Nucleic acid detection assays based on reverse transcriptase polymerase chain reaction (RT-PCR) are now widely available for RSV, often in a multiplexed panel for detection of multiple respiratory virus pathogens. These tests are typically more sensitive than any of the virus isolation or protein-based detection assays used in the past. This enhanced sensitivity is especially helpful when testing adults, who often shed virus in very low titers. Positive RT-PCR tests need to be interpreted in the context of the clinical scenario, since the tests can remain positive for prolonged periods of time after infection, well beyond the period during which infectious virus can be isolated. Since children may experience symptomatic respiratory infections every few weeks during the winter, caution must be used in interpretation of positive PCR tests, especially when multiple viruses are detected simultaneously in a sample. In the past nasopharyngeal aspirates or nasal washes were preferred to nasal or throat swabs but the increased sensitivity of RT-PCR-based assays has increased the usefulness of some kinds of swabs.
Three other diagnostic methods are still in use:
Enzyme immunoassay for the detection of viral antigen. Using appropriate monoclonal antibodies as capture and detector antibodies, enzyme immunoassay can detect as little as 10 ng of RSV antigen, and EIA methods are readily automated in large diagnostic or reference laboratories. Rapid antigen-detection kits are commercially available, reliable and sensitive, taking 15 to 20 minutes from start to finish.
Immunofluorescence (IF) on exfoliated cells. This method had been a mainstay of direct diagnosis of respiratory pathogens for many years, but requires laboratory equipment and expertise and thus is unsuitable for large numbers of samples.
Isolation of the virus in cell culture. The extreme lability of respiratory syncytial virus makes it essential that the specimen is taken early in the illness and added to cultured cells without delay and certainly without preliminary freezing. Human heteroploid cell lines such as HeLa or HEp-2 are the most sensitive, and a CPE generally becomes visible in three to five days. Fixation and staining generally reveal extensive syncytia containing acidophilic cytoplasmic inclusions, but some strains produce only rounded cells. An absence of hemadsorption distinguishes RSV from all the other paramyxoviruses. Isolates can be allocated into groups A and B, further subdivisible into various subgroups, by enzyme immunoassay using monoclonal antibodies to the G protein.
Epidemiological surveys can be undertaken by measuring serum antibodies using enzyme immunoassay with recombinant-derived antigens, but serology is not customarily used for diagnosis.