Abstract
Mycobacterium tuberculosis (M. tuberculosis) regions of difference (RD) encode proteins which are potentially useful as diagnostic reagents for tuberculosis (TB). In this study, 75 genes from M. tuberculosis RD1-RD16 were successfully cloned from which 68 proteins were expressed and purified. Three serum pools from patients with pulmonary TB (PTB), extra-pulmonary tuberculosis (EPTB) and healthy controls (HC) were used to preliminarily screen individual RD proteins. The OD630 ratio of the PTB or EPTB to the HC group ≥ 2-fold was positive. As a result, 29 proteins were obtained. The serological response to the identified antigens was further verified using 58 PTB samples with 38 sera from smear-positive PTB (PTB-SP) patients and 20 sera from smear-negative PTB (PTB-SN) patients, 16 EPTB samples, 42 latent M. tuberculosis infection samples and 40 HCs by indirect ELISA. With respect to the PTB diagnosis, receiver operating characteristic analysis showed that Rv0222 [area under the curve (AUC), 0.8129; 95% confidence interval (CI), 0.7280-0.8979] and Rv3403c (AUC, 0.8537; 95% CI, 0.7779-0.9294) performed better than ESAT6/CFP10 (AUC, 0.7435; 95% CI, 0.6465-0.8406). Rv0222 and Rv3403c demonstrated the highest diagnostic ability in the PTB-SP group (sensitivity, 86.8%; specificity, 80%), while Rv3403c demonstrated the highest diagnostic ability in the PTB-SN group (sensitivity, 70%; specificity, 80%). With respect to the EPTB diagnosis, Rv0222 exhibited the highest diagnostic value (AUC, 0.7523; sensitivity, 68.8%; specificity, 87.5%). In addition, the combination of Rv0222 and Rv3403c improved the test for PTB-SN. These results indicate that Rv0222 and Rv3403c would be potential diagnostic biomarkers for active TB serodiagnosis. Mouse experiments demonstrated that Rv0222 and Rv3403c elicited specific cellular and humoral responses which were characterized by production of IFN-γ, IgG1, and IgG2a, but a higher level of IgG1 than IgG2a.