Diphtheria , Corynebacterium diphtheria

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Diphtheria , Corynebacterium diphtheria

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Diphtheria , Corynebacterium diphtheria

  1. 1. DiphtheriaCorynebacterium diphtheria Kleb’s – Lofflers bacilli. Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 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 2
  3. 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 3
  4. 4. Corynebacterium diphtheria Dr.T.V.Rao MD 4
  5. 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 5
  6. 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 6
  7. 7. Classification McLeodClassified in to 3 Types1 Gravis2 Intermedius3.MitisGravis produce Most serious Hemorrhagic Paralytic complications - EpidemicIntermedius HemorrhagicMitis - obstructive complications, EndemicGeographic locations differTesting for toxigenicity is more important, Dr.T.V.Rao MD 7
  8. 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. MD Dr.T.V.Rao 8
  9. 9. Antigenic structure• Gravis 13,• Intermedius 4• Mitis 40• Bacteriophage typing 15 types Dr.T.V.Rao MD 9
  10. 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 10
  11. 11. Resistance• Can be killed at 58o c in 10 minute 100oc in 1 minuteSurvive in Blankets, Floor dust, toys inanimate objects Dr.T.V.Rao MD 11
  12. 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 12
  13. 13. Pharyngeal diphtheria• 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 Dr.T.V.Rao MD 13
  14. 14. Staining methods• Gram’s method• Alberts stain• Neissers stain• Panders 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 14
  15. 15. Cultural characters• Need enrichment Media• Containing• Blood, Serum or Egg 370 c ph. 7.4• Aerobic/Facultative anaerobic.• Commonly used medium• Loeffler serum slope,• Tellurite Blood agar, Dr.T.V.Rao MD 15
  16. 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 MediumCommonly used medium Tellurite blood agar Contains tellurite 0.04 tellurite Inhibits other bacteria Produce Grey/Black colonies. Dr.T.V.Rao MD 16
  17. 17. Biochemical Reactions• Acid production on Glucose,Galactose Maltose, DextrinDo not produce acid with Lactose, Mannitol, sucrose. All fermentation reactions tested in Hiss serum sugarsUrease test negative. Dr.T.V.Rao MD 17
  18. 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 18
  19. 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 19
  20. 20. Toxin ( Contd )• Toxin contain two components A 24,000 B 38,000A produce toxigenicity by proteolytic effectB Produce bindingToxin + Formalin = ToxoidWhat is Toxoid – Antigenic, not toxigenicTox + Corynephage Toxin production Dr.T.V.Rao MD 20
  21. 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 21
  22. 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 22
  23. 23. Diphtheria - Pseudo membrane• This may obstruct the airway and result in suffocation. Dr.T.V.Rao MD 23
  24. 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 Dr.T.V.Rao MD 24 form.
  25. 25. Elek plate for Demonstration of Toxin Dr.T.V.Rao MD 25
  26. 26. Animal testings (obsolete)
  27. 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 27
  28. 28. Dr.T.V.Rao MD 28
  29. 29. Pathogenicity• Incubation 3 – 4 days / one day• Faucal / Nasal /Laryngeal / Otic / Conjunctival,/Genital / Vulvae and Cutaneous ManifestationsDiphtheria is a toxemic condition. Malignant Sever toxemia ,Adenitis Bull neck Circulatory failureSeptic Gangrene , pseudo membrane. Dr.T.V.Rao MD 29
  30. 30. Pathogenicity• Hemorrhagic Epistaxis , Purpura General Bleeding tendencyAsphyxia , Acute circulatory failure,Paralysis Pneumonia, Septic shock, Otitis media. Toxemia, Necrotic changesDeath in Guinea pigs Dr.T.V.Rao MD 30
  31. 31. C. diphtheriaPathogenesis and ImmunityC. diphtheria occurs in therespiratory tract, in wounds, or onthe skin of infected persons ornormal carriers. It is spread bydroplets or by direct contact.Portal of entry: respiratory tract orskin abrasions. Dr.T.V.Rao MD 31
  32. 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 32
  33. 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 33
  34. 34. Systemic Toxigenic effects• Systemic toxigenic effects: necrosis in heart muscle, liver, kidneys and adrenals. Also produces neural damage. Dr.T.V.Rao MD 34
  35. 35. Dr.T.V.Rao MD 35
  36. 36. Complications• Asphyxia - causing mechanical obstruction.• May need tracheotomy• Circulatory failure.• Post Diphtheria paralysis Dr.T.V.Rao MD 36
