Published ahead of print on June 30, 2005, doi:10.1164/rccm.200502-315OC
© 2005 American Thoracic Society doi: 10.1164/rccm.200502-315OC Rhinovirus Viremia in Children with Respiratory InfectionsAllergy Department, Second Pediatric Clinic, University of Athens, Athens, Greece; and National Heart and Lung Institute, Imperial College, London, United Kingdom Correspondence and requests for reprints should be addressed to Nikolaos G. Papadopoulos, M.D., UPC Research Laboratories, 13 Levadias, 11527 Goudi, Greece. E-mail: ngp{at}allergy.gr
Rationale: Viremia has been implicated in many viral infections; however, viremia due to rhinovirus (RV; rhinoviremia) has been considered not to occur in normal individuals. Objective: To evaluate whether RV enters the bloodstream and identify the possible risk factors. Methods: Nasopharyngeal washes (NPWs) of 221 children with respiratory infections were examined for the presence of RV by reverse transcription–polymerase chain reaction. Blood from 88 children, whose NPW was RV-positive, and 31 of RV-negative control subjects was subsequently examined for the presence of RV in the blood by semi-nested reverse transcription–polymerase chain reaction. Rhinoviremia was then correlated with clinical characteristics of the disease.
Results: RV was detected in the blood of 10 out of 88 NPW RV-positive cases (11.4%): 7 of 28 children with asthma exacerbations (25.0%), 2 of 26 with common cold (7.7%), 1 of 25 with bronchiolitis (4.0%), and 0 of 9 with pneumonia (0%). All NPW RV-negative cases were negative in the blood. The proportion of rhinoviremia in children with asthma exacerbation was significantly higher compared with children suffering from the other diseases (25 vs. 5%, p = 0.01). Significant risk factors were: sampling Conclusions: Viremia may occur during RV respiratory infections in normal children and is rather common in the early course of acute asthma exacerbations, suggesting that rhinoviremia may be involved in asthma exacerbation pathogenesis.
Key Words: asthma • common cold • respiratory infections • rhinovirus • viremia Although the majority of rhinovirus (RV) infections produce mild disease, most frequently common colds, it is now well established that RVs are also involved in acute asthma exacerbations and other airway diseases (1). Almost half of asthma exacerbations in school-age children are associated with RV (2), whereas in adults, RV may account for up to half of asthma-related acute care visits (3). Despite the fact that RV has traditionally been considered an upper airway pathogen, efforts to explain the RV-related lower respiratory symptoms have led to evidence suggesting that RV is able to infect the lower airways and locally induce inflammation (4, 5). The mechanisms by which RV may reach the lower respiratory tract are not clear, although they may include direct inhalation of infected droplets or spread of the virus from the upper airway (6, 7). Viremia has been implicated in the spread of other viral infections (8–11); however, it has been considered that viremia does not occur during acute RV infection in normal individuals. Rhinoviremia has been described in case reports in five instances: in two cases of sudden, unexpected infant death (12); in two older children dying of acute respiratory disease (13, 14); and in one with concomitant adenovirus disease (15). Nevertheless, no study has attempted to assess rhinoviremia using the currently available, sensitive, diagnostic methodologies. We hypothesized that rhinoviremia may occur in normal children during respiratory infections. To evaluate this hypothesis, we assessed the presence of RV in blood, using polymerase chain reaction (PCR), in children with respiratory infections due to RV, and attempted to identify risk factors in this cohort. Some of the results of these studies have been previously reported in the form of abstract (16).
Subjects The study was performed in the Second Pediatric Clinic, at the "P&A Kyriakou" Children's Hospital, University of Athens, Greece, and was approved by the hospital's ethics committee; consent was obtained from all parents. A total of 221 children (116 female, 105 male), aged 1 to 14 years (mean age, 3.9 years; range, 6 months to 14 years), with acute respiratory symptoms (upper respiratory infection, asthma exacerbation, bronchiolitis, pneumonia) and a small number (n = 6) of healthy children were enrolled in the study. Diagnoses were based on well-defined clinical and laboratory criteria (see Materials and Methods, E2, in the online supplement). Patient demographic information, clinical symptoms and their duration, and personal and family history of atopy and/or asthma were also recorded by doctor-administered questionnaires (17, 18). Children with any chronic disease or those chronically receiving medications (with the exception of asthma and/or other allergic disease and related treatments) were excluded. Nasopharyngeal washes (NPWs) (19) and venous blood were obtained. After removal of red blood cells by centrifugation, cell-containing plasma was kept at –70°C until used (see Materials and Methods, E1, in the online supplement).
Virus Detection
The detection limit of semi-nested reverse transcription–PCR, determined by amplifying serial dilutions of an RV-containing plasmid, was 3 copies/µl of cDNA. RV cDNA from paired NPW and blood was also measured by Taqman PCR (Applied Biosystems, Foster City, CA), as previously described (21). In this assay, the lower limit of detection was 10 copies/µl of cDNA.
