Environmental factors associated with allergy in urban and rural children from the South African Food Allergy (SAFFA) cohort

Journal Pre-proof Environmental factors associated with allergy in urban and rural children from the South AFrican Food Allergy (SAFFA) cohort Michael E. Levin, MD PhD, Maresa Botha, MD, Wisdom Basera, MPH, Heidi E. Facey-Thomas, Dip. Nursing. RN. ACN (Allergy), Ben Gaunt, MD, Claudia L. Gray, MD PhD, Wanjiku Kiragu, MD, Jordache Ramjith, MSc, Alexandra Watkins, MA MSc, Jon Genuneit, MD PhD PII:

S0091-6749(19)31309-0

DOI:

https://doi.org/10.1016/j.jaci.2019.07.048

Reference:

YMAI 14207

To appear in:

Journal of Allergy and Clinical Immunology

Received Date: 26 April 2019 Revised Date:

21 June 2019

Accepted Date: 2 July 2019

Please cite this article as: Levin ME, Botha M, Basera W, Facey-Thomas HE, Gaunt B, Gray CL, Kiragu W, Ramjith J, Watkins A, Genuneit J, Environmental factors associated with allergy in urban and rural children from the South AFrican Food Allergy (SAFFA) cohort, Journal of Allergy and Clinical Immunology (2019), doi: https://doi.org/10.1016/j.jaci.2019.07.048. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology.

Environmental factors associated with allergy in urban and rural children from the South African Food Allergy (SAFFA) cohort

Rural

Urban

Farm animals Exposure to farm animals during pregnancy

Protection

Environmental tobacco smoke Environmental tobacco smoke

Antibiotic exposure Fermented milk

Fast foods/meat

Caesarean section Fast foods/meat

Risk

Protection

Risk

1 Environmental factors associated with allergy in urban and rural children from the South AFrican Food Allergy (SAFFA) cohort. Michael E Levin MD PhD, Maresa Botha MD, Wisdom Basera MPH, Heidi E. Facey-Thomas Dip. Nursing. RN. ACN (Allergy), Ben Gaunt MD, Claudia L. Gray MD PhD, Wanjiku Kiragu MD, Jordache Ramjith MSc, Alexandra Watkins MA MSc, Jon Genuneit MD PhD Affiliations Levin Division of Paediatric Allergy, Department of Paediatrics, University of Cape Town, Cape Town, South Africa and inVIVO Planetary Health, Group of the Worldwide Universities Network (WUN) Botha Division of Paediatric Allergy, Department of Paediatrics, University of Cape Town, Cape Town, South Africa Basera School of Public Health and Family Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa Facey Thomas Division of Paediatric Allergy, Department of Paediatrics, University of Cape Town, Cape Town, South Africa Gaunt Zithulele Hospital, Eastern Cape Department of Health, South Africa and Division of Primary Health Care, Health Sciences faculty, University of Cape Town, Cape Town, South Africa Gray Division of Paediatric Allergy, Department of Paediatrics, University of Cape Town, Cape Town, South Africa Kiragu Department of Paediatrics, Aga Khan University Hospital, Nairobi, Kenya. Ramjith Department for Health Evidence, Biostatistics Research Group, Radboud Institute for Health Sciences, Radboud University Medical Center, Nijmegen, The Netherlands. and Division of Epidemiology & Biostatistics, School of Public Health & Family Medicine, University of Cape Town, Cape Town, South Africa Watkins Division of Paediatric Allergy, Department of Paediatrics, University of Cape Town, Cape Town, South Africa Genuneit Institute of Epidemiology and Medical Biometry, Ulm University, Ulm, Germany and Center for Pediatric Research Leipzig, Hospital for Children and Adolescents, University of Leipzig Medical Center, Leipzig, Germany

2 Corresponding author: Michael E. Levin, Room 516, ICH building, Red Cross Hospital, Klipfontein Rd, Rondebosch 7700, South Africa. Email: [email protected], fax: +27 21 6891287, tel:+27 21 6585311 Funders: Medical Research Council of South Africa, Nestle, Mylan, ThermoFisher Word count: Conflicts of interest Michael E. Levin: Research funded by Medical Research Council of South Africa, Nestle, Mylan, ThermoFisher The rest of the authors have nothing to declare.

3 Author Contributions Michael E. Levin: Study design, data gathering, data analysis, writing up, article review. Maresa Botha: Study design, data gathering, data entry, data analysis, writing up, article review. Wisdom Basera: Study design, data entry, data analysis, article review. Heidi E. Facey-Thomas: Study design, data gathering, data entry, article review. Ben Gaunt: Study design, article review. Claudia L. Gray: Study design, article review. Wanjiku Kiragu: Study design, data gathering, data entry, article review. Jordache Ramjith: Study design, data analysis, article review. Alexandra Watkins: Study design, data gathering, data entry, data analysis, article review. Jon Genuneit: Study design, data analysis, article review.

ABSTRACT Background: The prevalence of allergic diseases differs in urban and rural populations. Objective: To assess associations between environmental and dietary factors with allergic diseases in urban and rural South African children. Methods: Toddlers aged 12-36 months were assessed for food- and aero-allergen sensitisation, atopic dermatitis, allergic rhinitis, asthma and challenge-proven food allergy. Information was collected on family history of allergic diseases, household size, socioeconomic status, delivery mode, antibiotic and probiotic use, exposure to fermented and unpasteurised milk, antihelminth treatment, sunlight exposure, pet and farm animal exposure, cigarette smoke and household cooking and heating fuels. Antenatal exposures to pets, livestock and cigarette smoke was assessed. A subsection completed questions on consumption of fruit/vegetables, fast foods, soft drinks/fruit juices and fried/microwaved meat. Results: Risk and protective factors differed between urban and rural settings. Exposure to farm animals in infants and their mothers during pregnancy was protective against allergic outcomes in the rural population. Consumption of unpasteurised milk is uncommon in this group of rural children and is unlikely to be an important factor in rural protection. In urban children birth by caesarean section is associated with food allergy and consumption of fermented milk products is associated with reduced asthma and atopic dermatitis. In both cohorts antenatal maternal smoking and environmental smoking exposure were predominantly associated with asthma and consumption of fast foods and fried meat were associated with allergy. Conclusion: In this rural environment exposure to livestock is the strongest protective factor. In urban communities, where animal contact is rare, risk factors include caesarian section and protective factors include consumption of fermented milk products. Modifiable risk factors urgently require interventions to prevent increasing allergies in countries undergoing rapid urbanization.

4 Key Points • In rural children exposure to farm animals is associated with reduced aeroallergen and food allergen sensitisation as well as food allergy. In rural settings, antenatal maternal farm animal exposure is associated with reduced rates of aeroallergen sensitisation, allergic rhinitis, food allergen sensitisation and food allergy. •

Consumption of unpasteurised milk is uncommon in this group of rural children and is unlikely to be an important factor in protection in the rural environment

•

In urban children consumption of fermented milk (amasi) is associated with reduced rates of asthma and atopic dermatitis

•

Birth by caesarean section is associated with higher rates of food allergy in urban communities.

