Anthrax teaching

Anthrax Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

� ~ 60 species; Gram-positive or Gram-variable bacilli• Large (0.5 x 1.2 to 2.5 x 10 um)

• Most are saprophytic contaminants or normal flora

• Bacillus anthracis is most important member

� Produce endospores

� Aerobic or facultatively anaerobic

� Bacillus spp. are ubiquitous• Soil, water, and airborne dust

• Thermophilic (< 75°C) and psychrophilic (>5-8°C)

• Can flourish at extremes of acidity & alkalinity (pH 2 to

10)

General Characteristics of Bacillus

Dr.T.V.Rao MD 2

Anthrax• From the Greek word anthrakos for coal

• Caused by spores

• Primarily a disease of domesticated & wild animals

– Herbivores such as sheep, cows, horses, goats

• Natural reservoir is soil

– Does not depend on an animal reservoir making it hard to eradicate

– Cannot be regularly cultivated from soils where there is an absence of endemic anthrax

– Occurs sporadically throughout US

– South Dakota, Arkansas, Texas, Louisiana, Mississippi, California recognized endemic areas

Dr.T.V.Rao MD 3

A Closer Look at Anthrax

• Anthrax is a disease of cattle, goats, and sheep

caused by a bacterium, Bacillus anthracis. It is rare

for humans to be infected. Most infections that do

occur are localized to small cuts in the skin whose

edges turn black (hence the name “anthracis”,

after anthracite coal). The disease is deadly for

humans because B. anthracis produces lethal

toxins.

Dr.T.V.Rao MD 4

Bacillus anthrax

Several landmarks

• 1st to Observe under Microscope

• 1st to communicable disease.

• 1st observe the spores ( Robert Koch )

• 1st to prepare for attenuated vaccine. ( Louis Pasteur)

Dr.T.V.Rao MD 5

Dr.T.V.Rao MD

20,000-100,000 cases estimated globally/yearhttp://www.vetmed.lsu.edu/whocc/mp_world.htm

Dr.T.V.Rao MD

Animal Transmission

• Most commonly infected by ingestion from

contaminated soil or contaminated feed or bone

meal

7

AnthraxAnthraxAnthraxAnthrax• An infectious,

Usually fatal disease

of warm-blooded

animals, especially of

cattle and sheep,

caused by the

bacterium Bacillus

anthracis.

Dr.T.V.Rao MD 8

B. anthracis�Gram-positive, spore-forming, non-motile bacillus

Dr.T.V.Rao MD 9

Anthrax Bacilli

Dr.T.V.Rao MD 10

Dr.T.V.Rao MD

Bacillus anthracisGeneral characteristics

• Bacillus anthracis

• Large, Gram positive, non-motile

rod

• Vegetative form

and spores

• Nearly worldwide distribution

• Over 1,200 strains

11

Bacillus anthracis• Gram + rod

• Facultative anaerobe

• 1 - 1.2µm in width x 3 - 5µm in length

• Belongs to the B. cereus family– Thiamin growth requirement– Glutamyl-polypeptide

capsule– Nonmotile

• Forms oval, centrally located endospores

Dr.T.V.Rao MD 12

The Spore

• Sporulation requires

– Poor nutrient conditions

– Presence of oxygen

• Spores

– Very resistant to extremes

– Survive for decades

– Taken up by host and germinate

• Lethal dose 2,500 to 55,000 spores

Dr.T.V.Rao MD 13

Endospore• Oxygen required for sporulation

• 1 spore per cell

• dehydrated cells

– Highly resistant to heat, cold,

chemical disinfectants, dry periods

• Protoplast carries the material for

future vegetative cell

• Cortex provides heat and radiation

resistance

• Spore wall provides protection from

chemicals & enzymes

Dr.T.V.Rao MD 14

Gram Stain Morphologyof B. anthracis

• Broad, gram-positive rod: 1–1.5 x 3–5 µ

• Oval, central to subterminal spores: 1 x 1.5 µ with no significant swelling of cell

• Spores usually NOT present in clinical specimens unless exposed to atmospheric O2

Dr.T.V.Rao MD 15

Mechanism of Infection

• Anthrax spores enter body

• Germinate & multiple in lymph nodes

• PA, EF, LF excreted from bacteria

• PA binds to TEM8.

