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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