Hypervariable region 1 quasispecies in hepatitis C virus genotypes 1b and 3 infected patients with normal and abnormal alanine aminotransferase levels.

The role of hepatitis C virus (HCV) heterogeneity in the severity of chronic hepatitis C infection remains unclear. Our aim was to study the hypervariable region 1 (HVR1) heterogeneity in patients with chronic hepatitis C infected with genotype 1b or 3 and with normal or abnormal alanine aminotransferase (ALT). HVR1 quasispecies were assessed by single strand conformational polymorphism (SSCP) in 67 patients with chronic hepatitis C, including 35 with persistently normal ALT and 32 with abnormal ALT. Sixty-two patients underwent a liver biopsy. Among the 67 patients, 40 were infected with genotype 1b and 27 with genotype 3. In univariate analysis, low heterogeneity (

Prospective study on anti-hepatitis C virus-positive patients with persistently normal serum alanine transaminase with or without detectable serum hepatitis C virus RNA.

A significant proportion of patients with detectable antibodies to hepatitis C virus have normal serum alanine transaminase levels. Our aim was to study the outcome of this group. Between 1992 and 1999, 135 consecutive anti-HCV-positive patients with persistently normal ALT were followed for 3.6 +/- 2.3 years (0.5 to 8.5 years), 108 had a liver biopsy at inclusion, and 24 had a second liver biopsy 3.5 +/- 1.0 years later. Serum HCV RNA was detectable with PCR in 94 patients (69%) and not detectable in 41 patients (31%). Patients with and without detectable serum HCV RNA had similar epidemiological characteristics. Serum ALT levels and anti-HCV ratio were lower (P =.001), and histological lesions had lower grade and stage in patients without detectable serum HCV RNA (P =.001). Liver HCV RNA was not detectable with PCR in the 12-serum HCV RNA-negative patients tested. During follow-up, all patients without detectable serum HCV RNA remained HCV RNA-negative and kept normal serum ALT; all patients with detectable serum HCV RNA remained HCV RNA-positive, 20 (21%) had a slight fluctuation of serum ALT above the upper limit of normal. No significant changes were observed in the liver lesions of the 24 patients who underwent a second liver biopsy. In anti-HCV-positive patients with persistently normal serum ALT, histological lesions are significantly lower in HCV RNA-negative than in HCV RNA-positive patients. During follow-up, the HCV RNA status of patients remained unchanged; 21% of the patients with detectable serum HCV RNA had slight increase in serum ALT levels, but histological lesions remained stable.

Cytokines as predictors for sustained response and as markers for immunomodulation in patients with chronic hepatitis C.

OBJECTIVES: (i) To characterize serum cytokine levels of tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL 6), IL 8 and IL 12 in non-cirrhotic patients with chronic hepatitis C, (ii) to correlate the levels of these cytokines with the degree of the disease at the basal level, (iii) to correlate these levels with the response to therapy, (iv) to compare profiles of cytokines in monotherapy (MT) versus combination therapy (CT), and (v) to compare the immunomodulatory effects of MT versus CT. DESIGN AND METHODS: 47 patients were enrolled in the study. The controls were 120 volunteers (recruited from students and staff) that did not present HCV RNA positive and were not known to suffer any other metabolic disease. Thirty patients formed the other group of controls, with alcoholic liver disease (ALD). Serum cytokine levels were assessed using enzyme-linked immunosorbent assay (ELISA). RESULTS: The sustained responders (SRs) have basal values much lower than relapsed responders (RRs) and non-responders (NRs) regardless of the therapy. CONCLUSIONS: Cytokines can be used as non-invasive markers for sustained response and as monitors for the outcome of therapy.

Assessment of viral loads in patients with chronic hepatitis C with AMPLICOR HCV MONITOR version 1.0, COBAS HCV MONITOR version 2.0, and QUANTIPLEX HCV RNA version 2.0 assays.

