Overview of Bioterrorism Agents Nicole Balmer M.D. 08/25/2006

Overview of Overview of Bioterrorism Bioterrorism

AgentsAgentsNicole Balmer M.D.Nicole Balmer M.D.

08/25/200608/25/2006

Lab Response NetworkLab Response Network

The Laboratory Response Network The Laboratory Response Network (LRN) was established by the (LRN) was established by the Department of Health and Human Department of Health and Human Services, Centers for Disease Control Services, Centers for Disease Control and Prevention (CDC). and Prevention (CDC).

Operational August 1999.Operational August 1999. Includes labs in all 50 states, UK, Includes labs in all 50 states, UK,

Canada, Australia and vet labs.Canada, Australia and vet labs.

Lab Response Network Lab Response Network

National Labs: Have unique resources to National Labs: Have unique resources to handle highly infectious agents and handle highly infectious agents and confirm diagnosisconfirm diagnosis

Reference Labs: Allows conclusive Reference Labs: Allows conclusive enough results to allow emergency enough results to allow emergency responseresponse

Sentinel Labs: “rule out and refer”Sentinel Labs: “rule out and refer” Denver Health; must meet certain standardsDenver Health; must meet certain standards

CDC Category A AgentsCDC Category A Agents

Category ACategory A Variola major: Variola major: SmallpoxSmallpox Bacillus anthracis: Bacillus anthracis: AnthraxAnthrax Yersinia pestis: Yersinia pestis: PlaguePlague Clostridium botulinumClostridium botulinum (botulinum toxins): (botulinum toxins):

BotulismBotulism Francisella tularensis: Francisella tularensis: TularemiaTularemia Filoviruses and Arenaviruses (e.g.Filoviruses and Arenaviruses (e.g., Ebola virus, , Ebola virus,

Lassa virusLassa virus): Viral hemorrhagic fevers): Viral hemorrhagic fevers

CDC Categories B & CCDC Categories B & C

CCategory Bategory B Coxiella burnetii: Coxiella burnetii: Q feverQ fever Brucella sppBrucella spp.: Brucellosis.: Brucellosis Burkholderia mallei: Burkholderia mallei: GlandersGlanders Burkholderia pseudomallei: Burkholderia pseudomallei: MelioidosisMelioidosis AlphavirusesAlphaviruses (VEE, EEE, WEEa): Encephalitis (VEE, EEE, WEEa): Encephalitis Rickettsia prowazekii: Rickettsia prowazekii: Typhus feverTyphus fever Toxins (e.g., Ricin, Staphylococcal enterotoxin B): Toxic syndromesToxins (e.g., Ricin, Staphylococcal enterotoxin B): Toxic syndromes Chlamydia psittaci: Chlamydia psittaci: PsittacosisPsittacosis Food safety threats (e.g.,Food safety threats (e.g., Salmonella spp Salmonella spp., ., Escherichia coliEscherichia coli O157:H7) O157:H7) Water safety threats (e.g.,Water safety threats (e.g., Vibrio cholerae Vibrio cholerae, Cryptosporidium parvum), Cryptosporidium parvum)

Category CCategory C Emerging threat agents (e.g., Emerging threat agents (e.g., Nipah virusNipah virus, hantavirus), hantavirus)

Biosafety LevelsBiosafety Levels

BSL-1: Microorganisms that are not known to BSL-1: Microorganisms that are not known to cause disease in healthy human humanscause disease in healthy human humans

BSL-2: Agents of moderate risk to personnel BSL-2: Agents of moderate risk to personnel and the environmentand the environment

BSL-3: Agents which may cause serious or BSL-3: Agents which may cause serious or potentially lethal diseases as a result of potentially lethal diseases as a result of exposure by the inhalation route exposure by the inhalation route

BSL-4: Dangerous and exotic agents that pose BSL-4: Dangerous and exotic agents that pose a high individual risk of aerosol-transmitted a high individual risk of aerosol-transmitted laboratory infections and life-threatening laboratory infections and life-threatening disease disease

Terror Bug #1Terror Bug #1

Tech screening gram stains says he sees “boxcar” gram positive Tech screening gram stains says he sees “boxcar” gram positive bacilli.bacilli.

Terror Bug # 1Terror Bug # 1

First decision:First decision:

“Boxcar” Gram positive rods

Aerobic Anaerobic


Bacteria is aerobicBacteria is aerobic Non-hemolytic on Non-hemolytic on

blood agarblood agar

Slighty convex grey-Slighty convex grey-white ground glass white ground glass colonies with colonies with irregular contoursirregular contours


Also known as Also known as “Medusa Head” “Medusa Head” coloniescolonies

Motility testMotility test

Organism in question in Non-motileOrganism in question in Non-motile

Bacillus anthracisBacillus anthracis


Large gram positive bacilli in short chains Large gram positive bacilli in short chains (boxcars) which look encapsulated.(boxcars) which look encapsulated.

