Bioterrorism Exercise ReviewOverview of B' anthracis

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Bioterrorism Exercise Review/Overview of B.
anthracis
Thomas Wisniewski, M.S. R.M. (AAM) Microbiology
Supervisor Clement J. Zablocki VA Medical
Center Milwaukee, Wisconsin David Warshauer,
Ph.D. Deputy Director Communicable Disease
Division Wisconsin State Laboratory of
Hygiene Madison, Wisconsin
November 14, 2007
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Bioterrorism Exercise Review/Overview of B.
anthracis

• Review of
• Bioterrorism Proficiency
• Educational Exercise

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Bioterrorism Exercise Review/Overview of B.
anthracis
Overview of WSLH Bioterrorism Proficiency Exercise

• Exercises provided twice a year (spring and
  autumn)
• A subset of laboratories is asked to follow
  notification/shipping protocols
• Purpose of exercises
• Practice rule out testing
• Identify gaps in emergency lab response system
• Practice and assess notification and transport
• Participation in exercises is voluntary, not
  connected to any regulatory agencies
• Individual results are shared only with the
  participating laboratory
• Each exercise is followed by an audioconference

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Bioterrorism Exercise Review/Overview of B.
anthracis
Overview of WSLH Bioterrorism Proficiency Exercise

• Two samples sent to 116 Wisconsin laboratories
• 105 reported test results (91)
• 11 did not report results
• 16 did not receive samples due to previous
  communication so they chose not to participate
• Sample BPE 07-2-1 Bacillus megaterium
• Sample BPE 07-2-2 Bacillus cereus
• Samples intended to simulate Bacillus anthracis
  in rule out testing

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Bioterrorism Exercise Review/Overview of B.
anthracis

• Results of Rule Out Tests

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Bioterrorism Exercise Review/Overview of B.
anthracis

• Results of Rule Out Tests (continued)

Expected reaction erroneously reported to be
positive in report
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Bioterrorism Exercise Review/Overview of B.
anthracis

• Results of Rule Out Tests (continued)

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Bioterrorism Exercise Review/Overview of B.
anthracis

• Results of Rule Out Tests (continued)

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Bioterrorism Exercise Review/Overview of B.
anthracis

• Results of Rule Out Tests (continued)

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Bioterrorism Exercise Review/Overview of B.
anthracis

• Results of Rule Out Tests (continued)

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Bioterrorism Exercise Review/Overview of B.
anthracis

• EMERGENCY LABORATORY RESPONSE EXERCISE
• Notification of WSLH via 24/7 Phone Number

Labs should notify WSLH 24/7 number per Wisconsin
Emergency Response Guide for Clinical
Laboratories protocol.
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Bioterrorism Exercise Review/Overview of B.
anthracis

• EMERGENCY LABORATORY RESPONSE EXERCISE
• Method of Transport

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Bioterrorism Exercise Review/Overview of B.
anthracis

• EMERGENCY LABORATORY RESPONSE EXERCISE
• Packaging

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Bioterrorism Exercise Review/Overview of B.
anthracis

• Overview of
• Bacillus anthracis

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Bioterrorism Exercise Review/Overview of B.
anthracis
Bacillus

• The genus is large, ca 70 species
• Aerobic, spore-forming, large, Gram-positive rods
  
• Motile or non-motile
• B. anthracis, B. cereus, and B. thuringiensis
  belong to the B. cereus group
• B. anthracis, the etiological agent of anthrax

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Bioterrorism Exercise Review/Overview of B.
anthracis
Terrorism Biological AgentsA List Examples

• Variola major
• Bacillus anthracis
• Yersinia pestis
• Clostridium botulinum toxin
• Francisella tularensis
• VHFs

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Bioterrorism Exercise Review/Overview of B.
anthracis
Biological AgentsA List - Highest Priority
ListNational Security Risk

• Easily disseminated or transmitted person to
  person
• High mortality - major P.H. impact
• Cause public panic, social disruption
• Special action for P.H. preparedness

