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

项目摘要 目前迫切需要为儿童开发基于非痰生物标志物的分诊和诊断测试 结核病（TB）。这对感染艾滋病毒的幼儿尤其重要，他们的结核病相关性很高， 死亡率，但通常不能产生痰，痰菌负荷较低。生物标志物的发现 儿童结核病需要超灵敏的平台来测量低丰度结核分枝杆菌（Mtb） 临床样品中的蛋白质和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确定结核分枝杆菌和/或宿主衍生的 可实现世界卫生组织（WHO）目标产品概况（TPP）的儿童生物特征 儿童结核病的非痰生物标志物分诊或诊断测试的准确性阈值。我们 假设结合联合收割机Mtb蛋白和宿主生物标记物生物特征具有功能性证据 与结核病发病机制或免疫相关性将具有最佳的诊断性能。为了评估这一假设， 我们将使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， 在乌干达、冈比亚和南非，在目标1中，我们将使用超- 灵敏的基于电化学发光（ECL）的免疫测定，以评估Mtb蛋白ESAT的存在， 6、CFP-10、MPT 64、MPT 32和Ag 85 B，来自100名儿童的库存血液和尿液样本的发现集 根据NIH共识定义，5岁以下确诊结核病和200例不太可能患有结核病（50%为HIV 两组的发病率）。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 通过体外模型和小鼠体内途径分析进行质谱功能评估 模型，以确定宿主蛋白质，翻译后修饰和代谢物，区分儿童与 确诊肺结核和不太可能的肺结核在目标3中，我们将使用目标1中鉴定的候选Mtb和宿主生物标志物 和2以获得具有至多10种分析物的生物特征，所述分析物仅由Mtb蛋白组成，仅由宿主生物标志物组成，以及 Mtb和宿主来源的生物标志物。发现集中符合WHO TPP标准的生物特征将1） 在来自300名5岁以下儿童的库存样本的独立测试集中进行了评估（100名确认患有 结核病，200人不太可能患有结核病;每组艾滋病毒感染率为50%），以验证诊断准确性并确定切割点。 2）使用预先选择的临界值，在350名5岁以下儿童的前瞻性队列中进行评价 以及微生物和临床参考标准。这些目标的实现将导致确定 有前途的生物特征，可以在大规模的实地研究中进一步验证，并转化为即时护理 对儿童结核病进行分诊和/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.