## 4 Discussion  
Our in-house RT-PCR assay targeting the _tcdB_ gene has a sensitivity of 10\({}^{4}\) CFU/mL (Supplementary Table S2) and showed no cross reactivity with any of the organisms included in the specificity panel (Supplementary Table S1). The correlation between DFS RT-PCR and GPP was excellent [k = 0.89, 95%CI (0.83, 0.94)]. By using DFS RT-PCR for comparison with GPP results, we found high positive and high negative agreement between the two tests for toxigenic _C. difficile_ detection. Our findings of a PPA of 97% and NPA of 99% align with previous studies comparing percent agreements for detection of _C. difficile_ toxins when conventional microbiological methods were used as reference standards for comparison with the GPP [23]. Despite the high agreements between the two assays, there was a significant difference in the results obtained by DFS RT-PCR and the GPP. Discordance between the tests was primarily observed in samples that tested GPP positive but DFS RT-PCR negative. Overall, 15 discordant samples (Figure 1) were identified, and these were alcohol shocked and enriched in C Diff Banana Broth\({}^{\text{TM}}\) to determine if the discordance observed was potentially due to differences in bacterial load. The results generated from the extracted DNA as template for PE RT-PCR showed an average difference of 12 cycles with lower Ct value using PE RT-PCR, suggesting an increase in target concentration of almost 4000-fold (Figure 2 and Supplementary Table S3). These findings supported that the alcohol shock followed by broth enrichment enhanced the culture of _C. difficile_[24; 25] resulting in the decrease of Ct values in the RT-PCR assay as compared with using stool samples directly (Figure 2). However, two samples did not follow the overall trend. The Ct value of Sample 9 tested using PE RT-PCR was similar to the Ct value of DFS RT-PCR after the alcohol shock enrichment treatment, whereas, for Sample 2, the PE RT-PCR Ct value was higher than the DFS RT-PCR by 2 (Ct 24.96 to Ct 27.11) (Figure 2 and Supplementary Table S3). This indicated that there was likely minimal effect of the alcohol shock enrichment on these two stool samples. Interestingly, both Sample 2 and Sample 9 tested negative by GPP and positive by DFS RT-PCR. These results further support the assumption that both results are true positive even though there were very minor differences in the RT-PCR results with direct-from-stool and enriched culture post alcohol shock treatment. As for Sample 15 which was GPP positive/DFS RT-PCR negative, it still tested negative by PE RT-PCR suggesting that it was a false positive by the GPP assay. Alternatively, this discordance between the GPP and DFS RT-PCR could be the result of sampling error used for the  
Figure 2: Comparison of Ct values for _tcdB_ amplification from DFS and PE of the same samples. shock enrichment or contamination during the GPP run [26]. Interestingly, the rate of false positivity of GPP we detected in our study (1/2641) was much lower than that of the false negative results generated by DFS RT-PCR (12/2641) suggesting that false positivity is less of an issue for the GPP assay targeting _C. difficile_. In this study, nucleic acid degradation during storage between testing by the GPP and DFS RT-PCR assays might have contributed to the discordance between the two assays. All the nucleic acid extracts were stored at -70 \({}^{\circ}\)C but unfortunately, they were not aliquoted as \"one-time-use\" samples and were subjected to freeze-thawed cycles for different testing. The freeze-thawed cycles may have affected the integrity of the nucleic acid and resulted in false negatives in the RT-PCR results. Based on the level of detection of the RT-PCR, the cutoff Ct value of the assay is at 35, and all the discordant samples except for samples 2 and 9, are considered as DFS RT-PCR negative/indeterminate as shown in Supplementary Table S3. However, the sensitivity of the in-house RT-PCR assay did improve after the alcohol shock-enrichment treatments.