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Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease.

Adult-onset neurodegenerative disorders, like Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), that share the accumulation of aggregated α-synuclein (αSynagg) as their hallmark molecular pathology are collectively known as α-synucleinopathies. Diagnosing α-synucleinopathies requires the post-mortem detection of αSynagg in various brain regions. Recent efforts to measure αSynagg in living patients include quantifying αSynagg in different biofluids as a biomarker for PD. We adopted the real-time quaking-induced conversion (RT-QuIC) assay to detect very low levels of αSynagg. We first optimized RT-QuIC for sensitivity, specificity, and reproducibility by using monomeric recombinant human wild-type αSyn as a substrate and αSynagg as the seed. Next, we exposed mouse microglia to αSyn pre-formed fibrils (αSynPFF) for 24 h. RT-QuIC assay revealed that the αSynPFF is taken up rapidly by mouse microglia, within 30 min, and cleared within 24 h. We then evaluated the αSyn RT-QuIC assay for detecting αSynagg in human PD, DLB, and Alzheimer's disease (AD) post-mortem brain homogenates (BH) along with PD and progressive supranuclear palsy (PSP) cerebrospinal fluid (CSF) samples and then determined protein aggregation rate (PAR) for αSynagg. The PD and DLB BH samples not only showed significantly higher αSynagg PAR compared to age-matched healthy controls and AD, but RT-QuIC was also highly reproducible with 94% sensitivity and 100% specificity. Similarly, PD CSF samples demonstrated significantly higher αSynagg PAR compared to age-matched healthy controls, with 100% sensitivity and specificity. Overall, the RT-QuIC assay accurately detects αSynagg seeding activity, offering a potential tool for antemortem diagnosis of α-synucleinopathies and other protein-misfolding disorders.

BackgroundThe definite diagnosis of human sporadic Creutzfeldt-Jakob disease (sCJD) largely depends on postmortem neuropathology and PrPSc detection in the brain. The development of prion RT-QuIC of cerebrospinal fluid (CSF) samples makes it possible for premortem diagnosis for sCJD. However the diagnostic potential of RT-QuIC of skin specimen for probable sCJD is not well researched. This study is to evaluate the diagnostic potential of RT-QuIC of skin specimen in human prion diseases.MethodsWe collected the paired skin and CSF samples from 29 recruited alive patients referred to Chinese CJD surveillance center, including 12 probable sCJD, 9 non-CJD, 3 genetic prion disease (gPrD) and 5 cases whose diagnoses still pending. The samples were subjected to RT-QuIC assays using recombinant hamster PrP protein rHaPrP90-231 as the substrate.ResultsAll 12 probable sCJD patients, 4 pending, and 1 T188K genetic CJD (gCJD) cases showed positive prion-seeding activity, while all 9 non-CJD patients were negative. CSF RT-QuIC positive seeding activity was only observed in 5 probable sCJD patients.ConclusionsOur preliminary data indicate high sensitivity and specificity of skin RT-QuIC in prion detection for Chinese probable sCJD and highlight that skin prion-seeding activity is a reliable biomarker for premortem diagnosis of human prion disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare and fatal neurodegenerative disorder with an incidence of 1.5 to 2 cases per million population/year. The disease is caused by a proteinaceous infectious agent, named prion (or PrPSc), which arises from the conformational conversion of the cellular prion protein (PrPC). Once formed, PrPSc interacts with the normally folded PrPC coercing it to undergo similar structural rearrangement. The disease is highly heterogeneous from a clinical and neuropathological point of view. The origin of this variability lies in the aberrant structures acquired by PrPSc. At least six different sCJD phenotypes have been described and each of them is thought to be caused by a peculiar PrPSc strain. Definitive sCJD diagnosis requires brain analysis with the aim of identifying intracerebral accumulation of PrPSc which currently represents the only reliable biomarker of the disease. Clinical diagnosis of sCJD is very challenging and is based on the combination of several clinical, instrumental and laboratory tests representing surrogate disease biomarkers. Thanks to the advent of the ultrasensitive Real-Time Quaking-Induced Conversion (RT-QuIC) assay, PrPSc was found in several peripheral tissues of sCJD patients, sometimes even before the clinical onset of the disease. This discovery represents an important step forward for the clinical diagnosis of sCJD. In this manuscript, we present an overview of the current applications and future perspectives of RT-QuIC in the field of sCJD diagnosis.

