Our world must take bio-security much more seriously [] It would be comparatively easy for terrorists to cause mass

death by using agents such as anthrax or weaponized smallpox. Lets not wait until something has gone terribly wrong to act collectively to meet this threat
`
`

Kofi Annan, UN Secretary General, February 13, 2005

BIOTERRORISM
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"Bioterrorism refers to the intentional release of biological agents or toxins for the purpose of harming or killing humans, animals or plants with the intent to intimidate or coerce a government or civilian population to further political or social objectives."
`

(Bioterrorism Incident Pre-planning and response guide, ICPO INTERPOL, 2007)

BIOTERRORISM
CLASSIFICATION OVERT Immediate Early recognition of event COVERT More challenging Clinical microbiologist & physician First to suspect the attack Delayed recognition & response

BIOLOGICAL WAREFARE
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The use of bacteria, viruses, fungi, or toxins to injure people, animal, or crops to gain a military advantage WWI: Germany
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Human:Vibrio cholerae & Yersenia pestis Animal: Bacillus antracis & Burkholderia mallei

Geneva protocol

Bio-safety Level (BSL)

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Classification of microorganism based on disease potential Combination of standard procedures and technique, safety equipment, and facilities designed to minimize the exposure of workers and the environment to infectious agents

Bio-safety Level (BSL)

Table 1. Bio-safety Level Classification BSL-1 Do not ordinarily cause disease in human BSL-2 Cause human disease But not readily transmitted BSL-3 Biological safety cabinet required [culture included] Transmission: respi route BSL-4 Transmission: respi route High risk of serious disease No available treatment Very strict precautions Ebola virus Congo Crimean hemorrhagic virus

Many are recovered Produce serious in clinical lab illness Bacillus subtillis HBV Samonella Bacillus anthracis [clinical specimen] Bacillus anthracis [grown in large concentration]

Bio-threat Level/Bio-terrorism Agent Category

` `

Category A Agents Highest Priority Agent ` Easily disseminated or transmitted person-toperson causing secondary and tertiary cases ` Cause high mortality with potential for major public health impact including the impact on health care facilities. ` May cause public panic and social disruption ` Require special action for public health preparedness.

Bio-threat Level/Bio-terrorism Agent Category

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Category B Agents 2nd highest priority

` ` `

Moderately easy to disseminate Cause moderate morbidity and low mortality Require specific enhancement of CDC's diagnostic capacity and enhanced disease surveillance

Bio-threat Level/Bio-terrorism Agent Category

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Category C Agents 3rd highest priority

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Emerging pathogens that could be engineered for mass dissemination Availability Ease of production and dissemination Potential for high morbidity and mortality and major health impact

` ` `

Bio-threat Level/Bio-terrorism Agents Category

Table 2. Bio-terrorism Agents Category Category A Variola major (small pox) Bacillus anthracis (anthrax) Yersinia pestis (plague) Clostridium botulinum toxin (botulism) Francisella tularensis (tularaemia) Filoviruses, Ebola hemorrhagic fever Marburg hemorrhagic fever Arenaviruses, Lassa (Lassa fever) Junin (Argentine hemorrhagic fever) And related viruses Category B
Coxiella burnetti (Q fever) Brucella species (brucellosis) Burkholderia mallei (glanders) Burkholderia pseudomallei (Melioidosis) *alphaviruses, Venezuelan encephalomyelitis Eastern and Western equine Encephalomyelitis Ricinus communis (Castor bearns) Clostridium perfringens Staphylococcal enterotoxin B Salmonella species Shigella dysenteriae Escherichia coli 0157:H7 Vibrio cholerae Cryptosporidium parvum *Rickettsia prowaken (Typhus fever) Chlamydia psittaci (Psittacosis)

Category C Nipah virus Hantaviruses Tickborne hemorrhagic fever viruses Tickborne encephalitis viruses Yellow Fever Multidrug resistant tuberculosis

General Characteristics of Bioterror Agent

` ` ` `

` `

Cost less than conventional and other weapon Culturing: No required training or expertise Mobile laboratories housed in large vans/semitrailers Produce large amount of bio-weapon in small laboratory, release the agent, and move on before the attack was noticed [COVERT] Wide scope of MOT Efficient transmission: Aerosol
` ` `

