Ultradeep sequencing of B and non-B HIV-1 subtypes: Viral diversity and drug resistance mutations before and after one month of antiretroviral therapy in naive patients

International audienc

Amplification and pyrosequencing of near-full-length hepatitis C virus for typing and Monitoring antiviral Resistant Strains

International audienceDirectly acting antivirals have contributed considerable progress to hepatitis C treatment, but they show variable activity depending on viral genotypes and subtypes. Therefore, accurate genotyping including recombinant form detection is still of major importance, as is the detection of resistance-associated mutations in case of therapeutic failure. To meet these goals, an approach to amplify the hepatitis C virus (HCV) near-complete genome with a single long-range PCR and sequence it with Roche GS Junior was developed. After optimization, the overall amplification success rate was 73% for usual genotypes (i.e. HCV1a, 1b, 3a and 4a, n = 16 of 22) and 45% for recombinant forms RF_2k/1b (n = 5 of 11). After pyrosequencing and subsequent de novo assembly, a near-full-length genomic consensus sequence was obtained for 19 of 21 samples. The genotype and subtype were confirmed by phylogenetic analysis for every sample, including the suspected recombinant forms. Resistance-associated mutations were detected in 7 of 13 samples at baseline, in the NS3 (n = 3) or NS5A region (n = 4). Of these samples, the treatment of one patient included daclatasvir and that patient experienced a relapse. Viral sequences from pre- and post-treatment samples of four patients who relapsed after sofosbuvir-based therapy were compared: the selected variants seem too far from the NS5B catalytic site to be held responsible. Although tested on a limited set of samples and with technical improvements still necessary, this assay has proved to be successful for both genotyping and resistance-associated variant detection on several HCV types

Ultradeep pyrosequencing of NS3 to predict response to triple therapy with protease inhibitors in previously treated chronic hepatitis C patients.

International audienceDespite the gain in sustained virological responses (SVR) provided by protease inhibitors (PIs), failures still occur. The aim of this study was to determine if a baseline analysis of the NS3 region using ultradeep pyrosequencing (UDPS) can help to predict an SVR. Serum samples from 40 patients with previously nonresponding genotype 1 chronic hepatitis C who were retreated with triple therapy, including a PI, were analyzed. Baseline UDPS of the NS3 gene was performed on plasma and peripheral blood mononuclear cells (PBMC). Mutations conferring resistance to PIs were sought. The overall diversity of the quasispecies was evaluated by calculating the Shannon entropy (SE). Resistance mutations were found in plasma and PBMC but were not discriminating enough to predict an SVR. NS3 quasispecies heterogeneity was significantly lower at baseline in patients achieving an SVR than in those not achieving an SVR (SE of 26.98 ± 16.64 × 10(-3) versus 44.93 ± 19.58 × 10(-3), P = 0.0047). With multivariate analysis, the independent predictors of an SVR were fibrosis of stage F ≤2 (odds ratio [OR], 13.3; 95% confidence interval [CI], 1.25 to 141.096; P < 0.03) and SE below the median (OR, 5.4; 95% CI, 1.22 to 23.87; P < 0.03). More than the presence of minor mutations at the baseline in plasma or in PBMC, the NS3 viral heterogeneity determined by UDPS is an independent factor for an SVR in previously treated patients receiving triple therapy that includes a PI

Ultradeep pyrosequencing of NS3 to predict response to triple therapy with protease inhibitors in previously treated chronic hepatitis C patients.

International audienceDespite the gain in sustained virological responses (SVR) provided by protease inhibitors (PIs), failures still occur. The aim of this study was to determine if a baseline analysis of the NS3 region using ultradeep pyrosequencing (UDPS) can help to predict an SVR. Serum samples from 40 patients with previously nonresponding genotype 1 chronic hepatitis C who were retreated with triple therapy, including a PI, were analyzed. Baseline UDPS of the NS3 gene was performed on plasma and peripheral blood mononuclear cells (PBMC). Mutations conferring resistance to PIs were sought. The overall diversity of the quasispecies was evaluated by calculating the Shannon entropy (SE). Resistance mutations were found in plasma and PBMC but were not discriminating enough to predict an SVR. NS3 quasispecies heterogeneity was significantly lower at baseline in patients achieving an SVR than in those not achieving an SVR (SE of 26.98 ± 16.64 × 10(-3) versus 44.93 ± 19.58 × 10(-3), P = 0.0047). With multivariate analysis, the independent predictors of an SVR were fibrosis of stage F ≤2 (odds ratio [OR], 13.3; 95% confidence interval [CI], 1.25 to 141.096; P < 0.03) and SE below the median (OR, 5.4; 95% CI, 1.22 to 23.87; P < 0.03). More than the presence of minor mutations at the baseline in plasma or in PBMC, the NS3 viral heterogeneity determined by UDPS is an independent factor for an SVR in previously treated patients receiving triple therapy that includes a PI