Transcript Overview of Bioterrorism Agents

Overview of
Bioterrorism Agents
Nicole Balmer M.D.
08/25/2006
Lab Response Network
 The Laboratory Response Network
(LRN) was established by the
Department of Health and Human
Services, Centers for Disease Control
and Prevention (CDC).
 Operational August 1999.
 Includes labs in all 50 states, UK,
Canada, Australia and vet labs.
Lab Response Network
 National Labs: Have unique resources to
handle highly infectious agents and
confirm diagnosis
 Reference Labs: Allows conclusive
enough results to allow emergency
response
 Sentinel Labs: “rule out and refer”
 Denver Health; must meet certain standards
CDC Category A Agents
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Category A
Variola major: Smallpox
Bacillus anthracis: Anthrax
Yersinia pestis: Plague
Clostridium botulinum (botulinum toxins):
Botulism
 Francisella tularensis: Tularemia
 Filoviruses and Arenaviruses (e.g., Ebola virus,
Lassa virus): Viral hemorrhagic fevers
CDC Categories B & C
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Category B
Coxiella burnetii: Q fever
Brucella spp.: Brucellosis
Burkholderia mallei: Glanders
Burkholderia pseudomallei: Melioidosis
Alphaviruses (VEE, EEE, WEEa): Encephalitis
Rickettsia prowazekii: Typhus fever
Toxins (e.g., Ricin, Staphylococcal enterotoxin B): Toxic syndromes
Chlamydia psittaci: Psittacosis
Food safety threats (e.g., Salmonella spp., Escherichia coli O157:H7)
Water safety threats (e.g., Vibrio cholerae, Cryptosporidium parvum)
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Category C
Emerging threat agents (e.g., Nipah virus, hantavirus)
Biosafety Levels
 BSL-1: Microorganisms that are not known to
cause disease in healthy human humans
 BSL-2: Agents of moderate risk to personnel
and the environment
 BSL-3: Agents which may cause serious or
potentially lethal diseases as a result of
exposure by the inhalation route
 BSL-4: Dangerous and exotic agents that pose
a high individual risk of aerosol-transmitted
laboratory infections and life-threatening
disease
Terror Bug #1
 Tech screening gram stains says he sees “boxcar” gram positive
bacilli.
Terror Bug # 1
First decision:
“Boxcar” Gram positive rods
Aerobic
Anaerobic
Terror Bug # 1
 Bacteria is aerobic
 Non-hemolytic on
blood agar
Slighty convex
grey-white ground
glass colonies with
irregular contours
Terror Bug # 1
 Also known as
“Medusa Head”
colonies
Motility test
 Organism in question in Non-motile
Bacillus anthracis
Bacillus anthracis
 Large gram positive bacilli in short chains
(boxcars) which look encapsulated.
 Non-hemolytic
 Medusa head colonies
 India Ink stain: + capsule (optional)
 Non-motile
 Notify FBI, state public health lab and
state public health department
Bacillus anthracis
Forearm lesion on Day 7—vesiculation and
ulceration of initial macular or papular
anthrax skin lesion.
Other Bacillus species
 Other
Bacillus
Terror Bug # 1
First decision:
“Boxcar” Gram positive rods
Aerobic
Anaerobic
Clostridium
 Anaerobic spore forming gram positive
bacilli
Clostridium botulinum
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Food-borne botulism
Wound botulism
Infant botulism
Adult intestinal colonization
Injection-related
Inhalational
Clostridium botulism
 Lethal foodborne intoxication with toxin types
A, B, E, or F; shorter incubation period -->poorer prognosis
 phage-mediated, systemic-acting A-B
neurotoxin (botulinum toxin = botulin) released
at cell lysis
 Mode of Action -- one of most extremely potent
neurotoxins known
 Lethal dose to humans, less than 1 mcg.
Clostridium botulism
 A-B toxin ingested, binds specific receptors on
peripheral cholinergic nerve endings
(neuromuscular junctions) where it blocks
release of presynaptic acetylcholine (excitatory
neurotransmitter) blocking muscle stimulation
and resulting in flaccid paralysis
 Early: nausea, vomiting, weakness, lassitude
(lack of energy), dizziness, constipation
 Later: double vision, difficulty in swallowing and
speaking
 Final: death due to respiratory paralysis
Clostridium botulism
 Acceptable specimens:
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Enema fluid
Nasal swab
Serum
Stool
 DH lab (and other sentinel labs) will only
accept suspected bioterrorism specimens to
send to Reference lab
 Any manipulation of specimen= BSL-3
 All work areas must be disinfected
Clostridium botulism
 Lab Identification
 Microscopic detection or Cx (culture) are
often unsuccessful (few organisms and
slow growing)
 Toxin detected and typed in lab via
toxicity and antitoxin neutralization tests
in mice or by ELISA
Clostridium botulism
 Colonies commonly
show some spreading
and have an irregular
edge. On egg yolk
medium, they usually
exhibit surface
iridescence when
examined by oblique
light. This luster zone,
often referred to as a
pearly layer, usually
extends beyond and
follows the irregular
contour of the colony.
