Reduced plasma concentrations of vitamin B6 and increased plasma concentrations of the neurotoxin 3-hydroxykynurenine are associated with nodding syndrome: a case control study in Gulu and Amuru districts, Northern Uganda

Introduction Nodding syndrome was first reported in Uganda in 2003 among internally displaced populations. Risk factors for the syndrome remain unknown. We therefore explored vitamin B6 deficiency and resulting high 3-hydroxykynurenine (3-HK) levels as risk factor for nodding syndrome in Northern Uganda. Methods Case-control study conducted in Gulu and Amuru districts. Cases were children/young adults with nodding syndrome. Healthy children/young adults were recruited as controls from same community as cases. Data on socio-demographic and other risk factors was collected using questionnaires. Whole blood was collected in EDTA tubes for assay of 3-HK and vitamin B6 using sandwich ELISA. Conditional logistic regression model was used to assess associations. Results 66 cases and 73 controls were studied. Factors associated with nodding syndrome were being positive for 3-HK (AOR=4.50, p=0.013), vitamin B6 concentration below mean (AOR=7.22, P=0.001), child being taken care of by mother only (AOR=5.43, p=0.011), child being taken care of by guardian (AOR=5.90, p=0.019) and child consuming relief food at weaning (AOR=4.05, p=0.021). Conclusion Having low vitamin B6 concentration which leads to a build up of 3-hydroxykynurenine concentration in cases as a main risk factor. Therefore, cases should be treated with vitamin B6 and community members should be sensitise to ensure adequate dietary intake of vitamin B6 so that the risk of nodding syndrome among children is averted. We encourage future prospective intervention study to be conducted to assess the effect of low vitamin B6 on the development of nodding syndrome via raised 3-HK concentration.


Introduction
Nodding syndrome was first described in Tanzania in the 1960s and similar conditions were reported in the 1980s in the southern part of Sudan (presently the Republic of South Sudan) [1,2]. It is currently unknown what causes nodding syndrome [3], which can be fatal or cause mental and physical disability in young children between the ages of 5 and 15 [4,5]. In Uganda, the syndrome is currently restricted to the northern region where about 5,000 cases have been detected and an estimated 300 children have succumbed to it [4]. Nodding syndrome was first reported in Uganda around 1997 among the population of internally displaced people's (IDP) camps [6]. The onset of symptoms in affected children is marked by the development of nodding head movements, which are reported to be provoked either by the sight of food or exposure to cold weather [4,7]. The nodding head movements have been documented to correspond with the onset of subclinical brain seizure activity on electroencephalography (EEG) [7]. Seizure activity is brief and halts when the child stops eating or feels warm again [4]. The syndrome is progressive [3,4] and is associated with cognitive impairment, stunting, lip changes and other physical deformities [7], mental retardation, behavioural disabilities and malnutrition [4,6]. Magnetic resonance imaging (MRI) scans have shown atrophy of cortical, cerebellar [3,7], hippocampal [3] and glial cells of cases [8]. There is to date no report of recovery from nodding syndrome; many cases have died from the syndrome [9]. Several etiological factors have been proposed, including infec¬tious, nutritional, environmental, and psychogenic causes [3,10]. Specific suspected causal exposures which were evaluated in previous studies include Onchocerca volvulus, bombs dropped during wartime, nutritional deficiencies, measles infection, consumption of monkey meat, and consumption of possibly contaminated relief seeds and relief food [11]. However, the exact cause and the pathophysiology of nodding syndrome remain unknown [1,4]. Low level of vitamin B6 was reported among cases and control in one study [10] and testing has failed to demonstrate associations between nodding syndrome and vitamin B6 [10,12]. We hypothesised that vitamin B6 deficiency resulting into increased production of neurotoxic 3-HK could predispose to nodding syndrome. This hypothesis was based on a previous study demonstrating that vitamin B6 deficiency leads to abnormal tryptophan metabolism, with the metabolite excreted in greatest quantity being 3-HK [13]. 3-HK, an endogenous tryptophan metabolite, is known to have toxic effects in the brain cells [14].
This study was therefore set in the context of this prevailing knowledge gap to explore vitamin B6 deficiency and resulting high 3-HK levels as a risk factor for nodding syndrome in the Acholi subregion of Northern Uganda.

