Eye Catching Advancement for Creutzfeldt–Jakob Disease Diagnostics

Prion diseases, including Creutzfeldt–Jakob disease (CJD), can be challenging to diagnose given nonspecific early symptoms such as insomnia, depression, headache, and weight loss. Whereas today a definitive diagnosis for nonfamilial cases still requires histopathological examination of biopsied or autopsied brain tissue, laboratory-based antemortem diagnostics have made considerable advances over the past half decade (1). CJD was first described in the 1920s by 2 German neurologists, Hans Gerhard Creutzfeldt and Alfons Maria Jakob, but it was not until the late 1970s and early 1980s that the causative prion protein (PrP) was characterized along with the hallmark histopathological findings of CJD. The first diagnostic guideline for CJD was developed in the 1970s, with antemortem diagnosis based on clinical symptoms such as rapid progressive dementia with additional symptoms such as myoclonus or pyramidal/extrapyramidal features. In 1998, the World Health Organization (WHO) included the first biofluid biomarker as part of the standard diagnostic criteria—elevated cerebrospinal fluid (CSF) 14-3-3 protein—in combination with clinical symptoms and electroencephalography. The next advancement came in the form of imaging biomarkers, when in 2009 characteristic magnetic resonance imaging features—abnormally high signal in the thalamus, basal ganglia, and cortical areas—were suggested. While these fluid and imaging biomarkers represented forward progress, they are not without their issues and controversies. Protein 14-3-3, for instance, is a nonspecific biomarker of neuronal injury and is released into the CSF in relatively high concentrations in the setting of rapid and extensive damage to brain tissue. Both sensitivity and specificity are a concern with CSF 14-3-3 and, as such, false negative (e.g., sampling during a slower phase of disease progression) and false positive (e.g., elevation due to non-CJD related brain injury) results must be considered when utilizing this biomarker clinically. Fortunately, a more sensitive and specific CSF biomarker for CJD has been subsequently identified: aggregated forms of the misfolded PrP (termed PrPSc). PrPSc in CSF is detected using highly-sensitive seed amplification assays, most commonly a version termed real-time quaking-induced conversion (RT-QuIC). Despite the logistical shortcomings of this approach (predominantly assay complexity, cost, and lengthy turnaround times), this technology has been adapted by prion surveillance centers in many countries to support the antemortem diagnosis of CJD (2). Recently, an examination of the seeding activity of tear fluid has provided preliminary evidence that PrPSc RT-QuIC testing may be adaptable to a fluid with considerably less-invasive collection requirements (3). RT-QuIC works by amplifying pathological PrPSc aggregates (i.e., seeds) present in the sample by supplying an exogenous source of PrPSc building blocks, i.e., monomeric PrP (4). Seeded amplification is then encouraged via heating and periodic shaking of the sample. Over time, the generation of larger aggregate structures is monitored via the amyloid-binding dye thioflavin T, which demonstrates enhanced fluorescence upon binding amyloid structures. For tear fluid, a CSF RT-QuIC assay was adapted by changing the monomeric source of PrP to a recombinant full-length form of the human PrP sequence with the E200K variant [noting that most contemporary RT-QuIC CSF assays use a truncated recombinant hamster PrP sequence (2)]. The E200K PrP variant is the most common familial CJD (fCJD) variant, with this particular familial form of the disease presenting similarly to sporadic CJD (sCJD). Data from in silico and in vitro studies suggest that this PrP variant increases the propensity of monomeric PrP to misfold into PrPSc. In the context of the tear fluid RT-QuIC assay, this modification may have been employed to improve the analytical sensitivity of the assay. The need for greater analytical sensitivity can also be inferred from the change in assay time of 50 hours for their CSF RT-QuIC assay to 150 hours for tear fluid. In general, RT-QuIC assay developers must strike a careful balance between promoting seeded amplification while not over encouraging spontaneous aggregation (i.e., false positive results from the monomeric PrP assay substrate forming PrPSc in the absence of an endogenous seed) (4). The other notable adaptation compared with the CSF assay is the specimen collection procedure. For the tear fluid assay, the sample is collected via a Schirmer test. The Schirmer test is an established, simple ophthalmology procedure to assess adequate tear production