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Bacillus anthracis Centers for Disease Control and Prevention

Anthrax: Background

"Anthrax:" Anthrax: Basics

Epidemiology of Anthrax in the 21st Century Agricultural, farm workers exposed to infected animals (rare) Non-industrial:  laboratorians through close contact with B. anthracis spores or civilians exposed to contaminated imported animal products (rare) Industrial:  processors of wool, hair, hides, bones, or other animal products (now rare) Intentional/bioterrorist:  inhalational and cutaneous exposure to B. anthracis spores through U.S. mail

"Cases of Anthrax in the..." Cases of Anthrax in the U.S., 1951–2000* (N = 409)

"Anthrax:" Anthrax: Current Issues in the U.S.

Anthrax Bioterrorism Issues (1) Surveillance for cutaneous and inhalational disease to identify attack Targeting prevention strategies Rapidly identify exposed populations Conduct epidemiologic investigation with environmental testing Supply postexposure prophylaxis Trace route of vehicle of exposure

Anthrax Bioterrorism Issues (2) Environmental assessment to determine exposures Decontamination Defining population at risk for pre-exposure immunization

Threat Assessment of Anthrax FBI and other law enforcement authorities  are investigating intentional exposures as criminal acts. Until source of exposures is eliminated, exposure to B. anthracis and subsequent clinical illness may continue. Clinicians and laboratorians should be vigilant for B. anthracis infection, particularly among mail handlers. CDC will provide updated information at www.bt.cdc.gov

Threat Assessment Clinical laboratorians should be alert to Bacillus species, particularly in specimens from previously healthy patients with rapidly progressive respiratory illness or cutaneous ulcer. If B. anthracis is suspected, laboratories should immediately notify the healthcare provider and local and state public health staff. For rapid identification of B. anthracis, state and local health departments should access the Laboratory Response Network for Bioterrorism (LRN).

Exposure Situation Management: B. anthracis in Envelope Antimicrobial prophylaxis for those potentially exposed Environmental samples Surface swabs Nasal swabs of potentially exposed persons (if <7 days) Refine list of potentially exposed persons Not exposed:  stop treatment Likely exposed:  continue treatment for      60 days total

Anthrax:  Case Definition Confirmed Case: Clinically compatible illness confirmed by isolation of B. anthracis or other laboratory  evidence based on at least two supportive laboratory tests Suspected Case: Clinically compatible illness with one supportive lab test or linked to a confirmed environmental exposure

Anthrax: Exposure Classification Exposure, laboratory-confirmed: Epidemiologically linked to a plausible environmental exposure, with laboratory evidence of B. anthracis from a nasal swab or other clinical sample Exposure, not laboratory-confirmed: Epidemiologically linked to a plausible environmental exposure, without laboratory evidence of B. anthracis

Anthrax: Clinical Information Cutaneous Inhalational Gastrointestinal

Anthrax: Cutaneous Begins as a papule, progresses through a vesicular stage to a depressed black necrotic ulcer (eschar) 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%

Anthrax: Inhalational (1) A brief prodrome 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)

Anthrax: Inhalational (2) Extremely rare in United States (20 reported cases in last century) Incubation period: 1–7 days (possibly ranging up to 42 days) Case fatality: Without antibiotic treatment—97% With antibiotic treatment—75%

Anthrax: Gastrointestinal Abdominal distress, usually accompanied by bloody vomiting or diarrhea, followed by fever and signs of septicemia Gastrointestinal illness sometimes seen as oropharyngeal ulcerations with cervical adenopathy and fever Develops after ingestion of contaminated, poorly cooked meat. Incubation period: 1–7 days Case-fatality: 25–60% (role of early antibiotic treatment is undefined)

"Anthrax:" Anthrax: Cutaneous

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"Anthrax:" Anthrax: Cutaneous

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Differential Diagnosis  of Cutaneous Anthrax Spider bite Ecthyma gangrenosum Ulceroglandular tularemia Plague Staphylococcal or streptococcal cellulitis Herpes simplex virus

Differential Diagnosis  of Inhalational Anthrax Mycoplasmal pneumonia Legionnaires’ disease Psittacosis Tularemia Q fever Viral pneumonia Histoplasmosis (fibrous mediastinitis) Coccidioidomycosis Malignancy

Differential Diagnosis of Gastrointestinal Anthrax Acute appendicitis Ruptured viscus Diverticulitis Diseases that cause acute cervical lymphadenitis or acute gastritis Dysentery

"Anthrax:" Anthrax: Diagnosis

"Anthrax:" Anthrax: Diagnosis

Anthrax: Diagnosis Gastrointestinal Blood cultures Oropharyngeal (OP) swab collection

Laboratory Criteria for Identification of B. anthracis (1) 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 Nonhemolytic

Laboratory Criteria for Identification of B. anthracis (2) 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)

Recommended Postexposure Prophylaxis to Prevent Inhalational Anthrax

Cutaneous Anthrax Treatment Protocol* for Cases Associated with Bioterrorist Events

Inhalational Anthrax Treatment Protocol* for Cases Associated with Bioterrorist Events (1)

Inhalational Anthrax Treatment Protocol* for Cases Associated with Bioterrorist Events (2)

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

Anthrax: Laboratory Information

Specimen Collection and Handling Primary isolation and Gram stain can be conducted at the hospital or clinical level Most clinical samples and suspect isolates will be handled via the Laboratory Response Network for Bioterrorism (LRN) and state public health laboratories (www.bt.cdc.gov) Triage of specimens at CDC by the Rapid Response and Advanced Technology (RRAT) Laboratory

Laboratory Response Network (LRN) LRN links state and local public health laboratories with advanced capacity laboratories ¾ including clinical, military, veterinary, agricultural, water, and food-testing laboratories. Laboratorians should contact their state public health laboratory to identify their local LRN representative.

