New Real-Time RT-PCR Assay with Single Copy Sensitivity for HIV-1 RNA in Plasma

Abstract

Throughout the course of untreated HIV-1 infection, there is extensive virus replication with release of virions into the blood leading to high-level viremia and a genetically diverse virus population. The level of HIV-1 RNA in plasma is an extremely valuable marker of viral RNA level replication, number of productively infected cells, risk of disease progression, and efficacy of antiretroviral therapy. FDA-approved assays that are used routinely to measure plasma HIV-1 RNA have quantification limits of 50-75 copies/ml, and antiretroviral therapy is initially considered successful if viremia can be reduced to below such at these limits. However, reduction of HIV-1 RNA to below 50- 75 copies/m1 does not guarantee long-term success and rebound of drug-resistant virus can occur implying that HIV-1 replication and evolution may be continuing. To investigate this possibility, an assay was developed that accurately measures HIV-1 RNA levels below 50 copies/ml with 50-fold greater sensitivity than the FDA-approved assays. This assay uses larger plasma sample volumes (7m1), improved nucleic acid isolation and purification techniques, and real-time RT-PCR, to accurately quantify HIV-1 in plasma samples with a limit of detection of 1 copy in 3.8m1 of plasma. Since the assay has a detection limit of 1 RNA copy, it is referred to as the single copy assay. To monitor the recovery of HIV-1 from patient plasma, samples are spiked with an internal standard, consisting of the replication-competent avian sarcoma-leukosis retrovirus vector RCAS BP(A), derived from an unrelated retrovirus based on the Rous sarcoma virus. Five important steps were required for the development of this assay: (1) Primer and probe construction in a conserved region of HIV-1 subtype B, in addition to transcripts within the same region for a standard curve; (2) Development and optimization of an RT-PCR assay for real-time PCR (TaqMan); (3) Primer and probe construction in a conserved region of the Rous-Sarcoma Virus (RSV), for the internal virion standard and creation of the transcripts of this same region for a standard curve; (4) Development of a viral RNA extraction method incorporating the internal virion standard; (5) Evaluation of assay sensitivity and reproducibility from start to finish. Access to the Los Alamos database (http://hiv-web.lanl.gov) enabled selection of primers and probe in a highly conserved coding sequence of gag for HIV-1 Subtype B and the RCAS internal virion standard. RNA transcripts were generated and a standard curve of a known quantity was used to develop and optimize a two-step RT-PCR assay for both HIV-1 and RCAS. Patient plasma (7m1/sample) samples with viral RNA concentrations known to be above 50 copies by current commercial methods were spiked with internal standard and used to establish a concentration and extraction method. Extracted samples were assayed in triplicate and the quantity of HIV-1 RNA measured via fluorescent probe technology. Separate analysis of the internal virion standard provided confirmation of the virion recovery and HIV-1 RNA extraction. To compare the performance of the single copy assay with other commonly used HIV-1 RNA assays, a low copy number panel (200 to 0.781 RNA copies/nil) was measured. The single copy assay consistently detected HIV-1 RNA in all samples (200 to 0.781 copies/ml), and the mean values from the 4 assays indicate that HIV-1 RNA could be quantified to 1 copy per ml, although the standard deviation increased at 1.56 and 0.781 copies/ml. To ensure the single copy assay primers and probe accurately quantified HIV-1 RNA, 22 samples from untreated HIV-infected patients with HIV RNA levels >100 copies/ml, were analyzed by both the bDNA and the single copy assay. The single copy assay demonstrated excellent concordance (r²=0.894) with the bDNA assay and 20 out of the 22 patient samples had similar viral RNA levels with both assays. Testing of plasma samples from 15 patients on antiretroviral therapy with HIV-1 RNA <75 copies/ml revealed persistent viremia in all 15 patients with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median of 13 copies/m1). Within 4 to 15 months after the initiation of antiretroviral treatment, four of the 15 patients showed stable, persistent HIV-1 RNA levels of 0.3-41 copies/ml which eventually reached a plateau. The sensitivity of the single copy assay allows for the quantification of persistent low-level viremia in patients whose viral loads are below the point of detection by commercial assays.