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

Ella Trembizki, Cameron Buckley, Basil Donovan, Marcus Chen, Rebecca Guy, John Kaldor, Monica Lahra, David Regan, Helen Smith, James Ward, David Whiley

Research output: Contribution to journalArticlepeer-review

29 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)3244-3249
Number of pages6
JournalJournal of Antimicrobial Chemotherapy
Issue number12
Publication statusPublished - 1 Dec 2015


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