LRN Criteria for Identification  of B. anthracis (1) LRN level A:  Rule-out and presumptive identification criteria From clinical samples, such as blood, CSF, skin lesion (eschar), or oropharyngeal ulcer:  encapsulated gram-positive rods From growth on sheep blood agar:  Large gram-positive rods Nonmotile Nonhemolytic on sheep blood agar

LRN Criteria for Identification  of B. anthracis (2) Many LRN laboratories use rapid screening assays (PCR for nucleic acid amplification and TRF immunoassay for antigen detection) on cultures and directly on clinical specimens. LRN confirmatory criteria for identification of B. anthracis is Capsule production and visualization and lysis by gamma-phage or Direct fluorescent antibody assays (DFA) for capsule antigen and cell wall-associated polysaccharide

Presumptive Identification of B. anthracis (1) Direct smears from clinical specimens Encapsulated broad rods in short chains, 2–4 cells. India ink will demonstrate capsule (Gram stain will not) B. anthracis not usually present in clinical specimens until late in course of disease

Presumptive Identification of B. anthracis (2) Smears from sheep blood agar or other routine nutrient medium Non-encapsulated broad rods in long chains Encapsulated bacilli grow only in nutrient agar supplemented with 0.8% sodium bicarbonate in presence of 5% CO2 (Note: this procedure is performed in Level B/LRN laboratories)

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Gram Stain Morphology of 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

"B." B. anthracis

Colony Characteristics of B. anthracis (1) After incubation on a blood agar plate for 12–24 hours at 35–37o C, well-isolated colonies are 2–5 mm in diameter; heavily inoculated areas may show growth in 6–8 hours Gray-white, flat or slightly convex colonies are irregularly round, with edges that slightly undulate, and have “ground glass” appearance Often have comma-shaped protrusions from colony edge (“Medusa head” colonies)

Colony Characteristics of B. anthracis (2) Tenacious consistency (when teased with a loop, the growth will stand up like beaten egg white) Nonhemolytic (weak hemolysis may be observed under areas of confluent growth in aging cultures and should NOT be confused with real β-hemolysis) Will not grow on MacConkey agar Nonmotile

Presumptive Identification Key for B. anthracis Nonhemolytic Nonmotile Encapsulated (requires India ink to visualize capsule) Gram-positive, spore-forming rod

Packaging and Transporting Protocol (1) Specimen packaging and labeling same as for any infectious substance If specimen is a dry powder or paper material, place in plastic self-sealing bag (e.g., Ziploc®) with biohazard label, and follow steps 1–4 (next slides) If specimen is a clinical specimen, place biohazard label on specimen receptacle, wrap receptacle with absorbent material, and follow steps 1–4 (next slides)

Packaging and Transporting Protocol (2) Place the bag or specimen receptacle into a leak-proof container (with tight cover) labeled “biohazard.” Place container into a second leak-proof container (with tight cover) also labeled “biohazard” and no larger than a one-gallon paint can. For a clinical specimen, place an ice pack (not ice) in the second container to keep specimen cold. For a nonclinical specimen (e.g., paper or powder) omit ice pack.

Packaging and Transporting Protocol (3) Place the second container into a third leak-proof container (with tight cover) labeled “biohazard” and no larger than a five-gallon paint can. Both the second and third containers should meet state and federal regulations for transport of hazardous material and be properly labeled.

Packing and Labeling Infectious Substances

Transporting Specimens to the DOH Public Health Lab Coordinate with DOH Public Health Lab and LRN Local FBI personnel may transport specimens if bioterrorism is suspected When specimens are shipped by commercial carrier, ship according to state and federal shipping regulations Contact shipping company, public health laboratory and local FBI

Disinfection and Disposal (1) Effective sporicidal solutions Commercially-available bleach, 0.5% hypochlorite (1 part household bleach to 9 parts water) Rinse off concentrated bleach to avoid caustic effects Approved sporicidal agents

Disinfection and Disposal (2) Surfaces and non-sterilizable equipment Wipe work surfaces before and after use with a sporicidal solution Routinely clean non-sterilizable equipment with a sporicidal solution Contaminated instruments (pipettes, needles, loops, micro slides) Soak in a sporicidal solution before autoclaving

Disinfection and Disposal (3) Accidental spills of material known or suspected to be contaminated with B. anthracis Contamination involving fresh clinical samples: Flood with sporicidal solution, soak for 5 minutes, then clean. Contamination involving lab samples (e.g., culture plates or blood cultures) or spills in areas below room temperature: Gently cover spill, then liberally apply sporicidal solution. Soak for 30 minutes, then clean. Autoclave or incinerate any soiled cleaning materials. Incinerate or steam sterilize cultures, infected material, and suspect material.

Summary Public health preparedness is needed. Early detection and response is critical. Communications networks (e.g., HAN, Epi-X, LRN) are key to success.