With the availability of very sensitive molecular techniques to detect viral pathogens, questions can be addressed about the causality between the presence of microbial nucleic acids and a clinical syndrome in individual patients. To aid a better understanding of the significance of a positive PCR test and to improve the interpretation of diagnostic results, it is imperative to evaluate the detection of pathogens in asymptomatic controls, in addition to persons with clinical disease. To our knowledge this is the first prospective study using real-time PCR that has compared the presence of a comprehensive panel of respiratory viruses between young children with respiratory illness and those without symptoms.
Our study showed that the prevalence of respiratory viruses is high in asymptomatic children (27%), particularly in infants (44%). In previous studies respiratory viruses were detected at varying frequencies in asymptomatic children, depending on the different virus species tested and the age of the patient population studied. For example, Winther et al. (29
) found that picornaviruses were found in only 5% of asymptomatic episodes, while Garcia-Garcia et al. (8
) found a 20% carriage rate of hBoV, RV, and AdV in their control group. Studies using a large panel of respiratory viruses have reported higher prevalences; Kusel et al. (15
) reported a prevalence of 25% in prospectively followed infants during periods without symptoms, and Jartti et al. (12
) found at least one virus in 45% of asymptomatic infants younger than 1 year old.
The prevalence of specific viral species differed considerably between symptomatic and asymptomatic children. Among symptomatic patients, RSV was the most prevalent virus, but this virus was rarely detected among controls. This is in agreement with other studies showing that RSV is infrequently detected in asymptomatic individuals (5
), suggesting that RSV infection is usually associated with clinical illness and should be regarded as the causative pathogen when detected in a patient with respiratory symptoms. The very low prevalence of hMPV and AdV in controls compared to relatively high prevalences in cases in our study suggests that the same may be true for these viruses. Previous studies have shown that hMPV is common in asymptomatic adults but indeed is rare in asymptomatic young children (26
). However, for AdV, asymptomatic carriage in children has been reported to be as high as 30% (20
). The low prevalence among controls in our study may be explained by the sampling method or by adenovirus serotype (2
). Our study and other studies showed a prevalence of AdV of ≤4% in asymptomatic individuals when using NW (8
), while a study using throat swabs revealed a prevalence of 33% (20
). This difference may be related to the latent presence of AdV DNA in human tonsil tissues (9
) and indicates the importance of taking into account specimen type for virological diagnosis. Further studies comparing different specimen types and adenovirus serotypes in symptomatic and asymptomatic individuals are clearly needed.
In contrast to RSV, hMPV, and AdV, viruses such as RV, hCoV, and hBoV were frequently found in asymptomatic children. This suggests that a causal inference based on the detection of these viruses in symptomatic patients should be made with caution. Assessment of viral load could potentially aid the interpretation of positive test results. Indeed, virus loads were higher in cases than in controls. The magnitude of these differences varied between different virus species. For RV, InfA, and hCoV, median viral loads differed significantly between cases and controls. A similar trend was observed for InfB and hBoV, but this trend failed to reach statistical significance, perhaps due to the limited numbers of positive samples for these species. Few studies have compared quantitative PCR results between symptomatic and asymptomatic children. For RV, Peltola et al. did not find statistical differences in RV load in symptomatic versus asymptomatic persons (18
). However, the methodology of this study differed from our study, as it compared both adults and children and analyzed samples taken at different time points of infection.
When differences in viral load between symptomatic and asymptomatic children are present, it should be feasible to define cutoff levels for the various viruses to aid in the clinical interpretation of test results. InfA was the only virus for which no overlap was noted between viruses in cases versus controls. One could speculate, therefore, that cutoff values for influenza virus could easily be determined, but larger sample sizes of InfA-positive cases and controls are clearly needed for this. For viruses that show overlap of viral loads amid cases and controls, ROC curves are useful to define cutoff values, as they demonstrate the effects of different cutoff levels on test specificity and sensitivity. By using the depicted ROC curve for RV, one could inform a clinician that for RV loads of ≥104.5 copies/ml, this virus is very likely to be the cause of the presenting illness, while doubts concerning the etiology may remain for cases with loads of <104.5 copies/ml.
There are obvious challenges in determining clinically relevant cutoff values for respiratory viruses in different patient groups, as these will likely be influenced by factors such as the timing of sampling during the course of illness and the presence of comorbidities. In addition, challenges exist with regard to sample quality and standardization of sampling methods. For example, sampling of the upper respiratory tract will always show some degree of interpatient variation. We used NWs, which will results in variations based on dilution of sampling. However, dilution of NWs is likely never greater than 10-fold, based on the amount of mucus excreted in asymptomatic individuals (4
). Therefore, semiquantitation is still applicable in samples that can reach up to 1010
viral particles per milliliter (6
Our observations point to the need for a more nuanced look at diagnostic results and the potential value of quantitative results in interpreting the clinical relevance of test results. Larger samples sizes in a variety of different patient groups are clearly needed to overcome the above-mentioned challenges and to design reliable ROC curves for infections with viruses that are also found frequently among controls.
Regarding the presence of a respiratory virus in the upper respiratory tract in the absence of symptoms, this observation may be explained by several causes. First, detectable virus could represent the period of incubation before the onset of symptoms. In our study we did not find a higher frequency of symptom development during the following week in asymptomatic children who were virus positive compared to those in whom no virus could be detected. However, reliable analyses could not be performed for the less-prevalent viruses in our study, like InfA, InfB, and hBoV. Second, the detection of virus could represent postinfectious shedding. Duration of viral shedding differs greatly between different studies, varying between several days from the start of symptoms to several weeks after resolution of symptoms. Duration of shedding likely depends on virus species, clinical background of the patient, and the detection method used (11
). In a prospective birth cohort study, about 25% of infants shed respiratory viruses (RV, hMPV, and hCoV or mixed infection) for 3 weeks or more after clinical illness (19
). We included young children as controls if they had been symptom-free for at least 7 days prior to inclusion; hence, it remains possible that a proportion of detectable viruses represented long-term postinfectious shedding. A third explanation could be subclinical infections. Studies of adult volunteers challenged with InfA or RSV showed that 23% to 33% do not develop clinically manifest symptoms (3
). Although such human volunteer studies have not been performed in children, it seems likely that asymptomatic infections also occur in children, especially in cases of less pathogenic viruses, like RV and hCoV.
The most important limitation of our report is the cross-sectional character of the study. Since controls were sampled once and clinical follow-up was done in only a proportion of controls, it is difficult to determine the exact explanation for positivity in individual participants (e.g., post-viral shedding, subclinical infection, or incubation before symptomatic infection). Larger longitudinal studies are needed to fully address the causes and implications of respiratory virus detection in asymptomatic persons. Such studies will also help to define cutoff values for specific viruses to aid in diagnostic interpretation. Our data indicate that defining such cutoff levels may be feasible and represent the next necessary step toward reliable laboratory diagnosis of viral respiratory infections.