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.

Chronic wasting disease (CWD) is a progressive and fatal spongiform encephalopathy of deer and elk species, caused by a misfolded variant of the normal prion protein. Horizontal transmission of the misfolded CWD prion between animals is thought to occur through shedding in saliva and other forms of excreta. The role of blood in CWD transmission is less clear, though infectivity has been demonstrated in various blood fractions. Blood-feeding insects, including ticks, are known vectors for a range of bacterial and viral infections in animals and humans, though to date there has been no evidence for their involvement in prion disease transmission. In the present study, we evaluated winter ticks (Dermacentor albipictus) collected from 136 North American elk (Cervus canadensis) in a CWD-endemic area for evidence of CWD prion amplification using the real time quaking-induced conversion assay (RT-QuIC). Although 30 elk were found to be CWD-positive (22%) postmortem, amplifiable prions were found in just a single tick collected from an elk in advanced stages of CWD infection, with some evidence for prions in ticks collected from elk in mid-stage infection. These findings suggest that further investigation of ticks as reservoirs for prion disease may be warranted.ImportanceThis study reports the first finding of detectable levels of prions linked to chronic wasting disease in a tick collected from a clinically infected elk. Using the real time quaking-induced conversion assay (RT-QuIC), evidence of amplifiable CWD prions was also found in ticks collected from elk in earlier stages of disease. Observed levels were at the lower end of our detection limits, though our findings suggest that additional research evaluating ticks collected from animals in late-stage disease may be warranted to further evaluate the role of ticks as potential vectors of chronic wasting disease.

Chronic wasting disease (CWD) is a prion-mediated, transmissible disease of cervids, including deer ( Odocoileus spp.), which is characterized by spongiform encephalopathy and death of the prion-infected animals. Official surveillance in the United States using immunohistochemistry (IHC) and ELISA entails the laborious collection of lymphoid and/or brainstem tissue after death. New, highly sensitive prion detection methods, such as real-time quaking-induced conversion (RT-QuIC), have shown promise in detecting abnormal prions from both antemortem and postmortem specimens. We compared RT-QuIC with ELISA and IHC for CWD detection utilizing deer retropharyngeal lymph node (RLN) tissues in a diagnostic laboratory setting. The RLNs were collected postmortem from hunter-harvested animals. RT-QuIC showed 100% sensitivity and specificity for 50 deer RLN (35 positive by both IHC and ELISA, 15 negative) included in our study. All deer were also genotyped for PRNP polymorphism. Most deer were homozygous at codons 95, 96, 116, and 226 (QQ/GG/AA/QQ genotype, with frequency 0.86), which are the codons implicated in disease susceptibility. Heterozygosity was noticed in Pennsylvania deer, albeit at a very low frequency, for codons 95GS (0.06) and 96QH (0.08), but deer with these genotypes were still found to be CWD prion-infected.

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein (αSyn). They include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In each disease, it has been proposed that aggregates of αSyn represent different conformational strains of αSyn, leading to self-propagation and spreading from cell to cell. It has been considered that αSyn aggregates grow by seeded polymerization mechanisms. Previously, the mechanism of seed conversion in prion protein aggregation has been exploited by real-time quaking-induced conversion (RT-QuIC) assay. It was further refined by incorporating the fluorescent dye thioflavin-T, which enabled the real-time monitoring of kinetic changes with a highly sensitive detection of seed aggregates present at an extremely low level. In an application for diagnostics, it has been reported that αSyn RT-QuIC exhibits specificity between 82% and 100%, while its sensitivity varies between 70% and 100%, on the basis of a study in which this assay was performed at multiple different laboratories. Furthermore, it has been suggested that the αSyn RT-QuIC method can be applied to study the biochemical characteristics of different αSyn strains among synucleinopathies. In this article, we describe the detailed protocols for αSyn RT-QuIC assays.

