OBJECTIVETo present the National Prion Disease Pathology Surveillance Centerʼs (NPDPSC’s) experience using CSF real-time quaking-induced conversion (RT-QuIC) as a diagnostic test, to examine factors associated with false-negative RT-QuIC results, and to investigate the impact of RT-QuICs on prion disease surveillance. METHODSBetween May 2015 and April 2018, the NPDPSC received 10,498 CSF specimens that were included in the study. Sensitivity and specificity analyses were performed on 567 autopsy-verified cases. Prion disease type, demographic characteristics, specimen color, and time variables were examined for association with RT-QuIC results. The effect of including positive RT-QuIC cases in prion disease surveillance was examined. RESULTSThe diagnostic sensitivity and specificity of RT-QuIC across all prion diseases were 90.3% and 98.5%, respectively. Diagnostic sensitivity was lower for fatal familial insomnia, Gerstmann-Sträussler-Scheinker disease, sporadic fatal insomnia, variably protease sensitive prionopathy, and the VV1 and MM2 subtypes of sporadic Creutzfeldt-Jakob disease. Individuals with prion disease and negative RT-QuIC results were younger and had lower tau levels and nonelevated 14-3-3 levels compared to RT-QuIC–positive cases. Sensitivity was high throughout the disease course. Some cases that initially tested RT-QuIC negative had a subsequent specimen test positive. Including positive RT-QuIC cases in surveillance statistics increased laboratory-based case ascertainment of prion disease by 90% over autopsy alone. CONCLUSIONSRT-QuIC has high sensitivity and specificity for diagnosing prion diseases. Sensitivity limitations are associated with prion disease type, age, and related CSF diagnostic results. RT-QuIC greatly improves laboratory-based prion disease ascertainment for surveillance purposes. CLASSIFICATION OF EVIDENCEThis study provides Class III evidence that second-generation RT-QuIC identifies prion disease with a sensitivity of 90.3% and specificity of 98.5% among patients being screened for these diseases due to concerning symptoms.