whereby a small strip of filter paper is placed inside the lower lid of the eye for a few minutes. For RT-QuIC testing, the filter paper was left on the eyelid for 8–10 minutes, and then removed and frozen until use. For sample processing, the filter paper was cut into pieces and incubated with the reaction buffer and then the liquid separated from the filter paper and subjected to the typical RT-QuIC workflow (2, 3). In this preliminary assessment of diagnostic performance, tear fluid was collected from individuals with sCJD, fCJD variants (including individuals symptomatic and asymptomatic at the time of sample collection) and controls (3). In the discovery and validation cohorts, the assay had a sensitivity in the range of 77.8% to 84.6%; however, as the sample sizes were small, these data should be interpreted with caution (sample sizes ranged from 6 to 17 individuals, with variable counts depending on whether one includes asymptomatic fCJD cases in the disease cohort or not). In the control groups (n = 26 discovery, and n = 68 validation), no samples were interpreted as positive. In the asymptomatic fCJD group, 4 of 5 in the discovery cohort and 2 of 3 in the validation cohort had positive RT-QuIC interpretations. While these are early and so far unreplicated findings, they have the potential to open new doors for CJD diagnostic testing and disease monitoring. For instance, noninvasive tear fluid collection would be particularly valuable in people presenting with the early signs and symptoms of CJD but where a lumbar puncture may not yet be under consideration or appropriate. It should be acknowledged, however, that a lumbar puncture is commonly performed for this clinical presentation. Routine CSF testing is helpful in identifying other causes of rapidly progressive neurological decline that may present similarly to CJD, such as immune-mediated, steroid-responsive, encephalopathy. Another important application of this testing is in the context of the investigation of pharmacotherapies for CJD, where a noninvasive specimen collection protocol would be ideal for screening individuals for trial participation and for longitudinal monitoring of drug efficacy. This new report on the PrPSc seeding capacity of tear fluid is consistent with previously published findings related to ophthalmological symptoms in CJD, disease transmission, and detection of PrPSc in eye tissues. Findings suggestive of prion pathology affecting eye structures include the presence of visual disturbances, such as diplopia, in 10%–20% of cases, and the development of blindness in 25%–42% of sCJD cases (5). More specifically, cases of iatrogenic transmission of prion disease due to corneal grafts from persons with CJD are well documented and is in part why the WHO “Infection Control Guidelines for Transmissible Spongiform Encephalopathies” ranks the eye in the high infectivity category along with brain and spinal cord tissue. Moreover, in cases of variant CJD (which arises from transmission of bovine spongiform encephalopathy), PrPSc has been detected via immunohistochemistry in select eye structures including the retina and optic nerve. With the advent of RT-QuIC, PrPSc has also been detected in eye structures in sCJD, with relatively strong seeding activity (as compared to CSF) in the retina and cornea (5). Beyond CSF and tear fluid, other tissues and fluids have been investigated in the context of RT-QuIC assays for proteinopathies including blood, skin, urine, saliva, and olfactory mucosa. While blood would be an obvious choice for a CJD biomarker test given its routine clinical collection, progress on blood-based PrPSc seeding assays has been slow (2). Compared to CSF, blood has a lower concentration of PrPSc and is a substantially more complex matrix, which is believed to be a source of interference in seed amplification assays. On the other hand, tear fluid, much like CSF, is a fluid with relatively lower protein content and complexity, and resides in close proximity to “high infectivity” tissues. To promote further development of tear fluid-based assays for CJD, ideal follow-up studies would include investigations in larger cohorts and longitudinal collections to investigate the correlation of RT-QuIC seeding activity with symptom onset and disease progression. The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list. Cyril Helbling (conceptualization—equal, writing—original draft-equal, writing—review, and editing—equal) and Mari DeMarco (conceptualization—equal, writing—original draft—equal, writing—review, and editing—equal). No authors declared any potential conflicts of interest. None declared. None declared.

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