ABSTRACT The global dissemination of ceftriaxone-resistant Neisseria gonorrhoeae threatens effective treatment and necessitates rapid diagnostic tools for surveillance. This study systematically evaluated three established molecular assays to identify practical and reliable approaches for detecting key resistance-associated penA markers, supporting evidence-based clinical decision-making. A total of 150 N. gonorrhoeae isolates were selected from an existing strain collection. Ceftriaxone minimum inhibitory concentrations (MICs) were determined by agar dilution, and full-length penA sequences were obtained by Sanger sequencing. High-resolution melting (HRM), loop-mediated isothermal amplification (LAMP), and real-time quantitative PCR (qPCR) were applied to genomic DNA from all isolates. Assay performance was assessed based on the detection of the A311V substitution, identification of the penA -60.001 allele, and classification of ceftriaxone-resistant isolates. Sanger sequencing revealed 34 distinct penA genotypes, confirming that 96 isolates carried the A311V substitution. While the dominant type was penA -60.001 ( n = 90), this mutation was also found in other types, including penA -37.001 and penA -294.001. For detecting penA -60.001, HRM and LAMP assays demonstrated high accuracy (sensitivity above 98%, specificity over 91%), significantly outperforming qPCR. The assays also showed substantially higher specificity when directly detecting the A311V substitution across all penA types. However, when assessed against the ceftriaxone resistance phenotype, the sensitivity of all methods dropped to approximately 90%, due to the presence of resistant strains lacking the A311V mutation. Both HRM and LAMP reliably detect A311V and penA -60.001. Given its high accuracy, straightforward interpretation, and minimal infrastructure requirements, LAMP is particularly suitable for routine clinical diagnostics and antimicrobial resistance surveillance. IMPORTANCE Rapid identification of antimicrobial-resistant Neisseria gonorrhoeae is essential for effective laboratory diagnostics and resistance surveillance. By systematically comparing three molecular assays across diverse penA genotypes, this study clarifies the strengths and limitations of commonly used approaches for detecting ceftriaxone resistance markers. The findings provide practical guidance for diagnostic laboratories seeking reliable and resource-appropriate methods to support antimicrobial resistance monitoring.
Zhao et al. (Wed,) studied this question.