Transcript Anthrax - AIIMS, New Delhi

ANTHRAX
• The anthrax bacillus, Bacillus anthracis, was the first
bacterium shown to be the cause of a disease- Koch’s
Postulate
• In 1877, Robert Koch grew the organism in pure culture,
demonstrated its ability to form endospores, and
produced experimental anthrax by injecting it into
animals.
• Anthrax is a disease of domesticated and wild animals
• Men suffer from anthrax occasionally due to close
contact with infected animal or animal products
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Bacillus anthracis
• Gram positive rods
• Capsulated ( Protein) – Capsule form in animal tissue and in special
laboratory condition ( 5% CO2)
• Forms endospore, centrally located, do not form in animal tissues
• MacFadyean ( Polychrome methylene blue) stain blue bacilli
with purple capsule
• Aerobic/ Facultative anerobe
• Grows on all ordinary medium (Medusa head appearance-uneven
wavy margin)
• Inverted fur tree appearance in liquid medium
• Biochemicals : Catalase +, reduces nitrate to nitrite, lecithinase+,
glucose, maltose, sucrose, trehalose fermented
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Robert Koch's original micrographs of the anthrax bacillus
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Bacillus anthracis. Gram stain. The cells have characteristic
squared ends. The endospores are ellipsoidal shaped and located
centrally in the sporangium. The spores are highly refractile to
light and resistant to staining.
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Bacillus cereus
Genotypically and
phenotypically it is very similar
to Bacillus cereus, which is
found in soil habitats around
the world
Bacillus thuringiensis.
Phase Photomicrograph
of vegetative cells,
intracellular spores (light)
and
parasporal crystals (dark).
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McFadyean's reaction showing short chains of Bacillus
anthracis cells lying among amorphous,disintegrated
capsular material. White blood cells can also be seen.
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Differential Characteristics of B. anthracis B. cereus and B. thuringiensis
Characteristic
B. anthracis
Differential Characteristics of B. anthracis B. cereus and B. thuringiensis
B. cereus and
B. thuringiensis
growth requirement for thiamin
+
-
hemolysis on sheep blood agar
-
+
glutamyl-polypeptide capsule
+
-
lysis by gamma phage
+
-
motility
-
+
growth on chloralhydrate agar
-
+
string-of-pearls test
+
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Physical properties ( methods for decontamination)
• SPORES SURVIVE FOR MANY YEARS ( DRY STATE AND SOIL )
• Moist heat kills – Vegetative cells 60 0 C X 30 minutes
Spores 100 0 C X 10 minutes
4% Formaldehyde kills spores
4% KMnO4 kills spores
Hypochlorite ( 0.5%) commercially available kills spores
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Epiedemiology
• Distribution worldwide
• Not common in West. Common in Africa ( Zimbabwe),
S.E. Asia, China, South America, Turkey, Pakistan, India
• Human to human or animal to animal transmission is rare
( not contagious)
•Grazing animals become infected through ingestion of
spores in the soil ( Carcasses become the source)
Epidemic : A. Spread to contiguous geographic areas by
infected animal
B. Non contiguous geographic areas by
- biting flies ( Zimbabwe)
- Vultures
- Contaminated surface water pool
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INDIA
Largest live stock population in the world
Incidence is not accurately known ( Sporadic cases reported)
