Table_1_Protein and Microbial Biomarkers in Sputum Discern Acute and Latent Tuberculosis in Investigation of Pastoral Ethiopian Cohort.xlsx

Differential diagnosis of tuberculosis (TB) and latent TB infection (LTBI) remains a public health priority in high TB burden countries. Pulmonary TB is diagnosed by sputum smear microscopy, chest X-rays, and PCR tests for distinct Mycobacterium tuberculosis (Mtb) genes. Clinical tests to diagnose LTBI rely on immune cell stimulation in blood plasma with TB-specific antigens followed by measurements of interferon-γ concentrations. The latter is an important cytokine for cellular immune responses against Mtb in infected lung tissues. Sputum smear microscopy and chest X-rays are not sufficiently sensitive while both PCR and interferon-γ release assays are expensive. Alternative biomarkers for the development of diagnostic tests to discern TB disease states are desirable. This study’s objective was to discover sputum diagnostic biomarker candidates from the analysis of samples from 161 human subjects including TB patients, individuals with LTBI, negative community controls (NCC) from the province South Omo, a pastoral region in Ethiopia. We analyzed 16S rRNA gene-based bacterial taxonomies and proteomic profiles. The sputum microbiota did not reveal statistically significant differences in α-diversity comparing the cohorts. The genus Mycobacterium, representing Mtb, was only identified for the TB group which also featured reduced abundance of the genus Rothia in comparison with the LTBI and NCC groups. Rothia is a respiratory tract commensal and may be sensitive to the inflammatory milieu generated by infection with Mtb. Proteomic data supported innate immune responses against the pathogen in subjects with pulmonary TB. Ferritin, an iron storage protein released by damaged host cells, was markedly increased in abundance in TB sputum compared to the LTBI and NCC groups, along with the α-1-acid glycoproteins ORM1 and ORM2. These proteins are acute phase reactants and inhibit excessive neutrophil activation. Proteomic data highlight the effector roles of neutrophils in the anti-Mtb response which was not observed for LTBI cases. Less abundant in the sputum of the LTBI group, compared to the NCC group, were two immunomodulatory proteins, mitochondrial TSPO and the extracellular ribonuclease T2. If validated, these proteins are of interest as new biomarkers for diagnosis of LTBI.

结核病（TB）与潜伏性结核感染（LTBI）的鉴别诊断在高结核负担国家仍为公共卫生的重点任务。肺结核的诊断主要通过痰涂片显微镜检查、胸部X光片和针对特定结核分枝杆菌（Mtb）基因的PCR测试。用于诊断LTBI的临床测试依赖于使用结核特异性抗原刺激血液中的免疫细胞，随后测量干扰素-γ的浓度。干扰素-γ是针对感染肺组织内Mtb的细胞免疫反应的重要细胞因子。痰涂片显微镜检查和胸部X光片在敏感性方面不足，而PCR和干扰素-γ释放试验的成本较高。因此，开发能够区分结核病状态的诊断测试的替代生物标志物是可取的。本研究旨在通过分析包括结核病患者、LTBI个体和来自埃塞俄比亚南奥莫省的阴性社区对照（NCC）在内的161名受试者的样本，从分析中发掘痰液诊断生物标志物候选者。我们分析了基于16S rRNA基因的细菌分类学和蛋白质组学图谱。痰液微生物群在α-多样性方面，比较各队列并无统计学上的显著差异。代表Mtb的属Mycobacterium仅在结核病组中被识别，与LTBI和NCC组相比，Rothia属的丰度也有所减少。Rothia是一种呼吸道共生菌，可能对Mtb感染产生的炎症环境敏感。蛋白质组数据支持了患有肺结核的受试者对病原体的天然免疫反应。铁蛋白，一种由受损宿主细胞释放的铁储存蛋白，在结核痰液中与LTBI和NCC组相比，其丰度显著增加，同时α-1-酸性糖蛋白ORM1和ORM2也有所增加。这些蛋白质是急性期反应蛋白，可抑制中性粒细胞的过度激活。蛋白质组数据突出了中性粒细胞在抗-Mtb反应中的效应作用，这在LTBI病例中并未观察到。与NCC组相比，LTBI组痰液中两种免疫调节蛋白——线粒体TSPO和细胞外核糖核酸酶T2含量较少。如果得到验证，这些蛋白质有望成为LTBI诊断的新生物标志物。（Mycobacterium tuberculosis, Mtb, Rothia, interferon-γ, α-diversity, 16S rRNA gene, proteomic profiles, neutrophils, immunomodulatory proteins, TSPO, ribonuclease T2）