In May of 2024, Mayo Clinic launched a new prion test, RT-QuIC Prion, CSF, which can distinguish prion disease from other causes of rapidly progressive dementia—particularly autoimmune forms of dementia and rapidly progressive forms of Alzheimer's disease.
"Rapidly progressive dementias are forms of dementia where the patient goes from the first symptom onset to loss of functional independence, usually in less than two years," says John Mills, Ph.D., co-director of Mayo Clinic's Clinical Neuroimmunology Laboratory.
In the early stages of rapidly progressive dementias, patients often exhibit nonspecific symptoms that make reaching a definitive diagnosis challenging. However, it's crucial to identify the correct cause as soon as possible, because some forms—such as those driven by autoimmune processes—may be treatable, while others are not.
"It's really important to be able to give the patient a quick diagnosis and an accurate diagnosis," says Dr. Mills. "Because if it's treatable, you're going to want to treat it right away."
Human prion disease, also called Creutzfeldt-Jakob disease (CJD), results from the misfolding of a normal prion protein—an essential protein present in everyone. The misfolded form of the protein, the cause of CJD, can emerge due to an underlying genetic mutation that predisposes the protein to misfold or spontaneous errors in protein folding.
The misfolded protein can trigger a self-perpetuating cycle by causing correctly folded prion proteins to adopt the pathogenic form. Over time, this process leads to the death of neurons. Unfortunately, there is no cure.
Until recently, lab testing for CJD has been limited to nonspecific neuronal degeneration markers. These tests can detect and confirm rapid destruction of the brain tissues is occurring, but they cannot confidently tell clinicians the underlying cause.
With the development of an RT-QuIC (or "real-time quaking-induced conversion") assay for prion disease, clinicians were able to, for the first time, get a diagnostic test result specific for prion disease with very high diagnostic accuracy. Before this test was available, it wouldn't be until an autopsy was complete that the cause could be confidently identified.
Making a dangerous agent safe to work with
RT-QuIC Prion, CSF (cerebral spinal fluid) is the only definitive antemortem clinical test of its kind that doesn't involve a brain biopsy.
"A lot of the labs that originally did this testing started with brain homogenates, and so when you talk about directly working with brain tissues, there's a clear risk of transmission in instances of needlesticks, for instance," says Dr. Mills.
Fortunately, RT-QuIC for prion disease was proven sensitive using CSF specimens, which is a low-risk specimen and can be manipulated in a standard clinical laboratory that operates at biosafety level 2 (BSL-2). But to ensure an additional layer of safety, the goal at Mayo Clinic was to perform this testing in a BSL-2+ laboratory, which meant a special lab space had to be built from the ground up.
"Creating the right space for this test was a tricky bit of doing," says Ellen Lexvold, technical specialist coordinator in the Neuroimmunology Lab, who helped build out the new test lab.
"It was like building an airplane as you're flying it. We had to tailor the workflow to special considerations, like engineered safety controls, seamless floor tiles, corrosion-resistant steel tables (bleach is one of the few substances that can inactivate pathogenic prion proteins), negative pressure airflow, and a lock-restricted area."
Grizzly bear country
Prion testing is so unique that, prior to Mayo's development of the RT-QuIC clinical assay, there was only one other lab in the U.S. offering the test and only a handful of laboratories performing the test worldwide.
The birthplace of RT-QuIC is Rocky Mountain Laboratories (RML), a research facility situated in the tiny Montana town of Hamilton, wedged between the Bitterroot and Sapphire Mountain ranges on a picturesque landscape.
"In 2010, the RML reported the use of a test called RT-QuIC in a landmark paper," says Dr. Mills.
"They demonstrated how this assay was able to detect very small amounts of pathogenic prion protein, and they could amplify those prion proteins in vitro, and detect them with very high sensitivity. This was the first time an assay that was specific to prion disease had enough sensitivity to be considered as a potential diagnostic test for prion disease."
In 2018, Dr. Mills, Lexvold, and Matt Roforth, a senior developer for Mayo Clinic's Department of Laboratory Medicine and Pathology, traveled to Hamilton to learn how to perform the test.
The team spent a week at RML, which, because of its research specialization in vector-borne diseases and emerging infectious diseases, is one of the few labs in the U.S. that has a BSL-4 rating, a requirement to house things like Ebola. RML is a National Institutes of Health (NIH) biomedical research facility.
"The facility is encircled in barbed-wire fencing, security gates, and before you can enter you have to have a background check," says Dr. Mills.
"Every morning when we walked in, we had to go through a security checkpoint. They inspect your belongings, you walk through a metal detector. There was very tight security for good reason as it houses highly contagious, dangerous pathogens."
