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| Journal of the New Zealand Medical Association,
26-January-2007, Vol 120 No 1248
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Endotoxin and indoor allergen levels in kindergartens
and daycare centres in Wellington, New Zealand
Karen Oldfield, Rob Siebers, Julian Crane
In New Zealand, a large majority of children attend daycare
centres and kindergartens early in life. New Zealand statistics in 1996 show
that 93.4% of 4-year-old children are involved in some form of early
education.1 This is often due to the work
commitments of the parents or guardians of the children.
Recent studies have shown that children who attend daycare
centres may be protected from asthma and atopy later in
life.2,3 Celédon et al followed 453
children from birth to 6 years and found that daycare attendance decreased the
prevalence of wheezing at age 6 years.2 This
was only associated with daycare in the first 6 months of life. If the child had
a maternal history of asthma, no significant protection was found. The authors
hypothesised those infants in daycare had an increased exposure to infectious
illnesses, thus leading to a decreased incidence of atopy and asthma later in
life.
In Tucson, USA, Ball et al followed 1035 children and
studied the relationship between asthma and daycare
attendance.3 They found that children who
attended daycare in the first 6 months of life had a decreased prevalence of
wheeze at age 6 years, especially those with an increased number of older
siblings. This study elicited an important finding that, at age 2, there were
many children who wheeze when attending daycare
centres. However, this was not always because
of asthma. Instead it was attributed to the many infectious respiratory
infections that children of that age are exposed to.
One hypothesised protective agent that is thought to cause a
decrease in atopy and asthma if exposed to it early in life is endotoxin.
Endotoxin is a lipopolysaccharide that is found in the cell walls of
Gram-negative bacteria and is able to modulate the immune system
strongly,4 favouring the Th1 immune response
that produces cytokines IL-12 and IF-g. These cytokines, in turn, down-regulate
the Th2 immune response that is thought to be responsible for asthma and atopy.
This concept (the hygiene hypothesis) proposes that the increase in allergic
disease is a result of decreased exposure to microbes in the
environment.5
To our knowledge there has only been one study that examined
levels of endotoxin in childcare centres and schools. A study in San Paolo,
Brazil found that daycare centres and preschools had higher levels of endotoxin
in comparison to elementary schools as well as all these sites studied having a
significantly greater amount of endotoxin than local
homes.6
Whilst early childhood exposure to high levels of endotoxin
is thought to be protective against the later development of atopy and asthma in
children, high levels of indoor allergens, such as those from house dust mites
and animals, when found together with high levels of endotoxin, are able to
exacerbate asthma symptoms.7,8 It is therefore
important to examine levels of these indoor biocontaminents in the daycare
environment as high levels may exacerbate symptoms in sensitised children.
As levels of endotoxin in New Zealand daycare centres or
kindergartens have not previously been determined—and there has been only
one study that examined levels of Der p 1 in childcare centres in New
Zealand,9 but not of other indoor
allergens—we measured levels of endotoxin and Der p 1 (house dust
mite), Fel d 1 (cat), Can f 1 (dog), and Bla g 2
(cockroach) indoor allergens in daycare centres and kindergartens in the
Wellington region and compared these to previously measured levels in Wellington
homes.
We hypothesised that there would be higher levels of
endotoxin in daycare centres, due to the increased amount of nappy changing in
children under the age of 3—as was originally proposed by Russo et
al.6
MethodsTwenty kindergartens identified in the Wellington city
region were contacted by phone and asked to participate in the study, which
involved a visit from the researcher with collection of dust samples and
answering a simple questionnaire. Eighteen of the kindergartens contacted agreed
to participate. Due to the high numbers of daycare centres in the region (120),
36 were randomly selected through random number generation and then contacted by
phone. Eighteen of these agreed to participate in the study. The age range of
children at the kindergartens was 3–5 years and the age range for children
at daycare centres was 0–6 years.
At the visit, the head teacher or manager was asked a
series of questions about the building (building characteristics, floor
coverings, cleaning habits, heating type, damp and mould, bedding and location,
shoes and pets) and its population (number of children enrolled, age-range,
adults present, and number of children less than 3 years old.
Dust samples were collected from both high-use areas
and sleeping areas. On floors covered with mats or carpets, a 1
m2 area was selected and dust collected with an
1100W Hitachi vacuum cleaner (Hitachi Ltd., Singapore) for 1 minute in a nylon
dust sock placed over the vacuum cleaner head.
