Working together to eliminate , konzo, tropical ataxic neuropathy (TAN) and .

Cassava Cyanide Diseases & Neurolathyrism Network (ISSN 1838-8817 (Print): ISSN 1838-8825 (Online) ______Issue Number 18, December 2011

Contents cyanoglycoside that is hydrolysable looked extremely poor and the only under certain conditions to release meal they had was boiled and raw Fatal food poisoning in hydrogen cyanide in a process cassava. In the second family, a Eastern Province, Kenya 1 called cyanogenesis. The hydrolysis child aged 5 died in Makueni District How to distinguish konzo from is facilitated by the enzyme Hospital while continuing with poliomyelitis 2 linamarase and hydroxynitrile lyase management. Both families On the molecular mechanisms of (found in cassava leaves). Toxic complained of headaches, hydrogen cyanide (HCN) generation abdominal pains and discomfort, konzo and neurolathyrism 2 takes place when cassava tissues vomiting, general body weakness Tropical spastic paraparesis are broken down1 and some fever. The Health Officer (konzo): a major neurologic Cassava is the fourth most important collected the cooked and uncooked problem in Caungula - Angola: food source worldwide after wheat, cassava and fresh samples from the first report in Angola 4 rice and maize2. Cassava roots have same plants where cassava was Control of konzo in DRC 4 total cyanide content of 1-1550 mg harvested. The cassava tasted bitter HCN equivalents/kg fresh weight as claimed by the family members. 3 (ppm) . The maximum safe cyanide The area had experienced drought level by WHO for cassava flour is for the last 3 years. CCDN Coordinator: 10 ppm.4 The cyanide content was Dr. J. Howard Bradbury Two families, a family of 6 from determined by a modified picrate EEG, Research School of Biology, 5 6 Makueni District and a family of 7 method. Chromatographic paper Australian National University from Kathonzweni District, all from was used instead of Whatman filter Canberra ACT 0200, Australia 5 Phone: +61-2-6125 0775 Eastern Province, Kenya were paper . Cyanide determinations E-mail: [email protected] affected after consuming raw and were done in triplicate. Fresh samples M1 and M2 were collected Coordinating Group: from the farm where cassava had J.P.Banea, Julie Cliff, Arnaldo Cumbana, been harvested, while sample M3 Ian Denton, Fernand Lambein, was raw cassava obtained from the N.L.V.Mlingi, Humberto Muquingue, Bala home of the affected family. M4 was Nambisan, Dulce Nhassico, S.L.N. Rao boiled cassava which had been

eaten by the family. Mean cyanide Country Contacts: Cameroon: E.E. Agbor; results (ppm) were M1 53.4, M2 D.R. Congo: D.Diasolua Ngudi and J. 47.8, M3 52.3, M4 46.0. The boiled Nsmire Chabwine; Indonesia: A. Hidayat; cassava, sample M4 had lower Kenya: R. Nungo; cyanide content than the uncooked Nigeria: M.N. Adindu and P.N. Okafor cassava, M3. This is attributed to the fact that boiling of cassava Website: reduced the cyanide content. www.anu.edu.au/BoZo/CCDN Samples C1 and C2 were collected cooked cassava brought from a from the farm where second family farmer and relative respectively in obtained the cassava that caused Fatal cassava food poisoning August and September 2011. A 4 death to one family member. C1 had in Eastern Province, Kenya year old child died in the first family, cyanide content of 73.2 ppm while see Figure, while the other members C2 was 56.4 ppm. It has been One of the cassava characteristics were taken to Makueni Hospital, shown that cyanoglycoside levels in is the presence of linamarin, a treated and discharged. The family 1 cassava root vary widely between In countries with known konzo, the cassava cultivars, plants of the reported poliomyelitis situation in On the molecular mechanisms same cultivars, different tissues of 2011 was the following: Angola and of konzo and neurolathyrism the same plant, roots of the same the Democratic Republic of Congo – plant, and even within the root re-established transmission of wild Konzo and neurolathyrism show 7 parenchyma. The samples also had poliovirus. very similar clinical symptoms while greatly exceeded the accepted level – importation from