Biological Terrorism

Anthrax

History
Caused by Bacillus anthracis Human zoonotic disease
Spores found in soil worldwide Primarily disease of herbivorous animals
Sheep, goats, cattle Many large documented epizootics

Occasional human disease

Epidemics have occurred but uncommon Rare in developed world
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Bioweapon Potential
Many countries have weaponized anthrax
Former bioweapon programs
U.S.S.R.,U.S.,U.K., and Japan

Recent bioweapon programs

Iraq

Attempted uses as bioterrorism agent

WW I: Germans inoculated Allied livestock WW II: Alleged Japanese use on prisoners
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Bioweapon Potential
Features of anthrax suitable as BT agent
Fairly easy to obtain, produce and store Spores easily dispersed as aerosol Moderately infectious High mortality for inhalational (86-100%)

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Bioweapon Potential
Aerosol method of delivery
Most likely method expected in BT attack Would cause primarily inhalational disease
Spores reside on particles of 1-5 m size Optimal size for deposition into alveoli Form of disease with highest mortality

Would infect the largest number of people

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Bioweapon Potential
Dispersed as powder
Frequent letter hoaxes since 1997 Recent letter deliveries
Highest risk is for cutaneous Inhalational theoretically possible
Particle size Likelihood of aerosolization

GI theoretically possible
Spores > hands > eating without handwashing
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Bioweapon Potential
Sverdlovsk, Russia 1979
Accidental release from anthrax drying plant 79 human cases
All downwind of plant 68 deaths Some infected with multiples strains

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Bioweapon Potential
Estimated effects of inhalational anthrax
100 kg spores released over city size of Washington DC
130,000 3 million deaths depending on weather conditions

Economic impact
$26.2 billion/100,000 exposed people

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Epidemiology
Three forms of natural disease
Inhalational
Rare (<5%) Most likely encountered in bioterrorism event

Cutaneous
Most common (95%) Direct contact of spores on skin

Gastrointestinal
Rare (<5%), never reported in U.S. Ingestion
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Epidemiology
All ages and genders affected Occurs worldwide Endemic areas - Africa, Asia True incidence not known
World 20,000-100,000 in 1958 U.S. 235 total reported cases 1955-1994
18 cases inhalational since 1900, last one 1976 Until 2001, last previous case cutaneous 1992
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Epidemiology
Mortality
Inhalational 86-100% (despite treatment)
Era of crude intensive supportive care

Cutaneous <5% (treated) 20% (untreated) GI approaches 100%

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Epidemiology
Incubation Period
Time from exposure to symptoms Very variable for inhalational
2-43 days reported Theoretically may be up to 100 days Delayed germination of spores

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Epidemiology
Human cases historical risk factors
Agricultural
Exposure to livestock

Occupational
Exposure to wool and hides Woolsorters disease = inhalational anthrax Rarely laboratory-acquired

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Epidemiology
Transmission
No human-to-human Naturally occurring cases
Skin exposure Ingestion Airborne

Bioterrorism
Aerosol (likely) Small volume powder (possible) Foodborne (unlikely)
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Epidemiology
Transmission
Inhalational
Handling hides/skins of infected animals Microbiology laboratory Intentional aerosol release Small volume powdered form
In letters, packages, etc Questionable risk, probably small

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Epidemiology
Transmission
Cutaneous
Handling hides/skins of infected animals Bites from arthropods (very rare) Handling powdered form in letters, etc. Intentional aerosol release
May see some cutaneous if large-scale

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Epidemiology
Transmission
Gastrointestinal
Ingestion of meat from infected animal Ingestion of intentionally contaminated food
Not likely in large scale Spores not as viable in large volumes of water

Ingestion from powder-contaminated hands Inhalational of spores on particles >5 Qm

Land in oropharynx
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Microbiology
Bacillus anthracis
Aerobic, Gram positive rod Long (1-10 m), thin (0.5-2.5 m) Forms inert spores when exposed to O2
Infectious form, hardy Approx 1 m in size

Vegetative bacillus state in vivo

Result of spore germination Non-infectious, fragile

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Microbiology
Colony characteristics
Large (4-5mm) Nonhemolytic Opaque white, gray Retain shape when manipulated (egg white) Forms capsule at 37 C, 5-20% CO2

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Microbiology
Classification
Same family: B. cereus, B. thuringiensis Differentiation from other Bacillus species
Non-motile Non -hemolytic on blood agar Does not ferment salicin

Note: Gram positive rods are usually labeled as contaminants by micro labs
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Microbiology
Environmental Survival
Spores are hardy
Resistant to drying, boiling <10 minutes Survive for years in soil Still viable for decades in perma-frost

