The Comparative Study Of Significance For The Diagnosis Of Tuberculous Pleurisy By Enzyme-linked Immunospot Assay

Background: In the country of high mobidity of tuberculosis, tuberculous pleurisy is the most common reason of pleural effusion. The gold standard of diagnosis of tuberculous pleurisy is the detect of Mycobacterium tuberculosis.The positive rate is very lower. The diagnostic technique of pathogeny and molecular biology,e.g MODS,PCR et al. have been developed fast, but they are stricted by sensitivity and specificity. Both of pleural biopsy and pleural endoscopy are invasive methods and can't used as usual. Recent years, 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. However, in the diagnosis of tuberculosis mainly concentrated in the application of Mycobacterium tuberculosis region of difference(RD1)-encoded early secreted antigen target (early secreting antigen target 6KD, ESAT-6) and culture filtrate protein (culture filtrate protein 10KD, CFP10) and its peptides.Only focused on peripheral blood mononuclear cells of active tuberculosis and latent tuberculosis infection,had high sensitivity and specificity. Compartmentalization reaction of tubercolous pleuritis suggested that the detection of cytokines of pleural effusion mononuclear cells (PEMC) is more significant to the diagnosis than peripheral blood mononuclear cells (PBMC). This experiment was to detect the pleural effusion-derived cytokine (e.g.IFN-γ)-secreting effector T lymphocytes cultured with the protein encoded by RD1 of Mycobacterium tuberculosis through ELISPOT assays and investigated the method in the diagnosis of tuberculous pleurisy.Methods: 98 subjects were selected in our study: 55 patients with tuberculous pleural effusion; 43 patients with malignancy pleural effusion. Separated the mononuclear cells from pleural effusion collected from the patients, then frozened. Resuscitated the Cells and cultured with ESAT-6 and Purified protein of ESAT-6/CFP-10 fusion protein, CFP - 10, Rv3873 and Rv3879c. Then detected the interferon-γreleasing T lymphocytes (also Spot Forming Cells, SFC) by ELISPOT.Results: The number of SFC cultured with ESAT-6, ESAT-6/CFP-10 fusion protein, CFP-10 , Rv3873 and Rv3879c showed much higher level in tuberculous pleurisy effusion group than malignant pleural effusion group. In tuberculous pleurisy, sensitivity stimulated by ESAT-6, ESAT-6/CFP-10 fusion protein and CFP-10 showed no significant difference and was of relatively high : 90.9%, 98.2% and 89.1%. but Rv3873 and Rv3879c showed no significant difference: 78.2% and72.7% and was lower than them. In malignancy pleural effusion, specificity stimulated by ESAT-6, ESAT-6/CFP-10 fusion protein, CFP-10,Rv3873 and Rv3879c showed no significant difference overall : 95.3%, 83.7% , 86.1% , 76.7% and 95.3% . but Rv3873 was lower than them.but the five Antigens showed no significant difference by Paired comparison. ESAT-6 ELISPOT had a higher application value for the diagnosis of tuberculous pleurisy than the other selected antigens.Conclusions: The detection of the pleural effusion-derived cytokine (e.g.IFN-γ)-secreting effector T lymphocytes stimulated by the high tuberculosis-specific antigens encoded by RD1 of Mycobacterium tuberculosis through ELISPOT assays had relatively high sensitivity and specificity in the diagnosis of tuberculous pleurisy. This method can help to identify tuberculous or malignant pleural effusion.