Impact of early life geohelminths on wheeze, asthma and atopy in Ecuadorian children at 8 years

Abstract Background Early‐life exposures to geohelminths may protect against development of wheeze/asthma and atopy. Objective To study the effect of maternal geohelminths and infections in children during the first 5 years on atopy, wheeze/asthma and airways reactivity/inflammation at 8 years. Methods Birth cohort of 2404 neonates followed to 8 years in rural Ecuador. Data on wheeze/asthma were collected by questionnaire and atopy by skin prick test (SPT) reactivity to 9 allergens. We measured airways reactivity to bronchodilator, fractional exhaled nitric oxide (FeNO) and nasal eosinophilia. Stool samples were examined for geohelminths by microscopy. Results 1933 (80.4%) children were evaluated at 8 years. Geohelminths were detected in 45.8% of mothers and 45.5% of children to 5 years. Frequencies of outcomes at 8 years were as follows: wheeze (6.6%), asthma between 5 and 8 years (7.9%), SPT (14.7%), airways reactivity (10%) and elevated FeNO (10.3%) and nasal eosinophilia (9.2%). Any maternal geohelminth was associated with reduced SPT prevalence (OR 0.72). Childhood Trichuris trichiura infections during the first 5 years were associated with reduced wheeze (OR 0.57) but greater parasite burdens with Ascaris lumbricoides at 5 years were associated with increased wheeze (OR 2.83) and asthma (OR 2.60). Associations between maternal geohelminths and wheeze/asthma were modified by atopy. Parasite‐specific effects on wheeze/asthma and airways reactivity and inflammation were observed in non‐atopic children. Conclusions Our data provide novel evidence for persistent effects of in utero geohelminth exposures on childhood atopy but highlight the complex nature of the relationship between geohelminths and the airways. Registered as an observational study (ISRCTN41239086).


| INTRODUC TI ON
Asthma is the most common chronic disease of childhood and is estimated to affect 358 million worldwide. 1 Asthma is increasing in prevalence in many low-and middle-income countries (LMICs). 2 Temporal trends of increasing asthma prevalence in LMICs are considered to be related to urbanization and loss of protective exposures associated with rural residence. 3 Recent years have seen increasing urbanization in LMICs, accompanied by reductions in poverty, improved access to basic services and transformation of the living environment. 3 Under such circumstances, the intensity of microbial exposures in early childhood is likely to have declined, affecting the maturation and regulation of the immune system and risk of inflammatory diseases including asthma. 4,5 Geohelminths (caused by Ascaris lumbricoides, Trichuris trichiura and hookworm) infect over 1 billion human worldwide 6 and are most prevalent among children living in conditions of poverty in tropical regions of LMICs. The most frequent geohelminths found in coastal Ecuador are Ascaris (A. lumbricoides) and Trichuris (T. trichiura) 7 that cause significant morbidity, particularly in children, through their effects on nutritional status, growth and cognition. 8 Geohelminths cause chronic infections that are associated with modulation of host Th2 inflammatory mechanisms. 8 The tight regulation of Th2 inflammatory responses may modulate inflammation associated with allergy and asthma. It has been suggested that the lower prevalence of asthma symptoms observed in rural compared to urban populations in tropical regions of LMICs 3 is explained by the immune modulatory effects of endemic geohelminth infections. 5 Epidemiological studies of the relationship between geohelminths and atopy or asthma have shown conflicting findings in cross-sectional analyses and intervention studies done largely in schoolchildren. [9][10][11][12][13][14][15] We hypothesized that in utero or early childhood exposures to geohelminths protect against the development of atopy and asthma in later childhood. To test this hypothesis, we followed an Ecuadorian cohort from birth to 8 years of age in an area of high endemicity. Previous analyses from the cohort showed a protective effect of maternal geohelminths on atopy to mite allergens at 3 years. 16 At 5 years, we observed that maternal infections increased the risk of wheeze while childhood infections protected against wheeze and atopy to perennial aeroallergens and raised the possibility that childhood infections might modulate wheeze through non-allergic mechanisms. 17 To understand better the effects of early childhood geohelminth exposures on wheeze, asthma and atopy, and whether the previously observed effects persist, we report findings at 8 years including novel measurements of airways inflammation and reactivity to provide further insights on effects of geohelminths on non-atopic wheezing illness and asthma.