•

Maternal smoking during pregnancy and parental smoking at the time of the study is associated with increased asthma in both urban and rural children.

•

High fast foods consumption (once a week or more) is associated with higher rates of atopic dermatitis in urban children and higher rates of aeroallergen sensitisation in rural children. Consumption of fried or microwaved meats is associated with higher rates of aeroallergen and food sensitisation in rural children and with the combined outcome measure of any asthma, allergic rhinitis or atopic dermatitis in urban children.

Key Words: Aeroallergen, aeroallergen sensitisation, Africa, allergic rhinitis, asthma, atopic dermatitis, eczema, food allergy, prevalence, food sensitisation, environmental exposures, fast foods, advanced glycation end products, smoking, probiotics, prebiotics, unpasteurised milk, antibiotics, antihelminthics, pets, farm animals, sunlight, delivery mode, caesarean section, cooking fuel, heating fuel, rural, urban, urbanisation Abbreviations used: AD – Atopic dermatitis AGE – Acute glycation end product AR - Allergic rhinitis AS – Allergic sensitisation FA – Food allergy FS – Food sensitisation OFC – Oral food challenge SAFFA study – South AFrican Food Allergy study SPT – Skin prick test

CAPSULE SUMMARY Farm animal exposure protects against allergy in rural settings, rendering other factors immaterial, however in urban settings, caesarean section and multiple antibiotic courses result in increased risk and consumption of fermented milk has a protective effect.

5 INTRODUCTION The significant increase in allergic conditions in various regions of the world has been too rapid to be explained by genetic changes. In addition, the differing prevalence of allergies in people with similar genetic origin living in various areas of the world suggests that environmental factors have an effect on modulating the risk of developing allergies. The hygiene hypothesis hypothesized that an over-hygienic environment and lack of infectious diseases contributed to immune diversion towards allergic diseases(1). Subsequently, the “old friends hypothesis”(2) and burgeoning data on the role of the normal skin(3) and gut(4) human microbiome in modulating immune responses led to the hypothesis that abnormalities in the composition and diversity of commensal microbial organisms could lead to immune dysregulation and allergic disease. A marked protective effects of “farm living” on asthma and allergies has been partly attributed to immune-modifying effects of bacterial endotoxins emitted by livestock and differences in consumption of unpasteurized cow’s milk(5), however different factors may be present in varied parts of the world(6–8). Nutritional factors have been associated with differing food allergy risk(9), particularly the timing of introduction of potentially allergenic foods, micronutrient intake during early life, the ingestion of pre- and pro-biotics by mothers and infants, the variety of foods ingested during the first year of life and the ingestion of fast foods, high in advanced glycation end products. The western dietary pattern is high in advanced glycation end-products (AGEs), found in cooked meat, oil, cheese and high-sugar diets, and this has been increasing in the last 3 decades in parallel with the allergy epidemic. Food allergy has been postulated to be effected by the consumption of foods high in advanced glycation end products (AGEs) (fried and microwaved meat and fast foods), foods that promote the formation of AGEs (sugar, especially fructose consumption, hence soft drinks and fruit juices) and foods protective against AGE formation (fresh fruit and vegetables).(10) The SAFFA (South AFrican Food Allergy) study investigated the food sensitisation and food allergy prevalence rates amongst children aged 1-3 years in urban Cape Town and the rural Eastern Cape regions of South Africa(11). We have previously reported on a significant difference in the prevalence of aeroallergen sensitisation (AS), eczema, asthma and allergic rhinitis as well as food sensitisation (FS) and food allergy (FA) between urban and rural South African children(12) and the interactions between nutritional status and infant feeding in this cohort(13). We assessed for differences in environmental factors between urban and rural communities and for associations with aeroallergen sensitisation, allergic diseases, FS and challenge proven FA. Environmental factors assessed included those that affect the microbiome (mode of delivery, exposure to livestock and pets, consumption of antibiotics and probiotics as well as antihelminth treatment) and dietary factors including consumption of unpasteurised milk, natural and medicinal probiotics, fast foods, fried and microwaved meats and fruit and vegetables.

METHODS Study design and setting 1185 urban and 398 rural toddlers aged 12-36 months were enrolled in the South AFrican Food Allergy (SAFFA) study. This study was conducted in the city of Cape Town and in the rural Eastern Cape province of South Africa. Study staff administered questionnaires with the parents or guardians of participants on self-reported allergic diseases, current environmental exposures and environmental exposures during pregnancy. All participants were examined for clinical signs of allergy and screened for food sensitisation and challenge proven food allergy. A subsection of participants was screened for aeroallergen sensitisation.

6 Sampling frame In Cape Town, registered early childhood development (ECD) centres were approached in random order and parents of all enrolled children aged 12 to 36 months were invited to participate. As ECD centres are rarely found in the rural area we conducted study days at 10 primary healthcare clinics recruiting all children of eligible age from the surrounding area. Sample size Sample size for the SAFFA study was calculated to determine and compare the prevalence of food sensitisation and food allergy in urban Cape Town and the rural Eastern Cape. Screening for aeroallergen sensitisation and questions about fast foods, soft drinks, fruit juices and fried meat etc were added into our assessment when we estimated an equal number of rural and urban participants would still be recruited and we aimed to enrol 400 participants in each group. Recruitment and assessment Researchers captured information on family history of allergic diseases, family size, household number, socioeconomic status (including parental employment and education) as well as information on delivery mode, antibiotic, probiotic and paracetamol use in the first year of life, exposure to pets and farm animals, exposure to amasi (fermented milk), antihelminth treatment, vaccination status, sunlight exposure, household cooking and heating fuels and cigarette smoke exposure. Mothers were assessed for exposures during pregnancy to smoking, probiotics, amasi and farm animals. A smaller subsection of participants completed questions on their consumption of fast foods, soft drinks and fruit juices, fried/microwaved meat as well as fruit and vegetable consumption. For each of these exposures we defined a high consumption category with high consumption of fast foods and fried or microwaved meat being once a week or more, high soft drink or fruit juice consumption being 3 or more times a week and high fruit and vegetables consumption being 4 portions or more consumed in the preceding 48 hours. Participants’ allergic diseases were determined by self-reported symptoms according to modified ISAAC criteria and through clinical examination for signs of allergic rhinitis (allergic shiners, nasal crease and Dennie’s lines) and atopic dermatitis (xerosis, visible skin lesions). Atopic dermatitis (AD) was diagnosed based on diagnostic criteria proposed by the American Academy of Dermatology including essential features of pruritus and eczematous rash with typical morphology and age-specific patterns, and a chronic or relapsing history(14). Skin prick tests (SPT) were performed for food allergens in the entire cohort and for aeroallergens in a smaller subsection. SPT were performed using ALK-Abello SPT solutions (ThermoFisher) to 7 common food allergens (egg white, peanut, cow’s milk, cod-fish, soya, wheat flour and hazelnut) and 4 aero-allergens (Dermatophagoides pteronyssinus, Blomia tropicales, German cockroach (Blatella germanica) and Grass mix containing Dactylis glomerata, Festuca eliator, Lolium perenne, Phleum pratense and Poa pratensis). In addition, fresh egg white, fresh peanut butter and fresh cow’s milk were used with histamine (10mg/ml) and saline as positive and negative controls respectively. SPTs were read at 15 minutes and were recorded as mean wheal diameter in millimetres. A mean wheal diameter of 3 mm for aeroallergens was considered positive. Participants with any reactive food SPT (≥1mm) and who were not on history tolerating an age appropriate portion of the specified food, qualified for an open oral food challenge (OFC) performed at Red Cross Children’s hospital in Cape Town and Zithulele Hospital in the Eastern Cape. This study was approved by the Human Research Ethics Committee of the University of Cape Town (HREC REF: 038/2012). Informed consent was obtained from parents or legal guardians of all participants. Statistical analysis Data was entered in a Microsoft Access database and analysed using STATA version 11.1 (Stata Corp, College Station, Texas). Outcome measures were assessed separately. Additionally subjects with any asthma, allergic rhinitis or atopic dermatitis were grouped as a combined outcome measure, referred to as “any asthma, allergic rhinitis or atopic dermatitis”.