Skin α-synuclein deposition is considered a potential biomarker for Parkinson’s disease (PD). Real-time quaking-induced conversion (RT-QuIC) is a novel, ultrasensitive, and efficient seeding assay that enables the detection of minute amounts of α-synuclein aggregates. We aimed to determine the diagnostic accuracy, reliability, and reproducibility of α-synuclein RT-QuIC assay of skin biopsy for diagnosing PD and to explore its correlation with clinical markers of PD in a two-center inter-laboratory comparison study. Patients with clinically diagnosed PD (n = 34), as well as control subjects (n = 30), underwent skin punch biopsy at multiple sites (neck, lower back, thigh, and lower leg). The skin biopsy samples (198 in total) were divided in half to be analyzed by RT-QuIC assay in two independent laboratories. The α-synuclein RT-QuIC assay of multiple skin biopsies supported the clinical diagnosis of PD with a diagnostic accuracy of 88.9% and showed a high degree of inter-rater agreement between the two laboratories (92.2%). Higher α-synuclein seeding activity in RT-QuIC was shown in patients with longer disease duration and more advanced disease stage and correlated with the presence of REM sleep behavior disorder, cognitive impairment, and constipation. The α-synuclein RT-QuIC assay of minimally invasive skin punch biopsy is a reliable and reproducible biomarker for Parkinson’s disease. Moreover, α-synuclein RT-QuIC seeding activity in the skin may serve as a potential indicator of progression as it correlates with the disease stage and certain non-motor symptoms.

Eforts 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, fuids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here 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 specifcity (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.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a human prion disease that is a relatively common differential diagnosis in dementia patients. Therefore it needs a good diagnostic tool. Brain autopsy is the golden standard for the diagnosis of CJD; however, a less invasive technique is 14-3-3 protein measurement in the cerebrospinal fluid (CSF). In this systematic review, we compared the diagnostic value of the 14-3-3 protein measurement to the newer RT-QuIC test and a variant of RT-QuIC where nasal brushing is used to collect the samples. The search via MeSH terms and quality assessment was carried out by two individual researchers. In 14-3-3 and RT-QuIC the sensitivity was comparable, respectively, 88% and 86%. Specificity however was higher in RT-QuIC 99.5% compared to 80% in 14-3-3. Nasal brushing showed the best results with a sensitivity of 97% and a specificity of 100%. Nasal brushing, despite being the best diagnostic tool according to the data, needs more study since there has only been a few studies regarding the technique. It is safe to say that due to the high specificity, RT-QuIC is superior to 14-3-3 testing.

Chronic wasting disease (CWD) is a prion disease circulating in wild and farmed cervid populations throughout North America (United States and Canada), Europe (Finland, Norway, Sweden), and South Korea. CWD is an immediate threat to cervid heritage and the disease is causing substantial economic losses across multiple sectors. In North America, hunting and farming industries focused on the processing and consumption of white-tailed deer (WTD) venison are particularly vulnerable to CWD, as millions of WTD are consumed annually. Real-time quaking-induced conversion (RT-QuIC) is a highly sensitive assay amplifying misfolded CWD prions in vitro and has facilitated CWD prion detection in a variety of tissues and excreta. To date, no study has comprehensively examined CWD prion content across bulk skeletal muscle tissues harvested from individual CWD infected WTD. Here, we use RT-QuIC to quantify prion-seeding activity in a variety of skeletal muscles from both wild and farmed CWD-positive WTD. We successfully detected CWD prions in muscles commonly used for consumption (e.g., backstrap, tenderloin, etc.) as well as within tongue and neck samples of WTD. Our results help to establish the utility of RT-QuIC for monitoring CWD prions in venison and suggest that the method is useful for preventing CWD prions from entering animal and human food chains. Moreover, our work indicates that CWD prions are more widely distributed across skeletal muscles of infected WTD than previously reported.

