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Effects of Helicobacter pylori, intestinal microflora and geohelminth infection on the
risk of allergic disease and sensitization in 3 year old Ethiopian children
Alemayehu Amberbir1, 2*, Girmay Medhin3, Woldaregay Erku3, Atalay Alem4, Rebecca Simms5,
Karen Robinson6, Andrew Fogarty2, 5, John Britton2, 5, Andrea Venn2, 5, Gail Davey1
Running Title: Helicobacter pylori, intestinal microflora, geohelminths and allergic disease
1. School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia.
2. Division of Epidemiology and Public Health, University of Nottingham, UK.
3. Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa,
Ethiopia.
4. Department of Psychiatry, Addis Ababa University, Addis Ababa, Ethiopia.
5. Respiratory Biomedical Research Unit, University of Nottingham, UK.
6. Center for Biomolecular Sciences, University of Nottingham, UK.
*Corresponding author, Email1: [email protected], Email2: [email protected]
School of Public Health, Addis Ababa University, PO Box 80596, Addis Ababa, Ethiopia
Tel: 00251-1-5157701, Fax: 00251-1-5517701
1
Abstract
Background: Epidemiological studies have suggested that gastro-intestinal infections including
Helicobacter pylori, intestinal microflora and geohelminths may influence the risk of asthma and
allergy but data from early life are lacking.
Objective: We aimed to determine the independent effects of these infections on allergic disease
symptoms and sensitization in an Ethiopian birth cohort.
Methods: In 2008/09, 878 children (87% of the 1006 original singletons in a population-based
birth cohort) were followed up at age three and interview data obtained on allergic symptoms
and potential confounders. Allergen skin tests to Dermatophagoides pteronyssinus and
cockroach were performed, levels of Der p 1 and Bla g 1 in the child’s bedding measured and
stool samples analyzed for geohelminths and, in a random subsample, enterococci, lactobacilli,
bifidobacteria and H. pylori antigen. The independent effects of each exposure on wheeze,
eczema, hay fever and sensitization were determined using multiple logistic regression.
Results: Children were commonly infected with H. pylori (41%; 253/616), enterococci (38.1%;
207/544), lactobacilli (31.1%; 169/544) and bifidobacteria (18.9%; 103/544) whereas
geohelminths were only found in 8.5% (75/866). H. pylori infection was associated with a
borderline significant reduced risk of eczema (adjusted OR 0.49, 95% CI 0.24 to 1.01, P=0.05)
and D. pteronyssinus sensitization (adjusted OR 0.42, 95% CI 0.17 to 1.08, P=0.07).
Geohelminths and intestinal microflora were not significantly associated with any of the
outcomes measured.
Conclusion: Among young children in a developing country, we found evidence to support the
hypothesis of a protective effect of H. pylori infection on the risk of allergic disease.
Key words: Helicobacter pylori, microflora, geohelminths, sensitization, eczema
2
Introduction
It is estimated that around 300 million people in the world have asthma and that it accounts for 1
in 250 deaths worldwide.[1] Epidemiological studies have shown a rise in the prevalence of
symptoms of asthma and associated allergic conditions in many countries,[1;2] and there is
consistent evidence that adoption of a western lifestyle and increased urbanization are associated
with increased risk of allergic diseases.[1-3] One hypothesis that has attracted much recent
interest is that organisms living in the gut, which become less prevalent with increased
urbanization and better hygiene, may play a protective role in the etiology of asthma and allergy.
In tropical areas, the first exposure to gastro-intestinal infections such as parasites typically
occurs at an early age, during a crucial period of immune development, however, to date, most
studies have been based on older children or adult populations.[4;5]
Particular interest has focused on the protective role of the stomach-colonizing bacterium
Helicobacter pylori. Rapid declines in the prevalence of H. pylori have been reported over the
last two decades, particularly amongst children in developed countries.[6-8] This trend is in
opposition to the rise in prevalence of allergy and asthma.[1;2] Furthermore, a range of
independent studies have reported a reduced risk of atopy and asthma in relation to H. pylori
infection,[9-13] with a protective effect reported in children,[9;10] and during adult life.[11-13]
This inverse relation also extends to objective markers of allergy.[11;12] Exposure to
geohelminth infection, particularly the systemic phase of hookworm and Ascaris lumbricoides
[14;15], and exclusively intestinal-lumen dwelling helminths of Trichuris trichiura [16] has been
implicated in the etiology of asthma and allergy. Our group’s work in Jimma in south west
Ethiopia showed a reduced risk of wheeze in relation to hookworm infection,[14] a finding
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replicated in a wider meta-analysis.[5] Our more recent work in Butajira, southern Ethiopia,
however, showed no significant protective effect of any geohelminth infection against wheeze or
asthma.[17] Intestinal microflora have also been hypothesized to play a role, with reports of
lower allergy prevalence in Estonian children who had high counts of lactobacilli, eubacteria and
enterococci, compared to Swedish children who had fewer of these microflora.[18-20]
The relationship between each of these gastro-intestinal infections and allergy remains uncertain,
since infection in developed countries is also potentially confounded by antibiotic therapy and
other medical interventions in early childhood. We have therefore studied these associations in a
birth cohort of three-year-old children living in Ethiopia, a developing country in which access to
medical services remains limited and asthma continue to emerge as a clinical problem.