7 The Mann-Whitney test was used to investigate differences between numerical variables and Fisher’s exact to assess associations between categorical variables. The z-test was used to investigate differences in proportions. A p-value of ≤0.05 was considered statistically significant. Univariate regression analysis was performed in the two study populations separately, as the markedly different exposures in the two poulations precluded combining them into a single population. We ran univariate logistic regression analysis on all exposure variables to calculate odds ratios for associations with allergy outcomes.For each AGE exposure variable we defined a “high consumption” category to create binary variables for univariate logistical regression analysis. Multivariate analysis was not performed as the numbers of subjects in many groups were too small to perform a meaningful analysis. RESULTS 1185 urban and 398 rural participants completed the study. 535 urban and 347 participants in the urban and rural cohorts respectively completed SPT for aeroallergens, (table 1) while 535 urban and 398 rural participants completed questions on AGE related food consumption. The median age of enrolment of the urban cohort was 26 months (IQR 22-32), significantly older than the rural cohort (21 months; IQR 17-28; p<0.001). More male than female participants were enrolled in both the urban and rural cohorts, however the proportions did not differ significantly between cohorts. The urban cohort reflected the ethnic composition of children under 5 in Cape Town. All children in the rural cohort were black African. Monthly household income was significantly higher in urban than rural participants (ZAR7000 vs. ZAR1380; p<0.001). The median rural household size was 6 (IQR 5-8), significantly larger than urban households of 4 (IQR 4-6) (p<0.001). Urban parents were significantly more likely to have completed tertiary education (43.3% vs. 5.0%; p<0.001) and were more likely to both be employed (73.1% vs. 8.3%; p<0.001). There were no significant differences between the smaller cohort who completed aeroallergen skin prick testing and the full cohort with regards to age, sex and socioeconomic status and personal and family history of atopy (table 1). Farming exposures Exposure to household pets in the rural cohort was markedly higher than in the urban cohort (78.6% vs. 42.1% p<0.001). Urban children with pets had higher rates of any allergy (48.7% vs. 42.4%; p=0.03; supplementary table 1) than those without pets. No significant associations were seen in the rural cohort. 2.5% of urban children had weekly contact with farm animals compared to 99.3% of rural children (p<0.001). Rural participants with no farm animal contact had significantly higher rates of aeroallergen sensitisation (50.0% vs. 3.5%; p=0.003), food sensitisation (33.3% vs. 5.1%; p=0.03), and food allergy (33.3% vs. 0.3%; p<0.001; supplementary table 1). Similarly, 2.4% of urban mothers had weekly contact with farm animals during pregnancy compared to 98.7% of rural mothers (p<0.001). In the rural cohort maternal farm animal exposure during pregnancy was associated with reduced rates of aeroallergen sensitisation (3.5% vs. 25.0%; p=0.03), food sensitisation at ≥3mm (2.5% vs. 20.0%, p=0.02) and food allergy (0.3% vs. 20.0%; p=0.03). In the rural cohort univariate logistical regression showed child’s exposure to farm animals to be associated with reduced rates of aeroallergen sensitisation (OR 0.04; p=0.02), food sensitisation (OR=0.05; p=0.02) and food allergy (OR 0.01; p=0.001) and maternal exposure during pregnancy was associated with reduced rates of food sensitisation (OR=0.1; p=0.05) and food allergy (OR=0.01; p=0.002). Sunlight exposure

8 Sunlight exposure was significantly higher in rural children during both summer and winter (table 2). In the urban cohort increased exposure to sunlight in summer was associated with less FS at 3mm (OR=0.8; 0.7-1.0; p=0.02) (supplementary table 2) and exposure in winter was associated with increased AR, AD and any allergy. Amasi (fermented milk) exposure was 64.8% in rural participants, significantly higher than the 41.2% of urban participants (p<0.001). Amongst urban black African participants, amasi exposure rates were 84.0% with 46.2% consuming amasi more than once a week compared to 6.6% of the rural cohort (p<0.001). The median age of first exposure was 12 months in all cohorts (Table 2). In the urban cohort consuming amasi was associated with lower rates of AR (16.8% vs. 31.3%; p<0.001), AD (21.3% vs. 28.6%; p=0.01) and self-reported asthma (5.3% vs. 11.5%; p=0.003). No significant associations were seen in the rural cohort (supplementary table 3). There was no significant difference in the consumption rates of unpasteurised milk between urban and rural children (2.6% vs. 1.8%; p=0.33). In the urban cohort children who had consumed unpasteurised milk had significantly lower rates of atopic dermatitis (9.7% vs. 26.1%; p=0.05). Delivery mode Caesarean section rate in the urban cohort was 40.5%, double that of the rural cohort of 18.8%. (p<0.001;Table 2). In the urban cohort, children born via caesarean section had double the food allergy rates of those born vaginally (3.3% vs. 1.6%; p=0.05). This difference however was not seen in the rural cohort (supplementary Table 4). Antibiotics and probiotic exposure Antibiotic exposure in the first year of life was 87.2% in the rural cohort, markedly higher than the 55.2% exposure rate in the urban cohort (p<0.001; Table 2). The median age of first exposure was 5 months in the rural cohorts, significantly younger than the median of 6 months in the urban cohort (p<0.001). Rural participants were more likely to have only used 1-2 courses (76.7% vs. 61.3%; p<0.001) while 8.3% of urban participants had more than 5 courses compared to 1.4% of rural participants (p<0.001). Antibiotic exposure in the black African urban children was lower than the Caucasian and mixed urban cohort (48.8% vs. 60.6%; p<0.001) and only 2% used 5 courses or more in the first year of life compared to 7.3% (p<0.001) Urban participants who received antibiotics in the first year of life had higher rates of allergic rhinitis (28.6% vs. 21.3%; p=0.004), self-reported asthma (12.7% vs. 4.5%; p<0.001) and any allergy (AR, AD and self-reported asthma combined 49.9% vs. 39.8%; p<0.001; supplementary table 5). There were no significant associations in the rural cohort. 14.9% of urban participants had probiotics in the first year of life at a median age of 6 months (IQR 4-9). Only 1 rural participant had probiotics. In the urban cohort probiotic use was associated with increased rates of AR (42.9% vs. 24.2%; p<0.001), AD (31.6% vs. 24.5%, p=0.05) and self-reported asthma (14.7% vs. 8.0%; p=0.004) as well as the combined variable of any allergy (65.0% vs. 41.7%; p<0.001; supplementary table 6). Antihelminth exposure Higher rates of antihelminth treatment were reported in rural participants (85.9% vs. 80.8%; p<0.001). Rural participants were also more likely to have been dewormed regularly (at least yearly) than urban participants (75.1% vs. 55.8%; p<0.001). In the urban cohort, children who had received antihelminth treatment had lower rates of food sensitisation at 1mm (10.2% vs. 15.6%; p=0.03) and 3mm (7.9% vs. 12.8%; p=0.03; supplementary table 5). Urban children who had been regularly dewormed (once a year or more) had lower rates of atopic dermatitis (22.7% vs. 28.1%; p=0.05) and any allergy (41.9% vs. 48.4%; p=0.03; supplementary table 7). There were no significant associations between antihelminth exposure and allergy outcome rates in the rural cohort. Cigarette smoke, household fossil fuel exposure 15.8% of urban mothers smoked whilst being pregnant compared to 2.3% of rural mothers (p<0.001). However black African urban mothers had similarly low rates of smoking during pregnancy (3.5%; p=0.28). Rural children whose

