C. difficile: Novel Gene Detection Method

Stephanie Angione PhD Candidate Brown University School of Engineering Center for Biomedical EngineeringMedicalResearch.com Interview with:
Stephanie Angione
PhD Candidate
Brown University School of Engineering
Center for Biomedical Engineering

MedicalResearch.com: What are the main findings of the study?

Answer: This study demonstrates the application of a novel nucleic acid detection platform to detect Clostridium difficile (C. difficile) in subjects presenting with acute diarrheal symptoms. This method amplifies three genes associated with C. difficile infection as well as genes associated with virulence attributed to the NAP1/027/BI strain. The novel PCR assay allows for simple and rapid detection of three C. difficile genes: tcdB, cdtB, and tcdC, which code for C. difficile toxin B, C. difficile binary toxin, and a protein suspected to regulate toxin production, which includes the NAP1/027/BI tcdC variant. Amplification of DNA from the tcdB, tcdC and cdtB genes can be carried out using a droplet sandwich platform that performs real-time polymerase chain reaction (PCR) in microliter droplets for the detection and identification of amplified fragments of DNA. Our technique of multiplex gene amplification provides a unique method that is both sensitive and specific to rapidly detect C. difficile in patient stool samples that can be adapted to point-of-care testing.

MedicalResearch.com: Were any of the findings unexpected?

Answer: Our identification of Clostridium difficile in a small pilot study of clinical samples was as expected, as we found 100% of the positive samples to contain both tcdB and tcdC. In a study of 32 total samples, 12 were negative for all C. difficile DNA, and 20 were C. difficle and tcdB positive. Of the positive samples, 11 contained the full gene fragment of tcdC, 7 contained the deletion of 18-bp, and 2 contained the deletion of 39-bp. The same 9 samples containing some deletion in tcdC were also positive for cdtB. Those strains that were found to be indicative of the NAP1/027/BI strain contained the 18-bp deletion in the tcdC gene segment, as well as the gene segment for cdtB. The 2 samples containing larger deletions in tcdC at 39-bp, as well as cdtB, were indicative of another hypervirulent strain; the 078 ribotype strain.

MedicalResearch.com: What should clinicians and patients take away from your report?

Answer: We have developed technology which we believe serves as the basis for a point-of-care C. difficile test that would rapidly differentiate hypervirulent strains from other C. difficile strains.  The latter finding may assist clinicians in treatment choices and possibly assist in patient placement based on the expected level of care needed for individual patients, particularly those with the NAP1/027/BI strain. This may help physicians to devise specific treatment regimens and may assist in contact isolation strategies necessary to address the challenge of C. difficile-associated diarrhea in healthcare facilities and community settings.

MedicalResearch.com: What recommendations do you have for future research as a result of this study?

Answer: The device and identification system are simple in design and can be integrated as a point-of-care test to help rapidly detect and identify Clostridium difficile strains that pose significant health threats in hospitals and other health care communities. Future research can be done to fully develop a point-of-care device for typing of Clostridium difficile and larger studies of clinical samples can be done utilizing the assay and device. As the rapid detection of these three important markers of infection not only indicates the presence of C. difficile, but also provides physicians with clinically relevant data regarding infection with a hypervirulent strain. Thus, the wider use of strain typing of C. difficile stool isolates can provide more detailed data regarding patient outcomes and symptoms as correlated with strain.

Citation:

A Novel Subtyping Assay for Detection of Clostridium difficile Virulence Genes
Journal of Molecular Diagnostics

Stephanie L. Angione, Aartik A. Sarma, Leonard A. Mermel, Anubhav Tripathi
Journal of Molecular Diagnostics, The
15 January 2014