1 Molecular Epidemiology Unit, Epidemiology and Health Services Research, School of Medicine, University of Leeds, Leeds, UK.
2 International Institute of Tropical Agriculture, Cotonou, Benin, West Africa.
3 London School of Hygiene & Tropical Medicine, London, UK.
Prof. CP Wild, Molecular Epidemiology Unit, Epidemiology and Health Services Research, Algernon Firth Building, School of Medicine, University of Leeds, Leeds, UK. E-mail: c.p.wild{at}leeds.ac.uk
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Abstract |
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Methods We conducted a cross-sectional study in Benin and Togo to investigate aflatoxin exposure in children around the time of weaning and correlated these data with food consumption, socioeconomic status, agro-ecological zone of residence, and anthropometric measures. Blood samples from 479 children (age 9 months to 5 years) from 16 villages in four agro-ecological zones were assayed for aflatoxin-albumin adducts (AF-alb) as a measure of recent past (23 months) exposure.
Results Aflatoxin-albumin adducts were detected in 475/479 (99%) children (geometric mean 32.8 pg/mg, 95% CI: 25.342.5). Adduct levels varied markedly across agro-ecological zones with mean levels being approximately four times higher in the central than in the northern region. The AF-alb level increased with age up to 3 years, and within the 13 year age group was significantly (P = 0.0001) related to weaning status; weaned children had approximately twofold higher mean AF-alb adduct levels (38 pg AF-lysine equivalents per mg of albumin [pg/mg]) than those receiving a mixture of breast milk and solid foods after adjustment for age, sex, agro-ecological zone, and socioeconomic status. A higher frequency of maize consumption, but not groundnut consumption, by the child in the preceding week was correlated with higher AF-alb adduct level. We previously reported that the prevalence of stunted growth (height for age Z-score HAZ) and being underweight (weight for age Z-score WAZ) were 33% and 29% respectively by World Health Organziation criteria. Children in these two categories had 3040% higher mean AF-alb levels than the remainder of the children and strong dose response relationships were observed between AF-alb levels and the extent of stunting and being underweight.
Conclusions Exposure to this common toxic contaminant of West African food increases markedly following weaning and exposure early in life is associated with reduced growth. These observations reinforce the need for aflatoxin exposure intervention strategies within high-risk countries, possibly targeted specifically at foods used in the post-weaning period.
Accepted 31 January 2003
Foetal and early childhood environment are considered critical for growth and disease risk in later life.1 Nutritional status of the mother during pregnancy and that of the infant following birth are both important in this respect.2 In developing countries, many individuals are not only malnourished but are also chronically exposed to high levels of toxic fungal metabolites (mycotoxins) in their diet. The heterogeneous distribution of mycotoxins within a given food commodity hinders accurate exposure measurement and hence associations between exposure and human health have been difficult to establish.3 One family of mycotoxins, the aflatoxins produced by Aspergillus spp., are proven carcinogens, immunotoxins, and cause growth retardation in animals.46 The development and use of aflatoxin-albumin (AF-alb) adducts as a biomarker of exposure revealed that over 90% of West African sera tested contained detectable AF-alb adducts, with exposure occurring throughout life, includingin utero and via breast milk.712 This biomarker has significantly contributed to our understanding of the role of aflatoxins in human hepatocellular carcinoma.13 The high prevalence and level of exposure throughout childhood suggests that child growth and development could be critically affected but to date little attention has been paid to understanding these effects. Aflatoxin exposure has been suggested as a causal or aggravating factor for Kwashiorkor in African children14,15 but problems in study design make interpretation difficult.5
In parts of West Africa, maize and groundnuts are major dietary staples. Aflatoxin exposure has been linked to the consumption of these crops with a marked seasonality in exposure,10,12 presumably related to crop management practices and hot, humid storage conditions, which facilitate fungal growth and mycotoxin production.16,17 Rural populations in Benin and Togo rely on maize as a staple whilst groundnuts are frequently includedin sauces and as a snack. Both commodities are stored under conditions that can promote Aspergillus growth and aflatoxin contamination. The level of aflatoxin in stored maize in this region has been shown to exceed 100 ppb in 50% of tested samples.17,18
This study was conducted to establish the geographical pattern and the influence of sociological factors, weaning, and patterns of weaning food consumption on aflatoxin exposure in Benin and Togo by measuring serum AF-alb in 479 children in 16 villages across four agro-ecological zones. Aflatoxin exposure has previously been shown to correlate with impaired growth in these children.19
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Material and Methods |
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Aflatoxin exposure assessment
A 5-ml blood sample was obtained from each child, and serum separated. One blood sample of the 480 was missing. The levels of AF-alb adduct were determined by albumin extraction, digestion and enzyme linked immunosorbent assay (ELISA) as previously described.20 The detection limit was 3 pg AF-lysine equivalents per mg of albumin (pg/mg). Controls included three positive and one negative control analysed alongside batches of samples. Samples were measured in quadruplicate on at least two occasions on separate days.
