Early Detection of Abnormal Prion Protein in Genetic Human Prion Diseases Now Possible Using Real-Time QUIC Assay

Abstract

IntroductionThe definitive diagnosis of genetic prion diseases (gPrD) requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau) protein in the cerebrospinal fluid (CSF), but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Sträussler-Scheinker syndrome (GSS). We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC)”, to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD) patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined.Method/Principal Findings56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N), and 24 cases of genetic CJD (gCJD), comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%), FFI (100%), gCJD E200K (87%), and gCJD V203I (100%). On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%), FFI (0%), gCJD E200K (73%), and gCJD V203I (67%). Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI.Conclusion/SignificanceRT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patient's family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.

Quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion

“…The ASA and RT-QuIC are generally analogous to sPMCA, but employ recombinant PrP C instead of brain homogenate as substrate for disease-specific, in vitro, amyloid formation (Atarashi et al, 2007(Atarashi et al, , 2011aColby et al, 2007). RT-QuIC offers the advantage of real-time assessment of amyloid fibril generation and has been successfully applied to many seed sources, some of which are available for antemortem sampling (Atarashi et al, 2011a, b;Elder et al, 2013;Haley et al, 2013;Henderson et al, 2013;McGuire et al, 2012;Orrú et al, 2011Orrú et al, , 2014Peden et al, 2011;Sano et al, 2013;Wilham et al, 2010).…”

Immediate and Ongoing Detection of Prions in the Blood of Hamsters and Deer following Oral, Nasal, or Blood Inoculations

“…f See reference 19. This is the first report of the detection of PrP C -CCA in the blood of animals within minutes of exposure to TSE inoculum. The use of PrP C -CCA to detect prions in tissues and bodily fluids of TSE-infected hosts has been shown to be as sensitive as a bioassay and has provided insight into a variety of prion diseases (15)(16)(17)(18). The immediate detection of the point-source inoculum was seen following exposure to mucosal surfaces in the nose and gut; the same immediate detection of PrP C -CCA was seen following i.v.…”

A new diagnostic test for Creutzfeldt-Jakob disease: Real-time quaking-induced conversion (RT-QuIC)

“…However, ongoing validation studies have demonstrated that the outcomes of RT-QuIC reactions depend on the exact conditions employed (biochemical, chemical and physical), as well as the nature of the sample and recombinant substrate. The protocol under evaluation by the National Microbiology Laboratory has been tested extensively for the detection of sporadic CJD (10,11,15,16 ). Using this protocol the Prion Laboratory Section achieved 100% sensitivity and 100% specificity in the evaluation of an external sporadic CJD infected CSF proficiency panel.…”