Performance of Version 2.0 of the Cobas AmpliPrep/Cobas TaqMan Real-Time PCR Assay for Hepatitis B Virus DNA Quantification

The detection and quantification of hepatitis B virus (HBV) DNA are essential for the diagnosis and treatment of chronic HBV infection. The use of real-time PCR assays for HBV DNA quantification is strongly recommended. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of version 2.0 (v2.0) of the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) assay, a fully automated platform for HBV DNA quantification in serum or in plasma with a claimed lower limit of detection of 20 IU/ml and a claimed upper limit of quantification of 1.7 × 10⁸ IU/ml. The specificity of the assay was 99% (95% confidence interval, 94.7 to 100%). Intra-assay and interassay coefficients of variation ranged from 0.21% to 2.67% and from 0.65% to 2.25%, respectively. The calibration of the assay was found to be satisfactory. Study of blood specimens from patients infected with HBV genotypes A to F showed good correspondence between HBV DNA levels measured by the CAP/CTM v2.0 assay, version 1.0 of the same assay, and the third-generation “branched DNA” assay. The CAP/CTM v2.0 assay quantified HBV DNA levels in serum or plasma from the same patients equally. In conclusion, the new version of the CAP/CTM assay is sensitive, specific, and reproducible. It accurately quantifies HBV DNA levels in patients chronically infected with HBV genotypes A to F. Improvements made to ensure equal quantification of HBV DNA in serum and plasma have been successful. Overall, the CAP/CTM assay, version 2.0, is well suited to monitoring clinical HBV DNA levels according to current clinical practice guidelines.Chronic hepatitis B virus (HBV) infection is associated with a large spectrum of liver diseases, ranging from a low-viremia inactive carrier state to chronic active hepatitis, which may subsequently evolve toward cirrhosis and hepatocellular carcinoma (HCC). Morbidity and mortality are linked to the persistence of viral replication, and hepatic complications develop in 15% to 40% of patients chronically infected with HBV. Overall, HBV-related end-stage liver disease and HCC are responsible for more than 750,000 deaths worldwide per year (5).The detection and quantification of HBV DNA are essential for diagnosing ongoing HBV infection and establishing the prognosis of related liver disease, influence the decision to treat, and are indispensable for monitoring the virological response to antiviral therapy and the emergence of resistance in order to tailor therapy (4). A number of HBV DNA detection and quantification assays are available. For many years, such methods were based on either hybrid capture, signal amplification by means of branched DNA (bDNA) technology, or classical PCR, all of which suffered from poor analytical sensitivity and a narrow range of HBV DNA quantification (3). More recently, assays based on real-time PCR quantification have been developed. Their use for the routine detection and quantification of HBV DNA is recommended, because of their excellent analytical sensitivity (lower limit of detection, 10 to 20 IU/ml), their specificity, their accuracy, and their broad dynamic range of linear quantification, which fully covers clinical needs (9). Among these assays, we recently evaluated the first-generation (V1.0) Cobas AmpliPrep/Cobas TaqMan (CAP/CTM; Roche Molecular Systems, Pleasanton, CA) assay. This assay was found to be sensitive, specific, and reproducible and to accurately quantify HBV DNA levels in patients chronically infected by HBV genotypes A to F (2). However, this assay could be used only for the quantification of HBV DNA in plasma.A second version of the CAP/CTM assay (v2.0) has been released recently. Several changes have been made; in particular, the assay can be used on both serum and plasma, and it requires 650 μl of sample instead of 850 μl. Its claimed dynamic range of quantification is 20 IU/ml to 1.7 × 10⁸ IU/ml (1.3 to 8.2 log₁₀ IU/ml). The goal of this study was to evaluate the intrinsic characteristics and clinical performance of the CAP/CTM v2.0 assay.     

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.     

Abbott RealTime Hepatitis C Virus (HCV) and Roche Cobas AmpliPrep/Cobas TaqMan HCV Assays for Prediction of Sustained Virological Response to Pegylated Interferon and Ribavirin in Chronic Hepatitis C Patients

