Table 1.

Methodologies for measuring the HIV-1 latent reservoir

Biomarker	Feature of the latent reservoir measured	Fraction of the reservoir included in measurement	Under/overestimation of replication competent reservoir size	Assay examples	Caveats	References
HIV-1 DNA	Either total or cell-associated HIV-1 DNA directly ex vivo by polymerase chain reaction (PCR)-based techniques	Both intact and defective HIV-1 DNA (except for IPDA); both inducible and non-induced viruses	Over	Droplet digital PCR (ddPCR); quantitative PCR (qPCR); Intact proviral DNA assay (IPDA); Q4PCR; SGS/NGS/near-full length genome sequencing (with or without integration site analysis)	With total HIV-1 DNA measurement, 2-LTR circles and episomal DNA are quantified along with proviral DNA; only IPDA and near-full length genome sequencing with integration site analysis measure intact provirus	[56, 179–182]
HIV-1 RNA induction	Cell-associated/cell-free RNA produced after ex vivo stimulation of infected cells	Both intact and defective (but transcription-competent) provirus; inducible viruses only	Over	Bulk CA-RNA PCR; Single cell RNA PCR; Single copy assay (SCA); SGS/NGS	A single round of maximal stimulation does not induce all transcription-competent proviruses	[183–188]
Induced HIV-1 protein production	Cell-associated HIV-1 proteins induced after a single round of ex vivo stimulation	Both intact and defective (but transcription-competent) provirus; inducible viruses only	Over	Flow/mass cytometry; FAST/digital microscopy; ELISA	A single round of maximal stimulation does not induce all transcription-competent proviruses; false positives associated with p24 quantitation	[189–192]
Viral outgrowth	Replication-competent virus that grows out after a single round of ex vivo stimulation of resting CD4+ T cells	Intact, inducible proviruses only	Under	Quantitative VOA (QVOA)	A single round of maximal stimulation does not induce all intact, inducible proviruses as some are in ‘deep’ latency	[5, 58, 60, 61]