Assessment of early virological response to antiviral therapy by comparing four assays for HCV RNA quantitation using the international unit standard: implications for clinical management of patients with chronic hepatitis C virus infection

WHO International Standards for nucleic acid tests are used widely to compare the different assays used in HCV RNA quantitation. The aim of the study was to assess the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. Twenty‐seven naïve patients infected chronically by HCV were treated with ribavirin plus PEG‐interferon‐alfa‐2b for 48 weeks. SVR was obtained for 16 patients (the other were non‐responders). For HCV RNA quantitation, four assays were undertaken: Versant HCV RNA 3.0 (Bayer), Real time PCR (TaqMan, Roche), LCx HCV RNA (Abbott), and Cobas Amplicor‐Monitor v2 (Roche). Considering a 2‐log decline at Week 12 after the beginning of therapy, discordant results were found with the four HCV RNA methods in predicting SVR or non‐response. At Week 4 and Week 12, significant differences were observed between Versant HCV RNA 3.0 versus PCR HCV Taqman, Versant HCV RNA 3.0 versus LCx HCV RNA, Cobas Monitor Amplicor HCV 2.0 versus LCx HCV RNA, and Cobas Monitor Amplicor HCV 2.0 versus PCR HCV Taqman (P < 0.001). The HCV RNA cutoff, given a 100% negative predictive value at Week 4 and Week 12, differed with the assays used to quantify HCV RNA, despite the use of the IU/ml units. Eighty‐nine percent of serum values for HCV RNA were concordant by the IU standard. All assays, however, failed to detect HCV RNA in some cases. Despite the use of the IU standard HCV‐infected patients might be monitored with only one assay. J. Med. Virol. 78:208–215, 2006. © 2005 Wiley‐Liss, Inc.     

Dynamic range of hepatitis C virus RNA quantification with the Cobas Ampliprep-Cobas Amplicor HCV Monitor v2.0 assay

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.     

Chemiluminescence enzyme immunoassay for monitoring hepatitis C virus core protein during interferon-alpha2b and ribavirin therapy in patients with genotype 1 and high viral loads

This study evaluated an updated chemiluminescence enzyme immunoassay (CLEIA) for hepatitis C virus (HCV) core protein for monitoring viral kinetics during treatment with interferon (IFN)-alpha and ribavirin. Using the CLEIA, serum levels of HCV core protein were measured in 17 patients with genotype 1 and high baseline viral loads during the first 4 weeks of combination therapy. HCV RNA was measured by the Amplicor Monitor test for comparison. At the start of therapy, the median HCV level (interquartile range) was 700 (540-940) kIU/ml of viral RNA and 11,310 (5,528-14,238) fmol/L of core protein. HCV RNA was above the upper limit of the linear range of the Amplicor Monitor test in 13 of the 17 patients, while the core protein level was within the linear range of the CLEIA in all patients. During therapy, the proportion of patients with HCV levels below the cutoff values at each time point was less with the Amplicor Monitor test than with CLEIA. Serum HCV core protein level decreased rapidly during the first 24 hr of therapy and more slowly thereafter, with median exponential decays of 1.08 and 0.046 log10/day, respectively. In the second phase, between day 1 and 28, the median decrease in HCV core protein level was higher in four patients with sustained virologic response (0.13 log10/day) than in 13 patients with no response (0.028 log10/day, P = 0.042). The wide linear range of the HCV core protein assay is appropriate for measuring viral loads during therapy with IFN-alpha and ribavirin.     

Detection and quantitation of HCV RNA using real-time PCR after automated sample processing

There is considerable evidence that the loss of hepatitis C virus (HCV) RNA during the first 3 months of treatment with pegylated interferon plus ribavirin is a prognostic marker of response to therapy. Real-time polymerase chain reaction (PCR) assays for quantifying HCV RNA in plasma or serum are now commercially available. The extraction of HCV RNA can also be automated. This report analyses the performance of the COBAS Ampliprep-COBAS Taqman 48 (CAP/CTM) real-time PCR assay and compares this new test with the COBAS Amplicor HCV Monitor v 2.0 assay (CAM). CAP/CTM was 100% specific. The assay was linear across a wide range of HCV RNA concentrations without sample dilution. The intra-assay variation was 0.3-3.3% and the interassay variation was 1.5-6.7%. A total of 118 clinical samples with different HCV genotypes were assayed using both methods. The results obtained using the two methods were well correlated (r = 0.89, P < 0.001). The mean difference [CAP/CTM-CAM] was 0.17 log IU/ml and it was not influenced by the HCV genotype or by the subtype. It is concluded that the new CAP/CTM system is adequate for quantifying HCV RNA in clinical practice.     