Nigeria of wild the apparent dietary determinants of 4 as recommended by WHO. This poliovirus. – cases of the two diseases are very different: could explain why the levels were vaccine derived poliomyelitis, see cassava cyanogens for konzo and a fatal to the two children in the two www.polioeradication.org. neuro-active amino acid in grass families and caused poisoning of the Thus four countries with known pea for neurolathyrism. This other family members. konzo have also reported similarity in clinical symptoms has Recommendations poliomyelitis, either wild or vaccine- been discussed before in this The communities in Makueni and derived, in 2011. Newsletter1 and at the Ghent Kathonzweni Districts, and other In the remote rural areas of Africa workshop.2 Could there be any areas of Eastern Kenya where where konzo is common, health similarity in the molecular etiology cassava is heavily consumed need service coverage is often low and with a possible junction of the two to be educated about toxic cyanide therefore vaccine coverage pathways? In this short contribution in cassava and on methods of and disease surveillance may be we summarize some facts and processing to reduce levels of poor. Where konzo is common and discuss some ideas. cyanide. There is a need to do an neglected, communities may see no From early studies on konzo, it was epidemiological study of the reason to report cases of acute considered that the presence of diseases caused by cassava . cyanogens in cassava was involved cyanide poisoning and following up The konzo areas may thus pose in the etiology of this disease, of affected families. High cyanide several challenges for polio perhaps the low protein intake from cassava roots should be processed eradication: low polio vaccine cassava roots as staple food in a before consumption and must not be coverage, poor disease surveillance, monotonous diet was an boiled and eaten, which is and possible confusion in the aggravating factor. More specifically, acceptable only for sweet (low diagnosis. the low content of essential amino cyanide) cassava roots. The following table shows the acids methionine and cysteine in the References differences and similarities between diet could reduce the formation of 1Ayer, G.S. (1985). Effect of the wetting of the two diseases glutathione, the central metabolite in cassava on product yield and cyanide konzo poliomyelitis the redox homeostasis, and induce detoxification. J. Food Tech., 20, 89 -96. diet of bitter yes coincidental 2Cock, J.M (1985). Cassava: New potential for the sensitivity to oxidative stress. In cassava a neglected crop. Boulder, CO, Westview 10 early studies on neurolathyrism, the 3Cardoso, AP., Mirione, E., Ernesto, M., more than often extremely presence of common vetch seeds one case in rare Massaza, F., Cliff, J., Haque, MR., Bradbury, (Vicia sativa) containing the toxic β- JH., (2005) Processing of cassava roots to the family remove cyanogens. J. Food Comp. Anal. 18, seasonal yes sometimes cyanoalanine found in most samples 451-460. predominant 3-15 years, under 15 of grass pea seed was blamed for 4FAO/WHO (1991). Food standards age groups adult years the toxicity. In 1964 a new non- programme. Codex Alimentarius Commission X11. Supplement 4. Rome, Italy. FAO/UN. women protein amino acid β-N-oxalyl-α,β- 5Bradbury, M.G.; Egan, S.V., Bradbury, J.H sudden yes yes diaminopropionic acid (β-ODAP) (1999). Determination of all forms of onset was discovered in grass pea seeds cyanogens in cassava roots and cssava type of spastic flaccid that was shown to have a specific products using picrate paper kits. J. Sci. Food paralysis Agric. 79, 593 - 601 action on nerve cells, and this 6 symmetry of symmetrical often Nyachoti, S.K. Njue W.M, Murugi, J.I, Wanjau, discovery started a long story to paralysis asymmetrical R.N, (2011). J.Kenya Chemical Society. ISSN prove that this was the cause of 1811 – 5934, Vol.6. No.1 visual often no 7De Brujin, G.H.