Review of Autumn 2006 WLRN Educational Proficiency Testing Exercise

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Review of Autumn 2006 WLRN Educational
Proficiency Testing Exercise

• November 8, 2006
• Carol Kirk
• Laboratory Network Coordinator
• Wisconsin State Laboratory of Hygiene
• Phone [phone]
• Email cjk_at_mail.slh.wisc.edu

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WLRN Proficiency Testing Notification/Shipping
Exercises

• Exercises provided twice a year subset of
  laboratories follow notification/shipping
  protocol
• Each exercise is followed by an audioconference
• Participation in exercises is voluntary, not
  connected to any regulatory agencies
• Individual results are not shared with anyone
  other than the participating laboratory
• Purpose of exercises
• Practice rule out testing
• Identify gaps in emergency lab response system
• Practice assess notification transport.

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Autumn 2006 WLRN Educational Proficiency Testing
Exercise

• 2 samples sent to 135 labs
• 107 reported test results (79)
• 15 chose not to participate
• 13 did not respond
• Sample BPE 06-2-1
• Acinetobacter baumannii
• Sample BPE 06-2-2
• Haemophilus influenzae
• Samples intended to simulate Francisella
  tularensis and Brucella spp. in rule out testing

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Results of Rule out Tests
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Results of Rule out Tests (cont.)
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Results of Rule out Tests
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Results of Rule out Tests
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Results of Rule out Tests
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Results of Rule out Tests
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Emergency Laboratory Response Exercise
Combining with PT seems to cause problem with
return
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Emergency Laboratory Response Exercise
Of participants each year - most notified WSLH
of intended referral
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Emergency Laboratory Response Exercise
Most participants each year used Dunham Express
for transport to WSLH
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Emergency Laboratory Response Exercise
Time from 24/7 notification to receipt of
sample 2004 15 notified 24/7 2005 20
notified 24/7 2006-1 12 notified 24/7 2006-2
11 notified 24/7
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Emergency Laboratory Response Exercise
Number of participants that used class 6.2
shipper for commercial or USPS transport.
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Emergency Laboratory Response Exercise
Number of participants that completely sealed
primary container for transport.
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Emergency Laboratory Response Exercise
Number of participants that addressed the package
to WSLH Rapid/Emergency Response
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Emergency Laboratory Response Exercise
Correct Address Label (per WERG
instructions) Rapid Response
BT/CT/Outbreak Wisconsin State Laboratory of
Hygiene -Communicable Diseases 465 Henry
Mall Madison, WI 53706 - Circle BT on the
label if bioterrorism-related, - Circle CT
on the label if chemical terrorism-related, -
Circle Outbreak on the label if
outbreak-related.
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Emergency Laboratory Response Exercise

• LHD Shipper Repository Use
• 4 labs used a shipper from a local health
  department (LHD) shipper repository
• Still concerns about after-hours access and ease
  of access.
• WSLH will provide emergency shippers to
  laboratories that indicated they want them on the
  survey.

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Thank you to all the participating laboratories!
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Dave Warshauer, Ph.D., D(ABMM) Deputy
Director Communicable Disease Division WSLH Phone
[phone] E-mail warshadm_at_slh.wisc.edu
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REMIND/REFRESH/THINK
Brucella spp. Francisella tularensis Bacillus
anthracis Yersinia pestis Others
RARE ISOLATES IN THE CLINICAL LABORATORY
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BRUCELLOSIS

• A zoonotic disease caused by any of 4 Brucella
  sp. abortus, melitensis, suis, and canis
• B. melitensis most common
• A systemic infection characterized by an undulant
  fever pattern
• But relatively rare in the U.S. with
  approximately 100 cases/yr

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• Brucellosis in the U.S.,1930-1990
• Public Health Success Story

1. National elimination program started
2. Strain 19 vaccine for cattle
3. Mandatory herd testing
4. Revitalization
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MMWR vol. 53, no. 53, 2006
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MMWR vol. 53, no. 53, 2006
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BRUCELLOSISTRANSMISSION

• Unpasteurized dairy products
• The most common mode of transmission
• Direct skin contact
• Occupational hazard for farmers, butchers,
  veterinarians, and laboratory personnel
• Aerosols
• Highly infectious (Infective Dose 10-100 organisms

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Infectious Dose
Bacteria Dose Route of Inoculation F.
tularensis 10 inhalation C.
burnetii 10 inhalation M.
tuberculosis lt10 inhalation Brucella
spp. 10-100 inhalation S. typhi 105
ingestion F. tularensis 108
ingestion
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Clinical Manifestations

• Fever
• Chills
• Weight loss
• Night sweats

• Headache
• Muscle aches
• Fatigue
• depression

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Brucella spp.Specimen Selection

• Blood or bone marrow
• Sources from which Brucellae are most often
  isolated
• Tissue (spleen, liver)
• Brucellae occasionally isolated
• Serum
• The diagnosis of brucellosis is frequently
  achieved by serology. An acute convalescent
  phase specimen should be collected (21d apart)

