![[ Visit Client Website ]](images/banner.gif)
Background: The incidence of antimicrobial resistant Neisseria gonorrhoeae continues to rise in Canada. Due to increased Nucleic Acid Amplification Testing (NAAT) for gonorrhea diagnosis, antimicrobial resistance data on ~70% of gonorrhea infections diagnosed by NAAT yearly is not available. A molecular method was developed to monitor antimicrobial resistance in N. gonorrhoeaeusing bacterial cultures and NAAT specimens.
Methods: N. gonorrhoeae multi antigen sequence type (NG-MAST) was determined and real-time (RT-) PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in six genes associated with extended-spectrum cephalosporin (ESC) or ciprofloxacin (CIP) resistance (ponA, mtrR, penA, gyrA, parC and porB) in 110 clinical NAAT specimens (106 APTIMA, 3 BD Viper, 1 neat urine) and matching bacterial cultures. RT-PCR results were compared to sequences determined by whole genome sequencing (WGS) and minimum inhibitory concentrations (MIC) for ESC and CIP were determined by agar dilution.
Results: SNP assay analysis and NG-MAST were successfully determined for 98 of 110 matched specimens. Decreased (MIC≥0.125µg/ml), moderate (MIC=0.032-0.063µg/ml), and full (MIC<0.032µg/ml) susceptibility to ESC (based on the number of SNPs) was observed in 11.2% (n=11), 12.2% (n=12) and 72.4% (n=71) of NAAT specimens, respectively, (4.1% (n=4) undetermined); and 9.2% (n=9), 26.5% (n=26) and 64.3% (n=63) of the bacterial cultures. CIP resistance was predicted in 22.4% (n=22) of NAAT specimens and 67.3% (n=66) were predicted susceptible, with 10.2% (n=10) undetermined; whereas 21.4% (n=21) of cultures were resistant and 78.6% (n=77) were susceptible. Agreement of SNPs detected between successful RT-PCR assays and WGS was 100% for ponA, mtrR, porB, gyrA D95, and parC; 98.8% for gyrA S91; and 97.7% for penA.
Conclusions: The utility of a RT-PCR assay for detection of known antimicrobial resistance markers was demonstrated to be useful to enhance surveillance by estimating antimicrobial resistance in situations where bacterial cultures of N. gonorrhoeae are no longer available.