Stability and Reproducibility Underscore Utility of RT-QuIC for Diagnosis of Creutzfeldt-Jakob Disease

Abstract

Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrP(Sc)). Recent studies applied the RT-QuIC methodology to cerebrospinal fluid (CSF) for diagnosing human prion diseases. However, to date, there has not been a formal multi-centre assessment of the reproducibility, validity and stability of RT-QuIC in this context, an indispensable step for establishment as a diagnostic test in clinical practice. In the present study, we analysed CSF from 110 prion disease patients and 400 control patients using the RT-QuIC method under various conditions. In addition, "blinded" ring trials between different participating sites were performed to estimate reproducibility. Using the previously established cut-off of 10,000 relative fluorescence units (rfu), we obtained a sensitivity of 85% and a specificity of 99%. The multi-centre inter-laboratory reproducibility of RT-QuIC revealed a Fleiss' kappa value of 0.83 (95% CI: 0.40-1.00) indicating an almost perfect agreement. Moreover, we investigated the impact of short-term CSF storage at different temperatures, long-term storage, repeated freezing and thawing cycles and the contamination of CSF with blood on the RT-QuIC seeding response. Our data indicated that the PrP(Sc) seed in CSF is stable to any type of storage condition but sensitive to contaminations with blood (>1250 erythrocytes/μL), which results in a false negative RT-QuIC response. Fresh blood-contaminated samples (3 days) can be rescued by removal of erythrocytes. The present study underlines the reproducibility and high stability of RT-QuIC across various CSF storage conditions with a remarkable sensitivity and specificity, suggesting RT-QuIC as an innovative and robust diagnostic method.

Keywords: Cerebrospinal fluid; Creutzfeldt-Jakob disease; Diagnostic test; Prion protein; Real-time quaking-induced conversion.

Fig. 1

Analysis of the sensitivity and specificity of the RT-QuIC assay. RT-QuIC response was measured in rfu over a period of 80 h. a RT-QuIC signalling response was evaluated as a diagnostic test by using receiver operating characteristic (ROC) curves. The ROC curve of the training dataset is shown suggesting a cut-off at 10,000 rfu. b Prion diseases exhibited a median RT-QuIC signal of 35,000 rfu, while controls remained under 10,000 rfu. c Considering >50 % positive RT-QuIC replicates as positive for prion disease, we obtained a specificity of 99 % and an averaged sensitivity of 85 %. d Two hundred CSF samples, retrospectively tested by RT-QuIC, are summarized

Fig. 2

Effect of various storage conditions on the RT-QuIC signal response. RT-QuIC reactions seeded with CSF from sCJD patients were analysed under short-term storage conditions (8 days) at room temperature (RT) and 4 °C (a, b), after 16 repeated freezing and thawing cycles (c) and after long-term storage (up to 9 years) (d). There was no significant trend for decreasing positivity rates over time when a storing at room temperature (p = 0.337), b storing at 4 °C (p = 0.713) or c after 16 freezing and thawing cycles (p = 0.537) and d over several years of storage at −80 °C. Numbers of positive replicates were calculated for each sample in percent. Standard deviation of the mean from n = 12 patients per group was indicated by error bars. For comparison between groups, we used ANOVAs

Fig. 3

Time course of prion seeding activity in the CSF of sCJD patients after defined storage conditions. RT-QuIC reactions seeded with CSF from sCJD patients were analysed under short-term storage conditions (8 days) at room temperature (RT) and 4 °C (a, b) after long-term storage (up to 9 years) (c) and after 16 repeated freezing and thawing cycles (d). The PrP^Sc seeding efficiency was not significantly changed under these conditions. Graphically displayed are means of all positive replicates per group at each point in time. The absolute values for rel. AUC and signal maximum were shown for each group, and the p values were calculated for each comparative analysis. p values were obtained using paired or unpaired t tests for comparisons of maximum values and rel. AUCs

Fig. 4

Influence of blood contamination on the RT-QuIC seeding response. CSF from sCJD patients (n = 6–8) was spiked with defined amounts of sonicated red blood cells. a The number of positive replicates was calculated and revealed that sCJD CSF samples spiked with more than 1250 cells/μL showed a false-negative RT-QuIC response. b CSF from sCJD (n = 8) patients was spiked with 5000 cells/μL and incubated at room temperature without sonication for 8 days showing that a significant inhibition via haemolysis of the RT-QuIC response started after 3 days. c, d Graphically displayed are means of all positive replicates per group at each point in time. Absolute values for rel. AUC and signal maximum were shown for each group, and the p values were calculated for each comparative analysis. All p values <0.05 were considered as significant