Nebraska Challenge Set Exercise February, 2012 1
Continuing Education Fax to 402.559.7799 Email to kstiles@unmc.edu 2
Resources recommended for Challenge: ASM Sentinel Guidelines Link found on • www.nphl.org BIOLOGICAL TERRORISM PROCEDURES 3
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BT Media Available from NPHL India Ink & wet prep for motility have been discontinued – not recommended by CDC Optional - Spot Indole now DMACA with cinnamaldehyde reagent Order in sets of 3 to include positive and negative controls with every test 5
Scenario 1 • Clinical History: 69 year old female • Source: Blood Cultures, 1 of 4 bottles positive • Note: Laboratory has received HAN from Nebraska DHHS issuing a warning of a potential Bacillus anthracis outbreak reported in multiple locations across Nebraska. 6
Direct Gram Stain Morphology 7 Images by NPHEL, St E’s and Alegent, Imperial
Gram Stain Comparisons Fall NCSE 2011 - Listeria NCSE 2012 - 01 unknown 8 Images by NPHL
Gram Stain Morphology from Colony 9 Images by Norfolk
Gram Stain Morphology from Colony 10 Images by Grand Island
Gram Stain Morphology from Colony 11 Images by NPHEL, FAMC, Alliance
Gram Stain Morphology at 72 hours 12
Colony Morphology Growth on SBA, Choc 13 Images by Alliance, FAMC, Bellevue (Offutt), Grand Island
NPHL Bench Guide 14
NPHL Bench Guide 15
Organism 1 Trigger Points • Sterile site • Large Gram Positive Rod in direct smear • Subterminal spores, cell not swelling • HAN warning from NE DHHS • Not Hemolytic on SBA • Catalase Positive (caution – test produces aerosol) 16
What did not fit? • Only 1 of 4 Blood Culture bottles positive • Not encapsulated in direct smear • Gram stain morphology not consistently chaining • Only 3 sentinel labs reported spores • Colony morphology more dry wrinkled • Growth was not 2-8 mm within 24 hr • Is the ground glass appearance and the comma shaped projections (medusa head) seen? • Did patient history or clinical presentation fit anthrax? 17 • Motility? Laboratories differed on results
Group Biochemical Results: NCSE 2012 - 01 Did you use any of the following to identify the organism? If yes, please indicate reaction: Positive Negative Not Applicable No Response Oxidase (2) (1) (19) 3 Catalase (12) (9) (1) 3 Indole (0) (3) (19) 3 Motility (2) (16) (4) 3 Nitrate (0) (0) (22) 3 Satellite (0) (0) (22) 3 Urease (0) (2) (20) 3 18
Motility Images 19 NCSE2012- Positive Negative 01 QC QC
Responses to Organism 1 NCSE 2012 - 01 1 Bacillus spp Unable to rule out B. anthracis 11 Bacillus spp., not B. 10 anthracis Non Bioterrorism Agent 20
What is the Best Answer for Organism 1? Bacillus spp Not B. anthracis Non Bioterrorism Agent?? Bacillus spp Unable to rule out B. anthracis Actual mimic organism sent: Bacillus spp Reference: • Organism was actual patient case which NPHL consulted with via STATPack • University of Minnesota College of Veterinary Medicine Fact Sheet 7/7/2006 21 • Gastrointestinal Anthrax in New Hampshire: A 2009 Case Report - Laboratory Medicine; June 2011 CE Update. http://labmed.ascpjournals.org/content/42/6/363
Morphology Comparisons 22 • Image by NPHEL at 5 days
Morphology Comparisons 23
Gram Stain Comparisons NCSE 2012 - 01 24 Images by NPHL, CDC PHIL image library
Worldwide anthrax outbreaks 2010-2012 http://healthmap.org/en/ 25
NSCE 2012 - 02 Clinical History: 2 year old male in ICU with aspiration pneumonia. Source: Endotracheal Note: Laboratory has isolated out multiple organisms including this organism. 26
Direct Smear Images 27 Images by Alegent, St E’s, Grand Island, North Platte,
Gram Stain Morphology from Colony Enlarged image to show “Bipolar” characteristics Images by Pender 28
Colony Morphology 29 Images by P Lab, Norfolk, Offutt, Grand Island
Colony Morphology @ 48 hours 30 Images by Norfolk, Imperial
Organism 2 Trigger Points • Gram Negative Rod – Bipolar Staining • Small growth at 24 hours? • Oxidase? 31
NPHL Bench Guide 32
Group Biochemical Results: NCSE 2012 -02 Did you use any of the following to identify the organism? If yes, please indicate reaction: Positive Negative Not Applicable No Response Oxidase (20) (2) (0) 3 Catalase (15) (5) (2) 3 Motility (15) (1) (6) 3 Indole (0) (19) (3) 3 Beta-lactamase (3) (1) (18) 3 Nitrate (3) (0) (19) 3 Satellite (0) (0) (22) 3 Urease (0) (14) (8) 3 33
Burkholderia Algorithm 34
NPHL Bench Guide Add Motility Negative 35
Responses to Organism 2 NCSE 2012 – 02 Burkholderia spp, unable to rule out B 1 pseudomallei 2 Burkholderia spp, 2 unable to rule out B pseudomallei or mallei Burkholderia 13 4 pseudomallei Yersinia spp, unable to rule out Y pestis Non Bioterrorism Agent 36
What is the Best Answer for Organism 2? • Burkholderia spp, unable to rule out B. pseudomallei Actual mimic organism sent: Burkholderia thailandensis Reference: Pneumonia and Septicemia Caused by Burkholderia thailandensis in the United States , Journal of Clinical Microbiology, Dec 2006, p 4601-4604 Comparison of Routine Bench and Molecular Diagnostic Methods in Identification of B. pseudomallei , Journal of Clincial Microbiology, March 2009, p 1578-1580. Burkholderia pseudomallei Misidentified by Automated System , Emerging Infectious 37 Diseases, Nov 2009, p 1799-1801.
Nebraska Challenge Set 2012 Next shipment date – July 16 Automatic Enrollment for 2013 BT Training needed? 38
APHL On Demand Course Bioterrorism Preparedness Training for LRN Sentinel Laboratories • Description • Each of these Intermediate-level, interactive online course modules review a component of the Laboratory Response Network (LRN) protocols for bioterrorism agent identification. The modules include case studies, real-life laboratory scenarios and links to resource information. Course materials may be used as part of a laboratory’s competency assessment program for terrorism preparedness. Updated for 2012, seven course modules are available, including: • · Anthrax (Bacillus anthracis) • · Brucella (Brucella spp) • · Burkholderia (Burkholderia spp) • · Plague (Yersinia pestis) • · Tularemia (Francisella tularensis) • · Laboratory Biosecurity 39 • · The Laboratory’s Role in Bioterrorism Preparedness (Laboratory Response Network, LRN)
STATPack Training – 3 CEU’s 40 http://training.statpack.org
Changes to Packaging & Shipping • New documentation requirements for couriers • Will affect how you send: • Shiga Toxin positive • MTB • Coccidiodes immitis • ROAR isolates (case by case basis) • Dates in June 2012 • June 5 - Omaha • June 7 - Kearney 41
Questions?? 42
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