Detection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative disorder originating from exposure to bovine-spongiform-encephalopathy-like prions. Prion infections are associated with long and clinically silent incubations. The number of asymptomatic individuals with vCJD prion infection is unknown, posing risk to others via blood transfusion, blood products, organ or tissue grafts, and contaminated medical instruments. We aimed to establish the sensitivity and specificity of a blood-based assay for detection of vCJD prion infection. METHODS: We developed a solid-state binding matrix to capture and concentrate disease-associated prion proteins and coupled this method to direct immunodetection of surface-bound material. Quantitative assay sensitivity was assessed with a serial dilution series of 10⁻7 to 10⁻¹° of vCJD prion-infected brain homogenate into whole human blood, with a baseline control of normal human brain homogenate in whole blood (10⁻6). To establish the sensitivity and specificity of the assay for detection of endogenous vCJD, we analysed a masked panel of 190 whole blood samples from 21 patients with vCJD, 27 with sporadic CJD, 42 with other neurological diseases, and 100 normal controls. Samples were masked and numbered by individuals independent of the assay and analysis. Each sample was tested twice in independent assay runs; only samples that were reactive in both runs were scored as positive overall. FINDINGS: We were able to distinguish a 10⁻¹° dilution of exogenous vCJD prion-infected brain from a 10⁻6 dilution of normal brain (mean chemiluminescent signal, 1·3×105 [SD 1·1×104] for vCJD vs 9·9×104 [4·5×10³] for normal brain; p<0·0001)­an assay sensitivity that was orders of magnitude higher than any previously reported. 15 samples in the masked panel were scored as positive. All 15 samples were from patients with vCJD, showing an assay sensitivity for vCJD of 71·4% (95% CI 47·8­88·7) and a specificity of 100% (95% CIs between 97·8% and 100%). INTERPRETATION: These initial studies provide a prototype blood test for diagnosis of vCJD in symptomatic individuals, which could allow development of large-scale screening tests for asymptomatic vCJD prion infection. FUNDING: UK Medical Research Council.

Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces.

Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental advance was the cell culture-based scrapie cell assay, allowing prion titres to be estimated more precisely and an order of magnitude faster than by animal bioassays. Here we describe a bioassay method that exploits the marked binding affinity of prions to steel surfaces. Using steel wires as a concentrating and sensitization tool and combining with an adapted scrapie cell endpoint assay we can achieve, for mouse prions, a sensitivity 100x higher than that achieved in standard mouse bioassays. The rapidity and sensitivity of this assay offers a major advance over small animal bioassay in many aspects of prion research. In addition, its specific application in assay of metal-bound prions allows evaluation of novel prion decontamination methods.

A standardized comparison of commercially available prion decontamination reagents using the Standard Steel-Binding Assay.

Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of mammals, such as variant Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Prions pose significant public health concerns through contamination of blood products and surgical instruments, and can resist conventional hospital sterilization methods. Prion infectivity binds avidly to surgical steel and can efficiently transfer infectivity to a suitable host, and much research has been performed to achieve effective prion decontamination of metal surfaces. Here, we exploit the highly sensitive Standard Steel-Binding Assay (SSBA) to perform a direct comparison of a variety of commercially available decontamination reagents marketed for the removal of prions, alongside conventional sterilization methods. We demonstrate that the efficacy of marketed prion decontamination reagents is highly variable and that the SSBA is able to rapidly evaluate current and future decontamination reagents.

Preclinical detection of infectivity and disease-specific PrP in blood throughout the incubation period of prion disease.

Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative disorder characterised by accumulation of pathological isoforms of the prion protein, PrP. Although cases of clinical vCJD are rare, there is evidence there may be tens of thousands of infectious carriers in the United Kingdom alone. This raises concern about the potential for perpetuation of infection via medical procedures, in particular transfusion of contaminated blood products. Accurate biochemical detection of prion infection is crucial to mitigate risk and we have previously reported a blood assay for vCJD. This assay is sensitive for abnormal PrP conformers at the earliest stages of preclinical prion disease in mice and precedes the maximum infectious titre in blood. Not only does this support the possibility of screening asymptomatic individuals, it will also facilitate the elucidation of the complex relationship that exists between the ensemble of abnormal PrP conformers present in blood and the relationship to infectivity.

Population screening for variant Creutzfeldt-Jakob disease using a novel blood test: diagnostic accuracy and feasibility study.

IMPORTANCE: Our study indicates a prototype blood-based variant Creutzfeldt-Jakob disease (vCJD) assay has sufficient sensitivity and specificity to justify a large study comparing vCJD prevalence in the United Kingdom with a bovine spongiform encephalopathy-unexposed population. In a clinical diagnostic capacity, the assay's likelihood ratios dramatically change an individual's pretest disease odds to posttest probabilities and can confirm vCJD infection. OBJECTIVES: To determine the diagnostic accuracy of a prototype blood test for vCJD and hence its suitability for clinical use and for screening prion-exposed populations. DESIGN, SETTING, AND PARTICIPANTS: Retrospective, cross-sectional diagnostic study of blood samples from national blood collection and prion disease centers in the United States and United Kingdom. Anonymized samples were representative of the US blood donor population (n = 5000), healthy UK donors (n = 200), patients with nonprion neurodegenerative diseases (n = 352), patients in whom a prion disease diagnosis was likely (n = 105), and patients with confirmed vCJD (n = 10). MAIN OUTCOME AND MEASURE: Presence of vCJD infection determined by a prototype test (now in clinical diagnostic use) that captures, enriches, and detects disease-associated prion protein from whole blood using stainless steel powder. RESULTS: The assay's specificity among the presumed negative American donor samples was 100% (95% CI, 99.93%-100%) and was confirmed in a healthy UK cohort (100% specificity; 95% CI, 98.2%-100%). Of potentially cross-reactive blood samples from patients with nonprion neurodegenerative diseases, no samples tested positive (100% specificity; 95% CI, 98.9%-100%). Among National Prion Clinic referrals in whom a prion disease diagnosis was likely, 2 patients with sporadic CJD tested positive (98.1% specificity; 95% CI, 93.3%-99.8%). Finally, we reconfirmed but could not refine our previous sensitivity estimate in a small blind panel of samples from unaffected individuals and patients with vCJD (70% sensitivity; 95% CI, 34.8%-93.3%). CONCLUSIONS AND RELEVANCE: In conjunction with the assay's established high sensitivity (71.4%; 95% CI, 47.8%-88.7%), the extremely high specificity supports using the assay to screen for vCJD infection in prion-exposed populations. Additionally, the lack of cross-reactivity and false positives in a range of nonprion neurodegenerative diseases supports the use of the assay in patient diagnosis.