Introduction
- Diphtheria is a serious infectious disease caused by the bacterium Corynebacterium diphtheriae.
- This pathogen primarily affects the respiratory tract, leading to the formation of a pseudomembrane in the throat, which can cause significant morbidity and mortality.
- Despite the effectiveness of vaccination programs, diphtheria has not been completely eradicated and can still cause outbreaks, particularly in under-vaccinated populations.
- Therefore, a thorough understanding of the laboratory methods for diagnosing diphtheria is essential for timely intervention and public health response.
Clinical Presentation of Diphtheria
Diphtheria manifests with a range of symptoms that may vary depending on the site of infection. The classic presentation includes:
- Respiratory Diphtheria: Characterized by:
- Sore throat
- Low-grade fever
- Swelling of the neck (bull neck) due to lymphadenopathy
- Formation of a grayish-white pseudomembrane that can obstruct the airway, leading to respiratory distress.
- Cutaneous Diphtheria: Involves lesions on the skin, particularly in tropical or underserved areas, often presenting as non-healing ulcers with a gray membrane.
- Other Forms: Rarely, diphtheria can affect other systems, including the cardiovascular system (myocarditis) and nervous system (neuropathy).
Sample Collection for Diagnosis
The primary specimens for laboratory diagnosis of diphtheria are derived from the site of infection.
- Throat Swabs
- Indications: Most commonly collected when respiratory diphtheria is suspected, especially if a pseudomembrane is present.
- Technique:
- Use a sterile swab with a plastic or wooden handle.
- Gently swab the tonsils’ surface, and the throat’s back, including any visible pseudomembrane.
- Avoid touching the tongue or other oral structures to reduce contamination.
- Nasal Swabs
- Indications: Useful when nasal diphtheria is suspected.
- Technique: Insert a sterile swab into the nasal cavity to collect secretions or material from the nasal mucosa.
- Biopsy of Pseudomembrane
- Indications: Rarely, a biopsy may be performed when the diagnosis is uncertain or when tissue characteristics need to be assessed.
- Technique: A small piece of the pseudomembrane can be excised for further analysis.
- Additional Specimens
- Serum Samples: In cases where systemic effects are observed (e.g., myocarditis), serum samples may be collected for serological analysis.
Laboratory Techniques for Diagnosis
The laboratory diagnosis of diphtheria involves a combination of culture techniques, microscopy, biochemical testing, and molecular methods.
Culture Methods
A. Media Selection
- Cystine-Tellurite Blood Agar: This selective medium inhibits the growth of competing organisms while allowing Corynebacterium diphtheriae to grow. On this medium, colonies of C. diphtheriae typically appear black due to tellurite reduction.
- Loeffler’s Serum Agar: An enriched medium that enhances the growth of diphtheria bacilli. Colonies can show characteristic morphology, aiding in identification.
B. Incubation Conditions
- Cultures should be incubated at 35–37°C for 24–48 hours in a CO₂-enriched environment to optimize growth conditions.
C. Colony Morphology
- After incubation, colonies suspected of C. diphtheriae are further examined for their characteristics, including size, shape, and color.
Microscopic Examination
A. Gram Staining
- A Gram stain of the swab can reveal the presence of Gram-positive bacilli. C. diphtheriae typically appears as pleomorphic rods, often arranged in V- or L-shaped formations reminiscent of Chinese letters.
B. Special Stains
- Albert’s Stain: Used to visualize metachromatic granules in C. diphtheriae, which can help confirm the organism’s identity.
- Methylene Blue Stain: It also highlights the presence of granules and can provide information on cell morphology.
Biochemical Tests
Catalase Test
- Corynebacterium diphtheriae is catalase-positive, which helps distinguish it from some other bacteria.
Urease Test
- C. diphtheriae is urease-negative, providing another differential criterion.
Other Tests
- Fermentation tests may be conducted to assess carbohydrate utilization, though these are less commonly performed due to the availability of molecular methods.
Molecular Methods
Polymerase Chain Reaction (PCR)
- PCR techniques are increasingly utilized for the rapid and specific detection of Corynebacterium diphtheriae and its toxin genes (tox).
- This method allows for quick identification, often within a few hours, and is particularly useful in cases where traditional culture methods may be slow or inconclusive.
Nucleic Acid Hybridization Techniques
- Techniques like RT-PCR can detect specific genetic markers associated with C. diphtheriae, enhancing diagnostic accuracy.
Serological Tests
- While less commonly used in routine practice, serological tests can detect antibodies to diphtheria toxin or other bacterium components. These tests may be more relevant in research settings or for epidemiological studies.
Interpretation of Results
- Culture Results
- Positive Culture: Isolation of Corynebacterium diphtheriae from throat or nasal swabs confirms the diagnosis. Black colonies on cystine-tellurite blood agar, alongside characteristic morphological and biochemical traits, substantiate this finding.
- Negative Culture: This may occur if the patient received antibiotics before sampling, leading to false negatives. Multiple specimens may need to be collected to increase detection chances.
- Microscopic Examination
- The presence of Gram-positive bacilli in characteristic arrangements supports a diagnosis of diphtheria, but culture isolation is required for confirmation.
- PCR Results
- Positive PCR: Indicates the presence of C. diphtheriae, confirming an active infection. This result is particularly valuable in cases where clinical symptoms suggest diphtheria but culture results are negative.
- Negative PCR: Suggests that the specific pathogen tested is absent; however, clinical correlation is necessary.
- Biochemical Tests
- Positive catalase and negative urease tests provide further confirmation of C. diphtheria.
Clinical Considerations
Prompt Diagnosis and Treatment
Due to the potential for rapid deterioration in patients with diphtheria, it is essential to start empirical treatment immediately upon suspicion of the disease, even before laboratory confirmation is obtained.
- Empirical Treatment: Administering diphtheria antitoxin (DAT) is critical for neutralizing the toxin and preventing complications. Antibiotics, such as penicillin or erythromycin, are also given to eliminate the bacteria and reduce transmission.
Follow-up and Public Health Surveillance
- Surveillance for diphtheria is critical, particularly in low vaccination rates. Public health measures, including vaccination campaigns and education, are essential to control outbreaks.
Outbreak Investigations
- Conducting laboratory surveillance, contact tracing, and possibly prophylactic treatment for exposed individuals is essential in an outbreak.