The most common cause of respiratory illness in children younger than 1 year of age is RSV infection.21 RSV infections most often occur during the late fall, winter, or early spring.22 Most children will have serologic evidence of RSV infection by 2 years of age.23
Clinical signs and symptoms of viral respiratory tract illness generally are not helpful in differentiating RSV-related from non–RSV-related LRIs.24,25 An accurate diagnosis of RSV infection depends on the ability to detect the virus in respiratory secretions. This article reviews the various methods currently available for the diagnosis of RSV, highlighting the advantages, disadvantages, and limitations of each method. A discussion of the possible effect that molecular amplification-based assays have on the seasonality of RSV is included.
A variety of laboratory methods are available to aid in the diagnosis of RSV infection in children and adults.20 These tests typically detect the presence of RSV in respiratory secretions. In general, children have higher concentrations of RSV in their secretions (several logs) than adults. Immunocompromised individuals and the elderly often have lower concentrations. Immunocompromised children may have intermittently high titers depending on their degree of immunosuppression, but tend to have prolonged shedding at lower titers.26,27 Currently available methods for diagnosis of RSV infections include cell (tissue) culture (CC); serology; and direct examination of respiratory secretions. This includes electron microscopy, indirect and direct immunofluorescent antibody tests, enzyme-linked immunoassays (EIAs), and nucleic acid amplification [eg, polymerase chain reaction (PCR)].20
For many years, CC was considered the gold standard for diagnosing respiratory viral infections. One advantage of CC is its ability to detect, although variably, viral coinfections. In addition, the isolated virus can be stored for future diagnostic studies. However, CC has disadvantages, including requirements for special specimen handling procedures to maintain viral viability, a long assay time, (average, 2–5 days), greater financial and labor costs per test, and relatively poor sensitivity compared with nucleic acid amplification testing (NAT). Even though recently developed diagnostic methods are more widely used, CC will remain important for analyzing a subset of each year's viral strains for genetic and antigenic change (eg, to look for new mutations that allow isolates to escape detection by antigen/PCR assays) and for discovering unknown viruses (eg, human metapneumovirus, Boca viruses, and new human coronaviruses). As the use of the CC assay decreases, so will the number of laboratories with the technical skills for tissue culture. Consequently, it may be necessary to develop regional centers for viral isolation to maintain the availability of technical skills and resources.
Serology usually is not helpful for diagnosis of acute respiratory virus infections because 10–30% of patients with documented respiratory viral infections will remain serologically negative.8,20 However, research-based serologic techniques do provide useful seroepidemiologic information.28
Most clinical laboratories use antigen detection assays to diagnose RSV infection, and several tests are available that will provide clinicians with the means for rapid diagnosis.24 Antigen-based assays, which have been available for more than 30 years, include indirect and direct immunofluorescent antibody tests, EIA, and optical immunoassays. Of the commercially available rapid diagnostic assays, EIAs usually have the best combination of sensitivity, specificity, and ease of use. However, the sensitivity and specificity of these tests vary according to the manufacturer, virus, and strain being detected, and adequacy of the specimen. No one specific assay has sufficient sensitivity and specificity to provide reliable results during the off-season (Table 1).29–39 Antigen-based methods are widely used because they are inexpensive, easy to perform, and easy to interpret. However, the assays currently approved by the Food and Drug Administration (FDA) continue to lack adequate sensitivity and specificity, especially when the prevalence of the virus is low (off-season) or in special populations, such as the immunocompromised or the elderly. Recent advances in laboratory diagnostic methods enable rapid multiplex point of care antigen detection for several respiratory viruses, including RSV. These methods allow the detection and differentiation of a number of respiratory viruses and have improved sensitivity (personal communication). However, antigen detection methods are often thousands of times less sensitive than molecular assays.
Molecular Assays (NAT).
Molecular assays have become the new gold standards for respiratory virus detection. They offer the advantage of being highly sensitive and highly specific (approaching 100%), when compared with CC or antigen assays.20,40 Studies comparing molecular assays to CC assays demonstrate significantly better sensitivity (12–50%) if a method for determining true positives is included in the assay (Fig. 3).
