Robust high-sensitivity real-time impedance analysis for neutralization drug screening and toxicity kinetics of Clostridioides difficile toxin TcdA.

Clostridioides difficile (C. difficile) 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 in vitro 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.