(1971). Etude du Caractere disturbances neurolathyrism. A major problem in Cynogenetique du Manioc (Manihot esculenta the research on both konzo and Crantz). Moded Land Hogesch Wageningen Recommendations in konzo areas neurolathyrism was the difficulty to 71(13), Wageningen. The Netherlands. produce an animal model mimicking 1. Teach health workers and Njue, WM., Mburu, F., Sauda, S., community leaders the difference the human condition of konzo or Chemistry Dept., Kenyatta University, Kenya between acute neurolathyrism. Especially the (suspected polio) and acute spastic sudden onset of both diseases is as How to distinguish konzo from paralysis (konzo). yet unexplained. Very young poliomyelitis 2. Make konzo a reportable disease. animals seemed to be more 3. Provide physical rehabilitation to susceptible to the neuro-excitant β- With poliomyelitis nearing all cases of paralysis. ODAP. eradication, surveillance for cases of 4. Ensure high polio vaccine While many papers on acute flaccid paralysis has been coverage. neurolathyrism claimed that grass stepped up. In areas where konzo pea contains high quality protein rich Cliff, J., occurs, the two diseases may be in lysine, it was overlooked that the Faculdade de Medecina, content of the sulfur amino acids confused, and konzo may be Universidade Eduardo Mondlane, reported as suspected poliomyelitis. Maputo, Mozambique. methionine and cysteine was the [email protected] lowest of all commercial legume 2 crops. Soybeans have twice as space between the pre-synaptic cell have been linked also to neuro- much of these essential sulfur and the post-synaptic cell. Normally degenerations with gradual onset,6 containing amino acids, needed for this glutamate is taken up by glial while the central role of glutathione the maintenance of the glutathione cells that are the energy suppliers of in neuro-degeneration has been level and the redox homeostasis in the neurons, and where glutamate is discussed by Nunn et al.7 the cells. Even in soybean the sulfur metabolized into glutamine that is Glutathione seems to be at the point amino acids are the limiting returned to the neuron and recycled. of confluence of the molecular essential amino acids and farmers The transporter of glutamate into the etiology of both konzo and feeding soybean to their cattle add neurons and glial cells is however neurolathyrism, and this seems to synthetic methionine as supplement inhibited by β-ODAP which be also the case for neuro- for better performance. An important increases the level of glutamate in degenerations with gradual onset similarity between the foodstuffs the synaptic cleft. This in turn where old age is the main risk causing konzo and neurolathyrism is increases the excitation of post- factor.6 thus the deficiency in sulfur synaptic neurons to potentially toxic Konzo and neurolathyrism share containing amino acids. Only after levels.4 Such excessive or several aspects of neuro-excito- consumption over an extended prolonged activation of glutamate toxicity: i) mitochondrial dysfunction period, the sudden onset of konzo or receptors gives rise to an increase with reduced energy supply to the neurolathyrism can occur, which of intracellular Ca2+ concentration, neurons,ii) oxidative stress that is makes it difficult for the victims to resulting from entry via Ca2+ enhanced by the deficiency of sulfur understand that a food that helped permeable glutamate receptors or amino acids in the staple diet, and them to survive droughts or even released from intracellular stores iii) the modulation of enzymes famine, can suddenly become toxic. such as mitochondria and the protecting against ROS and This gave rise to some popular endoplasmatic reticulum. The motor oxidative stress.4,8 Recently, a factor beliefs that konzo is caused by evil neurons have little buffering capacity increasing the incidence of spirits or that neurolathyrism is for Ca2+, and thus are more neurolathyrism symptoms in rats caused by walking through a field of vulnerable to such events. β-ODAP was identified as stress.9 The same flowering grass pea. also inhibits the cystine/glutamate authors also found transient The residual cyanogens present in antiporter on the membranes of glial hemorrhage in the lower part of the insufficiently treated cassava roots cells or neurons which lowers the spinal