Post on 23-Aug-2014
description
Diphtheria Corynebacterium diphtheria
Kleb’s – Loffler's bacilli.
Dr.T.V.Rao MD
Dr.T.V.Rao MD 1
Corynebacterium
• 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,
Dr.T.V.Rao MD 2
Corynebacterium diphtheria
• Slender rods
• Clubbing at both ends
• Pleomorphic
• Non capsulate / Acid fast Gram +
• Granules are composed of polymetapohosphate
• Staining with Loeffler’s methylene blue show bluish purple metachromatic granules. with polar bodies,
Dr.T.V.Rao MD 3
Corynebacterium diphtheria
Dr.T.V.Rao MD 4
Corynebacterium
• Classification – Corynebacterium diphtheria and
diphtheroids (look like C. diphtheria) are Gram- positive, club shaped rods.
• Some are saprophytic
• Some produce disease in animals.
• C. diphtheria is the most important pathogen in the group.
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Corynebacterium
• Morphology and cultural characteristics
–Small Gram positive Bacilli; arrangement=palisade or Chinese letters
–Growth on Blood agar – raised, translucent, gray colonies
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Classification McLeod
Classified in to 3 Types 1 Gravis
2 Intermedius
3.Mitis Gravis produce Most serious Hemorrhagic
Paralytic complications - Epidemic
Intermedius Hemorrhagic
Mitis - obstructive complications, Endemic
Geographic locations differ
Testing for toxigenicity is more important, Dr.T.V.Rao MD 7
Corynebacterium
–3 morphological types of C. diphtheria are found on tellurite containing media:
• Mitis – black colonies with a gray periphery
• Gravis – large, gray colonies
• Intermedius – small, dull gray to black.
• All produce an immunologically identical toxin.
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Antigenic structure
• Gravis 13,
• Intermedius 4
• Mitis 40
• Bacteriophage typing 15 types
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Cultural Characteristics
–Incubation -35-370 C for 24 hours.
–They prefer a pH of 7.8-8.0 for good growth.
–They require access to oxygen (poor AnO2 growth).
–Catalase +
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Resistance
• Can be killed at
58o c in 10 minute
100oc in 1 minute
Survive in Blankets, Floor dust,
toys inanimate objects
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What is Diphtheria • An infection of
local tissue of
Upper respiratory
tract with
production of toxin
which causes
systemic effects on
Heart and
Peripheral tissues,
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Pharyngeal diphtheria
• Insidious onset of exudative
pharyngitis
• Exudate spreads to form adherent
“pseudo membrane”
– can lead to respiratory obstruction
and death by asphyxiation
• Fever not high, but patient appears
toxic Dr.T.V.Rao MD 13
Staining methods
• Gram’s method
• Albert's stain
• Neissers stain
• Pander's stain
• On staining seen as
Pairs, Appear as v and L letters, resembling
Chinese letter pattern or also called
cuneiform arrangement. Dr.T.V.Rao MD 14
Cultural characters
• Need enrichment Media
• Containing
• Blood, Serum or Egg 370 c ph. 7.4
• Aerobic/Facultative anaerobic.
• Commonly used medium
• Loeffler serum slope,
• Tellurite Blood agar,
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Cultural characters
• Loeffler serum slope
Grows rapidly in 6 -8 hours,
Small white opaque disks
Turns to yellow
Tellurite blood agar Modified Mc Leod and Hoyle’s Medium
Commonly used medium
Tellurite blood agar Contains tellurite 0.04 tellurite Inhibits other bacteria
Produce Grey/Black colonies.
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Biochemical Reactions • Acid production on
Glucose,Galactose Maltose, Dextrin
Do not produce acid with
Lactose, Mannitol, sucrose.
All fermentation reactions tested in
Hiss serum sugars
Urease test negative.