  37. 37. Non toxigenic clinical manifestations• Bacteria can produce 1Endocarditis, 2Meingitis, 3 Cerebral abscess. 4 Osteoarthritis. Dr.T.V.Rao MD 37
  38. 38. Diagnosis of Diphtheria Dr.T.V.Rao MD 38
  39. 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 39
  40. 40. Collection of Specimens• Throat swabs• Smear examinations Gram s staining, Alberts, PondersImmunoflorescent methodsCultures on Loeffers serum slope Tellurite Blood agar, Blood agar. Dr.T.V.Rao MD 40
  41. 41. Dr.T.V.Rao MD 41
  42. 42. Growing the C.diptheria• Serum slope – Growth in 6 – 8 hours,• Stain with Neisses stain, Alberts stain• Bacilli have metachromatic granules,• Tellurite Blood agar takes two days for manifestation of colonies, Dr.T.V.Rao MD 42
  43. 43. Colonial morphologyBlack colonies on tellurite agar Dr.T.V.Rao MD 43
  44. 44. Virulence tests,• In Vivo and In Vitro• In Vivo in Animals• Subcutaneous tests Inject broth from culture into two Guinea pigs, 0.8 mlOne animal given 500 units of antitoxin beforeOther 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 44
  45. 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 45
  46. 46. Elek’s Test for In vitro toxigenicity Testing Dr.T.V.Rao MD 46
  47. 47. Schick Test• Injection of toxin Intradermal route• Produces redness/erythemati c in 2-4 days• No reaction – Protective immunity present. Dr.T.V.Rao MD 47
  48. 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 48
  49. 49. Prophylaxis• Immunization• Active – Passive• Both passive and Active.• Herd Immunity.• Schick test• Immunization with Antitoxin Dr.T.V.Rao MD 49
  50. 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 50
  51. 51. Passive Immunization• Given in Acute infections• Give Subcutaneously• 500 – 1000 Units of Antitoxin• Given as Horse Serum• Combined in Acute Infections ( Both Active Immunization with Toxoid and Antitoxin. Dr.T.V.Rao MD 51
  52. 52. Treatment• Antibiotic not useful in Acute infections,• Antitoxin a must and primary concern• Anti toxin obtained from horse serum• Mild 20,000 to 40,000• Moderate 40,000 to 60,000• Severe 80,000 to 1,00,000• Commonly used antibiotics,• Penicillin parentally,• Oral Erythromycin Dr.T.V.Rao MD 52
  53. 53. Treating Contacts•All contacts are advised to receive 500 mg Erythromycin 4 times a day. Dr.T.V.Rao MD 53
  54. 54. Summary• Diphtheria is still prevalent in many parts of the world – Risk of imported disease and outbreaks – Continuing need for vaccination• Toxin-mediated disease• Largely unexplored pathogenesis• Immense opportunities for research, especially in the post-genomic era
  55. 55. Other Corynebacterium• C.ulcerans• Like C.diptheria• Like Gravis type gelatin liquefied• Transmitted through cows Milk• Erythromycin effective.• Diphtheria antitoxin is protective. Dr.T.V.Rao MD 55
  56. 56. Diptheroids• Resembles C.diptheria• Commensals in throat, skin,• C.hofmani• C.xerosi• Propioniebacterium• P.acnes P.granulosum Dr.T.V.Rao MD 56
  57. 57. Other Corynebacterium SpeciesThey are ubiquitous in plants and animals. Many are found aspart of human normal flora and may cause opportunisticinfections, such as pneumonia, endocarditis, and soft tissueand bone infections, in immunocompromised patients. C. jeikeium: sepsis, endocarditis, wound infections, foreign body infections. C. urealyticum causes UT infections. It is a strong urease producer, infection of UT may lead to formation of stones. C. ulcerans is closely related to C. diphtheriae. May cause diphtheria-like disease.Resistant to many antibiotics. Treatment of bacteremia orendocarditis must be guided by antibiotic susceptibility tests. Dr.T.V.Rao MD 57
  58. 58. Other Corynebacterium Species• They are ubiquitous in plants and animals. Many are found as part of human normal flora and may cause opportunistic infections, such as pneumonia, endocarditis, and soft tissue and bone infections, in immunocompromised patients.• C. jeikeium: sepsis, endocarditis Dr.T.V.Rao MD 58
  59. 59. Other Corynebacterium Species• C. urealyticum causes UT infections. It is a strong urease producer, infection of UT may lead to formation of stones.• C. ulcerans is closely related to C. diphtheriae. May cause diphtheria-like disease.• Resistant to many antibiotics. Treatment of bacteremia or endocarditis must be guided by antibiotic susceptibility tests. Dr.T.V.Rao MD 59
  60. 60. • Programme Created byDr.T.V.Rao MD for Medicaland Paramedical Students Email doctortvrao@gmail.com Dr.T.V.Rao MD 60

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