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Bioterrorism Exercise Review/Overview of B.
anthracis
Anthrax Sent by MailSeptember/October 2001
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Bioterrorism Exercise Review/Overview of B.
anthracis
Epidemiology of Anthrax

• Acquired from contact with anthrax-infected
  animals or contaminated animal products
• Wool-sorters disease
• Most commonly seen in herbivores
• Cattle, sheep
• Ingestion of spores from the soil
• Vaccination programs have reduced mortality in
  animals

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Bioterrorism Exercise Review/Overview of B.
anthracis
Anthrax in Humans

• 3 major routes of infection
• Cutaneous
• Most common
• 2000 cases world
• In USA, 224 cases between 1944-1994. One in 2000
  Two in 2006
• Inhalation
• In USA, 18 cases of inhalation anthrax 1900-1976
• Goat hair mill or wool or tannery workers
• 2 laboratory acquired
• No naturally occurring cases since 1976

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Bioterrorism Exercise Review/Overview of B.
anthracis
Anthrax in Humans (continued)

• Gastrointestinal
• Outbreaks reported in Africa and Asia
• Ingestion of infected, insufficiently cooked
  contaminated meat with large numbers of
  vegetative cells

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Bioterrorism Exercise Review/Overview of B.
anthracis
Diseases

• Cutaneous
• Inhalational
• Gastrointestinal

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Bioterrorism Exercise Review/Overview of B.
anthracis
Cutaneous Anthrax

• Environmentally resistant spore enters a cut or
  abrasion
• Hands, forearms, head
• Incubation 12 hours to 12 days
• Initially itchy, leads to
• Papule
• Vesicle
• Eschar (1-3 cm black ulcer)
• Local edema lymphadenitis
• Death is rare if treated, but 5-20 mortality if
  untreated due to systemic or respiratory failure

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Bioterrorism Exercise Review/Overview of B.
anthracis
Cutaneous Anthrax
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Bioterrorism Exercise Review/Overview of B.
anthracis
Inhalation Anthrax

• Inhalation of B. anthracis spores leads to
  gradual, nonspecific onset
• Fever, malaise, and fatigue
• Nonproductive cough and mild chest discomfort
• A short period of improvement
• Abrupt, severe respiratory distress
• Shock and death usually rapidly occur despite
  treatment

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Bioterrorism Exercise Review/Overview of B.
anthracis
Gastrointestinal Anthrax

• Rare form, an acute inflammation of the GI tract
• Occurs in outbreaks from ingestion of meat from
  infected animals
• Symptoms
• Nausea/vomiting, anorexia, fever
• Abdominal pain, vomiting of blood, severe bloody
  diarrhea
• Difficult to diagnose, untreated mortality rate
  is 25-60

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Bioterrorism Exercise Review/Overview of B.
anthracis
Natural Anthrax Cases in the U.S.

• Cutaneous anthrax
• North Dakota farmer August 2000
• 67 yr old man disposing of five cows
• Wore leather gloves
• Developed small bump 4 days later on left cheek
• Afebrile
• Firm, nontender, superficial nodule with a 0.5 cm
  black eschar.

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Bioterrorism Exercise Review/Overview of B.
anthracis
Cutaneous Anthrax (continued)

• No cultures obtained
• Placed on ciprofloxacin for 14 days
• Paired serum obtained on September 22 and October
  5
• Positive antibody titers by ELISA to protective
  antigen confirmed B. anthracis infection
• Case was associated with anthrax epizootic in N.
  Dakota
• 32 farms were quarantined for anthrax in 2000
• 157 animals died on 31 farms

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Bioterrorism Exercise Review/Overview of B.
anthracis
Natural Anthrax Cases in the U.S.

