Real-time Quaking-induced Conversion Research Articles

Blood-Based Nanoparticle-Enhanced Quaking-Induced Conversion (Nano-QuIC): Inhibitor-Resistant Detection of Seeding Activity in Patients Diagnosed with Parkinson's Disease.

A hallmark of α-synucleinopathies (e.g., Parkinson's disease) is the misfolding and aggregation of α-synuclein in tissues and biological fluids. Protein amplification assays like real-time quaking-induced conversion (RT-QuIC) are sensitive yet currently limited to semi-invasive sample types such as cerebrospinal fluid because more accessible samples, such as blood, contain inhibitors. Here, we show that Nanoparticle-enhanced Quaking-induced Conversion (Nano-QuIC) can double the speed of reactions spiked with misfolded α-synuclein while increasing sensitivity 100-fold in human plasma. Nano-QuIC detected spike concentrations down to 90 pg/mL in lysed whole blood, while reactions without nanoparticles (RT-QuIC) failed to have any detection due to the presence of strong inhibitors. Moreover, Nano-QuIC showed increased seeding activity in plasma samples from Parkinson's patients (n = 4) versus healthy controls (n = 4). This sets the groundwork for the noninvasive diagnostic use of Nano-QuIC, potentially enabling early disease detection and management through blood-based testing.

Enhanced detection of chronic wasting disease in muscle tissue harvested from infected white-tailed deer employing combined prion amplification assays.

Zoonotic transmission of bovine spongiform encephalopathy or mad cow disease, by presumed consumption of infected beef, has increased awareness of the public health risk associated with prion diseases. Chronic wasting disease (CWD) affects moose, elk, and deer, all of which are frequently consumed by humans. Clear evidence of CWD transmission to humans has not been demonstrated, yet, establishing whether CWD prions are present in muscle tissue preferentially consumed by humans is of increasing interest. Conventional assays including immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) lack the sensitivity to detect low concentrations of prions presumed to be present outside neural or lymphatic tissues. Here we combined two prion amplification assays, the product of protein misfolding cyclic amplification (PMCA) applied directly into real-time quaking induced conversion (RT-QuIC) [denoted now as PQ] to demonstrate the presence of prion seeding activity (i.e. prions) in ~55% of hamstring muscles harvested from CWD-positive white-tailed deer. This compares to prion detection in only 10% of the same samples employing standard RT-QuIC. To determine the extent of CWD dissemination within muscle tissues commonly consumed we tested 7 additional muscles from a subset of deer by PQ. Tongue demonstrated the highest level of prions with ~92% positive. All negative controls remained negative in all PMCA and RT-QuIC assays. We conclude that the combination of PMCA with RT-QuIC readout permits detection of low prion concentrations present in muscle tissue of CWD-infected deer. These findings further demonstrate the utility of amplification assays as tools to detect very low levels of prion burden and supports their use to fill knowledge gaps in our understanding of CWD pathogenesis and zoonotic potential.

Inter-laboratory comparison of real-time quaking-induced conversion (RT-QuIC) for the detection of chronic wasting disease prions in white-tailed deer retropharyngeal lymph nodes.

The rapid geographic spread of chronic wasting disease (CWD) in white-tailed deer (WTD; Odocoileus virginianus) increases the need for the development and validation of new detection tests. Real-time quaking-induced conversion (RT-QuIC) has emerged as a sensitive tool for CWD prion detection, but federal approval in the United States has been challenged by practical constraints on validation and uncertainty surrounding RT-QuIC robustness between laboratories. To evaluate the effect of inter-laboratory variation on CWD prion detection using RT-QuIC, we conducted a multi-institution comparison on a shared anonymized sample set. We hypothesized that RT-QuIC can accurately and reliably detect the prions that cause CWD in postmortem samples from medial retropharyngeal lymph node (RPLN) tissue despite variation in laboratory protocols. Laboratories from 6 U.S. states (Michigan, Minnesota, Missouri, New York, Pennsylvania, Wisconsin) were enlisted to compare the use of RT-QuIC in determining CWD prion status (positive or negative) among 50 anonymized RPLNs of known prion status. Our sample set included animals of 3 codon 96 WTD genotypes known to affect CWD progression and detection (G96G, G96S, S96S). All 6 laboratories successfully identified the true disease status consistently for all 3 tested codon 96 genotypes. Our results indicate that RT-QuIC is a suitable test for the detection of CWD prions in RPLN tissues in several genotypes of WTD.

