A real-time PCR assay for direct characterization of the Neisseria gonorrhoeae GyrA 91 locus associated with ciprofloxacin susceptibility

Cameron Buckley, Ella Trembizki, Basil J Donovan, Marcus Y Chen, Kevin Freeman, Rebecca Jane Guy, Ratan Kundu, Monica M Lahra, David Regan, Helen V Smith, David Whiley

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13 Citations (Scopus)

Abstract

Objectives: The objective of this study was to develop a real-time PCR method for specific detection of the gonococcal GyrA amino acid 91 locus directly in clinical samples so as to predict Neisseria gonorrhoeae ciprofloxac insusceptibility.

Methods: The real-time PCR assay, GyrA91-PCR, was designed using two probes, one for detection of the WT S91 sequence and the other for detection of the S91F alteration. The performance of the assay was initially assessed using characterized N. gonorrhoeae isolates (n¼70), a panel of commensal Neisseria and Moraxella species(n¼55 isolates) and clinical samples providing negative results by a commercial N. gonorrhoeae nucleic acidamplification test (NAAT) method (n¼171). The GyrA91-PCR was then applied directly to N. gonorrhoeae NAAT-positive clinical samples (n¼210) from the year 2014 for which corresponding N. gonorrhoeae isolates with susceptibility results were also available.

Results: The GyrA91-PCR accurately characterized the GyrA 91 locus of all 70 N. gonorrhoeae isolates(sensitivity¼100%, 95% CI¼94.9%–100%), whereas all non-gonococcal isolates and N. gonorrhoeae NAAT negative clinical samples gave negative results by the GyrA91-PCR (specificity¼100%, 95% CI¼98.4%–100%). When applied to the 210 N. gonorrhoeae NAAT-positive clinical samples, the GyrA91-PCR successfully characterized 195 samples (92.9%, 95% CI¼88.5%–95.9%). When compared with the corresponding bacterial culture results, positivity by the GyrA91-PCR WT probe correctly predicted N. gonorrhoeae susceptibility to cipro-floxacin in 161 of 162 (99.4%, 95% CI¼96.6%–99.9%) samples.

Conclusions: The use of a PCR assay for detection of mutation in gyrA applied directly to clinical samples canpredict ciprofloxacin susceptibility in N. gonorrhoeae.
Original languageEnglish
Pages (from-to)353 - 356
Number of pages4
JournalJournal of Antimicrobial Chemotherapy
Volume71
Issue number2
DOIs
Publication statusPublished - 2016

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