# Emerging Diagnostics in _Clostridioides difficile_ Infection
## 3 Sampling the Community (Ribotyping and Mass Spectrometry)  
PCR-ribotyping has been used as the reference standard to investigate transmission in suspected CDI outbreaks for decades. The method analyzes the varying intergenic spacer regions (ISRs) between the 16S and 23S rRNA genes. PCR across the ISR generatesfragments of different lengths, the sizes of which can be resolved by gel or capillary electrophoresis [69]. The variability of the spacer regions between strains has been used to define hypervirulent lineages and trace epidemiological links, notably RT027 and RT078. However, PCR-ribotyping shows a low discriminatory capability in differentiating strains having ISRs of equal length but different nucleotide sequences [70]. Other _C. difficile_ typing methods include pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), whole-genome sequencing (WGS), multi-locus variable-number tandem repeat analysis (MLVA), and matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Mass spectrometry (MS) techniques such as gas chromatography-MS (GC-MS), high-resolution tandem MS (LC-MS/MS), and liquid chromatography-MS (LC-MS) are increasingly employed for ribotyping pathogenic bacteria in clinical samples. Soft ionization methods, including matrix-assisted laser desorption ionization (MALDI-TOF-MS) and electrospray ionization (ESI)-MS, allow the analysis of large molecules, whole cells, proteins, DNA, and, recently, the parasite _Plasmodium falciparum_ in human blood [71]. MALDI-TOF is particularly effective for directly analyzing bacterial isolates, delivering identification results in approximately 15-30 s. In clinical microbiology, MALDI-TOF MS produces a protein spectrum of a sample that is sourced to a database/reference library, where potentially new specific biomarkers are identified. This combinatorial approach facilitates microbial protein identification through peptide mass fingerprinting and the analysis of nucleic acid sequences and amplification products. In 2021, researchers evaluated the possibility of differentiating and classifying the different toxigenic _C. difficile_ strains found in 158 patient fecal samples using MALDI-TOF MS and two statistical classifying algorithm models (CAMs) [70]; the first CAM involved all 10 ribotypes, whereas the second one only involved 5 (PR1-PR5) ribotypes. Improved performance was achieved with the second CAM, showing a 100% recognition capability, 96.6% cross-validation accuracy, and 98.4% agreement (60 correctly typed strains, classified as PR1-PR5, out of 61 examined strains) with PCR-ribotyping results. The following year, the same group used MALDI-TOF MS to characterize an outbreak of 40 toxigenic _C. difficile_ DNA-positive patients in an Italian hospital [72]. In agreement with PCR, four strains were classified as RT 126 by T-MALDI. Of the 29 strains identified, 22 were linked to the outbreak, belonging to the same cluster (PRA). Further analysis revealed that 22/29 were tcdA+/tcdB-/cdt+, 4/29 were tcdA+/tcdB+/cdt+, and 3/29 strains were tcdA+/tcdB+/cdt-. The authors noted that the A+ B-phenotype is mostly encountered in food animals [15]. MALDI-TOF-MS is a typing method that is readily amenable to the epidemiological tracking of emerging and hypervirulent strains of _C. difficile_ and has been employed in the identification of numerous RTs such as RT001, RT017, RT027/RT176, and RT078/RT126 [73,74]. As well as CAMs, MALDI-TOF-MS was recently paired with four machine learning models and 65 isolates as a training set for species identification and the discrimination of hypervirulent (HV) from non-hypervirulent (NHV) ribotypes [75]. The authors showed that protein extract-based MALDI-TOF spectra coupled with ML could distinguish between HV and NHV ribotypes circulating in Europe (accuracy > 95%). Furthermore, subtyping of certain HVRTs (e.g., RT027/176 or RT023) was possible (100% accuracy, PLS-DA model).