Clostridium botulism
 Laboratory confirmation of toxin presence is via
a mouse bioassay, and identification of the
toxin type is performed by a mouse toxin
neutralization test.
 New methods of detection: In vitro methods of
detection, including polymerase chain reactionbased detection of clostridial genes and ELISA
identification of toxin, but these methods are
not widely available outside of research
institutions.
Use of Clostridium
botulism
 Japanese in World War II carried out human experiments on
prisoners in Manchuria.
 World War II, the British secretly used a botulism-impregnated
grenade in the assassination of a German Gestapo officer.
 The United States studied botulinum toxin as a military bioweapon
until President Nixon signed the Biological and Toxin Weapons
Convention in 1972,
 Iraq and the Soviet Union stockpiled neurotoxin, with Iraq
admitting to weaponizing thousands of liters of toxin in warheads
after the 1991 Gulf War.
 An attempt at terrorist use of Clostridium toxin in the early 1990s
by the Japanese Aum Shinryko cult against American military
targets was unsuccessful.
Clostridium botulism
 These were jars of contaminated
Jalapeño peppers involved in an
outbreak of botulism in Pontiac,
Michigan, April, 1977.
Francisella tularensis
 Faint staining, tiny,
pleomorphic gramnegative coccobacilli
that grows poorly on
blood agar, better on
chocolate
Francisella tularensis
 Oxidase negative
Francisella tularensis
 Catalase weakly positive
Francisella tularensis
 Beta lactamase positive
Francisella tularensis
 Satellite test negative
Francisella tularensis
 Urease negative
Francisella tularensis
 Thumb with skin ulcer of tularemia
Francisella tularensis
 Usually misidentified
using commercial ID
systems such as
Microscan.
 Usually ID’d as
H.influinzae (satellite
positive) or
Actinobacillus spp.
(beta lactamase
negative)
Brucella spp.
 Faintly staining
Gram-negative
coccobacillus
appearing as single
cells
 Cells are typically
larger than those of
F. tularensis
Brucella spp.
 Colony Characteristics
 Usually no visible or pinpoint at 24 hrs.
 Grows slowly on most standard lab media
including sheep blood, chocolate, and
TSA. Grows on Martin-Lewis and ThayerMartin agars.
 After 48 hrs. appears translucent, pinpoint and
smooth
 Non-hemolytic on sheep blood agar
 Some strains can grow on MacConkey agar
Brucella melitensis
Brucella abortus
Brucella spp.
 Non-Motile
 Catalase Positive
 Oxidase Positive (B. canis is
variable)
 Urease Positive (Strong, some with 5
min to 2 hrs)
Yersinia pestis
 “Fat” gram negative
rods arranged singly in
pairs and short chains
 Secondary infection
with Streptococcus
pneumoniae in
pneumonic plague.
 Bipolar “safety pin”
appearance, best seen
with Wright-Giemsa
(but can be seen with
other bugs)
Yersinia pestis
 Gray to white, light yellow, opaque,
pinpoint, nonhemolytic nonlactose
fermenting colonies.
 With age, cultures have fried egg
appearance
 Broth tube: 24 hrs. – “stalactite” growth
 48 hrs. “cotton fluff”
Yersinia pestis
Yersinia pestis
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Oxidase negative
Catalase positive
Urea negative
Indole negative
K/A
Nonmotile at 37C
Grows better at 28C
Indole
 Ability of a bacteria to breakdown the
amino acid trytophan
Yersinia pestis
Variola major
 This photograph of the left foot of a young
smallpox patient shows the typical smallpox
lesions located on the foot's plantar surface.
Smallpox
 Smallpox infection can be rapidly confirmed in the
laboratory by electron microscopic examination of
vesicular or pustular liquid or scabs.
 Definitive laboratory identification and characterization
of the virus involves growth of the virus in the cell
culture or on chorioallantoic egg membrane and
characterization of strains by use of biologic assays,
including the polymerase chain reaction (PCR),
restriction fragment-length polymorphism analysis
(RFLP) and ELISA. Confirmation using these methods
can be accomplished in a few hours.
Smallpox
 BSL-4
 Pharyngeal swab, scab matter, nasal
swab, serum
Virus may survive in scabs from patient for
several weeks.
(Note: Virons in scabs may remain viable
for years, but their being bound in fibrin
probably reduces their practical danger).
