Improved molecular detection of ceftriaxone resistance in Neisseria gonorrhoeae in a reference laboratory setting

BACKGROUND: Ceftriaxone resistance threatens the last remaining empirical monotherapy for Neisseria gonorrhoeae. Ceftriaxone-resistant N. gonorrhoeae is rare in England, but cases are increasing. Rapid detection of ceftriaxone resistance, commonly conferred by mutations in the penA gene, facilitates rapid risk assessment and public health intervention including enhanced follow-up of cases. OBJECTIVES: This study aimed to develop a real-time PCR for detection of the ceftriaxone resistance-associated penA-237.001 allele in N. gonorrhoeae, for use on both cultured isolates and residual clinical specimens. METHODS: A penA-237.001-specific assay was designed and evaluated using cultured isolates, residual clinical specimens and a broad exclusivity panel. Analytical sensitivity was assessed using serial dilutions of two penA-237.001-positive isolates prepared in specimen collection kit buffers and PBS. RESULTS: The assay demonstrated 100% specificity within the validation panel; all penA-237.001-positive isolates were correctly identified with no false-positive results. Detection was reliable to approximately 15 cfu/mL across all buffer types, with minimal cycle threshold variation. CONCLUSIONS: This assay enables rapid and specific detection of penA-237.001 in cultured isolates and residual molecular specimens. Used alongside existing molecular assays, it offers enhanced early detection of ceftriaxone-resistant N. gonorrhoeae and supports timely public health action.