Laboratory Diagnostics, Specimen Collection, and Biosafety Issues

1. Laboratory Diagnostics, Specimen Collection,and Biosafety Issues

2. Learning Objectives • Prepare and maintain collection kits • Collect and transport specimens safely and correctly • Manage laboratory specimens safely • Interpret laboratory data

3. The Specimen Collection Kit

4. Collection vials with VTM Polyester fiber-tipped applicators Sterile saline (0,85% NaCl) Sputum or mucus trap Tongue depressors Specimen collection cups or Petri dishes Transfer pipettes Secondary container Ice packs Items for blood collection Personal protective equipment Field collection forms A pen or marker for labeling samples Specimen Collection Kit

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 Samples

7. Clinical Specimen Sources Be prepared to collect specimens before you leave for the field • Suspected cases • Symptoms consistent with influenza • Contacts • Including people living or working with suspected cases

8. What to Collect Preferred specimens • Oropharyngeal swabs • Lower respiratory tract specimens • Bronchoalveolar lavage or tracheal aspirates Other specimens • Nasal swab • Nasopharyngeal swabs Collect the sample on several different days

9. When to Collect Respiratory Specimens • As soon as possible after symptoms begin • Before antiviral medications are administered • Even if symptoms began more than one week ago • Collect multiple specimens on multiple days

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

11. Field Data Collection Form • Patient name • Unique identification number • Patient demographic information • Patient’s health status

12. Specimen Tracking System Maintain a database to track: • Identification number • Subject information • Specimen collection date • Specimen collection location • Diagnostic test results

13. Specimen Storage, Handling, and Transportation

14. How to Store Specimens For specimens in VTM: • 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 ice or in refrigerator • Avoid freeze-thaw cycles • Better to keep on ice for a week than to have repeat freeze and thaw

15. How to Store Specimens For sera: • Store specimen at 4 °C • For both VTM specimens and sera, avoid repeated freeze-thaw cycles

16. Packing Specimens for Transportation • 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

17. Transporting Specimens • Initial testing at State Public Health Laboratory • Call CDC Hotline before sending specimens for reference testing 770-488-7100 • Send Priority Overnight • Include inventory sheet and CDC case ID number • Coordinate with the laboratory

18. Managing and Analyzing Laboratory Data

19. 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

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

21. Data Analysis Measures Prevalence The number of cases divided by the size of the population Sensitivity The number of cases identified by the test divided by the number of true positives which were identified by the gold standard technique Specificity The number of non-cases identified by the test divided by the number of true negatives that were identified by the gold standard technique

22. 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

23. Laboratory Tests

24. Laboratory Diagnosis for Avian Influenza Tests for respiratory samples: • PCR-based techniques • Virus isolation • Immunofluorescence • Rapid antigen detection Blood used for: • Measurement of specific antibodies (sera) • Viral isolation (whole blood) • PCR-based techniques (sera)

25. Laboratory Tests Virus Isolation • Gold standard for detection of avian influenza • Technically difficult and requires BSL-3 laboratory PCR Based Techniques • Sensitivity depends on the particular test, the influenza strain, and the type of specimen used • Sensitivity: 80 - 100% • Specificity: 90 - 100%

26. Serological Tests Paired serum samples are most useful Acute sample Within 7 days after symptom onset Convalescent sample More than 12 days after symptom onset

27. Interpreting Serology Tests

28. Summary • Maintain adequately stocked specimen collection kits and store them properly when they are not in use. • Oropharyngeal swabs and lower respiratory specimens are the best specimens to collect. • Collect multiple specimens (respiratory and blood) on multiple days.

29. Summary • Proper specimen storage, handling, and shipping is vital to a successful laboratory test. • Keep track of information on the specimens collected in a database or logbook. • When handling infectious materials in the laboratory or in the field, take safety precautions. • Properly dispose of any infectious material.

30. Summary • Most states will be able to perform some basic influenza identification tests on a clinical specimen, but more specific testing may need to be done at a higher level national or regional laboratory. • When you report on the progress of an outbreak investigation, share investigation results with local health officials and CDC.

31. Laboratory Practice Exercise

32. Glossary Centrifuge A machine that uses high-speed rotation to separate materials with different densities. Culture The growing of microorganisms in a nutrient-rich medium. Nucleic acid A component genetic material such as DNA or RNA found in all cells in humans, animals, bacteria, and viruses. Every species and organism has a unique pattern.

33. Glossary Pipette A glass or plastic tube used to measure or transfer small amounts of liquid. Saline A liquid solution made of salt and water. Viral Transport Medium (VTM) The preservative liquid in which specimens are stored until they are tested.

34. 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/guidelines/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/guidelines/humanspecimens/en/index.html