Non-hemolyticNon-hemolytic Medusa head coloniesMedusa head colonies India Ink stain: + capsule (optional)India Ink stain: + capsule (optional) Non-motileNon-motile Notify FBI, state public health lab and Notify FBI, state public health lab and

state public health departmentstate public health department


Forearm lesion on Day 7—vesiculation and Forearm lesion on Day 7—vesiculation and ulceration of initial macular or papular ulceration of initial macular or papular anthrax skin lesion.anthrax skin lesion.

Other Other BacillusBacillus species species

Other Other BacillusBacillus


First decision:First decision:

“Boxcar” Gram positive rods

Aerobic Anaerobic

ClostridiumClostridium

Anaerobic spore forming gram positive Anaerobic spore forming gram positive bacillibacilli

Clostridium botulinumClostridium botulinum

Food-borne botulism Food-borne botulism Wound botulismWound botulism Infant botulismInfant botulism Adult intestinal colonizationAdult intestinal colonization Injection-relatedInjection-related InhalationalInhalational

Clostridium botulismClostridium botulism

Lethal foodborne intoxication with toxin types Lethal foodborne intoxication with toxin types A, B, E, or F; shorter incubation periodA, B, E, or F; shorter incubation period --- --->>poorer prognosis poorer prognosis

phage-mediated, systemic-acting A-B phage-mediated, systemic-acting A-B neurotoxin (botulinum toxin = botulin) released neurotoxin (botulinum toxin = botulin) released at cell lysis at cell lysis

Mode of Action -- one of most extremely potent Mode of Action -- one of most extremely potent neurotoxins knownneurotoxins known Lethal dose to humans, less than 1 mcg. Lethal dose to humans, less than 1 mcg.


A-B toxin ingested, binds specific receptors on A-B toxin ingested, binds specific receptors on peripheral cholinergic nerve endings peripheral cholinergic nerve endings (neuromuscular junctions) where it blocks (neuromuscular junctions) where it blocks release of presynaptic acetylcholine (excitatory release of presynaptic acetylcholine (excitatory neurotransmitter) blocking muscle stimulation neurotransmitter) blocking muscle stimulation and resulting in flaccid paralysis and resulting in flaccid paralysis

Early: nausea, vomiting, weakness, lassitude Early: nausea, vomiting, weakness, lassitude (lack of energy), dizziness, constipation (lack of energy), dizziness, constipation

Later: double vision, difficulty in swallowing and Later: double vision, difficulty in swallowing and speaking speaking

Final: death due to respiratory paralysis Final: death due to respiratory paralysis


Acceptable specimens:Acceptable specimens: Enema fluidEnema fluid Nasal swabNasal swab SerumSerum StoolStool

DH lab (and other sentinel labs) will only DH lab (and other sentinel labs) will only accept suspected bioterrorism specimens to accept suspected bioterrorism specimens to send to Reference lab send to Reference lab

Any manipulation of specimen= BSL-3Any manipulation of specimen= BSL-3 All work areas must be disinfectedAll work areas must be disinfected


Lab Identification Lab Identification Microscopic detection or Cx (culture) are Microscopic detection or Cx (culture) are

often unsuccessful (few organisms and often unsuccessful (few organisms and slow growing) slow growing)

Toxin detected and typed in lab via Toxin detected and typed in lab via toxicity and antitoxin neutralization tests toxicity and antitoxin neutralization tests in mice or by ELISA in mice or by ELISA


Colonies commonly Colonies commonly show some spreading show some spreading and have an irregular and have an irregular edge. On egg yolk edge. On egg yolk medium, they usually medium, they usually exhibit surface exhibit surface iridescence when iridescence when examined by oblique examined by oblique light. This luster zone, light. This luster zone, often referred to as a often referred to as a pearly layer, usually pearly layer, usually extends beyond and extends beyond and follows the irregular follows the irregular contour of the colony. contour of the colony.


Laboratory confirmation of toxin presence is via Laboratory confirmation of toxin presence is via a mouse bioassay, and identification of the a mouse bioassay, and identification of the toxin type is performed by a mouse toxin toxin type is performed by a mouse toxin neutralization test. neutralization test.

New methods of detection: In vitro methods of New methods of detection: In vitro methods of detection, including polymerase chain reaction-detection, including polymerase chain reaction-based detection of clostridial genes and ELISA based detection of clostridial genes and ELISA identification of toxin, but these methods are identification of toxin, but these methods are not widely available outside of research not widely available outside of research institutions. institutions.

