Hepatitis C virus serologic and virologic tests and clinical diagnosis of HCV-related liver disease

The use of serological and virological tests has become essential in the management of hepatitis C virus (HCV) infection in order to diagnose infection, guide treatment decisions and assess the virological response to antiviral therapy. Virological tools include serological assays for anti-HCV antibody detection and serological determination of the HCV genotype, and molecular assays that detect and quantify HCV RNA and determine the HCV genotype. Anti-HCV antibody testing and HCV RNA testing are used to diagnose acute and chronic hepatitis C. Only patients with detectable HCV RNA should be considered for pegylated interferon alfa and ribavirin therapy and the HCV genotype should be systematically determined before treatment, as it determines the indication, the duration of treatment, the dose of ribavirin and the virological monitoring procedure. HCV RNA monitoring during therapy is used to tailor treatment duration in HCV genotype 1 infection, and molecular assays are used to assess the end-of-treatment and, most importantly the sustained virological response, i.e. the endpoint of therapy.     

Comparison of hepatitis C viral RNA and core antigen kinetics in the therapeutic follow up of hepatitis C virus and human immunodeficiency virus co-infected patients, treated by bitherapy interferon-ribavirin, within the framework of RIBAVIC protocol

AIM OF STUDY: The RIBAVIC protocol, established by ANRS in 2001 and closed in 2003, compared the efficacy and the tolerance of two bitherapy anti-Hepatitis C Virus for HIV-HCV co-infected patients: IFN-ribavirin and PEG-IFN-ribavirin for 48 weeks. Two hundred patients from protocol were tested for hepatitis C virus core antigen, to study this viral marker kinetics, before and under treatment, in comparison with hepatitis C virus RNA evolution. MATERIAL AND METHODS: The available samples for the 204 patients of our study were tested for RNA detection (COBAS AMPLICOR v2.0, Roche Diagnostics) and quantification (VERSANT HCV RNA v3.0, Bayer Diagnostics) and for quantification of core antigen (Ortho trak-C Assay, Ortho Clinical Diagnostics). The viral kinetics were established from samples quantified at D0, W2, W4, W12, W24, W48, W52, W72 (W =week), according to virological response assessed by PCR, six month after the end of treatment (non responders, sustained responders, relapsers et breakthroughs). RESULTS: We obtained, for each type of response, similar evolution of both viral markers. Trak-C assay show to be enough sensitive, with similar results whatever genotype of hepatitis C virus. The Pearson's correlation is excellent (R =0.94; P <0.001). The intergenotype correlation is correct too, whatever HCV genotype (1, 2, 3, 4). CONCLUSIONS: The HCV core antigen quantification by trak-C assay is a new tool for the follow-up of the treatment of patients with chronic hepatitis C and HIV co-infected.     

Performance of an automated system for quantitation of hepatitis C virus core antigen

The performance of an automated system for the HCV core antigen assay using the Lumispot LS-2000 automated analyzer was evaluated against that of the COBAS AMPLICOR HCV MONITOR Test, version 2.0 (COBAS HCM-2) for the testing of sera from 155 chronic hepatitis C patients. The within-run coefficient of variations (CVs) and the between-day CVs were <9.6 and <8.4%, respectively. The analytical detection limit of the HCV core antigen assay was 5.0 fmol/l and it was linear up to at least 45000 fmol/l. No blood elements interfered with the assay. HCV core antigen levels were significantly correlated with HCV RNA levels in both serogroup 1 and serogroup 2 (r=0.829, P<0.001). It is estimated that 100 fmol/l of HCV core antigen level was equivalent to approximately 30000 IU/ml of HCV RNA level. Three sera had HCV core antigen levels below the detection limit of the assay and their HCV RNA levels as determined by COBAS HCM-2 assay were very low at 1000, 1100 and 1700 IU/ml, respectively. In six IFN responders among seven patients, HCV core antigen levels were in parallel with HCV RNA levels. In conclusion, since this assay demonstrated good reproducibility, a favorable dynamic range and adequate sensitivity, it may be useful as an alternative direct marker of viral level monitoring in patients with hepatitis C.     

