Search Results (28)

Introduction and objective: Assessment of α-synuclein (αSyn) seed amplification assays (αSyn-SAA) accuracy in distinguishing Parkinson’s disease (PD) from controls using cerebrospinal fluid (CSF), blood, skin, extracellular vesicles (ECV), saliva, olfactory mucosa (OM), gastrointestinal tract (GIT), and submandibular gland (SMG). Methodology: PubMed was searched for articles from 2010 to January 2025. The quality assessment used robvis. Diagnostic values with a 95% confidence interval (CI) were obtained. Z-test, Wald CI, and ANOVA were performed. Diagnostic odds ratio (DOR) was used. Results: αSyn-SAAs showed strong diagnostic performance in distinguishing PD from controls across various tissue and fluid types. Overall, αSyn-SAAs demonstrated high sensitivity (86%) and specificity (92%). Among all biomatrices, CSF, skin, blood, and ECV yielded the highest diagnostic accuracy, with sensitivity and specificity approaching or exceeding 90%. In contrast, saliva, oral mucosa, and gastrointestinal tract samples showed more modest sensitivity, though specificity remained relatively high. ECV, CSF, skin, and blood matrices also demonstrated the highest DOR, supporting their potential clinical utility. Conclusions: ECV and blood warrant priority in αSyn-SAA for high accuracy and minimal invasiveness, while GIT, OM, and oral samples show limited utility; saliva and SMG need refinement. Full article

Disease monitoring informs the opportunities for intervention by natural resource agencies tasked with managing chronic wasting disease (CWD) in wild cervids. However, allocating funds toward testing can reduce those available for education, outreach, and disease reduction. Implementation of more efficient testing strategies can help meet both an expanding need by resource managers and a burgeoning demand from the hunting public in North America. Here, we evaluated the efficacy of pooled testing using the enzyme-linked immunosorbent assay (ELISA), the current screening test used by veterinary diagnostic laboratories in the United States, and real-time quaking-induced conversion (RT-QuIC), an amplification assay that is being evaluated by the U.S. Department of Agriculture but is not yet approved or commercially available. The samples used in this study consisted of medial retropharyngeal lymph nodes (RPLNs) routinely collected by the Iowa Department of Natural Resources during the 2019–2020 surveillance season. The test pools contained tissue from one positive deer diluted in tissue from an increasing number of undetected deer, with each individual contributing an equal tissue volume. ELISA remained positive with pooling thresholds of 1:1, 1:2, 1:4, and 1:9 at a standard volume of tissue homogenate, whereas RT-QuIC remained positive with pooling thresholds of 1:1, 1:2, 1:4, 1:9, 1:19, and 1:49 at a 0.02% tissue dilution. Our results suggest that pooled testing can reduce diagnostic costs multi-fold, and RT-QuIC can be a viable screening test compatible with current field collection standards. Full article

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. Full article

Background: 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. Methods: 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. Results: 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. Conclusions: 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. Full article

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. Full article

Real-time quaking-induced conversion (RT-QuIC) assays have become a common tool to detect chronic wasting disease (CWD) and are very sensitive provided the assay duration is sufficient. However, a prolonged assay duration may lead to non-specific signal amplification. The wide range of pre-defined assay durations in current RT-QuIC applications presents a need for methods to optimize the RT-QuIC assay. In this study, receiver operating characteristic (ROC) analysis was applied to optimize the assay duration for CWD screening in obex and retropharyngeal lymph node (RLN) tissue specimens. Two different fluorescence thresholds were used: a fixed threshold based on background fluorescence (T_stdev) and a max-point ratio (maximum/background fluorescence) threshold (T_MPR) to determine CWD positivity. The optimal assay duration was 33 h for obex and 30 h for RLN based on T_stdev, and 29 h for obex and 32 h for RLN based on T_MPR. The optimized assay durations were then evaluated for screening CWD in white-tailed deer from an affected farm. At a replicate level, using the optimized assay durations with T_Stdev and T_MPR, the level of agreement with enzyme-linked immunosorbent assay (ELISA) was significantly higher (p < 0.05) than that when using a 40 h assay duration. These findings demonstrate that the optimization of assay duration via a ROC analysis can improve RT-QuIC assays for screening CWD in white-tailed deer. Full article

