Optimization of the Real-Time Quaking-Induced Conversion Assay for Prion Disease Diagnosis

The real-time quaking-induced conversion (RT-QuIC) assay is a highly reproducible and robust methodology exhibiting an excellent pre-mortem diagnostic accuracy for prion diseases. However, the protocols might be time-consuming and improvement of the detection technology is needed. In the present study, we investigated the influence of a pre-analytical cerebrospinal fluid (CSF) treatment with proteinase K (PK) on the kinetic of the RT-QuIC signal response. For this purpose, we added PK at different concentrations in RT-QuIC reactions seeded with Creutzfeldt-Jakob disease (sCJD) CSF. We observed that a mild pre-analytical PK treatment of CSF samples resulted in an increased seeding efficiency of the RT-QuIC reaction. Quantitative seeding parameters, such as a higher area under the curve (AUC) value or a shorter lag phase indicated a higher conversion efficiency after treatment. The diagnostic accuracy resulting from 2 mu g/ml PK treatment was analyzed in a retrospective study, where we obtained a sensitivity of 89%. Additionally, we analyzed the agreement with the previously established standard RT-QuIC protocol without PK treatment in a prospective study. Here, we found an overall agreement of 94% to 96%. A Cohen's kappa of 0.9036 (95% CI: 0.8114-0.9958) indicates an almost perfect agreement between both protocols. In conclusion, the outcome of our study can be used for a further optimization of the RT-QuIC assay in particular for a reduction of the testing time

Effect of the micro-environment on alpha-synuclein conversion and implication in seeded conversion assays

Background: α-Synuclein is a small soluble protein, whose physiological function in the healthy brain is poorly understood. Intracellular inclusions of α-synuclein, referred to as Lewy bodies (LBs), are pathological hallmarks of α- synucleinopathies, such as Parkinson’s disease (PD) or dementia with Lewy bodies (DLB). Main body: Understanding of the molecular basis as well as the factors or conditions promoting α-synuclein misfolding and aggregation is an important step towards the comprehension of pathological mechanism of α- synucleinopathies and for the development of efficient therapeutic strategies. Based on the conversion and aggregation mechanism of α-synuclein, novel diagnostic tests, such as protein misfolding seeded conversion assays, e.g. the real-time quaking-induced conversion (RT-QuIC), had been developed. In diagnostics, α-synuclein RT-QuIC exhibits a specificity between 82 and 100% while the sensitivity varies between 70 and 100% among different laboratories. In addition, the α-synuclein RT-QuIC can be used to study the α-synuclein-seeding-characteristics of different α-synucleinopathies and to differentiate between DLB and PD. Conclusion: The variable diagnostic accuracy of current α-synuclein RT-QuIC occurs due to different protocols, cohorts and material etc.. An impact of micro-environmental factors on the α-synuclein aggregation and conversion process and the occurrence and detection of differential misfolded α-synuclein types or strains might underpin the clinical heterogeneity of α-synucleinopathies

Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt–jakob disease:An international study

Real-time quaking-induced conversion (RT-QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt\u2013Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT-QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160\u2013165

Studies of the self-propagating properties of the pathogenic prion protein in humans

