Respiratory Virus Direct Detection by Enzyme Immunoassay (EIA) and Direct Fluorescent Antibody (DFA) Tests


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Subject: Respiratory Virus Direct Detection by Enzyme Immunoassay (EIA) and Direct Fluorescent Antibody (DFA) Tests

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  • Tests for the direct detection of respiratory viral pathogens, like influenza, RSV and human metapneumovirus; they may provide results significantly sooner than results from respiratory virus culture or molecular diagnostic testing and serve a critical role in early patient management and infection control activities. EIA tests have only moderate sensitivity, but high specificity, for detection of influenza virus infection; they are commonly used for screening. DFA tests have high sensitivity and specificity compared to respiratory viral culture, and they may be a cost-effective definitive diagnostic technique.


  • EIA and DFA tests are commonly used to provide early screening for influenza virus infection. Assays vary in terms of sensitivity; specimens with negative results by EIA should be submitted for sensitive testing, like molecular assays, for patients at risk for complicated respiratory viral infection.

  • Method:

    • EIA: There are a number of kit formats for EIA tests. Antibodies directed at specific virus antigens are immobilized on the surface of a membrane of a test device. The specimen is added to the reaction surface, allowing virus antigen in the specimen to react with the bound antibodies. After washing, a second tagged virus-specific antibody reagent is added. After washing away excess detection antibody, a label-specific detection reagent is added and the test read as positive or negative.

    • DFA: Cells collected by nasopharyngeal swab or wash are fixed onto a microscopic slide. The slide is dried, fixed, and stained with a reagent containing labeled antibodies directed against specific virus antigens. The label is typically fluorogenic. Stained slides are examined by fluorescence microscopy using excitation and barrier filter appropriate for the specific fluorogenic label.

  • Special collection and transport instructions: Specimens are collected as recommended for samples for viral culture. Nasopharyngeal specimens, especially nasopharyngeal wash specimens, typically provide specimens with the greatest sensitivity for detection of infected patients.

  • Turnaround time: 24–48 hours. Some EIA kits allow testing with a turnaround time <4 hours.


  • Expected results: Negative.

  • Positive results:

    • EIA: The specificity of EIA assays is high; a diagnosis is established in patients with compatible signs and symptoms of influenza; additional diagnostic testing is generally not required.

    • DFA: Specimens showing a significant number of cells with 2+ or greater fluorescence are considered positive, establishing a diagnosis of influenza in patients with compatible signs and symptoms. Slides must be examined to ensure that the specimen contains enough respiratory epithelial cells to provide informative testing. Laboratories should establish a lower limit of cells present below which the test is considered uninterpretable. Specimens that demonstrate only few, faintly staining cells should be considered equivocal; repeat testing may provide clear positive or negative results.

  • Negative results:

    • EIA: A negative result does not exclude respiratory virus infection.

    • DFA: A cellular specimen without staining by the labeled reagent is reported as negative. Infection with the specific viral pathogen is unlikely.


  • The sensitivity for different commercially available EIA tests is variable. Sensitivities commonly range from 50% to 80%. Clinically, test performance depends on the type of specimen and quality of specimen collection. Only specimen types acceptable for the kit, collected according to kit instructions, should be accepted.

  • The PPV of antigen detection tests depends on the prevalence of viral pathogen in the region. Testing results should be interpreted with caution, if performed at all, during periods when there is a low prevalence of the pathogen circulating in the region.

  • Common pitfall: Poor test performance may be seen when specimens are submitted that have not been validated for the platform/kit used for testing. Anterior nasal swabs, for example, may be submitted instead of nasopharyngeal swabs, resulting in an increased number of false-negative results.