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.     

Performance characteristics of the COBAS Amplicor Hepatitis C Virus (HCV) Monitor, Version 2.0, International Unit assay and the National Genetics Institute HCV Superquant assay

The COBAS Amplicor Hepatitis C Virus (HCV) Monitor assay, version 2.0, which reports in international units per milliliter, was compared to the assay reported in copies per milliliter by analyzing dilution series and clinical plasma samples by both methods. In addition, the Amplicor international unit assay was compared to the National Genetics Institute HCV Superquant assay. The dilution series ranged from <100 to 5,000,000 HCV RNA copies/ml and consisted of 32 points, assayed in triplicate in each assay. Thirty clinical samples ranging from 1,000 to 1,000,000 HCV RNA copies/ml were assayed in duplicate. Deming regression analysis comparing the Amplicor HCV RNA international units-per-milliliter and copies-per-milliliter assays was calculated as follows: (Amplicor international units per milliliter) = 1.030(Amplicor copies per milliliter) - 0.392; R(2) = 0.981; n = 28; S(y/x) (standard error of the estimate) = 0.129. The linearity of the Amplicor international units-per-milliliter assay was as follows: observed = 0.886(expected) + 0.437; R(2) = 0.983; n = 30. The linearity of the Superquant assay was as follows: observed= 0.918 (expected) + 0.436; R(2) = 0.986; n = 32. Deming regression analysis comparing the Amplicor and Superquant assays was calculated as follows: Superquant = 1.066(Amplicor) - 0.0197; R(2) = 0.908; S(y/x) = 0.308; n = 28. The Amplicor and Superquant assays were linear through the range of 600 to 600,000 IU of HCV RNA/ml and approximately 300 to 5,000,000 HCV RNA copies/ml, respectively. The narrow range of the Amplicor assay means that some samples will require dilution and retesting for accurate quantification above 600,000 IU of HCV RNA/ml. The Amplicor and Superquant assays agreed well within the range of 600 to 600,000 IU of HCV RNA/ml (approximately 1,000 to approximately 1,000,000 HCV RNA copies/ml). Overall, the Amplicor and Superquant assays agree well, and results obtained in one assay could be expected to compare well with results from the other when reported in copies per milliliter.     

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.     

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 application of the Quantiplex HCV RNA 2.0 and Amplicor HCV Monitor assays for quantifying serum hepatitis C virus RNA

AIM: To compare the performance characteristics and clinical application of two different technologies for quantifying serum hepatitis C virus (HCV) RNA levels. METHODS: HCV RNA was quantified by Amplicor HCV Monitor assay (Amplicor) and Quantiplex HCV RNA 2.0 assay (bDNA-2) in 119 sera from 107 HCV infected patients. RESULTS: Both assays had similar sensitivity (79.4% for Amplicor; 86.0% for bDNA-2), acceptable coefficients of variation (5.3% in Amplicor; 2.6% in bDNA-2), and good linearity (r2 > or = 0.98). There was a positive correlation between quantification values of both methods (r = 0.683, p < 0.001). The Amplicor values were on an average 1.76 log lower than bDNA-2 results. Male subjects and HCV genotype 1b were significantly associated with higher viral load determined by Amplicor, but not with viral load measured by bDNA-2. In 70 chronic HCV infected patients treated with interferon alfa, mean (SD) pretreatment viral load in 27 complete responders (3.47 (0.84) logs for Amplicor, 5.63 (0.58) for bDNA-2) was significantly lower than in non-responders (4.43 (1.01) logs for Amplicor, 6.10 (0.67) logs for bDNA-2; p < 0.001). Cut off points of 3.9 logs for Amplicor and 5.8 logs for bDNA-2 were determined to be the best for predicting response to interferon alfa, giving acceptable sensitivity (70.4%, 74.1%), specificity (72.1%, 65.1%), and accuracy (71.4%, 68.6%), respectively. CONCLUSIONS: Both the Amplicor and bDNA-2 assays are clinically useful methods for HCV RNA quantification and are reliable for predicting the outcome of treatment, despite differences in absolute quantification values and in the correlation between HCV genotypes and viral load.     

