The past decade has seen rapid growth in the number and types of molecular assays used to measure the HIV reservoir[1], [2], [3], [4], [5], [6], [7], [8]. HIV viremia assay development has significantly improved analytical sensitivities and automated workflows[[2], [3], [4],9], and yet most individuals on antiretroviral therapy (ART) continue to have undetectable viral loads. Cell-associated HIV DNA assays constitute an important initial, albeit inflated, measurement of the HIV reservoir, while cell-associated HIV usRNA assays offer sensitive detection of transcriptionally competent integrated provirus[10,11]. 2-long terminal repeat (LTR) circle assays measure episomal HIV DNA that correlates with past active HIV replication, with varying kinetics[12], [13], [14]. Sensitive assays for these HIV-1 viral parameters may also be useful in diagnosing occult HIV-1 infection that might occur under certain circumstances[15].

New multiplex digital droplet PCR (ddPCR)-based HIV-1 DNA assays targeting multiple regions of the HIV-1 genome offer better measurements of the intact proviral reservoir, and have been confirmed via near full-length viral genome sequencing[14,16,17]. Readouts utilizing ddPCR have a number of advantages over classical quantitative PCR, including absolute multi-locus quantification[18] and higher tolerance for primer/probe mismatches and inhibitory substances[19]. These advantages are critical for the quantitation of cell-associated HIV parameters, given the profound sequence diversity of HIV[20], high levels of inactive proviruses with genomic deletions[21], and high cellular input required for these assays[15,22,23].

Comparatively few full clinical validations have been reported for HIV reservoir assays that meet CLIA and/or GCLP requirements for clinical testing and/or trials[1,15]. Our institution recently validated a multiplex intact HIV-1 proviral reservoir assay for use in clinical studies[17]. Here, we detail the clinical validation and evaluation of three ddPCR-based, cell-associated assays for HIV-1 DNA, unspliced RNA (usRNA), and 2-LTR circle quantitation for use in clinical trials.