4
Methods
Study population
The Butajira birth cohort is a population based birth cohort, full details of which have been
described elsewhere.[21;22] Briefly, the cohort is nested in the Butajira Demographic
Surveillance Site (DSS) which covers a sample of nine rural and one urban administrative units
in and around the town of Butajira in Southern Ethiopia.[23] Women aged between 15 and 49
years living in the DSS, and in the third trimester of pregnancy between July 2005 and February
2006, were recruited into the cohort. From July 2006, 1065 of these women gave birth to 1006
singleton infants, and mothers and children have been followed at various intervals by local
trained data collectors (Fig 1). Analyses of exposures collected at age one have been previously
reported. [21;22]The analysis reported here is based on cross sectional data from the singleton
children available at the three year follow up. Field data collection and laboratory analysis were
conducted from June 2008 to May 2009.
Data collection
The project data collectors visited the child at home and an interviewer-led questionnaire was
administered to the child’s mother. Questions on allergic disease symptoms were based on the
International Study of Asthma and Allergies in Children (ISAAC) core allergy and
environmental questionnaire,[24] and included wheeze ('In the last 12 months has your child had
wheezing or whistling in their chest?'), asthma ('In the last 12 months has your child had
asthma?'), hay fever (‘In the last 12 months has your child had problems with sneezing or
running nose (when not affected by cold or flu), or problems with itchy watery eyes?’) and
eczema ('In the last 12 months has your child ever had an itchy skin rash which has affected the
5
skin creases, eg, front of the elbow, behind the knees, the front of the ankles, around the neck, or
around the eyes?’). The questionnaire also asked about various potential confounders including
environmental factors (sanitation and water supply, household roof/wall/floor type, indoor
smoking, presence of animals, insecticide use, cooking facilities), asthma and allergy in the
family (see online supplement), household size, child’s sleeping place, number of siblings, birth
order, breast feeding and vaccination history at three years, and child’s use of medication (use of
paracetamol and antibiotics). Further socio-demographic characteristics including place of living,
child’s gender, maternal education and other economic variable were available from previous
follow up.
In addition to the questionnaire, allergen skin sensitization to D. pteronyssinus and cockroach
allergen (Biodiagnostics, Upton-upon-Severn, UK) were measured on each child using skinprick lancets. Glycerol saline and histamine dihydrochloride were used as negative and positive
controls, respectively. A positive test was defined as an average of two perpendicular wheal
diameters, one of which was the maximum measurable diameter, of at least 3mm greater than the
saline control response. Dust was sampled from child’s bedding and sleeping area by 2 min
suction through an ALK-Abello dust filter and transported to the UK for analysis. A stool sample
was collected from each child and transported to a local laboratory for analysis.
Laboratory analyses
The Der p 1 and Bla g 1 dust assays were performed using a standardized monoclonal antibodybased Enzyme-Linked Immunosorbent Assay (ELISA) method, developed in the Division of
Respiratory Medicine, University of Nottingham.
6
All faecal samples were analyzed for the geohelminth parasites Hookworm, Ascaris
lumbricoides, Trichuris trichiura, and other intestinal parasites. Samples were examined
qualitatively and quantitatively using the modified formol-ether concentration method, as at age
one.[25]
A random subsample of 544 faecal samples was taken for analysis of intestinal microflora and a
random subsample of 616 faecal samples taken for H. pylori antigen analysis (see online
supplement).
Statistical analysis
Data were double-entered into EpiData 3.1 (EpiData, Denmark). The datasets were cleaned,
coded and merged ready for analysis using Stata 11 (Statacorp, College Station, Texas, USA).
