Methods
Participants
This is a sub-cohort study of the Japan Environment and Children’s Study
(JECS). The study design and recruitment methods have been described in
detail previously.15,16 In brief, JECS is a
large-scale birth cohort study involving 100,000 pairs of parents and
children in 15 nationwide regions in Japan, wherein participants were
observed from the foetal period to the age of 13 years old. Participants
were recruited between January 2011 and March 2014; 103,060 pregnancies
were registered, of which 100,304 were live births. For the sub-cohort
study, 5,017 participants were randomly selected from those who met the
eligibility criteria and consented to participate in the sub-cohort
study.17 Data for this study were used from the
dataset released in October 2019 (dataset jecs-ta-20190930). A schematic
summarising the sample selection methodology is shown in Figure 1, and
detailed study methodology is provided in Supporting Information.
The JECS protocol was approved by the Institutional Review Board on
Epidemiological Studies of the Ministry of the Environment (IRB number:
100406001) and by the Ethics Committees of all participating
institutions. Written informed consent was obtained from all
participants.
Questionnaire
Questionnaires were sent to the participants by post at the age of 12,
18, 24, and 36 months old, and the parents filled them out and sent them
back. Those questionnaires were a modified version of ISAAC for children
aged 6–7 years, with translation validation in
Japanese.18-20 Okabe et al. investigated the validity
of asthma symptoms based on ISAAC questionnaires involving
0-60-month-old infants by using asthma diagnosis made by allergists as
criteria,21 wherein the sensitivity and specificity of
asthma symptoms were 0.91 and 0.64 in 0-24-month-old children and 0.88
and 0.68 in 36-60-month-old children, respectively. In our study, asthma
and wheezing were defined according to the definition by Okabe et
al..21,22 The incidence rates of wheezing and asthma
by age (0-12, 12-24, and 24-36 months old) were defined as a ratio of
participants initially judged as having wheezing/asthma at each age
compared to the that of all participants.
Information on lower respiratory tract inflammation and respiratory
syncytial virus (RSV) infection was obtained from the questionnaires at
the age of 12, 18, and 24 months. Information concerning the presence of
indoor pets and passive smoke after birth was obtained from the
questionnaires at 18 months old.
Blood and urinal examination of
mothers
Blood samples were obtained from mothers during the first trimester of
pregnancy. Total serum IgE titres, assayed using ImmunoCAP (Thermo
Fisher Scientific, Inc., Sweden), were analysed by an independent
clinical laboratory. Mothers with high levels of total serum IgE were
defined as those with a total IgE level of 214 IU/mL or
above.23Maternal spot urine was collected at mid-pregnancy, and the urinary
cotinine level was measured. Sample collection and measuring methods
were described in detail previously.24 The cut-off
values for active smoking and passive smoking during pregnancy were set
at 36.8 and 0.31 μg/g creatinine, respectively.24
Blood examination of children
The mothers and children visited the test site when their children were
24 months old; blood samples were obtained from children. Total serum
IgE titres, assayed using ImmunoCAP (Thermo Fisher Scientific, Inc.,
Sweden), were analysed by an independent clinical laboratory. As the
cut-off value of high levels of total serum IgE in 24-month-old children
is unclear, it was set at the upper tenth percentile of the total IgE
level.
Exposure assessment (mite
allergen/endotoxin)
Trained investigators visited homes when the children were 18 months old
and collected house dust from mattresses on which the children slept. A
50-cm × 100-cm frame exclusive for sampling was placed on the mattress,
and a vacuum cleaner (Model DC61, Dyson, Japan) was run over the area in
the frame for 2 minutes (Supp Info Figure 1). The collected dust was
frozen until analysis.Dermatophagoides pteronyssinus (Der p 1) andDermatophagoides farinae (Der f 1) in the dust were measured
using an enzyme-linked immunosorbent assay kit (Indoor Biotechnologies
Ltd, UK). Endotoxin levels in the dust were measured by kinetic
chromogenic limulus amebocyte lysate assays (Kinetic-QCL), obtained from
Lonza Japan (Tokyo). There is high structural similarity in the
corresponding allergen between Der p 1 and Der f 1. Once sensitised,
there is strong cross-reactivity even when sensitised with either
type.25 Thus, we used the total exposure to both mite
species Der 1 (Der p 1 + Der f 1) in the analysis of the association
with health outcomes.
Statistical analysis
Dust mite allergen and endotoxin concentrations were expressed as
‘amount per m2 of dust sampling area’ and ‘amount per
unit mass of dust’,26 and Spearman’s rank correlation
coefficient was calculated for these exposure indices. The correlation
coefficient for the ‘amount per gram of dust’ and ‘amount per
m2 dust sampling area’ of Der 1 was 0.86, and that in
endotoxin between the ‘amount per mg dust’ and ‘amount per
m2 dust sampling area’ was 0.79, demonstrating a high
correlation between these exposure indices. Thus, the ‘amount per
m2 dust sampling area’ was analysed in the main
analysis thereafter. Analysis of the ‘amount per unit mass of dust’ was
performed as a sensitivity analysis. If the data were lower than those
of the method detection limit, then a value of one-half was assigned. In
the analysis of the association with health outcomes, the participants
were classified into the first-to-fourth quartile (Q1-Q4) groups.
Regarding the association between the dust mite allergen or endotoxin
exposure level and health outcome, the odds ratios (ORs) of the exposure
level in the Q2-Q4 groups in comparison with the Q1 group were estimated
using a logistic regression model. Regarding health outcomes, the
incidence rates of asthma and wheezing by age (0-12, 12-24, 24-36, and
0-36 months old) were selected for the main analysis. Secondary analysis
was set to high levels of total serum IgE in children. High levels of
maternal total serum IgE, active smoking/passive smoking based on the
urinary cotinine level in mid-pregnancy, passive smoke after birth,
children’s sex, lower respiratory tract inflammation, RSV infection,
presence of indoor pets, and
climatic region were regarded as
covariates. Climatic region was classified into three areas based on
mean temperature and mean humidity (Supp Info Figure 2).
A trend test was then performed to confirm the quantitative relationship
of health outcomes with the exposure level. Regarding continuous
variable x giving 1, 2, 3, and 4 points to the Q1-Q4 groups as an
explanatory variable of the statistical model, the significance level,
p-value, of the OR of increase per unit x was calculated. All
statistical analyses were performed using SAS ver9.4 (SAS Institute
Inc., Cary, NC, USA).