Multisite Skin Biopsies vs Cerebrospinal Fluid for Prion Seeding Activity in the Diagnosis of Prion Diseases

Recent studies have revealed that autopsy skin samples from cadavers with prion diseases (PRDs) exhibited a positive prion seeding activity similar to cerebrospinal fluid (CSF). It is worthwhile to validate the findings with a large number of biopsy skin samples and compare the clinical value of prion seeding activity between skin biopsies and concurrent CSF specimens. To compare the prion seeding activity of skin biopsies and CSF samples and to determine the effectiveness of combination of the skin biopsies from multiple sites and numerous dilutions on the diagnosis for various types of PRDs. In the exploratory cohort, patients were enrolled from September 15, 2021, to December 15, 2023, and were followed up every 3 months until April 2024. The confirmatory cohort enrolled patients from December 16, 2023, to June 31, 2024. The exploratory cohort was conducted at a single center, the neurology department at Xuanwu Hospital. The confirmatory cohort was a multicenter study involving 4 hospitals in China. Participants included those diagnosed with probable sporadic Creutzfeldt-Jakob disease or genetically confirmed PRDs. Patients with uncertain diagnoses or those lost to follow-up were excluded. All patients with PRDs underwent skin sampling at 3 sites (the near-ear area, upper arm, lower back, and inner thigh), and a portion of them had CSF samples taken simultaneously. In the confirmatory cohort, a single skin biopsy site and CSF samples were simultaneously collected from a portion of patients with PRDs. The skin and CSF prion seeding activity was assessed using the real-time quaking-induced conversion (RT-QUIC) assay, with rHaPrP90-231, a Syrian hamster recombinant prion protein, as the substrate. In the exploratory cohort, skin samples were tested at dilutions of 10-2 through 10-4. In the confirmatory cohort, skin samples were tested at a dilution of 10-2. A total of four 15-μL wells of CSF were used in the RT-QUIC assay. Correlations between RT-QUIC results from the skin and CSF and the final diagnosis of enrolled patients. In the exploratory cohort, the study included 101 patients (mean [SD] age, 60.9 [10.2] years; 63 female [62.4%]) with PRD and 23 patients (mean [SD] age, 63.4 [9.1] years; 13 female [56.5%]) without PRD. A total of 94 patients had CSF samples taken simultaneously with the skin biopsy samples. In the confirmatory cohort, a single skin biopsy site and CSF sample were taken simultaneously in 43 patients with PRDs. Using an experimental condition of 10-2 dilution, the RT-QUIC positive rates of skin samples from different sites were comparable with those of the CSF (skin: 18 of 26 [69.2%] to 74 of 93 [79.6%] vs CSF: 71 of 94 [75.5%]). When tested at 3 different dilutions, all skin sample positivity rates increased to over 80.0% (79 of 93 for the near-ear area, 21 of 26 for the upper arm, 77 of 92 for the lower back, and 78 of 92 for the inner thigh). Combining samples from skin sites near the ear, inner thigh, and lower back in pairs yielded positivity rates exceeding 92.1% (93 of 101), significantly higher than CSF alone (71 of 94 [75.5%]; P =.002). When all skin sample sites were combined and tested at 3 dilution concentrations for RT-QUIC, the sensitivity reached 95.0% (96 of 101). In the confirmatory cohort, the RT-QUIC positive rate of a single skin biopsy sample was slightly higher than that of the CSF (34 of 43 [79.1%] vs 31 of 43 [72.1%]; P = .45). Results of this diagnostic study suggest that the sensitivity of an RT-QUIC analysis of a combination of 2 or more skin sites was superior to that of CSF in diagnosing PRDs.

C9orf72 Gene-Associated Frontotemporal Dementia Mimicking Autoimmune Pathology.