RML is quite particular about the use of the RT-QuIC technology and will only teach it onsite. Dr. Mills continues, "They want to know that whoever is doing the testing is doing it right. They walk you through their protocols, they want to watch you perform some of the testing so they're sure you know how to do it."
Right out of a movie
During the mornings and early afternoons at RML, the team learned cutting-edge technologies and established connections that would help them launch this test at Mayo. "Then there wasn't much to do with our free time," says Dr. Mills. "So we'd go hiking up the Bitterroot Mountains to check things out."
They also often ended up at a local diner, where everyone knew everyone and the owner doubled as the cook. The whole experience was "right out of a movie," according to Dr. Mills. In fact, parts of the TV series "Yellowstone" were filmed in Hamilton and the surrounding area.
Dr. Mills also recounts, "We met some renowned and well-regarded researchers who moved to Hamilton to work at RML, often from around the world. Not exactly the type of people you'd expect to be handy with a shotgun, but they had bears and all sorts of wild animals coming into their homes."
When the team returned to Rochester, they had to wait for a new lab space to be built before they could develop the test—a space they coined the "Bitterroot Lab" in honor of their Montana experience. It took more than two years (thanks to the pandemic interruption) to develop a clinically validated version of the prion test.
"We had to take something that was really meant for research and we had to update it, we had to scale it up to keep pace with the throughput workflow of a clinical space," says Lexvold.
"At the Rocky Mountain Labs, everything is done manually. So somebody is directly pipetting the sample. They're adding their agents into each well. It's a very manual assay. We did something different. We looked at different robotic, automated instrumentation that we maybe could use to do all that pipetting so it didn't have to be manual."
Dr. Mills adds, "We wanted a system that was small so that it could actually fit into a biosafety cabinet because we didn't want those samples being manipulated and mixed with all the reagents outside of the biosafety cabinet. So that was a challenge. But our lab staff really stepped up and embraced the challenge and believed in the vision of offering the test at Mayo Clinic."
Staff worked with a vendor that had a piece of equipment with a small footprint that, after some modifications, would work inside of a biosafety cabinet.
Jack Wu, Ph.D., now a clinical chemistry fellow, developed and validated the test. "Basically, you take the patient's CSF, mix it with a recombinant substrate prion protein that was made in-house at Mayo Clinic, and a fluorescent dye that labels the protein aggregates generated in the reaction," he says.
"You incubate this plate at a temperature of 55 degrees Celsius while shaking it with certain reagents. It measures for the fluorescence every 45 minutes and does this for 36 hours.
"The most critical component of this test is the production of high-quality substrate proteins, which sets the basis for the assay's superior performance in detecting some rare subtypes of human prion diseases. The RT-QuIC methodology is the first of its kind at Mayo."
Turnaround time for a test result is less than a week, which is no small feat considering how specialized the RT-QuIC is. "The laughing joke was that RT-QuIC wasn't so quick," says Dr. Mills. "But I think we are changing attitudes about this."
A definitive answer in place of a cure
Part of the reason Mayo went through all this trouble to get this test is, heretofore, there was only one other lab in the U.S. that performed a clinical version of the test: The National Prion Disease Pathology Surveillance Center (NPDPSC), a lab in Cleveland, Ohio, also trained by RML.
The NPDPSC was founded by the CDC as a result of the mad cow disease scare that happened many years ago, to monitor this disease threat in the future.
"The problem is they're not a traditional reference lab," says Dr. Mills. "They offer a prion test, but one of our concerns was that their turnaround time (for a test result) is long. They're not operating in the normal reference lab industry standards where you have 24/7 lab coverage, including holidays.
"We knew we could provide a faster turnaround time, innovate around the technology, and increase accessibility of this testing through Mayo Clinic Laboratories."
Dr. Mills continues, "In addition, Mayo Clinic strives to offer a one-stop shop for diagnostic testing. This test fits in nicely with that philosophy. Our patients were waiting a long time for diagnostic answers. And having to wait has a huge impact on them and their family."
Now, patients showing symptoms of a rapidly progressive form of dementia can get a definitive answer, quickly, with a highly sensitive test. This small comfort is what drove Mayo Clinic to send a team to Montana, build an entirely new lab space, and automate a complex research test for clinical use.
"There was a lot of heavy lifting that had to happen in the clinical lab, because this test is very unique from everything else we do," says Dr. Mills.
"But Mayo Clinic and our people have a history of supporting complex projects, even when they seem daunting to do, especially if there is an opportunity to improve patient care."
https://medicalxpress.com/news/2025-03-distinguishes-prion-disease-rapidly-dementia.html