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. Here 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.

Neurofilament light chain (NfL) and α-synuclein oligomeric seeds (α-syn-s) are promising biomarkers for patients with parkinsonism. We assessed their performance in discriminating Parkinson disease (PD) from atypical parkinsonisms (APDs) and evaluated the association between NfL levels and clinical measures of disease severity. We measured NfL in cerebrospinal fluid (CSF) and/or plasma by immunoassays and α-syn-s in CSF by real-time quaking-induced conversion (RT-QuIC) in patients with PD (n = 153), multiple system atrophy (MSA) (n = 80), progressive supranuclear palsy/cortico-basal syndrome (PSP/CBS) (n = 58), dementia with Lewy bodies (n = 64), isolated REM-sleep behaviour disorder (n = 19), and isolated autonomic failure (n = 30). Measures of disease severity included disease duration, UPDRS-III score, Hoehn and Yahr stage, orthostatic hypotension, MMSE score, and CSF amyloid-beta profile. Both CSF NfL (cNfL) and plasma NfL (pNfL) levels were markedly elevated in APDs, and allowed differentiation with PD (vs. APDs, cNfL AUC 0.96; pNfL AUC 0.95; vs. MSA cNfL AUC 0.99; pNfL AUC 0.97; vs. PSP/CBS cNfL AUC 0.94; pNfL AUC 0.94). RT-QuIC detected α-syn-s in 91.4% of PD, but only 2.5% of APDs (all MSA). In PD/PDD, motor scales significantly correlated with cNfL levels. Although pNfL and both cNfL and α-syn-s accurately distinguished PD from APDs, the combined assessment of CSF markers provided a higher diagnostic value (PD vs. APDs AUC 0.97; vs. MSA AUC 0.97; vs. PSP/CBS AUC 0.99) than RT-QuIC alone (p = 0.047 vs. APDs; p = 0.002 vs MSA; p = 0.007 vs PSP/CBS), or cNfL alone (p = 0.011 vs. APDs; p = 0.751 vs MSA; p = 0.0001 vs. PSP/CBS). The results support the use of these assays in specialised clinics.

Additional file 3. Biochemical analysis of ��Syn_RT-QuIC reaction products. Western blot images of ��Syn_RT-QuIC reaction products obtained at ITA-lab from OM samples of all individuals (PD = 13, MSA-P = 20, MSA-C = 10 and HS = 11) that were used for quantification analysis reported in Fig. 3e. Samples were digested with PK (2.5 mg/mL) and immunoblotted with the rabbit polyclonal ��-synuclein antibody Agrisera AS08 358 (1:1,000). PK resistant bands were observed in several ��Syn_RT-QuIC end products seeded with PD and MSA-P samples while they were not detected in all MSA-C and HS seeded samples. Vertical dashed lines in each blot indicate cropped images from separate gels (a). Dot blot images of ��Syn_RT-QuIC reaction products obtained at USA-lab from OM samples of all individuals (PD = 13, MSA-P = 20, MSA-C = 10 and HS = 11) that were used for quantification analysis reported in Fig. 2b and c. Total ��Syn signal from these products was present in all samples while ��Syn filament signal was present only in samples which induced ��Syn_RT-QuIC seeding activity (b).

