Laboratory Diagnosis of Influenza Virus

For the laboratory diagnosis of Influenza Virus, specimen should be taken as early as possible during the course of disease preferably within the first 72 hour after the onset of symptoms. Routine Specimens for Influenza Virus diagnosis include
A. Nasal Washings
B. Nasopharyngeal aspirate
C. Nasopharyngeal swab
D. Nasal and Throat swabs
For successful isolation, specimen should be placed on transport medium to stabilize the virus.

Isolation of Influenza Virus
a. Cell Culture
Madin-Darby Kidney Cell Line (MDCK) Supplemented with trypsin supports growth and multiple cycle of influenza A, B and C. Standard virus isolation is done in cell culture tubes seeded with MDCK Cells. The cultures are incubated at 34 degree centigrade either in stationary rack or in a roller machine. Some influenza viruses cause a distinct cytopathic effect in MDCK cells, several days after inoculation. Negative Cultures should be checked by a hemadsorption or hemagglutination test at two to three days interval.



b.Detection of Infected Cells in Clinical Specimens by IF
Demonstration of infected epithelial cells in Nasopharyngeal aspirate or Nasopharyngeal swab is a sensitive method (50-90% sensitive than virus isolation) for the diagnosis of influenza. Result can be obtained within a few hours after collection of the specimen.
Exfoliated cells in NPA and NPS are prepared, applied to microscopic slides and fixed. Polyclonal sera and/or monoclonal antibodies are used as reagent for the staining process.

c. Detection of Influenza Virus Antigens by Immunoassays
A variety of tests such as radioimmunoassay, EIA and fluoroimmunoassay have been developed for the detection of influenza virus antigens directly in clinical specimens or after amplification in cell culture.
Commonly antigens in clinical specimens or in cell culture material are captured by specific antibodies that have previously adsorbed to the solid phase. After incubation, unbound material is washed and the bound antigen is reacted with a secondary antibody and then incubated further with a labeled anti-species antibody.
d. Reverse Transcriptase PCR
The reverse transcriptase PCR has been applied to detect influenza virus in clinical specimens and in cell culture or egg grown material. The viral RNA is first transcribed into DNA, which is then amplified in a second step that uses suitable primers and DNA Polymerase.
Amplified DNA is electrophoresed in an agarose gel and DNA is detected by staining with ethidium bromide. Molecular weight markers are included to identify amplified DNA of the appropriate size. DNA can also be detected by molecular hybridisation with specific labeled probes either in Southern blots or by spot hybridization. Appropriate selection of primers permit type-specific identification of Influenza viruses A, B and C or subtype specific identification of Influenza Virus.

Serology
Although serologic methods seldom yield a result early enough to influence the patients treatment, they often established the diagnosis of influenza virus infection when virus is not detected by other methods. When the sensitivities of newly developed diagnostic tests are being evaluated, serology should be
considered instead of or in combination with virus isolation as the "Gold Standard": HAI, the neutralisation test (NT), And the complement fixation test (CFT) are traditional methods in serodiagnosis and seroepidemiologic studies of influenza, but during recent years, EIA has found wide application.
CF measures antibodies against the NP and thus allows type specific detection of antibodies to influenza A or B viruses. HAI and NT are more sensitive and measure antibodies against subtype and strain specific antigens.