Discussion:
We compared BAL cytology in children ≤ 36 months with recurrent wheezing whose symptoms were uncontrolled on ICS therapy. We characterized them by their Asthma Predictive Index status and evaluated BAL culture results. All of our patients were receiving the same dose range (low to moderate dose) of ICS independent of API status. We found no significant difference in any cell-type percentages between positive and negative API groups. Although neutrophilic inflammation was seen in both API groups, there was a trend for it to be higher in API +ve children (median of 42% vs 16%, p=0.09); however, this difference was not statistically significant probably due to the small sample size in this retrospective study.
In our analysis, evidence of infection was common. Almost 60% of children had positive bacterial cultures, Thus, a significant proportion of our patients had persistent bacterial bronchitis (PBB); i.e. the presence of a significant count of a bacterial pathogen along with elevated neutrophils, to explain their uncontrolled inflammation and persistence of respiratory symptoms despite ICS therapy. PBB has been proposed as an important cause of persistent wheezing in young children as well as potentially implicated in triggering acute wheezy episodes10. Bisgaard et al23demonstrated that bacterial colonization in healthy neonates was associated with an increased risk of subsequent recurrent wheezing and asthma. We demonstrate similar findings and believe that bacterial infection of the lower respiratory tract can be responsible for persistent wheezing and unresponsiveness to conventional therapy, regardless of API status. Our findings suggest that the diagnosis of PBB should be addressed by obtaining BAL or sputum sample for culture or by empiric antibiotic treatment before proceeding to a higher dose ICS or adjunctive therapy, given good adherence to therapy and proper inhalation technique. The concomitant presence of viruses in the infected BAL may suggest an initial viral infection with abnormal clearance of the respiratory secretions and subsequent infection by the most abundant respiratory bacterial pathogens, although Bisgaard et al10 showed 40% carriage rate of viruses in the respiratory tract of asymptomatic children. We found a strong correlation between positive viral and bacterial cultures in our analysis (p=0.02) in contrast to the findings by Bisgaard et al10.
To differentiate between colonization and infection, we have chosen a cut-off value for bacterial colony count of ≥104CFU/ml for each pathogen to define infection. To avoid contamination of BAL samples by nasopharyngeal species, we followed a strict technique of not using the suction channel before complete wedging in a sub-segmental airway. Furthermore, the examination of nasopharynx (as part of the patient’s comprehensive airway evaluation) was performed after obtaining BAL samples. An interesting group of patients are those who had sterile cultures and normal BAL cytology (n=18, 41%), and were uncontrolled on conventional asthma therapy, with no difference by API status. The etiology of their symptoms remains unclear.
Our results are similar to those of Krawiec et al11with a generalized inflammatory response marked by elevation in total cell counts with no dominance of any cell type in a group of similar age in which they excluded positive bacterial cultures, and viral studies were not performed. However, not all patients had received ICS. In contrast, Marguet et al12 and Le Bourgeois et al13 reported no correlation between neutrophil counts and BAL cultures in a similar age group. In the former’s study, viral detection was not performed, and not all patients received ICS. In the latter study, viral and bacterial culture studies were not performed on all BAL samples. Le Bourgeois et al13 found no difference between atopic and non-atopic children ≤ 36 months (all on ICS). In another study that included an older sample of patients (up to 11.9 years) Najafi et al14 showed neutrophilic inflammation both with and without bacterial infection in the airways of wheezy children, however, a correlation existed. Our study confirms a strong correlation between neutrophilic inflammation and positive bacterial (p<0.001) and viral cultures (p=0.01) that is independent of API status. We also report normal BAL cytology for children with non-infected BAL fluid who are unresponsive to asthma therapy regardless of API status. One potential explanation for that could be the observation made by Martinez et al24 that diminished airway function present shortly after birth predicts the recurrent lower respiratory tract infections and recurrent wheezing in the first three years of life. We did not have pulmonary function testing studies for the children in this study and thus cannot evaluate if the non-inflamed, culture negative group represents the transient early wheeze group described in the Tucson Children’s Respiratory Study.
