Review of September 2008 Educational Bioterrorism Proficiency Exercise Agent

2. 2 Objectives Describe the epidemiology, clinical manifestations, and treatment of Burkholderia mallei/pseudomallei infections. Discuss the public health and potential biothreat aspects of Burkholderia mallei/pseudomallei infections. Describe the laboratory detection and identification methods for Burkholderia mallei/pseudomallei.

3. Overview of WSLH September 2008 Educational Bioterrorism Proficiency Exercise Results

4. 4 Overview of WSLH Bioterrorism Proficiency Exercise Two samples sent to 115 Wisconsin laboratories 106 reported test results (92%) 11 did not report results Sample BPE 08-2-1: Burkholderia thailandensis Sample BPE 08-2-2: Burkholderia cepacia Samples intended to simulate Burkholderia mallei/pseudomallei in “rule out” testing

5. 5 Results of “Rule Out” Tests

6. 6 Results of “Rule Out” Tests (continued)

7. 7 Results of “Rule Out” Tests (continued)

8. 8 Results of “Rule Out” Tests (continued)

9. 9 Results of Organism ID

10. 10 Results of Organism ID (continued)

11. 11 If growth from a significant source takes at least 48 hours or more to get visible growth Use the following algorithm: Reminder of Laboratory Response Protocols

12. 12 Reminder of Laboratory Response Protocols

13. 13 Recommendations Consider developing the capability to perform “rule out” testing and incorporate it into your daily testing algorithms. Catalase Oxidase Indole Urease Motility

14. 14 Recommendations If you identify, or unable to rule out, an organism as Burkholderia mallei or Burkholderia pseudomallei Call the healthcare provider for more information about the patient Call local public health Call the WSLH and make arrangements to send the organism to the WSLH for confirmatory testing (24/7 Emergency # 608-263-3280)

16. History, Epidemiology, Clinical Manifestations, and Treatment of Burkholderia mallei/pseudomallei Infections

17. 17 History B. mallei Febrile illness seen typically in equines Horses, mules, donkeys Used as a biological weapon WWI Human infection due to animal exposure 1945---last naturally acquired infection in USA Lab acquired infection in 2000

18. 18 History B. pseudomallei Environmental organism Classified in the genus Pseudomonas 1912---Described by Whitmore and Krishnaswany Morphine addicts in Rangoon “Whitmore”s Disease Noted that it resembled glanders Melioidosis---Greek “melis”=“distemper in asses”

19. 19 Epidemiology B. mallei---Glanders Geographic distribution Eradicated from USA and W. Europe E. Europe, Middle East, Asia, and Africa Equines primary reservoir Found in other animals that eat infected horse meat (cats, etc)

20. 20 Epidemiology B. pseudomallei--melioidosis Endemic in tropical regions SE Asia, China, Malaysia, Singapore, N. Australia, Indian subcontinent Found in high concentrations in rice paddies Infections in USA imported from endemic areas Multiple cases of melioidosis in patients with no travel history----THINK BIOTERORISM ATTACK Laboratory acquired infections reported

21. 21 Clinical Manifestations: B. pseudomallei Melioidosis Asymptomatic---majority of those infected Acute Subacute Chronic

22. 22 Clinical Manifestations: B. pseudomallei Acute melioidosis Most commonly presents as pneumonia High fever, dyspnea, chest pain Purulent sputum, may be bloody Septicemic pneumonia most severe form 40% mortality Localized skin ulcers or abscesses Genitourinary infections Prostate Neurologic disease---brain stem encephalitis

23. 23 Clinical Manifestations: B. pseudomallei Subacute infection Mimics tuberculosis Low-grade fever, malaise, anorexia, weight loss over a period of months Nodular and cavitary lesions May be latent for years and reactivate “Vietnamese time bomb” Serologic evidence of infection in 225,000 Occasionally seen, but rare Most likely in immunosuppressed

24. 24 Clinical Manifestations: B. pseudomallei Chronic infection Similar to miliary TB Disseminated with granulomatous lesions in a variety of tissues May have minimal symptoms Most have fever, cough, and weight loss

25. 25 Risk Factors for Melioidosis

26. 26 Clinical Manifestations: B. mallei Glanders in humans Cutaneous or systemic disease Cutaneous—percutaneous inoculation Nodules with localized lymphadenitis Systemic Broncho- or lobar pneumonia Bacteremia with lesions in liver and spleen Fever, rigors, and malaise Often fatal without antimicrobial treatment

27. 27 Glanders/Meliodosis

28. 28 Transmission Glanders Person-to-person transmission Discharges from resp. tract and skin highly infectious Isolate patients Inhalation and percutaneous infection Melioidosis No person-to-person transmission Percutaneous infection most common Ingestion rare

