Corynebacterium Diphtheriae

What sample is needed for the diagnosis of Corynebacterium Diphtheriae?

• The swab is made from the throat or the suspected lesion.

What are the indications for Corynebacterium Diphtheriae?

• For the diagnosis of C. Diphtheriae (Diphtheria)

How will you discuss the microbiology of Corynebacterium Diphtheriae?

1. C. diphtheriae is a nonmotile, non-capsulated, club-shaped, Gram-positive bacillus.
   1. These are non-sporing.
   2. Highly pleomorphic-like Chinese letter appearance.
2. Habitat is the throat and nose of human beings.
3. These are pleomorphic gram-positive rods or clubs that divide in unique patterns, such as V, L, or W shapes, the so-called Chinese character shapes.
   1. These rods measure 3 x 0.3 µm.
4. Culture:
   1. These are aerobes and facultative anaerobes.
   2. The optimum temperature for the growth is 37 °C.
   3. Media containing blood or serum is needed.
   4. Selective media is needed for isolation.
   5. Most strains require nicotinic and pantothenic acids for growth; some also require thiamine, biotin, or pimelic acid.
5. The selective medium is used:
   1. Loeffler’s medium (coagulated blood serum.
      1. Take the growth after 12 hours and stain the growth with methylene blue, showing rod-shaped pleomorphic bacteria.
   2. Potassium tellurite medium.
      1. The colonies are black to gray within 24 hours of the culture.
6. The medium should be supplemented with amino acids for optimal production of diphtheria toxin.
7. Biochemical reactions:
   1. There is acid production from carbohydrates.
   2. Gravis strain can ferment starch and sugar.
   3. Intermedius and mitis can not ferment.
8. Toxigenic strains are lysogenic:
   1. These toxigenic strains cause the death of the experimental animal in 2 to 3 days,
   2. While antitoxin-protected animals can survive.
9. Classification based on colony morphology:
   1. Mitis.
   2. intermedius.
   3. Gravis.
10. For epidemiological studies, the C. diphtheriae typing is done by:
    1. Serotyping.
    2. Phage typing.
    3. Bacteriocin typing.

How will you discuss the pathogenesis of Corynebacterium Diphtheriae?

1. The asymptomatic nasopharyngeal carriage is common in regions where diphtheria is endemic.
2. Toxigenic strains cause disease in susceptible individuals by multiplying and secreting diphtheria toxin in the nasopharyngeal area or skin lesions.
3. There is often the formation of a pseudomembrane composed of fibrin, bacteria, and inflammatory cells.
4. The necrosis of the affected epithelium forms a pseudomembrane of fibrin, necrotic cells, and neutrophils.

5. After absorption into the blood, the toxin acts systemically on the myocardium, nervous tissue, and adrenal gland.
6. Only the motor nerves are affected.
7. Diphtheria toxin can be proteolytically cleaved into two fragments:
   1. Fragment A catalyzes the NAD+-dependent ADP-ribosylation.
   2. Fragment B binds to the cell surface receptor and facilitates the delivery of fragment A to the cytosol.
   3. The toxin can be inactivated with formaldehyde, and toxoids are formed.
      1. Toxoid is used for immunization.

What is the clinical presentation of Corynebacterium Diphtheriae?

1. C. diphtheriae infects the nasopharynx or skin.
2. Toxigenic strains secrete a potent exotoxin, which may cause diphtheria.
3. The symptoms of diphtheria include:
   1. Pharyngitis.
   2. Fever.
   3. Swelling of the neck or area surrounding the skin lesion.
4. Diphtheritic lesions:
   1. These are covered by a pseudomembrane.
5. The toxic outcome is that toxin is distributed to distant organs by the circulatory system and may cause:
   1. Paralysis.
   2. Congestive heart failure.
6. These are the clinical signs of nasopharyngeal diphtheria infection:
   1. A sore throat.
   2. Dysphagia.
   3. Bloody nasal discharge.
   4. Pseudomembrane formation.

How will you diagnose Corynebacterium Diphtheriae?

1. Clinical diagnosis depends upon culture-proven toxigenic C. diphtheriae infection of :
   1. The skin.
   2. Nose or throat.
2. Culture from the involved area is positive in 12 hours on Loffler’s medium and more slowly on blood agar of the toxin-producing strains.
   1. Always take nose culture.
3. Toxigenicity is identified by various in vitro (e.g., gel immunodiffusion, tissue culture) or in vivo (e.g., rabbit skin test, guinea pig challenge) methods.
4. This bacteria is Urease negative.
5. Schick test:
   1. This is a skin test to find immunity for circulating diphtheria antitoxin.
   2. This test indicates either previous immunization or infection.
   3. Inject intradermally on an anterior aspect of the forearm toxin.
   4. Also, a heat-inactivated toxin can be injected into another arm.
   5. Result: The positive test is erythema at the site of injection.
   6. Negative erythema on the heat-inactivated arm.
      1. The arm should be examined after 1 to 2 days.
      2. Again, examine after 5 to 7 days.
6. Special stain:
   1. Albert’s stain.
   2. Another stain used is Ponder’s stain.
7. Stained smear from the involved area is positive in >75% of the patients.

How will you stain the smear for Corynebacterium Diphtheriae?

1. Cover the fixed and dried slide with Loeffler’s methylene blue. Leave for 5 minutes.
2. Wash with water.
3. Decolorize with sulphuric acid with a dilution of 1/1000 immediately.
4. Acid should not stay for more than a few seconds.
5. Again, wash with water.
6. Treat with Gram’s iodine.
7. Again, wash with water.
8. Counterstain with 1% eosin for 30 seconds.
9. Wash with water and dry it.

What is the result of the staining procedure for Corynebacterium Diphtheriae?

• Metachromatic granules are seen on the smear.
• The body of the bacteria is pinkish, and the granules are blue-black.
  • Gram stain also shows gram-positive bacteria.

How will you treat Corynebacterium Diphtheriae?

1. WBCs are raised.
2. Albumin and urine casts are frequently present in the urine, and may rarely see RBCs in urine.
3. There may be moderate anemia.
4. Serum glucose is frequently decreased.
5. Give supportive treatment.
6. Give antitoxin, which will neutralize the diphtheria toxins. This will be effective in the early stages when the toxins have not reached the target organs.
7. Antibiotics like penicillin or erythromycin will kill the bacteria. It will make patients noncontagious.
   1.  Penicillin G kills the bacteria within 12 hours.
   2. Without any treatment, bacteria will disappear in 2 to 4 weeks.
8. The vaccine should be given because the patient doesn’t develop immunity to future infection.
   1. DPT (Diphtheria, pertussis, and tetanus) vaccine is given.
9. PCR gives rapid diagnosis.
10. Fluorescent antibody staining of suspected material gives a more rapid diagnosis and high positivity.

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