Cell cytotoxicity assay

Faecal samples were diluted with an equal volume of PBS, centrifuged and the supernatants were filtered through a 0.2 μm filter. The assay was performed by testing this sterile faecal filtrate in duplicates on a confluent monolayer of Vero cells after preincubation of cells with either PBS or C. difficile antitoxin. The final dilution of the stools was 1:40. For positive and neutralisation controls the Clostridium difficile toxin/antitoxin kit was used according to the instructions of the manufacturer (TechLab). A positive result was recorded if cell rounding was seen only in the unprotected cells after 24 or 48 h of incubation in a 37 °C CO₂-incubator.