High diagnostic value of second generation CSF RT-QuIC across the wide spectrum of CJD prions

Abstract

An early and accurate in vivo diagnosis of rapidly progressive dementia remains challenging, despite its critical importance for the outcome of treatable forms, and the formulation of prognosis. Real-Time Quaking-Induced Conversion (RT-QuIC) is an in vitro assay that, for the first time, specifically discriminates patients with prion disease. Here, using cerebrospinal fluid (CSF) samples from 239 patients with definite or probable prion disease and 100 patients with a definite alternative diagnosis, we compared the performance of the first (PQ-CSF) and second generation (IQ-CSF) RT-QuIC assays, and investigated the diagnostic value of IQ-CSF across the broad spectrum of human prions. Our results confirm the high sensitivity of IQ-CSF for detecting human prions with a sub-optimal sensitivity for the sporadic CJD subtypes MM2C and MM2T, and a low sensitivity limited to variant CJD, Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. While we found no difference in specificity between PQ-CSF and IQ-CSF, the latter showed a significant improvement in sensitivity, allowing prion detection in about 80% of PQ-CSF negative CJD samples. Our results strongly support the implementation of IQ-CSF in clinical practice. By rapidly confirming or excluding CJD with high accuracy the assay is expected to improve the outcome for patients and their enrollment in therapeutic trials.

Sporadic Fatal Insomnia in Europe: Phenotypic Features and Diagnostic Challenges

“…In virtually all patients in whom videopolysomnography was performed, we found a severe reduction of total sleep time and/or completely disorganized sleep with absence of the physiological polygrapic features of non-REM sleep stages N2 and N3 (spindles, K-complexes, and delta waves). 36,38 In contrast, in all cases who underwent CSF NfL analysis, we documented high values for this biomarker. 48,49 The present study provides, for the first time, the results of neurophysiological and biochemical evaluations of autonomic function in sFI patients.…”

Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples

“…Substrate selection is likewise an important factor to consider for successful amplification using the RT‐QuIC assay. One of the most widely employed substrates is a truncated form of the Syrian‐hamster prion protein (Cramm et al., 2016; Franceschini et al., 2017; Henderson et al., 2013; Orru et al., 2009; Schmitz et al., 2016; Wilham et al., 2010), although a number of other truncated and full‐length prion proteins have been used to successfully amplify a range of infectious prions (Cheng et al., 2016; Haley et al., 2017). Although careful handling of the recombinant protein is important for effective amplification, it is equally important to appropriately handle all buffers involved in the protein purification process (Support Protocol 2).…”

“…The RT-QuIC assay is the more recently developed of the two most common protocols for in vitro prion amplification (Atarashi et al, 2008;Wilham et al, 2010). The assay is under continual refinement, with occasional modifications made to improve both sensitivity and specificity reported over the years since its introduction (Denkers, Henderson, Mathiason, & Hoover, 2016;Franceschini et al, 2017;Vascellari et al, 2012). Unlike the PMCA assay (Basic Protocol 1), RT-QuIC amplification is dependent on recombinant prion protein commonly produced in bacterial cells, with expression and purification typically performed over the course of several days (Support Protocol 2).…”

“…The substrate for amplification in the RT-QuIC assay (Basic Protocol 2) is recombinant prion protein, most often produced in bacteria. Several different recombinant prion protein variants have been used in practice, most commonly a truncated Syrian-hamster protein that has been used by several laboratories to study various misfolded prions (Atarashi et al, 2007;Cramm et al, 2016;Franceschini et al, 2017;Haley et al, 2014;Orru, Favole, et al, 2015;Park et al, 2017;Peden et al, 2012). The researcher may find it easier to request aliquots of his or her preferred recombinant plasmid from established laboratories (Atarashi et al, 2007;Hwang, Greenlee, & Nicholson, 2017) than to develop them de novo, although they are not technically challenging to generate using either conventional DNA amplification or gene synthesis, plasmid cloning, and bacterial transformation techniques .…”

14-3-3 and enolase abundances in the CSF of Prion diseased rats

“…Definitive diagnosis of human prion disease involves direct detection of PrP CJD , typically from post-mortem brain samples. Ante-mortem methods such as CSF protein biomarkers, EEG, MRI, and RT-QuIC are used in combination with clinical symptoms to diagnose probable CJD [3–5]. The inclusion of MRI in the diagnostic criteria and development of direct methods for detecting PrP CJD , such as RT-QuIC, has reduced the dependence on indirect markers for diagnosis, however, 14-3-3 and NSE remains testable with the potential for prognostic utility.…”