Diagnosis Of Human Prion Diseases Research Articles

Establishment of a Special Platform for the Research of Prion and the Diagnosis of Human Prion Disease - China's Studies.

The studies of prions and prion disease usually need many special platforms and techniques that differ from those for classical microbes. Search of new biomarkers and establishment of new methods for the diagnosis of human prion diseases are priorities in the field of prion study.

The Use of Real-Time Quaking-Induced Conversion for the Diagnosis of Human Prion Diseases.

Prion diseases are rapidly progressive, invariably fatal, transmissible neurodegenerative disorders associated with the accumulation of the amyloidogenic form of the prion protein in the central nervous system (CNS). In humans, prion diseases are highly heterogeneous both clinically and neuropathologically. Prion diseases are challenging to diagnose as many other neurologic disorders share the same symptoms, especially at clinical onset. Definitive diagnosis requires brain autopsy to identify the accumulation of the pathological prion protein, which is the only specific disease biomarker. Although brain post-mortem investigation remains the gold standard for diagnosis, antemortem clinical, instrumental, and laboratory tests showing variable sensitivities and specificity, being surrogate disease biomarkers, have been progressively introduced in clinical practice to reach a diagnosis. More recently, the ultrasensitive Real-Time Quaking-Induced Conversion (RT-QuIC) assay, exploiting, for the first time, the detection of misfolded prion protein through an amplification strategy, has highly improved the “in-vitam” diagnostic process, reaching in cerebrospinal fluid (CSF) and olfactory mucosa (OM) around 96% sensitivity and close to 100% specificity. RT-QuIC also improved the detection of the pathologic prion protein in several peripheral tissues, possibly even before the clinical onset of the disease. The latter aspect is of great interest for the early and even preclinical diagnosis in subjects at genetic risk of developing the disease, who will likely be the main target population in future clinical trials. This review presents an overview of the current knowledge and future perspectives on using RT-QuIC to diagnose human prion diseases.

Validation and Application of Skin RT-QuIC to Patients in China with Probable CJD.

The definite diagnosis of human sporadic Creutzfeldt–Jakob disease (sCJD) largely depends on postmortem neuropathology and PrPSc 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.

The Latest Research on RT-QuIC Assays—A Literature Review

The misfolding of proteins such as the prion protein, α-synuclein, and tau represents a key initiating event for pathogenesis of most common neurodegenerative disorders, and its presence correlates with infectivity. To date, the diagnosis of these disorders mainly relied on the recognition of clinical symptoms when neurodegeneration was already at an advanced phase. In recent years, several efforts have been made to develop new diagnostic tools for the early diagnosis of prion diseases. The real-time quaking-induced conversion (RT–QuIC) assay, an in vitro assay that can indirectly detect very low amounts of PrPSc aggregates, has provided a very promising tool to improve the early diagnosis of human prion diseases. Over the decade since RT–QuIC was introduced, the diagnosis of not only prion diseases but also synucleinopathies and tauopathies has greatly improved. Therefore, in our study, we summarize the current trends and knowledge of RT–QuIC assays, as well as discuss the diagnosis of neurodegenerative diseases using RT–QuIC assays, which have been updated in recent years.

Clinical diagnosis of human prion disease.

Human prion disease may present in a non-specific way and is often diagnosed at a relatively late stage of the illness. Until recently, clinical diagnosis has been supported by tests that are mostly non-specific and, sometimes, insensitive. Recent laboratory developments have led to a variety of tests that rely on a disease-specific mechanism. One test, the CSF RT-QuIC (Real-Time Quaking-Induced Conversion) test is very sensitive and specific for sporadic CJD and is now used in routine clinical practice. Other tests, based on other tissues, including blood and urine, have been developed and potentially could improve both clinical diagnostic accuracy and lead to earlier diagnosis. While there are yet no proven treatments for prion disease, any treatment to be developed will almost certainly require earlier diagnosis if therapeutic success is to be realized.

Clinical Laboratory Tests Used To Aid in Diagnosis of Human Prion Disease.

