Detection of toxin production in Clostridium difficile strains by three different methods

Abstract

Objective: To compare two immunoassays for detection of toxins produced in vitro by isolates of Clostridium difficile with the standard tissue culture assay, to help in the diagnosis of C. difficile-associated diarrhoea.

Methods: Toxin production was investigated in 42 strains of C. difficile of various serotypes, ribotypes and S-protein types. These included strains from our laboratory collection, strains freshly isolated from stool specimens of patients suspected of suffering from C. difficile-associated disease or of carrying it asymptomatically, and one reference strain (NCTC 11223). Toxin was assayed by (i) a rapid slide immunoassay (C. difficile toxin A test, Clearview, Oxoid), (ii) an enzyme-linked microplate immunoassay (C. difficile toxin A/B test, Techlab), and (iii) a tissue culture assay. The rapid slide assay and the enzyme immunoassay were performed according to the manufacturers' recommendations. The tissue culture assay was performed using Vero cells.

Results: Thirty of the 42 strains (71%) were shown to be positive for toxin A by the slide immunoassay and 34 of the strains (81%) were found to be toxin A/B producers by the enzyme immunoassay. The same 34 strains that were positive in the enzyme immunoassay also produced toxin B (cytotoxin) in the tissue culture assay. The sensitivity, specificity, and positive and negative predictive values for the rapid slide immunoassay method were calculated to be 88.2%, 100.0%, 100.0% and 66.7%, respectively, when compared to tissue culture assay results as the reference method. These values for the enzyme immunoassay method were all 100.0%. In this study eight strains were found to be non-toxin-producing by all methods. It is possible that there were four strains that only produced toxin B (A- B+), and were missed by the rapid A-only assay.

Conclusions: We can recommend the use of the Techlab A + B enzyme immunoassay for the detection of toxin production by C. difficile strains because of its high sensitivity and specificity, its ease of use, and its capability of detecting both A- and B-type toxins.