Corynebacterium diphtheriae

microbiology

1. CORYNEBACTERIUM DIPHTHERIAE MAN BAHADUR RANA BPH ACAS,NEPAL

2. It is thin, slender rods. Non capsulated, non motile, non acid fast. Gram positive bacilli of varying length with an average size of 3x o.3 um. They frequently possess club-shaped swelling at one or both ends. When dividing, the bacilli snap and bend abruptly and appear as angle pairs or parallel rows of 3-4 bacilli (palisades) which resemble chinese letter. MORPHOLOGY

3. C. Diphtheria possess granules. These granules are known as metachromatic granules or volutin granules or Babes-Ernst bodies. Granules are composed of polymetaphosphate, and represent energy storage. With Albert stain, the granules stain bluish black and cytoplasm green. Contd....

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5. The source of infection are carriers who harbours the organism. There is no animal reservoir. Transmission occurs from person to person by respiratory droplet (aerosolised nasopharyngeal secretions) or skin contact. Cutaneous diphtheria also occurs. C. diphtheria is not an invasive organism and systemic manifestation occurs by production of toxin (exotoxin). Pathogenesis

6. C. Diphtheria that have been lysogenised with a bacteriophage (beta-phage) carrying the tox gene produce exotoxin. The toxin consist of two moieties: . Fraction A . Fraction B Toxin enters through receptor mediated endocytosis. Contd.....

7. Fraction A: the active moiety. Fraction B: the binding moiety that mediates the attachment of toxin to the cells via receptor. a). Fraction B – It has got a receptor binding region and a translocation region. Contd....

8. b). fractionB :

9. - subunit A then catalyses Adenosine diphosphate ADP- Ribolysation of EF-2 , a factor necessary for movement of nascent peptide chains on ribosomes. - One exotoxin molecule can inactivate the entire EF-2 content in a cell and thereby host cell protein, synthesis is completely blocked. Contd...

10. Toxin effect

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17. - Specific treatment against diphtheria (antitoxin and antibiotic) should be given immediately after clinical diagnosis without waiting for LABORATORY FINDINGS. SPECIFIC TREATMENT IS MORE IMPORTANT THAN LABORATORY DIAGNOSIS. LABORATORY DIAGNOSIS

18. Collection of specimen: - ( Throat swab, nose or larynx ) are taken from the site of lesion. - Two swabs are taken (one for smear examination and other for culture). Contd....

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20. Alberts staining: Procedure: - Flood the heat fixed smear with Alberts staining solution A for 5 minutes. - Drain the staining solution but do not wash. - Flood with solution B and keep for 1-2 minutes. - Wash carefully under running tap water,drain , blot dry and examine under oil immersion objective. SMEAR EXAMINATION

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22. For culture the swabs are inoculated on: - Loefflers serum slope. - Tellurite Blood Agar - Blood Agar CULTURE

23. Loefflers serum slope: - Growth appears in 6-8 hours. - If negative the media will have to be incubated for 24 hours. - Stain with Alberts Stain , will show the bacilli with metachromatic granules. Contd.....

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25. Tellurite Blood Agar: - It takes 2 days for manifestation of colonies. - The colonies show characteristics gray to black colour on tellurite agar. Contd....

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27. - The identification of an isolate as C. diphtheriae does not mean that the patient has diphtheria. - Diagnosis of diphtheria depends on showing that the isolate produce diphtheria toxin - This can be done either in : In vivo (animal inoculation) In vitro ( Eleks test) VIRULENCE TEST

28. Subcutaneous test: - Inject broth from culture into two guinea pigs 0.8 ml - One animal given 500 units of antitoxin before. - Others no antitoxin. - Animals not given antitoxin will die. LEADS TO LOSS OF ANIMALS. SO,WE DO NOT DO IT PRESENT. In Vivo (Animal Inoculation)

29. Eleks gel precipitation test: - Tested on the horse serum agar. - Filter paper impregnated with diphtheria antitoxin 1000 units/ml. - positive/Negative/Test strain tested for immunodiffusion. - Line of precipitation - Test positive. In Vitro

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31. Treatment

32. Immunization: A. Active immunization. B. Passive immunization. C. Combined immunization . . Schick test Prophylaxis

33. a) Single preparation: These include formol toxoid, alum precipitated toxoid (APT) and purified toxoid. b) Combined preparation: These consist of diphtheria toxoid in combination with tetanus toxoid and killed suspension of pertusis bacilli. Active Immunization

34. DPT - Triple Vaccine. - DPT and oral polio vaccine are given at the age of 6 weeks .(3 doses of DPT at least 4 weeks apart). - Booster dose is given at school entry at 5-6 years. - Given by intramuscular route. Contd....

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36. This is an emergency measure and to be employed when non immunized individuals are exposed to infection. It is given subcutaneously 500-1000 units of antitoxin or antidiphtheritic serum (ADS). Passive Immunization

37. Both active immunization with toxoid and antitoxin. Combined Immunization

38. Schick Test

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