Diagnostic tools to link Bacteroides fragilis toxin isotypes and human disease

描述（由申请方提供）：拟定研究的目标是开发一系列诊断检测方法，用于检测特定类型的产肠毒素脆弱类杆菌（ETBF）。ETBF已被证明会引起腹泻以及肠道炎症和结肠炎。实验证据表明ETBF诱导结肠肿瘤，并且一项人类研究报告了ETBF与人类结直肠癌（CRC）的关联。研究表明，ETBF菌株广泛分布于儿童和成人中，尽管在美国，ETBF在结肠炎疾病中的发病率和患病率以及在CRC风险人群中的携带率均未知。ETBF诱导的腹泻和肠道炎症是由于金属蛋白酶毒素B引起的。脆弱毒素（fragilis toxin，BFT），由ETBF分泌。BFT有3种已知的同种型，BFT-1、BFT-2和BFT-3，但不知道哪种BFT同种型在人类中引起最严重的疾病。目前可用于ETBF的测试是冗长、费力、昂贵的并且缺乏灵敏度。此外，没有可用的测试来可靠地确定哪些ETBF同种型存在于没有结合分子分析的厌氧粪便培养的患者中。它是定义疾病流行病学和理解临床结果的关键，以开发快速和特异性的检测，以确定ETBF的同种型是存在的。我们建议开发三种检测方法，每种方法都有能力识别严重肠道炎症、腹泻或结肠肿瘤患者的同种型特异性ETBF感染。第一种是基于PCR的测定，其将直接从粪便样品中定量检测和鉴定ETBF的特异性同种型。第二种是基于ELISA的检测，该检测将使用针对已知BFT同种型的单克隆抗体，直接从粪便样本中鉴定正在进行的感染是否是由于特定的同种型或同种型。这两种检测方法相结合将是一种比单独使用更强大的诊断工具，因为它们基于不同类型的大分子标签的存在。第三种也是基于ELISA的测定，其将检测人血清样品中对单个BFT同种型特异性的免疫球蛋白。腹泻病仍然是全世界发病率和死亡率的主要原因。长期后遗症包括肠易激综合征和儿童生长迟缓。结直肠癌是美国男性和女性癌症死亡的第二大原因。因此，确定产生不同BFT同种型的ETBF与人类结肠炎疾病、结肠炎和可能的结肠直肠癌之间的联系将影响公共卫生，并可能导致新疗法的开发。第一阶段申请的成功完成将为检测试剂盒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.