Laboratory Diagnostic Procedures for Influenza

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Laboratory Diagnostic Procedures for Influenza

• Dr. Rezvan Moniri
• Associate professor of Microbiology
• Kashan University of Medical Sciences

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Influenza viruses

• Spherical, Pleomorphic, 80-120 nm
• RNA(1), protein (73), lipid (20),
• Carbohydrate (6),
• Single-stranded RNA, segmented
• (8 molecules)
• Nine structural proteins, one nonstructural
• Contains viral hemagglutinin (HA) and
  neuraminidase (NA) proteins
• Genetic re-assortment is common

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Types, Subtypes, and Strains

• There are three types of influenza viruses A, B,
  and C (Nucleocapsid and matrix).
• Antigenic variations in the HA and NA, are used
  to subtype the virus.
• Only influenza A viruses has designated subtypes.
• Influenza A subtypes and B viruses are further
  classified by strains.

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Standard nomenclature

• Type
• Host of origin
• Geographic origin
• Strain number
• Year of isolation
• A/Hongkong/03/68(H3N2)
• A/swine/Iowa/15/30(H1N1)
• 15 subtypes of HA (H1-H15)
• Nine subtype of NA (N1-N9)
• For human Four HA(H1-H3,H5) and two NA(N1,N2)

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Variations of influenza viruses
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One of the billions of influenza viruses that
will be lying in wait for you this winter.

• One of the billions of influenza viruses that
  will be lying in wait for you this winter.

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Human Influenza Viruses and Avian Influenza A
Viruses

• Humans can be infected with influenza types A, B,
  and C viruses. Subtypes of influenza A that are
  currently circulating among people worldwide
  include H1N1, H1N2, and H3N2 viruses.
• Wild birds are the natural host for all known
  subtypes of influenza A viruses. Typically, wild
  birds do not become sick when they are infected
  with avian influenza A viruses.
• However, domestic poultry, such as turkeys and
  chickens, can become very sick and die from avian
  influenza, and some avian influenza A viruses
  also can cause serious disease and death in wild
  birds.

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Diagnosis of influenza

• Influenza illness can include any or all of these
  symptoms
• fever, muscle aches, headache, lack of energy,
  
• dry cough, sore throat, and possibly runny
  nose.
• The fever and body aches can last 3-5 days and
• the cough and lack of energy may last for 2
  or more weeks.
• Influenza can be difficult to diagnose based on
  clinical symptoms alone because the initial
  symptoms of influenza can be similar those caused
  by other infectious agents including, but not
  limited to, Mycoplasma pneumoniae, adenovirus,
  respiratory syncytial virus, parainfluenza
  viruses, rhinovirus, and Legionella spp.

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Diagnostic tests

• Diagnostic tests available for influenza include
  viral culture, serology, rapid antigen testing,
  polymerase chain reaction (PCR), and
  immunofluorescence assays.
• Sensitivity and specificity of any test for
  influenza might vary by the laboratory that
  performs the test, the type of test used, and the
  type of specimen tested.
• Among respiratory specimens for viral isolation
  or rapid detection, nasopharyngeal specimens are
  typically more effective than throat swab
  specimens.
• As with any diagnostic test, results should be
  evaluated in the context of other clinical and
  epidemiologic information available to
  health-care providers.

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Laboratory diagnosis

• Isolation of virus
• Identification of viral antigen
• Identification of viral nucleic acid in patients
  cell
• Demonstration of a specific immunologic response
  by the patient

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Appropriate samples for influenza testing

• nasopharyngeal or throat swab
• nasal wash
• nasal aspirates
• Samples should be collected within the first 4
  days of illness.
• Rapid influenza tests provide results within 24
  hours
• viral culture provides results in 3-10 days.

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Isolation and identification of virus

• Embryonated eggs and primary monkey kidney cells
• Cell cultures can be tested for the presence of
  virus by hemadsorption
• 3-5 days after inoculation, or the culture
  fluid can be examined for virus after 5-7 days by
  hemagglutination

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Isolation and identification of virus

• Viral isolates can be identified by
  hemagglutination inhibition, a procedure that
  permits rapid determination of the influenza type
  and subtype.
• To identify viral antigen directly in exfoliated
  cells in nasal aspirates using fluorescent
  antibodies.
• Detection of influenza RNA in clinical specimens
  using PCR.

