RT-QuIC is a more sensitive live animal test for detecting Chronic Wasting Disease (CWD) than immunohistochemistry (IHC) testing. RT-QuIC detected CWD an average of 2-6 months earlier than IHC in a study of infected deer. It also found CWD in 59.6% of samples that lacked follicles needed for IHC testing. While IHC is the approved live animal test, RT-QuIC could increase detection of CWD, especially in genotypes more resistant to disease. The USDA may need to recognize more resistant genotypes and newer tests as the deer industry shifts to promote resistance.

1. Advantages and Limitations of
Amplification Techniques for the
Antemortem Detection of CWD
2019 Missouri Deer Association
Annual Convention
Davin Henderson P.D.
Nicholas Haley D.V.M. PH.D.

3. Fecal CWD prions in 96GG and GS deer
96GG

4. Fecal CWD prions in 96GG and GS deer
96GS

5. RT-QuIC live animal CWD testing
• Next generation – more sensitive test for CWD
• Easily adapted to many tissue types and
excreta
• Can increase sensitivity and may be required
to testing for CWD in resistant genotypes
• May be able to determine the differences in
shedding between susceptible and resistant
genotypes
– Effective test in saliva, urine and feces.

6. USDA guidance on live animal testing
• Definitive diagnosis for a positive CWD case is
only made after an immunohistochemistry
test of the brain stem/obex or medial
retropharyngeal lymph node (RLN) by a USAD
facility
• Rectal biopsy test not approved for routine
regulatory testing
• Test results are reported as Positive or not
detected. No test can definitively be negative.

7. RT-QuIC and live animal testing
• RT-QuiC rectal biopsy testing is not an
approved test for CWD
• However, to be compliant with the USDA Herd
Protection Plan owners must report non-
negative unofficial test results.
“Animals with non-negative results on an
unofficial test are also considered to be CWD-
suspect animals and must be reported.“

8. CWD disease course
Asymptomatic phase
- Long multi-year
lymphoid replication
phase prior to
neuroinvasion
- CWD detection in RLN
only. No detectable prion
in brain
- Longer in resistant
genotypes
- Longer for lower dose
exposures
Symptomatic phase
- Neuroinvasion of the
obex
- Spread to other brain
regions
- Short duration (for all
genotypes?)
- IHC positive in both brain
and RLN
- Symptoms may be subtle

9. Direct Detection vs. Amyloid Amplification
Direct detection assays: ELISA, Western Blot, and
Immunohistochemistry
Amyloid Amplification: RT-QuIC, sPMCA
>10,000 fold more sensitive than ELISA or western blotting,
Dassanayake et al. Journal of General Virology (2016), 97, 803–812
Image: Shi et al. Acta Neuropathologica Communications 2013, 1:44

10. Live animal testing for CWD
IHC testing for CWD in deer rectal biopsies (Thomsen et. al
2012):
Diagnostic sensitivity
76% for 96GG deer
42% for 96GS deer.
36% for deer in the earliest stage of disease (obex grade 0)
100% for deer in the last 2 stages of preclinical disease (obex grades
3 and 4)

11. RT-QuIC Assay

12. RT-QuIC Assay
• 24-60 hour reaction time with no repeated tube transfers
• Real-time data with no western blots
• Rapid assay optimization
• High-throughput 96-well plate format
• Quantitative
• Potential for false positive reactions- less than 0.5% in 24 hour assay

13. Antemortem testing for CWD
Increased sensitivity over IHC on antemortem samples
Haley et al. 2016: RAMALT RT-QuIC correlated 96% with RAMALT IHC
ELK:
Recent elk study : RAMALT RT-QuIC correlated 97% with RAMALT IHC
83 positive samples by IHC and 131 by RT-QuIC (36.6% more
positives by RT-QuIC)
Study repeated in 3 separate labs >800 samples tested by
each group with near perfect correlation
Haley et al. 2016: RAMALT RT-QuIC correlated >95% with RAMALT IHC
Deer:
An additional 67 RT-QuIC positives were found in addition to
the 128 IHC positive deer. 195 total by RT-QuIC (34.3% more
by RT-QuIC)

14. HCP and CWD Resistant Genetics
“Herds with fewer than 50 percent GG
animals will not be permitted to use
ante-mortem RAMALT testing.”
USDA Priority:
Management for Easily Detectable CWD
Stake Holder Priority:
Management for Resistance to CWD

15. RT-QuIC antemortem testing
compared to postmortem IHC results
IHC Positive RT-QuIC Positive
total RLN Obex RAMALT RAMALT
96GG 130 123 113 94 118
96GS 174 132 86 31 68
96SS 42 19 0 0 6
96GG (130 total tested)
>100% detection of Obex + RLN
postmortem IHC positives
+20% more found with anti-mortem
tests. 94 IHC(+), 118 RT-QuIC(+)
96GS (174 total tested)
79% detection of Obex + RLN
postmortem IHC positives
+54% more found with anti-mortem
tests. 31 IHC(+), 68 RT-QuIC(+)
96SS (42 total tested)
31% detection of RLN only
postmortem IHC positives
100% more found with anti-mortem
tests. 0 IHC(+), 6 RT-QuIC(+)