Cerebrospinal fluid-based real-time quaking-induced conversion (CSF RT-QuIC) is currently the most prominent method for early detection of sporadic Creutzfeldt–Jakob disease (sCJD), the most common prion disease. CSF RT-QuIC delivers high sensitivity (>90%) and specificity (100%), which has been demonstrated by large ring-trial studies testing probable and definitive sCJD cohorts. Following the inclusion of CSF RT-QuIC in the revised European CJD Surveillance Network diagnostic criteria for sCJD, it has become a standard diagnostic procedure in many prion disease reference or surveillance centers around the world. In this study, we present the implementation of the second-generation CSF RT-QuIC (commonly known as Improved QuIC or IQ) at the Danish Reference Center for Prion Diseases (DRCPD). The method’s sensitivity and specificity were evaluated and validated by analyzing 63 CSF samples. These 63 samples were also analyzed at the National CJD Research and Surveillance Unit (NCJDRSU), based at the University of Edinburgh, UK; analysis was carried out using the first generation or previous CSF RT-QuIC method (PQ). The sensitivity and specificity of PQ during tests at the NCJDRSU were 92% and 100%, respectively. Using these 63 CSF samples, the agreement between the two RT-QuIC generations at DRCPD and NCJDRSU prion laboratories was 100%. Full article

Chronic wasting disease (CWD) is a disease affecting cervids and is caused by prions accumulating as pathogenic fibrils in lymphoid tissue and the central nervous system. Approaches for detecting CWD prions historically relied on antibody-based assays. However, recent advancements in protein amplification technology provided the foundation for a new class of CWD diagnostic tools. In particular, real-time quaking-induced conversion (RT-QuIC) has rapidly become a feasible option for CWD diagnosis. Despite its increased usage for CWD-focused research, there lacks a consensus regarding the interpretation of RT-QuIC data for diagnostic purposes. It is imperative then to identify a standardized and replicable method for determining CWD status from RT-QuIC data. Here, we assessed variables that could impact RT-QuIC results and explored the use of maxpoint ratios (maximumRFU/backgroundRFU) to improve the consistency of RT-QuIC analysis. We examined a variety of statistical analyses to retrospectively analyze CWD status based on RT-QuIC and ELISA results from 668 white-tailed deer lymph nodes. Our results revealed an MPR threshold of 2.0 for determining the rate of amyloid formation, and MPR analysis showed excellent agreement with independent ELISA results. These findings suggest that the use of MPR is a statistically viable option for normalizing between RT-QuIC experiments and defining CWD status. Full article

Genetic Creutzfeldt–Jakob disease (gCJD) associated with the V180I mutation in the prion protein (PrP) gene (PRNP) in phase with residue 129M is the most frequent cause of gCJD in East Asia, whereas it is quite uncommon in Caucasians. We report on a gCJD patient with the rare V180I-129V haplotype, showing an unusually long duration of the disease and a characteristic pathological PrP (PrP^Sc) glycotype. Family members carrying the mutation were fully asymptomatic, as commonly observed with this mutation. Neuropathological examination showed a lesion pattern corresponding to that commonly reported in Japanese V180I cases with vacuolization and gliosis of the cerebral cortexes, olfactory areas, hippocampus and amygdala. PrP was deposited with a punctate, synaptic-like pattern in the cerebral cortex, amygdala and olfactory tract. Western blot analyses of proteinase-K-resistant PrP showed the characteristic two-banding pattern of V180I gCJD, composed of mono- and un-glycosylated isoforms. In line with reports on other V180I cases in the literature, Real-Time Quaking Induced Conversion (RT-QuIC) analyses did not demonstrate the presence of seeding activity in the cerebrospinal fluid and olfactory mucosa, suggesting that this haplotype also may result in a reduced seeding efficiency of the pathological PrP. Further studies are required to understand the origin, penetrance, disease phenotype and transmissibility of 180I-129V haplotype in Caucasians. Full article

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. Full article

Various disease-associated forms or strains of α-synuclein (αSyn^D) can spread and accumulate in a prion-like fashion during synucleinopathies such as Parkinson’s disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA). This capacity for self-propagation has enabled the development of seed amplification assays (SAAs) that can detect αSyn^D in clinical samples. Notably, α-synuclein real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays have evolved as ultrasensitive, specific, and relatively practical methods for detecting αSyn^D in a variety of biospecimens including brain tissue, CSF, skin, and olfactory mucosa from synucleinopathy patients. However, αSyn SAAs still lack concordance in detecting MSA and familial forms of PD/DLB, and the assay parameters show poor correlations with various clinical measures. End-point dilution analysis in αSyn RT-QuIC assays allows for the quantitation of relative amounts of αSyn^D seeding activity that may correlate moderately with clinical measures and levels of other biomarkers. Herein, we review recent advancements in α-synuclein SAAs for detecting αSyn^D and describe in detail the modified Spearman–Karber quantification algorithm used with end-point dilutions. Full article