Favorable soil factors for spore viability

High moisture Organic content Alkaline pH High calcium concentration
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Microbiology
Virulence Factors
All necessary for full virulence Two plasmids
Capsule (plasmid pXO2)
Antiphagocytic

3 Exotoxin components (plasmid pXO1)

Protective Antigen Edema Factor Lethal Factor
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Microbiology
Protective Antigen
Binds Edema Factor to form Edema Toxin Facilitates entry of Edema Toxin into cells

Edema Factor
Massive edema by increasing intracellular cAMP Also inhibits neutrophil function

Lethal Factor
Stimulates macrophage release of TNF- , IL-1 Initiates cascade of events leading to sepsis
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Pathogenesis
Disease requires entry of spores into body Exposure does not always cause disease
Inoculation dose Route of entry Host immune status May depend on pathogen strain characteristics

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Pathogenesis
Forms of natural disease
Inhalational Cutaneous Gastrointestinal

Determined by route of entry

Disease occurs wherever spores germinate

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Pathogenesis
Inhalational
Spores on particles 1-5 Qm Inhaled and deposited into alveoli Estimated LD50 = 2500 55,000 spores
Dose required for lethal infection in 50% exposed Contained in imperceptibly small volume

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Pathogenesis
Inhalational
Phagocytosed by alveolar macrophages Migration to mediastinal/hilar lymph nodes Germination into vegetative bacilli
Triggered by nutrient-rich environment May be delayed up to 60 days
Factors not completely understood Dose, host factors likely play a role Antibiotic exposure may contribute Delayed germination after antibiotic suppression
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Pathogenesis
Inhalational
Vegetative bacillus is the virulent phase
Active toxin production Hemorrhagic necrotizing mediastinitis
Hallmark of inhalational anthrax Manifests as widened mediastinum on CXR

Does NOT cause pneumonia Followed by high-grade bacteremia

Seeding of multiple organs, including meninges
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Pathogenesis
Inhalational
Toxin production
Has usually begun by time of early symptoms Stimulates cascade of inflammatory mediators
Sepsis Multiorgan failure DIC

Eventual cause of death

Symptoms mark critical mass of bacterial burden Usually irreversible by this time Clearance of bacteria unhelpful as toxin-mediated Early research on antitoxin promising
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Pathogenesis
Cutaneous
Spores in contact with skin
Entry through visible cuts or microtrauma

Germination in skin Disease begins following germination

Toxin production
Local edema, erythema, necrosis, lymphocytic infiltrate No abscess or suppurative lesions

Eventual eschar formation

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Pathogenesis
Cutaneous
Systemic disease
Can occur, especially if untreated Spores/bacteria carried to regional lymph nodes
Lymphangitis/lymphadenitis Same syndrome as inhalational Sepsis, multiorgan failure

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Pathogenesis
Gastrointestinal
Spores contact mucosa
Oropharynx
Ingestion Aerosolized particles >5 Qm

Intestinal mucosa terminal ileum, cecum

Ingestion

Larger number of spores required for disease Incubation period 2-5 days
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Pathogenesis
Gastrointestinal
Spores migrate to lymphatics
Submucosal, mucosal lymphatic tissue Mesenteric nodes

Germination to vegetative bacilli Toxin production

Massive mucosal edema Mucosal ulcers, necrosis

Death from perforation or systemic disease

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Clinical Features
Symptoms depend on form of disease
Inhalational Cutaneous Gastrointestinal

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Clinical Features
Inhalational
Asymptomatic incubation period
Duration 2-43 days, ~10 days in Sverdlovsk

Prodromal phase
Correlates with germination, toxin production Nonspecific flu-like symptoms
Fever, malaise, myalgias Dyspnea, nonproductive cough, mild chest discomfort

Duration several hours to ~3 days Can have transient resolution before next phase
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Clinical Features
Inhalational
Fulminant Phase
Correlates with high-grade bacteremia/toxemia Critically Ill
Fever, diaphoresis Respiratory distress/failure, cyanosis Septic shock, multiorgan failure, DIC

50% develop hemorrhagic meningitis

Headache, meningismus, delirium, coma May be most prominent finding

Usually progresses to death in <36 hrs

Mean time from symptom onset to death ~3 days
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Clinical Features
Laboratory Findings
Gram positive bacilli in direct blood smear Electrolyte imbalances common

Radiographic Findings
Widened mediastinum
Minimal or no infiltrates

Can appear during prodrome phase

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Clinical Features
Cutaneous
Most common areas of exposure
Hands/arms Neck/head

Incubation period
3-5 days typical 12 days maximum

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Clinical Features
Cutaneous progression of painless lesions
Papule pruritic
24-36 hrs

