Accurate hepatitis C virus (HCV) RNA quantification is mandatory for the management of chronic hepatitis C therapy. HCV RNA level monitoring during antiviral treatment with pegylated alpha interferon (IFN-α) and ribavirin is key to assess virologic responses, guide treatment duration, and decide futility (1
). In patients infected with HCV genotype 1, treatment is now based on a triple combination of pegylated IFN-α, ribavirin, and one of two protease inhibitors, either telaprevir or boceprevir. A rapid virologic response (defined as an undetectable HCV RNA at week 4 of protease inhibitor administration) is a strong predictor of sustained viral eradication with this triple combination (3
). Futility rules have been established in order to prevent unnecessary exposure to the protease inhibitor and to avoid adverse events, the emergence of viral resistance, and useless costs. They are defined by an HCV RNA level of >1,000 international units (IU)/ml at week 4 or 12 or detectable at week 24 for telaprevir or by an HCV RNA level of ≥100 IU/ml at week 12 or detectable at week 24 for boceprevir (3
Numerous new direct-acting antiviral (DAA) drugs and host-targeted agents (HTA) that block the HCV life cycle have reached early to late clinical development. A number of trials are ongoing in treatment-naive and treatment-experienced patients infected with HCV genotypes 1 to 4, including (i) triple combinations of pegylated IFN-α, ribavirin, and a DAA or HTA, (ii) quadruple combinations of pegylated IFN-α, ribavirin, and two DAAs, or (iii) all-oral, IFN-free DAA/HTA-based regimens (10
). With these new therapies, HCV RNA level monitoring is and will remain the most useful parameter to assess treatment responses and guide treatment decisions.
Assays based on real-time PCR are recommended for HCV RNA detection and quantification by international Clinical Practice Guidelines and now widely used in clinical virology laboratories (1
). Among them, the Cobas AmpliPrep/Cobas TaqMan HCV test (CAP/CTM HCV; Roche Molecular Systems, Pleasanton, CA) is fully automated, making use of automated extraction with the Cobas AmpliPrep and automated real-time PCR amplification and quantification with the Cobas TaqMan device. The first-generation of this assay, CAP/CTM HCV, faced a number of technical issues (11
). In particular, we showed that approximately 30% of HCV genotype 4 infections were underestimated by >1 log10
IU/ml with this assay (12
). In addition, CAP/CTM HCV failed to detect HCV RNA in patients infected with HCV genotype 4 with high HCV RNA levels in other assays (11
). Sequence analysis of the 5′ untranslated region (5′UTR) of HCV genome, the region targeted by the assay primers and probe, allowed us to identify single nucleotide polymorphisms at 5′UTR positions 145 and 165 as the main causes of underestimation or lack of detection of HCV RNA in patients infected with HCV genotype 4 (14
). We confirmed this finding by generating in vitro
RNA transcripts from a plasmid containing either wild-type or mutated sequences and showing that the presence of one of these substitutions substantially reduced the HCV RNA level measured with CAP/CTM HCV, whereas the presence of both substitutions abolished HCV RNA detection (14
HCV genotype 4 is currently increasing in incidence and prevalence in the intravenous drug user community in Western Europe and many industrialized areas (16
). It now represents approximately 10% of cases in France (17
). HCV genotype 4 infection is difficult to treat and thus an important target of new HCV drug development. Therefore, accurate detection and quantification of HCV genotype 4 RNA is mandated in clinical trials with new HCV therapies and in clinical practice.
A second generation of the CAP/CTM assay for HCV RNA, the Cobas AmpliPrep/Cobas TaqMan HCV test, version 2.0 (CAP/CTM HCV v2.0), has been recently released. Several changes have been made, including a required volume of 650 μl instead of 850 μl in version 1, and the addition of a second probe to improve quantification of genotype 4 RNA. The aim of the present study was to evaluate the ability of the newly developed CAP/CTM HCV v2.0 assay to accurately quantify HCV RNA in a large series of patients infected with different subtypes of HCV genotype 4, frequently encountered in clinical practice.