Direct-acting antiviral (DAA) regimens provide a cure in >95% of patients with chronic HCV infection. However, in some patients in whom therapy fails, resistance-associated substitutions (RASs) can develop, limiting retreatment options and risking onward resistant virus transmission. In this study, we evaluated RAS prevalence and distribution, including novel NS5A RASs and clinical factors associated with RAS selection, among patients who experienced DAA treatment failure. SHARED is an international consortium of clinicians and scientists studying HCV drug resistance. HCV sequence linked metadata from 3,355 patients were collected from 22 countries. NS3, NS5A, and NS5B RASs in virologic failures, including novel NS5A substitutions, were examined. Associations of clinical and demographic characteristics with RAS selection were investigated. The frequency of RASs increased from its natural prevalence following DAA exposure: 37% to 60% in NS3, 29% to 80% in NS5A, 15% to 22% in NS5B for sofosbuvir, and 24% to 37% in NS5B for dasabuvir. Among 730 virologic failures, most were treated with first-generation DAAs, 94% had drug resistance in ≥1 DAA class: 31% single-class resistance, 42% dual-class resistance (predominantly against protease and NS5A inhibitors), and 21% triple-class resistance. Distinct patterns containing ≥2 highly resistant RASs were common. New potential NS5A RASs and adaptive changes were identified in genotypes 1a, 3, and 4. Following DAA failure, RAS selection was more frequent in older people with cirrhosis and those infected with genotypes 1b and 4. Drug resistance in HCV is frequent after DAA treatment failure. Previously unrecognized substitutions continue to emerge and remain uncharacterized. Although direct-acting antiviral medications effectively cure hepatitis C in most patients, sometimes treatment selects for resistant viruses, causing antiviral drugs to be either ineffective or only partially effective. Multidrug resistance is common in patients for whom DAA treatment fails. Older patients and patients with advanced liver diseases are more likely to select drug-resistant viruses. Collective efforts from international communities and governments are needed to develop an optimal approach to managing drug resistance and preventing the transmission of resistant viruses.

The advent of direct-acting antivirals (DAAs) has transformed the treatment landscape of hepatitis C

Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1–6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving >100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load >5 log₁₀ IU/mL. This genotype-independent sequencing method resulted in >99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.