Laboratory Diagnostic Procedures for Influenza

1. Laboratory Diagnostic Procedures for Influenza Dr. Rezvan Moniri Associate professor of Microbiology Kashan University of Medical Sciences

2. 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

3. 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.

4. 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)

5. Variations of influenza viruses

6. 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.

7. 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.

8. 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.

9. 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.

10. Laboratory diagnosis • Isolation of virus • Identification of viral antigen • Identification of viral nucleic acid in patient’s cell • Demonstration of a specific immunologic response by the patient

11. 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.

12. 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

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. Rapid tests • Most of the rapid tests that can be done in a physician's office are approximately >70% sensitive for detecting influenza and approximately >90% 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.

19. 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.

20. 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

21. ImmunofluorescenceDFA Antibody Staining • A & B • NP swab, nasal wash, bronchial wash, nasal aspirate, sputum • 2 - 4 hours

22. RT- PCR • A & B • NP swab, throat swab, nasal wash, bronchial wash, nasal aspirate, sputum • 1 - 2 days

23. Serology • A & B • paired acute and convalescent serum samples • >2 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.

24. Enzyme Immunoassay(EIA) • A & B • NP swab, throat swab, nasal wash, bronchial wash • 2 hours

25. Rapid Diagnostic Tests • Directigen Flu A (Becton-Dickinson) • Directigen Flu A+B (Becton-Dickinson) • Directigen EZ Flu A+B (Becton-Dickinson) • FLU OIA (Thermo Electron) • FLU OIA A/B (Thermo Electron) • XPECT Flu A & B (Remel) • NOW Influenza A & B (Binax) • QuickVue Influenza A+B Test (Quidel) • SAS Influenza A Test • SAS Influenza B Test • ZstatFlu (ZymeTx)

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