ArticlesDetection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay
Introduction
Prion disease encompasses various closely related and uniformly fatal neurodegenerative disorders affecting the CNS in human beings and animals. These disorders include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia and kuru in man, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease of deer and elk, and scrapie in sheep.1, 2 The emergence of variant CJD (vCJD) and the confirmation that it originates from exposure to BSE3 has raised a plethora of public health concerns affecting endoscopy, surgery, dentistry, organ transplantation, and blood transfusion.
Exposure of the UK population to BSE has been widespread, with more than 181 000 cases confirmed in cattle and estimates of total infections of 1–3 million.4 Although the number of clinical cases of vCJD has been fairly small, the number of infected individuals remains unclear.5 A retrospective study6 of archived surgical lymphoreticular specimens estimated a prevalence of infection in the UK population of 237 per million (95% CI 49–692 per million), which is far higher than the number of clinical cases of vCJD thus far. On the basis of this study, a prevalence estimate of one in 4000 was recommended by the Spongiform Encephalopathy Advisory Committee for management of public health risks.7, 8 Further investigations have not refined these estimates,9, 10 and a major caveat to these studies is the unknown sensitivity of the methods used to detect subclinical or preclinical cases of vCJD infection. The long incubation periods seen in natural human prion infections, which can exceed 50 years,11 and the existence of subclinical carrier states of prion infection in animal models12 offer a potential explanation for the discrepancy between estimates of infection prevalence and clinical cases.
Concern regarding secondary transmission of vCJD has had extensive effects on public health policy in the UK and elsewhere. Since the prevalence of preclinical or subclinical vCJD prion infection is poorly defined, the extent of future transfusion-transmission of vCJD cannot be accurately risk-assessed. Secondary infection from asymptomatic donors has already been confirmed in four recipients of blood transfusion.13, 14, 15 Although the number of transfusion recipients positively identified as having received packed red cells contaminated with vCJD is small, a much larger cohort of around 7000 recipients of contaminated plasma products have already been identified and notified of their at-risk status.16 Concern for this cohort has been heightened by post-mortem evidence of infection with vCJD prions in the spleen of an individual from this at-risk group with haemophilia.17
The infectious agents or prions responsible for transmission of disease are composed principally, if not entirely, of a misfolded form of host cellular prion protein (PrPC). This unique pathogenesis has posed major challenges to the development of diagnostic tests, which for infectious agents generally involves detection of a host humoral immune response or agent-specific nucleic acid, neither of which apply in vCJD. PrPC is expressed ubiquitously, although at highest concentrations in the CNS and cells of the immune system. When recruited during prion propagation, PrPC is remodelled to aggregated, detergent insoluble isoforms designated PrPSc, which are chemically identical but conformationally distinct from PrPC. Detection of PrPSc by the depletion of PrPC with proteinase K (PK) in CNS and lymphoreticular tissues generally correlates with the presence of prion infectivity18, 19 and remains wholly specific for prion disease. However, in recent years, researchers have found that additional PK-sensitive but disease-related abnormal isoforms of PrP might have an important role in prion disease pathogenesis,20, 21 and only 10% of abnormal PrP may be deposited in the form of PrPSc.22
Although the quantities of PrPSc deposited in neural tissues are sufficient during the symptomatic phase of illness for detection by conventional immunoassays such as western blotting and ELISA, concentrations in peripheral tissues are substantially lower.23 Quantification of infectious titre in rodent models has suggested that the levels of infectivity in blood are very low, with buffy coat fractions containing between 2 and 10 intracerebral LD50 units per mL during the asymptomatic phases of disease, rising to 100 LD50 units per mL during the clinical stage.24, 25
To successfully identify infection in blood, an assay has to be able to detect abnormal PrP in a range that is several orders of magnitude lower than the sensitivity of conventionally used immunoassays. Furthermore, the ratio of background PrPC (which is chemically identical to PrPSc) is higher in blood than in any other tissue, with the high lipid and protein content of blood potentially contributing to non-specific background signals.26 Conventionally, immunoassays for PrPSc have depended on protease pretreatment of tissues to degrade PrPC and other proteins, thereby reducing cross-reactivity.
The finding that prions can bind avidly to some surfaces, including metals,27 has been used to develop quantitative assays for infectivity28 that approach the high sensitivity needed to detect the low concentrations of prions and abnormal PrP associated with blood. We have adopted a similar approach for the capture and enrichment of abnormal PrP from whole blood using an optimised solid-state capture matrix derived from investigation of an extensive range of potential binding surfaces coupled with direct immunodetection of the surface-bound material. This approach avoids use of any proteolytic processing, ensuring that all abnormal PrP isoforms are available for detection. We aimed to establish the sensitivity and specificity of this test for detection of vCJD prion infection in blood.
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Setting
These studies were approved by the local research ethics committee of the UCL Institute of Neurology and the National Hospital for Neurology and Neurosurgery (NHNN; London, UK). Blood samples were obtained with informed consent from patients who were enrolled in the MRC PRION-1 trial29 or the National Prion Disease Monitoring Cohort study,30 or who were referred to the National Prion Clinic or Dementia Research Centre at the NHNN. Samples were collected in EDTA blood tubes and stored frozen at
Results
To establish the sensitivity of the assay compared with other methods, we analysed serial dilutions of vCJD brain homogenate diluted into whole blood. Exogenous spikes of vCJD-infected brain homogenate could clearly be distinguished from normal brain homogenate, even at a 1010-fold dilution (figure 1), a sensitivity more than four orders of magnitude higher than previously achieved for immunoassay of vCJD tissue.26 A chemiluminescent signal of mean 1·3×105 (SD 1·1×104) was obtained with 10−10
Discussion
Evidence shows that there is a risk of iatrogenic (secondary) vCJD prion infection from transfusion of blood and blood products13, 14, 15, 33 and, by inference, from many forms of surgical and dental interventions. Taken together with the existence of subclinical carrier states of prion disease12 and the long incubation periods for clinical disease,11 the implications for current and future public health could be substantial. Current risk reduction strategies in the UK, of uncertain efficacy or
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