Two commercial real-time PCR assays are currently available for sensitive hepatitis C virus (HCV) RNA quantification: the Abbott RealTime HCV assay (ART) and Roche Cobas AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM). We assessed whether the two real-time PCR assays were more effective than Roche Cobas Amplicor HCV Monitor test, v.2.0 (CAM) for prediction of the sustained virological response (SVR) to pegylated interferon (PEG-IFN) plus ribavirin (RBV) in chronic hepatitis C. Sixty patients chronically infected with HCV genotype 1b (37 males and 23 females, 53 ± 12 years of age) were treated with PEG-IFNα2b plus RBV for 48 weeks. Stored specimens at nine time points for each patient (at baseline, on treatment, and 24 weeks after treatment) were tested by the two real-time PCR assays and CAM. Twenty-six (43.3%) patients reached SVR. The positive predictive values (PPVs) for SVR of undetectable HCV RNA at week 12 by CAM, ART, and CAP/CTM were 74.3%, 88.0%, and 95.2%, respectively. An undetectable HCV RNA level by CAM, ART, and CAP/CTM correctly predicted SVR at week 4 in 100%, 100%, and 100% of patients, at weeks 5 to 8 in 91.7%, 100%, and 100% of patients, at weeks 9 to 12 in 55.6%, 75%, and 87.5% of patients, and at weeks 13 to 24 in 0%, 26.7%, and 40% of patients, respectively. Of 16 patients who relapsed after treatment, HCV RNA was detectable in 2 patients at the end of treatment by CAP/CTM but undetectable by ART and CAM. HCV RNA tests using ART and CAP/CTM are considered to be more effective at predicting SVR than CAM, and the PPV for SVR was slightly higher in CAP/CTM than in ART.     

Performance Comparison of the Versant HCV Genotype 2.0 Assay (LiPA) and the Abbott Realtime HCV Genotype II Assay for Detecting Hepatitis C Virus Genotype 6

The Versant HCV genotype 2.0 assay (line probe assay [LiPA] 2.0), based on reverse hybridization, and the Abbott Realtime HCV genotype II assay (Realtime II), based on genotype-specific real-time PCR, have been widely used to analyze hepatitis C virus (HCV) genotypes. However, their performances for detecting HCV genotype 6 infections have not been well studied. Here, we analyzed genotype 6 in 63 samples from the China HCV Genotyping Study that were originally identified as genotype 6 using the LiPA 2.0. The genotyping results were confirmed by nonstructural 5B (NS5B) or core sequence phylogenetic analysis. A total of 57 samples were confirmed to be genotype 6 (51 genotype 6a, 5 genotype 6n, and 1 genotype 6e). Four samples identified as a mixture of genotypes 6 and 4 by the LiPA 2.0 were confirmed to be genotype 3b. The remaining two samples classified as genotype 6 by the LiPA 2.0 were confirmed to be genotype 1b, which were intergenotypic recombinants and excluded from further comparison. In 57 genotype 6 samples detected using the Realtime II version 2.00 assay, 47 genotype 6a samples were identified as genotype 6, one 6e sample was misclassified as genotype 1, and four 6a and five 6n samples yielded indeterminate results. Nine nucleotide profiles in the 5′ untranslated region affected the performances of both assays. Therefore, our analysis shows that both assays have limitations in identifying HCV genotype 6. The LiPA 2.0 cannot distinguish some 3b samples from genotype 6 samples. The Realtime II assay fails to identify some 6a and all non-6a subtypes, and it misclassifies genotype 6e as genotype 1.     

Evaluation of an Upgraded Version of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Test for HIV-1 Load Quantification

