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Tuberculin skin test (TST) is used most widely for the detection of latent tuberculosis infection (LTBI), even though evidences suggest that it could be underreporting the prevalence of LTBI particularly in high disease-burden settings. We have explored whether  in vivo  (TST) and  in vitro  (cell-proliferative) T cell responses to PPD can serve as complementary measures. In addition, we also probed whether  in vitro  T cell response to cell-membrane antigens (Mem) of  Mycobacterium tuberculosis  (MTB) can serve as a biomarker for LTBI. Study subjects comprised 43 healthcare workers (HCWs), and 9 smear-positive TB patients served as ‘disease control’. To measure proliferative T cell responses, 0.1 ml blood (diluted 1:10) was incubated (5 days) with test or control antigen. Cells were stained with fluorescent antibodies to T cell (CD3+/CD4+/CD8+) surface markers and, after fixation and permeabilization, to nuclear proliferation marker Ki67. Data was acquired on a flow cytometer. HCWs who had an intimate exposure to MTB showed significantly higher TST positivity (85%) than the rest (43%), notwithstanding their BCG vaccination status. The proliferative responses of CD4+ and CD8+ subsets of T cells were comparable. Sixty seven and 100% TST-negative HCWs, respectively, were positive for proliferative T cell response to PPD and MTBMem. Cumulative positivity (TST or  in vitro ) was 86% with PPD and 100% with MTBMem indicating complementarity of the two responses. As standalone  in vitro  assay, MTBMem provided a significantly higher positivity (95%) than PPD (67%). T cell responses of TB patients were ‘generally’ depressed, having implications for the development of immunological assays for ‘progressive’ LTBI. Altogether, these results demonstrate that  in vivo  and  in vitro  T cell responses to PPD are complementary and  in vitro  response to MTBMem can be developed as a highly sensitive biomarker for LTBI.

plos one

Synergy between tuberculin skin test and proliferative T cell responses to PPD or cell-membrane antigens of Mycobacterium tuberculosis for detection of latent TB infection in a high disease-burden setting