Influenza Diagnostics

Laboratory Tests and Ancillaries

Seasonal Influenza  

Laboratory tests may be appropriate to confirm the diagnosis of seasonal influenza. The sensitivity of any test will depend on the laboratory that performs the test, the type of specimen used, and the type of test used.  

Persons to be considered for testing during the influenza season include the following: 

• Outpatient immunocompetent persons of any age at high risk for complications of influenza (eg hospitalization, death) presenting with acute febrile respiratory symptoms within 5 days of illness onset
• Outpatient immunocompromised persons of any age presenting with febrile respiratory symptoms irrespective of time from illness onset
• Hospitalized persons of any age (immunocompetent or immunocompromised) with fever and respiratory symptoms, including those with a diagnosis of community-acquired pneumonia (CAP) irrespective of time from illness onset
• Elderly persons and infants presenting with suspected sepsis or fever of unknown origin irrespective of time from illness onset
• Children with fever and respiratory symptoms presenting for medical evaluation irrespective of time from illness onset
• Persons that develop fever and respiratory symptoms after hospital admission irrespective of time from illness onset
• Immunocompetent persons with acute febrile respiratory symptoms but not at high risk for complications secondary to influenza infection may be tested for purposes of obtaining local surveillance data

Persons who should be tested at any time of the year include the following:

• Healthcare personnel and residents or visitors in an institution experiencing an influenza outbreak who present with febrile respiratory symptoms within 5 days of illness onset
• Persons who are epidemiologically linked to an influenza outbreak (eg household and close contacts of persons with suspected influenza, returned travelers from countries where influenza viruses may be circulating, participants in international mass gatherings, cruise ship passengers) within 5 days of illness onset

Immunofluorescence screening test may be direct fluorescent antibody staining (DFA) or indirect fluorescent antibody staining (IFA) for influenza antigen detection. The results are available within a few hours of specimen submission. Immunofluorescence has slightly lower sensitivity and specificity than viral culture and is highly dependent on laboratory expertise and the quality of the specimen collected (ie must include respiratory epithelium cells). 

Rapid viral tests are office-based tests that detect influenza A and B viruses. They can diagnose influenza A and B in 10-20 minutes and may help in clinical management and infection control decisions. The lack of sensitivity of these tests limits their use during the influenza season within a community. If results are negative, viral culture or reverse transcriptase-polymerase chain reaction (RT-PCR) may be indicated. 

Reverse transcriptase-polymerase chain reaction (RT-PCR) is the preferred test for specimens from persons with a history of exposure to animals with possible influenza illness. It is useful for quickly differentiating between influenza types and subtypes. It is more sensitive than standard viral culture in detecting influenza and may be used as a confirmatory test. Nevertheless, it is not widely available for clinical use and is expensive. A positive result does not exclude SARS-CoV-2 infection and vice versa. SARS-CoV-2 nucleic acid detection assay is recommended, especially if suspicion of COVID-19 is high (eg high SARS-CoV-2 community prevalence or recent close exposure to a person with COVID-19). 

Viral culture is the most accurate test; however, it is impractical since antiviral therapy needs to be instated within 48 hours of symptoms. The results of the viral culture take 2 to 10 days and it can also be costly. It is valuable for monitoring antiviral resistance, identifying strains that may possibly cause pandemics, and formulating vaccines for the following year.  

Avian Influenza

Specimens for non-ventilated patients include throat and nasal swab specimens which should be collected preferably before antiviral treatment. For mechanically ventilated patients, specimens from the throat, nasal cavity, bronchoalveolar lavage, and endotracheal aspirates should be collected. 

Conventional and real-time RT-PCR is the primary method for diagnosing H5N1 and H7N9 virus infections using respiratory specimens. Results are already available in less than 5 hours.  

Immunofluorescence is a rapid and sensitive method for directly detecting the presence of avian influenza antigens in clinical samples. Specific positive immunofluorescence staining is characterized by intense intracellular apple-green fluorescence. It has a sensitivity of 70-100% and a specificity of 80-100%. It is the test of choice when fairly rapid results are required since results are already available in less than 24 hours. 

Hemagglutination inhibition tests (HAI) are used to type the patient antibodies to avian influenza virus when standard avian influenza antigen is available as reference material. It is considered positive if there is a fourfold increase in the H5 antibody titer. Results are available in approximately 2 to 3 days. 

Microneutralization test is a sensitive and specific assay for detecting virus-specific antibodies to avian influenza A (H5N1) virus in the human serum, and potentially for detecting antibodies to other avian subtypes. It is considered positive if there is a fourfold increase in the H5 antibody titer. Results are available in approximately 3 days. 

Rapid detection of viral antigens is designed to be performed under field conditions, point-of-care of patients, or at the bedside by non-laboratory-trained persons. It is directly detected from infected cells shed in the patients’ specimens. The rapid tests take 1 to 2 hours to perform. It has limited sensitivity for the detection of human cases of avian influenza and is not recommended for routine detection of avian influenza viruses. 

Viral culture is the standard reference method for avian influenza virus diagnosis. It has a sensitivity and specificity of 100%. It should only be performed in approved biosafety level 3 enhanced laboratory conditions by experienced personnel wearing appropriate personal protective equipment. Positive results are available in a few days while negative results take approximately 10 to 14 days.  

WHO recommends that laboratories with no capacity for diagnosis of influenza A viruses should send representative specimens from suspect cases of influenza A to one of the WHO Collaborating Centers for Influenza.