Diagnostic Test for Diphtheria
The definitive diagnostic test for diphtheria requires isolation of Corynebacterium diphtheriae from a clinical specimen (typically throat or nasopharyngeal swab) combined with demonstration of toxin production, preferably by the Elek immunoprecipitation test or molecular detection of the tox gene by PCR 1.
Laboratory Diagnosis Algorithm
The diagnostic approach should proceed in two critical steps:
Step 1: Bacterial Isolation and Identification
- Collect throat and/or nasopharyngeal swabs from the adherent membrane or beneath it if present
- Culture on selective media (Tinsdale or tellurite agar) to isolate C. diphtheriae, C. ulcerans, or C. pseudotuberculosis
- Species identification can be performed by traditional biochemical methods or molecular techniques (real-time PCR targeting rpoB gene) 2, 3
Step 2: Toxigenicity Testing (Essential)
This is the most critical step, as only toxigenic strains cause diphtheria:
Primary method:
- Elek immunoprecipitation test remains the gold standard for detecting diphtheria toxin production 1, 4, 5. This test demonstrates toxin production by creating visible precipitation lines when bacterial toxin meets antitoxin in agar
Alternative/Complementary methods:
- Real-time PCR for tox gene detection provides rapid results (same day) and high sensitivity, detecting as few as 1.86 genome copies per reaction 2. The quadruplex qPCR can simultaneously detect the tox gene and differentiate species 2, 3
- Molecular methods are particularly valuable for weakly toxigenic strains, especially C. ulcerans, which may produce insufficient toxin for standard Elek test detection 5
Critical Pitfalls and Caveats
The Elek test has important limitations:
- Requires specialized expertise and antitoxin availability, which is declining in developed countries 4
- Standard protocols may miss weakly toxigenic isolates, particularly C. ulcerans strains 5
- Optimized protocols (adjusted antitoxin concentration, medium volume, and inoculum positioning) significantly improve detection of low-toxin producers 5
Important clinical considerations:
- Do not wait for laboratory confirmation to initiate treatment - diphtheria antitoxin and antibiotics must be started immediately based on clinical suspicion, as delays increase mortality 6
- Cutaneous diphtheria should not be reported as diphtheria per CDC guidelines 1
- Respiratory disease caused by non-toxigenic C. diphtheriae should still be reported as diphtheria 1
- All diphtheria isolates should be sent to reference laboratories for confirmation 1
Practical Implementation
For optimal diagnostic accuracy:
- Combine culture-based isolation with molecular tox gene detection when available 2, 3
- PCR provides faster turnaround (hours vs. days) and higher sensitivity than traditional methods 2
- If only culture is available, ensure proper Elek test technique or send isolates to reference laboratories 4, 5
- The combined real-time PCR approach targeting both tox and rpoB genes allows simultaneous detection of toxigenicity and species differentiation in a single assay 2, 3