The clinical diagnosis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), is challenging, especially at an early disease stage, due to the heterogeneous and often nonspecific clinical manifestations. The discovery of reliable specific markers for synucleinopathies would consequently be of great aid to the diagnosis and management of these disorders. Real-Time Quaking-Induced Conversion (RT-QuIC) is an ultrasensitive technique that has been previously used to detect self-templating amyloidogenic proteins in the cerebrospinal fluid (CSF) and other biospecimens in prion disease and synucleinopathies. Using a wild-type recombinant α-synuclein as a substrate, we applied RT-QuIC to a large cohort of 439 CSF samples from clinically well-characterized, or post-mortem verified patients with parkinsonism or dementia. Of significance, we also studied patients with isolated REM sleep behavior disorder (iRBD) (n = 18) and pure autonomic failure (PAF) (n = 28), representing clinical syndromes that are often caused by a synucleinopathy, and may precede the appearance of parkinsonism or cognitive decline. The results show that our RT-QuIC assay can accurately detect α-synuclein seeding activity across the spectrum of Lewy Body (LB)-related disorders (LBD), including DLB, PD, iRBD, and PAF, with an overall sensitivity of 95.3%. In contrast, all but two patients with MSA showed no α-synuclein seeding activity in the applied experimental setting. The analysis of the fluorescence response reflecting the amount of α-synuclein seeds revealed no significant differences between the clinical syndromes associated with LB pathology. Finally, the assay demonstrated 98% specificity in a neuropathological cohort of 101 cases lacking LB pathology. In conclusion, α-synuclein RT-QuIC provides an accurate marker of synucleinopathies linked to LB pathology and may have a pivotal role in the early discrimination and management of affected patients. The finding of no α-synuclein seeding activity in MSA seems to support the current view that MSA and LBD are associated with different conformational strains of α-synuclein.

The differential diagnosis of Creutzfeldt-Jakob disease (CJD) from other, sometimes treatable, neurological disorders is challenging, owing to the wide phenotypic heterogeneity of the disease. Real-time quaking-induced prion conversion (RT-QuIC) is a novel ultrasensitive in vitro assay, which, at variance with surrogate neurodegenerative biomarker assays, specifically targets the pathological prion protein (PrPSc). In the studies conducted to date in CJD, cerebrospinal fluid (CSF) RT-QuIC showed good diagnostic sensitivity (82–96%) and virtually full specificity. In the present study, we investigated the diagnostic value of both prion RT-QuIC and surrogate protein markers in a large patient population with suspected CJD and then evaluated the influence on CSF findings of the CJD type, and the associated amyloid-β (Aβ) and tau neuropathology. RT-QuIC showed an overall diagnostic sensitivity of 82.1% and a specificity of 99.4%. However, sensitivity was lower in CJD types linked to abnormal prion protein (PrPSc) type 2 (VV2, MV2K and MM2C) than in typical CJD (MM1). Among surrogate proteins markers (14-3-3, total (t)-tau, and t-tau/phosphorylated (p)-tau ratio) t-tau performed best in terms of both specificity and sensitivity for all sCJD types. Sporadic CJD VV2 and MV2K types demonstrated higher CSF levels of p-tau when compared to other sCJD types and this positively correlated with the amount of tiny tau deposits in brain areas showing spongiform change. CJD patients showed moderately reduced median Aβ42 CSF levels, with 38% of cases having significantly decreased protein levels in the absence of Aβ brain deposits. Our results: (1) support the use of both RT-QuIC and t-tau assays as first line laboratory investigations for the clinical diagnosis of CJD; (2) demonstrate a secondary tauopathy in CJD subtypes VV2 and MV2K, correlating with increased p-tau levels in the CSF and (3) provide novel insight into the issue of the accuracy of CSF p-tau and Aβ42 as markers of brain tauopathy and β-amyloidosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-017-1683-0) contains supplementary material, which is available to authorized users.

An early and accurate in vivo diagnosis of rapidly progressive dementia remains challenging, despite its critical importance for the outcome of treatable forms, and the formulation of prognosis. Real-Time Quaking-Induced Conversion (RT-QuIC) is an in vitro assay that, for the first time, specifically discriminates patients with prion disease. Here, using cerebrospinal fluid (CSF) samples from 239 patients with definite or probable prion disease and 100 patients with a definite alternative diagnosis, we compared the performance of the first (PQ-CSF) and second generation (IQ-CSF) RT-QuIC assays, and investigated the diagnostic value of IQ-CSF across the broad spectrum of human prions. Our results confirm the high sensitivity of IQ-CSF for detecting human prions with a sub-optimal sensitivity for the sporadic CJD subtypes MM2C and MM2T, and a low sensitivity limited to variant CJD, Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. While we found no difference in specificity between PQ-CSF and IQ-CSF, the latter showed a significant improvement in sensitivity, allowing prion detection in about 80% of PQ-CSF negative CJD samples. Our results strongly support the implementation of IQ-CSF in clinical practice. By rapidly confirming or excluding CJD with high accuracy the assay is expected to improve the outcome for patients and their enrollment in therapeutic trials.