Lung function and bronchodilator response in 4-year-old children
with different wheezing phenotypes
Ellie Oostveen, Sandra Dom, Kristine Desager, Margo Hagendorens,
Wilfried De Backer, Joost Weyler
Online Depository
Methods
Study design and study population
The Prospective Cohort on the Influence of Perinatal Factors on the Occurrence of
Asthma and Allergies (PIPO) study is a prospective birth cohort study involving
1128 children (1). Between June 1997 and December 2001, pregnant women in the
Antwerp region in Belgium were invited to participate by completing a screening
questionnaire. Data on demography, respiratory symptoms and risk factors for
asthma were collected through the use of postal questionnaires completed by the
parents: the first questionnaire covered the first year of life, with subsequent
questionnaires at 6-month intervals up to the age of 4 years. All questionnaires were
based on validated questionnaires from the International Study of Asthma and
Allergies in Childhood (ISAAC) (2). In addition to the questionnaires, the children
were invited to participate in a medical examination at the University of Antwerp
Hospital at the ages of 1 and 4 years. Both the parents and the participating child
were asked to provide a blood sample for allergy testing.
At the age of 4 years, the children were also invited for lung function testing by the
forced oscillation technique as part of the medical examination.
Atopic sensitisation
Total and specific IgE levels were assessed from blood samples from the children
and their parents, taken when the children were aged 1 and/or 4 years. IgE was
determined via a fluorescence enzyme immunoassay technique (CAP FEIA systems,
Phadia, Brussels, Belgium). In the 1-year-old children, specific IgE against
1
Dermatophagoides pteronyssinus, cat, dog, hen’s egg and cow’s milk were determined,
while in the 4-year-old children, specific IgE levels against birch pollen and timothy
grass pollen were also quantified. In the parents, specific IgE levels against 7
common inhalant allergens (D. pteronyssinus, cat, dog, birch pollen, timothy grass
pollen, mugwort pollen and Cladosporium herbarum) were quantified. Specific
IgE levels ≥ 0.35 kU.L-1 were considered positive. Atopic sensitization of the
child/mother was defined as at least one positive specific IgE at the age of 1 or
4 years.
Definitions of wheezing phenotypes:
Symptoms of wheeze were assessed through the answers to the core questions from
the International Study of Asthma and Allergies in Childhood questionnaire
(wheeze during the past 6 (or 12) months and the number of wheezing episodes) (2).
On the basis of the history of wheeze reported by the parents, the children were
divided into the following wheezing phenotypes (3), (4):
-
‘never wheeze’: children who had never wheezed during the first 4 years of life;
-
‘early-transient wheeze’: at least 1 wheezing episode during the first 3 years of
life, but no wheezing in the fourth year;
-
‘late-onset wheeze’: no wheeze in the first 3 years of life, and at least 1 episode
of wheeze in the fourth year.
-
‘persistent wheeze’: at least 1 episode of wheeze in the first 3 years, and at least
1 episode of wheeze in the fourth year.
2
Forced oscillation technique
A custom-made forced oscillation setup was used to measure the respiratory
impedance (Zrs) in the 4-year-old children (See Figure 1). The setup and Zrs
measurements met the requirements of the ATS and ERS recommendations (5, 6). A
loudspeaker generated a pseudorandom noise excitation pressure in the frequency
range 4 to 32 Hz ( 2 Hz) with 1 cmH2O of amplitude. Pressure and flow signals
were measured with identical Honeywell pressure transducers and a Fleisch
no. 1 pneumotachograph. Since the breathing circuit was continuously flushed with
a bias flow, no bacterial filter was used. The setup including the mouth piece had a
dead space of 35 mL. The measured impedance data were corrected for the
impedance of the pneumotachograph. Pressure and flow signals were low-passfiltered and recorded for 16 s, time-averaged with time blocks of 0.5 s, and Fourieranalyzed to obtain Zrs as the complex ratio between the pressure and the flow
signal. At least 5 measurements were collected, the child coming off the mouthpiece
in between the measurements. In the event of large within-test variation, Zrs data
were re-analyzed retrospectively. The average of 3-5 acceptable Zrs data was used
for further analysis. Reasons for the discarding of data were air leakage, swallowing,
glottis closure during the actual measurement, or outlying data relative to the
average data set.
Power analysis
In a large Antwerp epidemiological study performed some 10 years ago (7), it was
observed that 20% of the 5-7-year-old children had experienced “ever wheeze”, 50%
3
of whom had had complaints of wheeze in the past twelve months. Our
extrapolation of these findings leads us to expect that 10% of the 4-year-old children
had experienced wheeze in the past 12 months.
n1: total population
n2: group of children with wheeze
n2/n1 =  = 0.1
Furthermore, we anticipate that 4-year-old children with wheeze have a > 10%
higher airway resistance relative to the normals (4) |1- 2|: hypothetical expected
difference between the groups; |1- 2| = 0.10) and that the variation of airway
resistance in healthy preschool children is ~20% [(8); R5= 1.03 ± 0.23 kPa.s.L-1
(population standard deviation:  = 0.2)]. Power (1-) = 0.80.
