RT-QuIC detection of chronic wasting disease prion in platelet samples of white-tailed deer.

BACKGROUND: Chronic wasting disease (CWD) is a prion disease of captive and free-ranging cervids. Currently, a definitive diagnosis of CWD relies on immunohistochemistry detection of PrPSc in the obex and retropharyngeal lymph node (RPLN) of the affected cervids. For high-throughput screening of CWD in wild cervids, RPLN samples are tested by ELISA followed by IHC confirmation of positive results. Recently, real-time quacking-induced conversion (RT-QuIC) has been used to detect CWD positivity in various types of samples. To develop a blood RT-QuIC assay suitable for CWD diagnosis, this study evaluated the assay sensitivity and specificity with and without ASR1-based preanalytical enrichment and NaI as the main ionic component in assay buffer. RESULTS: A total of 23 platelet samples derived from CWD-positive deer (ELISA + /IHC +) and 30 platelet samples from CWD-negative (ELISA-) deer were tested. The diagnostic sensitivity was 43.48% (NaCl), 65.22% (NaI), 60.87% (NaCl-ASR1) or 82.61% (NaI-ASR1). The diagnostic specificity was 96.67% (NaCl), 100% (NaI), 100% (NaCl-ASR1), or 96.67% (NaI-ASR1). The probability of detecting CWD prion in platelet samples derived from CWD-positive deer was 0.924 (95% CRI: 0.714, 0.989) under NaI-ASR1 experimental condition and 0.530 (95% CRI: 0.156, 0.890) under NaCl alone condition. The rate of amyloid formation (RFA) was greatest under the NaI-ASR1 condition at 10-2 (0.01491, 95% CRI: 0.00675, 0.03384) and 10-3 (0.00629, 95% CRI: 0.00283, 0.01410) sample dilution levels. CONCLUSIONS: Incorporation of ASR1-based preanalytical enrichment and NaI as the main ionic component significantly improved the sensitivity of CWD RT-QuIC on deer platelet samples. Blood test by the improved RT-QuIC assay may be used for antemortem and postmortem diagnosis of CWD.

A microfluidic biosensor for the diagnosis of chronic wasting disease.

Cervids are affected by a neurologic disease that is always fatal to individuals and has population effects. This disease is called chronic wasting disease (CWD) and is caused by a misfolded prion protein. The disease is transmitted via contact with contaminated body fluids and tissue or exposure to the environment, such as drinking water or food. Current CWD diagnosis depends on ELISA screening of cervid lymph nodes and subsequent immunohistochemistry (IHC) confirmation of ELISA-positive results. The disease has proven to be difficult to control in part because of sensitivity and specificity issues with the current test regimen. We have investigated an accurate, rapid, and low-cost microfluidic microelectromechanical system (MEMS) biosensing device for the detection of CWD pathologic prions in retropharyngeal lymph nodes (RLNs), which is the current standard type of CWD diagnostic sample. The device consists of three novel regions for concentrating, trapping, and detecting the prion. The detection region includes an array of electrodes coated with a monoclonal antibody against pathologic prions. The experimental conditions were optimized using an engineered prion control antigen. Testing could be completed in less than 1 hour with high sensitivity and selectivity. The biosensor detected the engineered prion antigen at a 1:24 dilution, while ELISA detected the same antigen at a 1:8 dilution. The relative limit of detection (rLOD) of the biosensor was a 1:1000 dilution of a known strong positive RLN sample, whereas ELISA showed a rLOD of 1:100 dilution. Thus, the biosensor was 10 times more sensitive than ELISA, which is the currently approved CWD diagnostic test. The biosensor's specificity and selectivity were confirmed using known negative RPLN samples, a negative control antibody (monoclonal antibody against bovine coronavirus BCV), and two negative control antigens (bluetongue virus and Epizootic hemorrhagic disease virus). The biosensor's ability to detect pathogenic prions was verified by testing proteinase-digested positive RLN samples.