Biological Terrorism

1

Biological Terrorism Anthrax
2
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

3
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

4
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)

5
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 µm size
• Optimal size for deposition into alveoli
• Form of disease with highest mortality
• Would infect the largest number of people

6
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 gt hands gt eating without handwashing

7
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

8
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

9
Epidemiology

• Three forms of natural disease
• Inhalational
• Rare (lt5)
• Most likely encountered in bioterrorism event
• Cutaneous
• Most common (95)
• Direct contact of spores on skin
• Gastrointestinal
• Rare (lt5), never reported in U.S.
• Ingestion

10
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

11
Epidemiology

• Mortality
• Inhalational 86-100 (despite treatment)
• Era of crude intensive supportive care
• Cutaneous lt5 (treated) 20 (untreated)
• GI approaches 100

12
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

13
Epidemiology

• Human cases historical risk factors
• Agricultural
• Exposure to livestock
• Occupational
• Exposure to wool and hides
• Woolsorters disease inhalational anthrax
• Rarely laboratory-acquired

14
Epidemiology

• Transmission
• No human-to-human
• Naturally occurring cases
• Skin exposure
• Ingestion
• Airborne
• Bioterrorism
• Aerosol (likely)
• Small volume powder (possible)
• Foodborne (unlikely)

15
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

16
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

17
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 gt5 ?m
• Land in oropharynx

18
Microbiology

• Bacillus anthracis
• Aerobic, Gram positive rod
• Long (1-10µm), thin (0.5-2.5µm)
• Forms inert spores when exposed to O2
• Infectious form, hardy
• Approx 1µm in size
• Vegetative bacillus state in vivo
• Result of spore germination
• Non-infectious, fragile

19
Microbiology

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

20
Microbiology

• Classification
• Same family B. cereus, B. thuringiensis
• Differentiation from other Bacillus species
• Non-motile
• Non ß-hemolytic on blood agar
• Does not ferment salicin
• Note Gram positive rods are usually labeled as
  contaminants by micro labs

21
Microbiology

• Environmental Survival
• Spores are hardy
• Resistant to drying, boiling lt10 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

22
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

23
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-a, IL-1ß
• Initiates cascade of events leading to sepsis

24
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

25
Pathogenesis

• Forms of natural disease
• Inhalational
• Cutaneous
• Gastrointestinal
• Determined by route of entry
• Disease occurs wherever spores germinate

26
Pathogenesis

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

27
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

28
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

29
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

30
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

31
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

32
Pathogenesis

• Gastrointestinal
• Spores contact mucosa
• Oropharynx
• Ingestion
• Aerosolized particles gt5 ?m
• Intestinal mucosa terminal ileum, cecum
• Ingestion
• Larger number of spores required for disease
• Incubation period 2-5 days

33
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

34
Clinical Features

• Symptoms depend on form of disease
• Inhalational
• Cutaneous
• Gastrointestinal

35
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

36
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 lt36 hrs
• Mean time from symptom onset to death 3 days

37
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

38
Clinical Features

• Cutaneous
• Most common areas of exposure
• Hands/arms
• Neck/head
• Incubation period
• 3-5 days typical
• 12 days maximum

39
Clinical Features

• Cutaneous progression of painless lesions
• Papule pruritic
• Vesicle/bulla
• Ulcer contains organisms, sig. edema
• Eschar black, rarely scars

24-36 hrs
days
40
Clinical Features

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

41
Clinical Features

• Gastrointestinal
• Oropharyngeal
• Oral or esophageal ulcer
• Regional lymphadenopathy
• Edema, ascites
• Sepsis
• Abdominal
• Early symptoms - nausea, vomiting, malaise
• Late - hematochezia, acute abdomen, ascites

42
Diagnosis

• Early diagnosis is difficult
• Non specific symptoms
• Initially mild
• No readily available rapid specific tests

43
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

44
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 gt99 confirmation
• Available at Reference laboratories

45
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

46
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

47
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

48
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

49
Diagnosis
50
Differential Diagnosis

• Inhalational
• Influenza
• Pneumonia
• Community-acquired
• Atypical
• Pneumonic tularemia
• Pneumonic plague
• Mediastinitis
• Bacterial meningitis
• Thoracic aortic aneurysm

• Expect if anthrax
• Flu rapid diagnostic
• More severe in young pts
• No infiltrate
• No prior surgery
• Bloody CSF with GPBs
• Fever

51
Differential Diagnosis

• Cutaneous
• Spider bite
• Ecthyma gangrenosum
• Pyoderma gangrenosum
• Ulceroglandular tularemia
• Mycobacterial ulcer
• Cellulitis

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

52
Differential Diagnosis

• Gastrointestinal
• Gastroenteritis
• Typhoid
• Peritonitis
• Perforated ulcer
• Bowel obstruction

• Expect if anthrax
• Critically ill
• Acute abdomen
• Bloody diarrhea
• Fever

53
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

54
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

55
Treatment

• Hospitalization
• IV antibiotics
• Empiric until sensitivities are known
• Intensive supportive care
• Electrolyte and acid-base imbalances
• Mechanical ventilation
• Hemodynamic support

56
Treatment

• Antibiotic selection
• Naturally occurring strains
• Rare penicillin resistance, but inducible
  ß-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

57
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

58
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

59
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 gt8 yo and gt45 kg)
• Add one or two antibiotics for inhalational
• Weigh risks (arthropathy, dental enamel)

60
Treatment

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

61
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

62
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

63
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

64
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

65
Postexposure Prophylaxis

• Who should receive PEP?
• Anyone exposed to anthrax
• Not for contacts of cases, unless also exposed
• Empiric antibiotic therapy
• Vaccination

66
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

67
Postexposure Prophylaxis

• Antibiotic therapy
• Treat ASAP
• Prompt therapy can improve survival
• Continue for 60 days
• 30-45 days if vaccine administered

68
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

69
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

70
Prevention

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

71
Prevention

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

72
Prevention

• Vaccine
• Administration
• Dose schedule
• 0, 2 4 wks 6, 12 18 months initial series
• Annual booster
• 0.5 ml SQ

73
Prevention

• Vaccine Effective and Safe
• Efficacy
• gt95 protection vs. aerosol in animal models
• gt90 vs. cutaneous in humans
• Older vaccine that was less immunogenic
• Protection vs inhalational but too few cases to
  confirm

74
Prevention

• Vaccine
• Adverse Effects
• gt1.6 million doses given to military by 4/2000
• No deaths
• lt10 moderate/severe local reactions
• Erythema, edema
• lt1 systemic reactions
• Fever, malaise

75
Infection Control

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

76
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

77
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)

78
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

79
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

80
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

81
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

82
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

83
Outbreak Investigations 2001

• Florida
• Exposed
• Anyone at worksite for gt1 hour since 8/1
• 1/1075 nasal swabs , all given PEP
• Confirmed powder exposure from mail

84
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

85
Outbreak Investigations 2001

• New York City cutaneous cases
• Case1
• Vesicle fluid cx and Gram stain
• Eschar biopsy immunohistochemical stain
• Powder in letter confirmed anthrax spores
• Improving on oral ciprofloxacin

86
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

87
Outbreak Investigations 2001

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

88
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

89
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

90
Outbreak Investigations 2001
91
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

92
Anthrax Essential Pearls

• Single inhalational case is an emergency
• Contact Local Health Departments ASAP