Clinical validation of a quantitative HIV-1 DNA droplet digital PCR assay: Applications for detecting occult HIV-1 infection and monitoring cell-associated HIV-1 dynamics across different subtypes in HIV-1 prevention and cure trials

Background: In HIV-1-exposed infants, nucleic acid testing (NAT) is required to diagnose infection since passively transferred maternal antibodies preclude antibody testing. The sensitivity of clinical NAT assays is lowered with infant antiretroviral prophylaxis and, with empiric very early antiretroviral treatment of high-risk infants, thereby impacting early infant diagnosis. Similarly, adult HIV-1 infections acquired under pre-exposure prophylaxis may occur at low levels, with undetectable plasma viremia and indeterminate antibody tests, for which HIV-1 DNA testing maybe a useful adjunct. Cell-associated HIV-1 DNA concentrations are also used to monitor HIV-1 persistence in viral reservoirs with relevance to HIV-1 cure therapeutics, particularly in perinatal infections. Objectives: We clinically validated an HIV-1 DNA quantitative assay using droplet digital PCR (ddPCR), across different HIV-1 subtypes. Study Design: The analytical sensitivity and specificity of an HIV-1 DNA ddPCR assay was determined using serial dilutions of a plasmid containing HIV-1 LTR-gag spiked into peripheral blood mononuclear cells (PBMCs), with MOLT-4 cells or PBMCs infected with different HIV-1 subtypes (A, B and C), and U1 cells spiked into PBMCs. Inter- and intra-run variability were used to determine assay precision. Results: The HIV-1 LTR-gag ddPCR assay was reliable and reproducible, and exhibited high analytical specificity with sensitivity to near single copy level, across multiple HIV-1 subtypes, and a limit of detection of 4.09 copies/million PBMCs. Conclusions: This assay has applications for detecting occult HIV-1-infection in the setting of combination and long-acting regimens used for HIV-1 prevention, across different HIV-1 subtypes, in infants and adults, and in HIV-1 cure interventions.