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: • 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 Isolation of H5N1 VirusesGold Standard • Requires biosafety level 3 (BSL-3)- enhanced laboratory • Requires select agent approval from USDA (United States) • Allows antigenic & genetic characterization • Allows drug susceptibilities tests • Performed at WHO Influenza Centers • Antigenic characterization of viral isolates • Collect specimens for vaccine development

11. Immunofluorescence for H5N1 Testing • Requires H5 monoclonal antibody • Not part of WHO Reagentkit for seasonal influenza isolates • 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 • Quick results 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 human H5N1 virus infection

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 convalescent 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 more than one test to confirm results and use more than one sample from each patient

19. Review Question 1 Which of the following is the best technique to test respiratory samples for avian influenza? • PCR-based techniques • Virus isolation • Immunofluorescence • Rapid antigen detection Answers: a. PCR-based techniques

20. Review Question 2 Which of the following situations is most likely to lead to the 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 Dr. Terrence Tumpey examines reconstructed 1918 Pandemic Influenza Virus inside a specimen vial in a Biosafety Level 3-enhanced laboratory setting. Courtesy CDC/James Gathany.

22. Biosafety Laboratory Levels (BSL) • Risk of acquiring infections exists in laboratory • 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 enhanced = BSL-3 plus: • Respiratory protection and clothing change • Shower out of facility • HEPA filters

24. BSL Requirements for 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 appropriately packaged specimen to reference laboratory

25. WHO H5 Reference Laboratories • Collect specimens / virus isolates of A(H5N1) and other influenza subtypes • Provide timely surveillance data • Conduct antigenic and genetic analyses • Assist in developing methods for global surveillance and diagnosis of A(H5N1) • Participate in selecting, developing, and distributing candidate A(H5N1) vaccines • Develop and distribute reagents • Monitor susceptibility of emerging subtypes • Provide expertise and laboratory support to Member States

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 enhanced Answer: d. BSL-3enhanced 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 1-3 ml of VTM • VTM can be stored in a freezer at -20ºC to -40ºC until use • VTM 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 for Caring for H5N1 Patients • 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 to Collect Respiratory Specimens • As soon as possible after symptoms begin • Ideally before antiviral medicine administered • For multiple days • Sample multiple types of specimens

38. When to collect Serum Specimens Acute specimen As soon as possible, ideally within 7-10 days after symptom onset Convalescent specimen 2-3 weeks after the acute sample (> 21 days 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: Are nasopharyngeal swabs best for seasonal or avian influenza? Answer: Seasonal

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 • Beyond 48 hours store at -70 °C to -80 °C • Do notuse standard freezer; keep on ice or in refrigerator • Avoid freeze-thaw cycles

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 and before freezing

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 • If transporting specimens a long distance, send on dry ice. If transporting a short distance, ice is acceptable

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°C up to a week • They can be stored in a standard freezer Answer: a. Both sera and specimens in VTM can be stored for certain periods at 4°C for up to a week. Neither of these samples should be stored in a standard freezer, because the freeze-thaw cycle will destroy the virus