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Abstract Background Assays that specifically measure α‐synuclein seeding activity in biological fluids could revolutionize the diagnosis of Parkinson’s disease. Recent improvements in α‐synuclein real‐time quaking‐induced conversion assays of cerebrospinal fluid have dramatically reduced reaction times from 5‐13 days down to 1‐2 days. Objective To test our improved assay against a panel of cerebrospinal fluid specimens from patients with Parkinson’s disease and healthy controls from the MJ Fox Foundation/NINDS BioFIND collection. Methods Specimens collected from healthy controls and patients with clinically typical moderate‐to‐advanced Parkinson’s disease were tested without prior knowledge of disease status. Correlative analyses between assay parameters and clinical measures were performed by an independent investigator. Results BioFIND samples gave positive signals in 105/108 (97%) Parkinson’s disease cases versus 11/85 (13%) healthy controls. Receiver operating characteristic analyses of diagnosis of cases versus healthy controls gave areas under the curve of 95%. Beyond binary positive/negative determinations, only weak correlations were observed between various assay response parameters and Parkinson’s disease clinical measures or other cerebrospinal fluid analytes. Of note, REM sleep behavioral disorder questionnaire scores correlated with the reaction times needed to reach 50% maximum fluorescence. Maximum fluorescence was inversely correlated with Unified Parkinson's Disease Rating Scale motor scores, which was driven by the patients without REM sleep behavioral disorder. Conclusions Our improved α‐synuclein seed amplification assay dramatically reduces the time needed to diagnose Parkinson’s disease while maintaining the high‐performance standards associated with previous α‐synuclein seed assays, supporting the clinical utility of this assay for Parkinson’s disease diagnosis.

Abstract Objective Real‐time quaking‐induced conversion (RT‐QuIC) assays detect prion‐seeding activity in a variety of human biospecimens, including cerebrospinal fluid and olfactory mucosa swabs. The assay has shown high diagnostic accuracy in patients with prion disorders. Recently, advances in these tests have led to markedly improved diagnostic sensitivity and reduced assay times. Accordingly, an algorithm has been proposed that entails the use of RT‐QuIC analysis of both sample types to diagnose sporadic Creutzfeldt‐Jakob disease with nearly 100% accuracy. Here we present a multi‐center evaluation (ring trial) of the reproducibility of these improved “second generation” RT‐QuIC assays as applied to these diagnostic specimens. Methods Cerebrospinal fluid samples were analyzed from subjects with sporadic Creutzfeldt‐Jakob (n = 55) or other neurological diseases (n = 45) at multiple clinical centers. Olfactory mucosa brushings collected by multiple otolaryngologists were obtained from nine sporadic Creutzfeldt‐Jakob disease cases and 19 controls. These sample sets were initially tested blindly by RT‐QuIC by a coordinating laboratory, recoded, and then sent to five additional testing laboratories for blinded ring trial testing. Results Unblinding of the results by a third party indicated 98‐100% concordance between the results obtained by the testing of these cerebrospinal fluid and nasal brushings at the six laboratories. Interpretation This second‐generation RT‐QuIC assay is highly transferrable, reproducible, and therefore robust for the diagnosis of sporadic Creutzfeldt‐Jakob disease in clinical practice.

Abstract Objective The detection of prion seeding activity in CSF and olfactory mucosal brushings using real‐time quaking‐induced conversion assays allows highly accurate clinical diagnosis of sporadic Creutzfeldt–Jakob disease. To gauge transmission risks associated with these biospecimens and their testing, we have bioassayed prion infectivity levels in patients’ brain tissue, nasal brushings, and CSF, and assessed the pathogenicity of amplified products of real‐time quaking‐induced conversion assays seeded with Creutzfeldt–Jakob disease prions. Methods We obtained olfactory mucosal brushings and CSF from patients with a final diagnosis of sporadic Creutzfeldt–Jakob disease subtype MM1 (n = 3). Samples were inoculated intracerebrally into Tg66 transgenic mice that overexpress the homologous human 129M prion protein. The mice were evaluated for clinical, neuropathological, and biochemical evidence of prion infection. Results Patients’ brain tissue at 102 to 105 fold dilutions affected 47/48 Tg66 mice. In contrast, maximum acutely tolerable doses of insoluble pellets from their olfactory mucosa brushings caused evidence of prion disease in only 4/28 inoculated mice, and no effects were seen with 10‐fold dilutions. No clinical prion disease was observed in mice inoculated with antemortem CSF samples or prion‐seeded real‐time quaking‐induced conversion assay products. Interpretation Pellets from patients’ olfactory mucosa brushings had ≥10,000‐fold lower infectivity per unit volume than brain tissue, while CSF lacked detectable infectivity. Nonetheless, the results suggest that appropriate precautions may be warranted in surgical interventions involving the olfactory areas. The lack of pathogenic infectivity in the real‐time quaking‐induced conversion assay products provides evidence that the assay does not replicate biohazardous prions in vitro.