Otitis, rhinitis, and atopy in relation to domestic endotoxin and β-glucan exposure among children in Singapore
Environ Health Prev Med
Otitis, rhinitis, and atopy in relation to domestic endotoxin and b-glucan exposure among children in Singapore
Lynne Lim Hsueh Yee 0 1
Amilia Wee Li Yan 0 1
Ragnar Rylander 0 1
0 R. Rylander BioFact Environmental Health Research Center , Lerum , Sweden
1 L. L. H. Yee (&) A. W. L. Yan Department of Otolaryngology, National University of Singapore, National University Hospital , 5 Lower Kent Ridge Road , Singapore 119074 , Singapore
Background Exposure to microbial cell wall agents (MCWAs) has been related to the risk for atopy, otitis, and rhinitis. Objectives To relate domestic exposure to two important MCWAs-b-glucan and endotoxin-to the risk for otitis, rhinitis, atopy, and allergy in a sample of children from Singapore. Methods Subjects (n = 98) were recruited from July 2006 to December 2008. Blood samples were taken to determine nonspecific IgE and skin prick tests were performed. Dust samples were collected from the bedrooms of the subjects and analyzed for the content of b-glucan and endotoxin, using the Limulus method. Results Levels of IgE were significantly higher among children with rhinitis, and these children also had a larger proportion of atopics. There were no differences in b-glucan values between children with otitis, children with rhinitis, and controls. Endotoxin levels were lower in the homes of children with otitis, with a tendency for the levels to be lower in those with rhinitis. Among children with a high level of b-glucan, there was a higher proportion of those with high IgE values and atopy. Conclusions The results suggest that a low level of endotoxin is a risk factor for otitis and that a high level of b-glucan is a risk factor for atopic sensitisation. Reactions
to domestic indoor exposure are determined by several
indoor agents and their relative exposure levels.
Otitis Rhinitis Endotoxin b-Glucan
The early childhood disease panorama is dominated by
respiratory infections, rhinitis, and otitis. Ever since
Strachan published the theory on the hygiene hypothesis [
has become more and more apparent that exposure to
microbial cell wall agents (MCWAs) plays an important
role in the pathogenesis of these diseases. There are
numerous studies that demonstrate an influence of bacterial
endotoxin (lipopolysaccharide; LPS) on atopy, allergic skin
disease, and asthma [
]. Endotoxin as such causes an
inflammatory response in the airways and has been shown
to increase the frequency of wheeze and airways irritation
among children as well as adults. Several studies
demonstrate that exposure to environments with a higher level of
endotoxin in infancy will decrease the risk of atopy,
asthma, and atopic dermatitis [
3, 7, 8
Apart from endotoxin, the home environment comprises
exposure to other MCWAs, particularly b-glucan and
chitin from mould cell walls. b-Glucan in small doses has
been shown to depress immune function and sensitise the
host to a simultaneous exposure to endotoxin [
relationship between fungi and allergic rhinitis has been
], as well as a relationship between fungal
exposure and otitis [
]. The medical outcome of domestic
exposure is thus determined by the combined exposure to
different agents, and the prevailing disease will depend on
the relative proportions of the different agents present.
To further explore the relationships between domestic
exposure to MCWAs and common diseases in small
children, a study was undertaken where home levels of
endotoxin and b-glucan were related to the risk for rhinitis,
otitis, allergy, and atopic sensitisation among children in
Materials and methods
Subjects for the study (n = 98) were voluntarily recruited
consecutively from regular patients at the Otolaryngology
Outpatient Clinic at the National University Hospital in
Singapore. Based upon an examination by a clinical
practitioner, we recruited children with rhinitis (congestion,
sneezing, itching, and postnasal discharge), children with
otitis media (recurrent disease requiring myringotomy or
with flat tympanograms on at least two occasions lasting
3 months or longer after antibiotic treatment), and children
without these diseases as controls.
A questionnaire about the subjects’ living habits and
conditions was administered to the parents. Questions were
asked on the presence of allergy to food, animals, and
medication. The results were expressed as ‘‘allergy yes/no’’
for any type of allergy reported. Furthermore, consent was
taken for the collection of dust samples. The duration of the
stay in the flat/house was an average of 5.3 years with no
difference between children with disease and controls. The
study was approved by the Institutional Review Board at
the University of Singapore and adhered to the standards of
the Helsinki Declaration.
