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 (PrPC), to a misfolded and aggregate-prone structural conformer termed PrPSc (1). As the disease progresses, PrPSc acts as a template, recruiting newly synthesized PrPC to misfold and thus driving the pathological cascade (Fig. 1). The mature PrPSc 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 PrPC to PrPSc can be driven by high concentrations of PrP, low pH conditions, chemical denaturants, and the presence of PrPSc to seed polymerization. In vivo, drivers include mutations to the PRNP gene encoding PrP (familial forms), introduction of exogenous PrPSc (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 PrPSc has been observed including evidence of PrPSc 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 PrPSc. 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 PrPSc 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 PrPSc as a template for production or “amplification” of more PrPSc. Like the nucleotides supplied in a PCR reaction, PrPC substrate is supplied in PMCA in the form of either recombinant-PrPC or PrPC in the brain homogenate of transgenic animal models (Fig. 1). The ability of PrPSc to convert PrPC 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 35S-labeled recombinant hamster PrPC from uninfected tissue culture could be converted to 35S-PrPSc by adding a small amount of unlabeled-PrPSc purified from diseased hamster brains (2). Over the past 2 decades, this conversion property of PrPSc 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 PrPSc in the blood of individuals with vCJD (3). In this workflow, 250 μL of whole blood is mixed with a surfactant and then pelleted by centrifugation. The PMCA substrate of 10% transgenic mouse brain homogenate expressing human PrPC is added to the pellet. This mixture is intermittently sonicated, partially fragmenting any PrPSc present into a greater number of PrPSc subunits, followed by a period of rest to promote PrPC conversion and PrPSc elongation. PrPC substrate is repeatedly replenished after each PMCA cycle lasting 48 or 72 h. The detection of PrPSc 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 PrPSc but not PrPC to proteinase K, only samples containing PrPSc 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 10−10 dilution. For comparison, the amount of PrPSc in whole blood of symptomatic individuals with vCJD was estimated to be equivalent to a 10−9 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 PrPSc; however, major differences between the methods include the PrPC substrate, fragmentation technique, and detection method (Table 1). For the RT-QuIC assay, 20 μL of CSF is added to a reaction buffer containing thioflavin T and truncated recombinant hamster PrPC 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. Comparison of PMCA (3) and RT-QuIC (4) assays. Comparison of PMCA (3) and RT-QuIC (4) assays. 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 PrPSc 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 PrPSc, 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. variant Creutzfeldt–Jakob Disease sporadic CJD prion protein cerebrospinal fluid protein misfolding cyclic amplification real-time quaking-induced conversion.

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