Vesicle/bulla Ulcer contains organisms, sig. edema

days

Eschar black, rarely scars

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Clinical Features
Cutaneous
Systemic disease may develop
Lymphangitis and lymphadenopathy If untreated, can progress to sepsis, death

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Clinical Features
Gastrointestinal
Oropharyngeal
Oral or esophageal ulcer
Regional lymphadenopathy Edema, ascites Sepsis

Abdominal
Early symptoms - nausea, vomiting, malaise Late - hematochezia, acute abdomen, ascites
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Diagnosis
Early diagnosis is difficult
Non specific symptoms Initially mild No readily available rapid specific tests

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Diagnosis
Presumptive diagnosis
History of possible exposure Typical signs & symptoms Rapidly progressing nonspecific illness Widened mediastinum on CXR Large Gram+ bacilli from specimens
Can be seen on Gram stain if hi-grade bacteremia

Appropriate colonial morphology Necrotizing mediastinitis, meningitis at autopsy

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Diagnosis
Definitive diagnosis
Direct culture on standard blood agar
Gold standard, widely available Alert lab to work up Gram + bacilli if found 6-24 hours to grow Sensitivity depends on severity, prior antibiotic
Blood, fluid from skin lesions, pleural fluid, CSF, ascites Sputum unlikely to be helpful (not a pneumonia)

Very high specificity if non-motile, non-hemolytic Requires biochemical tests for >99% confirmation
Available at Reference laboratories
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Diagnosis
Definitive diagnosis
Rapid confirmatory tests
Role is to confirm if cultures are negative Currently available only at CDC
Polymerase Chain Reaction (PCR) Hi sensitivity and specificity Detects DNA Viable bacteria/spores not required Immunohistochemical stains Most clinical specimens can be used

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Diagnosis
Other diagnostic tests
Anthraxin skin test
Chemical extract of nonpathogenic B. anthracis Subdermal injection 82% sensitivity for cases within 3 days symptoms 99% sensitivity 4 weeks after symptom onset Not much experience with use in U.S. not used

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Diagnosis
Testing for exposure
Nasal swabs
Can detect spores prior to illness Currently used only as epidemiologic tool
Decision for PEP based on exposure risk May be useful for antibiotic sensitivity in exposed

Culture on standard media Swabs of nares and facial skin

Serologies
May be useful from epidemiologic standpoint Investigational only available at CDC
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Diagnosis
Environmental samples
Suspicious powders
Must be sent to reference laboratories as part of epidemiologic/criminal investigation Assessed using cultures, stains, PCR

Air sampling First responders

Handheld immunoassays
Not validated Useful for detecting massive contamination
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Diagnosis
Test
Culture Biochemical Skin test PCR ELISA

Availability Time
Most labs Large labs None Reference Reference

Sens

Spec
High High ? High High
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1-3 days Mod Hours N/A

1-2 days High Hours Hours High Mod

Differential Diagnosis
Inhalational
Influenza Pneumonia
Community-acquired Atypical Pneumonic tularemia Pneumonic plague

Expect if anthrax
Flu rapid diagnostic More severe in young pts No infiltrate

Mediastinitis Bacterial meningitis Thoracic aortic aneurysm

No prior surgery Bloody CSF with GPBs Fever

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Differential Diagnosis
Cutaneous
Spider bite Ecthyma gangrenosum Pyoderma gangrenosum Ulceroglandular tularemia Mycobacterial ulcer Cellulitis

Expect if anthrax
fever no response to 3 cephs painless, black eschar +/- lymphadenopathy usually sig. local edema

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Differential Diagnosis
Gastrointestinal
Gastroenteritis Typhoid Peritonitis Perforated ulcer Bowel obstruction

Expect if anthrax
Critically ill Acute abdomen Bloody diarrhea Fever

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Differential Diagnosis
Impact of suspected BT during flu season
Early disease mimics influenza Affects same population Increased role for rapid flu tests
Possible development of ER protocols
In settings of high suspicion for BT release Observation until flu test results obtained

Caveats
Possible addition of influenza to aerosol release False positives/negatives Must still use clinical judgement
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Treatment
Immediately treat presumptive cases
Prior to confirmation Rapid antibiotics may improve survival

Differentiate between cases and exposed

Cases
Potentially exposed with any signs/symptoms

Exposed
Potentially exposed but asymptomatic Provide Post-Exposure Prophylaxis
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Treatment
Hospitalization IV antibiotics
Empiric until sensitivities are known

