Antimicrobial resistance in Neisseria gonorrhoeae is a global public health emergency, and novel approaches for combating its emergence are needed. Resistance-guided therapy may alleviate selective pressure leading to drug resistance and improve cure rates. Ciprofloxacin susceptibility is an appealing target for resistance-guided therapy because high-level resistance is predicted by the absence of a single mutation at codon 91 of the gyrase A (gyrA) gene. Such approaches, however, are dependent on expensive and time-intensive laboratory equipment, which preclude implementation in low-resource settings where the prevalence of both Neisseria gonorrhoeae infection and antimicrobial resistance are high. Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK) technology, which uses CRISPR-Cas13a paired with isothermal amplification via recombinase polymerase amplification, can enable low-cost point-of-care assays with minimal laboratory infrastructure requirements. We aimed to develop a Cas13a-based Neisseria gonorrhoeae gyrA assay.
Methods
We developed SHERLOCK assays for ciprofloxacin resistance in Neisseria gonorrhoeae and evaluated their performance of the assay on 23 purified Neisseria gonorrhoeae isolates with known resistance profiles to ciprofloxacin: 3 susceptible isolates had minimum inhibitor concentrations (MIC) < 0.015, while 20 resistant isolates had an MIC between 8 and > 16. We further evaluated the analytic sensitivity of the assay via serial dilutions on synthetic DNA, and performed confirmatory gyrA genotyping by qPCR.
Results
We designed and evaluated a SHERLOCK assay that successfully distinguished wildtype and mutant gyrA genotype Neisseria gonorrhoeae using synthetic DNA. The figure shows the discrimination among purified Neisseria gonorrhoeae specimens. The assay could detect as few as 1,000 copies per microliter. All of the 20 specimens with mutant gyrA genotypes by qPCR were detected by the Cas13a assay, and 0 of the 3 wildtype isolates by qPCR were detected by the Cas13a assay, showing a 100% agreement.
Conclusion
Our novel Cas13a-based gyrA assay detects genetic markers in Neisseria gonorrhoeae linked to ciprofloxacin resistance, and reliably predicts phenotypic resistance. That platform has the capacity to be adapted into low-cost point-of-care testing, which could be impactful in low-resource settings.