Weight and height measurement
Child body weight and height were measured using accurately calibrated instruments (electronic scales: Soehnle, max wt 20 kg, accurate to 10 g; height measurement: SCHORR, Maryland, USA). Field workers, trained to maximize repeatability, made all height and weight measurements. Weight for age Z-score (WAZ), height for age Z-score (HAZ) and weight for height Z-score (WHZ) were calculated according to the median value of the international reference population recommended by National Center for Health Statistics (NCHS)/World Health Organization (WHO).21 Weight for height Z-score reflects body shape, a low value indicating wasting or thinness, whilst a low HAZ indicates stunting. A low WAZ is indicative of being underweight. According to the WHO criteria, a Z-value <2 for any of these criteria is recognized as malnutrition and <3 as severe malnutrition.
Statistical analysis
The AF-alb adduct data were not normally distributed, and were natural log transformed for statistical analysis. The difference between means was tested by t-test or ANOVA. Significant variables of age, agro-ecological zone, weaning status, and socioeconomic status were entered into a multivariable model to analyse effects on anthropometric data and AF-alb level. Initially, in univariate analysis both measures of socioeconomic status (mother and household) were compared with AF-alb levels but for the multivariable analysis, mothers socioeconomic status was used (see Subject recruitment). All the analysis was performed using SAS software. Geometric means for AF-alb with 95% CI are reported in the Tables and text unless otherwise stated.
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Results |
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Weaning status
Children were classified as either breastfed (i.e. still partially or wholly breastfed) or fully weaned (i.e. no breast milk). Amongst the breastfed children, the majority (98%) were being weaned onto a maize-based porridge and/or the family meal. In children 3 years of age, 158/302 were still being breastfed although only one child was exclusively breastfed. In children >3 years of age all but three children were fully weaned. The mean AF-alb level was approximately twofold higher in weaned children than in those breastfed for
3 years. After adjusting for age, socioeconomic status, and agro-ecological zone in a multivariable model there remained a highly significant (P = 0.0001) 1.8-fold difference in adduct levels between weaned and breastfed children (37.7 pg/mg; 95% CI: 30.846.1 and 21.1 pg/mg; 95% CI: 17.525.5, respectively) (Figure 1
). When children aged 13 years were divided into 6-month age groups there was a significant linear trend in the mean AF-alb level with age (P for trend < 0.001) (Figure 2
). Within a given age group there were marked differences in mean adduct levels by weaning status. For example, in age group 1.52.0 years the mean AF-alb level was 39.8 pg/mg for weaned (95% CI: 27.358.0) and 25.0 pg/mg for breastfed (95% CI: 20.131.0) children.
