Inter-laboratory comparison of real-time quaking-induced conversion (RT-QuIC) for the detection of chronic wasting disease prions in white-tailed deer retropharyngeal lymph nodes

Abstract

The rapid geographic spread of chronic wasting disease (CWD) in white-tailed deer (WTD; Odocoileus virginianus) increases the need for the development and validation of new detection tests. Real-time quaking-induced conversion (RT-QuIC) has emerged as a sensitive tool for CWD prion detection, but federal approval in the United States has been challenged by practical constraints on validation and uncertainty surrounding RT-QuIC robustness between laboratories. To evaluate the effect of inter-laboratory variation on CWD prion detection using RT-QuIC, we conducted a multi-institution comparison on a shared anonymized sample set. We hypothesized that RT-QuIC can accurately and reliably detect the prions that cause CWD in postmortem samples from medial retropharyngeal lymph node (RPLN) tissue despite variation in laboratory protocols. Laboratories from 6 U.S. states (Michigan, Minnesota, Missouri, New York, Pennsylvania, Wisconsin) were enlisted to compare the use of RT-QuIC in determining CWD prion status (positive or negative) among 50 anonymized RPLNs of known prion status. Our sample set included animals of 3 codon 96 WTD genotypes known to affect CWD progression and detection (G96G, G96S, S96S). All 6 laboratories successfully identified the true disease status consistently for all 3 tested codon 96 genotypes. Our results indicate that RT-QuIC is a suitable test for the detection of CWD prions in RPLN tissues in several genotypes of WTD.

Chronic wasting disease (CWD) is a universally fatal transmissible spongiform encephalopathy (TSE) in cervid species caused by an infectious, misfolded isoform of the cellular prion protein (PrP^C). ⁵⁷ Found in North America, Scandinavia, and South Korea, CWD spreads rapidly in both free-ranging and captive cervid herds due to its efficient pathogenetic and transmission mechanisms. Exposure to the infectious form of CWD prion protein (PrP^CWD) stimulates conversion of normal PrP^C to PrP^CWD, which accumulates and aggregates in the brain and lymphatic systems of an infected animal leading to neurologic damage and death. ⁵⁶ Prions are extremely stable and can remain infectious in the environment for decades.^16,33,35 Thus, even minor environmental contamination may be associated with a large transmission risk.^11,39 Control and management of CWD is dependent on efficient, early detection and prompt removal of infected animals from affected herds. ²⁹

A wide variety of tests have been developed to evaluate the presence and infectivity in tissues of PrP^CWD, the causative agent of CWD. Established tests include transgenic mouse bioassays, western blot, ELISA, and immunohistochemistry (IHC).^{5,12,13,18,24,44} To date in the United States, ELISA and IHC are the only federally approved tests for CWD; definitive diagnosis requires IHC testing at an approved university, state, or federal laboratory. ⁶⁰ These approved tests rely on detection of PrP^CWD in the obex section of the brain stem or medial retropharyngeal lymph node (RPLN). Both IHC and ELISA have high diagnostic sensitivity and specificity for obex and RPLN collected from animals in advanced stages of disease, but can have poor sensitivity when tested specimens are from animals early in disease progression.^{15,19,25,26,3,50-52}

Advances have led to the development of amyloid amplification assays including protein misfolding cyclic amplification (PMCA) and the real-time quaking-induced conversion (RT-QuIC) assay as emerging approaches to detect TSEs.^3,48 These methods leverage the amyloid-forming properties of PrP^CWD and measure the aggregation of amyloid fibrils initiated by the prion. Researchers have experimentally demonstrated increased sensitivity of PMCA and RT-QuIC relative to ELISA and IHC, notably during early preclinical stages of infection.^4,23,38,50 RT-QuIC is particularly well-suited for inclusion in routine CWD testing for a number of reasons (e.g., RT-QuIC does not require native mammalian brain substrate to function, the post-amplified test reactions are non-infectious, the assay does not require protease treatment [which can reduce signal] and does not generate additional infectious material).^2,7,46

A growing body of evidence gleaned through experimental comparisons of CWD prion detection with RT-QuIC, ELISA, and IHC tests shows that RT-QuIC performs equally well in diagnostic accuracy as the current federally approved tests.^25,50,54 To date, however, U.S. regulatory agencies consider RT-QuIC “experimental,” and U.S. federal approval to use RT-QuIC in addition to ELISA or IHC for CWD prion detection has not been granted. ¹⁷ Concerns inhibiting approval of RT-QuIC as a detection test for CWD prions include: 1) significant time required to complete bioassay confirmation of disease status; 2) high inter-laboratory variability in RT-QuIC protocols, analysis, and interpretation, including the potential for poor uniformity of recombinant substrate resulting from variable in-house synthesis protocols; and 3) the unknown impact of inter-laboratory variability on the final characterization of CWD prion status (i.e. reproducibility).

