Helminths are positively associated with atopy and wheeze in Ugandan fishing communities: results from a cross‐sectional survey

Abstract Background Parasitic helminths are potent immunomodulators and chronic infections may protect against allergy‐related disease and atopy. We conducted a cross‐sectional survey to test the hypothesis that in heavily helminth‐exposed fishing villages on Lake Victoria, Uganda, helminth infections would be inversely associated with allergy‐related conditions. Methods A household survey was conducted as baseline to an anthelminthic intervention trial. Outcomes were reported wheeze in last year, atopy assessed both by skin prick test (SPT) and by the measurement of allergen‐specific IgE to dust mites and cockroach in plasma. Helminth infections were ascertained by stool, urine and haemoparasitology. Associations were examined using multivariable regression. Results Two thousand three hundred and sixteen individuals were surveyed. Prevalence of reported wheeze was 2% in under‐fives and 5% in participants ≥5 years; 19% had a positive SPT; median Dermatophagoides‐specific IgE and cockroach‐specific IgE were 1440 and 220 ng/ml, respectively. S. mansoni, N. americanus, S. stercoralis, T. trichiura, M. perstans and A. lumbricoides prevalence was estimated as 51%, 22%, 12%, 10%, 2% and 1%, respectively. S. mansoni was positively associated with Dermatophagoides‐specific IgE [adjusted geometric mean ratio (aGMR) (95% confidence interval) 1.64 (1.23, 2.18)]; T. trichiura with SPT [adjusted odds ratio (aOR) 2.08 (1.38, 3.15)]; M. perstans with cockroach‐specific IgE [aGMR 2.37 (1.39, 4.06)], A. lumbricoides with wheeze in participants ≥5 years [aOR 6.36 (1.10, 36.63)] and with Dermatophagoides‐specific IgE [aGMR 2.34 (1.11, 4.95)]. No inverse associations were observed. Conclusions Contrary to our hypothesis, we found little evidence of an inverse relationship between helminths and allergy‐related outcomes, but strong evidence that individuals with certain helminths were more prone to atopy in this setting.

evading or modulating the host immune response. Evidence from animal models, and from in vitro studies on human samples, suggests that helminths can modulate the immune response not only to themselves, but also to unrelated pathogens, antigens and allergens (10,11). Helminths contain a range of molecules homologous to known allergens, but absent from mammals or markedly different from mammalian homologues. These induce IgE responses in mammalian hosts, and there is a strong evidence that this pathway is involved in protective immunity against helminths (12). Modulation of this atopic pathway is likely, therefore, to be particularly important for helminth survival, while concomitantly protecting against allergic disease (13).
Associations between helminths and allergy-related conditions have been investigated in a variety of settings [reviewed in (14)(15)(16)]. The majority of studies investigating helminthatopy associations have either reported an inverse association or no evidence of association. However, results for allergyrelated diseases such as asthma and eczema have been less consistent, varying both within and across helminth species. Meta-analyses have indicated that for asthma, Ascaris lumbricoides infection is positively associated and hookworm inversely associated, with borderline evidence of a positive association for Trichuris trichiura and no association for Strongyloides stercoralis (15). Very few studies have investigated associations with schistosomiasis, although those that have generally report an inverse association with atopy (17)(18)(19)(20).
Helminths may have an important role as modifiers of associations between markers of atopy and allergy-related diseases. It has been reported that IgE and SPT responses to house dust mite are positively associated in the absence of hookworm but unrelated among hookworm-infected individuals (21). Similarly, studies have found that positive associations between atopy and wheeze, and between atopy and eczema, exist only among individuals who do not have hookworm and not among those infected with hookworm (22,23). Finally, it has been reported that atopy and wheeze are only positively associated in the absence of concurrent Ascaris infection and not in its presence (24).
Intervention studies are an important tool for understanding the relationship between helminths and allergy-related diseases; however, findings of previous studies on the impact of anthelminthic treatment on atopy among children are conflicting (25)(26)(27)(28) and none showed effects on wheeze or eczema, although statistical power for these outcomes was usually limited. Variable findings for atopy may be a consequence of heterogeneity between study settings in helminth species; of note, no trial has yet investigated the effects of treatment of schistosomiasis on asthma, eczema and atopy.
We designed a cluster-randomized trial, the Lake Victoria Island Intervention Study on Worms and Allergy-related Diseases (LaVIISWA; ISRCTN47196031), to investigate the impact of intensive vs standard anthelminthic treatment over a 3-year period on allergy-related diseases, in a setting with heavy helminth burden, in particular schistosomiasis (29). We herein report the findings of a household survey, conducted as baseline, to investigate the hypothesis that helminth infections would be inversely associated with allergy-related conditions.

