The development of molecular techniques for the diagnosis and quantification of pathogens that cannot be cultured by traditional techniques has revolutionized the field of microbiology and infectious diseases. Certain organisms, such as RSV, can be grown in cell culture, yet the virus is relatively labile, and many investigators have begun to use RT-PCR to identify infection (30
). This is particularly true for adults, for whom viral culture is relatively insensitive (7
). Nested RT-PCR has been successfully used in epidemiologic studies of adults, with a sensitivity of 73% and specificity of 99% (6
). However, concern has arisen that new molecular techniques that detect minute quantities of viral RNA may not be associated with clinically meaningful illnesses and that residual nucleic acid may persist in the nasopharynx of infected individuals for long periods of time. Only one previous study has compared quantitative RT-PCR to viral culture for assessment of a respiratory virus over time (22
). In this investigation of six subjects with symptomatic influenza virus infection, virus could be detected for up to 7 days with PCR compared to 1 to 2 days with viral culture. The present study helps to define the meaning of quantitative RT-PCR in relation to viral culture for RSV and addresses the issue of low-level RNA persistence following infection.
The overall correlation of quantitative RT-PCR and viral titers was excellent. The pattern of viral shedding was quite similar by either method, although subjects tended to remain RT-PCR positive several days after virus was no longer isolated by culture. However, we did not find that subjects had detectable viral RNA for a prolonged period after becoming culture negative. Only one subject was RT-PCR positive on day 12 by quantitative PCR (limit of detection, 1.0 PFU). Even using the more sensitive method of nested RT-PCR (limit of detection, 0.1 PFU), only 4 of 12 subjects were positive on day 12, and all were negative on day 28. These results indicate that a low level of RNA does not persist in normal healthy adults. We conclude that the detection of viral RNA reflects active viral replication, although it will be important to study elderly and immunocompromised adults to determine if the same is true for these groups as well. Interestingly, 10 of 13 subjects were RT-PCR negative on day 1 by quantitative methodology, and 7 were negative by nested RT-PCR. These samples were collected only 24 h after inoculation of 104.7 TCID50, indicating the viral RNA can be rapidly cleared in the absence of virus replication.
Evaluation of subjects in a challenge study is very different from the evaluation of ill patients. In the setting of natural infection, specimens are rarely handled with precision, and transit times and conditions can be quite variable. In a previous study of patients infected with RSV who were ill for an average of 5 days at the time of evaluation, RT-PCR sensitivity was 73%, compared to 39% for culture (6
). Assuming a 3-day incubation period, subjects were typically cultured approximately 8 days after exposure. This time period corresponds to the days when the correlation between RT-PCR and culture declined in the present challenge study. Thus, it seems most likely that the discrepancy between culture and RT-PCR in natural infection is the result of both variable specimen processing and the time point during the course of illness when people seek medical attention. It is also possible that wild-type RSV isolates are more difficult to grow in cell culture than RSV A2, a tissue culture-adapted strain.
In vitro neutralization of virus by preexisting nasal antibody could be a factor in the difficulty in isolating virus in adults or children with RSV reinfection. Notwithstanding some variability in nasal IgA titer due to collection methods, it did not appear that high nasal IgA titers were not consistently associated with a discrepancy between culture and RT-PCR positivity. Correction of nasal IgA titers by total protein or urea nitrogen was not possible, because the nasal wash specimens were placed into viral transport medium, which contains protein.
Many questions about the pathophysiology of RSV disease in both children and adults exist. There are few studies of infants and none of adults that have addressed the issue of severity of illness and viral load, and those that do offer conflicting conclusions (1
). In infants, hospitalization for severe illness typically occurs at the point at which titers of culturable virus are declining, raising the possibility that disease is mediated by the immune response rather than viral cytotoxicity. Little data exist regarding the presence and quantity of RSV in the lower airways in adults or older children with severe disease. In the elderly, it is not clear whether severe disease is due to associated comorbid conditions or whether immunosenescence leads to prolonged and greater viral replication. Until now, many of these questions could not be addressed due to the difficulties of viral quantification by culture. Quantitative RT-PCR appears to be an excellent tool for future studies to address these important questions.
In summary, quantitative RT-PCR has an excellent correlation with viral titers, as measured by culture when specimens are obtained under optimal circumstances. Detection of RSV by RT-PCR indicates recent viral replication, and viral RNA does not persist for long periods following infection.