Research Of Susceptibility Markers And Serum Markers Of TB

Background: Tuberculosis(TB), a chronic respiratory infectious disease, is caused by Mycobacterium tuberculosis(MTB) and is hamful to human health seriously. The alarming resurgence of tuberculosis around the world is caused by Human Immunodeficiency Virus(HIV)/AIDS pandemic, the emergence of multi-drug resistant pulmonary tuberculosis(MDR-PTB) and the inadequate attention of TB from people. In order to improve the completion rate of TB treatment, the short course strategy of directly observed treatment has been expanded and substantly invested. However, one of the biggest obstacles for the globally TB control is to accurately diagnose the diseases. TB is one of the major infectious diseases that causes mortality and morbidity in the world. The improvement of patient’s detected rate and proper treatment are critical to the effective control of TB, and these are the most important measure for controlling the source of infection and stopping TB epidemic.In the present day, there were many types of clinical TB diagnostic methods, but they were unable to meet the needs of clinical auxiliary diagnosis and the guiding drug for various reasons. Therefore, it was urgent to find the new markers that were applied to detect patients and effectively prevent and control tuberculosis. Serum protein had important physiological functions, which included participation in the body’s immunity, metabolism, blood coagulation, exchange of material, transport, signal conditioning, etc. The composition and the changes of these low-abundance protein in serum were often related to pathology, which had significance for diagnosis the diseases and monitoring. In order to discover the blood markers of TB, we first needed to determine which blood markers existed in the serum of TB patients, especially PTB patients. For answering this question, the first step of this study was the system analysis of the literature about TB susceptible markers that were published in recent years. Studies had found that the genetic susceptibility of TB was determined by many genes. Molecular genetics of TB pathogenesis was a research hot spot. Among these genes, the correlation between Human Leucocyte Antigen(HLA) allele polymorphism and TB was a major concern. This study initially found the susceptible markers that were associated with TB in the blood through finding and analyzing the literature to explore the pathogenesis of its gene level.Gene was the carrier of genetic information, but the performer of life was protein, so protein markers may relate to the correlation of TB when it was compared to the susceptibie gene markers. As a new subject and the platform of proteomics research technology, the emergence of MALDI-TOF-MS provided a new train about the thought of finding serum markers of PTB. Proteomics and its relevant technology enabled people to understand all the expressed proteins by the cells and living organisms in a particular time and space. Therefore, the second and third parts of this study were using relative quantitative proteomics to discover and validate the serum proteins of sex hormone-binding globulin(SHBG) and Tubulin alpha-3E chain(TBA3E), which were valuable candidate markers of PTB and MDR-PTB respectively.Part I: Correlation analysisof susceptible markers of tuberculosisObjective: Meta-analysis was used for comprehensive analysis of the relevant and published literature about the relationship between HLA gene polymorphism and susceptible TB to find the susceptible genes for providing more scientific reference to the prevention and treatment of TB genes.Methods: Relevant studies were found from two databases Pub Med and EMBASE, and time period was January 1980 to in June 2014. Keywords: ‘‘Mycobacterium tuberculosis’ ’,‘‘tuberculosis’ ’,‘‘MTB”,‘‘TB”, ‘‘HLA- DRB1’’, ‘‘HLA-DQA1’’,‘‘HLA-DQB1’’,‘‘human leukocyte antigen’’,‘‘HLA antigen’’, ‘‘Polymorphism”,‘‘Polymorphisms”, ‘‘Genetic polymorphism”. Stata version 11.2 software package was used for data analysis.Results: DRB1*15, DRB1*08:03 and DQB1*0601 in TB patients had significantly higher frequencies than controls; but DRB1*03, DRB1*11, DRB1*11:03 and DRB1*12:02 had significantly lower frequencies in the total population. Subgroup analysis showed that DRB1*15, DRB1*08:03 and DQB1*0601 had significantly higher frequencies in Asian TB patients; but DRB1*03, and DRB1*07:01 had significantly lower frequencies in Asian TB patients. HLA-DQA1 polymorphism had no obvious correlation with the risk of tuberculosis.Conclusion: HLA-DRB1 and DQB1 allele polymorphism had strong associations with TB, which might be the susceptible markers of TB. DQA1 had no strong associations with TB, which might not be the susceptible markers of TB.Part II: