Real-time PCR tests are faster and more cost-effective methods that detect very low VL (10-15 IU/ml). Realtime PCR can accurately quantify HCV RNA levels over a linear range exceeding 6 logs (10 IU/mL to 100 million IU/mL). Therefore, a single test result serves the purpose of both quantitative and qualitative HCV NAT with similar sensitivity.
Minimal residual viremia might be predictive of post-treatment relapse. Rules for treatment duration and early discontinuation were mainly established with NAT assays with a detection limit of 50 IU/ml. Lower detection limits (undetectable VL defined as less than 15 IU/ml) did not significantly influence the SVR rates after shortened treatment duration, for patients with RVR (82% for G1 infected patients treated for 24 weeks and 95% for G2/3 infected patients treated for 16 weeks). The assay’s choice should be tailored to the dominant genotype in the study population, as some assays have been reported to substantially underestimate HCV RNA levels in certain genotypes. The same assay should be used for all samples from a single patient and, whenever possible, throughout the clinical development program.
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HCV genotyping
HCV genotyping should be performed in all HCV-infected persons prior to treatment initiation in order to plan for the duration of therapy and to estimate the likelihood of response. An assay based on viral population sequencing, reverse hybridization or real-time PCR, which has been validated for correct subtyping of at least subtypes 1a and 1b should be used. Two commercial assays are frequently used for HCV genotypes: – TruGene™ HCV Genotyping kit (Siemens Healthcare Diagnostics Division, Tarrytown, NY), based on direct sequence analysis of the 5’ UTR (untranslated region), – Versant™ HCV Genotype Assay LiPA (version I; Siemens Medical Solutions, Diagnostics Division, Fernwald, Germany), based on reverse hybridization analysis with genotype-specific oligonucleotide probes binding to the 5’ UTR. A second generation line probe assay (LiPA) contains probes targeting both the 5’ UTR and the core regions of the viral genome, improving the accuracy of discrimination between subtypes 1a and 1b. A new test which uses real-time PCR technology for HCV genotyping has recently been developed by Abbott Molecular.
HCV resistance monitoring. Like HIV and HBV, HCV has a high replication rate and replicates via an error-prone mechanism, generating resistance variants. In the near future, HCV resistance testing will most probably be part of the clinical monitoring algorithms. Assays based on viral population sequencing require a minimum VL of 1000 IU/mL and define the most common mutation patterns, without detecting the lowfrequency variants. Accurate determination of viral genotype/subtype is critical for resistance testing during the development of new direct-acting antivirals (DAAs).
Pretreatment samples are analyzed to detect known or novel predominant viral polymorphisms and to provide the comparator for mutations emerging at later time points, during or after treatment. On-treatment viremic samples are analyzed to determine specific changes associated with decreased susceptibility and virologic failure. Post-treatment samples are analyzed for persistence or loss of resistant variants and may help distinguish between re-infection and relapse.