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 关于耗时的细菌培养或无法定量的DNA测试，以检测是否存在少量 细菌。儿科结核病的管理尤其困难，因为目前基于生物体的方法 诊断和治疗监测对儿童来说是资源密集型、侵入性和不够敏感的， 容易患上少杆菌病。由于免疫学上的不成熟，年幼的儿童患上 疾病结核病进展迅速，发病率和死亡率很高。对他们治疗反应的评估 依赖主观措施，限制儿童获得急需的更短和更安全的结核病方案。更多 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……里面 为了解决目前儿科结核病管理中的局限性，我们建议开发一种快速诊断 与分枝杆菌分离无关的方法，用于定量低分子结核分枝杆菌(Mtb) 患者血液样本中的抗原(CFP-10和ESAT-6)和结核病相关宿主标记IP-10。ESAT-6 和CFP-10是检测活动性结核病及其治疗反应的理想生物标志物，IP-10对此有补充作用 结核分枝杆菌特有的蛋白质组模式将提供患者在治疗期间的病理生理变化的完整轮廓 抗结核治疗，同时也提高了诊断的敏感性。我们的策略是利用空洞的、聚焦于能量的 具有针对Mtb CFP-10和ESAT-6和主机IP-10的定制抗体的二氧化钛纳米壳功能化 多肽与高通量质谱学(MS)相结合，以提高诊断的敏感性和特异性。我们 评估了292名成人的仅Mtb抗原版本的纳米壳-MS平台(不含抗IP-10纳米壳) 102名儿科患者和对照，选自五个高度相关的队列(成人、儿科和潜伏性结核病， 艾滋病毒/结核病混合感染和非结核分枝杆菌感染)，来自我们的全球合作者。纳米壳质谱 对活动性结核(细菌学证实和临床诊断)的敏感性和特异性分别为， 成人为90.7%/97.7%，儿童为88.2%/100%，对培养阴性儿童结核的敏感性为78.6% 患者，大大超过世卫组织公布的该队列中4-15%的Xpert敏感度。纳米壳质谱结果 可在样本采集一小时内获得，而传统培养为4-6周，以及 在开始抗结核治疗的四天内，检测到血液中结核分枝杆菌抗原水平下降。在本建议书中 我们的目标是：1)利用纳米壳质谱技术建立活动性肺结核诊断的定量预测模型 大型、描述良好的儿童结核病前瞻性队列和2)决定了我们的方法在快速 治疗效果评价。我们的纳米壳-MS分析平台的优势是它使用了准确的， 高通量质谱学，已成为临床诊断的关键手段在许多地区 将我们的NanShell-MS平台与易于使用的“小型化”(鞋盒大小)集成在一起。 MS系统，也应该允许这种方法在资源有限的地区为儿科患者服务。

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.