Additional file 1. Detection of ��Syn_RT-QuIC seeding activity triggered by the brain homogenates of patients with PD, MSA-P, and MSA-C. We tested the performance of the new ��Syn_RT-QuIC protocol using the brain homogenates (BH) of autopsy-confirmed cases of PD (n = 1), MSA-P (n = 1), and MSA-C (n = 1) as sources of different ��SynD strains. The BH of a patient not affected by ��-synucleinopathy (NAS) was used as control. Immunohistochemical analysis revealed the presence of distinct ��SynD aggregates in the brain of tested patients, except for NAS. MSA-P and NAS images were taken using 10x magnification. MSA-C and PD images were taken using 40x magnification (a). Frozen brain samples (substantia nigra for PD, striatum for MSA-P, cerebellum for MSA-C, and frontal cortex for NAS) were homogenized at 10% in PBS (weight/volume) and diluted at 10��� 12. One ��l of each BH was added to 49 ��L of reaction mix and subjected to ��Syn_RT-QuIC analysis. The results indicate that PD (light green line), MSA-P (blue line), and MSA-C (grey line) induced ��Syn_RT-QuIC seeding activity before the time threshold set at 13 h while that of the patient with NAS (purple line) did not (b). Each sample was analyzed in quadruplicate. Curves represented in the graph were obtained by plotting the average fluorescence intensities of each sample against time.

Skin alpha-synuclein deposition is considered a potential biomarker for Parkinson's disease (PD). Real-time quaking-induced conversion (RT-QuIC) is a novel, ultrasensitive, and efficient seeding assay that enables the detection of minute amounts of alpha-synuclein aggregates. We aimed to determine the diagnostic accuracy, reliability, and reproducibility of alpha-synuclein RT-QuIC assay of skin biopsy for diagnosing PD and to explore its correlation with clinical markers of PD in a two-center inter-laboratory comparison study. Patients with clinically diagnosed PD (n = 34), as well as control subjects (n = 30), underwent skin punch biopsy at multiple sites (neck, lower back, thigh, and lower leg). The skin biopsy samples (198 in total) were divided in half to be analyzed by RT-QuIC assay in two independent laboratories. The a-synuclein RT-QuIC assay of multiple skin biopsies supported the clinical diagnosis of PD with a diagnostic accuracy of 88.9% and showed a high degree of inter-rater agreement between the two laboratories (92.2%). Higher alpha-synuclein seeding activity in RT-QuIC was shown in patients with longer disease duration and more advanced disease stage and correlated with the presence of REM sleep behavior disorder, cognitive impairment, and constipation. The alpha-synuclein RT-QuIC assay of minimally invasive skin punch biopsy is a reliable and reproducible biomarker for Parkinson's disease. Moreover, alpha-synuclein RT-QuIC seeding activity in the skin may serve as a potential indicator of progression as it correlates with the disease stage and certain non-motor symptoms.

Additional file 4. Morphological analsysis of ��Syn_RT-QuIC reaction products generated by MSA-P and MSA-C samples at ITA-lab. ��Syn_RT-QuIC reaction products were properly diluted and 10 ��L of the final dilutions was dropped onto 200-mesh Formvar-carbon coated nickel grids for 30 min and the remaining drop was blotted dry using filter papers. Samples were stained with 25% Uranyl Acetate Replacement (UAR, negative staining) for 10 min, the solution was removed using filter papers and the grids were air-dried for 15 min before the analyses. Images were recorded at 120 kV with a FEI Tecnai Spirit, equipped with an Olimpus Megaview G2 camera. Rec-��Syn amyloid fibrils were detected in all ��Syn_RT-QuIC products seeded with MSA-P samples, except for MSA-P_10 and MSA-P_11. In constrast, none of the MSA-C samples induced the formation of rec-��Syn fibrils (except for occasional, rare and short aggregates detected in MSA-C_1, MSA-C_3 and MSA-C_6). All TEM images were taken at the same magnification (scale bar: 200 nm).