Intensive supportive care

Electrolyte and acid-base imbalances Mechanical ventilation Hemodynamic support
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Treatment
Antibiotic selection
Naturally occurring strains
Rare penicillin resistance, but inducible -lactamase Penicillins, aminoglycosides, tetracyclines, erythromycin, chloramphenicol have been effective Ciprofloxacin very effective in vitro, animal studies Other fluoroquinolones probably effective

Engineered strains
Known penicillin, tetracycline resistance Highly resistant strains = mortality of untreated
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Treatment
Empiric Therapy
Until susceptibility patterns known Adults
Ciprofloxacin 400 mg IV q12 OR Doxycycline 100mg IV q12 AND (for inhalational) One or two other antibiotics

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Treatment
Other antibiotic considerations
Other fluoroquinolones possibly equivalent High dose penicillin for 2nd empiric agent
50% present with meningitis

Clindamycin for severe disease

May reduce toxin production

Chloramphenicol for known meningitis

Penetrates blood brain barrier
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Treatment
Empiric Therapy
Children
Ciprofloxacin 10-15 mg/kg/d IV q12, max 1 g/d OR Doxycycline 2.2 mg/kg IV q12 (adult dosage if >8 yo and >45 kg) Add one or two antibiotics for inhalational Weigh risks (arthropathy, dental enamel)

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Treatment
Empiric therapy
Pregnant women
Same as other adults Weigh small risks (fetal arthropathy) vs benefit

Immunosuppressed
same as other adults

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Treatment
Alternative antibiotics
If susceptible, or cipro/doxy not possible
Penicillin*, amoxicillin *FDA Approved Gentamicin, streptomycin Erythromycin, chloramphenicol

Ineffective antibiotics
Trimethoprim/Sulfamethoxazole Third generation cephalosporins

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Treatment
Susceptibility testing should be done
Narrow antibiotic if possible Must be cautious
Multiple strains with engineered resistance to different antibiotics may be coinfecting Watch for clinical response after switching antibiotic

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Treatment
Antibiotic therapy
Duration
60 days
Risk of delayed spore germination Vaccine availability Could reduce to 30-45 days therapy Stop antibiotics after 3rd vaccine dose

Switch to oral
Clinical improvement Patient able to tolerate oral medications
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Treatment
Other therapies
Passive immunization
Anthrax immunoglobulin from horse serum Risk of serum sickness

Antitoxin
Mutated Protective Antigen
Blocks cell entry of toxin Still immunogenic, could be an alternative vaccine Animal models promising
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Postexposure Prophylaxis
Who should receive PEP?
Anyone exposed to anthrax Not for contacts of cases, unless also exposed

Empiric antibiotic therapy Vaccination

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Postexposure Prophylaxis
Avoid unnecessary antibiotic usage
Potential shortages of those who need them Potential adverse effects
Hypersensitivity Neurological side effects, especially elderly Bone/cartilage disease in children Oral contraceptive failure

Future antibiotic resistance

Individuals own flora Community resistance patterns
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Postexposure Prophylaxis
Antibiotic therapy
Treat ASAP Prompt therapy can improve survival Continue for 60 days
30-45 days if vaccine administered

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Postexposure Prophylaxis
Antibiotic therapy
Same regimen as active treatment
Substituting oral equivalent for IV Ciprofloxacin 500 mg po bid empirically Alternatives
Doxycycline 100 mg po bid Amoxicillin 500 mg po tid

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Postexposure Prophylaxis
Antibiotic therapy
Children
Same dose adjustments as treatment Weigh benefits vs. risks Recommended switch if PCN-susceptible
Amoxicillin 80 mg/kg/day, max 500 mg tid

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Prevention
Vaccine
Anthrax Vaccine Adsorpbed (AVA) Supply
Limited, controlled by CDC Production problems
Single producer Bioport, Michigan Failed FDA standards None produced since 1998

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Prevention
Vaccine
Inactivated, cell-free filtrate Adsorbed onto Al(OH)3 Protective Antigen
Immunogenic component Necessary but not sufficient

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Prevention
Vaccine
Administration
Dose schedule
0, 2 & 4 wks; 6, 12 & 18 months initial series Annual booster

0.5 ml SQ

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Prevention
Vaccine Effective and Safe
Efficacy
>95% protection vs. aerosol in animal models >90% vs. cutaneous in humans
Older vaccine that was less immunogenic Protection vs inhalational but too few cases to confirm

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Prevention
Vaccine
Adverse Effects
>1.6 million doses given to military by 4/2000 No deaths <10% moderate/severe local reactions
Erythema, edema

<1% systemic reactions

Fever, malaise

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Infection Control
No person to person transmission Standard Precautions Laboratory safety
Biosafety Level (BSL) 2 Precautions

