Diphtheria Toxin Reference Testing

This service consists of the molecular detection of the Diphtheria Toxin (DT) tox gene by real-time PCR and, phenotypic detection of expressed diphtheria toxin by the modified Elek test.

Reference Service

Corynebacterium diphtheriae species complex

Special Bacteriology Unit

• Respiratory and cutaneous diphtheria

Pure cultures of Corynebacterium diphtheriae, C. ulcerans, C. pseudotuberculosis, C.rouxii, C. belfantii, and C. silvaticum

Slants (preferred) or plates of any suitable media and swabs in transport media are all acceptable. 

Store samples between room temperature and 37oC until shipped for testing. Ship actively growing cultures at room temperature.

Shipping of specimens shall be done by a TDG certified individual in accordance with TDG regulations. For additional information regarding classification of specimens for the purposes of shipping, consult either Part 2 Appendix 3 of the TDG Regulations or section 3.6.2 of the IATA Dangerous Goods Regulations as applicable.

Suspected diphtheria case with symptoms for respiratory diphtheria, throat diphtheria, cutaneous diphtheria, endocarditis, pharyngitis, and lymphadenitis.

Completed Special Bacteriology requisition form (most recent version of requisition is required), detailing all patient information and relevant clinical information. If possible, attach lab results that have already been done at local or provincial laboratories.

All patient and strain history must be included. For urgent samples, prior contact with the laboratory is required.

A multiplex real-time PCR is used to detect 2 targets within the fragment A of the DT tox gene, as well as a Corynebacterium diphtheriae species specific sequence of the rpoB gene. The phenotypic detection for the gene expression using the modified Elek test is ONLY performed on samples that are PCR positive for the tox gene. Strains with a positive PCR result but Elek negative (gene present but not expressed) are considered non-toxigenic (1). C. ulcerans and C. pseudotuberculosis are identified using sequence analysis of the rpoB gene.

9 calendar days.  For urgent samples, testing may be expedited if there is prior contact with the laboratory. In cases where staff or resources are limited final report may be delayed and status of request will be forwarded in a preliminary report.  Final report will be sent once all results from the molecular and phenotypic detection of diphtheria toxin as well as all other relevant tests are complete.

1. Efstratiou, A., KH Engler, CS Dawes, and D Sesardic. 1998. Comparison of Phenotypic and Genotypic Methods for Detection of Diphtheria Toxin among Isolates of Pathogenic Corynebacteria. J Clin Microbiol. 36(11): 3173-3177.
2. Efstratiou, A, and RC George. 1999. Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae and C. ulcerans. Communicable Disease and Public Health 2(4):250-257.
3. World Health Organization, 2021 WHO laboratory manual for the diagnosis of diphtheria and other related infections. ISBN 978-92-4-003805-9.
4. Public Health Agency of Canada.  2009.  Case description for Communicable Diseases under National Surveillance-Diphtheria. CCDR supple. Vol 35S2. Pg 63-64.
5. Engler, K. H., T. Glushkevick, I. K. Mazurova et al. 1997. A modified ELEK test for detection of toxigenic Corynebacteria in the diagnostic laboratory. J. Clin. Microbiol. 35:495-498.
6. Khamis, A, Raoult, D, La Scola, B. 2004. rpoB Gene Sequencing for Identification of Corynebacterium Species. JCM 42: 3925-3931
7. Schuhegger, R., Lindermayer, M., Kugler, R., Heesemann, J., Busch, U., & Sing, A. (2008). Detection of Toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans Strains by a Novel Real-Time PCR. J. Clin. Microbiol. 46(8), 2822-2823. doi:10.1128/jcm.01010-08.
8. Zoysa, A. D., Efstratiou, A., Mann, G., Harrison, T. G., & Fry, N. K. (2016). Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic corynebacteria. J. Med. Microbiol. 65(12), 1521-1527. doi:10.1099/jmm.0.000382
9. Badell, E., Hennart, M., Rodrigues, C., et al. 2020. Corynebacterium rouxii sp. nov., a novel member of the diphtheriae species complex. Res Microbiol. Apr-Jun;171(3-4):122-127. doi: 10.1016/j.resmic.2020.02.003. Epub 2020 Feb 28
10. Crestani, C., Arcari, G., Landier, A., et al. 2023. Corynebacterium ramonii sp. nov., a novel toxigenic member of the Corynebacterium diphtheriae species complex. Res Microbiol. Sep-Oct;174(7):104113. doi: 10.1016/j.resmic.2023.104113. Epub 2023 Aug 10.

Pathogen Safety Data Sheets: Infectious Substances – Diphtheroids

Pathogen Safety Data Sheets: Infectious Substances – Corynebacterium diphtheriae

For Health Professionals: Diphtheria

Diphtheria