Expanding the use of MALDI-TOF mass spectrometry in the microbiological laboratories: A rapid C. difficile toxin activity-based assay

ABSTRACT: Matrix-Assisted Laser Desorption/Ionization Time of Flight mass spectrometry (MALDI-TOF MS) has become a standard clinical tool for microbial identification. Its utilization in clinical microbiology laboratories keeps expanding from basic identifications towards detecting antibiotic resistance and microbial toxins. Clostridiodes difficile represents an emerging bacterial pathogen causing severe gastrointestinal infection associated with antibiotic therapy affecting normal gut microbiota. The virulence of C. difficile is mainly caused by two toxins, namely Toxin A and Toxin B. Established diagnostic methods, including PCR and enzyme-linked immunoassays, detect either the presence of the microorganism itself or the presence and concentration of the toxins, with only limited ability to gauge infection severity and evaluate the actual biochemical activity of the toxins and their potency to cause harm. This work addresses the infection severity using the functional activity detection of C. difficile toxins by MALDI-TOF MS assay performed on affinity MALDI chips. The MALDI chips are modified using ambient ion landing technology with an array of immobilized Neutralite Avidin (NeutrAvidin) spots that allow secondary immobilization of biotin-tagged recombinant RhoA, and this protein is a substrate for the Clostridiodes difficile toxin’s enzymatic activity. Monitoring mass shift due to the in-vitro glycosylation of the RhoA in the MALDI-TOF mass spectrum provides information about the Toxin’s B activity in the sample. The assay was used to analyze 20 samples of patients suspected of C. difficile infection and compared with the results obtained by standard methods. Observing the Toxin B activity, rather than the concentration, enhances diagnostic efficiency and offers valuable information about infection potency without the extensive resources required by reference methods like stool cytotoxicity assay and toxigenic culture. The presented assay is a representative example of analytical chemistry helping clinical microbiology achieve modern symptom-based diagnostics.

INSTRUMENT(S): autoflex

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Feces

SUBMITTER: Petr Pompach  

LAB HEAD: Petr Pompach

PROVIDER: PXD055622 | Pride | 2025-03-21

REPOSITORIES: Pride