Diphtheria Bacterial pharyngitis.pptx

MICROBIOLOGY OF DIPHTHERIA

1. Bacterial Pharyngitis

2. DIPHTHERIA 2

3. Introduction Diphtheria - highly infectious childhood disease caused by - Corynebacterium diphtheria . Primarily infects - throat and produces toxin (diphtheria toxin) - causes an exudative pharyngitis and membranous tonsillitis. 3

4. Introduction (Cont..) Genus Corynebacterium - gram-positive, noncapsulated , non- sporing , non-motile bacillus. Irregularly stained - shows club-shaped swellings. 4 Club-shaped bacilli in methylene blue-stained smear

5. Introduction (Cont..) 1. Chinese letter or cuneiform arrangement: Appear as V- or L-shaped in smear - bacterial cells divide and daughter cells tend to lie at acute angles to each other - snapping type of division . 2. Metachromatic granules: Present at ends or poles of the bacilli. 5 Gram-stained smear shows V- or L-shaped bacilli with cuneiform arrangement

6. Metachromatic Granules Also called polar bodies or Babes–Ernst bodies or volutin granules). Storage granules of the organism, composed of polymetaphosphates Granules - stained strongly gram-positive compared to remaining part of the bacilli. Granules take up bluish purple metachromatic color - stained with Loeffler’s methylene blue 6

7. Metachromatic Granules (Cont..) Better stained with special stains - Albert’s, Neisser’s and Ponder’s stain. Granules - well developed on enriched media - blood agar or Loeffler’s serum slope Volutin granules - also possessed by - Corynebacterium xerosis and Gardnerella vaginalis. 7

8. History Ancient disease, known since the time of Hippocrates. First recognized by Pierre Bretonneau (1826) - (Greek word diphtheros — meaning leather like) - leathery pseudomembrane formation over the tonsil First observed by Klebs (1883) First cultivated by Loeffler (1884) - Klebs-Loeffler bacillus . 8

9. Virulence Factors (Diphtheria Toxin) Toxin -polypeptide chain, comprises of two fragments—A (active) and B (binding) Fragment B binds to the host cell receptors - helps in entry of fragment A 9

10. Mechanism of Diphtheria Toxin (DT ) Fragment A -active fragment - causes ADP ribosylation of elongation factor 2 (EF-2) → leads to inhibition of EF-2 → leads to inhibition of translation step of protein synthesis. 10

11. Factors Regulating Toxin Production Phage coded: β- corynephage - carrying tox gene. Iron concentration: 0.1 mg per liter Other species: Also produced by C. ulcerans and C. pseudotuberculosis. 11

12. Toxoid is used for Vaccination Antigenic and antitoxins are protective in nature. However, as It is virulent - cannot be given directly for vaccination. Toxin - converted to toxoid - used for vaccination. Toxoid - form of toxin – virulence is lost, retaining its antigenicity 12

13. Toxoid is used for Vaccination (Cont..) Toxoid formation - promoted by formalin, acidic pH and prolonged storage Park William 8 strain - preparation of vaccine LF unit - Limit of flocculation (Lf) unit. 13

14. Pathogenicity and Clinical Manifestations Diphtheria is toxemia but never a bacteremia Bacilli are noninvasive - secrete the toxin - spreads via bloodstream to various organs Toxin responsible for all types of manifestations - local (respiratory) and systemic complications 14

15. Respiratory Diphtheria Most common form of diphtheria. Tonsil and pharynx ( faucial diphtheria) - most common sites followed by nose and larynx. Incubation period - 3–4 days. 15

16. Respiratory Diphtheria (Cont..) Faucial diphtheria: Diphtheria toxin elicits an inflammatory response - leads to necrosis of the epithelium and exudate formation and mucosal ulcers - lined by tough leathery greyish white pseudomembrane coat. 16 Pseudomembrane covering the tonsils classically seen in diphtheria

17. Respiratory Diphtheria (Cont..) Extension of pseudomembrane - into the larynx and bronchial airways - asphyxia. Bull-neck appearance - Tonsillar swelling and neck edema - foul breath, thick speech, and stridor (noisy breathing) 17 Bull neck appearance

