Before or within 1 week of initiating therapy, we obtained a sample of 30 mL of heparinized blood and used 7.5 × 106 peripheral blood mononuclear cells for ELISpot assays. We seeded precoated interferon-γ ELISpot plates (Mabtech AB, Stockholm, Sweden) with 2.5 × 105 peripheral blood mononuclear cells per well (17). Duplicate wells contained no antigen (negative control) or phytohemagglutinin (positive control) (ICN Biomedical, Aurora, Ohio) at 5 µg/mL. A further 13 pairs of duplicate wells each contained 1 of 13 peptide pools, which incorporated 5 to 7 overlapping 15-mer peptides. The assay on which T-SPOT.TB is based, defined as standard ELISpot in this study, comprises 35 such peptides assembled into 6 pools and spanning the length of early secretory antigenic target-6 and culture filtrate protein-10. Forty-five peptides from selected regions of Rv3873, Rv3878, and Rv3879c (Research Genetics, Huntsville, Alabama) were assembled into 7 pools (21). The ELISpotPLUS assay was defined as the 35 early secretory antigenic target-6 and culture filtrate protein-10 peptides, with 17 peptides from Rv3879c, assembled into 3 additional pools. The final concentration of each peptide was 10 µg/mL. After overnight incubation at 37 °C in 5% carbon dioxide, the plates were developed with preconjugated detector antibody (Mabtech AB) followed by chromogenic substrate (Moss, Pasadena, Maryland) (17). Spot-forming cells were counted by using an automated ELISpot reader (AID-GmbH, Strassberg, Germany). We predefined settings for the intensity and size of a counted spot and used the same settings throughout. We transferred mean readings from duplicate wells to a spreadsheet electronically and scored them as positive or negative by using a customized software program, ELISTAT (AID-GmbH). We scored responses as positive if test wells contained a mean of at least 5 spot-forming cells more than the mean of the negative control wells and were at least twice the mean of the negative control wells. This predefined cutoff point is the standard threshold used with our assay format in 9 studies including a total of 2506 participants (15–17, 25–30). Operators performing and reading the assays were blinded to tuberculin skin test results and personal identifiers. The ELISpot results were checked and countersigned before data entry by a scientist who did not perform the assays.