Quantification of Circulating Antigens for Pediatric TB Diagnosis and Treatment Monitoring

 描述（由申请人提供）：成人和儿童结核病（TB）诊断和治疗监测的常规方案严重依赖于耗时的细菌培养或无法定量的DNA检测方法，以检测是否存在少量细菌。对于儿童结核病，诊断和治疗特别困难，因为当前的临床方案对这些年轻患者的要求很高。同时感染人体免疫机能丧失病毒（艾滋病毒）和结核病的儿童虽然只占保健工作量的一小部分，但他们是最脆弱的群体之一，死亡率最高。我们选择这个具有挑战性的队列（由NIAID赞助的临床试验提供）来推动我们的技术平台可以做的事情。为了解决目前儿科TB临床管理中的局限性，我们开发了一种不依赖于分枝杆菌分离的快速血液诊断方法，以定量低分子量结核分枝杆菌（Mtb）抗原（CFP-10和ESAT-6）。ESAT-6和CFP- 10的特性使它们成为活动性TB诊断的理想生物标志物和TB疫苗开发的候选物。我们的策略结合了能量介导的多孔硅纳米盘（称为“pSiND”），其用对Mtb抗原肽具有高度特异性的定制抗体功能化，以及高通量质谱（Nanostructure-MS），用于灵敏度和特异性的双重增强。我们评估了我们的平台，其中292名成人和102名儿童患者和对照组选自5个高度相关的队列（活动性TB、HIV/TB合并感染、儿童TB、潜伏性TB和非TB分枝杆菌感染），这些队列由全球多家机构提供。成人和儿童活动性肺结核诊断的敏感性和特异性分别为90.7% / 97.7%和88.2% / 100%。循环抗原的绝对定量在检测抗分枝杆菌治疗开始后4天的治疗反应中是有用的。同样重要的是，我们可以在一个小时的样本到答案处理内做出准确的诊断，而不是等待典型的4-6周。在该提案中，我们的目标是：1）在cGMP设施中设计和开发符合FDA标准的按比例放大的纳米盘微制造方案; 2）使用来自患有HIV/TB的儿童的大队列的样品进行pSiND-MS的广泛临床验证; 3）确定我们的方法用于快速评估治疗功效的有效性;以及4）优化用于鉴定和定量CFP-10和ESAT-6的便携式pSiDN-miniMS系统的开发和临床验证。pSiND-MS技术平台具有额外的优势，因为高通量和准确的质谱分析已成为世界许多地区临床诊断的基本技术。“小型化和易于使用的MS系统在一个鞋盒大小的护理点应用的目的是服务于病人在资源有限的地区。我们所有目标的实现将显著改变全球结核病控制的临床管理策略，并可能通过量化病原体的循环抗原来改善其他传染病的诊断。

 DESCRIPTION (provided by applicant): Conventional protocols for adult and pediatric tuberculosis (TB) diagnosis and treatment monitoring rely heavily on time-consuming bacterial culture or unquantifiable DNA detection methods for the presence of small numbers of bacteria. For pediatric TB, diagnosis and treatment are particularly difficult because current clinical protocols demand much from these young patients. Although they comprise a small percentage of the health care caseload, children who are co-infected with human immunodeficiency virus (HIV) and TB represent one of the most vulnerable groups with one of the highest mortality rates. We choose this challenging cohort (provided by a NIAID-sponsored clinical trial) to push the boundaries of what our technology platform can do. In order to address the current limitations in clinical management of pediatric TB, we have developed a rapid blood-based diagnostic method independent of mycobacterial isolation to quantify the low molecular- weight Mycobacterium tuberculosis (Mtb) antigens (CFP-10 and ESAT-6). Characteristics of ESAT-6 and CFP- 10 make them ideal biomarkers for active TB diagnosis and candidates for TB vaccine development. Our strategy combines energy mediating porous silicon nanodisks (referred to as "pSiND"), functionalized with customized antibodies highly specific to Mtb antigen peptides, and high-throughput mass spectrometry (NanoDisk-MS) for dual enhancement of sensitivity and specificity. We evaluated our platform with 292 adult and 102 pediatric patients and controls chosen from five highly relevant cohorts (active TB, HIV/TB co- infection, pediatric TB, latent TB, and non-TB mycobacterial infection), provided by multiple institutes worldwide. Sensitivities and specificities of adults (90.7% / 97.7%) and children (88.2% / 100%) were achieved in active TB identification. Absolute quantification of circulating antigens was informative in detecting treatment response four days after anti-mycobacterial initiation. Just as important, we could render accurate diagnoses within one hour of sample-to-answer processing rather than wait the typical 4-6 weeks. In this proposal, we aim to: 1) design and develop the scale-up nanodisk microfabrication protocol with FDA compliant in cGMP facilities; 2) conduct an extensive clinical validation of pSiND-MS using samples from a large cohort of children with HIV/TB; 3) determine effectiveness of our approach for rapid evaluation of treatment efficacy; and 4) optimize development and clinical validation of a portable pSiDN-miniMS system for identification and quantification of CFP-10 and ESAT-6. The pSiND-MS technology platform has an added advantage in that high-throughput and accurate mass spectrometry has become a virtually essential technology for clinical diagnosis in many parts of the world. The "miniaturized and easy-to-use MS system at a shoebox size for point-of-care applications is aimed at serving patients in resource-limited areas. Achievement of all of our aims will significantly alter clinica management strategies for global TB control, and potentially improve diagnosis of other infectious diseases by quantifying circulating antigens of pathogenic organisms.