New assays for monitoring residual HIV burden in effectively treated individuals

Abstract

Purpose of review: Measurements of HIV burden have relied upon quantification of viral nucleic acids by real-time PCR (qPCR). To develop and test strategies for eradication, new methods are needed to better characterize residual cellular reservoirs in patients on suppressive antiretroviral therapy (ART). This review summarizes recent advances that may lead to clinically useful tests with improved sensitivity, reproducibility and throughput.

Recent findings: HIV DNA remains the most sensitive measure of residual infection, but its low levels are difficult to differentiate from assay noise by qPCR. Digital PCR has begun to improve the precision of existing real-time assays, but there remains a need to distinguish replication-competent proviruses. Rapid technological progress in single-cell analysis is beginning to offer new approaches, notably CyTOF and microengraving, which could provide vastly more information about the composition of the latent reservoir.

Summary: To investigate and assess therapies directed towards eradication, improved assays that simultaneously offer high sensitivity, precision and information content will be needed.

FIGURE 1

Signal and noise in standard assays. The figure illustrates the typical dynamics of total HIV RNA, HIV DNA and cell-associated infectivity [CAI, measured in infectious units per million cells (IUPM)] following initiation of ART in comparison with the standard error of measurement for each assay. The observed changes in all three quantities are small compared with the intrinsic assay noise. Only HIV RNA during the first few weeks of treatment can be resolved with high signal-to-noise ratio. ART, antiretroviral therapy.

FIGURE 2

Droplet digital PCR. (a) The PCR mixture, which includes the target template and fluorescent hydrolysis probe, is emulsified into approximately 15000 droplets. After thermal cycling, the number of fluorescent droplets is counted. The initial template concentration is then calculated, assuming random partitioning. (b) Replicate assay wells are plotted against one another to compare the precision of a digital PCR assay for HIV pol with an identical real-time PCR assay. For clinical samples (circles), the coefficient of variation was four-fold lower, on average. A larger reduction in the C.V., >20-fold, was seen for a 2-LTR assay in the same samples. C.V., coefficient of variation.