Comparison of Three Different FDA-Approved Plasma HIV-1 RNA Assay Platforms Confirms the Virologic Failure Endpoint of 200 Copies per Milliliter Despite Improved Assay Sensitivity

Abstract

Discrepancies between HIV-1 RNA results assayed by different FDA-approved platforms have been reported. Plasma samples collected from 332 randomly selected clinical trial participants during the second year of antiretroviral treatment were assayed with three FDA-approved platforms: UltraSensitive Roche Amplicor Monitor, v1.5 (Monitor), the Abbott RealTime HIV-1 test on the m2000 system (Abbott), and the Roche TaqMan HIV-1 test, v2.0 (TaqMan). Samples from 61 additional participants with confirmed HIV-1 RNA levels of >50 copies/ml during trial follow-up were also included. Endpoints were HIV-1 RNA quantification of ≤50 copies/ml versus >50 copies/ml at an individual-sample level (primary) and determination of confirmed virologic failure (VF) from longitudinal samples. A total of 389 participants had results obtained from all assays on at least one sample (median = 6). Proportions of results of >50 copies/ml were 19% (Monitor), 22% (TaqMan), and 25% (Abbott). Despite indication of strong agreement (Cohen's kappa, 0.76 to 0.82), Abbott was more likely to detect HIV-1 RNA levels of >50 copies/ml than Monitor (matched-pair odds ratio [mOR] = 4.2; modified Obuchowski P < 0.001) and TaqMan (mOR = 2.1; P < 0.001); TaqMan was more likely than Monitor (mOR = 2.6; P < 0.001). Despite strong agreement in classifying VF across assay comparisons (kappa, 0.75 to 0.92), at a 50-copies/ml threshold, differences in the probability of VF classification (in the same direction as primary) were apparent (all McNemar's P < 0.007). At a 200-copies/ml VF threshold, no differences between assays were apparent (all P > 0.13). Despite strong agreement among assays, significant differences were observed with respect to detecting HIV-1 RNA levels of >50 copies/ml and identifying VF at the 50-copies/ml threshold. This has important implications for the definition of VF in clinical trials and clinical practice.

Clinical Impact of Virological Failure and Resistance Analysis Definitions used in Pivotal Clinical Trials of Initial Antiretroviral Treatment: A Systematic Review

“…Current guidelines define VF as a confirmed viral load > 200 copies/ml, a threshold that eliminates most cases of apparent viremia caused by viral load blips or assay variability. 7 Therefore, at a minimum, genotyping should be attempted on all participants with confirmed VF at this threshold in clinical trials.…”

“…Furthermore, we specifically analyzed the rates of participants with confirmed VF and an HIV-1 RNA > 200 copies/mL even if this was not the predefined limit in the trial. This threshold has shown the highest degree of agreement between three US FDA-approved HIV-1 RNA assay platforms while minimizing differences observed between assays and has important implications for the definition of VF in clinical trials, guidelines, and clinical practice 6,7 .…”

Predictors of low‐level HIV viraemia and virological failure in the era of integrase inhibitors: A Spanish nationwide cohort

“…Virological failure was defined as pVL ≥ 50 copies/mL at week 48 and pVL ≥ 200 copies/mL at week 72 [as defined in most phase III randomized clinical trials of first‐line ART approved by the drug regulatory agencies (European Medicines Agency and US Food and Drug Administration)]. The threshold of 200 copies/mL eliminates most cases of apparent viraemia caused by pVL blips or technical assay variability [24].…”

Use of External Quality Control Material for HIV-1 RNA Testing To Assess the Comparability of Data Generated in Separate Laboratories and the Stability of HIV-1 RNA in Samples after Prolonged Storage

“…Establishing the integrity of the stored samples provides confidence in the ability to use stored samples to address scientific questions, such as in the study conducted by Lalama et al to evaluate the impact of different HIV-1 RNA cutoffs for evaluating virologic failure across HIV-1 RNA assay platforms (11). In that study, the virologic endpoints were evaluated by retesting stored plasma by three FDA-approved HIV-1 RNA assays, the Cobas, Roche Cobas AmpliPrep Cobas TaqMan, and Abbott RealTime HIV-1 RNA assays, and comparing the classifying confirmed virologic failures across the different platforms using either a 50-or 200-copy/ml cutoff.…”