bacterial agents of bioterroism
DESCRIPTION
Bacterial agents of bioterroism. Laboratory network for biological terrorism. Bacillus anthracis. Anthrax. Anthrax: Overview. Primarily disease of herbivores Humans usually infected by contact with infected animals or contaminated animal products Soil reservoir - PowerPoint PPT PresentationTRANSCRIPT
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Bacterial agents ofbioterroism
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Laboratory network forbiological terrorism
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Bacillus anthracis
Anthrax
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• Primarily disease of herbivores
• Humans usually infected by contact with infected animals or contaminated animal products
• Soil reservoir • Woolsorter’s disease
(inhalation anthrax)• No person-to-person
transmission of inhalational anthrax
Anthrax: Overview
CDC
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ANTHRAX
• Three forms of human anthrax occur:1. Cutaneous2. Gastrointestinal
•Oropharyngeal•Abdominal
3. Inhalation (Woolsorter’s Disease)
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Vesicle development, day 2Eschar formation, day 4
Cutaneous anthrax
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Inhalation Anthrax
• Infective dose = 8,000 - 15,000 spores
• Incubation period = 1-6 days
• Duration of illness = 3-5 days
• Fever, malaise, and fatigue
• Short period of improvement = up to 2 days
• Abrupt respiratory distress…death <24hrs
• Person to person transmission = no
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Anthrax: Specimen Selection
• Inhalation: Sputum and Blood
• Cutaneous: Vesicles and Eschar
• Gastrointestinal: Stool and Blood
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Bacillus anthracisKey Sentinel Lab Tests
• Gram stain• Growth characteristics on
agar• Sporulation, in air• Motility• Capsule by India Ink
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• Broad gram-positive rod: 1-1.5 X 3-5 µ
• Oval, central - subterminal spores: 1 X 1.5 µ with no significant swelling of cell
• Spores are NOT usually present in clinical specimens unless exposed to atmospheric O2
Bacillus anthracisBacillus anthracisGram Stain MorphologyGram Stain Morphology
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B. anthracis, Gram staindemonstrating spores
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• Colonial morphology of 18-24hr @ 35 C:– Well isolated colonies are 2-5 mm in
diameter– Flat or slightly convex, irregularly round– Edges: slightly undulate, often curly
tailing edges– Ground glass appearance– “Sticky” consistency….stands up like
beaten egg whites
B. B. anthracisanthracisColonial MorphologyColonial Morphology
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B. anthracis, colony on SBA
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““STICKY” consistency of STICKY” consistency of B. anthracis’B. anthracis’ colony on colony on SBASBA
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• Gram-positive, broad rod, catalase-positive, spore-positive, aerobe: Bacillus sp.
• Spores are oval and nonswelling with ground glass colony appearance: Bacillus morphology group 1, includes B. anthracis, B. cereus, B cereus var mycoides, and B. thuringiensis
Bacillus anthracisBacillus anthracisPresumptive IdentificationPresumptive Identification
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• Nonmotile: B anthracis and B cereus var mycoides (and B. megaterium)
• Nonhemolytic, forms capsule: Presumptive B. anthracis
• Refer to state lab for testing
Bacillus anthracisBacillus anthracisPresumptive Identification, con’tPresumptive Identification, con’t
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Yersinia pestis
Plague
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• Natural vector - Rodent flea • Mammalian hosts
– rats, squirrels, chipmunks, rabbits, and carnivores
• Enzootic or Epizootic
Plague: OverviewPlague: Overview
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Plague Epidemiology
• U.S. averages 13 cases/yr• 30% of cases are in Native Americans in
the Southwest. 15% case fatality rate• Most cases occur in summer and near the
patient’s residence– bubonic (infected lymph nodes)– septicemic (blood-borne organisms)– pneumonic (transmissible by aerosol;
deadliest)
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Yersinia pestisSpecimen Selection
• Specimen selection is important– Bubo - lymph node aspirate– Blood - organisms may be intermittent.