Hexaplex, the oldest commercial molecular assay for respiratory viruses, uses multiplex PCR to detect 7 or 8 of the most common respiratory viruses.41–44 Another assay, NRVA, (Nanogen Inc, San Diego, CA) is based on similar amplification strategies, primers, and probes, but uses a closed automated electronic microarray format to detect the 7 most common respiratory viruses.45
PCR technology has evolved to include novel chemistry and detection methods.20,40,46 Most of these methods are single- or dual-agent assays detected by gel analyses, enzyme hybridization, or assays that use novel fluorescent probes to enable real-time (RT) monitoring, such as Taqman, molecular beacon, eclipse, and scorpion probes (real-time RT-PCR).47 Although these assays are rapid (3–4 hours) and sensitive, their inability to be highly multiplexed (only 3 or 4 analytes) has limited their use for the diagnosis of common community-acquired respiratory viruses.
Recently, 3 highly multiplex assays were developed as research use only kits (EraGen Biosciences, TM Bioscience, and AutoGenomics Inc). All 3 assays rely on multiplex PCR for amplification of 16, 19, or 27 different respiratory viruses including RSV. EraGen Biosciences and TM Bioscience assays are partially open-format assays where different nucleic acid isolation platforms can be chosen if desired. Multiplex PCR followed by micro bead detection is performed on a cell sorting machine (Luminex Corporation). The technology is available, but additional work is required to create assays with improved sensitivity and specificity. Current research use only assays are being used for epidemiologic studies in many laboratories around the world but require an 8–9 hour turnaround time, use expensive equipment, and have sensitivities that are logarithmically less than those achieved with multiplex RT-PCR, enzyme hybridization, or single analyte real-time assays.
Factors Affecting Sensitivity and Specificity of the Assay
A number of factors may affect the sensitivity and specificity of an assay for viral detection. These include quality of the specimen, transport conditions of the specimen, quality of the reagents, laboratory technician experience, inter- and intralaboratory standardization, suitability of the assay to specific populations (eg, rapid antigen test in elderly versus young people) and prevalence of the agent in the community.
During the height of the RSV season, rapid antigen detection tests may be helpful in diagnosing RSV as the cause of lower respiratory tract disease, primarily bronchiolitis, among children. Among hospitalized children these tests may be useful adjuncts for infection control procedures. However, they are not routinely recommended by the American Academy of Pediatrics for use among children with bronchiolitis.48 The value and reliability of these tests for diagnosis among children with URIs is not clear. As previously noted, the advantages of these rapid antigen tests are their practicality and availability. They have good sensitivity and specificity, especially if used among normal children, and only if used during the peak of the virus season.
The value of RT-PCR as a detection method for RSV has now been demonstrated. However, there are no standardized FDA-approved assays, and variation among laboratories remains a problem. The sensitivity and specificity, as well as the predictive value of a positive or negative test must be established for each laboratory. Other disadvantages of RT-PCR include expense and its correlation with clinical findings. This is particularly problematic among immunocompromised patients. The correlation is better among hospitalized children presenting to the emergency department with an acute clinically compatible illness. However, the great sensitivity of the RT-PCR assays extends the time the virus may be detected in specimens over that by other methods. If a nonimmunocompromised child is RSV-positive by RT-PCR, it means that this child is acutely infected with RSV, has been ill recently with RSV, or is about to become ill with RSV. Almost all children are negative by RT-PCR after 14–21 days; but occasionally a child will remain positive for up to 4 weeks. During these longer periods of shedding detected by RT-PCR, the chance increases that another undetected viral infection may be present, especially among young children who have frequent viral infections during the respiratory season. Differentiation as to which is the cause of the acute illness may be aided in the future by the development of more readily available, practical, and cost- effective methods of quantitative RT-PCR.
Because of the small amount of viral antigen usually present in nasopharyngeal aspirates collected from RSV-infected adults, current antigen detection assays may lack sufficient sensitivity to detect RSV antigen. RT-PCR may be required to detect and diagnose RSV in adults.
Future developments in diagnostic tests should enhance our ability to rapidly, inexpensively, and easily determine the causative agent in suspected viral respiratory infections. Rapid diagnosis allows prompt implementation of the most effective and appropriate management to reduce morbidity, mortality, and healthcare costs. Until then, clinicians need to recognize the benefits and limitations of existing diagnostic tests.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
Respiratory syncytial virus; diagnosis of RSV; molecular assays; RT-PCR assays; antigen-based assays; serologic assays