Real-time Detection of Active TB in HIV Exposed Children on CustomizedNanotrap

 描述（由申请人提供）：结核病（TB），特别是多药耐药和广泛耐药结核病（M/XDR-TB），对艾滋病毒暴露儿童构成重大的全球性健康威胁，因为从感染到疾病的进展可能迅速且不可预测。目前儿童和成人结核病诊断和治疗监测的金标准严重依赖耗时的细菌培养或细菌DNA检测（例如GeneXpert MTB/RIF）方法。儿童结核病的临床诊断尤其具有挑战性，因为该疾病的少菌性（很少细菌）和难以获得临床相关标本。甚至样品收集本身也是一项巨大的任务，因为它通常需要侵入性程序（例如，胃抽吸、淋巴结活检和腰椎穿刺）。为了最大限度地减少这种传染病对易受艾滋病毒感染儿童造成的破坏和卫生保健负担，需要对活动性结核进行特异性和定量诊断检测，以早期识别和预防结核病传播。我们最近开发了一种生物标志物检测测定，其将二氧化硅纳米孔芯片（称为Nanotrap）的可调特性与我们的台式基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）的检测能力相结合，其利用了应用于MS的临床化学的最新进展，从而实现了迄今为止未见的不同生物标志物检测模式。我们的初步研究揭示了三个主要发现：（1）使用Nanotrap-MS平台，（CFP-10和ESAT-6）可以在循环中以极低的浓度检测到（低至1.0 fmol），在任何常规技术可以评估细菌负荷之前很好地标记可能的感染的存在;（2）我们使用该平台，仅通过检测患者血液中CFP-10和ESAT-6的存在，而不是执行繁琐的细菌分离程序，将51名HIV+/TB+儿童与27名HIV+/TB-儿童和18名健康对照区分开来，具有100%的特异性和94%的灵敏度;和（3）也许最引人注目的是，样品处理到回答或诊断之间的时间仅为半天，而不是常规方法所需的典型的4-6周时间！在该提案中，我们的目标是通过以下方式使Nanotrap-MS平台更接近于完全临床转化：a）建立简单但极其准确的方案，以定量少量血液等分试样中串联的CFP-10和ESAT-6的量; B）使用从TB感染儿童（也可能是HIV携带者）收集的大样本集，对Nanotrap-MS进行临床验证研究;以及c）研究该平台在监测抗结核疗法的（早期）治疗功效以及筛查可能出现的耐多药/广泛耐药结核方面的可行性。成功执行拟议的研究将有助于减少甚至预防结核病传播的可能性，并改善结核病的临床管理。此外，Nanotrap-MS平台不仅在结核病评估方面具有巨大的潜力，而且在几乎任何临床适应症中也具有广泛的应用，这些临床适应症表现为生物标志物在循环中的释放。

 DESCRIPTION (provided by applicant): Tuberculosis (TB), particularly multidrug and extensively drug-resistant TB (M/XDR-TB), poses a significant, worldwide health threat to HIV-exposed children, as progression from infection to disease can occur rapidly and unpredictably. The current gold standard for TB diagnosis and treatment monitoring in children as well as adults relies heavily on time-consuming bacterial culturing or bacterial DNA detection (e.g. GeneXpert MTB/RIF) methods. Clinical diagnosis of childhood TB is especially challenging because of the paucibacillary (few bacteria) nature of the disease and difficulties in obtaining clinically relevant specimens. Even sample collection is itself an enormous task because it often requires invasive procedures (e.g., gastric aspiration, lymph node biopsy, and lumbar puncture). To minimize the destruction and health care burdens resulting from this infectious disease in vulnerable HIV-infected children, a specific and quantitative diagnostic test for active TB is needed for early identification and prevention of TB transmission. We have recently developed a biomarkers detection assay that couples the tunable properties of silica nanopore chips (referred to as Nanotrap ) to the detection capabilities of our benchtop matrix-assisted laser desorption/ionization time-of flight mass spectrometry (MALDI-TOF MS), which capitalizes on recent advances in clinical chemistry applied to MS that enables a different biomarkers detection modality not seen to date. Our preliminary studies reveal three major findings: (1) with the Nanotrap-MS platform, two TB biomarkers (CFP-10 and ESAT-6) can be detected in circulation at an incredibly low concentration (as little as 1.0 fmol), flagging the presence of possible infection well before any conventional technique can assess bacterial load; (2) we have used the platform to differentiate 51 HIV+/TB+ children from 27 HIV+/TB- children and 18 healthy controls simply by detecting the presence of CFP-10 and ESAT-6 in patients' blood, rather than performing burdensome bacterial isolation procedures, with 100% specificity and 94% sensitivity; and (3) perhaps most strikingly, the time between sample processing to answer, or diagnosis, is only half a day rather than the typical 4-6 week period required for conventional methods! In this proposal, we aim to advance the Nanotrap-MS platform much closer to full clinical translation by: a) establishing a simple but extremely accurate protocol to quantify the amount of CFP-10 and ESAT-6 in tandem in a small blood aliquot; b) performing a clinical validation study for Nanotrap- MS, using a large sample set collected from TB-infected children who may also be HIV carriers; and c) investigating the platform's feasibility in monitoring the (early) treatment efficacy of anti-TB therapies, as well as screening for the possible emergence of M/XDR-TB. Successful execution of the proposed studies will help to diminish the likelihood of or even prevent TB transmission, and improve clinical management of TB. Furthermore, the Nanotrap-MS platform has enormous potential not just for TB disease assessment, but it also has broad applications in virtually any clinical indication that manifests in biomarker release int circulation.