Validation of a real-time quaking-induced conversion (RT-QuIC) assay protocol to detect chronic wasting disease using rectal mucosa of naturally infected, pre-clinical white-tailed deer (Odocoileus Virginianus)

Authors: PIEL, ROBERT - Washington State University; Veneziano, Susan; Nicholson, Eric; WALSH, DANIEL - Us Geological Survey (USGS); LOMAX, AARON - University Of Wisconsin; NICHOLS, TRACY - Animal And Plant Health Inspection Service (APHIS); SEABURY, CHRISTOPHER - Texas A&M University; Schneider, David

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/18/2024
Publication Date: 6/13/2024
Citation: Piel, R.B., Veneziano, S.E., Nicholson, E.M., Walsh, D.P., Lomax, A.D., Nichols, T.A., Seabury, C.M., Schneider, D.A. 2024. Validation of a real-time quaking-induced conversion (RT-QuIC) assay protocol to detect chronic wasting disease using rectal mucosa of naturally infected, pre-clinical white-tailed deer (Odocoileus Virginianus). PLOS ONE. 19(6): e0303037. https://doi.org/10.1371/journal.pone.0303037.
DOI: https://doi.org/10.1371/journal.pone.0303037

Interpretive Summary: Chronic wasting disease (CWD) is a fatal prion disease of cervids spreading across North America. Improving mitigation may require new methods that provide earlier live-animal detection, higher throughput, and less expense than the current standard of immunohistochemistry. Samples of the rectal mucosa from white-tailed deer were used to determine the diagnostic performance of a standardized protocol for the real-time quaking-induced conversion (RT-QuIC) assay and a new commercial source of the critical substrate. The protocol produced highly sensitive detection of CWD in white-tailed deer before the onset of clinical signs, and in a 96-well format in under 30 hours. The results support the potential use of this assay in management scenarios where sensitive live-animal diagnosis of CWD would be advantageous.

Technical Abstract: Chronic wasting disease (CWD) is a fatal prion disease of cervids spreading across North America. Improving mitigation may require expansion of the available toolkit to include new methods that provide earlier antemortem detection, higher throughput, and less expense than the current standard of immunohistochemistry (IHC). The rectal mucosa near the rectoanal junction is a site of early accumulation of CWD prions and is safely sampled in living animals by pinch biopsy. A fluorescence-based, 96-well format, protein-aggregation assay -- the real-time quaking-induced conversion (RT-QuIC) assay -- is capable of ultra-sensitive detection of CWD prions. The recombinant protein substrate is crucial to the assay's performance and is now commercially available. In this blinded independent study, the preclinical diagnostic performance of a standardized RT-QuIC protocol using a commercially sourced substrate (MNPROtein) and a laboratory-produced substrate was studied using mock biopsy samples of the rectal mucosa from 284 white-tailed deer. The samples were from a frozen archive of intact rectoanal junctions collected at depopulations of farmed herds positive for CWD in the United States. All deer were pre-clinical at the time of depopulation and infection status was established from the regulatory record, which evaluated the medial retropharyngeal lymph nodes (MRPLNs) and obex by CWD-IHC. A pre-analytic sample precipitation step was found to enhance the protocol's detection limit. Performance metrics were influenced by the choice of RT-QuIC diagnostic cut points (minimum number of positive wells and assay time) and by deer attributes (preclinical infection stage and prion protein genotype). The peak overall diagnostic sensitivities of the protocol were similar for both substrates (MNPROtein, 76.8%; laboratory-produced, 73.2%), though each was achieved at different cut points. Preclinical infection stage and prion protein genotype at codon 96 (G = glycine, S = serine) were primary predictors of sensitivity. The diagnostic sensitivities in late preclinical infections (CWD-IHC positive MPRLNs and obex) were similar, ranging from 96% in GG96 deer to 80% in xS96 deer (x = G or S). In early preclinical infections (CWD-IHC positive MRPLNs only), the diagnostic sensitivity was 64-71% in GG96 deer but only 25% in xS96 deer. These results demonstrate that this standardized RT-QuIC protocol for rectal biopsy samples using a commercial source of substrate produced stratified diagnostic sensitivities similar to or greater than those reported for CWD-IHC but in less than 30 hours of assay time and in a 96-well format. Notably, the RT-QuIC protocol used herein represents a standardization of protocols from several previous studies. Alignment of the sensitivities across these studies suggests the diagnostic performance of the assay is robust given quality reagents, optimized diagnostic criteria, and experienced staff.