Combination antiretroviral therapy (cART) has significantly reduced mortality and morbidity of those infected with HIV-1 [1, 2]. During cART, the plasma virus levels become undetectable by standard methods of detection [3-5]. Investigators have determined that the major cellular reservoir of latent HIV-1 proviral DNA is contained primarily within the central memory (TCM) and transitional memory (TTM) compartments of resting CD4+ T cells (rCD4)[6-8]. However, further analysis indicates that replication-competent virus is more readily detected in TCM than in TTM [9]. It is thought that the establishment of the latent reservoir occurs when activated CD4+ T cells become infected as they return to a resting memory state [2]. While the frequency of latently infected cells is extremely low, estimated to be 0.1-10 infectious units per million (IUPM) rCD4 in most patients on long-term therapy [7, 10, 11] during cessation of cART, these long-lasting rCD4 are believed to be the source of emerging virus [12, 13]. Therefore, to cure those infected with HIV-1, these latently infected rCD4 must be either eliminated or suppressed in their capacity to produce infectious HIV-1 upon therapy interruption [1, 14, 15]. However, due to the low frequency of the latently infected cells, lack of cell surface markers specific for latently infected cells, and presence of a replication defective viral genome in latently infected cells [16], it is extremely challenging to accurately quantify the size of the replication-competent reservoir, and hence, to determine the efficacy of any eradication strategy designed to eliminate the infected cells. There have been a number of reports that have identified associations with a few cell surface markers expressed on the CD4+ lymphocytes harboring latent HIV including the immune checkpoint molecules PD-1, LAG-3, TIGIT, and more recently CD32a [17]. Although all of these are important findings, it is unclear if the expression of these particular markers can be directly correlated with or are useful toward measuring of the size of the viral reservoir.

Quantification of the latent HIV-1 viral reservoir requires a highly sensitive assay capable of measuring the low frequency of rCD4 carrying functional provirus. Preferably, such an assay should be cost-effective and adaptable for utilization under highthroughput conditions and large-scale clinical applications. To date, the standard method for determining the size of the