A new RT-nestPCR sequencing analysis has been developed for hepatitis C virus (HCV) resistance mutations in the viral NS3 protease region. This simple and reproducible method can be useful in identifying natural prevalence of HCV in routine diagnostic in Nordic country.
The current standard of HCV treatment consists of Pegylated Inferferon-γ (PEG-IFN) and Ribavirin (RBV) has limited efficacy and causes significant side effects. Only ~ 50% of HCV genotype 1 infected patients achieve a sustained virologic response after treatment. Most efforts to develop new anti-HCV agents for patients who fail PEG-IFN+RBV based therapies have focused on inhibitors of key HCV enzymes such as the HCV NS3 protease and the NS5B polymerase.
However, the success for these new HCV specific compounds could be limited because of the appearance of drug resistance during treatment. Monitoring of resistance mutations during treatment should therefore be an important issue, as has already been the case in HIV HAART therapy.
Since HCV has extremely high sequence diversity, natural pre-existing resistance mutations have been found in untreated HCV patients. Recently, a significant prevalence (>1%) of known protease resistance mutations, e.g. R155K or V36M etc, in genotype 1a patients was reported. In the Nordic countries HCV subtype 1a is the predominant form, found in 25 - 31% of the patients. Since Medivir protease inhibitors TMC 435 have shown activity against genotype 1 – 4, other subtypes than 1a, e.g. 2b and 3a should be attractive to study with respect of R155K prevalence. In the Nordic countries there is a high prevalence of 2b (10-22 %) and 3a (20-33%).
To identify the prevalence of pre-existing mutations that confer drug resistance to HCV protease inhibitors in untreated patients, especially mutations involving R155K substitution. We have recently developed a RT-nestPCR method where we sequence the HCV protease gene and perform interpretation of resistance mutations. HCV RNA extraction was performed with easyMAG and nine microliters out of the 60-μl RNA was used for reverse transcription. Ten microliters of cDNA was then used in the nested PCR. PCR primers were designed from the protease region and tested in previously genotyped clinical samples (1 to 4 genotypes), which covers known 10 protease resistance mutations in NS3, including R155K and V36M. The method can amplify genotype-specific fragment, e.g. 1a, 2b and 3a. The PCR amplification products were sequenced. Sequences were then analyzed and aligned for the presence of previously identified resistance substitutions in the NS3 protease by Seqscape software. The sequencing assay was successful in samples containing >500IU/mL HCV RNA. The accuracy of this method has been validated by sequencing. Our approach represents an improvement over previous RT-PCR methods, since typing is reproducible, accurate and simpler.
Our method can be used as a routine diagnostic and should be useful to monitor resistance directly during treatment. The results will be integrated in discussions of therapeutic and diagnostic strategies in the Nordic regions. Such diagnostic method has yet been developed in Sweden.