Optimization of the Real-Time Quaking-Induced Conversion Assay for Prion Disease Diagnosis

Abstract

The real-time quaking-induced conversion (RT-QuIC) assay is a highly reproducible and robust methodology exhibiting an excellent pre-mortem diagnostic accuracy for prion diseases. However, the protocols might be time-consuming and improvement of the detection technology is needed. In the present study, we investigated the influence of a pre-analytical cerebrospinal fluid (CSF) treatment with proteinase K (PK) on the kinetic of the RT-QuIC signal response. For this purpose, we added PK at different concentrations in RT-QuIC reactions seeded with Creutzfeldt-Jakob disease (sCJD) CSF. We observed that a mild pre-analytical PK treatment of CSF samples resulted in an increased seeding efficiency of the RT-QuIC reaction. Quantitative seeding parameters, such as a higher area under the curve (AUC) value or a shorter lag phase indicated a higher conversion efficiency after treatment. The diagnostic accuracy resulting from 2 µg/ml PK treatment was analyzed in a retrospective study, where we obtained a sensitivity of 89%. Additionally, we analyzed the agreement with the previously established standard RT-QuIC protocol without PK treatment in a prospective study. Here, we found an overall agreement of 94% to 96%. A Cohen's kappa of 0.9036 (95% CI: 0.8114-0.9958) indicates an almost perfect agreement between both protocols. In conclusion, the outcome of our study can be used for a further optimization of the RT-QuIC assay in particular for a reduction of the testing time.

Clinical Use of Improved Diagnostic Testing for Detection of Prion Disease

“…Very low levels of prions may go undetected and sensitivity is decreased for FFI, sFI, VPSPr, GSS, and the VV1 and MM2 subtypes of sCJD likely due to strain (conformer) differences [ 84 ]. Additionally, RT-QuIC takes days to complete, though investigations into the optimization of RT-QuIC have shown promise in reducing time constraints [ 100 ]. Current diagnostic criteria do not make a distinction between second generation RT-QuIC (IQ-CSF) and first generation RT-QuIC, which has reduced diagnostic accuracy.…”

Optimization of the RT-QuIC in prion disease diagnostic

“…RT-QuIC assay mimics the seeded conversion process of PrP C in vitro, amplifying minuscule amounts of a PrP Sc derived from brain, CSF, olfactory mucosa (OM) brushings, or other tissues to an aggregated form of prion protein resistant to proteases (PrP Res ) 84,85 . phase, the area under the curve, and maximal signal intensity can be measured to evaluate the seeded conversion efficiency [86][87][88] . Finally, the 96-well plate format of RT-QuIC allows automatic analysis of multiple reactions (from more than 30 different samples in triplicates), thereby facilitating the use of RT-QuIC in routine diagnostics.…”