Test Method Validation

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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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Review of Spring 2007 WLRN Educational Testing
Proficiency Exercise

• May 2, 2007

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WLRN Testing Proficiency Notification/Shipping
Exercises

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

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Spring 2007 WLRN Testing Proficiency Exercise

• 2 samples sent to 118 labs
• 102 reported test results (86)
• 16 did not to participate
• Sample BPE 07-1-1
• Oligella urealytica
• Sample BPE 07-1-2
• Moraxella nonliquefaciens
• Samples intended to simulate 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
Most participants notified WSLH of intended
referral Labs should notify WSLH 24/7 number per
Wisconsin Emergency Response Guide for Clinical
Laboratories protocol. -Found error in answering
service protocol now corrected
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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
Number of participants that used class 6.2
shipper for transport, only required if
classified as Category A
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Emergency Laboratory Response Exercise

• All 18 samples were triple-packaged.
• 15 of 18 used Class 6.2 (Category A) shipper,
  But
• All 18 attached Infectious Substance/UN2814
  label for Category A substances
• Only 11 of laboratories attached a Dangerous
  Goods Form required for Category A substances in
  Commercial transport)
• Only 9 attached an itemized list to the secondary
  container (required for all Category A shipments
  and Category B shipments if transported by air or
  USPS (not if transported by ground)

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Emergency Laboratory Response Exercise

• Only 14 of 18 laboratories attached the name and
  telephone number of a person knowledgeable
  about the shipment to the outer container.
  (required for all Category A and Category B
  shipments)
• Only 15 of 18 laboratories included a
  requisition form with the sample

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Thank you to all the participating laboratories!

• Watch for WSLH audioconference reviewing the
  emergency response protocol in September.

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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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History of Brucella

• 1886---Sir David Bruce isolates Brucella
• Micrococcus melitensis
• Malta fever
• 1895---Bernhard Bang isolates B. abortus
• Bacillus abortus
• Bovine abortion
• 1918---Alice Evans demonstrates close similarity
  of the two organisms
• New genus proposed, Brucella,
• 1929---B. suis described by Huddleston
• Associated with aborted swine
• 1950s---Two additional species described
• B. ovis and B. neotomae
• 1968---B. canis identified as a cause of canine
  abortion

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Brucella species
From Wafa Al-Nassir, emedicine.com/topic248.htm
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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
• Relatively rare in the U.S. with approximately
  120 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. 54, no. 53, 2007
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MMWR vol. 54, no. 53, 2007
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TRANSMISSION
www.gsbs.utmb.edu/microbook/ch028.htm
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TRANSMISSION

• 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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Pathogenesis

• Patterns of illness similar in all forms of
  infection
• Brucella are intracellular organisms
• Survive and multiply within mononuclear
  phagocytes
• Become localized in reticuloendothelial system
  e.g. lymph nodes, liver, spleen, and bone marrow
• Illness reflects distribution of macrophages in
  bone, joints, brain, liver, spleen, and lung
• Abscesses and granulomas
• LPS is the major determinant of virulence

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Clinical ManifestationsAcute onset in 50 of
casesIncubation period of 5-60 days
Anorexia, asthenia,fatigue, weakness,
malaise Abdominal pain, constipation, diarrhea,
vomiting Anxiety, confusion, depression,
insomnia Paralysis, nuchal rigidity,
papilledema
From Wafa Al-Nassir, emedicine.com/topic248.htm
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Treatment

• Mortality 5 in untreated patients
• Usually from CNS infection and endocarditis
• Treatment
• Adults---Combination of doxycycline plus an
  aminoglycoside for 4 weeks followed by
  doxycycline and rifampin for 6 weeks
• Children---TMP-SMX plus rifampin 4-6 weeks

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Diagnosis

• Routine laboratory tests
• WBC---usually normal or depressed
• Anemia
• Thrombocytopenia
• Serology
• Serum agglutination test (SAT) gold standard
• B. abortus 1119 antigen
• Reacts with antibodies to B. melitensis and B.
  suis
• No single serum titer is diagnostic
• Most cases gt1160
• Acute and convalescent serum optimal for
  diagnosis
• Decrease in titer indicates good response to
  therapy
• Increase titer indicates relapse

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Diagnosis

• Culture
• 15-70 isolation rate depending on methods used
  and specimens cultured

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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