9 mothers smoked during pregnancy had significantly higher rates of self-reported asthma (11.1% vs. 0.8%; p=0.002; supplementary table 8). 52.0% of urban children had no smokers living in the house compared to 73.9% of rural children (p<0.001). 6.2% of urban children lived with ≥3 smokers compared to 0.8% of rural children (p<0.001). 23.1% urban mothers and 39.3% urban fathers smoked at the time of the study compared to 2.3% rural mothers and 22.6% rural fathers (both p<0.001). Smoking rates amongst urban black African mothers were low (4.0%), similar to that of black African rural mothers (p=0.14). Urban children whose mothers or fathers smoked had significantly higher rates of self-reported asthma than those who did not (13.1% vs. 7.7%; p=0.01 and 11.6% vs. 7.0% p<0.001). Maternal smoking was associated with higher rates of AR and any allergy in the urban cohort (32.1% vs. 23.2%; p=0.003, 50.4% vs. 43.4%; p=0.04). 99.5% of urban households used gas or electricity for cooking purposes and 28.7% paraffin for heating. Rural households mostly use paraffin (56.5%) and outside fires (76.6%) for cooking with 4% making fire inside to cook. Only 23.2% of households use electricity for cooking (table 2). In the urban cohort cooking with electricity or gas was associated with food allergy and cooking with paraffin was associated with lower risk of AR and any allergy (supplementary table 9) but numbers are too small for meaningful interpretation. In the urban community heating with paraffin was associated with decreased AR, AD and any allergy, heating with electricity was associated with increased AR, any allergy and food sensitisation at 1mm and heating with gas was associated with food sensitisation at 3mm. In rural communities heating with paraffin was associated with decreased AR,AD and any allergy supplementary table 10). Advanced glycation endproducts (AGE’s) in diet 31.2% of urban children consumed fast foods more than once a week compared to 9.5% of rural children (p<0.001; Table 2). Although urban black African children consumed fast food less frequently than the urban cohort in general (21.6% vs. 31.2%; p=0.01) this is still significantly more than the rural cohorts (p<0.001). Urban children with high fast food consumption rates had higher rates of AD than those with lower consumption rates (28.1% vs. 20.4%; p=0.05, OR 1.5 (1.0-2.3; p=0.05; supplementary table 11). This difference in AD was not seen in the rural cohort with low prevalence of AD, however high fast food consumption rates was associated with significantly higher rates of aeroallergen sensitisation (13.8% vs. 2.8%; p<0.01, OR 5.5; 1.6-19.1; p<0.01; supplementary table 11). Soft drink and/or fruit juice consumption rates were markedly higher in urban children than rural children with 68.8% of urban children consuming it ≥5 times a week compared to 24.9% of rural children (p<0.001). 74.0% of urban black African children had high consumption rates which was significantly more than their rural counterparts (p=0.03). In order to look at possible correlations with allergy outcomes we defined high consumption rates as ≥3 times per week. We found no significant association between higher consumption rates and allergy outcomes in either the urban or rural cohorts. 56.0% of urban children consumed fried or microwaved meats once a week or more compared to 7.5% in the rural cohort (p<0.001). Urban black African children had a significantly higher rate of consumption than rural children (46.0% vs. 7.5%; p<0.001). In the urban cohort children with high fried/microwaved meat consumption had higher rates of any allergy (43.5% vs. 35.3%; p=0.03). The rural cohort showed markedly higher rates of aeroallergen sensitisation rates (10.7% vs. 3.1%; p=0.05) and food sensitisation both at 1mm and 3mm in children with high fried meat consumption rates (SPT≥1mm: 16.7% vs. 4.4%; p<0.01 and SPT≥3mm: 10.0% vs. 2.2%; p=0.02). High consumption of fried/microwaved meat was positively associated in rural children with food sensitisation at 1mm and 3mm with odds ratios of 4.4 (CI 1.5-13.0; p=0.01) and 5.0 (CI 1.2-19.9; p=0.02) respectively. Fruit and vegetable consumption

