Introduction Klebs1883 discovered Loefflers1884 cultured Also known as

Introduction • • Klebs--1883 discovered Loefflers--1884 cultured Also known as KLB Emil von Behring- 1890 produced antitoxin • Awarded nobel prize Emil Von Behring

Morphology • Gram positive bacilli. 3 -6 μ x 0. 5 -0. 8 μ. • Chinese letter pattern, angular arrangement • Metachromatic granules. volutin granules, polymetaphosphate energy storage depots • Alberts stain – green and bluish black • Nonmotile noncapsulated, nonsporing

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

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

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

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

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

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.

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

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

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

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

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

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

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)

Elek's gel precipitation test

EPIDEMIOLOGY • • Formally important pediatric disease Developed countries - Eradicated Developing countries serious problem Rare in 1 st 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

Prevention • Active immunity- DPT dosage – 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

Prevention a) DTP (DPT) • Formal toxoid - Incubation of Toxin at p. H 7. 4 -7. 6 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.

b) Schedule i) primary immunization – - infants and children - 3 doses, 4 -6 weeks - 4 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)