1. Bioterrorism Agents – Plague Lesson 4 Laboratory

2. Objectives List appropriate specimensList appropriate specimens List tests available for diagnosisList tests available for diagnosis List laboratory criteria for diagnosisList laboratory criteria for diagnosis Describe safety proceduresDescribe safety procedures

3. Overview Critical for first casesCritical for first cases Collect immediately and before antibioticsCollect immediately and before antibiotics Store appropriatelyStore appropriately Presumptive diagnosesPresumptive diagnoses

4. Specimens Respiratory 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 amplificationTesting – Staining, culture, DNA amplification

5. Specimens Tissues Lung tissueLung tissue Lymph node tissueLymph node tissue SpleenSpleen LiverLiver Testing – Culture, Gram stainTesting – Culture, Gram stain

6. Specimens Tissues (Autopsy) LungLung LiverLiver SpleenSpleen Lymph nodesLymph nodes Skin lesionsSkin lesions KidneyKidney Testing – PCR (Specimens kept fresh frozen, unpreserved)Testing – PCR (Specimens kept fresh frozen, unpreserved)

7. Specimens Blood Baseline before antibioticsBaseline before antibiotics ConvalescentConvalescent –Minimum 14 days apart –3 to 4 weeks after symptom onset Two or more sequentially collected samples preferredTwo or more sequentially collected samples preferred

8. Specimen Handling Respiratory, Blood Respiratory/SputumRespiratory/Sputum –Sterile screw-capped containers –Room temperature (store 2  C- 8  C if 2-24 hrs) BloodBlood –Direct to lab at room temperature –Hold at ambient until incubator –Do not refrigerate

9. Specimen Handling Tissue 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 instructions

10. Specimens Tissue Handling Split into non-glass containersSplit into non-glass containers –4  C for live recovery –-20  C or -70  C for rapid molecular testing Formalin-fixedFormalin-fixed –Tests – Histopathology, HC, PCR –NOT frozen –Packaged separately from unpreserved

11. Laboratory Testing Pneumonic Plague Collect before initiating therapyCollect before initiating therapy Sputum, blood, lymph node aspirateSputum, blood, lymph node aspirate Gram Stain and cultureGram Stain and culture –Gram-negative coccobacillus –Bipolar (safety pin) staining Acute and convalescent bloods for public healthAcute and convalescent bloods for public health

12. Laboratory Testing Pneumonic 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

13. Laboratory Testing Pneumonic Plague Coagulation abnormalitiesCoagulation 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

14. Laboratory Case Classification Suspect Definition Clinical sx and exposure compatible ANDClinical sx and exposure compatible AND Small, Gram-negative and/or bipolar- staining, plump rods in singles, short chains, catalase (+) oxidase and urease (-)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

15. Laboratory Case Classification Probable Definition Clinical sx and exposure compatible ANDClinical sx and exposure compatible AND Specimen/Isolate positive by one of: DFA, PCR, IHCSpecimen/Isolate positive by one of: DFA, PCR, IHC OR single PHA/HI titer of  1:10 or positive ELISAOR single PHA/HI titer of  1:10 or positive ELISA Lab Tests – DFA, DNA, IHC, Serology

16. Laboratory Case Classification Confirmed Definition Positive DFA or DNA detection and bacteriophage lysis of recovered isolatePositive DFA or DNA detection and bacteriophage lysis of recovered isolate OR Paired sera 4-fold rise or fall by PHA/HI or ELISAOR Paired sera 4-fold rise or fall by PHA/HI or ELISA Tests – Bacteriophage lysis, Serology, IHC

17. Review Questions - Plague Lesson 4

18. Plague Review Question Lesson 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 identification?

19. Plague Review Question Lesson 4, Question 1 Which of the following is best for organism identification? A.Obtain initial blood specimen B.Obtain more than one blood specimen C.Get plasma or whole blood specimen on filter paper D.A smear or isolate from affected tissues

20. Plague Review Question Lesson 4, Question 1 Which of the following is best for organism identification? D. A smear or isolate from affected tissues

21. Plague Review Question Lesson 4, Question 2 Suspect pneumococcal plague patientSuspect pneumococcal plague patient Sputum sampleSputum sample What tests would your laboratory perform?

22. Plague Review Question Lesson 4, Question 2 What tests would your laboratory perform? A.PCR B.Gram stain and culture C.IHC

23. Plague Review Question Lesson 4, Question 2 What tests would your laboratory perform? B. Gram stain and culture

24. Plague Review Question Lesson 4, Question 3 Lab unable to rule out Y. pestisLab unable to rule out Y. 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 a confirmed case?

25. Plague Review Question Lesson 4, Question 3 At what point would public health rule them a confirmed case? A.Positive by DFA Staining B.Positive by PCR C.Isolate lysed at 22  C-25  C and 37  C D.Single PHA/HI titer <1:10

26. Plague Review Question Lesson 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

27. Plague Review Question Lesson 4, Question 4 About to submit blood specimensAbout to submit blood specimens Possible Y. pestisPossible Y. pestis What should you do first?

28. Plague Review Question Lesson 4, Question 4 What should you do first? A.Treat it as you would others B.Do not refrigerate C.Contact public health epidemiology D.Contact public health laboratory

29. Plague Review Question Lesson 4, Question 4 What should you do first? D. Contact public health laboratory