The C9orf72 mutation can manifest in diverse clinical ways, including rapid cognitive decline, parkinsonism, or late-life neuropsychiatric symptoms, sometimes mimicking autoimmune encephalitis. A 64-year-old female presented to the autoimmune neurology clinic with rapidly progressive dementia (RPD) associated with episodes of headache, confusion, auditory hallucinations, and abnormal electroencephalogram. She was treated empirically at an outside hospital for possible autoimmune encephalitis with intravenous methylprednisolone, but there was no improvement, and rapid cognitive decline continued. Family history was notable for RPD with akinetic mutism in her sister, sudden severe depression followed by parkinsonism with progressive dementia in her father in his 60s, and late-life gradually progressive dementia in her mother. Additional testing revealed a low titer positive contactin-associated protein-like 2 (CASPR2) immunoglobulin G (IgG) in the serum and elevated CSF 14-3-3 protein. CSF CASPR2 IgG and real-time quaking-induced conversion for Creutzfeldt-Jakob disease were negative. Brain MRI showed normal parenchymal volume. Genetic testing was conducted, which identified a heterozygous pathogenic hexanucleotide tandem repeat expansion in the C9orf72 gene. This case underscores the phenotypic variability of C9orf72 mutation and the importance of a detailed family history exploring young or atypical deaths and neuropsychiatric symptoms or behavioral changes. Genetic etiologies are crucial to consider in those with a family history concerning autosomal dominant inheritance patterns of early-onset dementia, parkinsonism, or late-onset psychiatric disease. Emphasis is placed on considering alternative etiologies early, particularly when there is no response to first-line immunomodulation for suspected autoimmune dementia.

Seeding Aggregation Assays in Lewy Bodies Disorders: A Narrative State-of-the-Art Review

Multiple system atrophy and Lewy body diseases (LBDs) such as Parkinson’s disease, dementia with Lewy bodies, and Parkinson’s disease with dementia, known as synucleinopathies, are defined neuropathologically by the accumulation and deposition of aberrant protein aggregates, primarily in neuronal cells. Seeding aggregation assays (SAA) have significant potential as biomarkers for early diagnosis, monitoring disease progression, and evaluating treatment efficacy for these diseases. Real-time quaking-induced conversion (RT-QuIC) and Protein Misfolding Cyclic Amplification (PMCA) assays represent two ultrasensitive protein amplification techniques that were initially tested for the field of prion disorders. Although the fundamental idea behind the creation of these two methods is very similar, their technical differences resulted in different levels of diagnostic accuracy for the identification of prion proteins, making the RT-QuIC assay the most trustworthy and effective instrument for the detection of suspected cases of LBDs and prion-like diseases.

Modeling Lewy body disease with SNCA triplication iPSC-derived cortical organoids and identifying therapeutic drugs.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

Rapid and sensitive determination of residual prion infectivity from prion-decontaminated surfaces.

Prion diseases are untreatable fatal transmissible neurodegenerative diseases that affect a wide range of mammals, including humans, and are caused by PrPSc, the infectious self-templating conformation of the host-encoded protein, PrPC. Prion diseases can be transmitted via surfaces (e.g., forceps, EEG electrodes) in laboratory and clinical settings. Here, we use a combination of surface swabbing and real-time quaking-induced conversion (RT-QuIC) to test for residual surface-associated prions following prion disinfection. We found that treatment of several prion-contaminated laboratory and clinically relevant surfaces with either water or 70% EtOH resulted in robust detection of surface-associated prions. In contrast, treatment of surfaces with sodium hypochlorite resulted in a failure to detect surface-associated prions. RT-QuIC analysis of prion-contaminated stainless steel wires paralleled the findings of the surface swab studies. Importantly, animal bioassay and RT-QuIC analysis of the same swab extracts are in agreement. We report on conditions that may interfere with the assay that need to be taken into consideration before using this technique. Overall, this method can be used to survey laboratory and clinical surfaces for prion infectivity following prion decontamination protocols.IMPORTANCEPrion diseases can be accidentally transmitted in clinical and occupational settings. While effective means of prion decontamination exist, methods for determining the effectiveness are only beginning to be described. Here, we analyze surface swab extracts using real-time quaking-induced conversion (RT-QuIC) to test for residual prions following prion disinfection of relevant clinical and laboratory surfaces. We found that this method can rapidly determine the efficacy of surface prion decontamination. Importantly, examination of surface extracts with RT-QuIC and animal bioassay produced similar findings, suggesting that this method can accurately assess the reduction in prion titer. We identified surface contaminants that interfere with the assay, which may be found in clinical and laboratory settings. Overall, this method can enhance clinical and laboratory prion safety measures.

