Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance

Trembizki, Ella, Buckley, Cameron, Donovan, Basil, Chen, Marcus, Guy, Rebecca, Kaldor, John, Lahra, Monica M., Regan, David G., Smith, Helen, Ward, James and Whiley, David M. (2015) Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance. Journal of Antimicrobial Chemotherapy, 70 12: 3244-3249. doi:10.1093/jac/dkv274


Author Trembizki, Ella
Buckley, Cameron
Donovan, Basil
Chen, Marcus
Guy, Rebecca
Kaldor, John
Lahra, Monica M.
Regan, David G.
Smith, Helen
Ward, James
Whiley, David M.
Title Direct real-time PCR-based detection of Neisseria gonorrhoeae 23S rRNA mutations associated with azithromycin resistance
Journal name Journal of Antimicrobial Chemotherapy   Check publisher's open access policy
ISSN 1460-2091
0305-7453
Publication date 2015-09-03
Year available 2015
Sub-type Article (original research)
DOI 10.1093/jac/dkv274
Open Access Status Not yet assessed
Volume 70
Issue 12
Start page 3244
End page 3249
Total pages 6
Place of publication Oxford, United Kingdom
Publisher Oxford University Press
Collection year 2016
Language eng
Formatted abstract
Objectives: 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.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
School of Medicine Publications
Child Health Research Centre Publications
 
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Created: Tue, 17 Nov 2015, 14:40:53 EST by Ella Trembizki on behalf of UQ Centre for Clinical Research