Hemmati et al. The structural states of the recombinant proteins were confirmed by CD spectroscopy. As shown in Figure 3, both ESAT-6 and ESAT-6/CFP-10 have high helical contents, whereas CFP-10 appears to have largely unstructured, random coil structure. ESAT-6/CFP-10 and commercial ESAT-6, showed positive reaction. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for recombinant proteins and PPD in ELISPOT assay were summarized in Table 2. Fig. 3: Far-UV circular dichroism spectra obtained from solution of CFP-10 (A), ESAT-6 (B), and ESAT- 6/CFP-10 (C). The samples from healthy individuals were ana- lyzed to calculate mean and standard deviation (SD) and then cutoff point for positive samples was set at mean + 2SD.6 The distribution of responses to ESAT- 6, CFP-10, ESAT-6/CFP-10, PPD and commercial ESAT-6 was shown in Figure 4. Results are mean values determined by independent observers, given as spots per million PBMCs. Numbers of IFN-γ SFC against ESAT-6, CFP-10 , ESAT-6/CFP-10 and commercial ESAT-6 were significantly higher in TB patients than in BCG-vaccinated healthy donors (Fig- ure 4). All patients participated in this study were previously vaccinated with BCG, nevertheless 25% of them showed negative skin test. All the patients in our IFN-γ ELISPOT analysis for ESAT-6, CFP-10 Discussion Late diagnosis and treatment of tuberculosis increases the risk of disease dissemination and decreases the survival of some subgroups of patients.16-18 Thus new advancements in techniques, which facilitate rapid diagnosis, are required to control TB successfully. Using ESAT-6 and CFP-10, two specific antigens encoded in the RD1 locus of the bacterial genome, which distinguish M. tuberculosis from other Myco- bacteria, has increased specificity and sensitivity of IFN-γ ELISPOT assay.14 This study reports the re- sults of the first evaluation of ex vivo INF-γ based assay (TSPOTTB) for the diagnosis of TB in adults by recombinant antigens of native M. tuberculosis C as the standard strain of Iran. The antigens chosen for the present study have been found to be strong targets for human B- and T-cell responses.19 Overlapping peptides of ESAT-6 and CFP-10 offer increased spec- ificity over the PPD skin test when they were used in ELISPOT assay for the diagnosis of M. tuberculo- sis.20,21 Arend et al. have shown that ESAT-6 and CFP-10 in combination with T-cell assays can pro- vide the level of specificity and sensitivity necessary to detect exposure to TB.22 According to our results from ELISPOT assay, these recombinant proteins could discriminate TB patients from healthy BCG- vaccinated individuals with p values <0.001. Accord- ing to values of sensitivity, specificity, PPV and NPV for all recombinant produced proteins, commercial ESAT-6 and PPD, and considering that 25% of TB patients were negative in their TST result, we suggest that ESAT-6 and ESAT-6/CFP-10 proteins could provide high level of confidence for application in Table 2: Values of sensitivity, specificity, PPV and NPV Antigens Sensitivity Recombinant ESAT-6 93 Recombinant CFP-10 Recombinant ESAT-6/CFP-10 Commercial ESAT-6 PPD 90 100 93 76 Specificity 100 96 100 100 85 PPV 100 96 100 100 90 NPV 100 100 100 100 80 560 WWW.ircmj.com Vol 13 August 2011