| Background:Tuberculosis(TB)is caused by Mycobacterium tuberculosis(MTB)infection.It is estimated that about a quarter of people in the world are infected with Mycobacterium tuberculosis,including active tuberculosis(ATB)and latent tuberculosis infection(LTBI).Nearly 20%of the general populations in China have LTBI,and these populations have a 5%-10%chance of developing ATB in their lives.Therefore,fast and accurate identification of ATB and LTBI is essential for accurate anti TB treatment,and is of great importance in reducing the incidence rate of tuberculosis.At present,the diagnosis of tuberculosis is difficult.It is of great clinical significance to differentiate ATB and LTBI by adding new antigens or detecting a variety of cytokines.Objective:To evaluate the accuracy of MTB antigen-specific cellular immune response in differentiating ATB and LTBI by detecting the frequency and proportion of specific T cells secreted by MTB latent associated antigen rv1733c,rv1733c SLP and MTB specific antigen ESAT-6 and CFP-10 by Fluorospot.Methods:This study is divided into two parts,both of which are case-control studies.In the first part,patients with ATB confirmed by pathogens hospitalized in Peking Union Medical College Hospital and Beijing chest hospital from January to December 2017 were included as case group,while patients with latent TB infection in the same period were included as control group.FluoroSpot was used to detect the frequency and ratio of T cells that secret IFN-?/IL-2 after stimulation by MTB latent associated antigen to assess the accuracy in the differential diagnosis of ATB and LTBI.In the second part,patients with ATB etiologically and clinically diagnosed in Peking Union Medical College Hospital and Beijing Chest Hospital from April 2020 to April 2021 were included as the case group,and patients with LTBI during the same period as well as the uninfected people served as control groups.FluoroSpot was used to detect the frequency and ratio of T cells that secret IFN-?/IL-2/TNF-? after stimulation by MTB specific antigen ESAT-6 and CFP-10 to assess the accuracy in the differential diagnosis of ATB and LTBI.Results:In the first part,57 ATB cases and 36 LTBI cases were included.Following stimulation with Rv1733c and Rv1733c SLP,it was found that the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP had the largest area under the ROC curve,and the area under the ROC curve was 0.766(95%CI,0.662-0.870).With a cutoff value of 1(SFCs/2.5×105 PBMCs)for frequency,sensitivity and specificity of distinguishing between ATB and LTBI were 72.2%(95CI 54.8%-85.8%)and 73.7%(95CI 60.3%-84.5%).ESAT-6&CFP-10-FluoroSpot detected the frequency and proportion of single IFN-?-secreting T cells,the sensitivity and specificity of differential diagnosis of ATB and LTBI were 82.5%(95CI 70.1%-91.3%)and 66.7%(95CI 49.0%-81.4%),respectively.Combining with the frequency of single IL-2-secreting T cells stimulated by Rv1733c SLP on the basis of ESAT-6&CFP-10-FluoroSpot,the sensitivity and specificity were increased to 84.2%(95CI 72.1%-92.5%)and 83.3%(95CI 67.2%-93.4%)respectively.In the second part,50 cases of ATB,87 cases of LTBI,and 71 cases of uninfected patients were enrolled.FluoroSpot(IFN-?/IL-2/TNF-?)and T-SPOT.TB detected a significant correlation(p<0.0001)between the frequency of IFN-? secreting T cells after stimulation with MTB-specific antigens(ESAT-6/CFP-10).The ROC curve was drawn by the frequency of IFN-?+or IL-2+(IFN-?+orlL-2+)secreting T cells after stimulation with ESAT-6&CFP-10 antigen.The maximum AUROC was 0.980(95CI 0.963-1.003),and when the diagnostic threshold was 7(SFCs/2.5×105 PBMCs),the sensitivity and specificity for differential diagnosis of ATB and uninfected patients were 100%and 87.3%.Among the 50 ATB patients,the positive rate of T-SPOT.TB was 82%,and Fluorospot could be increased to 90%.Among the 25 ATB patients diagnosed with the pathogen,the positive rate of T-SPOT.TB was 92%,and Fluorospot could be increased to 100%.Among 25 ATB patients diagnosed with clinical criteria,the positive rate of T-SPOT.TB was 72%,and Fluorospot could be increased to 80%.The sensitivity and specificity of T-SPOT.TB were 48.8%and 85.7%.The frequency and proportion of IFN-?+/IL-2-/TNF-?',IFN-?'/IL-2+/TNF-?',IFN-?'/IL-2'/TNF-?+,IFN-?+/IL-2+/TNF-?',IFN-?'/IL-2-/TNF-?+ and IFN-?'/IL-2+/TNF-?+ secreting specific T cells stimulated by ESAT-6&CFP-10 antigen were used as independent variables to conduct binary logistic regression analysis and draw ROC curve.When the AUROC was 0.858(95 CI 0.787-0.929)and the diagnostic cut-off value was 0.3,the sensitivity and specificity of differential diagnosis of ATB and LTBI were 76.7%and 84.3%.Conclusion:Rv1733c SLP,in combination with ESAT-6&CFP-10,has the potential to be used as a candidate antigen for differentiate between ATB and LTBI.The application of Fluorospot(IFN-?/IL-2/TNF-?)to detect the MTB specific antigen multi cytokine immune response has better accuracy in the differential diagnosis of ATB and LTBI than T-SPOT.TB. |
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