Replicate Aptima Assay for Quantifying Residual Plasma Viremia in Individuals on Antiretroviral Therapy

Abstract

Detection of residual plasma viremia in antiretroviral therapy (ART)-suppressed HIV-infected individuals is critical for characterizing the latent reservoir and evaluating the impact of cure interventions. Ultracentrifugation-based single-copy assays are sensitive but labor intensive. Fully automated replicate testing using a standard clinical viral load assay was evaluated as a high-throughput alternative for the quantification of low-level viremia. Four plasma samples from blood donors with acute HIV-1 infection and one viral culture supernatant were serially diluted into 25-ml samples to nominal viral loads ranging from 39 to <0.5 copies (cp)/ml. Each dilution was tested with 45 replicates (reps) using 0.5 ml/rep with the Aptima HIV-1 Quant assay. The nominal and estimated viral loads based on the single-hit Poisson model were compared, and a hybrid Poisson digital model for calibrated viral load estimation was derived. Testing performed using 45 reps on longitudinal plasma samples from 50 ART-suppressed individuals in the Reservoir Assay Validation and Evaluation Network (RAVEN) study cohort (range of 1 to 19 years of continuous ART suppression) showed a median viral load of 0.54 cp/ml (interquartile range [IQR], 0.22 to 1.46 cp/ml) and a 14% (95% confidence interval [CI], 9% to 19%) decline in viral load for each additional year in duration suppressed. Within the RAVEN cohort, the expected false-negative rate for detection at lower rep numbers using 9 and 18 reps was 26% and 14%, respectively. Residual plasma viremia levels positively correlated with cell-associated HIV RNA and DNA. The performance characteristics of the replicate Aptima assay support its use for quantifying residual plasma viremia to study the latent HIV reservoir and cure interventions.

Keywords: human immunodeficiency virus; latent reservoir; quantification; residual RNA; ultrasensitive viral load; viremia.

FIG 1

Replicate Aptima testing results for serially diluted HIV culture supernatant and donor plasma samples. For each nominal viral load in cp/ml (on the x axis), the total number of replicates and the number of positive replicates are shown at the top of each graph, as well as the individual replicate results in cp/ml (on the y axis). ND, not detected.

FIG 2

Comparison of nominal and estimated viral load based on the single-hit Poisson model. Data for each of the 5 individual standards are shown in blue. The overall calibration factor (black open circle) was calculated as the geometric mean of the 5 individual standard calibration factors. Error bars, 95% confidence interval.

FIG 3

Modeling the effect of lower replicate numbers on the false-negative rate of detection (A) and precision of the viral load estimate (B).

FIG 4

Application of the replicate Aptima assay (45 reps) to the RAVEN cohort of 50 ART-suppressed individuals with 1 to 6 collections within a 2-year period (A), stratified by timing of ART initiation (B), and in longitudinal samples as a function of years of continuous ART suppression (C). Viral load estimates are indicated by symbols in panel A, with error bars indicating upper and lower confidence intervals around the estimate. Lines and error bars in panel B indicate median VL and IQR, respectively. LNR, the lowest nonzero result. Circles and squares in panel C indicate ART initiation during early and chronic infection, respectively. Open symbols in panel C indicate undetectable VL.