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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
0.0001mg can kill guinea pig weighing 250 grams
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C. diphtheria toxin
• Toxin enters
through receptor
mediated
endocytosis
• Acidification of
endocytic vesicle
allows A to
dissociate from B
• A enters
cycoplasm Dr.T.V.Rao MD 19
Toxin ( Contd ) • Toxin contain two components
A 24,000
B 38,000
A produce toxigenicity by proteolytic effect
B Produce binding
Toxin + Formalin = Toxoid
What is Toxoid – Antigenic, not toxigenic
Tox + Corynephage Toxin production
Dr.T.V.Rao MD 20
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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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 - Pseudo
membrane • This may obstruct the airway and result in
suffocation.
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Corynebacterium
–To prove that an isolate can cause diphtheria, one must demonstrate toxin production. • This is most often done on an Elek plate:
– The organism is streaked on a plate containing low iron.
– A filter strip containing anti-toxin antibody is placed perpendicular to the streak of the organism.
– Diffusion of the antibody into the medium and secretion of the toxin into the medium occur.
– At the zone of equivalence, a precipitate will form.
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Elek plate for Demonstration
of Toxin
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Animal testings (obsolete)
Pathogenicity
• Bacteria Invade, Colonise,Proliferate
• Bacteria are lysogenized by Beta
phage
• Produce toxin,
• Kills epithelial and Neutrophils,
• Produce Pharyngitis and cutaneous
lesions.
Dr.T.V.Rao MD 27
Dr.T.V.Rao MD 28
Pathogenicity
• Incubation 3 – 4 days / one day
• Faucal / Nasal /Laryngeal / Otic /
Conjunctival,/Genital / Vulvae
and Cutaneous Manifestations
Diphtheria is a toxemic condition.
Malignant Sever toxemia ,Adenitis Bull
neck Circulatory failure
Septic Gangrene , pseudo membrane.
Dr.T.V.Rao MD 29
Pathogenicity • Hemorrhagic
Epistaxis ,
Purpura
General Bleeding tendency
Asphyxia , Acute circulatory failure,
Paralysis Pneumonia, Septic shock, Otitis media. Toxemia, Necrotic changes
Death in Guinea pigs
Dr.T.V.Rao MD 30
C. diphtheria Pathogenesis and Immunity
C. diphtheria occurs in the
respiratory tract, in wounds, or on
the skin of infected persons or
normal carriers. It is spread by
droplets or by direct contact.
Portal of entry: respiratory tract or
skin abrasions. Dr.T.V.Rao MD 31
Diphtheria bacilli produce Toxin
• Diphtheria bacilli
colonize and grow on
mucous membranes,
and start to produce
toxin, which is then
absorbed into the
mucous membranes,
and even spread by
the bloodstream.
Dr.T.V.Rao MD 32
Toxicity of Diphtheria Toxin
• Local toxigenic effects: elicit
inflammatory response and
necrosis of the faucial mucosa
cells-- formation of "pseudo-
membrane“ (composed of
bacteria, lymphocytes, plasma
cells, fibrin, and dead cells),
causing respiratory obstruction.
Dr.T.V.Rao MD 33
Systemic Toxigenic effects
• Systemic
toxigenic effects:
necrosis in heart
muscle, liver,
kidneys and
adrenals. Also
produces neural
damage.
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Complications
• Asphyxia - causing
mechanical obstruction.
• May need tracheotomy
• Circulatory failure.
• Post Diphtheria paralysis
Dr.T.V.Rao MD 36
Non toxigenic clinical
manifestations
• Bacteria can produce
1Endocarditis,
2Meingitis,
3 Cerebral abscess.
4 Osteoarthritis.
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Diagnosis of Diphtheria
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Laboratory 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
• Throat swabs
• Smear examinations
Gram s staining,
Alberts, Ponders
Immunoflorescent methods
Cultures on Loeffers serum slope
Tellurite Blood agar,
Blood agar.