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Serology

• Antibodies to several viral proteins (
  hemagglutinin, neuraminidase, nucleoprotein, and
  matrix ) are produced during infection.
• Routine serodiagnostic tests in used are based on
  hemagglutination inhibition test (HI) and ELISA.
• HI reveals the strain of virus.
• Neutralization test are the most specific
• ELISA test is more sensitive than other assays.

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Serum samples

• Serum samples also can be tested for influenza
  antibody to diagnose recent infections.
• Two samples should be collected per person one
  sample within the first week of illness and a
  second sample 2-4 weeks later.
• If antibody levels increase from the first to
  the second sample, influenza infection likely
  occurred. Because of the length of time needed
  for a diagnosis of influenza by serologic
  testing, other diagnostic testing should be used
  if a more rapid diagnosis is needed.

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Commercial rapid diagnostic tests

• Commercial rapid diagnostic tests are available
  that can detect influenza viruses within 30
  minutes.
• These rapid tests differ in the types of
  influenza viruses they can detect and whether
  they can distinguish between influenza types.
• Different tests can detect
• 1) only influenza A viruses
• 2) both influenza A and B viruses, but not
  
• distinguish between the two types
• 3) both influenza A and B and distinguish
  between
• the two.

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Rapid tests

• The specificity and, in particular, the
  sensitivity of rapid tests are lower than for
  viral culture and vary by test. Because of the
  lower sensitivity of the rapid tests, physicians
  should consider confirming negative tests with
  viral culture or other means because of the
  possibility of false-negative rapid test results,
  especially during periods of peak community
  influenza activity.
• In contrast, false-positive rapid test results
  are less likely, but can occur during periods of
  low influenza activity.
• Therefore, when interpreting results of a rapid
  influenza test, physicians should consider the
  positive and negative predictive values of the
  test in the context of the level of influenza
  activity in their community.

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Rapid tests

• Most of the rapid tests that can be done in a
  physician's office are approximately gt70
  sensitive for detecting influenza and
  approximately gt90 specific.
• Thus, as many as 30 of samples that would be
  positive for influenza by viral culture may give
  a negative rapid test result.
• And, some rapid test results may indicate
  influenza when a person is not infected with
  influenza.

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Outbreaks of Respiratory illness

• During outbreaks of respiratory illness when
  influenza is suspected, some samples should be
  tested by both rapid tests and by viral culture.
• The collection of some samples for viral culture
  is essential for determining the influenza
  subtypes and strains causing illness, and for
  surveillance of new strains that may need to be
  included in the next year's influenza vaccine.
• During outbreaks of influenza-like illness, viral
  culture also can help identify other causes of
  illness when influenza is not the cause.

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Viral Culture

• Influenza Types Detected A B
• Acceptable Specimens NP swab, throat swab, nasal
  wash, bronchial wash, nasal aspirate, sputum
• Time for Results 5-10 days

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Immunofluorescence DFA Antibody Staining

• A B
• NP swab, nasal wash, bronchial wash, nasal
  aspirate, sputum
• 2 - 4 hours

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RT- PCR

• A B
• NP swab, throat swab, nasal wash, bronchial
  wash, nasal aspirate, sputum
• 1 - 2 days

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Serology

• A B
• paired acute and convalescent serum samples
• gt2 weeks
• A fourfold or greater rise in antibody titer from
  the acute (collected within the 1st week of
  illness) to the convalescent-phase (collected 2-4
  weeks after the acute sample) sample is
  indicative of recent infection.

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Enzyme Immunoassay(EIA)

• A B
• NP swab, throat swab,
• nasal wash, bronchial wash
• 2 hours

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Rapid Diagnostic Tests

• Directigen Flu A (Becton-Dickinson)
• Directigen Flu AB (Becton-Dickinson)
• Directigen EZ Flu AB (Becton-Dickinson)
• FLU OIA (Thermo Electron)
• FLU OIA A/B (Thermo Electron)
• XPECT Flu A B (Remel)
• NOW Influenza A B (Binax)
• QuickVue Influenza AB Test (Quidel)
• SAS Influenza A Test
• SAS Influenza B Test
• ZstatFlu (ZymeTx)

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