To our knowledge, this is the first study to compare the lung function of school-age children with a history of severe bronchiolitis associated with viral coinfection vs. single-viral infection. Our results show that the coinfection group had higher lung function values (FEV1, z-score FEV1 and z-score FEV1/FVC values) than the single-infection one at 7-9 years, considered as a whole. However, when analyzing the viral subgroups, this difference holded only in cases of single-HRV infection, but not in the single-RSV ones. In fact, when lung function of patients with viral coinfections were compared with those with single-RSV infections, no significant differences could be found. In contrast, almost all pulmonary function values of the single-HRV group, both pre and post bronchodilator, were significantly lower than those of coinfections. Also, the likelihood of having FEV1< 80% was up to 15 times higher in the single-HRV group compared to the single-RSV one and up to 8 times higher than in the coinfection group.
Previous studies have analysed the association between RSV bronchiolitis and pulmonary function sequelae, although there has been little standardization between studies, with contradictory results [8–10, 27–30]. A recent systematic review  including 31 studies, whose primary outcome was the evaluation of long-term pulmonary sequelae measured by pulmonary function test, in children with previously RSV respiratory infection during the first 3 years of life, found no association between RSV infection and abnormal pulmonary function in 13 studies, while 16 did reported this association. Many of the studies included in that review enrolled small numbers of participants, only five studies included more than 100 patients, many of them used different pulmonary function techniques and reported different pulmonary function indices. But nevertheless, their results reflect varying abnormal pulmonary function when compared with infants without bronchiolitis, being the most commonly described, obstruction to airflow or obstructive airways disease with or without bronchodilator reversibility. Most studies included in that systematic review compared pulmonary function in children with previously RSV bronchiolitis with a control group of healthy infants. This is not the case in our study, in which the aim was to compare lung function in children admitted for bronchiolitis with single versus doble or multiple viral infection and not versus a control group of healthy children. We report here, for the first time, that children hospitalized for single-HRV bronchiolitis have significant lower lung function values, at 7-9 years, than those admitted for bronchiolitis with viral coinfection or with single RSV-infection. In fact, single-HRV-bronchiolitis was the only risk factor independently associated with FEV1 < 80% The sample size of our study was calculated to compare the lung function of viral-coinfection bronchiolitis with single-infections, but not to compare the different single-infections with each other. For this reason, other single respiratory infections could not be compared and larger studies are needed.
Guilbert et al.  in a longitudinal cohort of children at risk of asthma, prospectively explored the relationships among early life virus-specific wheezing, childhood lung function, and asthma from 4 to 8 years of age. They provided novel evidence that early HRV-wheezing illnesses in high-atopy risk infants are related to lower lung function in childhood, with significantly lower FEV1%, FEV0.5, FEF25-75, FEV1/FVC and FEV0.5/FVC compared with children without HRV-wheezing illnesses. In contrast, children with RSV-wheezing illnesses did not have significant differences in any of the measured spirometric indices when compared with children who did not wheeze with RSV. These relationships were less pronounced but were still significantly different after administration of bronchodilator and thus, are not likely explained by increased airway tone alone. Our results also evidence that children with early single-HRV bronchiolitis have significantly lower FEV1 (%), lower z-score FEV1, both pre- and post-bronchodilator compared with single-RSV and with viral coinfections. However, whereas children included in Gilbert´s study where part of a birth cohort of high-risk atopic children followed-up on an outpatient basis, with mostly mild respiratory infections in the first 3 years of age, all of our patients were hospitalized infants < 24 months of age and therefore, with more severe bronchiolitis. As Guilbert et al.  argued, although they did not find evidence that RSV-wheezing illnesses were associated with reduced lung function, few of their study participants had severe illness requiring hospitalization. It is possible that more severe RSV illnesses could lead to significant reductions in lung function. In our case, as previously published, similar severity regarding the acute episode of bronchiolitis was found between coinfections and single infections, with no significant differences in oxygen therapy requirement or length of hospital stay. Therefore, the different respiratory morbidity observed at 7-9 years of age cannot be attributed to greater or lesser severity of acute bronchiolitis and according to our results, could be more related to the different viral etiology of the acute episode.
