Direct quantitation of the circulating Mtb-peptidome for pediatric TB management

摘要 成人和儿童结核病（TB）诊断和治疗监测的传统方法严重依赖于 耗时的细菌培养或无法定量的DNA检测，以检测是否存在少量的 细菌儿科结核病管理特别困难，因为目前基于微生物的方法 诊断和治疗监测是资源密集型的，侵入性的，对儿童不够敏感， 他们往往患有少杆菌病。由于他们的免疫不成熟，幼儿处于高风险， 结核病进展迅速，发病率和死亡率显著。治疗反应评估 依赖于主观措施，限制了儿童获得急需的更短和更安全的结核病治疗方案。更 因此，儿童迫切需要快速和严格的结核病诊断和治疗监测方法。在 为了解决目前儿科结核病管理的局限性，我们建议开发一种快速诊断方法， 方法，独立于分枝杆菌分离，定量低分子量M。结核病（Mtb） 抗原（CFP-10和ESAT-6）和TB相关宿主标志物IP-10。ESAT-6 和CFP-10是检测活动性TB及其治疗反应的理想生物标志物， 结核病特异性蛋白质组模式将提供一个完整的档案，患者的病理生理变化， 抗结核治疗，同时提高诊断敏感性。我们的策略是利用中空的，能量集中 TiO 2 NanoShells用Mtb CFP-10和ESAT-6和宿主IP-10特异性的定制抗体功能化 通过高通量质谱（MS）检测多肽，以提高诊断灵敏度和特异性。我们 使用292名成人评估了该NanoShell-MS平台的仅Mtb抗原版本（无抗IP-10 NanoShells） 和102名儿童患者和对照，选自5个高度相关的群组（成人、儿童和潜伏性TB， 艾滋病毒/结核病合并感染和非结核分枝杆菌感染）。NanoShell-MS 对活动性结核（细菌学确诊和临床诊断）的敏感性和特异性分别为， 成人中为90.7% / 97.7%，儿童中为88.2% / 100%，培养阴性儿童TB的敏感性为78.6% 患者，大大超过WHO公布的该队列中4-15%的Xpert灵敏度。NanoShell-MS结果 与常规培养的4-6周相比，可以在样品收集的1小时内获得，并且 在抗TB治疗开始的四天内检测到血液Mtb抗原水平的降低。本提案中 我们的目标是：1）使用NanoShell-MS分析来开发一种定量预测模型，用于在以下情况下诊断活动性结核病： 大型的、描述良好的前瞻性儿童结核病队列，2）确定我们的方法在快速治疗结核病方面的实用性， 治疗效果评价。我们的NanoShell-MS分析平台的优势在于它使用准确， 高通量质谱已成为临床诊断的重要手段， 同时将我们的NanoShell-MS平台与易于使用的“小型化”（鞋盒大小） MS系统也应该允许这种方法为资源有限地区的儿科患者提供服务。

ABSTRACT Conventional methods for adult and pediatric tuberculosis (TB) diagnosis and treatment monitoring rely heavily on time-consuming bacterial culture or unquantifiable DNA assays to detect the presence of small numbers of bacteria. Pediatric TB management is particularly difficult because current organism-based methods for diagnosis and treatment monitoring are resource-intensive, invasive and inadequately sensitive for children, who tend to have paucibacillary disease. Due their immunological immaturity, young children are at high risk of rapid disease TB progression, with significant morbidity and mortality. Assessment of their treatment response relies on subjective measures, limiting children's access to much needed shorter and safer TB regimens. More rapid and rigorous methods of TB diagnosis and treatment monitoring are thus critically needed for children. In order to address current limitations in pediatric TB management, we propose to develop a rapid diagnostic method, independent of mycobacterial isolation, that quantifies the low molecular-weight M. tuberculosis (Mtb) antigens (CFP-10 and ESAT-6) and the TB-associated host marker IP-10 in patient blood samples. ESAT-6 and CFP-10 are ideal biomarkers to detect active TB and its treatment response, and addition of IP-10 to this Mtb-specific proteomic pattern will provide an integrated profile of patients' pathophysiological changes during anti-TB treatment, while also improving diagnostic sensitivity. Our strategy utilizes hollow, energy-focusing TiO2 NanoShells functionalized with custom antibodies specific for Mtb CFP-10 and ESAT-6 and host IP-10 peptides with high-throughput mass spectrometry (MS) to increase diagnostic sensitivity and specificity. We evaluated an Mtb-antigen-only version of this NanoShell-MS platform (no anti-IP-10 NanoShells) with 292 adult and 102 pediatric patients and controls chosen from five highly relevant cohorts (adult, pediatric and latent TB, HIV/TB co-infection, and non-TB mycobacterial infection), from our global collaborators. NanoShell-MS sensitivity and specificity for active TB (bacteriologically confirmed and clinically diagnosed) were, respectively, 90.7% / 97.7% in adults and 88.2% / 100% in children, with 78.6% sensitivity in culture-negative pediatric TB patients, greatly exceeding the WHO's published 4-15% Xpert sensitivity in this cohort. NanoShell-MS results can be obtained within one hour of sample collection compared to 4-6 weeks for conventional culture, and detected decreases in blood Mtb antigen levels within four days of anti-TB treatment initiation. In this proposal we aim to 1) use NanoShell-MS profiling to develop a quantitative prediction model for active TB diagnosis in large, well-described prospective pediatric TB cohorts and 2) determine the utility of our approach for rapid evaluation of treatment efficacy. Our NanoShell-MS assay platform has the advantage that it uses accurate, high-throughput mass spectrometry, which has become a critical means for clinical diagnosis in many parts of the world, while integration of our NanoShell-MS platform with an easy-to-use “miniaturized” (shoebox size) MS system which should also allow this approach to serve pediatric patients in resource-limited areas.