Laboratory Diagnosis of Influenza
Laboratory diagnosis of influenza is based on virus isolation and identification.
Nasopharingeal washings and throat swabs are used for the examination. The specimens should be taken within 3 days after the disease onset.
Rapid methods comprise immunofluorescence assay of nasal swabs for viral antigen detection and the identification of viral nucleic acid in patient’s infected cells by reverse transcription PCR (RT-PCR).
For viral cultivation the 9-10-day-old embryonated chicken eggs and primary monkey kidney cells are used. Inoculation is produced into the media, supplemented with antibiotics to suppress the concomitant bacterial flora, and with trypsin, which activates viral binding to the cell culture.
After three or four days of culture influenza virus is detected by hemagglutination test (virus indication). If the result is negative, a second passage through the fresh culture is performed.
Virus identification is accomplished by hemagglutination inhibition test with subtype-specific reference antisera to most prevalent viral strains. Also it can be made by neutralization of viral cytopathic effect.
Serological method is used for retrospective influenza infection diagnostics to confirm the identification data of viral type and subtype. Antibodies to H-, N-, or M-proteins and viral nucleoprotein NP are produced in patients with influenza.
For diagnosis confirmation paired sera tests are required because the patients can maintain some anti-influenza antibodies level due to previous influenza infections. Hemagglutination inhibition test and ELISA are mainly used. Positive test relies upon the fourfold rise of specific antibody titers.
For identification of a new type of influenza virus the antibodies to viral NP antigens are evaluated. The previously mentioned reactions, as well as complement fixation test and neutralization test are used.