Diagnostic tools to link Bacteroides fragilis toxin isotypes and human disease

描述(由申请人提供)：拟议研究的目标是开发一系列诊断分析方法，以检测特定类型的产肠毒素脆弱类杆菌(ETBF)。ETBF已被证明会导致腹泻、肠炎和结肠炎。实验证据表明，ETBF会诱发结肠癌，一项人类研究报告称，ETBF与人类结直肠癌(CRC)有关。研究表明ETBF菌株在儿童和成人中广泛分布，尽管ETBF在腹泻疾病中的发病率和流行率以及在美国结直肠癌高危人群中的携带率都不清楚。ETBF引起的腹泻和肠炎是由ETBF分泌的一种金属蛋白酶毒素--脆弱芽孢杆菌毒素(BFT)引起的。已知的BFT有3种亚型，即BFT-1、BFT-2和BFT-3，但目前尚不清楚哪种BFT亚型(S)会导致人类最严重的疾病。目前可用于ETBF的测试费时、费力、昂贵且缺乏敏感度。此外，目前还没有可靠的测试来确定在没有结合分子分析的厌氧粪便培养的患者中存在哪些ETBF亚型。对于定义疾病流行病学和了解临床结果，开发快速和特异的分析以确定存在哪种ETBF亚型是至关重要的。我们建议开发三种检测方法，每种方法都能够识别出现严重肠炎、腹泻或结肠肿瘤的患者中的同型特异性ETBF感染。第一种是基于聚合酶链式反应的检测，它将直接从粪便样本中定量检测和鉴定ETBF的特定同种类型。第二种是基于ELISA的检测，它将使用针对已知的BFT同型的单抗，直接从粪便样本中识别持续感染是由特定的一个或多个同型引起的。这两种分析结合起来将是一种比单独使用更强大的诊断工具，因为它们是基于不同类型的大分子标签的存在。第三种也是一种基于酶联免疫吸附试验的检测方法，它将检测人类血清样本中针对单个BFT亚型的免疫球蛋白。腹泻病仍然是全世界发病率和死亡率的主要原因。长期的后遗症包括肠易激综合征和儿童生长迟缓。在美国，结直肠癌是导致男性和女性癌症死亡的第二大原因。因此，确定ETBF产生不同的BFT亚型与人类腹泻、结肠炎和可能的结直肠癌之间的联系将影响公众健康，并可能导致新疗法的开发。成功完成这一第一阶段的应用将为测试分析的灵敏度和有效性奠定基础，这将是第二阶段项目的一部分。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop a series of diagnostic assays that will detect specific types of enterotoxigenic Bacteroides fragilis (ETBF). ETBF has been shown to cause diarrhea as well as bowel inflammation and colitis. Experimental evidence indicates that ETBF induces colon tumors and one human study reported an association of ETBF with human colorectal cancer (CRC). Studies have shown that ETBF strains are widely distributed in both children and adults, although neither the incidence and prevalence of ETBF in diarrheal diseases nor carriage in those at risk for CRC in the United States are known. ETBF- induced diarrhea and bowel inflammation is due to a metalloprotease toxin, B. fragilis toxin (BFT), secreted by ETBF. There are 3 known isotypes of BFT, BFT-1, BFT-2 and BFT-3, but it is unknown which BFT isotype(s) cause the most severe disease in humans. Current available tests for ETBF are lengthy, laborious, expensive and lack sensitivity. Furthermore, there are no tests available to determine reliably which isotypes of ETBF are present in patients without anaerobic stool culture combined with molecular analyses. It is critical for defining disease epidemiology and understanding clinical outcomes to develop fast and specific assays to identify which isotype of ETBF is present. We propose to develop three assays that will each have the capability of identifying isotype-specific ETBF infections in patients presenting with severe bowel inflammation, diarrhea or colon neoplasia. The first is a PCR-based assay that will detect and identify, quantitatively, specific isotypes of ETBF directly from stool samples. The second is an ELISA-based assay that will use monoclonal antibodies directed against the known isotypes of BFT, to identify directly from stool samples whether ongoing infections are due to a specific isotype or isotypes. These two assays will be a more powerful diagnostic tool in combination than either alone, because they are based on the presence of different types of macromolecular tags. The third is also an ELISA-based assay, which will detect immunoglobulins specific to individual BFT isotypes in human serum samples. Diarrheal diseases remain a leading cause of morbidity and mortality throughout the world. Long term sequelae include irritable bowel syndrome and growth delay in children. Colorectal cancer is the second leading cause of cancer death in men and women in the United States. Therefore determining the link between ETBF producing differing BFT isotypes and diarrheal diseases, colitis and possibly colorectal cancer in humans will influence public health and may lead to the development of novel therapeutics. Successful completion of this Phase I application will lay the groundwork for testing the sensitivity and efficacy of the assays, which will be part of the Phase II project.