Diphtheria – Culture, Reference Isolates Identification Confirmation & Toxin Production Confirmation

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Background
This page provides testing information for:

Confirmation of identification of potential diphtheria-causing bacterial isolates as well as the presence of the diphtheria toxin gene and toxin production.
Detection of Corynebacterium diphtheria and/or Corynebacterium ulcerans from clinical specimens including toxin testing at Public Health Ontario (PHO).

The causative agent(s) of diphtheria include toxigenic Corynebacterium diphtheriae and rarely Corynebacterium ulcerans and Corynebacterium pseudotuberculosis, however not all isolates of these species produce diphtheria toxin.

The following testing options are available when investigating potential cases of diphtheria or identification/confirmation of potential diphtheria toxin producing isolates at Public Health Ontario’s (PHO) laboratory.

Bacterial cultures – Aerobic – Reference ID/Confirmation

Updates
As of June 9, 2026: Updated specimen collection requirements

Added specimens that are not acceptable for testing. Refer to Acceptance/Rejection Criteria section.
Added additional collection kits suitable for diphtheria culture. Refer to the Specimen Requirements table.

* Copie d'impression non contrôlée. Valide uniquement le jour de l'impression : 14 juin 2026.

Testing Indications

All clinical isolates identified as Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis must be tested to determine if they are toxigenic, regardless of specimen source. The combination of culture, molecular and phenotypic methods to detect and confirm toxin production are needed for confirmation of a diagnosis of diphtheria.

Diphtheria is primarily a respiratory infection. Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis can also be cultured from wounds and other sources. Appropriate specimens should be collected during the acute phase of illness.

For suspected outbreak cases, contact your local public health unit prior to collecting specimens.

Acceptance/Rejection Criteria

Swabs and other specimens are not acceptable for testing if received more than 3 days after collection date and time.
Specimens in viral transport media and dry swabs are NOT acceptable for testing.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Confirmation of Identification of Corynebacterium diphtheriae, Corynebacterium ulcerans or Corynebacterium pseudotuberculosis and toxin testing	Reference Bacteriology Requisition	Pure viable subculture of organism on appropriate media that supports the growth (e.g. Blood Agar) or a swab of a pure subculture in Amies transport medium that could be from any specimen source (e.g. respiratory, wound, blood culture etc.).	N/A	Bacterial culture media that supports growth of the organism
Diphtheria Culture	General Test Requisition	Swab collected from: Throat Nose Nasopharynx Nasopharyngeal Membranous material Tonsillar fossae, Posterior pharynx or Uvula (see specific notes 1 and 2 below)	N/A	Swab in Amies charcoal transport media E-Swab in Amies transport media Swab in other transport media supporting growth of the organism (i.e. Stuart’s, Loeffler’s)
Diphtheria Culture	General Test Requisition	Skin lesion: Aspirate Swab Membranous material (see specific note 3 below)	N/A	Swab in Amies charcoal transport media E-Swab in Amies transport media Swab in other transport media supporting growth of the organism (i.e. Stuart’s, Loeffler’s)

Submission and Collection Notes

Swabs should be taken from inflamed areas of the throat, nose and nasopharynx in symptomatic patients. If present, membranous material should also be submitted.

For detection of asymptomatic carriers, nasopharyngeal and throat swabs (tonsillar fossae, posterior pharynx and uvula) should be collected.

Material should be taken from the affected area of the skin, either by aspirate or swab. Pseudomembrane material may also be cultured. Remove any crusted material and swab the base of the lesion(s).

Complete all fields of the requisition form. Include, the patient’s full name, date of birth, Health Card Number (must match the specimen label), enter the name of the test requested, source of specimen, date of onset, date of collection, outbreak/investigation number, physician name and address, and clinical diagnosis.

Label the specimen(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.

Timing of Specimen Collection

Specimens should be collected during the acute phase of illness and delivered to the laboratory as soon as possible.

Storage and Transport

Specimens should be stored at 2-8°C following collection and shipped to PHO’s laboratory on ice packs within 3 days of collection. All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Specimens received are set up daily (Monday to Friday) for Aerobic bacteria Identification confirmation, and diphtheria culture at PHO’s laboratory-Toronto. Turnaround time is up to 5 days from receipt.

For confirmed Corynebacterium diphtheriae, Corynebacterium ulcerans or Corynebacterium pseudotuberculosis, diphtheria toxin gene detection by PCR is performed every Tuesday and Thursday at PHO’s laboratory –Toronto. Turnaround time is 3 days after identification confirmation of the isolate.

Confirmation of diphtheria toxin production (via modified Elek test) of isolates is performed at National Microbiology Laboratory (NML) at Winnipeg, Manitoba. Turnaround time is 9 calendar days from the date the isolate was received at NML.

Stat and Critical Specimens Testing

STAT testing is not available but if urgent testing is required (i.e. critical samples) notify PHO’s Laboratory Customer Service to ensure the sample is on the next available run.

Test Methods

Swabs, aspirate, skin, or membranous material (pseudomenbrane) are set up on culture media for bacterial isolation.
Isolates are identified/confirmed by gram stain morphology, colonial morphology and use of Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF MS).
All isolates identified as Corynebacterium diphtheriae, Corynebacterium ulcerans or Corynebacterium pseudotuberculosis are tested by real-time polymerase chain reaction (PCR) for detection of Diphtheria Toxin gene.
Isolates that are positive for Diphtheria Toxin gene by PCR are submitted to National Microbiology Laboratory for confirmation by real-time PCR and phenotypic detection of expressed diphtheria toxin by modified Elek test.

Interpretation

Test	Result
Confirmation of Identification	Isolate identified as [Genus species]
Diphtheria Culture	C. diphtheriae (or C. ulcerans) culture: Not isolated C. diphtheriae (or C. ulcerans) culture: Isolated
Diphtheria Toxin Gene PCR	Diphtheria toxin gene by real time PCR: Detected ( Presumptive) Comment: This is a real-time PCR assay that has been verified at the Public Health Ontario Laboratory for clinical use. It has not been cleared or approved by Health Canada. The specimen has been forwarded to NML for phenotypic toxin detection testing (modified Elek testing).
Diphtheria Toxin Gene PCR	Diphtheria toxin gene by real time PCR: Indeterminate Comment: This is a real-time PCR assay that has been verified at the Public Health Ontario Laboratory for clinical use. It has not been cleared or approved by Health Canada. The specimen has been forwarded to NML for phenotypic toxin detection testing (modified Elek testing).
Diphtheria Toxin Gene PCR	Diphtheria toxin gene by real time PCR: Not detected Comment: This is a real-time PCR assay that has been verified at the Public Health Ontario Laboratory for clinical use. It has not been cleared or approved by Health Canada.

Reporting

Results are reported to the ordering physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Toxin producing C. diphtheriae or C. ulcerans specimens are reported to the Medical Officer of Health as per Health Protection and Promotion Act.

References

McTaggart, L. R., Scione, A., Hillier, K., Brown, E., Wilson, S. E., & Kus, J. V. (2025). Toxigenic diphtheria rarely detected amid rising cases of nontoxigenic Corynebacterium diphtheriae infections in Ontario, Canada, 2011-2023. Journal of clinical microbiology, 63(12), e0145225. https://doi.org/10.1128/jcm.01452-25