Association between allergic responses and Schistosoma mansoni infection in residents in a low-endemic setting in Brazil

Introduction: Schistosomiasis is endemic in 76 countries and territories. Several studies have found an inverse correlation between parasitic disease and the development of allergies. The purpose of the present study was to determine whether infection with Schistosoma mansoni in subjects with a low parasite load is protective against allergy. The fi nal sample consisted of 39 S. mansoni-positive and 52 S. mansoni-negative residents of a small community in northeastern Brazil. Methods: All subjects were submitted to the Kato-Katz test, anti-S. mansoni IgG measurement, the prick test for aeroallergens, eosinophil counts and serum IgE measurement. Results: Subjects who reacted to one or more antigens in the prick test were considered allergic. Only 7 S. mansoni-positive subjects (17.9%) reacted to one or more antigens, whereas 20 S. mansoni-negative subjects (38.5%) tested positive for allergy. Conclusions: Our fi ndings suggest that, in areas of low endemicity, infection with S. mansoni signifi cantly reduces the risk of the development of allergy in subjects with a low parasite load.

Schistosomiasis is endemic in 76 countries and territories.An estimated 200-250 million people are currently infected with schistosomiasis, and another 600-780 million people are at risk of infection 1,2 .
This disease is caused when granulomatous infl ammatory lesions form around host tissues infected by Schistosoma mansoni eggs 3 .Eggs deposited in the liver stimulate cluster of differentiation 4+ (CD4+) T cells, which in turn activate macrophages and induce late hypersensitivity reactions leading to the formation of granulomas 4 .Initially, the immune response consists of an acute Th1 cell response directed at the adult worm; then, when eggs are deposited, the response is gradually shifted to a Th2 response.Failure to develop an effective Th2 response results in Th1-and Th17-mediated exacerbation of granulomatous infl ammation 5 .
During the acute stage, the immune response to helminth infection is mediated by Th1 cells.This type of response generates heightened production of interferon-γ (IFN-γ) 6 and tumor necrosis factor-α (TNF-α), which activate macrophages and induce the immunoglobulin G (IgG)-mediated opsonization and phagocytosis of antigens 7 .In the chronic stage, the immune response is mostly mediated by Th2 cells, leading to elevated interleukin 4 (IL-4) and interleukin 5 (IL-5) levels and interleukin 10 (IL-10) secretion and the subsequent reduction of IFN-γ 8 .
Several researchers have reported an inverse correlation between parasitic disease and the development of allergic diseases, leading to the hypothesis that parasites may have a protective (immunomodulatory) effect against atopy 9 .For instance, a study conducted in Equador reported an inverse correlation between helminth infection and allergy skin test results in children 10 .
Recent studies have also shown that asthma and allergic diseases are less prevalent in rural settings 11 , possibly due to protective effects associated with low socioeconomic status refl ected in life style, food habits, large families, the prevalence of helminth infection and lack of good hygiene 12 .Likewise, it has been suggested that allergic diseases are less prevalent in developing countries because populations are more likely

METHODS
to be exposed to bacteria and helminth infections, which are associated with poverty and the lack of basic sanitation.Helminth infections and allergic diseases are both associated with Th2 cytokines and high levels of immunoglobulin E (IgE) and eosinophilia.Although the two conditions appear to involve similar immune responses, a negative correlation has been demonstrated between helminth infection and allergic diseases 13 .
Many epidemiologists investigating the increasing prevalence of allergic diseases subscribe to the hygiene hypothesis 14 , which asserts that the lack of exposure to infectious agents and parasites, improvements in hygiene and the use of vaccines and antibiotics (especially in developed countries) can compromise the ability of the immune system to respond adequately to certain challenges, conceivably due to an imbalance between the Th1 and Th2 cell-mediated immune responses 15 .
The lower frequency of positive allergic tests and decreased asthma severity in populations infected with helminths may be due to several factors, such as enhanced polyclonal IgE production, reduced levels of allergen-specifi c IgE, high concentrations of antigen-specifi c IgG4, the activation of regulatory cells and the production of regulatory cytokines.For instance, IL-10 can inhibit the release of histamine and other mast cell mediators.Because infection with S. mansoni is associated with increased IL-10 production, this may be the main mechanism by which allergic response is suppressed in infected individuals 16 .
In general, studies evaluating the relationship between helminth infection, sensitivity to allergy skin tests and symptoms of allergic diseases have shown that the allergic response is reduced following infection with Schistosoma sp.Likewise, cross-sectional studies indicate that subjects infected with S. mansoni and Schistosoma haematobium are less frequently positive for aeroallergens on skin testing 10 .It should be noted that these studies were conducted in regions where schistosomiasis is considered highly endemic.The purpose of the present study was to determine whether infection with S. mansoni in subjects with a low parasite load is also protective against allergic diseases.

