Abstract
We wish to address several methodologic and interpretive concerns with JVDI readership regarding the recent paper: Ralston BA, et al., “Comparison of the detection of the chronic wasting disease prion by real-time quaking-induced conversion (rtQuIC) and silica nanoparticle–rtQuIC (nano-rtQuIC) assays in retropharyngeal lymph nodes of white-tailed deer, mule deer, and Rocky Mountain elk” (JVDI 2026;38(2):174–183). 5
This study builds on a collective body of work to expand testing for chronic wasting disease (CWD) using rtQuIC. We strongly support continued investigation into rtQuIC assays and their derivatives, as this is essential if we are to integrate these tools into our diagnostic toolkits for managing CWD. Our concern is that some of the methodologic choices and presentation of the work in the article may diminish the value of rtQuIC as an effective diagnostic tool for CWD. Thus, in the spirit of collaborative advancement, we identify those methodologic concerns in this letter. These nuances are critical for readers to consider, given that they may influence the perceived sensitivity of these assays and inform future test standardization.
First, direct comparison of this study’s technique with those of previously published nano-rtQuIC studies1,2 (termed Nano-QuIC in previous studies) is complicated by differences in sample preparation and assay conditions. In the Ralston study, 5 lymph node homogenates were centrifuged, and supernatant was used as seed in the assays, whereas previous studies used whole homogenate. This difference may alter prion concentration and assay performance. Additionally, differences in key assay parameters—such as incubation temperature and run duration—and approaches to account for background fluorescence inherent to nanoparticle-based assays would also be expected to influence outcomes. For instance, the addition of nanoparticles substantially affects baseline fluorescence, and consistent treatment of background signal is critical for achieving standardized results.
Second, regarding assay parameters, within the investigators’ own experiments, they found their rtQuIC protocol was sensitive to incubation temperature. Although most of their experiments were performed at 50°C for 24 h, an additional experiment using white-tailed deer ELISA-positive retropharyngeal lymph node samples was conducted at 44°C for 36 h under standard rtQuIC conditions. This experiment demonstrated 100% agreement with ELISA results, compared with 80% agreement under the 50°C/24 h conditions. Given this improvement, it would have been valuable to evaluate the 44°C/36 h condition across all species and samples for a more complete comparison of assay performance, particularly as lower incubating temperatures (often 42°C) are more commonly used for detecting CWD prions in lymph tissues by rtQuIC.3,6 –8 This key finding suggests that the reported lower sensitivity may be the result of the chosen protocol parameters, rather than a limitation of the assay itself, especially given that previous studies have demonstrated equal or higher sensitivity of rtQuIC at 42°C compared with ELISA.3,6 –8
Third, the authors used different numbers of technical replicates for the 2 assays: rtQuIC reactions were performed in quadruplicate wells whereas nano-rtQuIC reactions were performed in triplicate. Because positivity was defined as >50% of wells exceeding the RFU threshold, this difference could potentially produce discordant classifications between assays (e.g., 2/4 wells = negative versus 2/3 wells = positive), even when underlying signals are similar. We do not believe this strongly affected the results in this study but it is a methodologic consideration.
Finally, we note 2 issues related to statistical analysis. Receiver operating characteristic (ROC) analysis was conducted using individual-well maximum-slope values, which assumes independence among observations. However, wells originating from the same sample, which it appears the investigators used, are not fully independent, potentially influencing the ROC threshold estimation. 4 We do not believe this strongly influenced results, but it is an important statistical limitation to consider. In addition and more importantly, sensitivity and specificity estimates were reported without 95% CIs. Without CIs, assessing the precision of these estimates or determining if the differences between assays are significant is difficult.
We offer these considerations to support the authors’ valuable work and to ensure clarity for the scientific and regulatory communities in accurately interpreting the data regarding rtQuIC and nano-rtQuIC assay performance.
