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-l 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.

Origin of the HIV-1 group O epidemic in western lowland gorillas

“…All gorilla fecal samples were first tested for the presence of HIV-1 cross-reactive antibodies using the INNO-LIA HIV I/II score confirmation test (Innogenetics) as previously described (9,16). An RT-qPCR assay, previously shown to detect HIV-1 groups M, N, O, and P; SIVcpzPtt; SIVcpzPts; and SIVgor in fecal samples (18), was used to screen all antibody-positive, as well as some antibody-negative, fecal samples. Viral RNA was extracted using the NucliSens Magnetic Extraction Kit (BioMerieux) (17).…”

“…In addition to antibody detection, we tested all INNO-LIApositive and -negative specimens from the same nesting and/or feeding site for the presence of SIVgor viral RNA using a realtime quantitative PCR (RT-qPCR) assay (18). Viral RNA was detected in 33 (58%) of 57 antibody-positive samples but also in 15 (7.4%) of 204 antibody-negative samples.…”

Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

“…The time spent for analysis and the costs are an issue, especially if other viral or bacterial pathogens need to be screened and the frequency of positive results is Ͻ10% (45). The development of generic assays for virus identification in clinical samples is one strategy to address this problem (49,50). To date, such an approach for NoVs has not been feasible due to their genetic diversity.…”

Field evaluation of an open and polyvalent universal HIV-1/SIVcpz/SIVgor quantitative RT-PCR assay for HIV-1 viral load monitoring in comparison to Abbott RealTime HIV-1 in Cameroon

“…We recently developed and described an ‘in house’ quantitative RT-PCR assay, called HIV-1/SIVcpz/SIVgor RT-qPCR assay, that detects and quantifies a wide diversity of HIV-1 group M variants, the divergent HIV-1 group O, as well as the simian precursors of HIV-1: SIVcpz Ptt , SIVcpz Pts and SIVgor from chimpanzees and gorillas (Etienne et al, 2013). The test was initially compared with a previously validated Generic HIV Viral Load assay (Biocentric, Bandol, France) (Rouet et al, 2007, 2010) on a panel of plasma samples known to represent a large diversity of HIV-1 group M subtypes and CRFs.…”

“…The VLs were highly correlated (r= 0.95) and the analytical specificity and sensitivity were 100% and 97.4%, respectively. Moreover, HIV-1 group O plasma samples were better quantified compared to the Abbott RealTi m e HIV-1 (Abbott Molecular, IL, USA) (Etienne et al, 2013). In the present study, we further evaluated the performance of this recently developed assay for its capacity to detect and quantify HIV-1 variants with the aim to implement the assay use in RLCs.…”