Corynebacterium diphtheria
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CorynebacteriumCorynebacteriumDiphtheria Diphtheria
Dr.T.V.Rao MD.Dr.T.V.Rao MD.
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Diphtheria
• Greek diphtheria (leather hide)
• Caused by Aerobic Gram +ve rods
• Corynebacterium diphtheria
• Exotoxin production only if infected by virus phage infected carrying toxin gene
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CorynebacteriumCorynebacterium• 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,
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What is Diphtheria• An infection of
local tissue of URT with production of toxin which causes systemic effects on Heart and Peripheral tissues,
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DefinitionDefinition
• Diphtheria is an acute, toxin-mediated disease caused by toxigenic Corynebacterium diphtheria.
• It’s a very contagious and potentially life-threatening bacterial disease.
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Definition • It’s a localized infectious disease,
which usually attacks the throat and nose mucous membrane
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Etiology
• C. diphtheriae is an aerobic gram-positive bacillus.
–Pleomorphic, club-end–Non-spore-forming–Non-acid-fast–Non-motile
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Etiology• The major virulence determinant is
an exotoxin, diphtheria toxin. After binding to the host cells, the active subunit will interrupt the protein synthesis of the target host cell and results in cell death.
• Toxoid made from diphtheria toxin can be used as vaccine.
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Etiology• There are three biotypes —
gravis, Intermedius, and mitis. The most severe clinical type of this disease is associated with the gravis biotype, but any strain may produce toxin.
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Pathogenesis • Entry ------ the bacilli multiply locally in the
throat and elaborate a powerful exotoxin ----- produce local and systemic symptoms.
Local lesions : • Exotoxin causes necrosis of the epithelial cells
and liberates serous and fibrinous material which forms a grayish white pseudo membrane
• The membrane bleeds on being dislodged• Surrounding tissue is inflamed and edematous
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Fauces ( throat )
Fauces : - two pillars of mucous membrane. Anterior : known as the palatoglossal arch and Posterior : the palatopharyngeal archBetween these two arches is the palatine tonsil.
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Typical Presentation of Bull Neck
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Local manifestation Local manifestation
Depend on the site of lesion:
Nasal diphtheria : • Unilateral or bilateral
serosanguineous ( blood and serous fluid ) discharge from the nose
• Excoriation of upper lip • Toxemia is minimal
Faucial diphtheria : • Redness and swelling
over Fauces• Exudates on the
tonsils coalesces to form grayish white pseudo membrane
• Regional lymph nodes are inflamed
• Sore throat and • dysphagia
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Corynebacterium diphtheriaCorynebacterium diphtheria
• Slender rods• Clubbing at both ends• Pleomorphic• Non capsulate / Acid fast Gram +• Granules are composed of
polymetapohosphate• Staining with Loffler's methylene blue
show bluish purple metachromatic granules. with polar bodies,
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Staining methods• Grams method• Albert's stain
• Neissers stain
• Ponders stain
• On staining seen as
Pairs, Appear as v and L letters, resembling Chinese letter pattern or also called cuneiform arrangement.
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Cultural charactersCultural characters• Need enrichment Media
• Contain
• Blood, Serum or Egg 37 c ph 7.4
• Aerobic/Facultative anaerobic.
• Commonly used medium
• Loeffler serum slope,
• Tellurite Blood agar,
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Gram +ve Bacilli and Colonies
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• Selective & differential medium
• Corynebacterium are resistant to tellurite– Reduced to tellurium
• Forms deposit in colonies– Colonies appear dark
• Biotypes– gravis, Intermedius, mitis
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Culturing Culturing
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Growing on Culture Plates• Loffler serum slope Grows rapidly in
6 -8 hours, Small white
opaque disks Turns to yellow Tellurite blood agar
Modified Mac Leod
Hoyles medium.Dr.T.V.Rao MD 21
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Commonly used medium
• Tellurite blood agar Contains tellurite 0.04 tellurite Inhibits other bacteria
• Produce Grey/Black colonies.
