Methods and Compositions for the Detection of Functional Clostridium Difficile Toxins

Market and Background: Clostridium difficile (C. difficile) is the leading identifiable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. This bacterium has been implicated as the causative organism for 10-25% of the reported cases of antibiotic-associated diarrhea, 50-75% of antibiotic-associated colitis, and 90- 100% of pseudomembranous colitis. The toxigenic strains of C. difficile produce two notable proteins, toxin A and toxin B, which are important virulent factors in the pathogenesis of C. difficile. Both toxins have the same enzymatic cleavage activity and are cytotoxic to cultured cells; however, toxin B is 100 to 1,000-fold more potent than toxin A in most cell lines.

Competitors and Current Problems: Current clinical methods for diagnosing C. difficile infections are mostly qualitative tests that detect either the bacteria or the toxins. Current tests rely on a combination of at least two techniques, which may include culture isolation, PCR detection of the toxin-encoding genes, the Cyto-assay, and immunological detection of the toxins (ELISA). These tests require a second confirmatory assay to determine if a strain is pathogenic. The alarming emergence of hypervirulent strains of C. difficile with increased toxin production, severity of disease, morbidity, and mortality emphasizes the need for a culture method that permits simultaneous isolation and detection of virulent strains. C. difficile strains can either be toxin-producing (toxigenic) or non-toxin producing (non-toxigenic). Only toxin A and/ or toxin B-producing strains cause disease. Current culture methods do not differentiate toxigenic and non-toxigenic strains, because they are unable to detect toxin activity.

The Technology: Dr. Herbert DuPont and colleagues at UTHealth have developed a quantitative C. difficile activity assay that enables cost-efficient, sensitive, quantitative measurement of the cleavage activities of toxin A and toxin B of C. difficile in both a culture supernatant and a selective and differential agar-based assay. The plate assay enables identification of toxin-producing C. difficile without the need for additional toxin-confirmatory tests by taking advantage of unique cleavage characteristics of the toxins to differentiate activity simultaneously with detection.