We found no increase in eosinophils in the BAL of uncontrolled wheezy preschool children in either API groups. Our definition of high eosinophil percentage was ≥2%. The role of eosinophilic inflammation in preschool children with asthma and wheezing has not been completely clarified, although it is considered important to guide anti-inflammatory therapy. Elevated BAL eosinophils are a common finding in adults and older children with asthma, which makes this finding in our study somewhat surprising. It is interesting that no difference was found comparing the API groups. We are unaware of any study looking at young preschool children reporting eosinophilic inflammation in their BAL. One study by Thavagnanam et al25 reported increased eosinophils in the BAL of children ≤ 36 months who later developed wheezing using a lower cut-off value to define high eosinophils (1.5%) that we consider normal rather than increased. Marguet et al12 found no eosinophils in the BAL of wheezy infants (regardless of ICS therapy), however, eosinophil percentages were high in the BAL of older wheezy children (median age 7 years). A similar finding was reported by Najafi14 et al for children who were not receiving anti-inflammatory therapy. Ferreira et al26 didn’t find elevated eosinophils comparing atopic and non-atopic children (mean age 4.7 years), half of them receiving ICS. Looking at children ≤ 36 months of age, Le Bourgeois et al 13 reported no eosinophils in BAL of atopic and non-atopic wheezy children unresponsive to ICS. When looking at endobronchial biopsies rather than BAL fluid cytology, Sagalani et al27 reported eosinophilic inflammation in the biopsies from wheezy preschool children (mean age 29 months, 62% on ICS) compared to their controls. Other studies have documented an increase in eosinophil cationic protein (ECP) in BAL of a younger pediatric population, although this did not correlate with atopy28. Whether the lack of eosinophils is due to the confounding effect of inhaled corticosteroid therapy, especially in severe uncontrolled wheezing, or is related to the method of sampling the airways (BAL sampling compared to bronchial biopsies) is unknown. However, it suggests that PBB rather than uncontrolled eosinophilic inflammation was responsible for continued symptoms in the API+ve group.
The utility of LLM as a marker of aspiration has been controversial since they can be elevated in several inflammatory conditions. However, similar to several studies 29, we found no correlation between LLM percentages and neutrophils. LLM didn’t correlate with other cell types in the BAL of our patients. LLM percentages didn’t differ by API status, culture status, or age.
Our study is limited by its small size and retrospective nature. Its small size leaves it underpowered to demonstrate a significant difference in neutrophil counts between the API groups, where interestingly, a trend toward higher counts in the API positive group was seen. The study population included only difficult-to-control children on ICS therapy independent of API status and may not reflect findings in steroid naive children. However, for ethical reasons, this will remain a challenge for future investigators and until a well standardized, non-invasive method to study airway inflammation in infants and young children is developed. No control group was evaluated in our study and findings were compared to normal values published in pediatric pulmonary literature10. All children were on mild-moderate doses of inhaled steroids, the effect of which cannot be controlled for given the ethical considerations that preclude stopping or delaying treatment in this group especially. Some data suggests no effect of ICS on BAL cytology comparing the treated and untreated children11,30. Also, molecular viral detection techniques such as RT-PCR were not used, which are more sensitive than viral cultures.
In conclusion, protracted bacterial bronchitis (PBB) plays an important role in the persistence of respiratory symptoms for children ≤36 months of age unresponsive to ICS therapy regardless of API status. This should be considered prior to increasing anti-inflammatory therapy in this group of children. There was no difference in BAL cell cytology comparing API negative and positive children ≤ 36 months old with recurrent respiratory symptoms that are unresponsive to inhaled corticosteroids. Neutrophilic inflammation correlates strongly with BAL viral and bacterial infections and is absent in non-infected lavages. Until a more standardized non-invasive method to investigate airway inflammation in young children is adopted, utilizing BAL fluid cytology will continue to be an important tool in the management of children with chronic recurrent wheeze.
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