For smooth floors, a 2
m2 area was selected and vacuumed for 2
minutes. Dust samples were sifted through a 425 µm steel mesh sieve; total
dust weight recorded, and stored at –20°C until analysis.
For endotoxin levels, 200 mg of sifted dust was
extracted with 5 ml of endotoxin-free water containing 0.05% Tween 20, shaken
for 30 minutes at 250 rpm at room temperature and then centrifuged for 10
minutes at 1000g.
Aliquots of supernatants were stored in endotoxin-free
glass tubes at –20° until analysis.
Endotoxin activity was measured by a kinetic amebocyte lysate assay on 1:500
dilutions of the supernatants (BioWhittaker Inc., Walkersville, MD, USA) and
analysed by four-parameter curve fitting.
For indoor allergen levels, 100 mg of sifted dust was
extracted with 1 ml of phosphate-buffered saline for 30 minutes at room
temperature, centrifuged for 10 minutes at 3,000g and supernatants stored at
–4°C. Der p 1 and Fel d 1 were measured by double
monoclonal ELISAs, and Can f 1 and Bla g 2 by
monoclonal/polyclonal antibody ELISA (Indoor Biotechnologies, Cardiff, UK).
Our research group has used these analytical methods
for the last decade and between-batch precision for endotoxin and allergens are
about 20% and 10% respectively.10,11
As endotoxin and indoor allergen data were skewed, they
were log-transformed and results expressed as geometric means with 95% CI as in
previous studies from our research group.9-11
Differences between types of childcare centres and measured parameters were
analysed by two-tailed Student t-test. Statistical significance was set at the
p<0.05 level. Due to the relatively small study size we did not conduct
multivariate analyses.
The Central Regional Ethics Committee approved the
study and informed written consent was obtained from all participating childcare
centres.
ResultsOn average there were 39 (range 25–45) children per
session in kindergartens, and 25 (range 16–50) children per session in
daycare centres. In daycare centres there were on average 12 (range 2–23)
children per session under the age of 3 years.
Of the buildings visited, 35 were wooden and 1 was brick; 35
centres had carpet or mats on their floor, while 1 had a wooden floor. There
were 9 synthetic carpets, 16 wool carpets, and 6 mixed carpets (carpet type was
unknown in 3 centres).
Twenty-six centres had pets that were kept in cages, and 25
reported that they commonly had cats coming onto the property. All centres were
cleaned daily. This involved mopping and vacuuming the entire area used by the
children.
All 60 dust samples collected were analysed for endotoxin
and 57 samples were analysed for indoor allergens (3 samples had sufficient dust
only for endotoxin determination). Table 1 shows the results for endotoxin and
indoor allergen levels.
Table 1. Endotoxin and indoor allergens in
kindergartens and daycare centres in the Wellington region (n=36)
There was no significant difference in endotoxin levels
between kindergartens and the other daycare centres (p=0.38), nor for Fel d
1 (p=0.35), Can f 1 (p=0.51), or Bla g 2 (p=0.78).
However, there were significant differences in Der p 1 with daycare
centres having higher Der p 1 levels than kindergartens; geometric
means were 0.30 µg/g (95% CI: 0.09–2.33) and 0.03 µg /g (95% CI:
0.008–0.22) respectively; p=0.0055).
DiscussionThis study found that levels of endotoxin in kindergartens
and daycare centres are of similar levels to those found in Wellington
homes.10 Wickens et al studied 77 Wellington
homes in New Zealand where the geometric mean floor endotoxin was 22,700 EU/g,
comparable with a geometric mean of 29,206 EU/g in this study.
Our results differ from Brazil, where the endotoxin levels
(EU/g) in daycare centres and preschools were three-fold higher than in local
homes.6 However, this comparison with Brazil
must be treated with caution as extraction and analytical methods were different
from the current study and may also be due to batch differences in LAL
reagents.12
Endotoxin is though to be protective for the development of
atopy and asthma if children are exposed in the first year of
life.4 This is one of the reasons why
attendance at daycare centres may be protective against atopy and asthma.
However, our study shows that there is little difference in endotoxin levels
between daycare centres and kindergartens, and homes in New Zealand, unlike
Brazil. Also, a previous study in New Zealand has shown that daycare attendance
is not protective for the development of
asthma.13 However, exposure to higher endotoxin
levels than found in our study early in life could be protective against the
development of asthma and atopy in childhood.
