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-log(10) difference) and precision (standard deviation of 0.04 to 0.22 log(10)). 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, R(2) = 0.88; R(2) = 0.94 without 105 genotype 4 samples). Paired plasma and serum samples showed similar performance (n = 25, R(2) = 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.

Accuracy to 2nd International HIV-1 RNA WHO Standard: assessment of three generations of quantitative HIV-1 RNA nucleic acid amplification tests.

BACKGROUND: Standardization of quantitative HIV-1 tests to a global primary standard is required by regulatory authorities to ensure comparability of test results across different assays and platforms of different manufacturers. OBJECTIVES AND STUDY DESIGN: Three generations of quantitative HIV-1 tests, the COBAS(®) AMPLICOR(®) HIV-1 Monitor Test, v1.5 (HIV-1 Monitor test v1.5); the COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 Test (HIV-1 TaqMan(®) test v1.0); and the dual-target-based COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) HIV-1 Test, v2.0 (HIV-1 TaqMan(®) test v2.0), were assessed for accuracy to World Health Organization (WHO) 2nd International Standard for human immunodeficiency virus 1 (HIV-1) RNA (NIBSC code 97/650) at concentration levels below 1667 IU/mL including relevant medical decision points. RESULTS: With the 2nd WHO Standard the mean difference across all concentrations was -0.07 log(10) for the HIV-1 Monitor test v1.5; +0.12 log(10) for the HIV-1 TaqMan(®) test v1.0; and +0.09 log(10) for the HIV-1 TaqMan(®) test v2.0. Linearity, including concentrations below the claimed limit of quantitation, was demonstrated for HIV-1 TaqMan(®) test v2.0. The HIV-1 TaqMan(®) test v1.0 showed a trend towards higher quantitation at very low concentration levels and the HIV-1 Monitor test v1.5 had a tendency towards lower quantitation at low concentration levels. CONCLUSIONS: All three assays are closely traceable to the primary WHO HIV-1 RNA standard for in vitro diagnostic IVD assays. Compared to the other two assays, the HIV-1 TaqMan(®) test v2.0 showed better linearity around the limit of detection and below.

Improved HIV-1 RNA quantitation by COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 using a novel dual-target approach.

BACKGROUND: HIV-1 RNA viral load is a key parameter for reliable treatment monitoring of HIV-1 infection. Accurate HIV-1 RNA quantitation can be impaired by primer and probe sequence polymorphisms as a result of tremendous genetic diversity and ongoing evolution of HIV-1. A novel dual HIV-1 target amplification approach was realized in the quantitative COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2.0 (HIV-1 TaqMan test v2.0) to cope with the high genetic diversity of the virus. OBJECTIVES AND STUDY DESIGN: The performance of the new assay was evaluated for sensitivity, dynamic range, precision, subtype inclusivity, diagnostic and analytical specificity, interfering substances, and correlation with the COBAS AmpliPrep/COBAS TaqMan HIV-1 (HIV-1 TaqMan test v1.0) predecessor test in patients specimens. RESULTS: The new assay demonstrated a sensitivity of 20 copies/mL, a linear measuring range of 20-10,000,000 copies/mL, with a lower limit of quantitation of 20 copies/mL. HIV-1 Group M subtypes and HIV-1 Group O were quantified within +/-0.3 log(10) of the assigned titers. Specificity was 100% in 660 tested specimens, no cross reactivity was found for 15 pathogens nor any interference for endogenous substances or 29 drugs. Good comparability with the predecessor assay was demonstrated in 82 positive patient samples. In selected clinical samples 35/66 specimens were found underquantitated in the predecessor assay; all were quantitated correctly in the new assay. CONCLUSIONS: The dual-target approach for the HIV-1 TaqMan test v2.0 enables superior HIV-1 Group M subtype coverage including HIV-1 Group O detection. Correct quantitation of specimens underquantitated in the HIV-1 TaqMan test v1.0 test was demonstrated.

Fully automated quantification of hepatitis C virus (HCV) RNA in human plasma and human serum by the COBAS AmpliPrep/COBAS TaqMan system.

BACKGROUND: HCV RNA is commonly recognized as key parameter for reliable diagnosis and treatment monitoring of HCV infection. Determination of blood HCV RNA concentrations reduces the pre-seroconversion period in the diagnosis of HCV infection and supports management of interferon alpha-based therapies of chronic HCV infection. OBJECTIVES AND STUDY DESIGN: The COBAS AmpliPrep/COBAS TaqMan HCV Test combines automated extraction of nucleic acids on the COBAS AmpliPrep Instrument with real-time PCR on the COBAS TaqMan Analyzer, thus greatly reducing hands-on time during sample preparation and amplification/detection. The test, which is calibrated to the 1st International HCV WHO Standard, was evaluated for sensitivity, dynamic range, precision, matrix equivalence, genotype inclusivity, interfering substances, diagnostic and analytical specificity, as well as for correlation with two other commercial tests for HCV RNA quantification. RESULTS: The COBAS AmpliPrep/COBAS TaqMan HCV Test demonstrated a >6-log dynamic range of 43-6.90 E+7 IU/mL, a sensitivity (95% hit rate) of at least 15 IU/mL for HCV WHO Standard and a comparable quantification of genotypes 1-6. HCV quantification results were in good correlation with those obtained by the COBAS AMPLICOR HCV MONITOR Test v2.0 and the VERSANT HCV RNA 3.0 test. CONCLUSIONS: The fully automated COBAS AmpliPrep/COBAS TaqMan HCV Test excellently accomplishes the requirements for highly sensitive detection and reliable quantification of HCV in clinical samples and thus improves therapy monitoring and management of HCV infection.

Comparison of conventional PCR with real-time PCR and branched DNA-based assays for hepatitis C virus RNA quantification and clinical significance for genotypes 1 to 5.

The key parameter for diagnosis and management of hepatitis C virus (HCV) infection is HCV RNA. Standardization of HCV RNA assays to IU is mainly based on genotype 1 panels. Little is known about the variability of commercially available HCV RNA assays for quantification of different genotypes. Two real-time reverse transcription (RT)-PCR assays (COBAS TaqMan HCV Test for use with the High-Pure System [HPS/CTM] and COBAS Ampliprep/COBAS TaqMan HCV Test [CAP/CTM]), one standard RT-PCR assay (COBAS Amplicor HCV Monitor 2.0 [CAM]), and one signal amplification assay (Versant Quantitative 3.0 [branched DNA [bDNA]]) were compared for quantification of genotypes 1 to 5 (n = 108). Using CAM as a reference assay for genotype 1-infected patients, the mean interassay differences compared with CAP/CTM, HPS/CTM, and bDNA were 0.16, -0.13, and -0.48 log(10) IU/ml HCV RNA, respectively. Comparison of CAM with CAP/CTM, HPS/CTM, and bDNA for the remaining genotypes showed the following results, respectively: 2a/c, -0.24, -0.78, and -0.49; 2b, -0.21, -0.18, and -0.64; 3a, 0.13, -1.04, and -0.55; 4, -0.52, -1.51, and -0.05; and 5, -0.28, -1.00, and -0.24 log IU/ml HCV RNA. A correct decision for treatment discontinuation in genotype 1 patients at week 12 was possible only when the same assay was used at baseline and week 12. Comparison of CAM with the CAP/CTM assay showed equal quantifications of genotype 1, 2, 3, and 5 samples, while genotype 4 samples were slightly underestimated. For the HPS/CTM assay, a significant underestimation of the HCV RNA concentrations of genotypes 2a/c, 3, 4, and 5 was observed. For the bDNA assay, a constant lower quantification of genotypes 1 to 3 was detected.