Table 2.
Characteristics of Participating Assays, Including Starting Material, Number of Replicates Assayed, Culture Conditions, and Culture Monitoring Readout
| Assays by Assay Type | Starting Material | Dilution Replicates, No. | Dilutions and Cell Input at Each Dilution | Stimulation | Target Cells | ARV Culture Additions | Culture Duration | HIV Target to Monitor HIV-1 Outgrowth Culture Supernatant | Assay to Monitor HIV-1 Outgrowth Culture Supernatant | Measurement |
|---|---|---|---|---|---|---|---|---|---|---|
| Classic QVOA | ||||||||||
| QVOA M [15] | 120 mL of WB; 300 × 106 PBMCs | 6 | 3-Fold 1 × 106–1 × 102 rCD4+ | PHA plus γ-irradiated PBMCs | CD8-depleted PBMCs | None | 2 wk | p24 | PerkinElmer HIV-1 p24 ELISA | IUPM |
| QVOA S [7, 13] | ≥100mL of WB; 400 × 106 PBMCs | 5–22 | 5-Fold 1 × 106–0 rCD4+ | PHA plus γ-irradiated PBMCs | MOLT-4/ CCR5 | None | 3 wk | p24 | PerkinElmer HIV-1 p24 ELISA | IUPM |
| QVOA SR [7, 13] | ≥100 mL of WB; 400 × 106 PBMCs | 5–22 | 5-Fold 1 × 106–0 rCD4+ | PHA plus γ-irradiated PBMCs | CD8-depleted PBMCs | None | 20 d | p24 | PerkinElmer HIV-1 p24 ELISA | IUPM |
| usQVOA | ||||||||||
| QVOA Simoa [7, 13, 16] | ≥100 mL of WB; 400 × 106 PBMCs | 5–22 | 5-Fold 1 × 106–0 rCD4+ | PHA plus γ-irradiated PBMCs | CD8-depleted PBMCs | None | 20 d | p24 | Quanterix p24 Simoa | IUPM |
| QVOA RNA [5, 9] | 100 mL of WB; 7–20 × 106 rCD4+ | 6 | 3-Fold 1 × 106–1372 rCD4+ | Plate-bound αCD3/ αCD28 | MOLT-4/ CCR5 | None | 9–16 d | gag | RT-PCR | IUPM |
| Next-generation QVOA | ||||||||||
| TILDA [17] | 10–20 mL of WB; 10 × 106 PBMCs | ≤24 | 4-Fold 18 000 –1000 CD4+ | PMA/ ionomycin | None | Zidovudine, efavirenz, raltegravir | 18 h | Tat/rev msHIV caRNA | Nested RT-PCR | Inducible msHIV RNA |
| iCARED [5, 9] cfRNA, caRNA1, caRNA2 | 100 mL of WB; 7–20 × 106 rCD4+ | 6–12 | 3-Fold 5 × 105–229 rCD4+ | Plate-bound αCD3/ αCD28 | None | Raltegravir | 3 d | Cell-associated and cell-free gag, tat-rev RNA | dd-PCR | Transcription- competent virus, single round infection |
Abbreviations: ARV, antiretroviral therapy; caRNA, cell-associated RNA; caRNA1, cell-associated human immunodeficiency virus (HIV) gag RNA; caRNA2, cell-associated HIV tat-rev RNA; cfRNA, cell-free HIV RNA; dd-PCR, digital droplet polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; iCARED, inducible cell-associated RNA expression in dilution; IUPM, infectious units per million; MOLT-4, T-lymphoblastic cell line; msHIV, multiply spliced HIV; PBMCs, peripheral blood mononuclear cells; PHA, phytohemagglutinin; PMA, Phorbol 12-myristate 13-acetate; QVOA, quantitative viral outgrowth assay; QVOA M, QVOA by University of Pittsburgh; QVOA RNA, QVOA by University of California, San Diego, with HIV RNA readout; QVOA S, QVOA by Johns Hopkins University; QVOA Simoa, QVOA by Southern Research using Simoa readout; QVOA SR, QVOA by Southern Research; rCD4+, resting CD4+; RT-PCR, reverse-transcription polymerase chain reaction: TILDA, tat/rev-induced limiting dilution assay; usQVOA, QVOA with ultrasensitive detection HIV-1 outgrowth in culture supernatant; WB, whole blood.