Development of a TaqMan assay for the six major genotypes of hepatitis C virus: comparison with commercial assays

A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 10(2.6)-10(6.5) IU/ml. In addition, the inter- and intra-assay precision were approximately 3% CV and <2% CV, respectively. Comparison with results obtained by commercially available HCV RNA Nucleic Acid Technology kits (Versant HCV RNA 3.0 b-DNA and Amplicor HCV Monitor), that also employ the WHO standard, allowed validation of the TaqMan assay against all major HCV genotypes. Both commercial methods detected HCV RNA over a wide dynamic range, but showed a consistent difference of about 0.3 log10 when evaluating samples of different HCV genotypes. The genome titers obtained with the three methods correlated with the infectivity titers previously determined for the HCV reference strains. TaqMan assays have become an essential tool to follow viral load in clinical samples and cell culture-based experiments and this technology offers significant advantages in linear dynamic range, sensitivity and customization.     

Comparison of different HCV viral load and genotyping assays.

BACKGROUND: We report an interlaboratory comparison of methods for the determination of hepatitis C virus (HCV) serum load and genotype between a recently, established molecular laboratory at the Alaska Native Medical Center (ANMC) and two independent laboratories using different assays. At ANMC, a Real-time quantitative RT-PCR amplification methodology (QPCR) has been developed in which HCV viral loads are determined by interpolation of QPCR results to those of standards calibrated to the World Health Organization (WHO) First International Standard for HCV. HCV genotype is subsequently determined by direct sequencing of the DNA fragment generated from the QPCR assay. OBJECTIVES AND STUDY DESIGN: The above methods were statistically compared to results obtained for the same patient sera by two independent laboratories using different commercially available viral load assays; Quantiplex HCV RNA (Bayer Diagnostics) and Amplicor HCV Monitor (v 2.0) (Roche Molecular Systems), as well as two different genotyping assays; restriction fragment length polymorphism (RFLP) and INNO-LiPA HCV II (Innogenetics). RESULTS: ANMC's Real-time QPCR HCV viral load results compared moderately well with those obtained by the Quantiplex HCV RNA method (R2=0.3813), and compared quite well with recent lot numbers of Amplicor HCV Monitor in which viral loads are derived in IU/ml (R2=0.6408), but compared poorly with earlier lot numbers of Amplicor HCV Monitor in which viral loads were derived in copies/ml (R2=0.0913). The ANMC direct sequencing method for genotype determination compared moderately to very well with both the RFLP (84-86%) and INNO-LiPA (85-97.5%) methods. CONCLUSIONS: These viral load comparisons highlight the discrepancies that may occur when patient HCV viral loads are monitored using different types of assays. Comparison of HCV genotype by different methods is more reliable statistically and important clinically for predicting probability of response to antiviral therapy. However, viral loads are important for monitoring response once therapy has begun.     

Evaluation of the NucliSens EasyQ assay in HIV-1-infected individuals in South Africa

We compared the performance of the NucliSens EasyQ assay (bioMerieux) combined with the manual NucliSens miniMag extraction methodology to the Roche Cobas Ampliprep/Standard Amplicor Monitor methodology (Roche Diagnostics) for HIV-1 RNA quantitation in HIV-1-infected individuals in South Africa. Plasma samples (284) from HIV sero-positive patients at different stages of infection were analyzed. The distribution of results was typical of the clinical samples received at the laboratory where 20% have viral load results <400 copies/ml (2.6 log) and 18% have viral load results >750000 copies/ml (5.8 log) using the Roche Amplicor Monitor standard assay. All statistical analyses were performed using log10-transformed values for all the variables in the analyses, i.e. log10EasyQIU/ml, and log10RNA (log10 copies/ml, Amplicor). Roche values were converted from RNA copies per ml to IU/ml by multiplying the Roche value by 0.51. HIV RNA levels quantitated by the NucliSens EasyQ assay correlated significantly with those of the Roche Cobas Amplicor Monitor assay (r=0.874, p<0.0001). Reproducibility of the NucliSens EasyQ assay in the log6IU range yielded CV variance of 1.3-2.84% for two well-trained technologists. In addition, a retrospective evaluation of the performance of the NucliSens EasyQ assay in 102 runs (2448) samples was conducted in the laboratory over a 4-month interval. Factors considered during this evaluation included time taken to perform the assay, volume requirements, number of required repeats, potential for contamination.     

