Detection by flow cytometry of ESAT-6- and PPD-specific circulating CD4+T lymphocytes as a diagnostic tool for tuberculosis

Background: The identification of mycobacteria represents the gold standard in the diagnosis of tuberculosis ( TB), but it is not applicable in all patients, and immunological tests, such as the tuberculin skin test (TST), are not specific and sensitive enough. Methods: By flow cytometry, we measured the CD4T-cell response to purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6) protein using the intracellular cytokine staining technique on whole blood samples obtained from active TB (n = 16), latent TB (n = 17), Bacille Calmette-Guerin (BCG)-vaccinated (n = 11) and healthy ( n = 10) donors. All the patients were also tested with conventional TST. Results: The identification by flow cytometry of PPD-specific T lymphocytes upon antigen stimulation of whole blood enables the discrimination of active TB, latent TB and BCG-vaccinated subjects from healthy individuals, whereas the ESAT-6 response discriminated active TB from healthy and BCG-vaccinated individuals. Moreover, this test enables identification of active TB patients who were negative on TST and to distinguish between TB and non- typical mycobacteria TB infections. Conclusions: The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than TST, and could represent a further tool in the diagnosis of TB infection.