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. 2017 Apr 28;215(Suppl 3):S134–S141. doi: 10.1093/infdis/jiw648

Table 1.

Advantages and Limitations of Strategies to Quantify and Characterize the Human Immunodeficiency Virus Reservoir

Approach Advantages Limitations
Total and integrated HIV DNA by quantitative PCR (eg, real-time, digital PCR) Highly sensitive, able to be performed on tissues and cells; inexpensive; high throughput The majority of HIV DNA is defective and does not constitute replication-competent virus; variation in measurements across assays and laboratories; PCR inhibitors exist in various biofluids
Cell-associated HIV RNA by quantitative PCR Highly sensitive, can be used to detect the entire spectrum of HIV transcript species within cells; inexpensive; high throughput Usually measured from bulk cell extracts; sensitive to time of sampling and time from sampling to processing; variation in measurements across assays and laboratories; PCR inhibitors exist in various biofluids
Ultrasensitive measurement of residual plasma HIV RNA Highly sensitive, may reflect the “active” HIV reservoir Remains to be determined if replication-competent virus is exclusively characterized
qVOA Provides measurement of replication-competent HIV reservoir Expensive; time consuming; requires large numbers of cells; variation in results across assays and laboratories; primarily used on cells obtained from peripheral blood; challenging to obtain sufficient viable cells from tissues; some genetically intact viruses may not grow in culture
Inducible measures of HIV reactivation (eg, TILDA) Provides measure of the percentage or number of cells in which HIV reactivates upon maximal stimulation; relatively high throughput; requires fewer cells than traditional qVOA Does not provide measures of replication competence as RNA can be generated from defective viral genomes; challenges with downstream isolation and characterization of genomic DNA or mRNA from individual cells
Viral protein quantification (eg, HIV p24) Measures virus with sufficient genetic integrity to drive transcription, translation, and downstream processing May overestimate viral reservoir size as replication-incompetent viruses may still generate protein
PET-based imaging/nuclear medicine Has the potential to survey the whole-body HIV reservoir in various tissues and anatomical locations In development; requires expression of viral protein and may lack sensitivity required to detect latently infected cells or low levels of viral transcriptional and translational activity in antiretroviral-treated individuals; potential for low signal to noise ratios; expensive; involves in vivo radiation exposure
Single-cell HIV reservoir characterization Potential to lead to a greater understanding of the genomic and transcriptional differences between actively infected, latently infected and uninfected cells Commercially available platforms are expensive and lack the throughput to characterize millions of cells that may be required given low frequency of latently infected CD4 T cells in individuals on ART; higher throughput assays still in development
Measurement of anti-HIV immune responses (indirect marker) Titer and avidity of HIV antibodies may represent whole-body, tissue-based HIV persistence and be useful in predicting HIV recrudescence following ATI Heterogeneous responses; larger longitudinal and cross-sectional studies required to rigorously associate immune responses with reservoir size and HIV rebound dynamics

Abbreviations: ART, antiretroviral therapy; ATI, analytical treatment interruptions; HIV, human immunodeficiency virus; mRNA, messenger RNA; PCR, polymerase chain reaction; PET, positron emission tomography; qVOA, quantitative viral outgrowth assay; TILDA, Tat/Rev-induced limiting dilution assay.

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