Old nodes, new tricks: optimized methods for chronic wasting disease prion detection in preserved retropharyngeal lymph nodes

Chronic wasting disease (CWD) is a highly transmissible, global prion disease of captive and wild cervids. The approved tests for the detection of the infectious CWD prion (PrP^CWD) are ELISA and immunohistochemistry (IHC), but both have limitations and can yield discrepant results. ELISA requires a fresh or frozen medial retropharyngeal lymph node (RPLN); IHC requires the RPLN to be formalin-fixed and paraffin-embedded, thus requiring 2 different storage protocols. Real-time quaking-induced conversion (rtQuIC) is a sensitive amplification technique used commonly in research to detect prions. Here, we optimized a rtQuIC assay for CWD prion detection in formalin-fixed (FF) and formalin-fixed paraffin-embedded tissues (FFPE) from white-tailed deer in Pennsylvania with various prion protein (PRNP) genotypes. Our proposed protocol differs from other publications because it does not require hazardous solvents such as xylene. We found that FF RPLN samples can be tested by rtQuIC with up to 96% sensitivity and 100% specificity, and FFPE RPLN samples can be tested by rtQuIC with up to 98% sensitivity and 99% specificity for PrP^CWD detection. Our results are comparable to testing fresh or frozen tissue, and we did not find an effect of prion protein genotype on assay performance. This application of rtQuIC has the potential to improve surveillance and disease management in regions with limited access to cold storage and allow testing of RPLNs with discrepant PrP^CWD results when tissue of sufficient quality is unavailable.