title: Rapid diagnosis of Clostridium difficile infection by multiplex real-time PCR.
abstract: The gold standards for the diagnosis of Clostridium difficile infections (CDIs) are the cytotoxicity assay and the toxigenic culture. However, both methods are time-consuming and the results are not available before 24-48 h. We developed and evaluated a multiplex in-house real-time polymerase chain reaction (PCR) assay for the simultaneous detection of toxigenic strains of C. difficile and the presumptive identification of the epidemic NAP1/027/BI strain from stools. Amplifications were performed using specific primers for tcdB and tcdC on an ABI Prism 7300 (Applied Biosystems). The detection of amplicons was done using TaqMan probes. The analytical sensitivity of the multiplex real-time PCR for detecting tcdB was estimated to 10 CFU/g of stools. This assay was assessed from 881 consecutive unformed stools from patients suspected of having CDI. The gold standard was the toxigenic culture for the diagnosis of CDI and PCR ribotyping for the identification of the NAP1/027/BI strain. The prevalence of positive toxigenic culture was 9.31%. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 86.59%, 97.43%, 78.02%, and 98.57%, respectively, for the real-time PCR and 70.73%, 100%, 100%, and 97.08%, respectively, for the cytotoxicity assay.
authors.affiliation: National Reference Laboratory for C. difficile, Paris VI University, Paris, France. frederic.barbut@sat.aphp.fr
authors.forename: F
authors.initials: F
authors.lastname: Barbut
authors.forename: M
authors.initials: M
authors.lastname: Monot
authors.forename: A
authors.initials: A
authors.lastname: Rousseau
authors.forename: S
authors.initials: S
authors.lastname: Cavelot
authors.forename: T
authors.initials: T
authors.lastname: Simon
authors.forename: B
authors.initials: B
authors.lastname: Burghoffer
authors.forename: V
authors.initials: V
authors.lastname: Lalande
authors.forename: J
authors.initials: J
authors.lastname: Tankovic
authors.forename: J-C
authors.initials: JC
authors.lastname: Petit
authors.forename: B
authors.initials: B
authors.lastname: Dupuy
authors.forename: C
authors.initials: C
authors.lastname: Eckert
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D016360:Clostridioides difficile / Q000235:genetics / Q000302:isolation & purification*; D003015:Clostridium Infections / Q000175:diagnosis* / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000382:microbiology; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012097:Repressor Proteins / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D015345:Oligonucleotide Probes; D012097:Repressor Proteins; C105615:TcdC protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile