A Simpler and More Sensitive Single-Copy HIV-1 RNA Assay for Quantification of Persistent HIV-1 Viremia in Individuals on Suppressive Antiretroviral Therapy

Abstract

A real-time quantitative reverse transcriptase PCR assay with single-copy sensitivity targeting the integrase region of HIV-1 (integrase single-copy assay [iSCA] v1.0) has been widely used to quantify plasma viremia in individuals on antiretroviral therapy (ART). iSCA v1.0 requires the use of an ultracentrifuge, and only about half of the nucleic acid extracted from plasma is assayed for HIV-1 RNA. We sought to simplify sample processing using microcentrifugation and improve assay sensitivity by testing more than 75% of the total extracted nucleic acid for HIV-1 RNA (iSCA v2.0). We evaluated the limit of detection (LoD) of iSCA v2.0 by testing replicates of low-copy plasma HIV-1 RNA standards. By probit analysis, the 95% LoD was 1 copy of HIV-1 RNA per milliliter for a 5-ml plasma sample. To compare the sensitivity of iSCA v1.0 and v2.0, we tested plasma samples with both assays from 60 participants on ART with HIV-1 RNA below 20 cps/ml. Of the 31 samples that had no detectable HIV-1 RNA by iSCA v1.0, 17 (55%) were detectable by v2.0 with an HIV-1 RNA mean value of 3.5 cps/ml. Twenty-nine samples were detectable with both assay versions, but average values of HIV-1 RNA cps/ml were 2.7-fold higher for v2.0 than v1.0. These results support the adoption of a new, more sensitive and simpler single-copy HIV-1 RNA assay (iSCA v2.0) to quantify residual viremia on ART and to assess the impact of experimental interventions designed to decrease HIV-1 reservoirs.

Differences in HIV Markers between Infected Individuals Treated with Different ART Regimens: Implications for the Persistence of Viral Reservoirs

“…Unquantifiable VLs can thus be categorized as undetectable or detectable. Using ultrasensitive RT-qPCR assays, most individuals with VLs below the limit of quantification of commercial assays still have detectable HIV RNA in plasma, averaging around 1-3 copies/mL [48][49][50][51][52][53][54]. A number of excellent reviews on persistent residual viremia (RV) in ART-treated individuals have been published [55][56][57][58][59][60][61][62][63], and a mathematical model of its origins has been put forward [64].…”

Enhancing care for people living with HIV: current and future monitoring approaches

“…So far, guidelines do not clarify how to use TD/ TND data, but this parameter has been recently used in clinical trials to evaluate efficacy of 2-drug combinations and triple therapies [14,15]. On the other hand, despite recent improvements, the use of investigational single-copy assays to quantify residual HIV RNA well below the threshold of the routine assays is likely to remain in the research setting, mainly due to cost, current lack of standardization, and the large volume of blood required [16].…”

Persistent HIV-1 Viremia on Antiretroviral Therapy: Measurement and Mechanisms

“…In the most recent iteration of a single-copy assay (iSCA v2.0), ultracentrifugation is replaced with tabletop centrifugation and a greater proportion (∼80%) of the total extracted nucleic acid is tested for HIV-1 RNA. Importantly, when equal volumes of the same donor plasma were tested using versions of iSCA, 55% of the samples that had no HIV-1 RNA detected by iSCA v1.0 had HIV-1 RNA detected by iSCA v2.0 (Tosiano et al, 2019a).…”