Prion diseases are a group of rapidly progressive and always fatal neurodegenerative disorders caused by misfolded prion protein in the brain. Although autopsy remains the gold-standard diagnostic tool, antemortem laboratory testing can be performed to aid in the diagnosis of prion disease. This review is meant to help laboratory directors and physicians in their interpretation of test results. Laboratory assays to detect both nonspecific biomarkers of prion disease and prion-specific biomarkers can be used. The levels of nonspecific biomarkers in cerebrospinal fluid (CSF) are elevated when rapid neurodegeneration is occurring in the patient, and these markers include 14-3-3, tau, neuron-specific enolase, S100B, and alpha-synuclein. These markers have various sensitivities and specificities but are overall limited, as the levels of any of these analytes can be elevated in nonprion disease that is causing rapid damage of brain tissue. Prion-specific assays used in clinical laboratory testing are currently limited to two options. The first option is second-generation real-time quaking-induced conversion (RT-QuIC) performed on CSF, and the second option is Western blotting of a brain biopsy specimen used to detect protease-resistant prion protein. Although both tests have exquisite specificity, RT-QuIC has a sensitivity of 92 to 97.2% in symptomatic individuals, compared to the brain biopsy Western blot sensitivity of 20 to 60%. RT-QuIC was added to the Centers for Disease Control and Prevention's diagnostic criteria for prion disease in 2018. Other caveats of laboratory testing need to be considered, as sporadic, genetic, and acquired forms of prion disease have different clinical and laboratory presentations, and these caveats are discussed. Laboratory testing plays an important role in the diagnosis of prion disease, which is often challenging to diagnose.

Are We Ready for Detecting α-Synuclein Prone to Aggregation in Patients? The Case of "Protein-Misfolding Cyclic Amplification" and "Real-Time Quaking-Induced Conversion" as Diagnostic Tools.

The accumulation and deposition of α-synuclein aggregates in brain tissue is the main event in the pathogenesis of different neurodegenerative disorders grouped under the term of synucleinopathies. They include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. To date, the diagnosis of any of these disorders mainly relies on the recognition of clinical symptoms, when the neurodegeneration is already in an advanced phase. In the last years, several efforts have been carried out to develop new diagnostic tools for early diagnosis of synucleinopathies, with special interest to Parkinson's disease. The Protein-Misfolding Cyclic Amplification (PMCA) and the Real-Time Quaking-Induced Conversion (RT-QuIC) are ultrasensitive protein amplification assays for the detection of misfolded protein aggregates. Starting from the successful application in the diagnosis of human prion diseases, these techniques were recently tested for the detection of misfolded α-synuclein in brain homogenates and cerebrospinal fluid samples of patients affected by synucleinopathies. So far, only a few studies on a limited number of samples have been performed to test PMCA and RT-QuIC diagnostic reliability. Neverthless, these assays have shown very high sensitivity and specificity in detecting synucleinopathies even at the pre-clinical stage. Despite the application of PMCA and RT-QuIC for α-synuclein detection in biological fluids is very recent, these techniques seem to have the potential for identifying subjects that will be likely to develop synucleinopathies.

Amplification and Detection of Minuscule Amounts of Misfolded Prion Protein by Using the Real-Time Quaking-Induced Conversion.

A characteristic feature of transmissible spongiform encephalopathies (TSE) is the progressive accumulation of protein aggregates in the brain in a self-propagation manner. Based on this mechanism, in vitro protein amplification systems (such as real-time quaking-induced conversion (RT-QuIC)) for the detection of misfolded prion protein scrapie (PrPres) in CSF were a major step in pre-mortem diagnosis of human prion diseases. Here, we describe a protocol of the RT-QuIC assay to detect PrPres in CSF of prion disease patients. This methodology depends on prion seeds that induce misfolding and aggregation of a substrate by cycles of incubation and quaking. Besides diagnostics, further applications of the RT-QuIC appear to be promising for discrimination between different PrP subtypes or strains, understanding the mechanism of protein misfolding and pre-screening of anti-prion drugs. The technique can be further developed to be used to study characteristics of misfolded proteins in other "prion like" diseases, such as tauopathies, synucleinopathies, or amyloidopathies.

Biomarkers for human prion disease : Results from the creutzfeldt–jakob disease surveillance committee in Japan

Background: The differential diagnosis of human prion disease (PrD) from other, sometimes treatable, neurological disorders is challenging, owing to the wide phenotypic heterogeneity of the disease. Real-time quaking-induced prion conversion (RT-QuIC) is a novel ultrasensitive in vitro assay, which, at variance with surrogate neurodegenerative biomarker assays, specifically targets the pathological prion protein (PrPSc).

