Standardization of Data Analysis for RT-QuIC-Based Detection of Chronic Wasting Disease

Abstract

Chronic wasting disease (CWD) is a disease affecting cervids and is caused by prions accumulating as pathogenic fibrils in lymphoid tissue and the central nervous system. Approaches for detecting CWD prions historically relied on antibody-based assays. However, recent advancements in protein amplification technology provided the foundation for a new class of CWD diagnostic tools. In particular, real-time quaking-induced conversion (RT-QuIC) has rapidly become a feasible option for CWD diagnosis. Despite its increased usage for CWD-focused research, there lacks a consensus regarding the interpretation of RT-QuIC data for diagnostic purposes. It is imperative then to identify a standardized and replicable method for determining CWD status from RT-QuIC data. Here, we assessed variables that could impact RT-QuIC results and explored the use of maxpoint ratios (maximumRFU/backgroundRFU) to improve the consistency of RT-QuIC analysis. We examined a variety of statistical analyses to retrospectively analyze CWD status based on RT-QuIC and ELISA results from 668 white-tailed deer lymph nodes. Our results revealed an MPR threshold of 2.0 for determining the rate of amyloid formation, and MPR analysis showed excellent agreement with independent ELISA results. These findings suggest that the use of MPR is a statistically viable option for normalizing between RT-QuIC experiments and defining CWD status.

Optimization of RT-QuIC Assay Duration for Screening Chronic Wasting Disease in White-Tailed Deer

“…Nonetheless, our findings demonstrated the effectiveness of using ROC and AUC analyses for optimizing RT-QuIC assay durations in screening CWD as an alternative to ELISA. As many factors may affect RT-QuIC performance [ 9 , 27 , 28 ], it is expected that the optimal assay duration may vary for different sample matrixes, with different substrates and reagents, and using different fluorometers. In addition, with ROC curves, there are multiple methods to interpret the optimal threshold cut-offs, such as the Youden index and the point closest to the (0,1) method [ 31 ].…”

“…Determination of optimal assay duration time for T MPR was based on the same ThT data from the above RT-QuIC reactions that were seeded with the control CWD+ and CWD− brain and RLN tissue homogenates. MPR was defined as the ratio of maximum RFU to background (4th cycle) RFU by Rowden et al [ 9 ]. In this study, MPR was calculated for each cycle using the maximum accumulated RFU, from the 4th cycle (52 min of the assay) to the 224th cycle (65 h), using the maximum accumulated RFU within the corresponding cycles, and thus, 221 MPRs were calculated from each reaction.…”

“…The classification of specimens tested by RT-QuIC was carried out using the Mann–Whitney U-test and the probability test using T stdev [ 16 ], as well as the Welch’s t -test and the probability test using T MPR [ 9 ]. Cycle threshold or MPR from quadruplicate RT-QuIC reactions on each tissue specimens were compared with the negative controls with the Mann–Whitney U-test and Welch’s t -test using R. For the probability test, tissue specimens were classified as negative if no replicates surpassed the threshold, positive if all 4 replicates surpassed the threshold, and suspect if at least 1 out of 4 replicates surpassed the threshold.…”

“…Within the past decade, real-time quaking-induced conversion (RT-QuIC) assays have become common and have shown potential to be used for the routine detection of CWD in cervids [ 9 ]. Many RT-QuIC test protocols have been developed and used for the detection of PrP CWD in a range of biological tissues and environmental samples [ 5 , 6 , 7 , 10 , 11 , 12 ].…”

“…Additionally, the Mann–Whitney U-test has been used to determine CWD positivity by comparing the cycle thresholds (Ct) or rates (the reciprocals of cycle thresholds) of replicate reactions of a specimen with those of the negative controls [ 6 ]. Recently, the use of max-point ratios (the maximum fluorescence/background fluorescence, MPR) has been proposed to improve the consistency of RT-QuIC analyses [ 9 , 12 ]. With the MPR, specimens were classified as CWD-positive using Welch’s analysis of variance (ANOVA) by comparing the MPR values of unknown specimens against those of a known negative control.…”