The prevalence of each outcome (wheeze, eczema, hay fever, and sensitization to D.
pteronyssinus and cockroach allergen) was computed and the relation between symptoms and
sensitization assessed. As the prevalence of asthma was small (only two), this outcome was not
analyzed further. The association of each exposure variable (H. pylori, enterococci, lactobacilli,
bifidobacterium, Hookworm, A. lumbricoides and T. trichiura infection) on each outcome was
determined by computing odds ratios with 95% confidence intervals, adjusted for a priori
confounders place of residence, child’s gender and maternal education, using multiple logistic
regression. The impact of further controlling for other potential confounders listed in the
methods, and other intestinal helminths/protozoan, was explored and any that altered the odds
ratios for the exposures of interest by more than 10% were retained in the model.
Study power
7
For an outcome with 8% prevalence, our sample of 878 children provided approximately 80%
power at the 5% significance level to detect an odds ratio of 0.45 for an exposure for which 40%
are positive.
Ethics
The study was approved by the ethics committee of Nottingham University, United Kingdom
and the ethics committee of the Ethiopian Science and Technology Ministry. Any child found to
be positive for any geohelminth parasites was treated with antihelmintics as per the national
protocol.
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Results
The birth cohort at age three
Of the 1006 singleton babies born in the cohort, 81 (8.1%) children had died, 37 (3.7%) had outmigrated, eight (0.8%) declined to participate in the study and two (0.2%) were missing, leaving
878 (87%) children with data at age three years (Fig 1). Just over half were male (50.6%) and the
majority (87.0%) were from rural areas. All but two children provided responses to the allergic
symptom outcomes and 864 (98%) provided skin sensitization data. In fewer than 2% of the
children the stool sample was not adequate for laboratory analysis (Fig 1).
Prevalence of allergic diseases and sensitization
Wheeze was reported in 9.1% (80/876) of children, eczema in 6.3% (55/876) and hay fever in
5.0% (44/876) (Table 1). 5.6% (48/864) and 4.2% (36/864) of the children were sensitized to D.
pteronyssinus and cockroach allergen respectively, and sensitization to any allergen was found in
8.7% (75/864) of the children (Table 1). Wheeze and eczema were reported slightly more
frequently, and hay fever slightly less frequently in urban than rural children, although
differences were not statistically significant. Almost identical proportions of urban and rural
children were sensitized to cockroach and D. pteronyssinus allergen (Table 1). Wheeze, eczema
and hay fever were not significantly associated with sensitization to D. pteronyssinus, cockroach
or both. As only a child with any sensitization reported allergic symptoms, no further adjusted
analysis could be carried out.
Relation between potential confounders and allergic outcomes
Allergic disease symptoms were not significantly related to gender or socio-economic status as
measured by maternal education (Table 2). The risk of all three allergic symptoms was increased
9
in those with maternal allergic history, significantly so for hay fever only. The risk of wheeze
and eczema was significantly increased in relation to paternal allergic history; parental allergic
history was unrelated to sensitization (Table 2). Use of paracetamol and antibiotics in the child
were both significantly associated with an increased risk of wheeze, whereas paracetamol use
was negatively associated with D. pteronyssinus sensitization. Der p 1 and Bla g 1 allergen levels
in bedding were not significantly related either to allergic symptoms or sensitization (Table 2).
Effects of H. pylori and intestinal microflora on allergic diseases and sensitization
The most common intestinal microflora detected were enterococci 38.1% (207/544) followed by
lactobacilli 31.1% (169/544) and bifidobacteria 19.0% (103/544). H. pylori was identified in
41.1% (253/616) of children (Table 3). The presence of enterococci, lactobacilli and
bifidobacteria in stool tended to be associated with increased risk of allergic symptoms and
decreased risk of allergic sensitization, but no associations were significant (Table 3). However,
infection with H. pylori was associated with a borderline significant decreased risk of eczema
(adjusted OR 0.49, 95% CI 0.24 to 1.01, p=0.054) and D. pteronyssinus sensitization (adjusted
OR 0.42, 95% CI 0.17 to 1.08, p=0.071) (Table 3), and the magnitude of these effects changed
little following adjustment for other potential confounders listed in Table 2 and for geohelminth
and microflora exposure.
