Soil‐transmitted helminth parasites and allergy: Observations from Ecuador

Summary There is considerable interest as to potential protective effects of soil‐transmitted helminths (STH) against allergy and allergic diseases. Here, we discuss findings of studies done of the effects of STH parasites on atopy and allergic diseases in Ecuador. While cross‐sectional studies have consistently shown a reduced prevalence of allergen skin prick test (SPT) reactivity among infected schoolchildren, the removal of these infections by repeated deworming did not affect SPT prevalence over the short‐term (ie, 12 months) but may have increased SPT prevalence over the long‐term (ie, 15‐17 years). In the case of allergic symptoms, cross‐sectional studies have generally not shown associations with STH and intervention studies showed no impact on prevalence. However, a birth cohort suggested that early STH infections might reduce wheeze by 5 years. Allergic sensitization to Ascaris, however, explained a significant proportion of wheezing among rural schoolchildren. Studies of the effects of STH on immune and inflammatory responses indicated a potential role of STH in contributing to more robust regulation. The effects of STH on allergy are likely to be determined by history of exposure over the life‐course and by interactions with a wide variety of other infectious and non‐infectious factors.


| INTRODUC TI ON
Over the past 20 years we have conducted a programme of research in Ecuador exploring how helminths may affect the development of allergy among children. Our research followed observations made in Africa 1 and a highly influential series of studies published during the 1990s by Lynch and colleagues in Venezuela. [2][3][4][5] The Venezuelan studies and others that followed elsewhere [6][7][8] showed variable but measurable effects of soil-transmitted helminth (STH) or geohelminth infections on allergen skin test reactivity and asthma symptoms, and were done in the context of a growing interest in the hygiene hypothesis and potential protective effects against allergy of childhood infections. 9 In this review, we present an overview of the findings of epidemiological and immunological studies done in Ecuador to understand better the effects of STH parasites on allergy during childhood. We will present current evidence for effects of STH parasites on atopy and allergic symptoms, focussing largely on the findings of studies done in Ecuador, and referring where relevant to the wide body of research that has been done elsewhere in Latin America and other endemic regions for STH parasites. The review will show that evidence for a protective effect of STH parasites against allergy in children remains fragmentary and inconsistent and that, while STH parasites do modulate the host anti-parasite immune response, there is limited evidence that they play a critical role in modulating the host inflammatory responses that may lead to allergic diseases in humans ( Figure 1).

| SOIL-TR AN S MIT TEDHELMINTH S
Infections with the common STH parasites (Ascaris lumbricoides, Trichuris trichiura and hookworm) are estimated to infect 1.5 billions worldwide, 10 primarily in poor tropical and sub-tropical

Summary
There is considerable interest as to potential protective effects of soil-transmitted helminths (STH) against allergy and allergic diseases. Here, we discuss findings of studies done of the effects of STH parasites on atopy and allergic diseases in Ecuador.
While cross-sectional studies have consistently shown a reduced prevalence of allergen skin prick test (SPT) reactivity among infected schoolchildren, the removal of these infections by repeated deworming did not affect SPT prevalence over the short-term (ie, 12 months) but may have increased SPT prevalence over the longterm (ie, 15-17 years). In the case of allergic symptoms, cross-sectional studies have generally not shown associations with STH and intervention studies showed no impact on prevalence. However, a birth cohort suggested that early STH infections might reduce wheeze by 5 years. Allergic sensitization to Ascaris, however, explained a significant proportion of wheezing among rural schoolchildren. Studies of the effects of STH on immune and inflammatory responses indicated a potential role of STH in contributing to more robust regulation. The effects of STH on allergy are likely to be determined by history of exposure over the life-course and by interactions with a wide variety of other infectious and non-infectious factors.
regions among populations living in poverty and inadequate sanitation. STH infections have been associated with significant disability and mortality, particularly in children. 10 The World Health Assembly (WHA) in 2001 endorsed a strategy for the control of these parasites through periodic treatments of schoolchildren with anthelmintic drugs. 11 In line with this resolution, WHO, national governments and donor organisations have prioritised the control of STH infections through periodic treatments with anthelmintic drugs to school-age children. Subsequently, recommendations for preventive chemotherapy have been expanded to include other high-risk groups, namely pre-school children and women of childbearing age. 12,13 The prevalence of STH infections is likely to have declined significantly in many low and middle-income countries (LMICs) over the past 10-20 years through reductions in extreme poverty, access to improved sanitation and increasing coverage rates for preventive chemotherapy for high-risk groups. Nevertheless, regions of high STH prevalence are likely to persist among populations that continue to suffer high levels of poverty and which are geographically isolated. A recent national survey of STH prevalence in Ecuador that studied a representative sample of schoolchildren estimated a national prevalence of 28%, 14 considerably lower than previous estimates, reflecting substantial progress over the previous 10 years in achieving the millennium development goals. However, the Amazon region of Ecuador continued to have high prevalence rates (59%) reflecting the relative isolation of this population that continues to suffer high levels of poverty 14,15

