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 fibertipped 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
Target region
for seasonal
influenza
Target
region for H5N1
detection
Image obtained from www.nlm.nih.gov
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
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•
•
•
•
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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
Packing Specimens for
Transportation
• 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
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/
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Microsoft Excel and Access
Oracle
MySQL
Filemaker Pro
How to Present Results
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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 Influenza
Tests for respiratory samples:
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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 sample
Within 7 days after symptom onset
Convalescent sample
2 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/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