Effects of geohelminths on allergic diseases and sensitization
Hookworm, A. lumbricoides and T. trichiura were detected in 4.9% (42/866), 4.3% (37/866) and
0.1% (1/866) of the children, respectively, with an overall prevalence of any infection of 8.5%
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(75/866) (Table 4). Hookworm infection was associated with decreased risks of all outcomes
except wheeze which was slightly increased, but confidence intervals were wide and no
associations were statistically significant (Table 4). All three allergic disease outcomes were
reduced in relation to A. lumbricoides infection, whereas the risk of sensitization was increased,
but again no effects were statistical significant (Table 4).
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Discussion
In this population-based birth cohort of young Ethiopian children, we have found a borderline
significant decreased risk of eczema symptoms and D. pteronyssinus sensitization in relation to
H. pylori infection, but no evidence to support an etiological role for the microflora enterococci,
lactobacilli or bifidobacteria. The former observation is a novel one in children of this age from a
developing country with a high prevalence of early H. pylori infection, and is consistent with our
a priori hypothesis that H. pylori infection reduces the risk of allergic disease.
The strengths of this study are that the data come from a population based birth cohort with high
response rate and good retention (95% of surviving mother-child pairs retained between birth
and three years), thereby minimizing selection bias. We have also used the same standardized
allergy questions administered in the one-year follow up of this birth cohort [21;22] and which
are based on the widely used and validated ISAAC questions.[24] Some measurement error
however is possible with reliance on self-reported allergic symptoms, for example other itchy
skin manifestations such as scabies may be misclassified as ‘eczema symptoms’. We have
previously shown in young Ethiopian children that the ISAAC criteria for eczema have a high
negative predictive value (91%), taking clinical examination as a reference, but a lower positive
predictive value (49%), suggesting the positive responses to the eczema question are not always
true eczema.[26]. However analysis of risk factors for eczema in the same survey population
resulted in similar findings (direction and size of effects) regardless of whether eczema was
defined using the ISAAC question or clinical examination.[27]. Wheezing symptoms in children
in the study area are described using a well-known, onomatopoeic local term.
12
It is also possible, however at this age that, some reported wheeze and possibly hay fever may be
of a more infection-related phenotype [28] The associations of these outcomes with parental
allergy suggests that some of these are markers of allergy, on the other hand, the positive
association with paracetamol and antibiotic use is consistent with an infection-related transient
phenotype. We also have found that reported outcome measures were unrelated with
sensitization to dust mite and cockroach allergens. These may be due to the low prevalence of
reported symptoms and sensitization in the study or may reflect a true dissociation caused by
other factors such as early infections, as previously reported in other developing country
settings.[14;16]
Another strength of the study was the inclusion of multiple gastro-intestinal organisms (H.
pylori, intestinal microflora and geohelminths), controlled for in the multivariate analysis. The
collection of various potential confounders including markers of socio-economic status and dust
allergens levels allowed an adjusted analysis to be explored. We have also made use of H. pylori
stool antigen testing, widely accepted as the most reliable means to determine current infection
status in young children.[29] The intestinal microflora in our bacteriological analysis were
identified mostly to the genus level presumptively using a microscope, but no identification to
the species level was made, and analysed qualitatively, so bias relating to either missing or
misclassifying the species may have arisen. Also, measurement of geohelminth intensity level
using the sedimentation method may miss eggs. We have no information on the exposure status
of the children younger than three years, particularly for H. pylori and microflora, and it is
possible that considering all infections (incident and prevalent) as incident has led to an
underestimation of the effects of these infections.
13
The high prevalence of H. pylori infection in our study population (41.1%) of three year old
children, can be contrasted with a prevalence of 6.1% in six year old children living in a
developed country.[10] However, our findings are comparable to a previous study in Butajira,
where H. pylori infection was found in 48% of 2-4 year old children, and higher prevalence in
older than younger children.[30] Our H. pylori prevalence estimate is also consistent with other
estimates in African children including an estimate of 37.5% amongst under-3 year old children
in Cameron[31] and 46% in 1-3 year old children in Uganda.[32]
Our findings from a developing country population-based birth cohort provide further evidence
for an effect of H. pylori on eczema and D. pteronyssinus sensitization, both of which have been
reported to be causally linked to the risk of childhood asthma.[33;34] Meta analysis of the
available prospective birth cohort studies on eczema and asthma in young children showed that
eczema in early life is associated with an increased risk of childhood asthma.[34] Studies have
also reported that exposure to indoor allergens, particularly D. pteronyssinus allergen, increases
the risk of development of asthma and other allergic diseases.[14;17;33] Studies have also shown
consistent associations between H. pylori infection and reduced risk of allergy and asthma.[9-13]
The links between H. pylori and allergic diseases were reviewed by Blaser et al,[4] who reported
a consistent inverse relationship from the cross-sectional studies, but concluded that there was no
clear significant trend from the case-control studies; possibly as a consequence of the small
numbers of individuals studied.[4]
Alternative explanations, such as reverse causation, for the association seen in this study, are
unlikely to explain the effects of H. pylori on eczema and D. pteronyssinus sensitization. Though
14
age at acquisition was not assessed in this study, the fact that H. pylori infection occurs in early
childhood[30;35] and persists for life,[6] makes it unlikely that eczema and D. pteronyssinus
sensitization precede H. pylori infection. Our findings were controlled for markers of socioeconomic status which may have confounded the previous study.[36] The positive association
with hay fever, however, could be due to the outcome not being related to an allergic etiology.
