Bioterror agents

types of bioterrorist evens

announce: overt

unannounced: covert

category A bioterror agents

Highest concern

variola major

bacillus anthracis

yersinia pestis

clostridium botulinum toxin

francisella tularensis

hemorrhagic fever viruses (filoviruses and arenaviruses)

category B bioterror agents

coxiella buretti

brucella spp.

burkholderia mallei

staphylococcal enterotoxin B

food and waterborne agents

ricin toxin

alphaviruses

category C bioterror agents

nipah virus

hantavirus

yellow fever virus

tickborne encephalitis virus

multidrug resistant mycobacterium tuberculosis

BSL-1

organism that do not cause disease in healthy humans and are of minimal potential hazard to lab personnel and the environment (ex. B. subtilis)

standard microbiology safety practices

no special safety equipment required

laboratory clothing recommended

sink for hand washing, open bench top resistant and impervious to water

BSL-2

organisms associated with human disease and pose moderate potential hazard (ex. shigella)

BSL-1 safety practices plus limited access to lab and extreme precautions with contaminated sharps

class I or II biological safety cabinet

appropriate personal protective equipment

BSL-1 facility requirements plus autoclave and eye wash

BSL-3

organism which pose serious or potentially lethal disease when inhaled (ex. M. tuberculosis)

BSL-2 safety practices plus controlled access to lab and all procedures conducted in biological safety cabinet

Class I or II biological safety cabinet

appropriate personal protective equipment

BSL-2 facility requirements plus negative airflow, air exhaust to outside, self-closing double doors

BSL-4

organisms with life threatening potential and transmission by aerosol or of unknown risk of transmission (ex. Ebola)

maximum containment, special clothing shower upon exit, separate building, special engineering design

route of infection- food

potentially significant route of delivery

secondary to either purposeful or accidental exposure to aerosol

route of infection- water

capacity to affect large numbers of people

dilution factor

water treatment may be effective in removal of agents

route of infection- respiratory

inhalation of spores, droplets and aerosols

aerosols are most effective delivery method

advantages of biologic as weapons

infectious via aerosol, organisms fairly stable in environment, susceptible civilian populations, high morbidity and mortality, person to person transmission (smallpox, plague, VHF), difficult to diagnose and/or treat, easy to obtain, inexpensive to produce, potential for dissemination over large geographic area, creates panic, can overwhelm medical services, perpetrators escape easily

level A laboratory

BSL-2 lab with a certified class II biological safety cabinet, BSL-1 microbiology practices, directed by competent scientists, personnel specifically trained in handling pathogenic agents.

role is to rule out critical biological agents and refer to higher level laboratory

if announced: notify FBI, and the PHL, based on consultation, test and refer

if unannounced: rule out, if unable to rule out call the nearest level B lab

laboratory risk for bioterrorism agents

B. anthracis: BSL-2, low risks

Y. pestis: BSL-2, medium risk

brucella spp: BSL-2/3, high risk

F. tularensis: BSL-2/3, high risk

botulinum toxin: BSL-2, medium risk

smallpox: BSL-4, high risk

viral hemorrhagic fever: BSL-4, high risk

plague epidemiology

US averages 13 cases per year

30% of cases are in Native Americans in the southwest.

15% case fatality

most cases occur in summer

bubonic, septicemic, and pneumonic

Yersinia pestis specimen selection

bubonic: bubo, lymph node aspirate

septicemic: blood, obtain three sets 10-30 min apart

pneumonic: sputum, bronchial washings

yersinia pestis specimen inoculation

inoculate routine plating media and make thin smear for DFA

use Wayson only if DFA is unavailable, Wayson stain is not diagnostic must confirm by DFA and mouse inoculation

yersinia pestis characteristics

small, gram negative bipolar coccobacilli

Wayson stain is pink-blue cells with a closed safety pin looks

BHI broth will have little chunks in it

Botulism

Diagnosis of botulism is made clinically

Health care providers suspecting botulism should contact their State Health Department

Infective dose: 0.001 µg/kg

Incubation period: 18 - 36 hours

Dry mouth, double vision, droopy eyelids, dilated pupils n Progressive descending bilateral muscle weakness & paralysis. Respiratory failure and death

Mortality 5-10%, up to 25%

Level A Procedures for Botulism Event

Properly collected specimens are to be referred to designated testing laboratories

Prior to the shipment of any botulism associated specimen, the designated laboratory must be notified and approved by the State Health Department

Clinical specimens to be collected: Serum, Gastric contents or vomitus, Feces or return from sterile water enema, Wound tissue

Botulism toxins are extremely poisonous

Minute quantities acquired by ingestion, inhalation, or by absorption can cause death

All materials suspected of containing toxin must be handled with CAUTION

anthrax Epidemiology

Primarily a disease of herbivorous animals such as sheep, cattle, goats, and horses

Humans acquire the infection accidentally in agricultural or industrial setting, During processing of hides or animal hair, gains access through cuts or inhalation

Anthrax Clinical Manifestations

Cutaneous anthrax begins 2 to 5 days after inoculation of spores (95%), lesion starts as an erythematous papule that progresses into an ulcerative black eschar or “malignant pustule”

Pulmonary anthrax (rare) is acquired by inhalation of spores, malaise, mild fever, nonproductive cough follows

Gastrointestinal (very rare)

Anthrax Laboratory Diagnosis

Large gram-positive bacilli in short chains

Nonhemolytic, white to gray on sheep’s blood agar

"Medusa head” appearance

Lack of motility

Penicillin inhibition zone

Capsule formation

“STICKY” consistency on SBA

catalase-positive

Aerobic spore formation

Inhalational 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

No person-to-person transmission

Anthrax Specimen Selection

Inhalation: Sputum and Blood

Cutaneous: Vesicles and Eschar

Gastrointestinal: Stool and Blood

Francisella tularensis

Plague-like disease in rodents (California), Deer-fly fever (Utah), Glandular tick fever (Idaho and Montana) Market men’s disease (Washington, DC), Rabbit fever (Central States), O’Hara’s disease (Japan)

Poorly staining, tiny Gram-negative coccobacilli

Fastidious, requires cysteine for robust growth: Cysteine Heart Agar (CHA) is ideal, BYCE can be used

tularemia

Contagious --- no

Infective dose --- 10-50 organisms

Incubation period --- 1-21 days (average=3-5 days)

Duration of illness --- ~2 weeks

Mortality --- treated : low, untreated: moderate

Persistence of organism ---months in moist soil n Vaccine efficacy --- good, ~80%

Brucellosis

Zoonotic disease caused by any of 4 Brucella spp.: abortus, melitensis, suis, and canis

Systemic infection characterized by an undulant fever pattern

Relatively rare in the U.S. with approximately 100 cases/year

The most commonly reported laboratory-associated bacterial infection

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

No person-to-person transmission

Mortality: < 5% n Stable organisms

Brucellosis transmission

Ingestion: The most common mode of transmission

Direct skin contact/puncture: Occupational hazard for farmers, butchers and veterinarians

Aerosols: Highly infectious

Brucella spp. Specimen Selection

Serum

Blood or bone marrow

Tissue (spleen, liver)

Brucella spp. Key Level A Lab Tests

Colonial morphology on SBA, Fastidious ¨Visible growth may take 48 - 72 hrs, Small (0.5-1.0mm), convex, glistening, Non-hemolytic and non-pigmented

Oxidase

Urea hydrolysis: B. suis & B. canis ~15 min, B. abortus & B. melitensis ~24hr