Validation for clinical use of a novel HIV-2 plasma RNA viral load assay using the Abbott m2000 platform

Abstract

Background: Optimal care of persons infected with human immunodeficiency virus type 2 (HIV-2) requires an accurate assessment of HIV-2 plasma viral load (VL), but no clinically approved quantitative HIV-2 RNA VL assay exists.

Objectives: To validate a novel quantitative HIV-2 RNA assay for clinical and research use.

Study design: The Abbott m2000sp/rt platform was adapted for quantification of HIV-2 RNA in plasma. Amplification targeted a region of the long terminal repeat conserved in Group A and B HIV-2. Electron microscopy-counted-HIV-2 standards, the WHO/NIBSC HIV-2 International Standard and clinical specimens (N=162) were used to determine the precision, sensitivity, specificity, linear range, accuracy, and clinical performance of the assay.

Results: The quantitative linear range of the HIV-2 RNA assay was 10-1,000,000 copies/mL (R(2)>0.99), with a limit of detection of 8 copies/mL (95% CI, 5-18 copies/mL). The assay did not cross-react with HIV-1, and quantification of HIV-2 RNA was not affected by the presence of >5 log(10)HIV-1 RNA copies/mL. The total standard deviation (SD) and intra- and inter-run SD were 0.095, 0.093 and 0.162, respectively, at nominal inputs of 3.7, 1.7 and 1.0 log(10)HIV-2 RNA copies/mL. The HIV-2 WHO/NIBSC International Standard (1000 IU) was shown to contain 152 RNA copies/mL (95% CI 141-163). Overall, HIV-2 RNA was quantified at ≥10 copies/mL from 86 (53%) clinical specimens (median, 2.24 log(10) copies/mL; range 10-16,870), and nine specimens (6%) had HIV-2 RNA detected at <10 copies/mL.

Conclusions: We developed and validated a highly sensitive HIV-2 VL assay that is suitable for clinical and research use.

Figure 1

Representative data demonstrating the linear range of the HIV-2 VL assay. Viral RNA levels were measured in HIV-seronegative plasma samples that were spiked with EM-counted HIV-2 NIH-Z at final concentrations of 10, 50, 100, 500, 5000, 100,000, 500,000 and 1,000,000 RNA copies/ml. Slope and Y-intercept values for the resultant trend line were 0.995 and 0.056, respectively, as determined by linear regression (R² = 0.997). Each sample was assayed in triplicate.

Figure 2

HIV-2 viral loads in clinical plasma samples (crosses) and in HIV-2 NIH-Z and EHO standards (open squares) plotted against the corresponding cycle number (CN) for the internal RNA control. Clinical plasma samples included HIV-seronegative, HIV-seropositive, hepatitis B and hepatitis C– infected patients.

Figure 3

Effect of interfering substances on measurements of HIV-2 RNA in plasma. Each box shows the highest, lowest and mean RNA values in triplicate samples. Panel 1, EDTA plasma; Panel 2, Hemolyzed plasma Low (0.5g/dL hemoglobin); Panel 3, Hemolyzed plasma Mid (1.0g/dL hemoglobin); Panel 4 Hemolyzed plasma Hi (1.95g/dL hemoglobin); Panel 5, Heparin plasma (19.85 USP/mL heparin); Panel 6, Lipemic plasma (785 mg/dL triglycerides); Panel 7, Icteric plasma (15mg/dL bilirubin). Mean RNA values were compared between the interfering substance and EDTA plasma using generalized linear models and Bonferroni corrections. A similar pattern of inhibition was observed for the IC CN for each panel (data not shown).