Our observation therefore suggests a possible role of H. pylori in the etiology of allergic diseases
that calls for further investigation through a well-powered prospective birth cohort study.
Commensal microflora contain various microbial antigens that may contribute to individual risk
of allergy. The important contenders among intestinal microflora species believed to induce
immune stimulation of young children, are enterococci, lactobacilli and bifidobacteria.[18-20;37]
However, despite high prevalence of microflora colonization, we found no evidence to support
an etiological role for them among young Ethiopian children. The three pioneering small scale
studies comparing allergy prevalence in Estonian and Swedish infants reported differences in the
microflora composition between allergic and non allergic Estonian and Swedish children,
independent of antibiotic use.[18-20] This finding, however, has not been replicated in several
large scale studies,[10;37] and altogether the evidence is either inconclusive or inconsistent.
The hypothesis that geohelminth infection may reduce the risk of allergic disease and
sensitization was proposed four decades ago, and has continued to be a focus of research in
developing countries. Our group’s work in Jimma, Ethiopia, demonstrated apparent protection
against allergic disease,[14;15] with dissociation of the risk of allergic sensitization from the risk
of wheeze due to the protective effect of high intensity geohelminth infection common in rural
15
areas.[14] Similarly a prospective cohort study in an urban setting in Brazil showed that early
heavy infections with T. Trichiura were associated with reduced risk of allergen skin test
reactivity in later childhood.[16] In contrast to this, however, this study of young children
showed no evidence of the protective effect of geohelminth parasites on allergic diseases or
allergic sensitization. This finding is consistent with our previous work in the same population,
in Butajira, Ethiopia.[17] The lower prevalence of geohelminths in our children may be due to a
mass de-worming intervention started in 2005 in under-five children,[25] and may account for
the lack of any effect of geohelminths on allergic diseases and sensitization. Further follow up of
the birth cohort will clearly be helpful to clarify the associations seen in this study.
In conclusion, this study from a developing country birth cohort provides modest further
evidence for a protective role of H. pylori on allergic disease, which is unlikely to be explained
by reverse causation, socio-economic status or use of antibiotics. We found no evidence to
support the etiological role of the intestinal microflora enterococci, lactobacilli and bifidobacteria
on allergic diseases or sensitization, and power to assess effects on geohelminths was limited.
We continue to follow up this birth cohort, in order to explore these relationships as the children
reach school age.
Acknowledgements
We are very much indebted to the mothers and their children who participated in the study and
the data collectors who followed the cohort throughout the field work. We also thank the
technicians of the Butajira health center for examining the stool samples. We are highly grateful
to Haile Alemayehu for conducting the quality controls during analysis of bacteriological
16
samples. The study was funded by Asthma UK project grant (07/036), with additional funding
from the Wellcome Trust and the Faculty of Medicine and Health Sciences Research Strategy
Group of Nottingham University.
Competing interests
We declare that we do not have any conflict of interest.
17
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21
Figure legends
Fig. 1 The study cohort
1065 women recruited
16 had multiple births
40 had stillbirth
2 migrated out of area
1 died in pregnancy
1006 singleton babies born
alive
64 infants died
10 out-migrated before one
year of age
932 singleton babies
available at one year follow-up
17 child deaths
27 out migrated
8 refused to participate
2 unknown
878 singleton babies available at
three years follow-up
876 reported symptom
outcome data of whom:
862 had geohelminth data
542 had microflora data
614 had H. pylori data
864 provided skin test data of
whom:
858 had geohelminth data
537 had microflora data
609 had H. pylori data
22