Single real-time reverse transcription-PCR assay for detection and quantification of genetically diverse HIV-1, SIVcpz, and SIVgor strains

Abstract

Although antiretroviral treatment availability has improved, the virological monitoring of patients remains largely uneven across regions. In addition, viral quantification tests are suffering from human immunodeficiency virus type 1 (HIV-1) genetic diversity, fueled by the emergence of new recombinants and of lentiviruses from nonhuman primates. We developed a real-time reverse transcription-PCR (RT-PCR) assay that is relatively inexpensive and able to detect and quantify all circulating forms of HIV-1 and its simian immunodeficiency virus (SIV) precursors, SIVcpz and SIVgor. Primers and a probe were designed to detect all variants of the HIV-1/SIVcpz/SIVgor lineage. HIV-1 M plasma (n = 190; 1.68 to 7.78 log(10) copies/ml) representing eight subtypes, nine circulating recombinant forms, and 21 unique recombinant forms were tested. The mean PCR efficiency was 99%, with low coefficients of intra- and interassay variation (<5%) and a limit of quantification of <2.50 log(10) copies/ml, with a 200-μl plasma volume. On the studied range, the specificity and the analytical sensitivity were 100 and 97.4%, respectively. The viral loads were highly correlated (r = 0.95, P < 0.0001) with the reference method (generic HIV assay; Biocentric) and had no systematic difference, irrespective of genotype. Furthermore, 22 HIV-1 O plasmas were screened and were better quantified compared to the gold-standard RealTime HIV-1 assay (Abbott), including four samples that were only quantified by our assay. Finally, we could quantify SIVcpzPtt and SIVcpzPts from chimpanzee plasma (n = 5) and amplify SIVcpz and SIVgor from feces. Thus, the newly developed real-time RT-PCR assay detects and quantifies strains from the HIV-1/SIVcpz/SIVgor lineage, including a wide diversity of group M strains and HIV-1 O. It can therefore be useful in geographical areas of high HIV diversity and at risk for the emergence of new HIV variants.

Fig 1

(A and B) Phylogenetic relationships between HIV-1 group O strains (A) and SIVcpz/SIVgor viruses (B), tested by our RT-qPCR assay with reference strains. (A) Phylogenetic tree derived from an env region (gp41; 394 bp). Gray arrows highlight strains from our panel. Some strains were not sequenced because of material limitations and others were available only in pol region. (B) The tree is derived from a small env region (gp41; 248 bp), constructed by BioNJ (66). White arrows highlight strains from our fecal sample panel (except from CR4112 amplified in the pol region only, and CR6278, -6466, -6495, -6534, and -6682 amplified in a small 195-bp region of gp41), and black arrows highlight strains from plasma samples. All of the sequences were retrieved from the HIV database (http://www.hiv.lanl.gov/).

Fig 2

HIV-1 group M RNA viral load quantified by the generic HIV-1 viral load Biocentric kit and our new RT-qPCR assay. (A) Samples (n = 185) detected with both techniques were plotted on this linearity plot. The solid line represents the fitted regression. Pearson correlation r, 0.95 (P < 0.0001). The 2.50-log₁₀ copies/ml limit of detection is represented by gray dashed lines. (B) The 185 samples detected with both techniques were plotted on this Bland-Altman difference plot. The vertical axis indicates the difference between the Biocentric and the new RT-qPCR assay viral load, and the horizontal axis shows the mean viral load between the two techniques. The mean bias on the difference (solid line, ∂ = 0.023 log₁₀ copies/ml) and limits of agreement (dashed lines) are shown on the graphic. On the right vertical axis are represented two main limits: the ±0.5 log₁₀ copies/ml limit, and the 95% CI interval (−0.027 to 0.072 log₁₀ copies/ml).