With respect to atopy, in the prospective multicentre observational EuroPrevall-iFAAM birth cohort study, the median prevalence of allergic rhinitis in 10 563 school-age children was 13.3%. Studies on the prevalence of atopy among children with history of lower respiratory viral infection (LRTI) have shown contradictory results. Some studies have found an increased risk of allergic sensitization after LRTI in childhood , other reported protection against allergic sensitization through the stimulation of Th-1 cytokine production , whereas other studies did not find any influence of childhood viral LRTI on the risk of subsequent atopy . The results of a recent systematic review and meta-analysis suggest that there is no association between LRTI at < 5 years and positivity of skin prick test or atopic dermatitis. In contrast, the overall analyses showed that there was a higher frequency of allergic rhinoconjunctivitis in children with history of LRTI in the first 5 years of age (OR = 1.7 [95%CI = 1.1–2.9]) . Ho et al.  also found that bronchiolitis before the age of two years, was independently associated with an increased risk of allergic rhinitis in 6–8-year-old children in Taipei. Our results show a global prevalence of allergic rhinitis of 34% in children previously hospitalized for bronchiolitis, considerably higher than the observed in 9623 6-7-year-old Spanish children in the GAN Phase I survey, with figures ranging from 12.5 to 18.8% , suggesting that there is undoubtedly an association between the two entities, which should be further studied. On the other hand, we also describe different prevalence of allergic rhinitis depending on the viral etiology of the acute bronchiolitis, with single-HRV infections having the highest rate of allergic rhinitis (52%) at 7-9 years of age, compared with single-RSV (31%, p=0.07) or with coinfections (17%, p=0.004). It is worth noting that single-HRV patients had the highest rate of allergic rhinitis, and that the likelihood of current asthma at 7-9 years in our cohort was 4 times higher in children with allergic rhinitis. Also, the lowest values of pulmonary function at 7-9 years of age were also found in single-HRV infected children. All these data strongly suggest that children with severe single-HRV-bronchiolitis have the highest risk of presenting allergic rhinitis and lower lung function values at 7-9 years of age when compared with single-RSV infections or with coinfections.
The mechanism of the association of bronchiolitis, mainly that associated with single-HRV infection, with allergic rhinitis is not yet well known. Korppi et al.  in a prospective post-bronchiolitis follow-up, showed that IL33 rs1342326 polymorphism was independently associated with allergic rhinitis and could also be associated with severe childhood asthma at school age. Our group, in a previous study , detected nasal IL-33 cytokine secretion in infants < 2 years of age hospitalized with bronchiolitis, with significantly higher detection rate in patients with HRV-infection, who also showed higher nasal concentrations of IL-33 than non-HRV-infants. In other recent study by our group, we also found high type 2 innate lymphoid cells (ILC2), and higher nasal levels of IL-33, TLR3, IFNG, IL10 and FLG mRNA in infants hospitalized with bronchiolitis . IL-33 plays an important role in T helper (Th) type 2 immunity activating, for example, Th2-type lymphocytes, mast cells and eosinophils leading finally to allergic diseases such as allergic rhinitis . IL-33 have been constantly found in nasal and lung epithelium and in serum, sputum and bronchoalveolar lavage of adults with asthma . In view of all these data, it could be hypothesized that early viral lower respiratory tract infections, mainly HRV-infections, could enhance the release of different cytokines, such as IL-33, that activates the innate type-2 cells (ILC2) and type-2 helper (Th2) cells through IL-1RL1, leading to amplified production of cornerstone type-2 cytokines and could partly explain the higher rate of allergic rhinitis and current asthma found in our single-HRV-bronchiolitis patients.