29. 29 Treatment Melioidosis Acute or chronic infection Imipenem, meropenem, or ceftazidime for 2-4 weeks followed by oral amoxicillin-clavulanate or combination of doxycycline and SXT for 3-6 months No data on prophylactic agents B. pseudomallei frequently resistant to penicillin, ampicillin, 1st and 2nd generation cephalosporins, fluoroquinolones, and aminoglycosides

30. 30 Treatment Glanders No experience with modern antimicrobials Likely same therapy as for melioidosis would be effective Lab acquired case responded to imipenem and doxycycline for 2 weeks, then oral doxycycline and azithromycin

31. 31

32. 32 Laboratory Detection and Identification Methods for Burkholderia mallei/pseudomallei

33. 33 Select Agents - CDC Category B Category B Are moderately easy to disseminate; Result in moderate morbidity rates and low mortality rates; and require specific enhancements of CDC's diagnostic capacity and enhanced disease surveillance. Brucella spp. (Brucellosis) Epsilon toxin Clostridium perfringens Food threats (Salmonella spp. E. coli 0157, Shigella spp.) Burkholderia mallei (Glanders) Burkholderia pseudomallei (Melioidosis) Chlamydia psittaci (Psittacosis) Coxiella burnetii (Q Fever) Ricin toxin from Ricinus communis (castor bean) Staphylococcal enterotoxin B Rickettsia prowazeki (Typhus Fever) Viral encephalitis (VEE, EEE, WEE) Water threats (Vibro cholerae), Cryptosporidium parvum)

34. 34 Biosafety Personal protective equipment includes gloves, gown, mask, and protective faceshield. Primary patient cultures must be manipulated in a Class ll biological safety cabinet (BSL2). Biosafety level 2 (BSL-2) Appropriate for handling moderate-risk agents that cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. All cultures must be taped shut during incubation.

35. 35 Rapid Test to Rule Out B. mallei and B. pseudomallei Gram Stain Grow on nonselective and selective media Growth at 42°C (P. aeruginosa will also grow) B. mallei - No growth at 48 hours, faint growth at 72 hours B. pseudomallei – heavy growth at 48 hours. B. pseudomallei is not difficult to isolate, but B. mallei is less robust Motility Catalase Oxidase Indole Colistin/Polymixin B Colistin is Polymixin E

36. 36 Motility Motility semisolid medium with 2,3,5-triphenyltetrazolium chloride (TTC) indicator Wet mount motility method is NOT RECOMMENDED May be difficult to read, especially if not routinely performed B. mallei is nonmotile, and B. pseudomallei is motile

37. 37 CATALASE B. mallei and B. pseudomallei are Catalase positive Perform if the isolate is not growing well on MacConkey agar in 48 hours

38. 38 Oxidase

39. 39 Indole B. mallei and B. pseudomallei are Indole negative Some Crysobacterium and Vibrio are Indole positive

40. 40 Colistin/Polymixin B Resistance Place a colistin (10 µg) or polymyxin B (300 U) disk in 1st or 2nd quadrant of sheep blood agar plate B. mallei and B. pseudomallei are Resistant to polymyxin B and colistin. Burkholderia, Chromobacterium violaceum, some Vibrio, and Ralstonia are Resistant; most Pseudomonas species are Susceptible. An alternative method is to inoculate PC agar (Bile salts, Crystal violet, Polymixin B, Ticarcillin) or modified Thayer Martin (Vancomycin, Nystatin, Colistin) Lack of growth on this media may be due to other additives and should be confirmed with a disk test

41. 41 Burkholderia pseudomallei

42. 42 B. pseudomallei Suspect when: General Unusual number of clinical specimens received from patients with similar symptoms Unusual isolates from more than one patient Oxidase positive GNR that will not ID on automated systems. ID does not make sense. High clinical suspicion

43. 43 B. pseudomallei (cont.) Suspect when: Specific to B. pseudomallei Travel history Recent history of travel to the region of endemicity (Southeast Asia, the Philippines, the Indian subcontinent, or the northern coast of Australia Unusual Susceptibility pattern Gentamycin and colistin Resistant. Amoxicillin/clavulate (augmentin) Sensitive. If B. pseudomallei infection is suspected, all samples from sites with a normal flora should be cultured on PC agar in addition to routine culture media (if available). Additional plate may be incubated at 42°C

44. 44 Burkholderia pseudomalleiPresumptive Identification

45. 45 Burkholderia pseudomalleiPresumptive Identification (cont.)

