Dietary cyanogen exposure and early child neurodevelopment: An observational study from the Democratic Republic of Congo

Background Dietary cyanogen exposure from ingesting bitter (toxic) cassava as a main source of food in sub-Saharan Africa is related to neurological impairments in sub-Saharan Africa. We explored possible association with early child neurodevelopmental outcomes. Methods We undertook a cross-sectional neurodevelopmental assessment of 12–48 month-old children using the Mullen Scale of Early Learning (MSEL) and the Gensini Gavito Scale (GGS). We used the Hopkins Symptoms Checklist-10 (HSCL-10) and Goldberg Depression Anxiety Scale (GDAS) to screen for symptoms of maternal depression-anxiety. We used the cyanogen content in household cassava flour and urinary thiocyanate (SCN) as biomarkers of dietary cyanogen exposure. We employed multivariable generalized linear models (GLM) with Gamma link function to determine predictors of early child neurodevelopmental outcomes. Results The mean (SD) and median (IQR) of cyanogen content of cassava household flour were above the WHO cut-off points of 10 ppm (52.18 [3279]) and 50 (30–50) ppm, respectively. Mean (SD) urinary levels of thiocyanate and median (IQR) were respectively 81781 (47459) and 688 (344–1032) μmole/l in mothers, and 61749 (44948) and 688 (344–688) μmole/l in children reflecting individual high levels as well as a community-wide cyanogenic exposure. The concentration of cyanide in cassava flour was significantly associated with early child neurodevelopment, motor development and cognitive ability as indicated by univariable linear regression (p < 0.05). After adjusting for biological and socioeconomic predictors at multivariable analyses, fine motor proficiency and child neurodevelopment remained the main predictors associated with the concentration of cyanide in cassava flour: coefficients of -008 to -.15 (p < 001). We also found a significant association between child linear growth, early child neurodevelopment, cognitive ability and motor development at both univariable and multivariable linear regression analyses coefficients of 1.44 to 7.31 (p < 001). Conclusion Dietary cyanogen exposure is associated with early child neurodevelopment, cognitive abilities and motor development, even in the absence of clinically evident paralysis. There is a need for community-wide interventions for better cassava processing practices for detoxification, improved nutrition, and neuro-rehabilitation, all of which are essential for optimal development in exposed children. This paper was prepared according to the STROBE guidelines for reporting of observational studies [14] (S1 File). The study was conducted in Kahemba, which is a severely konzo-affected zone in the Bandundu province located south-west in the DRC, bordering Angola. Kahemba has an area of 20 000 km2 and an estimated population of 250 000 inhabitants relying mostly on subsistence cassava farming. Bitter cassava is the staple crop grown, and it is processed for food consumption by women. The processing consists of soaking the cassava roots in water for a recommended period of three to four nights, then drying them in the sun for one to two days before pounding them to make flour. The flour is subsequently mixed with boiling water to make a soft dough, which is eaten with gravy. In times of intense cassava trading and/or agro-ecological crises such as drought, shortcuts in cassava processing are common [7, 15], and signs of intoxication occur as residual amounts of cyanogenic compounds are left in the roots [16]. Over the last decades, Kahemba has been the scene of repeated outbreaks of konzo and its prevalence in certain villages is reportedly up to 20% [15, 17]. We carried out a cross-sectional study within the cohort of the longitudinal study (parent study) on the neuropsychological effects of cassava in school-aged children [12]. From this parent study, we have recruited households (mothers/caretakers) with 12–48 month old children with and without konzo that have consented to participate. Children with a medical history of illnesses possibly affecting the CNS, such as epilepsy, cerebral palsy, and acute malnutrition, were excluded from the study. After consent, we were able to enrol recruit a convenient sample of 61 and 53 households with and without konzo, respectively. At the time of the study, none of the eligible children was listed as being a konzo subject in the health zone incidence registry. Local health workers conducted structured interviews with the mothers/caregivers to gather information on sociodemographic, socioeconomic, and home environment during home visits. Parental level of education was scaled from 0 (no education) to 4 (university level). Socioeconomic status was ranked using a generated wealth index based on assets, quality of housing (type of floor, roofing, toilet facilities, water source, electricity, etc.). This method has been used previously as a proxy in the same setting [12]. The short version of the Caldwell Home Observation for Measurement of the Environment (HOME) (S1 Table), which has been adapted for the African context [18], was used to assess parenting style and the child’s level of stimulation and the learning opportunities offered by the home environment. This tool has been previously used in the DRC and have been shown to be a useful measure of mother-child interaction [12]. We only conducted mothers/caregivers 18 items interviews without any home observation due to financial and time constraints. All children were systematically screened for signs of the disease through a clinical examination that included a neurological examination. Anthropometric measurements were taken according to standard procedures [19]. Mid-upper arm circumference (MUAC) was used as a standard measurement of nutritional status according to WHO recommendation for children 6 to 60 months of age. The cut-off value of 115 mm indicates severe acute malnutrition [20, 