The subjects underwent regular standard diagnostic
procedures for otitis and rhinitis. Three milliliters of blood was
taken in ethylenediaminetetraacetate (EDTA) tubes for the
determination of IgE. Due to the young age group of the
subjects, blood was only taken from subjects undergoing
surgery for tonsillitis or adeno hypertrophy.
Atopy was determined with a skin prick test using a
battery of 15 common allergens after confirming that
antihistamine drugs were not consumed. A multiple skin
test applicator (Multi-Test II; Lincoln Diagnostics,
Decatur, IL, USA) was used to prick the skin. The distance
between each needle was 2 cm. One drop of extract was
applied to the marked positions. Histamine hydrochloride
was used as the positive control and phenolated
glycerolsaline was used as the negative control. The extracts were
left on the skin for 20 min, after which the skin was
swabbed with alcohol to remove the extracts. A pen was
used to circle the wheals and a ruler was used to measure
the diameter. A positive result was recorded when a
3 9 3 mm wheal was present. Atopy was defined as a
positive skin prick test to any allergen tested.
Determination of endotoxin and ß-glucan
Floor dust from the bedrooms of the children was collected
using a handheld vacuum cleaner (Pelican; Philips,
Singapore). Coffee filters (Boncafe Filter Bags; Boncafe,
Singapore) were fitted into the vacuum cleaner to collect
dust samples. Parents were instructed not to vacuum-clean
the floor for a day. A one-by-one meter surface area was
vacuumed during 5 min. The dust was weighed, suspended
in 10 mL of LAL-reagent water, and shaken for 15 min.
One milliliter of the extract was withdrawn from the
solution for endotoxin analysis. b-Glucan was extracted
from the same sample by adding 1 mL of 3 N NaOH to the
suspension (9 mL). The sample was then shaken for
15 min, after which 1 mL was withdrawn for the b-glucan
Serial dilutions were prepared of the two extracts using
LAL-reagent water. The amount of endotoxin was
determined using the Limulus Amebocyte Lysate assay
(Pyrocrome; Cape Cod Incorporated, East Falmouth, MA, USA).
The amount of b-glucan was determined using a
glucanspecific version of the Limulus Amebocyte Lysate assay
(Glucatell; Cape Cod Incorporated). For both endotoxin
and b-glucan the kinetic procedure was used. The analyses
were performed at Mycometer A/S in Copenhagen,
Denmark. For technical reasons endotoxin was only determined
in a limited number of samples (n = 55).
The amounts of endotoxin and b-glucan were compared for
the groups with different diagnoses and for those with
atopy/no atopy and allergy/no allergy. Statistical
significance was assessed using the Mann–Whitney test and
Fisher’s exact test, with P = 0.05 as the threshold for
significance. There are minor differences in the numbers of
children in different disease groups in the Tables as all tests
could not be done on all children due to technical
The final cohort comprised 98 subjects (24 controls, 56
with rhinitis and 18 with otitis). The average age was 6.5
years (SD 3.2) with no difference between genders. Age
and gender distributions were very similar in the different
There was no relationship between the levels of b-glucan
and endotoxin in the homes. The material was analyzed in
relation to the presence of air conditioners in the homes of
the children; there were no differences in b-glucan or
endotoxin values. In homes with air conditioners, the
average IgE values were higher (169 vs. 83 mg/L), but the
difference was not statistically significant.
The clinical data for children with different diseases are
reported in Table 1.
Among children with rhinitis, the levels of IgE and the
proportion of atopics were higher than in the other groups
(P = 0.016 Mann–Whitney and P = 0.003 Fisher’s exact
test) and there was a tendency to a higher proportion of
children with allergy (P = 0.06).
The amounts of endotoxin and b-glucan in relation to
the different diseases are reported in Table 2.
The endotoxin level was lower in the homes of children
with otitis (P = 0.019), with a tendency for lower levels in
the homes of children with rhinitis (P = 0.105).
The results from the skin prick testing demonstrated that
71 children (74%) were atopic. Of those, 10 (14%) were
atopic to moulds. The levels of endotoxin and b-glucan in
relation to atopy are reported in Table 3.
