Molecular characterisation of human prion amplification in cell-free systems and diagnostic applications

Prion diseases are a group of fatal neurodegenerative diseases associated with proteopathy occurring in humans and other mammals. The mechanism of prion replication is thought to be based on the induced misfolding of the host encoded prion protein and can be emulated in vitro by methodologies termed cell-free conversion (CFC) systems. Currently, the two most common implementation of CFC systems are the Protein Misfolding Cyclic Amplification (PMCA) reaction and the Real Time Quaking Induced Conversion (RT-QuIC) reaction. The overarching aim of this thesis is to describe and compare the human prion amplification in vitro by PMCA and RTQuIC and to extend the diagnostic applicability of the latter, which is currently employed for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). Therefore, in this thesis, PMCA and RT-QuIC differential amplification abilities were systematically investigated and, for the first time, PMCA and RT-QuIC reaction kinetics compared using a specifically defined set of conditions. In addition, the research performed in this thesis has shown that the sCJD cerebrospinal fluid and brain samples seed the conformational change of the full-length hamster recombinant prion protein in a similar way in RT-QuIC, and that the RT-QuIC analysis of sCJD urine samples is possible, however, it has low sensitivity and, therefore, a limited diagnostic potential

Concordance of cerebrospinal fluid real‐time quaking‐induced conversion across the European Creutzfeldt–Jakob Disease Surveillance Network

Background and purpose: Cerebrospinal fluid (CSF) real-time quaking-induced conversion (RT-QuIC) has a high degree of sensitivity and specificity for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) and this has led to its being included in revised European CJD Surveillance Network diagnostic criteria for sCJD. As CSF RT-QuIC becomes more widely established, it is crucial that the analytical performance of individual laboratories is consistent. The aim of this ring-trial was to ascertain the degree of concordance between European countries undertaking CSF RT-QuIC. Methods: Ten identical CSF samples, seven from probable or neuropathologically confirmed sCJD and three from non-CJD cases, were sent to 13 laboratories from 11 countries for RT-QuIC analysis. A range of instrumentation and different recombinant prion protein substrates were used. Each laboratory analysed the CSF samples blinded to the diagnosis and reported the results as positive or negative. Results: All 13 laboratories correctly identified five of the seven sCJD cases and the remaining two sCJD cases were identified by 92% of laboratories. Of the two sCJD cases that were not identified by all laboratories, one had a disease duration >26 months with a negative 14-3-3, whilst the remaining case had a 4-month disease duration and a positive 14-3-3. A single false positive CSF RT-QuIC result was observed in this study. Conclusions: This study shows that CSF RT-QuIC demonstrates an excellent concordance between centres, even when using a variety of instrumentation, recombinant prion protein substrates and CSF volumes. The adoption of CSF RT-QuIC by all CJD surveillance centres is recommended. Keywords: Creutzfeldt-Jakob disease; cerebrospinal fluid; prion; real-time quaking-induced conversion

Concordance of cerebrospinal fluid real‐time quaking‐induced conversion across the European Creutzfeldt–Jakob Disease Surveillance Network

Background: Cerebrospinal fluid (CSF) Real Time-Quaking Induced Conversion (RT-QuIC) has a high degree of sensitivity and specificity for the diagnosis of sporadic Creutzfeldt-Jakob Disease (sCJD) and this has led to it being included in revised European CJD Surveillance network diagnostic criteria for sCJD. As CSF RT-QuIC becomes more widely established, it is crucial that the analytical performance of individual laboratories is consistent. The aim of this ring-trial was to ascertain the degree of concordance between European countries undertaking CSF RT-QuIC. Methods: Ten identical CSF samples, seven from probable or neuropathologically confirmed sCJD and three from non-CJD cases, were sent to 13 laboratories from 11 countries for RT-QuIC analysis. A range of instrumentation and different recombinant prion protein substrates were used. Each laboratory analysed the CSF samples blinded to the diagnosis and reported the results as positive or negative. Results: All 13 laboratories correctly identified five of the seven sCJD cases and the remaining two sCJD cases were identified by 92% of laboratories. Of the two sCJD cases that were not identified by all laboratories; one had a disease duration greater than 26 months with a negative 14-3-3, whilst the remaining case had a 4 month disease duration and a positive 14-3-3. A single false positive CSF RT-QuIC result was observed in this study. Conclusions: This study shows that CSF RT-QuIC demonstrates an excellent concordance between centres, even when using a variety of instrumentation, rPrP substrates and CSF volumes. We recommend the adoption of CSF RT-QuIC by all CJD surveillance centres