We evaluated the performance of the prototype Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0, using prospective and archived clinical samples initially underquantitated by the Cobas AmpliPrep/Cobas TaqMan HIV-1 test. The performance of the new test was significantly improved, and the majority of the underquantitation observed with the first-version test was eliminated.Plasma HIV viral load quantification is a validated tool for monitoring human immunodeficiency virus type 1 (HIV-1) infection (7). In 2005, Abbott Molecular (Rungis, France) and Roche Diagnostics GmbH (Mannheim, Germany) launched CE-marked (European Community) tests with the automated extraction of nucleic acids coupled to real-time PCR, giving a broader linear range of quantification and fewer time-consuming manipulations (2, 6, 9). For accurate viral load quantitation by PCR, HIV genetic diversity poses a major difficulty, especially in patients infected by non-B subtypes (1, 4, 6, 10, 11). In France, the frequency of naïve chronically infected patients with non-B subtypes reached 42% in 2006 to 2007 (D. Descamps, M. L. Chaix, A. Storto, F. Barin, S. Pakianather, A. G. Marcellin, M. Wirden, B. Masquelier, F. Brun-Vezinet, D. Costagliola, and the ANRS AC11 Resistance Group, presented at the XVI Conference on Retroviruses and Opportunistic Infections, Montreal, Canada, 8 to 11 February 2009).Recently, we reported significant HIV-1 viral load discrepancies between the Cobas AmpliPrep/Cobas TaqMan HIV-1 test (TaqMan 1; also termed CAP/CTM v1.0) and the Cobas Amplicor HIV-1 Monitor test, version 1.5 (reference assay; also termed CA/HIM v1.5). TaqMan 1 underquantified not only divergent subtypes like CRF-02 but also subtype B isolates (5). Reports on underquantitation spurred the development of a second-generation real-time PCR assay, the Cobas AmpliPrep/Cobas TaqMan HIV-1 test, version 2.0 (TaqMan 2; also termed CA/CTM v2.0), which simultaneously amplifies and detects two targets of the HIV-1 genome.The aim of this study was to evaluate the performance of this new prototype side by side with TaqMan 1. Results for both tests were compared to data for the reference assay.Two ANRS (Agence Nationale de Recherche sur le SIDA) member virology laboratories in Paris, France (Bichat-Claude Bernard and Necker Hospitals), performed the evaluation using a panel of archived samples and a prospective panel of routine clinical plasma samples from the two ANRS virology laboratories. The archived panel included 25 plasma samples, stored at −80°C, for which the TaqMan 1 results previously had deviated from the reference assay results by ≥0.5 log₁₀ (5). The prospective panel consisted of 263 routine plasma samples with detectable HIV-1 viral load. All specimens were diluted in HIV-1-negative plasma to generate about 5 ml, were aliquoted, and were stored frozen until single use. Reagents were provided by Roche Molecular Diagnostics.Analyses for the TaqMan tests were performed in the two French laboratories, and the reference assay results were provided by Roche Molecular Diagnostics. Agreement between the three assays was evaluated by Bland-Altman plots (3). Underquantitation was defined as a greater-than −0.5 log₁₀-titer deviation from the reference test.Among the 25 archived samples, the HIV-1 subtype distribution was the following: A (n = 3), B (n = 9), F (n = 1), G (n = 1), CRF02 (n = 10), and CRF06 (n = 1). The mean viral load was 2.94, 3.94, and 3.77 log₁₀ copies/ml for TaqMan 1, TaqMan 2, and the reference assay, respectively (Fig. ​(Fig.1A,1A, B, C, and Table ​Table1).1). Seventeen of the 25 specimens were confirmed as underquantitated by more than 0.5 log₁₀ in TaqMan 1 compared to results for the reference test (>1 log₁₀, n = 10; −0.5 to −1 log₁₀, n = 7). Among these samples, all 17 differed from the reference by less than −0.5 log₁₀ in the new test. Five out the 25 samples were underquantitated in a range of −0.3 to −0.5 log₁₀, and the underquantitation previously described was not reproducible in this study for three samples (5).Open in a separate windowFIG. 1.Bland-Altman plots for comparisons between CAP/CTM v1.0, CAP/CTM v2.0, and CA/HIM v1.5 in a panel of 25 archived plasma samples. (A) Bland-Altman plot for the comparison between CAP/CTM v1.0 and CA/HIM v1.5 in 25 archived samples. The broken line indicates the mean log₁₀ difference, and the dotted lines represent the borders of the 95% confidence intervals (−2.29 and 0.62). (B) Bland-Altman plot for the comparison between CAP/CTM v2.0 and CA/HIM v1.5 in 25 archived samples. The broken line indicates the mean log₁₀ difference, and the dotted lines represent the borders of the 95% confidence intervals (−0.79 and 1.12). (C) Bland-Altman plot for the comparison between CAP/CTM v1.0 and CAP/CTM v2.0 in 25 archived samples. The broken line indicates the mean log₁₀ difference, and the dotted lines represent the borders of the 95% confidence intervals (−0.43 and 2.42).

TABLE 1.

Deming regression and Bland-Altman plots for measurement of agreement between CAP/CTM v1.0, CAP/CT v2.0, and CA/HIM v1.5^a

Evaluation of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 Test and Identification of Rare Polymorphisms Potentially Affecting Assay Performance

