Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis.

Bovine tuberculosis (bTB) is a common zoonotic disease, caused by Mycobacterium bovis (M. bovis), responsible for significant economic losses worldwide. Its diagnosis is based on the detection of cell mediated immunity under the exposure to protein purified derivative tuberculin (PPD), a complex and poorly characterized reagent. The cross-reactivity to non-tuberculous mycobacterium species (false-positive results) has been crucial to develop a more proper antigen. In the present study, we selected six M. bovis Open Reading Frames (Mb1992, Mb2031c, Mb2319, Mb2843c, Mb2845c and Mb3212c) by in-silico analysis and evaluated them in experimental and natural infection; none of these antigens had been previously assessed as diagnostic antigens for bTB. The reactivity performance was tested in animals with both positive and negative Tuberculin Skin Test (TST) results as well as in cattle infected with Mycobacterium avium subesp. paratuberculosis (MAP). The six recombinant antigens individually induced an IFN-γ response, with overall responder frequency ranging from 18.3 to 31%. Mb2845c was the most valuable antigen with the potential to discriminate TST-positive cattle from either TST-negative or MAP infected animals. Mb2845c showed similar performance to that observed with ESAT-6 and PPD-B among TST and MTC specific-PCR positive animals, although this result needs to be proven in further studies with a higher sample size. Our data confirm the feacibility to implement bioinformatic screening tools and suggest Mb2845c as a potential diagnostic antigen to be tested in protein cocktails to evaluate their contribution to bTB diagnosis.

Serodiagnosis of tuberculosis: specific detection of free and complex-dissociated antibodies anti-mycobacterium tuberculosis recombinant antigens.

The diagnostic test characteristics of detecting free and complex-dissociated IgG to three recombinant antigens of Mycobacterium tuberculosis (38-kDa, Ag16 and Ag85B), singly and in combination, were evaluated in sera from 161 tuberculous patients [smear-positive pulmonary TB (50), smear-negative pulmonary TB (pTBsm-) (60) and extrapulmonary TB (51)) and 214 control patients (mycobacteriosis (14), mycoses(14), leprosy(4), other underlying diseases (82) and healthy people (100)]. The individual antigens ranged from 25% to 42% in sensitivity and from 93% to 96% in specificity, while considering free IgG response. Addition of complex-dissociated antibodies against each individual antigen improved the sensitivity up to 55%. The number and levels of specific antibodies varied greatly from individual to individual. Combination of individual results for free and complex-dissociated IgG to 38-kDa, Ag16 and Ag85B offered 76% sensitivity and 83% specificity. When the three antigens were placed in the same well, the sensitivity was lower than that expected on the basis of single antigen (63%) but with a good specificity (95%), even in the group of mycobacteriosis or mycoses. The highest contribution of complex-dissociated IgG results to free IgG results was seen for the diagnosis of pTBsm- patients. In conclusion, although neither single recombinant antigen was reactive with most sera from TB patients even after the measurement of both free and complex-dissociated antibodies, the use of multi-antigen cocktails improved the diagnostic utility of the ELISA assay, allowing the identification of almost 70% of pTBsm-, with a high level of specificity; the use of additional, well selected antigens should lead to the detection of almost all patients with TB.

Serodiagnosis of tuberculosis: specific detection of free and complex-dissociated antibodies anti-mycobacterium tuberculosis recombinant antigens

The diagnostic test characteristics of detecting free and complex-dissociated IgG to three recombinant antigens of Mycobacterium tuberculosis (38-kDa, Ag16 and Ag85B), singly and in combination, were evaluated in sera from 161 tuberculous patients [smear-positive pulmonary TB (50), smear-negative pulmonary TB (pTBsm-) (60) and extrapulmonary TB (51)) and 214 control patients (mycobacteriosis (14), mycoses(14), leprosy(4), other underlying diseases (82) and healthy people (100)]. The individual antigens ranged from 25 percent to 42 percent in sensitivity and from 93 percent to 96 percent in specificity, while considering free IgG response. Addition of complex-dissociated antibodies against each individual antigen improved the sensitivity up to 55 percent. The number and levels of specific antibodies varied greatly from individual to individual. Combination of individual results for free and complex-dissociated IgG to 38-kDa, Ag16 and Ag85B offered 76 percent sensitivity and 83 percent specificity. When the three antigens were placed in the same well, the sensitivity was lower than that expected on the basis of single antigen (63 percent) but with a good specificity (95 percent), even in the group of mycobacteriosis or mycoses. The highest contribution of complex-dissociated IgG results to free IgG results was seen for the diagnosis of pTBsm- patients. In conclusion, although neither single recombinant antigen was reactive with most sera from TB patients even after the measurement of both free and complex-dissociated antibodies, the use of multi-antigen cocktails improved the diagnostic utility of the ELISA assay, allowing the identification of almost 70 percent of pTBsm-, with a high level of specificity; the use of additional, well selected antigens should lead to the detection of almost all patients with TB.