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Discussion |
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The AF-alb level increased with age for children <3 years old, after which the level reached a plateau. It is important to understand whether this is due to age per se or a change in diet from breast milk to solid food. In a multivariable model including children aged 3 years, weaning status was significantly associated with AF-alb with an approximately twofold higher mean adduct level in those no longer being breastfed. In this model, age was not significantly associated with AF-alb and the original association probably reflected the changing pattern of weaning status with age. When children, aged 13 years, were divided into 6-month age groups (1.01.5, 1.52.0, 2.02.5, 2.53.0 years) AF-alb levels were higher in weaned than breastfed children within each group where the comparison could be made (Figure 2
). This again suggests weaning status, rather than age, to be the major determinant of aflatoxin exposure level in young children. The lower AF-alb levels associated with breastfeeding almost certainly reflect lower levels of aflatoxin in breast milk than weaning and family foods and even lower levels of exposure might be expected in wholly breastfed infants. In addition, the majority of aflatoxins in milk are in the form of the less toxic and carcinogenic AFM1 hydroxylated metabolite rather than the highly toxic AFB1 predominantly found in foods.4,11,22
Both maize and groundnuts are susceptible to Aspergillus spp. growth and aflatoxin contamination. This is particularly problematic in countries such as Benin and Togo because maize is the principal staple food in the area and groundnuts are frequently consumed in snacks and sauces. The frequency of maize consumption by the child showed only a relatively weak positive correlation with the AF-alb level. This probably reflects a number of factors including: the absence of an estimate of quantity of maize consumption; the effects of sorting and food preparation on aflatoxin levels; the contribution of aflatoxin from other dietary components; and the fact that the AF-alb marker integrates exposure over a period longer than the one week to which the questionnaire relates. There was no significant association between reported groundnut consumption and AF-alb. Groundnuts are often consumed as a snack and children may have consumed these away from the home unbeknown to the mother, leading to underreporting. However, despite the lack of association, subjects from SGS had the highest reported frequency of groundnut consumption and the highest AF-alb levels. In addition, when comparing Lainta and Gbatope, the two villages with the extremes of aflatoxin exposure, the most notable difference is the frequency of groundnut consumption; Lainta having the higher consumption and the highest mean AF-alb level. Thus it is possible that an effect of groundnut consumption on AF-alb level may have been masked by the more frequent consumption of aflatoxin-contaminated maize or missed due to underreporting and the other limitations discussed above for maize.
We have previously reported the striking associations between aflatoxin and impaired growth in these children.19 In animal models, aflatoxin is reported to reduce growth rates,6 but this had not been previously studied in human populations. These adverse growth effects are strongly correlated with the change from breastfeeding to solid foods, including maize, which is used in ground form as the basis for porridge for weaning purposes. Whether the effects of weaning foods and associated reduced growth are a direct result of aflatoxin exposure cannot be confirmed from the current cross-sectional study. For example, the AF-alb biomarker may be reflecting an infants consumption of fungus-affected food, which as a consequence is of poor nutritional quality. However, as aflatoxins are secondary fungal metabolites and different species of Aspergillus produce different amounts of aflatoxin, toxin level is not always correlated with the fungal load. In this context, socioeconomic status might also be expected to correlate with poor food quality and higher AF-alb levels but no strong effect was observed. This absence of effect notwithstanding, the difficulties in assessing socioeconomic status in these populations should be noted. An alternative explanation of our observations could be that malnutrition affects albumin level or turnover and consequently disrupts the relationship between aflatoxin exposure and AF-alb. However, serum albumin levels were measured in this study and were within the normal physiological range (data not shown).
This study suggests that extended breastfeeding results in a period of lower aflatoxin exposure, in a population whose primary weaning foods are at high-risk of aflatoxin contamination. Weaning results in a marked increase in exposure, as aflatoxin-contaminated household foods begin to be consumed, and this change in diet is associated with growth faltering, particularly stunting. The aflatoxin contamination of maize used in weaning foods may be a major source of exposure but a contribution from groundnuts cannot be ruled out from this study. Future work is needed, first to confirm this effect in longitudinal studies in children; second, to understand the mechanism by which aflatoxin affects growth; and third, to address the possible associated health consequences. The observations also emphasize the need for aflatoxin exposure intervention strategies within high-risk countries, possibly targeted specifically at the post-weaning period.16
KEY MESSAGES
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Acknowledgments |
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References |
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