| Tuberculosis is a global problem of public health. Accurate and rapid detection of TB is essential for TB control. In developing countries, tuberculin skin test (TST), the only available immune-based diagnostic test for clinical use, is not specific enough due to the cross reaction between the antigens from M. tuberculosis and other mycobacteria. It underscores the need for identifying M. tuberculosis specific antigens and developing rapid, specific as well as cost-effective diagnostic tests that detect both active TB and LTBI (latent TB infection) from BCG-vaccinated individuals.Antigens encoded in the region of differentiation (RD) of M. tuberculosis constitute a potential source of specific antigens for immuno-diagnosis and vaccine development. In the present study, seven M. tuberculosis proteins from RD2 and RD11, Rv1978, NrdF1, MPT64, CFP21, Rv1985c, PPE57 and PPE59, were successfully cloned, expressed in Escherichia coli, purified and investigated for their potentially diagnostic value for TB infection among the BCG-vaccinated individuals. T-cell response to the seven proteins was evaluated by IFN-γELISPOT in 58 tuberculosis patients, 21 LTBI and 32 BCG-vaccinated controls in comparison with the commercial T-SPOT. TB kit. Humoral response to Rv1978, NrdF1, CFP21 and Rv1985c was evaluated by ELISA in 117 TB patients, 45 LTBI and 67 BCG-vaccinated controls, including all those who had T-cell assay. in comparison with a commercial PATHOZYME-MYCO IgG kit.Data showed that T-cell IFN-γreleasing ELISPOT of all seven purified recombinant proteins could be used to distinguish TB patients and LTBI from BCG-vaccinated healthy controls. ELISPOT of Rv1985c, Rv1978, NrdF1, MPT64, CFP21, PPE57 and PPE59 achieved sensitivities of 63%, 59%, 60%, 82%, 48%, 59% and 47% respectively in the detection of active TB and specificities of 97%, 94%, 90%, 76%, 93%, 100% and 93% respectively in BCG-vaccinated healthy controls. Surprisingly, the response to Rv1978 in LTBI group was significantly higher than that in TB group, suggesting Rv1978 may be involved in TB latent infection. Although PPE57, NrdF1 and Rv1985c had lower sensitivity in active TB detection than ESAT-6 and only equivalent to CFP-10, combination of PPE57 or NrdF1 or Rv1985c with early secreted antigen target 6 (ESAT-6) or 10-kDa culture filtrate protein (CFP-10) in the IFN-γreleasing ESLIPOT assay could or had the potential to increase the sensitivities in detecting active TB, for ESAT-6 from 82.1% to 85.7% or 92.9% or 92.9%, respectively, and for CFP-10 from 67.9% to 78.6% or 83.9% or 83.9%, respectively.In serodiagnosis, data demonstrated that only Rv1985c was specifically recognized by both TB and LTBI groups compared with healthy controls. Rv1978 was only specifically recognized by TB group whereas NrdfF1 together with CFP21 had no difference at all in recognition among the groups. Totally, Rv1985c IgG-ELISA achieved 52% and 62% in sensitivity and both 97% in specificity when detecting latent and active tuberculosis respectively. The sensitivity in detecting active TB was significantly higher than that of PATHOZYME-MYCO kit (34%).In conclusion, Rv1985c is identified as a novel antigen which can be used to immunologically diagnose TB infection individuals among BCG-vaccinated population. NrdF1 and PPE57 are also discovered as potential T-cell antigens in TB diagnosis for their high sensitivities, specificities and promising antigenic combination potential.
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