10 Fruit and vegetable consumption in the 24 hours preceding the study was higher in urban participants with 79.1% consuming 4 portions or more compared to 11.3% of rural children (p<0.001). Urban black African children had similar high consumption rates (74.8%; p<0.001). In the urban cohort high consumption rates of fruit and vegetable were associated with significantly higher rates of any allergy (42.8% vs. 28.6%; p=0.03, OR=1.9; CI 1.2-2.9; p<0.01). In the rural cohort high consumption of fruit and vegetables was associated with significantly higher rates of selfreported asthma (5.7% vs. 3.6%; p<0.001), food sensitisation at ≥3mm (8.9% vs. 2.0%; p=0.01) and food allergy (4.4% vs. 0.0%; p<0.001). High fruit and vegetable consumption were positively associated with food sensitisation ≥3mm (OR=4.8; 1.4-17.2; p=0.02). DISCUSSION Early observations of hayfever prevalence being inversely associated with household size, and that only older siblings but not younger siblings conferred protection led to the proposal of the hygiene hypothesis(1). Additional support for the idea of microbial exposures being protective against allergy in urban environments stem from epidemiological studies showing the protective effects of day care attendance and pet (particularly dog) ownership, as well as mechanistic studies showing the protective effects of the indoor microbiome as well as surrogate markers of bacterial and fungal exposure(15–17). Farming or urban/rural exposure studies have been performed in multiple disparate environments, documenting and adding detail to the protective effects seen in rural settings(12,13,18–27). In most studies, contact with farm animals such as livestock was protective(21–24,28–31), and in others a role for the ingestion of unpasteurised cow’s milk was protective(32–34). The role of each of these effects in disparate communities across the globe, and the interactions between these effects and others such as family history of allergy and asthma, family size, maternal smoking, diet, physical activity, vitamin D and sunlight exposure, antibiotic exposure, caesarean section, pro- and pre-biotics remains controversial. Pet and farm animal exposure In this rural cohort, farm animal exposure was associated with decreased aeroallergen sensitisation, food sensitisation and food allergy, and maternal farm animal exposure was associated with lower aeroallergen sensitisation, allergic rhinitis, food sensitisation and food allergy. The magnitude of the effect was the largest of any single variable, however the number of participants without farm animal exposure was very low. The magnitude of this effect may vary with setting, and depend on critical timing of exposures prenatally(21), early in life(22) and even up to the teen years(24), however once present the effect may be sustained during life(35). Unpasteurised milk and amasi (fermented milk) A protective effect for the ingestion of unpasteurized milk has been found in some urban/rural studies(32–34). Contrary to what we expected, urban children were more likely to have been exposed to unpasteurized milk than rural children where exposure rates were extremely low. We subsequently found that livestock are infrequently milked in the rural study site, reportedly due to fears of brucellosis as a consequence of a failure of government cattle immunization programmes. In the urban cohort, consumption of unpasteurized milk was associated with decreased atopic dermatitis. In the rural cohort consumption of unpasteurised milk was associated with decreased food sensitisation and decreased food allergy however numbers of participants consuming unpasteurised milk were so low that the large-scale protective effect in this rural cohort cannot be related to the consumption of unpasteurised milk. Although amasi was traditionally a product made from unpasteurized milk by individual households this has almost completely ceased as it is now made commercially using pasteurized milk and is widely available in both urban and rural communities. Consumption of amasi was strongly associated with decreased atopic dermatitis, asthma and any allergy, however this effect was confined to the urban population. Amasi is a fermented milk rich in probiotics, which may contribute to gut microbial diversity, possibly compensating for deficient gut microbial diversioty that may be present in urban children(36).

11 Antenatal (maternal) exposures and delivery mode The rural cohort had higher self-reported asthma in participants whose mothers smoked during pregnancy. Prior data on smoking rates amongst urban pregnant South African women showed low rates amongst black African women (4% in 1997, 12.4% in 2006) whereas very high rates were recorded amongst women of mixed ancestry (47% in 1997, 49.5% in 2006)(37,38). This pattern seems to persist in this cohort (urban overall 15.8%, urban mixed ancestry 27.2%, urban Caucasian 18.8%, urban black African 3.5%, rural black African 2.3%). Data on current smoking in the mothers was much higher (urban overall 23.1%, urban mixed ancestry 41.4%, urban Caucasian 29.4%, urban black African 4.0%, rural black African 2.3%) suggesting modest declines in smoking amongst women in the last 10 years but large success in reducing smoking during pregnancy over that time. Maternal smoking during pregnancy and parental smoking at the time of the study was associated with increased self-reported asthma in both urban and rural children, representing a significant modifiable factor. Caesarian section rates were 40.5% in the urban cohort and 18.8% in the rural cohort. Caesarian section delivery was associated with food allergy in the offspring, but only in the urban cohort. Hospital-based deliveries, rather than home births are the norm in both urban and rural South Africa. The caesarean section rate in the public health sector is 26% and 60% in women delivering in private health care facilities(39). The higher Caesarean section rate in our urban cohort likely reflects increased access to private healthcare and the ability to choose this delivery mode in the absence of clinical indications. This may be an important factor in the difference in allergy between the two cohorts and represents a truly modifiable factor in reduction of allergy risk. Antibiotic and probiotic exposure Antibiotic exposure was associated with multiple allergic outcomes in the urban but not the rural cohort. An unexpected finding was the high rate of antibiotic use in the rural community. This may reflect the majority of emergency care being provided by nurse-led primary care facilities where antibiotic stewardship policies are poor and antibiotics may be prescribed unnecessarily for minor viral infections. In the urban communities, however, there was a higher prevalence of multiple and prolonged antibiotic use(40). This may either reflect a true increase in infectious diseases (apart from tuberculosis) in the urban community, or the tendency of doctors in urban settings to prescribe multiple doses rather than refer for further management. It is possible that the associations between antibiotic and probiotic use and increased prevalence of allergies is due to reverse causation, with antibiotics used as (inappropriate) treatment for allergies being mistaken for such conditions. Helminths Prior data in black African subjects in urban Cape Town has shown a positive association between ascaris sensitisation and aeroallergen sensitisation but not allergic diseases and raised the possibility that the correlation may be due to reverse causation with no marked proallergic or protective effect of ascaris infestation.(41). In this study, however, exposure to anti-helminthic medication was associated with decreased allergy outcomes in the urban cohort. Public health policy mandates 6 monthly mebendazole from the age of 6 months for all children regardless of suspected helminthic infection. Rural children had higher rates of deworming, earlier first deworming and more regular deworming that the urban cohort. This may reflect higher clinical infestation requiring additional treatment as in a subset of this study, stool samples revealed positive kato-katz in 6.7% of urban non-allergic infants and 10.5% of rural non-allergic subjects(42). Sunlight exposure There is no significant difference in latitude between the urban (Cape Town 33.9◦S) and rural (Mqanduli 31.9◦S) sites. There was a significantly longer exposure to sunlight in the rural cohort. It is unknown whether this translates into higher levels of Vitamin D. The data was gathered as sunlight exposure in winter and summer separately to allow a more accurate assessment of exposure by mothers having to average out seasonal variation. The data is not strong, with confidence intervals that approach 1.0. In addition, longer sunlight exposure may also be a proxy for exposure to the outdoor environment including exposure to outdoor animals and livestock.

