PEDIATRICS Vol. 105 No. 1 January 2000, pp. 79-83

Eosinophilia at the Time of Respiratory Syncytial Virus Bronchiolitis Predicts Childhood Reactive Airway Disease

Daryl R. Ehlenfield, MD, Kathleen Cameron, and Robert C. Welliver, MD

From the Department of Pediatrics, State University of New York at Buffalo, School of Medicine and Biomedical Sciences, and the Division of Infectious Diseases, Children's Hospital of Buffalo, Buffalo, New York.

	ABSTRACT

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Objective.  Bronchiolitis in infancy is viewed as a risk factor for childhood asthma, but factors predicting which infants will have persistent wheezing have not been identified. In addition, the nature of the association between the 2 conditions is uncertain. We wished to determine whether eosinophil counts at the time of acute bronchiolitis predicted the presence of wheezing in later childhood.

Methods.  We retrospectively identified infants hospitalized with bronchiolitis, determined peripheral blood eosinophil counts at the time of bronchiolitis, and then contacted their families when they had reached 7 years of age.

Results.  Eosinophil counts at the time of bronchiolitis were greater in subjects who would have wheezing at 7 years of age (median: 98 cells/mm³) than in infants who would have no recurrent wheezing (median: 0 cells/mm³) or transient wheezing only up to 3 years of age (median: 0 cells/mm³). When the effects of family history of asthma, gender, and passive exposure to cigarette smoke were examined, only eosinophilia at the time of bronchiolitis demonstrated a statistically significant relationship to the presence of wheezing at 7 years of age.

Conclusions.  Eosinophilia at the time of bronchiolitis generally predicts the development of wheezing persisting into later childhood. Therefore, the association of bronchiolitis and childhood asthma seems more likely to be attributable to an immunologic anomaly that precedes the development of, or is induced by, bronchiolitis rather than to structural damage to the airway as a result of bronchiolitis.  Key words:  respiratory syncytial virus, bronchiolitis, childhood asthma, eosinophils.

Respiratory syncytial virus (RSV) bronchiolitis in infancy is frequently complicated by the development of recurrent wheezing in later childhood.¹ No factors have been identified that can predict accurately which infants will have recurrent wheezing. Also, it has not been clearly determined whether bronchiolitis in infancy causes long-term airway changes, which result in childhood asthma, or whether the occurrence of bronchiolitis indicates the presence of an underlying predisposition to wheezing both in infancy and in later childhood.

A previous study from this institution evaluated peripheral blood eosinophil counts in infants over 2 months of age with RSV infection.² That study demonstrated that infants of this age with upper respiratory tract illness alone at the time of RSV infection, and most infants with RSV bronchiolitis, had suppression of eosinophil counts in comparison to healthy infants and those with noninfectious illnesses. However a subset of infants with bronchiolitis, particularly males, had eosinophilia during acute bronchiolitis. Because elevated eosinophil counts are associated with childhood asthma, we undertook the present study to determine whether those infants who had detectable eosinophil counts during bronchiolitis were more likely to develop wheezing that persisted into later childhood.

	PATIENTS AND METHODS

Study Subjects

The study was approved by the institutional review board of the Children's Hospital of Buffalo. The population studied consisted of 43 infants hospitalized in the RSV seasons of 1991 and 1992 with acute bronchiolitis. Because infants <2 months of age generally have elevated eosinophil counts and maintain the elevation regardless of the form of RSV infection,² only infants 2 to 18 months of age were included. Infants with chronic underlying illnesses (including but not restricted to bronchopulmonary dysplasia, cystic fibrosis, congenital heart disease, and immunodeficiency states) were excluded from analysis, as were infants receiving corticosteroid therapy. All subjects were experiencing their first episode of wheezing, and all had RSV infection confirmed by the detection of RSV antigen in nasopharyngeal secretions using commercial direct immunofluorescence assays. Demographic features of study subjects are summarized in Table 1. Peripheral blood eosinophil counts were determined on the day of hospitalization. Both the percentage of eosinophils and the total numbers of eosinophils (percentage of eosinophils multiplied by the total white blood cell count) were recorded. In most cases, only a single estimation of eosinophil counts was available. In 4 cases, a second count was obtained, and varied by <10% from the original count.

