Quantification of Circulating Antigens for Pediatric TB Diagnosis and Treatment Monitoring

 描述(申请人提供)：成人和儿童结核病(TB)诊断和治疗监测的传统方案严重依赖耗时的细菌培养或无法量化的DNA检测方法来检测少量细菌的存在。对于儿童结核病，诊断和治疗尤其困难，因为目前的临床方案对这些年轻患者要求很高。虽然他们只占卫生保健工作量的一小部分，但同时感染人类免疫缺陷病毒(艾滋病毒)和结核病的儿童是死亡率最高的最脆弱群体之一。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能够做的事情的界限。为了解决目前儿童结核病临床治疗的局限性，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，以定量检测低分子结核分枝杆菌(Mtb)抗原(CFP-10和ESAT-6)。ESAT-6和CFP-10的特性使它们成为活动性结核病诊断和结核病疫苗开发的理想生物标志物。我们的策略结合了能量介导型多孔硅纳米盘(简称pSiND)和高通量质谱仪(NanDisk-MS)，以提高灵敏度和特异度。我们评估了我们的平台，包括292名成人患者和102名儿科患者和对照，这些患者和对照是从全球多个研究所提供的五个高度相关的队列(活动性结核病、艾滋病毒/结核病合并感染、儿童结核病、潜伏性结核病和非结核分枝杆菌感染)中挑选出来的。成人(90.7%/97.7%)和儿童(88.2%/100%)诊断活动性肺结核的灵敏度和特异度分别为90.7%和88.2%。循环抗原的绝对定量在检测抗分枝杆菌开始治疗4天后的治疗反应中是有用的。同样重要的是，我们可以在样本到答案处理的一个小时内提供准确的诊断，而不是等待典型的4-6周。在这项建议中，我们的目标是：1)在cGMP设施中设计和开发符合FDA的放大纳米盘微制造协议；2)使用来自大量艾滋病毒/结核病儿童队列的样本对pSiND-MS进行广泛的临床验证；3)确定我们用于快速评估疗效的方法的有效性；以及4)优化便携式pSiDN-Minims系统的开发和临床验证，以用于CFP-10和ESAT-6的识别和定量。PSiND-MS技术平台还有一个额外的优势，那就是高通量和准确的质谱学已经成为世界许多地区临床诊断的基本技术。这个鞋盒大小的小型易于使用的MS系统旨在为资源有限地区的患者提供服务。我们所有目标的实现将显著改变Clinica全球结核病控制的管理策略，并可能通过量化病原体的循环抗原来改进对其他传染病的诊断。

 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.