Performance evaluation of the Aptima HIV-1 RNA Quant assay on the Panther system using the standard and dilution protocols

Abstract

Background: Currently, FDA-approved HIV-1 viral load (VL) assays use venipuncture-derived plasma. The Hologic Panther system uses 0.7 mL total volume for the Aptima HIV-1 Quant Assay standard (APT-Quant-std) and dilution (APT-Quant-dil) protocols. However, smaller plasma volumes from fingerstick whole blood (FSB) collected in EDTA-microtainer tubes (MCT) could provide an easier sample collection method for HIV-1 VL testing.

Objectives: To evaluate the performance of the APT-Quant-std compared to the Roche CAP/CTM and Abbott m2000RT VL assays and an alternative APTQuant 1:7 dilution protocol, the latter using 100 μL of MCT-derived plasma from FSB.

Study design: Linearity was determined using commercial HIV-1 RNA plasma controls. Dilutions ranging 1.56-2.95 log10 copies/mL were prepared to determine the APT-Quant-dil Limit of Quantitation (LOQ) using Probit analysis. Specificity of APT-Quant-std was calculated using 326 HIVnegative samples. To evaluate agreement, 329 plasma specimens were tested with APT-Quant-std, CAP/CTM, and m2000RT. Forty-seven matched venipuncture and MCT-derived plasma specimens were tested with APT-Quant-std and APT-Quant-dil.

Results: Among the RNA controls, specificity was 99.69 % for APT-Quant-std. The R2 values were 0.988 (APT-Quant-std/CAP/CTM), 0.980 (APT-Quant-std/ m2000RT), and 0.997 (APT-Quant-std/APT-Quant-dil). The APT-Quant-dil LOQ was estimated at 2.7 log10 copies/mL (500 copies/mL) (95 %CI 2.62-2.87). At 2.3 log10 copies/mL (200 copies/mL), the overall agreement was 91.0 % for APT-Quant-std/CAP/CTM, 85.7 % for APT-Quant-std/m2000RT, and 82.9 % for APT-Quant-std/APT-Quant-dil. Quantified APT-Quant-std results were on average 0.2 log10 copies/mL higher than CAP/CTM and m2000RT and 0.14 log10 copies/mL higher than APT-Quant-dil.

Conclusion: APT-Quant showed similar performance compared to the CAP/CTM and m2000RT assays and remains sensitive and accurate using the dilution protocol.

Keywords: Aptima HIV-1 quant; HIV diagnosis; Microtainer preparation tubes; Self-collection; Viral load.

Published by Elsevier B.V.

Figures

Figure 1:. Collection, shipment and processing of MCTs during the Engagement study from FSB and viral load testing using the 1:7 APT-Quant-dil protocol.

MCTs: Microtainer Tubes. FSB: Fingerstick whole blood.

Figure 2:. Linear regression analysis using commercial HIV-1 RNA controls

(A) Mean VL values of seven Linearity Panel (LP) replicates and triplicate AcroMetrix Low, Mid and High controls with APT-Quant-std were compared to known CAP/CTM and m2000RT assays results. (B) Mean VL of three replicates of APT-Quant-dil compared to APT-Quant-std results using LP and AcroMetrix High and Mid controls.

Figure 3:. Bland-Altman plots of quantified clinical samples

Quantified VL results (in log10 copies/mL) to show agreement from (A) APT-Quant-std and CAP/CTM (n=174), (B) APT-Quant-std and m2000RT (n=184) and (C) APT-Quant-std and APT-Quant-dil (n=24). The average VL values of paired samples are plotted on the X-axis, and the differences between the VL values are plotted on the Y-axis. The solid horizontal lines represent mean difference (bias) and the two horizontal dashed lines represent the 95% confidence intervals (CI), corresponding to the mean ± 1.96 SD.