• EF and/or LF binds

• Complex internalized by endocytosis

• Acidification of endosome

• LF or EF crosses into cytosol via PA mediated ion-conductive channels

Dr.T.V.Rao MD 16

Cultural

characteristics

• Aerobe, facultative anaerobe

• Grows between 12 -45 c

• 2-3 mm colonies

• Edge like matted hair

• Medusa head appearance

• Blood agar Hemolytic colonies

Dr.T.V.Rao MD 17

Appearance of Anthrax

• String of pearl

appearance with

Pencillin

• Differentiates Anthrax

and Cereus

Dr.T.V.Rao MD 18

SELECTIVE MEDIUM

PLET

• Contain

• 1 polymyxins

• 2 Lysozyme

• 3 Ethylene dioxide

• 4 Tetra acetic acid

Contains EDTA

Dr.T.V.Rao MD 19

Biochemical Reactions

•Gelatin –

Inverted fir

tree

appearance

Dr.T.V.Rao MD 20

Biochemical

Reactions

• Glucose, Maltose

and Sucrose

fermented with

acid but no gas

• Catalase positive

Dr.T.V.Rao MD 21

Sterilization of environments Floor

space/shed/vehicle

• Preliminary disinfection using 10% formaldehyde; (1-1.5 It/ sq.m.) or 4%

• Gluteraldehydes for at least 2 hours

• Cleaning - by washing or scrubbing with hot water

• Final disinfection by one of the following disinfectants applied for at least 2 hours.

• 10% formaldehyde

4% Gluteraldehydes

• 3% hydrogen peroxide or

• 1% per acetic acid

Dr.T.V.Rao MD 22

Wool and Hair

• By duckering process (five stages) i.e.

• lmmersion in 0.25-0.3% soda liquor

• Immersion in soap liquor;

• Two immersions in 2% formaldehyde solution; and

• Rinsing in water

Dr.T.V.Rao MD 23

Antibiotics

• Pencillin

• Erythrocin

• Tetracycline

• Chloramphenicol

• Occasional strains

resistant to penicillin

are encountered

Dr.T.V.Rao MD 24

TransmittedTransmittedTransmittedTransmitted• The disease can be

transmitted to humans through contact with contaminated animal substances, such as hair, feces, or hides, and is characterized by ulcerative skin lesions

Dr.T.V.Rao MD 25

Criteria in Transmission

• Skin: direct skin contact with spores; in nature, contact with infected animals or animal products (usually related to occupational exposure)

• Respiratory tract: inhalation of aerosolized spores

• GI: consumption of undercooked or raw meat products or dairy products from infected animals

• NO person-to-person transmission of inhalation or GI anthrax

Dr.T.V.Rao MD 26

Anthrax CycleAnthrax CycleAnthrax CycleAnthrax Cycle

Dr.T.V.Rao MD 27

Pathogenesis

• The infectious dose of B. anthracis in humans by any route is not precisely known.

– Rely on primate data

– Minimum infection dose of ~ 1,000-8,000 spores

– LD50 of 8,000-10,000 spores for inhalation

• Virulence depends on 2 factors

– Capsule

– 3 toxins http://www.kvarkadabra.net/index.html?/biologija/teksti/biolosko_orozje.htm

Dr.T.V.Rao MD 28

Anthrax:

Clinical Presentation

Cutaneous

Inhalational

Gastrointestinal

Dr.T.V.Rao MD 29

Epidemiology

of Anthrax in

Animal and

Human Hosts

Dr.T.V.Rao MD 30

Three forms of Anthrax

• Cutaneous anthrax

– Skin

–Most common

– Spores enter to skin through small lesions

• Inhalation anthrax

– Spores are inhaled

• Gastrointestinal (GI) anthrax

– Spores are ingested

– Oral-pharyngeal and abdominal

Dr.T.V.Rao MD 31

Dr.T.V.Rao MD

Cutaneous Anthrax

• 95% of all cases globally

• Incubation: 2-3 days (up to 12 days)