18. Cutaneous Diphtheria Punched-out ulcerative lesions with necrosis, due to the organism itself and is not toxin-mediated. Also - caused by nontoxigenic strains. Increasing incidence, especially in vaccinated children. 18

19. Systemic Complications Neurologic manifestations - Toxin mediated non-inflammatory demyelinating disorder presented with: Cranial nerve involvement & Peripheral neuropathy Ciliary paralysis Myocarditis Typically associated with arrhythmias and dilated cardiomyopathy. 19

20. Laboratory diagnosis of Diphtheria Specimen: Throat swab and a portion of pseudomembrane Direct smear Gram stain: Club shaped gram-positive bacilli with Chinese letter arrangement Albert’s stain: Green bacilli with bluish black metachromatic granules 20

21. Laboratory diagnosis of Diphtheria (Cont..) Culture media Enriched medium: Blood agar, chocolate agar and Loeffler’s serum slope Selective medium: Potassium tellurite agar and Tinsdale medium, produces black colonies 21 Loeffler’s serum slope; Potassium tellurite agar shows black colonies.

22. Laboratory diagnosis of Diphtheria (Cont..) Identification Biochemical tests such as sugar fermentation tests using Hiss’s serum sugar media Automated identification systems such as MALDI-TOF or VITEK 22

23. Laboratory diagnosis of Diphtheria (Cont..) Diphtheria toxin demonstration In vivo tests (Guinea pig inoculation): Subcutaneous and intracutaneous tests In vitro tests: Elek’s gel precipitation test Detection of tox gene-by PCR Detection of toxin-by ELISA or ICT Cytotoxicity on cell lines. 23

24. Laboratory diagnosis of Diphtheria (Cont..) Diphtheria toxin demonstration In vivo tests (Guinea pig inoculation): Subcutaneous and intracutaneous tests In vitro tests: Elek’s gel precipitation test Detection of tox gene-by PCR Detection of toxin-by ELISA or ICT Cytotoxicity on cell lines. 24

25. Elek’s gel precipitation test 25

26. Elek’s gel precipitation test (Cont..) Isolates 1 to 4 are toxigenic strains Isolates 1 and 2: Precipitation bands crossed over – toxins are not-identical - strains are unrelated Isolate 2 and 3: Partial fusion of precipitation bands - strains are partially related to each other Isolates 3 and 4: Precipitation bands fused with each other - strains are completely related Isolate 5 : non-toxigenic strain (no precipitation band is formed). 26

27. Typing of C. diphtheriae Useful for epidemiological studies, to know the relatedness between the isolates. Biotyping - use in the past - four biotypes —gravis, intermedius, mitis and belfanti . Vary in virulence and toxin production -gravis100% toxigenic and more virulent. 27

28. Epidemiology Incidence decreasing – widespread vaccination coverage Source of infection - Carriers (95%) & cases (5%) Carriers (0.1% to 5%) Temporary (persist for a month) or chronic (persist for a year). Nasal carriers (more dangerous - frequent shedding) & throat carriers 28

29. Epidemiology (Cont..) Transmission - respiratory droplets or rarely by contact with infected skin Reservoir - Humans are the only reservoir Age - Common age affected - 1–5 years. Shift from preschool to school age with immunization Newborns - maternal antibodies protective Global situation - Due to wide spread immunization, cases were drastically declined by >95% over last 3 decades. 29

30. Epidemiology (Cont..) Resurgence of diphtheria In 2019, outbreaks – Tamil Nadu, Kerala and Karnataka and few other states Majority (>70%) of cases - children 5–10 years or more - low coverage of diphtheria vaccine especially the booster doses - primary cause of its resurgence Waning immunity in adults - minor cause contributes to adult diphtheria. 30

31. Treatment of Diphtheria Treatment - started immediately on clinical suspicion of diphtheria. Antidiphtheritic serum or ADS (antitoxin): Passive immunization - antidiphtheritic horse serum - neutralizes the toxin. A test dose should be given to check for hypersensitivity It is given either IM or IV and the dose depends on stage of illness: 31

32. Treatment of Diphtheria (Cont..) Early stage (< 48 hours): 20,000–40,000 units If pharyngeal membranes present: 40,000–60,000 units Late stage (> 3 days, with bull neck): 80,000–120,000 units. Human antitoxin therapy is under development. 32