Study area and population
The study was carried out in Planalto do Cajueiro, a locality in the municipality of Maranguape (State of Ceará, Northeastern Brazil).The prevalence of schistosomiasis in the region rose from 8.53% in 2006 to 13.76% in 2007.
Located 30km from Fortaleza (the State capital), Maranguape covers an area of 591km 2 and has 113,561 inhabitants 17 .All members of the community (n=903) were invited to participate in the study, but only 357 agreed to undergo parasite examination, and 250 were submitted to serological tests.Subjects fulfi lling the following criteria were excluded from the study: I) not a permanent resident of Planalto do Cajueiro, II) using anti-allergic drugs, such as corticoids or immunosuppressants, III) a history of immunodepression, IV) pregnant, V) a history of anaphylactic shock, VI) needle or skin test phobia, or VII) under 2 years of age.

Study groups
The study group included 39 subjects who tested positive for S. mansoni (Sm + ) by the Kato-Katz technique and enzymelinked immunosorbent assay (ELISA).The control group consisted of 52 randomly selected subjects who tested negative for S. mansoni (Sm -) by the Kato-Katz technique and ELISA.Thus, 91 participants were included in the analysis.
To identify subjects with and without allergy, all participants (both Sm + and Sm -) were submitted to the prick test (PT).Thus, four groups were established: Schistosoma mansoni-positive and allergic (Sm + PT + ); Schistosoma mansoni-positive and not allergic (Sm + PT -); Schistosoma mansoni-negative and allergic (Sm -PT + ) and Schistosoma mansoni-negative and not allergic (Sm -PT -).

Egg detection (Kato-Katz technique):
Egg detection was performed according to the Kato-Katz technique 18 , using a Helm-Test ® kit.To increase the sensitivity of the test, three slides of each fecal sample (rather than one slide, as recommended by the manufacturer) were used for the detection of eggs from S. mansoni and other helminths.
Anti-Schistosoma mansoni IgG antibodies: Using adult worm antigen in conjunction with a protocol slightly adapted from Colley et al. 19 , an ELISA was performed to test for anti-Schistosoma mansoni IgG antibodies.The optical density was measured with an automatic ELISA reader (BioTeck®) using a 490nm fi lter.
Reactions with an optical density above 0.283 were considered positive.This cut-off value was based on the average optical density plus two standard deviations of 35 control serum samples collected from S. mansoni-negative individuals from a region where schistosomiasis is not endemic.
Prick test: the preferred site for the prick test is the inner forearm.Following antisepsis with 70% alcohol, a 25µL aliquot of each allergenic extract was introduced into the skin surface with a standard disposable pricker according to a predefi ned grid 20 .
The allergens included three species of dust mites (Dermatophagoides pteronissimus, Dermatophagoides farinae and Blomia tropicalis), cockroaches (mix) and fungi (mix).The reaction was measured after 20 min of exposure.
Hemogram: a complete hemogram was performed with an automated hematology analyzer (Sysmex KX-21N).To quantify eosinophils, a smear of the sample was submitted to rapid panoptic staining for a differential leukocyte count under the microscope.
Serum IgE concentrations: the total serum IgE concentration was measured with a commercially available kit (Monobind IgE AccuLite™ CLIA), following the manufacturerʼs instructions.The reading was performed by ELISA, at 450nm.

