Impaired growth in rural Gambian infants exposed to aflatoxin: a prospective cohort study

Undernutrition and its consequences, including faltered growth, is a major contributor to high mortality rates in children under the age of five years [1]. It can also lead to impaired cognitive ability and reduced school performance, leading to reduced productivity in adult life and economic losses for the country [2]. Although reasonable progress has been made to reduce the global burden of undernutrition in children under five years of age (from 39·6% in 1990 to 23·8% in 2014) the rate of reduction in Africa has progressed more slowly than other regions [3]. Undernutrition is a multifactorial condition; hence, in order to facilitate effective prevention in Africa all underlying risk factors should be identified and targeted.

Exposure to aflatoxin, a mycotoxin produced by fungi that contaminate major cereal crops worldwide, with highest occurrence in hot and humid climates, is a major public health concern due to its carcinogenic [4], immunosuppressive [5] and growth suppressing effects [6]. Populations at highest risk of exposure are those from rural subsistence farming-communities in developing regions, such as in sub-Saharan Africa, where maize and groundnuts are dietary staples and diet variety is poor. In such settings, this high risk of exposure is further compounded by difficulties in the avoidance of contaminated food supplies, limited enforcement of regulatory food standards, and inadequate food storage conditions.

Aflatoxin exposure during foetal development, infancy and early childhood, particularly during the weaning stage when children are gradually introduced to family food, has been associated with low birth weight [7, 8], micronutrient deficiencies [9], growth faltering [10–12], liver damage [13], and immunosuppression [14]. The evidence supporting a causal association between aflatoxin exposure and impaired growth however, is limited. Only a small number of longitudinal studies covering the first 24 months following birth, a critical time period for linear growth, have been conducted in settings where both undernutrition and aflatoxin exposure are prevalent, and the findings among these studies are inconsistent [7, 12, 15].

A number of possible mechanisms by which aflatoxin exposure may cause stunted growth have been proposed, including reduced intestinal absorption of nutrients, and reduced levels of insulin-like growth factor-1 (IGF-1), a peptide hormone that stimulates growth. Aflatoxin causes liver toxicity, which may result in reduced levels of IGF-1 for which the liver is the main site of production [16].

This study aimed to examine the relationship between aflatoxin exposure and growth in Gambian infants from birth to two years of age, and to test the hypothesis that reductions in IGF-axis proteins could be a mechanism for growth impairment. The ENID trial [17] is a randomised trial of nutritional supplementation during pregnancy and infancy on infant immune development. As part of the trial protocol, detailed data on infant growth, feeding practices and morbidity were collected, providing an opportunity to explore how aflatoxin exposure, nutrition and infection interact to reduce growth in children living in an area with high aflatoxin exposure [7, 14, 18].

The results from this study embedded within the ENID Trial are reported in accordance with STROBE guidelines.

Study population

The ENID trial (ISRCTN49285450) primarily examined whether early immune development can be improved through pre-natal and infant nutritional repletion. The trial followed pregnant women and their infants up to one year of age. The ENID-Growth study was an extension of the ENID trial that continued to follow the infants to two years of age. The ENID trial protocol has been described in detail elsewhere [17]. In brief, pregnant women (< 20 weeks gestation) from rural subsistence-farming villages located within The Gambia were recruited in early 2010, and randomised to one of four supplementation groups until delivery: 1) Iron-folate = standard care, 2) multiple micronutrients (MMN), 3) protein-energy (PE) + iron-folate, or 4) PE + MMN. Their infants were then randomised from 6 to 18 months of age to one of two supplementation groups: 1) lipid-based nutritional supplementation (LNS) + MMN, or 2) LNS only.

Infants in the main ENID trial were born between August 2010 and February 2014. For the current sub-study, infants born between May 2011 and December 2012, where plasma samples were available, were included (Fig. 1). All infants received the Expanded Programme on Immunisation as per Gambian government protocol.

The results of this study have confirmed the negative impact of aflatoxin exposure on child growth in children up to 2 years old. We hypothesised based on our previous findings in older children [20] that a reduction in IGF levels could contribute to the mechanism by which this occurs. However, in this population we did not see reduced IGF1 or IGFBP3 associated with aflatoxin exposure.

Growth faltering is common in low and middle income countries [21, 22], and is multifactorial in aetiology. Inadequate dietary intake, infection, early breastfeeding cessation and poverty have all been identified as factors that contribute to faltered growth [23]. The results from this study conducted in The Gambia, covering the period from birth to two years of age, are consistent with the hypothesis that aflatoxin exposure may also be an important factor.

In this population growth faltering and aflatoxin exposure occurred simultaneously. For instance, from 6 to 24 months of age the amount of infants with stunted growth increased fivefold, the amount of wasting doubled and the amount of infants classified as underweight increased threefold. This infant growth pattern is consistently observed within this sub-Sahara African community [24]. Approximately half of the infants’ blood samples had detectable AF-alb concentrations when aged 6 months, and almost all the samples had detectable concentrations when aged 12 and 18 months. These prevalence rates are comparable to those reported in other sub-Sahara African countries, where AF-alb is typically detected in ~ 95% of collected blood samples [25].

