The Significance Of Enzyme-linked Immunospot Assay In The Diagnosis Of Active Tuberculosis And Latent Tuberculosis Infection

Background: there is no reliable measures of detecting active tuberculosis except for smear positive tuberculosis. Tuberculin skin test (TST) is currently the routine method to detect the cellular immune response to M. tuberculosis infection,but the antigen- purified protein derivative(PPD) is a complex of more than 200 tuberculous components and has poor accuracy, therefore we can not identify the M. tuberculosis infection by this method. ESAT-6 and CFP-10 are tuberculosis-specific antigens encoded by RD1 of M. tuberculosis complex as well as absent from BCG and most environmental mycobacteria, and they can stimulate PBMC of active tuberculosis and latent tuberculosis infection to secrete high level IFN-γ. Enzyme-linked immunospot assay (ELISPOT) is a kind of sensitive and reliable method which enumerated peripheral blood-derived cytokine (e.g.IFN-γ)-secreting effector T lymphocytes responding to antigen epitopes and it has been extensively used in many medical research fields including tuberculosis, tumor, HIV infection and so on. Lalvani A diagnosed active tuberculosis and latent tuberculosis by this method at first time in 2001,identified the high sensitivity and specificity of ELISPOT. This experiment was to investigate the in-vitro antigen specific ELISPOT based on different M. tuberculosis antigens in the diagnosis of active tuberculosis and latent tuberculosis infection.Methods: 286 participants were recruited in our study: 132 patients with active tuberculosis(55 patients with smear positive tuberculosis, 50 patients with smear negative tuberculosis, 27 patients with tuberculous pleurisy),;90 healthy control participants (25 TST negative healthy control, 19 TST positive healthy control, 46 TST negative control patients with non-tuberculosis pulmonary diseases); 9 cases with history of tuberculosis treatments; 55 healthy cases with tuberculosis exposure history(divided into different groups according to tuberculosis-exposed degree). The PBMC of all participants were extracted and co-cultured with the following antigens: purified protein derivative (PPD), early secreting antigen target 6KD,(ESAT-6)and ESAT-6/CFP-10 fusion protein. And the interferon-γreleasing T lymphocytes (also Spot Forming Cells, SFC) were enumerated by ELISPOT. TST was also administered to healthy cases with tuberculosis exposure history.Results: SFC count after stimulation withPPD, ESAT-6/CFP-10 fusion protein and ESAT-6 showed much higher level in active tuberculosis than that in control cases. ESAT-6/CFP-10 fusion protein ELISPOT was of relatively high sensitivity (90.3%) and specificity (84.4%) in the diagnosis of all active tuberculosis, PPD-ELISPOT was of poor specificity (68.9%) due to BCG vaccination. In active tuberculosis, SFC count and sensitivity stimulated by different antigens showed no significant difference among smear positive tuberculosis, smear negative tuberculosis and tuberculous pleurisy. SFC count detected by ESAT-6/CFP-10 fusion protein ELISPOT correlated with duration of therapy.In the healthy cases with tuberculosis exposure history, ESAT-6/CFP-10 fusion protein ELISPOT correlated significantly more closely with M. tuberculosis exposure, and didn't associated with BCG vaccination, while TST mainly correlated with BCG vaccination. SFC count after stimulation with ESAT-6/CFP-10 fusion protein and ESAT-6 showed much higher level in active tuberculosis than that in healthy cases with tuberculosis exposure history,and SFC count showed much higher level in healthy cases with tuberculosis exposure history than in healthy control.Conclusion:ESAT-6/CFP-10 fusion protein-ELISPOT is a more accurate approach for the diagnosis of active tuberculosis, offers some basis for the diagnosis of smear negative tuberculosis and tuberculous pleurisy, and acts as a marker of antituberculous chemotherapy. ESAT-6/CFP-10 fusion protein-ELISPOT can also be used for the identification of individuals who have latent tuberculosis infection, and successfully distinguished active tuberculosis and latent tuberculosis infection according to the different SFC count.