| ALLERGY
Asthma and eczema, considered to be caused by inflammatory mechanisms, are among the commonest of all chronic diseases of childhood in industrialized countries. Asthma alone is estimated to affect more than 300 millions worldwide 16 and has emerged as an important public health problem in many non-industrialized regions, particularly in growing cities and among urbanizing populations. [16][17][18] Temporal trends of increasing allergic disease prevalence are thought to be explained by a changing living environment, perhaps related to a decline in the incidence of childhood infectious diseases and reduction in the diversity of environmental microbiota. 19 Rural residence has consistently been shown to be protective against allergy 18,20 but it is not clear which rural exposures may mediate this protection. Numerous studies in traditional farming settings in Europe have shown farming to be strongly protective against atopy and allergic diseases, 21 particularly if farming exposures occur in early life. In such settings, protection appears to be partly dependent on exposures to a diverse range of microorganisms in the environment. 22 Protection against allergy is thought to be strongest when protective exposures occur during early life 23,24 while the immune system is still developing and may allow the developing immune response to develop a greater capacity to regulate inflammation. Such exposures are likely to be multiple, are likely to vary according the environment in which a child is raised and may include helminths.

| S THPAR A S ITE SANDALLERGY
The apparently low prevalence of allergy in the rural tropics may be explained partly by endemic helminth infections. In order to survive for periods of years in their mammalian hosts, helminth parasites have developed immune mechanisms that modulate the inflammatory responses that the host has evolved to kill them. 25 Such inflammatory responses include key elements of Th2-mediated allergic inflammation (eg, IgE, mast cells, eosinophils, Th2 cells, etc).
Helminths may suppress not only these killing and expulsion mechanisms but also shared inflammatory pathways that lead to atopy and allergic diseases. A protective role for STH and other helminth parasites against allergy has been incorporated into an extended hygiene hypothesis that attributes protection against allergy not only to early childhood infections (eg, bacteria and viruses) but also parasites and a more diverse host microbiota. 26  infection and species of infecting parasite. We will now consider the findings of epidemiological studies done in Ecuador examining the effects of STH parasites on atopy and allergic diseases.

