title: Determination of Neisseria gonorrhoeae susceptibility to ciprofloxacin in clinical specimens from men using a real-time PCR assay.
abstract: A real-time PCR (RT-PCR) assay was modified to simultaneously detect Neisseria gonorrhoeae and to determine gonococcal susceptibility to ciprofloxacin using clinical samples. The modified RT-PCR assay was validated using DNA extracted from 40 linked isolates and urethral swabs, 24 of which had linked first-pass urine samples, obtained from men presenting with urethral gonorrhoea. The RT-PCR assay enabled amplification of N. gonorrhoeae dcmH, gyrA and parC genes. The quinolone resistance-determining regions (QRDRs) of the isolates' gyrA and parC genes were sequenced. Following successful validation, 33 first-pass urine-derived DNA extracts, obtained from men with gonorrhoea, were tested with the assay and results were compared with blinded ciprofloxacin susceptibility data. Gonococcal susceptibility to ciprofloxacin correlated perfectly with gyrA amplicon generation. No gyrA amplicons were detected for gonococcal infections due to ciprofloxacin-intermediate/resistant organisms. Amplification of parC correlated less well with ciprofloxacin susceptibility phenotypes. Simultaneous non-generation of gyrA and parC amplicons consistently predicted the presence of ciprofloxacin-resistant gonococci. Characteristic point mutations in the gyrA/parC QRDRs were found in DNA amplified from those extracts that failed to produce gyrA/parC amplicons. The RT-PCR assay performed well with DNA extracted from first-pass urine specimens and results correlated perfectly with ciprofloxacin susceptibility phenotypes. In conclusion, the modified RT-PCR assay can detect N. gonorrhoeae in DNA extracted from first-pass urine specimens of men with urethral gonorrhoea and accurately predicts gonococcal susceptibility to ciprofloxacin. This molecular assay provides a useful tool for surveillance and patient management in settings where fluoroquinolones can still be used for treatment of gonorrhoea.
authors.affiliation: Centre for HIV and STIs, National Institute for Communicable Diseases, National Health Laboratory Service, 1 Modderfontein Road, Sandringham 2192, South Africa.
authors.forename: M P
authors.initials: MP
authors.lastname: Magooa
authors.forename: E E
authors.initials: EE
authors.lastname: Müller
authors.forename: L
authors.initials: L
authors.lastname: Gumede
authors.forename: D A
authors.initials: DA
authors.lastname: Lewis
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005798:Genes, Bacterial; D005838:Genotype; D006069:Gonorrhea / Q000382:microbiology*; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D002939:Ciprofloxacin