Benefit of hepatitis C virus core antigen assay in prediction of therapeutic response to interferon and ribavirin combination therapy

A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) and ribavirin. HCV core Ag negativity could predict SVR on day 1 (sensitivity = 100%, specificity = 85.0%, accuracy = 86.4%), whereas RNA negativity could predict SVR on day 7 (sensitivity = 100%, specificity = 87.2%, accuracy = 88.6%). None of the patients who had detectable serum core Ag or RNA on day 14 achieved SVR (specificity = 100%). The predictive accuracy on day 14 was higher by RNA negativity (93.2%) than that by core Ag negativity (75.0%). The combined predictive criterion of both viral load decline during the first 24 h and basal viral load was also predictive for SVR; the sensitivities of Lumipulse-Ag and Amplicor-M were 45.5 and 47.6%, respectively, and the specificity was 100%. Amplicor-M had better predictive accuracy than Lumipulse-Ag in 2-week disappearance tests because it had better sensitivity. On the other hand, estimates of kinetic parameters were similar regardless of the detection method. Although the correlations between Lumipulse-Ag and Amplicor-M were good both before and 24 h after IFN administration, HCV core Ag seemed to be relatively lower 24 h after IFN administration than before administration. Lumipulse-Ag seems to be useful for detecting the HCV concentration during IFN therapy; however, we still need to understand the characteristics of the assay.     

A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma

Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined.     

Japanese reference panel of blood specimens for evaluation of hepatitis C virus RNA and core antigen quantitative assays

An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits.     

Performance evaluation of the Artus hepatitis C virus QS-RGQ assay

Accurate determination of hepatitis C virus RNA level is essential for evaluating the response to antiviral therapy, to determine the duration of treatment, and to predict treatment outcome. Currently, two real-time based polymerase chain reaction assays are used widely to monitor the hepatitis C RNA level: the Abbott RealTime HCV assay and the Cobas Taqman HCV assay. Recently, a third assay has become commercially available: the Artus HCV QS-RGQ assay, which uses the QIAsymphony SP/AS platform for sample preparation and PCR-setup, and the Rotor-Gene Q for amplification and detection. In this study, the performance of the Artus HCV QS-RGQ assay was tested on 105 plasma samples and compared to that of the Cobas Taqman HCV assay. Linear regression analysis showed a good agreement between the two assays. A slightly better sensitivity was observed with the Cobas Taqman assay, while higher hepatitis C viral RNA levels were measured by the Artus HCV QS-RGQ assay in samples positive for hepatitis C genotypes 4. Taken together, the data suggest that the Artus HCV QS-RGQ assay is useful in a diagnostic setting. The combination with the versatile QIAsymphony SP/AS system may represent a major advantage for clinical virological laboratories aiming at optimizing their workflow.     

Comparative evaluation of the total hepatitis C virus core antigen,branched-DNA,and amplicor monitor assays in determining viremia for patients with chronic hepatitis C during interferon plus ribavirin combination therapy

An assay prototype designed to detect and quantify total hepatitis C virus [HCV] core antigen (HCV core Ag) protein in serum and plasma in the presence or absence of anti-HCV antibodies has been recently developed by Ortho-Clinical Diagnostics. The aim of the study was to evaluate the sensitivity, specificity, and reproducibility of the Total HCV core Ag assay in comparison with two quantitative assays for HCV RNA: Quantiplex HCV RNA 2.0 (bDNA v2.0) or Versant HCV RNA 3.0 (bDNA v3.0) assays and the Cobas Amplicor HCV Monitor version 2.0 (HCM v2.0) test. We have studied samples of a well-characterized panel and samples from patients with chronic hepatitis C treated with interferon alone or with ribavirin. We have also compared the kinetics of HCV core Ag and HCV RNA in the follow-up of treated patients. The HCV core Ag assay exhibited linear behavior across samples from different genotypes. The coefficients of variation for intra- and interassay performance were 5.11 and 9.95%, respectively. The specificity of the assay tested in blood donors was 99.5%. Samples from HCV-infected patients showed that the correlation between the HCV core Ag and the two HCV RNA quantitative assays (bDNA and HCM v2.0) was 0.8 and 0.7, respectively. This correlation was maintained across different genotypes of HCV (r(2) = 0.64 to 0.94). Baseline HCV core Ag values were significantly lower in sustained responders to interferon (IFN) than in other groups of patients (5.31 log(10) [10(4) pg/ml] versus 5.99 log(10) [10(4) pg/ml]; P < 0.001). In patients treated with IFN or combination therapy, we found an association between a decrease of more than 2 log IU/ml in viral load, undetectable HCV core Ag, and sustained response. Among sustained responders to IFN alone or combination therapy and among relapsers after IFN alone, 84 out of 101 (83.2%) had undetectable HCV core Ag, and 76 out of 96 (79.2%) had a viral load decrease of >/=2 log IU/ml, after 1 month of treatment. In conclusion, the Total HCV core Ag assay is a new useful test for the detection of HCV viremia and the monitoring of patients treated with IFN alone or in combination with ribavirin.     

