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 ϭ 35) or group B (n ϭ 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 log 10 copies/PCR and 27.02% at 0.78 log 10 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 ϭ 0.68; 95% confidence interval [CI], 0.4 to 0.8; P Ͻ 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.

Performance evaluation of a laboratory developed PCR test for quantitation of HIV-2 viral RNA

“…Although the samples provided were noted as serologically reactive for HIV-2 by HIV type differentiation assays, 42% of the samples when tested in duplicate did not demonstrate detectable HIV-2 RNA by our assay. Nevertheless our results are comparable with those reported for other laboratory developed HIV-2 RNA quantitative assays which showed that plasma HIV-2 RNA levels in viremic individuals tend to be low (2.8 log 10 copies/ml on average) with only 6% of viremic individuals over 1,000 copies/ml, and below level of detection in 28.0 to 46.5% of confirmed HIV-2 infections [28,29,31,32]. The high proportion of undetectable viral load among the clinical samples tested is similar to that reported in other studies in Europe and Africa.…”

“…The performance of a dual-target assay for HIV-2 viral load is reported by Bertine et.al. (29). HIV-2 Group B isolates are very rare in the United States and we were only able to include two B isolates in our study.…”

First-line Raltegravir/Emtricitabine/Tenofovir Combination in Human Immunodeficiency Virus Type 2 (HIV-2) Infection: A Phase 2, Noncomparative Trial (ANRS 159 HIV-2)

“…Ultrasensitive pVL quantification was performed at W0, W24, and W48 using real-time reverse-transcription PCR assay with a threshold between 5 and 10 copies/mL depending on the volume of plasma available (Biocentric, Bandol, France) [ 33 ].Total HIV-2 DNA quantification was performed at the same time points using a real-time PCR assay with a threshold of 6 copies developed by the ANRS AC-11 HIV Quantification group [ 34 ].…”

Total HIV DNA: a global marker of HIV persistence

“…The same technology has been also developed for HIV-2 infected patients, having usually low reservoir levels. A new assay for HIV-2 DNA quantification based on the same technology has also been developed [ 9 ].…”