• Spores enter skin through open wound or abrasion

• Papule progresses to black Escher

• Severe edema

• Fever and malaise

32

Anthrax:Cutaneous

� Begins as a papule, progresses through a vesicular stage to a depressed black necrotic ulcer (Escher)

� Edema, redness, and/or necrosis without ulceration may occur

� Form most commonly encountered in naturally occurring cases

� Incubation period: 1–12 days� Case-fatality:

� Without antibiotic treatment—20%� With antibiotic treatment—1%

Dr.T.V.Rao MD 33

Anthrax:Inhalational

� A brief prodromal resembling a “viral-like”

illness, characterized by myalgia, fatigue,

fever, with or without respiratory

symptoms, followed by hypoxia and

dyspnea, often with radiographic evidence

of mediastinal widening.

� Meningitis in 50% of patients

� Rhinorrhea (rare)

Dr.T.V.Rao MD 34

Capsule• Glycocalyx

– Sticky, gelatinous polymer external to cell wall

• pX02 plasmid

• Made up of D-glutamic acid

• Non-toxic on its own

• Only encapsulated B. anthracisvirulent

• Most important role during establishment of disease

– Protects against phagocytosis & lysis during vegetative state

Dr.T.V.Rao MD 35

Virulence Factors

• 1 Capsular polypeptide

• Anthrax Toxin

• Both are coded by separate plasmid

• The capsular polypeptide aids virulence by

inhibiting phagocytosis, loss of plasmid loss of

virulence

• How the live attenuated anthrax spore vaccine (

Sterne strain )

Dr.T.V.Rao MD 36

Anthrax Toxin

The toxin is a three factions

The edema factor, (OF Factor I )

The protective antigen factor ( PA or

Factor II )

The lethal factor ( LF or Factor III )

Individually they are not toxic

Dr.T.V.Rao MD 37

How Toxicity Manifests

• They are not toxic indivually but whole complex

produces local edema and generalisaed shock.

• PA I which is the fraction which binds to the target

cell surface and in turn provides attachment sites

for OF or LF facilitating .their entry into the cell

Dr.T.V.Rao MD 38

TOXIGENICITY• OF island adenyl cyclase which is activated only inside

the target cells leading to the intracellular accumulation of cyclic AMP

• Responsible for edema and other biological effects of toxin.

• Entry of LF toxin into the target cell causes cell death.

• Loss of plasmid which encodes anthrax toxin renders the strain avirulant.

• Sterne vaccine strain devoid of Plasmid coding for the capsule polysaccharide.

Dr.T.V.Rao MD 39

Pathogenesis• Anthrax spores enter body• Germinate & multiple in lymph nodes• PA, EF, LF excreted from bacteria• PA binds to TEM8. • PA nicked by protease furin– 20-kDa segment off leaving 63-kDa peptide– Heptamer forms

• EF and/or LF binds• Complex internalized by endocytosis• Acidification of endosome• LF or EF crosses into cytosol via PA mediated ion-conductive

channels• LF cleaves MAPKK 1 & 2• EF stimulates cAMP

Dr.T.V.Rao MD 40

Clinical Presentation of Anthrax Cutaneous Anthrax

� 95% human cases are cutaneous infections

� 1 to 5 days after contact

� Small, pruritic, non-painful papule at inoculation site

� Papule develops into hemorrhagic vesicle & ruptures

� Slow-healing painless ulcer covered with black Escher

surrounded by edema

� Infection may spread to lymphatic's w/ local adenopathy

� Septicemia may develop

� 20% mortality in untreated cutaneous anthrax

Dr.T.V.Rao MD 41

Cutaneous Anthrax

CDC, Cutaneous Anthrax—Vesicle Development

Dr.T.V.Rao MD 42

Left, Forearm lesion on day 7—vesiculation and ulceration of initial macular

or papular anthrax skin lesion. Right, Eschar of the neck on day 15 of

illness, typical of the last stage of the lesion. From Binford CH, Connor DH,

eds. Pathology of Tropical and Extraordinary Diseases. Vol 1. Washington,

DC: AFIP; 1976:119. AFIP negative 71-1290–2.

