Citreoviridin and Acute Cardiac Beriberi occurrence in Maranhão State, Brazil

ROSA, C.A.R.; KELLER, K.; OLIVEIRA, A. A.; XAVIER, T. A.; KELLER, L.A.C.; MARASI, A.C.; KRUGER, C.D.; ASTORECA, A.; CAVAGLIERI, L. R.; DIREITO, G.M.; EIFERT, E.C. ; LIMA, T.A.S.; MODERNELL, K.G.; NUNES, F.I.B. ; LUZ, M.S.; OLIVEIRA,D.C.N.

Amsterdam

Congreso; The World Mycotoxin conference; 2008

Beriberi is a disease caused by a deficiency of thiamine (vitamin B1) that affects many systems of the body, including the muscles, heart, nerves, and digestive system. This word means "I can not, I can not" in Singhalese, which reflects the crippling effect it has on its victims. It is common in parts of southeast Asia, where white rice is the main food. It can also be seen in chronic alcoholics with an inadequate diet. A substance able to produce a similar disease to beriberi is citreoviridin (CTV), a metabolite of Penicillium citreonigrum Dierckx thought to be the cause of yellow rice poisoning, manifested in humans and experimental animals by respiratory and circulatory failure, paralysis, convulsions and death. Identical to beriberi, originally considered to be a thiamin nutritional deficiency. Since May 2006, there were 1028 cases of beribéri in southwestern of Maranhão State (Brazil) with 32 deaths. These cases were described as beribéri dry (38%), wet (18%), cardiac (40%) and Wernicke-Korsakoff syndrome (4%). The Shoshim-Kakke (cardiac beriberi) in Japan, the Keshan in China and Southeast Asian countries, are related to the consumption of molded rice and yellow (Yellow Rice Disease), contaminated with the mycotoxin CTV. This mycotoxin and its derivatives, described as a-pirones  compounds, are mycotoxins produced from soil fungi, such as Penicillium citreonigrum Dierckx (P. citreoviride Biourge), Eupenicillium ocrhosalmoneum (P. ochrasalmoneum Udagawa) DB Scott & Stolk, who rapidly grow during storage and storage after rice and maize harvest. Citreoviridin was first isolated from metabolites of P. citreonigrum strains associated with cases of acute berberi (Shoshin-kakke). In Japan, yellow rice disease has not been reported since the decades that the yellow rice was excluded altogether from the Japanese market (Saito et al, 1971, Ueno, 1972, 1986). Keshan in China has been linked to CTV, which is often found in samples of rice and maize (Dean et al, 2004, ShuQui, et al, 2004). Datta & Ghosh, (1981) considered CTV as a anti-thiamine factor. They argued that CTV is a potent inhibitor of mitochondrial ATpases, as the adenosine-trifosfatase. The addition of thiamine diphosphate (TPP) produced the reversal of the inhibitory effect of ATpases. These findings suggested the relationship between CTV, the induction of beriberi and its antithiamine effect. The literature that describes the change in eating habits, with the inclusion of other sources of thiamine, has reduced the occurrence of Shoshin-kakke, and has eliminated the occurrence of deaths. Penicillium citreonigrum strains and citreoviridin were isolated and detected from 419 samples of rice farms in areas where there was at least one case of diagnosed beribéri in the Maranhão State, Brazil (Rosa et al., 2007ab). The regional rice production is rudimentary in small farms which uses rice cultivar called cateto. The fungal colony forming units (cfu/g) methodology was done by serial dilution in peptone water (1%) and aliquots of 0.1 mL from 10-2 to 10-4 dilutions in Dichloran Rose Bengal Chloranfenicol agar (DRBC) and Dichloran Glycerol 18% agar (DG18) were placed. Fusarium spp were isolated from Nash-Snyder agar. Plates were incubaded at 28o C for 7 days and identification was done in accordance with the appropriate keys. The CTV levels were determined by HPLC (Schotwel et al., 1988). The rice samples were extracted with acetonitrile:water (86:14, v/v) and the extracts were concentrated under N2. The CTV was measured using normal phase of liquid chromatography. The mobile phase was ethyl acetate:n-hexane (80:20, v/v), and a flow of 1.5 ml/min. The detection was made by fluorescence with 388 nm excitement and 480 nm emission. The standard solution of CTV (SIGMA Co., St. Louis-U.S.) in