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Classification of McLeodClassified in to 3 Types1 Gravis2 Intermedius3.MitisGravis produce Most serious Hemorrhagic Paralytic complications - Epidemic Intermedius Hemorrhagic Mitis - obstructive complications, Endemic Geographic locations differTesting for toxigenicity is more important,
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Biochemical ReactionsBiochemical Reactions
• Acid
Glucose,Galactose Maltose, Dextrin
Do not produce acid withDo not produce acid with Lactose, Mannitol, sucrose.All fermentation reactions tested in Hiss serum sugarsUrease test negative.Proteolytic
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Toxin 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 0.0001 can kill guinea pig
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Diphtheria toxin: Part A
• Active site• Enzyme
• Blocks protein synthesis– ADP-ribosyl transferase– elongation factor 2 (EF2)
• Specific for mammalian cells– Prokaryotes have different EF2
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Diphtheria Toxin: Part BDiphtheria Toxin: Part B• Binding Site• Binds to cell receptor• Bound receptor
internalized• Endosome
– Hydrolyzed by protease
– Disulfide broken– Part A released
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Activation of Diphtheria Toxin
A
A
B
B
A
B
A
B
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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
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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,
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Antigenic structureAntigenic structure
• Gravis 13,
• Intermedius 4
• Mitis 40
• Bacteriophage typing 15 types
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Resistance Resistance
• Can be killed at
580 c in 10 mt
1000 c in 1 mt
Survive in Blankets, Floor dust, toys inanimate objects
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PathogenicityPathogenicity
• Bacteria Invade, Colonise,Proliferate
• Bacteria are lysogenized by Beta phage
• Produce toxin,
• Kills epithelial and Neutrophils,
• Produce Pharyngitis and cutaneous lesions.
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Pathogenicity Pathogenicity
• Incubation 3 – 4 days / one day
• Faucal / Nasal /Laryngeal / Otic / Conjunctiva,/Genital / Vulvae
Coetaneous
Diphtheria is a toxemic condition.
Malignant Sever toxemia ,Adenitis Bull neck Circulatory failure
Septic Gangrene , pseudo membrane.Dr.T.V.Rao MD 34
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Pathogenicity • Hemorrhagic Epistaxis , Purpura General Bleeding tendencyAsphyxia , Acute circulatory failure,Paralysis Pneumonia, Septic shock, Otitis
media. Toxemia, Necrotic changesDeath in Guinea pigs
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Diphtheria• Nasopharyngeal
diphtheria– Pharyngeal – Laryngeal
• Cutaneous diphtheria
• Systemic complications
DIAGNOSIS MUST BE CLINICAL!!!!Dr.T.V.Rao MD 36
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Clinical features 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,
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Diphtheria Clinical FeaturesIncubation period 2-5 days
(range, 1-10 days)May involve any mucous membraneClassified based on site of infectionanterior nasalpharyngeal and tonsillarlaryngealcutaneousoculargenital
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Diphtheria Clinical FeaturesDiphtheria Clinical Features
Incubation period 2-5 days (range, 1-10 days)
May involve any mucous membraneClassified based on site of infectionanterior nasalpharyngeal and tonsillarlaryngealcutaneousoculargenital
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Thick MembraneThick Membrane
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Pseudo membrane
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Skin LesionsSkin Lesions
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Pathogenicity
1 Faucial Diphtheria very common,
• Malignant or Hyper toxic toxemia Marked adenitis, circulatory failure,
• Paralytic sequale
2 Septic ulceration cellulitis, gangrene Epistaxis Bleeding tendency,
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Complications
• Asphyxia - causing mechanical obstruction.
• May need tracheotomy
• Circulatory failure.
• Post Diphtheria paralysis Dr.T.V.Rao MD 45
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Non toxigenic clinical Non toxigenic clinical manifestationsmanifestations
• Bacteria can produce
1. Endocarditis,
2.Meingitis,
3 Cerebral abscess.
4 Osteoarthritis.
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Laboratory DiagnosisLaboratory Diagnosis
• Specific treatment is more important than Laboratory Diagnosis.