Pondicherry ( JIPMER) - 30 human cases reported ( Mostly Cutaneous,
Septicemic or Meningeal)
Vellore ( CMC)- 49 human cases
Chittor ( Rajasthan)- 30 human cases
Tirupati ( Andhrapradesh)- 25 human cases
Midnapur ( WB)- 22 human cases
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Pathogenesis
Endospores
(Abrasion, inhalation, ingestion)
Death
Introduced
Septicemia
Phagocytosed by Macrophages
10 7 to 10 8/ml
Regional LNs
Blood stream
Multiply in Lymphatics
Germinate inside Macrophages
Release
Vegetative Forms
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Clinically three forms of Human anthrax occur
A. Cutaneous anthrax
B. Pulmonary anthrax
C. Intestinal anthrax
Broadly can be classified into
Non Industrial/Agricultural ( Through infected animals):
Cutaneous anthrax
Rarely intestinal anthrax
Industrial Anthrax ( Through animal products):
Mostly through animal products( wools, hair, hides, bones)
Likely to develop Cutaneous and pulmonary anthrax ( inhalation)
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Cutaneous Anthrax
• Mainly in professionals( Veterinarian, butcher, Zoo keeper
• Spores infect skin- a characteristic gelatinous edema develops at the
site (Papule- Vesicle-Malignant Pustule- Necrotic ulcer)
• 80-90% heal spontaneously ( 2-6wks)
• 0-20% progressive disease – develop septicemia
• 95-99% of all human anthrax occur as cutaneous anthrax
Intestinal Anthrax
• Due to in ingestion of infected carcasses
• Mucosal lesion to the lymphatic system
• Rare in developed countries
• Extremely high mortality rate
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PULMONARY ANTHRAX
• Require very high infective dose ( > 10,000 spores)
• Acquired through inhalation of spores ( Bioterrorism - aerosol)
• Present with symptoms of severe respiratory infection( High fever &
Chest pain)
• Haemorrhagic mediastinitis
• Progress to septicemia very rapidly
• 10 7 to 10 9 bacilli/ ml of blood at the time of death
• Mortality rate is very high > 95%
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DIFFERENTIAL DIAGNOSIS OF ANTHRAX
CUTANEOUS ANTHRAX
Boils, Erysipelas, Cutaneous TB, Leprosy, Plague, Vaccinia, Rickettsial pox,
tularemia
INTESTINAL ANTHRAX
Typhoid fever, Acute Gastroenteritis, Tularemia, Peritonitis, Peptic ulcer,
Mechanical obstruction
PULMONARY ANTHRAX
Viral pneumonia, Mycoplasma. Psittacosis, Legionnaires disease, Q fever,
Histoplasmosis, Coccidiodomycosis, Silicosis, Sarcoidosis
Meningeal Anthrax : Sometime manifest as meningitis
D/D : Bacterial meningitis
Aseptic meningitis
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VIRULENCE FACTORS
Anthrax Toxin – Complex of proteins ( all the components thermolabile)
A. Protective antigen
B. Edema factor
C. Lethal Factor
Protein capsule – Poly D Glutamic acid capsule
- Inhibits phagocytosis ( Unencapsulated strains –
nonpathogenic)
Anthrax Toxin
Protective antigen : Binds plasma membrane of target cells
Cleaved to 2 fragments ( cellular trypsin or proteases)
Larger fragment is attached to cell surface – binding domain for LF & EF
Specific receptor mediated endocytosis of LF & EF
EDEMA FACTOR
( Edema Factor + Protective Ag = Edema toxin)
Calmodulin dependent adenyl cyclase
Increased cellular cAMP
Edema
Impaired Neutrophil function
Depletes ATP from Macrophages
LETHAL FACTOR
( Lethal Factor + Protective Ag = Lethal toxin)
Zinc metallo proteases that inactivates protein kinases
Stimulates Macrophages – TNF alpha and IL – 1 beta – Shock & Death
Death due to oxygen depletion, secondary shock, increased vascular
permeability, respiratory failure and cardiac failure.
Sudden and unexpected.