Dilution may occur in food/water Person-to-person spread Use of SPORES [Disadvantage: Out-of-target]

Laboratory Response Network (LRN), 1999

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Established by CDC, Association of Public Health Laboratories (APHL), Federal Bureau of Investigation (FBI) & USAMRIID Goal: To decentralize testing capabilities and to link state and local laboratories with advanced-capacity clinical, military, veterinary, agricultural, water, and food testing laboratories

Laboratory Response Network (LRN), 1999

National Labs

Definitive characterization

Confirmatory testing

Reference Labs

Recognize Rule out Refer

Sentinel Labs Figure 1. The structure of LRN

Bio-terror Agents
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Bacillus anthracis Yersenia pestis Francisella tularensis Brucella Species Burrholderia Species Coxiella burnetii Variola Virus Viral hemorrhagic Fevers Clostridium botulinum Toxin Staphylococcal Enterotoxis Ricin

Bacillus anthracis
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Anthrax
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Aerobic Gram positive bacilli Disease of herbivores Cutaneous anthrax lesion [common] Gastrointestinal anthrax nausea & vomiting Inhalation of spores

Forms
` ` `

Figure 1. Bacillus anthracis in Grams stain

Bacillus anthracis
Figure 3. Necrotic lesion of cutaneous anthrax characterized by black eschar

Figure 4. Inhalation of anthrax spores develops into mediastinitis shown in chest radiograph

Bacillus anthracis
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Specimen Collection
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Cutaneous = Vesicular fluid GI = blood; {culture} stool & rectal swab Inhalation = blood Gram positive bacilli; may be evident for capsule Rapid growth (8 hours) Nonhemolytic colonies (SBA) Medusa-head colonies

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Direct Examination
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Culture
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Yersenia pestis
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History Pandemic Plague

1. 2.

EGYPT: 541AD ASIA: 1330 & EUROPE: 1347 BLACK DEATH

`
` ` `

1894: AlexandreYersin Bubonic plague

1st to describe the agent Xenopsylla cheopis (rat flea) - vector Rattus rattus (black rat) most responsible in urban outbreak

Yersenia pestis
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Bubonic plague (most common form)

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Bite of infected flea (MOT) S&S: Fever, chills, headache, malaise Multiply in the lymph node = BUBO Leads to disseminated intravascular coagulation (DIC) with petichae & gangrene

Figure 5. Clinical manifestation of Yersenia pestis: Bubo on the leg (left) & Gangrene (right). Media from Center for Disease Control and Prevention.

Yersenia pestis
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Specimen Collection
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Sputum, Bronchial wash, Tracheal aspirate (specimen of choice) w/ sepsis & fever: blood w/ bubo: aspirated lesion Gram negative bacilli, singly or short chain (long in liquid med.) Bipolar staining characteristic/ Safety pin appearance Slow (48 hours) Nonhemolytic (SBA) Fried egg appearance
Figure 6. Yersenia pestis in Grams stain. Safety pin appearance. Media from Center for Biologic Counterterrorism and Emerging Diseases.

Direct Examination
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Culture
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Francisella tularensis
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1911: 1st isolated & described Outbreak of ground squirrels in Tulare County, CA Tularemia/Oharas disease
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Accidental contact on the infected organism Common reservoir: Several tick species Most common mammal-associated: Rabbit

Recognized as potential bioweapon

Francisella tularensis
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Ulceroglandular tularemia (most common)

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Inoculation on the dermis/Bite of infected organism Incubation: 3-10 days = febrile w/ chills, headaches, cough, chest pain May manifest bubonic plague-like buboes Develops among outdoor-related work Inhalation Fever and LRTI (influenza-like illness)
Figure 7. Cutaneous lesion of tularemia on the right hand. Media from Center for Disease Control and Prevention

Pneumonic tularemia
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Francisella tularensis
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Specimen Collection
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Blood & Biopsies Pleomorphic gram negative coccobacilli Growth not rapid (36-48 hours) Cystine-dependent Grows well CAP, Thayer-Martin, Buffered charcoal yeastextraxt

