Bioterrorism:Overview with a Focus on Laboratory Medicine

1. Bioterrorism:Overview with a Focus on Laboratory Medicine

2. History of Biologic Warfare • 1495 - Spaniards place blood from leprosy patients in wine of French • 1763 - Sir Jeffery Amherst and Henry Bouquet deliberately attempt to infect native Americans with smallpox at Fort Pitt using blankets • 1939-1945 - Japanese Unit 731 used plague and other BW against Chinese

3. US Biologic Weapons Program • Began during FDR administration • Ended by Nixon in 1969-72 • 1972 Nixon signed BW convention and Geneva Protocol • Ratified in 1975

4. Soviet BW Program • 1979 Accidental release of Anthrax at Soviet center at Sverdlovsk • 66 deaths out of 77 confirmed cases (86%)

5. BW Manufacturing CapacitiesMinistry of Defense • Sverdlovsk facility--anthrax • 100+ tons stockpiled • Production capacity > 1000 tons annually • Kirov facility--plague • 20 tons stockpiled • Production capacity ~ 200 tons annually • Zagorsk facility--smallpox • 20 tons stockpiled • Production capacity ~ 100 tons annually

6. Rogue Nations BW Program • 17 nations suspected of BW capability or research • Iran, Iraq, Libya, Syria, North Korea, Taiwan, Israel, Egypt, Vietnam, Laos, Cuba, Bulgaria, India, South Korea, South Africa, China and Russia

7. Rogue Nations BW Program • 1995 UN documents Iraq conducted research and development work on anthrax, botulinum toxins, Clostridium perfringens, aflatoxins, wheat cover smut, and ricin • Iraq produced 19,000 liters of concentrated botulinum toxin (nearly 10,000 liters filled into munitions), 8,500 liters of concentrated anthrax (6,500 liters filled into munitions) and 2,200 liters of aflatoxin (1,580 liters filled into munitions).

8. Bioterrorism • Aum Shinrikyo cult • March 20, 1995 Sarin gas attack Tokyo subway • experimented with botulin toxin, anthrax, cholera, and Q fever • In early 1994, cult doctors were quoted on Russian radio as discussing the possibility of using Ebola as a biological weapon • attempted several apparently unsuccessful acts of biological terrorism in Japan between 1990 and 1995

9. Partial Listing of Known Biological Weapons Agents

10. Biological Weapons Being Developed--Late ‘80s/Early ‘90s GENETICALLY ENGINEERED STRAINS Antibiotic-resistant (AR) plague AR tularemia AR anthrax Antibiotic- and sulfonamide-resistant glanders Immune system-overcoming (IO) plague IO tularemia IO anthrax Smallpox with VEE genes inserted

11. BSL-1 BSL-2 Most clinical labs agents of minimal to moderate hazard access to lab limited use biological safety cabinets or hoods BSL-3 BSL-4 specialized facilities very hazardous agents USAMRIID CDC Laboratories - Biosafety Levels

12. Laboratory Response Network for Bioterrorism LEVEL D: CDC LEVEL C: Typing Labs, Public Health Labs LEVEL B: Public Health Labs LEVEL A: Clinical Labs

13. Level A Laboratory:Definition • BSL-2 Laboratory with a certified Class II biological safety cabinet • BSL-1 microbiology practices plus • Directed by competent scientists • Personnel specifically trained in handling pathogenic agents • Biological safety cabinet, Class II • Access limited by lab director

14. Level A Laboratory:Definition, con’t • BSL-2 Laboratory with a certified Class II biological safety cabinet • Physical containment practices to minimize infectious aerosols • “Sharps”precautions • PPE (lab coat, gloves, face shield) • Biohazard warning signs • Biosafety manual defining waste/ S&H/ decontamination/surveillance policy/ CC

15. Role of the Level A Laboratory • Rule out critical biological agents • Refer to higher level laboratory

16. Bioterrorism Agents:Laboratory Risk AgentBSLLab Risk B. anthracis 2 low Y. pestis 2 medium F. tularensis 2/3 high Brucella spp. 2/3 high Botulinum toxin 2 medium Smallpox 4 high Viral Hemorrhagic fever 4 high

17. Epidemiology • Zoonotic disease in cattle, sheep and horses • Spores present in soil • Wool sorters and handlers of infected animals or animal products

18. aerobic gram-positive spore-forming nonmotile Bacillus species vegetative cell 1-8m spores 1m nonhemolytic on 5% SBA at 35°C (18-24 h) Microbiology

19. Bacillus anthracis

20. Bacillus anthracis Color Atlas and Textbook of Diagnostic Microbiology JB Lippincott1983 page 331.

21. Anthrax: Laboratory Diagnosis • Diagnostic testing only useful in the setting of clinical illness: • Smear/culture: nares, wound, blood, CSF • PCR: blood, CSF, pleural fluid • Serology: usually only useful retrospectively • Nares culture not recommended for those without symptoms • unless recommended by public health authorities as part of outbreak investigation

22. Nasal Culture: Why? • Therapeutic: works better than Xanax but are we misleading patients? • Diagnostic: almost certainly of no value • Epidemiologic: of real use to determine the boundaries of exposure

