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.

Performance evaluation of the new Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 for quantification of human immunodeficiency virus type 1 RNA.

Despite FDA approval and CE marking of commercial tests, manufacturer-independent testing of the technical aspects of newly developed tests is important. To evaluate the analytical performance and explore the clinical applicability of the new Roche COBAS AmpliPrep COBAS TaqMan HIV-1 test, version 2.0 (CAP/CTM v2.0), platform comparison was performed with the Roche CAP/CTM test, version 2.0, the COBAS Amplicor HIV-1 Monitor Test, version 1.5 (CAP/CA v1.5), the COBAS AmpliPrep COBAS TaqMan HIV-1 Test (CAP/CTM v1.0), and the Abbott m2000 RealTime HIV-1 assay on panels and diagnostic samples. Specificity was tested for HIV-2 samples. Furthermore, samples from HIV-1-seropositive individuals with CAP/CA v1.5-measured viral loads below 50 HIV-1 RNA copies per ml (cp/ml) and replicates of HIV-1-seronegative plasma were tested in a checkerboard analysis. CAP/CTM v2.0 is HIV-1 specific, with broad genotype inclusivity and no serious underquantification of viral load relative to the other assays used. Low viral loads below the threshold of quantification for CAP/CA v1.5 are observed with CAP/CTM v2.0. A CAP/CTM v2.0-measured viral load of >50 copies/ml in these samples correlated with therapy failure. In conclusion, CAP/CTM v2.0 is an accurate and reliable test for HIV-1 viral load measurement relative to the other assays used with respect to specificity, sensitivity, and genotype inclusivity.

First transmission of human immunodeficiency virus Type 1 by a cellular blood product after mandatory nucleic acid screening in Germany.

BACKGROUND: In February 2007, a 63-year-old man underwent surgery. Retrospective testing with nucleic acid testing (NAT) showed that the patient was human immunodeficiency virus Type 1 (HIV-1) positive 10 days after transfusion. The transfusion-transmitted infection had been identified by a donor-related lookback started in April 2007 after anti-HIV seroconversion. METHODS: Sequence analysis was performed in the gag-pol region as well as in the V3 loop env region. Archived plasma from the transmitting donation was investigated for the individual-donation NAT with the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Roche CAP/CTM HIV-1 test) and for HIV antigen/antibody combination testing (Abbott Architect). Additional testing was done on the donor's follow-up sample and on the recipient's sample. RESULTS: The Roche CAP/CTM HIV-1 test failed to detect viral RNA by minipool NAT in the index donation (April 2007) as well as in the donation that caused the infection (January 2007). Phylogenetic analysis showed a very high genetic similarity among viral sequences from both donor and recipient, proving the HIV-1 transmission by sequence data. CONCLUSION: This case represents the first documented HIV-1 transmission by transfusion of red blood cells after mandatory introduction of HIV-1 NAT for blood screening in Germany. Low viral load and mismatches in the primer/probe region might explain the detection failure of the NAT screening assay. A certain risk remains that new virus variants contain mutations at positions critical for amplification or detection of viral genomes. An option to reduce the risk of a detection failure by NAT is the simultaneous use of several conserved regions as amplification targets.

Fully automated quantification of human immunodeficiency virus (HIV) type 1 RNA in human plasma by the COBAS AmpliPrep/COBAS TaqMan system.

BACKGROUND: HIV-1 RNA is a key parameter for reliable diagnosis and treatment of HIV-1 infection. The determination of HIV-1 RNA reduces the pre-seroconversion period in the diagnosis of HIV-1 infection and supports clinical management of HIV-1-infected patients. OBJECTIVES AND STUDY DESIGN: The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test combines automated extraction of total 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 was evaluated for sensitivity, dynamic range, precision, subtype inclusivity, interfering substances, diagnostic and analytical specificity, as well as correlation with three other commercial tests for HIV-1 RNA quantification. RESULTS: The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test demonstrated an assay sensitivity of 40 copies/mL, a greater than 5 log(10) measuring range of 40-1.0E+07 copies/mL (1.6-7.0 log(10)) and a reliable determination of HIV-1 group M and N subtypes in EDTA plasma. Quantification results were highly correlated with those obtained by the COBAS AMPLICOR HIV-1 MONITOR Test v1.5, the COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR Test v1.5 and the VERSANT HIV-1 RNA 3.0 assay. CONCLUSIONS: The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test excellently satisfies the requirements for reliable quantification of HIV-1 RNA in clinical specimens by a broad linear measuring range and a fully automated quantification procedure. It is highly appropriate for therapy monitoring and routine management of HIV-1 infection.

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.