Nebraska Challenge Nebraska Challenge Set Exercise Set Exercise November 2011 November, 2011 1
Continuing Education Fax to 402.559.7838 Email to kstiles@unmc.edu 2
Resources recommended for Challenge: ASM ASM Sentinel Guidelines Link found on • www.nphl.org BIOLOGICAL TERRORISM PROCEDURES 3 3
4
BT Media Available from NPHL India Ink & wet prep for motility have been India Ink & wet prep for motility have been discontinued – not recommended Optional ‐ Spot Indole now DMACA with Optional Spot Indole now DMACA with cinnamaldehyde reagent Order in sets of 3 to include positive and negative controls with every test 5
Scenario 3 Scenario 3 Clinical History: 17 year old male Source: Blood 2 of 2 bottles positive 6
Gram Stain Morphology 7 7 Images by Fremont
Gram Stain Morphology 8 8 Images by North Platte
Gram Stain Morphology 9 9 Images by North Platte
Colony Morphology Growth on SBA Choc MacConkey Growth on SBA, Choc, MacConkey 10 10 Images by Imperial, Fremont, Bellevue
NPHL Bench Guide NPHL Bench Guide 11 11
Organism 3 Trigger Points g gg • Gram Negative Rod • Slow growth at 37°C • Growth on MacConkey – Non Fermenter • Growth on MacConkey – Non Fermenter • Oxidase Negative • Catalase Positive (caution – test produces aerosol) • Spot Indole Negative • Urease Positive • Not Francisella or Brucella ‐ GNR, grows on MAC 12 12 • Not Burkholderi a – Oxidase negative
NPHL Bench Guide NPHL Bench Guide 13 13
Group Biochemical Results: Did you use any of the following to identify the organism? If yes, please indicate reaction: y y g y g y , p Positive Negative Not Applicable No Response Oxidase (3) (10) (0) 1 Catalase Catalase (12) (12) (1) (1) (1) 1 (1) 1 Indole (0) (11) (1) 1 Motility Motility (0) (0) (7) (7) (5) (5) 3 3 Nitrate (0) (1) (9) 5 Satellite (0) (1) (9) 5 Urease (7) (6) (1) 1 14 14
Urease at 24 hours 15 15 Images by Fremont
Responses to Organism 3 Responses to Organism 3 NCSE ‐ 03 CS 03 Yersinia spp, unable to 1 rule of Yersinia pestis 6 4 Yersinia spp., not Yersinia pestis Non Bioterrorism Agent 3 Other 16 16
What is the Best Answer for Organism 3? Unable to rule out Yersinia pestis , refer Yersinia spp Not Yersinia pestis Yersinia spp Not Yersinia pestis Non Bioterrorism Agent Actual mimic organism sent Actual mimic organism sent: Yersinia pseudotuberculosis Reference: Reference: • Two Cases of Human Plague — Oregon, 2010 ; MMWR / February 25, 2011 / Vol. 60 / No. 7 Fatal Laboratory ‐ Acquired Infection with an Attenuated Yersinia pestis Strain; MMWR Weekly / • Vol. 60 / No. 7; Feb 25, 2011 • Persistence of Yersinia pestis in Soil Under Natural Conditions; Emerging Infectious Diseases 17 17 www.cdc.gov/eid , Vol. 14, No. 6, June 2008
Are there any trigger points? y gg p • Oxidase ( ‐ ) Oxidase ( ) • Glucose (+) • Fits Enterobacteriaceae pattern Fits Enterobacteriaceae pattern • Without patient history hard to create a differential • Typically tested on commercial ID systems 18 18
Yersinia pestis – Commercial ID Systems • Included in the data base of MicroScan, Vitek, , , and API 20E.. etc. • True accuracy not yet determined 19 19
Yersinia pestis – Commercial p ID Systems • If ID as Y. pestis – send to NPHL • May ID as Shigella ssp, H 2 S ‐ negative 2 Salmonella , Acinetobacter , or Y. pseudotuberculosis • Biochemically inert nature of organism NCSE Results: NCSE Results: • 2 MicroScan users reported = Acinetobacter 98 ‐ 99% • 1 MicroScan users reported = Shigella spp 97% 20 20 • 2 Vitek users reported = Yersinia pseudotuberculosis 96 ‐ 99% • 1 API user reported = Yersinia pseudotuberculosis 95%
Yersinia pestis –C ommercial ID Systems • How do we know when to question ID results?? • Colony morphology • Colony morphology • Inpatient/outpatient status ‐ nosocomial infection infection • Source & history • Confirm by 2 nd methodology – API or • Confirm by 2 methodology – API or conventional biochemicals 21 21
Yersinia pestis Gram stain Yersinia pestis Gram stain • Small, gram negative rods negative rods • Classic morphology is morphology is bipolar or “safety ‐ pin” ONLY IF in Wright ‐ Giemsa http://www.ph.ucla.edu/epi/bioter/plagueapha_id.html 22 22 • Not predictable