Additional file 5. Dye-binding assay and epitope mapping of ��Syn_RT-QuIC products generated by MSA-P (n = 20) and MSA-C (n = 10) samples at ITA-lab. Thirty-five ��L of ��Syn_RT-QuIC products, generated without the use of ThT, were incubated with three different fluorescent dyes: (i) ThT [10 ��M], (ii) Congo Red [5 ��M] and (iii) Amytracker 630 (purchased from Ebba Biotech and diluted 1:800) for 30 min in the dark at room temperature. Fluorescent signals were measured with the appropriate wave-lengths (448 exc/482 emi for ThT, 544 exc/620 emi for Congo red and 510 exc/635 emi for Amytracker 630) using the BMG LabTech CLARIOSTAR microplate reader. Regardless of the probe used, all MSA-P samples (except for MSA-P_10 and MSA-P_11) showed strong fluorescent signals while the MSA-C did not. These differences were statistically significant in the case of (i) ThT (t-test p = 0.0063), (ii) Congo red (t-test, p = 0.0018) and (iii) Amytracker 630 (t-test, p = 0.0051). Graphs show the mean values (�� SEM) of MSA-P and MSA-C fluorescent signals obtained from (i) ThT (graph on the left), (ii) Congo red (graph in the middle) and (iii) Amytracker 630 (graph on the right) analysis (a). Eight ��L of ��Syn_RT-QuIC products generated by MSA-P and MSA-C samples were treated with PK [2.5 mg/mL] for 1 h at 37 ��C under shaking (500 rpm) and immunoblotted with antibodies directed against three different epitopes of ��-synuclein: (i) clone 5C2 (epitopes 61���95, Novus Biologicals), (ii) AB5038 (epitopes 111���131, Chemicon international), and (iii) clone 42 (epitopes 15���123, BD Bioscience). No signals were observed in samples immunoblotted with the AB5038 and the clone 5C2, while with the clone 42, PK resistant bands were specifically detected in ��Syn_RT-QuIC products seeded with MSA-P but not in those seeded with MSA-C samples (b).

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein (αSyn). They include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In each disease, it has been proposed that aggregates of αSyn represent different conformational strains of αSyn, leading to self-propagation and spreading from cell to cell. It has been considered that αSyn aggregates grow by seeded polymerization mechanisms. Previously, the mechanism of seed conversion in prion protein aggregation has been exploited by real-time quaking-induced conversion (RT-QuIC) assay. It was further refined by incorporating the fluorescent dye thioflavin-T, which enabled the real-time monitoring of kinetic changes with a highly sensitive detection of seed aggregates present at an extremely low level. In an application for diagnostics, it has been reported that αSyn RT-QuIC exhibits specificity between 82% and 100%, while its sensitivity varies between 70% and 100%, on the basis of a study in which this assay was performed at multiple different laboratories. Furthermore, it has been suggested that the αSyn RT-QuIC method can be applied to study the biochemical characteristics of different αSyn strains among synucleinopathies. In this article, we describe the detailed protocols for αSyn RT-QuIC assays.

An amendment to this paper has been published and can be accessed via the original article.

he article Ultrasensitive RT‑QuIC assay with high sensitivity and specificity for Lewy body‑.

Chronic wasting disease (CWD) is a transmissible prion disease of the cervidae family. ELISA and IHC tests performed postmortem on the medial retropharyngeal lymph nodes (RPLN) or obex are considered diagnostic gold standards for prion detection. However, differences in CWD transmission, stage of infection, pathogenesis, and strain can limit performance. To overcome these uncertainties, we used Bayesian statistics to assess the accuracy of RT-QuIC, an increasingly used prion amplification assay, to diagnose CWD on tonsil (TLN), parotid (PLN) and submandibular lymph nodes (SMLN), and ELISA/IHC on RPLN of white-tailed deer (WTD) sampled from Minnesota. Dichotomous RT-QuIC and ELISA/IHC results from wild (n = 61) and captive (n = 46) WTD were analyzed with two-dependent-test, one-population models. RT-QuIC performed on TLN and SMLN of the wild WTD population had similar sensitivity (median range (MR): 92.2–95.1) to ELISA/IHC on RPLN (MR: 91.1–92.3). Slightly lower (4–7%) sensitivity estimates were obtained from farmed animal and PLN models. RT-QuIC specificity estimates were high (MR: 94.5–98.5%) and similar to ELISA/IHC estimates (MR: 95.7–97.6%) in all models. This study offers new insights on RT-QuIC and ELISA/IHC performance at the population level and under field conditions, an important step in CWD diagnosis and management.