Whilst the above evidence shows that aflatoxin exposure and infant growth faltering are both prevalent in this cohort, it does not imply causality; it is possible that both are the consequence of the same circumstances, including poverty and insufficient food intake. Nevertheless, when further analyses were conducted, there was some evidence to suggest that aflatoxin exposure may lie on the causal pathway. For instance, aflatoxin exposure was inversely related to WLZ, WAZ and LAZ scores between 6 and 18 months of age after adjusting for a range of confounders. Furthermore, aflatoxin exposure appeared to temporally proceed impaired infant growth, as infants with higher aflatoxin exposure measured at 6 months of age, demonstrated less gain in WLZ scores between 6 and 12 months, and similarly infants with higher aflatoxin exposure at 12 months of age demonstrated less gain in length and LAZ scores between 12 and 18 months.

Although the size of the effect observed here was not great, these findings contribute to the body of evidence from existing cross-sectional [10, 14, 20, 26–28] and longitudinal studies [7, 12, 29] that have also tested this hypothesis, and have found similar inverse relationships between aflatoxin exposure and LAZ [7, 10, 12, 27–29], WLZ [10, 27, 29] and WAZ [10, 14, 26] scores. There is no doubt that understanding the possible contribution of aflatoxin exposure to child growth impairment is complex, with many other potential contributing factors. In our longitudinal study conducted in Tanzania (infants aged 6–14 months) [15], a trend in lower growth over 12 months in children with higher AF-alb did not reach statistical significance, but we did find a significant inverse association between exposure to another mycotoxin that contaminates maize, fumonisin, and LAZ scores. Mean AF-alb concentrations observed over the three sampling time points in that particular study, however, were lower than those observed in this current study, consistent with higher levels of exposure that have been associated with groundnut intake (more common in Gambia) versus maize intake, elsewhere [30].

Notably, the associations between aflatoxin and growth impairment observed in the current study remained after controlling for a range of important confounders including mother’s household quality, an indicator of SES. Low SES is related to inadequate dietary intake and infectious diseases, which consequently can lead to impaired linear growth during childhood. There is also evidence to suggest its association with higher aflatoxin exposure. For instance, Leroy et al. [31] found an array of socioeconomic determinants associated with lower aflatoxin exposure levels in rural Kenyan women, including higher levels of education, land ownership, food security, higher household expenditure and use of fertiliser. While the current study did not find a significant association between mother’s household quality and aflatoxin exposure, we have not tested how well these measurements of household quality are associated with wealth.

To further elucidate the relationship between aflatoxin exposure and child growth impairment it is essential that the molecular and biological mechanisms by which aflatoxin exposure causes impaired growth be identified. It has been proposed that alteration of the growth hormone-IGF system by aflatoxin exposure, possibly due to protein synthesis, liver toxicity or DNA methylation, could be a potential pathway. In an earlier study of older Kenyan children [20], it was estimated that 16% of the effect of aflatoxin being associated with reduced child height could be explained by reduced IGF1/IGFBP-3 levels. In this current study there were no significant associations between IGF1 and AF-alb concentrations, but an inverse relationship was observed between AF-alb levels at 6 months of age and IGFBP-3 concentrations at age 12 months. There are a number of differences between the two studies, most notably that the prior study was cross-sectional, the population were adolescents and that a different method was used to quantify the IGF-axis proteins. Further research is, therefore, warranted to determine if alteration in IGF-axis by aflatoxin exposure is a mechanistic pathway for growth stunting, particularly during the critical 24 months after birth.

The strengths of this study include the large representative sample that is well characterised owing to the data collected longitudinally on morbidity, feeding practices and infant growth. Also objective measurements of aflatoxin exposure and growth were used which enhances the validity of the results. The main limitation of this research is that an observational study design was used, which is susceptible to potential bias, including response bias (specifically social desirability bias) for the household quality questionnaire and recall bias for the morbidity and feeding questionnaires. Since outcomes are measured as sectional events and initiation of exposure before outcome onset cannot be clearly established there is also potential temporality bias. Although this sub-study was from within a nutrition intervention trial, the population sampled was representative of the local population as all women within the area who became pregnant during the trial period were available for recruitment (with specific eligibility exceptions [17]), and were randomised to the intervention on recruitment. Furthermore, the WAZ, LAZ and WLZ scores (6, 12, 18 and 24 months) did not significantly differ according to infant supplementation group, so we are confident that the nutrition intervention did not influence our findings.

This study of Gambian infants, covering the first two years following birth, found that aflatoxin exposure was associated with impaired growth. The findings contribute to a body of evidence that suggests aflatoxin may be an underlying determinant of impaired child growth. Further research, however, is required to identify the biological mechanistic pathways, and to design and implement intervention studies that target aflatoxin exposure alongside child undernutrition.