Longitudinal detection of prion infection in preclinical sheep blood samples compared using 3 assays

AbstractVariant Creutzfeldt-Jakob disease (vCJD) is a devastating disease caused by transmission of bovine spongiform encephalopathy to humans. Although vCJD cases are now rare, evidence from appendix surveys suggests that a small proportion of the United Kingdom population may be infected without showing signs of disease. These “silent” carriers could present a risk of iatrogenic vCJD transmission through medical procedures or blood/organ donation, and currently there are no validated tests to identify infected asymptomatic individuals using easily accessible samples. To address this issue, we evaluated the performance of 3 blood-based assays in a blinded study, using longitudinal sample series from a well-established large animal model of vCJD. The assays rely on amplification of misfolded prion protein (PrPSc; a marker of prion infection) and include real-time quaking-induced conversion (RT-QuIC), and 2 versions of protein misfolding cyclic amplification (PMCA). Although diagnostic sensitivity was higher for both PMCA assays (100%) than RT-QuIC (61%), all 3 assays detected prion infection in blood samples collected 26 months before the onset of clinical signs and gave no false-positive results. Parallel estimation of blood prion infectivity titers in a sensitive transgenic mouse line showed positive correlation of infectivity with PrPSc detection by the assays, suggesting that they are suitable for detection of asymptomatic vCJD infection in the human population. This study represents, to our knowledge, the largest comparison to date of preclinical prion detection in blood samples from a relevant animal model. The outcomes will guide efforts to improve early detection of prion disease and reduce infection risks in humans.

Right anterior insula ASL hypoperfusion as a diagnostic biomarker of prodromal and mild dementia with Lewy bodies: preliminary evidence using a Bayesian approach.

Identifying and validating a biomarker with high specificity in early-stage dementia with Lewy bodies (DLB) using a feasible method is crucial to enhance the current suboptimal diagnostic procedure. Previous research revealed abnormalities, including hypoperfusion in the right anterior insular cortex at group level, in prodromal DLB. Exploring hypoperfusion of the right anterior insula, at an individual-level and assessing its relevance as a potential imaging biomarker in early DLB, has, to our knowledge, not been investigated. Our preliminary study aims to assess the feasibility of the technique and to provide a methodological framework for further investigation. We assessed the feasibility and accuracy of the hypoperfusion of the right anterior insula per arterial spin labelling magnetic resonance imaging (ASL-MRI) as a diagnostic biomarker in early DLB and provided rough estimates of its sensitivity and specificity. Defining the region of interest based on previous research, we established the biomarker as the hypoperfusion of the right anterior insula. Discriminative and analytical performances were assessed in comparison to a control group of treatment-resistant depression patients. Bayesian diagnostic reasoning was employed to assess the biomarker diagnostic usability in early DLB in two scenarios: healthy elderly controls and mild cognitive impairment. Additionally, we updated probabilities by integrating data from the Mayo-clinic cognitive fluctuations scale and real-time quaking-induced conversion (RT-QuIC) α-synuclein data. Lastly, a whole-brain perfusion analysis of DLB patients was conducted to identify further brain regions with discriminative abilities. We successfully replicated the right anterior insular hypoperfusion (RAI-Hypo) in all DLB patients at the individual level. The overall sensitivity of the biomarker was 96%, and the specificity was 92%. Bayesian testing revealed the biomarker's highest performance in early-stage DLB with cognitive fluctuations, showcasing a diagnostic potential associated with a high precision and moderate accuracy. In a cognitively non-impaired population, the RAI-Hypo showed a limited usability and lacked in selectivity to qualify as a screening tool. The exploratory whole-brain analysis revealed perfect discriminative capacities in the bilateral anterior insulae and the left inferior parietal lobule. Further studies are needed to confirm our preliminary results. If performance is maintained in subsequent studies and is compared to a more suitable control population, the proposed biomarker may be eventually sufficient to discriminate early-stage DLB from non-DLB.

Transmission of Norwegian reindeer CWD to sheep by intracerebral inoculation results in an unusual phenotype and prion distribution

Chronic wasting disease (CWD), a prion disease affecting cervids, has been known in North America (NA) since the 1960s and emerged in Norway in 2016. Surveillance and studies have revealed that there are different forms of CWD in Fennoscandia: contagious CWD in Norwegian reindeer and sporadic CWD in moose and red deer. Experimental studies have demonstrated that NA CWD prions can infect various species, but thus far, there have been no reports of natural transmission to non-cervid species. In vitro and laboratory animal studies of the Norwegian CWD strains suggest that these strains are different from the NA strains. In this work, we describe the intracerebral transmission of reindeer CWD to six scrapie-susceptible sheep. Detection methods included immunohistochemistry (IHC), western blot (WB), enzyme-linked immunosorbent assay (ELISA), real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA). In the brain, grey matter vacuolation was limited, while all sheep exhibited vacuolation of the white matter. IHC and WB conventional detection techniques failed to detect prions; however, positive seeding activity with the RT-QuIC and PMCA amplification techniques was observed in the central nervous system of all but one sheep. Prions were robustly amplified in the lymph nodes of all animals, mainly by RT-QuIC. Additionally, two lymph nodes were positive by WB, and one was positive by ELISA. These findings suggest that sheep can propagate reindeer CWD prions after intracerebral inoculation, resulting in an unusual disease phenotype and prion distribution with a low amount of detectable prions.