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.     

Performance of the New Bayer VERSANT HCV RNA 3.0 assay for quantitation of hepatitis C virus RNA in plasma and serum: conversion to international units and comparison with the Roche COBAS Amplicor HCV Monitor, Version 2.0, assay

We have evaluated the VERSANT HCV RNA 3.0. Assay (HCV 3.0 bDNA assay) (Bayer Diagnostics, Berkeley, Calif.), which is an improved signal amplification procedure for the HCV 2.0 bDNA assay for the quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The HCV 3.0 bDNA assay has a linear dynamic range of 2.5 x 10(3) to 4.0 x 10(7) HCV RNA copies per ml (c/ml). The performance of the HCV 3.0 bDNA assay was evaluated using three different test panels. An overall specificity of 96.8% relative to the detection limit of the HCV 3.0 bDNA assay was found. The intra- and interrun reproducibilities for both the dilution panel and the NAP (AcroMetrix, Benicia, Calif.) panel were consistent with coefficients of variation of less than 9%. Quantitation with the HCV 3.0 bDNA assay was linear over the entire range of both panels (ranges of 4.4 x 10(3) to 3.5 x 10(6) c/ml and 5 x 10(3) to 2 x 10(6) IU/ml, respectively), with correlation coefficients of 0.999, slopes close to one, and intercepts close to zero. The regression equation indicated that 1 IU corresponded to about 4.8 copies of HCV RNA. A correlation coefficient of 0.941 was found for HCV RNA values (in international units per milliliter) obtained from the HCV 3.0 bDNA assay and the HCV Monitor version 2.0 assay (HCV Monitor 2.0 assay) (Roche Diagnostic Systems, Branchburg, N.J.). Quantitative results obtained close to the lower limit of the HCV 3.0 bDNA assay might imply that its lower limit should be reconsidered and raised, if necessary. It appeared that quantitation values obtained from the HCV Monitor 2.0 assay of between 5 x 10(2) and 10(5) IU/ml were in general higher than those obtained from the HCV 3.0 bDNA assay, whereas values obtained from the HCV Monitor 2.0 assay were underestimated for samples with HCV RNA levels above 10(5) IU/ml.     

Quantification of hepatitis C virus by TaqMan PCR: comparison with HCV Amplicor Monitor assay.

The quantitation of serum levels of hepatitis C virus RNA in chronic hepatitis C has been regarded as one of the most important indicators for the outcome of interferon therapy. A new method was used for quantitating the copy number of hepatitis C virus RNA using TaqMan polymerase chain reaction and for comparing the ability and usefulness of this assay with Amplicor Monitor assay in 138 patients. The detection range of hepatitis C virus RNA by TaqMan polymerase chain reaction was from 2 x 10(3) to 2 x 10(8) copies/ml. Hepatitis C virus RNA was detectable in 128 cases (92.8%) and undetectable in 10 cases (7.2%) by this method. The RNA levels measured by Amplicor Monitor assay correlated significantly with those measured by TaqMan polymerase chain reaction assay and the sensitivity of the two assays was almost equal. Thus, TaqMan polymerase chain reaction assay appears sufficiently sensitive for the evaluation of hepatitis C virus RNA and would be useful for the diagnosis and management of hepatitis C virus infection.     

Quantitative and cost comparison of ultrasensitive human immunodeficiency virus type 1 RNA viral load assays: Bayer bDNA quantiplex versions 3.0 and 2.0 and Roche PCR Amplicor monitor version 1.5

