Confirmation of Mycobacterium tuberculosis infection by flow cytometry after ex vivo incubation of peripheral blood T cells with an ESAT‐6‐derived peptide pool

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Abstract
Background The presence of a T‐cell response to the early secretory antigenic target‐6 (ESAT‐6) indicates previous infection with or exposure to Mycobacterium tuberculosis. Measuring this response is useful for identifying individuals infected with M. tuberculosis. It was also reported that the frequencies of ESAT‐6‐specific T cells correlate with disease state. Established procedures measure secreted T‐cell cytokines following whole blood stimulation with recombinant ESAT‐6 protein or use Elispot as a read‐out. Methods A single ESAT‐6‐ spanning pool of overlapping peptides (15 amino acids length with 11 overlaps) was used for overnight stimulation of peripheral blood mononuclear cells (PBMCs) from 15 patients infected with Mycobacterium tuberculosis and 11 healthy controls. T‐cell responses were rated positive if interferon‐γ (IFN‐γ)‐producing T cells were identified above background level, using 4‐color cytokine flow cytometry. Results Thirteen of 15 (87%) patients, but none of the healthy controls, had a positive CD4 T‐cell response to the ESAT‐6 spanning peptide pool. The frequencies of IFN‐γ‐producing cells varied between 1 and 167 per 10,000 CD4 T cells. The test performed as well as the tests described in the literature. Conclusions Cytokine flow cytometry following PBMC stimulation with an ESAT‐6 spanning peptide pool is a useful laboratory test for ESAT‐6‐specific T cells combining precise counting and multi‐parameter phenotyping.

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