# Emerging Diagnostics in _Clostridioides difficile_ Infection
## 5 Ultra-Sensitive Immuonassays and Real-Time Analysis  
In an attempt to discern CDI from asymptomatic carriage, many laboratories often utilize the sensitivity of an NAAT in combination with the specificity of an EIA in a multistep algorithm [107]. However, multistep algorithms are expensive and are often out of reach in rural India and Brazil [87], where a single inexpensive test is often employed. However, the current requirements of high specificity and ultra-sensitivity suggest that only a few single-molecule counting platforms, such as the single-molecule array (SIMOA) and Singulex Clarity measurement systems [108], can achieve the required level of performance. In a recent study, the Singulex Clarity _C. diff_ toxin A/B assay measured TcA and TcdB in stool samples using the Singulex Clarity system. Upon evaluating 311 patient stool samples, the Singulex Clarity _C. diff_ toxin A/B assay yielded 97.7% sensitivity and 100% specificity in less than 40 min. Moreover, the automated assay exhibited a limit of detection (LOD) of 2.0 pg/mL (TcdA) and 0.7 pg/mL (TcdB), several orders of magnitude higher (800 to 2500 pg/mL) than current EIAs. Another molecular platform is the \"single-molecule array\" (SIMOA), which isolates and detects individual proteins on paramagnetic beads in femtoliter-sized wells, enabling more sensitive immunoassay than conventional EIAs. Research employing SIMOA demonstrated a limit of detection (LOD) of 0.6 and 2.9 pg/mL in pretreated fecal aliquots for toxins A and B, with an intra-assay coefficient of variation below 10% [109]. The platform also detected toxins in 24% more samples with laboratory-defined CDI than the high-performing toxin EIA (95% (63/66) versus 71% (47/66)). However, further research by the previous group indicated that toxin concentration alone was not definitive for symptomatic CDI using SIMOA, suggesting that a real-time approach may be necessary [110]. As highlighted by [PERSON] et al., a simmering concern regarding the applicability of these standalone ultra-sensitive tests is the preanalytical loss of toxin activity and the inability to distinguish colonized patients from infected ones [111]. A recent study by [PERSON] et al. set out to address this clinical dichotomy by comparing the performance of the Singulex Clarity (SC) _C. diff_ toxins A/B with a multistep algorithm using a GDH-and-toxin EIA and toxins A and B arbitrated by a semiquantitative CCNA [112]. Upon evaluation, the researchers reported that Clarity had 96.2% negative agreement with GDH\(-\)/toxin\(-\) samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with  
Figure 4: Recommended test algorithms for the diagnosis of CDI incorporating EIA, GDH, and NAAT tests; (+) positive, (\(-\)) negative. Modified with permission [36]. GDH+/toxin-/CCNA- samples. In conclusion, the authors suggested that the Clarity toxin assay may offer an accurate, standalone solution for CDI diagnostics. The real-time cell analysis (RTCA) system stands out as a label-free, real-time, and non-invasive technique with high sensitivity and specificity, and it has been employed in previous studies to quantify functional and toxical tcdB concentrations in fecal samples [113]. A recent study [114] using RTCA highlighted the significant differences in functional and toxical tcdB levels in the supernatant of _C. difficile_ colonized (CDC) and CDI group samples, even though the total levels of TcdB protein were similar, 490.00 \(\pm\) 133.29 ng/mL and 439.82 \(\pm\) 114.66 ng/mL. Interestingly, co-culturing tcdB with CDC samples resulted in a significant reduction in toxin concentration, suggesting the RTCA process may require an extra degree of refinement (increase in ultracentrifugation speeds, additional filtration) so that potential interferents such as bile acids, outer membrane vesicles (OMVs), cellular exosomes, and C-terminal combined repetitive oligopeptides (CROPs) are excluded from assay measurements [115].