Article In Brief
Using an assay, investigators found evidence of prions in eye tissues, particularly in the retinas, of deceased patients with sporadic Creutzfeldt-Jakob (sCJD) disease. They are working on research to determine if novel dyes can detect prions in the retina for early sCJD detection.
A highly sensitive assay has found minute quantities of prions in compartments of postmortem eye tissues tested, including muscle, according to the largest case series to date involving sporadic Creutzfeldt-Jakob disease (sCJD).
Using the real-time quaking-induced conversion (RT-QuIC) assay, the study found prion seeding in all retinal samples. Some patients had levels in the retina as high as those typically found in the brain. The study also found prion seeding in all other ocular tissues, although at levels up to four log fold lower in some tissues, particularly the vitreous, lens, and cornea, than in the brain or retina.
Standard immunohistochemical staining, while good enough to detect prions in the retina, failed to find them in the other ocular tissues.
Prions have previously been detected with RT-QuIC in patients who had been suspected of having sCJD prior to death in postmortem samples of skin, cerebrospinal fluid (CSF), and cribriform plate nasal tissue. While it was expected that they would also be found in the cornea, based on cases of iatrogenic prion disease caused by corneal transplant, the observation that they could be detected in all other ocular tissue, and so high in the retina, was surprising, said one of the paper's co-authors, Michael D. Geschwind, MD, PhD, FAAN, professor of neurology and Michael J. Homer Chair in Neurology at the UC San Francisco (UCSF) Memory and Aging Center.
“Just because we detected prions in all regions of the eye using RT-QuIC does not mean that the prions were at high enough levels to be transmissible from these regions,” however, Dr. Geschwind said, “with the possible exception of the very high levels found in the retina. Further investigation is needed to assess the transmissibility of prions from exposure to various eye regions, particularly focusing on regions that are more commonly accessed during common ophthalmological procedures.”
In related work, Dr. Geschwind said he is trying to develop a minimally invasive way of detecting prions in the eyes of suspected sCJD patients and is working on a National Institutes of Health grant with the senior author of the paper, Christina J. Sigurdson, DVM, PhD, professor of pathology at UC San Diego's Institute for Neurodegenerative Diseases. They are partnering with a company, Amydis, “to see if novel fluorescent dyes that bind various amyloids might be able to detect prions in the retina,” Dr. Geschwind said. The target group would be people at risk for prion disease but not symptomatic, such as those who carry a prion protein gene mutation or may have had an iatrogenic exposure.
The RT-QuIC assay was first described in 2011 by Japanese researchers in Nature Medicine. This assay amplifies misfolded proteins in a manner similar to how polymerase chain reaction (PCR) amplifies nucleic acid. RT-QuIC uses normal prion proteins (from various sources, including recombinant, hamster, or bank vole) as a substrate to amplify tiny amounts of prion proteins in a sample into amyloid fibrils that can be detected by adding thioflavin T, which binds amyloid and fluoresces, to the mixture. The “quaking” referred to in the assay's name is nothing more than vigorous intermittent shaking of the solution, using a dedicated shaker device, which is believed to facilitate and speed the reaction.
For a study in mBio, Dr. Sigurdson and colleagues analyzed the eyes from 11 cases of pathologically confirmed sCJD representing three subtypes, as well as six controls. The sCJD patients were enrolled in the UCSF Memory and Aging Center clinical prion research program.
Although prions were detected in all 11 postmortem samples, only three of the patients had experienced visual disturbances while alive such as transient monocular blindness and blurry or double vision, while three had impaired visuospatial skills. In addition, four of the 11 cases had visual signs, including nystagmus, slow ocular pursuit, and increased saccade latency, but these were not considered visual symptoms. No patients had visual field deficits.
Prion levels in the retina were “remarkably high and approached levels in [the] brain” in more than half of the cases, the paper reported. “The VV2 subtype showed the highest prion seeding activities in the retina and visible deposits in the inner and outer plexiform layers.”
It has been known for years that the cornea of infected patients must contain prions, because corneal grafts from such people have resulted in two probable and three possible cases of prion transmission. Yet until now the distribution and extent of prion seeding has been unclear, and so the true risk of transmission through ophthalmic procedures has been unknown. And with 185,576 corneal transplantations performed in 116 countries in 2012, the need to minimize risk is clear.
RT-QuIC of CSF is currently in use as a diagnostic test for prion disease in several countries including the U.S. In May of 2018, a study of 214 patients enrolled into the UK National Prion Monitoring Cohort Study — all of whom had postmortem confirmed sCJD — compared them to 50 patients with an alternative final diagnosis. Diffusion-weighted magnetic resonance imaging (MRI) was positive in 92 percent of patients with CJD compared to just 2 percent of the others.
