| Background:Tuberculosis(TB)is a chronic disease caused by Mycobacterium Tuberculosis(Mtb)infection potentially affecting various organs and tissues.According to World Health Organization(WHO)’s global tuberculosis report,only 55%of the pulmonary TB is diagnosed microbiologically in China.Discriminating Active tuberculosis(ATB)from non-ATB in febrile patients remains challenging when etiological evidence is absent.Immunological examination is a hot issue in the field of tuberculosis diagnosis,and it has great significance for differentiating ATB from LTBI by adding new antigens or detecting a variety of cytokines.Objectives1.To compare QuantiFERON-TB Gold Plus(QFT-Plus)and T-SPOT.TB for diagnosing ATB in febrile patients,investigate the influencing factors of positive results and verify the potential value of QFT-Plus in identification of ATB and non-active TB infection.2.To explore the differences of Interferon gamma(IFN-y),Tumor necrosis factor-α(TNF-α),Interferon-2(IL-2),Interferon-6(IL-6),Interferon-5(IL-5),Interferon-10(IL-10),Interferon-inducible protein-10(IP-10),Interleukin-1 receptor antagonist(IL-1Ra),chemokine(C-X-C)Ligand-1(CXCL-1)and Macrophage chemoattractant protein 1(MCP-1)in ATB,inactive tuberculosis(Inactive TB),latent tuberculosis infection(LTBI)and Healthy Control(HC)after QFT-Plus,Early secreting antigen target-6(ESAT-6),Culture filtrate protein-10(CFP-10),Rv2028c and synthetic long peptides derived from Rv1733c(Rv1733c SLP)antigen stimulation,and to screen antigens and cytokines with potential to discriminate MTB infection status.MethodsPart 1:Comparison of QFT-Plus and T-SPOT.TB in diagnosis of active tuberculosis in febrile patientsThe first part applied a case-control study design.Febrile patients diagnosed with ATB either clinically or microbiologically in Peking Union Medical College Hospital and Beijing Chest Hospital from April 2020 to July 2021 were enrolled into case group,while excluded from ATB during the same period were enrolled into the control group.QFT-Plus and T-SPOT.TB were tested simultaneously to evaluate the consistency of the results and compare the sensitivity,specificity,predictive values and likelihood ratios for diagnosing ATB.To evaluate the accuracy of QFT-Plus for differentiating ATB from non-active TB infection.Multivariable Logistic regression analysis was used to analyze the influencing factors of positive results.Part 2:Mycobacterium tuberculosis-specific multiple cytokine response in different tuberculosis infection status:an exploratory studyMicrobiologically confirmed ATB patients in Peking Union Medical College Hospital and Beijing Chest Hospital from February to April,2022 were selected,while LTBI,Inactive TB and HC were recruited in health persons during the same period.Whole blood was stimulated with QFT-Plus TB1,TB2 antigen,ESAT-6,CFP-10 antigens and Latency Antigens Rv1733c,Rv2028c.The secretion of IFN-γ,TNF-α,IL-2,IL-6,IL-10,IL-5,IP-10,IL-1Ra,CXCL-1 and MCP-1 in Plasma were measured by Luminex Assays.ResultsPart 1:Comparison of QFT-Plus and T-SPOT.TB in diagnosis of active tuberculosis in febrile patients1.The proportion of Indeterminate results(ITRS)in QFT-Plus and T-SPOT.TB were 3.3%and 0,respectively.2.The consistency between the results of the QFT-Plus and T-SPOT.TB was substantial(kappa=0.61,p<0.001).3.The area under the Receiver operating characteristic(ROC)curve of the QFT-Plus and T-SPOT.TB for diagnosing ATB was 0.792 and 0.849(p=0.07),respectively.The sensitivity of differentiating microbiologically confirmed ATB from non-ATB was 93.1%in QFT-Plus versus 96.6%in T-SPOT.TB.4.The influencing factors of T-SPOT.TB positive result were male,(odds ratio(OR)2.33,95%confidence interval(CI)1.27-4.26,p=0.006),evidences of previous TB(OR 11.36(95%CI 4.62-27.94),p<0.001)while the those of QFT-Plus positive result were male(OR 3.17(95%CI 1.73-5.84),p<0.001),use of Immunosuppressive drugs(OR 0.49(95%CI 0.26-0.94),p=0.03)and evidences of previous TB(OR 7.58(95%CI 3.60-15.98),p<0.001).5.The AUC of QFT-Plus TB2-TB1 differentiating ATB from non-active TB infection was 0.576(95%CI 0.477-0.674,p=0.138).Part 2:Mycobacterium tuberculosis-specific multiple cytokine response in different tuberculosis infection status:an exploratory study1.After stimulation with QFT-Plus TB1 or TB2 antigens,the level of IFN-γ in ATB and LTBI groups was significantly higher than that in HC group(p<0.05),while there was no significant difference between ATB and LTBI groups(p>0.05).Cytokines including IL-2,IL-1Ra,CXCL-1 have the similar secretion patterns to IFN-γ.After stimulation with TB1 antigen,the level of IP-10 in ATB group was significantly higher than that in LTBI,previous TB and HC groups(p<0.05).After stimulation with TB2 antigen,the level of IL-6 in ATB group was significantly higher than that in LTBI and HC groups(p<0.05).There was no significant difference in cytokines secretion levels in QFT-Plus specific CD8 response(TB2-TB1)in most groups.2.After stimulation with ESAT-6 antigen,the secretion levels of IL-6,IP-10,IL-1Ra and CXCL-1 in ATB group were significantly higher than those in LTBI,Inactive TB and HC groups(p<0.05).After stimulation with CFP-10 antigen,the secretion levels of IL-6,IP-10,IL-1Ra,CXCL-1 and MCP-1 in ATB group were significantly higher than those in Inactive TB and HC groups(p<0.05).3.After stimulation with Rv1733c SLP antigen,the secretion levels of IFN-γ and IL-2 in ATB group were significantly lower than those in LTBI group,while the levels of IL-6 and CXCL-1 were significantly higher than those in LTBI group(p<0.01).After stimulation with Rv2028c antigen,the secretion levels of IFN-γ and IL-2 in ATB group were significantly lower than those in LTBI and previous TB groups,while the level of CXCL-1 was significantly higher than that in LTBI group(p<0.05).Conlusions1,There was no significant difference between the QFT-Plus and T-SPOT.TB in differential diagnosis of ATB and non-ATB in febrile patients.QFT-Plus is prone to ITRS.2.The influencing factors of T-SPOT.TB positive result were male,evidences of previous TB,while the those of QFT-Plus positive result were male,use of Immunosuppressive drugs.3.In addition to IFN-γ,the detection of IL-2,IL-1Ra,CXCL-1 by QFT-Plus may have the potential to detect tuberculosis infection.Combined detection of IP-10 and IL-6 levels may have the potential to differentiate ATB from LTBI.4.When combining Mtb virulence factors ESAT-6&CFP-10 with Rv1733cSLP/Rv2028c,detection of multiple cytokines including IFN-γ,IL-2,IL-6,IP-10,IL-1Ra and CXCL-1 may have the potential to identify different status of MTB infection. |
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