Take three specimens 10-30 minutes apart– Pneumonic
•Sputum/throat - use Wayson stain•Bronchial washings - Wayson stain
• Inoculate routine plating media
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Sentinel Lab Procedures
Yersinia pestis
• Gram stain
• Wayson stain
• Growth characteristics on agar
• Growth characteristics in broth
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Yersinia pestisGram stain
• Small, gram-negative coccobacilli
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Yersinia pestisWayson Stain
• Used for rapid assessment– when it is a part of the identification process
• Best with tissue, sputum, blood• Stains of pure culture isolates tend to lose
bipolarity• Pink-blue cells with polar granules (safety pin
appearance)
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Yersinia pestisWayson Stain
Wayson stain alone is not diagnostic
• Pink-blue cells with a closed safety pin look
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Y.pestis
48 h culture on SBA
72 h culture on SBA
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• Small Gram-negative, poorly staining rods from blood, lymph node aspirate, or respiratory specimens
• Safety pin appearance in Gram, Wright, Giemsa, or Wayson stain
• More than one patient in a short, specified period with fever, lymphadenopathy
• Refer to state lab
Yersinia pestisYersinia pestisTechnical HintsTechnical Hints
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Francisella tularensisFrancisella tularensis
Tularemia
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Tularemia: Overview
• Disease of Northern Hemisphere• In U.S., most cases associated with
rabbits/hares and ticks • About 200 cases/year in U.S.
– most in South central and Western states– majority of cases in summer, some in
winter
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Reported Cases of Tularemia Reported Cases of Tularemia - 1990-1998- 1990-1998
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• Low infectious dose
–1 to 10 organisms by aerosol or intradermal route
• No person-to-person transmission
Tularemia: Overview (cont’d)
• Several forms of human tularemia exist:
- Ulceroglandular, glandular, oculoglandular,
oropharyngeal, intestinal, pneumonic, and
typhoidal
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Tularemia: Specimen Selection
• Serum - acute and convalescent
• Blood cultures
• Sputum
• Swab – ulcer or eye
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Sentinel Lab ProceduresFrancisella tularensis
• This is a dangerous, highly virulent organism and it should not be manipulated at the bench. Laboratory-acquired infections can occur easily.
• Gram stain• Growth characteristics in broth• Growth characteristics in agar
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Francisella tularensis• Poorly staining, tiny Gram-negative coccobacilli
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Francisella tularensisGrowth Characteristics
• Fastidious, requires cysteine for robust growth: Cysteine Heart Agar (CHA) is ideal– Enriched chocolate agar + 9% sheep blood +
cysteine– Not part of Sentinel Lab routine procedures– BCYE (for Legionella) also works
• Will grow initially on sheep and chocolate blood agar and Thayer-Martin agar, but poorly or not at all on passage
• Grows slowly at 35oC, poorly at 28oC
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Francisella tularensisGrowth Characteristics
• 24 hours– gray-white, translucent colonies– usually too small to be seen individually
• 48 hours– Sheep Blood Agar - <1 mm, gray-white,
opaque, no hemolysis
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Francisella tularensis
Sheep blood agar Chocolate agar Cysteine heart agar
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Francisella tularensisTechnical Hints
• Tiny, Gram-negative coccobacilli from blood, lymph node aspirate, or respiratory specimens
• Blood isolates that will grow slowly on chocolate agar but poorly or not at all on blood agar in 24 hours
• Faint growth in thio; requires cysteine in other broth
• Refer to state lab
If you see:
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Brucella spp.
Brucellosis
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BRUCELLOSIS
• A zoonotic disease caused by any of 4 Brucella sp.: abortus, melitensis, suis, and canis
• A systemic infection characterized by an undulant fever pattern
• But relatively rare in the U.S. with approximately 100 cases/yr
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BRUCELLOSIS:TRANSMISSION
• Unpasteurized dairy products– The most common mode of
transmission
• Direct skin contact– Occupational hazard for farmers,
butchers, veterinarians, and laboratory personnel
• Aerosols– Highly infectious
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•Infective dose = 10 -100 organisms
•Incubation period = 5 days - > 6 months
•Duration of illness = weeks to months
•Fever, profuse sweating, malaise, headache and muscle/back pain.