Prediction of the efficacy of antiviral therapy for hepatitis C virus infection by an ultrasensitive RT-PCR assay

The efficacy of interferon therapy for hepatitis C virus (HCV) infection improved remarkably. However, virologic relapse occurs in a substantial proportion of patients with virologic response (defined as an HCV RNA level below 50 IU/ml at the end-of-treatment). A highly sensitive RT-nested PCR assay capable of detecting almost a single copy of HCV RNA and a real-time RT-PCR assay to quantify HCV RNA down to 120 copies per ml were developed. The RT-nested PCR assay showed that 1 IU of HCV RNA is equivalent to 12.2 copies. For 28 patients with virologic response (12 relapsers and 16 sustained virologic responders), week-4 and end-of-treatment plasma samples were retested. At week 4, HCV RNA was detected by the RT-nested PCR and qualitative COBAS Amplicor HCV version 2.0 in 8/9 (89%) and 6/9 (67%) samples from relapsers, and in 4/16 (25%) and 2/16 (13%) samples from sustained virologic responders, respectively. End-of-treatment samples with HCV-negative by the qualitative COBAS Amplicor were positive by the present assay in 4/12 (25%) of relapsing patients and 0/16 (0%) of sustained virologic responders. The viral levels detected by the present assay in the Amplicor-negative samples were 3.5-17.3 copies/ml, which is below the detection limit of COBAS Amplicor. In conclusion, the highly sensitive RT-nested PCR assay can predict sustained virologic response at week 4 and virologic relapse at the end-of-treatment more accurately than COBAS Amplicor, suggesting its usefulness in monitoring antiviral therapy for HCV infection.     

Response prediction and treatment tailoring for chronic hepatitis C virus genotype 1 infection

We monitored early viral response during the treatment of hepatitis C virus (HCV) infection with the aim of identifying predictors of treatment outcome. We studied 53 patients with genotype 1 infection who received 180 microg/week pegylated interferon alfa-2a and 1,000 or 1,200 mg/day ribavirin depending on body weight and serially assessed HCV RNA in serum, using the Cobas TaqMan assay. Thirty-one patients (58%) achieved sustained viral response (SVR). SVR was obtained in 100% (10/10) of patients with pretreatment viremia concentrations below 400,000 IU/ml, in 100% (14/14) of patients with more than 1.5 log reduction of HCV RNA after 4 days of treatment, and in 95% (22/23) of patients with a rate of decline in viremia higher than 0.70 log units/week during the second phase. Non-SVR was seen in all patients with a second-phase decline rate lower than 0.35 log units/week. Patients with slopes between 0.50 and 0.80 log units/week achieved SVR (4/4) unless the treatment dose was modified (3/3). We conclude that the second-phase slope appears to be an accurate and useful predictor of treatment response. On the basis of these findings, we propose a model of tailored treatment which takes into account the second-phase slope and the amount of HCV RNA after 21 days of treatment.     

Second-Generation Cobas AmpliPrep/Cobas TaqMan HCV Quantitative Test for Viral Load Monitoring: a Novel Dual-Probe Assay Design

Hepatitis C virus (HCV) RNA viral load (VL) monitoring is a well-established diagnostic tool for the management of chronic hepatitis C patients. HCV RNA VL results are used to make treatment decisions with the goal of therapy to achieve an undetectable VL result. Therefore, a sensitive assay with high specificity in detecting and accurately quantifying HCV RNA across genotypes is critical. Additionally, a lower sample volume requirement is desirable for the laboratory and the patient. This study evaluated the performance characteristics of a second-generation real-time PCR assay, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2.0 (CAP/CTM HCV test, v2.0), designed with a novel dual-probe approach and an optimized automated extraction and amplification procedure. The new assay demonstrated a limit of detection and lower limit of quantification of 15 IU/ml across all HCV genotypes and was linear from 15 to 100,000,000 IU/ml with high accuracy (<0.2-log₁₀ difference) and precision (standard deviation of 0.04 to 0.22 log₁₀). A specificity of 100% was demonstrated with 600 HCV-seronegative specimens without cross-reactivity or interference. Correlation to the Cobas AmpliPrep/Cobas TaqMan HCV test (version 1) was good (n = 412 genotype 1 to 6 samples, R² = 0.88; R² = 0.94 without 105 genotype 4 samples). Paired plasma and serum samples showed similar performance (n = 25, R² = 0.99). The sample input volume was reduced from 1 to 0.65 ml in the second version. The CAP/CTM HCV test, v2.0, demonstrated excellent performance and sensitivity across all HCV genotypes with a smaller sample volume. The new HCV RNA VL assay has performance characteristics that make it suitable for use with currently available direct-acting antiviral agents.     