• Inhalation Anthrax
• February 16, 2006 drummer collapsed
• Admitted to local Pennsylvania hospital
• Chest x-ray
• Bilateral infiltrates and pleural effusions
• February 17, transferred to tertiary care center
  with worsening respiratory status
• 4 BCs grew gram-positive rods
• Confirmed at SPHL as B. anthracis on February 21
• Serology positive for protective antigen antibody

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Bioterrorism Exercise Review/Overview of B.
anthracis
Inhalation Anthrax

• Epidemiology
• Patient made traditional African drums
• Used hard-dried animal hides
• Goat hides from Africa
• Scraped hair off with a razor, generating
  aerosols
• Did not wear gloves or mask
• Environmental samples
• Home, van, workspace
• All positive by culture and PCR

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Bioterrorism Exercise Review/Overview of B.
anthracis
Inhalation Anthrax (continued)

• Four persons received postexposure prophylaxis
• Had been in workspace during procedures that
  generated aerosols
• No additional cases identified

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Bioterrorism Exercise Review/Overview of B.
anthracis
Specimen Collection for Suspected Cases

• Cutaneous
• Vesicle, Eschar fluid (blood if systemic)
• Inhalational
• Lower respiratory secretions, Blood
• Gastrointestinal
• Stool, Blood

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Bioterrorism Exercise Review/Overview of B.
anthracis
Specimen Collection for Suspected Cases
(continued)

• Postmortem
• Blood
• Hemorrhagic fluids
• Avoid contamination, do not necropsy
• Note that putrefactive microorganisms rapidly
  overgrow B. anthracis, hindering postmortem
  recovery (Carcasses contain spores for years!)

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Bioterrorism Exercise Review/Overview of B.
anthracis
Routine Clinical Specimens

• Blood
• CSF
• Other Sterile fluids
• Lower Respiratory Specimens
• Skin Wounds
• Other Unlikely Sites

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Bioterrorism Exercise Review/Overview of B.
anthracis
Biosafety Considerations

• Person to person transmission is rare aerosol
  generating activities are the primary risk
• Overt
• Sub-culturing Blood Cultures
• Centrifuging
• Grinding
• Vortex-mixing
• Covert
• Hot looping
• Sniffing plates

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Bioterrorism Exercise Review/Overview of B.
anthracis
Biosafety Considerations

• Use BSL-2 practices to process any clinical
  specimen suspected to contain B. anthracis
• Need to Consider Treating All Specimens as
  Potential Source
• PPE and a class I or II biosafety cabinet for
  aerosol-generating activities
• Use BSL 2.5 when processing suspected B.
  anthracis cultures
• BSL-3 practices and containment equipment in a
  BSL-2 facility
• All procedures with PPE in a I/II biosafety
  cabinet

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Bioterrorism Exercise Review/Overview of B.
anthracis
A combination of two tests is recommended for
Rule Out
WSLH LabTAG Bench Guide, 1st edition
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Bioterrorism Exercise Review/Overview of B.
anthracis
Gram Stain Morphology

• Blood
• Short chains of 2-4 cells
• Negative staining capsule
• Large encapsulated Gram positive bacilli
• No spores (high CO2 level inhibit)

• Blood Agar
• Chains of Gram positive bacilli
• oval, central-to-subterminal spores without
  significant cell wall distention (No tennis
  racquets)
• Capsule not easily induced in vivo

Either finding presumptive B. anthracis
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Bioterrorism Exercise Review/Overview of B.
anthracis
Gram Stain
Single cells or short chains
Long chains
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Bioterrorism Exercise Review/Overview of B.
anthracis
Colony Characteristics

• After incubation of SBA plate for 16-24 hr at
  35C, colonies
• Are 2-5 mm in diameter
• Are irregularly round, flat or slightly convex
  with wavy edges
• Have a ground-glass appearance
• Have comma-shaped projections from the colony
  edge, producing the "Medusa-head" form
• Typically have a tenacious consistency (i.e.,
  stands up like beaten egg white)

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Bioterrorism Exercise Review/Overview of B.
anthracis
Colony Morphology
B. anthracis
B. cereus
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Bioterrorism Exercise Review/Overview of B.
anthracis
Motility