There was a slightly higher level of b-glucan in the
homes of children with atopy, but the difference was not
statistically significant. Among those with a b-glucan level
above the first quartile (12.0 ng/mg), there was a
significantly (P = 0.049) larger number of children with IgE
values above 305 (3rd quartile) and a tendency to a higher
proportion of atopic children (54 out of 71–76% as
compared to 16 out of 24 (67%) in the lower exposure group
(not significant; NS).
Allergy was reported for 73 children (78%). The levels
of endotoxin, b-glucan, and IgE in relation to reported
allergy are shown in Table 4.
There was a significantly lower level of endotoxin in the
homes of children with allergy (P = 0.025). When the group
with high levels of endotoxin (3 quartile, [6.0 ng/mg) in
their homes was analyzed, there was a lower proportion of
children with allergy in this group (3 out of 8 as compared to
21 out of 26 in the lower exposure group. P = 0.031). There
was no difference in levels of IgE.
The material was finally analyzed with regard to
smoking habits of the parents. In homes with smoking
parents there were slightly higher values of endotoxin and
b-glucan (NS). Among control children, those with rhinitis,
and those with otitis, 35%, 24.2%, and 33% of the parents
were smokers, respectively. Among nonatopic children
20% of the parents were smokers as compared to 29%
among the atopics. Among children with reported allergy,
20% of the parents were smokers as compared to 47%
among those with no allergy (P = 0.085).
The major results from the study regarding the MCWAs
studied were a lower proportion of children with allergy in
homes with high levels of endotoxin, a higher risk of otitis
in homes with a low level of endotoxin, and a higher
proportion of children with high levels of IgE and atopy in
homes with high levels of b-glucan. The higher level of
IgE among children with rhinitis was expected, as well as
the higher level of IgE among atopic subjects.
The study has some shortcomings. Determinations of
exposure were made using floor dust, which is different
from the airborne dust that is inhaled. A number of
previous studies have, however, used floor dust and found
relationships with various endotoxin-induced diseases. The
analysis of endotoxin and b-glucan was made using the
Limulus assay, which, although very sensitive, is difficult
to handle. This is illustrated by the large variations
obtained when the same sample was tested in different
]. Due to technical reasons endotoxin levels
were not determined in all homes, which makes the data
basis for this variable small. On the other hand, the lower
levels of endotoxin in the homes of children with allergy
and otitis are consistent with results from previous studies.
The shortcomings in the exposure determination probably
mean that the relation between exposure and different
effects would have been stronger if more precise exposure
methods had been used.
It has previously been reported that the population
attributable fraction of atopy varied from 0 to 71% among
54,178 children in 22 countries [
]. The proportion was
higher in affluent countries, which agrees with the
incidence reported here (74%) and reflects the high standard of
living in Singapore.
The lower levels of endotoxin in the homes of children
with allergy support the concept of endotoxin as a
protective agent, elaborated in many previous studies [
3, 7, 8,
]. The results also suggest that a low level of
endotoxin is a risk factor for otitis, which was not characterized
by increased levels of IgE. The underlying mechanism for
the protective effect of endotoxin is probably a Th1-driven
inflammatory response, induced by the effect of endotoxin
on macrophages and induced by the TLR-4 receptor in the
innate immune system [
Exposure to b-glucan was related to a higher level of
IgE and a tendency to an increased risk of atopy. This
agrees with previous findings where an increased
prevalence of atopy was reported among children in schools
where airborne moulds were found, compared to control
schools with no mould damage [
]. Among preschool
children, a higher odds ratio for skin prick test reactivity
was related to dampness and visible mould in the home
. Furthermore, an association was found between
Aspergillus spores and atopy in school children [
Among children at risk for allergy, exposure to Aspergillus
at home was related to a lower content of Th1 cytokines,
such as interferon c (IFNc), tumor necrosis factor a
(TNFa), and interleukin 2 secreted from T cells [
effects reflect the immunosupressive effect of non-soluble
b-glucan in small doses [
In conclusion, the results from the study demonstrate the
importance of exposure to endotoxin and the mould cell
wall agent b-glucan for the risk for rhinitis, otitis, allergy,
and atopy among children.
The results suggest that both endotoxin and b-glucan/
mould exposure need to be considered in risk evaluations
of indoor environments.
Acknowledgments The study was supported by a research grant
from the National Medical Research Centre Start-Up Grants.
Conflict of interest statement None of the authors have any
disclosures to make and none have financial or other relationships with
suppliers of equipment or utensils used in the study.
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