1. Corynebacterium diphtheriae

2. Introduction Klebs--1883 discovered Loefflers--1884 cultured
Also known as KLB
Emil von Behring produced antitoxin
Awarded nobel prize
Emil Von Behring

3. Morphology Gram positive bacilli. 3-6 μ x 0.5-0.8 μ.
v or k or L shape.
Chinese letter pattern, angular arrangement
Metachromatic granules. volutin granules, polymetaphosphate energy storage depots
Alberts stain – green and bluish black
Nonmotile noncapsulated, nonsporing
pleomorphic

5. Cultivation Loefflers serum slope– creamy white colonies in 6-8 hrs
Potassium tellurite medium—black colonies
Blood agar

7. Biochemical reactions
Hiss serum
Ferments glucose ,maltose with acid only
Lactose, sucrose, mannitol not fermented
Urease negative
Starch –only gravis type, not intermedius or mitis

8. Pathogenicity Produces exotoxin Lysogenic conversion with beta phage
Toxin – heat labile protein
A and B fraction
Toxicity- disease
Antigenicity- immunity
Toxoid – toxin without toxicity

9. Toxin Mechanism of action
-B subunit binds to receptor -toxin molecule cleaved at protease sensitive site between
A-B portion (remain bonded by disulfide link) -receptor-toxin complex englufed into vesicle of host cell -reduction of disulfide bond, A subunit released into cytoplasm -Necrosis/neuotoxic effects on organism by exotoxin

10. Biotypes
1)gravis(13 types)-most serious disease Colonies: large, irregular, gray
2)mitis(40 types)-mild illness colonies: small, round, convex, black
3)intermedius(4 types)-intermediate severity Colonies: small, flat and gray

11. Diphtheria Children, fatal if not treated in time
Exclusively human disease
Droplet infection- fomites
Fever, cervical lymphadenopathy, pseudomembrane
Myocarditis arrhythmia fatal
Polyneuropathy, paltine paralysis
Rare in adults.

12. Pseudomembrane formation
Thick membrane lining throat that interferes with eating, drinking and breathing Contents: WBC, RBC, bacteria, disintegrating epithelial cells Cause: fibrinous exudate from local necrosis by exotoxin (removal of PF may cause bleeding) Localization: faucial, laryngeal, nasal, otitic, conjunctival, genital, cutaneous

13. Septic: Ulceretic: Faucial:
Signs: sore throat, fever complications: prostration, dyspnea, asphyxia, arhythmia,
difficulties of speech, vision, swallowing, movement
Septic:
Ulceretic:

14. Clinical classification
i) Malignant (hypertoxic) diphtheria
Signs: severe toxemia and adenitis, lymph glands
swelling in the neck Complications: death-circulatory failure, paralytic
sequelae
ii) Septic diphtheria: Signs: ulceration with pseudomembrane formation and cellulites (gangrene around pm)
iii) Hemorrhagic diphtheria Signs: local and general bleeding from edge of psudomembrane, conjunctival, epistaxis and purpura

15. Complications
1)Asphyxia-obstruction of resp tract 2)Acute circulatory failure 3)kidney failure 4)paralysis-soft palate, eye muscles, extremities (3rd-4th week) 5)septic sequelae-pnemonia, otitis media

16. Laboratory diagnosis
Sample collection: Throat swab or swab from membrane
Microscopy: Gram stain and Alberts stain
Culture: Loefflers and PT
Biochemicals
Virulence test in vivo and in vitro

17. Virulence test contd In vivo In vitro Eleks gel precipitation test
Guinea pig– subcutaneous
intradermal
Test and control animals to be used
In vitro
Eleks gel precipitation test

18. Toxigenicity test (virulence test)
i) Animal inocculation -bacteria culture emulsified in water and 0.8 ml injected into 2 guinea pigs GP A-has dipht antitoxin (injected 2 hours before) GP B-Doesn't have antitoxin
Result: Guinea pig B dies.
ii) Elek's gel precipitation test -filter paper saturated with antitoxin is placed on agar plate with 20% horse serum -bacterial culture streaked at right angles to filter paper
iii) tissue culture test -incorporation of bacteria into agar overlay of eukaryotic cell culture monolayers. Result: toxin diffuses into cells and kills them
iv) PCR assays -test presence of specific bacteriophate gene (tox)

19. Elek's gel precipitation test

20. EPIDEMIOLOGY Formally important pediatric disease
Developed countries - Eradicated
Developing countries serious problem
Rare in 1st year – maternal antibodies
Peak 1-5 years, 5-10 years- decreases there after
Immunity – sub clinical infections
Carriers outnumber cases – throat & nasal
Rarely spread through milk

21. Prevention Active immunity- DPT dosage Passive immunity- ADS
Antitoxin level – 0.01/ ml protective
Passive immunity- ADS
When susceptibles are exposed
500 – 1000 units sc of Anti Diphtheritic Serum ( ADS)
Combined immunisation
First dose of adsorbed toxoid on one arm
ADS on another arm
To be continued with full course of active immunization

22. Prevention
DTP (DPT)-
Formal toxoid - Incubation of Toxin at pH for 3 – 4 weeks at 37 °C
Adsorbed toxoid – purified toxoid adsorbed on to aluminum phosphate or hydroxide
triple vaccine given to children.
trivalent preparation
Diphtheria toxoid, Tetanus toxoid and Pertussis vaccine
Td- contains absorbed tetanus and ten-fold smaller dose of diphtheria toxoid.

23. i) primary immunization –
Schedule
i) primary immunization –
- infants and children doses, 4-6 weeks th dose after a year booster at school entry
ii) Booster immunization
- adults Td toxoids used (traveling adults may need more)
Shick test-to test sensitivity (allergic reaction)

24. Treatment Antitoxin – 20,000 to 1 lakh units
Antibiotic therapy – Penicillin
Carriers - Erythromycin

25. Patch in the throat Diphtheria Streptococcal tonsillitis
Oral thrush– Candida albicans
Spirochete- Borrelia vincenti

26. Other Corynebacteria C. ulcerans: 2. C.minutissimum- 3. C. tenuis:
Diphtheria like lesions
Spread thru milk
Antitoxin protective, erythromycin
2. C.minutissimum-
Erythrasma
3. C. tenuis:
Trichomycosis axillaris

27. 4. C.pseudotuberculosis:
Veterinary importance
5. C.parvum:
Immunomodulator
6. A. haemolyticum:
pharyngitis, skin ulcers
7. C.jakieum:
Skin inf/ BSI in IC. Rx- Vancomycin.

28. Diphtheroids
Morphologically resembling diphtheria but do not cause any disease.
Present as commensals in throat, skin
Do not contain metachromatic granules.
Do not produce any toxin ie virulence test is negative.
C.hofmani, C.xerosis, C.pseudodiphtheriticum

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