How is respiratory syncytial virus (RSV) testing performed?

RSV Testing: Diagnostic Methods and Specimen Collection

Nucleic acid amplification testing (NAAT/RT-PCR) performed on a nasopharyngeal swab or nasal aspirate is the gold standard for diagnosing RSV infection and should be used as the first-line test in all patient populations. 1, 2

Optimal Diagnostic Method

• NAAT/RT-PCR is the most sensitive diagnostic modality available, with sensitivity exceeding that of both viral culture and antigen detection methods. 1, 2
• Multiple FDA-cleared multiplex PCR platforms can simultaneously detect RSV along with influenza and other respiratory viruses, including FilmArray Respiratory Panel, ePlex Respiratory Pathogen Panel, and Xpert Flu/RSV XC. 1
• Real-time RT-PCR, multiplex microarray competitive DNA hybridization, nested multiplex RT-PCR, isothermal nucleic acid amplification, and loop-mediated isothermal DNA amplification are all acceptable NAAT methodologies. 1

Specimen Collection for Immunocompetent Patients

• Nasopharyngeal swab or nasopharyngeal aspirate/washing are the optimal upper respiratory specimens for RSV detection in patients with upper respiratory symptoms. 1, 2
• Throat swabs combined with nasal swabs are acceptable alternatives when nasopharyngeal collection is not feasible. 1
• Nasal swabs alone demonstrate nearly equivalent sensitivity to nasopharyngeal swabs when tested by RT-qPCR (93-95% detection rates with almost perfect agreement, κ > 0.8). 3
• Specimens should be placed in sterile containers or viral transport medium and transported at room temperature within 2 hours. 1

Critical Consideration for Immunocompromised Patients

In immunocompromised adults—particularly hematopoietic stem cell transplant recipients—lower respiratory tract specimens (bronchoalveolar lavage fluid or endotracheal aspirate) are significantly more sensitive than upper respiratory samples and must be obtained. 1, 2

• Lower respiratory tract testing sensitivity in immunocompromised adults: BAL 88.9%, endotracheal aspirate 71.4%, versus nasal wash only 15%. 1
• Upper and lower respiratory tract specimens show high discordance rates in adult HSCT recipients for most respiratory viruses, though RSV demonstrates 92% concordance—still, lower tract sampling remains superior. 1
• Upper respiratory tract testing alone frequently misses RSV infections in immunocompromised patients, making lower tract sampling mandatory when pneumonia is suspected. 2

Alternative Diagnostic Methods (When NAAT Unavailable)

Rapid Antigen Detection Tests

• Rapid antigen detection methods (direct fluorescent antibody stain or rapid immunoassay) provide results within 4 hours but have significantly lower sensitivity than NAAT. 1
• Antigen tests have poor sensitivity in adults (40-80%) due to lower viral loads compared to children and should not be used as the sole diagnostic tool. 2
• Sensitivity of rapid antigen tests in pediatric studies ranges from 83.5-91% with specificity 94.3-98.2%, but these figures do not translate to adult populations. 4, 5
• Negative rapid antigen tests should be confirmed by NAAT before ruling out RSV infection. 2, 6

Viral Culture

• Viral culture was historically considered the gold standard but requires 2-5 days for results and has been largely replaced by NAAT. 1
• Culture requires a dedicated virology laboratory and is less sensitive than NAAT, though it has higher clinical specificity for active disease. 1, 7
• Specimens for culture should be transported in sterile containers or viral transport medium at room temperature or ideally on wet ice within 2 hours. 1

When to Test for RSV

• Test adults presenting with new-onset respiratory symptoms (cough, nasal congestion/discharge, shortness of breath, sore throat) during RSV season, typically December through March in the Northern Hemisphere. 2
• Prioritize testing in high-risk populations: patients with chronic cardiac or pulmonary disease, immunocompromised individuals (especially transplant recipients), and residents of long-term care facilities. 2
• Test febrile infants ≤60 days old being evaluated for serious bacterial infection, as a positive RSV result reduces (but does not eliminate) the risk of concurrent bacterial infection. 8
• Test infants receiving palivizumab prophylaxis who develop bronchiolitis to determine if breakthrough RSV infection occurred, which would warrant discontinuation of further prophylaxis. 8

Common Diagnostic Pitfalls to Avoid

• Never rely on clinical features alone to distinguish RSV from influenza or other viral infections—laboratory confirmation is essential because clinical presentations overlap significantly. 2
• Do not accept negative rapid antigen tests as definitive in adults or when clinical suspicion remains high—confirm with NAAT. 2, 6
• In critically ill or immunocompromised patients, do not rely on single upper respiratory tract specimens alone—obtain lower respiratory samples for higher diagnostic yield. 2
• Avoid using serology for acute diagnosis of RSV infection, as high seroprevalence from prior exposure makes it useful only for epidemiological studies, not clinical diagnosis. 2
• Do not delay testing beyond 48 hours of symptom onset when possible, as viral shedding is highest early in illness and test sensitivity is optimal. 6