•Person to person transmission = no
•Mortality = <5%
•Persistence of organism = very stable
BRUCELLOSISBRUCELLOSIS
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Brucella spp.Specimen Selection
• Serum– The diagnosis of brucellosis is
frequently achieved by serology. An acute & convalescent phase specimen should be collected (21d apart)
• Blood or bone marrow– Sources from which Brucellae are most
often isolated
• Tissue (spleen, liver)– Brucellae occasionally isolated
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• Brucellosis is THE most commonly reported laboratory-associated bacterial infection.
• Cases have occurred in clinical laboratory settings by “sniffing” cultures, direct skin contact with cultures, and aerosol generating procedures
Brucella Brucella spp.spp.Biosafety AlertBiosafety Alert
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• Colonial morphology on SBA
• Gram stain morphology
• Oxidase positive
• Urea hydrolysis positive
Sentinel Lab TestsSentinel Lab TestsBrucellaBrucella spp. spp.
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Colonial morphology on SBA
–Fastidious
–Visible growth may take 48 - 72 hrs
–Small (0.5-1.0mm), convex, glistening
– Non-hemolytic and non-pigmented
BrucellaBrucella spp. spp.Key Sentinel Lab TestsKey Sentinel Lab Tests
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B. melitensis on sheep blood agar
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Gram Stain Morphology
–Tiny (very)
–Faintly staining
–Gram-negative coccobacilli
–0.5 - 0.7 x 0.6 - 1.5
BrucellaBrucella spp. spp.Key Sentinel Lab TestsKey Sentinel Lab Tests
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• Tiny, faintly staining, gram-negative coccobacilli from blood or bone marrow
• Slow growth on Sheep Blood Agar, 2-3 days for colony appearance
• Oxidase +
• Urease +
• Handle plates with care
• Refer to state lab
Brucella spp.Brucella spp.Review of Key TestsReview of Key Tests
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Clostridium botulinum
Botulism
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• Caused by toxin from Clostridium botulinum– toxin types A, B, E, most commonly
associated with human disease– most potent lethal substance known to man
(lethal dose 1ng/kg)
• C. botulinum spores found in soil worldwide• Approximately 100 reported cases/year in the
U.S.– infant most common (72%)– food borne not common
• No person-to-person transmission
Botulism: OverviewBotulism: Overview
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• Infective dose: 0.001 g/kg
• Incubation period: 18 - 36 hr (6hr to 10 d)
• Dry mouth, double vision, droopy eyelids, dilated pupils
• Generalized, progressive descending bilateral muscle weakness & paralysis
• Respiratory failure and death
• Mortality usually 5 – 10%
FOODBORNE BOTULISMFOODBORNE BOTULISM
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FOODBORNE BOTULISM
• Among 309 persons with clinically diagnosed botulism reported to CDC from 1975 to 1988:– Stool cultures for C. botulinum: 51% +– Serum botulinum toxin testing: 37% +– Stool botulinum toxin testing: 23% +
• Overall, at least one of the above tests was positive for 65% of all patients
• Diagnosis is primarily clinical
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Sentinel Lab Procedures
for Botulism Event• Properly collected specimens are to be
referred to designated testing laboratories
• Prior to the shipment of any botulism-
associated specimen, testing must be arranged with MDCH laboratory
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Clinical specimens to be collected:
1. Serum
2. Feces
3. Food samples
Autopsy specimens:
1. Serum
2. Gastric and intestinal contents
Sentinel Lab ProceduresSentinel Lab Proceduresfor Botulism Eventfor Botulism Event
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Materials suspected of containing botulism toxin must be handled:
–Biological Safety Cabinet (Class II)
–Laboratory Coats
–Disposable surgical gloves
–Face shield (as needed)
BotulismBotulismBiosafety AlertBiosafety Alert
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BOTULISM
• The diagnosis of botulism is made clinically, i.e., based on the patient’s case history and physical findings
• Health care providers suspecting botulism should contact the Michigan Department of Community Health
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Botulism
Referral Lab Procedures
• Mouse bioassay
• Isolation of C. botulinum