A low complexity rapid molecular method for detection of Clostridium difficile in stool

• Published in: PloS one 2014-01, Vol.9 (1), p.e83808-e83808
• Main Authors: McElgunn, Cathal J, Pereira, Clint R, Parham, Nicholas J, Smythe, James E, Wigglesworth, Michael J, Smielewska, Anna, Parmar, Surendra A, Gandelman, Olga A, Brown, Nicholas M, Tisi, Laurence C, Curran, Martin D
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0083808

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.