cord, potentially indicating can be detoxified in the liver by the availability of glutathione precursors disturbance in the blood-brain enzyme rhodanese. Hereby the free and increases oxidative stress.4 barrier (BBB). Occurrence of konzo cyanide is transformed into the less Besides being an inhibitor of the and neurolathyrism is mostly toxic thiocyanate (SCN-) that can be cystine/glutamate antiporter, β- restricted to poor and often illiterate removed via urine. This ODAP is also a substrate for this people of low socio-economic detoxification needs one molecule of transporter and can enter the cells, status. Social stress in combination sulfur amino acid per molecule of where it can affect the mitochondrial with malnutrition and physiological cyanide. In case of deficiency of respiration. In particular, oxidative stress during extended methionine and cysteine, the mitochondrial complex-1 seemed to periods may increase the fragility of detoxification is not complete and be inhibited.5 the BBB and the susceptibility for cyanate is formed. This cyanate The mitochondria are also the target konzo and neurolathyrism. The very (OCN-) can activate the same for cyanide toxicity, affecting the high incidence of neurolathyrism in a AMPA-receptors on the nerve cell electron chain and the formation of forced labor camp during the second membranes that in the case of ATP. The effect of cyanide or β- worldwar10 may corroborate this neurolathyrism are activated by the ODAP on the mitochondria not only hypothesis. grass pea metabolite β-ODAP (β-N- causes depletion of ATP, it also References oxalyl-L-α,β-diaminopropionic acid), increases the production of reactive 1 Lambein F., Defoort B., Kuo Y.-H. (2004) and is known to cause oxygen species (ROS). The cell Konzo or Neurolathyrism: is there a difference? 3 CCDN News 3, 2-3. neurodegeneration. normally can protect itself against 2 Bradbury J.H., Lambein, F. (2011), Konzo While two physiological important these aggressive ROS by a battery and neurolathyrism: similarities and factors, i) the deficiency in sulfur of enzymes such as superoxide dissimilarities between these crippling neurodegenerative diseases of the poor. Food amino acids that hampers the dismutase, catalase and glutathione Chem. Toxicol. 49, 537-538. formation of glutathione and ii) the peroxidase that act in concert to 3 Tor-Agbidye, J., Palmer, VS ; Lasarev, MR ; excitation of AMPA-receptors, are maintain the redox homeostasis. Craig, AM ; Blythe, LL ; Sabri, MI ; Spencer, potential factors in the etiology of This can however be jeopardized by PS., (1999) Bioactivation of cyanide to cyanate in sulfur amino acid deficiency: Relevance to both konzo and neurolathyrism, the the reduced availability of neurological disease in humans subsisting on grass pea metabolite β-ODAP has glutathione (GSH). The activity of cassava. Toxicol. Sci. 50, 228-235 4 many additional effects in the cyanide or β-ODAP as described Van Moorhem M., Lambein F., Leybaert L. (2011) Unraveling the mechanism of β-N- nervous system. The best known above, in addition to the deficiency oxalyl-α,β-diaminopropionic acid (β-ODAP) effect of β-ODAP is the excitation of of sulfur amino acids contribute to induced excitotoxicity and oxidative stress, the AMPA-receptors, a subset of this low availability of GSH. This relevance for neurolathyrism prevention Food Chem. Toxicol. 49 (3), 550-555. glutamate receptors, which may also results in the formation of mixed 5 Sriram K., Shankar S.K., Boyd M.R., result in excessive activation of post- protein-glutathione disulfides Ravindranath V., (1998) Thiol oxidation and synaptic neurons. β-ODAP is also (PrSSG) that normally are reduced loss of mitochondrial complex I precede known to stimulate the release of by GSH. Dysfunction of excitatory amino acid-mediated neurodegeneration. J. Neurosci.18, 10287- glutamate into the synaptic cleft, the mitochondria and oxidative stress 10296 3

