Enhanced Creutzfeldt-Jakob disease surveillance in the older population: Assessment of a protocol for screening brain tissue donations for prion disease.

Human prion diseases, including Creutzfeldt-Jakob disease (CJD), occur in sporadic, genetic, and acquired forms. Variant Creutzfeldt-Jakob disease (vCJD) first reported in 1996 in the United Kingdom (UK), resulted from contamination of food with bovine spongiform encephalopathy. There is a concern that UK national surveillance mechanisms might miss some CJD cases (including vCJD), particularly in the older population where other neurodegenerative disorders are more prevalent. We developed a highly sensitive protocol for analysing autopsy brain tissue for the misfolded prion protein (PrPSc ) associated with prion disease, which could be used to screen for prion disease in the elderly. Brain tissue samples from 331 donors to the Edinburgh Brain and Tissue Bank (EBTB), from 2005 to 2022, were analysed, using immunohistochemical analysis on fixed tissue, and five biochemical tests on frozen specimens from six brain regions, based on different principles for detecting PrPSc . An algorithm was established for classifying the biochemical results. To test the effectiveness of the protocol, several neuropathologically confirmed prion disease controls, including vCJD, were included and blinded in the study cohort. On unblinding, all the positive control cases had been correctly identified. No other cases tested positive; our analysis uncovered no overlooked prion disease cases. Our algorithm for classifying cases was effective for handling anomalous biochemical results. An overall analysis suggested that a reduced biochemical protocol employing only three of the five tests on only two brain tissue regions gave sufficient sensitivity and specificity. We conclude that this protocol may be useful as a UK-wide screening programme for human prion disease in selected brains from autopsies in the elderly. Further improvements to the protocol were suggested by enhancements of the in vitro conversion assays made during the course of this study.

Temperature-Enhanced mcr-1 Colistin Resistance Gene Detection with Electrochemical Impedance Spectroscopy Biosensors.

Antibiotic resistance is now one of the biggest threats humankind is facing, as highlighted in a declaration by the General Assembly of the United Nations in 2016. In particular, the growing resistance rates of Gram-negative bacteria cause increasing concerns. The occurrence of the easily transferable, plasmid-encoded mcr-1 colistin resistance gene further worsened the situation, significantly enhancing the risk of the occurrence of pan-resistant bacteria. There is therefore a strong demand for new rapid molecular diagnostic tests for the detection of mcr-1 gene-associated colistin resistance. Electrochemical impedance spectroscopy (EIS) is a well-suited method for rapid antimicrobial resistance detection as it enables rapid, label-free target detection in a cost-efficient manner. Here, we describe the development of an EIS-based mcr-1 gene detection test, including the design of mcr-1-specific peptide nucleic acid probes and assay specificity optimization through temperature-controlled real-time kinetic EIS measurements. A new flow cell measurement setup enabled for the first time detailed real-time, kinetic temperature-controlled hybridization and dehybridization studies of EIS-based nucleic acid biosensors. The temperature-controlled EIS setup allowed single-nucleotide polymorphism discrimination. Target hybridization at 60 °C enhanced the perfect match/mismatch (PM/MM) discrimination ratio from 2.1 at room temperature to 3.4. A hybridization and washing temperature of 55 °C further increased the PM/MM discrimination ratio to 5.7 by diminishing the mismatch signal during the washing step while keeping the perfect match signal. This newly developed mcr-1 gene detection test enabled the direct, specific label, and amplification-free detection of mcr-1 gene harboring plasmids from Escherichia coli.

Susceptibility of Human Prion Protein to Conversion by Chronic Wasting Disease Prions.

Chronic wasting disease (CWD) is a contagious and fatal neurodegenerative disease and a serious animal health issue for deer and elk in North America. The identification of the first cases of CWD among free-ranging reindeer and moose in Europe brings back into focus the unresolved issue of whether CWD can be zoonotic like bovine spongiform encephalopathy. We used a cell-free seeded protein misfolding assay to determine whether CWD prions from elk, white-tailed deer, and reindeer in North America can convert the human prion protein to the disease-associated form. We found that prions can convert, but the efficiency of conversion is affected by polymorphic variation in the cervid and human prion protein genes. In view of the similarity of reindeer, elk, and white-tailed deer in North America to reindeer, red deer, and roe deer, respectively, in Europe, a more comprehensive and thorough assessment of the zoonotic potential of CWD might be warranted.