Yersinia images by CDC Growth 24 hr @ 37° Growth 24 hr @ 25° Yersinia pestis at 72 hr. 23 23 Tenacious when touched by inoculation loop – 48 hours
Yersinia pestis ‐ Direct Culture Growth on Agar Plate: 24 2
Scenario 4 Scenario 4 Clinical History: 44 year old male seen in ER i Source: Ascitis Fluid 25 25
STATPack Images 26 26 Images by NPHL
STATPack Images 27 27 Images by Grand Island
STATPack Images 28 28 Images by Grand Island – “Soft beta colony”
STATPack Images 29 29 Images by Imperial
Organism 4 Trigger Points • Growth on Blood and Chocolate Agar Growth on Blood and Chocolate Agar • No Growth on MacConkey • Gram Stain: Gram Positive Rod Gram Stain: Gram Positive Rod • Slight β hemolysis *** • Tube Catalase Positive • Motility variable • Growth rate 1 ‐ 2 mm colonies at 48 hours • Can Bacillus be ruled out? 30 30
Gram Stain Comparisons Gram Stain Comparisons 31 31 Images by NPHL Images by NPHL
Group Biochemical Results: Did you use any of the following to identify the organism? If yes, please indicate reaction: y y g y g y , p Positive Negative Not Applicable No Response Oxidase (1) (4) (9) 1 Catalase Catalase (15) (15) (0) (0) (0) (0) 0 0 Motility (7) (6) (1) 1 Indole Indole (0) (0) (3) (3) (10) 2 (10) 2 Beta ‐ lactamase (0) (0) (10) 5 Nitrate (0) (1) (8) 6 Satellite (0) (0) (10) 5 Urease (0) (5) (7) 3 32 32
Motility at 25° C Motility at 25 C 33 33
Responses to Organism 4 Responses to Organism 4 NCSE – 04 CS 0 1 Non Bioterrorism Agent 4 Bacillus sp., not B. 10 anthracis Bacillus sp. unable to rule out B. anthracis 34 34
Responses to Organism 4 Responses to Organism 4 • Non BT Agent – Continue with routine Non BT Agent Continue with routine identification • Commercial systems: • Commercial systems: • 2 laboratories used MicroScan = L. monocytogenes 99% L 99% • 3 laboratories used Vitek = L. seeligeri 99% • Rapid kits ?? • Recommend sending to NPHL for 35 35 confirmation
What is the Best Answer for Organism 4? • Non Bioterrorism Agent Non Bioterrorism Agent Actual mimic organism sent: Listeria seeligeri Li t i li i Reference: Spontaneous bacterial peritonitis due to Listeria monocytogenes , Infectious Diseases in Clinical Practice, Volume 17, Number 1, 2009 Listeriosis due to Infection with a Catalase ‐ Negative Strain of Listeria monocytogenes Listeriosis due to Infection with a Catalase Negative Strain of Listeria monocytogenes , 36 36 Journal of Clincial Microbiology, May 2006, p 1917 ‐ 18
Specimen Collection Recommendations CDC Enteric Bacteriology Discussion List SALM ‐ USA@LISTSERV.CDC.GOV CDC Enteric Bacteriology Discussion List SALM USA@LISTSERV.CDC.GOV We are not recommending stool culture for Listeria, especially not for the ‘worried well.’ If a person is symptomatic, then fluid from a normally sterile site, such as blood, should be cultured for Listeria. Stool culture is not sensitive enough (a negative does not rule out carriage) and a positive result can be ambiguous as well i d l i ) d i i l b bi ll because Listeria are very common bacteria that are ubiquitous in the environment so carriage of non ‐ pathogenic variants occurs at a fairly high rate (estimates range from 1 – 15%). ) Kind regards, Cheryl Cheryl L Tarr Ph D Cheryl L. Tarr, Ph.D. Chief, Listeria , Yersinia , Vibrio and Enterobacteriaceae Reference Laboratories 1600 Clifton Road, Mailstop C ‐ 03 National Center for Emerging and Zoonotic Infectious Diseases 37 37 Centers for Disease Control and Prevention Atlanta GA 30329
Nebraska Challenge Set 2012 Nebraska Challenge Set 2012 Automatic Enrollment Automatic Enrollment Dates to avoid: CAP LPX April & September p p CAP D 1 ‐ 9 March & August API ? Dates to schedule 2012 NCSE February & October? May & November? May & November? 38 38
Fall 2011 ROAR Workshops Fall 2011 ROAR Workshops • December 16 December 16 Hastings Hastings • December 19 Norfolk 39 39
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