A Novel Diagnostic Target in the Hepatitis C Virus Genome

Background
Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5′-noncoding region (5′-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.

Methods and Findings
In this study we determined by de novo sequencing that the 3′-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10−9 IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5′-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.

Conclusion
This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.

Citation: Drexler JF, Kupfer B, Petersen N, Grotto RMT, Rodrigues SMC, et al. (2009) A Novel Diagnostic Target in the Hepatitis C Virus Genome. PLoS Med 6(2): e1000031. doi:10.1371/journal.pmed.1000031

Academic Editor: Paul Klenerman, Oxford University, United Kingdom

Received: September 8, 2008; Accepted: December 24, 2008;

Published: February 10, 2009

Copyright: © 2009 Drexler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The Brazilian study was partially supported by a donation of reagents from Qiagen, Germany. The German study was supported by the German Ministry of Health (BMGS) as a part of funding of the National Reference Centre for Tropical Infections at the Bernhard Nocht Institute and by European Union contract number SSPE-CT-2005–022639. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: bDNA, branched DNA; HCV, hepatitis C virus; IU, international units; LANL, Los Alamos National Laboratory; LOD, limit of detection; NAT, nucleic acid testing; NCR, noncoding region; RT-PCR, reverse transcriptase PCR; SD, standard deviation; X-tail RT-PCR, X-tail real-time RT-PCR assay.

* To whom correspondence should be addressed. E-mail: drosten@virology-bonn.de