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Laboratory Diagnosis of Brucellosis
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J. Infect. Dis. 153 122-125 1986
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CASTEÑADA BOTTLES
Proc. Soc. Exp. Biol. Med. 64 114-115 1947
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RADIOMETRIC
Parameter Recovery Detection
Arnow et al.a 100 4-8 days
Kolman et al.b 60.0 14 days
aJAMA. 251 505-507 1984
bEur. J. Clin. Microbiol. Infect. Dis. 10
647-648 1991
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ISOLATOR
Parameter Recovery Detection
Navas et al.a 100.0 3.1 days
Kolman et al.b 55.0 3.5 days
aDiagn. Microbiol. Infect. Dis. 16 79-81 1993
bEur. J. Clin. Microbiol. Infect. Dis. 10
647-648 1991
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NON-RADIOMETRIC -- I
Parameter Recovery Detection
Navas et al.a 85.7 20.6 days
Yagupskyb 78.8 7 days
aDiagn. Microbiol. Infect. Dis. 16 79-81 1993
bJ. Clin. Microbiol. 32 1899-1901 1994
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NON-RADIOMETRIC -- II
Parameter Recovery Detection
Yagupsky et al.a 95.0 3 days
Yagupskyb 78.8 7 days
aJ. Clin. Microbiol. 35 1382-1384 1997
bJ. Clin. Microbiol. 32 1899-1901 1994
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CONTINUOUS vs. ISOLATOR
J. Clin. Microbiol. 35 1382-1384 1997
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MEDIA
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CELLULAR MORPHOLOGY
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COLONY MORPHOLOGY
Slow growing resembles inoculum _at_ 24 hours
Tiny, white, convex, non-hemolytic, moist,
glistening at 48 hours
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CONFUSION
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MISIDENTIFICATION OF Brucella
Moraxella phenylpyruvica (API 20NE)
Haemophilus influenzae biotype IV (MicroScan HNID)
Clin. Infect. Dis. 17 1068-1069 1993
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BENCH-TOP/HOOD-TOP REAGENTS
Unable to rule out Brucella species refer to
state public health laboratory for confirmation
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LABORATORY ACQUISITION
Biosafety in Microbiological and Biomedical
Laboratories, 5th ed.
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PRACTICES
Biosafety Level (BSL)-2 Standard microbiology
practice Limited access Biohazard warning
signs Sharps precautions Biosafety manual
defining decontamination, surveillance
BSL-3 BSL-2 practice Controlled
access Decontamination of all waste Baseline
serum Decontamination of laboratory clothing
before laundering
Biosafety in Microbiological and Biomedical
Laboratories, 5th ed.
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1 BARRIERS SAFETY EQUIPMENT
BSL-2 Class I or II biological safety cabinet
for manipulations of agents that cause splashes
or aerosols of infectious materials Laboratory
coats gloves face protection as needed
BSL-3 Class I or II biological safety cabinet
for all open manipulations of agents Protective
laboratory clothing gloves respiratory protectio
n as needed
Biosafety in Microbiological and Biomedical
Laboratories, 5th ed.
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FACILITIES (2 BARRIERS)
BSL-2 Open bench, sink required Autoclave
available
BSL-3 BSL-2 facilities Physical separation
from access corridors Self-closing, double-door
access Exhaust air not recirculated Negative
airflow into laboratory
Biosafety in Microbiological and Biomedical
Laboratories, 5th ed.
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SUSCEPTIBILITY TESTING
MIC method
BSL-3 practices
Gentamicin Tetracycline Streptomycin Doxycycline
Trimethoprim-sulfamethoxazole
? aminoglycoside, ? tetracycline MIC
for capnophilic strains
CLSI. M100-S17 136-137 2007
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SEROLOGY
Standard tube agglutination (serum agglutination
test)
Standardized stable commercial B. abortus smooth
antigen Untreated dilutions of patient sera
2-mercaptoethanol Incubate 48 hours in 37C
water bath Endpoint is highest dilution with
complete clumping and resultant clearing of fluid
Am. J. Clin. Pathol. 24 496-498 1954
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SEROLOGY
Standard tube agglutination (serum agglutination
test)
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SEROLOGY
Standard tube agglutination (serum agglutination
test)
-- Cross-reactivity Francisella
tularensis Vibrio cholerae Yersinia
enterocolitica -- Rough B. canis antigen does
not precipitate with agglutinins from smooth
organisms
Rev. Infect. Dis. 13 359-372 1991
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SEROLOGY
Standard tube agglutination (serum agglutination
test)
Enzyme immunoassay
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BRUCELLA ENZYME IMMUNOASSAY
Clin. Infect. Dis. 14 131-140 1992
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BRUCELLA ENZYME IMMUNOASSAY
89 persistently IgG
69 persistently IgA
26 persistently STA
25 persistently IgM
Clin. Infect. Dis. 14 131-140 1992
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SEROLOGY
Standard tube agglutination (serum agglutination
test)
Enzyme immunoassay
Coombs test
Useful in diagnosis of chronic brucellosis Signs
and symptoms are vague and indefinite
Lancet. ii 1181-1183 1966
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SEROLOGY
Standard tube agglutination (serum agglutination
test)
Enzyme immunoassay
Coombs test
Rose Bengal test
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ROSE BENGAL TEST
Rapid screening test performed on card or tile
Antigen is rose bengal-stained suspension of
smooth attenuated strain of B. abortus
Sometimes confirmed with complement fixation
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SEROLOGY
Standard tube agglutination (serum agglutination
test)
Enzyme immunoassay
Coombs test
Rose Bengal test
Recent advances
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18 kD CYTOPLASMIC ANTIGEN
J. Clin. Microbiol. 31 2141-2145 1993
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DIPSTICK SEROLOGY
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DIRECT DETECTION
Microscopy not sufficiently sensitive
No laboratory protocol for DFA
Molecular (back to Dr. Warshauer)
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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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Select Agent Reporting

• Contact CDC by telephone
• 404-498-2255
• Within 7 days of identification
• Transfer to a registered entity
• Destroy---Autoclave, incinerate, bleach
• Documentation
• CDC Form 4 (www.cdc.gov/od/sap)
• Maintain a copy of the record for 3 years

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