Differences between microbiome species and genes in children from different regions of the Democratic Republic of Congo (DRC) may affect the release of cyanide after poorly processed cassava is consumed, and impact on development of the neurological disorder konzo, according to a study in 180 children, carried out by Children’s National Hospital researchers.

Konzo—a severe, irreversible motor neuron disease that results in paralysis—can result after consuming poorly processed Cassava (Manihot esculenta Crantz) a manioc root that represents a food security crop for over half a billion people in the developing world. The newly reported research indicates that children living in high-risk konzo areas have high glucosidase (linamarase) microbes and low rhodanese microbes in their gut, which could mean greater susceptibility to and less protection against konzo.

From left to right: Vincent Kambale and Dr. Matthew Bramble Here, liquid nitrogen is being replenished into the tank containing samples en route to Kinshasa from Kahemba. [Brambel et al. Children’s National Hospital, Nature Communications (2021)]

The researchers suggest that their newly reported study is the first to shed light on the gut microbiome of populations that rely on toxic cassava as their primary food source. “Knowing who is more at risk could result in targeted interventions to process cassava better or try to diversify the diet,” said Eric Vilain, MD, PhD, director of the Center for Genetic Medicine Research at Children’s National. “An alternative intervention is to modify the microbiome to increase the level of protection. This is, however, a difficult task which may have unintended consequences and other side effects.”

Vilain is co-senior and co-corresponding author of the team’s published paper in Nature Genetics, which is titled, “The gut microbiome in konzo,” and in which the authors concluded, “Our findings indicate that the gut microbiome structure is highly variable depending on region of sampling, but most interestingly, we identify unique enrichments of bacterial species and functional pathways that potentially modulate the susceptibility of konzo in prone regions of the Congo.”

The DRC is one of the least developed countries in the world, and a high percentage of individuals rely on a monotonous cassava diet for basic survival. However, poorly processed cassava contains linamarin, a cyanogenic compound. Enzymes with glucosidase activity act to convert starch to simple sugars, but they also break down linamarin, which then releases cyanide into the body. “Consumption of improperly processed food derived from bitter cassava, which harbors high levels of cyanogenic compounds such as linamarin, can result in an irreversible nonprogressive motor neuron disease known as konzo, that predominantly manifests in children and women of childbearing age,” the authors explained. “The occurrence of konzo is strongly associated with the consumption of improperly processed bitter cassava coupled with malnutrition and environmental stressors such as drought and turbulent times, leading to irreversible spastic paralysis and neurocognitive deficit.”

But while certain risk factors, such as food insecurity, chronic malnutrition, and particularly a lack of sulfur-containing amino acids, are associated with outbreaks of konzo, “ … the exact biological mechanisms underlying disease susceptibility and severity have remained poorly understood, “the team continued.

To understand the influence of a detrimental subsistence on the gut flora and its relationship to this debilitating multifactorial neurological disease, the researchers compared the gut microbiome profiles in 180 children from the DRC using shotgun metagenomic sequencing. This approach evaluates bacterial diversity and detects the abundance of microbes and microbial genes in various environments.

The samples were collected in Kinshasa, an urban area with diversified diet and without konzo; Masi-Manimba, a rural area with predominant cassava diet and low prevalence of konzo; and Kahemba, a region with predominant cassava diet and high prevalence of konzo. “Few populations in the world rely exclusively on toxic foods for survival, making the DRC a unique country to query the influence of a detrimental subsistence on the gut flora and its relationship to this debilitating multifactorial neurological disease,” the scientists further noted.

They first looked at the structure of the gut microbiome with respect to urban versus rural contexts, and found that regardless of region, all the study groups on average harbored >450 unique species. Also, the investigators wrote, “While all groups appear to harbor diverse microbiome structure, numerous differences were detected when comparing the profiles of individuals from Kinshasa to those residing in either Masi-Manimba or Kahemba.” The results indicated that for their study populations there were “regional specifications and influences” that contributed to the overall structure of the gut flora, “outside of a simple urban versus rural context.”

While the findings indicated an enrichment of bacteria capable of hydrolyzing linamarin, as well as genes coding for ß-D-glucosidase, among children in Kahemba, the authors also stressed that the development of konzo is multifactorial in nature with numerous environmental variables and stressors. “As such, the gut microbiome cannot be the sole cause of disease, but rather a required modulator, as without a functioning gut microbiome, linamarin, and other cyanogenic glucosides would pose little to no risk to humans,” they concluded.

Matthew S. Bramble, PhD, staff scientist at Children’s National, commented, “While the gut microbiome is not the sole cause of disease given that environment and malnourishment play a role, it is a required modulator. Simply stated, without gut microbes, linamarin and other cyanogenic glucosides would pose little to no risk to humans.”

Neerja Vashist is learning how to make fufu. Fufu is a traditional food made from cassava flour, and the cassava flour used in the making of the fufu here has gone through the wetting method to further remove toxins from the cassava flour prior to consumption. [Brambel et al. Children’s National Hospital, Nature Communications (2021)]

“This study overcame many challenges of doing research in low-resource settings,” said Desire Tshala-Katumbay, MD, MPH, PhD, FANA, co-senior author and expert scientist at Institut National de Recherche Biomédicale in Kinshasa, DRC, and professor of neurology at Oregon Health & Science University. “It will open novel avenues to prevent konzo, a devastating disease for many children in Sub-Saharan Africa.”

For next steps, the researchers aim to study sibling pairs from konzo-prone regions of Kahemba where only one sibling is affected with the disease. “Studying siblings will help us control for factors that cannot be controlled otherwise, such as the cassava preparation in the household,” said Neerja Vashist, a research trainee at Children’s National. “In this work, each sample had approximately 5 million DNA reads each, so for our follow-up, we plan to increase that to greater than 40 million reads per sample and the overall study cohort size. This study design will allow us to confirm that the trends we observed hold on a larger scale, while enhancing our ability to comprehensively characterize the gut microbiome.”

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