Exposure to high endotoxin in infancy leads to increased
wheezing in childhood14 and also causes
wheezing in house dust mite sensitised asthmatics.15
Thus, the levels of endotoxin in our study may be of relevance to
wheezing in children attending kindergartens and child-care centres. Wheezing is
a hallmark of asthma and New Zealand has a high prevalence of asthma.
Indoor allergens levels in kindergartens and daycare centres
were much lower than in Wellington homes.11 We
have previously measured Der p 1 from carpets in Wellington homes with a
geometric mean of 25.5 µg/g.11 This is
much higher than the levels found in kindergartens and daycare centres, where
the levels were 0.03 µg/g and 0.30 µg/g respectively. These results
are similar to those found in public places in New Zealand
results.9 In that study, 17 childcare centres
were included where the geometric mean of Der p 1 was 0.22 µg/g.
In comparison, geometric Der p 1 levels in daycare centres and
preschools in Brazil were much higher at 2.6 µg/g and 6.3 µg/g
respectively.6
It is important to note that in some of the daycare centres
and kindergartens in our study there were high levels of Der p 1, with
the highest recorded level of 103.8 µg/g. Der p 1 levels greater
than 10 µg/g can exacerbate symptoms in atopic
patients,8 thus in these childcare centres an
increase in the occurrence of symptoms in house dust mite sensitised children
may be expected.
The geometric mean of Fel d 1 (1.24 µg/g)
found in kindergartens and childcare centres is lower than the proposed
exacerbation threshold of 8 µg/g.8 In one
study, levels found in 224 Wellington homes varied between those with or without
cats present.7 However, in those homes with a
cat (40.8 µg/g) and those without (3.3 µg/g), Fel d 1 levels
were higher than in kindergartens and daycare centres.
In another study we found that Fel d 1 levels in
public places were lower than in homes, equal to those found in our
study.16 Cat allergen is most likely
transferred into places such as kindergartens on the clothes of children who own
pets,17 but it also important to note that 25
of the centres in our study reported that they had cats straying onto the
property, which may account for some of the higher levels found (highest: 13.9
µg/g).
The Can f 1 and Bla g 2 levels in
kindergartens and daycare centres (geometric means: 0.43 µg/g and 0.028
µg/g respectively) are similar to levels found in a study of Tokelauan
family homes in Wellington, New Zealand.18 In
that study geometric mean levels of Can f 1 and Bla g 2 were
0.62 µg/g and 0.03 µg/g
respectively.16
The low levels of indoor allergens found in the
kindergartens and daycare centres may be attributed to the rigorous daily
cleaning regime that the centres undergo. Each centre was mopped and vacuumed at
least one a day, with some also being cleaned in the weekend. A previous study
found that daily vacuuming for five weeks consecutively reduces Der p 1
in carpet dust samples by about 60% and returns to initial levels when switched
to weekly vacuuming.19
Study limitations include the small number of kindergarten
and daycare centres in the study, which may not be representative of all
childcare facilities in New Zealand. For instance, domestic Der p 1
levels are about four-fold lower in Dunedin than in
Wellington.20 Also, only a small area was
sampled for endotoxin and indoor allergens. However, we have previously shown
that a central area is representative for the whole room for Der p 1
and Fel d 1.21
In conclusion, daycare centres and kindergartens in the
Wellington region have similar levels of endotoxin as homes in the region, and
much lower levels of indoor allergens. However, due to similar levels of
endotoxin in daycare centres and domestic dwellings in Wellington, it would be
interesting to see if higher levels of endotoxin would be protective against the
development of atopy and asthma later in life, as has been shown in overseas
studies.
Another implication is that those children who are already
sensitised to allergens before attending a daycare centre or kindergarten are
unlikely to be at risk from exacerbation of symptoms, except in some centres
where there were high levels of Der p 1 and Fel d 1.
Conflict of interest statement: The
authors have no conflicts of interest.
Author information: Karen Oldfield, Trainee
Intern; Rob Siebers, Senior Research Fellow; Julian Crane, Professor; Wellington
Asthma Research Group, Wellington School of Medicine and Health Sciences,
Wellington South
Acknowledgements: Karen Oldfield was
supported by a summer studentship from the Wellington Medical Research
Foundation. We also thank all the participating childcare facilities for their
help and cooperation.
Correspondence: R Siebers, Wellington
School of Medicine and Health Sciences, PO Box 7343, Wellington South. Email: rob.siebers@otago.ac.nz
References:
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