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REVIEW OF THE WLRNBIOTERRORISM
PROFICIECYEXERCISE
November 8, 2006
Rule Out or Refer - Brucella spp.
- Franciscella tularensis
Raymond P. Podzorski, Ph.D., D(ABMM) Clinical
Microbiologist Department of Pathology Oconomowoc
Memorial Hospital Waukesha Memorial Hospital
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Culture Brucella spp.
Blood Incubate for 10 days in automated systems
and perform terminal subcultures at 7 days (hold
plates for 7 days, seal plates) Blood Incubate
for 21 days in non-automated broth blood Cultures
with blind subcultures every 7 days (hold plates
for 7 days, seal plates) Tissue inoculate BAP,
Choc, and MacConkey (EMB) and incubate for 7
days at 35? C with 5-10 CO2, in humidity (seal
plates)
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Growth Characteristics Brucella spp.
Sheep Blood Agar, 35? C 24 Hours - colonies very
small, haze in area of original
inoculum 48 Hours - colonies are tiny, white,
convex, non-hemolytic, moist, and
glistening Grows on Chocolate agar, Martin-Lewis
agar, Thayer- Martin agar, BCYE, and some strains
grow on MacConkey agar
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Brucella spp. on Sheep Blood Agar at 48 Hours
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Brucella spp. Gram Stain
fine sand appearance on Gram stain
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Brucella spp. Handling
Working with Suspected Specimens Do all work
with specimen in a hood using BSL-2
practice Culture plates should be
sealed Working with Suspected Isolates Do all
work with the culture in a hood using BSL-3
practice Do not attempt to set up automated or
semi-automated ID You dont want to be
sniffing the plates Culture plates should be
sealed
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Screening Colonies for Brucella spp.
Gram stain - tiny gram negative (fine sand)
coccobacilli Growth sheep blood agar (poor),
chocolate agar X V Factors not required for
growth on SBA Oxidase Positive Catalase
Positive Urease Positive
Cannot rule out Brucella spp. CONTACT WSLH OR
REFER TO LRN REFERENCE LABORATORY
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LRN Reference Laboratory Tests for Brucella spp.
Conventional culture and biochemical
identification PCR based identification Time-res
olved fluorescence Tbilisi phage susceptibility
test Slide agglutination Serological testing of
the suspected patient
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Francisella tularensis
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Reported Cases of Tularemia-1990-1998
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MMWR vol. 53, no. 53, 2006
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Francisella tularensis---Epidemiology

• Summer peak
• June - August
• December peak
• Occupational risks
• Laboratory workers
• farmers
• veterinarians
• hunters
• meat handlers
• 75 male

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Clinical Manifestations

• Ulceroglandular 70-80
• Papule developing into a tender ulcer
• Localized lymphadenopathy
• Glandular 3-20
• No ulcer present
• Oculoglandular 1
• Oropharyngeal 0-12
• Pneumonic 7-20
• Typhoidal (Septicemic) 5-30

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Ulceroglandular Disease
Ulcer on scalp
Hand Ulcer
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Transmission

• Handling carcasses or skin of infected animals
• Rodents,
• Rabbits
• Beavers and muskrats
• Insect vectors
• Ticks
• Deerflies
• Waterborne

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F. tularensis
F. tularensis most common (F.
philomiragia) Specimen exposure/risk skin
lesion exudates, respiratory
secretions, CSF, tissue Have been identified as
Haemophilus influenzae Actinobacillus
spp. Pasteurella multocida
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Culture F. tularensis
Serological testing is the most common method for
diagnosing Tularemia. Media most strains
require cysteine or cystine and the preferred
media was glucose-cysteine blood agar. Skin
lesions, respiratory specimens, tissue
inoculate BAP, Chocolate, and MacConkey
(EMB) Incubate for 7 days at 35? C with 5-10
CO2, in humidity (seal plates)
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Growth CharacteristicsFranciscella tularensis
Sheep Blood Agar - 35? C, fastidious, grows
poorly or not at all, if it does grow
on SBA, small colonies, gray-white,
non- or ?-hemolytic may grow
initially on SBA, but fail to grow on
a SBA subculture Chocolate Agar - 24-48 hours,
if growth, colonies very small, haze in
area of original inoculum - 72 hours,
colonies are 1-2 mm, blue-gray to, light
gray, flat, smooth, and shiny
Grows on Thayer-Martin agar, BCYE
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Francisella tularensis on sheep blood agar at 48
hours
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Franciscella tularensis on chocolate agar at 72
hours
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Gram stain of Francisella tularensis
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F. Tularensis Handling
Working with Suspected Specimens Do all work
with specimen in a hood using BSL-2
practice Plates should be sealed Working with
Suspected Isolates Do all work with the culture
in a hood using BSL-3 practice Do not attempt to
set up automated or semi-automated ID You dont
want to be sniffing the plates Plates should
be sealed
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Screening Colonies for F. tularensis
Gram stain tiny, faintly staining, gram
negative coccobacilli Growth
sheep blood agar (no growth or poor growth)
chocolate agar, BCYE, cysteine-supplemented
agar after 48 to 72 hours X V
Factors not required for growth on SBA Oxidase
Negative Catalase Weak Positive Urease
Negative
Cannot rule out F. tularensis CONTACT WSLH OR
REFER TO LRN REFERENCE LABORATORY
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LRN Reference Laboratory Tests for F. tularensis
Conventional culture and biochemical
identification PCR based identification Time-res
olved fluorescence Direct fluorescent antibody
testing of isolate Slide agglutination Serologic
al testing of the suspected patient
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Organism Test catalase
oxidase urea XV gram stain F.
tularensis (w) - -
- tiny ccb Brucella spp.
- tiny ccb Pasteurella
spp. v -
rod, ccb Acinetobacter sp. -
v - fat ccb Psychrobacter phenyl
pyruvicus -
fat ccb Actinobacillus spp. v
- rod, ccb Oligella
sp. -
tiny ccb Bordetella bronchiseptica
- rod,
ccb Haemophilus influenzae v
v ccb
wweak, ccbcoccobacilli, vvariable
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Brucella/Franciscella
KEEP THESE ORGANISMS IN MIND VERY UNCOMMON IN
WISCONSIN THREE IN ONE WEEK? SOMETHING IS
UP RULE OUT OR REFER DO NOT WORK UP THESE
ORGANISMS