We undertook a multi-institution comparison of the efficacy of RT-QuIC for the detection of CWD prions in RPLN tissues of white-tailed deer (WTD; Odocoileus virginianus) among university research and state veterinary diagnostic laboratories using different RT-QuIC protocols. We hypothesized that different RT-QuIC detection protocols can identify CWD prions in the RPLN tissues of WTD. Our objectives were to conduct anonymized verification tests in 6 regional laboratories to: 1) determine the efficacy of using RT-QuIC to detect CWD prions; 2) determine whether the 5 different RT-QuIC protocols produced reproducible results; and 3) investigate whether WTD genotype influenced test results.

Materials and methods

Participating laboratories

Participating laboratories included: Michigan State University (East Lansing, MI); University of Minnesota (St. Paul, MN); Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri (Columbia, MO); New York State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Cornell University (Ithaca, NY); Pennsylvania Veterinary Laboratory, Pennsylvania Animal Diagnostic Laboratory System (Harrisburg, PA); and University of Wisconsin–Madison (Madison, WI). Laboratories in MI, MO, NY, and PA contributed samples and performed laboratory analyses. The laboratory at MN performed laboratory analyses and maintained the shared sample library and dataset. The laboratory at WI contributed samples that were analyzed in MN.

Anonymized sample set preparation

Participating laboratories sourced RPLN samples of hunter-harvested WTD tissue from their archives. Prior to archival, diagnosticians had determined the true disease status (CWD-positive and CWD-nondetect) on RPLN samples by ELISA, followed by confirmatory IHC for any sample found to contain CWD prions.

Researchers at MI, MO, and PA randomly selected from their in-state archives 5 specimens known to be CWD-positive and 5 specimens known to be CWD-nondetected. Researchers at WI selected archived samples with known CWD status (n = 5 each, CWD-positive and CWD-nondetect) that originated in Texas and Wisconsin (n = 5, each). As NY is not known to harbor CWD, NY contributed 10 CWD-nondetect samples. Researchers at each contributing laboratory then prepared their 10 RPLN tissue homogenates per their in-house standard protocols to ensure consistent sample input for RT-QuIC between laboratories (Suppl. Material: Appendix A). Homogenates and associated metadata were transferred to the centralized materials repository overseen by researchers at MN. Researchers from MN received the homogenates and associated metadata from each laboratory, aggregated the samples into a project-wide set, prepared aliquots of each submitted homogenate, and distributed the anonymized set of 50 samples to each participating laboratory for RT-QuIC testing. Due to MN also participating in laboratory analysis of the sample set, different staff were involved in the preparation and distribution of samples and management of their metadata from those staff involved in the laboratory analysis to ensure anonymization.

Testing the blinded sample set

RT-QuIC

Diagnosticians at each participating laboratory received the complete anonymized sample set frozen on dry ice, then tested each sample for CWD per their own in-house RT-QuIC laboratory protocol and analysis pipeline (Suppl. Material: Appendix B). Diagnosticians then reported the results of CWD status (CWD-positive, CWD-nondetect, CWD-suspect) for each of the 50 specimens to the centralized (project-wide) dataset at MN.

Genotyping

To evaluate the effect of deer genotype at sites of known variability in the PRNP locus, which may affect amyloid amplification test outcomes, we performed 2 different genotyping tests. First, we obtained genotype data for PRNP codon 96 using standard protocols deployed by the USDA for validation testing (Tracy Nichols, USDA, pers. comm., Aug 2022). Briefly, researchers in NY extracted DNA from all 50 homogenates with a modified protocol (DNeasy blood and tissue kit; Qiagen). Briefly, modifications of the manufacturer’s protocol were incorporated at lysis and elution. Lysis was performed by combining 20 μL of homogenate with 180 μL of buffer ATL, and 20 μL of proteinase K; the lysis mixture was incubated for 1 h at 56°C with rotational agitation at 200 rpm. Elution was performed in 200 μL of buffer AE with 5 min of incubation at room temperature. DNA extracts were submitted to Gene Check (Greeley, CO, USA) for genotyping at codon 96 of the PRNP locus. Genotype results were received from Gene Check by NY and transferred to the centralized dataset.