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Decontamination
Highest risk of infection at initial release
Duration of aerosol viability
Several hours to one day under optimal conditions Covert aerosol long dispersed by recognition 1st case

Risk of secondary aerosolization is low

Heavily contaminated small areas
May benefit from decontamination

Decontamination may not be feasible for large areas

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Decontamination
Skin, clothing
Thorough washing with soap and water Avoid bleach on skin

Instruments for invasive procedures

Sterilize, e.g. 5% hypochlorite solution

Sporicidal agents
Sodium or calcium hypochlorite (bleach)
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Decontamination
Suspicious letters/packages
Do not open or shake Place in plastic bag or leakproof container If visibly contaminated or container unavailable
Gently cover paper, clothing, box, trash can

Leave room/area, isolate room from others Thoroughly wash hands with soap and water Report to local security / law enforcement List all persons in vicinity
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Decontamination
Opened envelope with suspicious substance
Gently cover, avoid all contact Leave room and isolate from others Thoroughly wash hands with soap and water Notify local security / law enforcement Carefully remove outer clothing, put in plastic Shower with soap and water List all persons in area
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Outbreak Investigations 2001

Case definitions
Confirmed case
Clinically compatible syndrome +culture or 2 +non-culture diagnostics

Presumptive case
Clinically compatible syndrome 1 +non-culture diagnostic or confirmed exposure

Exposures
Confirmed exposure
May be aided by nasal swab cultures, serology

Asymptomatic
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Outbreak Investigations 2001

Florida (Palm Beach)
1st U.S. case since 1976 reported 10/4/01 1st ever cases of intentional infection Inhalational Index Case
63yo man presented with fever and altered MS Preceding flu-like symptoms Reported by astute clinician
Noticed GPBs in CSF on 10/2 Lab confirmation by State and CDC on 10/4

Rapid deterioration, died on 10/5

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Outbreak Investigations 2001

Florida Case #2
73yo man Admitted 10/1 for pneumonia Nasal swab culture + on 10/5 PCR+ on pleural fluid, serology + Responding to antibiotics, still in hospital

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Outbreak Investigations 2001

Florida
Exposed
Anyone at worksite for >1 hour since 8/1 1/1075 nasal swabs +, all given PEP

Confirmed powder exposure from mail

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Outbreak Investigations 2001

New York City - cutaneous cases
Case #1 38 yo woman, NBC employee
Handled suspicious letter with powder marked 9/18 9/25 developed raised skin lesion on chest
Progressive erythema, edema over 3 days

9/29 malaise and HA, lesion painless 10/1 5cm oval, raised border, satellite vesicles
Left cervical LAD Black eschar over next few days

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Outbreak Investigations 2001

New York City cutaneous cases
Case#1
Vesicle fluid cx and Gram stain Eschar biopsy +immunohistochemical stain Powder in letter confirmed anthrax spores Improving on oral ciprofloxacin

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Outbreak Investigations 2001

New York City cutaneous cases
Case #2 7 month old son of ABC worker
Visited worksite on 9/28 9/29 large weeping skin lesion left arm
Nontender, massive edema Progressed to ulcerative with black eschar Initial Dx- spider bite Complicated by hemolytic anemia, thrombocytopenia

10/12 anthrax considered

10/2 blood PCR+, 10/13 skin bx IHC stain+

No source identified, improving with ciprofloxacin

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Outbreak Investigations 2001

New York City
Exposures by nasal/facial swab cxs
Police officer transporting the NBC sample 2 lab techs processing NBC sample

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Outbreak Investigations 2001

Washington, D.C.
Letter sent to Senator Daschle
Originated from Trenton, NJ 28 Senate staff confirmed exposure Evacuation of Senate then House

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Outbreak Investigations 2001

Trenton, New Jersey
2 confirmed inhalational cases
Postal workers in distribution center Others with symptoms, results pending

2 suspicious deaths
Probable inhalational anthrax

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Outbreak Investigations 2001

As of 10/22/01

FL 2 0 2 6 1

NY 0 4 4 3 0

NJ 4 0 4 ? 2

DC 0 1 1 29 0

Inhalational Cutaneous Total Cases Exposure Deaths

(all inhalational)

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Anthrax Essential Pearls

Rapidly fatal flu-like illness in previous healthy Widened mediastinum on Chest X-ray Painless black skin ulcer Non-motile gram positive bacilli in specimens Diagnosis primarily by routine culture No person-to-person transmission Rx prior to prodrome essential for survival Empiric therapy - ciprofloxacin
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Anthrax Essential Pearls

Single inhalational case is an emergency
Contact Local Health Departments ASAP

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