Evaluation of the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test and identification of rare polymorphisms potentially affecting assay performance

Abstract

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 approximately 1.7 and 7.0 log(10) 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 log(10) copies/ml lower than CAP/CTM and Amplicor averaged 0.427 log(10) copies/ml lower than CAP/CTM for the subtype panel. Correlation with samples previously tested by Amplicor was excellent (R(2) = 0.884; average difference [Amplicor value minus CAP/CTM value], 0.008 log(10) copies/ml). Of the 305 HIV samples tested, 7 samples generated CAP/CTM titers between 1.0 and 2.75 log(10) 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.

FIG. 1.

ValiQuant panel. The members of the AcroMetrix ValiQuant HIV-1 RNA quantification panel were tested in triplicate on each of 3 days. All replicates of the negative panel member were “not detected.” The data were analyzed by Deming regression, shown by the heavy line. The light line represents the line of identity.

FIG. 2.

Subtype panel. The members of the HIV RNA genotype performance panel PRD202 (SeraCare Life Sciences, Milford, MA) were diluted 3-fold in Basematrix and tested in triplicate by the Amplicor, Versant, and CAP/CTM assays. The subtype designations and average log₁₀ copies/ml are shown. The error bars represent 1 standard deviation. No HIV-1 RNA was detected by any assay for the negative-control panel member. For the pairs marked with an asterisk, P > 0.05; all other pairs, P < 0.05.

FIG. 3.

Correlation samples. A total of 218 samples previously tested in the Amplicor assay were tested in the CAP/CTM assay. The data were analyzed by the method of Bland and Altman (1). The bold line indicates the average difference, and the dashed lines indicate the average ± 2 standard deviations.