Background Diagnosis of Neisseria (N.) gonorrhoeae is dependent on nucleic acid amplification testing (NAAT), which is not available in resource-limited settings where the prevalence of infection is highest. Recent advances in molecular diagnostics leveraging the high specificity of CRISPR enzymes can permit field-deployable, point-of-care lateral flow assays. We previously reported on the development and in vitro performance of a lateral flow assay for detecting N. gonorrhoeae. Here we aimed to pair that assay with point-of-care DNA extraction techniques and assess the performance on clinical urine specimens. Methods We collected an additional urine specimen among individuals enrolling in an ongoing clinical trial at the Massachusetts General Hospital Sexual Health Clinic who presented with symptoms of urethritis or cervicitis (urethral or vaginal discharge, dysuria, or dyspareunia). We then assessed thermal, detergent, and combination DNA extraction conditions, varying the duration of heat at 95 C and concentration of Triton X. We assessed the efficacy of the various DNA extraction methods by quantitative polymerase chain reaction (qPCR). Once an extraction method was selected, samples were incubated for 90 minutes to permit isothermal recombinase polymerase amplification. We then assessed the performance of lateral flow Cas13a-based detection using our previously designed porA probe and primer system for N. gonorrhoeae detection, comparing lateral flow results with NAAT results from clinical care. Results We assessed DNA extraction conditions on 3 clinical urine specimens. There was no consistent significant difference in copies per microliter of DNA obtained using more or less heat. On average, we noted that 0.02% triton combined with 5 minutes of heating to 95 C resulted in the highest DNA yield, however, 0.02% triton alone resulted in a quantity of DNA that was above the previously determined analytic sensitivity of the assay. Given that detergent-based extraction is more easily deployable, we selected that as our method for extraction. Twenty three clinical specimens treated with 0.02% triton were added to the Cas13a detection system. Lateral flow detections were run in duplicate. The Cas13a-based assay detected 8 of 8 (100%) positive specimens, and 0 of 15 negative specimens. Conclusion Using point-of-care DNA extraction, isothermal amplification, and Cas13a-based detection, our point-of-care lateral flow N. gonorrhoeae assay correctly identified 23 clinical urine specimens as either positive or negative. Further evaluation of this assay among larger samples and more diverse sample types is warranted.

P.C.S. is a co-founder of, shareholder in, and consultant to Sherlock Biosciences and Delve Bio, as well as a board member of and shareholder in Danaher Corporation. J.E.L previously served as a consultant to SHERLOCK Biosciences. J.B. has received research funding from Analog Devices Inc., Zeus Scientific, Immunetics, Pfizer, DiaSorin and bioMerieux, and has been a paid consultant to Flightpath Biosciences, Tarsus Pharmaceuticals, T2 Biosystems, DiaSorin, and Roche Diagnostics. J.J. has received in-kind research support from binx health. The remaining authors have nothing to disclose.

This work was supported in part by the Massachusetts General Hospital Department of Medicine Innovation Program grant to R.H.G, NIH NIAID U19AI110818 to P.C.S, grants 2019123 and 2021287 from the Doris Duke Charitable Foundation to J.E.L., and a small project award from the American Sexually Transmitted Disease Association to J.J.

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The Massachusetts General Brigham Institutional Review Board gave ethical approval for this work, as well as the secondary use of clinical specimens.

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