OBJECTIVES: Surveillance for @entity6368 @entity4580 resistance is of growing importance given increasing use of @entity2756 and @entity4580 dual therapy for gonorrhoea treatment. In this study, we developed two real-time PCR methods for direct detection of two key @entity6368 23S rRNA mutations associated with @entity4580 resistance. METHODS: The real-time PCR assays, 2611-PCR and 2059-PCR, targeted the @entity2954 23S rRNA @entity68148 and @entity40782 mutations, respectively. A major design challenge was that @entity2954 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 @entity2954 (n=70) and non- @entity2954 (n=28) Neisseria species. Analytical specificity was further assessed by testing @entity6368 nucleic acid amplification test (NAAT)-negative clinical samples (n=90), before being applied to @entity6368 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- @entity2954 species. For the @entity6368 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 @entity2954 2611 and 2059 23S rRNA @entity2036 were both successfully characterized in 266/306 (87%) of the @entity6368 NAAT-positive clinical specimens. CONCLUSIONS: Real-time PCR detection of @entity2954 23S rRNA mutations directly from clinical samples is feasible and may enhance culture- and non-culture-based @entity6368 resistance surveillance.

Direct real-time PCR-based detection of XXXX 23S rRNA mutations associated with @entity4580 resistance.

@entity2954 :: ('MESH:D006069', 'Disease') :: ['gonococcal'] @entity2756 :: ('MESH:D002443', 'Chemical') :: ['ceftriaxone'] @entity6368 :: ('485', 'Species') :: ['Neisseria gonorrhoeae', 'N. gonorrhoeae'] @entity2036 :: ('MESH:D009711', 'Chemical') :: ['nucleotides'] @entity4580 :: ('MESH:D017963', 'Chemical') :: ['azithromycin'] @entity40782 :: ('c|SUB|A|2059|G', 'DNAMutation') :: ['A2059G'] @entity68148 :: ('c|SUB|C|2611|T', 'DNAMutation') :: ['C2611T']
@entity6368 :: (485,Species) :: ['Neisseria gonorrhoeae']
