Rapid Phenotypic Methods for the Detection of Diphtheria Toxin Amongst Clinical Isolates of Corynebacteria

Clinical Microbiology and Antimicrobial Chemotherapy. 2001; 3(2):156-162

Journal article


Two rapid, phenotypic methods were developed for the detection of diphtheria toxin amongst clinical isolates of corynebacteria, an enzyme immunoassay (EIA) and an immunochromatographic strip (ICS) test. Both assays use equine polyclonal antitoxin as the capture antibody. The detecting antibody is a monoclonal antibody, specific for fragment A of the diphtheria toxin molecule, labelled with alkaline phosphatase for use in the EIA and colloidal gold for the ICS. The assays are rapid, sensitive and specific: a final result is available within 3h of colony selection and toxigenicity can be detected from isolates grown on a diverse range of culture media, including selective agars. The limits of detection of the EIA are 0,1 ng/ml and of the ICS test 0,5 ng/ml diphtheria toxin. Toxin detection using the EIA was compared to the Elek test and PCR detection of fragment A of the diphtheria toxin (tox) gene, using 245 isolates of corynebacteria. The results for the EIA were in complete concordance with the Elek test: 87 toxigenic and 158 non-toxigenic isolates. Ten of the phenotypically non-toxigenic strains were found to contain fragment A of the tox gene but did not express the toxin protein. These isolates were found to be non-toxigenic in the Vero cell tissue culture cytotoxicity assay and were therefore, non-toxi-genic for diagnostic purposes. The use of the ICS test, in comparison with the Elek test, for detection of toxigenicity was evaluated in field trials in countries of the former USSR, using 488 isolates of various Corynebacterium spp. The results for the ICS test were in complete concordance with the Elek test (243 toxigenic and 245 non-toxigenic isolates). The ICS test was also evaluated for direct detection of toxigenicity from throat swabs. One hundred and twelve throat swabs from suspected diphtheria cases and carriers were examined by conventional culture and direct ICS. The results showed 98% concordance (110112) and the sensitivity and specificity of the ICS was 95 and 99%, respectively.

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