Next, to evaluate PRNP genotype at additional codons that also may affect test outcomes, researchers at MSU extracted DNA from all 50 homogenates (DNeasy blood & tissue kit; Qiagen) per the manufacturer’s protocol, then performed PCR testing (ProFlex PCR system; Applied Biosystems) on the stable region of the PRNP (90-230). ³¹ The protocol included a final concentration of 0.1 μM of each primer in a 20-μL final reaction volume: CWD-13 (5′-TTTTGCAGATAAGTCATCATGGTGAAA-3′; forward) and CWD-LA (5′-AGAAGATAATGAAAACAGGAAGGTTGC-3′; reverse); amplification was performed using 95°C for 15 min of initial denaturation, 35 cycles of denaturation at 95°C for 45 s, annealing at 57°C for 45 s, and extension at 72°C for 90 s, with final extension for 1 cycle at 72°C for 10 min. Purified PCR products (Exo-SAP-IT; Applied Biosystems) were sequenced at the MSU genomics core facility (Research Technology Support Facility, E. Lansing, MI, USA). Sequences were aligned against a WTD reference (GenBank AF156185) on MEGA11 software (https://www.megasoftware.net/) to identify polymorphic loci. Amino acids were analyzed for polymorphisms identified at the following codon locations: 95 (glutamine Q/histidine H), 96 (glycine G/serine S), 116 (alanine A/glycine G), 197 (lysine K/glutamic acid E), 215 (glutamine Q/lysine K), 226 (glutamine Q/lysine K). Finally, results were transferred to the centralized (project-wide) dataset.

Unanonymizing and analysis of data

Researchers at MN obtained the diagnostic results of CWD status from each of the participating laboratories, the genotyping results from Gene Check, and the genotyping results from MI, appended the results to the centralized (project-wide) dataset, then unanonymized the data. Samples with a disease status of “suspect” were reclassified as “nondetect” for statistical analysis to adopt the most conservative approach to binary classification.

Project statisticians conducted 8 statistical tests to answer the following questions about RT-QuIC in WTD RPLN:

1. Do RT-QuIC results agree with the true status of CWD?

2. Do differing RT-QuIC protocols produce CWD results that agree with each other?

3. 3–5. Do RT-QuIC results agree with the true status of CWD in: 3) homozygous (GG) specimens, 4) homozygous (SS) specimens, and 5) heterozygous (GS) specimens, respectively?

4. 6–8. Do differing protocols produce CWD results that agree in: 6) homozygous (GG) specimens, 7) homozygous (SS) specimens, and 8) heterozygous (GS) specimens, respectively?

For each test, we used a chi-square goodness-of-fit test to investigate whether observed binary diagnostic results differed. ⁴² We downgraded the type I error rate (α) with the Bonferroni correction to experiment-wise α′. ⁴² We further computed the confusion matrix to obtain sensitivities and specificities of the protocols used by each participating laboratory. ⁵⁵ All computational analyses were performed in R v.4.3.1. ⁴ We used a downgraded α′ of 0.0031[0.05/(2 × 8)] to conduct all statistical tests.

Results

Sample set CWD status and genotype

We obtained a set of 50 WTD RPLN samples from archives in MI, MO, NY, PA, TX, and WI. Per the unanonymized results from ELISA and/or IHC, the dataset contained 20 of 50 (40%) CWD-positive samples and 30 of 50 (60%) CWD-negative samples (Table 1). Per Gene Check, at codon 96, 34 of 50 (68%) of samples were homozygous GG, 10 of 50 (20%) were heterozygous GS, 5 of 50 (10%) were homozygous SS, and the genotype of the remaining 1 of 50 (2%) could not be determined (Table 1). At codon 95, 40 of 50 (80%) samples were homozygous QQ, 3 of 50 (6%) were heterozygous QH, and the genotype of the remaining 7 of 50 (14%) could not be determined (Suppl. Material: Appendix C). At codon 123, 42 of 50 (84%) samples were homozygous AA, the genotype of 7 of 50 (14%) could not be determined, and we identified a unique polymorphism that has not been described in the literature in a single sample (AT). No genetic variation was observed in any other tested codons.

Table 1.

Results of using the real-time quaking-induced conversion (RT-QuIC) analysis on retropharyngeal lymph node tissues from white-tailed deer.