methanol (100 ng/mL) contained approximately 96% purity and after calibration showed a 6:4 ratio (m/m) of the mixture citreoviridina/isocitreoviridina (iCTV). The detection limit was 1 ng/g. Fungal counts in DRBC ranged from 2.5 x 10 to 4.8 x 106 CFU.g-1 and in DG18 from 1.0 x 10 to 1.0 x 107 CFU.g-1. Fusarium species were found at 1.7 x 102 CFU.g-1 average. Analyzed rice samples from tropical climate area exhibited high fungal counts. Moreover, 335 Aspergillus strains and their teleomorphs (70.2%) were isolated, followed by Penicillium spp and teleomorphs (13.8%), Cladosporium spp (8.7%), Fusarium spp. (2.9%) and Mucor spp (2.2%). Penicillium citrinum was prevalent (12.8%), followed by A. flavus (10.4%), Eurotium sp (9.9%) and A. niger (8.3%). Penicillium citreonigrum (4.3%) was isolated from eleven samples, with only one strain not able to produce CTV. The performed HPLC analyses of rice showed five samples contaminated with CTV at levels ranging from 12 to 96.7 ng/g. The iCTV was detected in two of the five positive samples. Four positive samples were from cateto rice harvested in 2006, agulhão rice harvested in 2007 and two samples of rice bran. All positive samples came from the region of beribéri occurrence, in the southwestern State of Maranhao, Brazil. The methodology used in this study is being optimized. However, it proved to be effective to detect citreoviridin/isocitreoviridin with a 86% recovery in samples fortified with 100 ng/g. This is the first occurrence of Cardiac Beriberi caused by citreoviridin intoxication in West and the first human micotoxicose outbreak described in the Americas. The administration of thiamine to sick patients in the Maranhão State decreased  and stopped mortality and reversed the vast majority of beriberi cases. These data point to the immediate adoption of Good Agricultural Practices, appropriate hygienic-sanitary measures to be applied in regional production and consumption of rice as well as health education targeting the population of the region. References Datta, Ghosh (1981). Production and purification of Penicillium citreoviride toxin and its effect on TPP-dependent liver transketolase. Folia Microbiol (Praha). 26, 408-12 Li DeAn, Sun ShuQiu, Li, X. D., Li XiaoDan, Qing XueMei (2004). Determination of citreoviridin in crops by high performance liquid chromatography. Chinese Journal of Endemiology, 23, 160-162. Rosa, C.A.R.; Keller, K.; Oliveira, A.A.; Xavier, T. A.; Marasi, A.C.; Astoreca, A.; Eifert, E.C.; Lima, T.A.S.; Modernell, K.G.; Nunes, F.I.B.; Luz, M.S.; Oliveira,D.C.N. (2007a). Micobiota toxígena isolada de amostras de arroz coletadas em áreas de ocorrência de Beriberi no sudoeste do Maranhão, Brasil. – Resultados Preliminares. Anais do Congresso Brasileiro de Toxicologia, 15. Buzios, RJ. Revista Brasileira de Toxicologia . Rosa, C.A.R.; Direito, G.M.; Eifert, E.C.; Lima, T.A.S.; Modernell, K.G.; Nunes, F.I.B.; Luz, M.S.; Oliveira,D.C.N. (2007b). Detecção de Citreoviridina por CLAE em amostras de arroz coletadas em áreas de ocorrência de Beriberi no sudoeste do Maranhão, Brasil. – Resultados Preliminares. Anais do Congresso Brasileiro de Toxicologia, 15. Buzios, RJ. Revista Brasileira de Toxicologia. Saito, M., M. Enomoto, and T. Tatsuno.. Yellowed rice toxins: luteroskyrin and related compounds, chlorine-containing compounds and citrinin. In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. VI: fungal toxins. Academic Press, New York, N.Y. p. 299–380, 1971. Sun ShuQiu, Li DeAn, Hui Yang, Hui, Y., Li XiaoDan, Li, X. D. (2004). Survey on citreoviridin level in market cereals of 13 counties of Heilongjiang province. Chinese Journal of Endemiology 23, 367-368. Ueno, Y. Production of citreoviridin, a neurotoxic mycotoxin of Penicillium citreo-viride. In: I. F. H. Purchase (Editor). Mycotoxins. Elsevier 1974, Amsterdam. P283. Ueno, Y. (1986). Toxicology of microbial toxins. Pure & Applied. Chem., Vol. 58, No. 2, pp. 339-350, 9. Yang Qiuhui, Zhou Hongbo, LI Qunwei (1999) Liquid chromatograph method for determination of citreoviridin in cereal, Institute of KBD, Harbin Medical University, Harbin 150001. S432.44 1000- 4955 02-0107-09