| STHandatopy
We have repeatedly observed, in a series of cross-sectional studies in tropical and sub-tropical regions of the Ecuadorian Provinces of Esmeraldas and Pichincha, an inverse association between the microscopic presence of STH parasites in stool samples and atopy measured using skin test reactivity to environmental allergens. [26][27][28][29][30] These studies were done in schoolchildren with high prevalence rates for STH (~70%), where the dominant parasites were A. lumbricoides and T. trichiura, and where the background prevalence of SPT was variable (~10% in Esmeraldas 30 vs ~20% in Pichincha [26][27][28][29] ).
The findings are summarized in Table 1. These studies showed inverse associations between STH infections and SPT with odds ratios of ~0.7 and independent effects of both A. lumbricoides and or T. trichiura on SPT with evidence for greater inverse effects at higher parasite burdens for both parasites (Table 1). Further, the presence of anti-Ascaris IgG4 antibodies, used as a marker for chronic STH infection, was inversely associated with SPT. 26 Based on these findings, we hypothesized that STH were actively suppressing SPT and that this suppression might be reversed by curative chemotherapy. To test this hypothesis, we did a clusterrandomized study in which schoolchildren living in endemic communities were randomized to receive albendazole given every 2 months for a year vs no treatment. 29 A placebo was not used because of difficulties in blinding mothers to treatment allocation. The study showed no effect on atopy after 12 months of periodic anthelmintic treatment. 29 The study had several limitations: (a) a period of 12 months may be insufficient to allow reversal of the immunologic mechanism by which STH parasites suppress SPT; (b) single doses of 400 mg of albendazole have limited efficacy against T. trichiura infection; and (c) effects on SPT may be parasite species-specific (eg, the prevalence of hookworm was relatively low [15%] in our study 29 ). Randomized intervention studies conducted elsewhere among schoolchildren have shown inconsistent effects on atopy [31][32][33] ; a cluster-randomized study in Indonesia was unable to demonstrate an effect on SPT following periodic anthelmintic treatments given over 21 months 33 while randomized studies in Vietnam (12 months follow-up 32 ) and Gabon (30 months follow-up 31 ) provided evidence for an increase in the prevalence 32 or incidence of SPT. 31 The findings of these studies do suggest that 12 months of periodic chemotherapy is probably sufficient to show an effect 32 if one truly exists and that hookworm prevalence may not be a critical factor 32,33 -hookworm prevalence in the Indonesian study was >60%.
To look at long-term effects of anthelmintic treatments on SPT, we analysed a population in Esmeraldas Province where annual or twice-annual treatments with the broad-spectrum anthelmintic drug ivermectin had been distributed at a community level for the control of onchocerciasis over the previous 15-17 years. 34,35 SPT and STH prevalence was compared between communities that had received ivermectin compared with communities that had not received any mass drug administrations (MDA). We observed a greater prevalence of SPT in treated communities and this effect was partially explained by a lower prevalence of T. trichiura infection but not A. lumbricoides. 34 Periodic anthelmintic treatments given over 15 or more years may cut transmission levels and reduce community prevalence of STH and thus attenuate the induction of immune regulatory mechanisms associated with chronic infections. It is possible that T. trichiura may be particularly important in mediating an SPTsuppressive effect in some populations. The higher incidence of SPT in Gabon following anthelmintic treatment was explained partly by effects on T. trichiura prevalence, 31 and a prospective study in Brazil indicated that suppressive effects against SPT in childhood were related to having higher parasite burdens with T. trichiura earlier in childhood. 36 An explanation for our observations that periodic anthelmintic treatments of schoolchildren over 12 months did not affect SPT prevalence 29 but that periodic mass treatments of communities over 15 years did seem to have an effect, 34  Environmental exposures that have been associated with protection against allergy such as farming have stronger effects when exposure occurs in utero and in early childhood. 23,24 In the case of STH infections, early exposures could occur through trans-placental passage of parasite antigens from the mother to foetus in utero, through breast milk in the infant or through early childhood exposures to STH parasites in the home. Such early exposures could cause immunological sensitization or tolerization to parasite antigens leading to down-regulation of host anti-parasite responses and reduced allergy through immunological cross-reactivity between parasite and environmental allergens or bystander suppression. 37 Certainly, antigen-specific T cell responses to Ascaris antigens are measurable in newborns of infected mothers indicating in utero sensitization. 38 To explore the potential role of early-life exposures to STH parasites on the development of allergy, we followed a birth cohort, the ECUAVIDA cohort, in a rural District in Esmeraldas Province.
We measured the presence of STH infections in mothers and their children during the first 5 years of life and monitored the effects of these early exposures on the development of SPT. 39 Analyses of the cohort to date have shown no significant effects on SPT overall.
However sub-group analyses showed a reduced prevalence of SPT to mite or perennial allergens among 3-year-old children who had infected mothers 26 and among 5-year-old children who had STH infections during the first 3 years of life. 40  species or by parasite burden were not seen. Ongoing analyses of the cohort at 8 years of age are trying to discern the significance of these sub-group findings over the longer term.
If we are to ignore the findings of sub-group analyses within the birth cohort which require replication, it would seem overall that, in