Discussion
Data from our study indicates that cases were much older than controls and some had lived with the syndrome for over ten years. This is consistent with other studies done on similar groups [10,11]. Nodding syndrome is neurological disorder that becomes more severe with time as reported by other investigators [20,21].
This explains why cases were much older than controls because cases had lived with the syndrome for many years. Majority of cases were born at home compared with majority of controls who were born at health facilities. At the peak of the insurgency in northern Uganda, majority of the population were displaced living in internally displaced persons' camps where most health facilities were closed due to insecurity [22]. Majority of controls were born at health facilities because when government health facilities were inaccessible, relief agencies had to provide make-shift health facilities within the IDP camps to serve the IDP population [23]. We found the number of children with nodding syndrome onset to be higher in 2008 than any other year. This corresponds with the time government was trying to disband the IDP camps in northern Uganda [24]. In our study, children who were being taken care of isonazid, penicillamine), smoking, alcoholism and consumption of the mushrooms Gyromitra esculenta and Agaricus bisporis [25].
None of the cases were on any of the above drugs or indicated mushrooms as one of their sources of food at the time of study.
Smoking and drinking alcohol are not practiced among children and young adult as it is against the cultural norms among the Acholi community of northern Uganda where this study was conducted.
However, further analysis of data showed an inverse correlation between vitamin B6 and source of food consumed at weaning. Also, children whose parents indicated that they were fed on relief food at weaning were more likely to experience nodding syndrome than camps [6] and when most people were relying on relief food. Our study shows an inverse correlation between vitamin B6 and 3-HK and this is consistent with other study [27]. Cases were more likely to have decreased level of vitamin B6 concentration than controls and elevated level of the neurotoxic 3-HK in their plasma than controls. This is consistent with other studies which have shown that vitamin B6 deficiency leads to a build up of 3-HK concentration in human body [13,28,29].
Therefore it is important to note whether plasma levels of 3-HK can reflect brain levels of 3-HK. Studies in animal have demonstrated that peripheral 3-HK is actively transported from the circulation through the blood brain barrier [30]. Increased plasma 3-HK levels therefore can lead to an increased brain levels of 3-HK. In other pathological disorders like Parkinson's disease, Alzheimer's disease (AD) and acquired immunodeficiency syndrome dementia [31][32][33], the concentration of 3-HK have been found to be greatly increased.
These diseases are known to be associated with neuronal cell death thereby causing dysfunction in neuronal circuitry [14]. The neurotoxicity of 3-HK on brain cells could explain why other studies using MRI scan had reported brain atrophy among nodding Page number not for citation purposes 5 syndrome patients [3,7,8]. Our study had the following limitations: Recall bias since most of the parents/guardians were asked about their past events. Parent/guardians of cases may recall better past event for their children than parents/guardians of controls because of the problem they are currently faced with. We also could not compare the level of Vitamin B6 and 3-HK both in cases and control at the point when cases started experiencing nodding syndrome due to the case-control design of the study. Cases were older than controls due to the fact that we match cases to controls based on the age range for nodding syndrome patients as had been reported by other studies to be between 5-15 years [10,11,20,21]. Further more, we did not match cases and control for sex since previous study had shown that excretion of 3-HK is not affected by gender [34]. Also, 52% of the cases did not have raised 3-HK in their plasma and this could be attributed to nutritional supplement including multivitamins which were being given to nodding syndrome cases. Management of malnutrition is one of the key activities in the guidelines for managing nodding syndrome in Uganda as proposed by Ministry of Health [21]. A study has revealed that the concentration of 3-HK was found to dramatically decreased following supplementation with pyridoxine [13]. Other study has shown that vitamin B6 is required as a coenzyme in the catabolism of 3-HK in human body [35]. Thus nutritional supplementation with multivitamins would have provided vitamin B6 which is required to catabolise 3-HK in the cases. This would then lead to depletion of 3-HK in the plasma of cases thereby testing negative for raised 3-HK in plasma. Our findings need to be replicated and expanded in future studies. Despite these limitations, this investigation provides new information in addition to those already published on nodding syndrome.

Conclusion
We identified that having low vitamin B6 concentration which leads to a build up of 3-HK concentration in cases as a main risk factor. Therefore, cases should be treated with vitamin B6 and community members be sensitised on adequate dietary intake of food rich in vitamin B6 so that the risk of nodding syndrome among children is averted. We encourage future prospective intervention study to be conducted to assess the effect of low vitamin B6 on the development of nodding syndrome via raised 3-HK concentration.

What is known about this topic
• Nodding syndrome is association with Onchocerca volvulus detected by skin snip or serologic analysis; • Magnetic resonance imaging (MRI) scans have shown atrophy of cortical, cerebellar, hippocampal and glial cells of cases; • Head nodding is triggered by sight of food or exposure to cold weather.

What this study adds
• Cases were having reduced vitamin B6 concentration in their plasma than controls and this is significantly associated with nodding syndrome; • Cases were having raised concentration of 3hydroxykynurenine in their plasma than controls and this is significantly associated with nodding syndrome; • Feeding children on relief food at weaning is significantly associated with nodding syndrome in our study.

Competing interests
The authors declare that there is no conflict of interests regarding the publication of this paper.  Table 1: Socio-demographic characteristics of the study participants