Study design and procedures
LaVIISWA is being conducted in 26 fishing villages on the Lake Victoria islands of Koome subcounty, Mukono district, Uganda, a remote setting accessible in 2-3 h from Entebbe by powered canoe. Full details of the trial design are described elsewhere (29). The baseline household survey was conducted between October 2012 and July 2013, across all trial villages, immediately preceding intervention roll-out. All households in participating villages were eligible for inclusion in the survey. Available household listings were checked and updated by the research team, and simple random samples of 45 households were selected from each village. In selected households, all members were eligible for inclusion in the survey.
Questionnaires were completed regarding household features and individual social-demographic characteristics. Information regarding asthma, eczema and allergy symptoms was obtained using questions from the International Study on Allergy and Asthma in Children (ISAAC) questionnaire, with supplementary questions from the UK diagnostic criteria for atopic eczema (30,31).
A general history and examination, including height, weight and hepatosplenomegaly, was performed. All individuals were examined for visible flexural dermatitis: for this, all team members were trained in the standardized approach described in (32). SPTs were performed on participants aged ≥1 year, using standard methods, with three allergens [Dermatophagoides mix, Blomia tropicalis and German cockroach (Blatella germanica)] and positive and negative controls (ALK-Abell o; supplied by Laboratory Specialities (Pty) Ltd., Randburg, South Africa). Each participant was asked for one stool sample; mid-stream urine samples were requested from all participants in the 15 villages surveyed from February 2013 onwards. Blood samples of 14 ml were obtained from individuals ≥13 years, 10 ml from children 5-12 years and 6 ml from children 1-4 years. Individuals were offered HIV counselling and testing in collaboration with local health service providers.
Ethical approval was granted by the Research and Ethics Committee of the Uganda Virus Research Institute, the Uganda National Council for Science and Technology, and the London School of Hygiene and Tropical Medicine. Individual written informed consent (for adults ≥18 years and emancipated minors, and for children by a parent or guardian) and assent (for children 8-17 years) was sought for survey participation.

Laboratory methods
Two slides from each stool sample were examined (by different technicians) using the Kato-Katz method (33). The remaining sample was suspended in ethanol and stored at À80°C to allow further investigation for Necator americanus and Strongyloides  , and, among a subset of 200 participants, for Ancylostoma duodenale, using real-time polymerase chain reaction (RT-PCR) (34). Quality control for PCR assays was conducted at St Elisabeth's Hospital, Tilburg, NL. The Uganda results were comparable for N. americanus and A. duodenale, but had a lower detection rate for S. stercoralis. The presence of circulating cathodic antigen (CCA) of S. mansoni in urine was assessed (Rapid Medical Diagnostics, Pretoria, South Africa). Infection intensity based on Kato-Katz results was classified using WHO-recommended cut-offs (35). For PCR results, there are no standard cut-offs for categorizing infection intensity; however, based on results from Verweij et al. (34), individuals with C t > 30 would have parasite loads difficult to detect by microscope. Mansonella perstans infection was determined by a modified Knott's method (36); malaria was determined by thick blood film.
IgE specific to Dermatophagoides and cockroach allergens was measured by ELISA (29). The lower detection limit for our in-house ELISA was 15.6 ng/ml. We used 20-fold diluted plasma samples in our assay; hence, the lower detection limit in undiluted plasma was calculated as 312 ng/ml. This was used as a cut-off to create binary variables for detectable vs undetectable responses for each allergen.