46. 46 Gram Stain – B. pseudomallei Small gram negative rods 1-3 um x 0.3 um. May show bi-polar staining Irregularly arranged May be arranged in long bundles

47. 47 Colony Morphology – B. pseudomallei Growth on BAP, CHOC, MacConkey/EMB Typical specimens include blood culture, respiratory, urine, wound/abscess material Transport specimens at room temperature 35–37°C, ambient atmosphere (CO2 incubation is acceptable). Keep plates for 5 days Selective media Ashdown’s media Place a colistin disk or polymyxin B disk in initial inoculation area of BAP. Burkholderia will grow up to disk Poor growth at 24 hours Smooth, creamy to white colonies at 24 hrs., may become dry (looks like Pseudomonas stutzeri) or mucoid at 48 hrs.

48. 48

49. 49 Other Characteristics - B. pseudomallei Strong, musty or dirt-like odor (“Sniffing” of plates containing B. pseudomallei is dangerous and should not be done. However, the odor will be apparent without sniffing) Recovery of B. pseudomallei from blood culture within the first 24 hours of incubation indicates fulminant sepsis, which has a very high (90%) mortality rate Triple Sugar Iron (TSI) Agar No change(Pink/Pink) or slight oxidation (Yellow/Pink) Decontamination 0.5% hypochlorite solution, prepared within 24 hours.

50. 50 Susceptibility Do Not Perform - Refer to State Lab. Requires BSL-3 containment and personnel precautions You may not know until after susceptibility performed Consult ID physician Comments B. pseudomallei Resistant to penicillin, ampicillin, first- and second-generation cephalosporins, gentamicin, tobramycin, and streptomycin Susceptible to ceftazidime, imipenem, meropenem, piperacillin, amoxicillin-clavulanate, ceftriaxone, and cefotaxime. B. mallei Like B. pseudomallei, except gentamicin, clarithromycin, and azithromycin are susceptible

51. 51 Burkholderia mallei

52. 52 B. mallei Suspect when: General Unusual number of clinical specimens received from patients with similar symptoms Unusual isolates from more than one patient Oxidase positive GNR that will not ID on automated systems. ID does not make sense. High clinical suspicion Specific for B. mallei Travel history Slow growing gram negative rod

53. 53 Burkholderia malleiPresumptive Identification

54. 54 Burkholderia malleiPresumptive Identification (cont.)

55. 55 Gram Stain – B. mallei Small, straight or slightly curved gram-negative coccobacilli or rods, arranged in pairs, parallel bundles, or the “Chinese letter” forms.

56. 56 Colony Morphology – B. mallei Growth on BAP, and CHOC. May not grow on MacConkey/EMB. Typical specimens include blood culture, respiratory, urine, wound/abscess material Transport specimens at room temperature 35–37°C, ambient atmosphere (CO2 incubation is acceptable). Keep plates for 5 days Selective media Ashdown’s media Poor growth at 24 hours – All GNR that are pinpoints at 24 hours should be taped and worked up in a BSC. Place a colistin disk or polymyxin B disk in initial inoculation area of BAP. Burkholderia will grow up to disk Colonies are gray, translucent, and have no pigment or distinctive odor.

57. 57 Survey Results

58. 58 Sample BT-01 Results

59. 59 Sample BT-01 Results (Cont.)

60. 60 Sample BT-01 Results (Cont.)

61. 61 The Results for BT-01 – Burkholderia thailandensisGoal – BT Vs. Non BT Isolate

62. 62 BT-01 - Burkholderia thailandensis

63. 63 Sample BT-02 Results

64. 64 Sample BT-02 Results (Cont.)

65. 65 The Results for BT-02 - Burkholderia cepaciaGoal – ID or Refer

66. 66 BT-02 - Burkholderia cepacia

67. 67 References SENTINEL LABORATORY GUIDELINES FOR SUSPECTED AGENTS OF BIOTERRORISM. Burkholderia mallei and B. Pseudomallei. American Society for Microbiology, Feb. 2008. http://www.asm.org/ASM/files/LeftMarginHeaderList/DOWNLOADFILENAME/000000002739/BpseudomalleiBmalleiRevision2008.pdf Centers for Disease Control and Prevention Website. http://www.cdc.gov/ Biosafety in Microbiological and Biomedical Laboratories. 5th Ed. 2007. http://cdc.gov/od/ohs

68. 68 References SENTINEL LABORATORY GUIDELINES FOR SUSPECTED AGENTS OF BIOTERRORISM. Burkholderia mallei and B. Pseudomallei. American Society for Microbiology, Feb. 2008. http://www.asm.org/ASM/files/LeftMarginHeaderList/DOWNLOADFILENAME/000000002739/BpseudomalleiBmalleiRevision2008.pdf Centers for Disease Control and Prevention Website. http://www.cdc.gov/ Biosafety in Microbiological and Biomedical Laboratories. 5th Ed. 2007. http://cdc.gov/od/ohs