• Motility rules out B. anthracis
• A rapid slide motility test may be used, but if
  negative, confirm with overnight tube motility
  medium

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Bioterrorism Exercise Review/Overview of B.
anthracis
Additional Test - India Ink

• Not Highly Recommended or Commonly Done
• India ink may visualize the capsule of B.
  anthracis in body fluids
• Problems
• Who has India ink anymore?
• Insensitive and non-specific
• Use cautiously results are presumptive

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Bioterrorism Exercise Review/Overview of B.
anthracis
Additional Test - Penicillin Sensitivity

• Not Highly Recommended or Commonly Done
• Pen sensitivity was once a cornerstone of B.
  anthracis rapid ID, but has fallen out of favor.
• Why?
• No CLSI standards what is Sensitive?
• B. anthracis possesses beta-lactamases poorly
  expressed in vitro
• Potentially engineered strain penicillin
  resistant?
• Growth up to a Pen disk suggests resistance
  interpret with caution

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Bioterrorism Exercise Review/Overview of B.
anthracis
Packaging - Infectious Substances

• Triple packaging
• Several other specific requirements must be met
  for packaging

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Bioterrorism Exercise Review/Overview of B.
anthracis
Referring Possible BT Agent

• Refer to the Wisconsin Emergency Response Guide
  for Clinical Laboratories
• Contact the State Lab, call 24/7 Pager number
  [phone]
• Ship isolate as Infectious Substance to WSLH via
  Dunham Express or your courier

Wisconsin State Laboratory of Hygiene Communicable
Diseases Rapid Response
?
?
6.2 Infectious Substance Shipper
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Bioterrorism Exercise Review/Overview of B.
anthracis
Reference Laboratory Testing

• Real-time PCR
• Gamma phage susceptibility
• DFA stains
• Capsule
• Cell wall-associated polysaccharide
• Capsule production
• MFadyean stain

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Bioterrorism Exercise Review/Overview of B.
anthracis
Lysis by Gamma Phage
Susceptibility of suspected isolate to lysis by
gamma phage offers a rapid and reliable means of
distinguishing B. anthracis from other Bacillus
spp.
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Bioterrorism Exercise Review/Overview of B.
anthracis
Gamma Phage Test
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Bioterrorism Exercise Review/Overview of B.
anthracis
Capsule Visualization Using MFadyean Stain
Capsule appears as pink area around blue bacilli.
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Bioterrorism Exercise Review/Overview of B.
anthracis
Select Agent Program and Clinical Labs

• What are diagnostic/clinical labs required to do
  if they encounter a select agent?
• Select Agent Regulations cover specific
  guidelines regarding
• Notification
• Select agent handling protocol
• Documentation
• Final rule published in the Federal Register
• 7 CFR Part 331 and 9 CFR Part 121
• http//www.cdc.gov/od/sap/final_rule.htm

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Bioterrorism Exercise Review/Overview of B.
anthracis
Select Agent Reporting

• Contact CDC by telephone
• [phone]
• Within 7 days of identification
• Transfer to a registered entity
• Destroy---Autoclave, incinerate,
• Documentation
• CDC Form 4 (www.cdc.gov/od/sap)
• Maintain a copy of the record for 3 years

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Bioterrorism Exercise Review/Overview of B.
anthracis
Summary Specimen Collection

• Cutaneous
• Vesicle/Eschar fluids (blood)
• Inhalational
• Lower respiratory secretions
• Blood
• Gastrointestinal
• Stool
• Blood
• Routine Clinical Specimens

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Bioterrorism Exercise Review/Overview of B.
anthracis
Summary Key Sentinel Lab Tests

• Gram stain
• Capsule (direct)
• Spores (culture)
• Colonial morphology
• Non-hemolytic
• Irregular, white, ground glass, Medusa Head
• Tenacious
• Isolate findings
• Catalase positive
• Non-motile

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Bioterrorism Exercise Review/Overview of B.
anthracis
Thank You to All the Participating
Laboratories!