12 AGE’s and fruit and vegetable consumption Advanced glycation end products are the products of proteins binding with reducing sugars in an enzyme independent manner. They are found in high concentrations in the modern urban diet, predominantly in animal proteins and fats that are cooked at high temperatures, whether fried or microwaved, but less with boiling. Increased consumption of sugars, especially fructose, increase the formation of endogenously produced AGEs(43). The consumption of natural antioxidants have an anti-glycation effect, predominantly from polyphenols present in fresh fruits and vegetables(44). AGEs function as alarmins through signaling via the High Mobility Box 1 alarmin receptor, the receptor for Advanced Glycation End-products(43). AGEs have been postulated to be a risk factor for the development of food allergy and other allergic and inflammatory diseases. Increased exposure to fast foods and sugary drinks have been described as risk factors for food allergy as well as asthma, rhinoconjunctivitis and eczema (43). The ISAAC study phase 3 showed that eating fast foods three or more times a week was associated with a 39% increased risk of severe asthma in adolescents and a 27% increase risk among children, as well as an increased risk of severe rhinoconjunctivitis and severe eczema(45). On the other hand, intake of fruit more than 3 times weekly was associated with an 11% decrease in asthma(45). Fructose consumption from fruit juices and soft drinks has been associated with increased risk of asthma(46). Other dietary factors such as fibre and macro- and micronutrient ingestion, may be correlated with the factors measured and may have direct effects on the gut microbiome.Foods with high AGE levels were consumed more frequently by urban than rural children. In addition “AGE-protective” foods (fresh fruit and vegetables) was also higher in the urban setting. In the rural cohort there was an interaction between food consumption patterns possibly attributable to socioeconomic class, with higher fruit and vegetable intake being associated with frequent fast food ingestion (p<0.001), but this association was not present in the urban cohort. In the urban cohort increased fried/microwaved meats was associated with (any of) asthma, AR and AD and high fast food consumption with AD. In the rural cohort, increased fast food consumption and increased fried/microwaved meat consumption was associated with aeroallergen sensitization. High fruit and vegetable consumption was associated in the urban cohort with (any of) asthma, AR or AD and in the rural cohort with asthma, AD and food allergy. In the rural cohort this may be attributed to an interaction with the consumption of high AGE containing foods. A subsection of the rural cohort (47 children) were included as non-atopic controls in a study (36,47) comparing the diet and intestinal microbiome in Black African children with and without atopic dermatitis. Significantly lower sugar and saturated fat consumption was reported in the diets of rural children without AD than in their atopic counterparts and a significant association was found between higher consumption of sugar and AD. Increased daily intake of sugar was associated with lower diversity of the gut microbiome and increased relative abundance of Prevotella copri which in turn was inversely associated with AD(36). Study limitations The relatively smaller sample size of the rural cohort and their lower prevalence of allergies limited our ability to show clearer correlations between exposures and allergy outcomes. This may also partly account for the different patterns of association between risk factors and allergy outcomes between the rural and urban cohorts. Univariate regression analysis was performed in the two study populations separately, as the markedly different exposures in the two populations precluded combining them into a single population. For some exposure and outcome measures, numbers of participants in either the urban, rural cohort or both, precluded meaningful analysis and multivariate analysis was therefore not performed. Questionnaires were administered by trained researchers familiar in the participants’ first language as self-completed questionnaires have been shown to be susceptible to language differences in this community(19,48,49). Within the urban population, the subcohort that had aeroallergen skin prick tests had slightly lower rates of asthma and allergic rhinitis than those that did not have skin tests performed. There were no statistically significant differences in the rural subcohorts with and without skin testing. In addition to the markedly different exposures in the urban and rural communities and the markedly different outcome variables, there were also markedly different rates of family history of allergy between urban and rural participants. Because sampling was random in both

13 settings, it is unlikely that selection bias within one or both of the communities is responsible for this. Rather this may reflect multi-generational protection effects within the rural environment affecting not only the study participants but also their parents. Summary Risk and protective factors differed markedly between urban and rural settings, but urban black African participants closely resembled the rest of the urban cohort rather than the black African rural cohort. This supports other research indicating that despite some differences existing between populations(50,51), environment rather than ethnicity, is the single most important factor in allergy risk and prevention in this setting(52). Antenatal and childhood farm animal exposure, rather than the consumption of unpasteurised milk, is the strongest factor associated with protection against multiple forms of allergy in this rural setting. It is likely that these effects are modulated through exposure to microbes or microbial products promoting protective immune responses. It is interesting to hypothesize whether this mechanism could explain the difference in risk and protective effects seen in these urban and rural settings. It may be that the rural environment’s multiple environmental exposures (but most markedly exposure to farm animals) cause strong protective epigenetic modification mediated via microbiome diversity that is sufficient to render other risk factors immaterial. In the urban setting, however, where these protective factors are not present, risk factors such as caesarean section and multiple antibiotic courses may be unmasked as causing further deleterious effects on the microbiome resulting in increased risk. In addition the consumption of fermented milk products has no effect in this rural environment, but a marked protective effect in seen in the urban cohort. This may imply that the bacterial diversity that is lacking in the urban environment can be partially abrogated by the consumption of fermented milk products, but that this intervention is unnecessary in rural settings. Priorities in children’s environmental health in South African address the effects of rural underdevelopment and poverty including lack of access to clean water and sanitation, as well as the effects of rapid urbanisation including increased exposure to indoor fuels, vehicular pollution, industrial toxins (e.g. lead, arsenic) and pesticides. Climate change is likely to amplify these negative environmental effects and adds to the urgency of setting guidelines to holistically address environmental exposures including those that are protective in the rural environment (53). Our data support the hypotheses that protection from allergy may be associated with an environment rich in exposure to a variety of bacteria and endotoxins, a higher exposure rate to minor infections (as evidenced by higher early antibiotic usage), a diet rich in natural probiotics and reduced intake of fast foods. Increased outside time and reduced fast food intake are major advantages in rural environments. Loss of such rural protective factors with urbanization, particularly farm animal exposure, may expose an innate allergy risk(54,55) which had been kept in check by protective factors. Many of these risk factors are modifiable. Although we cannot replicate the rural environment, discouraging fast food culture and inappropriate caesarian sections, increasing exposure to fruit and vegetables and encouraging increased healthy outdoor time, with exposure to animals, “green spaces” and healthy soil are interventions that are feasible in all settings. Such interventions need to be addressed urgently to avoid continued increase of allergies in several African countries running in parallel with the process of urbanization(7,8).

