Laboratory Diagnostics and Specimen Collection
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
The Specimen Collection Kit
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
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
How to Safely And Correctly Collect Specimens
Image obtained from www.nlm.nih.gov
Target regionfor seasonalinfluenza
Target region for H5N1 detection
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
When to Collect Respiratory Specimens
• As soon as possible after symptoms begin
• Ideally before antiviral medications are administered
• Collect multiple specimens on multiple days
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:
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;
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.
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
Personal Protective Equipment
• Gloves
• Mask
• Gown
• Eye protection
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
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
Specimen Storage, Handling, and Transportation
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
• 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
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
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
Managing Laboratory Data
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
Computer Software
• Epi-info (CDC) – Free, at http://www.cdc.gov/epiinfo/
• Microsoft Excel and Access
• Oracle
• MySQL
• Filemaker Pro
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
Laboratory Tests
Laboratory Diagnosis for InfluenzaTests 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)
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
Serological Samples
Paired serum samples are most useful
Acute sampleWithin 7 days after symptom onset
Convalescent sample2 to 4 weeks after acute sample
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.
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.
Laboratory Practice Exercise
May, 2007
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
