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.• 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

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 diseaseColonies: large, irregular, gray

2)mitis(40 types)-mild illnesscolonies: small, round, convex, black

3)intermedius(4 types)-intermediate severityColonies: 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 breathingContents: WBC, RBC, bacteria, disintegrating epithelial cellsCause: 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, fevercomplications: 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 sequelaeii) Septic diphtheria:

Signs: ulceration with pseudomembrane formation and cellulites (gangrene around pm)

iii) Hemorrhagic diphtheriaSigns: local and general bleeding from edge of psudomembrane, conjunctival, epistaxis and purpura

Complications

1)Asphyxia-obstruction of resp tract2)Acute circulatory failure3)kidney failure 4)paralysis-soft palate, eye muscles, extremities (3rd-4th 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 vivoGuinea 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 pigsGP 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

EPIDEMIOLOGYEPIDEMIOLOGY

• Formally important pediatric diseaseFormally important pediatric disease• Developed countries - EradicatedDeveloped countries - Eradicated• Developing countries serious problemDeveloping countries serious problem• Rare in 1Rare in 1stst year – maternal antibodies year – maternal antibodies• Peak 1-5 years, 5-10 years- decreases there afterPeak 1-5 years, 5-10 years- decreases there after• Immunity – sub clinical infectionsImmunity – sub clinical infections• Carriers outnumber cases – throat & nasalCarriers outnumber cases – throat & nasal• Rarely spread through milkRarely spread through milk

Prevention• Active immunity- DPT dosage

– Antitoxin level – 0.01/ ml protectiveAntitoxin level – 0.01/ ml protective

• Passive immunity- ADS– When susceptibles are exposedWhen susceptibles are exposed– 500 – 1000 units sc of Anti Diphtheritic Serum ( ADS)500 – 1000 units sc of Anti Diphtheritic Serum ( ADS)

• Combined immunisation– First dose of adsorbed toxoid on one armFirst dose of adsorbed toxoid on one arm– ADS on another armADS on another arm– To be continued with full course of active To be continued with full course of active

immunizationimmunization

Preventiona) DTP (DPT)- • Formal toxoid - Incubation of Toxin at pH 7.4 -7.6 for 3 – Formal toxoid - Incubation of Toxin at pH 7.4 -7.6 for 3 –

4 weeks at 37 4 weeks at 37 °°C C • Adsorbed toxoid – purified toxoid adsorbed on to Adsorbed toxoid – purified toxoid adsorbed on to

aluminum phosphate or hydroxidealuminum 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 - 4th 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)

Treatment

• Antitoxin – 20,000 to 1 lakh unitsAntitoxin – 20,000 to 1 lakh units

• Antibiotic therapy – PenicillinAntibiotic therapy – Penicillin

• Carriers - ErythromycinCarriers - Erythromycin

Patch in the throat

• Diphtheria

• Streptococcal tonsillitis

• Oral thrush– Candida albicans

• Spirochete- Borrelia vincenti

Other Corynebacteria

1. C. ulcerans:

– Diphtheria like lesions

– Spread thru milk

– Antitoxin protective, erythromycin

2. C.minutissimum- – Erythrasma

3. C. tenuis:- Trichomycosis axillaris

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.

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