Sample	Anonymized truth		CWD test result
	True CWD status	True genotype	MI*	MN†	MO†	NY†	PA1‡	PA2*
1	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
2	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
3	Pos	GS	Pos	Pos	Pos	Pos	Pos	Pos
4	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
5	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
6	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
7	Neg	GS	Neg	Neg	Neg	Neg	Neg	Neg
8	Neg	GS	Neg	Neg	Neg	Neg	Neg	Neg
9	Neg	GG	Neg	Neg	Pos	Neg	Neg	Neg
10	Neg	§	Neg	Neg	Neg	Neg	Neg	Neg
11	Neg	GS	Neg	Neg	Neg	Neg	Neg	Neg
12	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
13	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
14	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
15	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
16	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
17	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
18	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
19	Pos	GS	Pos	Pos	Pos	Pos	Pos	Pos
20	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
21	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
22	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
23	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
24	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
25	Neg	SS	Neg	Neg	Neg	Neg	Neg	Neg
26	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
27	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
28	Neg	GS	Neg	Neg	Neg	Neg	Neg	Neg
29	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
30	Pos	GS	Pos	Pos	Pos	Sus	Pos	Pos
31	Pos	GS	Neg	Pos	Pos	Neg	Pos	Pos
32	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
33	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
34	Neg	GS	Neg	Neg	Neg	Neg	Neg	Neg
35	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
36	Neg	GS	Neg	Neg	Neg	Neg	Neg	Neg
37	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
38	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
39	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
40	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg
41	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
42	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
43	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
44	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
45	Pos	GG	Pos	Pos	Pos	Pos	Pos	Pos
46	Neg	SS	Neg	Neg	Neg	Neg	Neg	Neg
47	Neg	SS	Neg	Neg	Neg	Neg	Neg	Neg
48	Neg	SS	Neg	Neg	Neg	Neg	Neg	Neg
49	Neg	SS	Neg	Neg	Neg	Neg	Neg	Neg
50	Neg	GG	Neg	Neg	Neg	Neg	Neg	Neg

True chronic wasting disease (CWD) status = ELISA followed by immunohistochemistry (IHC); Neg = CWD-nondetect; Pos = CWD-positive. True genotype is based on codon 96 results from Gene Check; GG = glycine G/glycine G, SS = serine S/serine S, GS = glycine G/serine S. MI = Veterinary Medical Center, Michigan State University; MN = College of Veterinary Medicine at University of Minnesota; MO = Veterinary Medical Diagnostic Laboratory, Division of Animal Sciences, University of Missouri; NY = New York State Animal Health Diagnostic Laboratory, Cornell University; PA = Pennsylvania Veterinary Laboratory, Pennsylvania Animal Diagnostic Laboratory System. Values in boldface indicate discrepancies between RT-QuIC result and true disease status.

^*Sample homogenates diluted to 10^–3 and tested in triplicate with MNPROtein rPrP substrate (PA2).

^†Sample homogenates diluted to 10^–3 and tested in quadruplicate with MNPROtein rPrP substrate (PA2).

^‡Sample homogenates diluted to 10^–3 and tested in duplicate with CWD Evolution rPrP substrate (PA1).

§ True genotype could not be determined.

Statistical analysis

We found that the binary CWD results generated by all participating laboratories’ RT-QuIC protocols were true to the extrinsic status of disease (or presence of prion) in the RPLN tissues (χ²_df=11 = 0.203; p = 1.00). Binary CWD disease status was consistent across laboratories (χ²_df=11 = 0.175; p = 1.00). We did not find significant differences between true disease status and reported test results for any genotype (Table 2). We did not find significant differences in reported disease status between laboratories for any genotype (Table 2). For completeness, we included in Appendix B the confusion matrix of test performance for each research laboratory.

Table 2.

Results of chi-square goodness-of-fit test to compare binary real-time quaking-induced conversion (RT-QuIC) chronic wasting disease (CWD) prion test results to current gold standard tests (IHC/ELISA) and among participating laboratories.

Test	χ2 df  = 11	p	Comparison	Genotype, codon 96	Interpretation
1	0.203	1.00	RT-QuIC vs. IHC/ELISA	All	RT-QuIC results matched the true disease status.
2	0.175	1.00	Inter-laboratory result concordance	All	RT-QuIC results were consistent across labs.
3	0.058	1.00	RT-QuIC vs. IHC/ELISA	GG	RT-QuIC results matched the true disease status.
4	0.000	1.00	RT-QuIC vs. IHC/ELISA	SS	RT-QuIC results matched the true disease status.
5	0.955	1.00	RT-QuIC vs. IHC/ELISA	GS	RT-QuIC results matched the true disease status
6	0.049	1.00	Inter-laboratory result concordance	GG	RT-QuIC results were consistent across labs.
7	0.000	1.00	Inter-laboratory result concordance	SS	RT-QuIC results were consistent across labs.
8	0.627	1.00	Inter-laboratory result concordance	GS	RT-QuIC results were consistent across labs.

