title: Enhancing gonococcal antimicrobial resistance surveillance: a real-time PCR assay for detection of penicillinase-producing Neisseria gonorrhoeae by use of noncultured clinical samples.
abstract: With increasing concerns regarding diminishing treatment options for gonorrhea, maintaining the efficacy of currently used treatments and ensuring optimal Neisseria gonorrhoeae antimicrobial resistance surveillance are of the utmost importance. Penicillin is still used to treat gonorrhea in some parts of the world. In this study, we developed and validated a real-time PCR assay for the detection of penicillinase-producing N. gonorrhoeae (PPNG) in noncultured clinical samples with the aim of enhancing penicillin resistance surveillance. The assay (PPNG-PCR2) was designed to be an indirect marker of penicillinase activity, by targeting a region of sequence predicted to be conserved across all N. gonorrhoeae plasmid types harboring the beta-lactamase gene while not specifically targeting the actual beta-lactamase-encoding sequence. The assay was evaluated by using a total of 118 N. gonorrhoeae clinical isolates and 1,194 clinical specimens, including 239 N. gonorrhoeae-positive clinical samples from which N. gonorrhoeae cells were isolated and for which phenotypic penicillinase results are available. Overall, the PPNG-PCR2 assay provided 100% sensitivity and 98.7% specificity compared to bacterial culture results for the detection of PPNG in clinical specimens. PPNG-PCR2 false-positive results, presumably due to cross-reactions with unrelated bacterial species, were observed for up to 1.3% of clinical samples but could be distinguished on the basis of high cycle threshold values. In tandem with phenotypic surveillance, the PPNG-PCR2 assay has the potential to provide enhanced epidemiological surveillance of N. gonorrhoeae penicillin resistance and is of particular relevance to regions where penicillin is still used to treat gonorrhea.
authors.affiliation: Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital & Health Service District, Herston Road, Herston, Queensland 4029, Australia.
authors.forename: Namraj
authors.initials: N
authors.lastname: Goire
authors.forename: Kevin
authors.initials: K
authors.lastname: Freeman
authors.forename: John W
authors.initials: JW
authors.lastname: Tapsall
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Lambert
authors.forename: Michael D
authors.initials: MD
authors.lastname: Nissen
authors.forename: Theo P
authors.initials: TP
authors.lastname: Sloots
authors.forename: David M
authors.initials: DM
authors.lastname: Whiley
mesh_terms: D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D006069:Gonorrhea / Q000382:microbiology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009344:Neisseria gonorrhoeae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D010405:Penicillinase / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D010405:Penicillinase