Ultra-efficient Amplification of Abnormal Prion Protein by Modified Protein Misfolding Cyclic Amplification with Electric Current

Prion diseases are clinically diagnosed and confirmed upon post-mortem histopathological examination of brain tissue. The only reliable molecular marker for prion diseases is abnormal prion protein (PrPSc), a pathologically conformed prion protein that primarily accumulates in the central nervous system and to a lesser extent in lymphoreticular tissues. However, the use of PrPSc as a marker for preclinical diagnoses is limited because the concentration of PrPSc in easily accessible body fluids is extremely low. Hence, one of the most promising approaches would be the development of an efficient in vitro amplification method for PrPSc. Indeed, protein misfolding cyclic amplification (PMCA) has become an important diagnostic tool for prion diseases. Here, we first describe a new superior PMCA device that employs electricity (referred to as ePMCA) to amplify PrPSc. The ePMCA device markedly improved the detection limit for PrPSc by amplifying trace amounts of pathogenic prion protein by applying electricity to improve PMCA. To increase the cavitation of sonication, a glass sample tube was used, and the upper side of the horn was shaped such that it had a curved cross-section. The ePMCA device enabled PrPSc to be amplified even from a sample seeded with 10–28-fold diluted 263K scrapie-infected brain homogenates with recombinant hamster prion protein (rHaPrP). In addition, the efficiency of prion amplification was best when 50 mM HEPES and 1% Triton X-100 were used as a PMCA conversion buffer in the various conditions that we applied. These results indicate that ePMCA would be very valuable for the rapid and specific diagnosis of human prion diseases and, thus, may provide a practically improved method for antemortem diagnoses using the body fluids of patients and animals with prion disease.

Methods for Molecular Diagnosis of Human Prion Disease.

Human prion diseases are associated with a range of clinical presentations, and they are classified by both clinicopathological syndrome and etiology, with subclassification according to molecular criteria. Here, we describe updated procedures that are currently used within the MRC Prion Unit at UCLto determine a molecular diagnosis of human prion disease. Sequencing of the PRNP open reading frame to establish the presence of pathogenic mutations is described, together with detailed methods for immunoblot or immunohistochemical determination of the presence of abnormal prion protein in the brain or peripheral tissues.

Diagnosis of Human Prion Disease Using Real-Time Quaking-Induced Conversion Testing of Olfactory Mucosa and Cerebrospinal Fluid Samples

Early and accurate in vivo diagnosis of Creutzfeldt-Jakob disease (CJD) is necessary for quickly distinguishing treatable from untreatable rapidly progressive dementias and for future therapeutic trials. This early diagnosis is becoming possible using the real-time quaking-induced conversion (RT-QuIC) seeding assay, which detects minute amounts of the disease-specific pathologic prion protein in cerebrospinal fluid (CSF) or olfactory mucosa (OM) samples. To develop an algorithm for accurate and early diagnosis of CJD by using the RT-QuIC assay on CSF samples, OM samples, or both. In this case-control study, samples of CSF and OM were collected from 86 patients with a clinical diagnosis of probable (n = 51), possible (n = 24), or suspected (n = 11) CJD and 104 negative control samples (54 CSF and 50 OM). The CSF and OM samples were analyzed using conventional RT-QuIC. The CSF samples underwent further testing using improved RT-QuIC conditions. In addition, the diagnostic performance of a novel, easy-to-use, gentle flocked swab for sampling of OM was evaluated. Data were collected from January 1 to June 30, 2015. Correlations between RT-QuIC results and the final diagnosis of recruited patients. Among the 86 patients (37 men [43%] and 49 women [57%]; mean [SD] age, 65.7 [11.5] years) included for analysis, all 61 patients with sporadic CJD had positive RT-QuIC findings using OM or CSF samples or both for an overall RT-QuIC diagnostic sensitivity of 100% (95% CI, 93%-100%). All patients with a final diagnosis of non-prion disease (71 CSF and 67 OM samples) had negative RT-QuIC findings for 100% specificity (95% CI, 94%-100%). Of 8 symptomatic patients with various mutations causing CJD or Gerstmann-Sträussler-Scheinker syndrome, 6 had positive and 2 had negative RT-QuIC findings for a sensitivity of 75% (95% CI, 36%-96%). A proposed diagnostic algorithm for sporadic CJD combines CSF and OM RT-QuIC testing to provide virtually 100% diagnostic sensitivity and specificity in the clinical phase of the disease.

Quality evaluation for the surveillance system of human prion diseases in China based on the data from 2010 to 2016

ABSTRACTThe surveillance of CJD or human prion diseases (PrDs) has been conducted for 10 y in China. To evaluate the quality of China CJD surveillance system, the collections of the clinical and epidemiological information, the sampling, the clinical examinations and laboratory tests and follow-up survey were separately analyzed based on the data from 2010 to 2015. The obtaining rates of clinical-information table, epidemiological-information table, sample inspection sheet and medical record of the referring patients from reporting units to the center of CJD surveillance maintained or reached at very high levels, being close to 100% in the past 3 y. 93.82%, 85.23%, 96.21% and 94.70% of the reported cases had the data of MRI, EEG, CSF 14-3-3 and PRNP sequencing, respectively. Follow-up surveys were conducted in about 50% cases in 2010 and 2011, 93.39% cases in 2012 and 100% cases in the last 3 y. High obtaining rates of the clinical and epidemiological data, high conducting rates of the relevant clinical examinations and laboratory tests, high performing rates of follow-up survey for every referring case reflect a good implemental capacity in China CJD surveillance system, which supplies solid basis for recognition and diagnosis of human prion diseases and guarantees good quality of China CJD surveillance system.

