Characteristics of hepatitis C virus resistance in an international cohort after a decade of direct-acting antivirals

Abstract

Background & aims: 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.

Methods: 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.

Results: 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.

Conclusions: Drug resistance in HCV is frequent after DAA treatment failure. Previously unrecognized substitutions continue to emerge and remain uncharacterized.

Lay summary: 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.

Keywords: DAA; DAA, direct-acting antiviral; DCV, daclatasvir; DSV, dasabuvir; GT, genotype; HCV; LDV, ledipasvir; NI, nucleoside; NNI, non-nucleoside; NS5A; NS5AI, NS5A replication complex inhibitor; OR, odds ratio; PI, NS3 protease inhibitor; PIB, pibrentasvir; RAS; RASs, resistance-associated substitutions; SHARED, The Surveillance of Hepatitis C Antiviral Resistance, Epidemiology and methoDologies; SOF, sofosbuvir; SVR, sustained virologic response; VEL, velpatasvir; aOR, adjusted odds ratio; sFC, substitution frequency change; virologic failure.

Graphical abstract

Fig. 1

Prevalence of resistance-associated substitutions in DAA-naïve and -experienced patients with HCV. HCV sequences from virologic failures treated with combination regimens containing a PI (SIM, GZR, PAR/r, GLE, ASV, VOX, BOC, TVR), an NS5AI (LDV, DCV, EBR, VEL, OMB, PIB), or an NI (SOF)/NNI (DSV) were evaluated for the prevalence of RASs in NS3, NS5A, NS5B_SOF, and NS5B_DSV, respectively. HCV DAA-naïve patients were included to estimate the natural RAS prevalence. Patients who harbored ≥1 RAS variant listed in the 2020 EASL recommendations on treatment of hepatitis C were counted. ASV, asunaprevir; BOC, boceprevir; DAA, direct-acting antiviral; DCV, daclatasvir; DSV, dasabuvir; EBR, elbasvir; GLE, glecaprevir; GT, genotype; GT1-other, any GT1 subtypes except GT1a and GT1b; GZR, grazoprevir; LDV, ledipasvir; NA, not available; NI, nucleoside inhibitor; NNI, non-nucleoside inhibitor; NS5AI, NS5A inhibitor; OMB, ombitasvir; PAR/r, paritaprevir/ritonavir; PI, protease inhibitor; PIB, pibrentasvir; RAS(s), resistance-associated substitution(s); SIM, simeprevir; SOF, sofosbuvir; TVR, telaprevir; VEL, velpatasvir; VOX, voxilaprevir.

Fig. 2

Micro-representation of direct-acting antiviral class resistance after failing first-line therapies. A subset of virologic failures (n = 730) with sequences available from all 3 drug target genes (NS3, NS5A, and NS5B) were included for the analyses. Amino acid substitutions at all positions of the respective genes listed in the 2020 EASL recommendations on treatment of hepatitis C were included for the evaluation. PI RAS, NS5AI RAS, and NI RAS represent RASs selected in the HCV NS3, NS5A, and NS5B genes, respectively. Each circle represents the number of patients with detectable RAS in each drug class: purple, PI; green, NS5AI; and orange, NI. The size of the circle is proportional to the number of patients with detectable RASs. The intersecting regions represent dual- or triple-class resistance. NS5AI, NS5A inhibitor; NI, nucleoside inhibitor (sofosbuvir); PI, protease inhibitor; RASs, resistance-associated substitutions.

Fig. 3

NS5A resistance patterns in different genotypes. NS5A amino acid sequences obtained from patients treated with NS5A inhibitor-containing regimens before (DAA: -) and after (DAA: +) treatment were grouped according to their genotypes. The number of sequences before/after DAA treatment was 697/424 in GT1a, 262/451 in GT1b, 479/497 in GT3, 108/108 in GT4. The number of variant amino acids relative to the reference at each indicated position was enumerated and normalized to the total number of sequences in the respective genotypes. The amino acids are colored according to their side-chain biochemical properties: green = hydrophobic, purple = polar uncharged, orange = positively charged, red = negative charged, blue = special cases. The percentages of patients with detectable RASs in genotypes 1a, 1b, 3, and 4 are shown. The NS5AI-naïve sequences contained subtypes 3a/b/g/h and 4a/d/f/l/o/q/r/v. The NS5AI-exposed sequences contained subtypes 3a/b/g/h/k and 4a/b/d/f/g/k/n/o/q/r/v. DAA, direct-acting antiviral; GT, genotype; RASs, resistance-associated substitutions.

Fig. 4

Number of NS5A substitutions after direct-acting antiviral failure in different genotypes. NS5A sequences collected from patients at the follow-up visits after failing NS5AI-treatment were stratified according to their genotypes. There were 424 GT1a, 451 GT1b, 23 GT2, 497 GT3, 108 GT4, and 7 GT6. Amino acid variants relative to the genotype-specific reference strains were evaluated at positions 24, 26, 28, 29, 30, 31, 32, 38, 58, 62, 92, and 93 within NS5A. The number of detectable RASs for each sample was binned into the respective substitution subgroups. The number of patients in each substitution subgroup was summed and normalized to the total number in each genotype (percentage of patients, y-axis). GT, genotype; RASs, resistance-associated substitutions.

Fig. 5

Substitution frequency change before and after treatment in NS5A. The sFC relative to the reference amino acids before and after treatment was calculated independently at each of the first 200 amino acids within NS5A. The dotted line represents the mean sFC across the 200 amino acids in each genotype. The gray bar across the amino acids depicts a "genetic drift corridor" (2 SD from the mean sFC) to exclude variants with usual genetic drifts. Amino acid positions with a significant sFC before and after treatment are shown. A "positive" sFC represents an increase in the substitution levels (or decrease in the reference amino acid levels) after treatment; a "negative" sFC represents a decrease in the substitution levels (or increase in the reference amino acids) after treatment. Amino acid positions with a high-frequency change but without a specific substitution (i.e., only a mixture of substitutions) are not indicated. The amino acids of interest identified by the pairwise comparison are also noted (ˆ) for comparison. GT, genotype; sFC, substitution frequency change.