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

TLDR

More sensitive assays for HIV‑1 RNA are needed to detect, quantify, and characterize persistent viremia in patients on antiretroviral therapy whose plasma HIV‑1 RNA levels are suppressed to less than 50–75 copies/ml. The study developed an internally controlled real‑time reverse transcriptase‑initiated PCR assay capable of quantifying HIV‑1 RNA down to 1 copy per ml of plasma. The assay employs HIV‑1 RNA transcripts ranging from 1 to 10^6 copies per reaction to produce a reproducible linear regression of input copy number versus threshold cycle, and was benchmarked against the ultrasensitive AMPLICOR HIV‑1 MONITOR assay using a low‑copy panel of 200 to 0.781 copies/ml. The single‑copy assay demonstrated superior sensitivity, detecting HIV‑1 RNA as low as 0.781 copies/ml and revealing persistent viremia in all 15 antiretroviral‑treated patients with levels ranging from 1 to 32 copies/ml (median 13 copies/ml).

Abstract

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.