Since its initial application to patients with sporadic Creutzfeldt-Jakob disease (sCJD), significant technical improvements have been made to the real time-quaking-induced conversion (RT-QuIC) assay in CSF, making it much faster and more accurate.^{Reference Orrú, Groveman and Hughson1} CSF analysis by second-generation RT-QuIC Improved QuIC conditions yielded a sensitivity of 95% and a specificity of around 100%, as only a few false-positive cases have been reported out of thousands of CSF samples tested.^{Reference Slota, Myskiw and Peterson2} In this retrospective study, 2667 CSF samples were collected from individuals with suspected prion disorder from 2016 to 2024 across Canada provinces.^{Reference Green3} These samples were analyzed for 14.3.3 and Tau levels and by end-point QuIC (EP-QuIC). This assay identified 535 positive CSF samples, with sCJD pathological confirmation in 284 CJD cases. The overall sensitivity was 95%, with a single false-positive result from a patient with immune-mediated encephalopathy.^{Reference Green3} These results are in line with the extensive studies in CSF of sCJD patients and confirm the high-fidelity and diagnostic strength of the QuIC assay, also in sCJD subtypes, showing a higher sensitivity in MM2 and VV mixed compared to previous studies.^{Reference Hermann, Schmitz and Cramm4} Paradoxically, a higher number of EP-QuIC-negative CSF samples belonged to the MM1 subtype, which is usually characterized by a typical disease phenotype. The negative results were related to blood contamination. However, it has been demonstrated that diluting and retesting these false-negative CSF samples can further increase the sensitivity of the assay.^{Reference Fiorini, Iselle and Perra5} CSF biomarkers, such as 14-3-3 and tau protein, were also determined quantitatively, and their levels were related to the extent of disease progression. The high diagnostic accuracy of RT-QuIC assays has, of course, radically changed clinicians’ attitudes toward sCJD diagnosis worldwide, fostered by reproducibility among laboratories. Notably, prior to the introduction of RT-QuIC assays, the detection of 14-3-3 in CSF supported a CJD diagnosis, but the low specificity of this test resulted in uncertainty and the pursuit of additional, unnecessary diagnostic investigations. This study provides further confirmation of the success of RT-QuIC assays in sCJD diagnosis. Although RT-QuIC does not detect genetic prion diseases (i.e., Fatal Familial Insomnia and Gerstmann-Straussler-Scheinker) or variant CJD with the same accuracy as sCJD, family history or genetic testing can confirm the final diagnosis. A single patient with variant CJD and MV at codon 129 has been reported, which could be confused with sCJD.^{Reference Mok, Jaunmuktane and Susan6} However, the intensive surveillance system for CJD has made variant CJD an exhausting disease.