Anthrax: Cutaneous

Dr.T.V.Rao MD 43

Anthrax: Cutaneous

Healing after treatment

Dr.T.V.Rao MD 44

Inhalation Anthrax • The infection begins with the inhalation

of the anthrax spore.

• Spores need to be less than 5 microns (millionths of a meter) to reach the alveolus.

• Macrophages lyse and destroy some of the spores.

• Survived spores are transported to lymph nodes.

• At least 2,500 spores have to be inhaled to cause an infection.

Inhalation Anthrax, Introduction, DRP, Armed Forces Institute of PathologyDr.T.V.Rao MD 45

Inhalation Anthrax • Disease immediately follows germination.

• Spores replicate in the lymph nodes.

• The two lungs are separated by a structure called the mediastinum, which contains the heart, trachea, esophagus, and blood vessels.

• Bacterial toxins released during replication result in mediastinal widening and pleural effusions (accumulation of fluid in the pleural space).

Inhalation Anthrax, Introduction, DRP, Armed Forces Institute of PathologyDr.T.V.Rao MD 46

�Mediastinal widening

JAMA 1999;281:1735–1745

Anthrax: Inhalational

Dr.T.V.Rao MD 47

Mediastinal Widening and Pleural Effusion on Chest X-Ray in Inhalational

Anthrax

Dr.T.V.Rao MD 48

Gastrointestinal Anthrax

• GI anthrax may follow after the consumption of contaminated, poorly cooked meat.

• There are 2 different forms of GI anthrax:

1) Oral-pharyngeal

2) Abdominal

• Abdominal anthrax is more common than the oral-pharyngeal form.

http://science.howstuffworks.com/anthrax1.htmDr.T.V.Rao MD 49

Clinical Presentation of AnthraxInhalation Anthrax

�Virtually 100% fatal (pneumonic)

�Meningitis may complicate cutaneous

and inhalation forms of disease

�Pharyngeal anthrax• Fever

• Pharyngitis

• Neck swelling

Dr.T.V.Rao MD 50

Clinical Presentation of Anthrax Gastrointestinal (Ingestion) Anthrax

�Virtually 100% fatal

�Abdominal pain

�Hemorrhagic ascites

�Paracentesis fluid may reveal

gram-positive rods

Dr.T.V.Rao MD 51

Anthrax:Diagnosis

Cutaneous

� Gram stain, polymerase chain reaction

(PCR), or culture of vesicular fluid,

exudate, or eschar

� Blood culture if systemic symptoms

present

� Biopsy for immunohistochemistry,

especially if person taking

antimicrobialsDr.T.V.Rao MD 52

Dr.T.V.Rao MD

Diagnosis in Humans

• Isolation of B. anthracis

– Blood, skin

– Respiratory secretions

• Serology

• ELISA

• Nasal swabs

– Screening tool

53

Dr.T.V.Rao MD

Diagnosis in Humans

• Anthrax quick ELISA test

– New test approved by FDA on June 7th, 2004.

– Detects antibodies produced during infection with

Bacillus anthracis

– Quicker and easier to interpret than previous antibody

testing methods

• Results in less than ONE hour

54

Clues to diagnosis

• Aerobic blood culture growth of large, gram-positive bacilli provides preliminary identification of Bacillus species

Dr.T.V.Rao MD 55

Laboratory Criteria for Identification of B. anthracis

• From clinical samples, such as blood,

cerebrospinal fluid (CSF), skin lesion

(eschar), or oropharyngeal ulcer

–Encapsulated gram-positive rods on Gram

stain

• From growth on sheep blood agar:

– Large gram-positive rods

–Nonmotile

–NonhemolyticDr.T.V.Rao MD 56

Anthrax:Diagnosis

Inhalational

� Chest X-ray—widened mediastinum,

pleural effusions, infiltrates, pulmonary

congestion

� Affected tissue biopsy for

immunohistochemistry

� Any available sterile site fluid for Gram

stain, PCR, or culture

� Pleural fluid cell block for

immunohistochemistryDr.T.V.Rao MD 57

B. anthracis:

Confirmatory Identification

Isolate

Phage

lysis

Capsule

Bicarbonate

media

Horse

blood(M’Fadyean

Stain)

(M’Fadyean stain

India ink stain)

DFA

Capsule antigen

Cell wall

Dr.T.V.Rao MD 58

Isolate on SBA

Colony morphology

Hemolysis

Motility

Spores Gram stain

Malachite green

B. anthracis:Presumptive Identification

Clinical specimen (blood, CSF, etc.)

Gram stain

Capsule production

Dr.T.V.Rao MD 59


• From clinical samples, such as blood,

cerebrospinal fluid (CSF), skin lesion

(eschar), or oropharyngeal ulcer

–Encapsulated gram-positive rods on Gram

stain

• From growth on sheep blood agar:

– Large gram-positive rods

–Nonmotile

–NonhemolyticDr.T.V.Rao MD 60


• Rapid screening assay (PCR- and antigen-detection based) for use on cultures and directly on clinical specimens

• Confirmatory criteria for identification of B. anthracis

– Capsule production

– Lysis by gamma-phage

– Direct fluorescent antibody assay (DFA)

Dr.T.V.Rao MD 61

PCR Assay • Detection time:

- PCR only takes several hours

ex) Rapid-cycle RT-PCR can be finished within 1-2 hours

• Can start early treatment of Anthrax

• There are many different types of PCR assays for the detection of Anthrax such as multiplex PCR, enter bacterial repetitive intragenic consensus-PCR (ERIC-PCR), and long-range repetitive element polymorphism-PCR.

• Rapid diagnostic methods provide answers in minutes or hours instead of days.

Dr.T.V.Rao MD 62

Cautions on Treatment

• Obtain specimens for culture BEFORE initiating antimicrobial therapy.

• Do NOT use extended-spectrum cephalosporins or trimethoprim/sulfamethoxazole because anthrax may be resistant to these drugs.

Dr.T.V.Rao MD 63

Treatment & Prophylaxis

� Treatment• Penicillin is drug of choice

• Erythromycin, chloramphenicol acceptable alternatives

• Doxycycline now commonly recognized as prophylactic

� Vaccine (controversial)� Laboratory workers

� Employees of mills handling goat hair

� Active duty military members

� Potentially entire populace of U.S. for herd immunity

Dr.T.V.Rao MD 64

Dr.T.V.Rao MD

Treatment

• Penicillin

– Has been the drug of choice

– Some strains resistant to penicillin and doxycycline

• Ciprofloxacin

– Chosen as treatment of choice in 2001

– No strains known to be resistant

• Doxycycline may be preferable

65

Treatment• Before 2001, 1st line of treatment was

penicillin G– Stopped for fear of genetically

engineered resistant strains• 60 day course of antibiotics• Ciprofloxacin

– fluoroquinolone– 500 mg tablet every 12h or 400 mg IV

every 12h – Inhibits DNA synthesis

• Doxycycline– 6-deoxy-tetracycline– 100 mg tablet every 12h or 100 mg IV

every 12h – Inhibits protein synthesis

• For inhalational, need another antimicrobial agent– clindamycin – rifampin – chloramphenico

hrax.html

Dr.T.V.Rao MD 66

Trends on Vaccine• BioThrax/Anthrax vaccine absorbed

– Made by Bioport

– Route of exposure not important

• Administered subcutaneously

– .5mL at 0, 2, and 4 weeks, and at 6, 12, & 18 months, & booster doses at 1 yr intervals

• PA from attenuated, nonencapsulated Sterne strain absorbed onto aluminum hydroxide

– Contains no dead or live bacteria in the preparation

– Antibodies to PA prevent binding to the target cell & confer protection from anthrax.

