| Summary: | Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be treated with readily available antibiotics, but patients in low-resource settings often do not return to the clinic for results. As current NG diagnostic gold standards suffer from slow turnaround time to result, a rapid, sensitive molecular diagnostic would help increase appropriate treatment at the point-of-care. Here, we report on the design and development of a minimally-instrumented paper-fluidic POC diagnostic that incorporates patient swab sample lysis, isothermal nucleic acid capture, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and lateral flow visual detection. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and device clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept sensitivity and specificity study. This proof-of-principle paper-fluidic diagnostic, which can be completed within 80 minutes, selectively amplifies and detects NG in multiplex with NGIC. It has a clinically relevant limit of detection of 1000 NG cells per device. In urethral swab sample trials (N=20), the device approaches current gold standard NG diagnostic capabilities with 90% sensitivity and 100% specificity and vaginal swab sample trials (N=20) surpass the gold standard with 100% sensitivity and 100% specificity.
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