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.     

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.     

Comparison of two quantitative CMV PCR tests, Cobas Amplicor CMV Monitor and TaqMan assay, and pp65-antigenemia assay in the determination of viral loads from peripheral blood of organ transplant patients.

BACKGROUND: Quantitative PCR assays have become the most common methods in the determination of viral load during cytomegalovirus (CMV) infection of transplant patients. However, usually these tests are still quite time-consuming and labor-intensive which diminishes their utility of these tests in routine diagnostic laboratories. Objectives: The objective of this study was to develop a quantitative CMV PCR test which is time-saving and easy to perform for the detection and monitoring of CMV infection of transplant patients. STUDY DESIGN: The quantitative real time CMV PCR assay using TaqMan chemistry and an automated sample preparation system, MagNA Pure LC, was developed. The designed quantitative CMV test was compared to commercial quantitative PCR test, Cobas Amplicor Monitor, in the determination of CMV DNA loads in plasma samples of liver and kidney transplant patients. The results were also correlated with the CMV pp65-antigenemia test. The clinical material of 270 blood specimens of transplant patients were tested using these two PCR methods and pp65-antigenemia test in parallel. Plasma samples were used for PCR assays and leucocytes for the antigenemia test. RESULTS: The TaqMan assay described was easy to perform, it was rapid (3-4 h) and hands-on time needed for performing the test was short. The detection limit of the assay was 250 copies/ml (cps/ml) plasma and the linear range up to 25,000,000 cps/ml. TaqMan assay was the most sensitive test detecting 92% of the CMV positive findings. Cobas Monitor detected 80% and pp65 test 88% of the positive findings. The correlations between TaqMan and antigenemia assays, and between Cobas Amplicor and antigenemia were statistically significant and high, R = 0.84 (P < 0.0001) and R = 0.80 (P < 0.0001), respectively. Also correlation between two PCR tests was statistically significant (R = 0.64, P < 0.0001). Of the 27 patient studied, 19 demonstrated CMV antigenemia and DNAemia in their blood during the post transplant monitoring. Thirteen of these patients developed a symptomatic CMV infection and were treated with ganciclovir. The peak viral loads of symptomatic patients were statistically higher by all three methods than those of asymptomatic patients. CONCLUSIONS: The developed real time TaqMan assay was rapid and easily performed and could be the best alternative for the diagnosis of CMV infection and monitoring of liver and kidney transplant patients.     

Performance of an automated system for quantification of hepatitis C virus RNA

The Amplicor HCV Monitor test for quantitative determination of serum or plasma hepatitis C virus (HCV) RNA was modified recently and introduced onto the Cobas Amplicor instrument to automate fully amplification, detection and calculation of results. This new version (v2.0) was evaluated in a routine diagnostic laboratory. The sensitivity and reproducibility were assessed on well-characterized panels (Eurohep) and clinical samples. HCV RNA levels measured by the v2.0 Monitor test were about 1log(10) higher than those detected by the previous version test, and genotypes 1 and 3 were quantified with equal sensitivity. Within the linear dynamic range of 10(3) to 10(6) copies/ml, the coefficients of variation for both intra- and inter-assay reproducibility ranged from 1.9 to 2.95%. This test system was found to be a reliable and labor saving assay for the quantification of HCV RNA.     

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.     

The Cobas AmpliPrep/Cobas TaqMan HCV Test,Version 2.0, Real-Time PCR Assay Accurately Quantifies Hepatitis C Virus Genotype 4 RNA