Hemorrhagic Fever
Viruses
 The two viruses considered to be the
greatest bioterrorism threats are Ebola
and Marburg, the two members of the
filovirus family
Hemorrhagic Fever
Viruses
 This was the local Red
Cross team in Kikwit,
Zaire, during the Ebola
VHF outbreak in 1995.
This team went to the
homes in the area to
bring patients with
suspected Ebola viral
hemorrhagic fever (VHF)
to the Kikwit hospital, as
well as to remove
corpses.
Hemorrhagic Fever
Viruses
 BSL-4
 Antigen-Capture ELISA, RT-PCR (most
useful clinically)
 IgM by Antibody-Capture ELISA, Viral
isolation
 Acute and convalescent IgG serologies in
survivors (only helpful retrospectively)
References
 1. http://www.bt.cdc.gov/Agent/Agentlist.asp
 2. Gaido, L. Denver Health Microbiology
Procedure Manual-Bioterrorism Agents, 2004.
 3. Arnon SS et al. Botulinum toxin as a
biological weapon: Medical and Public Health
Management. JAMA. 2001; 285(8): 1059-70.
 4. Dennis DT et al. Tularemia as a biological
weapon: Medical and Public Health
Management. JAMA. 2001; 285(21): 2763-73
References
 5. Ingelsby TV et al. Plague as a biological
weapon: Medical and Public Health
Management. JAMA. 200o; 283(17): 2281-90.
Dr. Elmer Koneman’s contribution (August 26, 2006):
I have read through Nicole Balmer's Power Point presentation on Bioterrorism agents posted on the Web. A
couple of years ago I worked with Jim Beebe, Head of the Colo Dept. Health Micro Lab, in publishing a
bioterrorism CD.
Dr. Balmer leads one through the in-laboratory identification of Francisella tularensis, including the use of an
automated system. Dr. Beebe and I, in creating the algorithm for the identification of this highly contagious
microbe, indicated that any slow-growing isolate, growing poorly if at all on blood agar but with tiny colonies
on chocolate agar, that appear as poorly staining tiny gram negative cocco-bacilli on gram stain, that are
cytochrome oxidase-negative and are non-motile in a direct mount preparation should be sent immediately to
the state laboratory with no attempt to make an in-laboratory identification. We essentially have established a
similar algorithm for the direct preliminary exam for Brucella, leading to immediate referral to the state lab of
any suspicious isolate, again to prevent any chance for a laboratory acquired infection resulting from a full inhouse work up. I would like to bring this precaution to your attention.
I believe regulations mitigate that the agents of bioterrorism should be definitively worked up only in a Level
3 microbiology laboratory. This may not be true for all the agents, but I know it is for F. tularensis and
Brucella species. The chances are high that laboratory-acquired infections may occur from handling these
isolates and work up in Level-2 laboratories or below elevates this possibility. Most hospital or even
university labs don't meet this criteria; therefore, any time F. tularensis or Brucella species is suspected, the
State Lab should be consulted and the cultures forwarded under their guidelines.
Next are two slides onto which I transcribed the presumptive identification algorithms of F. tularensis and
Brucella sp. These were taken from the CD: "The Bacterial Agents of Bioterrorism", authored by James
Beebe, Elmer Koneman, and Christie Grueser, with the copyright being held by CACMLE. The full disk is
available from CACMLE, and is their self-study product is #96A05CD. You might consider acquiring this disk
for additional information and future reference (call 303 321 1734 for details). Thank you.
Elmer W. Koneman, M. D.
HINTS FOR RAPID IDENTIFICATION OF
FRANCISELLA TULARENSIS
Small pin-point colonies on sheep blood agar
Good growth on chocolate, cystine heart and BCYE agars
Growth enhanced under CO2 atmosphere
Very tiny, poorly-staining, Gram-negative coccobacilli
Spot cytochrome oxidase negative
Bacterial cells non-motile in direct mount preparation
Suspect Francisella tularensis
Refer immediately to public health laboratory
:
Beebe J, Koneman EW, Grueser C The Bacterial Agens of Bioterrorism, CACMLE, SS #96A05DC
HINTS FOR RAPID IDENTIFICATION OF
BRUCELLA SPECIES
Slow growth of tiny, gray-white, shiny colonies on BA
Very pale-staining gram-negative coccobacilli on Gram stain
Spot cytochrome oxidase positive
Rapid hydrolysis of Urea: Positive reaction
in 1-4 Hours on Christensen’s urea agar
Suspect BRUCELLA SPECIES
Consult Public Health Laboratory Immediately
Beebe J, Koneman EW, Grueser C: The Bacterial Agens of Bioterrorism, CACMLE, SS#96A05DC