结核病（Tuberculosis, TB）是由结核分枝杆菌（Mycobacterium tuberculosis, M.tb）感染人体所引起的慢性传染病，因常侵染人体肺部，多称为肺结核。由于其高感染率和高死亡率，TB超过艾滋病成为单一感染源致死的主要原因。结核病人多集中在东亚地区，其中中国结核感染率居世界第三位。M.tb感染人体时会导致潜伏结核感染（Latent tuberculosis infection, LTBI）发生，且约5%-10%的LTBI存在发展为活动性结核（Active Tuberculosis, ATB）的潜在风险，对该人群的疏忽也是结核广泛传播的原因之一。但目前ATB和LTBI人群早期快速诊断的灵敏度和检出率均较低，且临床上没有可以有效区分LTBI、ATB和其他非结核病人群的检验方法因此，迫切需要新的结核病生物标志物，建立针对ATB和LTBI的快速诊断方法。

代谢组学是发现新的生物标志物以提高诊断效率、优化治疗方案的有力技术之一。尿液相较其他体液而言更易获得和操作，是临床研究的理想生物样本。因此，本研究采用超高效液相色谱质谱联用技术及生物信息学分析等方法，对90例ATB患者、LTBI和非结核病对照组（NC）的尿液样本进行了非靶向代谢组分析，并对差异表达代谢物及涉及的代谢途径进行分析。样本经过色谱质谱分析后，得到ATB、LTBI和NC的代谢谱。分析图谱在阴离子模式下共获得3744种代谢物成分，在阳离子模式下共获得4086种代谢物成分。经代谢物数据库搜索比对后，分别得到762和559个代谢物。应用多变量统计学分析发现偏最小二乘判别分析（Partial Least Squares Discriminant Analysis, PLS-DA）和正交偏最小二乘判别分析（Orthogonal Partial Least Squares Discriminant Analysis, OPLS-DA）方法分析模型图中ATB、LTBI、NC三组存在明显的分组趋势。根据变量投影重要度值大于1.5，差异倍数大于2，T test检验P值<0.05为标准对鉴定得到的代谢物进行筛选，发现不同模式下显著变化的分别有196和138个，一些代谢物在多组比较中均表现出显著差异。将差异代谢物应用于聚类分析（Hierarchical clustering analysis, HCA）结果显示，阳离子模式和阴离子模式下LTBI与NC组之间存在较为明显的区分，阳离子模式下LTBI与ATB组间存在明显的区分。通过KEGG分析发现差异代谢物涉及的途径包括花生四烯酸循环、精氨酸代谢通路、亮氨酸、异亮氨酸和缬氨酸代谢通路等。多变量统计分析显示，三组比较间有统计学意义（P<0.05）的差异代谢物共有6种，其中谷胱甘肽和组胺的ROC AUC>0.75，可以作为潜在分子标识进行后续分析。采用酶联免疫吸附试验（Enzyme Linked Immunosorbent Assay, ELISA）对另48例尿液样本（每组16例）中谷胱甘肽和组胺的水平进行了检测，发现ATB、LTBI和NC样本中的谷胱甘肽和组胺含量均有不同程度的变化，且进行T-test分析得到显著差异结果，可以作为ATB和LTBI的潜在分子诊断标识。

本课题研究表明尿液小分子代谢物在不同状态下的结核病人中存在较大差异，且这些差异表达的代谢物大多参与了机体免疫应答途径、M.tb感染导致的多中心能量转化以及尿素、尿酸等代谢循环。ELISA验证检测结果提示谷胱甘肽和组胺可作为潜在的结核生物标志物，为建立更方便、快捷、灵敏且无创的临床结核诊断检测提供了科学基础。

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 ESI- mode 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.