Development of a LH-PCR Based Diagnostic for Inflammatory Bowel Disease

描述(申请人提供)：炎症性肠病(IBD)，即溃疡性结肠炎(UC)和克罗恩病(CD)，是一种慢性、终生、反复发作的疾病，影响近100万美国人，每年给美国医疗保健系统造成约20亿美元的损失。IBDS的病因不明，无法治愈，发病率正在上升。我们发现IBD患者黏膜相关或管腔壁粘附菌群的多样性降低。我们还确定了可能与IBD相关的模式的黄体生成素-聚合酶链式反应指纹中的推测峰。此外，我们的黄体生成素-聚合酶链式反应指纹图谱与克隆序列的链接表明，在IBD患者的黏膜菌群中存在特定的细菌群。这些发现使我们假设，在IBD中，存在附着在回结肠壁上的细菌的保护性生物膜，以及管腔细菌对这一保护性生物膜的入侵(生物失调)。我们的初步结果表明，沿着结肠和回肠的这种保护性生物膜几乎没有变化。我们的初步观察还表明，肠腔/粪便微生物区系可能指示疾病状态。我们的目标是确定通过对患者采集的直肠拭子和粪便样本进行指纹分析来监测生物失调的可行性。我们的第二个目标是通过对粘膜、肠腔、直肠拭子和粪便样本进行焦磷酸测序来充分表征这些微生物群落。如果我们证明粪便卡片和直肠拭子是管腔和粘膜细菌模式的可靠代表，那么我们的诊断测试将是评估失调的最可行、非侵入性和容易的测试。患者可以将粪便放在卡片上(或者医生可以在直肠检查后将粪便放在卡片上)，并将其邮寄到我们的中心实验室进行细菌指纹鉴定。目标是在临床表现出来之前识别发作，从而防止有症状的发作并损害肠道。这可以明显提高生活质量，防止手术并发症的发生。利用环境微生物学中强大的技术来描述人类微生物区系的研究可以在理解这些疾病方面带来重大进展。人们越来越认识到微生物区系对健康和疾病的重要性，不仅限于IBD，而且在其他领域也是如此，如结直肠癌发生、肠道感染、乳糜泻、肠易激综合征、益生菌和益生菌治疗。这项提议涉及首次在胃肠道微生物区系研究中使用一种复杂且高度重复性的细菌指纹分析工具--黄体生成素-聚合酶链式反应。为这项建议组建的团队是跨学科的，并结合了三组非常有价值的专业知识，以解决GI微生物区系的复杂性，即临床IBD专业知识、环境微生物学专业知识和生物信息学专业知识，以分析复杂的数据集。基于黄体生成素-聚合酶链式反应的新诊断工具可作为该提议的直接结果用于常规实时临床使用。这样的工具可以用来帮助确定疾病的存在/缺失，并预测疾病的各种表型表现、病程和治疗效果。如果对照的微生物区系与IBD之间的明显差异被定义为建议的那样，那么实施具有重大商业潜力的IBD的临床诊断也是可行的。如果我们证明粪便卡片是粘膜附着细菌模式的可靠代表，而直肠拭子是粘膜活检的替代方法，那么我们的诊断试验将是评估生物失调最可行、无创和容易的试验。患者可以将粪便放在卡片上(或者医生可以在直肠检查后将粪便放在卡片上)，并将其邮寄到我们的中心实验室进行细菌指纹鉴定。此外，患者(或初级保健医生)可以安全地将棉签插入直肠，并将其摩擦到直肠黏膜上，获得样本，然后将其发送到我们的中心实验室进行细菌指纹鉴定。

DESCRIPTION (provided by applicant): Inflammatory Bowel Diseases (IBDs), namely ulcerative colitis (UC) and Crohn's disease (CD), are chronic, lifelong, relapsing illnesses, affecting close to 1 million Americans and costing approximately 2 billion dollars/year to the US healthcare system. IBDs are of unknown cause, have no cure and are increasing in incidence. We have found that the diversity of the mucosa associated or luminal wall adherent microbial populations is decreased in IBD. We also identified putative peaks in LH-PCR fingerprints that may indicate patterns associated with IBD. Furthermore, linkage of our LH-PCR fingerprints to clone sequences show that certain bacterial groups are present in the mucosal flora of IBD patients. These findings have led us to hypothesize that there is a protective biofilm of bacteria adhering to the Ileocolonic wall and an invasion of this protective biofilm by lumen bacteria in IBD (dysbiosis). Our initial results indicate that there is little variation in this protective biofilm along the colon and ileum. Our initial observations also indicate that the lumen/stool microflora may be indicative of the disease state. Our goal is to determine the feasibility of monitoring dysbiosis by fingerprinting rectal swabs and stool samples collected by the patient. Our second aim is to fully characterize these microflora communities by pyrosequencing mucosal, lumen, rectal swab, and stool samples. If we demonstrate that stool cards and rectal swabs are a reliable representation of the lumen and mucosal bacterial patterns, then our diagnostic test will be the most feasible, noninvasive and easy test to assess dysbiosis. Patients can place their stool on the cards (or physicians can place the stool on the card after rectal examination) and mail them to our central laboratory for bacterial fingerprinting. The goal is to identify flare up before it becomes clinically apparent and thus prevent the symptomatic flare-ups with damage to the intestinal track. This could clearly improve quality of life and prevent the complication of surgery. Studies that characterize microflora in humans using powerful techniques from environmental microbiology can bring about significant advances in the understanding of these illnesses. There is a growing recognition of the importance of microflora in health and disease not limited to IBD but also in other areas such as colorectal carcinogenesis, intestinal infections, celiac disease, irritable bowel syndrome, prebiotic and probiotic therapies. This proposal involves the first time use of a sophisticated and highly reproducible bacterial fingerprinting tool, LH-PCR, in the study of microflora in the GI tract. The team assembled for this proposal is interdisciplinary and combines three very valuable sets of expertise to address the complexity of the GI microflora, namely clinical IBD expertise, environmental microbiology expertise, and bioinformatics expertise to analyze complex sets of data. New diagnostic tools based on LH-PCR can be utilized for routine real-time clinical use as direct result of this proposal. Such a tool can then be used to help determine the absence/presence of disease; and predict the various phenotypic presentations of the disease, the disease course, and treatment effects. If distinct differences between the microflora of controls and IBD are defined as proposed, it would be also feasible to implement a clinical diagnostic for IBD with major commercial potential. If we demonstrate that stool card is a reliable representation of the mucosal attached bacterial pattern and rectal swabs are a surrogate for mucosal biopsies, then our diagnostic test will be the most feasible, noninvasive and easy test to assess dysbiosis. Patients can place their stool on the cards (or physicians can place the stool on the card after rectal examination) and mail them to our central laboratory for bacterial fingerprinting. Furthermore, patients (or primary care physicians) can safely insert the swab in the rectum and rub it against the rectal mucosa and obtain the sample and then send it to our central laboratory for bacterial fingerprinting.