Prionkrankheiten sind übertragbare, tödliche neurodegenerative Erkrankungen beim Menschen und bei Tieren. Sie basieren auf der Konversion des zellulären Prion-Proteins (PrPC) in seine pathogene Form (PrPSc). Durch diese Konversion sind Prionkrankheiten pathogen und übertragbar. Bis heute ist weder der dem zugrunde liegende Mechanismus verstanden noch eine Behandlung gefunden worden. Die sichere Diagnose einer sporadischen Prionkrankheit ist ausschließlich mittels Gehirnbiopsie möglich, weswegen zu Lebzeiten des Patienten häufig nur die Diagnose einer wahrscheinlichen Prionkrankheit erfolgt. Zusammen mit der klinischen Heterogenität der Prionkrankheiten weisen neueste Erkenntnisse auf das Vorhandensein mehrerer humaner Prionstämme hin. Für die Suche nach Medikamenten fehlt ein geeigneter Wirkstoff-Suchtest, der auf der humanen Pathogenese basiert, für einen hohen Durchsatz geeignet und gut reproduzierbar ist. Die real-time quaking-induced conversion (RT-QuIC), eine neu entwickelte in vitro-Methode, erlaubt den Nachweis von bisher nicht messbaren Mengen an PrPSc in humanem Liquor cerebrospinalis (Liquor). Dazu werden die selbst-replizierenden Eigenschaften des PrPSc genutzt. Erste Untersuchungen weisen auf distinkte Eigenschaften humaner Prionkrankheiten in der RT-QuIC hin. Zum Einsatz in Diagnostik und Forschung bedarf es jedoch einer umfassenden Validierung der Methode für die Anwendung mit humanem Liquor. In dieser Arbeit beträgt die Sensitivität der RT-QuIC 85,5 % und die Spezifität 99,5 % für humane Prionkrankheiten. Die Reproduzierbarkeit im Ringversuch ist gut bis exzellent. Die Kurzzeitlagerungen der Liquorproben bei Raumtemperatur und +4°C sowie die Langzeitlagerung bei −80°C und das wiederholte Einfrieren und Auftauen haben keinen Einfluss auf die Testergebnisse. Jedoch führt die Kontamination mit Blut zu falsch-negativen Resultaten. Diese Ergebnisse weisen auf eine Eignung der RT-QuIC zur sicheren Diagnose von Prionkrankheiten zu Lebzeiten der Patienten hin. Zur Charakterisierung des Reaktionspotentials möglicher humaner Prionstämme wurden Liquorproben von verschiedenen humanen Prionkrankheiten wie bspw. der sporadischen und der genetischen Form mittels RT-QuIC untersucht. Die Auswertung der Daten zeigt distinkte Eigenschaften des PrPSc im Liquor, die moduliert werden durch die Form der Prionkrankheit, den Prnp Codon 129-Genotyp und die Krankheitsdauer. Diese Ergebnisse zeigen das Potential der RT-QuIC, die selbst-replizierenden Eigenschaften des PrPSc im Liquor zu untersuchen, womit erstmals eine Methode zur Verfügung steht, um diese Effekte in Patienten während der symptomatischen Phase zu studieren. Zur Nutzung der RT-QuIC als neuartige Methode zur Wirkstoffsuche wurde die Wirkung mehrerer Stoffe auf die RT-QuIC-Reaktion untersucht. Doxyzyklin inhibiert diese Reaktion sowohl in Korrelation mit der Dosis als auch mit dem Zeitpunkt der Zugabe. Diese Ergebnisse weisen auf eine Eignung der RT-QuIC zur Suche von Stoffen hin, die den PrP-Konversionsprozess inhibieren und zeigen die inhibierende Wirkung von Doxyzyklin auf die in-vitro-Amplifikation von PrPSc

How To Build an Integrated Neighborhood Approach to Support Community-Dwelling Older People?

<p class="p1"><span class="s1"><strong>Background:</strong> Although the need for integrated neighborhood approaches (INAs) is widely recognized, we lack insight into strategies like INA. We describe diverse Dutch INA partners’ experiences to provide integrated person- and population-centered support to community-dwelling older people using an adapted version of Valentijn and colleagues’ integrated care model. Our main objective was to explore the experiences with INA participation. We sought to increase our understanding of the challenges facing these partners and identify factors facilitating and inhibiting integration within and among multiple levels.</span> <p class="p1"><span class="s1"><strong>Methods:</strong> Twenty-one interviews with INA partners (including local health and social care organizations, older people, municipal officers, and a health insurer) were conducted and subjected to latent content analysis. </span> <p class="p1"><span class="s1"><strong>Results:</strong> This study showed that integrated care and support provision through an INA is a complex, dynamic process requiring multilevel alignment of activities. The INA achieved integration at the personal, service, and professional levels only occasionally. Micro-level bottom-up initiatives were not aligned with top-down incentives, forcing community workers to establish integration 'despite' rather than because of meso- and macro-level contexts. </span> <p class="p1"><span class="s1"><strong>Conclusions:</strong> Top-down incentives should be better aligned with bottom-up initiatives. This study further demonstrated the importance of community-level engagement in integrated care and support provision.</span

To vaccinate or not to vaccinate? Perspectives on HPV vaccination among girls, boys, and parents in the Netherlands: a Q-methodological study