The formula (where a one-tailed = 0.05 was assumed)
(n1+n2) ≥ ((1+ )2/ ) × (z0.05 + z)2 × 2 /(1- 2)2 + (z0.052)/2
≥ ((1 + 0.1)2/ 0.1) × (z0.05 + z)2 × 2 /(1- 2)2 + (z0.052)/2
≥ 12.1 × 6.19 × 4 + 1.4
≥ 300
indicates that a total sample size of about 300 children should be sufficient to detect
differences in lung function between children with and without wheeze.
Data analysis
Exclusion of Zrs data
In some children, huge variations between the individual measurements were
observed in Zrs, and especially in Rrs. We arbitrarily defined a Zrs measurement as
4
reliable when the coefficient of variation (CoV= SD/mean value) of the Rrs data
points was < 15% in more than half of the data points of that particular test. With
this reliability criterion, we excluded the Zrs data from 6 children at baseline; postbronchodilator Zrs data were rejected in another 10 children.
Results
The success rate of FOT in these 4-year-old children was high: acceptable Zrs data
were attained in 95% of the children. The coefficients of variation (CoVs) of the
baseline respiratory resistance at 4, 6 and 8 Hz (R4, R6 and R8) for the overall cohort
(n=535) were on average 8.6% (SD 4.3), 7.7% (3.1) and 7.7 % (3.1), respectively. The
average CoVs of R4, R6 and R8 for the groups of children with different wheezing
phenotypes were not meaningfully different from each other and were always
below 10%. The within-test variabilities of the baseline reactance data at 4, 6 and 8
Hz (X4, X6 and X8) were significantly lower than those of R4, R6 and R8,
respectively (p<0.001 at all frequencies, paired t-test).
In more than one-third of the children who underwent lung function testing (see
Figure 2), the wheezing phenotype of the child could not be determined because
questionnaires had not been adequately completed or had not been returned. In our
study design, postal questionnaires were sent out every 6 months. Despite the small
numbers of children in the different wheezing phenotype groups, significant
differences were observed both in baseline respiratory function and in the
5
bronchodilator response between the children with different wheezing phenotypes.
At 4 years of age, the children with early-transient wheeze yielded higher baseline
resistance values as compared with those who never wheezed. The children with
persistent wheeze displayed a poorer baseline lung function than that of the children
who never wheezed or those with early-transient wheeze, and their bronchodilator
response was larger.
Adjustment for potential confounders
Mean resistance and reactance values at 4, 6 and 8 Hz and the area under the
reactance curve (AX, see text) were estimated by multiple linear regression (MLR)
analysis with the sex, age, weight, height, education level of the mother, atopy of the
mother, presence of siblings, exposure to parental smoking, pets, eczema, rhinitis,
atopy of the child, lower respiratory tract infections and antibiotics use at 4 years of
age as potential confounders. These analyses did not alter the results. Exclusion of
the children who used inhaled corticosteroids at 4 years of age (n=8) or who
presented with common cold at the test day (n=45) did not alter the results of the
analyses either. We also investigated whether atopy per se explains the group
differences in lung function. The separate MLR analysis where the atopy of the child,
the wheeze phenotype and the lung function indices were taken into account,
revealed that the associations between all the lung function indices and the wheeze
phenotype became even stronger on the addition of atopy to the model, except for
fres and fres, where the association was left unchanged.
