Abstracts of the 2010 International Congress of the
European Association of Poisons Centres and Clinical
Toxicologists, 11-14 May 2010, Bordeaux, France
Clinical Toxicology
March 2010, Vol. 48, No. 3 , Pages 240-318 (doi:10.3109/15563651003740240)
34. Molecular Identification of Lepiota brunneoincarnata: Application to the Clinical Setting
Iturralde MJ,1 Ballesteros S,2 Marín Serra J,3 Martín MP.4
1
Servicio Biología, Instituto Nacional Toxicología y Ciencias Forenses, Madrid; 2Servicio Información Toxicológica,
Instituto Nacional Toxicología y Ciencias Forenses, Madrid; 3 Servicio de Pediatría, Hospital Dr Pexet, Valencia;
4
Real Jardín Botánico, CSIC, Madrid, Spain
Objective:Amatoxin poisoning is ascribed to 35 amatoxin containing species belonging to the genera Amanita,
Galerina and Lepiota.1 The high degree of polymorphisms within the ITS nrDNA region has been used in the
identification of fungal species and separation of species, as well as to establish the limits between very closely
related taxa. This is the first description of the application of the study of the regions ITS-1 and ITS-2 to the
resolution of a mushroom poisoning caused by Lepiota brunneoincarnata
.Methods:Mycological identification of
the mushroom fragments observed in the meal was made by botanical classification with macroscopic and
microscopic characteristics. ITS-1 and ITS-2 regions were studied by PCR and subsequent sequencing analysis.2
Sequencing data were compared with homologous sequences located at public databases (Blast search, NCBI).
Searching for amanitins was made by High Performance Thin-Layer
Chromatography (HPTLC) applied to the
cooked mushrooms.Results:Three patients from a family suffered from hepatotoxic poisoning after eating a stew
of mushrooms. The samples were picked from a lawn in a public park in the city of Valencia. The Spanish Poison
Center was contacted 18 hours after ingestion. The main complaints at this moment were vomiting, colic,
abdominal pain and diarrhea. Hepatic transaminases were slightly elevated. Macroscopic examination of the
mushroom fragments led to no conclusive result. Ellipsoid smooth thick-walled hyalin spores were seen in the
sample. The staining of spores with Melzer´s iodine reagent turned reddish (dextrinoid reaction). Both results
suggested the presence of a member of the genus Lepiota. The blast search of the ITS1 and ITS2 nrDNA
sequences showed 99% similarity with a sequence of Lepiota brunneoincarnata. Alpha
and beta amanitins were
detected by HPTLC.Conclusion:Molecular methods can be used to develop diagnostic tests for fungi.
Comparative analysis against GenBank database was useful in the identification of this
species.References:1.
Roux X, Labadie P, Morand C, et al Mushroom poisoning by brunneoincarnata: about two cases. Ann Fr Anesth
Reanim 2008; 27:450–2. 2. Iturralde MJ, Ballesteros S, Ramoacuten MF, et al. DNA Profiling: A promising tool for
mushroom poisoning diagnosis. Clin Toxicol 2004; 42:535.