Poor performance of hepatitis C antibody tests in hospital patients in Uganda

Most hepatitis C testing in Uganda is performed using commercial rapid strip assays (RSA) to detect antibodies to hepatitis C virus (anti‐HCV), rather than enzyme immunoassays (EIA). The prevalence of hepatitis C antibodies in a Ugandan hospital population was determined using both methods to test their accuracy using nucleic acid testing (NAT) as a reference. Sera from 380 consecutive hospitalized Ugandan patients were tested for anti‐HCV using an RSA in Uganda, with subsequent automated third‐generation EIA testing in the United States, followed by NAT. Recombinant immunoblot assays (RIBA) were used as a supplementary test to detect anti‐HCV epitopes. Overall, anti‐HCV was detected in 48/380 (13%) by one or both antibody tests. Anti‐HCV was detected in 19 (5.0%) patients by RSA and in 33 (8.7%) patients by EIA; only four patients were anti‐HCV positive by both methods. Fourteen of the 48 anti‐HCV positive patients had detectable serum HCV RNA, 7 each by bDNA assay or by PCR. RSA detected only 7 of 14 HCV RNA positive sera. Of 29 RNA negative but anti‐HCV positive patients tested by RIBA, only two were anti‐HCV positive; 27 were anti‐HCV negative or indeterminate. Anti‐HCV testing by RSA and/or EIA was neither sensitive nor specific for detection of ongoing HCV infection in hospitalized Ugandan patients. Our findings underscore the importance of confirmatory nucleic acid testing, which, despite its increased cost, appears essential to manage African patients with HCV. J. Med. Virol. 82:1371–1378, 2010. © 2010 Wiley‐Liss, Inc.     

Simultaneous detection of hepatitis C virus (HCV) core antigen and anti-HCV antibodies improves the early detection of HCV infection

To evaluate whether a new enzyme immunoassay developed for the simultaneous detection of hepatitis C virus (HCV) core antigen (Ag) and anti-HCV antibodies (anti-HCV Ab) (Monolisa HCV Ag/Ab ULTRA; Bio-Rad) could improve the early detection of HCV infection, we compared its sensitivity to that of anti-HCV, HCV core Ag, and HCV RNA assays. The populations studied included 12 blood donor samples positive for HCV RNA and HCV core Ag but negative for anti-HCV antibodies and 23 hemodialysis patients who developed anti-HCV Ab (seroconversion) during the follow-up. From these 23 individuals, 83 samples sequentially collected prior to seroconversion and 108 samples collected after seroconversion were tested. Six of 12 blood donations were positive by the HCV Ag/Ab assay. In the hemodialysis cohort, the 24 HCV RNA-negative samples were negative by the HCV Ag/Ab assay and 23 of the 59 HCV RNA-positive samples (39%) were positive. The HCV Ag/Ab assay detected HCV infection on average 21.6 days before the most sensitive antibody assay. The HCV Ag/Ab assay did not detect HCV infection as early as the HCV RNA assay (mean delay, 30.3 days) or HCV Ag assay (mean delays, 27.9, and 16.3 days by the HCV core Ag quantification assay and the HCV Ag blood screening assay, respectively). This new assay provides a notable improvement for the early detection of HCV infection during the so-called window period compared with anti-HCV Ab assays and could be a useful alternative to HCV RNA detection or HCV core Ag assays for diagnosis or blood screening when nucleic acid technologies or HCV core Ag detection are not implemented.     

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.     

