Detection of Prions

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

1) 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. We performed a multicenter evaluation (ring trial) of the reproducibility of these improved second generation RT-QuIC assays as applied to these diagnostic specimens. Cerebrospinal fluid samples were analyzed from subjects with sporadic Creutzfeldt-Jakob (n=55) or other neurological diseases (n=45) disease at multiple clinical centers. Olfactory mucosa brushings collected by multiple otolaryngologists were obtained from 9 sporadic Creutzfeldt-Jakob disease cases and 19 controls. These sample sets were initially tested blindly by RT-QuIC by our laboratory, recoded, and then sent to 5 additional testing laboratories for blinded ring-trial testing. 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. We conclude that this second-generation RT-QuIC assay is highly transferrable, reproducible, and therefore robust for the diagnosis of sporadic Creutzfeldt-Jakob disease in clinical practice. 2) Assays that specifically measure -synuclein seeding activity in biological fluids could revolutionize the diagnosis of Parkinsons disease. Our recent improvements in -synuclein real-time quaking-induced conversion assays of cerebrospinal fluid have dramatically reduced reaction times from 5-13 days down to 1-2 days. We tested our improved assay against a panel of cerebrospinal fluid specimens from patients with Parkinsons disease and healthy controls from the MJ Fox Foundation/NINDS BioFIND collection. Specimens collected from healthy controls and patients with clinically typical moderate-to-advanced Parkinsons disease were tested without prior knowledge of disease status. Correlative analyses between assay parameters and clinical measures were performed by an independent investigator. BioFIND samples gave positive signals in 105/108 (97%) Parkinsons 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 Parkinsons 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. Our improved -synuclein seed amplification assay dramatically reduces the time needed to diagnose Parkinsons disease while maintaining the high-performance standards associated with previous -synuclein seed assays, supporting the clinical utility of this assay for Parkinsons disease diagnosis. 3) We also continued to help clinical centers in Italy and The Netherlands to perform -syn RT-QuIC analysis of CSF samples from subjects with mild cognitive impairment (MCI) and subjective cognitive impairment (SCI). -syn RT-QuIC was applied to 289 CSF samples obtained from two independent cohorts, including 81 patients with probable MCI-LB (70.76.6 y, 13.6% F, MMSE 26.12.4), 120 with probable MCI-AD (68.67.4 y, 45.8% F, MMSE 25.52.8), and 30 with unspecified MCI (65.49.3 y, 30.0% F, MMSE 27.03.0). Fifty-eight individuals with no cognitive decline or evidence of neurodegenerative disease and 121 individuals lacking brain -syn deposits at the neuropathological examination were used as controls. RT-QuIC identified MCI-LB patients against cognitively unimpaired controls with 95% sensitivity, 97% specificity, and 96% accuracy, and showed 98% specificity in neuropathological controls. The accuracy of the test for MCI-LB was consistent between the two cohorts (97.3% vs. 93.7%). Thirteen percent of MCI-AD patients also had a positive test; of note, 44% of them developed one core or supportive clinical feature of dementia with Lewy bodies (DLB) at follow-up, suggesting an underlying LB co-pathology. These findings indicate that CSF -syn RT-QuIC is a robust biomarker for prodromal DLB. Further studies are needed to fully explore the added value of the assay to the current research criteria for MCI-LB. 4) Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. We have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids. 5) Sporadic Creutzfeldt-Jakob Disease (sCJD) rarely affects women of childbearing age. There is currently no evidence of vertical transmission, but one mouse transmission study demonstrated infectivity of the placenta. Given the biosafety implications of performing Caesarean sections (C-section) in these patients, we helped clinicians at the University of Alberta by testing for the infectious prion protein (PrPSc) in products of gestation using our ultrasensitive real time quaking-induced conversion (RT-QuIC) assay. A 35-year-old woman with sCJD presented in her 10th gestational week with an eight month history of progressive cognitive impairment. Amniotic fluid, cord blood and placental tissue were collected and analyzed using modified RT-QuIC protocols. The patients diagnosis of sCJD, MM2 subtype, was confirmed at autopsy. There were borderline positive results in one sampled area of the placenta, but otherwise the cord blood and amniotic fluid were negative on our RT-QuIC assays. A healthy baby was delivered via C-section at 36 weeks 3 days gestation, with no evidence of neurological disease to date. Using an adapted RT-QuIC protocol, low levels of PrPSc were detected in the placenta, but only in a minority of tissue areas sampled. We conclude that precautions should be taken with products of gestation, but the level of PrPSc is extremely low.