- Levy, Claire N;
- Hughes, Sean M;
- Roychoudhury, Pavitra;
- Reeves, Daniel B;
- Amstuz, Chelsea;
- Zhu, Haiying;
- Huang, Meei-Li;
- Wei, Yulun;
- Bull, Marta E;
- Cassidy, Noah AJ;
- McClure, Jan;
- Frenkel, Lisa M;
- Stone, Mars;
- Bakkour, Sonia;
- Wonderlich, Elizabeth R;
- Busch, Michael P;
- Deeks, Steven G;
- Schiffer, Joshua T;
- Coombs, Robert W;
- Lehman, Dara A;
- Jerome, Keith R;
- Hladik, Florian
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.