005.1 Real-time pcr and melt curve analysis targeting gyra gene for prediction of ciprofloxacin resistance in clinical neisseria gonorrhoeae isolates. (13th September 2015) - Hemarajata, P
Abstract : Introduction: Increasing antimicrobial resistance in Neisseria gonorrhoeae has been a major problem worldwide, limiting effective empirical therapeutic options in patients infected with multi-drug resistant strains. Guidelines from U.S. CDC no longer recommend treatment with fluoroquinolones (FQs) due to emergence of resistance nationally. However, current prevalence of resistance in the US is still low at 12% and treatment with FQs may be a viable option for susceptible isolates. A rapid molecular test predicting FQ susceptibility in N. gonorrhoeae isolates would help physicians in determining an effective treatment plan for each patient. Methods: Twenty-three ciprofloxacin (CIP)-susceptible and 77 CIP-resistant clinical N. gonorrhoeae isolates were obtained from Neisseria Reference Laboratory at University of Washington and grown on chocolate agar plates. To determine the association between mutations in gyrA gene and CIP resistance in those isolates, we extracted DNA from culture and performed real-time PCR on a Lightcycler 480 based on the HybProbe system targeting gyrA gene followed by melt curve genotyping. Results: Melt curve genotyping analysis demonstrated wild-type melt patterns in all (100%) 23 CIP-susceptible isolates, while all 77 (100%) CIP-resistant isolates demonstrated mutant melt patterns. Conclusion: There was a 100% concordance between PCR melt genotypes and CIP susceptibility among all clinical isolates tested. The assay is currently being validated for testing on DNA extracted directly from clinical specimens ultimately to be offered for use in clinical practice. Disclosure of interest statement: This study was funded in part by NIH R21AI109005.