The Bonferroni-corrected significant p-value is 0.0031.

Discussion

We found the differing RT-QuIC methods used by 6 veterinary diagnostic laboratories to be robust for the detection of CWD prions in WTD RPLNs from WTD. Across 50 anonymized samples, there was no significant difference between the CWD status determined by RT-QuIC and the true CWD status determined by ELISA and IHC. Additionally, we found no significant difference in the CWD status determined using RT-QuIC methods between laboratories despite laboratory-specific variations in the protocols used for amyloid amplification and data analysis.

Inter-laboratory variations in RT-QuIC experimental protocols included: 1) choice of sodium salt (NaCl vs. NaI), 2) concentration of reaction components, 3) sample replication, 4) reaction temperature, 5) shaking and resting cycle duration, and 6) reaction duration (Appendix B). Additional variation included intra-laboratory variability in PA testing 2 different sources of recombinant substrate (CWD Evolution and MNPROtein) named PA1 and PA2, respectively. Despite this variability, binary disease classifications were not concordant among participating laboratories for only 3 samples. These included one positive sample classified as “suspect” by NY (classified as “positive” by MI, MN, MO, PA1, PA2), which is a classification that triggers repeat confirmatory RT-QuIC testing in standard laboratory protocols. However, as our study was designed to evaluate RT-QuIC results for a single test per sample, repeat RT-QuIC testing was not performed. The observed variability in CWD classification within the anonymized RPLN sample set may be due to several factors including differential probability of amyloid seeding among the reaction conditions tested or altered ratio of RT-QuIC inhibitors to available PrP^CWD within sample homogenates after freeze-thaw cycles. All samples were treated identically following selection for our study but may have undergone an unknown number of freeze-thaw cycles prior to selection; loss of seeding in RT-QuIC has been reported following thermal instability in other sample types.^36,53

Although our study was intended to evaluate the robustness of RT-QuIC to inter-laboratory variation, future development and adoption of standardized protocols are necessary to advance CWD detection in RPLN by RT-QuIC methods. Use of standardized testing protocols is a founding principle of the National Animal Health Laboratory Network (NAHLN), and development of a consensus laboratory protocol for RT-QuIC is needed before deployment among NAHLN laboratories. ⁶⁰ In addition to universal laboratory protocols, adoption of standardized analytical pipelines and an interpretation framework can help ensure repeatability of results. ⁴⁷ Following standardized protocols, completion of a true ring test can provide final evidence that RT-QuIC methods are ready for CWD prion detection in WTD RPLN, as has been done for RT-QuIC detection of human TSE agents.^37,41

We evaluated the effect of host genetics on the efficacy of RT-QuIC because, as an amyloid amplification assay, the conversion rate of PrP^C to PrP^CWD may be affected by the sequence of the tested PRNP gene. ¹ All lab protocols worked equally well for determining the true status of CWD using RPLN tissue from each of the 3 possible genotypes at codon 96 (GG, GS, SS). In addition to codon 96, we genotyped all samples at other codons known or suspected to affect the rate of amyloid aggregation including 95, 116, 197, 215, and 226.^{32,34,40,43,58} However, due to the lack of observed variability, we were unable to statistically test the genotype-specific efficacy of RT-QuIC at these sites.

We observed that 1 of 3 detected Q95H samples failed to achieve concordant CWD disease characterization among all laboratories. Given that our sample size of Q95H animals was too small to permit statistical analysis, this observation highlights the importance of considering deer genetics in future evaluations of CWD test diagnostic sensitivity and specificity. We recommend rigorous genotyping of samples in future test-development efforts.

Genotyping identified a novel polymorphism at codon location 123, where an alanine to threonine conversion was observed. We speculate that this polymorphism may be associated with altered CWD susceptibility due to its close proximity to codon 129, which confers susceptibility or resistance to sporadic Creutzfeldt-Jakob disease.^45,49 The codon 123 polymorphism was found in a CWD-nondetect animal; future study is needed to determine if the polymorphism is associated with altered CWD susceptibility.

