New Real-Time Reverse Transcriptase-Initiated PCR Assay with Single-Copy Sensitivity for Human Immunodeficiency Virus Type 1 RNA in Plasma

Abstract

More sensitive assays for human immunodeficiency virus type 1 (HIV-1) RNA are needed to detect, quantify, and characterize persistent viremia in patients who are receiving antiretroviral therapy and whose plasma HIV-1 RNA levels are suppressed to less than 50 to 75 copies/ml. We therefore developed an internally controlled real-time reverse transcriptase-initiated PCR assay that quantifies HIV-1 RNA concentrations down to 1 copy per ml of plasma. This assay with single-copy sensitivity (the single-copy assay) generates a reproducible linear regression plot of input copy number versus threshold cycle by using HIV-1 RNA transcripts at copy numbers ranging from 1 to 10 6 per reaction mixture. The single-copy assay was compared to the ultrasensitive AMPLICOR HIV-1 MONITOR assay and a more sensitive modification of the ultrasensitive assay by repeatedly testing a low-copy-number panel containing 200 to 0.781 copies of HIV-1 RNA per ml of plasma. This comparison showed that the single-copy assay had a greater sensitivity than the other assays and was the only assay that detected HIV-1 RNA at levels as low as 0.781 copies/ml. Testing of plasma samples from 15 patients who were receiving antiretroviral therapy and who had <75 HIV-1 RNA copies/ml revealed persistent viremia in all 15 patients, with HIV-1 RNA levels ranging from 1 to 32 copies/ml (median, 13 copies/ml). The greater sensitivity of the single-copy assay should allow better characterization of persistent viremia in patients who are receiving antiretroviral therapy and whose HIV-1 RNA levels are suppressed to below the detection limits of present assays.

Antiviral drug-resistant HBV: Standardization of nomenclature and assays and recommendations for management

“…Single genome sequencing has been used in studying antiviral-resistant HIV mutations. 27,28 This method is tedious and does not provide information on the absolute frequency of the mutation. In addition, given the high rate of spontaneous mutations during HBV replication, the clinical significance of mutants that may be present in Ͻ0.1% of the viral population is uncertain.…”

Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy

“…The SCA for HIV-1 RNA detection was performed as described in ref. 10, starting with Ϸ3 ml of plasma, leading to a typical lower limit of quantitation for these samples of 0.63 copies per ml. For each sample, three separate aliquots of the cDNA product were assayed for HIV-1 RNA and two aliquots for the recombinant avian retrovirus internal standard RNA using real-time PCR of conserved sequences within gag as described in ref.…”

“…For each sample, three separate aliquots of the cDNA product were assayed for HIV-1 RNA and two aliquots for the recombinant avian retrovirus internal standard RNA using real-time PCR of conserved sequences within gag as described in ref. 10. For five patients, an archived pretherapy sample was unavailable and a postbaseline value was obtained during the first 2 weeks of treatment.…”

One Percent Tenofovir Applied Topically to Humanized BLT Mice and Used According to the CAPRISA 004 Experimental Design Demonstrates Partial Protection from Vaginal HIV Infection, Validating the BLT Model for Evaluation of New Microbicide Candidates

“…Infection of BLT mice with HIV-1 was monitored in peripheral blood as follows: by determining plasma levels of viral antigenemia using a p24 enzyme-linked immunosorbent assay ([ELISA] assay sensitivity, 7.8 pg/ml; Coulter); by determining levels of viral RNA in plasma using either an Amplicor (Roche) assay or one-step reverse transcriptase real-time PCR (ABI custom TaqMan Assays-by-Design) according to the manufacturer's instructions (with primers 5Ј-CATGTTTTCAGCATTATCA GAAGGA-3Ј and 5Ј-TGCTTGATGTCCCCCCACT-3Ј and MGB-probe 5Ј-FAM-CCACCCCACAAGATTTAAACACCATGCTAA-Q-3Ј, where FAM is 6-carboxyfluorescein [37]; assay sensitivity of 400 RNA copies per ml in both assays); and by determining levels of viral DNA in peripheral blood cells (realtime PCR analysis; assay sensitivity of 10 copies) as previously described (11,12,45,53). The single-cell suspension preparation from the female reproductive tract (FRT) was adapted from a published protocol (22).…”