Detection of Host Response In Clostridium Difficile Infection

 描述（由申请人提供）：本研究申请的目的是为一种全球重要但诊断不佳的医疗保健相关传染病（即艰难梭菌感染（CDI））的诊断检测开发范式转变。CDI是由肠内C.艰难梭菌，一种革兰氏阳性、孢子形成、专性厌氧细菌。它能引起从中度腹泻到严重结肠炎（毒性）的各种疾病 巨结肠），死亡风险高。在过去的二十年中，CDI在医疗机构中重新出现，死亡率增加了近10倍，医疗成本显著增加。目前的CDI诊断仅限于结合临床症状检测生物体和/或其毒素产物，并且不能区分感染患者与简单定殖患者，从而导致不准确的诊断以及抗生素误用/过度使用。为了克服这些局限性和解决不确定性的CDI诊断，一个突破性的概念CDI诊断将开发和评估在本研究项目。该项目的基本原理是C。艰难梭菌毒素B的作用机制导致结肠上皮细胞的细胞骨架破坏，释放非肌肉原肌球蛋白（Tm）。我们的假设是，患者粪便中Tm（结肠上皮细胞骨架中的一种主要蛋白质）水平的增加表明了一种特异性的 宿主对CDI的应答，可作为诊断C.很难应用目的是开发和评价粪便标本中的Tm检测作为CDI临床显著表现的生物标志物。为了实现这一目标，我们将追求两个具体目标：1）确定临床上适当的粪便Tm检测的可行性。这一目标将通过执行多项任务来实现-生成Tm特异性抗体、优化粪便标本中的Tm回收方法、利用模拟CDI阴性和阳性标本的质控品开发和优化Tm检测方案以及检测潜在干扰。2)评价粪便Tm作为从临床不同患者人群中获得的标本的CDI特异性宿主应答标志物。为此，将使用CDI阳性和阴性临床标本进行Tm检测，并将数据与相同标本的临床和微生物学记录进行比较。由于获得和使用了临床微生物实验室的剩余样本，因此可以获得这些记录。CDI诊断的特异性将在来自其他肠道细菌病原体所致腹泻患者的粪便标本上进行验证（E。大肠杆菌、沙门氏菌、志贺氏菌和弯曲杆菌）、寄生虫、腹泻相关性腹泻（非感染所致）和肠道疾病（如炎症性肠病）。这一建议的概念具有很大的潜力，允许开发一种新的诊断应用CDI。它将作为下一代传染病诊断的一个例子，测量宿主反应，而不是检测病原体或其产物。我们的长期目标是通过与行业合作伙伴（如TechLab（r），Inc.）合作开发宿主反应CDI测试，作为一种商业化的检测方法，可以接受FDA的许可试验。

 DESCRIPTION (provided by applicant): The aim of this research application is to develop a paradigm shift in diagnostic testing for a globally important, yet poorly diagnosed, healthcare-associated infectious disease, namely Clostridium difficile infection (CDI). CDI results from infection of the bowel by C. difficile, a Gram-positive, spore-forming, obligate anaerobic bacterium. It can cause (diarrheal) illness ranging from moderate diarrhea to severe colitis (toxic megacolon) with a high risk of death. For the last two decades CDI has re-emerged in healthcare facilities with nearly a 10- fold increase in mortality and significant excess healthcar cost. Current CDI diagnostics is limited to detection of the organism and/or its toxin product(s) i conjunction with clinical symptoms, and does not differentiate infected from simply colonized patients, thus leading to inaccurate diagnosis as well as antibiotic mis/overuse. To overcome these limitations and resolve uncertainty of CDI diagnosis, a ground-breaking concept for CDI diagnosis will be developed and evaluated during this research project. The project rationale is that C. difficile toxin B mechanism of action causes cytoskeletal disruption of colonic epithelial cells with release of non- muscle tropomyosin (Tm). Our hypothesis is that increased levels of Tm, a major protein in colonic epithelial cytoskeleton, in the patient's stool indicates a specific host response to CDI and can be used as a diagnostic target signifying active infection from C. difficile. The application objective is to develop and evaluate Tm detection in stool specimens as a biomarker for clinically significant manifestations of CDI. To accomplish it, we will pursue two specific aims: 1) Determine the feasibility of clinically appropriate fecal Tm detection. This aim will be accomplished by performing multiple tasks - generation of Tm specific antibodies, optimization of Tm recovery method from stool specimens, Tm detection protocol development and optimization utilizing controls mimicking CDI negative and positive specimens, and testing for potential interferences. 2) Evaluate fecal Tm as a CDI specific host response marker on specimens obtained from clinically diverse patient populations. For that, CDI positive and negative clinical specimens will be utilized for Tm detection and data comparison to clinical and microbiological records of the same specimens. The records are available due to access and use of remnant samples from the clinical microbiology laboratory. The specificity of CDI diagnostics will be validated on stool specimens from patients with diarrhea due to other enteric bacterial pathogens (E. coli, Salmonella, Shigella spp, and Campylobacter), parasites, antibiotic-associated diarrhea (not due to infection), and bowel disorders like inflammatory bowel disease. This proposed concept has great potential to allow development of a novel diagnostic application for CDI. It will serve as an example for the next generation of infectious disease diagnostics measuring host response instead of detecting pathogens or their products. Our long term goal is to improve patient care by delivering a host response CDI test ready for development with an industry partner, such as TechLab(r), Inc., as a commercial assay that can undergo FDA clearance trials.