Potential markers in PTB serum found by using the combination of i TRAQ labeling and MALDI-TOF/MS technologyObjective: Differentially expressed proteins were identified in sera from PTB patients; comparing them with the controls by using relative quantitative proteomics technology and validated, which provided valuable candidate markers in serum for the prevention and control of PTB.Methods:Serum proteins of five samples were analyzed by i TRAQ that combined with MALDI-TOF-MS and identified by Proteinpilot software; including differential expressed proteins from 7 cases of MDR-PTB, 30 cases of smear-negative pulmonary tuberculosis(SNP-TB), 10 cases of smear-positive pulmonary tuberculosis(SPP-TB), 30 cases of healthy controls, and 10 cases of pneumonia. Bioinformatics analysis was used to analyze the function of differentially expressed proteins. Similar as potential biomarkers of PTB, SHBG was validated by IHC, ELISA, and Western blot. IHC validation included 40 cases of PTB and 10 cases of control group; ELISA validation included 35 cases of MDR-PTB, 70 cases of SNP-TB, 55 cases of SPP-TB, 24 cases of healthy controls, and 15 cases of pneumonia; Western Blot validation included 7 cases of MDR-PTB, 30 cases of SNP-TB, 10 cases of SPP-TB, 30 cases of healthy controls, and 10 cases of pneumonia. According to the result of ELISA, receiver operating characteristic curve(ROC) was drawn and used to analyze the related indicators.Results: Relative quantitative proteomics identified and selected 26 differentially expressed proteins in PTB serum, which included 16 overexpressed proteins and 10 downregulated proteins. Sex hormone binding globulin(SHBG) was found to be significantly elevated among PTB patients when compared with the controls that was examined by IHC, ELISA and Western blot. This result was consistent with the result of proteomics screening. ROC curve achieved AUC of 0.981, the accuracy of 91.07%, the sensitivity of 84.6% and the specificity of 94.6% in diagnosing PTB according to serum SHBG expression level that was examined by ELISA.Conclusion: The platform of proteomics screening was established to detect proteins in PTB serum. Serum SHBG protein had a high accuracy, sensitivity and specificity when distinguishing PTB, pneumonia, and healthy controls, which might be a valuable candidate marker in the prevention and control of PTB.Part III: Potential markers in MDR-PTB serum found by using the combination of i TRAQ labeling and MALDI-TOF/MS technologyObjective: Differentially expressed proteins were identified in sera from MDR-PTB patients and compared with the controls by using relative quantitative proteomics technology and validated by ELISA and multiple reaction monitoring(MRM).Methods:Serum proteins of four samples were analyzed by i TRAQ method that combined with MALDI-TOF-MS and identified by proteinpilot software, which included differential expressed proteins from 7 cases of MDRPTB, 30 cases of SNP-TB, 10 cases of SPP-TB, 30 cases of healthy controls, and 10 cases of pneumonia. Application of bioinformatics analysis was ued to analyze cellular elements, molecular function, and biological process of significantly differentially- expressed proteins; potential biomarkers of MDRPTB, Fibronectin 1(FN1) and Vitronectin(VTN) were validated next. ELISA validation included 13 cases of MDR-PTB, 22 cases of SNP-TB, 19 cases of healthy controls, and 15 cases of pneumonia; MRM validation included 10 cases of MDR-PTB, 10 cases of SNP-TB, 10 cases of healthy controls, and 10 cases of pneumonia. ROC was drawn and used to analyze the related indicators.Results: Comparing to the controls, 17 differentially expressed proteins were recognized among MDR-PTB patients, which included 9 overexpressed proteins and 8 downregulated proteins by i TRAQ-MALDI-TOF-MS. Tubulin alpha-3E chain(TBA3E) was found to be significantly elevated among MDRPTB patients when compared with the controls that were examined by ELISA and MRM. This result was consistent with the results of proteomics screening, and it was consistent with i TRAQ result as well. ROC curve examined serum TBA3 E by ELISA achieved AUC of 0.949, the accuracy of 78.74%, the sensitivity of 75.6%, and the specificity of 91.5% when diagnosing MDR-PTB. FN1 and VTN were found to be significantly decreased that were examined by MRM, and this outcome was consistent with the result of proteomics screening. However, the result from ELISA of VTN was not consistent with the result from proteomics screening.Conclusion: TBA3 E was suggested to be a possible serum biomarker for MDR-PTB to distinguish PTB, pneumonia, and healthy controls with high accuracy, sensitivity, and specificity. For becoming potential biological markers of MDR-PTB, FN1 and VTN were needed for further research.