New sensitive assay could potentially improve detection of prion diseases
An advanced assay that potentially has better sensitivity than currently available tests for detecting a prion disease affecting elk has been described by a group of researchers in The Journal of Molecular Diagnostics.
Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, affect both humans and animals. They are rare, progressive neurodegenerative illnesses that have potential to pass from animals to humans, highlighted by the case of bovine spongiform encephalopathy. Surveillance of TSEs is important for both public health and food safety.
“The significance of TSEs on human health was not entirely realized until cases of variant Creutzfeldt-Jakob disease (vCJD) in humans had been discovered in the years following the BSE outbreak in the UK. These vCJD cases were associated with consuming meat products contaminated with BSE prions,” explained lead investigator Stefanie Czub of the Canadian BSE Reference Lab, Canadian Food Inspection Agency, Lethbridge Laboratory (Alberta,Canada).
“The result is that many countries have enacted TSE surveillance programs, aiming to eradicate livestock-related TSEs.”
In order to detect infections early, surveillance programs are reliant on highly sensitive diagnostic methods. Researchers have developed a method of measuring prion protein conversion time (from normal cellular form to prion form), by combining statistical analyses to yield a prion-detecting amyloid seeding assay (ASAs) with a known degree of confidence, thus addressing the need to define steadfast analytical performance criteria for prion ASAs.
The sensitivity of the new assay technique, the timed prion seeding assay (tASA), was compared with other currently available tests.
To assess the sensitivity of the timed prion seeding assay, test samples were generated utilising elk brains infected experimentally with chronic wasting disease (CWD), a prion disease that affects hoofed ruminants in the deer family. Determination of TSE-positive and TSE-negative states was possible as the investigators were able to define clear cut-off criteria.
ASAs also have the potential to detect and measure TSE infection in blood, saliva and urine, unlike TSE rapid tests. This presents clinical advantages, such as the ability to sample blood instead of having to rely on more invasive tissue biopsy and also to screen blood donations for any contamination.
“We found that the tASA was at least as sensitive as two rodent bioassays and up to 16 times more sensitive than three different TSE rapid tests,” noted lead author John G Gray Canadian BSE Reference Lab, Canadian Food Inspection Agency.
“This study should further advance ASAs as recognized prion detection systems. We believe this methodology represents the future for prion diagnostics, especially concerning human health, for example in screening blood donations.”
The conjectured mechanism of prion propagation in vivo is mimicked by the in vitro tTSA method. Recombinant prion-related protein is used as a substrate by the assay and conversion is detected via changes in fluorescence.
Specifications to help avoid false-positive results (30 hours) or false-negative results in weakly-positive samples (48 hours) are described in the report, along with the number of replications necessary for adequate sensitivity (2–12).
“This study represents an important first step for the tASA diagnostic protocol to gain regulatory approval for its use in TSE surveillance programs targeting CWD in cervids,” commented Holger Wille of the University of Alberta Department of Biochemistry and Centre for Prions and Protein Folding Diseases in Edmonton, (Alberta, Canada).
“Additional work will also be needed to fine-tune and test tASA for the detection of prions in peripheral organs and environmental samples, which represent a substantial unmet need to track the spread of CWD prions among North America cervids as well as in the environment.”
Sources: New assay offers improved detection of deadly prion diseases; Gray JG, Graham C, Dudas S, Paxman E, Vuong B, Czub S. Defining and assessing analytical performance criteria for transmissible spongiform encephalopathy – detecting amyloid seeding assays. The Journal of Molecular Diagnostics doi: 10.1016/j.jmoldx.2016.01.005 (2016).
