ABSTRACT
BACKGROUND: There is a growing need for sensitive high-throughput cytomegalovirus (CMV) PCR tests due to the increasing number of immunocompromised patients requiring monitoring for active CMV infection. OBJECTIVES: To compare the fully automated COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) (CAP/CTM) CMV test (this test is currently under development and not commercially available) for EDTA-plasma to the reference method COBAS(®) AMPLICOR CMV MONITOR. STUDY DESIGN: A prospective feasibility study with parallel analysis of 433 EDTA-plasma samples from 277 patients on both systems was carried out after the analytical performance of the new system had been assessed. RESULTS: The new system has a wide linear range from 2.0 to 7.3 log(10) CMV-DNA copies/ml EDTA-plasma and a detection limit of 46 copies/ml with excellent accuracy and precision. When testing clinical samples, the CAP/CTM CMV test compared extremely well with the COBAS(®) AMPLICOR CMV MONITOR (R(2)=0.93, p<0.001) with increased sensitivity and linear range. Discrepant samples all contained low titers of CMV-DNA. In two of the study patients, CMV-DNAemia was detected by the CAP/CTM CMV test up to eight weeks earlier than by COBAS(®) AMPLICOR CMV MONITOR. CONCLUSION: An IVD/CE marked version of the CAP/CTM CMV test will enable laboratories to provide a sensitive, fully automated high-throughput CMV PCR.
Subject(s)
Cytomegalovirus Infections/virology , Cytomegalovirus/genetics , DNA, Viral/genetics , Polymerase Chain Reaction/methods , Biological Assay/methods , Cytomegalovirus Infections/blood , Feasibility Studies , Female , Humans , Immunocompromised Host/immunology , Male , Middle Aged , Prospective StudiesABSTRACT
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.
Subject(s)
HIV Infections/diagnosis , HIV Infections/virology , HIV-1/genetics , Polymerase Chain Reaction/methods , RNA, Viral/analysis , Reagent Kits, Diagnostic , Viral Load , HIV-1/isolation & purification , Humans , Sensitivity and SpecificityABSTRACT
BACKGROUND: Viral load quantification is established in the clinical management of chronic Hepatitis B virus (HBV) infection for assessing efficacies and resistance developments in anti-viral drug treatment. OBJECTIVES: The fully automated COBAS AmpliPrep/COBAS TaqMan HBV Test, v2.0 was evaluated for the linear measuring range and the inclusivity of HBV genotype determination in EDTA plasma and serum samples. STUDY DESIGN: Two kit lots of the test were used to determine the linear measuring range as well as linearity and limit of detection applying different concentration levels of specimens representing HBV genotypes A to H along with a pre-core mutant and the WHO Standard. RESULTS: The COBAS AmpliPrep/COBAS TaqMan HBV Test, v2.0 displayed a linear measuring range of seven log(10) steps from 20IU/mL (lower limit of quantification) to 2.3E+08IU/mL (upper limit of quantification) yielding similar results for EDTA plasma and serum. Inclusivity was shown by reliable quantification of HBV genotypes A to H at different concentration levels. The >or=95% hit rate LOD was 15IU/mL for genotypes C, D, F, G, the pre-core mutant and the WHO Standard and 20IU/mL for genotypes A, B, E and H matching the test's lower limit of quantification. 95% PROBIT analysis yielded concentrations of 8.9IU/mL for the WHO Standard and of 6.0-16.4IU/mL for the genotypes. CONCLUSIONS: The COBAS AmpliPrep/COBAS TaqMan HBV Test, v2.0 provides genotype inclusivity for accurate viral load monitoring in serum and EDTA plasma samples and supports clinical routine in the management of HBV infection.
