Saturday, August 13, 2011

Laboratory diagnosis of Influenza virus

• Collection of samples, viral isolation and cell culture
Nasal washings, gargles, and throat swabs are the best specimens for viral isolation and should be obtained within 3 days after the onset of symptoms. Classically, embryonated eggs and primary monkey kidney cells have been the isolation methods of choice for influenza viruses, although some continuous cell lines may be used such as Madin Darby canine kidney with addition of trypsin (Lamb and Krug, 2001).
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. If the results are negative; a passage is made into fresh cultures. This passage may be necessary, because primary viral isolates are often fastidious and grow slowly. Viral isolates can be identified by HI, a procedure that permits rapid determination of the Influenza type and subtype. To do this, reference sera to currently prevalent strains must be used. Hemagglutination by the new isolate will be inhibited by antiserum to the homologous subtype. For rapid diagnosis, cell cultures on coverslips in shell vials may be inoculated and stained 1- 2 days later with pools of MAbs to respiratory agents. Positives are confirmed by use of single FA (Wright and Webster, 2001).
• Antigen detection
It is possible to identify viral Ag directly in exfoliated cells in nasal aspirates using FA. This test is rapid but is not as sensitive as viral isolation, does not provide full details about viral strain (Lamb and Krug, 2001).
• Serology
Abs to several viral proteins (hemagglutinin, neuraminidase and matrix) are produced during infection with Influenza virus. Routine serology is based on HI and ELISA. Paired acute and convalescent sera are necessary, because normal individuals usually have Influenza Abs. A fourfold or greater increase in titer must occur to indicate Influenza infection. The HI test reveals the strain of virus responsible for infection only if the correct Ag is available for use. Neutralization tests are the most specific and the best predictor of susceptibility to infection but are more unwieldy and more time-consuming to perform than the other tests. ELISA test is more sensitive than other assays (Reid, 1999).
• Nucleic acid detection
Rapid tests based on detection of Influenza RNA in clinical specimens using PCR are also possible. It will be useful for diagnostic testing and for identification of these viruses in laboratory viral cultures (Horimoto and Kawaoka, 2005).

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