Standardizing Statistical Tests for CWD Detection with “Tricky” samples for RT-QuIC

A concerning disease of North American cervids is caused by an infectious protein that results in a fatal, neurodegenerative disease known as Chronic Wasting Disease (CWD). As CWD continues to spread rapidly across North America, new detection methods are needed more than ever to assess the prevalence of CWD in live animals and across the landscape. Detecting CWD prions in the excreta of cervids remains difficult, as inhibitors within feces and intermittent prion shedding result in lower sensitivity and specificity with the amplification assay real-time quaking induced conversion (RT-QuIC) compared to its use with other tissue types. To overcome these barriers, we propose a modification to previous fecal homogenization methods to enhance sensitivity and specificity by scraping the outer surface region from white-tailed deer fecal pellets. As feces pass through the intestinal tract, they will contact the intestinal mucosa, where prions are likely shed, therefore allowing more intestinal mucosal cells to be incorporated into the fecal homogenate. Our results show improved sensitivity (83%) and specificity (85%) using this method. While our sensitivity and specificity are not perfect, we also propose the investigation of different statistical tests that may help account for unique variables specific to fecal RT-QuIC. Using this approach fecal RT-QuIC is possible without prior use of protein cyclic amplification assay (PMCA). By effectively detecting CWD prions in feces, testing methods using feces in a non-invasive antemortem manner or from the environment could be implemented along with CWD diagnostics to enhance surveillance and improve management decisions.