Molecular typing and characterisation of antimicrobial resistance determinants of Neisseria gonorrhoeae among symptomatic individuals in the Eastern Cape, South Africa

Background. Antimicrobial resistance in Neisseria gonorrhoeae threatens gonorrhoea treatment, while surveillance is constrained by limited culture capacity. We evaluated multi-antigen sequence typing (NG-MAST) and targeted real-time PCR for molecular epidemiology and detection of antimicrobial resistance-associated determinants directly from clinical specimens. Methods. We analysed 235 N. gonorrhoeae-positive stored DNA extracts from symptomatic individuals in South Africa. NG-MAST sequence types were obtained directly or from whole-genome sequences (WGS) of paired cultured isolates. Real-time PCR targeted quinolone resistance-associated mutations (RAMs) in parC and gyrA and macrolide RAMs in the 23S rRNA gene. Results were compared with WGS and with phenotypic susceptibility results from paired isolates. Results. NG-MAST sequence types were obtained for 200/235 (85.1%) specimens and were diverse, with ST21253 predominating (n=26). At least one gyrA or parC mutation was detected by real-time PCR in 110/193 (57.0%) specimens. Compared with WGS, positive agreement was 81.2% for parC and 97.0% for gyrA, and negative agreement 100% and 95.8%, respectively. Among 65 ciprofloxacin-resistant isolates, RAMs were detected in 60 (92.3%) by real-time PCR and 61 (93.8%) by WGS; four had no RAM detected by either method. The 23S rRNA C2611T mutation was detected by both methods in one isolate (azithromycin MIC, 8 mg/L); no A2059G mutations were detected. Conclusions. Gonococcal sequence types were diverse, quinolone RAMs were common, and the assessed macrolide RAMs were rare. Real-time PCR applied directly to specimen DNA agreed well with WGS, but the targeted markers did not fully predict ciprofloxacin susceptibility, supporting complementary molecular and phenotypic approaches.