Prion disease detection, PMCA kinetics, and IgG in urine from sheep naturally/experimentally infected with scrapie and deer with preclinical/clinical chronic wasting disease.

Prion diseases, also known as transmissible spongiform encephalopathies, are fatal neurodegenerative disorders. Low levels of infectious agent and limited, infrequent success of disease transmissibility and PrP(Sc) detection have been reported with urine from experimentally infected clinical cervids and rodents. We report the detection of prion disease-associated seeding activity (PASA) in urine from naturally and orally infected sheep with clinical scrapie agent and orally infected preclinical and infected white-tailed deer with clinical chronic wasting disease (CWD). This is the first report on PASA detection of PrP(Sc) from the urine of naturally or preclinical prion-diseased ovine or cervids. Detection was achieved by using the surround optical fiber immunoassay (SOFIA) to measure the products of limited serial protein misfolding cyclic amplification (sPMCA). Conversion of PrP(C) to PrP(Sc) was not influenced by the presence of poly(A) during sPMCA or by the homogeneity of the PrP genotypes between the PrP(C) source and urine donor animals. Analysis of the sPMCA-SOFIA data resembled a linear, rather than an exponential, course. Compared to uninfected animals, there was a 2- to 4-log increase of proteinase K-sensitive, light chain immunoglobulin G (IgG) fragments in scrapie-infected sheep but not in infected CWD-infected deer. The higher-than-normal range of IgG levels found in the naturally and experimentally infected clinical scrapie-infected sheep were independent of their genotypes. Although analysis of urine samples throughout the course of infection would be necessary to determine the usefulness of altered IgG levels as a disease biomarker, detection of PrP(Sc) from PASA in urine points to its potential value for antemortem diagnosis of prion diseases.

A novel method for preclinical detection of PrPSc in blood.

In this study, we demonstrate that a moderate amount of protein misfolding cyclic amplification (PMCA) coupled to a novel surround optical fibre immunoassay (SOFIA) detection scheme can be used to detect the disease-associated form of the prion protein (PrP(Sc)) in protease-untreated plasma from preclinical and clinical scrapie sheep, and white-tailed deer with chronic wasting disease, following natural and experimental infection. PrP(Sc), resulting from a conformational change of the normal (cellular) form of prion protein (PrP(C)), is considered central to neuropathogenesis and serves as the only reliable molecular marker for prion disease diagnosis. While the highest levels of PrP(Sc) are present in the central nervous system, the development of a reasonable diagnostic assay requires the use of body fluids that characteristically contain exceedingly low levels of PrP(Sc). PrP(Sc) has been detected in the blood of sick animals by means of PMCA technology. However, repeated cycling over several days, which is necessary for PMCA of blood material, has been reported to result in decreased specificity (false positives). To generate an assay for PrP(Sc) in blood that is both highly sensitive and specific, we have utilized limited serial PMCA (sPMCA) with SOFIA. We did not find any enhancement of sPMCA with the addition of polyadenylic acid nor was it necessary to match the genotypes of the PrP(C) and PrP(Sc) sources for efficient amplification.

High occupancy increases the risk of early death or readmission after transfer from intensive care.

OBJECTIVE: To determine whether a lack of intensive care unit beds was leading to premature patient discharge from the intensive care unit and subsequent early readmission or death. DESIGN: Prospective cohort study. SETTING: A single Canadian tertiary care teaching hospital. PATIENTS: All intensive care unit admissions between January 1, 1989 and December 31, 1996 were collected prospectively for inclusion in a registry database. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: There was a positive correlation between early readmission or death and average quarterly intensive care unit percent occupancy (p = .001). During the study period, 8693 patients experienced 10,185 admissions to intensive care. Of the 8222 patients remaining under active treatment (patients under palliative care were excluded), there were 455 (5.5%) adverse events (431 intensive care unit readmissions and 24 deaths) in the first 7 days post intensive care unit discharge. Patients requiring a new surgical intervention with postoperative intensive care unit admission were not considered readmissions. In a multivariate analysis, significant risk factors for an adverse event included age >35 yrs, particular diagnoses (respiratory diagnoses, sepsis, neurosurgery, thoracic surgery, and gastrointestinal diagnoses), Acute Physiology and Chronic Health Evaluation II score, and intensive care unit length of stay. Discharge from the intensive care unit at a time of no vacancy was also a significant risk factor for intensive care unit readmission or unexpected death with an adjusted relative risk of 1.56 (95% confidence interval 1.05, 2.31). CONCLUSIONS: Increased patient occupancy within an intensive care unit is associated with an increased risk of early death or intensive care unit readmission post intensive care unit discharge. Overloading the capacity of an intensive care unit to care for critically ill patients may affect physician decision-making, resulting in premature discharge from the intensive care unit.

Surround optical fiber immunoassay (SOFIA): an ultra-sensitive assay for prion protein detection.

We describe the development of a new technology (SOFIA) and demonstrate its utility by establishing a sensitive and specific assay for PrP(Sc). SOFIA is a surround optical fiber immunoassay which is comprised of a set of specific monoclonal antibodies and comprehensive capture of high energy fluorescence emission. In its current format, this system is capable of detecting less than 10 attogram (ag) of hamster, sheep and deer recombinant PrP. Approximately 10 ag of PrP(Sc) from 263 K-infected hamster brains can be detected with similar lower limits of PrP(Sc) detection from the brains of scrapie-infected sheep and deer infected with chronic wasting disease. These detection limits allow protease treated and untreated material to be diluted beyond the point where PrP(C), non-specific proteins or other extraneous material may interfere with PrP(Sc) signal detection and/or specificity. This not only eliminates the issue of specificity of PrP(Sc) detection but also increases sensitivity since the possibility of partial PrP(Sc) proteolysis is no longer a concern. SOFIA will likely lead to early antemortem detection of transmissible encephalopathies and is also amenable for use with additional target amplification protocols. SOFIA represents a sensitive means for detecting specific proteins involved in disease pathogenesis and/or diagnosis that extends beyond the scope of the transmissible spongiform encephalopathies.

Simple and specific detection of abnormal prion protein by a magnetic bead-based immunoassay coupled with laser-induced fluorescence spectrofluorometry.

Transmissible spongiform encephalopathies (TSEs), also termed prion diseases, are fatal neurodegenerative conditions that affect both humans and animals. The transmissibility and fatal nature of TSEs necessitate their rapid and accurate diagnosis. Laser-induced fluorescence (LIF) spectrofluorometry is useful for obtaining measurements on fluorescence-labeled targets with a high degree of sensitivity. In the present study, we applied this technology to the immunological detection of abnormal prion protein, PrPSc, which is a universal diagnostic marker for TSEs. The assay format consists of a magnetic bead-based sandwich immunoassay utilizing a biotin-conjugated capture antibody and a fluorophore-labeled detector antibody. By using one pair of anti-PrP monoclonal antibodies (MAbs), PrPSc in brain homogenates from various experimental and natural TSEs can be easily detected with high specificity. Furthermore, the assay proved to be applicable for the detection of PrPSc in the lymph nodes from deer with TSE. The sensitivity of the assay was shown to be comparable to standard immunoblotting, but has several advantages over conventional tests, in terms of flexibility, simplicity, specificity, and run time. These results provide an important basis for the development of an early diagnostic test with potential for multi-sample analysis.