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Amplification of Misfolded Prion Proteins in Blood and Cerebrospinal Fluid for Detection of Creutzfeldt-Jakob Disease.

The most widely recognized human prion disease, or transmissible spongiform encephalopathy is variant Creutzfeldt--Jakob disease (vCJD) [2]. vCJD is related to consumption of beef from cattle infected with bovine spongiform encephalopathy (BSE) colloquially referred to as mad cow disease. Identified in 1996, vCJD was so termed because of its "variant" presentation relative to the fatal neurodegenerative disease first described by Creutzfeldt and Jakob in the 1920s. Unlike the late-onset sporadic-CJD (sCJD, mean onset = 65 years), vCJD had a considerably younger age of onset (mean onset = 26 years) and neuropathology comparable to BSE. New cases of vCJD peaked in 2000 in the United Kingdom and sharply declined with the epidemiological and experimental connection with BSE and introduction of new animal health control measures. Prion disease can be divided into the following 3 major groups: acquired (including vCJD, Kuru, and iatrogenic cases), familial, and sporadic. Although vCJD garners perhaps the greatest attention, it belongs to the group with the smallest incidence proportion (<1%), with sCJD being the most common form of the disease (85% cumulative incidence).

What makes prion disease distinctive is the peculiar nature of the infectious agent. The disease is characterized by a change in conformation of an endogenous cellular glycoprotein, the prion protein ([PrP.sup.C]), to a misfolded and aggregate-prone structural conformer termed [PrP.sup.Sc] (1). As the disease progresses, [PrP.sup.Sc] acts as a template, recruiting newly synthesized [PrP.sup.C] to misfold and thus driving the pathological cascade (Fig. 1). The mature [PrP.sup.Sc] aggregates are characterized by their resistance to proteinase K digestion, apple-green birefringence when stained with Congo red, and their ability to enhance the fluorescence emission of the dye thioflavin T. In vitro, conversion of [PrP.sup.C] to [PrP.sup.Sc] can be driven by high concentrations of PrP, low pH conditions, chemical denaturants, and the presence of [PrP.sup.Sc] to seed polymerization. In vivo, drivers include mutations to the PRNP gene encoding PrP (familial forms), introduction of exogenous [PrP.sup.Sc] (e.g., contaminated neurosurgical equipment), and other unknown causes (sporadic forms). While highly infectious tissues and fluids are found in the central nervous system, in vCJD, wider tissue distribution of [PrP.sup.Sc] has been observed including evidence of [PrP.sup.Sc] in the appendix, tonsil, and spleen. Wider tissue distribution combined with the variable age of onset and disease duration of vCJD has raised concern regarding blood and tissue donation from asymptomatic individuals.

The gold standard for diagnosis of CJD is neuropathological examination of brain tissue by brain biopsy or, most commonly, at autopsy. Definite diagnosis includes demonstration of the aggregation of PrP molecules and formation of [PrP.sup.Sc]. The former assessment can be made by immunohistochemical analysis and the latter by proteinase K treatment followed by Western blot analysis for PrP or visualization of [PrP.sup.Sc] fibrils. With the exception of the rare familial forms, a definite antemortem diagnosis is not possible. Historically, antemortem laboratory investigations of human prion diseases have relied on 2 nonspecific cerebrospinal fluid (CSF) biomarkers: tau and 14-3-3. In the symptomatic phase of the disease, the concentrations of tau and 14-3-3 proteins in CSF dramatically rise because of the rapid destruction of neurons. These protein biomarkers have modest specificity and sensitivity for prion diseases and may be increased in any condition causing rapid neuronal destruction including stroke, vascular dementia, subarachnoid hemorrhage, and central nervous system tumors. To support antemortem diagnosis, biomarkers with greater specificity are needed.

Fortunately, the field has taken major steps toward implementation of assays with higher specificity for antemortem diagnosis of CJD with the evolution of the protein misfolding cyclic amplification (PMCA) technique. PMCA of prions, analogous to the PCR for DNA, uses endogenous [PrP.sup.Sc] as a template for production or "amplification" of more [PrP.sup.Sc]. Like the nucleotides supplied in a PCR reaction, [PrP.sup.C] substrate is supplied in PMCA in the form of either recombinant-[PrP.sup.C] or [PrP.sup.C] in the brain homogenate of transgenic animal models (Fig. 1). The ability of [PrP.sup.Sc] to convert [PrP.sup.C] has long been used in the field to characterize properties related to conversion and transmission of the disease. In one of the seminal in vitro prion conversion studies, Kocisko et al. demonstrated that [sup.35]S-labeled recombinant hamster PrPC from uninfected tissue culture could be converted to [sup.35]S-[PrP.sup.Sc] by adding a small amount of unlabeled-[PrP.sup.Sc] purified from diseased hamster brains (2). Over the past 2 decades, this conversion property of [PrP.sup.Sc] has been exploited to develop sensitive biofluid diagnostics for both vCJD (3) and sCJD (4).