Direct Examination
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Culture
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Brucella Species
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Malta fever, undulant fever, Mediterranean fever, Cyprus fever, Bangs disease
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Associated with human

` ` ` ` ` ` `

Brucella melitensis (sheep & goats) Brucella suis (swine) Brucella abortus (cattle)
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Not associated with human

Brucella ovis (sheep) Brucella neotomae (desert wood rat) Brucella canis (dogs) Potential aerosol waepon

Brucella Species
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Brucellosis
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MOT: Breaks in skin, ingestion of contaminated food, inhalation

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Respiratory tract or Occupational exposure

Hematogenous dissemination (seeding of multiple organ)

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Lung, Liver, Spleen, CNS

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Malaise, Night swear, Relapsing fever, Chills, Myaglia Relapse may occur after medication

Figure 8. Exposure to infected animals may transmit Brucella virus to human. Media from ADAM

Brucella Species
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Specimen Collection
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Biosafety cabinet Blood, BM, tissues and fluid from affected organ, abscess Small Aerobic Pleomorphic Gram negative coccobacilli Blood culture bottles: retain up to 10 days (slow) Grows in aerobic - CAP & SBA (48 -72 hours)
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` `

Direct Examination
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Culture
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Small, circular, smooth, convex, nonpigmented, nonhemolytic Catalase, Oxidase, Nitrate reduction, Urease positivity, Motility

Burkholderia Species
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2 potential agent:

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B. mallei (Glanders) B. pseudomallei (Meliodosis)

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Endemic: SE Asia, South Pacific, Africa, India, Middle East

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Found in soil & water Equids (horses, mules, donkeys) Direct contact with infected animals
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2Cases (human-human transmission): Sexual or direct contact

S&S: Fever, Myaglia, Headache, Chest pain

Brucella Species
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Specimen Collection
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Blood, BM, Sputum, Bronchial alveolar lavage, Abscess, Urine, Sputum Gram negative coccobacilli (B. mallei); positive (B. pseudomallei) Incubation: 35C, 5%CO2 5 days SBA: small, circular, butyrous colony after 24-48 hours MAC

` `

Direct Examination
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Culture
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Coxiella burnetii
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Q(uery) fever
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Outbreak: slaughterhouse workers, Australia 1935 Reservoir: cattle, sheep, goats, dogs, cats, deer, fowl (asymptomatic) Risk: Occupational exposure Develops to PNEUMONIA or HEPATITIS (chronic)

Figure 9. A possible reservoir of Coxiella brunetii, a goat.

Coxiella burnetii
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Specimen Collection
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Blood, Serum, Tissue, Body fluids Obligate intracellular Gram negative coccobacilli Grow in the cell monolayer (not in plated media) High titer: Phase 1 Phase II

Direct Examination
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Culture
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Serological testing
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Variola Virus
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Family Poxviridae, Genus Othropoxviridae [smallpox] Brick-shaped double-stranded DNA virus MOT: Respiratory droplets/Direct contact/Fomite Replicates at oropharynx/respiratory mucosa then to lymph nodes
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TRANSIENT ASYMPOTMATIC VIREMIA

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Fever & malaise, influenza-like syndrome in 810 days Oral lesions Light macular rash > Vesicular rash

Viral hemorrhagic Fevers

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All viruses: single stranded RNA viruses Ebola Marburg viruses (family Fioviridae) Lassa fever virus (family Arenaviridae) Crimean-Congo hemorrhagic fever virus Rift Valley virus Hantaviruses (family Bunyaviridae) Dengue Yellow fever Omsk hemorrhagic fever viruses (family Flaviviridae)

Viral hemorrhagic Fevers

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Incubation: 2-3 weeks

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Fever, Rash, Myglia, Arthalgia, Nausea, Conjunctivitis, Diarrhea, CNS symptoms {not all viruses} Infection:Varying degree of bleeding Ranging from DIC, petichiae, hemorrhage of mucous membrane, to blood in urine and vomitus Should not collect from patient with suspected viral hemorrhagic fever until after consultation [compatible to disease] Serum, Heparinized plasma. Whole blood, Respiratory aspirates, Tissue & Urine

