Diphtheria Corynebacterium diphtheria Kleb’s – Loffler's bacilli.

1. DiphtheriaCorynebacterium diphtheriaKleb’s – Loffler's bacilli. Dr.T.V.Rao MD Dr.T.V.Rao MD

2. Corynebacterium • Gram + Non Acid fast, Non motile, • Irregularly stained with granules, • Club shaped swelling at one or both ends so the name • Important Pathogen Corynebacterium diphtheria, Diptheros meaning leather, Dr.T.V.Rao MD

3. Corynebacterium diphtheria • Slender rods • Clubbing at both ends • Pleomorphic • Non capsulate / Acid fast Gram + • Granules are composed of polymetapohosphate • Staining with Loeffler’s methylene blue show bluish purple metachromatic granules. with polar bodies, Dr.T.V.Rao MD

4. Corynebacterium diphtheria Dr.T.V.Rao MD

5. Corynebacterium • Classification • Corynebacterium  diphtheria  and diphtheroids (look  like  C. diphtheria)  are  Gram- positive, club  shaped  rods.   • Some  are saprophytic • Some   produce  disease   in   animals.   • C. diphtheria is the most important pathogen in the group. Dr.T.V.Rao MD

6. Corynebacterium • Morphology and cultural characteristics • Small Gram positive Bacilli; arrangement=palisade or Chinese letters • Growth on Blood agar – raised, translucent, gray colonies  Dr.T.V.Rao MD

7. Classification McLeod Classified in to 3 Types 1 Gravis 2 Intermedius 3.Mitis Gravis produce Most serious Hemorrhagic Paralytic complications - Epidemic Intermedius Hemorrhagic Mitis - obstructive complications, Endemic Geographic locations differ Testing for toxigenicity is more important, Dr.T.V.Rao MD

8. Corynebacterium • 3 morphological  types of C. diphtheria are  found on tellurite containing media: • Mitis – black colonies with a gray  periphery • Gravis– large,  gray  colonies • Intermedius – small, dull gray  to black. • All produce an immunologically identical toxin. Dr.T.V.Rao MD

9. Antigenic structure • Gravis 13, • Intermedius 4 • Mitis 40 • Bacteriophage typing 15 types Dr.T.V.Rao MD

10. Cultural Characteristics • Incubation -35-370 C for 24 hours. • They prefer a pH of 7.8-8.0 for good growth. • They require access to oxygen (poor AnO2 growth). • Catalase + Dr.T.V.Rao MD

11. Resistance • Can be killed at 58oc in 10 minute 100oc in 1 minute Survive in Blankets, Floor dust, toys inanimate objects Dr.T.V.Rao MD

12. What is Diphtheria • An infection of local tissue of Upper respiratory tract with production of toxin which causes systemic effects on Heart and Peripheral tissues, Dr.T.V.Rao MD

13. Insidious onset of exudative pharyngitis Exudate spreads to form adherent “pseudo membrane” can lead to respiratory obstruction and death by asphyxiation Fever not high, but patient appears toxic Pharyngeal diphtheria Dr.T.V.Rao MD

14. Staining methods • Gram’s method • Albert's stain • Neissers stain • Pander's stain • On staining seen as Pairs, Appear as v and L letters, resembling Chinese letter pattern or also called cuneiform arrangement. Dr.T.V.Rao MD

15. Cultural characters • Need enrichment Media • Containing • Blood, Serum or Egg 370c ph. 7.4 • Aerobic/Facultative anaerobic. • Commonly used medium • Loeffler serum slope, • Tellurite Blood agar, Dr.T.V.Rao MD

16. Cultural characters • Loeffler serum slope Grows rapidly in 6 -8 hours, Small white opaque disks Turns to yellow Tellurite blood agar Modified Mc Leod and Hoyle’s Medium Commonly used medium Tellurite blood agar Contains tellurite 0.04 tellurite Inhibits other bacteria Produce Grey/Black colonies. Dr.T.V.Rao MD

17. Biochemical Reactions • Acid production on Glucose,Galactose Maltose, Dextrin Do not produce acid with Lactose, Mannitol, sucrose. All fermentation reactions tested in Hiss serum sugars Urease test negative. Dr.T.V.Rao MD

18. Toxin • Pathogenicity associated with Toxin • Gravis/Intermedius 95-99% are toxigenic • Mitis 80 – 85% • Some abundant others poorly • Toxin production park William 8 • Toxin M W 62,000 0.0001mg can kill guinea pig weighing 250 grams Dr.T.V.Rao MD

19. C. diphtheria toxin • Toxin enters through receptor mediated endocytosis • Acidification of endocytic vesicle allows A to dissociate from B • A enters cycoplasm Dr.T.V.Rao MD

20. Toxin ( Contd ) • Toxin contain two components A 24,000 B 38,000 A produce toxigenicity by proteolytic effect B Produce binding Toxin + Formalin = Toxoid What is Toxoid – Antigenic, not toxigenic Tox + Corynephage Toxin production Dr.T.V.Rao MD

21. Toxin ( contd ) • Need iron 0.1 mg/liter. • Toxin inhibits protein synthesis • Fragment A catalyzes the transfer of ADP ribose from the Nicotinamide adenine dinucleotide ( NAD ) to the eukaryotic elongation factor 2 /(Fragment A inhibits polypeptide chain elongation in the presence of Nicotinamide adenine dinucleotide by inactivating elongation factor • Causes involvement with affinity. Myocarditis, Adrenals Nerve endings, Dr.T.V.Rao MD

22. Clinical features • Malaise, Sore throat, Fever • Adherent grey pseudo membrane • Nasal ulcers, • Obstruction of larynx and lower airways, • Difficulty in swallowing • Lead to Myocarditis, Peripheral neuritis, • Paralysis of limbs, Dr.T.V.Rao MD