Abstract Background Despite the introduction of Human papillomavirus (HPV) vaccination in national immunization programs (NIPs), vaccination rates in most countries remain relatively low. An understanding of the reasons underlying decisions about whether to vaccinate is essential in order to promote wider spread of HPV vaccination. This is particularly important in relation to policies seeking to address shortfalls in current HPV campaigns. The aim of this study was to explore prevailing perspectives concerning HPV vaccination among girls, boys, and parents, and so to identify potential determinants of HPV vaccination decisions in these groups. Method Perspectives were explored using Q-methodology. Forty-seven girls, 39 boys, and 107 parents in the Netherlands were asked to rank a comprehensive set of 35 statements, assembled based on the health belief model (HBM), according to their agreement with them. By-person factor analysis was used to identify common patterns in these rankings, which were interpreted as perspectives on HPV vaccination. These perspectives were further interpreted and described using data collected with interviews and open-ended questions. Results The analysis revealed four perspectives: “prevention is better than cure,” “fear of unknown side effects,” “lack of information and awareness,” and “my body, my choice.” The first two perspectives and corresponding determinants of HPV vaccination decisions were coherent and distinct; the third and fourth perspectives were more ambiguous and, to some extent, incoherent, involving doubt and lack of awareness and information (perspective 3), and overconfidence (perspective 4). Conclusions Given the aim of publically funded vaccination programs to minimize the spread of HPV infection and HPV-related disease and the concerns about current uptake levels, our results indicate that focus should be placed on increasing awareness and knowledge, in particular among those in a modifiable phase

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Performed CSF tests and surveillance dat

Data from: Validation and utilization of amended diagnostic criteria in Creutzfeldt-Jakob Disease surveillance

Objective: To validate an amended protocol for clinical diagnosis of sporadic Creutzfeldt-Jakob Disease (sCJD) including Real-Time Quaking-induced Conversion (RT-QuIC) and to observe its utilization in CJD surveillance. Methods: In the framework of a prospective epidemiological study, all neuropathological confirmed sCJD cases that received CSF RT-QuIC analysis during diagnostic work up (n=65) and a control group of non- CJD cases (n=118) were selected to investigate the accuracy of an amended diagnostic protocol. The patients had been referred to the German National Reference Center for Transmissible Spongiform Encephalopathies. The influence of the amended protocol on incidence figures was evaluated in the context of three years of surveillance activity (screened cases using 14-3-3 test: n=18.789, highly suspicious cases of CJD: n=704). Annual incidences were calculated using current criteria and the amended protocol. Results: The amended protocol showed a sensitivity of 97% and a specificity of 99%. When it was applied to all suspected cases that were referred to the reference center, the assessed incidence of CJD increased from 1.7 to 2.2 per million in 2016. Conclusion: CJD surveillance remains challenging as information from external healthcare institutions can be limited. RT-QuIC shows excellent diagnostic accuracy when applied in the clinical setting to symptomatic patients. Data for RT-QuIC alone, when applied as a general screening test, are not available yet. We propose an amended research protocol which improves early and accurate clinical diagnosis of sCJD during surveillance activities. The utilization of this protocol will probably lead to a significant increase of the incidence rate

Stability and Reproducibility Underscore Utility of RT-QuIC for Diagnosis of Creutzfeldt-Jakob Disease.

Real-time quaking-induced conversion (RT-QuIC) allows the amplification of miniscule amounts of scrapie prion protein (PrP(Sc)). Recent studies applied the RT-QuIC methodology to cerebrospinal fluid (CSF) for diagnosing human prion diseases. However, to date, there has not been a formal multi-centre assessment of the reproducibility, validity and stability of RT-QuIC in this context, an indispensable step for establishment as a diagnostic test in clinical practice. In the present study, we analysed CSF from 110 prion disease patients and 400 control patients using the RT-QuIC method under various conditions. In addition, "blinded" ring trials between different participating sites were performed to estimate reproducibility. Using the previously established cut-off of 10,000 relative fluorescence units (rfu), we obtained a sensitivity of 85 % and a specificity of 99 %. The multi-centre inter-laboratory reproducibility of RT-QuIC revealed a Fleiss' kappa value of 0.83 (95 % CI: 0.40-1.00) indicating an almost perfect agreement. Moreover, we investigated the impact of short-term CSF storage at different temperatures, long-term storage, repeated freezing and thawing cycles and the contamination of CSF with blood on the RT-QuIC seeding response. Our data indicated that the PrP(Sc) seed in CSF is stable to any type of storage condition but sensitive to contaminations with blood (>1250 erythrocytes/μL), which results in a false negative RT-QuIC response. Fresh blood-contaminated samples (3 days) can be rescued by removal of erythrocytes. The present study underlines the reproducibility and high stability of RT-QuIC across various CSF storage conditions with a remarkable sensitivity and specificity, suggesting RT-QuIC as an innovative and robust diagnostic method.peerReviewe