Dynamics of HCV genotype 4 resistance-associated variants during virologic escape with pIFN/RBV+daclatasvir: a case study using ultra deep pyrosequencing.

Background: Daclatasvir (DCV) is an approved NS5A inhibitor with potent anti-HCV activity and broad genotype coverage. DCV resistance-associated variants (RAVs) have been described for patients infected with genotype (GT) 1, but increased GT4 prevalence in European countries as a result of immigration has boosted interest in this genotype.

Objectives: Establishment of NS5A variability in treatment-naive patients with HCV genotype 4 infection and a case study of the dynamics of resistance-associated variants in a virologic failure receiving pIFN/RBV+DCV, as assessed by ultra-deep sequencing.

Study Design: Five treatment-naïve GT4 patients (GT4a [n = 1], GT4d [n = 3], GT4o [n = 1]) were evaluated for inclusion in the COMMAND-4 study and treatment with pIFN/RBV±DCV.

Results: Patient (Pt) 1 received pIFN/RBV; Pts2-4 received pIFN/RBV + DCV; Pt5 was a screening failure. Pt1 relapsed; Pt2 experienced breakthrough at Wk4; Pts3 and 4 achieved a sustained virologic response. No substitutions associated with DCV-resistance were detected at baseline. In terms of viremic time points for Pts1 and 2, the extent of NS5A diversity pre-treatment was not significantly related to viral load (r = -0568; p = 0.035). In Pt2, multiple substitutions associated with DCV-resistance were observed after breakthrough at NS5A amino acid positions 28, 31 and 93. These substitutions were frequently observed on the same haplotype (L28S + M31I = 55.52, 82.50, and 99.36% at Wk4, 8 and 9; L28S + M31I + Y93H = 11.77, 5.01 and <0.6% at Wk4, 8 and 9).

Conclusions: This is the first report to describe DCV-resistance in patients infected with GT4d, supporting a possible role for a recently described RAV (L28S), and presenting the dynamics of HCV quasispecies during therapy failure, with indications of changes of diversity and association of mutations.