# Emerging Diagnostics in _Clostridioides difficile_ Infection
## 4 Established and New Immunological and Enzyme Assays  
At the start of germination, multiple metal storage mechanisms at the host epithelial interface act to starve bacteria of nutrient metals in a process known as nutritional immunity [91]. As a countermeasure, the anaerbe releases a battery of toxins and digestive enzymes, the degree to which (depending on the ribotype) present in the host's or patient's stool. Detection of these proteins can be achieved through various methods, such as nucleic acid amplification tests (NAATs), glutamate dehydrogenase (GDH) enzyme immunoassay(EIAs) for toxins A and B and/or toxinogenic culture (TC), and a cell cytotoxicity neutralization assay (CCNA). Regarded as the gold standards for diagnosing CDI (_Clostridioides difficile_ infection), in the first stage of TC, toxinogenic isolates are identified via media such as modified CCFA (cyclo-serine-cefoxitin-fructose agar with biliary salts and egg yolks) and ChromID C. _difficile_ agar (bioMerieux, Grenoble, France). Studies show that ChromID yields a higher 24 h recovery (sensitivity, 92%) than CCFA (sensitivity, 22%) [92; 93; 94]. Another study confirmed that ChromID C. _difficile_ agar outperforms CCFA, cyclo-serine-cefoxitin-egg yolk agar, and tryptone soy agar with sheep blood [95]. In step two, isolates are re-cultured in broth, and the supernatant is filtered and added to a cell line culture. The cytopathic effect (CPE) is evaluated and neutralized by antitoxins. For CCNA, CPE is observed as cell rounding; some strains may induce protrusions, known as a sordelli-like CPE. If the CPE is reversed by antitoxins, the test is positive for _C. difficile_ toxins. Various cell lines have been used for toxin detection, including African green monkey kidney, McCoy, MRC-5, primary rhesus monkey kidney, and Vero cells [96]. Although TC and CCNA have demonstrated high sensitivities (90-95%), they are cumbersome, costly, and time-consuming, typically requiring 3-5 days to complete. In addition, the former test is subject to pre-analytic factors and user experience; thus, occasional clinical cases of CDI may be missed [97]. Faster response times can be achieved with cheaper EIA tests, which offer 96-98% specificities but have relatively low sensitivities of 52-75% for toxins A and/or B. Similar response times are possible with GDH tests, which exhibit widely varying sensitivities (50-99%) and specificities (70-100%) [98]. In a zoonotic study, GDH sensitivity (89.4%) values have been achieved with immunomodtoragraphic tests for _C. difficile_ in dog stool samples [99]. Currently, several commercial toxin enzyme immunoassay(toxin EIAs) such as ProSpecT Toxin A/B (Remel Products, Thermo Fisher Scientific, Lenexa, KS, USA) and _C. difficile_ Tox A/B II (TechLab Inc., Radford, VA, USA) as well as glutamate dehydrogenase (GDH) EIAs like _C. Diff_ Chek-60 and _C. Diff_ Quik Chek (TechLab Inc., Radford, VA, USA) are commercially available. These products generally have a low cost per test. While GDH EIAs offer high sensitivity of up to 90%, they cannot differentiate between CDI and asymptomatic colonization or the presence of non-toxic strains. An attempt to remedy the GDH diagnostic quandary was recently proposed by [PERSON] et al. [100], in which optimized diagnostic algorithms incorporating the cost-effectiveness of GDH with high specificity and increased sensitivity were evaluated. Using 39 TC-positive and 147 TC-negative samples, the researchers assessed the performance of two algorithms, GDH-AB and GDH-NAAT. The former algorithm showed good concordance with TC in detecting toxigenic _C. difficile_ (kappa = 0.82), while the sensitivity (48.7%) of the GDH-AB algorithm was too low to meet the demand for CDI diagnosis. Several improvements in assay design have sought to address the low sensitivity of GDH-AB and the inability of standalone tests to discern asymptomatic carriage from an active infection. Of note is the recent work by [PERSON] et al.