br Background To treat chronic

Background To treat chronic HCV genotype (GT) 1-infected patients, telaprevir (TLP) or boceprevir (BOC), first generation protease inhibitors (PIs), were used in combination with pegylated-interferon (PEG-IFN) and ribavirin (RBV) triple therapy [1], [2]. The main weaknesses of the first-generation PIs are their low genetic barrier to resistance and the fact that their effectiveness is limited to GT-1 patients [2], [3], [4], [5]. A second-wave of first generation PIs has a higher barrier to resistance; better activity against multiple genotypes, except GT-3 [6]; more convenient dosing schedules; and improved safety and tolerance. Second-generation PIs are broadly active against all genotypes and against viral isolates that carry resistance mutations for first-generation PIs [7]. In combination with PEG-IFN and RBV, the new PIs appear to achieve greater sustained virologic response (SVR) rates than the first-generation PIs [8]. IFN-free options combining several direct-acting antiviral (DAA) regimens progressively replaced the use of PEG-IFN with a highly sustained virological response [9], [10]. Not surprisingly, resistance has been demonstrated for DAAs in clinical tests, specifically for PIs and NS5A polymerase inhibitors [11]. To our knowledge, the possibility of the long-term persistence of NS3 resistance mutations and their impact on PI re-treatment is unknown. Ultra-deep pyrosequencing (UDPS) techniques with a cut-off below the standard 20% threshold of Sanger sequencing may provide additional information regarding the tranylcypromine sale and importance of low-level variants for the prediction of the viral response to HCV inhibitors [12].
Objectives
Results
Discussion In our study, the most prevalent minority mutations were V36M, R155K and A156T, and all of these mutations were detected with a relatively high mutant viral load. Several studies have demonstrated the presence of natural minority mutations associated with resistance to BOC and TLP among treatment-naïve HCV infected individuals [21], [22], [23]. TLP or BOC-RAVs (V36M, R155K/T or A156T) were detected after therapy cessation in 18% of patients (4/22) at a range of 1.2–99% of the viral population. The results from a previous study demonstrated that the frequency of TLP RAVs declined after treatment failure to low or undetectable levels and were no longer detectable in the majority of patients during follow-up using deep-sequencing [24]. Our study extends these results, as we demonstrated the presence of persistent variants in 18% of patients (4/22) after 1 year, as measured using the UDPS technology. Nevertheless, there are some limitations to our study that need to be considered. At VF, the results from three patients (patient 12, 16 and 20) were excluded from the analysis due to unsuccessful amplification or UDPS assay limitations. Although the persistence of NS3/4A variants was detected up to 11 months, additional follow-up data are necessary to determine the true duration or persistence of NS3/4A resistance. Moreover, to detect low frequency RAVs, it would be necessary to concentrate the RNA from higher volumes of plasma during extraction and to use a larger quantity of RNA for PCR to preserve viral diversity before amplification. The key PIs resistance mutations, V36M, R155K or R155T, in GT 1a-infected patients were detected in baseline or follow-up samples [19], [25]. This result suggests that in patients 8, 10, 11 and 19, the persistent variants sequenced during follow-up are naturally occurring variants that arose after treatment or VF. Previous studies have suggested that certain types of mutations, such as V36A/M or R155K/T/Q, could have an impact on TLP or BOC treatment failure (Table 4) [12], [19], [23], [24], [25]. In our study, the V36M, R155K, R155T and A156T baseline relative mutant viral loads of patients with persistent RAVs was significantly higher (P<0.001) than those of patients without persistent RAVs. The presence of RAVs in this cohort significantly impacted the persistence of the RAVs.