Real-Time Quaking-Induced Conversion Analysis for the Diagnosis of Sporadic Creutzfeldt-Jakob Disease in Korea.

Background and PurposeThe level of 14-3-3 protein in the cerebrospinal fluid (CSF) is increased in Creutzfeldt-Jakob disease (CJD) patients, which has led to it being used as a clinical biomarker for the ante-mortem diagnosis of human prion diseases. However, the specificity of the 14-3-3 protein is less reliable for CJD diagnosis. Newly developed assays including real-time quaking-induced conversion (RT-QuIC) have made it possible to detect the PrPSc-like abnormal prion isoform with a high sensitivity in animal and human specimens that might contain a minute amount of PrPSc due to in vitro prion replication.MethodsThis study applied a highly sensitive RT-QuIC assay using recombinant human PrP to detect PrPSc in the CSF of 81 patients with sporadic CJD (sCJD) in Korea.ResultsRT-QuIC analysis of the CSF samples based on the expression levels of 14-3-3 and total tau proteins revealed positivity in 62 of 81 sCJD patients (sensitivity of 76.5%) but no positive results in the 100 non-CJD patients.ConclusionsThe sensitivity of the RT-QuIC in this study was similar to that in some previous reports, and the specificity of RT-QuIC was higher than that of 14-3-3 in CSF, suggesting that RT-QuIC analysis can complement the weakness of the specificity of 14-3-3 for the diagnosis of sCJD. These results indicate that RT-QuIC might be very useful for the rapid and specific diagnosis of sCJD and provide a practical novel method for the ante-mortem diagnosis of human prion diseases.

Metabolic patterns in prion diseases: an FDG PET voxel-based analysis

Clinical diagnosis of human prion diseases can be challenging since symptoms are common to other disorders associated with rapidly progressive dementia. In this context, (18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET) might be a useful complementary tool. The aim of this study was to determine the metabolic pattern in human prion diseases, particularly sporadic Creutzfeldt-Jakob disease (sCJD), the new variant of Creutzfeldt-Jakob disease (vCJD) and fatal familial insomnia (FFI). We retrospectively studied 17 patients with a definitive, probable or possible prion disease who underwent FDG PET in our institution. Of these patients, 12 were diagnosed as sCJD (9 definitive, 2 probable and 1 possible), 1 was diagnosed as definitive vCJD and 4 were diagnosed as definitive FFI. The hypometabolic pattern of each individual and comparisons across the groups of subjects (control subjects, sCJD and FFI) were evaluated using a voxel-based analysis. The sCJD group exhibited a pattern of hypometabolism that affected both subcortical (bilateral caudate, thalamus) and cortical (frontal cortex) structures, while the FFI group only presented a slight hypometabolism in the thalamus. Individual analysis demonstrated a considerable variability of metabolic patterns among patients, with the thalamus and basal ganglia the most frequently affected areas, combined in some cases with frontal and temporal hypometabolism. Patients with a prion disease exhibit a characteristic pattern of brain metabolism presentation in FDG PET imaging. Consequently, in patients with rapidly progressive cognitive impairment, the detection of these patterns in the FDG PET study could orient the diagnosis to a prion disease.

Prion protein aggregation assays in the diagnosis of human prion diseases

ABSTRACT Sporadic Creutzfeldt–Jakob disease (sCJD) is the most common form of human prion disease and is associated with a progressive cognitive decline and death usually occurs within 6 months. Neuropathologically these diseases are characterized by the deposition of an abnormal form (PrPSc) of a normally expressed protein PrPC. At present there are no disease-specific diagnostic tests for prion diseases. Therefore, a test that will enable accurate and earlier diagnosis is needed. The ability of PrPSc to convert native PrPC into PrPSc has been exploited in a variety of protein aggregation assays such as protein misfolding cyclic amplification (PMCA), and real-time QuIC (RT-QuIC). Cerebrospinal fluid RT-QuIC is rapidly growing in acceptance as a reliable and accurate diagnostic test for sCJD.