K E Y W O R D S
asthma, atopy, Ecuador, geohelminths, wheeze

G R A P H I C A L A B S T R A C T
Maternal geohelminths protect 8-year-old children from the development of allergen skin prick test reactivity with strongest effects observed in infected children of infected mothers. Early childhood infections with Trichuris trichiura protect against wheeze at 8 years. Effects of geohelminths on wheeze/asthma and airways reactivity and inflammation at 8 years vary by parasite species and atopy.

| Study design, setting and participants
A prospective study from birth was done in the District of Quininde in Esmeraldas Province, Ecuador, as described. 18 The District serves a population of approximately 150,000 with limited access to basic services. The District is largely rural with economic activities based mainly on agriculture. The District includes 3 towns of 10,000 or more inhabitants that contain within municipal urban boundaries, rapidly expanding peri-urban populations representing the poorer segment of the population living in precarious circumstances with limited access to basic services. Neonates were recruited at a public hospital between November 2005 and December 2009. Follow-up evaluations and sample collections were done at 13 months and at 2, 3, 5 and 8 years of age.

| Study procedures
A questionnaire was used to collect data on sociodemographic factors, family history of allergy, and home environment by interview of the child's mother around the time of birth. Questionnaires were repeated periodically for wheeze and asthma symptoms. Wheeze was defined as any episode of wheeze during the previous 12 months at 8 years. Asthma was defined as wheeze during the previous 3 years plus one or both of parentally reported wheeze up to 5 years and a doctor diagnosis of asthma ever.
Stool samples to detect geohelminths were collected from mothers before birth and from children periodically from birth. Samples were examined using a combination of saline mounts, modified Kato-Katz, formol-ether concentration, and carbon-coproculture methods. 19 A positive sample was defined by the presence of at least one egg or larva from any of the four detection methods. Ascaris and Trichuris infection intensities were expressed as eggs per gram (epg) of faeces.
Spirometry was done at 8 years using a MicroLoop spirometer (CareFusion, UK) before and after 200 μg salbutamol administered.
A positive test for airways reactivity was an increase in FEV 1 of ≥12%. Fractional exhaled nitric oxide was measured in parts per billion using NObreath (Bedfont Scientific, UK). Nasal wash samples were collected at 8 years as described.

| Statistical analysis
To measure effects of geohelminths on asthma prevalence with >80% power at significance level of 0.05, we estimated that we would need to follow up 1725 children to detect a difference in asthma prevalence of ≥6% with infection risks of 50% among mothers and 35% among children. Primary exposures were maternal and childhood geohelminth infections, and primary outcomes were wheeze, asthma, and SPT to any allergen. Exploratory analyses addressed the effects of geohelminth species and infection intensities on primary outcomes, and effects of geohelminths on airways reactivity and airways inflammation. Univariable and multivariable logistic regression were used to estimate associations. Potential confounders are shown in Table 1. Urban-rural residence was defined by municipal geographic boundaries. A socio-economic status (SES) index was created using principal component analysis of 7 socioeconomic variables. 16 A conservative analytic approach was used for all adjusted analyses in which potential confounders included were those with p < .05 in univariable analyses for any of the primary outcomes. All statistical analyses were done using Stata 11 (StataCorp, College Station, Tex).

| Ethical considerations
Study protocols were approved by ethics committees in Ecuador (Hospital Pedro Vicente Maldonado, Universidad San Francisco de Quito, and Universidad Internacional del Ecuador) and UK (London School of Hygiene and Tropical Medicine). The study is registered as an observational study (ISRCTN41239086). Informed written consent was obtained from the child's mother, and minor assent was obtained from the child at 8 years. Anthelmintic treatment was provided to mothers and children with positive stools for geohelminths as recommended. 21

| Cohort participants
Analyses at 8 years of age were done using data from 1933 (80.4%) children of 2404 newborns initially recruited and for whom complete data were available on primary exposures and outcomes ( Figure 1).
Frequencies of potential confounders for children included in and excluded from the analysis were similar (Table S1).