Rapid on-site amplification and visual detection of misfolded proteins via microfluidic quaking-induced conversion (Micro-QuIC)

Protein misfolding diseases, such as prion diseases, Alzheimer’s, and Parkinson’s, share a common molecular mechanism involving the misfolding and aggregation of specific proteins. There is an urgent need for point-of-care (POC) diagnostic technologies that can accurately detect these misfolded proteins, facilitating early diagnosis and intervention. Here, we introduce the microfluidic quaking-induced conversion (Micro-QuIC), a novel acoustofluidic platform for the rapid and sensitive detection of protein misfolding diseases. We demonstrate the utility of our technology using chronic wasting disease (CWD) as a model system, since samples from wild white-tailed deer are readily accessible, and CWD shares similarities with human protein misfolding diseases. Acoustofluidic mixing enables homogeneous mixing of reagents in a high-Reynolds-number regime, significantly accelerating the turnaround time for CWD diagnosis. Our Micro-QuIC assay amplifies prions significantly faster than the current gold standard, real-time quaking-induced conversion (RT-QuIC). Furthermore, we integrated Micro-QuIC with a gold nanoparticle-based, naked-eye detection method, which enables visual discrimination between CWD-positive and CWD-negative samples without the need for a bulky fluorescence detection module. This integration creates a rapid, POC testing platform capable of detecting misfolded proteins associated with a variety of protein misfolding diseases.

Skin α-Synuclein Seeding Activity in Patients with Type 1 Gaucher Disease.

Patients with type 1 Gaucher disease (GD1) have a significantly increased risk of developing Parkinson's disease (PD). The objective of this study was to evaluate skin α-synuclein (αSyn) seeding activity as a biomarker for GD1-related PD (GD1-PD). This single-center study administered motor and cognitive examinations and questionnaires of nonmotor symptoms to adult patients with GD1. Optional skin biopsy was performed for skin αSyn seed amplification assay (αSyn SAA) using real-time quaking-induced conversion assay. Forty-nine patients were enrolled, and 36 underwent skin biopsy. Two study participants had PD. Ten participants were αSyn SAA positive (27.8%), 7 (19.4%) were intermediate, and 19 (52.8%) were negative. Positive αSyn seeding activity was observed in the single GD1-PD case who consented to biopsy. αSyn SAA positivity was associated with older age (p = 0.043), although αSyn SAA positivity was more prevalent in patients with GD1 than historic controls. Longitudinal follow-up is required to determine whether skin αSyn seeding activity can be an early biomarker for GD1-PD. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Evaluation and Limitations of the Novel Chemiluminescent Enzyme Immunoassay Technique for Measuring Total Tau Protein in the Cerebrospinal Fluid of Patients with Human Prion Disease: A 10-Year Prospective Study (2011-2020).

