Title: Comparative evaluation of the performance of proposed diagnostic tests for vCJD in preclinical blood samples.
Funder: Department of Health and Social Care

Abstract: Background vCJD was first reported in the UK in 1996, and since then, 178 deaths from probable or definite vCJD have occurred, with three cases related to transfusion of contaminated blood. A number of risk reduction measures aimed at prevention of human-to-human transmission have been implemented, including leucoreduction of labile blood products, importation of plasma, blood donor deferral, and limitations on re-use of surgical instruments. The necessity for many of these precautionary measures would be removed if there was a sensitive and specific blood test that could reliably identify infected individuals during the long asymptomatic incubation period before clinical signs develop. Aims Recently, a number of sensitive methods for the detection of PrPSc (a specific marker of prion diseases such as vCJD) have been applied to blood, and other body fluids. These include methods to amplify PrPSc, such as protein misfolding cyclic amplification (PMCA) and real-time quaking induced conversion (RT-QuIC), or to selectively concentrate PrPSc, as in the direct detection assay (DDA). Some of these assays have shown high levels of sensitivity and specificity when tested on blood samples from patients with vCJD. However, these tests would be most useful if they could also reliably detect preclinical infection, but their ability to do so is difficult to assess due to the very limited numbers of preclinical blood samples available from preclinical vCJD patients. The aim of this study is to compare the performance of PMCA, DDA and RT-QuIC assays in detecting preclinical infection, using an archive of blood samples collected from sheep experimentally infected with BSE. Research plan Blood samples that will be tested in this study were collected from BSE-infected sheep used in a previous DH-funded project as a model to study the risks of transmission of vCJD by transfusion of labile blood components. We have longitudinal series of blood samples collected from a total of 50 sheep infected by oral or intravenous (transfusion) routes, at regular intervals from pre-infection until the development of clinical disease (up to 17 time points per animal). Methods developed for detection of PrPSc in human blood samples (PMCA, DDA, RT-QuIC) will be adapted and optimised, where necessary, for use in BSE-infected sheep. These methods will then be applied to identical panels of sheep blood samples from early, middle and late preclinical time points to compare their sensitivity at all stages of the infection, and the degree of inter-indivdual variation in results. The results of the assays will be correlated with data on infectivity titres in blood at the same time points, which will be measured using a sensitive transgenic mouse bioassay. Research team The lead applicant pioneered the use of sheep as a model for studying blood-borne transmission of prions, and is responsible for the previous DH sheep blood transfusion project (007/0162), which generated the sample archive proposed for use in this study. The team is further strengthened by collaborations with world-leading prion researchers, including Olivier Andreoletti (expertise in blood-borne prion infectivity and PMCA), Daisy Bougard (development of PMCA as a blood-based diagnostic assay for vCJD) and Alison Green (development of RT-QuIC as a diagnostic assay for human prion diseases). Potential impact The results of this research would inform the choice of test to be applied to human blood samples, e.g. in prevalence studies, or screening of donated blood. In future, new tests could also be evaluated on the same sample panels for comparison. It will also provide data on how positive test results correlate with the ability to transmit infection by blood transfusion, which could help to refibe current risk assessment models used as an aid to public health policy decisions.

Lay abstract: This project aims to improve methods for diagnosis and control of infectious brain diseases, known as prion diseases. Prion diseases include variant Creutzfeld-Jakob disease (vCJD) in humans, which resulted from eating meat from cattle affected with bovine spongiform encephalopathy (BSE). Individuals infected with prions can remain healthy for years before developing signs/symptoms of brain disease. They may even remain disease-free during their natural lifespan. In the human population they pose a potential risk to others through blood and organ donation. There have been several vCJD cases attributed to known exposure to infected blood products. There are currently no tests that can easily detect individuals with these &ldquo;silent&rdquo; infections, known as preclinical or subclinical infections. However, new blood/urine tests have recently been developed, which can accurately identify patients who have symptoms of vCJD. These tests would be much more useful if they could also detect preclinical infection, but there are not enough suitable human samples to test them on. We have previously used sheep infected experimentally with BSE as a model to study the risk of transmitting prion infection by transfusion of blood components that are commonly used in humans, such as red cells and plasma. These studies were done because sheep infected with BSE show many similarities to human vCJD patients. We collected and froze blood samples from infected sheep at regular intervals from the time they were infected until they developed signs of brain disease several years later. The aim of our research project is to use these samples to compare three different blood tests for vCJD and find the one that is best able to detect preclinical infection. This would help to decide which test should be taken forward for use in humans, for example, to test donated blood for vCJD.

Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: NEUROLOGICAL