Journal Article

A Low Complexity Rapid Molecular Method for Detection of Clostridium difficile in Stool

Parmar, Surendra A., Gandelman, Olga A., McElgunn, Cathal J., Brown, Nicholas M., Curran, Martin D., Wigglesworth, Michael J., Smielewska, Anna, Tisi, Laurence C., Pereira, Clint R., Smythe, James E., Parham, Nicholas J.

2014

Overview

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB(+), n = 111; tcdB(-), n= 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours. In a further study (tcdB(+), n = 47; tcdB(-), n= 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB(+), n = 25; tcdB(-), n= 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Biology / Clostridium difficile / Clostridium difficile - genetics / Clostridium difficile - isolation & purification / Complexity / Cytotoxicity / Diarrhea / Elution / Feces - microbiology / Gene Dosage / Gene expression / Health surveillance / Hospitals / Humans / Laboratories / Medicine / Methods / Models, Biological / Molecular Diagnostic Techniques - methods / Public health / Reagent Kits, Diagnostic / Real-Time Polymerase Chain Reaction / Reference Standards / Sensitivity / Sensitivity and Specificity / Silica / Silicon dioxide / Test procedures / Toxin A / Toxin B / Toxins