# Emerging Diagnostics in _Clostridioides difficile_ Infection
## 2 Identification of Resistant and Viable Spores
used a modified version of RS, namely laser tweezer Raman spectroscopy (LTRS), to examine the resilience of three strains (DS1813, CD630, R20291) to sodium hypochlorite. LTRS generates a spectral fingerprint of the trapped object, enabling the detection of chemical differences between treated and untreated spores and distinctions within vegetative cells. A key indicator of internal spore damage is a loss of structural integrity, which presents a reduction in the intensity of the CaDPA Raman peaks. Upon exposure to 0.5% hypochlorite (aq) (hospital surface decontamination requirements, 4 log reduction), CD630 and R20291 exhibited 2.2 \(\pm\) 0.1 and 0.8 \(\pm\) 0.1 log reductions compared to controls. The researchers attributed the loss in sensitivity to mechanistic differences and reduced surface area (spore aggregation). Such a loss in R20291 sensitivity was unexpected, with authors citing the overuse of chlorine-releasing agents (CRAs) during the pandemic as a potential cause worthy of future investigation. DPA leakage and its chelating potential can also be assessed using turbidity, absorbance, scattering, and fluorescence spectroscopic measurements [47; 48; 49]. For example, surface-enhanced Raman scattering (SERS) was recently combined with functionalized silver-terbium nanoparticles specific to DPA in the simple and sensitive detection of spores [50]. Another approach employed the enhanced luminescence from a terbium-lanthanide-fluorescent polyfluorene (PFO) semiconductor complex upon exposure to CaDPA, exhibiting a detectable concentration range of 2.5 to 25 nM for the chelating agent [51]. An alternative trivalent ion to terbium is europium, which was recently incorporated with a metal-organic gel (MOG), producing an enhanced electrochemiluminescence (ECL) signal upon exposure to DPA with a detection limit of 7.35 nm [52]. Terbium and europium are expensive; thus, divalent substitutes like magnesium and copper are also employed. Recent work using a catechol-substituted monostyryl boradiazainadecene (BODIPY) by [PERSON] et al. [53] to sequentially detect Cu\({}^{2+}\) ions and dipicolinic acid (DPA) permitted the detection of low concentrations of spores 1.0 \(\times\) 10\({}^{5}\)/mL in solution. For those researchers interested in investigating the strain-specific properties of bacterial spores via LTRS and other optical spectroscopic techniques, the review by [PERSON] et al. is suggested [54]. Apart from DPA leakage, the structural viability and adherence of resistant spores on organic and inorganic surfaces can be assessed via various forms of microscopy [55]. Understanding the properties that enhance spore adherence is crucial for reducing transmission and preventing infection recurrence. In a recent study [32], the viability of _C. difficile_ spores (DS1748, R20291, and DS1813), seeded on a range of clinically relevant surfaces such as isolation gowns, stainless steel, and floor vinyl, was assessed after exposure to a high-strength biocide (sodium dichloroisocyanurate). All strains remained viable on the clinical surfaces after exposure to the recommended disinfection concentration, demonstrating ineffectual sporicidal action. An interesting observation from this study was the easy exchange of spores between hydrophobic surfaces, such as fluid-resistant gowns and stainless steel. Of notable concern was the inability of single-use gowns to trap spores within their fibers and prevent onward transmission. In addition to surface adherence, _C. difficile_ spores can survive in abiotic environments by embedding themselves in mono- or mixed-species biofilms [56]. These biofilms serve multiple roles that may contribute to recurrence. They can act as reservoirs for spores and vegetative cells, shielding them from antibiotic activity and promoting persistent cell formation.