21]. Anthropometrics Z-scores and body mass index-for-age were calculated and used as continuous variables. Maternal depression and anxiety symptoms were assessed through a structured interview using Hopkins symptoms checklist-10 (HSCL-10) and Goldberg Depressive Anxiety Scale (GDAS) (S1 Table). HSCL-25 is a well-known and widely used screening instrument [22]. We used the short version HSCL-10 [23], which is considered a good screening instrument in primary health care settings, and research [24, 25]. GDAS is an easy to administer scale [26] that has been validated in the DRC [27]. All derived scores from HSCL-10 and GDAS were analysed as continuous variables. Early child development and cognition were measured through clinical observation and interviews with caregivers/parents. The tools selected to measure early child development and cognition in the present study, are presented in S1 Table. The Mullen Scales of Early Learning (MSEL) [28] is easy to administer and has been found useful for assessing child development and cognition in low resource settings. It is a quick and reliable assessment tool for childhood development measuring cognitive ability and motor development [28]. It has been translated into French [18]. The study team was trained by a psychologist (MJB) and MSEL instructions were administered in the local language. The Gensini Gavito Scale (GGS) is specifically validated in DRC, and was used to cross-validate findings on early child neurodevelopment, cognitive ability and motor development outcomes. It is a neurodevelopmental scale developed for the assessment of psychomotor development and growth of children in low-income settings [29]. The scale is widely used and has been adapted locally. The Ten Questions Questionnaire (TQQ) [30] is a screening tool for child disability that has been developed for use in resource-limited settings, and was used to gather information on child development and disabilities as perceived by the mother (S1 Table). It is the most widely used tool for child disability assessment in low- and middle-income countries [31, 32]. At the local health station, three neuropsychiatrists who did not have access to the health zone registry of konzo patients received the mother-child dyad. They performed the clinical evaluation of the child, and gathered all medical information from pregnancy, milestones, growth, and breastfeeding practices. For quality assurance purposes, two doctors performed the same tasks, and one conducted the parent interview. Each child was evaluated according to a standardised format in the same setting and in presence of the child’s primary caregiver. The sample collection and storage methods have been previously described by colleagues [33]. Briefly, a team of laboratory technicians collected samples of urine and cassava flour in each household on the day of the clinical examination of the child, and measured the concentration using the SCN picrate kit D1 and B2 protocols [34]. Mothers were given instructions on how to collect the child’s urine using a clean jar for younger children. In the present study, we only measured the cyanide-yielding capacity in household cassava flour, and thiocyanate (SCN, metabolite of cyanide) in urine from the mother-child dyads as the sole source of exposure. Exposure from cassava leaves is unlikely or minimal due to the cooking process prior to the consumption of cassava leaves. Protocols to determine the total cyanide-yielding capacity in cassava products (Kit B2) and thiocyanate in urine (Kit D1) are available on line: (http://biology-assets.anu.edu.au/hosted_sites/CCDN/five.html). Early child development, cognitive ability and motor development were the main outcomes, whereas dietary cyanogen was the main exposure variable considered in the analysis. Means, medians and interquartile ranges were used to summarize the distributions of continuous variables, whereas proportions were calculated for dichotomous variables. Linear regression, generalized linear model (GLM) analyses with Gamma link function were used for univariable and multivariable adjusted analyses. Linear regression was used to explore associations between MSEL and GGS scores, and dietary cyanogen concentration in household cassava flour and urine. Regression models first examined each explanatory variable for association with the main outcomes, with regression parameters providing unadjusted estimates of the association. Second, multivariable adjusted models were built to include the main exposure variables while adjusting for relevant sociodemographic and biological factors: age, sex, child’s anthropometric characteristics, the HOME score, socioeconomic status, maternal depression/anxiety, child nutritional status, and maternal risk factors during pregnancy. Main outcomes and exposure variables were analyzed as continuous variables. Other predictors such as maternal depression/anxiety and child nutritional status were also analysed as continuous variables. Data concerning maternal smoking and alcohol consumption during pregnancy, as well as current information including introduction of solid food before six months of age, and the presence of previous morbidities such as epilepsy, sickle cell anemia, or cerebral palsy in the child were analyzed as dichotomous variables. Limited backward elimination was performed: explanatory variables that were not significant were removed from the models only when they did not change the estimates of other effects. Lastly, we dichotomized the household variable in two groups of konzo-affected and unaffected households to explore whether there were differences between the two groups. All tests were 2-tailed and conducted at 0·05 level of significance. Analyses were done with the statistical package Stata 14 (www.stata.com). The Institutional Review Board of the Ministry of Health in DRC number CE/368/2014 approved the study. Signed or fingerprint informed consent was obtained from each mother (caregiver) / guardian.