Proof-of-concept MALDI-TOF-MS assay for the detection of Toxin B enzymatic activity in <i>Clostridioides difficile</i> infection

The diagnostics of <i>Clostridioides difficile</i> infection is usually based on the identification of the bacterial pathogen and/or on the detection of the Toxins A and B. Due to the variance in Toxins A and B activity across species, the toxin concentration determined by standard methods does not necessarily correlate with the severity of the disease. Assays that would target toxins' enzymatic activity are not routinely used because the requirements are unsuitable for clinical laboratories. In this study, we demonstrate a new approach that determines the presence and potency of Toxin B indirectly by determining its enzymatic activity rather than its concentration. This is performed by detecting mass difference due to glycosylation of RhoA substrate by Toxin B, which is then determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented proof-of-concept assay thus offers the possibility to quickly determine the activity of <i>C. difficile</i> toxins directly in the stool samples without pathogen cultivation.