Laboratory Testing for H5N1 in Humans

Laboratory Testing for Avian Influenza in Humans

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General Considerations

• Tests for influenza include: viral culture, polymerase chain reaction (PCR), rapid antigen testing, and immunofluorescence.
• Laboratory tests are widely used to identify influenza virus at the genus level (influenza A/B) or at the H-type level (H1, H3, and H5).
• H subtype-specific tests must be used to identify potential avian strains, including H5N1.
• The World Health Organization (WHO) recommends forwarding all H5, H7, and H9-positive isolates to a designated influenza reference laboratory for confirmation and N-typing (see References: WHO: Guidelines for global surveillance of influenza A/H5; WHO: Recommended laboratory tests to identify avian influenza A virus in specimens from patients with an influenza-like illness).
• Serologic tests have been used to diagnose infection retrospectively.
• During a pandemic alert period for an avian influenza virus, patients who meet certain criteria (such as influenza symptoms and recent travel to an area affected by a novel strain) should be considered for laboratory testing.
• During a pandemic (involving an avian strain or other strain), recommendations for laboratory testing may be somewhat unique and dependent upon factors such as: (1) availability of reagents and laboratory surge capacity, (2) the presence or absence of other influenza strains in the community, (3) level of influenza activity in the community, and (4) treatment considerations.
• The sensitivity and specificity of laboratory tests appears to vary with the involved strain, which has implications for avian influenza and other emerging influenza variants (see References: Weinberg 2005).
• Laboratory tests are required for specific identification of avian influenza. The most likely ways that an avian influenza strain would be detected in the human population are:
  • Outbreak investigations or investigation of unexplained death in a previously healthy individual
  • Influenza surveillance with laboratory testing
  • Investigation of unusual laboratory findings
  • Testing of persons with influenza-like symptoms who meet certain exposure criteria

• Laboratory-based influenza surveillance networks
  • WHO Global Influenza Surveillance Network (see References)
  • CDC National Respiratory and Enteric Virus Surveillance System (NREVSS) (see References)
  • State or local surveillance health department surveillance networks

Specimen Collection

The following information is taken from a field operations guide for H5N1 influenza that was released by WHO in early November 2006 (see References: WHO 2006: Collecting, preserving, and shipping specimens for the diagnosis of avian influenza A [H5N1] virus infection). Information also was taken from the HHS Pandemic Influenza Plan where noted (see References: HHS 2005: Pandemic influenza plan [Part 2, Supplement 2]).

Specimens to collect from suspect cases

• Upper respiratory tract
  • Posterior-pharyngeal (throat) swabs (provide the highest yield)
  • Nasal swabs with nasal secretions (from the anterior turbinate areas) or nasopharyngeal aspirates or swabs (these specimens are more appropriate for seasonal influenza and the yield may be lower for avian influenza)
• Lower respiratory tract:
  • A tracheal aspirate or bronchoalveolar lavage specimen (if the patient is intubated)
• Blood:
  • Serum (acute and convalescent if possible)
• Secondary specimens:
  • Plasma in EDTA (for detection of viral RNA)
  • Rectal swab (for patients with diarrhea)
  • Spinal fluid (if meningitis is suspected and a spinal tap is performed for diagnostic purposes)
  • Pleural tap fluid (referred to in the HHS Plan)
  • Autopsy specimens (referred to in the HHS Plan)

When to collect specimens from suspect cases

• Ideally, a throat swab should be taken within 3 days after illness onset; if initial specimens are negative, but a high index of suspicion remains, testing should be repeated as soon as possible. (According to the HHS Plan, specimens optimally should be collected within 4 days after illness onset.)
• Virus may be detected in tracheal aspirates from onset of lower respiratory symptoms until the second or third week of illness.
• An acute phase serum sample should be taken 7 days or less after symptom onset and a convalescent sample should be taken 3 to 4 weeks following illness onset.
• Single serum samples should be collected 14 days or later after symptom onset.
• Serum or plasma for detecting viral RNA should be obtained during the first 7 to 9 days after symptom onset.
• Ideally specimens should be collected before antiviral therapy, but treatment should not be delayed in order to take specimens.
• Specimens should be collected from deceased patients as soon as possible after death.

Specimen collection and transport

• Detailed methods for specimen collection and transport are provided in the WHO field guide (see References: WHO 2006: Collecting, preserving, and shipping specimens for the diagnosis of avian influenza A [H5N1] virus infection).
• Infection control precautions should be consistently observed during specimen collection.
• Only sterile dacron or rayon swabs with plastic shafts should be used. Calcium alginate or cotton swabs or swabs with wooden sticks should not be used (or used only when appropriate swabs are not available).
• Viral transport media (VTM) should be used for nasopharyngeal and oropharyngeal swabs and, according to the HHS Plan, specimens should be maintained at refrigerator temperature (4ºC to 8^oC) until testing is performed. Freezing at 70ºC is best for maintaining viability during extended storage.
• According to the HHS Plan, with regard to autopsy specimens, large airways have the highest yield for immunohistochemistry (IHC) tests. Eight blocks or fixed-tissue specimens from each of the following sites should be obtained. Fixed tissue should be transported at room temperature (not frozen); fresh unfixed tissue should be frozen.
  • Central (hilar) lung with segmental bronchi
  • Right and left primary bronchi
  • Trachea (proximal and distal)
  • Representative pulmonary parenchyma from right and left lung