23. Dixon TC et al. Anthrax. NEJM 1999;341:815-26.

24. CutaneousAnthrax • Bacterium or spore enters skin abrasion/cut • Begins as raised itchy bump/papule • resembles an insect bite • 1-2 days  fluid filled vesicle  ulcer • vesicle contains numerous organisms • Characteristic black necrotic center • Pronounced edema  edema toxin • local/regional lymphadenopathy often present

25. Cutaneous anthrax

26. Cutaneous Anthrax

27. Progression of Cutaneous Anthrax Dixon TC et al. Anthrax. NEJM 1999;341:815-26.

28. Cutaneous Anthrax • 20% of untreated cases result in death • systemic spread, resp distress from edema • Deaths rare following appropriate therapy • Lesions sterilize in 24 hours, resolve within several weeks

29. Gastrointestinal Anthrax • Follows consumption of contaminated meat • Acute inflammation of GI tract • nausea, anorexia, vomiting, fever, followed by: • abdominal pain, hematemesis, severe diarrhea • Mortality rate 25-60% • Least common clinical manifestation

30. Inhalation Anthrax • Results from inhaling anthrax spores • 1-5 micron diameter, can reach alveoli • Disease onset often gradual, nonspecific • incubation period 1-6 days, up to 60? • fever, malaise, fatigue, nonproductive cough • short period of improvement followed by: • Abrupt onset of severe respiratory distress, cyanosis, shock and death within 24-36 hrs

31. Inhalation Anthrax Anthrax as a Biological Weapon. (JAMA. 1999;281:1735-1745).

32. Anthrax: Hemorrhagic Lymphadenitis Mediastinal lymph node in a patient with inhalation anthrax.

33. Vaccine • Cell free filtrate  protective Ag + alum • 93% effective in protecting against cutaneous anthrax in humans • animal models: efficacy against aerosol challenge • 6 injections (0,2,4 weeks; 6,12,18 months) • annual boosters required • local rxns in 30%, systemic rxns in <0.2% • Only 1 US manufacturer (BioPort Corp) • currently not available to public • Mandated for all active and reserve-duty US military personnel

34. Therapy • Almost all antibiotics, except TMP/SMX and cephalosporins, are effective against naturally occurring strains • Penicillin resistance and tetracycline resistance engineered by Russian scientists • Creating quinolone resistance is reportedly difficult, ergo the Cipro recommendation • Duration of up to 60 days may be required (animal work and Sverdlovsk)

35. Therapy Prophylaxis • Ciprofloxacin 500 mg po bid X 60 days • Doxycycline 100 mg po bid X 60 days • Cipro or Amoxicillin recommended for pregnant women and children • Once susceptibilities of the organism is known, may use penicillin or amoxicillin

36. Infection Control • No person to person spread • Standard hospital barrier isolation • Clean surfaces with hypochlorite solution • Proper burial or cremation • cremation preferred to reduce embalming risk

37. Decontamination • Primary aerosolization • last hours to 1 day • Spores on environmental surfaces • little or no risk of re-aerosolization • hypochlorite solution • Exposure to “suspect” substance • Wash with soap and water • antibiotics till proven not to be anthrax

38. Smallpox

39. History • First used as a weapon in French and Indian Wars (1754-67) • Declared eradicated by WHO in 1977 • Vaccinations stopped in 1980 • Stocks of virus stored in two places • CDC in Atlanta • Institute of Virus Preparations in Moscow • WHO recommended destruction of remaining stocks in 1999

40. Epidemiology • Variola major (30% mortality) and Variola minor (1% mortality) • Spread person to person by droplet nuclei/aerosols, by direct contact or contaminated clothing/bedding • Transmission doesn’t occur until onset of rash • Most infectious for first 7-10 days of rash. As scabs form infectivity decreases

41. Smallpox Family Tree Family: Poxviridae Subfamily: Chordopoxvirinae Genus: Orthopoxvirus Species: variola Subspecies: major and minor Related Species: vaccinia cowpox monkeypox ectromelia camelpox taterapox raccoonpox Uasin Gishu Related Genera: Avipoxvirus Capripoxvirus Leporipoxvirus Parapoxvirus Suipoxvirus Tanapox Molluscum contagiosum

42. Clinical Features • Oropharynx - infectious dose only a few virions • Asymptomatic viremia on day 3-4 • Secondary viremia on day 8 • High fever, malaise, headache and backache on day 12-14 • Maculopapular rash begins on mouth face and forearms then spreads to trunk and legs

43. Smallpox Pustules

44. Diagnosis • Lab confirmation • specimen collection wearing gloves and mask • vesicular/pustular fluid (open with blunt end of scalpel) • collect fluid on cotton swab • deposit in vacutainer and seal with tape then place in second durable watertight container • contact CDC for shipping instructions (requires BSL-4 ) • EM / culture / PCR with RFLP

45. Vaccine • Routine vaccination in US stopped in 1972 • Immune status of people vaccinated 30 years ago uncertain (probably susceptible) • Vaccine by Wyeth Lab. in storage enough for 6-7 million persons • No manufacturers now equipped to produce large quantities of vaccine (about 3 years to ramp up)

46. Therapy • Vaccination within 1-3 days after exposure may prevent or ameliorate illness • No antiviral substances have proven effective • Recent studies on mice and a few monkeys suggest cidofovir may prevent infection if given within 1-2 days of exposure • must be given IV • frequent serious renal toxicity

50. December 15