Dr.T.V.Rao MD 40
Dr.T.V.Rao MD 41
Growing the C.diptheria
• Serum slope – Growth in 6 – 8 hours,
• Stain with Neisse's stain, Albert's
stain
• Bacilli have metachromatic granules,
• Tellurite Blood agar takes two days
for manifestation of colonies,
Dr.T.V.Rao MD 42
Colonial morphology
Black colonies on tellurite agar
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Virulence tests, • In Vivo and In Vitro
• In Vivo in Animals
• Subcutaneous tests
Inject broth from culture into two Guinea pigs, 0.8 ml
One animal given 500 units of antitoxin before
Other no Antitoxin.
Animal not given antitoxin will die
Leads to Loss of Animals. So we do not do it at present
Dr.T.V.Rao MD 44
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.
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Elek’s Test for In vitro
toxigenicity Testing
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Schick Test
• Injection of toxin
Intradermal route
• Produces
redness/erythemati
c in 2-4 days
• No reaction –
Protective
immunity present.
Dr.T.V.Rao MD 47
Epidemiology
• Eradicated in developed nations,
• Children between 2 – 5 years.
• A symptomatic carriers
• Person to person contact.
• Carriers spread.
• Prolonged contact.
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Prophylaxis • Immunization
• Active – Passive
• Both passive and Active.
• Herd Immunity.
• Schick test
• Immunization with Antitoxin
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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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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 • Antibiotic not useful in Acute infections,
• Antitoxin a must and primary concern
• 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
Dr.T.V.Rao MD 52
Treating Contacts
•All contacts are
advised to receive
500 mg
Erythromycin 4 times
a day. Dr.T.V.Rao MD 53
Summary • Diphtheria is still prevalent in many
parts of the world
– Risk of imported disease and outbreaks
– Continuing need for vaccination
• Toxin-mediated disease
• Largely unexplored pathogenesis
• Immense opportunities for research, especially in the post-genomic era
Other Corynebacterium
• C.ulcerans
• Like C.diptheria
• Like Gravis type gelatin liquefied
• Transmitted through cows Milk
• Erythromycin effective.
• Diphtheria antitoxin is protective.
Dr.T.V.Rao MD 55
Diptheroids
• Resembles C.diptheria
• Commensals in throat, skin,
• C.hofmani
• C.xerosi
• Propioniebacterium
• P.acnes P.granulosum
Dr.T.V.Rao MD 56
Other Corynebacterium Species They are ubiquitous in plants and animals. Many are found as
part of human normal flora and may cause opportunistic
infections, such as pneumonia, endocarditis, and soft tissue
and bone infections, in immunocompromised patients.
C. jeikeium: sepsis, endocarditis, wound infections,
foreign body infections.
C. urealyticum causes UT infections. It is a strong urease
producer, infection of UT may lead to formation of stones.
C. ulcerans is closely related to C. diphtheriae. May cause
diphtheria-like disease.
Resistant to many antibiotics. Treatment of bacteremia or
endocarditis must be guided by antibiotic susceptibility tests. Dr.T.V.Rao MD 57
Other Corynebacterium Species
• They are ubiquitous in plants and animals.
Many are found as part of human normal
flora and may cause opportunistic
infections, such as pneumonia,
endocarditis, and soft tissue and bone
infections, in immunocompromised
patients.
• C. jeikeium: sepsis, endocarditis Dr.T.V.Rao MD 58
Other Corynebacterium Species
• C. urealyticum causes UT infections. It is a
strong urease producer, infection of UT may
lead to formation of stones.
• C. ulcerans is closely related to C.
diphtheriae. May cause diphtheria-like
disease.
• Resistant to many antibiotics. Treatment of
bacteremia or endocarditis must be guided by
antibiotic susceptibility tests. Dr.T.V.Rao MD 59
• Programme Created by
Dr.T.V.Rao MD for Medical
and Paramedical Students
doctortvrao@gmail.com
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