In a recent publication, Concha-Marambio et al. describe a PMCA technique for detection of [PrP.sup.Sc] in the blood of individuals with vCJD (3). In this workflow, 250 [micro]L of whole blood is mixed with a surfactant and then pelleted by centrifugation. The PMCA substrate of 10% transgenic mouse brain homogenate expressing human [PrP.sup.C] is added to the pellet. This mixture is intermittently sonicated, partially fragmenting any [PrP.sup.Sc] present into a greater number of [PrP.sup.Sc] subunits, followed by a period of rest to promote [PrP.sup.C] conversion and [PrP.sup.Sc] elongation. [PrP.sup.C] substrate is repeatedly replenished after each PMCA cycle lasting 48 or 72 h. The detection of [PrP.sup.Sc] mimics the established procedures used for definitive diagnosis of CJD from brain tissue, whereby an aliquot of the PMCA product after each cycle is digested with proteinase K and subjected to Western blot analysis for detection of PrP. Given the resistance of [PrP.sup.Sc] but not [PrP.sup.C] to proteinase K, only samples containing [PrP.sup.Sc] are positive by Western blot analysis.

This PMCA technique was applied to whole blood collections from individuals with vCJD (n = 14), sCJD (n = 6), other neurodegenerative disorders (n = 60), and nondegenerative neurological disorders (n = 26), as well as in healthy controls (n = 49) (3). After 3-5 cycles of PMCA, all vCJD samples were positive; after 5 cycles, no control samples were positive, demonstrating high sensitivity and specificity for vCJD. vCJD brain homogenate was serially diluted to assess detection sensitivity of the PMCA assay; a positive result was obtained down to a [10.sup.-10] dilution. For comparison, the amount of [PrP.sup.Sc] in whole blood of symptomatic individuals with vCJD was estimated to be equivalent to a [10.sup.-9] dilution of vCJD brain homogenate on the basis of the number of PMCA cycles required for detection of a majority of the vCJD blood samples tested. Challenges for implementation of this technique include a lengthy and laborious assay workflow. A 5-cycle PMCA run requires >11 days for just the sonication phase, not including proteinase K digestion and Western blot analysis. However, the potential for antemortem diagnosis of vCJD and application to subclinical disease and asymptomatic carriers outweighs concerns regarding current workflow limitations.

While the PMCA assay demonstrates specificity for vCJD, there is an alternate approach that has broader prion disease specificity including detection of sCJD--the real-time quaking-induced conversion (RT-QuIC) assay (4). RT-QuIC, like PMCA, makes use of the conversion properties of [PrP.sup.Sc]; however, major differences between the methods include the [PrP.sup.C] substrate, fragmentation technique, and detection method (Table 1). For the RT-QuIC assay, 20 [micro]L of CSF is added to a reaction buffer containing thioflavin T and truncated recombinant hamster [PrP.sup.C] as the conversion substrate. The mixture is then incubated in a shaking plate reader for up to 24 h with periodic shaking and measurement of thioflavin T fluorescence. The performance of the assay was tested using CSF specimens from individuals with sCJD (n = 48), other neurological disorders (n = 30), and nonneurological controls (n = 9). In total, 46 of 48 sCJD specimens were positive by RT-QuIC, and all other specimens tested were negative. Numerous large-scale prospective and retrospective diagnostic accuracy studies have since been conducted, demonstrating comparable to improved performance characteristics.

