The In-vitro Interferon-gamma Release Assay For The Diagnosis Of Latent Tubercular Infections And Tuberculosis

Objective: To investigate the in-vitro interferon-gamma(IFN- Y )release assay based on different Mycobacterium tuberculosis(M.tb) antigens in the diagnosis of tuberculosis and of latent tubercular infections, and to compare it with the tuberculin skin test(TST). To compare the assay based on the peripheral blood mononuclear cells (PBMC) with that based on the whole blood. Methods: All subjects were divided into three groups: group 1, patients with tuberculosis (n=62); group 2, healthy (n=27); group 3, patients with cancer (n=29). The PBMC were co-cultured for 5 days with different antigens: purified protein derivitives (PPD) of tuberculin, early secretary antigenic target 6kDa protein (ESAT-6), 38kDa antigen. The protein levels of IFN- y were detected by ELISA. Total RNA was extracted from the PBMC after 5 days culture. The expression of the IFN-r mRNA was detected by RT-PCR. The two methods based on the PBMC or based on the whole blood were done at the same time in 10 subjects. Results: For healthy control, the TST is positive related to the history of BCG vaccination and the contact degree with sputum-positive TB patients (P=0.047, P=0.041 respectively). But the ESAT-6 based IFN-r release assay is only significantly related to the contact degree with sputum-positive TB patients, and there is no significant positive relation with the history of BCG vaccination. The in-vitro IFN- Y release level is highest in TB patients arid is lowest in cancer patients. The sensitivity and the specificity of the IFN- y release assay based on 38kDa are 64.9% and 89.3% for the diagnosis of tuberculosis. The expression of IFN-r mRNA was detected only in part of subjects using RT-PCR, and it did not correlated with the production of the IFN-r protein. The two methods based on PBMC and whole blood correlate well (r=0.835, P<0.001)Conclusions: The IFN-r release assay based on ESAT-6 may be better than TST when diagnosis of the latency of M.tb. The IFN-r release assay based on 38kDa may be a promising reagent when diagnosis to the tuberculosis. Expression of the mRNA for IFN-r does not parallel the production of the IFN-r protein. The assay based on the whole blood may be replace the assay based on PBMC.