Prion diseases are progressive and irreversible neurodegenerative disorders with a low incidence (1.5–2 cases per million per year). Genetic (10–15%), acquired (anecdotal) and sporadic (85%) forms of the disease have been described. The clinical spectrum of prion diseases is very varied, although the most common symptoms are rapidly progressive dementia, cerebellar ataxia and myoclonus. Mean life expectancy from the onset of symptoms is 6 months. There are currently diagnostic criteria based on clinical phenotype, as well as neuroimaging biomarkers (magnetic resonance imaging), neurophysiological tests (electroencephalogram and polysomnogram), and cerebrospinal fluid biomarkers (14-3-3 protein and real-time quaking-induced conversion (RT-QuIC)). The sensitivity and specificity of some of these tests (electroencephalogram and 14-3-3 protein) is under debate and the applicability of other tests, such as RT-QuIC, is not universal. However, the usefulness of these biomarkers beyond the most frequent prion disease, sporadic Creutzfeldt–Jakob disease, remains unclear. Therefore, research is being carried out on new, more efficient cerebrospinal fluid biomarkers (total tau, ratio total tau/phosphorylated tau and neurofilament light chain) and potential blood biomarkers (neurofilament light chain, among others) to try to universalize access to early diagnosis in the case of prion diseases. Full article

Variably protease-sensitive prionopathy is an exceedingly rare, likely underestimated, sporadic prion disease that is characterized by heterogeneous and often non-specific clinical and pathological features posing diagnostic challenges. We report the results of a comprehensive analysis of three emblematic cases carrying different genotypes at the methionine (M)/valine (V) polymorphic codon 129 in the prion protein gene (PRNP). Clinical, biochemical, and neuropathological findings highlighted the prominent role of the host genetic background as a phenotypic modulator. In particular, the PRNP codon 129 showed a remarkable influence on the physicochemical properties of the pathological prion protein (PrP^Sc), especially on the sensitivity to proteinase K (PK) digestion (VV > MV > MM), which variably affected the three main fragments (i.e., of 19, 17, and 7 kDa, respectively) comprising the PrP^Sc profile after PK digestion and immunoblotting. This, in turn, correlated with significant differences in the ratio between the 19 kDa and the 7 kDa fragments which was highest in the MM case and lowest in the VV one. The relative amount of cerebral and cerebellar PrP mini-plaques immunohistochemistry showed a similar association with the codon 129 genotype (i.e., VV > MV > MM). Clinical manifestations and results of diagnostic investigations were non-specific, except for the detection of prion seeding activity by the real-time quaking-induced conversion assay in the only cerebrospinal fluid sample that we tested (from patient 129VV). Full article

Parkinson’s disease (PD) and multiple system atrophy (MSA) are caused by two distinct strains of disease-associated α-synuclein (αSyn^D). Recently, we have shown that olfactory mucosa (OM) samples of patients with PD and MSA can seed the aggregation of recombinant α-synuclein by means of Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC). Remarkably, the biochemical and morphological properties of the final α-synuclein aggregates significantly differed between PD and MSA seeded samples. Here, these aggregates were given to neuron-like differentiated SH-SY5Y cells and distinct inflammatory responses were observed. To deepen whether the morphological features of α-synuclein aggregates were responsible for this variable SH-SY5Y inflammatory response, we generated three biochemically and morphologically distinct α-synuclein aggregates starting from recombinant α-synuclein that were used to seed αSyn_RT-QuIC reaction; the final reaction products were used to stimulate SH-SY5Y cells. Our study showed that, in contrast to OM samples of PD and MSA patients, the artificial aggregates did not transfer their distinctive features to the αSyn_RT-QuIC products and the latter induced analogous inflammatory responses in cells. Thus, the natural composition of the αSyn^D strains but also other specific factors in OM tissue can substantially modulate the biochemical, morphological and inflammatory features of the αSyn_RT-QuIC products. Full article

The definite diagnosis of human sporadic Creutzfeldt–Jakob disease (sCJD) largely depends on postmortem neuropathology and PrP^Sc detection in the brain. The development of real-time quaking-induced conversion (RT-QuIC) of cerebrospinal fluid (CSF) samples makes it possible for premortem diagnosis for sCJD. To test the diagnostic potential of RT-QuIC of skin specimens for probable sCJD, we collected the paired skin and CSF samples from 51 recruited living patients referred to the Chinese CJD surveillance center, including 34 probable sCJD, 14 non-CJD, and 3 genetic prion disease (gPrD). The samples were subjected to RT-QuIC assays using recombinant hamster PrP protein rHaPrP90-231 as the substrate. Using skin RT-QuIC assay, 91.2% (31/34) probable sCJD patients, and 1 T188K genetic CJD (gCJD) cases showed positive prion-seeding activity, while 85.7% (12/14) non-CJD patients were negative. CSF RT-QuIC positive seeding activity was only observed in 14 probable sCJD patients. Analysis of the reactivity of 38 positive skin RT-QuIC tests revealed that the positive rates in the preparations of 10⁻², 10⁻³ and 10⁻⁴ diluted skin samples were 88.6% (39/44), 63.6% (28/44), and 25.0% (11/44), respectively. Eleven probable sCJD patients donated two skin specimens collected at different sites simultaneously. Although 95.5% (21/22) skin RT-QuIC elicited positive reaction, the reactivity varied. Our 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. Full article