A fully integrated assay and platform for detecting Clostridium difficile

DESCRIPTION (provided by applicant): Antibiotic treatment of infections with harmful bacteria can destroy the normal colonic flora, and increase the patient's susceptibility to Clostridium difficile-associated disease (CDAD). Pathogenic strains of C. difficile commonly produce two large clostridial toxins, A (TcdA) and B (TcdB), responsible for CDAD. The TcdC gene is a negative regulator of the TcdA and TcdB genes, and the most highly virulent strain of C. difficile, BI/NAP1, has a single point mutation in this gene that results in a premature stop codon. The lack of a functional TcdC protein in these strains results in an over expression of the TcdA and TcdB toxins. This strain is associated with more severe morbidity and mortality in patients. Simple assays that are robust and specific for CDAD and in particular for the BI/NAP1 strain are needed to facilitate better diagnosis and treatment of C. difficile infected patients. The current, widely used, EIA tests are rapid but lack sensitivity whereas the more sensitive tests (cytotoxicity assay or C. difficile culture) require one or more days to complete. There is also clearly a need for a reliable, sensitive, and expeditious method for testing of environmental contamination. A sensitive assay that is capable of processing and detecting C. diff spores would substantially protect the health of the patient as well as reduce the risk of spread of CDAD in hospitals, nursing homes, and rehabilitation facilities. Most cases of remission are due to re-infection. Decontamination is very difficult and currently not supported by a reliable and sensitive method of verification. In this Phase I, we propose to develop a simple assay system for the detection of the TcdA gene, the TcdB gene and the TcdC point mutation using proprietary helicase-dependent amplification (HAD ) technology. HDA reactions will detect TcdA for the presence and load of Clostridium difficile and TcdC D117 mutation for the presence of the highly virulent strain of C. difficile. We will develop a point-of-care (POC) instrument that will extract DNA from stool samples and environmental swabs. The sample processing methods selected for this plan are robust and simple, avoiding the need for wash steps, elution buffers, or centrifugation. This will be accomplished through the development of subsystem modules, each designed in a cartridge that is independently functional to its purpose, is disposable after one use, and can also be snapped together to the other modules for integrated function. This sample processing system will be integrated with the fluorimeter, a real-time fluorescent detection system that has been previously used with the HDA assay method. PUBLIC HEALTH RELEVANCE: There is a clear clinical need for a rapid but sensitive test for C. difficile infection. The current, widely used, EIA tests are rapid but lack sensitivity whereas the more sensitive tests (cytotoxicity assay or C. difficile culture) require one or more days to complete. There is also clearly a need for a reliable, sensitive and expeditious method for testing for environmental contamination. A sensitive assay that is capable of processing and detecting C. diff spores would substantially protect the health of patients and reduce the cost of healthcare in hospitals, nursing homes and rehabilitation facilities. The goal of this project is to develop a point-of-care system for the detection of C. difficile.