Bioterrorism Agents

1
Bioterrorism Agents PlagueLesson 4

• Laboratory

2
Objectives

• List appropriate specimens
• List tests available for diagnosis
• List laboratory criteria for diagnosis
• Describe safety procedures

3
Overview

• Critical for first cases
• Collect immediately and before antibiotics
• Store appropriately
• Presumptive diagnoses

4
SpecimensRespiratory

• Pharyngeal swabs
• Tracheal washes or aspirates
• Sputum specimens
• Trans-thoracic lung aspirates
• Pleural fluid collection
• Testing Staining, culture, DNA amplification

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SpecimensTissues

• Lung tissue
• Lymph node tissue
• Spleen
• Liver
• Testing Culture, Gram stain

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SpecimensTissues (Autopsy)

• Lung
• Liver
• Spleen
• Lymph nodes
• Skin lesions
• Kidney
• Testing PCR (Specimens kept fresh frozen,
  unpreserved)

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SpecimensBlood

• Baseline before antibiotics
• Convalescent
• Minimum 14 days apart
• 3 to 4 weeks after symptom onset
• Two or more sequentially collected samples
  preferred

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Specimen HandlingRespiratory, Blood

• Respiratory/Sputum
• Sterile screw-capped containers
• Room temperature (store 2?C-8?C if 2-24 hrs)
• Blood
• Direct to lab at room temperature
• Hold at ambient until incubator
• Do not refrigerate

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Specimen HandlingTissue

• Sterile container
• 1 to 2 drops normal saline
• Transport at room temperature
• Chill if delay
• Contact public health for detailed instructions

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SpecimensTissue Handling

• Split into non-glass containers
• 4?C for live recovery
• -20?C or -70?C for rapid molecular testing
• Formalin-fixed
• Tests Histopathology, HC, PCR
• NOT frozen
• Packaged separately from unpreserved

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Laboratory TestingPneumonic Plague

• Collect before initiating therapy
• Sputum, blood, lymph node aspirate
• Gram Stain and culture
• Gram-negative coccobacillus
• Bipolar (safety pin) staining
• Acute and convalescent bloods for public health

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Laboratory TestingPneumonic Plague
Leukocyte Count Elevated
Differential Neutrophil predominance, including immature forms
Platelets May be normal or low
WBCs 15,000-25,000/µl, left shift
AST, ALT, bilirubin Elevated
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Laboratory TestingPneumonic Plague

• Coagulation abnormalities
• Fibrin split products
• DIC possible
• Increased fibrin degradation products
• Hypofibrinogenemia
• Prolongation of PT and PTT

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Laboratory Case ClassificationSuspect

• Definition
• Clinical sx and exposure compatible AND
• Small, Gram-negative and/or bipolar-staining,
  plump rods in singles, short chains, catalase ()
  oxidase and urease (-)
• Lab Tests Gram stain, differential stain,
  culture, catalase, oxidase urease

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Laboratory Case ClassificationProbable

• Definition
• Clinical sx and exposure compatible AND
• Specimen/Isolate positive by one of DFA, PCR,
  IHC
• OR single PHA/HI titer of ?110 or positive ELISA
• Lab Tests DFA, DNA, IHC,
• Serology

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Laboratory Case ClassificationConfirmed

• Definition
• Positive DFA or DNA detection and bacteriophage
  lysis of recovered isolate
• OR Paired sera 4-fold rise or fall by PHA/HI or
  ELISA
• Tests Bacteriophage lysis, Serology, IHC

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Review Questions - Plague

• Lesson 4

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Plague Review QuestionLesson 4, Question 1

• Suspect pneumonic plague
• Sending for labs before antibiotics
• Which of the following is best for organism
  identification?

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Plague Review QuestionLesson 4, Question 1

• Which of the following is best for organism
  identification?
• Obtain initial blood specimen
• Obtain more than one blood specimen
• Get plasma or whole blood specimen on filter
  paper
• A smear or isolate from affected tissues

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Plague Review QuestionLesson 4, Question 1

• Which of the following is best for organism
  identification?
• D. A smear or isolate from affected tissues

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Plague Review QuestionLesson 4, Question 2

• Suspect pneumococcal plague patient
• Sputum sample
• What tests would your laboratory perform?

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Plague Review QuestionLesson 4, Question 2

• What tests would your laboratory perform?
• PCR
• Gram stain and culture
• IHC

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Plague Review QuestionLesson 4, Question 2

• What tests would your laboratory perform?
• B. Gram stain and culture

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Plague Review QuestionLesson 4, Question 3

• Lab unable to rule out Y. pestis
• Specimens sent to public health
• Patient now suspect case
• At what point would public health rule them a
  confirmed case?

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Plague Review QuestionLesson 4, Question 3

• At what point would public health rule them a
  confirmed case?
• Positive by DFA Staining
• Positive by PCR
• Isolate lysed at 22?C-25?C and 37?C
• Single PHA/HI titer lt110

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Plague Review QuestionLesson 4, Question 3

• At what point would public health rule them a
  confirmed case?
• C. Isolate lysed at 22?C-25?C and 37?C

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Plague Review QuestionLesson 4, Question 4

• About to submit blood specimens
• Possible Y. pestis
• What should you do first?

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Plague Review QuestionLesson 4, Question 4

• What should you do first?
• Treat it as you would others
• Do not refrigerate
• Contact public health epidemiology
• Contact public health laboratory

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Plague Review QuestionLesson 4, Question 4

• What should you do first?
• D. Contact public health laboratory