Use of Use of Clostridium Clostridium botulismbotulism

Japanese in World War II carried out human experiments on Japanese in World War II carried out human experiments on prisoners in Manchuria. prisoners in Manchuria.

World War II, the British secretly used a botulism-impregnated World War II, the British secretly used a botulism-impregnated grenade in the assassination of a German Gestapo officer.grenade in the assassination of a German Gestapo officer.

The United States studied botulinum toxin as a military bioweapon The United States studied botulinum toxin as a military bioweapon until President Nixon signed the Biological and Toxin Weapons until President Nixon signed the Biological and Toxin Weapons Convention in 1972,Convention in 1972,

Iraq and the Soviet Union stockpiled neurotoxin, with Iraq Iraq and the Soviet Union stockpiled neurotoxin, with Iraq admitting to weaponizing thousands of liters of toxin in warheads admitting to weaponizing thousands of liters of toxin in warheads after the 1991 Gulf War. after the 1991 Gulf War.

An attempt at terrorist use of An attempt at terrorist use of ClostridiumClostridium toxin in the early 1990s toxin in the early 1990s by the Japanese Aum Shinryko cult against American military by the Japanese Aum Shinryko cult against American military targets was unsuccessful. targets was unsuccessful.


These were jars of contaminated These were jars of contaminated Jalapeño peppers involved in an Jalapeño peppers involved in an outbreak of botulism in Pontiac, outbreak of botulism in Pontiac, Michigan, April, 1977.Michigan, April, 1977.

Francisella tularensisFrancisella tularensis

Faint staining, tiny, Faint staining, tiny, pleomorphic gram-pleomorphic gram-negative coccobacilli negative coccobacilli that grows poorly on that grows poorly on blood agar, better on blood agar, better on chocolatechocolate


Oxidase negativeOxidase negative


Catalase weakly positiveCatalase weakly positive


Beta lactamase positiveBeta lactamase positive


Satellite test negativeSatellite test negative


Urease negativeUrease negative


Thumb with skin ulcer of tularemiaThumb with skin ulcer of tularemia


Usually misidentified Usually misidentified using commercial ID using commercial ID systems such as systems such as Microscan.Microscan.

Usually ID’d as Usually ID’d as H.influinzaeH.influinzae (satellite (satellite positive) or positive) or ActinobacillusActinobacillus spp. spp. (beta lactamase (beta lactamase negative)negative)

Brucella spp.Brucella spp.

Faintly staining Faintly staining Gram-negative Gram-negative coccobacillus coccobacillus appearing as single appearing as single cells cells

Cells are typically Cells are typically larger than those of larger than those of F. tularensis F. tularensis


Colony CharacteristicsColony Characteristics Usually no visible or pinpoint at 24 hrs. Usually no visible or pinpoint at 24 hrs. Grows slowly on most standard lab media Grows slowly on most standard lab media

including sheep blood, chocolate, and TSA. including sheep blood, chocolate, and TSA. Grows on Martin-Lewis and Thayer-Martin Grows on Martin-Lewis and Thayer-Martin agars. agars.

After 48 hrs. appears translucent, pinpoint and After 48 hrs. appears translucent, pinpoint and smooth smooth

Non-hemolytic on sheep blood agar Non-hemolytic on sheep blood agar Some strains can grow on MacConkey agar Some strains can grow on MacConkey agar

Brucella melitensisBrucella melitensis

Brucella abortusBrucella abortus


Non-MotileNon-Motile Catalase PositiveCatalase Positive Oxidase Positive (B. canis is Oxidase Positive (B. canis is

variable)variable) Urease Positive (Strong, some with 5 Urease Positive (Strong, some with 5

min to 2 hrs)min to 2 hrs)

Yersinia pestisYersinia pestis

““Fat” gram negative Fat” gram negative rods arranged singly in rods arranged singly in pairs and short chainspairs and short chains

Secondary infection Secondary infection with Streptococcus with Streptococcus pneumoniae in pneumoniae in pneumonic plague.pneumonic plague.

Bipolar “safety pin” Bipolar “safety pin” appearance, best seen appearance, best seen with Wright-Giemsa with Wright-Giemsa (but can be seen with (but can be seen with other bugs)other bugs)


Gray to white, light yellow, opaque, Gray to white, light yellow, opaque, pinpoint, nonhemolytic nonlactose pinpoint, nonhemolytic nonlactose fermenting colonies.fermenting colonies.

