A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir

Cell Rep Med. 2021 Apr 12;2(4):100243. doi: 10.1016/j.xcrm.2021.100243. eCollection 2021 Apr 20.


Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4+ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

Keywords: HIV cure; HIV reservoir; IPDA; digital PCR; genital mucosa; intact proviral DNA assay; intestinal mucosa; multiplexing; rectal; viral outgrowth assay.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Viral / analysis
  • HIV Infections / genetics*
  • HIV Seropositivity / genetics
  • HIV-1 / genetics*
  • Humans
  • Polymerase Chain Reaction* / methods
  • Proviruses / genetics*
  • Viral Load / methods
  • Virus Latency / genetics


  • DNA, Viral