Table 4.

Measuring the Latent Reservoir.

Technique	Description	Advantages	Limitations
ddPCR (droplet digital PCR) (reviewed [135])	Target molecules are emulsified into thousands of nanoliter droplets and amplified by PCR using a primer-probe set. Droplets containing HIV genomic material that fluoresces above a certain threshold will be considered positive. The ratio between the positive and negative droplets is used to calculate the absolute number of starting molecules using a Poisson distribution.	Very accurate and versatile Can be used to measure both viral RNA and DNA Does not need a standard curve Produces absolute quantification More reproducible than standard qPCR	Technically difficult and thresholds are set by the user High false-positive rate
IPDA (intact proviral DNA assay) [136]	Uses two amplicons covering the packaging signal (ψ) and env region and ddPCR to designate deleted proviruses as defective. In parallel, multiplex PCR is performed with two unique primer-probe sets targeting ψ and env with unique labeling probes and measured by ddPCR. Primer-probe sets amplify validated, highly conserved regions of the genome. Droplets are scored based on expression of combinations of probe fluorescent patterns.	Primers are pre-designed to recognize most HIV sequences Can quickly determine if a provirus or RNA genome is likely to be intact or defective Primer-probe sets can determine the level of intactness or defectiveness of most proviral and RNA genomes	The entire viral genome is not sequenced and can therefore mis-classify HIV genomes as intact Over-estimates the number of intact viral genomes Requires ddPCR
TILDA(Tat/Rev induced limiting dilution assay) [137]	CD4+ T cells are stimulated in vitro (PMA and ionomycin) to maximally produce tat/rev transcripts. Cells are serially diluted as replicates. Real-time PCR with primer-probe pairs are used to quantify inducible viral RNA. The frequency of cells with inducible HIV RNA can then be calculated from the number of positive wells at each dilution by the maximum likelihood method.	Measures latent reservoir from total CD4+ T cells Viral transcripts detected without RNA extraction Requires small blood sample Less labor and time required compared to similar protocols	Only measures genomes capable of being reactivated Not all latent infections can be reactivated and measured [65] Assumes multiply spliced genomes indicate replication-competence
QVOA (quantitative viral outgrowth assay) [138,139]	Culture method to quantify the replication-competent viral reservoir. HIV(+) donor rCD4+ T cells are cultured with irradiated PBMCs and CD4+ T cells from an HIV(-) donor and stimulated (PHA; IL-2). Replication -competent virus can spread to HIV(-) CD4+ T cells, amplifying the infection, allowing detection and quantification of viral outgrowth.	Detects and quantifies replication-competent virus Measures both pre- and post-integration latency Well-established and widely used protocol	Only measures genomes capable of being reactivated Not all latent infections can be reactivated and measured Underestimates the size of the latent reservoir Requires allogenic donor lymphoblasts for spreading infection
mQVOA (Modified quantitative viral outgrowth assay) [140]	A more sensitive adaptation of the gold-standard assay; CD4+ T cells from patients are serially diluted and stimulated (αCD3/CD28 antibodies). MOLT-4/CCR5 cells are co-cultured with the primary cells. HIV RNA is extracted, and RT-qPCR is performed to amplify pol. The number of wells positive for HIV RNA at each dilution level are used to determine the infection frequency by maximum likelihood estimate.	Does not require allogenic donor lymphoblasts for spreading infection qPCR used for quantitative measurement of the viral RNA products	Only measures genomes capable of being reactivated Not all latent infections can be reactivated and measured Underestimates the size of the latent reservoir
dQVOA (differentiation culture quantitative viral Outgrowth assay) [141]	Measures the impact of TEM differentiation on induction and outgrowth of replication-competent HIV. rCD4+ T cells from patients are activated through culture with a differentiation cytokine cocktails to drive cells towards the TEM terminally differentiated subset. Cells are distributed at limiting dilutions and cultured in differentiation cytokines, then activated. Titer measured by p24 ELISA.	Higher viral titer induction rate compared to QVOA Higher frequency of latent cell activation over QVOA Does not require allogenic donor lymphoblasts for spreading infection	Only measures genomes capable of being reactivated Not all latent infections can be reactivated and measured Underestimates the size of the latent reservoir