Diagnostics research

What did adding amplification assays to the criteria achieve?

Watson and colleagues validated the revised international surveillance criteria for sporadic CJD on 501 cases and 146 non-cases from the United Kingdom, France, Germany and Italy. In-life sensitivity rose to 92.2 percent, from 77.5 percent under the previous criteria, with specificity essentially unchanged at about 81 percent. In the subset investigated with the full panel, sensitivity rose to 97.8 percent from 76.2 percent. The two additions responsible were cortical ribboning on MRI, with 67.9 percent sensitivity and 86.5 percent specificity, and RT-QuIC, with 91.6 percent sensitivity and 100 percent specificity in that cohort.

The 2026 Viruses review places RT-QuIC as the most informative cerebrospinal fluid test, with specificity around 99 percent and sensitivity above 90 percent for second generation assays, and notes it can also be run on olfactory mucosa brushings. It describes CSF 14-3-3 and total tau as elevated but lacking specificity, diffusion-weighted MRI as the most sensitive imaging test with early scans sometimes normal, and periodic sharp wave complexes on EEG in about two thirds of sporadic cases. For the full diagnostic sequence, see how prion disease is diagnosed.

Which cases do the assays miss?

Two recent studies set the limits. Myskiw and colleagues compared substrates in a retrospective Canadian and United States panel and found that a deer mouse recombinant prion protein substrate read at end point detected cases that truncated hamster RT-QuIC did not. In their Table 2, fatal familial insomnia with D178N was detected in 12 of 13 by the deer mouse end point assay and in 0 of 13 by truncated hamster RT-QuIC; Gerstmann-Straussler-Scheinker with P102L in 10 of 10 against 4 of 10. The paper carries internal inconsistencies that a reader should note: the abstract cites 50 inherited cases where Table 2 lists 40 plus 10 atypical sporadic cases, and the abstract’s sensitivity and specificity denominators do not match the tables. The authors list no independent validation, no inter-laboratory reproducibility testing and no protocol harmonisation as outstanding.

Cheng and colleagues reported an end point format on 91 retrospective cerebrospinal fluid samples, 45 confirmed CJD of which 43 were sporadic and 2 genetic, against 46 clinically confirmed non-CJD. End point quaking-induced conversion gave 98 percent sensitivity and 91 percent specificity, RT-QuIC on the same samples 96 percent and 98 percent, and 14-3-3 84 percent and 70 percent. The ratio cut-offs were set by receiver operating characteristic analysis on the same panel, and the authors note that prospective analyses would be needed to establish performance in clinical use. The side by side figures are collected in comparing the tests.

Which laboratories hold the reference methods?

Reference capability is concentrated in a small number of national centres, and what each offers is specific.

The Italian National CJD Registry at the Istituto Superiore di Sanita runs PrP RT-QuIC on cerebrospinal fluid and genetic analysis for physicians, with separate consent forms for diagnostic and research use of genetic, CSF, urine, olfactory mucosa and saliva samples. Its service page states that it no longer performs the CSF 14-3-3 test, and it lists no prices.

The Australian National CJD Registry, established in October 1993 and based at The Florey under contract to the Commonwealth Department of Health, provides national surveillance together with diagnostic testing for sporadic, familial and iatrogenic CJD, including a CJD biomarker panel of 14-3-3 protein ELISA and RT-QuIC described as NATA and RCPA accredited, tissue biopsy and PRNP genetic testing. It arranges brain-only autopsies at no cost to the family or referring institution, with neuropathology results taking three to twelve months.

The laboratory medicine service at the Istituto Besta in Milan runs seeded amplification assays, RT-QuIC and protein misfolding cyclic amplification for brain biomarkers in suspected prion disease inside a biosafety level 3 laboratory.

Neuropathology at Universitaetsklinikum des Saarlandes in Homburg describes a different method set: a membrane adsorption assay on native tissue, Western blot typing that distinguishes prion type 1 from type 2 by fragment size and intensity after enzyme digestion, the paraffin-embedded tissue blot for the distribution of prion aggregates, and immunohistochemistry. The same group applies the tissue blot and a microfiltration assay to synuclein aggregates in Parkinson disease, dementia with Lewy bodies and multiple system atrophy.

On the animal side, the Ottawa Animal Health Laboratory is one of five Canadian Food Inspection Agency animal health laboratories. It diagnoses transmissible spongiform encephalopathies including bovine spongiform encephalopathy, chronic wasting disease and scrapie, genotypes animals to assess resistance, holds ISO/IEC 17025 accreditation, and serves as a Canadian reference laboratory for scrapie and chronic wasting disease among other diseases.

What is being asked of diagnostics beyond diagnosis?

The 2026 Lancet Neurology review by Zerr, Hermann, Mok and Mead states that blood-based diagnostic biomarkers are now close to clinical application, that research is moving towards presymptomatic mutation carriers and non-invasive sampling in blood, tear fluid and urine, and that a disease staging system based on biomarkers should be evaluated in future clinical studies. That shift, from classifying a patient to measuring where a patient sits in a disease course, is what makes these assays relevant to trials. It is covered in biomarkers.

The containment these methods require, and their use in instrument and donor tissue work, sit in transmission and biosafety. The diseases the assays are asked to separate are described in disease research.