Screening Of Antigen And Biomarker Of New Type Of Tuberculosis And Its Application In Diagnosis Of Tuberculosis

A major breakthrough in the diagnosis of infection with Mycobacterium tuberculosis has been the development of in vitro assays that measure the production of interferon (IFN)-γ in response to stimulation with M. tuberculosis-specific antigens (IFN-y release assay (IGRA) tests). The IGRA tests have been extensively examined and current evidence suggests that both tests have a low false-positive rate. Compared with TST, the IGRAs have a low false-positive rate in BCG-vaccinated individuals and are better correlated with risk factors for infection with M. tuberculosis. However, there is a concern that the diagnostic sensitivity is still suboptimal and the tests perform with more indeterminate results, particularly in patients with severe TB or immunosuppressive status. The detection rate of IGRA could potentially be enhanced with the addition of further M. tuberculosis-specific antigens, by improving the incubation step or by measuring alternative or additional biomarkers for IFN-y.Antigens encoded in the region of differentiation (RD) of Mycobacterium tuberculosis constitute a potential source of specific antigens for immunodiagnosis and vaccine development. In the present study, we firstly evaluated the diagnostic potential of specific T-cell epitopes selected from two recently identified immunodominant antigens Rv1985c and Rv3425from RD2and RD11based on eptiopes mapping in tuberculosis (TB) patients and M.bovis BCG-vaccinated healthy individuals. Using a whole blood IFN-y release assay, we analyzed the T-cell epitopes on the sequence of the whole antigens with overlapping peptides. The epitopes on Rv1985c and Rv3425was found to be distributed throughout the molecule in TB patients. Though both Rv1985c and Rv3425had peptides that could induce IFN-gamma release in healthy controls, but the cross-reactive epitopes focused on only a few peptides compared to TB patients.Then the specific epitopes that can discriminate TB infection and BCG-vaccination were carefully selected. The diagnostic potential of the peptide cocktails were evaluated and combined with QFT. The most promising peptide pools from Rv1985c showed a sensitivity of53.9%and specificity of95.5%, which was comparable to the peptide pools from ESAT-6. Upon combing the novel specific peptides with QFT, the sensitivity increased from86.4%to96.2%without affecting the specificity. These results identify specific peptides from Rv1985c and Rv3425and confirm the diagnostic value for TB infection in BCG-vaccinated population.Next, we sought to identify biomarker responses to tuberculosis specific antigens which could improve the diagnosis of tuberculosis infection and allow the differentiation of active and latent infections. We recruited28subjects with active tuberculosis,34with latent tuberculosis and26healthy controls and used the Bio-plex Array to evaluate27biomarkers in the supernatant of whole blood samples following overnight stimulation using the Quantiferon Gold In-Tube kit. IFN-γ, IP-10, IL-2, MCP-1, IL-1RA and IL-15response was significantly higher in TB-infected (active or latent) subjects compared to the uninfected group (p<0.0001). Among the27parameters, baseline expression levels of VEGF, IL-12, IP-10, IFN-γ, MCP-1and TB antigen-stimulated IFN-y and PDGF were found to be significantly higher in the active tuberculosis group compared to the latent tuberculosis group. When combined, VEGF, IL-12and PDGF accurately identified79.4%of active and92.9%of latent infections. These results suggest that active TB may be accurately differentiated from LTBI utilizing adaptations of the commercial QFT test that includes measurement of VEGF, IL-12, IP-10, PDGF or IFN-γ in supernatants from QFT assays. This approach holds promise for development as a rapid diagnostic test for active TB.Since IP-10and IL-2was the most promising biomarker that could distinguish TB infection, then we evaluated the diagnostic performance of interferon-gamma (IFN-γ) inducible protein10(IP-10) and IL-2for distinguishing ATB and LTBI from BCG-vaccinated population.66active TB patients,73household contacts and77healthy controls were recruited. P-10and IL-2were determined by enzyme-linked immunosorbent assay from whole blood stimulated with Mtb-specific antigens. The positive rates of QFT, IP-10and IL-2test were86.4%,89.4%and86.4%for the ATB group;38.4%,57.5%and39.7%in HHC group and17.1%,18.4%and14.5%in HC group, respectively. By combining QFT, IP-10and IL-2tests, the detection rate increased to95.5%in ATB group and60.3%in HHC group without a significant increase in positive responders in HC group and the indeterminate rate decrease to0.4%(1/215). The IP-10and IL-2responses were strongly associated with long Mtb exposure time and the degree of smear-positivity of the index cases. The IL-2/IFN-γ ratio stimulated by TB antigens could discriminate LTBI from ATB with sensitivity of77.2%and specificity of87.2%. The results demonstrated that IP-10and IL-2stimulated by Mtb specific antigens may be used for detecting both ATB and LTBI. Combining IP-10and IL-2with QFT could increase the detection accuracy of active TB and reduce the indeterminate results compared with the current IGRAs based on QFT alone.In conclusion, in our study we identify specific peptides from antigen Rv1985c and Rv3425which could be used for detect TB infection in BCG-vaccinated population. The combination of VEGF, IL-12and PDGF responses was accurate in distinguishing persons with active tuberculosis from persons with latent tuberculosis. IP-10and IL-2responses to Mycobacterium tuberculosis-specific antigens could be used to diagnose TB infection. Combining IP-10, IL-2and IFN-γmay be a simple approach to increase the detection rate of the Mtb-specific in vitro tests.