Mycopathologia 155: 183–189, 2001. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 183 Allergenic evaluation of Malassezia furfur crude extracts R.F. Gandra 1 , T.A. Melo 1 , F.E. Matsumoto 1 , M.F.C. Pires 2 , J. Croce 3 , W. Gambale 1 & C.R. Paula 1 1 Departamento de Microbiologia, Universidade de Sao Paulo; 2 Instituto Adolpho Lutz, 3 Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brasil Received 6 December 2001; accepted in revised form 22 June 2002 Abstract Crude extracts of the lipophilic yeast Malassezia furfur were obtained from 2, 6, 10 and 28 day old cultures. The in vitro cultivation periods corresponded, respectively, to the lag phase, middle of the log phase, end of log phase and the decline phase of the growth curve, which was based on viable cell counts obtained with a fluorescent viability test. Biochemical analyses showed that the protein and carbohydrate contents were greater in day 10 extracts. Seventy patients with different allergic manifestations and 30 healthy volunteers were skin prick tested using the extracts. Of these, thirteen (18.57%) patients gave positive responses. SDS PAGE gradient electrophoretic profiles of the preparations indicated that the 28 day extracts contained the greatest number of protein bands with molecular weights ranging mostly between 30 and 94 kDa. Immunoblots incubated with individual patient sera showed that four IgE binding M. furfur allergens of approximately 88, 61, 52 and 39 kDa were present in the 28 day extracts. The components identified could be used for detecting IgE mediated responses to M. furfur among individuals affected with different allergic conditions. Key words: Malassezia furfur, allergens, prick test, immunoblotting. Introduction The lipophilic yeast Malassezia furfur is a common saprophyte of the normal human skin [1]. This organ- ism has complex lipid requirements for growth and has been isolated from the upper trunk, head and face from sebum-rich areas of the body [2]. Two distinct classes of M. furfur allergens are known to induce type I hypersensitivity reactions. One corresponds to cell wall or cytoplasmatic pro- tein and the other to polysaccharides or glycoproteins, probably mannans or mannoproteins. Their relative frequencies may vary according to the length of the in vitro growth period of the yeast [3]. Several studies have demonstrated the involvement of fungi in allergic conditions [4–7] and M. furfur specific IgE antibod- ies have been detected in both healthy individuals and patients with skin allergies mainly atopic dermatitis [2, 8, 9–16]. Crude preparations of fungi are often used in skin prick and intradermic tests. However, the * Published in 2002. characterization and standardization of the allergens is fundamental to understanding the allergenic profile of M. furfur. The aim of the present study was to obtain crude extracts of M. furfur derived from different growth phases of the yeast. Then to analyze their protein and carbohydrate contents, evaluate their in vivo allergenic potential and characterize the allergens by immun- oblotting. Sera would be used from patients with various allergic manifestations, including not only the commonly investigated atopic dermatitis but also dis- orders in frequently associated with M. furfur, such as rhinitis, asthma, bronchitis and urticaria. Materials and methods Identification of Malassezia furfur strains We identified the M. furfur isolates according to the method of Guillot et al. [17]. Briefly, M. furfur yeast suspensions were adjusted at about 10 5 cells/mL and