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Table 1 Methodologies for measuring the HIV-1 latent reservoir

From: Addressing an HIV cure in LMIC

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, 179,180,181,182]

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

[183,184,185,186,187,188]

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

[189,190,191,192]

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]