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Accurate quantification of hepatitis C virus (HCV) RNA is needed in clinical practice to decide whether to continue or stop pegylated interferon-alpha-ribavirin combination therapy at week 12 of treatment for patients with chronic hepatitis C. Currently the HCV RNA quantification assay most widely used worldwide is the Amplicor HCV Monitor v2.0 assay (Roche Molecular Systems, Pleasanton, Calif.). The HCV RNA extraction step can be automated in the Cobas Ampliprep device. In this work, we show that the dynamic range of HCV RNA quantification of the Cobas Ampliprep/Cobas Amplicor HCV Monitor v2.0 procedure is 600 to 200,000 HCV RNA IU/ml (2.8 to 5.3 log IU/ml), which does not cover the full range of HCV RNA levels in infected patients. Any sample containing more than 200,000 IU/ml (5.3 log IU/ml) must thus be retested after dilution for accurate quantification. These results emphasize the need for commercial HCV RNA quantification assays with a broader range of linear quantification, such as real-time PCR-based assays.

The detection and quantification of hepatitis B virus (HBV) genomes in molecular biology-based assays appear to be the most reliable methods for monitoring HBV infection and assessing responses to antiviral treatment. The aim of this study was to evaluate the performance of three HBV-DNA detection and quantification assays currently used for the management of HBV-infected patients: a solution-hybridization assay based on hybrid-capture (Digene Hybrid-Capture, Murex Diagnostics, Dartford, UK); a signal-amplification assay based on 'branched-DNA' (bDNA) technology (Quantiplex HBV DNA, Bayer Diagnostics, Emeryville, CA); and a target-amplification assay based on competitive polymerase chain reaction (Amplicor HBV Monitor, Roche Molecular Systems, Pleasanton, CA). The Monitor assay was significantly more sensitive than both the hybrid-capture and bDNA methods. This better sensitivity appeared to be clinically relevant. The linear ranges of quantification in the hybrid-capture, bDNA and Monitor methods were 6.5-9 log10 genome copies/ml, 6.5-9.5 log10 genome equivalents/ml, and 3-5.5 log10 genome copies/ml, respectively. However, the HBV-DNA units used in the three assays were not comparable. The specificity of the hybrid-capture, bDNA and Monitor assays was 99.2% (95% confidence interval: 97.7-100.0%), 99.2% (97.7-100.0%), and 97.8% (95.3-100%), respectively. Their within-run coefficients of variation and log10 SDs were 5.5% (+/- 0.025 log10 copies/ml), 6.7% (+/- 0.029 log10 Eq/ml) and 21.0% (+/- 0.093 log10 copies/ml), respectively. Between-run coefficients of variation ranged from 4.4-39.1%, 5-39.5%, and 17.8-96.1%, respectively. The competitive PCR-based Monitor assay appears to be significantly more sensitive but slightly less specific and reproducible than the hybrid-capture and bDNA methods. Given their respective performance, these three assays should be used in complementary fashion in the management of HBV-infected patients.

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.

HCV exists within its host as pools of related genetic variants referred to as quasispecies. The hypervariable region 1 (HVR1) of the E2 envelope gene is subjected to strong selective pressure from neutralizing antibodies. The genetic complexity of this region is defined as the total number of genetic variants within the quasispecies population. The genetic complexity of the HVR1 region was examined in patients with chronic hepatitis C and its relationship with the epidemiology of HCV infection, and its influence on liver disease and the response to interferon treatment were determined in 114 patients with chronic hepatitis C. The genetic complexity of the HVR1 major variants was measured before treatment by using a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) technique, and was compared with epidemiological, clinical, virological and histological features. The patients were treated with 3 megaunits of interferon (IFN) alfa for 3 to 6 months and the response to treatment was assessed at 3, 6 and 12 months. The HVR1 could be studied in 101 of the 114 patients (89%). Genetic complexity was significantly higher in patients infected through blood transfusion than intravenous drug use (mean complexity index: 5.7 +/- 2.3 vs. 4.7 +/- 1.5, respectively; P = 0.04). This relationship was independent of age and the estimated time since infection. No significant relationship was found with other parameters of infection or liver disease. In univariate analysis, the genetic complexity of HVR1 major variants did not affect the rates of ALT normalization at months 3 and 6 of IFN treatment. HVR1 genetic complexity was lower in patients with a sustained virological response than in non-responders (4.0 +/- 1.7 vs. 5.4 +/- 2.0, respectively; P = 0.07). In multivariate analysis of pretreatment parameters associated with a sustained virological response to treatment, three parameters appeared to be independent predictors of such a response: a low viral load (P < 0.04), a low anti-HCV core IgM titer (P = 0.03) and a low genetic complexity of HVR1 major variants (P < 0.04). In conclusion, the HVR1 of HCV has a quasispecies distribution in infected individuals. Its genetic complexity is significantly higher in transfusion recipients than in intravenous drug users, suggesting that the size of the initial inoculum affects the later emergence and development of viral quasispecies. The genetic complexity of HVR1, together with viral load and the anti-HCV IgM titer, are independent predictors of a sustained virological response to IFN alfa in patients with chronic hepatitis.

