An ultrasensitive quaking-induced conversion assay for all forms of sporadic and inherited human prion diseases

Introduction: Quaking‐induced conversion assays to detect prion amyloid seeding in cerebrospinal fluid (CSF) are highly accurate for antemortem testing of patients with human prion disorders such as Creutzfeldt-Jakob disease (CJD). Most diagnostic laboratories use real-time quaking induced conversion (RT-QuIC) that tracks seeding over time. Insensitivity of detection for inherited prion diseases has been noted, including fatal familial insomnia (FFI), Gerstmann-Straussler-Scheinker (GSS) syndrome, as well as for rare subtypes of CJD, and the atypical human prion disease, variably protease-sensitive prionopathy (VPSPr).

Objectives: To evaluate the North American deer mouse (Peromyscus maniculatus) recombinant prion protein as substrate in CSF QuIC assays.

Methods: In Canada, a simpler iteration of the RT-QuIC test has been developed for the diagnostic laboratory. This uses an alternative method of mechanical agitation to initiate seeding and aggregation of recombinant prion protein, and a single endpoint (EP) fluorescence reading. The accuracy of EP-QuIC aligns with the second-generation RT-QuIC test used worldwide. Here we substitute recombinant hamster prion protein substrate with that of deer mouse and validate for a wide variety of sporadic and inherited human prion diseases.

Results/Discussion: The novel recombinant prion substrate demonstrated almost 100% sensitivity and specificity in EP-QuIC including IPDs and subtypes previously found to be refractory to testing, including FFI, GSS, the VV1 subtype, and VPSPr. 

Conclusion: The EP-QuIC assay requires no specialized laboratory equipment and is amenable to accreditation as part of a diagnostic workflow. Inclusion of the deer mouse substrate provides universal, ultrasensitive detection of prion seeding for diagnostic and potentially, prognostic use.