Creutzfeldt-Jakob disease: clinical and diagnostic aspects

In this article, authors analyzed a modern approach to the diagnosis of Creutzfeldt-Jakob disease (CJD) based on the clinical signs, cerebrospinal fluid markers, electroencephalography and magnetic resonance imaging. It was demonstrated for the first time that patients with late-onset CJD differed from younger CJD patients with respect to MRI profiles and initial clinical presentation. To date, cerebrospinal fluid (CSF) analysis, particularly protein 14-3-3 testing, presents an important approach to the identification of disease cases. A spectrum of differential diagnosis of rapid progressive dementia varied from neurodegenerative dementias to dementia due to acute neurological conditions. Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein. Recent studies applied the RT-QuIC methodology to CSF for the diagnosis of human prion diseases.

Factors intrinsic and extrinsic to blood hamper the development of a routine blood test for human prion diseases.

Development of numerous advanced techniques in recent years have allowed detection of the pathological prion protein (PrP(TSE)), the unique marker of transmissible spongiform encephalopathies (TSEs, or prion diseases), in the blood of animals and humans; however, an ante mortem screening test that can be used for the routine diagnosis of human prion diseases remains unavailable. A critical, analytical review of all the diagnostic assays developed to date will allow an evaluation of progress in this field and may facilitate the identification of the possible reasons for this delay. Thus, in this review, I provide a detailed overview of the techniques currently available for detecting PrP(TSE) and other markers of the disease in blood, as well as an analysis of the significance, feasibility, reliability and application spectrum for these methods. I highlight that factors intrinsic and extrinsic to blood may interfere with the detection of PrP(TSE)/prions, and that this is not yet taken into account in current tests. This may inspire researchers in this field to not only aspire to increase test sensitivity, but also to adopt other strategies in order to identify and overcome the limitations that hamper the development of a successful routine blood test for prion diseases.

髄液によるプリオン病の診断と鑑別診断

Hsich et al reported 14-3-3 protein in CSF supports a diagnosis of human prion disease, and 14-3-3 protein is one of supportive diagnostic criteria on WHO (1998). In the presence of 14-3-3 protein and total tau protein is widely used as a surrogate marker in the pre-mortem diagnosis of human prion disease and other rapidly progressive dementia. Most recent research report that the sensitivity of 14-3-3 protein was 43%-100%, and the specificity of 14-3-3 protein was 47%-97%. And the sensitivity of total tau protein was 43%-100%, and the specificity of total tau protein was 47%-97%. In other hand we recently developed a new in vitro amplification technology, designated "RT-QUIC assay", for the detection of PrP(Sc) in CSF of sCJD. Other group reported that CSF RT-QUIC method analysis has the potential to be a more specific diagnostic test for sCJD than current CSF tests, but their method is different from our method and their study was not enough. Now we are analyzing for the sensitivity and specificity of biomarkers and RT-QUIC method in CSF of definite cases of human prion disease, the number of definite cases of human prion disease is larger than that of other studies.

High sensitivity of an ELISA kit for detection of the gamma-isoform of 14-3-3 proteins: usefulness in laboratory diagnosis of human prion disease

BackgroundThe gamma-isoform of the 14-3-3 protein (14-3-3 gamma) is expressed in neurons, and could be a specific marker for neuronal damage. This protein has been reported as a detectable biomarker, especially in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients by Western blotting (WB) or enzyme-linked immunosorbent assays (ELISAs). Western blotting for 14-3-3 gamma is not sensitive, and the reported data are conflicting among publications. An ELISA specific for 14-3-3 gamma is not available.MethodsCJD patients (n = 114 sporadic CJD patients, 7 genetic CJD, and 3 iatrogenic CJD) and 99 patients with other neurodegenerative diseases were examined in this study. The CSF samples obtained were analyzed by Western blotting for 14-3-3 gamma, and by ELISA for total tau protein. We evaluated the sensitivity and specificity of the newly developed sandwich ELISA for 14-3-3 gamma.ResultsThe cut-off value of the 14-3-3 gamma ELISA was > 1, 683 AU/ml; and sensitivity was 95.2%, with 72.7% specificity. This specificity was the same for the total tau protein ELISA. Seven CJD cases were negative by WB but positive using the 14-3-3 gamma ELISA, indicating that the ELISA is more sensitive. All 21 cases of early stage CJD could be diagnosed using a combination of the 14-3-3γ ELISA and diffusion weighted MR imaging (DWI-MRI).ConclusionThe 14-3-3 gamma ELISA was more sensitive than conventional WB, and was useful for laboratory diagnosis of CJD, similar to the ELISA for the tau protein. Using DWI-MRI and these ELISA tests on CSF, diagnosis of CJD will be possible even at early stages of the disease.