We evaluated the FDA-approved Roche Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) HIV-1 viral load assay for sensitivity, reproducibility, linearity, HIV-1 subtype detection, and correlation to the Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor). The limit of detection calculated by probit analysis was 23.8 copies/ml using the 2nd International WHO Standard and 30.8 copies/ml using Viral Quality Assurance (VQA) standard material. Serial dilutions of six patient samples were used to determine inter- and intra-assay reproducibility and linearity, which were very good (<8% coefficient of variation [CV]; between ∼1.7 and 7.0 log₁₀ copies/ml). Subtype detection was evaluated in the CAP/CTM, Amplicor, and Bayer Versant HIV-1 bDNA 3.0 (Versant) assays using a commercially available panel. Versant averaged 0.829 log₁₀ copies/ml lower than CAP/CTM and Amplicor averaged 0.427 log₁₀ copies/ml lower than CAP/CTM for the subtype panel. Correlation with samples previously tested by Amplicor was excellent (R² = 0.884; average difference [Amplicor value minus CAP/CTM value], 0.008 log₁₀ copies/ml). Of the 305 HIV samples tested, 7 samples generated CAP/CTM titers between 1.0 and 2.75 log₁₀ copies/ml lower than those for Amplicor. Three of these samples revealed primer and probe mismatches that could account for the discrepancies. Otherwise, the CAP/CTM assay exhibits excellent sensitivity, dynamic range, reproducibility, and correlation with Amplicor in an automated format.Measurement of HIV-1 RNA in the plasma of infected patients is critical for guiding treatment. Over the past decade, several commercial quantitative HIV-1 RNA assays have become available that utilize endpoint PCR, isothermal amplification, or signal amplification techniques. Most recently, Abbott Molecular (Des Plaines, IL) and Roche Molecular Systems (Branchburg, NJ) received FDA approval for their real-time PCR-based systems, the RealTime HIV-1 assay and Cobas AmpliPrep/Cobas TaqMan HIV-1 Test (CAP/CTM), respectively. Each assay has its own advantages and disadvantages in terms of sensitivity, equipment requirements, throughput, dynamic range, subtype detection, and cost (1a, 4, 6, 7, 8, 11, 13, 15, 16).The CAP/CTM test includes a “docked” version that permits automated “sample in—results out” analysis of specimens without user intervention. We evaluated this configuration and compared its performance to those of the Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor), and the Bayer Versant HIV-1 bDNA 3.0 assay (Versant). Seven samples which gave discrepant Amplicor versus CAP/CTM results were further evaluated by sequencing analysis.     

Comparison of Abbott RealTime HCV Genotype II with Versant Line Probe Assay 2.0 for Hepatitis C Virus Genotyping

Genotyping and subtyping of 225 samples with hepatitis C virus (HCV) genotype 1, 2, 3, or 6 infection were done with Versant LiPA 2.0 and Abbott RealTime HCV Genotype (GT) II by using direct sequencing of the NS5B and 5′ untranslated regions as the reference standards. The concordance rates were >99.2% for genotypes and 96.1% for subtypes 1a and 1b. Both the Abbott RealTime and Versant LiPA assays can accurately determine hepatitis C virus genotypes. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00979979","term_id":"NCT00979979"}}NCT00979979.)     

Establishment of a Novel Quantitative Hepatitis D Virus (HDV) RNA Assay Using the Cobas TaqMan Platform To Study HDV RNA Kinetics

