title: A new rapid method for Clostridium difficile DNA extraction and detection in stool: toward point-of-care diagnostic testing.
abstract: We describe a new method for the rapid diagnosis of Clostridium difficile infection, with stool sample preparation and DNA extraction by heat and physical disruption in a single-use lysis microreactor (LMR), followed by a rapid PCR amplification step. All steps can be accomplished in <20 minutes overall. Gel electrophoresis is currently used to detect the amplification product, pending real-time availability with an ultra-rapid thermocycler. Compared with the dual enzyme immunoassay (EIA) screening test (C. diff Quik Chek Complete; Techlab, Blacksburg, VA), the novel LMR/PCR assay showed complete concordance with all glutamate dehydrogenase (GDH) results (GDH(+)/toxin(+), n = 48; GDH(-)/toxin(-), n = 81). All 69 stool samples with discordant EIA results (GDH(+)/toxin(-)) were tested by both the LMR/PCR assay and the loop-mediated isothermal amplification test (LAMP) (Illumigene C. difficile; Meridian Bioscience, Cincinnati, OH). In 64/69 EIA-discordant samples, LAMP and LMR/PCR results matched (both positive in 29 sample and both negative in 35 samples); in the remaining 5 samples, results were discrepant between the LAMP assay (all five negative) and the LMR/PCR assay (all 5 positive). Overall, LMR/PCR testing matched the current algorithm of EIA and/or LAMP reflex testing in 193/198 (97.5%) samples. The present proof-of-concept study suggests that the novel LMR/PCR technique described here may be developed as an inexpensive, rapid, and reliable point-of-care diagnostic test for C. difficile infection and other infectious diseases.
authors.affiliation: Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA. afreifeld@unmc.edu
authors.forename: Alison G
authors.initials: AG
authors.lastname: Freifeld
authors.forename: Kari A
authors.initials: KA
authors.lastname: Simonsen
authors.forename: Christine S
authors.initials: CS
authors.lastname: Booth
authors.forename: Xing
authors.initials: X
authors.lastname: Zhao
authors.forename: Scott E
authors.initials: SE
authors.lastname: Whitney
authors.forename: Teresa
authors.initials: T
authors.lastname: Karre
authors.forename: Peter C
authors.initials: PC
authors.lastname: Iwen
authors.forename: Hendrik J
authors.initials: HJ
authors.lastname: Viljoen
mesh_terms: D001431:Bacteriological Techniques; D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification*; D003015:Clostridium Infections / Q000175:diagnosis*; D004761:Enterocolitis, Pseudomembranous / Q000175:diagnosis*; D005243:Feces / Q000382:microbiology*; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D019095:Point-of-Care Systems; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D005969:Glutamate Dehydrogenase