Follow-up

Infants with RSV bronchiolitis were initially identified by consulting records of the virology laboratory. Medical records pertaining to the initial hospitalization were then reviewed to confirm the diagnosis of bronchiolitis and to determine eosinophil counts at the time of bronchiolitis. A total of 104 patients were identified who met the age criteria, and in whom complete blood counts and differentials had been performed. Once the subjects had reached 7 years of age, we attempted to contact parents or guardians by telephone. Responses were obtained from 43 (41.3%) families, the other 61 families having moved away or being otherwise unavailable. Parents then were asked whether the study subject had experienced any wheezing after the initial episode of bronchiolitis and at what age these episodes of recurrent wheezing had taken place. Offices of private physicians or emergency department records were consulted to confirm parental reports. Passive exposure to cigarette smoke and family history of asthma were documented in the medical record at the time of hospitalization for bronchiolitis and were determined again by telephone contact at the 7-year follow-up. All parents who reported smoking at the time of bronchiolitis had continued to smoke until the time of the follow-up telephone call, except in 1 case.

Definitions

Bronchiolitis was defined as the occurrence of wheezing of new onset at the time of RSV infection. All subjects were examined a minimum of 3 times during the hospitalization for bronchiolitis. Bronchiolitis also was considered to be present in the absence of wheezing if infants had hypoxia (oxygen saturation <97% as determined by oximetry) and hyperinflation on chest radiographs. A positive family history of asthma was defined as the presence of an immediate family member who had received bronchodilator treatment for asthma. Exposure to cigarette smoke was defined as having taken place if someone smoked at least 3 cigarettes per day in the home of the study subject. Exposure to cigarette smoke in day care centers could not be evaluated. Recurrent wheezing was classified as either transient or persistent; in all cases, recurrent wheezing was accepted as having occurred only if the subject had received bronchodilators for the wheezing episode. Transient wheezing after bronchiolitis was defined as wheezing occurring as late as 3 years of age but not later. Persistent wheezing was defined as the occurrence of wheezing within the year preceding the telephone call.

Statistical Analysis

Because eosinophil counts were not randomly distributed, comparisons of total eosinophil counts among groups were conducted using the nonparametric Mann-Whitney U test. Comparisons of groups with any or no eosinophilia (actually 1% vs <1% eosinophilia) and other comparisons of nominal variables were made using the Fisher's exact test. Statistical comparisons were completed using StatView 4.5 for Macintosh (Abacus Concepts, Berkeley, CA).

	RESULTS

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Determinants of Eosinophil Counts During Bronchiolitis

Peripheral blood eosinophil counts at the time of bronchiolitis are analyzed by family history of asthma, gender, and passive exposure to cigarette smoke in Fig 1. Infants from asthmatic families had a median of 49 eosinophils/mm³ (range: 0-352), whereas infants from nonasthmatic families had a median of 0 eosinophils/mm³ (range: 0-628; P = .52). Male infants had a median of 84 eosinophils/mm³ (range: 0-628), whereas females had a median eosinophil count of 0 cells/mm³ (range: 0-146; P = .021). Infants exposed to cigarette smoke also had a median eosinophil count of 49 cells/mm³ (range: 0-628), whereas infants not exposed had a median count of 0 cells/mm³ (range: 0-410; P = .52).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Factors associated with peripheral blood eosinophilia during bronchiolitis. Bars indicate median eosinophil counts in infants with bronchiolitis. Male infants with bronchiolitis had higher eosinophil counts (median: 84/mm3) than female infants (median: 0 cells/mm3). Eosinophil counts did not differ significantly in infants with a family history of asthma, compared with infants from nonasthmatic families. Eosinophil counts in infants exposed to cigarette smoke in the home were not different from those of infants from nonsmoking families.

Relationship of Eosinophil Counts and Recurrent Wheezing After Bronchiolitis

Figure 2 illustrates the eosinophil counts at the time of bronchiolitis in subjects who would later have either no wheezing episodes after bronchiolitis, transient wheezing through 3 years of age but not thereafter, or persistent wheezing through 7 years of age. Most infants who had no additional wheezing had no eosinophilia at the time of bronchiolitis. Only 5 of 23 (21.7%) of these subjects had eosinophilia, with a median of 0 cells/mm³. Of the 5 subjects who would have transient wheezing, 2 (40%) had eosinophilia with bronchiolitis (median: 0 cells/mm³). Fifteen subjects had persistent wheezing at 7 years of age; 9 (60%) of these had eosinophilia at the time of bronchiolitis (median: 98 cells/mm³). Eosinophil counts at the time of bronchiolitis were greater in those subjects who would go on to have persistent wheezing than in those who would have no wheezing after bronchiolitis (P = .030). In addition, eosinophil counts were greater in subjects with persistent wheezing than in the group of subjects who were not wheezing at 7 years of age (groups with no wheezing and transient wheezing combined; P = .046).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   The relationship of eosinophil counts during bronchioilitis to recurrent wheezing after bronchiolitis. Eosinophil counts were greater in infants who would manifest persistent wheezing through 7 years of age (median: 98 cells/mm3; far right column) than in infants with no recurrent wheezing (median: 0 cells/mm3; P = .030).

