## 3 Results  
In the specificity panel for _tcdB_ RT-PCR assay only _C. difficile_ was positive for the _tcdB_ gene and no cross reactivity was observed with the other organisms in the specificity panel (Supplementary Table S1). The results from the sensitivity panel indicated that the limit of detection is \(10^{4}\) colony forming units (CFU)/mL and we established the Ct cutoff value of our assay at 35 (Supplementary Table S2).  
GPP testing and DFS RT-PCR were performed on 2641 clinical stool samples. Of the 2641 stool samples tested by GPP, 74 (2.8%) toxin-positive _C. difficile_ samples were detected based on the presence of the _tcdB_ target. Sixty-one (2.3%) samples were both GPP positive and DFS RT-PCR positive for _tcdB_, while 13 (0.5%) samples were GPP positive for _tcdB_ but were negative by DFS RT-PCR. Conversely, 2565 samples (97.2%) were both GPP negative and DFS RT-PCR negative for _tcdB_, and two samples (0.1%) were GPP negative for _tcdB_ but were tested positive for _tcdB_ by DFS RT-PCR (Figure 1). In total, fifteen (0.6%) of the samples tested yielded discordant results between the GPP and DFS RT-PCR. Discordance was more likely to be GPP positive/DFS RT-PCR negative than GPP negative/DFS RT-PCR positive.  
The agreement between DFS RT-PCR and GPP was excellent [k = 0.89, 95% CI (0.83, 0.94)]. The PPA between DFS RT-PCR and GPP was 96.8% [95% CI (89.0%, 99.6%)], while the NPA between DFS RT-PCR and GPP was 99.5% [95% CI (99.1%, 99.7%)]. McNemar analysis comparing the results of the GPP and DFS RT-PCR demonstrated that the difference between the two tests was significant (\(p\) = 0.0098).  
With the addition of a culture enrichment and repeat RT-PCR, twelve of the thirteen (92.3%) GPP positive/DFS RT-PCR negative discordant samples were resolved after enrichment by culture, while one sample (7.7%) remained negative (Table 2). Both of the discordant GPP negative/DFS RT-PCR positive samples tested positive following culture enrichment (Table 2 and Figure 1). After the discordant samples were resolved using culture-enriched RT-PCR, we determined that the analytical sensitivity and specificity of GPP was 97.3% (95% CI [90.7%, 99.7%] and 100% (95% CI [99.8%, 100%] and of DFS RT-PCR was 84.0% (95% CI [73.7%, 91.4%]) and 100% (95% CI [99.9%, 100%], respectively.  
Overall, PE RT-PCR Ct values among the discordant samples were improved as compared to the DFS RT-PCR Ct values before culture enrichment (Figure 2, Supplementary Table S3).  
Only discordant samples that produced Ct values in both DFS RT-PCR and PE RT-PCR were included (\(n\) = 13). The cut off Ct values of the RT-PCR assay is at 35. All samples Ct values showed an average decrease of 12 cycles following PE RT-PCR except sample 2\({}^{\blacklozenge}\) (dashed line) and sample 9\({}^{\blacklozenge}\) (dotted line), which showed minimal changes in the Ct values. These 2 samples were tested negative for GPP assay but positive for DFS RT-PCR before culture enrichment.  
There was no significant difference in terms of Ct values of DFS RT-PCR between 40 participants with acute gastroenteritis (median 27.6, interquartile range: 26.6-30.9) and the 22 without gastroenteritis (median 28.3, interquartile range: 25.2-31.6). The Ct values of DFS RT-PCR of the participants <2 years were significantly lower than those \(\geq\)2 years, (median 27.6 interquartile range: 26.2-30.9 and median 32.6, interquartile range: 31.8-33.4, respectively, \(p\) < 0.001). The frequency of concordance samples did not differ significantly between children age <2 and \(\geq\)2 years [99.3% (1597/1607) vs. 99.7% (1029/1032); \(p\) = 0.18].  
\begin{table}
\begin{tabular}{c c c c} \hline
**GPP** & **DFS RT-PCR** & **PE RT-PCR** & **No. Samples (\(n\) = 15)** \\ \hline Positive & Negative & Positive & 12 \\ Negative & Positive & Positive & 2 \\ Positive & Negative & Negative & 1 \\ \hline \end{tabular} PE RT-PCR refers to results for _tcdB_ after culture enrichment.  
\end{table}
Table 2: Discordant results for _tcdB_ between the GPP and DFS RT-PCR resolved by PE RT-PCR.