Statistical methods
This was a cross-sectional analysis of survey data. Outcomes were reported wheeze in the last 12 months for children <5 years and for participants ≥5 years; visible flexural dermatitis; atopy defined as positive SPT response to any allergen for participants ≥1 year; atopy assessed as concentration of asIgE and analysed both as a continuous outcome and as detectable/nondetectable using the cut-off of 312 ng/ml. Exposures for the analysis were helminth infections. The following variables were considered as potential confounders: individual socio-demographic characteristics (age, sex, birth order, number of siblings, area of birth, area resided in for first 5 years, preschool attendance; occupation, maternal tribe, paternal tribe); behavioural and clinical characteristics (hand-washing behaviour, BCG scar, maternal or paternal allergy/asthma/ eczema, immunization history, breastfeeding, exposure to anthelminthic treatment in utero, anthelminthic treatment in last 12 months, artemisinin combination treatment for malaria in last 12 months, malaria infection, HIV infection); and household characteristics (crowding, animal ownership, asset score, indoor cooking, toilet access, drinking water source, washing water source, malaria control measures).
Assuming an average of 2.8 people per household, we expected that sampling 45 households per village would yield at least 3250 participants. For common exposures (prevalence ≥20%), assuming a design effect of 1.5 and outcome prevalence of 10%, the study would have over 80% power to detect risk ratios ≥1.5.
All analyses employed the 'svy' survey commands in Stata to allow for clustering of respondents within villages using linearized standard errors (37) and for variable village sizes using weights. Village-level weights were calculated based on the numbers of included and total households in each village. For binary outcomes, univariable and multivariable logistic regressions were used to obtain crude and adjusted odds ratios (OR) and 95% confidence intervals (CI). P-values were calculated using Wald tests. Adjustment was made for any potential confounder for which there was evidence of crude association with the outcome or which was considered to have a possible role, a priori.
Raw asIgE responses were skewed. Therefore, we used simple and multiple linear regressions to examine the association between helminth infections and log 10 levels of asIgE, and back transformed results to obtain geometric mean ratios (GMRs) and 95% CIs. For all outcomes, the role of S. mansoni infection intensity was assessed using the test for trend.
The population attributable fraction (PAF) for reported wheeze due to atopy was estimated as p'(ORÀ1)/OR with p' the prevalence of a positive SPT response among individuals with reported wheeze and OR the odds ratio for the wheeze-SPT association. We prespecified that we would examine whether helminth infections modified the associations between the atopy and the wheeze outcomes, by fitting interaction terms in multivariable logistic regression models. We also undertook a series of additional exploratory interaction analyses between helminth infections for each outcome, in an attempt to understand the primary association findings. Finally, as most previous studies have been performed in children, we conducted an exploratory investigation into whether associations between allergy-related outcomes and between helminths and allergy-related outcomes differed by age group (<16 vs ≥16 years).

Study participant characteristics
Of 1170 households selected, 144 (median per village 5, range 0-13) were excluded, because nobody was available to take part (n = 74), household members refused (n = 34), household was unoccupied (n = 28), household members' main place of residence was another selected household (n = 6), and household members were ill (n = 2). From the remaining 1026 households, 2316 individuals were surveyed. Characteristics of the participating individuals are shown in Table 1, with further details described elsewhere (29).
Reported wheeze in the last 12 months was rare (Table 1) but increased with age (Fig. 1A); 15 participants (0.7%) had visible flexural eczema (four satisfied the UK criteria for atopic eczema). Nineteen per cent of participants were atopic based on SPT with cockroach the most common allergen to elicit a response. Prevalence of positive SPT response to cockroach peaked in school-aged children while for other allergens, prevalence increased with age (Fig. 1A). Median (IQR) asIgE was 1440 (170-3990) ng/ml for Dermatophagoides and 220 (70-650) ng/ml for cockroach, with 73% of participants having detectable levels of Dermatophagoides IgE, 41% having detectable levels of cockroach IgE and 80% having detectable asIgE for either allergen.
The numbers and survey design-adjusted percentages of individuals infected with each helminth are shown in Table 1.
S. mansoni was most commonly detected, with infections peaking in prevalence and intensity among school-aged children (Fig. 1B), followed by N. americanus, S. stercoralis, T. trichiura, M. perstans and A. lumbricoides (Table 1). We did not detect any A. duodenale among the subgroup of 200 participants investigated. A third of those infected with S. mansoni based on Kato-Katz had heavy infections. For both T. trichiura and A. lumbricoides, all but seven infected individuals had light infections; therefore, we were not powered to look for associations between intensities of these helminths and the study outcomes. Infection intensities for N. americanus and S. stercoralis were generally light, with median (IQR) C t values of 35.7 (33.0, 39.1) and 34.2 (31.9, 37.1), respectively. Based on urine CCA, 72% of 917 individuals tested were infected with S. mansoni (compared to 48% classified as infected by Kato-Katz in this subgroup). Of the 421 individuals who were S. mansoni uninfected based on Kato-Katz and for whom CCA results were available, 218 (52%) were positive on CCA and could be considered as having 'very light' infections not detected with Kato-Katz analysis of a single stool sample.