Specimen Collection
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Clostridium botulinum Toxin

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Botox/Botulinum toxin Recovered from soil species throughout the world Food-borne botulinum: ingestion of toxin Incubation: (vary) 2 hours 8 days
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Develop trouble in speaking, swallowing, and seeing Respiratory paralysis = Death

Specimen Collection
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Feces, gastric aspirate/vomitus, Serum, Tissues/exudates & food specimen >>> LRN reference laboratory

Staphylococcal Enterotoxins
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Small molecular wt. polypeptides

`

BACTERIAL SUPERANTIGEN family

Mild exposure resembles CMI response Approx. 18 staphylococcal enterotoxins identified Symptoms vary: enterotoxin & route of entry SEB: Staphylococcal enterotoxin B
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Category B bioterrorism agent Fever, Respiratory complaints (cough, dyspnea, and chest pain), GI symptoms Severe: Pulmonary edema, respiratory distress syndrome, shock, & possibly death

Ricin
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Category B bioterrorism agent From castor beans (Ricinus communis)

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Potent biologic toxin that inhibits protein synthesis

Figure 10. Castor beans

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Inhalation: mist/powder Ingestion: Profuse vomiting & diarrhea Multisystem organ failure Possibly death within 36 -72 hours

Figure 11. Image of Georgi Markov, a Bulgarian writer & journalist in London, died after injected with ricin pellet.

Outbreak
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CDC:

` Occurrence

of more cases of disease than normally expected within a specific place or group of people over a given period of time

Outbreak vs Epidemic vs Pandemic

Outbreak / Epidemic
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Pandemic
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Epidemiologist same meaning Epidemic > Outbreak

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Widespread disease epidemic Global

Serious connotation

Why investigate???
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To understand & ultimately control and prevent spread of disease

` ` `

Collecting information Identifying the source Make recommendations

To facilitate development of new vaccines, drugs and changes in human behavior as well as legislation for the improvement of public health

STEPS in OUTBREAK INVESTIGATION

1. Prepare for field work ` Investigators should be: FAMILIAR WITH THE DISEASE SHOULD HAVE A PLAN OF ACTION Supplies needed Division of tasks among members Administrative and Travel arrangement

STEPS in OUTBREAK INVESTIGATION

2. Establish the existence of an outbreak
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Investigators can examine health department surveillance records, hospital records, and other disease registries.

***IF INFORMATION IS UNAVAILABLE: Interview with the doctor or people within the community

STEPS in OUTBREAK INVESTIGATION

3.Verify the diagnosis ` Review clinical findings and lab tests ` Determine the specific nature of disease
For example: In infectious disease outbreaks, additional lab tests may be necessary to determine the specific strain of microbe implicated in the outbreak.

STEPS in OUTBREAK INVESTIGATION

4. Define and identify cases ` Investigator is responsible for establishing what constitutes a case. -Information about disease -Characteristics of the patient -Information about the location -Specific range in time

STEPS in OUTBREAK INVESTIGATION

5. Describe and orient the data in terms of time, place and person. ` Investigator understand more about the outbreak by compiling a comprehensive description of its trens over time, place, and kinds of people (age, race, sex) affected by the disease.

STEPS in OUTBREAK INVESTIGATION

6. Develop hypotheses ` Making educated guess about the source of disease, mode of transmission, and/or exposures that caused the disease based on available information

STEPS in OUTBREAK INVESTIGATION

7. Evaluate hypotheses ` Evaluation of hypotheses by looking at the facts ` Crunching numbers to get actual statistics

STEPS in OUTBREAK INVESTIGATION

8. Refine hypotheses and carry out additional studies ADDITIONAL STUDIES: Lab tests Environmental studies

STEPS in OUTBREAK INVESTIGATION

9. Implement control and prevention measures ` Control and prevention methods are usually aimed toward: Source of disease Interrupting transmission Limiting exposure

STEPS in OUTBREAK INVESTIGATION

10. Communicate findings ` Findings of the investigation should be communicated to local health authorities -IMPLEMENATION OF CONTROL MEASURES
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Make written that will provide legal record of the findings and contribute to public awareness