Accurate hepatitis C virus (HCV) RNA quantification is mandatory for the management of chronic hepatitis C therapy. The first-generation Cobas AmpliPrep/Cobas TaqMan HCV test (CAP/CTM HCV) underestimated HCV RNA levels by >1-log₁₀ international units/ml in a number of patients infected with HCV genotype 4 and occasionally failed to detect it. The aim of this study was to evaluate the ability of the Cobas AmpliPrep/Cobas TaqMan HCV test, version 2.0 (CAP/CTM HCV v2.0), to accurately quantify HCV RNA in a large series of patients infected with different subtypes of HCV genotype 4. Group A comprised 122 patients with chronic HCV genotype 4 infection, and group B comprised 4 patients with HCV genotype 4 in whom HCV RNA was undetectable using the CAP/CTM HCV. Each specimen was tested with the third-generation branched DNA (bDNA) assay, CAP/CTM HCV, and CAP/CTM HCV v2.0. The HCV RNA level was lower in CAP/CTM HCV than in bDNA in 76.2% of cases, regardless of the HCV genotype 4 subtype. In contrast, the correlation between bDNA and CAP/CTM HCV v2.0 values was excellent. CAP/CTM HCV v2.0 accurately quantified HCV RNA levels in the presence of an A-to-T substitution at position 165 alone or combined with a G-to-A substitution at position 145 of the 5′ untranslated region of HCV genome. In conclusion, CAP/CTM HCV v2.0 accurately quantifies HCV RNA in genotype 4 clinical specimens, regardless of the subtype, and can be confidently used in clinical trials and clinical practice with this genotype.     

An OPTIMIZE Study Retrospective Analysis for Management of Telaprevir-Treated Hepatitis C Virus (HCV)-Infected Patients by Use of the Abbott RealTime HCV RNA Assay

Protease inhibitor (PI)-based response-guided triple therapies for hepatitis C virus (HCV) infection are still widely used. Noncirrhotic treatment-naive and prior relapser patients receiving telaprevir-based treatment are eligible for shorter, 24-week total therapy if HCV RNA is undetectable at both weeks 4 and 12. In this study, the concordance in HCV RNA assessments between the Roche High Pure System/Cobas TaqMan and Abbott RealTime HCV RNA assays and the impacts of different HCV RNA cutoffs on treatment outcome were evaluated. A total of 2,629 samples from 663 HCV genotype 1 patients receiving telaprevir/pegylated interferon/ribavirin in OPTIMIZE were analyzed using the High Pure System and reanalyzed using Abbott RealTime (limits of detection, 15.1 IU/ml versus 8.3 IU/ml; limits of quantification, 25 IU/ml versus 12 IU/ml, respectively). Overall, good concordance was observed between the assays. Using undetectable HCV RNA at week 4, 34% of the patients would be eligible for shorter treatment duration with Abbott RealTime versus 72% with the High Pure System. However, using <12 IU/ml for Abbott RealTime, a similar proportion (74%) would be eligible. Of the patients receiving 24-week total therapy, 87% achieved a sustained virologic response with undetectable HCV RNA by the High Pure System or <12 IU/ml by Abbott RealTime; however, 92% of the patients with undetectable HCV RNA by Abbott RealTime achieved a sustained virologic response. Using undetectable HCV RNA as the cutoff, the more sensitive Abbott RealTime assay would identify fewer patients eligible for shorter treatment than the High Pure System. Our data confirm the <12-IU/ml cutoff, as previously established in other studies of the Abbott RealTime assay, to determine eligibility for shortened PI-based HCV treatment. (The study was registered with ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01241760","term_id":"NCT01241760"}}NCT01241760.)     

Transient reappearance of serum hepatitis C virus RNA observed by real-time PCR during antiviral therapy with peginterferon and ribavirin in patients with HCV genotype 1b

BackgroundThe “response-guided therapy” based on response of hepatitis C virus (HCV) during antiviral combination therapy with peginterferon and ribavirin is important for patients with HCV genotype 1. However, the sensitivity of previous assays for serum HCV RNA is limited.ObjectivesWe evaluated the changes in serum HCV RNA during the combination therapy using a novel method for measurement based on real-time PCR.Study designChanges in serum HCV RNA during the combination therapy were reanalyzed using TaqMan PCR assay in 144 patients with chronic HCV genotype 1b infection who underwent the therapy under HCV RNA monitoring with the Amplicor Monitor assay. Treatment duration was elongated from 48 weeks to 72 weeks in 17 patients based on the time when serum HCV RNA became negative.ResultsIn 9 of 144 (6.3%) patients, serum HCV RNA transiently appeared again on the TaqMan PCR assay after having previously become negative. At the point of reappearance, the Amplicor Monitor assay gave a negative result in all patients, and no flare of alanine aminotransferase activity was observed. Each of the 9 patients achieved an end-of-treatment response but relapsed after the end of treatment, including 3 patients in whom the treatment duration was elongated to 72 weeks.ConclusionsAttention should be paid to this phenomenon in the antiviral treatment for patients with HCV infection. The transient reappearance of HCV RNA in the serum indicates a high likelihood of relapse, and is likely to be missed without frequent measurements by a sensitive detection method.