Laboratory Diagnostics and Specimen Collection

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Laboratory Diagnostics and Specimen Collection
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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

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The Specimen Collection Kit
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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

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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

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

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When to Collect Respiratory Specimens

• As soon as possible after symptoms begin
• Ideally before antiviral medications are
  administered
• Collect multiple specimens on multiple days

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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

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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
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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.
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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

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Personal Protective Equipment

• Gloves
• Mask
• Gown
• Eye protection

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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

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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

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Specimen Storage, Handling, and Transportation
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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 ice or
  in refrigerator
• Avoid freeze-thaw cycles
• Better to keep on ice for a week than to have
  repeat freeze and thaw

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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

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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

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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

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Managing Laboratory Data
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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

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Computer Software

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

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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

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Laboratory Tests
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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)

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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

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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

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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.

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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.

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Laboratory Practice Exercise
May, 2007
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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/gui
  delines/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/gui
  delines/humanspecimens/en/index.html