M. tuberculosis exosome detection for pediatric TB diagnosis

项目概要/摘要 每年有100多万新的结核病病例和239 000例与结核病有关的死亡发生在儿童中。 幼儿，特别是感染艾滋病毒的幼儿，更有可能出现播散性或肺外结核 和少菌性疾病，经常被呼吸道采样遗漏。无痰、基于生物标志物的诊断工具 为了在儿童中快速发现结核病并作出治疗反应，迫切需要使用容易获得的标本。 外泌体是一种小的（30-100 nm）膜性细胞外囊泡（EV）， 隔室;由M分泌的那些。结核病（Mtb）或Mtb感染的巨噬细胞似乎发挥了重要作用， 在结核病发病机制中的作用。我们的合作者开发了一种快速灵敏的纳米等离子体增强 散射（nPES）测定，其直接从少至IyL的血清中检测Mtb-外来体（Mtb-EV）。证据- 用Mtb标志物LpqH（19-kDa Mtb脂蛋白）和LAM进行的概念nPES测定区分了 来自高危患者和正常对照的成人结核病，以及儿童结核病病例（包括HIV+）和对照 具有高灵敏度和特异性。 我们建议使用来自儿科紧急开始HAART（PUSH）研究的存档标本和临床数据 （NCT 02063880）和一个新提出的疑似结核病儿童的前瞻性队列， 评价nPES检测的Mtb-EV用于儿科TB诊断（目的1）、治疗反应（目的 2），以及即时护理平台的评价（目标3）。除了评估常规诊断外， 性能指标，我们建议使用先进的流行病学方法（贝叶斯潜在类分析） 在不完美的背景下在成人结核病患者和健康对照中进行的额外评价 包括家庭接触者（成人和儿童）和最近接种卡介苗的未患结核病的婴儿。 我们假设nPES检测到的Mtb-EV将1）具有与参考相似的诊断性能， Xpert/培养在确诊结核病的儿童中进行，无需痰液，并确定其他儿童 呼吸道样本遗漏，2）将提供治疗反应的有用替代标志物， 成功治疗期间的定量水平，以及3）将保持床旁平台的性能。 使用来自接受强化结核病治疗的HIV感染儿童的特征性队列的冷冻保存样本 对艾滋病毒感染率高的疑似结核病儿童进行评估和前瞻性队列研究， 用于有效评价一种具有潜在临床影响的新型诊断方法，以改善儿科结核病诊断。

PROJECT SUMMARY/ABSTRACT Over one million new cases of tuberculosis (TB) and 239,000 TB-related deaths occur in children each year. Young children, especially those with HIV, are more likely to present with disseminated or extrapulmonary TB and paucibacillary disease, often missed by respiratory sampling. Non-sputum, biomarker-based diagnostic tools for rapid TB detection and treatment response in children, using easily obtained specimens are urgently needed. Exosomes are small (30-100 nm) membranous extracellular vesicles (EVs) originating from endosomal cell compartments; those secreted by M. tuberculosis (Mtb) or Mtb-infected macrophages appear to play a significant role in Mtb pathogenesis. Our collaborators have developed a rapid and sensitive nanoplasmon-enhanced scattering (nPES) assay which directly detects Mtb-exosomes (Mtb-EVs) from as little as 1 L of serum. Proof- of-concept nPES assays performed with Mtb markers LpqH (19-kDa Mtb lipoprotein) and LAM distinguished adult TB from at-risk patients and normal controls, and among pediatric TB cases (including HIV+) and controls with high sensitivity and specificity. We propose using archived specimens and clinical data from the Pediatric Urgent Start of HAART (PUSH) Study (NCT02063880) and a new proposed prospective cohort of children suspected of TB with high HIV prevalence to evaluate performance of nPES detected Mtb-EVs for pediatric TB diagnosis (Aim 1), treatment response (Aim 2), and evaluation of a point-of-care platform (Aim 3). In addition to assessing conventional diagnostic performance measures, we propose to use advanced epidemiologic methods (Bayesian latent class analysis) given the context of an imperfect reference. Additional evaluation in adult TB patients and healthy controls including household contacts (adults and children) and recently BCG-vaccinated infants without TB is proposed. We hypothesize nPES detected Mtb-EVs will 1) have similar diagnostic performance to the reference of Xpert/culture among children with confirmed TB without the need for sputum, and identify additional children missed by respiratory sample, 2) will provide a useful surrogate marker of treatment response with decline in quantitative levels during successful treatment, and 3) will maintain performance with a point-of-care platform. Using cryopreserved samples from a well-characterized cohort of children with HIV who underwent intensive TB evaluation and a prospective cohort of children suspected of TB with high HIV prevalence provides opportunity for efficient evaluation of a novel diagnostic with potential for clinical impact to improve pediatric TB diagnosis.