New Highly Sensitive Real-Time PCR Assay for HIV-2 Group A and Group B DNA Quantification

Abstract

HIV-2 infection is characterized by a very low replication rate in most cases and low progression. This necessitates an approach to patient monitoring that differs from that for HIV-1 infection. Here, a new highly specific and sensitive method for HIV-2 DNA quantification was developed. The new test is based on quantitative real-time PCR targeting the long terminal repeat (LTR) and gag regions and using an internal control. Analytical performance was determined in three laboratories, and clinical performance was determined on blood samples from 63 patients infected with HIV-2 group A (n = 35) or group B (n = 28). The specificity was 100%. The 95% limit of detection was three copies/PCR and the limit of quantification was six copies/PCR. The within-run coefficients of variation were between 1.03% at 3.78 log₁₀ copies/PCR and 27.02% at 0.78 log₁₀ copies/PCR. The between-run coefficient of variation was 5.10%. Both manual and automated nucleic acid extraction methods were validated. HIV-2 DNA loads were detectable in blood cells from all 63 patients. When HIV-2 DNA was quantifiable, median loads were significantly higher in antiretroviral-treated than in naive patients and were similar for groups A and B. HIV-2 DNA load was correlated with HIV-2 RNA load (r = 0.68; 95% confidence interval [CI], 0.4 to 0.8; P < 0.0001). Our data show that this new assay is highly sensitive and quantifies the two main HIV-2 groups, making it useful for the diagnosis of HIV-2 infection and for pathogenesis studies on HIV-2 reservoirs.

Keywords: DNA; HIV-2; PCR; quantification.

FIG 1

Standard curve of the HIV-2 DNA real-time PCR assay (n = 22 runs). The cycle threshold (C_T) is the number of cycles at which fluorescence passes a fixed limit (time to positivity). Median values and 25% and 75% interquartile ranges of the C_T are indicated (logarithmic scale).

FIG 2

Comparison of manual and automated extractions from blood cell pellets and whole blood. (a) HIV-2 DNA was quantified in PBMCs (peripheral blood mononuclear cells [lymphocytes plus monocytes]) isolated from whole blood using Ficoll (n = 15). (b) HIV-2 DNA was quantified in leukocytes from whole blood, including PBMCs and polynuclear cells (n = 11).

FIG 3

Clinical performance. (a) Correlation between HIV-2 DNA and HIV-2 RNA loads (r = 0.68; 95% confidence interval, 0.4 to 0.8; P < 0.0001 for the whole group). An arbitrary value of 1.60 log₁₀ copies/ml was attributed to samples with an undetectable plasma viral load. (b) Distribution of HIV-2 DNA based on HIV-2 RNA concentration strata: <40, 40 to 500, 500 to 5,000, and 5,000 to 50,000 copies/ml.