bioterrorism agents – plague lesson 4 laboratory

Bioterrorism Agents – PlagueBioterrorism Agents – PlagueLesson 4Lesson 4

LaboratoryLaboratory

ObjectivesObjectives

• List appropriate List appropriate specimensspecimens

• List tests available for List tests available for diagnosisdiagnosis

• List laboratory criteria for List laboratory criteria for diagnosisdiagnosis

• Describe safety Describe safety proceduresprocedures

OverviewOverview

• Critical for first casesCritical for first cases

• Collect immediately and before antibioticsCollect immediately and before antibiotics

• Store appropriatelyStore appropriately

• Presumptive diagnosesPresumptive diagnoses

SpecimensSpecimensRespiratoryRespiratory

• Pharyngeal swabsPharyngeal swabs

• Tracheal washes or aspiratesTracheal washes or aspirates

• Sputum specimensSputum specimens

• Trans-thoracic lung aspiratesTrans-thoracic lung aspirates

• Pleural fluid collectionPleural fluid collection

• Testing – Staining, culture, DNA Testing – Staining, culture, DNA amplificationamplification

SpecimensSpecimensTissuesTissues

• Lung tissueLung tissue

• Lymph node tissueLymph node tissue

• SpleenSpleen

• LiverLiver

• Testing – Culture, Gram stainTesting – Culture, Gram stain

SpecimensSpecimensTissues (Autopsy)Tissues (Autopsy)

• LungLung• LiverLiver• SpleenSpleen• Lymph nodesLymph nodes• Skin lesionsSkin lesions• KidneyKidney• Testing – PCR (Specimens kept fresh Testing – PCR (Specimens kept fresh

frozen, unpreserved)frozen, unpreserved)

SpecimensSpecimensBloodBlood

• Baseline before antibioticsBaseline before antibiotics

• ConvalescentConvalescent– Minimum 14 days apartMinimum 14 days apart– 3 to 4 weeks after symptom onset3 to 4 weeks after symptom onset

• Two or more sequentially collected Two or more sequentially collected samples preferredsamples preferred

Specimen HandlingSpecimen HandlingRespiratory, BloodRespiratory, Blood

• Respiratory/SputumRespiratory/Sputum– Sterile screw-capped containersSterile screw-capped containers– Room temperature (store 2Room temperature (store 2C-C-

88C if 2-24 hrs)C if 2-24 hrs)

• BloodBlood– Direct to lab at room Direct to lab at room

temperaturetemperature– Hold at ambient until incubatorHold at ambient until incubator– Do not refrigerateDo not refrigerate

Specimen HandlingSpecimen HandlingTissueTissue

• Sterile containerSterile container

• 1 to 2 drops normal saline1 to 2 drops normal saline

• Transport at room temperatureTransport at room temperature

• Chill if delayChill if delay

Contact public health for detailed Contact public health for detailed instructionsinstructions

SpecimensSpecimensTissue HandlingTissue Handling

• Split into non-glass Split into non-glass containerscontainers– 44C for live recoveryC for live recovery– -20-20C or -70C or -70C for rapid C for rapid

molecular testingmolecular testing

• Formalin-fixedFormalin-fixed– Tests – Histopathology, HC, Tests – Histopathology, HC,

PCRPCR– NOT frozenNOT frozen– Packaged separately from Packaged separately from

unpreservedunpreserved

Laboratory TestingLaboratory TestingPneumonic PlaguePneumonic Plague

• Collect before initiating Collect before initiating therapytherapy

• Sputum, blood, lymph node Sputum, blood, lymph node aspirateaspirate

• Gram Stain and cultureGram Stain and culture– Gram-negative coccobacillusGram-negative coccobacillus– Bipolar (safety pin) stainingBipolar (safety pin) staining

• Acute and convalescent Acute and convalescent bloods for public healthbloods for public health


Leukocyte CountLeukocyte Count ElevatedElevated

DifferentialDifferential Neutrophil Neutrophil predominance, including predominance, including immature formsimmature forms