6 Lin M.T., Beal M.F. (2006) Mitochondrial related to consumption of cassava consumed with beans or some other dysfunction and oxidative stress in with a high concentration of cyanide. flavouring. neurodegenerative diseases. Nature 443, 787- 795. The women accepted the wetting 7 Bettencourt, MS., Paquisse, MM., Zangulo, A., Nunn P.B., Lyddiard J.R.A., Perera K.P.W.C. Resende, I. method willingly and found that it (2011) Brain glutathione as a target for Universidade Agostinho Neto, Luanda, Angola. produced much tastier fufu than that aetiological factors in neurolathyrism and produced from untreated flour, which konzo. Food Chem. Toxicol. 49, 662-667 [Ed: This is the abstract of a poster 8 was bitter due to the presence of Kassa R.M., Kasensa N.L., Monterroso V.H. given at the 20th World Congress of Kayton R.J. Klimek J.E., David L.L., Lunganza bitter linamarin, and the sweet fufu Neurology, Marrakesh, Morocco, K.R., Kayembe K.T., Bentivoglio M., Juliano could be stored for longer (2 days) S.L., Tshala-Katumbay D. (2011) On the (2011). than fufu from untreated flour. The biomarkers and mechanisms of konzo, a http://wcn.kenesapp.com/WCN_268/ distinct upper motor neuron disease total cyanide content of the cassava poster_16595/program.aspx . An associated with food (cassava) cyanogenic flour after the wetting treatment was exposure. Food Chem. Toxicol. 49 (3), 571- initial report from the same source reduced to less than 10 ppm, the 578. was given in CCDN News No 16, 3- 9 FAO/WHO limit for cassava flour. Kusama-Eguchi K., Yamazaki Y., Suda A., 4, (2010). Ueda T., Hirayama Y., Ikegami F., Watanabe The mean urinary thiocyanate Dr Julie Cliff reports that Dr Miguel K., May M., Lambein F. and Kusama T..(2010) content of 100 school children Hind-limb paraparesis in a rat model for Bettancourt stated to the Angola (which measures the total cyanide neurolathyrism associated with apoptosis and Press Agency that the region of an impaired vascular endothelial growth factor intake over the previous week or so) Caungula continued to report system in the spinal cord. J. Comparat. Neurol. was reduced from 332 µ mole/L to a 518, 928-942. innumerable cases of konzo, and 10 safe level of 130 µ mole/L. The Lambein F., Ngudi D.D., Kuo Y.H. (2001) that the disease was a continuing number of urinary thiocyanate Vapniarca revisited: Lessons from an inhuman serious public health problem.] human experience. Lathyrus Lathyrism samples that exceeded a dangerous

Newsletter 2, 5-7. level of 350 µ mole/L decreased Control of konzo in DRC Lambein, F. [email protected], from 26 at the beginning of the

Diasolua Ngudi D. [email protected], Van intervention (March 2010) to zero by Moorhem M., Kuo Y.-H. Konzo is an irreversible paralysis of May 2011. No new cases of konzo IPBO, Ghent University, Ghent, Belgium. the legs that occurs mainly in occurred in Kay Kalenge during the children and young women after Tropical spastic paraparesis intervention (March 2010 to childbirth, due to intake of large (konzo): a major neurologic September 2011) which included amounts of cyanide containing problem in Caungula - Angola: 1.2 two dry seasons when incidence of compounds from bitter cassava. It 3 konzo normally peaks, due to large first report in Angola was first discovered by Dr Trolli in intakes of total cyanide over the 1938 in Popokabaka Health Zone, A particular kind of spastic cassava harvest. Bandundu Province, Belgian Congo, paraparesis was first reported in The success of this method in now the Democratic Republic of Republic of Zaire about 80 years preventing konzo is due to (1) Congo (DRC) and has been brought ago. Since then other reports were education of all women to under control for the first time in the made in other tropical countries 4 understand that konzo comes from a same location. such as Mozambique, Tanzania and poison (cyanide) present in cassava In Kay Kalenge village in Cameroon, related to cyanide intake flour and is not due to witchcraft, (2) Popokabaka Health Zone there were of cassava. The typical clinical training in use of the wetting method 34 cases of konzo in a population of picture is spastic paraparesis with to remove cyanide compounds, 1250. The women of the village abnormality of gait while walking or helped by use of laminated posters were taught that konzo is due to a running with onset less than a week in the local language