# Emerging Diagnostics in _Clostridioides difficile_ Infection
## 1 Introduction  
First isolated in 1935 from the stools of newborn infants, the environmentally ubiquitous anaerobic _Clostridioides difficile_ (_C. difficile_) is a spore-forming Gram-positive bacillus with toxigenic potential [1]. The rod-shaped pathogen secretes two toxins, enterotoxin A (tcdA) and cytotoxin B (tcdB), which are primarily responsible for the pathogenicity observed in individuals with _C. difficile_ infection. In addition to tcdA and tcdB, there is a third binary toxin (CDTa and CDTb), denominated CDT, which is thought to upregulate tcdA and tcdB production [1, 2]. The role of CDT is unclear, although initial experiments suggest it may enhance pathogenic virulence by suppressing the protective colonic eosinophil in the host [3]. Other virulence factors include surface-layer (Cwp84 and 66) and flagella (HiC and FiD) proteins associated with bacterial adherence and migration [4]. The inflammatory role of these toxins and associated regulators (tcdC) is well documented and is beyond the scope of this review [1]. _C. difficile_ infections (CDIs) can be characterized as either community-acquired (CA-CDI) or healthcare-acquired (HA-CDI), depending on whether symptoms present in the first 3 days upon hospitalization or later [5]. Delineating HA-CDI from CA-CDI transmission remains challenging; initial studies involving 957 patients identified 333 isolates with 2 single-nucleotide variants (SNVs) and 488 with 10 SNVs. Of the 333 individuals, 35% had a clonal relationship with the same ribotype, whilst 120 exhibited no identifiable epidemiological link [6]. Furthermore, isolates with the same genotype showed substantial genetic diversity, suggesting that many unknown reservoirs (asymptomatic carriers and hospital staff) may have contributed to hospital transmission. Asymptomatic carriage can be defined as the presence or detection of _C. difficile_ toxins in the absence of known disease pathologies. In addition [7] to asymptomatic carriers, other studies showed that a large percentage (27%) of HA infections and returning (previously _C. difficile_-negative) patients were community-acquired and subsequently imported into the hospital [8]. The risk factors for HA-CDI and CA-CDI in adults include prior antibiotic use [9, 10], chronic inflammatory diseases [11], gastric acid medications, nutrient availability, and reduced microbial diversity [12]. Notably, 31% of younger, healthy UK females (median age: 50-51) without prior antibiotic exposure were diagnosed with CA-CDI [13]. Recent reports suggest that family exposure is a significant risk factor for increased incidence [14]. Younger family members are particularly susceptible, with prevalence in children (<16 years) approaching 40% and 53% in neonates and infants (2-12 months), with many being asymptomatic [15]. Recurrence in children mirrors adults at 20-30%. Factors contributing to neonatal susceptibility include nutritional deficiencies, reduced colonization resistance, and inadequate anamnestic immune responses [16; 17]. In addition to human hosts, whole-genome sequencing (WGS) data indicate that farm animals, such as pigs and chickens, can act as reservoirs for _C. difficile_, facilitating zoonotic bidirectional transfer (ZBT) [18]. _C. difficile_ is also found in rivers, lakes, soil, household environments, and the nasal-oral airways of pets like dogs and cats [19; 20; 21; 22]. Phylogenetic analysis shows extensive clustering of human, animal pathogenic, and antibiotic-resistant _C. difficile_ strains, highlighting the role of farm and food networks in long-distance transmission [23]. Sequence type 11 (ST11) ribotypes, including hyperviulent RT027, have multiple tetM-associated clonal expansions [24]. Of note are the resistant alleles, tetM 10 and tetM 16, shared among pathogenic and zoonotic species [25; 26]. As of 2020, over 800 C. _diffific_ ribotypes have been reported, with several frequently identified in the USA [27]. _C.