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The Journal of Molecular Diagnostics, Vol. 18, No. 3, May 2016
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COMMENTARY The Detection of Infectious Prions In Vitro Conversion Assays Change the (Folding) Landscape Holger Wille From the Department of Biochemistry, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada CME Accreditation Statement: This activity (“JMD 2016 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2016 CME Program in Molecular Diagnostics”) for a maximum of 36 AMA PRA Category 1 Credit(s). Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.
The uncontrollable spread of chronic wasting disease (CWD) in cervids in the United States, Canada, and South Korea makes the detection of prions, the infectious agent that causes prion diseases [alias transmissible spongiform encephalopathies (TSEs)] an important animal health problem with implications for human food safety. Gray et al1 describe a novel diagnostic protocolda timed amyloid seeding assay (tASA)dthat achieves a sensitivity matching that of bioassays in laboratory rodents and surpassing any commercially available TSE rapid test. Clear cutoff criteria are defined for the sensitive detection of elk CWD prions in brain tissue, allowing a robust determination of TSEþ and TSE states. The study represents an important first step for the tASA diagnostic protocol to gain regulatory approval for its use in TSE surveillance programs targeting CWD in cervids.
The Study Design The tASA diagnostic protocol achieves its sensitivity by using an in vitro conversion approach in which recombinant prion protein (recPrP) is seeded with brain homogenate containing infectious prions.1 Under controlled conditions, recPrP is converted into an amyloid state, which is detected by an increase in thioflavin T fluorescence. By introducing a time limit (a 30-hour cutoff) for the conversion reaction,
false-positive results (type I errors) arising from the spontaneous conversion of recPrP into amyloid were avoided. A 48-hour time point was found to be an appropriate cutoff to suggest retesting of weakly positive samples. To avoid false-negative results (type II errors), up to 24 parallel assays were used to maximally boost the sensitivity. Because it is not practical to use 24 parallel assays in a realistic testing scenario, statistical arguments were used to correlate sensitivity levels with the number of assay replicates. Depending on sample dilution and the wanted sensitivity level, between 2 and 12 replicates seem to be required to surpass the sensitivity of the commercially available TSE rapid tests. In vitro conversion assays were first developed with brain-derived cellular prion protein as a substrate and prions2 or synthetic peptides3 as seeds to convert cellular prion protein into a prion-like conformation in vitro that was detected by its partial protease resistance. Later, the ASA combined the seeding of the conversion reaction with the use of recPrP as a substrate and thioflavin T fluorescence as
Accepted for publication March 16, 2016. Disclosures: None disclosed. Address correspondence to Holger Wille, Ph.D., Department of Biochemistry, Centre for Prions and Protein Folding Diseases, University of Alberta, 204 Brain and Aging Research Bldg, Edmonton, AB, Canada T6G 2M8. E-mail: wille@ualberta.ca.
Copyright ª 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2016.03.001
Wille a readout for a sensitive and rapid detection system.4 Because the ASA did not rely on the partial protease resistance of converted recPrP for the detection, it was capable of detecting protease-sensitive forms of the infectious prion protein5 and achieved a higher sensitivity.
Prion Diagnostics An alternative diagnostic assay, protein misfolding cyclic amplification (PMCA), uses repeated rounds of sonication to trigger the in vitro fibrillization of brain-derived prion proteins.6 The PMCA assay has found widespread use in research settings, but is limited by its use of cellular prion protein as a conversion substrate and by its reliance on protease resistance as a readout for conversion. A related assay, real-time quaking-induced conversion, replaced the repeated sonication with shaking and can use recombinantly sourced prion proteins as a substrate.7 Furthermore, real-time quaking-induced conversion also uses thioflavin T fluorescence to detect the in vitro conversion of recPrP. The ability to use a recPrP as a substrate and to detect conversion via thioflavin T fluorescence gives real-time quaking-induced conversion a definitive advantage over PMCA with respect to standardization, substrate availability, throughput, and sensitivity.
Conclusions The timed ASA-based assay described by Gray et al1 is the first of the ultrasensitive prion conversion assays to define binary cutoff values for the detection of CWD prions. By defining rigorous criteria for the diagnosis of CWD infection, tASA has taken a significant first step
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toward regulatory approval as a diagnostic tool. In the future, testing of blinded samples that were collected under real-world conditions will be necessary to define the diagnostic sensitivity and specificity of this assay. Additional work will also be needed to fine-tune and test tASA for the detection of prions in peripheral organs and environmental samples, which represent a substantial unmet need to track the spread of CWD prions among North American cervids as well as in the environment.
References 1. Gray JG, Graham C, Dudas S, Paxman E, Vuong B, Czub S: Defining and assessing analytical performance criteria for Transmissible Spongiform Encephalopathyedetecting amyloid seeding assays. J Mol Diagn 2016, 18:454e467 2. Kocisko DA, Come JH, Priola SA, Chesebro B, Raymond GJ, Lansbury PT, Caughey B: Cell-free formation of protease-resistant prion protein. Nature 1994, 370:471e474 3. Kaneko K, Peretz D, Pan KM, Blochberger TC, Wille H, Gabizon R, Griffith OH, Cohen FE, Baldwin MA, Prusiner SB: Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform. Proc Natl Acad Sci U S A 1995, 92:11160e11164 4. Colby DW, Zhang Q, Wang S, Groth D, Legname G, Riesner D, Prusiner SB: Prion detection by an amyloid seeding assay. Proc Natl Acad Sci U S A 2007, 104:20914e20919 5. Safar J, Wille H, Itri V, Groth D, Serban H, Torchia M, Cohen FE, Prusiner SB: Eight prion strains have PrP(Sc) molecules with different conformations. Nat Med 1998, 4:1157e1165 6. Saborio GP, Permanne B, Soto C: Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 2001, 411:810e813 7. Atarashi R, Satoh K, Sano K, Fuse T, Yamaguchi N, Ishibashi D, Matsubara T, Nakagaki T, Yamanaka H, Shirabe S, Yamada M, Mizusawa H, Kitamoto T, Klug G, McGlade A, Collins SJ, Nishida N: Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat Med 2011, 17:175e178
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