title: Evaluation of a rapid molecular screening approach for the detection of toxigenic Clostridium difficile in general and subsequent identification of the tcdC Δ117 mutation in human stools.
abstract: We have developed and validated a rapid molecular screening protocol for toxigenic Clostridium difficile, that also enables the identification of the hypervirulent epidemic 027/NAP1 strain. We describe a multiplex real-time PCR assay, which detects the presence of the tcdA and tcdB genes directly in stool samples. In case of positive PCR results, a separate multiplex real-time PCR typing assay was performed targeting the tcdC gene frame shift mutation at position 117. We prospectively compared the results of the screening PCR with those of a cytotoxicity assay (CTA), and a rapid immuno-enzyme assay for 161 stool samples with a specific request for diagnosis of C. difficile infection (CDI). A total of 16 stool samples were positive by CTA. The screening PCR assay confirmed all 16 samples, and gave a PCR positive signal in eight additional samples. The typing PCR assay detected the tcdC Δ117 mutation in 2/24 samples suggesting the presence of the epidemic strain in these samples. This was confirmed by PCR ribotyping and sequencing of the tcdC gene. Using CTA as the "gold standard", the sensitivity, specificity, positive predictive value, and negative predictive value, for the screening PCR were 100%, 94.4%, 66.7%, and 100%, respectively. In conclusion, PCR may serve as a rapid negative screening assay for patients suspected of having CDI, although the low PPV hamper the use of PCR as a standalone test. However, PCR results may provide valuable information for patient management and minimising the spread of the epidemic 027/NAP1 strain.
authors.affiliation: Department of Research & Development, Laboratory for Infectious Diseases, Groningen, The Netherlands. r.de.boer@infectielab.nl
authors.forename: R F
authors.initials: RF
authors.lastname: de Boer
authors.forename: J J
authors.initials: JJ
authors.lastname: Wijma
authors.forename: T
authors.initials: T
authors.lastname: Schuurman
authors.forename: J
authors.initials: J
authors.lastname: Moedt
authors.forename: B G
authors.initials: BG
authors.lastname: Dijk-Alberts
authors.forename: A
authors.initials: A
authors.lastname: Ott
authors.forename: A M D
authors.initials: AM
authors.lastname: Kooistra-Smid
authors.forename: Y T H P
authors.initials: YT
authors.lastname: van Duynhoven
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D016360:Clostridioides difficile / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology; D005243:Feces / Q000382:microbiology*; D016368:Frameshift Mutation; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012097:Repressor Proteins / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; C105615:TcdC protein, Clostridium difficile