# Emerging Diagnostics in _Clostridioides difficile_ Infection
## 5 Ultra-Sensitive Immuonassays and Real-Time Analysis
In order to identify the suspected interferents, the authors recommended conducting a study on a comparative proteome and metabolomics. Additional efforts to overcome the limitations (inherent matrix-associated cytotoxicity (MAC) and subjective interpretation of cytopathic effects of classical CCNA), resulting in improvements in the diagnostic accuracy of CDI, were recently addressed by [PERSON] et al. [116] in which an automated version of CCNA with a luminescent readout was used to analyze clinically relevant stool samples for infection. In the first instance, a viability threshold was established by determining a viability/fold that offered 98% specificity with 99.98% confidence to discern the effects of toxin B from those generated by MAC on Vero cells. With the automated assay, positive cut-offs using diarral samples from donors classified as CDI-negative demonstrated a minimum of 98% assay specificity. In conclusion, the authors suggested that automated CCNA should be combined with a highly sensitive molecular test and employed as either a prescreen or confirmatory test, providing a distinct advantage in detecting early infections or mild cases where the pathogen load is below the detection threshold of standard tests [117]. A selection of traditional and emerging detection methods is shown in Table 1.  
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Test Method** & **Type** & **Sensitivity** & **Specificity** & **Time to Result** & **Cost** & **Comments** \\ \hline PCR & Molecular & 80–100\% & 87–99\% & Hours & High & \begin{tabular}{c} Detects toxin genes, may \\ over diagnose due to \\ asymptomatic carriage. \\ Detects toxins A and/or B, \\ low sensitivity. \\ \end{tabular}  \\ EIA & Immunological & 52–75\% & 96–98\% & Hours & Low & \begin{tabular}{c} Detects toxin genes, may \\ asymptomatic carriage. \\ Detects toxins A and/or B, \\ low sensitivity. \\ \end{tabular}  \\ GDH-EIA & Enzymatic & 50–99\% & 70–100\% & Hours & Low & \begin{tabular}{c} Detects toxin genes, may \\ asymptomatic carriage. \\ Detects toxins A and/or B, \\ low sensitivity. \\ \end{tabular}  \\ TC & Microbiological & 90–95\% & 90–95\% & 3–5 Days & High & \begin{tabular}{c} Detects toxin genes, may \\ over diagnose due to \\ asymptomatic carriage. \\ Detects toxins A and/or B, \\ low sensitivity. \\ \end{tabular}  \\ CCNA & Cell-based & 90–95\% & 95–98\% & 24–48 h & High & \begin{tabular}{c} Gold standard, \\ detection, time-consuming. \\ Rapid identification of \\ strains, effective for \\ epidemiological studies. \\ High sensitivity, simpler \\ than PCR, suitable for \\ point-of-care testing. \\ Automated, detects \\ individual molecules, high \\ performance in identifying \\ true CDI. \\ Automated, high specificity \\ and sensitivity, suitable for \\ standalone CDI diagnostics. \\ \end{tabular}  \\ MALDI-TOF & Mass & 90–100\% & 90–100\% & Minutes & Medium & \begin{tabular}{c} Detects toxin genes, may \\ over diagnose due to \\ asymptomatic carriage. \\ Detects toxins A and/or B, \\ low sensitivity. \\ \end{tabular}  \\ LAMP & Molecular & 90–100\% & 94.5–99.1\% & \begin{tabular}{c} Minutes \\ 0-100\% \\ 0-100\% \\ 0-100\% \\ 0-100\% \\ \end{tabular}  \\ SIMOA & Molecular & 97.7\% (tcdA) & 100\% & \begin{tabular}{c} Minutes \\ to Hours \\ 0-100\% \\ 0-100\% \\ \end{tabular}  \\  & \ \begin{tabular}{c} High \\ 100\% \\ 0-100\% \\ \end{tabular}  \\  &
\begin{tabular}{c} High \\ 100\% \\ 100\%Clearly, the cost of USI may limit its broader application to research settings and clinical trials involving immunocompromised and immunosenescent cohorts where low levels of toxin can be significant, particularly in periods of endemicity, where high-throughput screening is required [118; 119; 120]. Moreover, automated USI could also enhance elderly monitoring (care settings) by accounting for atypical symptoms and comorbidities [121], thereby further reducing the risk of CDI transmission and overdiagnosis.