High frequency of multiclass HCV resistance-associated mutations in patients failing direct-acting antivirals: real-life data.

BACKGROUND: Direct-acting antiviral (DAA) therapy has dramatically increased sustained virological response rates in HCV-infected patients. However, resistance-associated substitutions (RAS) interfering with NS3- and NS5A-targeted therapy, still emerge. This real-life study analysed the type and frequency of RAS in rare cases of patients failing DAA regimens in 12 clinical centres in Israel. METHODS: Blood samples and clinical data from 49 patients who failed various DAAs were collected. RAS identified in the NS3 and NS5A regions by population (Sanger) and next-generation sequencing (NGS) were compared by treatment regimen and HCV subtypes. RESULTS: The majority (71.4%, 35/49) of patients were infected with the genotype (GT)1b strain, while 12.2% (6/49) carried GT1a and 14.3% GT3a/b (7), GT4a (1) and GT1b/GT3a. RAS were identified in 85.7% (42/49) of failures, of which 90.5% (38/42) were clinically relevant RAS (known to be associated with a specific GT and DAA in patients failing therapy or those with more than twofold change in in vitro replicon assays). The most abundant RAS were 168A/E/Q/G/N/V (32.6%, 16/49) identified in NS3, and 93H/N (61.2%, 30/49), 31I/M/V (34.7%, 17/49) and 30R/H/K (12.2%, 6/49), identified in NS5A. Significantly more clinically relevant RAS were identified in NS5A (82.2%, 37/45) than in NS3 (35.7%, 10/28; P<0.01). While RAS were identified in all GT1a, GT3b and GT4a failures (100%, 10/10), only 71.8% (28/39) of GT1b or GT3a failures had RAS (P=0.09). In four cases, NGS identified additional clinically relevant RAS and in one patient, NGS deciphered coexistence of GT3a and GT1b infections. CONCLUSIONS: Our findings, together with additional real-life data, will contribute to the optimization of retreatment in DAA failure, when cost-related and suboptimal regimens must be employed.

Characterization of hepatitis B and delta coinfection in Israel.

BACKGROUND: Characteristics of hepatitis B (HBV) and delta (HDV) coinfection in various geographical regions, including Israel, remain unclear. Here we studied HDV seroprevalence in Israel, assessed HDV/HBV viral loads, circulating genotypes and hepatitis delta antigen (HDAg) conservation. METHODS: Serological anti HDV IgG results from 8969 HBsAg positive individuals tested in 2010-2015 were retrospectively analyzed to determine HDV seroprevalence. In a cohort of HBV/HDV coinfected (n=58) and HBV monoinfected (n=27) patients, quantitative real-time PCR (qRT-PCR) and sequencing were performed to determine viral loads, genotypes and hepatitis delta antigen (HDAg) protein sequence. RESULTS: 6.5% (587/8969) of the HBsAg positive patients were positive for anti HDV antibodies. HDV viral load was >2 log copies/ml higher than HBV viral load in most of the coinfected patients with detectable HDV RNA (86%, 50/58). HDV genotype 1 was identified in all patients, most of whom did not express HBV. While 66.6% (4/6) of the HBV/HDV co-expressing patients carried HBV-D2 only 18.5% (5/27) of the HBV monoinfections had HBV-D2 (p=0.03). Higher genetic variability in the HDAg protein sequence was associated with higher HDV viral load. CONCLUSIONS: The overall significant prevalence of HDV (6.5%) mandates HDV RNA testing for all coinfected patients. Patients positive for HDV RNA (characterized by low HBV DNA blood levels) carried HDV genotype 1. Taken together, the significant HDV seroprevalence and the lack of effective anti-HDV therapy, necessitates strict clinical surveillance especially in patients with higher HDV viral loads and increased viral evolution.

HCV genotype-1 subtypes and resistance-associated substitutions in drug-naive and in direct-acting antiviral treatment failure patients.

BACKGROUND: Direct-acting antiviral (DAA) treatment regimens and response rates of patients with HCV genotype-1 (GT1) are currently considered subtype-dependent. Identification of clinically relevant resistance-associated substitutions (RASs) in the NS3 and NS5A proteins at baseline and in DAA failures, may also impact clinical decisions. METHODS: In a multicentre cohort study (n=308), NS3 or NS5B sequencing (n=248) was used to discriminate between GT1 subtypes. The correlation between baseline NS3 and NS5A RASs on the 12-week sustained virological response (SVR12) rates of 160 of the patients treated with second-generation DAAs was also assessed. Post-treatment resistance analysis was performed on samples from 58 patients exhibiting DAA virological failure. RESULTS: GT1a, GT1b and GT1d subtypes were identified in 23.0%, 75.4% and 1.2% of tested samples. GT1b was most prevalent (97.7%, 128/131) among patients born in the former Soviet Union. The Q80K NS3 RAS was identified in 17.5% (10/57) of the GT1a carriers, most of whom were Israeli-born. NS3 and NS5A baseline RASs showed a negligible correlation with SVR12 rates. Treatment-emergent RASs were observed among 8.9% (4/45) and 76.9% (10/13) of first- and second-generation DAA failures, respectively, with D168V/E (NS3), Y93H and L31M (NS5A) being the most prevalent mutations. CONCLUSIONS: NS3 sequencing analysis can successfully discriminate between GT1 subtypes and identify NS3 amino acid substitutions. While pre-treatment NS3 and NS5A RASs marginally affect second-generation DAA SVR12 rates, post-treatment resistance analysis should be considered prior to re-therapy.