Bioterrorism Agent Case Studies: Manuel's Mystery and Loretta's Lymph

1. Bioterrorism Agent Case Studies Beth Schweitzer MT(ASCP), SM Microbiology Specialist Website: www.nphl.org NPHL Nebraska Public Health Lab

2. Case Format • Lack of pertinent patient information • Travel history • Occupation • Symptoms • Duration of Illness

3. Case Format • Cultures are set according to NPHL Operating Procedures • BAP • Chocolate • MacConkey • Thioglycolate Broth

4. Zoonotic Diseases • Naturally occurring • Cases in NE • Safety concerns for laboratorian

5. Manuel’s Mystery • Blood Cultures drawn on 16 y/o Hispanic male • Instrument flags bottles positive at day 4 • Gram stain: NOS • Bottles blind subbed and placed back on machine • Bottles discarded on day 5 (standard operating procedure)

6. Con’t • BAP and Choc show a haze of growth at 48 hours ( 6th day of original Blood culture) • Gram stain: tiny gram- negative coccobacilli • What organisms should you be thinking!!!

7. Haemophilus • Pasturella • Oligella • Bordetella • Brucella • Francisella • Acinetobacter • Psychrobacter

8. Think Biosafety • In a biosafety cabinet you perform following: • Identification for Haemophilus • Rapid NH • Quad/Tri Plate • Staph streak

9. Think Biosafety • Reactions do not fit the pattern for Haemophilus!! • What now?

10. Think Biosafety • Do not perform automated commercial identification on slow growing-GNCB that do not grow on MAC/EMB. • The risk of aerosols is too high!

11. Think Biosafety • In a biosafety cabinet you perform following: • Urease slant (Pos in 2 hours) • Tube motility (Neg at 48 hours)

12. TEST Oxidase Motility Urease Nitrate BAP X/V Brucella + 95% - + + + - Bordetella+ + + + + - Acinetobacter- - V - + - Psychrobacter + - + V + - Oligella + V + + + - Haemophilus V - V + - +

13. What organism can’t we rule out? Brucella spp.

16. Brucella spp. - Biosafety Alert: • Brucellosis was the most commonly reported laboratory-associated bacterial infection. • Cases have occurred in clinical laboratory settings by “sniffing” cultures, direct skin contact with cultures, and aerosol generating procedures (e.g. culture manipulation)

17. Brucellosis: • 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% • Persistence of organism: very stable

18. Brucella spp.-Specimen Selection: • Serum • The diagnosis of brucellosis is frequently achieved by serology. An acute & convalescent phase specimen should be collected (21 days apart) • Blood or bone marrow • Most often isolated from these • Tissue (spleen, liver) • Occasionally isolated

19. Brucella spp. – Technical hints • Oxidase positive, rapid urease positive, non-motile, slow growing gram negative-coccobacillus REFER

20. Naturally occurring • Patient was a visitor from Mexico and had ingested unpasteurized goat cheese

21. Loretta’s Lymph • Lymph node aspirate from a 35 y/o female • Gram stain: Moderate WBC’s Few Gram- negative rods

22. Growth at 24 hours • Tiny (1-2 mm) grey colonies on BAP and Choc • Non-lactose fermenter on Mac

23. Biochemical tests • Oxidase (-) • Indole (-) • Use commercial ID system

24. What organism could it be? • Fits Enterobacteriaceae pattern • Without patient history hard to create a differential

25. Y. pestis – commercial ID systems • Included in the data base of MicroScan, Vitek, and API 20E.. etc True accuracy not yet determined 60% of California’s Y. pestis ID’s are from commercial systems

26. Y. pestis – commercial ID systems • If ID as Y. pestis – send to NPHL • May ID as Shigella ssp, H2S- negative Salmonella, Acinetobacter, or Y. pseudotuberculosis • Biochemically inert nature of organism

27. Y. pestis –commercial ID systems • How do we know when to question ID results?? • Source • Colony morphology • H2S neg Salmonella • Shigella characteristics • Inpatient/outpatient status-nosocomial infection.

29. Plague Epidemiology: • During 1988-2002, 112 human cases were reported from 11 western states • 30% of cases are in Native Americans in the Southwest. • 15% case fatality rate • 80% of cases occur in summer and near the patient’s residence- FLEAS

30. Y. pestis -Specimen Selection: • Specimen selection is important • Bubonic • Bubo - lymph node aspirate • Septicemic - blood - organisms may be intermittent. Take three specimens 10-30 minutes apart • Pneumonic • Sputum/throat • Bronchial washings

31. Y. pestis - Gram stain from sputum • Small, gram-negative rods

32. Y. pestis - Direct Culture Growthon Agar Plate:

33. Y. pestis - Growth in Broth: • Thioglycolate Broth (may see from original specimen set-up) • Do not shake tubes • Observe suspended flocculent clumps on side and bottom of tube. Broth remains clear • May also happen with Y. pseudotuberculosis or S. pneumoniae

34. Y. pestis – Growth in Broth culture: Y.pestis Y. pseudotuberculosis

35. Y. pestis - Technical Hints: • Small gram-negative, poorly staining rods from blood, lymph node aspirate, or respiratory specimens, BLOODY SPUTUM Refer

36. Naturally Occurring • Couple from New York City infected by flea bites on trip to New Mexico • Returned home to NYC before becoming ill

37. Bob’s Booboo • Aerobic culture is collected from wound on the hand of a 45 y/o male • Gram stain: NOS Mod WBC

38. Con’t • At 48 hours there is slight haze growth on the BAP and Choc • Gram stain: faintly staining gram- negative coccobacilli • What organisms should you be thinking!!!

39. Haemophilus • Pasturella • Oligella • Bordetella • Brucella • Francisella • Acinetobacter • Psychrobacter • Actinobacillus

40. Think Biosafety • Do not perform automated commercial identification on slow growing-GNCB that do not grow on MAC/EMB. • The risk of aerosols is too high!

41. Think Biosafety • In a biosafety cabinet growth you perform the following: • Oxidase (Negative) • Manual commercial identification system • No identification • Reisolate to BAP and Choc

42. No growth on BAP after reisolation • What to do now???!?!?!

43. Haemophilus is a likely organism • Haemophilus quad plate is set for identification • No growth is seen • Its NOT Haemophilus… What Now?

44. In a biosafety cabinet the following tests are performed: • Motility (Negative at 24 hours) • Urease (Negative at 24 hours) • B-lactamase (+)

45. What organism can’t we rule out? Francisella

47. F. tularensis – Gram stain: Poorly staining, tiny Gram-negative coccobacilli

48. Francisella/E. coli

49. F. tularensis –growth characteristics: • Will grow initially on sheep blood agar, chocolate agar and Thayer-Martin agar, but poorly or not at all on sub • Grows slowly at 35oC, poorly at 28oC • 24 hours on BAP and Choc • gray-white, translucent colonies • usually too small to be seen individually

50. F. tularensis –growth characteristics: • Thayer Martin is a good selective media from a mixed culture