| Frequencies of exposures and outcomes
Almost half (45.8%) the children had an infected mother (Ascaris 27.6%, Trichuris 28.9%, hookworm 5.6% and Strongyloides stercoralis 4.0%). Geohelminth infections during the first 5 years were observed TA B L E 1 Frequencies of maternal and childhood geohelminth infections to 5 years of age and potential confounders and associations with wheeze and allergen skin test (SPT) reactivity to any allergen at 8 years and asthma between 5 and 8 years

| Maternal geohelminth protect against atopy
Before adjustment for potential non-helminth confounders, both maternal geohelminths and childhood geohelmiths were associated with a significant reduction in SPT positivity, but not wheeze or asthma (  Figure 2).

| Childhood trichuriasis protects against wheeze but ascariasis increase risk of wheeze and asthma
Geohelminth infections to 13 months, 2 and 3 years of age were not significantly associated with primary outcomes (Table S2). Maternal geohelminth parasite species or parasite burden were not associated with primary outcomes (Figure 3 and Table S3). The presence of any Note: SPT-allergen skin prick test reactivity to any of 9 allergens. Odds ratios (OR) and 95% confidence intervals (95% CI) for univariable associations were estimated using logistic regression. p < .05 are shown in bold. Ethnicity 'other' represents: 1417 Mestizo/8 Indigenous. Numbers of missing values (brackets) were as follows: maternal allergy (14) and pneumonia during first 13 months (85). Other helminths: mother (S. stercoralis, 4.0%; Hymenolepis spp., 0.5%); child (hookworm, 1.1%; S.stercoralis, 1.5%; Hymenolepis spp., 4.2%). a Socio-economic status (SES) represents tertiles of z scores obtained using a factor analysis with 1 representing the lowest and 3 the highest SES. b Household overcrowding is defined as the number of people living in the household per sleeping room.

| Strongest protective effects against SPT were seen among infected children of infected mothers
Chronic exposures to childhood geohelminth infections were as-  (Table S4).

| Children of mothers with greater ascariasis infection intensities have greater levels of FeNO
There were no significant associations of geohelminths with airways reactivity, FeNO and nasal eosinophilia ( Figure 2 and Table S5). When considering parasite species and burden, elevated FeNO was associated with moderate/heavy parasite burdens with Ascaris both in TA B L E 2 Adjusted analyses for associations between maternal and childhood geohelminth infections to 5 years of age or potential confounders and associations with wheeze and allergen skin prick test (SPT) reactivity to any allergen at 8 years and asthma between 5 and 8 years

| SPT modifies association between maternal geohelminths and wheeze/asthma
SPT reactivity was strongly associated with wheeze (adj. 4.13, 95% CI 2.80-6.08, p < .001) and asthma (adj. OR 2.32, 95% CI 1.57-3.42, p < .001). We explored if effects of geohelminths on outcomes might vary by SPT ( Figure 2 and Table S6). Although interactions were seen for SPT on geohelminth-outcome associations, they were not highly significant. However, overall associations between maternal geohelminths and wheeze/asthma were negative among atopic but positive among non-atopic children.

| Maternal geohelminth parasite species are associated with childhood wheeze/asthma and airways reactivity and inflammation among nonatopics
Among non-atopic children, maternal geohelminths were positively as-  (Figure 4 and  (Table S7). Neither any maternal nor any childhood geohelminth infections were associated with airways reactivity, elevated FeNO or nasal eosinophilia irrespective of atopy (Table S8).
Analyses by parasite species and burden showed effects among nonatopic children (Table S9)

