M. tuberculosis exosome detection for pediatric TB diagnosis

项目摘要/摘要 每年有100多万新的结核病病例和239,000例与结核病相关的死亡病例发生在儿童中。 幼儿，特别是那些携带艾滋病毒的儿童，更有可能出现播散性或肺外结核病。 以及少杆菌病，经常被呼吸道样本遗漏。基于生物标志物的非痰诊断工具 对于儿童结核病的快速检测和治疗反应，迫切需要使用容易获得的标本。 外切小体是小的(30-100 nm)膜性细胞外小泡(EVS)，起源于内胚体细胞 那些由结核分枝杆菌(Mtb)或感染Mtb的巨噬细胞分泌的间隔似乎发挥着重要的作用 在结核分枝杆菌发病机制中的作用。我们的合作者已经开发出一种快速而灵敏的纳米等离子增强型 散射(NPES)法可直接从低至1的L血清中检测结核分枝杆菌外切体(MTB-EVS)。证明- 用Mtb标记LpqH(19-kDa Mtb脂蛋白)和LAM区分进行概念外NPE分析 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+)和对照的成人结核病 具有很高的敏感性和特异性。 我们建议使用HAART儿科紧急启动(PUSH)研究的存档样本和临床数据 (NCT02063880)和一项新的拟议的艾滋病毒高流行率疑似结核病儿童的预期队列 评价NPES检测结核分枝杆菌EVS在儿童结核病诊断(AIM 1)、治疗反应(AIM)中的性能 2)，以及评价护理点平台(目标3)。除了评估常规诊断 绩效衡量，我们建议使用先进的流行病学方法(贝叶斯潜在类别分析) 考虑到一个不完美的引用的背景。成人结核病患者和健康对照的额外评估 建议包括家庭接触者(成人和儿童)和最近接种卡介苗的无结核病婴儿。 我们假设检测到的NPE将1)具有与参考的相似的诊断性能 在不需要痰的确诊结核病儿童中进行专门/培养，并确定其他儿童 被呼吸道样本遗漏，2)将提供治疗反应的有用替代标记物 在成功治疗期间的量化水平，以及3)将通过护理点平台保持性能。 使用冷冻保存的样本，这些样本来自一组特征良好的艾滋病病毒携带者儿童，他们经历了重症结核病 艾滋病毒高流行率的疑似结核病儿童的评估和前瞻性队列提供了机会 为了有效地评估一种具有潜在临床影响的新诊断方法，以提高儿童结核病的诊断水平。

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.