Factors Associated With Persistent Wheezing at 7 Years of Age

Figure 3 compares the effects on wheezing at 7 years of age of several factors recognized to be associated with childhood asthma. Neither passive smoking (P = .47) nor gender (P > .99) were associated with wheezing at 7 years of age. Of 12 infants from asthmatic families, 7 (58%) had wheezing at 7 years of age, whereas 8 (29%) of 31 children from nonasthmatic families also had wheezing at 7 years of age, a difference that falls just short of statistical significance (P = .074). The only factor that was associated in a statistically significant fashion with wheezing at 7 years of age was the presence of eosinophilia at the time of bronchiolitis. That is 9 of 16 (56%) subjects who had eosinophilia with bronchiolitis had persistent wheezing, whereas only 6 of 27 (22%) subjects without detectable eosinophilia during bronchiolitis had persistent wheezing at 7 years of age (P = .045).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   The relationship of various factors to the persistence wheezing to 7 years of age. Neither passive exposure to cigarette smoke nor gender was associated with wheezing in childhood. Of infants with positive family histories of asthma, 58% had persistent wheezing through 7 years of age, whereas 29% of infants from nonasthmatic families had persistent wheezing (P = .074). Of all infants with eosinophilia during bronchiolitis, 56% had wheezing at 7 years of age, whereas only 22% of infants without detectable eosinophilia had wheezing at 7 years of age (P = .045).

Predictive Value of Eosinophilia for Persistent Wheezing

The positive predictive value of eosinophilia for wheezing at 7 years of age was 56%. The negative predictive value of the absence of eosinophilia for the absence of wheezing at 7 years of age was 78%. The sensitivity of eosinophilia during bronchiolitis for detection of subjects who would have persistent wheezing was 60%, whereas the specificity was 75%.

	DISCUSSION

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The results of this study demonstrate that infants who have peripheral blood eosinophilia at the time of RSV bronchiolitis are more likely to have wheezing that persists until 7 years of age than are infants who are capable of suppressing eosinophilia during bronchiolitis. The association of eosinophilia with persistent wheezing could not be explained based on gender, family history of asthma, or exposure to cigarette smoke in the home, each of which has been associated with the development of childhood asthma. Eosinophilia with bronchiolitis was not explained by a family history of asthma. The risk for persistent wheezing was determined using any eosinophilia (in fact, 1% eosinophilia) as the cutoff for analysis. This corresponded to an eosinophil count of 84 cells per mm³, the lowest calculated count observed.

Three previous studies have evaluated the relationship of the quantity of eosinophil-cationic protein (ECP), a product of eosinophil degranulation, in serum at the time of bronchiolitis to the development of wheezing immediately after bronchiolitis with conflicting results. Reijonen and colleagues³ followed infants for 16 weeks after an acute episode of bronchiolitis and found that recurrent wheezing during this brief interval was more common in infants who had higher serum ECP concentrations at the time of bronchiolitis. Koller et al⁴ followed infants with bronchiolitis for 12 months after acute bronchiolitis and similarly found an association of greater ECP concentrations at the time of bronchiolitis with more frequent recurrences of wheezing during this limited interval. Neither of these studies followed subjects into later childhood. This is important in that many infants who have recurrent wheezing shortly following acute bronchiolitis will no longer have wheezing after 2 to 3 years of age.⁵ Thus, the relationship of ECP concentrations during bronchiolitis to childhood asthma could not be assessed in these studies of ECP. In a third study, Oymar and Bjerknes⁶ found no association of serum ECP at the time of bronchiolitis to the presence of recurrent wheezing in patients followed for an average of 3 years. The longer period of follow-up in the present study indicates that the association of eosinophil activation during bronchiolitis and recurrent wheezing continues at least through 7 years of age.

