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

描述（由申请人提供）：炎症性肠病（IBD），即溃疡性结肠炎（UC）和克罗恩病（CD），是慢性、终身、复发性疾病，影响近100万美国人，美国医疗保健系统每年花费约20亿美元。IBD病因不明，无法治愈，发病率正在增加。我们已经发现，IBD中粘膜相关或腔壁粘附微生物种群的多样性降低。我们还确定了LH-PCR指纹中可能表明IBD相关模式的推定峰。此外，我们的LH-PCR指纹与克隆序列的连接表明某些细菌群存在于IBD患者的粘膜植物群中。这些发现使我们假设存在粘附于回结肠壁的细菌的保护性生物膜以及IBD中管腔细菌对该保护性生物膜的入侵（生态失调）。我们的初步结果表明，在这种保护性生物膜沿着结肠和回肠变化不大。我们的初步观察还表明，管腔/粪便微生物菌群可能是疾病状态的指示。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识别来监测微生态失调的可行性。我们的第二个目标是通过焦磷酸测序粘膜、管腔、直肠拭子和粪便样本来充分表征这些微生物群。如果我们证明粪便卡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生态失调的最可行，最无创和最简单的测试。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分析。目的是在临床表现明显之前识别发作，从而防止有症状的发作伴肠道损伤。这可以明显改善生活质量，防止手术并发症。使用环境微生物学的强大技术来表征人类微生物区系的研究可以在理解这些疾病方面取得重大进展。越来越多的人认识到微生物群落在健康和疾病中的重要性，不仅限于IBD，而且在其他领域，如结直肠癌发生，肠道感染，乳糜泻，肠易激综合征，益生元和益生菌疗法。该提案涉及首次使用一种复杂的和高度可重复的细菌指纹工具，LH-PCR，在胃肠道微生物菌群的研究。为该提案组建的团队是跨学科的，并结合了三套非常有价值的专业知识来解决GI微生物群落的复杂性，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来分析复杂的数据集。基于LH-PCR的新诊断工具可用于常规实时临床使用，作为该提议的直接结果。然后，这种工具可以用于帮助确定疾病的存在/不存在;并预测疾病的各种表型表现、疾病过程和治疗效果。如果对照和IBD的微生物区系之间的明显差异被定义为拟定的，则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是粘膜附着细菌模式的可靠代表，直肠拭子是粘膜活检的替代品，那么我们的诊断测试将是最可行的，非侵入性的和简单的测试来评估生态失调。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分析。此外，患者（或初级保健医生）可以安全地将拭子插入直肠并在直肠粘膜上摩擦，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分析。

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.