Table 2. Characteristics of assays.

Assay	Assay characteristics	Sample size	Assay input	Primersa	Unit of comparison	Limit of detectionb (LOD)	Dynamic rangec (logs)	Normalization method	Explanation for negative values	Ref.
Viral outgrowth	95% CI for individual determinations = ±0.7 logs based on Poisson statistics and 2 replicates/cell number; coefficient of variation = 0.95d	120–180 ml blood	resting CD4+ T cells	none	infectious units per 106 (IUPM) resting CD4+ T cells	0.02	2.7	none	frequency<LOD	5,6,9, 10,33
Droplet digital PCR for HIV-1 DNA	coefficient of variation depends on template number (Strain et al., submitted); accuracy superior to kinetic PCR (5 fold at low copy numbers)	variable, 5×106 cells in this study	resting CD4+ T cells or PBMC	primers: 2539→2562, 2659→2634. probe: 2589→2604	copies per 106 cells	2	3.2	normalized using RNAse P to quantify host genomic DNA	frequency<LOD; primer mismatch	53
Alu PCR for integrated HIV-1 DNA	detects individual integrated HIV-1 genomes with standard curve to correct for sites too far from an Alu sequence to be detected; coefficient of variation = 0.20	variable, 5×106 cells in this study	resting CD4+ T cells or PBMC	primers, outer: Alu primer, 800→782; inner: 525→542, 619-599. probe: 559→596	copies per 106 cells	3	3.0	normalized by [DNA] assuming 1 µg = 150,000 cells	all assays above LOD in this study	39, 40, 55, 80
qPCR for HIV-1 DNA rectal biopsies	coefficient of variation for 10 copy standard = 0.18	up to 30 3 mm biopsies	cells from biopsy	primers: 522→543, 640→626. probe: 581→559.	HIV-1 genomes per 106 CD4+ T cells	0.05	5.3	normalized by [DNA] and %CD4+ T cells (assuming 1 ug = 160,000 cells and all virus in CD4+ T cells)	all assays above LOD in this study	44
Droplet digital PCR for 2-LTR circles	coefficient of variation depends on template number (Strain et al., submitted); accuracy superior to kinetic PCR (>10 fold at low copy numbers)	variable, 5×106 cells in this study	resting CD4+ T cells or PBMC	primers: 9588→9607, 48→28. probe: 556→530	copies per 106 cells	2	1.1	normalized using RNAse P to quantify host genomic DNA	frequency<LOD; primer mismatch	53
Single copy assay for residual viremia	sensitivity superior to approved clinical assay; internal control for virus recovery	8 ml plasma	plasma	primers: 1299→1323	copies/ml of plasma	0.2	1.1	none	viral RNA<LOD; primer mismatch	46,47, 67

^aHXB2 coordinates.

^bBased in standard sample size. Except for residual viremia, LOD is expressed as infectious units or copies per 10⁶ cells. For residual viremia, the LOD is 0.2 copies/ml of plasma.

^cDynamic range is reported here as the difference in log units between the highest value measured in these study patients and the limit of detection of the relevant assay.

^dBased on repeat measurements in the same patient as reported in reference 10 and assuming no decay in the reservoir.