Associations between allergy-related outcomes
Key associations between allergy-related outcomes and between helminths and allergy-related outcomes are summarized in Fig. 2. Individuals with a positive SPT response to any allergen were more likely to report wheeze [OR 2.49 (95% CI: 1.43, 4.33), P = 0.002]; the PAF for reported wheeze associated with atopy based on SPT was 19.9%. This association was seen consistently for both under-and overfives and for each of the three allergens used for SPT, and was stronger as the number of allergens for which participants had a positive SPT increased (P-value for trend test <0.001). Individuals with higher Dermatophagoides-specific IgE were more likely to have a positive SPT response to Dermatophagoides [OR for each unit increase in log Dermatophagoides-specific

Associations between helminths and allergy-related outcomes
For reported wheeze in under-fives, we were only able to examine associations with S. mansoni and T. trichiura (as for  Table 2 summarizes associations between helminth infections and wheeze in over-fives, and atopy based on SPT.

Investigation of effect modification by helminths and age group
The positive association between Dermatophagoides IgE level and wheeze was only seen among those infected with S. The positive associations between T. trichiura and SPT response and between A. lumbricoides and wheeze in overfives were enhanced by concurrent infection with S. sterco-ralis (interaction P = 0.004 and P < 0.001, respectively; Table 4) and the former was also enhanced by concurrent hookworm (interaction P = 0.05; Table 4). M. perstans infection was associated with higher levels of Dermatophagoidesspecific IgE in the absence of concurrent S. mansoni infection (interaction P = 0.007). There was no evidence of effect modification between age group and helminths for any outcome.