Recently, the investigation of cerebrospinal fluid (CSF) biomarkers for diagnosing human prion diseases (HPD) has garnered significant attention. Reproducibility and accuracy are paramount in biomarker research, particularly in the measurement of total tau (T-tau) protein, which is a crucial diagnostic marker. Given the global impact of the coronavirus disease pandemic, the frequency of measuring this protein using one of the world's fully automated assays, chemiluminescent enzyme immunoassay (CLEA), has increased. At present, the diagnosis and monitoring of neurological diseases mainly rely on traditional methods, but their accuracy and responsiveness are limited. There is limited knowledge of the accuracy of CLEA in tau measurements. We aimed to measure T-tau protein using CLEA and to elucidate its merits and limitations. We randomly selected 60 patients with rapidly progressive dementia, using ELISA and CLEA analysis of cerebrospinal fluid specimens. Additionally, we used Western blotting to detect the presence of 14-3-3 protein and employed real-time quaking-induced conversion (RT-QuIC) assays to analyze the same set of samples. Furthermore, we examined the correlation coefficient between ELISA and CLEA results in a subset of 60 samples. Moreover, using CLEA, we evaluated the diurnal reproducibility, storage stability, dilutability, and freeze-thaw effects in three selected samples. In 172 patients, 172 samples were extracted, with each patient providing only one sample, and a total of 88 (35 men and 53 women) tested positive for HPD in the RT-QuIC assay. In contrast, all CSF samples from the remaining 84 patients without HPD (50 men and 34 women) tested negative in the RT-QuIC assay. Both ELISA and CLEA showed perfect sensitivity and specificity (100%) in measuring T-tau protein levels. In addition, ELISA and CLEA are similar in terms of measurement sensitivity and marginal effect of detection extrema. CLEA analysis exhibited instability for certain samples with T-tau protein levels exceeding 2000 pg/mL, leading to low reproducibility during dilution analysis. Our findings indicate that CLEA outperforms ELISA in terms of diurnal reproducibility, storage stability, and freeze-thaw effects. However, ELISA demonstrated superior performance in the dilution assay. Therefore, it is imperative to develop innovative approaches for the dilution of biomarker samples for CLEA measurements during clinical trials.

A minimally Invasive Biomarker for Sensitive and Accurate Diagnosis of Parkinson's Disease.

ImportanceParkinson′s disease (PD), the second most common neurodegenerative disease, is pathologically characterized by intraneuronal deposition of misfolded alpha-synuclein aggregates (αSynD). αSynDseeding activities in CSF and skin samples have shown great promise in PD diagnosis, but they require invasive procedures. Sensitive and accurate αSynDseed amplification assay (αSyn-SAA) for more accessible and minimally invasive samples (such as blood and saliva) are urgently needed for PD pathological diagnosis in routine clinical practice.ObjectiveTo develop a sensitive and accurate αSyn-SAA biomarker using blood and saliva samples for sensitive, accurate and minimally invasive PD diagnosis.Design, Setting, and ParticipantsThis prospective diagnostic study evaluates serum and saliva samples collected from patients clinically diagnosed with PD or healthy controls (HC) without PD at an academic Parkinson′s and Movement Disorders Center from February 2020 to March 2024. Patients diagnosed with non-PD parkinsonism were excluded from this analysis. A total of 124 serum samples (82 PD and 42 HC) and 131 saliva samples (83 PD and 48 HC) were collected and examined by αSyn-SAA. Out of the 124 serum donors, a subset of 74 subjects (48 PD and 26 HC) also donated saliva samples during the same visits. PD patients with serum samples had a mean age of 69.21 years (range 44-88); HC subjects with serum samples had a mean age of 66.55 years (range 44-81); PD patients with saliva samples had a mean age of 69.58 years (range 49-87); HC subjects with saliva samples had a mean age of 64.71 years (range 30-81).Main Outcomes and MeasuresSerum and/or saliva αSynDseeding activities from PD and HC subjects were measured by αSyn-SAA using the Real-Time Quaking-Induced Conversion (RT-QuIC) platform. These PD patients had extensive clinical assessments including MDS-UPDRS. For a subset of PD and HC subjects whose serum and saliva samples were both collected during the same visits, the αSynDseeding activities in both samples from the same subjects were examined, and the diagnostic accuracies for PD based on the seeding activities in either sample alone or both samples together were compared.ResultsRT-QuIC analysis of αSynDseeding activities in the 124 serum samples revealed a sensitivity of 80.49%, a specificity of 90.48%, and an accuracy of 0.9006 (AUC of ROC, 95% CI, 0.8472-0.9539, p<0.0001) for PD diagnosis. RT-QuIC analysis of αSynDseeding activity in 131 saliva samples revealed a sensitivity of 74.70%, a specificity of 97.92%, and an accuracy of 0.8966 (AUC of ROC, 95% CI, 0.8454-0.9478, p<0.0001). When aSynDseeding activities in the paired serum-saliva samples from the subset of 48 PD and 26 HC subjects were considered together, sensitivity was 95.83%, specificity was 96.15%, and the accuracy was 0.98 (AUC of ROC, 95% CI, 0.96-1.00, p<0.001), which are significantly better than when αSynDseeding activities in either serum or saliva were used alone. For the paired serum-saliva samples, when specificity was set at 100% by elevating the αSyn-SAA cutoff values, a sensitivity of 91.7% and an accuracy of 0.9457 were still attained. Detailed correlation analysis revealed that αSynDseeding activities in the serum of PD patients were correlated inversely with Montreal Cognitive Assessment (MoCA) score (p=0.04), positively with Hamilton Depression Rating Scale (HAM-D) (p=0.03), and weakly positively with PDQ-39 cognitive impairment score (p=0.07). Subgroup analysis revealed that the inverse correlation with MoCA was only seen in males (p=0.013) and weakly in the ≥70 age group (p=0.07), and that the positive correlation with HAM-D was only seen in females (p=0.04) and in the <70 age group (p=0.01). In contrast, αSynDseeding activities in the saliva of PD patients were inversely correlated with age at diagnosis (p=0.02) and the REM sleep behavior disorder (RBD) status (p=0.04), but subgroup analysis showed that the inverse correlation with age at diagnosis was only seen in males (p=0.04) and in the <70 age group (p=0.01).Conclusion and RelevanceOur data show that concurrent RT-QuIC assay of αSynDseeding activities in both serum and saliva can achieve high diagnostic accuracies comparable to that of CSF αSyn-SAA, suggesting that αSynDseeding activities in serum and saliva together can potentially be used as a valuable biomarker for highly sensitive, accurate, and minimally invasive diagnosis of PD in routine clinical practice. αSynDseeding activities in serum and saliva of PD patients correlate differentially with some clinical characteristics and in an age and sex-dependent manner.

