Abstract
Introduction Every year, Neisseria gonorrhoeae (Ng) causes 106 million new gonorrhoea infections worldwide and has recently joined the fast-expanding group of multi-drug resistant superbugs. Treatment is often empirical/delayed as culture-based antibiotic susceptibility tests in current use can take several days. A Point-of-Care Test (POCT) for antimicrobial susceptibility would change this; for example, no longer recommended antibiotics such as ciprofloxacin could be used in certain regions where high percentages of isolates remain susceptible. The objective of the present study was to develop and validate a portfolio of diagnostic primer pairs for application in a Nucleic Acid Amplification-based POCT for the concurrent diagnosis of Ng and ciprofloxacin susceptibility. Methods A bioinformatics analysis of >30,000 bacterial genomes identified 9 unique signature sequences specific to Ng FA1090 that were used as targets in a SYBR green-based qPCR assay. 9 diagnostic primer pairs were evaluated for specificity and sensitivity on 271 Ng and non-gonococcal isolates. Two primer pairs targeting ciprofloxacin resistance-conferring single nucleotide polymorphisms in gyrA were tested on 200 resistant and susceptible Ng isolates. qPCR was performed by an Applied Biosystems StepOnePLus TM qPCR system. Results Out of the 9 diagnostic primer pairs tested for Ng identification, one probe failed to detect 28 positive samples of Ng out of a total of 234 isolates. Another primer pair, which amplifies the DR9 repeat region used in the COBAS4800 was not able to detect one Ng isolate collected from Hong Kong. The remaining 7 primer pairs showed 100% specificity in terms of Ng detection and were highly sensitive in detecting Ng DNA in concentrations as low as 0.00001 ng/ul. A multiplexed assay using ciprofloxacin susceptibility-determining primer pairs distinctly differentiated between resistant and susceptible isolates based on melt curve analysis. Conclusion A POCT-adaptable assay has been developed for the simultaneous identification of N. gonorrhoeae and its ciprofloxacin susceptibility status. Disclosure of interest statement The present work was supported by Grand Challenges Canada ((#S5 398). No grants were received from any company in the development of this study.
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