Abstract
Aims: A diagnostic test for prion diseases that can reliably detect preclinical infection in easily accessible biological samples, such as blood, would be an invaluable tool to protect against potential spread of disease in humans and animals. Yet the development of such a test has remained challenging; partly due to the extremely low concentrations of disease-associated prion protein (PrPSc) present in blood during preclinical stages of infection.
To determine which existing technologies are best able to detect preclinical infection, we have exploited an extensive archive of preclinical blood samples from sheep experimentally infected with BSE (as a model of vCJD) to compare three in vitro tests for prion disease, including two based on the ‘protein misfolding cyclic amplification’ assay (PMCA), and one based on ‘real time quaking-induced conversion’ (RT-QuIC). By comparing the outcome of these tests with infectivity titres (as determined by bioassay) we also investigated whether in vitro seeding activity correlated with prion infectivity in blood.
Material and Methods: Three blinded panels, comprising a longitudinal series of n = 139 blood samples from BSE-infected sheep collected at regular time points throughout the course of infection (alongside appropriate negative controls) were independently tested using two previously established PMCA methods, ‘microplate-based PMCA’ (mb-PMCA) [1] and ‘plasminogen bead-capture PMCA’ (capture-PMCA) [2], and a novel RT-QuIC assay which incorporates capture of PrPSc from whole blood on iron oxide beads. A subset of blood samples represented in the panels was inoculated intracerebrally into transgenic (tgBov) mice overexpressing bovine PrP, to allow estimation of BSE infectivity titres.
Results: By comparing assay performance on equivalent blinded panels, we demonstrated that: (i) all three tests (mb-PMCA, capture-PMCA, RT-QuIC) showed 100% specificity, (ii) both PMCA assays were significantly more sensitive than RT-QuIC in detecting prion infected samples, (iii) the route of infection was a major factor influencing test results, as a substantially higher proportion of samples from intravenously infected sheep tested positive, compared to samples from orally infected animals. Furthermore, by comparing these data with bioassay results, we showed that in vitro seeding activity correlated with detectable prion infectivity in blood, and that infectivity titres were positively correlated with PrPSc concentration.
Conclusions: The in vitro tests evaluated in this study represent accurate and sensitive diagnostic tools for detection of preclinical prionemia. It is hoped that the outcome of this study will improve methods for early detection of prion diseases and guide policies to reduce the risk of prion transmission through blood donation.
To determine which existing technologies are best able to detect preclinical infection, we have exploited an extensive archive of preclinical blood samples from sheep experimentally infected with BSE (as a model of vCJD) to compare three in vitro tests for prion disease, including two based on the ‘protein misfolding cyclic amplification’ assay (PMCA), and one based on ‘real time quaking-induced conversion’ (RT-QuIC). By comparing the outcome of these tests with infectivity titres (as determined by bioassay) we also investigated whether in vitro seeding activity correlated with prion infectivity in blood.
Material and Methods: Three blinded panels, comprising a longitudinal series of n = 139 blood samples from BSE-infected sheep collected at regular time points throughout the course of infection (alongside appropriate negative controls) were independently tested using two previously established PMCA methods, ‘microplate-based PMCA’ (mb-PMCA) [1] and ‘plasminogen bead-capture PMCA’ (capture-PMCA) [2], and a novel RT-QuIC assay which incorporates capture of PrPSc from whole blood on iron oxide beads. A subset of blood samples represented in the panels was inoculated intracerebrally into transgenic (tgBov) mice overexpressing bovine PrP, to allow estimation of BSE infectivity titres.
Results: By comparing assay performance on equivalent blinded panels, we demonstrated that: (i) all three tests (mb-PMCA, capture-PMCA, RT-QuIC) showed 100% specificity, (ii) both PMCA assays were significantly more sensitive than RT-QuIC in detecting prion infected samples, (iii) the route of infection was a major factor influencing test results, as a substantially higher proportion of samples from intravenously infected sheep tested positive, compared to samples from orally infected animals. Furthermore, by comparing these data with bioassay results, we showed that in vitro seeding activity correlated with detectable prion infectivity in blood, and that infectivity titres were positively correlated with PrPSc concentration.
Conclusions: The in vitro tests evaluated in this study represent accurate and sensitive diagnostic tools for detection of preclinical prionemia. It is hoped that the outcome of this study will improve methods for early detection of prion diseases and guide policies to reduce the risk of prion transmission through blood donation.
| Original language | English |
|---|---|
| Pages | 235-236 |
| Number of pages | 1 |
| DOIs | |
| Publication status | Published - 13 Sept 2022 |
| Event | Prion 2022 - Georg-August University of Göttingen, Göttingen, Germany Duration: 13 Sept 2022 → 16 Sept 2022 https://prion2022.org/ |
Conference
| Conference | Prion 2022 |
|---|---|
| Country/Territory | Germany |
| City | Göttingen |
| Period | 13/09/22 → 16/09/22 |
| Internet address |