A positive CSF RT-QuIC test was observed in 89 percent of CJD cases compared with none of the others. Dr. Geschwind noted that in their cohort at UCSF, RT-QuIC in CSF only showed a sensitivity of 60 percent, lower than several other studies. The specificity of this test in CSF, he noted however, is about 98 percent. Thus, he contends that a positive test, particularly in a patient with a clinical history and MRI suggestive of CJD is very useful diagnostically, but a negative RT-QuIC does not help and should be repeated if clinical suspicion is high.
Prior studies have used a similar protein amplification method, called the protein misfolding cyclic amplification (PMCA) technique, to detect prions in both blood and urine samples from patients with variant CJD, transmitted by consumption of beef from cattle infected with mad cow disease. In sporadic CJD, however, prions have yet to be detected in either blood or urine.
Simpler diagnostic tests for patients suspected of having sCJD may be in the offing. In 2017 prion seeding activity and infectivity was detected using both RT-QuIC and an animal-based bioassay in postmortem skin samples of patients with sporadic CJD, suggesting that a skin test might be a feasible antemortem measure. The first author of that paper is also the first author of the new paper: Christina D. Orrú, PhD, a researcher who is seeking to develop antemortem diagnostic tests for CJD at the Rocky Mountain Laboratories of the National Institute of Allergy and Infectious Diseases in Hamilton, MT.
In May 2018 a study of 214 patients enrolled in the UK National Prion Monitoring Cohort Study, all of whom had postmortem-confirmed sporadic CJD, compared them with 50 patients with an alternative final diagnosis. Diffusion-weighted magnetic resonance imaging was positive in 92 percent of patients with CJD compared with just 2 percent of the others. A positive cerebrospinal fluid RT-QuIC test was observed in 89 percent of CJD cases, compared with no incidents in the others.
Still, the possibility of using the RT-QuIC test in patients, whether before or after onset of suspicious symptoms, raises vexing ethical questions, said Dr. Geschwind.
“We do not know when sCJD, or even genetic prion disease, begins relative to symptom onset,” he said. Even if the test can detect prions prior to symptom onset, “detection does not mean transmissibility,” he said. “We would not want persons with mutations for genetic prion disease to be denied needed medical procedures because an ultrasensitive test detects prions without knowing the actual implications for transmissibility. Additionally, depending on the specific prion protein gene mutation, they have variable penetrance, and thus some persons carrying a mutation might never develop prion disease.”
Luisa Gregori, PhD, principal investigator in the Office of Blood Research and Review at the US Food and Drug Administration (FDA), said in a written statement that while the finding of prions in the eye was not surprising, “the value of this paper is in the relatively large number (for a rare disease) of sCJD cases tested and the extensive investigation of the different areas of the eye in all those cases. The results also highlight another application of RT-QuIC.”
The development of a screening test for CJD in living patients, she said, “could enhance the safety of certain at-risk donated biological products, such as cornea and dura mater. However, these tests should be FDA-approved, suitable for a clinical laboratory setting, and show high sensitivity.”
She noted that RT-QuIC has also been used with CSF and nasal brushing. The latter test materials “are easier to obtain,” she said, “and are already being investigated for antemortem diagnostic purposes.” While the paper demonstrates that ocular testing with RT-QuIC is feasible, she concluded, “whether this test can realistically be implemented in routine donor screening of eye donors remains to be assessed.”
The new results further support the likelihood that prions are present in virtually all organs and tissues, said Wen-Quan Zou, MD, PhD, associate professor of pathology at Case Western Reserve University and associate director of the National Prion Disease Pathology Surveillance Center in Cleveland.
“It will be very important to set up a procedure to screen tissues for transplantation,” said Dr. Zou, the senior author of the skin study in 2017. “CSF is used for testing when the patient already has clinical signs. But for preclinical testing, the more readily accessible specimens, such as skin and perhaps other peripheral tissue, are going to be better, given that blood and urine have been shown to have poor sensitivity for prion detection in patients with sCJD.”
Claudio Soto, PhD, professor of neurology and director of the George and Cynthia W. Mitchell Center for Alzheimer's Disease and Other Brain Related Illnesses at the University of Texas' McGovern Medical School in Houston, said: “I was glad to see the study was able to detect prions in three subtypes of sCJD. In some of the sporadic forms, prions are much less present in the periphery.”
Dr. Soto's group in Houston is currently trying to determine if prions due to sCJD can be detected in the blood and urine. “The amount in blood would be orders of magnitude less than what is found in the eyes or brain,” he said. “But we've always thought it would be there. It's just a matter of finding it.”
His group is also working with the company Amprion to develop the protein misfolding cyclic amplification (PMCA) and RT-QuIC tests for prion detection for diverse applications including patient diagnosis, blood safety, and to screen biological materials for clinical use. “We are going to the FDA for approval,” Dr. Soto said.
Dr. Geschwind has consulted for Quest Diagnostics, Inc., receives NIH funding for work in prion disorders, and is part of an NIH small business grant with Amydis, a company that is developing fluorescent compounds for detection of prions and other amyloid proteins. Dr. Gregori had no financial disclosures.