The aim of this study was to evaluate, in patients with chronic hepatitis C, 1) the prevalence and the epidemiological characteristics of GB virus C (GBV-C) infection, 2) the influence of GBV-C on hepatitis C virus (HCV) infection, 3) the pathogenicity of GBV-C in the absence of treatment and under interferon therapy, and 4) the effect of interferon alfa on GBV-C and HCV replications. One hundred fifteen patients with chronic hepatitis C were studied. Before treatment, they were tested for GBV-C RNA by PCR and GBV-C genotype was determined for positive samples. Pretreatment information was collected, including age, gender, source of HCV, estimated duration of HCV infection, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score on liver biopsy, HCV genotype, HCV viral burden and anti-HCV core IgM antibodies. The genetic complexity of the hypervariable region 1 (HVR1) of HCV was studied by PCR-Single Strand Conformation Polymorphism. All patients were treated with 3 to 9 mega units of interferon alfa-2a three times per week for 3 to 6 months. The influence of GBV-C on the evolution of ALT and HCV replication during and after treatment was studied, and GBV-C and HCV RNA were monitored monthly by PCR during this period. Eighteen patients (16%) were GBV-C RNA-positive. Among 11 samples studied, GBV-C genotype 2a was present in 9 cases, 2b in one case and type 3 in one case. GBV-C RNA-positive patients were significantly younger than GBV-C RNA-negative ones (38.4 +/- 11.5 vs. 47.4 +/- 14.0, P = 0.012), a result independent of the route of transmission and the disease duration. No difference between GBV-C RNA-positive and -negative patients was found for other epidemiological parameters (e.g. gender, risk factor for parenteral viral infections, disease duration and HCV genotypes), or for the characteristics of HCV infection and related liver disease (e.g. HCV RNA level, genetic complexity of the HVR1, anti-HCV core IgM, alanine aminotransferase and gamma-glutamyl transpeptidase activities, cirrhosis and Knodell's score). GBV-C did not influence the rates of ALT normalization at months 3, 6 and 12 and of sustained hepatitis C virological response at month 12 of treatment follow-up. During treatment, GBV-C viremia became undetectable in 12 patients (67%) but relapse occurred after treatment withdrawal in all the nine patients with sufficient follow-up. In the remaining six patients (33%), GBV-C resisted interferon. Whatever the effect of interferon on GBV-C replication, the ALT levels correlated with the presence of HCV RNA. In conclusion, GBV-C infection is frequent in patients with chronic hepatitis C, who are mainly, but not exclusively, infected by GBV-C genotype 2a. GBV-C positive patients are significantly younger than GBV-C negative ones. GBV-C does not seem to affect HCV replication, liver disease and responses of HCV infection and liver disease to interferon therapy. GBV-C is sensitive to 3 mega units of interferon alfa administered three times per week in two-thirds of the patients, but relapse is constant with this dosage after treatment withdrawal.

