Standardization of hepatitis C virus RNA quantification.

It was recently recommended that hepatitis C virus (HCV) RNA quantification be used to tailor the duration of combined interferon alfa (IFN-alpha)/ribavirin therapy in patients infected by HCV genotypes 1, 4, and 5. This recommendation has been difficult to implement in the absence of standardized quantitative units for HCV RNA. The aim of this work was to define clinically relevant HCV RNA loads in standardized international units (IU), for use in routine clinical and research applications based on standardized quantitative assays. Two hepatitis C virus RNA quantitative assays were used: (1) the Superquant assay (National Genetics Institute, Los Angeles, CA), for which possibly relevant thresholds were established; and (2) the semi-automated Cobas Amplicor HCV Monitor assay version 2.0 (Cobas v2.0, Roche Molecular Systems, Pleasanton, CA) that measures HCV RNA loads in IU/mL. Quantification in the Cobas v2.0 assay was linear over the entire range of values tested, including viral loads higher than 850,000 IU/mL after 100-fold dilution. The accuracy and precision of the measures in IU/mL were satisfactory with Cobas v2.0. The results obtained with Superquant and Cobas v2.0 correlated (r =.932; P <.0001). A value of 2,000,000 copies/mL (6.3 log(10) copies/mL) with Superquant was converted to nearly 800,000 IU/mL (5.9 log(10) IU/mL). In conclusion, all HCV RNA quantitative assays should give HCV RNA loads in international units and be validated with appropriate calibrated panels; 800,000 IU/mL in any of these assays should be used as the decision threshold to tailor the IFN-alpha/ribavirin treatment duration in patients infected by HCV genotypes 1, 4, and 5.

Routine detection and quantification of hepatitis B virus DNA in clinical laboratories: performance of three commercial 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.

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.

What strategy should be used for diagnosis of hepatitis C virus infection in clinical laboratories?

The aim of this study was to determine a cost-effective strategy for the diagnosis of hepatitis C virus (HCV) infection in clinical laboratories. Anti-HCV antibodies were sought in 3,014 consecutive unselected samples with two different enzyme-linked immunosorbent assays (ELISA). An immunoblot-based confirmatory assay (RIBA3.0) was performed in the samples with at least one ELISA positive or weakly positive. HCV RNA was evaluated using HCV polymerase chain reaction (PCR) in the samples with a weakly positive ELISA, discrepant results of the two ELISAs, or an indeterminate RIBA3.0 pattern. The two ELISAs gave concordant results in 2,957 (98.1%) of the 3,014 samples (negative in 87.9%, positive in 11.8%, and weakly positive in 0.3%), and discrepant results in 57 (1.9%). RIBA3.0 was positive in 338 of the 350 ELISA-positive samples (96.6%) and indeterminate in 12. Six of them were PCR-positive. Among the 8 weakly positive samples, 1 was RIBA3.0-positive, 6 were RIBA3.0-indeterminate, and 1 was RIBA3.0-negative; all were PCR-negative. Among the 57 samples with discrepant ELISA results, 4 were RIBA3.0-positive (none were PCR-positive), 22 were RIBA3.0-indeterminate (1 was PCR-positive), and 31 were RIBA3.0-negative (6 were PCR-positive). In these cases, the clinical context and PCR detection of HCV RNA allowed for definitive classification. In conclusion, one single ELISA determination is necessary for diagnosis of HCV infection in clinical laboratories, and confirmation of positive or weakly positive ELISAs with immunoblot-based confirmatory assays is no longer needed. HCV-RNA detection by PCR helps to resolve weakly positive or negative ELISA results when the clinical context is compatible with hepatitis C.

Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C.

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.

GB virus C (GBV-C) infection in patients with chronic hepatitis C. Influence on liver disease and on hepatitis virus behaviour: effect of interferon alfa therapy.

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.

Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay.

In patients with chronic hepatitis C, determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules. The suitabilities of two versions of a serological, so-called serotyping assay (Murex HCV Serotyping Assay version 1-3 [SA1-3] and Murex HCV Serotyping Assay version 1-6 [SA1-6]; Murex Diagnostics Ltd.), based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes were studied. The results were compared with those of a molecular biology-based genotyping method (HCV Line Probe Assay [INNO-LiPA HCV]; Innogenetics S.A.), based on hybridization of PCR products onto genotype-specific probes designed in the 5' noncoding region of the genome, obtained with pretreatment serum samples from 88 patients with chronic hepatitis C eligible for interferon therapy. Definitive genotyping was performed by sequence analysis of three regions of the viral genome in all samples with discrepant typing results found among at least two of the three assays studied. In all instances, sequence analysis confirmed the result of the INNO-LiPA HCV test. The sensitivity of SA1-3 was 75% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 92% of the samples typeable by SA1-3. The sensitivity of SA1-6 was 89% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 94% of the samples typeable by SA1-6. Overall, SA1-6 had increased sensitivity relative to SA1-3 but remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivities between different HCV genotypes could be responsible for the mistyping of 8 (SA1-3) and 6% (SA1-6) of the samples. Subtyping of 1a and 1b is still not possible with the existing peptides, but discriminating between subtypes may not be necessary for routine use.

