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

Despite 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.

Prevalence and impact of occult hepatitis B infection in chronic hepatitis C patients treated with pegylated interferon and ribavirin.

The prevalence of occult hepatitis B, defined by absence of HBsAg and HBV DNA, ranges widely in patients with hepatitis C. This may influence the treatment of hepatitis C and the severity of liver disease. Sensitive and specific real-time PCR techniques are available commercially and can detect more reliably low HBV DNA levels. The aim of this study was to determine the prevalence of occult hepatitis B virus infection using the COBAS Taqman assay (Roche Diagnostics, Meylan, France) in the serum and liver of HBsAg negative patients with chronic hepatitis C and to evaluate its clinical consequences on liver pathology and its impact on the response to treatment with peg-IFNalpha and Ribavirin. HBV DNA detection was assessed retrospectively on 140 sera and 113 liver biopsies of HCV positive/HBsAg negative patients before treatment. A 4.4% (5/113) prevalence of occult hepatitis B was recorded in liver samples and in none of the sera. Anti-HBc was not detected in one, three of whom were sustained virological responders to treatment, one was relapsed responder and one was non-responder. Furthermore, in this cohort composed of 12% anti-HBs negative/anti-HBc positive and 20% anti-HBs positive/anti-HBc positive patients, anti-HBc was not associated with pre-therapeutic viral load, ALT serum levels, and histological activity or fibrosis. Using a commercial real-time PCR assay, we observed a low prevalence of occult B hepatitis. This, just as anti-HBC status, had no clinical impact in a large cohort of hepatitis C patients. It therefore does not appear useful to screen for occult hepatitis B in these patients with this test before beginning HCV treatment.

Ribavirin quantification in combination treatment of chronic hepatitis C.

Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C(18)-bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra- and interassay accuracies, precision, and stability at 4 degrees C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 +/- 1.06 micro g/ml (n = 24) at week 12 of treatment (W12) and 3.24 +/- 1.35 micro g/ml (n = 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n = 11) than in patients with treatment failure (n = 13), with significant intergroup differences at W12 (P = 0.030) and W24 (P = 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.