Our findings corroborate previous work that reported comparable diagnostic sensitivity with RT-QuIC compared to ELISA or IHC.^{10,21,23,25,50,54} Here, sensitivity of RT-QuIC on WTD RPLN was 90–100% among participating laboratories and the specificity was 97–100%. Crucially, these results demonstrate high repeatability between laboratories indicating that RT-QuIC is broadly effective for the detection of CWD prions in WTD RPLN. This demonstrated efficacy is promising for future realization of key downstream benefits of RT-QuIC approval including 1) expansion of the testing toolkit for CWD, and 2) potential increase in efficiency of the approval process for future advancements in CWD prion detection tools, which may improve diagnostic sensitivity or expand the pool of available sample types.

Approval of RT-QuIC for CWD detection in RPLN would rapidly advance CWD management in free-ranging WTD by adding testing capacity with an additional tool that can integrate into existing surveillance plans. Testing of RPLNs is the current “gold standard” and many wildlife management agencies have comprehensive RPLN sample collection protocols built into their surveillance plans. ⁵⁹ Agencies are thus ideally positioned to quickly leverage an additional tool to supplement their CWD surveillance plans and meet the increasing demand for testing as CWD continues to spread.

The approval of RT-QuIC for detecting CWD in WTD RPLN has potential to enhance the efficiency of federal agencies in approving future RT-QuIC advancements. Approval of a new or modified laboratory test requires comparison to the existing “gold standard”; for a test to be approved, it must demonstrate comparable or improved diagnostic sensitivity and specificity to current “gold standard” diagnostics. RT-QuIC presents a challenge to regulating agencies because the prediction of increased sensitivity compared to the current IHC “gold standard” makes it challenging to correctly classify the type I error (false-positives) rate; if RT-QuIC detects PrP^CWD within samples that are below the limit of detection for IHC, the results may be incorrectly classified as a false-positive. Clarification of the true type 1 error rate is critical because false-positive results have potential to lead to management decisions including culling and can require expenditure of significant financial and personnel resources. To address this challenge, ongoing evaluations of RT-QuIC for CWD detection in WTD RPLN are leveraging transgenic mouse bioassays as the “gold standard” for comparison (Tracy Nichols, pers. comm., Aug 2022). These bioassays have increased sensitivity but are time and resource intensive. Should RT-QuIC receive approval, future test advances have potential to be compared against this new, more efficient test. Crucially, this may provide improved agility for the CWD testing “toolkit” to expand adaptively as technologic developments and improved understanding of disease progression are achieved.

Many experimental modifications to RT-QuIC methods have shown potential to further refine the test. For example, sample-specific selection of the sodium salt used in the RT-QuIC reaction can decrease the lag time for amyloid seeding, providing faster results. ²⁷ Additionally, a 2023 study demonstrated that inclusion of nanoparticles in the RT-QuIC reaction can significantly decrease detection time and increase sensitivity by overcoming the effects of inhibitors present in complex sample matrices. ⁹ Incorporation of the best possible methods for CWD detection is crucial to give wildlife managers and stakeholders the greatest opportunity for successful disease management outcomes.

Other future improvements in CWD testing include modification of sample selection. Better sensitivity by capturing the variability in the progression of disease between animals could be accomplished with protocols that test multiple lymphoid tissues per animal. ⁶⁰ In addition to the potential for improved diagnostic sensitivity, RT-QuIC may specifically improve detection of PrP^CWD in RPLN samples that are poorly suited for testing via IHC. With IHC, there is spatially explicit sample preparation for obex, and sensitivity declines when prepared RPLN samples lack lymphoid follicles. ²³

In experimental applications, RT-QuIC has successfully detected PrP^CWD in myriad tissues (i.e. RPLN, brain, ear pinnae, muscle, third eyelid, skin), excreta (saliva, urine, feces), and environmental samples.^{6,8,10,14,20,22,28,30,36} Although the kinetics of prion shedding vary between animals, and antemortem tests with high diagnostic sensitivity have remained elusive, now that we have shown RT-QuIC to be robust in proven samples across laboratories, further progress can be made on antemortem tests that may require the improved sensitivity and tissue flexibility that RT-QuIC may provide over other approved tests.

Our findings clearly show that the RT-QuIC method has high reproducibility for detection of CWD prions in WTD RPLN across 5 laboratories, even when reaction conditions and analytical pipelines differed among laboratories. Prompt development of standardized RT-QuIC laboratory protocols and federal approval as a CWD detection test have power to add a valuable and simple detection assay for CWD managers throughout the United States.