1 Isolation of Diphtheria bacilli.
2.Testing for toxigenicity,
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Collection of Specimens Collection of Specimens
• Throat swabs • Smear examinations Gram s staining, Albert's, PondersImmunoflorescent methodsCultures on Loeffers serum slope Tellurite Blood agar, Blood agar.
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Isolation of C.diptheria
• Serum slope – Growth in 6 – 8 hours,
• Stain with Neissers stain Albert's stain
• Bacilli have metachromatic granules,
• Tellurite Blood agar takes two days for manifestation of colonies,
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Virulence tests,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 antitoxinOther no Vaccine.Animal not given antitoxin will die Loss of Animals. Restricts its testing.
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Intracutaneous MethodIntracutaneous Method
• One animal given 500 units before toxin
• Other 50 units after Toxin
• So the Animals can be saved
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In Vitro Testing 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 55
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Toxigenicity Tests
In Vitro Elek test
In Vivo Animal inoculationrabbit skin test-
necrosis
guinea pig challenge test- lethal
low [Fe 2+] induces toxinDr.T.V.Rao MD 56
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Schick TesSchick Test ( Out dated )t ( Out dated )–Schick test: It is an intradermal test,
the test is carried out by injecting intradermally into the skin of forearm 0.2 ml of diphtheria toxin, while into the opposite arm is injected as a control, the same amount of toxin which has been inactivated by heat.
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Interpretation• Negative reaction: If a person had immunity to diphtheria,
no reaction will be observed on either arm.
• Positive reaction: An area of in duration 10-15 mm in diameter generally appears within 24-36 hours reaching its maximum development by 4-7 days, the control arm shows no change. The person is susceptible to diphtheria.
• False positive reaction: A red flush develops in both arms, the reaction fades very quickly, and disappears by 4th day. This is an allergic type of reaction found in certain individuals
• Combined reaction: the control arm shows pseudo positive reaction and the test arm is true +ve reaction, susceptible and need vaccinationDr.T.V.Rao MD 59
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Schick TestSchick Test
• Injection of toxin I D
• Produces redness/erythematic in 2-4 days
• No reaction – Protective immunity present.
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EpidemiologyEpidemiology
• Eradicated in developed nations,
• Children between 2 – 5 years.
• A symptomatic carriers
• Person to person contact.
• Carriers spread.
• Prolonged contact.
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ProphylaxisProphylaxis
• Immunization
• Active – Passive
• Both passive and Active.
• Herd Immunity.
• Schick test
• Immunization with AntitoxinDr.T.V.Rao MD 63
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Active Immunization.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.
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Prevention
Vaccination: Immunisation with diphtheria toxoid, combined with tetanus and pertussis toxoid (DTP vaccine), should be given to all children at two, three and four months of age. Booster doses are given between the ages of 3 and 5 .
The child is given a further booster vaccine before leaving school and is then considered to be protected for a further 10 years (16 – 18 years).
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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.
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Treatment Treatment • Antibiotic not useful in Acute infections,• Antitoxin a must.• 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
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Diphtheria EpidemiologyDiphtheria EpidemiologyReservoir Human carriers
Usually asymptomatic
Transmission Respiratory Skin and fomites rarely
Temporal pattern Winter and spring
Communicability Up to several weekswithout antibiotics
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Treating ContactsTreating Contacts
• All contacts are advised to receive
500 mg Erythromycin 4 times a day.
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Other CorynebacteriumOther Corynebacterium
• C.ulcerans
• Like C.diptheria
• Gravis type gelatin liquefied
• Transmitted through cows Milk
• Erythromycin effective.
• Diphtheria antitoxin is protective.Dr.T.V.Rao MD 71
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Diptheroids
• Resembles C.diptheria
• Commensals in throat, skin,
• C.hofmani
• C.xerosi
• Propioniebacterium
• P.acnes P.granulosum Dr.T.V.Rao MD 72
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• Programme Created by Dr.T.V.Rao Programme Created by Dr.T.V.Rao MD for Medical and Paramedical MD for Medical and Paramedical Students in Developing World Students in Developing World
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