| S THANDALLERG I CSYMP TOMS
We examined associations between STH parasites and allergic symptoms (wheezing, eczema and rhinitis) in a series of crosssectional studies in Ecuador. 27,30,41 The findings are summarized in Table 2. Overall, we observed no associations between STH parasites and allergic symptoms and no effects of individual parasite species or by parasite burden. 27,30 However, in an analysis of schoolchildren in rural Esmeraldas Province we did observe an inverse association between higher parasite burdens with T. trichiura infection and atopic wheeze (defined by recent wheeze in the presence of SPT). 40 Periodic anthelmintic treatments with albendazole over 12 months did not affect the prevalence of allergic symptoms in schoolchildren in Pichincha Province. 29 These findings are consistent with previous randomized intervention studies done in schoolchildren. 32,33 Analysis of allergic symptoms among schoolchildren living in communities that had received annual or twice-annual anthelmintic treatments over the previous 15-17 years was not associated with a significant difference in wheeze or rhinitis symptoms, but there was an increased risk of eczema symptoms among children living in treated communities. 34 To investigate if early exposures to STH parasites might affect the development of allergic diseases, we evaluated the ECUAVIDA cohort. 39 Analyses of the cohort to 3 years did not show an effect of maternal STH infections on wheeze or eczema although in subgroup analyses maternal ascariasis was associated with an increased risk of eczema in children. 26 However, by 5 years of age there was an increased risk of wheeze associated with maternal STH infections while childhood STH, particularly those acquired during the third year of life, were associated with a reduced risk of wheeze and asthma, an effect that was only seen among non-atopic children. 40 These observed effects were not associated with specific STH parasites or were affected by parasite burden although burdens were low in young children. 40 How might these contradictory findings be explained? We have shown previously in the same cohort that maternal ascariasis is associated with increased immune responsiveness to Ascaris antigens in newborns. 38 A. lumbricoides infections have been shown to increase the risk of wheeze/asthma in older children and adults 7,[42][43][44] and this effect may be "transmitted" from mother to child through in utero sensitization to Ascaris antigens. Contrasting effects of STH on wheeze/asthma in younger vs older subjects may depend on history of STH exposures and type of inflammatory lung response. Earlier infections may modulate anti-parasite inflammatory responses in highly endemic populations 37 allowing protective effects against wheeze/asthma to appear. Protective immunity to helminth parasites is age-dependent and non-sterile in endemic populations. 45 With continued exposures and immune maturation (eg, around school age), more effective anti-parasite 45 the association between anti-Ascaris IgE and wheeze could be explained by cross-reactivity between IgE epitopes of parasites and aeroallergens. 56 Mite atopy appeared to explain a greater proportion of wheeze in urban compared to rural schoolchildren 53 and in a population of asthmatics with a low prevalence of STH, the association between anti-Ascaris IgE and acute asthma disappeared after controlling for mite IgE. 54 This is in contrast to a previous rural study that showed that controlling for mite IgE did not affect the association between anti-Ascaris IgE and wheeze. 55 Rural wheeze, much of which was explained by anti-Ascaris IgE (and not mite IgE), was a mild self-limiting illness not requiring maintenance therapy while that in the urban population was more severe, required maintenance therapy more frequently, and was more strongly associated with mite atopy. These data may indicate, therefore, that endemic exposures to ascariasis attenuate asthma severity and that urban populations with a lower prevalence of STH parasites and stronger mite atopy, may suffer more severe disease.
Soil-transmitted helminths parasites might affect the expression and severity of wheeze/asthma through the attenuation of the association with atopy. There is evidence from other regions, that STH parasites may weaken the association between atopy and allergic symptoms. 57,58 In case-control studies done among rural and urban schoolchildren in Esmeraldas Province, we showed that mite atopy (measured by IgE) was more strongly associated with recent wheeze in urban compared to rural children and that the association between atopy and wheeze was attenuated by markers of STH infection. 53 Overall, data from a series of cross-sectional studies conducted

| IMMUNOLOG IC ALMECHANIS MS MED IATINGS THEFFEC TONHOS T INFL AMMATORYRE S P ONS E
Our observations to date of effects of STH parasites on atopy and allergic diseases in Ecuador indicate a complex relationship. The inverse association between STH parasites and SPT has been interpreted to suggest an active suppressive mechanism but our observations indicate, that if real, this relationship may take many years of living in a parasite-free or less endemic environment to be reversed. 34 Observations from Europe indicate that emergence of atopy, once a protective farming-related exposure has been removed, may occur over a period of years and is largely independent of age. 59 Findings of studies investigating immunological mechanisms are summarized in Table 3. Although STH parasites induce strong Th2 response in humans, 60 the intensity of the response seems to be modulated by concurrent STH infections 61 and increases following effective treatment. 62 Th2-mediated inflammatory responses are required to kill and expel parasites and the ability of the parasites to modulate the severity of these responses has clear implications for parasite survival. 25 The mechanisms by which STH parasites modulate host inflammatory responses are uncertain. The antiinflammatory cytokine, IL-10, has a powerful immune modulatory role in tissue helminth infections such as filarial and schistosome parasites, 63 but its role during STH infections is less clear. 25,60 The suppression of SPT associated with STH parasites may be explained by a reduction in the responsiveness of cells that bear the high-affinity receptor for IgE (ie, mast cells and basophils) to activation signals including cross-linking of specific IgE. An effect of this suppression is the marked disassociation between the presence of specific IgE and SPT to specific allergens that has been reported in numerous STH endemic populations including in Ecuador. 55 Explanations for the disassociation include immunological suppression through immune mediators such as IL-10 64 and the presence of IgE of low biological activity directed against cross-reactive protein and carbohydrate structures in STH parasites. 65 We have explored potential mechanisms by which STH infections might suppress SPT and were unable to identify a role for parasite-induced IL-10 66 in suppressing SPT or the disassociation between specific IgE and skin test reactivity to specific allergens, 66 reported previously for schistosomiasis. 64  of exposures associated with poverty and poor hygiene such as household hygiene, 72 exposure to farm animals 59,73 and household overcrowding. 28 Thus, the helminth effect on SPT might not be mediated by STH but by exposures with which they are associated or the effect might be one for which STH are sufficient but not necessary. As mentioned above, it appears unlikely that STH-specific IL-10 has a key role in modulatory effects of STH on non-parasite inflammatory responses such as that measured by SPT.
Soil-transmitted helminths infections have been associated with a stronger regulation of the immune response that can be measured in vitro either at homeostatis (ie, that measured in un-