title: A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility.
abstract: OBJECTIVE
  The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxacin susceptibility.
  
  
  METHODS
  The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (n = 70), a panel of commensal Neisseria and Moraxella species (n = 55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acid amplification test (NAAT) method (n = 171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (n = 210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available.
  
  
  RESULTS
  The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates (sensitivity = 100%, 95% CI = 94.9%-100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT-negative clinical samples gave negative results by the GyrA91-PCR (specificity = 100%, 95% CI = 98.4%-100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CI = 88.5%-95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to ciprofloxacin in 161 of 162 (99.4%, 95% CI = 96.6%-99.9%) samples.
  
  
  CONCLUSIONS
  The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples can predict ciprofloxacin susceptibility in N. gonorrhoeae.
authors.affiliation: Queensland Paediatric Infectious Diseases Laboratory, Queensland Children's Medical Research Institute, Brisbane, Queensland 4029, Australia University of Queensland Child Health Research Centre, Brisbane, Queensland 4029, Australia.
authors.forename: Cameron
authors.initials: C
authors.lastname: Buckley
authors.affiliation: Queensland Paediatric Infectious Diseases Laboratory, Queensland Children's Medical Research Institute, Brisbane, Queensland 4029, Australia University of Queensland Child Health Research Centre, Brisbane, Queensland 4029, Australia.
authors.forename: Ella
authors.initials: E
authors.lastname: Trembizki
authors.affiliation: Kirby Institute, UNSW Australia, Sydney, New South Wales 2052, Australia.
authors.forename: Basil
authors.initials: B
authors.lastname: Donovan
authors.affiliation: Melbourne Sexual Health Centre, Alfred Health, Carlton, Melbourne, Victoria 3053, Australia Central Clinical School, Monash University, Melbourne, Victoria 3181, Australia.
authors.forename: Marcus
authors.initials: M
authors.lastname: Chen
authors.affiliation: Microbiology Laboratory, Pathology Department, Royal Darwin Hospital, Darwin, Northern Territory, Australia.
authors.forename: Kevin
authors.initials: K
authors.lastname: Freeman
authors.affiliation: Kirby Institute, UNSW Australia, Sydney, New South Wales 2052, Australia.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Guy
authors.affiliation: WHO Collaborating Centre for STD, Microbiology Department, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, New South Wales 2031, Australia.
authors.forename: Ratan
authors.initials: R
authors.lastname: Kundu
authors.affiliation: WHO Collaborating Centre for STD, Microbiology Department, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, New South Wales 2031, Australia.
authors.forename: Monica M
authors.initials: MM
authors.lastname: Lahra
authors.affiliation: Kirby Institute, UNSW Australia, Sydney, New South Wales 2052, Australia.
authors.forename: David G
authors.initials: DG
authors.lastname: Regan
authors.affiliation: Public Health Microbiology, Communicable Disease, Queensland Health Forensic and Scientific Services, Archerfield, Brisbane, Queensland, Australia.
authors.forename: Helen
authors.initials: H
authors.lastname: Smith
authors.affiliation: Queensland Paediatric Infectious Diseases Laboratory, Queensland Children's Medical Research Institute, Brisbane, Queensland 4029, Australia University of Queensland Child Health Research Centre, Brisbane, Queensland 4029, Australia UQ Centre for Clinical Research (UQCCR), The University of Queensland, Brisbane, Queensland 4029, Australia d.whiley@uq.edu.au.
authors.forename: David M
authors.initials: DM
authors.lastname: Whiley
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D005838:Genotype; D006801:Humans; D009016:Moraxella / Q000187:drug effects / Q000235:genetics; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000900:Anti-Bacterial Agents; D002939:Ciprofloxacin; D027081:DNA Gyrase