What technique should be used for routine detection and quantification of HBV DNA in clinical samples?

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.

Factors affecting treatment responses to interferon-alpha in chronic hepatitis C.

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.

Significance of indeterminate third-generation hepatitis C virus recombinant immunoblot assay.

Indeterminate hepatitis C virus (HCV) third-generation recombinant immunoblot assay (RIBA3.0; Ortho Diagnostic Systems) patterns were arbitrarily defined by the manufacturer as the detection of only one antibody out of the four that were sought, namely, c100 (NS4 encoded), c22 (core encoded), c33c (NS3 encoded), and NS5 (NS5 encoded). The aims of the present study were (i) to determine the prevalence of indeterminate RIBA3.0 patterns in patients consecutively tested for anti-HCV antibodies in a university hospital; (ii) to evaluate the significance of these patterns in terms of viral replication, liver disease, and risk factors for HCV; and (iii) to get an insight into the mechanism underlying this peculiar immune response. Among 3,074 serum samples consecutively tested for anti-HCV antibodies, 588 were found to be positive by screening assays. Fifty-nine of them (10%) were RIBA3.0 indeterminate and were compared with 59 RIBA3.0-positive ones. Thirty-one RIBA3.0-indeterminate and 53 RIBA3.0-positive serum samples were HCV RNA positive by PCR (53 versus 90%; P < 10(-6). RIBA3.0-indeterminate and RIBA-3.0-positive patients with positive PCR results were not significantly different for the prevalence of risk factors for HCV infection and elevated serum alanine aminotransferase activities. Immunosuppression, attributable to coexisting human immunodeficiency virus infection, organ transplantation, or the administration of immunosuppressive drugs, was significantly more frequent in PCR-positive, RIBA3.0-indeterminate patients than in PCR-negative, RIBA3.0 indeterminate patients (P < 0.001) and PCR-positive patients with a positive RIBA3.0 result (P < 0.01). The distribution of HCV genotypes did not differ significantly between HCV RNA-positive patients with indeterminate or positive RIBA3.0 results. In conclusion, the prevalence of indeterminate RIBA3.0 patterns in virology laboratories is about 10%; in about half of these patients HCV replication is detected by PCR; the main factor responsible for indeterminate RIBA3.0 patterns could be immunosuppression, whereas HCV genotypes do not seem to play major role.

Significance of anti-hepatitis C virus core IgM antibodies in patients with chronic hepatitis C.

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.

Influence of hepatitis C virus (HCV) genotypes on HCV recombinant immunoblot assay patterns.

Ninety-six patients with chronic hepatitis C were studied. A second-generation recombinant immunoblot assay detected anti-NS4 antibodies significantly more often in patients infected by hepatitis C virus genotype 1 than in patients infected by other types. By a third-generation recombinant immunoblot assay, the prevalences of the four antibodies measured did not differ according to the hepatitis C virus genotype.

Is the detection of anti-hepatitis C virus core IgM influenced by the presence of serum rheumatoid factor?

Rheumatoid factor (RF) induces false-positive results in the detection of serum antibodies, especially of the IgM type. About 70% of the patients with chronic hepatitis C have abnormal levels of serum RF. The aim of this study was to determine whether the presence of serum RF could influence the detection of anti-HCV core IgM, using an assay designed not to pick up RFs by the addition of goat antibodies directed against human IgG in the sample diluent. Serum anti-HCV core IgM antibodies and RF were sought in 60 patients with chronic hepatitis C. Serum anti-HCV IgG antibodies and anti-HCV core IgM antibodies were also sought in 101 patients with high levels of RF. Anti-HCV core IgM antibodies were found in 45% and serum RF in 72% of the patients with chronic hepatitis C. Neither the prevalence nor the levels of RF differed significantly between IgM positive and negative patients. Eight percent of the 101 patients with raised RF had anti-HCV antibodies and two of them had anti-HCV core IgM antibodies. No patient without anti-HCV antibodies had anti-HCV core IgM antibodies. These results show that: a) the detection of anti-HCV core IgM in patients with chronic hepatitis C is independent of the presence of serum RF; b) high titers of serum RF are not responsible for false-positive results of anti-HCV IgM tests. The study suggests that the test used could be a confident tool for studies on the significance of anti-HCV core IgM antibodies in chronic hepatitis C.