Preliminary Evaluation Of Novel Biomarkers For Active Tuberculosis Diagnosis

Tuberculosis(TB)is still a major cause of morbidity and mortality worldwide.The disease is caused by infection of the acid-fase bacillus Mycobacterium tuberculosis.The global problem of TB is currently worsening with the emergence of drug-resitant MTB stains and the HIV/AIDS crisis.Identification of the acid-fast bacilli in sputum is the most widely used diagnostic test for active TB,but sputum smear microscopy has low sensitivity,and Mycobacterium tuberculosis culture requires a long time.IGRAs have been introduced into clinical practice for MTB infection diagnosis,and can differentiate MTB infection from BCG vaccination,but are unble to discriminate active TB patients from latent infected subjects.Xpert MTB/RIF,an automated molecular test for MTB and resistance to rifampin,has provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum.However,for extra-pulmonary TB,the collection of extra-pulmonary material is difficult and the number of bacteria in the specimens is low;thus,the sensitivity of the Xpert MTB/RIF test is often inadequate.The immune response to MTB is complex and incompletely characterized,which hampers the development of new immunoassays.Therefore,in this thesis,we focused on the important topics in the field of clinical diagnosis of active TB patients.The thesis was consisted of three parts of study.The first study was designed to investigate the differences between cytokines and chemokines in whole blood among active TB,LTBI and healthy participants stimulated by ESAT6 and CFP10.In this study,seventy-one soluble cytokines and chemokines were tested using Luminex liquid array-based multiplexed immunoassays.For the 71 examined factors,our results indicated that the unstimulated levels of IL-8 Nil,IP-10Nil,MIP-laNil,and sIL-2RaNil and the antigen stimulated levels of IL-8(Ag-Nil),VEGF(Ag-Nil),and MCP-3(Ag-Nil)were potential biomarkers for differentiating between active TB and LTBI,with AUCs of 0.8,0.86,0.755,0.845,0.825,0.812 and 0.75,respectively.The G-CSF(Ag-Nil),GM-CSF(Ag-Nil),IL-la(Ag-Nil),IL-2(Ag-Nil),IP-10(Ag-Nil),BCA-1(Ag-Nil)and Eotaxin-l(Ag-Nil)responses were significantly higher in patients with active TB and LTBI compared with healthy participants(p<0.05),with AUCs of 0.922,0.902,0.908,1.0,0.937,0.919 and 0.935,respectively.The diagnostic effects of immune factors were future validated in the subsequent expreiments can be repeated except for G-CSF(Ag-nil).The second study was designed to evaluat the diagnostic performance of antigen specific IL-6 in active TB patients.Rv0183 protein,the only monoglyceride lipase identified in mycobacteria,was used to stimulated freshly heearin-treated whole blood.The Rv0183-specific IL-6 response was significantly higher in aTB patients(n = 128)than in those with non-TB lung disease(n = 64)and in healthy individuals(n = 327)(p<0.0001),and not affected by latent TB infection.In IGRA+ suspected active TB patients,the sensitivity,specificity,PPV and NPV of IL-6 response(with cutoff of 235.2 pg/ml)were 85.7%,100%,100%and 51.5%for diagnosing aTB,respectively.While in IGRA-ones,they were 87.5%,80.5%,60.9%and 95.0%with 174.2 pg/ml IL-6 response as cutoff,respectively.At the same time,Rv0183 specific IL-6 can also be used for the diagnosis of extrapulmonary tuberculosis.The preliminary findings also found that Rv0183 specific IL-6 has the pontetial to be as biomarkers for treatment response.In this study,we have developed an automated IL-6 quantification assay based on chemiluminesence magnetic particle method,and it will be widely applied.The third part of this study was designed to evaluat the diagnostic performance of DUSP3 protein in active TB patients.In this study,we first found that the level of DUSP-3 protein in peripheral blood can be used for the diagnosis of active TB.The expression level of DUSP3 in peripheral blood of active TB patients was significantly higher than that of old tuberculosis and non-tuberculosis lung disease.The diagnostic sensitivity was 77.5%and the specificity was 75.76%.Although DUSP3 cannot be used for the detection of extrapulmonary tuberculosis,which can be used as a detection marker achieve POCT detection,and can greatly improve the ability of tuberculosis diangsosis.In conclusion,our preliminary data found a variety of host biomarkers that can be used for the diagnosis of active TB.Firstly,ESAT6/CFP10-based study has suggested cytokines and chemokines could be used as host biomarkers for diagnosting active TB as well as MTB infection.In addition,our results clearly show that the Rv0183 antigen-specific IL-6 response has the potential to be used as an immune-diagnosis test for active TB in clinical pracitic.In this parper,we also proved the important application value of DUSP3 protein in the diagnosis of active pulmonary tuberculosis,whice provides an important reference for the correct diagnosis of active TB.