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 ϳ1.7 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.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. MATERIALS AND METHODS Limit of detection.To determine the limit of detection in the CAP/CTM assay, nine dilutions of the WHO reference material (HIV-1 RNA, 2nd International Standard, 97/650; NIBSC, Potters Bar, United Kingdom) or Viral Quality Assurance (VQA) standard (Rush University Medical Center, Chicago, IL) were prepared in Basematrix diluent (SeraCare Life Sciences, Milford, MA). Fourteen replicates a...

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

“…The Cobas Ampliprep/Cobas TaqMan 48 analyzer (Roche Diagnostics, Ltd., Rotkruez, Switzerland) was used for automated extraction, amplification, and quantification following the Roche manufacturer's standard guidelines. The methodology has been previously published (20,29) and is briefly described below.…”

Does chronic alcohol use by HIV-infected patients on d4T/3TC/NVP drug regimen effect the HIV viral load and what is the therapeutic window of the drugs, CD4+ count and WBC count in patients with high viral load during the 9 months period of follow up?

“…The plasma samples in the cryovials were stored at -70°C prior to viral load analysis. The HIV viral load (number of copies/ml) were determined using Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor), Roche Diagnostics GmbH using the Roche (2006) and Pyne et al (2010) methods. 32,33 The print-out of each sample was made.…”

“…The HIV viral load (number of copies/ml) were determined using Roche Amplicor HIV-1 monitor test, version 1.5 (Amplicor), Roche Diagnostics GmbH using the Roche (2006) and Pyne et al (2010) methods. 32,33 The print-out of each sample was made. All the HIV-infected patients with very high HIV viral load ≥400 copies/ml during the study, their plasma drug concentrations of d4T, 3TC and NVP; CD4 + count and WBC counts were analyzed to determine if these have any correlation with the observed high HIV viral load.…”

Single Real-Time Reverse Transcription-PCR Assay for Detection and Quantification of Genetically Diverse HIV-1, SIVcpz, and SIVgor Strains

“…Some recent generic or "in-house" tests have designed their assay to target a wide variety of HIV-1 strains and could validate their technique on multiple samples from different geographic areas (reviewed in reference 24). However, the main limit of the majority of these tests is that their designed primers and probe have numerous mismatches with HIV-1 groups O, N, and P (alignment with HIV-1 sequences from the HIV.lanl database [data not shown]), implying the possible nondetection or underquantification of these circulating strains (25). On the other hand, a real-time PCR assay developed by Gueudin and coworkers specifically quantifies HIV-1 group O but does not detect HIV-1 group M strains (22,26).…”