poison present in cassava flour that and irreversible course. No reports (http://online.anu.edu.au/BoZo/CCD causes konzo and how to treat the have been made before in Angola. N/) and (3) full commitment to use of cassava flour to remove the poison In order to clarify an unusual 5,6 the wetting method on a regular by using the wetting method. as frequency of unknown disease at the basis. This result was achieved over follows: The cassava flour was site, we went to Caungula, which is the period of the intervention by placed in a bowl and a mark made a small rural village in the northeast visits every 4 months by the full on the inside at the level of the dry of Angola near the border with the team, interspersed with visits every flour. Water was mixed in until the Democratic Republic of Congo. We month by the Caritas team from wet flour came up to the same mark. observed 20 patients affected with Popokabaka. The same The wet flour was spread out on a spastic paraparesis with different methodology is now being used in a basket in a thin layer for about 2 degrees of gait limitation. In 10 of 7 one year intervention in three hours in the sun or for about 5 them we collected blood samples for villages where konzo is prevalent in hours in the shade. The enzyme cyanide, HIV and HTLV virus Boko Health Zone, Bandundu (linamarase) present in the flour analyses. The patients were from 5 Province, supported by funding from decomposed the cyanide compound to 34 years old, mostly female. From the Australian Agency for (linamarin) with production of those whose blood was collected, all International Development (AusAID) hydrogen cyanide gas. The wetting the blood samples were non- and further interventions to prevent method is truly a gift from God to reactive to HIV and HTLV 1 and 2, konzo in other villages are being these people. The damp flour was but all of them had high levels of planned. then cooked in the traditional way, cyanide. by mixing with boiling water to References We conclude that tropical spastic 1 Cliff, J., Martensson, J., Lundquist, P., produce a thick porridge called fufu, paraparesis (konzo) in Angola is Rosling H., Sorbo, B., (1985). Association of 4 high cyanide and low sulphur intake in cassava induced spastic paraparesis. Lancet 11, 1211- 1213. 2 Howlett, W.P., Brubaker, G.R., Mlingi, N., Rosling, H.,(1990). Konzo, an epidemic upper motor neuron disease studied in Tanzania. Brain 113, 223–235. 3 Trolli, G.(1938) Paraplegie spastique epidemique, “Konzo” des indigenes du Kwango. In: Trolli, G., ed. Resume des observations reunies, au Kwango, au sujet de deux affections dʼorigine indeterminee. Brussels: Fonds reine Elisabeth, 1-36. 4 Banea, JP,, Nahimana, G., Mandombi, C., Bradbury, JH., Denton, IC., Kuwa, N., (2012) Prevention of konzo in DRC using the wetting method on cassava flour. Food Chem. Toxicol., submitted for publication. 5 Bradbury, J.H., (2006). Simple wetting method to reduce cyanogen content of cassava. J. Food Comp. Anal. 19, 388-393. 6 Cumbana, A., Mirione, E., Cliff, J., Bradbury, J.H., 2007. Reduction of cyanide content of cassava flour in Mozambique by the wetting method. Food Chem. 101, 894-897. 7 Bradbury, J.H., Denton, I.C., 2010. Rapid wetting method to reduce cyanogen content of cassava flour. Food Chem., 121, 591-594.

Banea, JP,a Nahimana, G.,a Mandombi, C.,b Bradbury, JH.,c Denton, IC,c Kuwa, N.a aProgramme National de Nutrition (PRONANUT), Kinshasa, DRC, b Hopitall General de Reference, Zone de la Sante de Popokabaka, DRC, c EEG, Research School of Biology, Australian National University, Canberra, Australia.

CCDN News will consider for publication short articles and letters (1-3 pages A 4 double spaced) in English. Because CCDN News is a newsletter, full-size original papers or reviews cannot be considered for publication. Material published in CCDN News may be freely reproduced, but always indicate that it comes from CCDN News. Please send all correspondence to the CCDNN Coordinator, Dr J Howard Bradbury, EEG, Research School of Biology, Australian National University, Canberra, ACT 0200, Australia.

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