Ring trial of 2nd generation RT‐QuIC diagnostic tests for sporadic CJD

Abstract

Objective: Real-time quaking-induced conversion (RT-QuIC) assays detect prion-seeding activity in a variety of human biospecimens, including cerebrospinal fluid and olfactory mucosa swabs. The assay has shown high diagnostic accuracy in patients with prion disorders. Recently, advances in these tests have led to markedly improved diagnostic sensitivity and reduced assay times. Accordingly, an algorithm has been proposed that entails the use of RT-QuIC analysis of both sample types to diagnose sporadic Creutzfeldt-Jakob disease with nearly 100% accuracy. Here we present a multi-center evaluation (ring trial) of the reproducibility of these improved "second generation" RT-QuIC assays as applied to these diagnostic specimens. Methods: Cerebrospinal fluid samples were analyzed from subjects with sporadic Creutzfeldt-Jakob (n = 55) or other neurological diseases (n = 45) at multiple clinical centers. Olfactory mucosa brushings collected by multiple otolaryngologists were obtained from nine sporadic Creutzfeldt-Jakob disease cases and 19 controls. These sample sets were initially tested blindly by RT-QuIC by a coordinating laboratory, recoded, and then sent to five additional testing laboratories for blinded ring trial testing. Results: Unblinding of the results by a third party indicated 98-100% concordance between the results obtained by the testing of these cerebrospinal fluid and nasal brushings at the six laboratories. Interpretation: This second-generation RT-QuIC assay is highly transferrable, reproducible, and therefore robust for the diagnosis of sporadic Creutzfeldt-Jakob disease in clinical practice.

Detection of Prions in Brain Homogenates and CSF Samples Using a Second-Generation RT-QuIC Assay: A Useful Tool for Retrospective Analysis of Archived Samples

“…The second-generation RT-QuIC, also known as the IQ assay (Improved QuIC), is a version of the assay with increased sensitivity and significantly shorter time of detection [10]. Almost all human studies utilizing second-generation RT-QuIC have used CSF as the analyte [8][9][10][11][15][16][17][18][19][20][21]. In our retrospective study, we evaluated the second-generation RT-QuIC assay for the analysis of archival post-mortem BH and CSF samples within a TSE patient cohort typical of the Czech Republic [22,23].…”

Structurally distinct external solvent-exposed domains drive replication of major human prions

“…Evidence accumulated with prions and more recently with a growing list of other proteins indicate that the misfolded conformers can be amplified with recombinant substrates in a seeded reaction in vitro— a prerequisite step for misfolding propagation in the brain [ 22 , 24 , 29 , 41 , 46 , 63 – 65 ]. At initial 10 −6 dilution of purified prions, we observed lower ThT fluorescence, and in MM2 prions an extended lag phase after 200ms irradiation ( Fig 3 ).…”

Sporadic Creutzfeldt-Jakob disease: Real-Time Quaking Induced Conversion (RT-QuIC) assay represents a major diagnostic advance

“… 113 Overall, these characteristics support the choice by WHO to include the CSF RT-QuIC test in the diagnostic criteria for sCJD. 39 , 99 , 101 As previously mentioned, only few specialized laboratories have adopted the RT-QuIC technology. However, the assay is relatively easy to learn and can be rapidly used by trained personnel, thus consenting its widening in other centers specialized in the diagnosis of neurodegenerative diseases associated with protein misfolding.…”

“…55,56,83,92,[95][96][97][98] In 2020, an international trial confirmed the robustness and reliability of the second generation RT-QuIC for the diagnosis of sCJD. 99 Furthermore, a recent article by Fiorini et al 100 demonstrated that through the combined RT-QuIC analysis of the CSF and OM collected from the same sCJD patient it is possible to reach a diagnostic accuracy of 100%. For these reasons, the RT-QuIC has been introduced among the diagnostic criteria of some surveillance centers.…”