Clinical benefit of HCV core antigen assay in patients receiving interferon and ribavirin combination therapy

A highly sensitive second generation HCV core antigen assay has recently been developed. We compared viral disappearance and kinetics data between commercially available core antigen assays, Lumipulse Ortho HCV Ag, and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor Test, Version 2 to estimate the predictive benefit of sustained viral response (SVR) and non-SVR in 59 patients treated with interferon and ribavirin combination therapy. We found a good correlation between HCV core Ag and HCV RNA level regardless of genotype. Although the sensitivity of the core antigen assay was lower than PCR, the dynamic range was broader than that of the PCR assay, so that we did not need to dilute the samples in 59 patients. We detected serial decline of core Ag levels in 24 hrs, 7 days and 14 days after interferon combination therapy. The decline of core antigen levels was significant in SVR patients compared to non-SVR as well as in genotype 2a, 2b patients compared to 1b. Core antigen-negative on day 1 could predict all 10 SVR patients (PPV = 100%), whereas RNA-negative could predict 22 SVR out of 25 on day 14 (PPV = 88.0%). None of the patients who had detectable serum core antigen on day 14 became SVR(NPV = 100%), although NPV was 91.2% on RNA negativity. An easy, simple, low cost new HCV core antigen detecting system seems to be useful for assessing and monitoring IFN treatment for HCV.     

Total HCV core antigen assay. A new marker of HCV viremia and its application during treatment of chronic hepatitis C

The present study assesses the clinical usefulness of the hepatitis C core antigen assay for monitoring of patients being treated for chronic hepatitis C virus (HCV) infection. Eighty-six serum samples were selected at random from 16 patients and levels of HCV RNA and HCV core antigen were determined simultaneously and in parallel to compare both techniques. The data obtained were compared by Pearson’s correlation and the coefficients calculated by Fisher transformation and by calculating the difference and standard error. A good linear correlation was observed between both techniques. Maximum correlation, with significant difference, was found between patients infected with the 1a genotype and other genotypes. In conclusion, the HCV core antigen assay is useful for the diagnosis of early infection; however, its use for determining the exact timing of viral elimination during treatment is clearly unsuitable.     

Evaluation of the core antigen assay as a second-line supplemental test for diagnosis of active hepatitis C virus infection

The British Columbia Center for Disease Control laboratory performs approximately 95% of all hepatitis C virus (HCV) antibody tests for the province's 4 million inhabitants. In 2002, the laboratory tested 96,000 specimens for anti-HCV antibodies, of which 4,800 (5%) were seroreactive and required confirmation of active infection. Although HCV RNA assays with a sensitivity of 50 IU/ml or less are recommended for the confirmation of active HCV infection, given the large number of seroreactive specimens tested annually, we evaluated the Ortho trak-C assay (OTCA) as a second-line confirmatory test and determined its limit of detection (LoD). Of 502 specimens from treatment-na?ve anti-HCV-positive individuals, 478 had sufficient volumes for evaluation by the OTCA and HCV RNA tests. Core antigen was not detected in 147 of 478 (30.8%) of these specimens, of which 37 of 147 (25.2%) were shown to be viremic by the VERSANT HCV (version 3.0) (branched-DNA) assay and/or the VERSANT HCV qualitative assay. Testing of 144 replicates of a World Health Organization standard dilution series indicated that the LoD of OTCA was approximately 27,000 IU/ml. This LoD is consistent with the inability of OTCA to detect core antigen in clinical specimens with low viral loads. We conclude that OTCA has limited value as a confirmatory test for the diagnosis of active HCV infection because 37 of 367 (10%) of viremic specimens had undetectable core antigen. Qualitative HCV RNA testing remains the present standard for the confirmation of active HCV infection in the diagnostic setting.     

Utility of HCV core antigen ELISA in the screening for hepatitis C virus infection in patients on hemodialysis

An enzyme immuno assay for hepatitis C core antigen was recently developed and its performance was compared with that of the hepatitis C virus (HCV) RNA in the screening of HCV infection in patients on hemodialysis. One hundred and eleven chronic renal failure patients undergoing haemodialysis between May 2003 and October 2004 were included in the study. All the patients were tested for anti HCV antibody, core antigen and RNA. Fifteen patients were anti HCV antibody positive, three patients were positive for HCV core antigen and RNA, three patients were positive for HCV RNA, while two patients were positive only for core antigen but negative for RNA. In anti HCV antibody positive patients, the core antigen was negative while the viral RNA continued to be present. Hence, relying solely on a single HCV core antigen assay may not be useful for a definite diagnosis of early HCV infection. The sensitivity and specificity of the assay were 60% and 83% respectively, while the positive predictive value was 14.3%, negative predictive value was 97.7% and the efficiency was 81.9%.