• 95% of vaccinated Rhesus monkeys survived lethal doses of inhaled anthrax

• A December 22, 2003 ruling temporarily halted the Department of Defense’s anthrax vaccination program

– Lifting of that injunction on January 7, 2004

Dr.T.V.Rao MD 67

Immune Protection Against Anthrax

• Live cellular vaccines

– "Sterne" type live spore (toxigenic, noncapsulating)

– Former USSR STI live spore (toxigenic, non-capsulating)

– "Pasteur" type (mixed culture, reduced virulence)

• Sterile, acellular vaccines

– US "anthrax vaccine adsorbed" (AVA)—not licensed for use in civilian populations

– UK "anthrax vaccine precipitated" (AVP)• Recombinant PA research vaccines

– AI3+; Freund’s; Saponin, Monophosphoryl lipid A; Ribi

Dr.T.V.Rao MD 68

Dr.T.V.Rao MD

Vaccination

• Cell-free filtrate

• Licensed in 1970

• At risk

–Wool mill workers

– Veterinarians

– Lab workers

– Livestock handlers

–Military personnel

69

Dr.T.V.Rao MD

Vaccine Schedule

• 3 injections at two-week intervals

• 3 injections 6 months apart

• Annual booster

70

Recommended Post exposure Prophylaxis to Prevent Inhalational Anthrax

Initial Therapy Duration

Adults Ciprofloxacin 60 days

(including pregnant 500 mg PO BID

women and OR

immunocompromised) Doxycycline

100 mg PO BID

Children Ciprofloxacin* 60 days

10–15 mg/kg PO Q 12 hrs. Change to

OR amoxicillin

Doxycycline: if susceptible

>8 yrs. and >45 kg: 100 mg PO BID

>8 yrs. and <45 kg: 2.2 mg/kg PO BID

<8 yrs.: 2.2 mg/kg PO BID

*Ciprofloxacin not to exceed 1 gram daily in children

Patient information sheets at www.bt.cdc.govDr.T.V.Rao MD 71

Precautions to Health care Workers

• Standard contact

precautions. Avoid

direct contact

with wound or

wound drainage.

Dr.T.V.Rao MD 72

Anthrax Has Been Used As a

Bioweapon• Because it is deadly, noncontagious, and dispersed

by spores, anthrax has always been considered a

good candidate for a bioweapon (table 3). Late in

2001, this possibility became a reality. Letters

containing anthrax spores were sent to several

news reporters and two United States Senators.

Five people died of inhalational anthrax as a result

of exposure to these spores.

Dr.T.V.Rao MD 73

Weaponization & Bacillus Anthracis:

Why is this Agent Considered to be the Department of Defense’s

Number-One/Two Biological Threat?

A sample of anthrax bacteria at the National School

of Biological Sciences, Mexico City

Dr.T.V.Rao MD 74

Analysis of the 2001 US Anthrax Attacks

� Above anthrax-containing envelopes

postmarked September 18th, 2001

� Above anthrax-containing envelopes

postmarked October 9, 2001

*Also believed to be three or more other envelopes that were never foundDr.T.V.Rao MD 75

Dr.T.V.Rao MD

Anthrax Cases, 2001

• 22 cases

– 11 cutaneous

– 11 inhalational

• 5 deaths (all inhalational)

– Index case in Florida

– 2 postal workers in Maryland

– Hospital supply worker in NYC

– Elderly farm woman in Connecticut

76

Analysis of the 2001 US Anthrax Attacks

• Anthrax in Envelopes

– Concentration of about 1 trillion spores per gram– 2 grams anthrax per envelope– Each letter contained ~200 million times average LD50

– All anthrax was unmilled, contained a certain type of silica to reduce electrostatic charges and was of the Ames strain – all characteristic of US weapons-grade anthrax

Dr.T.V.Rao MD 77

Created by Dr.T.V.Rao MD for “ e

“ learning resources in Developing

World• Email

[email protected]

Dr.T.V.Rao MD 78

Anthrax teaching

Education