Multiple international ring trials have been completed for detection of sCJD by RT-QuIC. In 2 such trials, 25 CSF specimens were analyzed by a total of 11 testing centers, each using their own version of the RT-QuIC assay (including variations in the type of conversion substrate and instrumentation used) (5). There was 1 false negative by a single laboratory and no false positives, yielding an overall sensitivity of 85.7% to 100% and a specificity of 100%. While the assay requires CSF, a lumbar puncture is routinely performed in such cases and thus CSF is a commonly available specimen type for this patient population. Nonetheless, the RT-QuIC assay has been adapted for use with nasal brushings with promising results. CSF RT-QuIC has been clinically implemented for sCJD based on its advantages over the traditional CSF biomarkers tau and 14-3-3. In North America, both prion surveillance centers--the Public Health Agency of Canada and the US National Prion Disease Pathology Surveillance Center--offer laboratory-developed test versions of the RT-QuIC assay.

Antemortem diagnosis for prion diseases is critical on several fronts. From the perspective of the development of new disease-modifying therapeutics, early diagnosis enables testing of novel interventions before there is significant and irreversible damage to the brain. From the perspective of routine care, confident antemortem diagnosis of CJD would aid in the delivery of supportive care and initiation of appropriate procedures associated with collection and handling of tissues and fluids from affected individuals.

A potential application for both the RT-QuIC and PMCA assays was recently illustrated in a case of prion disease that by neuroimaging resembled sCJD; however, neuropathological examination identified a [PrP.sup.Sc] form consistent with vCJD. This represented the first documented case of vCJD in an individual heterozygous for methionine(M)/valine(V) at residue 129 of PrP. All previous cases of vCJD had been associated with M/M homozygotes. In vitro, the M variant more readily polymerizes to form [PrP.sup.Sc], and in vivo M/M individuals have higher susceptibility to both sCJD and vCJD. The emergence of this first case of M/V vCJD, more than 15 years after the peak of vCJD in the United Kingdom, has reignited concerns over potential latent vCJD cases in the population. Diagnostic tools applicable to both the symptomatic and asymptomatic phases of CJD are thus needed.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contribution to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.


(1.) DeMarco ML, Daggett V. From conversion to aggregation: protofibril formation of the prion protein. Proc Natl Acad Sci USA 2004;101:2293-8.

(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: 471-4.

(3.) Concha-Marambio L, Pritzkow S, Moda F, Tagliavini F, Ironside JW, Schulz PE, Soto C. Detection of prions in blood from patients with variant Creutzfeldt-Jakob disease. Sci Transl Med 2016;8:370ra183.

(4.) Orru CD, Groveman BR, Hughson AG, Zanusso G, Coulthart MB, Caughey B. Rapid and sensitive RT-QuIC detection of human Creutzfeldt-Jakob disease using cerebrospinal fluid. MBio 2015;6:e0245114.

(5.) McGuire LI, Poleggi A, Poggiolini I, Suardi S, Grznarova K, Shi S, et al. Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic Creutzfeldt-Jakob disease: an international study. Ann Neurol 2016;80:160-5.

Mari L. DeMarco [1] * ([dagger])

[1] Department of Pathology and Laboratory Medicine, University of British Columbia and St Paul's Hospital, Providence Health Care, Vancouver, Canada.

* Address correspondence to the author at: St Paul's Hospital, 1081 Burrard St, Vancouver, Canada, V6Z1Y6. Fax 604-806-8815; e-mail

([dagger]) Member of the Society for Young Clinical Laboratorians (SYCL) ( community/sycl).

Received April 3, 2017; accepted May 25, 2017.

Previously published online at DOI: 10.1373/clinchem.2017.272229

[2] Nonstandard abbreviations: vCJD, variant Creutzfeldt-Jakob Disease; sCJD, sporadic CJD; PrP, prion protein; CSF, cerebrospinal fluid; PMCA, protein misfolding cyclic amplification; RT-QuIC, real-time quaking-induced conversion.

Caption: Fig. 1. Mechanism (A) and kinetics (B) of prion protein misfolding and aggregation.
Table 1. Comparison of PMCA (3)and RT-QuIC (4)assays.

Property             PMCA                    RT-QuIC

Prion disease        vCJD                    Sporadic, familial,
specificity                                  and acquired

[PrP.sup.C]          Transgenic mouse        Truncated recombinant
substrate            brain homogenate        hamster [PrP.sup.C]
                     expressing human        (2nd generation assays)

Fragmentation        Sonication              Shaking

Fluids and tissues   Blood, urine            CSF, nasal brushings

Detection            Western blot            Thioflavin T

Turnaround time      [congruent to]11 days   <24 h

Currently in         No                      Yes
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Title Annotation:Perspective
Author:DeMarco, Mari L.
Publication:Clinical Chemistry
Article Type:Essay
Date:Nov 1, 2017
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