Sequencing
Viral Culture
Statistical Analysis
Presence of Rhinoviremia A considerable proportion of children with respiratory symptoms were infected with RV (88/221, 39.8%). There were 28 of 43 children with asthma exacerbation (65.1%), 26 of 56 with upper respiratory tract infection (46.4%), 25 of 112 with bronchiolitis (22.3%), and 9 of 21 with pneumonia (42.8%). RV was present in the blood of 10 out of 88 cases (11.4%): 7 out of 28 of children with asthma exacerbations (25.0%), 2 of 26 with upper respiratory tract infection (7.7%), 1 of 25 with bronchiolitis (4.0%), and 0 of 9 with pneumonia (0%) were found to be positive for RV in both nose and plasma. These were further confirmed with Bgl I restriction digestion (20) and four positive pairs of NPW and blood samples were further confirmed with sequence analysis. Blood from all 31 children, whose NPW was negative for RV, and from all six healthy children was also RV-negative. Furthermore, RV was not detected in the sera of RV plasma-positive patients. Finally, single-round and Taqman PCRs were not able to detect RV in blood; plasma-positive samples could not be cultured in Ohio-HeLa cells.
Association of Rhinoviremia with Clinical Parameters
Age, presence of fever, family, and personal history of atopy and inhaled corticosteroid treatment were not significantly associated with the presence of RV in blood. The risk of rhinoviremia was decreased by 85% in cases sampled later than 24 hours from symptom initiation (> 24 vs.
Further analysis was performed in the subgroup of children with asthma exacerbations: children with rhinoviremia had more severe disease, assessed according to the Global Initiative for Asthma criteria (see Table E1) (22) and expressed as a significantly increased rate of moderate–severe exacerbations (86 vs. 33%, p = 0.03).
The data of this study suggest that viremia is not an uncommon event during RV respiratory infections in normal children, and is even more common during acute asthma exacerbations. This is the first study assessing rhinoviremia using currently available PCR-based methodologies, which have been validated and show high sensitivity and specificity (20, 23). In the small number of cases reported in the literature, rhinoviremia has been observed in children dying of acute respiratory diseases (12, 14, 24) or in those who died suddenly and unexpectedly (12). In these cases, viremia was assessed by virus culture in serum and lung tissues at necropsy. In our study, RV was detected in plasma but not serum of normal children, suggesting that the virus is intracellular. Rhinoviremia is likely of short duration, as it was mainly present during the first 24 hours from symptom initiation, suggesting that viremia probably occurs soon after infection. Among factors that could affect the antiviral response to RV and, consequently, viremia are disease localization (upper vs. lower respiratory system), viral load (which may correlate with disease severity [25]), or age. Although the numbers of patients in each group were small, the lack of difference in rhinoviremia incidence between cases of severe lower respiratory disease in infants (bronchiolitis) and cases of upper respiratory disease in older children suggests that disease localization and age are not important factors in determining risk of viremia. In contrast, rhinoviremia was found to be considerably more frequent in acute asthma exacerbations than in other respiratory diseases, as well as in children with a history of asthma, whether or not they were included in the study for this reason. This interesting association warrants further investigation: the possibility of increased susceptibility of individuals with asthma to viral, in particular RV, infections has been a subject of debate (26, 27). Of course, causality cannot be inferred by the results of this study. It is possible that the presence of RV in the bloodstream may activate immune cells and induce a systemic immune response, because it has been shown that RV enters and activates monocytes (28) or modulates expression of costimulatory molecules (29, 30), resulting in an acute asthma exacerbation. Alternatively, individuals with defective immune responses, permissive to asthma exacerbations, may also be more susceptible to RV infections. In the same line of reasoning, the finding that rhinoviremia was more frequent in severe rather than mild exacerbations may suggest that increased susceptibility to viral infection plays a role in inducing a severe exacerbation (25) and at the same time permits viral "leakage" to the bloodstream. Alternatively, subjects with increased susceptibility to the virus, possibly due to a defect in antiviral immunity may also have increased susceptibility to severe exacerbations. In both the above cases, it is possible that the presence of RV in the bloodstream may affect the outcome of viral infection, which could help explain the high prevalence of this agent in asthma exacerbations (2). The unique presence of viremia in the boys observed in our study requires confirmation, because it may be due to the small number of subjects. Viremia has also been implicated in the spread of many viral infections from the upper to the lower respiratory tract (9–11). Considering the possibility that this mechanism may occur in RV infections as well, of four methods used to detect the virus in this study, only the most sensitive semi-nested reverse transcription–PCR was able to detect RV in the blood. This suggests that only a very low number of copies are present; furthermore, it cannot be excluded that the genetic material detected may represent RNA molecules in the process of elimination and not live, replicating virus in peripheral blood. In summary, viremia occurs occasionally in normal children after respiratory infections due to RV, whereas it is detected significantly more frequently during acute asthma exacerbations and is related to exacerbation severity. The presence of RV in the bloodstream could be related to impaired antiviral immunity in asthma or may simply be a marker of increased severity. The possibility that rhinoviremia may play a role in the pathogenesis of acute asthma exacerbations requires further investigation.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Conflict of Interest Statement: None of the authors have a financial interest with a commercial entity that has an interest in the subject of this manuscript. Received in original form February 28, 2005; accepted in final form June 27, 2005
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ABSTRACT
METHODS
24 hours from symptom initiation, personal history of asthma, and male sex. Age, fever, family, and personal history of atopy did not affect the presence of RV in the blood. 
