Abstract
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. 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). Cross-reactions with commensal Neisseria strains remained evident for both assays; however, cycle threshold (Ct) values were significantly delayed, indicating reduced sensitivity for ...Continue Reading
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