23. Diphtheria - Pseudo membrane • This may obstruct the  airway  and result  in suffocation. Dr.T.V.Rao MD

24. Corynebacterium • To prove  that an isolate can cause diphtheria, one  must demonstrate  toxin  production. • This is most often done  on  an  Elek plate: • The  organism is streaked on a plate containing low iron. • A filter strip containing anti-toxin antibody is placed perpendicular to the streak of the organism. • Diffusion of the antibody into the medium and secretion of the toxin into the medium occur. • At the zone of equivalence, a precipitate will form. Dr.T.V.Rao MD

25. Elek plate for Demonstration of Toxin Dr.T.V.Rao MD

26. Animal testings (obsolete)

27. Pathogenicity • Bacteria Invade, Colonise,Proliferate • Bacteria are lysogenized by Beta phage • Produce toxin, • Kills epithelial and Neutrophils, • Produce Pharyngitis and cutaneous lesions. Dr.T.V.Rao MD

28. Dr.T.V.Rao MD

29. Pathogenicity • Incubation 3 – 4 days / one day • Faucal / Nasal /Laryngeal / Otic / Conjunctival,/Genital / Vulvae and Cutaneous Manifestations Diphtheria is a toxemic condition. Malignant Sever toxemia ,Adenitis Bull neck Circulatory failure Septic Gangrene , pseudo membrane. Dr.T.V.Rao MD

30. Pathogenicity • Hemorrhagic Epistaxis , Purpura General Bleeding tendency Asphyxia , Acute circulatory failure, Paralysis Pneumonia, Septic shock, Otitis media. Toxemia, Necrotic changes Death in Guinea pigs Dr.T.V.Rao MD

31. C. diphtheria Pathogenesis and Immunity C. diphtheria occurs in the respiratory tract, in wounds, or on the skin of infected persons or normal carriers. It is spread by droplets or by direct contact. Portal of entry: respiratory tract or skin abrasions. Dr.T.V.Rao MD

32. Diphtheria bacilli produce Toxin • Diphtheria bacilli colonize and grow on mucous membranes, and start to produce toxin, which is then absorbed into the mucous membranes, and even spread by the bloodstream. Dr.T.V.Rao MD

33. Toxicity of Diphtheria Toxin • Local toxigenic effects: elicit inflammatory response and necrosis of the faucial mucosa cells-- formation of "pseudo-membrane“ (composed of bacteria, lymphocytes, plasma cells, fibrin, and dead cells), causing respiratory obstruction. Dr.T.V.Rao MD

34. Systemic Toxigenic effects • Systemic toxigenic effects: necrosis in heart muscle, liver, kidneys and adrenals. Also produces neural damage. Dr.T.V.Rao MD

35. Dr.T.V.Rao MD

36. Complications • Asphyxia - causing mechanical obstruction. • May need tracheotomy • Circulatory failure. • Post Diphtheria paralysis Dr.T.V.Rao MD

37. Non toxigenic clinical manifestations • Bacteria can produce 1Endocarditis, 2Meingitis, 3 Cerebral abscess. 4 Osteoarthritis. Dr.T.V.Rao MD

38. Diagnosis of Diphtheria Dr.T.V.Rao MD

39. Laboratory Diagnosis • Specific treatment is more important than Laboratory Diagnosis. 1 Isolation of Diphtheria bacilli. 2.Testing for toxigenicity, Dr.T.V.Rao MD

40. Collection of Specimens • Throat swabs • Smear examinations Gram s staining, Alberts, Ponders Immunoflorescent methods Cultures on Loeffers serum slope Tellurite Blood agar, Blood agar. Dr.T.V.Rao MD

41. Dr.T.V.Rao MD

42. Growing the C.diptheria • Serum slope – Growth in 6 – 8 hours, • Stain with Neisse's stain, Albert's stain • Bacilli have metachromatic granules, • Tellurite Blood agar takes two days for manifestation of colonies, Dr.T.V.Rao MD

43. Colonial morphology Black colonies on tellurite agar Dr.T.V.Rao MD

44. Virulence tests, • In Vivo and In Vitro • In Vivo in Animals • Subcutaneous tests Inject broth from culture into two Guinea pigs, 0.8 ml One animal given 500 units of antitoxin before Other no Antitoxin. Animal not given antitoxin will die Leads to Loss of Animals. So we do not do it at present Dr.T.V.Rao MD

45. In Vitro Testing • Elek’s Gel precipitation testing • Filter paper impregnated with Diphtheria antitoxin 1000 Units / ml • Tested on the horse serum agar • Positive / Negative /Test strains tested for Immunodiffusion • Line of precipitation – test positive • Other methods testing in Tissue cultures. Dr.T.V.Rao MD

46. Elek’s Test for In vitro toxigenicity Testing Dr.T.V.Rao MD

47. Schick Test • Injection of toxin Intradermal route • Produces redness/erythematic in 2-4 days • No reaction – Protective immunity present. Dr.T.V.Rao MD

48. Epidemiology • Eradicated in developed nations, • Children between 2 – 5 years. • A symptomatic carriers • Person to person contact. • Carriers spread. • Prolonged contact. Dr.T.V.Rao MD

49. Prophylaxis • Immunization • Active – Passive • Both passive and Active. • Herd Immunity. • Schick test • Immunization with Antitoxin Dr.T.V.Rao MD

50. Active Immunization. • Toxoid – Toxin treated with Formaldehyde • Absorbed Toxoid • Given by Intramuscular route • Given in DTP –Triple Vaccine • Primary Immunization • Three Doses of DPT at least 4 weeks apart. • Non vaccinated • Three doses of Toxoid four weeks apart • One dose after One Year. Dr.T.V.Rao MD