Nebraska Challenge Nebraska Challenge Set Exercise Set Exercise - - PowerPoint PPT Presentation

Nebraska Challenge Nebraska Challenge Set Exercise Set Exercise

November 2011 November, 2011

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Continuing Education

Fax to 402.559.7838 Email to kstiles@unmc.edu

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Resources recommended for Challenge:

ASM ASM Sentinel Guidelines

Link found on

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• www.nphl.org

BIOLOGICAL TERRORISM PROCEDURES

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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

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Scenario 3 Scenario 3

Clinical History: 17 year old male Source: Blood 2 of 2 bottles positive

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Gram Stain Morphology

7 Images by Fremont 7

Gram Stain Morphology

8 Images by North Platte 8

Gram Stain Morphology

9 Images by North Platte 9

Colony Morphology

Growth on SBA Choc MacConkey Growth on SBA, Choc, MacConkey

10 Images by Imperial, Fremont, Bellevue 10

NPHL Bench Guide NPHL Bench Guide

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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

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• Not Burkholderia – Oxidase negative

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NPHL Bench Guide NPHL Bench Guide

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Group Biochemical Results:

Did you use any of the following to identify the organism? If yes, please indicate reaction:

Oxidase Catalase (3) (12) (10) (1) (0) 1 (1) 1

y y g y g y , p

Positive Negative Not Applicable No Response

Catalase Indole Motility (12) (0) (0) (1) (11) (7) (1) 1 (1) 1 (5) 3 Motility Nitrate Satellite (0) (0) (0) (7) (1) (1) (5) 3 (9) 5 (9) 5 Urease (7) (6) (1) 1 14 14

Urease at 24 hours

15 Images by Fremont 15

Responses to Organism 3 Responses to Organism 3

NCSE ‐ 03 CS 03

6 4 1 Yersinia spp, unable to rule of Yersinia pestis Yersinia spp., not 3 Yersinia pestis Non Bioterrorism Agent 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

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www.cdc.gov/eid , Vol. 14, No. 6, June 2008

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Are there any trigger points?

• Oxidase (‐)

y gg p

Oxidase ( )

• Glucose (+)
• Fits Enterobacteriaceae pattern

Fits Enterobacteriaceae pattern

• Without patient history hard to create a

differential

• Typically tested on commercial ID systems

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Yersinia pestis – Commercial ID Systems

• Included in the data base of MicroScan, Vitek,

, , and API 20E.. etc.

• True accuracy not yet determined

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Yersinia pestis – Commercial p ID Systems

• If ID as Y. pestis – send to NPHL
• May ID as Shigella ssp, H2S‐negative

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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%

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• 2 Vitek users reported = Yersinia pseudotuberculosis 96‐99%
• 1 API user reported = Yersinia pseudotuberculosis 95%

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Yersinia pestis –Commercial 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 2nd methodology – API or
• Confirm by 2

methodology – API or conventional biochemicals

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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

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http://www.ph.ucla.edu/epi/bioter/plagueapha_id.html

• Not predictable

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Yersinia images by CDC Growth 24 hr @ 37° Growth 24 hr @ 25° 23 Yersinia pestis at 72 hr. 23 Tenacious when touched by inoculation loop – 48 hours

Yersinia pestis ‐ Direct Culture Growth on Agar Plate:

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Scenario 4 Scenario 4

Clinical History: 44 year old male i seen in ER Source: Ascitis Fluid

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STATPack Images

26 Images by NPHL 26

STATPack Images

27 Images by Grand Island 27

STATPack Images

28 Images by Grand Island – “Soft beta colony” 28

STATPack Images

29 Images by Imperial 29

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?

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Gram Stain Comparisons Gram Stain Comparisons

31 Images by NPHL 31 Images by NPHL

Group Biochemical Results:

Did you use any of the following to identify the organism? If yes, please indicate reaction:

Oxidase Catalase (1) (15) (4) (0) (9) 1 (0)

y y g y g y , p

Positive Negative Not Applicable No Response

Catalase Motility Indole (15) (7) (0) (0) (6) (3) (0) (1) 1 (10) 2 Indole Beta‐lactamase Nitrate (0) (0) (0) (3) (0) (1) (10) 2 (10) 5 (8) 6 Satellite Urease (0) (0) (0) (5) (10) 5 (7) 3 32 32

Motility at 25° C Motility at 25 C

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Responses to Organism 4 Responses to Organism 4

NCSE – 04 CS

4 1 Non Bioterrorism Agent 10 Bacillus sp., not B. 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 99%

• L. monocytogenes 99%
• 3 laboratories used Vitek = L. seeligeri 99%

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• Rapid kits ??
• Recommend sending to NPHL for

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confirmation

What is the Best Answer for Organism 4?

• Non Bioterrorism Agent

Non Bioterrorism Agent Actual mimic organism sent: Li t i li i Listeria seeligeri

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

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Listeriosis due to Infection with a Catalase Negative Strain of Listeria monocytogenes, Journal of Clincial Microbiology, May 2006, p 1917‐18

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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 i d l i ) d i i l b bi ll negative does not rule out carriage) and a positive result can be ambiguous as well 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 37 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 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?

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• May & November?

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Fall 2011 ROAR Workshops Fall 2011 ROAR Workshops

• December 16

Hastings December 16 Hastings

• December 19

Norfolk

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APHL On Demand Course

Bi t i P d T i i f LRNS ti l 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) t l f bi t i t id tifi ti Th d l i l d 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. U d t d f 2010 d l il bl i l di Updated for 2010, seven course modules are available, including:

• ∙ Anthrax (Bacillus anthracis)
• ∙ Brucella (Brucella spp)
• ∙ Burkholderia (Burkholderia spp)
• ∙ Burkholderia (Burkholderia spp)
• ∙ Plague (Yersinia pestis)
• ∙ Tularemia (Francisella tularensis)
• ∙ Laboratory Biosecurity

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Laboratory Biosecurity

• ∙ The Laboratory’s Role in Bioterrorism Preparedness (Laboratory

Response Network, LRN)

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STATPack Training – 3 CEU’s STATPack Training 3 CEU s

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http://training.statpack.org

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Changes to Packaging & Shipping Changes to Packaging & Shipping

• Watch for updates on NPHL packaging & shipping

Watch for updates on NPHL packaging & shipping

• New documentation requirements for couriers
• Will affect how you send:
• Shiga Toxin positive
• Shiga Toxin positive
• MTB
• Coccidiodes immitis

Coccidiodes immitis

• ROAR isolates (case by case basis)

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Questions??

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