Net Emergence of Substitutions at Position 28 on NS5A of Hepatitis C Virus Genotype 4 in Patients Failing Direct-Acting Antivirals by Next-Generation Sequencing.

BACKGROUND: More data on resistance of HCV genotype (GT) 3 and 4 to Direct-Acting Antivirals (DAAs) are still needed. We investigated presence of Resistance-Associated Substitutions (RASs) pre- and post-treatment and their emergence under DAAs in HCV GT3 and GT4 infected patients failing DAA regimens by next-generation sequencing (NGS).

METHODS: Sanger sequencing and NGS were performed on NS5B and NS5A for plasma samples prior- and post-treatment of 13 patients. Positions implicated in resistance to anti-NS5A and anti-NS5B in literature were analysed.

RESULTS: No baseline RASs was detected on NS5B but one GT4r virus developed the mutation S282T at failure. On NS5A, we detected pre-existing RASs or polymorphisms in viruses of 6/10 patients (L28M for a GT4a, M28V for a GT4r, L30R for a GT4a, 2 GT4d,and 1 GT4r, T58P for a GT4d) by Sanger sequencing and in viruses of 7/10 patients by NGS. Additional baseline minority substitutions detected by NGS were Y93H in a GT3a, L28M in a GT4a and a GT4d, and L28F in a GT4d virus. At failure, these substitutions were found at frequency of 100%. The Y93H was detected alone at baseline while the L28M and L28F were accompanied by polymorphisms L30R or L30R + T58P.

CONCLUSIONS: The use of NGS in patients failing DAAs and infected by HCV GT3 and GT4 revealed the emergence of specific patterns of substitutions on NS5A and NS5B, in particular substitutions at position 28 on NS5A in GT4 virus, highlighting the need to list these substitutions in guidelines for resistance interpretation.