Advancing prion diagnostics: full-length human E200K RT-QuIC substrate facilitates prion detection in tear fluid and improves sensitivity in cerebrospinal fluid

Abstract The real-time quaking-induced conversion (RT-QuIC) assay has revolutionized prion disease diagnosis by detecting amyloidogenic PrP conformers in different tissue types and body fluids. Recently, we achieved a breakthrough by detecting amyloidogenic PrP conformers in tear fluid (TF), a non-invasive biofluid, warranting further evaluation. We refined our RT-QuIC protocol to assess seeding conversion efficiency using various recombinant substrates, including full-length human (FL Hu), and mutant versions linked to genetic prion diseases, such as E200K and D178N, in both CSF and TF samples. Our study included patients with sporadic and familial prion diseases. FL Hu E200K showed the highest seeding efficiency in cerebrospinal fluid (CSF), with sensitivity increasing from 78 to 93% for symptomatic Creutzfeldt–Jakob disease (CJD) and from 19 to 75% for fatal familial insomnia (FFI) compared with the hamster-sheep substrate. For tear fluid, diagnostic sensitivity was 85% for sCJD and 64% for symptomatic genetic prion diseases, the assay was also able to detect amyloidogenic PrP in 57% of the healthy mutation carriers (HMC, persons at risk without symptoms). We validated the diagnostic accuracy of the TF RT-QuIC from our previous study (cohort 1) in a second independent study. Additionally, we confirmed that TF RT-QuIC requires FL Hu E200K substrates since hamster-sheep failed in detection of seeding activity in tears. Notably, only 1 out of 184 controls without prion disease tested positive. Studies comparing different disease stages (early vs. late) showed that later stages produced a stronger signal response. Our study demonstrates that the FL Hu E200K rec PrP substrate improves RT-QuIC sensitivity in CSF diagnostics and validates the reliable detection of seeding activity in TF in two cohorts as well as in HMC.