Statistical analysis
Demographic, clinical and laboratory data were organized using Microsoft Offi ce Excel 2007.Using GraphPad Prisma software, the data distribution was analyzed for normality, and the differences were analyzed with Fisherʼs exact test, the Kruskal-Wallis test followed by Dunnʼs multiple comparison test and the Mann-Whitney test.A sample of 250 residents from a community with an 11.2% prevalence of schistosomiasis was examined.Thirty-nine of these subjects were S. mansoni-positive by Kato-Katz and ELISA.According to the results of ELISA for anti-S mansoni IgG antibodies, 118 samples (47.2%) were positive and 132 (52.8%) were negative.A positive result by Kato-Katz or ELISA indicates current infection and/or an antibody immune response to the parasite, respectively.The 91 selected participants (39 Sm + and 52 Sm -) were submitted to the prick test, and those reacting to one or more antigens were considered allergic.Only 7 Sm + subjects (17.9%) reacted to one or more allergens.In contrast, 20 Sm -subjects (38.5%) tested positive for allergy (Figure 1).When analyzed with Fisherʼs exact test, the fi ndings shown in Figure 1 yielded the following results: p=0.0394, relative risk=0.5185,IC95%=0.2619-1.027(confi dence interval at 95%) and odds ratio=0.350,IC95%=0.130-0.9426.The fact that 18 of the 91 participants presented co-infection with other helminths or protozoa is not likely to have affected the results, considering the diversity of species and the negligible incidence of each.
Since 1976, when interventions to control schistosomiasis began, the epidemiological profi le of the disease has changed in many regions of Brazil.In particular, control measures have had widely documented benefi cial impacts on disease prevalence, severity and morbidity.However, these infections are still transmitted throughout Ceará state, causing mostly mild infections 21 .
In tropical regions, chronic helminth infection and allergic diseases are known to be inversely correlated 22 .Helminth infection is an environmental factor that may skew the T cell response to reduce the risk of allergic disease 23 , and S. mansoni has been associated with this protective effect 24 .Interleukins play an important role in immune defense against this parasite.In particular, IL-4 stimulates the production of helminth-specifi c IgE antibodies, which cover the worms as a result of antibodydependent cytotoxicity, while IL-5 activates eosinophils that attach to the IgE-covered helminths through γ heavy chainspecifi c Fc receptors 25 .To evaluate the influence of S. mansoni infection on the allergic response, we submitted our subjects to the prick test using environmental allergens.According to the literature, in regions where schistosomiasis is highly endemic and the parasite load is high, reactivity to skin allergy tests tends to be reduced.In our study, only 7 Sm + subjects (17.9%) reacted to one or more of the allergens tested, whereas 20 Sm -subjects (38.5%) tested positive for one or more allergens.These results support the hypothesis that helminth infection has a protective effect on the development of allergic diseases.Similar fi ndings were reported by Catapani et al. 26 in a study on allergic diseases in a Brazilian region where schistosomiasis is endemic, as these authors also found a lower prevalence of asthma among infected individuals.This result may be explained by the ability of chronic helminth infection to induce immunomodulatory responses associated with increased IL-10 or transforming growth factor-β (TGF-β) production by several regulatory cell types, thereby suppressing the mechanisms responsible for the development of allergy 27 .Based on the prick test results in our study, subjects with and without schistosomiasis were further divided into allergic and non-allergic categories, thereby defi ning four groups (Sm + PT + , Sm + PT -, Sm -PT + and Sm -PT -).Considering the important role IgE plays in both allergic responses and helminth infections, the total serum IgE concentration was measured.As expected, these levels were higher in S. mansonipositive and/or prick test-positive subjects than in their negative counterparts 28 .Intragroup differences in optical density may be due to low responsiveness or variation in genetic characteristics of certain individuals and/or unknown allergies.

Eosinophils
Our results are supported by those of Cooper et al. 29 , who found a positive correlation between the inhibition of skin reactivity to aeroallergens in subjects infected with helminths and increased total IgE levels.Likewise, Moraes et al. 30 reported that total serum IgE levels were higher in patients with atopy than in patients without this condition.
Eosinophils are proinflammatory cells associated with allergic diseases and parasite infections 31 and are believed to play an important role in the immune response to S. mansoni infection 32 .This hypothesis is based primarily on the histopathological evidence of eosinophils clustering around worms in biopsied tissues 33 as well as the observation that, in the presence of antibodies and/or the complement system, eosinophils mediate the destruction of schistosomula in vitro 34 .
When the Kruskal-Wallis test, followed by Dunnʼs multiple comparison test, was used to analyze the differences in eosinophil counts between the four groups, signifi cantly higher numbers were observed for S. mansoni-positive subjects.However, there was no signifi cant association between high eosinophil counts and prick test reactivity, likely because eosinophilia is a common reaction to infections and allergies alike 35 .
Given the heterogeneity of exposure and the diversity of parasite species, helminth infection is not invariably protective against aeroallergens 36 .However, the results of the present study suggest that, in areas of low endemicity, low-level S. mansoni infection signifi cantly reduces the risk of the development of allergy.
The study protocol had been previously approved by the Research Ethics Committee of the School of Medicine of the Federal University of Ceará [Universidade Federal do Ceará (UFC)] on March 4, 2010 (fi led under #329/09).