Specific Detection of Naturally Occurring Hepatitis C Virus Mutants with Resistance to Telaprevir and Boceprevir (Protease Inhibitors) among Treatment-Naïve Infected Individuals

Abstract

The use of telaprevir and boceprevir, both protease inhibitors (PI), as part of the specifically targeted antiviral therapy for hepatitis C (STAT-C) has significantly improved sustained virologic response (SVR) rates. However, different clinical studies have also identified several mutations associated with viral resistance to both PIs. In the absence of selective pressure, drug-resistant hepatitis C virus (HCV) mutants are generally present at low frequency, making mutation detection challenging. Here, we describe a mismatch amplification mutation assay (MAMA) PCR method for the specific detection of naturally occurring drugresistant HCV mutants. MAMA PCR successfully identified the corresponding HCV variants, while conventional methods such as direct sequencing, endpoint limiting dilution (EPLD), and bacterial cloning were not sensitive enough to detect circulating drug-resistant mutants in clinical specimens. Ultradeep pyrosequencing was used to confirm the presence of the corresponding HCV mutants. In treatment-naïve patients, the frequency of all resistant variants was below 1%. Deep amplicon sequencing allowed a detailed analysis of the structure of the viral population among these patients, showing that the evolution of the NS3 is limited to a rather small sequence space. Monitoring of HCV drug resistance before and during treatment is likely to provide important information for management of patients undergoing anti-HCV therapy.

Hepatitis C virus genetic variability and evolution

“…Moreover, these technologies are becoming rapidly accessible all around the world which will likely revolutionise the field of molecular epidemiology [110] . It is worth mentioning that these technologies offer, as already discussed, several advantages over conventional methods, such as consensus sequencing, bacterial cloning, and endpoint limiting dilution [107] . Furthermore, as the development of a variety of software and algorithms capable of handling the massive amount of data generated by NGS platforms is increasing in parallel with the advances in these technologies, it will likely expedite the implementation of such approaches in a variety of settings in the near future [110] .…”

“…This method provides a good idea of the major sequences present, but unfortunately it cannot detect minor variants that are present at a frequency below 20%-25% [106] . With the development of deep sequencing technologies, detection of drug-resistant variants became more sensitive allowing the identification of variants present at very low frequencies (about 0.1%-1%) [107][108][109] . For HCV, the deep sequencing method was first used to detect emergence of NS3 mutants.…”

“…This way it was clearly demonstrated that de novo telaprevir-resistant NS3 mutants arose in mice injected with wild-type HCV only 2 wk after the beginning of treatment [108] . Deep sequencing was also used to confirm results of naturally occurring drug-resistant HCV mutants detected by a novel mismatch amplification mutation assay polymerase chain reaction [107] .…”

“…In this case NGS was used to determine intra-host viral genetic variation by deep sequencing the HCV hypervariable region, which allowed a detailed analysis of the structure of the viral quasispecies in the patients' population under study [110] . NGS approaches are powerful methods that allow a rather comprehensive analysis of the intra-host viral genetic variation [107] . Moreover, these technologies are becoming rapidly accessible all around the world which will likely revolutionise the field of molecular epidemiology [110] .…”

Hepatitis C virus resistance to new specifically-targeted antiviral therapy: A public health perspective

“…In addition, in single-and 3-d multiple-ascending-dose studies in HCV genotype 1a-or 1b-infected patients, a predominant pre-existing NS3 baseline polymorphism (NS3-Q80K) had ambiguous effects, but no clinically-relevant resistance-associated variants emerged in these clinical studies [53] . Finally, the large turnover and population size, together with the high mutation rate of the virus implicates that HCV variants resistant to new DAAs may be present as minority species (not detectable by direct population sequencing) within the complex pool of viral genomes circulating in a single patient [33,54,55] . Pre-existing variants resistant to Boceprevir or Telaprevir may impair virologic response before treatment is initiated [56,57] .…”