The correlation between response to antiviral therapy and pretreatment viral load in patients with chronic hepatitis C has prompted the development of quantitative assays to measure viral load. The aim of our study was to assess the clinical relevance of the newly developed semiautomated PCR system COBAS HCV MONITOR version 2.0 in comparison with (i) the AMPLICOR HCV MONITOR version 1.0 assay, which underestimates RNA concentration of hepatitis C virus (HCV) genotypes 2 to 6, and (ii) the QUANTIPLEX HCV RNA version 2.0 assay, which achieves equivalent quantification for each HCV genotype, with samples from 174 patients diagnosed with chronic hepatitis C before therapy. The level and range of quantification measured with AMPLICOR HCV MONITOR version 1.0 were 1 log lower than when measured with the COBAS HCV MONITOR version 2.0, at 0.261 x 10(6) RNA copies/ml (range, 0.001 x 10(6) to 2.50 x 10(6) RNA copies/ml) and 4.032 x 10(6) RNA copies/ml (range, 0.026 x 10(6) to 72.6 x 10(6) RNA copies/ml), respectively. The two assays showed a poor correlation (r(2) = 0.175). The level and range of quantification were similar when measured with the COBAS HCV MONITOR version 2.0 and QUANTIPLEX HCV RNA version 2.0 assays, at 3.03 x 10(6) RNA copies/ml (range, 0.023 x 10(6) to 72.6 x 10(6) RNA copies/ml) and 4.91 Meq/ml (range, 0.200 to 49.5 Meq/ml), respectively. The two assays showed a strong correlation (r(2) = 0. 686) for each HCV genotype. The duration of treatment (6 or 12 months) is modulated according to HCV genotype and viral load. Our results indicate that COBAS HCV MONITOR version 2.0 and QUANTIPLEX HCV RNA version 2.0 assays showing an equal dynamic range for each HCV genotype are suitable tools to assess patients before therapy.

A new step toward standardization of serum hepatitis C virus-RNA quantification in patients with chronic hepatitis C.

The need to improve efficacy of antiviral therapy for chronic hepatitis C has prompted the development of quantitative assays, which allows the assessment of viral load before therapy. The aim of our study was to evaluate the clinical relevance of 3 serum HCV-RNA quantitative assays in 87 patients with chronic hepatitis C, the noncommercially available SuperQuant assay (National Genetic Institute), recently used in large international controlled trials, the most early and widely used Quantiplex HCV RNA v2.0 assay (branched DNA [bDNA] v2.0; Bayer Diagnostics, Puteaux, France), and the new generation Cobas Amplicor HCV Monitor assay (COBAS v2.0; Roche Diagnostics Systems, Meylan, France), which is a semiautomated reverse transcription-polymerase chain reaction (RT-PCR) assay. The level and range of quantification were similar between all assays and a strong correlation was observed over all HCV genotypes among the assays. Recent publications have suggested that the baseline cut-off level of 2 x 10(6) copies/mL, as determined by the SuperQuant assay, is able to discriminate between patients with low viral load from those with high viral load and can be used to predict responses to therapy. Because all 3 assays use different testing technologies we examined how many of our patients fell above this defined cut-off level when tested by the other assays; of 22 patients measured below 2 x 10(6) copies/mL with the SuperQuant assay, 17 of 22 and 19 of 22 patients were eligible with the bDNA v2. 0 and the COBAS v2.0 assays, respectively (P >.05). Our results indicate that the 2 commercial assays can be used to determine treatment schedules in patients with chronic hepatitis C, providing a flexibility in multicenter controlled trials by offering better accessibility of test results.

Quantification of hepatitis C virus RNA in serum by branched DNA-based signal amplification assays.

