Single-copy Assay Research Articles

A Comparison of Viral Loads between HIV‐1–Infected Elite Suppressors and Individuals Who Receive Suppressive Highly Active Antiretroviral Therapy

Elite suppressors are untreated individuals with human immunodeficiency virus type 1 (HIV-1) infection who maintain viral loads <50 copies/mL. Using a single-copy assay, we show that there is no statistically significant difference between the proportions of elite suppressors and patients receiving suppressive highly active antiretroviral therapy who have viral loads of <1 copy/mL.

Valproic acid without intensified antiviral therapy has limited impact on persistent HIV infection of resting CD4+ T cells

Valproic acid and intensified antiretroviral therapy may deplete resting CD4+ T-cell HIV infection. We tested the ability of valproic acid to deplete resting CD4+ T-cell infection in patients receiving standard antiretroviral therapy. Resting CD4+ T-cell infection was measured in 11 stably aviremic volunteers twice prior to, and twice after Depakote ER 1000 mg was added to standard antiretroviral therapy. Resting CD4+ T-cell infection frequency was measured by outgrowth assay. Low-level viremia was quantitated by single copy plasma HIV RNA assay. A decrease in resting CD4+ T-cell infection was observed in only four of the 11 patients. Levels of immune activation and HIV-specific T-cell response were low and stable. Valproic acid levels ranged from 26 to 96 microg/ml when measured near trough. Single copy assay was performed in nine patients. In three patients with depletion of resting CD4+ T-cell infection following valproic acid, single copy assay ranged from less than 1-5 copies/ml. Continuous low-level viremia was observed in three patients with stable resting CD4+ T-cell infection (24-87, 8-87, and 1-7 copies/ml respectively) in whom multiple samples were analyzed. The prospective addition of valproic acid to stable antiretroviral therapy reduced the frequency of resting CD4+ T-cell infection in a minority of volunteers. In patients in whom resting CD4+ T-cell infection depletion was observed, viremia was rarely detectable by single copy assay.

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

Residual viremia can be detected in most HIV-1-infected patients on antiretroviral therapy despite suppression of plasma RNA to <50 copies per ml, but the source and duration of this viremia is currently unknown. Therefore, we analyzed longitudinal plasma samples from 40 patients enrolled in the Abbott M97-720 trial at baseline (pretherapy) and weeks 60 to 384 by using an HIV-1 RNA assay with single-copy sensitivity. All patients were on therapy (lopinavir/ritonavir, stavudine, and lamivudine) with plasma HIV RNA <50 copies per ml by week 96 of the study and thereafter. Single-copy assay results revealed that 77% of the patient samples had detectable low-level viremia (>/=1 copy per ml), and all patients had at least one sample with detectable viremia. A nonlinear mixed effects model revealed a biphasic decline in plasma RNA levels occurring over weeks 60 to 384: an initial phase of decay with a half-life of 39 weeks and a subsequent phase with no perceptible decay. The level of pretherapy viremia extrapolated for each phase of decay was significantly correlated with total baseline viremia for each patient (R(2) = 0.27, P = 0.001 and R(2) = 0.19, P < 0.005, respectively), supporting a biological link between the extent of overall baseline viral infection and the infection of long-lived reservoirs. These data suggest that low-level persistent viremia appears to arise from at least two cell compartments, one in which viral production decays over time and a second in which viral production remains stable for at least 7 years.

ART Suppresses Plasma HIV-1 RNA to a Stable Set Point Predicted by Pretherapy Viremia

Current antiretroviral therapy is effective in suppressing but not eliminating HIV-1 infection. Understanding the source of viral persistence is essential for developing strategies to eradicate HIV-1 infection. We therefore investigated the level of plasma HIV-1 RNA in patients with viremia suppressed to less than 50–75 copies/ml on standard protease inhibitor- or non-nucleoside reverse transcriptase inhibitor-containing antiretroviral therapy using a new, real-time PCR-based assay for HIV-1 RNA with a limit of detection of one copy of HIV-1 RNA. Single copy assay results revealed that >80% of patients on initial antiretroviral therapy for 60 wk had persistent viremia of one copy/ml or more with an overall median of 3.1 copies/ml. The level of viremia correlated with pretherapy plasma HIV-1 RNA but not with the specific treatment regimen. Longitudinal studies revealed no significant decline in the level of viremia between 60 and 110 wk of suppressive antiretroviral therapy. These data suggest that the persistent viremia on current antiretroviral therapy is derived, at least in part, from long-lived cells that are infected prior to initiation of therapy.

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

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.