Detection of Chronic Wasting Disease Prions in Prairie Soils from Endemic Regions.

Chronic wasting disease (CWD) is a contagious prion disease that affects cervids in North America, Northern Europe, and South Korea. CWD is spread through direct and indirect horizontal transmission, with both clinical and preclinical animals shedding CWD prions in saliva, urine, and feces. CWD particles can persist in the environment for years, and soils may pose a risk for transmission to susceptible animals. Our study presents a sensitive method for detecting prions in the environmental samples of prairie soils. Soils were collected from CWD-endemic regions with high (Saskatchewan, Canada) and low (North Dakota, USA) CWD prevalence. Heat extraction with SDS-buffer, a serial protein misfolding cyclic amplification assay coupled with a real-time quaking-induced conversion assay was used to detect the presence of CWD prions in soils. In the prairie area of South Saskatchewan where the CWD prevalence rate in male mule deer is greater than 70%, 75% of the soil samples tested were positive, while in the low-prevalence prairie region of North Dakota (11% prevalence in male mule deer), none of the soils contained prion seeding activity. Soil-bound CWD prion detection has the potential to improve our understanding of the environmental spread of CWD, benefiting both surveillance and mitigation approaches.

Seeding activity of human superoxide dismutase 1 aggregates in familial and sporadic amyotrophic lateral sclerosis postmortem neural tissues by real-time quaking-induced conversion

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease with average lifespan of 2–5 years after diagnosis. The identification of novel prognostic and pharmacodynamic biomarkers are needed to facilitate therapeutic development. Metalloprotein human superoxide dismutase 1 (SOD1) is known to accumulate and form aggregates in patient neural tissue with familial ALS linked to mutations in their SOD1 gene. Aggregates of SOD1 have also been detected in other forms of ALS, including the sporadic form and the most common familial form linked to abnormal hexanucleotide repeat expansions in the Chromosome 9 open reading frame 72 (C9ORF72) gene. Here, we report the development of a real-time quaking-induced conversion (RT-QuIC) seed amplification assay using a recombinant human SOD1 substrate to measure SOD1 seeding activity in postmortem spinal cord and motor cortex tissue from persons with different ALS etiologies. Our SOD1 RT-QuIC assay detected SOD1 seeds in motor cortex and spinal cord dilutions down to 10–5. Importantly, we detected SOD1 seeding activity in specimens from both sporadic and familial ALS cases, with the latter having mutations in either their SOD1 or C9ORF72 genes. Analyses of RT-QuIC parameters indicated similar lag phases in spinal cords of sporadic and familial ALS patients, but higher ThT fluorescence maxima by SOD1 familial ALS specimens and sporadic ALS thoracic cord specimens. For a subset of sporadic ALS patients, motor cortex and spinal cords were examined, with seeding activity in both anatomical regions. Our results suggest SOD1 seeds are in ALS patient neural tissues not linked to SOD1 mutation, suggesting that SOD1 seeding activity may be a promising biomarker, particularly in sporadic ALS cases for whom genetic testing is uninformative.

