Biomarkers

What do the seeding assays measure?

Seeded amplification detects the misfolded prion protein by using it to template conversion of a recombinant or brain-derived substrate. The 2026 Viruses review gives RT-QuIC on cerebrospinal fluid specificity around 99 percent and sensitivity above 90 percent for second generation assays, and notes application to olfactory mucosa brushings. The Istituto Besta laboratory medicine service runs seeded amplification assays, RT-QuIC and protein misfolding cyclic amplification for brain biomarkers in suspected prion disease inside a biosafety level 3 laboratory, with Fabio Moda among its senior health staff. The Australian National CJD Registry at The Florey offers a CJD biomarker panel of 14-3-3 protein ELISA and RT-QuIC, described as NATA and RCPA accredited, alongside PRNP genetic testing.

Substrate choice changes which cases are detected. In the Myskiw and colleagues panel, a deer mouse recombinant prion protein substrate read at end point detected fatal familial insomnia with D178N in 12 of 13 samples where truncated hamster RT-QuIC detected 0 of 13, and Gerstmann-Straussler-Scheinker with P102L in 10 of 10 against 4 of 10. The same paper has internal inconsistencies between its abstract and its tables in both the inherited case count and the reported sensitivities, and the authors list independent validation, inter-laboratory reproducibility and protocol harmonisation as still outstanding. A seeding assay result is therefore conditional on the substrate and protocol that produced it. The comparison across formats is in comparing the tests.

How far do neurofilaments reach?

Steinacker and colleagues studied 103 patients from Ulm and Goettingen, including 43 with CJD, of which 33 were sporadic and 9 genetic, against 40 non-demented and 20 demented controls. Serum neurofilament light averaged 317 pg per ml in sporadic and 223 pg per ml in genetic CJD, against roughly 26 to 49 pg per ml in controls; cerebrospinal fluid values were about 8,100 and 8,800 pg per ml against roughly 1,500 to 1,600. At a 44.7 pg per ml cut-off, serum neurofilament light gave 100 percent sensitivity and 85.5 percent specificity; CSF neurofilament light at 2,156 pg per ml gave 97.3 percent and 86.5 percent.

The presymptomatic claim rests on one person. A single P102L carrier was sampled before symptoms and again at onset. CSF neurofilament light and phosphorylated neurofilament heavy were already above the control 75th percentile while CSF tau was normal, and CSF RT-QuIC was negative before symptoms and positive at onset. The authors describe this as preliminary evidence and call neurofilaments promising tools for predicting onset. Cut-offs in that study were chosen to maximise the Youden index on the same data, which tends to overstate accuracy, and differential diagnosis against other rapidly progressive dementias was not tested. The 2026 Viruses review still classifies neurofilament light and phosphorylated neurofilament heavy as mainly research tools.

Which glial markers are being examined?

Two targets have moved from mouse pathology into human samples, and both carry small human group sizes.

Caredio and colleagues, reporting in Nature Communications in 2026 from the Aguzzi group with the Polymenidou laboratory and the Goettingen group, describe a microglial state in terminally sick prion-infected mice marked by high Gpnmb expression and phagocytic and lysosomal gene signatures, concentrated in regions with heavy cell death. They report that soluble factors released by dying cells, rather than protein aggregates, drive this state. The preprint version reported raised GPNMB in cerebrospinal fluid from sporadic CJD patients. The human tissue comparison involves five sporadic CJD patients against five matched controls, which is too small to support a diagnostic claim, and GPNMB is also being developed as a pharmacodynamic marker of PIKfyve inhibition in amyotrophic lateral sclerosis, so a raised value is not specific to prion disease.

Vanni and colleagues measured SERPINA3 in 128 human post-mortem frontal cortex samples. Messenger RNA was about 40 times control levels in sporadic CJD, 39 times in genetic and in variant CJD, 53 times in fatal familial insomnia and 347 times in iatrogenic CJD, against about 7 times in Alzheimer disease, a figure the authors note may be overestimated because their Alzheimer group was older than the available controls. Protein was about six times control levels by Western blot, with staining in most residual cortical neurons and many reactive astrocytes. The biofluid result closes the diagnostic door: SERPINA3 messenger RNA was absent from blood, and SERPINA3 protein in cerebrospinal fluid did not differ significantly between Alzheimer disease and sporadic CJD. Its interest is as a target rather than a marker, which is taken up in sporadic CJD research.

What would a trial endpoint require?

The 2026 Lancet Neurology review states that blood-based diagnostic biomarkers are now close to clinical application, that work is moving towards presymptomatic 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. It also raises the interpretive problem created by detecting misfolded prion protein in healthy mutation carriers before symptoms, and how that relates to other molecular signals detectable before onset.

Trial design is where this is being tested. The CJD Foundation drug development page lists the Broad Institute divalent siRNA programme under trial registration NCT07444580, with a presymptomatic arm opened according to a statement dated 14 October 2026, and an Ionis antisense oligonucleotide programme under NCT06153966. Prion protein concentration in cerebrospinal fluid is the measurement these programmes use to show target engagement. Whether any marker can also show clinical benefit is unsettled. The sponsor-side view of this question is in biomarkers and endpoints.

The assay methods themselves are described in diagnostics research, and the diagnostic sequence in a patient in how prion disease is diagnosed.