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Table 1. Baseline resistance and reactance values and the absolute and relative
changes with respect to the baseline after bronchodilation in the groups of children
with different wheezing phenotypes
Baseline
R4
(hPa.s.L-1)
R6
(hPa.s.L-1)
R8
(hPa.s.L-1)
X4
X6
X8
AX
fres
(hPa.s.L-1)
(hPa.s.L-1)
(hPa.s.L-1)
(hPa.L-1)
(Hz)
Bronchodilation
R4
R6
R8
R4
R6
R8
X4
X6
X8
AX
fres
X4|
X6|
X8|
AX|
(hPa.s.L-1)
(hPa.s.L-1)
(hPa.s.L-1)
(% baseline)
(% baseline)
(% baseline)
(hPa.s.L-1)
(hPa.s.L-1)
(hPa.s.L-1)
(hPa.L-1)
(Hz)
(% baseline)
(% baseline)
(% baseline)
(% baseline)
Never
Early
Persistent
n=144
n=127
n=54
mean
(95% CI)
mean
(95% CI)
10.3
(9.9 , 10.7)
11.0
(10.5 , 11.5) *
11.9
(11.1 , 12.7)
***, †
9.8
(9.5 , 10.2)
10.5
(10.1 , 11.0) *
11.5
(10.7 , 12.2)
***, ††
9.6
(9.2 , 9.9)
10.1
(9.7 , 10.6)
11.0
(10.3 , 11.7)
***, †
-4.1
(-4.4 , -3.9)
-4.5
(-4.8 , -4.2)
-4.8
(-5.3 , -4.3)
*
-2.6
(-2.7 , -2.4)
-3.0
(-3.2 , -2.7) *
-3.3
(-3.7 , -2.9)
**
-1.5
(-1.7 , -1.4)
-2.0
(-2.3 , -1.8) ** -2.3
(-2.8 , -1.9)
**
19.9
(17.6 , 22.1)
27.2
(23.4 , 31.1) ** 32.6
(25.8 , 39.3)
***
16.2
(15.5 , 17.0)
18.2
(17.3 , 19.1) ** 19.5
(17.9 , 21.0)
***
n=121
n=53
n=139
mean
(95% CI)
-2.3
(-2.6 , -2.0)
-2.6
(-3.1 , -2.2)
-3.4
(-4.1 , -2.7)
*, †
-2.1
(-2.4 , -1.9)
-2.5
(-2.9 , -2.2)
-3.2
(-3.8 , -2.5)
**
-2.2
(-2.4 , -1.9)
-2.4
(-2.8 , -2.1)
-3.1
(-3.7 , -2.5)
**, †
-21.5
(-23.8, -19.2)
-22.1
(-24.6, -19.5)
-26.0
(-30.1, -25.9)
-20.7
(-22.9, -18.4)
-22.3
(-25.0, -19.7)
-25.4
(-29.4, -21.3) *
-21.4
(-23.7, -19.1)
-22.6
(-25.2, -20.0)
-26.1
(-30.0, -22.2)
1.0
(0.9 , 1.2)
1.2
(1.0 , 1.5)
1.4
(1.0 , 1.8)
0.9
(0.8 , 1.0)
1.1
(1.0 , 1.3)
1.4
(1.0 , 1.8)
*
0.9
(0.7 , 1.0)
1.2
(1.0 , 1.4)
1.5
(1.1 , 1.9)
**
-9.8
(-11.6 , -8.1)
-16.3
-4.4
(-5.1 , -3.7)
-5.5
(-6.3 , -4.8) *
-6.8
(-8.2 , -5.3)
23.0
(19.8, 26.1)
24.2
(21.2, 24.1)
25.2
(19.9, 30.4)
33.2
(29.6, 36.9)
35.2
(31.6, 38.7)
36.1
(28.8, 43.4)
57.2
(46.7, 67.7)
60.1
(48.2, 72.0)
63.7
(44.8, 82.6)
43.0
(37.2, 48.8)
50.9
(47.0, 54.9)
53.9
(46.5, 61.4)
**
(-19.6 , -13.0) ** -21.2
(-27.4 , -15.1) ***
**
*
R4, R6, R8 and X4, X6, X8: resistance and reactance, respectively, at 4, 6 and 8 Hz.
7
AX: area under the reactance curve. fres: resonance frequency. CI: confidence
interval. *, **, ***: p< 0.05, 0.01 and 0.001, respectively, relative to children who never
wheezed. †, ††: p< 0.05 and 0.01, respectively, relative to children with earlytransient wheeze.
8
Table 2. Cutoff values for a significant bronchodilator response based on the 5th
(95th) percentile in the group of children who never wheezed.
Absolute change
Relative change
|R4|
5.5
(hPa.s.L-1)
43
%baseline
|R6|
5.0
(hPa.s.L-1)
41
%baseline
|R8|
5.2
(hPa.s.L-1)
43
%baseline
|X4|
2.7
(hPa.s.L-1)
51
%baseline
|X6|
2.3
(hPa.s.L-1)
62
%baseline
|X8|
2.4
(hPa.s.L-1)
120
%baseline
|AX|
31
(hPa.L-1)
81
%baseline
9
BF
PN
MP
LS
PT
Figure 1. Forced oscillation setup for measurement of respiratory impedance.
MP: mouth piece, PN: pneumotachograph, PT: pressure transducers, BF: bias flow,
LS: loudspeaker.
10
Invited for medical
examination and LF
testing (n=1128)
Medical examination
performed (n=577)
No permission for
LF testing (n=10)
Permission given for
LF testing (n=567)
FOT successful
(n=543)
Included in study
Excluded from study:
(n=535)
medication use (n=2)
FOT not successful (n=24)
- equipment failure (n=5)
- reluctance of the child (n=8)
- artifacts during measurement (n=11)
unreliable data (n=6)
Study population
(n=332)
Incomplete
questionnaires
(n=203)
Figure 2: Flow chart of the process of data collection in the children participating in
the medical examination and lung function (LF) testing at 4 years of age.
11
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