Prion Seeding Activity in Plant Tissues Detected by RT-QuIC.

Prion diseases such as scrapie, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) affect domesticated and wild herbivorous mammals. Animals afflicted with CWD, the transmissible spongiform encephalopathy of cervids (deer, elk, and moose), shed prions into the environment, where they may persist and remain infectious for years. These environmental prions may remain in soil, be transported in surface waters, or assimilated into plants. Environmental sampling is an emerging area of TSE research and can provide more information about prion fate and transport once shed by infected animals. In this study, we have developed the first published method for the extraction and detection of prions in plant tissue using the real-time quaking-induced conversion (RT-QuIC) assay. Incubation with a zwitterionic surfactant followed by precipitation with sodium phosphotungstate concentrates the prions within samples and allows for sensitive detection of prion seeding activity. Using this protocol, we demonstrate that prions can be detected within plant tissues and on plant surfaces using the RT-QuIC assay.

Sporadic Creutzfeldt-Jakob disease in adults over 80years: a 10-year review of United Kingdom surveillance.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rapidly progressive neurodegenerative disease with public health implications. Mean age of onset is 68years. Age-specific incidence declines after 80years. This may arise from under-ascertainment or other biological features of the disease. Accurate characterisation of late-onset sCJD is important for early diagnosis, avoiding unnecessary investigations and improving ascertainment for public health purposes. To phenotype the clinical features and investigation profile of sCJD in adults >80years. We analysed all probable and definite sCJD cases identified by the UK National CJD Research & Surveillance Unit over a 10-year period (2011-2021). Individuals were grouped by age of onset. Clinical features and investigation profiles were compared. 10.3% (123/1196) had an age of onset over 80. Median survival was shorter (3.2 vs 4.3months; P < 0.001). Pyramidal signs (48.3% vs 34.2%; P = 0.008) and akinetic mutism (55.1% vs 33.2%; P < 0.001) were more frequent. Psychiatric symptoms (26.3% vs 39.6%; P = 0.01) and cerebellar signs (65.4% vs 78.6%, P = 0.007) were less frequent. Cognitive impairment and myoclonus were highly prevalent regardless of age. Between age groups, the diagnostic sensitivity of cerebrospinal fluid real-time quaking-induced conversion (CSF RT-QuIC) (92.9% vs 91.9%, P = 0.74) was comparable, electroencephalography was superior (41.5% vs 25.4%; P = 0.006) and MRI was inferior (67.8% vs 91.4%; P < 0.001). Late-onset sCJD has distinct clinical features, shorter survival and a different profile of investigation sensitivity. CSF RT-QuIC, MRI brain and specialist CJD review is recommended in older adults with a rapidly progressive neurological disorder. Autopsy is valuable when the cause remains elusive.

α-Synuclein: A Promising Biomarker for Parkinson's Disease and Related Disorders.

Mutations in the SNCA gene, which encodes α-synuclein (α-syn), play a key role in the development of genetic Parkinson's disease (PD). α-Syn is a major component of Lewy bodies in PD and glial cytoplasmic inclusions in multiple system atrophy (MSA). Rapid eye movement sleep behavior disorder patients often progress to PD, dementia with Lewy bodies, or MSA, which are collectively known as α-synucleinopathies. The loss of dopaminergic neurons with Lewy bodies precedes motor dysfunction in these diseases, but the mechanisms of neurodegeneration due to α-syn aggregation are poorly understood. Monitoring α-syn aggregation in vivo could serve as a diagnostic biomarker and help elucidate pathogenesis, necessitating a simple and accurate detection method. Seed amplification assays (SAAs), such as real-time quaking-induced conversion and protein misfolding cyclic amplification, are used to detect small amounts of abnormally structured α-syn protofibrils, which are central to aggregation. These methods are promising for the early diagnosis of α-synucleinopathy. Differences in α-syn filament structures between α-synucleinopathies, as observed through transmission electron microscopy and cryo-electron microscopy, suggest their role in the pathogenesis of neurodegeneration. SAAs may differentiate between subtypes of α-synucleinopathy and other diseases. Efforts are also being made to identify α-syn from blood using various methods. This review introduces body fluid α-syn biomarkers based on pathogenic α-syn seeds, which are expected to redefine α-synucleinopathy diagnosis and staging, improving clinical research accuracy and facilitating biomarker development.