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17

18

TABLE 1. Characteristics of the study population by study site TOTAL SAFFA COHORT

Urban n=1185

n

% or median

ENROLMENT AGE 1185 26 (MONTHS) SEX Male 622 52.5% Female 563 47.5% Ethnicity Black African 549 46.3% Mixed Ancestry 551 46.5% Caucasian 85 7.2% SOCIOECONOMIC STATUS Household income 1184 7000 (Median in ZAR) Total number of people living in the household 1184 4 Parental education Secondary education 674 56.9% Tertiary education 511 43.1% Parental employment 1120 • Both parents unemployed 41 3.7% • One parent employed/studying 260 23.2% • Both parents employed/studying 819 73.1% Family History of atopy (first degree relatives) Any allergy 578 48.8% Atopy/Allergy Asthma 106 9.0% Allergic rhinitis 300 25.3% Atopic dermatitis 303 25.6% Food sensitisation 107 9.0% Food allergy 27 2.5%

Total cohort and aeroallergen cohort Urban vs urban; Rural vs rural

SUB-COHORT WITH AEROALLERGEN RESULTS

Rural n=398

Urban n=535

95% CI or IQR

n

22-32

398

21

17-28

50.5-56.3 44.6-50.4

225 173

56.5% 43.5%

51.5-61.5 38.5-48.5

43.5-49.2 43.6-49.4 5.8-8.8

398 0 0

100% 0 0

300016250

398

4-6

Rural n=347

n

% or median

95% CI or IQR

n

% or median

95% CI

pvalue

535

26

21-31

437

21

16-27

<0.001

0.162 0.162

287 259

51.6% 48.4%

49.3-57.9 44.1-52.7

198 149

57.1% 42.9%

51.7-62.3 37.7-48.3

0.11

0.73;0.87 0.73; 0.87

n/a

n/a

250 273 12

46.7% 51.0% 2.2%

42.2-51.1 46.7-55.3 1.2-3.9

347

100%

n/a

n/a

0.97; na 0.06; na <0.001; na

1380

990-2390

<0.001

534

7000

350015000

347

1450

990-2390

<0.001

397

6

5-8

<0.001

534

5

4-6

346

7

5-9

<0.001

54.0-59.7 40.3-46.0

378 20 388

95.0% 5.0%

92.4-96.9 3.1-7.7

<0.001 <0.001

305 225

57.0% 42.1%

52.7-61.3 37.8-46.4

274 18

79.0% 5.2%

74.3-83.1 3.1-

<0.001

2.6-4.9

190

49.9%

43.9-54.1

<0.001

19

3.7%

2.1-5.5

162

47.9%

41.3-52.1

20.7-25.8

165

42.5%

37.6-47.6

<0.001

136

26.8%

21.7-29.3

147

43.4%

37.1-47.8

70.4-75.7

33

8.5%

5.9-11.7

<0.001

353

69.5%

61.8-70.0

29

8.6%

4.5-9.4

45.9-51.6

30

7.5%

4.9-10.1

<0.001

265

50.0%

45.3-53.8

23

6.6%

4.0-9.3

<0.001

0.65; 0.54

7.4-10.7 22.8-27.8 23.1-28.1 7.5-10.8 1.5-3.7

4 13 8 11 2

1.0% 3.3% 2.0% 2.8% 0.5%

0.3-2.6 1.5-5.0 0.6-3.4 1.4-4.9 0.1-1.8

<0.001 <0.001 <0.001 <0.001 <0.006

28 108 122 43 12

5.2% 20.2% 22.8% 8.0% 2.2%

3.3-7.1 16.8-23.6 9.2-26.4 5.7-10.4 0.9-3.5

2 13 5 11 2

0.6% 3.8% 1.4% 3.3% 0.6%

0.2-1.4 1.7-5.8 0.2-2.7 1.3-5.0 0.2-1.4

<0.001 <0.001 <0.001 0.002 0.04

0.01; 0.54 0.02; 0.71 0.213; 0.53 0.50; 0.72 0.71; 0.85

% or median

95% CI

p-value

p-value

0.52;0.60 0.70;0.90

1.0; 0.785 <0.001

0.11; 0.978 0.30; 0.912

TABLE 2. DESCRIPTION OF EXPOSURES IN URBAN AND RURAL COHORTS

19

URBAN n=1185

URBAN Black African n=551

RURAL n=398

urban: rural

Urban BA: rural BA

n

% or median

95% CI or IQR

n

% or median

95% CI or IQR

n

% or median

95% CI or IQR

p-value

p-value

PETS Own cat Own dog Own cat/dog

117 416 499

14.9% 35.1% 42.1%

8.2-11.7 32.4-37.9 39.3-45.0

44 124 147

8.0% 22.5% 26.7%

6.3-11.4 19.1-26.2 24.9-32.9

146 288 313

36.7% 72.4% 78.6%

31.9-41.6 67.7-76.7 74.3-83.6

<0.001 <0.001 <0.001

<0.001 <0.001 <0.001

FARM ANIMALS

30

2.5%

1.7-3.6

12

2.2%

1.1-3.8

395

99.3%

97.8-99.8

<0.001

<0.001

Probiotic use during pregnancy

34

2.9%

2.0-4.0

35

6.4%

4.5-8.7

0

0

0

<0.001

<0.001

Amasi consumption during pregnancy

117

9.9%

8.2-11.7

109

19.8%

16.5-23.4

242

60.9%

55.8-65.6

<0.001

<0.001

Farm animal exposure during pregnancy

28

2.4%

1.6-3.4

21

3.8%

2.4-5.8

393

98.7%

97.1-99.6

<0.001

<0.001

Maternal smoking during pregnancy

187

15.8%

13.8-18.0

19

3.5%

2.3-5.8

9

2.3%

1.0-4.3

<0.001

0.27

Caesarean section rate

480

40.5%

37.7-43.4

206

37.4%

33.3-41.6

75

18.8%

15.1-23.0

<0.001

<0.001

0.9 2.2

0.5-1.6 1.3-3.2

1.0 2.1

0.5-1.7 1.3-3.3

4 6

2-7 4-8

<0.001 <0.001

<0.001 <0.001

48.8%

44.6-53.1

87.2%

83.5-90.3

<0.001

<0.001

7.0

4-9

5.0

3.9-8.0

<0.001

FARM ENVIRONMENT EXPOSURES

ANTENATAL EXPOSURES & DELIVERY MODE

SUNLIGHT EXPOSURE (hours per day) Winter Summer

ANTIBIOTIC AND PROBIOTIC EXPOSURE IN THE FIRST YEAR OF LIFE ANTIBIOTIC EXPOSURE

654

55.2%

52.3-58.1

6.0

4.0-9.0

531 420 166 57

44.8% 35.4% 14% 4.8%

42.0-47.7 32.7-38.2 12.1-16.1 3.7-6.2

282 197 54 11

51.2% 35.5% 9.8% 2.0%

46.9-55.4 31.8-39.9 7.5-12.6 1.0-3.5

51 267 75 5

12.8% 67.1% 18.8% 1.3%

9.7-16.5 62.2-71.7 15.1-23.0 0.1-2.9

<0.001 <0.001 0.02 0.002

<0.001 <0.001 <0.001 0.38

Antibiotics used in the last 2 months

447

37.7%

35.0-40.1

151

27.4%

23.7-31.3

142

35.7%

31.0-40.6

<0.08

0.01

PROBIOTIC EXPOSURE

177

14.9%

13.0-17.1

35

6.4%

4.5-8.7

1

0.3%

0.01-1.4

<0.001

<0.001

6.0

4-9

8

4-9

8.0

8-8

0.78

22 8 4

64.7% 23.5% 11.8%

Age of first exposure (months) Total number of courses none 1-2 3-5 >5

Median age of first exposure (months) Total number of days probiotics given < 10days 10-20 days >20 days

