Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance.
OBJECTIVE
Surveillance for Neisseria gonorrhoeae azithromycin resistance is of growing importance given increasing use of ceftriaxone and azithromycin dual therapy for gonorrhoea treatment. In this study, we developed two real-time PCR methods for direct detection of two key N. gonorrhoeae 23S rRNA mutations associated with azithromycin resistance.


METHODS
The real-time PCR assays, 2611-PCR and 2059-PCR, targeted the gonococcal 23S rRNA C2611T and A2059G mutations, respectively. A major design challenge was that gonococcal 23S rRNA sequences have high sequence homology with those of commensal Neisseria species. To limit the potential for cross-reaction, 'non-template' bases were utilized in primer sequences. The performance of the methods was initially assessed using a panel of gonococcal (n = 70) and non-gonococcal (n = 28) Neisseria species. Analytical specificity was further assessed by testing N. gonorrhoeae nucleic acid amplification test (NAAT)-negative clinical samples (n = 90), before being applied to N. gonorrhoeae NAAT-positive clinical samples (n = 306).


RESULTS
Cross-reactions with commensal Neisseria strains remained evident for both assays; however, cycle threshold (Ct) values were significantly delayed, indicating reduced sensitivity for non-gonococcal species. For the N. gonorrhoeae NAAT-negative clinical samples, 7/21 pharyngeal samples provided evidence of cross-reaction (Ct values >40 cycles); however, the remaining urogenital and rectal swab samples were negative. In total, the gonococcal 2611 and 2059 23S rRNA nucleotides were both successfully characterized in 266/306 (87%) of the N. gonorrhoeae NAAT-positive clinical specimens.


CONCLUSIONS
Real-time PCR detection of gonococcal 23S rRNA mutations directly from clinical samples is feasible and may enhance culture- and non-culture-based N. gonorrhoeae resistance surveillance.
Queensland Paediatric Infectious Diseases (QPID) Laboratory, Queensland Children's Medical Research Institute, Brisbane, Queensland 4029, Australia UQ Child Health Research Centre, School of Medicine, The University of Queensland, Brisbane, Queensland 4029, Australia elina.trembizki@uq.net.au.
Ella
E
Trembizki
Queensland Paediatric Infectious Diseases (QPID) Laboratory, Queensland Children's Medical Research Institute, Brisbane, Queensland 4029, Australia UQ Child Health Research Centre, School of Medicine, The University of Queensland, Brisbane, Queensland 4029, Australia.
Cameron
C
Buckley
Kirby Institute, UNSW Australia, Sydney, New South Wales 2052, Australia Sydney Sexual Health Centre, Sydney Hospital, Sydney, New South Wales 2000, Australia.
Basil
B
Donovan
Melbourne Sexual Health Centre, Alfred Health, Carlton, Victoria 3053, Australia Central Clinical School, Monash University, Melbourne, Victoria 3800, Australia.
Marcus
M
Chen
Kirby Institute, UNSW Australia, Sydney, New South Wales 2052, Australia.
Rebecca
R
Guy
Kirby Institute, UNSW Australia, Sydney, New South Wales 2052, Australia.
John
J
Kaldor
WHO Collaborating Centre for STD, Microbiology Department, South Eastern Area Laboratory Services, Prince of Wales Hospital, Sydney, New South Wales 2031, Australia.
Monica M
MM
Lahra
Kirby Institute, UNSW Australia, Sydney, New South Wales 2052, Australia.
David G
DG
Regan
Public Health Microbiology, Public and Environmental Health, Queensland Health Forensic and Scientific Services, Archerfield, Queensland 4107, Australia.
Helen
H
Smith
South Australian Health and Medical Research Institute, Adelaide, South Australia 5000, Australia.
James
J
Ward
Queensland Paediatric Infectious Diseases (QPID) Laboratory, Queensland Children's Medical Research Institute, Brisbane, Queensland 4029, Australia UQ Child Health Research Centre, School of Medicine, The University of Queensland, Brisbane, Queensland 4029, Australia.
David M
DM
Whiley
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017963:Azithromycin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D060005:Genotyping Techniques / Q000379:methods*; D006069:Gonorrhea / Q000175:diagnosis / Q000382:microbiology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D009344:Neisseria gonorrhoeae / Q000187:drug effects* / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
D000900:Anti-Bacterial Agents; D012338:RNA, Ribosomal, 23S; D017963:Azithromycin