The objective of the study was to compare the clinical sensitivity and specificity of versions 1.0 and 2.0 of the branched DNA (bDNA)-based hepatitis C virus (HCV) RNA quantification assay, and also to compare the values yielded by the two versions according to the HCV genotype. Serum samples from 268 patients tested routinely by a non-quantitative HCV RNA PCR assay (group A) were tested with version 2.0 of the bDNA assay. Samples from 342 HCV PCR-positive patients with chronic hepatitis C eligible for interferon treatment (group B) were tested with both version 1.0 and version 2.0 of the bDNA assay. Version 2.0 had a clinical sensitivity of 92% (95% confidence interval (CI): 87-97%) in group A and 89% (86-92%) in group B. In group B, the gain in sensitivity with bDNA 2.0 was 16% relative to bDNA 1.0 (P < 0.001). The log values of the two assays correlated with samples positive by both assays (r = 0.83, P < 0.0001), but the distribution of values was larger in samples containing HCV genotypes 2 and 3. The mean ratio of assay 2.0/assay 1.0 values was 1.69 +/- 1.44 (range: 0.33-13.43). The mean ratio was close to 1 with samples containing genotype 1 or 4, but ranged from 0.33 to more than 5. The mean ratio was close to 3 with samples containing genotype 2 or 3, and ranged from 0.5 to more than 13. HCV RNA levels were significantly lower in samples containing genotype 4 than in those containing other genotypes. Sera from 200 anti-HCV-negative, HCV RNA PCR-negative blood donors (group C), and from 164 anti-HCV-negative patients with symptoms of chronic liver disease (group D) were used to assess the clinical specificity of bDNA 2.0. In addition, samples with an HCV RNA titer between 0.2 (assay cutoff) and 0.5 MEq/ml from a group of 546 patients tested routinely for HCV RNA load by bDNA 2.0 (group E) were retested by bDNA 2.0 and by qualitative PCR. The specificity of bDNA 2.0 was 100% (98-100%) in group C and 99% (97-100%) in group D. Among the 41 samples from group E, 38 were positive by bDNA 2.0 retesting (36 were PCR-positive) and three were negative by bDNA 2.0 retesting (all were PCR-positive). It is concluded that version 2.0 of the bDNA assay is markedly more sensitive than version 1.0 and has a good specificity. In contrast with version 1.0, version 2.0 is not influenced by the HCV genotype. The relationship between values obtained with assays 1.0 and 2.0 on clinical specimens is not linear, indicating that HCV RNA titers cannot reliably be calculated from the results of version 1.0.

Pretreatment serum hepatitis C virus RNA levels and hepatitis C virus genotype are the main and independent prognostic factors of sustained response to interferon alfa therapy in chronic hepatitis C.

The aim of the study was to determine the respective influence of pretreatment serum hepatitis C virus (HCV) RNA levels and HCV genotype on the response to interferon (IFN) alfa in patients with chronic hepatitis C. We retrospectively studied 141 patients with chronic hepatitis C included in two consecutive controlled trials of IFN alfa. A sustained response was observed in 28, a response followed by relapse in 43, and no response in 70 patients. Pretreatment serum HCV RNA quantitation with the branched DNA (bDNA) assay and HCV genotyping with reverse hybridization assay (LiPA) were performed in all patients. Seventy-four percent of the patients had detectable serum HCV RNA (43%, 77% and 84%) in the three groups of patients with sustained response, relapse, and no response, respectively (P = .005). Mean serum HCV RNA level were 1.4 +/- 6 x 10(6), 4.8 +/- 6 x 10(6), and 3.9 +/- 5 x 10(6) genomes/mL in patients with sustained response, response and relapse, and no response, respectively (P < .01). Genotype 1b was found in 7%, 47%, and 46% of the patients in the three response groups, respectively. By univariate analysis, age, source, and duration of HCV infection, serum HCV RNA levels, and HCV genotypes were significantly different in the three response groups. By multivariate analysis, the only independent factors associated with sustained response were low serum HCV RNA levels and HCV genotype other than 1b. Pretreatment serum HCV RNA levels and HCV genotype are the main and independent factors associated with sustained response to IFN therapy.