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Virulence of Anthrax bacillus is due to presence of two plasmids
px01 – Toxin encoding plasmid
- 110 megadalton
- temperature-sensitive plasmid
px02 - Capsule encoding plasmid ( 3 genes - cap A, cap B, cap C)
- 60 megadalton plasmid
- synthesis of poly glutamic acid capsule
Both plasmids are required for virulence
- loss of either - attenuation
- genes expressed only in vegetative state
Pasteur strain - Encapsulated
Sterne strain – Non encapsulated
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LABORATORY DIAGNOSIS
Few points to remember
• Anthrax is not highly contagious
• Cutaneous anthrax is not lethal and is readily treated with
common antibiotics
• ID for human pulmonary / intestinal infection is > 10,000 spores
SPECIMEN TO COLLECT ( HUMAN ANTHRAX)
Disposable gloves, masks, overalls, boots, head gear and dust mask
Disposable items – Autoclave and incinerate
Cutaneous anthrax: Vesicular exudate – swabs and capillary tube aspirate
Intestinal anthrax: - Stool sample - isolate – guinea pig inoculation
- Blood( venipuncture) smear examination for bacilli
- Peritoneal fluid for culture
- Paired sera for Ab
Pulmonary anthrax: If mild disease ( No sample)
Severely ill – Blood , sputum, serum samples for Ab
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SAMPLES FROM ANIMAL
Sudden death of animal in areas where anthrax was reported earlier
Carcasses 1 or 2 day old
Aspirate blood - MacFadyean stain for bacilli
Direct demonstration by IFA
Direct plating on blood agar
Putrefying carcasses
Blood, tissue and hide
Culture on selective medium
Soil sample from the areas where the carcass as lying
Serological assay
ELISA: based on anthrax toxin ( PA, LF and EF) for routine confirmation and
vaccine response)
Molecular techniques ( Only in the referral laboratories):
- RFLP
- PCR Fingerprinting
Animal Inoculation: Guinea pig and mice inoculation
Culture is confirmed by gamma phage lysis ( PlyG lysin enzyme- g phage)
IMMUNITY TO ANTHRAX
Resistance against anthrax vary from species to species
- Human are partially immune to anthrax
Resistance can be of two types
- Resistance to the establishment of infection but sensitive to toxin
- Resistance to toxin but susceptible to infection
Animals surviving naturally acquired anthrax are immune to reinfection
Protective antibodies against the anthrax toxin and against the capsule
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Resistance to Anthrax vary from species to species
Animal Infectio
mod
us
el
dose
Toxic dose
causing
death
Bacteria per ml
blood at time
death
Mouse
5 cells
1000 units/kg
107
Monkey
3000
cells
2500 unit/kg
107
Rat
106 cells
15 units/kg
105
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TREATMENT
Antibiotics should be given to unvaccinated individuals exposed to inhalation
anthrax.
Penicillin, tetracyclines and fluoroquinolones are effective if administered before the
onset of lymphatic spread or septicemia
Antibiotic treatment is effective in cutaneous anthrax
Inhalation anthrax can be effectively treated with antibiotics administered prior to
lymphatic spread or septicemia
INITIAL THERAPY
OPTIMAL THERAPY
Adults
Ciproflox
( 400mg iv BDX60days)
Penicillin G 4 mu iv qdsX60days
Doxycycline 100mg iv BDX60 days
Children
Ciproflox
20-30mg/kgbodywt ivX60days
Penicllin G 50,000 u/kg X 60 days
Alternatives – Amox, Tetracycline, Chloramphenicol, Erythromycin, Streptomycin
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Vaccine against Anthrax
Killed bacilli and/or capsular antigens produce no significant immunity.
A nonencapsulated toxigenic strain (Sterne Strain) has been used effectively in
livestock.
Vaccine for humans: ( avirulent and nonencapsulated) sublethal amounts of the toxin
produced
Licensed in the U.S. is a preparation of the protective antigen (PA)
Dose:
A. 3 doses subcutaneously at the interval of 2 wks
B. Followed by three additional doses at 6,12 and 18 months
C. Annual booster dose
Who are to be vaccinated
- Professionals ( Veternarians, butcher, Zoo keeper, Wild life workers, Forest guards)
- Military personnels
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Anthrax and Biological Warfare
Countries > 10 countries in the world
Clouds of spores of Anthrax bacilli – aerosol ( war heads filled with anthrax spores)
- Through dried spores in envelops
September 9/11 WTO attack
Postal workers affected – Inhalation anthrax ( 40% mortality)
US – Columbia, Florida, New Jersey, N. York
Other parts of the world
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