| DISCUSS ION
We tested the hypothesis that early-life exposures to geohelminthsthrough an infected mother during pregnancy or early childhood, or both-protect against wheeze/asthma and atopy at school age.
To do this, we followed a birth cohort study to measure effects of maternal and early childhood geohelminths on the development of atopy (measured as SPT), wheeze/asthma and airways reactivity and inflammation(measured by elevated FeNO and nasal eosinophilia) at 8 years. Our findings indicate that maternal geohelminths have persistent protective effects against childhood SPT but that this effect was strongest among children of infected mothers who also acquired infections. A maternal effect on increased wheeze F I G U R E 2 Adjusted associations between maternal and childhood geohelminths and study outcomes among all children and stratified by atopy (SPT). Shown are adjusted ORs and 95% CIs. Air React., airways reactivity; FeNO, fractional exhaled nitric oxide; Nas eosin, nasal eosinophilia; SPT, allergen skin prick test reactivity and airways inflammation was seen among non-atopic children, the dominant phenotype in non-affluent societies. 22 29 Our data showed positive associations between greater parasite burdens with Ascaris in mothers and risk of asthma ( Figure 4) and markers of airways inflammation (Table S9) in non-atopic children, while Ascaris in children was associated with elevated FeNO (Table S9).
Our observation that maternal infections protect against atopy ( Figure 2) is consistent with observations of inverse associations between geohelminths and SPT from cross-sectional studies of schoolchildren. 22,25,34 A protective effect of maternal geohelminth (against mite) was present from 3 years of age. 16 SPT prevalence in schoolchildren. 36 Long-term ivermectin started before most children were born, likely resulted in reduced geohelminth infections in mothers. 36 We have shown previously in this population that newborns of mothers infected with Ascaris have evidence of sensitization of CD4+ T cells to Ascaris antigens. 37 The same is likely to be true for T. trichiura that has an intimate relationship with the mucosal immune system. 8 Certainly, geohelminth antigens are present in the blood 38 of infected mothers and can cross the placenta to sensitize the foetus. Decreased responsiveness could be associated with tolerization to parasite allergens including those that are cross-reactive with aeroallergens. Extensive cross-reactivity has been demonstrated between helminth parasites and aeroallergens, 39 and such cross-reactivity can mediate cross-sensitization in immediate hypersensitivity skin reactions in murine models. 40 The suppressive effect of maternal geohelminths on SPT (Figure 2) in children could occur through tolerization to cross-reactive allergens.
Differences in the life cycle of the two principal geohelminth species present in the study setting could explain parasite species-specific effects among children acquiring infections during childhood. Trichuris is exclusively enteric and has an intimate relationship with the host mucosa-it inserts its anterior end into the mucosa where it feeds-and has strong regulatory effects on mucosal inflammatory responses. 8 Such an effect could explain the modulatory effect of early-life trichuriasis on wheeze symptoms ( Figure 3). In contrast, Ascaris has a phase of larval migration through the lungs where it can induce strong inflammatory responses. 8 Childhood infections with Ascaris might be expected to increase eosinophilic inflammation in the airways and might explain elevated FeNO (Table S9) to be more specific than recent wheeze but likewise may be subject to misclassification.

| CON CLUS IONS
Evidence of a protective effect of STH parasites against allergy in children remains fragmentary and inconsistent. Our data indicate that maternal geohelminths protect children from the development of allergen SPT but increase the risk of wheeze, and airways reactivity and inflammation. The latter effects were attributable to specific parasite species. Early childhood Trichuris appeared to protect against wheeze. Overall, our findings indicate that in utero exposures to geohelminths through maternal infections may have long-lasting effects on allergic inflammation and airways disease. These effects extended to school age and were modified by childhood infections, parasite species and atopy.

ACK N OWLED G EM ENTS
We thank the ECUAVIDA study team for their dedicated work and the cohort mothers and children for their enthusiastic participation.
We acknowledge also the support of the Directors and staff of the Hospital 'Padre Alberto Buffoni' in Quininde, Esmeraldas Province.
The study forms part of the SCAALA (Social Changes, Asthma and Allergies in Latin America) programme of research. The study was funded by the Wellcome Trust (grant 088862/Z/09/Z)

CO N FLI C T O F I NTE R E S T
None of the authors had any conflict of interest.