Childhood ‘Omics’ and Mycobacterium tuberculosis-derived BiOsignatures (COMBO) for TB diagnosis in high HIV prevalence settings

项目总结 迫切需要开发基于非痰生物标记物的儿童分类和诊断测试 结核病(TB)。这对感染艾滋病毒的幼儿尤其重要，因为他们与结核病有关。 死亡率较低，但往往不能产痰，痰菌负担较低。生物标记物的发现 儿童结核病需要超灵敏平台来检测低丰度结核分枝杆菌(Mtb) 临床样本中的蛋白质和对宿主蛋白质的更多研究，蛋白质的翻译后修饰， 以及比上游RNA表达更有可能反映宿主-病原体相互作用的代谢物 导致结核病。拟议项目的总体目标是查明结核分枝杆菌和/或宿主衍生的 能够达到世界卫生组织(WHO)目标产品简介(TPP)的儿童生物签名 儿童结核病非痰生物标志物分诊或诊断试验的准确度阈值。我们 假设结合结核分枝杆菌蛋白和宿主生物标记物的生物特征与功能的证据 与结核病发病机制或免疫相关的指标将具有最好的诊断性能。为了评估这一假设， 我们将利用三口井的样本进行生物标志物发现和初步临床验证研究- 描述了乌干达、冈比亚和南非的儿科结核病队列。在目标1中，我们将使用一种超 灵敏的电化学发光(ECL)免疫分析法检测结核分枝杆菌蛋白的存在 6、CFP-10、MPT64、MPT32和Ag85B在100名儿童的血液和尿液样本中的发现集 根据NIH共识定义，5岁以下确诊结核病和200名不太可能患有结核病的儿童(50%是艾滋病毒 两组中的患病率)。在目标2中，我们将使用相同的发现集来执行定向和非定向 通过通径分析进行功能评估的质谱学，体外模型和体内小鼠 识别宿主蛋白、翻译后修饰和代谢产物的模型，以区分儿童 确诊的结核病与不太可能的结核病。在目标3中，我们将使用目标1中确定的候选结核分枝杆菌和宿主生物标记物 和2获得最多10个分析物的生物特征，这些分析物仅由Mtb蛋白、宿主生物标志物组成，以及 结核分枝杆菌和宿主衍生的生物标记物。在发现集中符合WHO TPP标准的生物签名将是1) 在来自300名5岁以下儿童的银行样本的独立测试集中进行评估(100名确诊儿童 结核病，200例不太可能患有结核病；每组艾滋病毒流行率为50%)，以验证诊断的准确性并建立切割点 在350名5岁以下儿童的前瞻性队列中，使用预先选择的截止值进行评估 以及微生物学和临床参考标准。完成这些目标将导致确定 有希望的生物签名可以在大规模现场研究中进一步验证并转化为护理点 儿童结核病的分类和/或诊断测试。

PROJECT SUMMARY There is an urgent need to develop non-sputum biomarker-based triage and diagnostic tests for childhood tuberculosis (TB). This is particularly important for young children with HIV infection, who have high TB-related mortality but often cannot produce sputum and have lower sputum bacillary burden. Biomarker discovery for childhood TB requires ultra-sensitive platforms to measure low-abundant Mycobacterium tuberculosis (Mtb) proteins in clinical samples and greater investigation of host proteins, post-translational modifications of proteins, and metabolites that are more likely than upstream RNA expression to reflect the host-pathogen interactions that lead to TB disease. The overall objective of the proposed project is to identify Mtb- and/or host-derived biosignatures in children that can achieve World Health Organization (WHO) target product profile (TPP) accuracy thresholds for a non-sputum biomarker-based triage or diagnostic test for childhood TB. We hypothesize that biosignatures that combine Mtb proteins and host biomarkers with evidence of functional relevance to TB pathogenesis or immunity will have the best diagnostic performance. To assess this hypothesis, we will conduct biomarker discovery and initial clinical validation studies using samples from three well- characterized pediatric TB cohorts in Uganda, The Gambia and South Africa. In Aim 1, we will use an ultra- sensitive electrochemoluminescence (ECL)-based immunoassay to assess the presence of Mtb proteins ESAT- 6, CFP-10, MPT64, MPT32, and Ag85B in a discovery set of banked blood and urine samples from 100 children under 5 years old with confirmed TB and 200 with unlikely TB per NIH consensus definitions (50% HIV prevalence in both groups). In Aim 2, we will use the same discovery set to perform targeted and untargeted mass spectrometry with functional assessment through pathway analysis, in vitro models and in vivo mouse models to identify host proteins, post-translational modifications and metabolites that distinguish children with confirmed versus unlikely TB. In Aim 3, we will use the candidate Mtb and host biomarkers identified in Aims 1 and 2 to derive biosignatures with up to 10 analytes consisting of Mtb proteins only, host biomarkers only, and both Mtb- and host-derived biomarkers. Biosignatures that meet WHO TPP criteria in the discovery set will 1) be evaluated in an independent test set of banked samples from 300 children under 5 years old (100 with confirmed TB, 200 with unlikely TB; 50% HIV prevalence in each group) to verify diagnostic accuracy and establish cut- offs and 2) be evaluated in a prospective cohort of 350 children under 5 years old using the pre-select cut-offs and both microbiological and clinical reference standards. Completion of these aims will result in identification of promising biosignatures that can be further validated in large-scale field studies and translated into point-of-care triage and/or diagnostic tests for childhood TB.