Improved single-copy assays for quantification of persistent HIV-1 viremia in patients on suppressive antiretroviral therapy

Abstract

A quantitative real-time PCR (qRT-PCR) assay with single-copy sensitivity targeting HIV-1 gag RNA (the gag single-copy assay [gSCA]) has been used widely to quantify plasma viremia below the limit of detection of clinical assays in patients on effective antiretroviral therapy (ART), but viral RNA in 15 to 30% of samples amplifies inefficiently because of primer/probe mismatches. We sought to develop improved single-copy assays with increased sensitivity by improving nucleic acid recovery, designing qRT-PCR primers and a probe for a highly conserved region of integrase in the HIV-1 pol gene (the integrase single-copy assay [iSCA]), and increasing the plasma volume tested (Mega-iSCA). We evaluated gSCA versus iSCA in paired plasma samples from 10 consecutive patients with viremia of >1,000 copies/ml and 25 consecutive patients on suppressive ART. Three of 10 viremic samples amplified inefficiently with gSCA compared to the Roche Cobas Ampliprep/TaqMan 2.0, whereas all 10 samples amplified efficiently with iSCA. Among 25 samples from patients on suppressive ART, 8 of 12 samples that were negative for HIV-1 RNA by gSCA had detectable HIV-1 RNA by iSCA, and iSCA detected 3-fold or higher HIV-1 RNA levels compared to gSCA in 10 of 25 samples. Large-volume plasma samples (>20 ml) from 7 patients were assayed using Mega-iSCA, and HIV-1 RNA was quantifiable in 6, including 4 of 5 that were negative by standard-volume iSCA. These improved assays with superior sensitivity will be useful for evaluating whether in vivo interventions can reduce plasma viremia and for assessing relationships between residual viremia and other virologic parameters, including the inducible proviral reservoir.

FIG 1

Proportion of nucleotides in 200-bp fragments that match the HIV-1 2012 subtype B consensus sequence. A consensus sequence was generated from a total of 675 aligned full-length subtype B sequences from the Los Alamos HIV Sequence Database, and the number of bases in 200-bp fragments that matched the consensus sequence was calculated. The most highly conserved regions are present where the original gSCA primers were designed and in the 3′ region of pol, where the iSCA primers and probe target.

FIG 2

Comparison of sequence logos for the forward (A), reverse (B), and probe (C) binding regions for iSCA and gSCA from 675 subtype B sequences from the Los Alamos HIV Sequence Database. The iSCA system has fewer mismatches than the gSCA system, suggesting that the primers and probe from the iSCA system would efficiently amplify HIV-1 RNA in a higher proportion of patient samples compared to the gSCA system.

FIG 3

Relationship between plasma HIV-1 RNA measured with gSCA versus iSCA. (A) There is no significant correlation between HIV-1 RNA values measured by gSCA and iSCA in the 10 samples from viremic patients because of the 3 samples with inefficient amplification by gSCA. The dashed lines indicate the limit of quantification of Roche TM2.0 (20 cps/ml). (B) There is a nonsignificant trend toward a correlation between HIV-1 RNA values measured by iSCA versus gSCA in the 25 plasma samples from patients on suppressive ART. The dotted lines indicate the limit of detection of iSCA and gSCA (0.6 cps/ml). (C) HIV-1 RNA values are significantly correlated between iSCA and gSCA for all 35 patients evaluated in this study. The dashed lines indicate the limit of quantification of Roche TM2.0 (20 cps/ml), and the dotted lines indicate the limit of detection of iSCA and gSCA (0.6 cps/ml).