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

Issue: Aug 2010 Volume 48, Number 8

Diana Koletzki 1,
Stéphanie Dumont 1,
Hans Vermeiren 1,a,
Bart Fevery 1,
Pieter De Smet 2,b,
Lieven J. Stuyver 1,

1 Virco BVBA, Mechelen, Belgium
2 Tibotec-Virco, Mechelen, Belgium
a Present address: Galápagos, Mechelen, Belgium.
b Present address: MIPS NV, Zwijnaarde, Belgium.

Corresponding author: Dr. Diana Koletzki, Virco BVBA, Generaal De Wittelaan L11 B3, 2800 Mechelen, Belgium Phone: +32 15 461 356, Fax: +32 15 461 955,

Citation Information. Clinical Chemistry and Laboratory Medicine. Volume 48, Issue 8, Pages 1095–1102, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2010.236, Available online: 28/06/2010, August 2010

Publication History: Received: 14/8/2009; accepted: 11/3/2010; published online: 28/06/2010

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Clin Chem Lab Med 2010;48:1095–102.

Keywords hepatitis C virus, NS5B, sequencing, subtype

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