Martinez and colleagues⁷ studied peripheral blood eosinophil counts in infants at the time of their first episode of lower respiratory illness (LRI). These infants were then followed through 6 years of age. As in the present study, higher eosinophil counts at the time of initial LRI were associated with wheezing persisting to 6 years of age, but not with wheezing that ceased at 3 years of age. Therefore, the findings of the present study confirm those of Martinez and colleagues and extend them in several ways. First, only approximately two thirds of the subjects of the study by Martinez et al were diagnosed with bronchiolitis at the time of first LRI. Other subjects had an initial diagnosis of croup (10%-12%), pneumonia (4%-8%), bronchitis (6%-10%), or other respiratory conditions (3.5%-7%). Second, the study by Martinez et al did not determine whether eosinophilia was related to a family history of atopy or to gender. Third, their subjects were seen as outpatients and had a variety of viral agents identified as the cause of their initial LRI episodes. The subjects in the present study all had bronchiolitis attributable to RSV infection as the initial respiratory illness, and all were hospitalized. Therefore, the association of eosinophilia and persistent wheezing holds even in those infants with more severe forms of bronchiolitis, in which persistent airway damage as a result of the infection itself was perhaps more likely to occur.

The mechanism by which eosinophilia is maintained in those infants with bronchiolitis who will have persistent wheezing has not been investigated. Secretion of increased amounts of interleukin- (IL-) 4 and IL-5, which are chemotactic for eosinophils,^8,9 represents 1 possible mechanism. However quantities of these 2 cytokines do not seem to be increased in bronchiolitis.^10-13 In fact, the results of a study from this institution suggested that interferon- (IFN-), which is believed to oppose the activity of IL-4 and IL-5, was the predominant cytokine released into the airway during RSV-induced wheezing illnesses.¹³ In a more recent study (manuscript submitted), we found that macrophage inflammatory protein-1 (MIP-1), a chemokine that is also chemotactic for eosinophils, is present in higher concentrations in the respiratory secretions of infants with RSV bronchiolitis than in secretions from healthy infants or those with upper respiratory illness alone attributable to RSV infection. The release of MIP-1 is enhanced by IFN-. Therefore, certain infants may respond to RSV infection by releasing particularly greater quantities of IFN-, and subsequently, MIP-1. These individuals might then have higher eosinophil counts during bronchiolitis. It is also possible that eosinophils might be absent from the blood of infants with bronchiolitis because they have migrated into the lung or airway lumen. This is unlikely, because eosinophils have not been observed in lung tissues of infants with fatal RSV infection¹⁵ or in samples of nasopharyngeal secretions examined by ourselves (unpublished observations) or others.¹⁶ A third possible explanation for our findings is that infants with bronchiolitis have a reduced capacity for suppressing eosinophil counts during viral infections. There is no evidence available currently to confirm or dispute this possibility. An alternative explanation might be that infants with eosinophilia at the time of bronchiolitis have a reduced capacity for suppressing eosinophil counts during viral infections.

The association of eosinophilia with recurrent wheezing after bronchiolitis provides a general measure for predicting which infants with comparatively severe forms of bronchiolitis (those hospitalized with the initial bronchiolitis episode) will have persistent wheezing in later childhood. The study by Martinez and colleagues⁷ involved mostly outpatients, suggesting that the association is true for infants with milder forms of bronchiolitis also. More importantly the results seem to indicate that an immunologic mechanism, which causes the association of eosinophilia and wheezing, is already present at the time of infantile bronchiolitis, as it is at the time of childhood asthma. Although additional proof is required, these findings suggest that the association of bronchiolitis in infancy with childhood asthma is not attributable to damage to the airway as a result of RSV infection, but rather to a common immunologic anomaly that is either induced by RSV infection or which was present before RSV infection occurred. These findings, therefore, add to our knowledge of how asthma develops in childhood.

	FOOTNOTES

Received for publication May 18, 1999; accepted Sep 29, 1999.

Reprint requests to (R.C.W.) Children's Hospital, 219 Bryant St, Buffalo, NY 14222. E-mail: rwelliver{at}upa.chob.edu

	ABBREVIATIONS

RSV, respiratory syncytial virus; ECP, eosinophil-cationic protein; LRI, lower respiratory illness; IL, interleukin; IFN-, interferon-; MIP-1, macrophage inflammatory protein-1.

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