With age, cultures have fried egg With age, cultures have fried egg appearanceappearance

Broth tube: 24 hrs. – “stalactite” growthBroth tube: 24 hrs. – “stalactite” growth 48 hrs. “cotton fluff”48 hrs. “cotton fluff”



Oxidase negativeOxidase negative Catalase positiveCatalase positive Urea negativeUrea negative Indole negativeIndole negative K/AK/A Nonmotile at 37CNonmotile at 37C Grows better at 28CGrows better at 28C

IndoleIndole

Ability of a bacteria to breakdown the Ability of a bacteria to breakdown the amino acid trytophan amino acid trytophan


Variola majorVariola major

This photograph of the left foot of a young This photograph of the left foot of a young smallpox patient shows the typical smallpox smallpox patient shows the typical smallpox lesions located on the foot's plantar surface.lesions located on the foot's plantar surface.

SmallpoxSmallpox

Smallpox infection can be rapidly confirmed in the Smallpox infection can be rapidly confirmed in the laboratory by electron microscopic examination of laboratory by electron microscopic examination of vesicular or pustular liquid or scabs.vesicular or pustular liquid or scabs.

Definitive laboratory identification and characterization Definitive laboratory identification and characterization of the virus involves growth of the virus in the cell of the virus involves growth of the virus in the cell culture or on chorioallantoic egg membrane and culture or on chorioallantoic egg membrane and characterization of strains by use of biologic assays, characterization of strains by use of biologic assays, including the polymerase chain reaction (PCR), including the polymerase chain reaction (PCR), restriction fragment-length polymorphism analysis restriction fragment-length polymorphism analysis (RFLP) and ELISA. Confirmation using these methods (RFLP) and ELISA. Confirmation using these methods can be accomplished in a few hours. can be accomplished in a few hours.

SmallpoxSmallpox

BSL-4BSL-4 Pharyngeal swab, scab matter, nasal Pharyngeal swab, scab matter, nasal

swab, serum 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 Hemorrhagic Fever VirusesViruses

The two viruses considered to be the The two viruses considered to be the greatest bioterrorism threats are greatest bioterrorism threats are EbolaEbola and and Marburg,Marburg, the two members of the the two members of the filovirusfilovirus family family


This was the local Red This was the local Red Cross team in Kikwit, Cross team in Kikwit, Zaire, during the Ebola Zaire, during the Ebola VHF outbreak in 1995. VHF outbreak in 1995. This team went to the This team went to the homes in the area to homes in the area to bring patients with bring patients with suspected Ebola viral suspected Ebola viral hemorrhagic fever (VHF) hemorrhagic fever (VHF) to the Kikwit hospital, as to the Kikwit hospital, as well as to remove well as to remove corpses. corpses.


BSL-4BSL-4 Antigen-Capture ELISA, RT-PCR (most Antigen-Capture ELISA, RT-PCR (most

useful clinically)useful clinically) IgM by Antibody-Capture ELISA, Viral IgM by Antibody-Capture ELISA, Viral

isolationisolation Acute and convalescent IgG serologies in Acute and convalescent IgG serologies in

survivors (only helpful retrospectively) survivors (only helpful retrospectively)

ReferencesReferences

1. 1. http://www.bt.cdc.gov/Agent/Agentlist.asphttp://www.bt.cdc.gov/Agent/Agentlist.asp 2. Gaido, L. 2. Gaido, L. Denver Health Microbiology Denver Health Microbiology

Procedure Manual-Bioterrorism AgentsProcedure Manual-Bioterrorism Agents, 2004., 2004. 3. Arnon SS et al. Botulinum toxin as a 3. Arnon SS et al. Botulinum toxin as a

biological weapon: Medical and Public Health biological weapon: Medical and Public Health Management. JAMA. 2001; 285(8): 1059-70.Management. JAMA. 2001; 285(8): 1059-70.

4. Dennis DT et al. Tularemia as a biological 4. Dennis DT et al. Tularemia as a biological weapon: Medical and Public Health weapon: Medical and Public Health Management. JAMA. 2001; 285(21): 2763-73 Management. JAMA. 2001; 285(21): 2763-73

ReferencesReferences

5. Ingelsby TV et al. Plague5. Ingelsby TV et al. Plague as a biological as a biological weapon: Medical and Public Health weapon: Medical and Public Health Management. JAMA. 200o; 283(17): 2281-90. 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 in-house 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 tularensisRefer 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 SPECIESConsult Public Health Laboratory Immediately

Beebe J, Koneman EW, Grueser C: The Bacterial Agens of Bioterrorism, CACMLE, SS#96A05DC

Documents

Overview of Bioterrorism Agents Nicole Balmer M.D. 08/25/2006