Abstract
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.
Highlights
Clostridium difficile is a Gram positive bacterium that is the most frequent cause of infectious bacterial diarrhea worldwide [1]
Typical Heat Elution (HE)-Loopmediated Isothermal Amplification (LAMP)-Bioluminescent Assay in Real-Time (BART) results The LAMP-BART results from one of the runs from the comparison with Illumigene are presented in Figure 2 and Table 2
If the Inhibitor Control peak time was later than 35 minutes a retest was necessary if the C. difficile test for that sample was negative
Summary
Clostridium difficile is a Gram positive bacterium that is the most frequent cause of infectious bacterial diarrhea worldwide [1]. An epidemic of CDI with continuously increasing rates was seen in the USA, Canada and most of Europe starting around 2000– 2002. This epidemic was mostly associated with the hypervirulent ribotype 027/NAP1 B1 strain. Mandatory surveillance was introduced in April 2007 and confirms this fall in cases This may be due to the emergence and subsequent decline of hypervirulent strains of C. difficile (e.g., ribotype 027/NAP1, responsible for 55% of isolates ribotyped in 2007/08, but only 12% of isolates in 2010/11) [9]. The 027/NAP1 strain has an expanded repertoire of antibiotic resistance elements, produces greater quantities of toxins, and gives rise to more severe disease with higher mortality rates and an increased probability of relapse following clinical treatment [10,11]
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