Discussion
In these remote fishing communities of Lake Victoria where helminths are highly prevalent, atopy was more common in individuals infected with Trichuris, schistosomiasis, M. perstans or Ascaris, and reported wheeze in the last year was more common among those with Ascaris infection. Our findings are in contrast to the hypothesis that chronic helminth infections protect against atopy and allergy-related diseases.
We explored various explanations for why our findings might differ from those reported by others. Concurrent infection with other helminths or pathogens could be a factor: we found some evidence that the association between Trichuris infection and SPT response was enhanced by concurrent infection with S. stercoralis or hookworm (Table 4); these results could also be interpreted as indicating that S. stercoralis and hookworm infections were inversely associated with atopy, in the absence of Trichuris co-infection. The fact that our survey was not restricted to children (unlike most other studies) is unlikely to be the explanation: although we found that the inverse association between cockroach IgE and reported wheeze was not seen in children, there was otherwise no evidence for effect modification by age. Our findings were consistent for both cockroach and dust mite allergens;   thus, differences between studies in allergens used is unlikely to explain our different findings. Another possible explanation that we cannot exclude is that individuals in our study setting may have suffered more long-term and chronic infections compared to those in other studies. Finally, although hookworm infection as detected by PCR was fairly common, it was generally of low intensity; hence, this may have reduced our ability to detect associations for this helminth. We estimate that in this setting, the population fraction of reported wheeze attributable to atopy is around 20%, lower than reported in most high-income settings, but similar to many other LICs (9,38). Consistent with findings from other developing country settings (21), hookworm infection appeared to suppress associations between IgE and SPT for cockroach responses; however, schistosomiasis had the opposite effect, promoting associations between atopy and wheeze for both dust mite and cockroach allergens.
Helminth infections could promote atopy either by nonspecifically driving the antigen presentation-T-cell-to-B-cell immune response axis towards greater production of IgE, or by inducing cross-reactive IgE. Regarding the latter, asthma severity has been shown to be related to Ascaris IgE levels, which correlate with mite-specific IgE (39), cross-reactivity has been demonstrated for selected Ascaris and mite antigens (40), and immunization of rabbits with Ascaris antigens induces IgE which cross-reacts with house dust mite (41). This accords with our observed association between Ascaris and elevated dust mite-specific IgE. Likewise, molecular modelling indicates that S. mansoni contains cysteine proteases homologous to the house dust mite antigen Der p 1 (42) and S. mansoni infection was associated with elevated house dust mite-specific IgE in our study. Cross-reactive IgE can also occur to molecules such as tropomyosin which are highly conserved between invertebrates (including nematodes, schistosomes, mites and cockroach) (43) and, consistent with cross-reactivity, the prevalence of positive SPT responses to cockroach and the heaviest S. mansoni infections both peaked in school-aged children in our study.
Mechanisms for a positive association between helminths and wheeze could include exacerbation of allergen-specific atopic responses or a direct response to the allergen-like helminth proteins experienced during larval migration through the lungs. The former might explain our finding that concurrent S. mansoni infection strengthens the association between allergen-specific IgE and wheeze. The latter may explain the association between Ascaris and wheezethe long-recognized L€ offler's syndrome (44)although cross-reactivity between Ascaris and mite allergens may also contribute, as discussed above.
For each of these possible mechanisms, concurrent infection with a helminth species that down-modulates allergyrelated immune responses might modify the association between pro-allergenic helminth species and allergy-related outcomes in the same way that hookworm has been observed to modify the link between allergen-specific IgE and histamine release (21). However, our interaction analyses showed little evidence of such effects: the only result consistent with this hypothesis was that S. mansoni infection modified the association between M. perstans and house dust mite-specific IgE production.
Despite the positive associations observed between helminths and allergy-related outcomes in this study, the overall prevalence of wheeze, eczema and SPT positivity was low compared with developed countries and urban settings in low or middle-income countries (8, 45). This suggests that factors other than current active helminth infection in these communities have important protective effects against allergy-related outcomes. These factors could include prenatal exposure to helminths (22,46), exposures to a myriad of infectious, xeno- M. perstansmalaria infection; A. lumbricoidesmaternal history of asthma. For associations with SPT (any and allergen-specific), all ORs were also adjusted for number of siblings, area of birth, and helminth-specific ORs were additionally adjusted as follows: S. mansoni -T. trichiura infection; hookworm -S. mansoni infection, S. stercoralis infection, T. trichiura infection. For associations with Dermatophagoides SPT, helminth-specific ORs were additionally adjusted as follows: S. mansonimaternal tribe, hand-washing behaviour; hookwormhousehold toilet access; T. trichiuramaternal tribe, household toilet access; M. perstansmaternal tribe. For associations with cockroach SPT, helminth-specific ORs were additionally adjusted as follows: hookwormhousehold asset score; T. trichiurahousehold toilet access; S. stercoralishousehold asset score. For associations with Blomia tropicalis SPT, ORs for S. mansoni and hookworm were additionally adjusted for handwashing behaviour.    (47,48), and a range of lifestyle factors (49), which await further investigation in this setting.
We found high levels of allergen-specific IgE. This could be a consequence of immunological cross-reactivity between helminth allergens and aeroallergens or a result of nonspecific stimulation of IgE production resulting from intense helminth exposure (as discussed above), or an artefact of our inhouse assay. Further studies to determine the characteristics of the IgE present in this population are in progress.
Our study had some limitations. We could only evaluate helminth infections endemic to our study setting: for example, for hookworm, we could investigate associations between N. americanus and allergy outcomes, but not A. duodenale, as the latter was not found. The cross-sectional design of this survey means that, strictly speaking, we cannot tell the relative timing of allergen sensitization and helminth exposure. However, our age-prevalence profiles show that both prenatal helminth exposure and infection in infancy and early childhood are likely in this setting so prior exposure to helminths, or con-current exposure to helminths and allergens, is likely to have occurred. Although the use of reported wheeze in the last 12 months has been validated as a proxy measure for asthma in many settings (50), there is no direct translation of the word 'wheeze' in the local language; thus, this outcome is likely to be subject to misclassification. Indeed, the increasing prevalence of reported wheeze with age that we observed could indicate that the phenotype being captured was to some extent related to chronic bronchitis rather than asthma. We investigated the use of a video questionnaire for wheeze in the study and found that agreement between the two approaches was fairly low although participants reporting wheeze had, on average, reduced levels of lung function parameters (29). Although we collected data on reported allergic rhinitis (a common disease caused by the allergens tested in our study, in some settings) in our survey and investigated it as an exploratory outcome, we found it to be uncommon and there was no evidence of association with any helminth; however, it is possible that this outcome was subject to misclassification, for similar reasons to wheeze. An additional source of misclassification is that we used single stool samples to assess helminth infection status (51,52). Indeed for the subgroup of participants who underwent urine CCA testing, the prevalence of S. mansoni was found to be much higher than when tested using Kato-Katz of the single stool sample. Also, the PCR method used had limited sensitivity for Strongyloides. This could have led to underestimation of the size of any true association. The study involved a large number of statistical tests, for which we made no formal adjustment; however, the consistent patterns of positive associations are unlikely to be explained by chance. Findings from our interaction analyses should be treated with caution. Although not all of these analyses were preplanned, we felt they were important to try to shed light on our unexpected findings.
In conclusion, we found that certain helminth infections were positively associated with allergy-related outcomes in this setting, with inverse associations only being seen in subgroup analyses. The LaVIISWA trial is currently ongoing and the impact of intensive vs standard anthelminthic treatment will be investigated in a further cross-sectional household survey in 2016. At that time, we will be able to assess not only the direct impact of worm removal on allergyrelated outcomes, but also the effect of the trial interventions on the associations reported herein. If there is a causal relationship underlying the observed associations, then the allergy-related outcome prevalence might reduce with the removal of helminth infection, rather than increasing as initially hypothesized.