Sequence analyses to accelerate the development of rapid resistance assays targeting the Neisseria gonorrhoeae gyrA gene

PURPOSE: Mutations in the Neisseria gonorrhoeae (NG) gyrA codons 91 and 95 are known to confer resistance to ciprofloxacin, but questions remain over whether all the various permutations of these codons are needed for optimal molecular detection of NG ciprofloxacin susceptibility. Here, we used publicly available sequence data to answer this question to help inform the design of next-generation NG gyrA assays. METHODS: NG gyrA alleles (n = 73) were downloaded from the NG-STAR platform, as were all NG sequences and associated metadata from Pathogenwatch (n = 38,367 records; n= 31,114 following de-duplication). Analyses focused on understanding the frequency of NG gyrA 91 and 95 alterations and the applicability of assay designs to detect ciprofloxacin resistance. RESULTS: While the 73 NG-STAR alleles reflect considerable gyrA sequence diversity, just four amino acid combinations at codons 91 and 95 (wild-type; S91F/D95A; S91F/D95G; S91F/D95N) comprised > 99% of isolates. Also, 91-only and 95-only mutants are rare, comprising less than 1% of sequences, and moreover were typically sporadic temporally and genetically (e.g., within and across different MLSTs). The results also indicate that certain NG-STAR alleles (e.g., gyrA_42.0, gyrA_53.0, and gyrA_60.0) have high sequence homology with other commensals, including Neisseria meningitidis, and hence warrant careful consideration during assay design to avoid off-target detection. CONCLUSION: The results highlight that 91-only and 95-only mutants do occur at relatively low frequency, and hence assays should ideally detect both SNPs independently, with 91 as the priority. However, an assay targeting either codon alone, or both codons collectively without independently discriminating each codon, would still correctly classify approximately 99% of resistant alleles. The 0.47% of single-codon mutants, if not accounted for by an assay, nonetheless carry a real, if small, risk of false-susceptible results at the individual patient level. Local validation against contemporary sequence diversity, ideally integrated with the European Centre for Disease Prevention and Control (ECDC) Euro-GASP framework, remains essential for any assay design and deployment decision.