PlateletsPlatelets May be normal or lowMay be normal or low

WBCsWBCs 15,000-25,000/µl, left 15,000-25,000/µl, left shiftshift

AST, ALT, bilirubinAST, ALT, bilirubin ElevatedElevated


• Coagulation abnormalities Coagulation abnormalities

• Fibrin split productsFibrin split products

• DIC possibleDIC possible

• Increased fibrin degradation productsIncreased fibrin degradation products

• HypofibrinogenemiaHypofibrinogenemia

• Prolongation of PT and PTTProlongation of PT and PTT

Laboratory Case ClassificationLaboratory Case ClassificationSuspectSuspect

DefinitionDefinition• Clinical sx and exposure compatible ANDClinical sx and exposure compatible AND• Small, Gram-negative and/or bipolar-Small, Gram-negative and/or bipolar-

staining, plump rods in singles, short staining, plump rods in singles, short chains, catalase (+) oxidase and urease (-)chains, catalase (+) oxidase and urease (-)

Lab Tests – Gram stain, differential stain, Lab Tests – Gram stain, differential stain, culture, catalase, oxidase ureaseculture, catalase, oxidase urease

Laboratory Case ClassificationLaboratory Case ClassificationProbableProbable

DefinitionDefinition• Clinical sx and exposure Clinical sx and exposure

compatible ANDcompatible AND• Specimen/Isolate positive by Specimen/Isolate positive by

one of: DFA, PCR, IHCone of: DFA, PCR, IHC• OR single PHA/HI titer of OR single PHA/HI titer of 1:10 1:10

or positive ELISAor positive ELISA

Lab Tests – DFA, DNA, IHC, Lab Tests – DFA, DNA, IHC, SerologySerology

Laboratory Case ClassificationLaboratory Case ClassificationConfirmedConfirmed

DefinitionDefinition

• Positive DFA or DNA detection and Positive DFA or DNA detection and bacteriophage lysis of recovered isolatebacteriophage lysis of recovered isolate

• OR Paired sera 4-fold rise or fall by OR Paired sera 4-fold rise or fall by PHA/HI or ELISAPHA/HI or ELISA

Tests – Bacteriophage lysis, Serology, IHCTests – Bacteriophage lysis, Serology, IHC

Review Questions - PlagueReview Questions - Plague

Lesson 4Lesson 4

Plague Review QuestionPlague Review QuestionLesson 4, Question 1Lesson 4, Question 1

• Suspect pneumonic plagueSuspect pneumonic plague

• Sending for labs before antibioticsSending for labs before antibiotics

Which of the following is best for organism Which of the following is best for organism identification?identification?



A.A. Obtain initial blood specimenObtain initial blood specimen

B.B. Obtain more than one blood specimenObtain more than one blood specimen

C.C. Get plasma or whole blood specimen on Get plasma or whole blood specimen on filter paperfilter paper

D.D. A smear or isolate from affected tissuesA smear or isolate from affected tissues



D. A smear or isolate from affected tissuesD. A smear or isolate from affected tissues


• Suspect pneumococcal plague patientSuspect pneumococcal plague patient

• Sputum sampleSputum sample

What tests would your laboratory perform?What tests would your laboratory perform?



A.A. PCRPCR

B.B. Gram stain and cultureGram stain and culture

C.C. IHCIHC



B. Gram stain and cultureB. Gram stain and culture


• Lab unable to rule out Lab unable to rule out Y. pestisY. pestis

• Specimens sent to public healthSpecimens sent to public health

• Patient now suspect casePatient now suspect case

At what point would public health rule them At what point would public health rule them a confirmed case?a confirmed case?



A.A. Positive by DFA StainingPositive by DFA Staining

B.B. Positive by PCRPositive by PCR

C.C. Isolate lysed at 22Isolate lysed at 22C-25C-25C and 37C and 37CCD.D. Single PHA/HI titer <1:10Single PHA/HI titer <1:10



C. Isolate lysed at 22C. Isolate lysed at 22C-25C-25C and 37C and 37CC


• About to submit blood specimensAbout to submit blood specimens

• Possible Possible Y. pestisY. pestis

What should you do first?What should you do first?

Plague Review QuestionPlague Review QuestionLesson 4, Question 4Lesson 4, Question 4What should you do first?What should you do first?

A.A. Treat it as you would othersTreat it as you would others

B.B. Do not refrigerateDo not refrigerate

C.C. Contact public health epidemiologyContact public health epidemiology

D.D. Contact public health laboratoryContact public health laboratory

Plague Review QuestionPlague Review QuestionLesson 4, Question 4Lesson 4, Question 4What should you do first?What should you do first?

D. Contact public health laboratoryD. Contact public health laboratory

bioterrorism agents – plague lesson 4 laboratory

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laboratory slide

unpreserved slide

gram stain slide

public health slide

specimens tissue handling

bilirubin elevated slide

dna amplification slide

specimens blood baseline