title: Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic corynebacteria.
abstract: Toxigenic corynebacteria are uncommon in the UK; however, laboratory confirmation by the national reference laboratory can inform public health action according to national guidelines. Standard phenotypic tests for identification and toxin expression of isolates can take from ≥24 to ≥48 h from receipt. To decrease the time to result, a real-time PCR (qPCR) assay was developed for confirmation of both identification of Corynebacterium diphtheriae and Corynebacterium ulcerans/Corynebacterium pseudotuberculosis and detection of the diphtheria toxin gene. Target genes were the RNA polymerase β-subunit-encoding gene (rpoB) and A-subunit of the diphtheria toxin gene (tox). Green fluorescent protein DNA (gfp) was used as an internal process control. qPCR results were obtained within 3 to 4 h after receipt of isolate. The assay was validated according to published guidelines and demonstrated high diagnostic sensitivity (100 %), high specificity (98-100 %) and positive and negative predictive values of 91 to 100 % and 100 %, respectively, compared to both block-based PCR and the Elek test, together with a greatly reduced time from isolate receipt to reporting. Limitations of the qPCR assay were the inability to distinguish between C. ulcerans and C. pseudotuberculosis and that the presence of the toxin gene as demonstrated by qPCR may not always predict toxin expression. Thus, confirmation of expression of diphtheria toxin is always sought using the phenotypic Elek test. The new qPCR assay was formally introduced as the front-line test for putative toxigenic corynebacteria to inform public health action in England and Wales on 1 April 2014.
authors.affiliation: Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England, Microbiology Reference Services, National Infection Service, London, UK.
authors.forename: Aruni
authors.initials: A
authors.lastname: De Zoysa
authors.affiliation: Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England, Microbiology Reference Services, National Infection Service, London, UK.
authors.forename: Androulla
authors.initials: A
authors.lastname: Efstratiou
authors.affiliation: Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England, Microbiology Reference Services, National Infection Service, London, UK.
authors.forename: Ginder
authors.initials: G
authors.lastname: Mann
authors.affiliation: Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England, Microbiology Reference Services, National Infection Service, London, UK.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Harrison
authors.affiliation: Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England, Microbiology Reference Services, National Infection Service, London, UK.
authors.forename: Norman K
authors.initials: NK
authors.lastname: Fry
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D003352:Corynebacterium / Q000235:genetics* / Q000302:isolation & purification*; D003354:Corynebacterium Infections / Q000175:diagnosis* / Q000382:microbiology*; D003353:Corynebacterium diphtheriae / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D016925:Corynebacterium pseudotuberculosis / Q000235:genetics / Q000302:isolation & purification*; D004165:Diphtheria / Q000175:diagnosis* / Q000382:microbiology; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000235:genetics; D004739:England; D006801:Humans; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D004167:Diphtheria Toxin