16. 96GS and 96SS deer had more deer in
an earlier stage of disease
96GG deer 92% CWD Positive in
BOTH RLN and Obex
96GS deer 65% CWD Positive in
BOTH RLN and Obex
96SS deer 0% CWD Positive in
BOTH RLN and Obex
IHC Post-mortem Detection
96GG deer >100% Detection
in deer RLN+ and Obex+
96GS deer 79% Detection
in deer RLN+ and Obex+
96SS 31% detection of RLN only
postmortem IHC positives
RT-QuIC Ante-mortem Detection

17. Antemortem RAMALT sensitive
genotypes
IHC PositiveRT-QuIC Positive

18. Antemortem RAMALT resistant
genotypes
Few IHC PositiveMany RT-QuIC Positive

19. USDA only recognizes resistance at
codon 96 in deer and 132 in elk
• Other clearly resistant genotypes are not
recognized
• The industry is shifting fast and USDA will be
forced to respond
– Harder to detect CWD when there is less of it
– Harder to detect CWD in traditional assays if the
prion form is less stable (95H)
– Possibility of new strains emerging

20. Herd Plan with Ante-mortem Testing:
• Known codon 96 genotype
• Serial sampling with 90% of the herd with
testable samples
• First whole herd test within 24 months of known
exposure
– Second test at least 6 months after first test and 3
years after exposure. >70% 96GG
– Second test at least 6 months after first test and 3.5
years after exposure. Between 50% and 70% 96GG
– Less than 50% 96GG not permitted to have ante-
mortem testing in herd plan

21. Herd Plan with RT-QuIC testing to
supplement IHC testing
IHC Testing RAMALT
with RT-QuIC testing
IHC Testing RAMALT
Year 1

22. Herd Plan with RT-QuIC testing to
supplement IHC testing
IHC Testing RAMALT
with RT-QuIC testing
IHC Testing RAMALT
Year 2

23. Herd Plan with RT-QuIC testing to
supplement IHC testing
IHC Testing RAMALT
with RT-QuIC testing
IHC Testing RAMALT
Year 3

24. Study Design
White-tailed deer inoculated by mouth with low doses of CWD
Tissues evaluated for PrPCWD by:
1) immunohistochemistry
2) RT-QuIC with and without NaPTA precipitation
3 6 1290
Collection of fluids and biopsies every 3 months
5 96 GG deer
5 96GS deer
30

25. RT-QuIC detection of CWD precedes
detection by IHC in many cases

26. Time course of detection: IHC
compared to RT-QuIC

27. Time course of detection: IHC
compared to RT-QuIC

28. RT-QuIC detection of CWD precedes
detection by IHC in many cases
Avg. Avg. GG Avg. GS
QuIC (+) T 15.55 12.50 19.20
QuIC (+) R 17.18 14.00 21.00
NAPTA QuIC (+) T 13.09 10.50 16.20
NAPTA QuIC (+) R 11.45 10.00 13.20
IHC T (+) 18.00 14.00 22.80
IHC R (+) 19.00 16.50 24.00
Delta Tonsil -2.45 -1.50 -3.60
Delta RAMALT -3.67 -2.50 -6.00
Delta T NAPTA -4.91 -3.50 -6.60
Delta R NAPTA -6.33 -3.50 -8.00

29. RT-QuIC can increase the number of
testable samples from a herd sampling
Samples taken for IHC may not contain a suitable
number of follicles for an accurate identification of CWD
CWD Detection in samples with no follicles:
28 of 47 (59.6%) tested positive by RT-QuIC
Deer that had a previous IHC positive result that had a
sample with no follicles: 18/18 (100%) tested positive by
RT-QuIC

30. Summary of longitudinal biopsy study
• 11 of 16 deer (68.9%) RT-QuIC detection
preceded IHC immunoreactivity, whether in
tonsil or RAMALT
• 18/18 samples expected to be CWD(+) that
had no follicles for IHC analysis tested positive
by RT-QuIC
• 82 samples that tested positive by IHC were
also positive by RT-QuIC

31. Conclusions:
• RT-QuIC is a next generation technologies with
increased sensitivity for antemortem detection of
CWD
• Amplification methods may be key for detection of
CWD in resistant deer or elk that may persist in an
early disease state for longer periods of time
• RT-QuIC can increase the percentage of samples that
can be tested over IHC due to lack of follicles.
• IHC detection for live animal testing is well established
and widely available. No false positive tests but is not
as sensitive as amplification tests