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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.

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