{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T06:07:18Z","timestamp":1788934038546,"version":"build-2803163510"},"reference-count":56,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>\n                    <jats:italic>Clostridioides difficile (C. difficile<\/jats:italic>\n                    ) is the main cause of nosocomial infections. It is responsible for mild to life-threatening severe gastrointestinal infections whose typical symptoms are fluid loss caused by Toxin A (TcdA) and Toxin B. In cell cultures, the two exotoxins provoke cell rounding, a morphological change that is visible under the microscope. This characteristic has been widely exploited in\n                    <jats:italic>in vitro<\/jats:italic>\n                    assays to evaluate their cytotoxic effect as well as the neutralizing activity of anti-toxin compounds. However, the evaluation of morphological changes relies on subjective scoring, introducing a risk for bias, limiting reproducibility and sensitivity. In the present study, we demonstrate, optimize and validate the applicability of cell-based impedance with real-time cell analysis (CBI-RTCA) for quantifying the effects of TcdA on Vero cells. CBI-RTCA can be used to monitor changes in cell adhesion and proliferation, which are measured as Cell Index (CI). We assessed the effect of TcdA, and a TcdA-neutralizing antibody, using CBI-RTCA and compared the results with visual scoring readouts from multiple operators. The addition of TcdA to Vero cells caused a dose-dependent decrease in CI with a TcdA detection limit of 0.15 pM. CBI-RTCA was more sensitive than visual morphology assessment, detecting reduced cell adhesion within 1 h of TcdA treatment, compared with 5 h by microscopy. TcdA was neutralized by AH3_Fc antibody and using CBI-RTCA which enabled kinetic measurements of the neutralizing drug. The kinetic readouts reported significantly lower variability than morphological inspection, thereby showing higher reproducibility. In addition, CBI-RTCA was utilized for TcdA quantification. The generation of a dose-response curve which correlated the percentage of cell viability to TcdA concentrations allowed the quantification of the low TcdA concentration (12.5 pM) giving 10% of cell viability. The study demonstrates that CBI-RTCA enables continuous, kinetic measurement of toxic and drug-neutralizing effects throughout the experiment with minimal operational effort, eliminating labor-intensive image sampling and readout bias. Moreover, CBI-RTCA is well suited for the detection and quantification of TcdA, even at low concentrations, making it ideal for drug screening and high-through-put applications and has the potential to be adapted and validated for further toxins.\n                  <\/jats:p>","DOI":"10.3389\/fmicb.2026.1939062","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T05:50:08Z","timestamp":1788933008000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Robust high-sensitivity real-time impedance analysis for neutralization drug screening and toxicity kinetics of Clostridioides difficile toxin TcdA"],"prefix":"10.3389","volume":"17","author":[{"given":"Marta","family":"Braghetto","sequence":"first","affiliation":[{"name":"Biotherapeutic Glycoengineering and Immunology, Section for Medical Biotechnology, Department of Biotechnology and Biomedicine, Technical University of Denmark","place":["Kgs Lyngby, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah Line","family":"Skovbakke","sequence":"additional","affiliation":[{"name":"Biotherapeutic Glycoengineering and Immunology, Section for Medical Biotechnology, Department of Biotechnology and Biomedicine, Technical University of Denmark","place":["Kgs Lyngby, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lasse Ebdrup","family":"Pedersen","sequence":"additional","affiliation":[{"name":"Biotherapeutic Glycoengineering and Immunology, Section for Medical Biotechnology, Department of Biotechnology and Biomedicine, Technical University of Denmark","place":["Kgs Lyngby, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steffen","family":"Goletz","sequence":"additional","affiliation":[{"name":"Biotherapeutic Glycoengineering and Immunology, Section for Medical Biotechnology, Department of Biotechnology and Biomedicine, Technical University of Denmark","place":["Kgs Lyngby, Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,9]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1146\/annurev-micro-090816-093458","article-title":"Clostridium difficile toxin biology.","volume":"71","author":"Aktories","year":"2017","journal-title":"Annu. 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