Neisseria gonorrhoeae and its increasing antimicrobial resistance have raised global health concerns, creating an urgent need for more advanced diagnostics to contain the spread. We conducted a preclinical trial using an ultrasensitive enzyme-linked immunosorbent assay (ELISA) with Thio-nicotinamide-adenine dinucleotide (NAD) cycling for the detection of N. gonorrhoeae . Three independent researchers conducted the initial experiments to obtain calibration curves. The inter-assay reproducibility across the 3 investigators yielded coefficients of variation of 11% when measuring 500000 CFU/mL, confirming the consistent performance of the ultrasensitive ELISA. Specificity against common clinical microorganisms was also assessed. Performance was first evaluated in simulated urine, vaginal fluid, and saliva. A comparative evaluation of 90 clinical isolates was then performed using a conventional ELISA method. The ultrasensitive ELISA produced consistent calibration curves ( R 2 = 0.99) with limits of detection of 1.1 × 10 4 to 1.8 × 10 4 CFU/mL. No cross-reactivity to common pathogens was observed. The assay results revealed linearity in simulated fluids, with a modestly reduced sensitivity in urine and vaginal fluid. At 50000 CFU/mL, the ultrasensitive ELISA generated signals >50-fold stronger than the p -nitrophenyl phosphate ( p NPP) ELISA. For clinical isolates, the ultrasensitive ELISA identified 78.9% as positive compared with 30.0% by p NPP ELISA ( p = 0.0005). The ultrasensitive ELISA detected N. gonorrhoea with high sensitivity and specificity across sample types, consistently outperforming the conventional ELISA, and demonstrating the potential of this approach as a cost-effective diagnostic alternative.
Chen et al. (Thu,) studied this question.