The fraction of exhaled nitric oxide has been suggested as a non-invasive biomarker of eosinophilic inflammation . Although some authors found that FeNO measurements in infants with recurrent wheezing episodes were associated with persistence of wheezing through age 3 years , others like Mikalsen et al.  found no differences between 11-year-old children hospitalized for bronchiolitis and the control group. FeNO, in the study of Mikalsen et al. was associated with atopy, but not with asthma in both groups. Our results also showed no differences regarding FeNO levels between coinfection and single infection groups. By contrast, children with allergic sensitization had significantly higher FeNO values than not sensitized ones. No child with asthma but without allergic sensitization had elevated FeNO. These results, in line with those obtained by Mikalsen et al.  suggest that only children who develop atopic asthma have eosinophilic airway inflammation, translated by elevated levels of FeNO.
According to our results, the probability of developing asthma at age 7-9 years was almost twice as high in children with coinfection (31%) than in those with single-infection considered as a whole (18%), being coinfection an independent risk factor for current asthma at school age. In addition, patients with viral coinfection showed greater respiratory morbidity, with higher rate of admissions for asthma, higher rate of treatment with the combination inhaled corticosteroid/long-acting β2-agonist (ICS/LABA), usually prescribed for moderate/severe asthma, and persistence of symptoms in the intercrisis periods. All these data strongly suggest that coinfections seem to be associated with increased respiratory morbidity, at least until the age of 7-9 years, when compared with single-infections. However, as was observed for pulmonary function, when coinfections were compared only with single-HRV infections, no significant differences were found in terms of frequency of current asthma or chronic anti-asthma treatment, although the likelihood of been admitted for asthma were almost 3 times higher in the coinfection group. In contrast, the frequency of current asthma in single-RSV children was 5 times lower than in children with coinfection and 4 times lower than in children with single-HRV infection. Our data suggest that bronchiolitis associated with single-HRV or viral coinfection pose a significantly higher risk, not only for the development of asthma, but also for more severe asthma, than single-RSV bronchiolitis. Hyvärinen et al.  reported in 2005 for the first time, the link from HRV- induced wheezing in infancy to emergence of childhood asthma. Since then, the role of HRV in bronchiolitis and the subsequent development of recurrent wheezing and asthma has been described [48, 49] and this association still remained significant in children ≥10 years . Recently, Hasegawa et al.  found that the risk of developing recurrent wheeze differed by the causative virus of the bronchiolitis, with higher risk for children with rhinovirus C infection compared to RSV. As previously mentioned, we could not compare the role of different types of HRV because our sample size was not calculated to achieve this goal. On the other hand, Lukkarinen et al. , in a follow up study in 127 children, found that HRV-induced first wheezing episode predicts atopic but not nonatopic asthma at school age. In contrast, in our cohort, single-HRV bronchiolitis remained associated with asthma even in children without allergic sensitization.
Our study has potential limitations. Firstly, the study did not have information from healthy controls because our objective was to compare the role of different viral etiologies in early infancy on the development of asthma among infants with severe bronchiolitis. Also, the sample size was calculated to compare the lung function at 7-9 years in children previously admitted for bronchiolitis with single infection or co-infection and therefore, some of our findings, not related with this goal, should be confirmed in larger studies with enough statistical power. Finally, the number of single infections with viruses other than RSV or HRV is not very large, due to the high rate of coinfections among some viruses like HBoV.
The strengths of the study are the prospective design and longitudinal follow-up. Only hospitalized patients with bronchiolitis younger than 2 years were included. At admission, RT-PCR to identify 16 respiratory viruses were performed in nasopharyngeal aspirate and clinical data were prospectively recorded. At follow-up, a standardized and validated clinical questionnaire (ISAAC questionnaire) was used and lung function as well as FeNO measurement could be performed in a high percentage of patients. On the other hand, specific tests (SPT) were done to confirm allergic sensitization.
In summary, according to our results, the respiratory morbidity at 7-9 years after severe bronchiolitis associated with viral coinfection or single-HRV infection is significantly higher than single-RSV infections. Single-HRV bronchiolitis is independently associated with lower lung function values. Clinical studies attempting to identify the young children at increased risk for HRV- bronchiolitis are critical to the development of therapeutic and prevention strategies.