Laboratory Diagnostics, Specimen Collection, and Biosafety Issues

1. Laboratory Diagnostics, Specimen Collection,and Biosafety Issues

2. Learning Objectives • Describe avian influenza-related laboratory procedures • Define laboratory safety • List elements of specimen collection kit • Explain how to collect & transport specimens • Describe infectious waste disposal • Explain how to manage laboratory data

3. Session Overview • Laboratory diagnosis of human cases of avian influenza • Laboratory safety • Specimen collection and transportation • Specimen storage, handling, packaging, and transportation • Waste disposal and decontamination • Managing and analyzing laboratory data

4. Laboratory Diagnosis of Human Cases of Avian Influenza

5. Laboratory Diagnostics for Avian Influenza • Tests on respiratory samples: • PCR-based techniques • Virus isolation • Immunofluorescence • Rapid antigen detection • Tests on serum: • Measure specific antibodies • PCR-based techniques

6. H5N1 Virus Found in Other Human Specimens • H5N1 virus infection of cerebrospinal fluid documented in a fatal case with seizures and coma • H5N1 virus has also been found in: • Rectal swab specimens and stool of fatal cases with diarrhea • Serum and plasma of fatal cases • All respiratory secretions and bodily fluids of H5N1 patients should be considered potentially infected with H5N1 virus

7. PCR-based Techniques • Respiratory samples • Diagnostic assays and sequencing • Sensitivity depends on: • Particular assay • Influenza strain • Type of the specimen • Quality of the specimen

8. PCR: H5N1 Virus Testing Primary method: detection of H5N1 viral RNA by reverse-transcription polymerase chain reaction (RT-PCR) • Conventional RT-PCR • Real-time RT-PCR (RT-RT-PCR) • Highly sensitive and specific

9. Interpretation of Real-time RT-PCR Results Test reactions

10. Virus IsolationGold Standard • Biosafety level 3 (BSL-3) laboratory • Antigenic & genetic characterization • Drug susceptibilities tests • WHO Influenza Centers • Antigenic characterization of viral isolates • Collect specimens for vaccine development

11. Immunofluorescence • Requires H5 monoclonal antibody • Not part of WHO Reagentkit • Interpretation difficult • Results: • Presence of influenza virus • Subtype identification

12. Indirect IF Staining of Cells From Tracheal Aspirate Anti-H5 Anti-H3 Anti-A/NP Anti-B Taken from: World Bank Training by Alexander Klimov, CDC

13. Rapid Antigen Tests • Fast and commercially available • Low sensitivity • False negatives and false positives • Identifies viral type (type A or B) NOT subtype (H5, H3, H1) • Clinically useful to guide treatment, but must test for H5 with another assay RAPID TESTS NOT RECOMMENDED For Detection of H5N1 virus

14. Using Serology Delay for anti-H5 levels to rise Requires acute and convalescent sera, 3 weeks apart Not useful for clinical management Can confirm epidemic cases, if respiratory specimens not available

15. Influenza Serology Tests • Micro-neutralization assays • Western Blot • Enzyme immunoassays Require acute and convalescent sera (serum obtained >21 days from onset)

16. Interpreting Serology Tests * four-fold or greater increase between acute and convalescent sera

17. Serological Specimens • Paired serum specimens most useful • 4-fold rise in antibody titer • Single serum specimens: • Useful in outbreak investigations of novel human influenza viruses • If prevalence unknown, case serum specimen compared to age-matched control specimen

18. How to Avoid Misinterpretation of Diagnostic Tests • Use appropriate controls • Understand • What is test identifying? • What are limitations of assay? • Use multiple tests to confirm results

19. Review Question #1 Name two ways to test respiratory samples for avian influenza. Possible Answers: • PCR-based techniques • Virus isolation • Immunofluorescence • Rapid antigen detection

20. Review Question #2 Which of the following is NOT a way to avoid misinterpretation of diagnostic tests? • Use appropriate controls for each assay • Understand what the test is identifying • Understand the limitations of each assay • Use only one test if you are sure that it is correct Answer: d. Instead, you should use multiple tests to confirm results.

21. Laboratory Safety

22. Biosafety Laboratory Levels (BSL) • Risk of acquiring infections • Laboratory safety guidelines evolved • Safe work sites result from: • Engineering controls • Management policies • Work practices and procedures • Medical interventions (occasional) Increasing biosafety levels = increasing levels of personnel & environmental protection http://www.cdc.gov/OD/ohs/symp5/

23. BSL-2 and BSL-3 • BSL-2 • Good microbiological technique • Protective clothing • Biohazard sign • BSL-3 = BSL-2 plus: • Controlled access • Directional air flow • BSL-3 (+) = BSL-3 plus: • Respiratory protection and clothing change • HEPA filters

24. Potential H5N1 Influenza Specimens • BSL2 lab with BSL3 work practices needed for: • Aliquoting human specimens • Nucleic acid extractions • All Diagnostic testing except viral culture • BSL3 safety measures and guidelines necessary to culture H5N1   If laboratory does not meet BSL2 requirements, ship specimen to reference laboratory