Quantification of human immunodeficiency virus type 1 (HIV-1) RNA as a measure of viral load has greatly improved the monitoring of therapies for infected individuals. With the significant reductions in viral load now observed in individuals treated with highly active anti-retroviral therapy (HAART), viral load assays have been adapted to achieve greater sensitivity. Two commercially available ultrasensitive assays, the Bayer Quantiplex HIV-1 bDNA version 3.0 (bDNA 3.0) assay and the Roche Amplicor HIV-1 Monitor Ultrasensitive version 1.5 (Amplicor 1.5) assay, are now being used to monitor HIV-1-infected individuals. Both of these ultrasensitive assays have a reported lower limit of 50 HIV-1 RNA copies/ml and were developed from corresponding older generation assays with lower limits of 400 to 500 copies/ml. However, the comparability of viral load data generated by these ultrasensitive assays and the relative costs of labor, disposables, and biohazardous wastes were not determined in most cases. In this study, we used matched clinical plasma samples to compare the quantification of the newer bDNA 3.0 assay with that of the older bDNA 2.0 assay and to compare the quantification and costs of the bDNA 3.0 assay and the Amplicor 1.5 assay. We found that quantification by the bDNA 3.0 assay was approximately twofold higher than that by the bDNA 2.0 assay and was highly correlated to that by the Amplicor 1.5 assay. Moreover, cost analysis based on labor, disposables, and biohazardous wastes showed significant savings with the bDNA 3.0 assay as compared to the costs of the Amplicor 1.5 assay.     

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.     

Use of Molecular Assays in Diagnosis and Monitoring of Cytomegalovirus Disease following Renal Transplantation

We compared two commercial molecular assays (the Murex Hybrid Capture CMV DNA assay [HCA], version 2, and the Roche Amplicor plasma PCR assay) with a standard shell vial assay in detecting and predicting cytomegalovirus (CMV) disease in a group of renal transplant patients and assessed the role of viral load measurements (using the HCA) in their management. The sensitivity of the HCA and Amplicor assay in terms of disease detection was 100%, compared to 71% for the shell vial assay. Both the HCA and the PCR assay detected all cases of disease, at medians of 11 and 12.5 days before the onset of symptoms, respectively. Significantly higher viral loads were detected in those patients with symptoms (7.9 x 10(5) copies/ml) than in patients without symptoms (7.9 x 10(4) copies/ml; P < 0.0001). There was also a trend towards higher viral loads in those patients with primary infections (7.8 x 10(5) copies/ml) than in those patients with reactivations of CMV disease or reinfections. Successful treatment with ganciclovir was associated with a >90% reduction in viral load. Both of these new assays are sensitive and easy to use. A comparison of accurate quantitation is also useful in monitoring responses to antiviral therapy.     

Comparison of the COBAS TaqMan HBV test with the COBAS Amplicor monitor test for measurement of hepatitis B virus DNA in serum

Quantitation of low hepatitis B virus (HBV) DNA levels in patients with chronic hepatitis B is important for monitoring natural history of disease and treatment efficacy. This study aimed to compare the quantitation range and analytical sensitivity of the newly developed COBAS TaqMan HBV test (TaqMan test) with the COBAS Amplicor HBV Monitor Test (Amplicor test), using the Eurohep HBV reference plasma and serum samples from patients. Serial dilutions (2.7x10(1)-2.7x10(8) copies/ml) of the Eurohep HBV reference plasma and 50 serum samples from chronic hepatitis B patients were tested by both assays. The TaqMan test could detect seven (2.7x10(2)-2.7x10(8) copies/ml) of eight dilutions of the reference plasma, while the Amplicor test could only detect three of them (2.7x10(3)-2.7x10(5) copies/ml). The HBV DNA values measured by the TaqMan test correlated very well with the theoretical Eurohep standard values (r=0.998, P<0.001). There were good correlations between the HBV DNA levels measured by the two assays on both the Eurohep reference plasma (r=0.993, P<0.001) and serum samples from patients (r=0.904, P<0.001). Compared to the Amplicor test, the TaqMan test had a higher sensitivity (50 vs. 300 copies/ml), shorter assay time (6 vs. 10 hr), and wider dynamic range (8 vs. 3 logs), and was more cost-effective in a clinical setting. These data indicate that the TaqMan test is an excellent tool for HBV DNA quantitation.     

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.     

Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.     

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.