencing using nested primers specific to the 5'UTR and non-structural 5B (NS5B) region. Bioinformatics tools play an important role in identifying HCV-1 subtypes by predicting the size of the amplicon; determining the specific restriction enzyme to cut a given nucleic acid sequence; viewing and editing the electropherogram; aligning nucleotide sequences with prototypes; searching for identical sequences; and understanding the evolution and relationships of various subtypes. The HCV nucleotide sequences reported in this study have been deposited to GenBank. Overall, this information can be utilized to generate molecular diagnostic tests in the future.
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    ABSTRACT: We compared Hepatitis C virus (HCV) genotyping by direct sequencing of the non-structural 5b region (NS5b) and a commercial PCR/hybridization method based on the conserved 5´-untranslated region (5'UTR). One hundred twenty HCV containing plasma samples were analyzed by NS5b sequencing with focus on samples with undetermined results or 1b subtype identification in the used combination of Cobas® AmpliPrep/Cobas® TaqMan96® PCR and subsequent Versant® HCV Genotype 2.0 Assay (LiPA). There was 100% concordance between the two methods for genotyping but only 83% for subtyping. Seventeen samples were designated 1b by hybridization but subtype 1a by NS5b sequencing. This is a general 5'UTR problem as the discordant results were additionally confirmed by 5'UTR sequencing. Thus our routine combination not only misclassified 38.6% of subtype 1a isolates as 1b but in contrast to NS5b sequencing was The applied 5'UTR methods allow the rapid determination of HCV genotypes but failed to correctly identify the subtype in many samples. This has implications for epidemiological studies or forensic evaluation of chains of infection and NS5b sequencing therefore is our method of choice under those circumstances.
    Minerva medica 08/2012; 103(4):293-7. · 0.91 Impact Factor
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