Rapid Cas13a-based <i>pen</i> A genotyping for cefixime susceptibility in <i>Neisseria gonorrhoeae</i>

ABSTRACT Antimicrobial resistance in Neisseria gonorrhoeae is an urgent public health threat. Resistance-guided therapy can assure appropriate treatment and reintroduce alternative therapeutic options by identifying genetic predictors of resistance. Mosaicism at codons 375–377 of the pen A gene is associated with cefixime resistance. Rapid, field-deployable assays for predicting cefixime susceptibility are lacking. We used a machine-learning algorithm to develop a CRISPR Cas13a-based assay to detect the absence of mosaicism at codons 375–377 of the pen A gene, combined with isothermal amplification in a single reaction. We integrated the assay onto a portable fluorescence-based platform. We evaluated performance using cultured isolates and compared results with PCR genotyping and phenotypic antimicrobial susceptibility testing. We also assessed the feasibility of reagent lyophilization of an existing por A N. gonorrhoeae detection assay, which can support cold-chain-independent deployment. Among 40 N. gonorrhoeae isolates, the Cas13a pen A assay demonstrated 100% concordance with PCR genotyping and 92.5% (95% confidence interval 79.6%–98.4%) concordance with phenotypic cefixime susceptibility. Median time to detection of the pen A mosaic assay was 12 min (interquartile range [IQR] 5 min). The lyophilized por A N. gonorrhoeae detection system detected all 12 isolates with a median time to detection of 45 min (IQR 40–45) compared to 45 min (IQR 35–50) for the positive aqueous control, although peak fluorescence was higher for the aqueous control ( P &lt; 0.01). The Cas13a assay was rapid and demonstrated strong correlation with genotypic and phenotypic cefixime susceptibility in N. gonorrhoeae , while a lyophilized assay retained functionality. IMPORTANCE Antimicrobial resistance in Neisseria gonorrhoeae is an urgent threat to public health. Recent reports have highlighted the continued rise in ceftriaxone resistance, our last-line empiric treatment option. Molecular assays that detect the genetic determinants of resistance can improve antibiotic stewardship and increase the therapeutic options for cases of gonorrhea. As a result, those tests can reduce the pressure toward the emergence of ceftriaxone resistance. However, most molecular assays cannot be deployed in low-resource settings due to a lack of infrastructure. We report on the development of a point-of-care assay for predicting resistance to an oral first-line treatment option, cefixime. The assay provided results in under 30 min and demonstrated strong correlation with phenotypic and genotypic resistance. Furthermore, we report on the proof-of-concept freeze-drying of assay reagents, which could permit cold-chain-independent use. Collectively, our study provides the groundwork for developing and deploying such molecular resistance assays in low-resource settings.