25. WHO H5N1 Laboratory Network • Reference laboratories responsible for surveillance and vaccine preparation • Perform detailed antigenic and genetic characterization • Report positive samples to WHO • Submit positive samples to WHO laboratory for characterization • Include background clinical information with the shipment • WHO results shared with original laboratory

26. Review Question #3 What Biosafety Level needs to be in place in order to culture H5N1? • BSL-1 • BSL-2 • BSL-3 • BSL-3+ Answer: c. BSL-3 safety measures and guidelines should be used for culturing potential H5N1 viruses

27. Specimen Collection

28. Specimen Collection Kit • Personal protective equipment (PPE) • Viral transport medium (VTM) collection vials • Swabs: throat, nasal, & nasopharyngeal • Tongue depressors • Nasal Wash Equipment • Transfer pipettes • Secondary container • Ice packs • Items for blood collection • Field collection forms • Labels and pen or marker Store kit in a dry, cool place Keep kit accessible for after hours

29. Viral Transport Medium (VTM) • Used to store & transport specimens • Isolates & maintains virus integrity • Prevents bacteria and fungi growth • Can be made in a lab or purchased • Different types of VTM: • Animal specimen collection • Viral isolation of human specimens

30. Storing VTM • Sterile collection vials containing 2-3 ml of VTM • Vials can be stored in a freezer at -20ºC to -40ºC until use • Vials can be stored for short periods of time at 4 - 8 ºC

31. Polyester Fiber-Tipped Applicator • Should ideally be dacron, rayon, or polyester-fiber swabs Remember! Use throat swabs for avian influenza and nasopharyngeal swabs for seasonal influenza

32. Personal Protective Equipment • Masks (N-95 or N/P/R-100) • Gloves • Protective eye wear (goggles) • Hair covers • Boot or shoe covers • Protective clothing (gown or apron)

34. Clinical Specimen Sources Prepare to collect specimens before you leave for the field • Persons meeting trigger criteria • Includes WHO suspected and probable cases • Symptomatic Contacts • Symptomatic people living/working with suspected cases

35. What to Collect From an Ambulatory patient • Throat swab (priority) and • Nasopharyngeal swab • If necessary, collect into same VTM From an Intubated patient • Tracheal aspirate

36. When to Collect Specimens

37. When Do I Collect Respiratory Specimens? • As soon as possible after symptoms begin • Before antiviral medicine administered • For multiple days • Sample multiple types of specimens

38. When to collect Serum Specimens Acute specimen Within 7 days after symptom onset Convalescent specimen 2-3 weeks after the acute sample (> 21 days weeks after symptom onset)

39. Oropharyngeal (Throat) Swab

40. Nasopharyngeal Swab http://www.nlm.nih.gov/medlineplus/ency/imagepages/9687.htm

41. Nasopharyngeal Aspirate (Nasal Wash)Collection Process • Attach mucus trap to vacuum source • Place catheter into nostril parallel to palate • Apply vacuum • Slowly remove catheter while slightly rotating it • Repeat with other nostril using same catheter • After collection, flush catheter with 3 ml VTM and return VTM to a plastic vial

42. Labeling Specimens Use pre-printed barcode labels: • On specimen container • On field data collection form • In log book Label each specimen with: • Subject’s unique identification number

43. Field Data Collection Forms

44. Review Question #4 True or False: If you collect a respiratory specimen (throat swab, nasopharyngeal swab), you should collect acute and convalescent specimens. Answer: False. Acute and convalescent specimens are only needed for serological tests. However, you should collect respiratory specimens on multiple days.

45. Specimen Storage, Handling, Packaging and Transportation

46. How to Store Specimens For specimens in VTM: • Transport to laboratory as soon as possible • Within 48 hours store at 4 °C to 8 °C before and during transportation • Beyond 48 hours store at -70 °C to -80 °C • Do notuse standard freezer; keep on ice or in refrigerator • Avoid freeze-thaw cycles • Keep on ice for a week instead of freezer

47. How to Store Specimens For sera: • Store specimen at • 4 °C to 8 °C for short periods of time • - 20 °C to - 40 °C for long term storage • Avoid freeze-thaw cycles • Centrifuge blood and aliquot serum to another container before shipping

48. Packing Specimens for Transportation Goal: protect specimens during transportation • Use three packaging layers • Use water tight first layer • Use absorbent material in all layers • <500mL of liquid in specimen collection container

49. Transporting Specimens • WHO guidelines for the safe transport http://www.who.int/csr/emc97_3.pdf • Follow local regulations • Coordinate with the laboratory

50. Review Question #5 Which of the following is true about storing clinical specimens (sera or specimens in VTM)? • They can be stored at 4-8 °C • They can be stored in a standard freezer Answer: a. Both sera and specimens in VTM can be stored for certain periods at 4-8 °C. Neither of these samples should be stored in a standard freezer, because the freeze-thaw cycle will destroy the virus