1. Laboratory Diagnostics and Specimen Collection

2. Learning Objectives Know the basic components of specimen collection kits for use with suspect avian influenza cases Know what specimens to collect, how to collect them, store them, and transport them to the laboratory Discuss diagnostic tests and laboratory data management

3. The Specimen Collection Kit

4. Specimen Collection Kit Personal protective equipment Collection vials with VTM Polyester fiber- tipped applicators Tongue depressors Items for blood collection Secondary container/ cooler Ice packs Suspect case forms A pen or marker for labeling samples Labels

5. How to Manage Kits Store specimen collection kits in a dry, cool place Store specimen collection kit where it will be accessible after hours and on weekends

6. How to Safely And Correctly Collect Specimens Image obtained from www.nlm.nih.gov Target region for seasonal influenza Target region for H5N1 detection

7. What to Collect Preferred specimens Oropharyngeal swabs Lower respiratory tract specimens –Bronchoalveolar lavage or tracheal aspirates Other specimens Nasopharyngeal swabs Nasal swabs and aspirates Acute and convalescent sera Sputum specimens Collect samples on several different days

8. When to Collect Respiratory Specimens As soon as possible after symptoms begin Ideally before antiviral medications are administered Collect multiple specimens on multiple days

9. Updated Interim Guidance for Laboratory Testing of Persons with Suspected Infection with Avian Influenza A (H5N1) Virus in the United States Testing for avian influenza A (H5N1) virus infection is recommended for a patient who has an illness that: requires hospitalization or is fatal; AND has or had a documented temperature of ≥100.4° F; AND has radiographically confirmed pneumonia, acute respiratory distress syndrome (ARDS), or other severe respiratory illness for which an alternate diagnosis has not been established; AND has at least one of the following potential exposures within 10 days of symptom onset:

10. A) History of travel to a country with influenza H5N1 documented in poultry, wild birds, and/or humans, AND had at least one of the following potential exposures during travel: direct contact with (e.g., touching) sick or dead domestic poultry; direct contact with surfaces contaminated with poultry feces; consumption of raw or incompletely cooked poultry or poultry products; direct contact with sick or dead wild birds suspected or confirmed to have influenza H5N1; close contact (approach within 1 meter [approx. 3 feet]) of a person who was hospitalized or died due to a severe unexplained respiratory illness;

11. B) Close contact (approach within 1 meter [approx. 3 feet]) of an ill patient who was confirmed or suspected to have H5N1; or C) Worked with live influenza H5N1 virus in a laboratory.

12. Case by Case Considerations! Mild or atypical disease (hospitalized or ambulatory) with one of the exposures listed above Severe or fatal respiratory disease whose epidemiological information is uncertain, unavailable, or otherwise suspicious but does not meet the criteria above

13. Personal Protective Equipment Gloves Mask Gown Eye protection

14. Suspect Case Form Patient name Unique identification number Patient symptoms and date of onset Specimens and collection date Whether or not patient is hospitalized Patient contact information Patient demographic information

15. Specimen Tracking System Maintain a database to track: Identification number Subject information Type of specimen Specimen collection date Specimen collection location Date of laboratory receipt of specimen Date tested Diagnostic test results

16. Specimen Storage, Handling, and Transportation

17. How to Store Specimens For specimens in VTM and Sera: Transport to laboratory as soon as possible Store specimens at 4 °C before and during transportation within 48 hours Store specimens at -70 °C beyond 48 hours Do not store in standard freezer – keep on dry ice or in refrigerator Avoid freeze-thaw cycles –Better to keep on ice for a week than to have repeat freeze and thaw

18. All diagnostic specimens should be shipped on dry ice. –Double-bag specimens if dry ice is used. For short distances can keep specimens at 4 ºC –Fill a cooler with ice packs or coolant packs –Double-bag specimens if you use dry ice Include an itemized list of specimens with identification numbers and laboratory instructions Packing Specimens for Transportation

19. Transporting Specimens Initial testing at State Public Health Laboratory Call CDC Hotline before sending specimens for reference testing 770-488-7100 Send overnight or using appropriate means based on recommendations from the State Department of health or CDC Include inventory sheet and CDC case ID number Coordinate with the laboratory

20. Laboratory Biosafety Laboratory testing of suspected novel influenza viruses should be conducted in biosafety level 3 (BSL-3) laboratories –Infectious agents that may be transmitted via the airborne route –PPE for laboratory personnel may include respirators

21. Managing Laboratory Data

22. Data Management Rules Double check data entry accuracy Include unique identification numbers Keep subject names confidential Track testing dates and results Back up the database

23. Computer Software Epi-info (CDC) –Free, at http://www.cdc.gov/epiinfo/http://www.cdc.gov/epiinfo/ Microsoft Excel and Access Oracle MySQL Filemaker Pro

24. How to Present Results Report: Time and place of the outbreak Prevalence of infection Clinical information about cases Epidemic curve Share results with local health officials and CDC

25. Laboratory Tests

26. Laboratory Diagnosis for Influenza Tests for respiratory samples: PCR-based techniques Virus isolation Immunofluorescence Rapid antigen detection Blood used for: Measurement of specific antibodies (Most common for influenza, sera is used) Viral isolation (whole blood if viremia is a consideration) PCR-based techniques (sera)

27. Laboratory Tests for Avian Influenza A (H5N1) PCR Based Techniques- recommended –Sensitivity depends on the particular test, the influenza strain, and the type of specimen used Virus Isolation –Technically difficult –Requires a BSL-3 laboratory with enhancements

28. Serological Samples Paired serum samples are most useful Acute sample Within 7 days after symptom onset Convalescent sample 2 to 4 weeks after acute sample

29. Summary When handling infectious materials in the laboratory or in the field, take safety precautions. Proper specimen storage, handling, and shipping are vital to successful laboratory tests. Maintain adequately stocked specimen collection kits and store them properly when they are not in use.

30. Summary Oropharyngeal swabs and lower respiratory specimens are the best specimens to collect for avian influenza A (H5N1). Collect multiple specimens (respiratory and blood) on multiple days. Keep track of information on the specimens collected in a database or logbook. Properly dispose of any infectious material.

31. Laboratory Practice Exercise May, 2007

32. References and Resources Recommended laboratory tests to identify avian influenza A virus in specimens from humans. World Health Organization, June, 2005. http://www.who.int/csr/disease/avian_influenza/guide lines/avian_labtests2.pdf http://www.who.int/csr/disease/avian_influenza/guide lines/avian_labtests2.pdf WHO guidelines for the collection of human specimens for laboratory diagnosis of avian influenza infection, 12 January 2005. http://www.who.int/csr/disease/avian_influenza/guide lines/humanspecimens/en/index.html http://www.who.int/csr/disease/avian_influenza/guide lines/humanspecimens/en/index.html