Detection of hepatitis B virus (HBV) DNA in serum allows monitoring of HBV replication and assessing responses to antiviral treatment. HBV DNA quantification measures virus replication and can be used as a prognosis indicator of liver disease and an index of response to antiviral drugs. The aim of this study was to compare the performances of three HBV DNA detection and/or quantification techniques for assessing HBV replication. Three hundred unselected sera with a request for HBV DNA detection and quantification were tested with a molecular hybridisation technique without amplification (Digene Hybrid-Capture, Murex Diagnostics Ltd), a signal amplification assay based on branched DNA technology (Quantiplex HBV DNA, Chiron diagnostics), and an 'in-house' qualitative, non quantitative target amplification assay based on the polymerase chain reaction (PCR) with primers located in the S gene of the HBV genome. Hybrid-capture and branched DNA gave concordant results in 278 cases (93%). In the 128 samples positive by both assays, DNA titres in pg/ml were related significantly (r = 0.70, P < 0.0001). but branched DNA titres increased more rapidly than hybrid-capture titres when the amount of HBV DNA in the sample increased. Twenty-two sera (7%) were negative by hybrid-capture, but positive in branched DNA (detection rate gain: 15%). In these 22 patients, DNA titres were low, HBsAg was present in all instances and alanine aminotransferase activity was elevated in 18 patients (82%); HBeAg was present in seven patients (32%) and anti-HBe antibodies in 18 patients (82%); liver biopsy, undertaken in 18 patients, revealed chronic active hepatitis in all instances, associated with cirrhosis in eight cases. Qualitative, non-quantitative HBV DNA PCR was positive in 75 (50%) of the 150 hybrid-capture-negative, branched DNA-negative samples, including a significant proportion of patients without evidence of ongoing HBV-related liver disease. The results show that in general, the branched DNA assay detects HBV DNA in more patients than hybrid-capture and that this improved detection rate is relevant clinically and genome equivalents/ml are preferred to pg/ml to quantify HBV DNA in clinical specimens and finally qualitative, non-quantitative polymerase chain reaction can detect HBV DNA in patients without evidence of active HBV-related liver disease. This study emphasizes the need for more sensitive, university standardised quantitative HBV DNA assays and for the definition of clinically relevant cutoffs with these assays.

Parameters have been studied to predict responses to interferon (IFN) therapy for chronic hepatitis C, but the definition of a response, the times at which responses were assessed, and the pretreatment parameters considered differ markedly from study to study. Thus, 113 patients with chronic hepatitis C were treated 3-6 months with 3 MU of IFN-alpha 2a three times a week and assessed for pretreatment parameters predictive of responses to IFN. In a multivariate analysis, a biochemical response (normal aminotransferase activity) at the end of treatment was significantly associated with low body weight, normal gamma-glutamyl transpeptidase activity, and a pretreatment hepatitis C virus (HCV) genotype other than 1. Six months after the end of treatment, a low virus burden and a lack of anti-HCV IgM core antibodies were independently associated with sustained virologic response (i.e., normal aminotransferase activity and HCV RNA negativity). Therefore, these pretreatment parameters should be taken into account when individual treatment protocols are designed.

Antihepatitis C virus (HCV) IgM antibodies were found in patients with both acute and chronic hepatitis C. The aims of the study were to determine the significance, in terms of liver disease and virological parameters, of anti-HCV core IgM antibodies in the serum of patients with chronic hepatitis C, and the possible relationship between the presence of these antibodies before treatment and biochemical and virological responses to interferon therapy. Sixty-one patients with chronic hepatitis C were studied. Tests for serum anti-HCV core IgM antibodies were carried out before treatment. The patients received 3 mega units of interferon alpha-2a subcutaneously thrice weekly for at least 3 months (6 months when alanine aminotransferase activity was normal at month 3). A biochemical response to interferon therapy was defined as normal alanine aminotransferase activity at the end of treatment (month 6: biochemical response) and 6 months later (month 12: sustained biochemical response). A sustained virological response was defined as serum HCV RNA negativity by a polymerase chain reaction-based detection method (PCR) in patients with normal alanine aminotransferase at month 12. Anti-HCV core IgM antibodies were detected in 28 of the 61 patients (46%). The prevalence of these antibodies was significantly higher in patients infected with HCV genotype 1 (including subtypes 1a and 1b) than in patients infected with other genotypes (including 2a and 3a) (57% vs. 17%; P < 0.01). No significant difference was found between IgM-positive and IgM-negative patients as regards the mean age, sex ratio, serum alanine aminotransferase and gamma-glutamyl transpeptidase activities, the prevalence of cirrhosis in liver biopsy specimens, detection of HCV RNA by PCR, and quantitation by branched DNA assay. At month 6 of interferon therapy, normal alanine aminotransferase activity was significantly more frequent in IgM-negative than in IgM-positive patients (52% vs. 21%, respectively; P < 0.02). At month 12, normal alanine aminotransferase activity and PCR negativity were significantly more frequent in IgM-negative than in IgM-positive patients (18% vs. 0%, P < 0.04). It is concluded that anti-HCV core IgM antibodies in serum are significantly more frequent in patients infected by HCV type 1 than by other types. This suggests that their overall prevalence in patients with chronic hepatitis C in industrialized countries, where HCV type 1 accounts for the majority of infections, would be of the order of 50%, that anti-HCV core IgM antibodies are not associated with characteristic features of liver disease, and that their presence before treatment is associated with a failure of interferon alpha therapy to clear the virus.