33. Treatment of Diphtheria (Cont..) Antibiotics: Penicillin or erythromycin is the drug of choice. If given early (<6 h of infection), before the toxin release Prevent further release of toxin by killing the bacilli Treatment of cutaneous diphtheria Treatment of carriers: Drug of choice is erythromycin. 33

34. Prophylaxis - Post-exposure Prophylaxis For close contacts (e.g. household), booster dose of diphtheria vaccine + penicillin G (single dose) or erythromycin (7–10 days) is recommended. 34

35. Prophylaxis - Vaccination Protective titer >0.01 Unit/mL of antitoxin Vaccine not effective for - cutaneous diph . & carrier state Types of Vaccine: Single vaccine : Diphtheria toxoid (alum or formal precipitated) Combined vaccine : 35

36. Prophylaxis – Vaccination (Cont..) Combined vaccine : DPT : Contains DT (diphtheria toxoid), Pertussis (whole cell) & TT DaPT : Contains DT, TT and acellular pertussis ( aP ) Td: Contains tetanus toxoid and adult dose (2 Lf ) of diphtheria toxoid Pentavalent vaccine: DPT + hepatitis B and Haemophilus influenzae type b. 36

37. Prophylaxis – Vaccination (Cont..) Administration of Diphtheria Vaccine: Schedule: Under National Immunization Schedule (NIS) of India 2020 (Chapter 20, Table 20.4): Children: Total seven doses are given. Three doses at 6, 10 and 14 weeks of birth Booster doses at 16–24 months & 5 years 37

38. Prophylaxis – Vaccination (Cont..) Site: deep intramuscularly (IM) at anterolateral aspect of thigh Thiomersal (0.01%) - preservative Storage: - kept at 2–8 o C; if accidentally frozen - discarded Dose: The usual dose (given to children) - 25 Lf units, adult dose - 2 Lf units 38

39. Prophylaxis – Vaccination (Cont..) Adult immunization: Td vaccine recommended for adults >18 years who have completed their primary vaccination schedule - booster dose once in every 10 years till the age of 65 Adults who have not completed their primary vaccination schedule: 3 doses of Td given at 0, 1 month, and 1 year. 39

40. Adverse Reactions following DPT Administration Mild - Fever and local reaction (swelling and indurations) Severe: Whole cell killed vaccine of B. pertussis is encephalitogenic . Hence, DPT is not recommended after 7 years of age Absolute contraindication to DPT Hypersensitivity to previous dose Progressive neurological disorder 40

41. DIPHTHEROIDS 41

42. DIPHTHEROIDS Also called – Coryneform bacteria Nondiphtherial corynebacteria Normal commensals in throat, skin, conjunctiva Invasive disease in immunocompromised patients 42

43. DIPHTHEROIDS (Cont..) Differentiated from C. diphtheriae by many features: Stains more uniformly than C. diphtheriae Palisade arrangement: Arranged in parallel rows rather than cuneiform pattern Absence of metachromatic granules (except C. xerosis ). 43

44. DIPHTHEROIDS (Cont..) Coryneforms that are rarely pathogenic to man are: C. ulcerans and C.pseudotuberculosis produce diphtheria toxin and cause localized ulcerations in throat C. ulcerans causes infections in cows. Human infections may occur through cow's milk C. pseudotuberculosis ( Preisz – Nocard bacillus) – Animal pathogen. Human infection very rare. 44

45. DIPHTHEROIDS (Cont..) Coryneforms that are rarely pathogenic to man are (Cont..): C. Minutissimum : Localized infection of skin (axilla and groin) ‘ erythrasma ’ Wood’s lamp - emit coral red color 45

46. DIPHTHEROIDS (Cont..) Coryneforms that are rarely pathogenic to man are (Cont..): C. Jeikeium : Lipophilic, colonizes skin of hospitalized patients Can cause bacteremia , endocarditis and meningitis, especially in immunocompromized Usually multidrug resistant, responds only to vancomycin 46

47. DIPHTHEROIDS (Cont..) Coryneforms that are rarely pathogenic to man are (Cont..): C. Urealyticum Skin commensal, rarely causes urinary tract infection (pyelonephritis) & alkaline encrusted cystitis (struvite stones in alkaline urine) in immunocompromized 47