Diagnostic tools to link Bacteroides fragilis toxin isotypes and human disease

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 presenceof 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 throughoutthe 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. PUBLIC HEALTH RELEVANCE: Colitis and bowel inflammation affect millions of people (both children and adults) each year in the United States. One cause of these diseases is ETBF (Enterotoxigenic Bacteroides fragilis). This application will develop diagnostic assays to detect ETBF in stool from patients with colitis and bowel inflammation and also determine whether patients were exposed to ETBF in the past.