Research On Molecular Diagnostic Markers Of Tuberculosis Based On Urine Metabolomics

Tuberculosis(TB)is a chronic infectious disease caused by the infection of Mycobacterium tuberculosis(M.tb).Because of its high infection rate and high mortality rate,TB surpasses AIDS to become a leading cause of death from a single infectious agent.Most TB patients are concentrated in East Asia.the infection rate of tuberculosis in China ranks third in the world.It is likely to lead to latent tuberculosis infection(LTBI)when M.tb infects the human body.About 5%of 10%of LTBI has a potential risk of developing active tuberculosis(ATB).Negligence of this population is also one of the reasons for widespread tuberculosis.However,the sensitivity and detection rate of early rapid diagnosis in ATB and LTBI population are low,and there is no effective test method to distinguish LTBI,ATB and other non-TB population.Therefore,there is an urgent need for new tuberculosis biomarkers to establish a rapid diagnostic method for ATB and LTBI.The metabolomic analysis is a powerful technique for discovering new biomarkers to improve diagnosis,prognosis and treatment.Easier to obtain and operate than other body fluids,urine is an ideal biological sample for clinical research.In this study,90 urine samples of patients with ATB,LTBI and non-tuberculosis control(NC)group were analyzed by ultra high performance liquid chromatography mass spectrometry(UPLC-MS/MS)and bioinformatics analysis,and then the differentially expressed metabolites and metabolic pathways involved were analyzed.The results showed that the metabolic spectra of ATB,LTBI and NC were obtained after UPLC-MS/MS analysis.A total of 3744 metabolites were obtained in the ESImode and 4086 metabolites were obtained in the ESI+mode.After searching and matching the online metabolites database,762 and 559 metabolites were obtained respectively.By using multivariate statistical analysis,it was found that there was an obvious grouping trend among ATB,LTBI and NC in partial least squares discriminant analysis(PLS-DA)and orthogonal partial least squares discriminant analysis(OPLS-DA).The identified metabolites were screened according to the variable importance in projection(VIP)value greater than 1.5 and the fold change more than 2 with P-value<0.05.It was found that 196 and 138 metabolites changed significantly,respectively and some metabolites showed significant differences in multiple comparisons.Significant differential metabolites were applied to hierarchical cluster analysis(HCA).The results showed that there was a clear distinction between LTBI and NC group in both ESI+and ESI-mode,and between LTBI and ATB group in ESI+ mode.Based on KEGG pathway analysis,the pathways involved in differential metabolites are concerned with immune regulation,energy metabolism and humoral circulation,which included arachidonic acid cycle,arginine metabolic pathway,leucine,isoleucine and valine metabolic pathway.Multivariate statistical analysis showed that there were six different metabolites among the three comparisons,in which the ROC AUC of glutathione(GSH)and histamine was more than 0.75.They could be used as potential molecular markers for follow-up analysis.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of GSH and histamine in 48 urine samples(16 cases in each group).It was found that the contents of glutathione and histamine in ATB,LTBI and NC samples changed in different degrees and verified by T-test with significant differences,which provided the possibility for the study of non-invasive biomarkers of TB.This study shows that there are great differences in urinary small molecular metabolites in different states of TB patients,and most of these differentially expressed metabolites are involved in immune response pathways,polycentric energy conversion caused by M.tb infection and metabolic cycles such as urea and uric acid.The results of ELISA suggest that GSH and histamine can be used as potential biomarkers of TB,which provides a scientific basis for the establishment of a more convenient,rapid,sensitive and non-invasive clinical diagnosis and detection of tuberculosis.