title: A low complexity rapid molecular method for detection of Clostridium difficile in stool.
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.
authors.affiliation: Lumora Ltd, Ely, Cambridgeshire, United Kingdom.
authors.forename: Cathal J
authors.initials: CJ
authors.lastname: McElgunn
authors.affiliation: Lumora Ltd, Ely, Cambridgeshire, United Kingdom.
authors.forename: Clint R
authors.initials: CR
authors.lastname: Pereira
authors.affiliation: Public Health England, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Parham
authors.affiliation: Lumora Ltd, Ely, Cambridgeshire, United Kingdom.
authors.forename: James E
authors.initials: JE
authors.lastname: Smythe
authors.affiliation: Lumora Ltd, Ely, Cambridgeshire, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Wigglesworth
authors.affiliation: Public Health England, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Anna
authors.initials: A
authors.lastname: Smielewska
authors.affiliation: Public Health England, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Surendra A
authors.initials: SA
authors.lastname: Parmar
authors.affiliation: Lumora Ltd, Ely, Cambridgeshire, United Kingdom.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Gandelman
authors.affiliation: Public Health England, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Brown
authors.affiliation: Lumora Ltd, Ely, Cambridgeshire, United Kingdom.
authors.forename: Laurence C
authors.initials: LC
authors.lastname: Tisi
authors.affiliation: Public Health England, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge, Cambridgeshire, United Kingdom.
authors.forename: Martin D
authors.initials: MD
authors.lastname: Curran
mesh_terms: D016360:Clostridioides difficile / Q000235:genetics* / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology*; D018628:Gene Dosage; D006801:Humans; D008954:Models, Biological; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D012015:Reference Standards; D012680:Sensitivity and Specificity
chemical_list: D011933:Reagent Kits, Diagnostic