109 27 42

61.2% 15.2% 23.6%

269

347

1 -

100%

1.0 0.002 <0.001

<0.001 0.02 0.09

20 EXPOSURE TO FERMENTED MILK (AMASI) Proportion of children ever exposed to amasi

488

41.2%

38.4-44.1

12

9-15

Median age at first exposure Frequency of amasi intake < once per month 1-4 times per month >4 times per month

115 155 221

23.4% 31.6% 45%

UNPASTEURISED MILK EXPOSURE

31

2.6%

958

ANTI-HELMINTH EXPOSURE Children ever exposed to antihelminth treatment Median age at first deworming (months) Median age at last deworming (months) Children who received regular deworming (once yearly)

463

84.0%

(80.7-87.0)

12

9-16

258

64.8%

82.2-90.2

<0.001

12

10-12

0.75

<0.001

<0.001 <0.001 <0.001

0.88 <0.001 <0.001

0.7-3.6

0.33

0.04

85.9%

82.1-89.2

<0.001

0.16

12 18

12-12 12-24

<0.001 <0.001

0.002 <0.001

299

75.1%

70.6-79.3

<0.001

<0.001

2.7-6.5 3-5

9

2.3% 2

1.0-4.3 2-3

<0.001 <0.001

0.14 0.14

3.5%

2.3-5.8

9

2.3%

1.0-4.3

<0.001

0.28

171

31.0% 5

29.4-37.8 3-10

90

22.6% 5

18.6%-27.0 3-6

<0.001 <0.001

0.004 0.12

392 120 26 10

71.5% 21.9% 4.7% 1.8%

294 90 11 3

73.9% 22.6% 2.8% 0.8%

<0.001 0.04 <0.001 <0.001

0.58 0.76 0.13 0.17

99.5% 0.9% 0.3% 0.0%

23.2% 56.5% 4.0% 76.6%

<0.001 <0.001 <0.001 <0.001

41.4% 6.1% 28.7% 4.1% 24.3%

4.8% 1.5% 43% 51.0% 6.3%

<0.001 <0.001 <0.001 <0.001 <0.001

99 149 213

21.5% 32.3% 46.2%

1.8-3.7

23

4.2%

80.8%

78.5-83,1

455

12 18

12-18 18-24

651

55.8%

52.1-55.8

274

23.1% 5

187

73 167 17

28.4% 65.0% 6.6%

2.9-6.7

7

1.8%

82.6%

79.2-85.7

342

12 22

12-17.5 18-24

300

55.7%

54.3-63.0

20.8-25.6 4-10

22

4.0% 4

15.8%

13.8-18.0

19

466

39.3% 7

36.5-42.2 5-10

615 329 165 73

52.0% 27.8% 14.0% 6.2%

CIGARETTE SMOKE and FOSSIL FUEL EXPOSURE MOTHERS smoking Median number of cigarettes mothers smoke per day Proportion of mothers who smoked during pregnancy Proportion of fathers smoking Median number of cigarettes fathers smoke per day Number of smokers in the house 0 1 2 ≥3 COOKING FUEL Gas/Electricity Paraffin Fire inside Fire outside HEATING FUEL Electricity Gas Kerosene/Paraffin Wood/coal fire None

21 AGE EXPOSURE FAST FOOD CONSUMPTION LOW (less than once per week)

368

68.8%

64.7-72.7

196

78.4%

72.8-83.3

360

90.5%

87.3-93.2

<0.001

<0.001

HIGH (once per week or more)

167

31.2%

27.3-35.3.0

54

21.6%

16.7-27.2

38

9.5%

6.9-12.9

<0.001

<0.001

Low consumption (<3 times a week)

108

20.2%

16.9-23.8

65

26.0%

20.7-31.9

236

59.3%

54.3-64.2

<0.001

<0.001

High consumption (≥3 times a week)

427

79.8%

76.2-83.1

185

74.0%

68.1-79.3

162

40.7%

(35.8-45.7)

<0.001

0.03

Low consumption (less than once a week)

241

45.1%

40.8-49.4

135

54.0%

47.6-60.3

368

92.5%

89.4-94.9

<0.001

<0.001

High consumption (Once a week or more)

294

55.0%

50.6-59.2

115

46.0%

39.7-52.4

30

7.5%

(5.1-10.6)

<0.001

<0.001

SOFT DRINKS/FRUIT JUICE CONSUMPTION

FRIED OR MICROWAVED MEAT CONSUMPTION

HIGH FRUIT AND VEGETABLE PORTIONS CONSUMPTION (BINARY) Low consumption (<4 portions per day)

112

20.9%

17.6-24.6

63

25.2%

19.9-31.1

353

88.7%

85.2-91.6

<0.001

<0.001

High consumption (≥4 portions per day)

423

79.1%

75.4-82.4

187

74.8%

68.9-80.1

45

11.3%

8.4-14.8

<0.001

<0.001

22

↓

↓

Unpasteurised milk exposure Antenatal exposures Maternal smoking during pregnancy Maternal probiotic use Maternal Amasi consumption Maternal farm animal exposure

Food allergy

Atopic dermatitis

↓ ↓

↓

↓

V

↓

V

↓

RURAL

Farming exposures Pet exposure Farm animal exposure Amasi exposure

Food sensitisation

URBAN

Asthma

Allergic Rhinitis

Any AR, AD, asthma

Aeroallergen sensitisation

Food allergy

Food sensitisation

Atopic dermatitis

Asthma

Allergic Rhinitis

Aeroallergen sensitisation

Exposure

Any AR, AD, asthma

Table 3: Summary of EXPOSURE EFFECTS:

↓ V ↑

V

Delivery mode Caesarean Section Antihelminth/Antibiotic/Probiotic Exposure Antibiotic exposure first year of ↑ life Probiotic Exposure first year of ↑ life Anti-helminth exposure (ever) Antihelminth exposure V (regularly) Sunlight exposure (average hours per day) Summer Winter Cigarette smoke and fossil fuel exposure Cigarette smoke (mom) Λ

V

Λ ↑

↑

↑

↑

↑ V V

V ↑

↑

Cigarette smoke (dad)

Λ

↑

↑ ↑

↑

Cooking Fuels Electricity Paraffin Fire in the house

Λ ↓

↓

↓

↓

Heating Fuels Electricity

↑

↑

Gas Paraffin

↓

↓

AGE’s High Fast Foods consumption High Soft Drinks/Fruit Juices consumption High Fried /microwaved meats consumption High Fruit and veg consumption Λ: Increased risk: p<0.5 V:Decreased risk: p<0.5 ↑: Increased risk: p<0.1 ↓: Decreased risk: p<0.1 ↑: Increased risk: p<0.001 ↓: Decreased risk: p<0.001

Λ ↓ Λ

Λ ↑

↓

↑

Λ

Λ

↑

↑

↑