Methods
Study population
The COCOA (COhort for Childhood Origin of Asthma and Allergic diseases)
comprised the general Korean population and aimed to investigate the
causal contribution of genetics, perinatal environment, maternal
lifestyle, and psychosocial stress of the mother and child on pediatric
susceptibility to allergic diseases.(14) (See “study population” in
the Online Repository)
Of the 2,846 enrolled infants, 488 were lost to follow-up; children with
incomplete questionnaire data about AD and antibiotic exposure until 5
years of age were excluded. The remaining 1,637 children were included
in this study.
DNA Collection and SNP Genotyping
Genomic DNA was extracted from the cord blood mononuclear cells of each
child and genotyped for IL-13 (rs20541) polymorphisms using the
TagMan assay (ABI, Foster City, CA, USA). The endpoint fluorescent
readings were measured on an ABI 7900HT Sequence Detection System (ABI,
Foster City, CA, USA). The details of the methods are described in a
previous study.(15) Duplicate samples and negative controls were
included to ensure genotyping accuracy.
Physician’s assessment of allergic diseases and antibiotic
exposure
The presence of AD was clinically diagnosed by pediatric allergists on
the basis of the Hanifin and Rajka criteria.(16) Presence of AD was
defined as physician-diagnosed AD in the preceding 12 months during each
follow-up annually, and the incidence of AD was defined as the number of
new cases of physician-diagnosed AD that developed since birth over a
defined period. At consecutive visits, AD patients were assessed by
pediatric allergists using the SCORing Atopic Dermatitis (SCORAD).
Higher numbers indicate greater severity, and the scale ranges from 0 to
103.(17) AD severity was categorized as mild (<15) and
moderate to severe (≥15) according to the objective components of the
index (clinical signs and disease extent).(18)
Early antibiotic treatment was defined as exposure to antibiotics for
more than 3 days within the first 6 months of life, regardless of
whether the infant was hospitalized or not, and pediatric allergists
obtained the information about the antibiotics at each visit after
birth.
Outcomes
Our primary outcome of interest was AD incidence at the age of 6 months
and 1 to 3 years according to the frequency of antibiotic exposure
within 6 months as assessed by pediatric allergists. Other primary
outcomes were the influence of antibiotic exposure on AD phenotypes and
investigator-reported clinical signs (SCORAD).
The secondary outcomes were AD incidence at the age of 6 months and 1 to
3 years according to IL-13 genetic variations. Other secondary outcomes
were combined effects of IL-13 genetic variations and antibiotic
exposure on the AD incidence and phenotypes. Two transition periods were
considered, namely, age from 6 months to 2 years and from 2 to 5 years,
to classify AD phenotypes. We defined four different phenotypes of AD:
the early-transient phenotype, with AD onset within 2-years of age and
no further symptoms later; the early-persistent phenotype, with onset
within 2 years of age and symptoms do not improve within less than 2
years; the late phenotype, with onset after 2 years of age; and the
non-AD.(19)
Statistical analysis
Data are presented as frequencies and proportions for categorical
variables. Chi-square test and
Fisher’s exact test were performed
to evaluate the associations between the incidence of AD at the age of 1
year and the variables. To assess effect modification according toIL-13 polymorphisms and early-life antibiotic exposure, subjects
were divided into 4 groups according to environmental factors
(antibiotic use within 6 months for more than 3 days) and genetic
background (genotypes) and multivariable logistic regression models were
performed with the data to estimate adjusted odd ratios (aOR) and the
corresponding 95% confidence intervals (95% CI) for a comparison of AD
occurrence risk by relevant covariates after adjusting for potential
confounders: sex, maternal education level, family history of allergic
diseases, history of breastfeeding, and mode of delivery. Multinomial
logistic regression analysis was also used to identify the effects of
the use of antibiotics within 6 months of age and the combined effect of
the use of antibiotics and IL-13 genetic variations on AD phenotypes.
Finally, we tested for trends regarding the effect of the risk factors
(early-life environmental factors and genetic polymorphisms) on the
development of AD by two-factor
analysis of variance (two-way ANOVA). All statistical tests were
two-sided, and significance levels of p values were set at
<0.05. Statistical analyses were performed using SPSS
Statistics, version 23.0 (IBM SPSS Statistics, Inc., Chicago, IL).