Determination of hepatitis D virus (HDV) viremia represents the “gold standard” for the diagnosis of HDV infection. Hepatitis B virus (HBV)-HDV coinfection frequently leads to end-stage liver disease and hepatocellular carcinoma. No commercial assay for HDV RNA quantification that includes automated nucleic acid extraction is available, and in-house PCR tests are not well standardized. However, knowledge of HDV RNA levels may give important information for patient management and could be a useful tool for monitoring the response to antiviral therapies. One platform that is widely used for HBV DNA or HCV RNA quantification is the Cobas Ampliprep/TaqMan system. Using the utility channel of this platform, we established a novel protocol for TaqMan-based HDV RNA quantification after automatic extraction of RNA by the Ampliprep system. The assay was specific and showed linearity over a wide range from 3 × 10² to 10⁷ copies/ml. Reproducibility was demonstrated by determination of the interrun and intrarun variabilities, which were similar to those achieved with the commercially available Cobas TaqMan assays for HCV RNA and HBV DNA. HDV RNA levels were stable in whole blood (n = 4), plasma (n = 3), and serum (n = 3) samples at room temperature for up to 6 days. Importantly, HDV RNA viremia showed only minor fluctuations, with the log₁₀ coefficient of variation being between 1.3 and 11.2% for hepatitis delta patients studied every 2 weeks for up to 3 months (n = 6), while a rapid viral decline was observed early during treatment with pegylated alfa-2a interferon (n = 6). In conclusion, this novel automated HDV RNA assay is a useful tool for monitoring HDV-infected patients both before and during antiviral therapy.Hepatitis delta is the most severe form of chronic viral hepatitis in humans. The hepatitis delta virus (HDV) is a defective RNA virus which requires the hepatitis B virus (HBV) surface antigen (HBsAg) for complete replication and transmission, although the full extent of the HBV helper function has not been unexplored (27, 34). The HDV genome is a small, 1,678-nucleotide single-stranded RNA with a circular configuration that can form a rod-like structure with at least 70% paired bases (16, 35). The HDV RNA encodes small and large hepatitis delta antigens as the sole proteins (34).Hepatitis delta occurs only in HBsAg-positive individuals either as an acute coinfection or as a superinfection in patients with chronic hepatitis B (9). Several studies have shown that chronic HDV infection leads to more severe liver disease than chronic HBV monoinfection, with the course of progression of the fibrosis being accelerated, the risk of hepatocellular carcinoma being increased, and early decompensation occurring in the setting of established cirrhosis (9, 12, 13, 36). Simultaneous HBV and HDV infection has also been shown to be more severe than infection with HBV alone in chimpanzees (6).The current treatment options for patients with delta hepatitis are very limited, as alfa interferon is able to clear HDV only in a minority of patients (23). High doses of alfa interferon have been associated with a beneficial long-term outcome in a small cohort of Italian hepatitis delta patients (10, 11). Pegylated alfa interferon has also been used in small trials to treat delta hepatitis, and the sustained virological response rates were about 20% (2, 7, 21). The nucleoside and nucleotide analogues used for the treatment of HBV infection are ineffective against HDV (22, 23, 39, 40, 42). No study has systematically investigated the effect of tenofovir or entecavir, two potent HBV polymerase inhibitors that have recently been approved for use for the treatment of hepatitis B, on HDV replication (5, 8).HDV RNA determination enables the diagnosis of active viremia in case of the detection of anti-HDV in patients with chronic hepatitis B. Moreover, a possible correlation between HDV RNA levels and disease activity has been proposed (38); however, that correlation was not confirmed by others (43). In addition, HDV RNA quantification represents a useful tool for monitoring the response to treatment in patients with delta hepatitis receiving antiviral therapy. Several in-house quantitative HDV RNA PCR assays have been developed (2, 17, 38). However, these assays are not well standardized and quantitative values are difficult to compare.HBV DNA or HCV RNA quantification is usually performed with commercial, fully automated PCR-based or branched DNA-based assays. These commercial systems also include automated nucleic acid extraction. One of those is the Cobas Ampliprep/TaqMan assay. Until recently, it was not possible to run in-house-based PCRs on that platform. By using AMPLILINK software (version 3.2) and utility channel applications (version 3.0), it is now possible to design in-house PCR protocols. The aim of the work described here was to (i) establish a protocol for TaqMan-based HDV RNA quantification with optimized primers after the automatic extraction of RNA by the Ampliprep system that can be used for routine diagnostics and (ii) evaluation of the protocol for longitudinal HDV RNA determinations in both untreated patients and patients receiving pegylated alfa-2a interferon.     

Evaluation of Quantification of HIV-1 RNA Viral Load in Plasma and Dried Blood Spots by Use of the Semiautomated Cobas Amplicor Assay and the Fully Automated Cobas Ampliprep/TaqMan Assay,Version 2.0, in Kisumu,Kenya

In Kenya, HIV-1 viral load monitoring is commonly performed with the Cobas Amplicor using plasma specimens. Interest is growing in transitioning to real-time PCR (RT-PCR), such as the Cobas Ampliprep/Cobas TaqMan (CAP/CTM), using dried blood spots (DBS). Before implementation, direct evaluation of the two assays using DBS field specimens is required. This study compares the sensitivity, specificity, negative and positive predictive values (NPV and PPV, respectively), concordance, and agreement between HIV-1 viral load measurements using plasma and DBS specimens obtained from 512 HIV-1-infected pregnant females enrolled in the Kisumu Breastfeeding Study and tested with the Cobas Amplicor and CAP/CTM assays. The sensitivity and NPV of viral load detection in DBS specimens were higher with CAP/CTM (sensitivity, 100%; 95% confidence interval [CI], 99.1 to 100.0%; NPV, 100%; 95% CI, 59.0 to 100.0%) than the Cobas Amplicor (sensitivity, 96.6%; 95% CI, 94.3 to 98.1%; NPV, 58.8%; 95% CI, 40.7 to 75.4%). The PPVs were comparable between both assays when using DBS. The specificity of viral load detection in DBS specimens was lower with CAP/CTM (77.8%; 95% CI, 40.0 to 97.2%) than that of the Cobas Amplicor (95.2%; 95% CI, 76.2 to 99.9%). Good concordance and agreement were observed when paired plasma and DBS specimens were tested with both assays. Lower specificity with the CAP/CTM is likely due to proviral HIV-1 DNA amplification and lower detection limits with RT-PCR. However, the CAP/CTM has better sensitivity and higher throughput than the Cobas Amplicor. These findings suggest that DBS may be a suitable alternative to plasma when using RT-PCR, which could increase access to viral load monitoring in resource-limited settings.