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Quantitative Culture of Malassezia Species from Different Body Sites Of Medical Mycology 2001, 39, 243±251 Accepted 28November 2000 Quantitativeculture of Malassezia species fromdifferent body sites ofindividuals with or without dermatoses A.K. GUPTA*, , Y. KOHLI , ,R.C. SUMMERBELL§,$, J. FAERGEMANN } y y z *Divisionof Dermatology, Department ofMedicine, Sunnybrook Health Science’s Center and Women’s CollegeHospital (Sunnybrooksite) and University ofToronto, Canada; MediprobeLaboratories, Toronto, Ontario, Canada; Department of y z Microbiology,The Hospital forSick Children,Toronto, Ontario, Canada; §Centraalbureau voorSchimmelcultures, Baarn, the Downloaded from https://academic.oup.com/mmy/article/39/3/243/946776 by guest on 27 September 2021 Netherlands; $Department ofLaboratoryMedicine and Pathobiology,University ofToronto, Toronto, Ontario, Canada; }Sahlgrenska University Hospital, Gothenburg, Sweden Quantitativecultures were obtained using contact platesto determinewhether the quantityand composition of Malassezia speciesat a givenanatomic site in normal individualsdiffers from that of patientswith variouscutaneous dermatoses.The sampleincluded 20 clinically healthy individuals (without anydermatosis) and 110patients with dermatoses(including 31 with atopicdermatitis [AD], 28with psoriasis[PS], 28with seborrheicdermatitis [SD] and23 with pityriasisversicolor [PV]). Contact plates lledwith specialculture medium were used to obtain a quantitativeculture from vebody sites(scalp, forehead, arm, trunk andleg) of everyindividual. The number of cfu wererecorded for everyplate that grew Malassezia yeasts,and 3– 5 colonieswere isolated for identication to specieslevel usingmicroscopic, physiological and molecular characteristics. The mean cfu counts observedamong patients with AD,PS andSD was signicantly lower than normal control subjects( P <0 05).The mean cfu counts from PVpatientswas not different ¢ from that of healthycontrol subjects.Overall, for allconditions consideredtogether, the meancfu counts inlesional sites were signi cantly lower than innon-lesional sites (P <0 05).Furthermore, the meancfu counts from lesionalsites in patients ¢ with ADandPS weresigni cantly lower than the correspondingvalue in patients with PV (P <0 05). Six Malassezia specieswere recovered from the different ¢ dermatoses. Malasseziasympodialis was the most common speciesassociated with ADand PV patientsand healthy control subjects,while M. globosa was most frequentlyisolated from PSandSD patients.More than one Malassezia species was recoveredat any given anatomic site from both controls aswellas individualswith dermatoses. M. globosa wasequally likely to berecovered from scalp,forehead and trunk, but lesslikely to derivefrom armsand legs. M.restricta and M. sloofae were recoveredmore frequently from the upper body (scalpand forehead) than from the lowerbody. Among normal individuals and for patientswith ADand PV, M. sympodialis was signicantly more likely to affectthe foreheadthan the legs. Keywords contact plates,dermatoses, Malassezia species,quantitative culturing Introduction The lipophilic yeast Malassezia furfur and related species are members of the normal human cutaneous ora of Correspondence:Aditya K. GuptaM.D., Suite 6, 490Wonderland RoadSouth, London, Ontario, Canada N6K 1L6. skin and produce clinical disease under conditions that Tel.: 1519657 4222; fax: 1519657 4233; permit massive growth of the fungus. Prior to 1996, the ‡ ‡ e-mail:[email protected] genus Malassezia was thought to contain only three ã 2001 ISHAM,ISHAM Medical Mycology , 39, 243±251 244 Gupta et al. species, namely, M. furfur (Robin) Baillon 1889, objective of identifying Malassezia species according to M. pachydermatis (Weidman) Dodge 1935 and current techniques and determining their possible M. sympodialis Simmons and Gue´ ho 1990. Recently, association with various skin conditions. Malassezia was revised to include four new taxa, This study was undertaken with the following objec- M. globosa, M.restricta , M. obtusa and M. sloofae, in tives: (i)to quantify cultures of Malassezia species from addition to the three former taxa [1]. The etiological role different body sites in humans and (ii) to determine of Malassezia yeasts in tinea (pityriasis) versicolor, whether the quantity and composition of Malassezia Malassezia folliculitis and seborrheic dermatitis (SD) species at agiven anatomic site in normal individuals has been shown inseveral reports [2]. Under the current differs fromthat of patients with various cutaneous classication system, however, the specic clinical dermatoses. Downloaded from https://academic.oup.com/mmy/article/39/3/243/946776 by guest on 27 September 2021 association of each of these species is not clearly understood. Materialsand methods Malassezia yeasts can be cultured fromalmost all adults. Roberts [3,4] reported that 97% of clinically Preparation ofcontact plates healthy people carry the fungus on their scalp and 92% BBL1 RODAC1 (Beckton Dickinson, Cockeysville, carry it on their trunk. Since Malassezia species can be MD, USA) contact plates were used for sampling. This cultured fromalmost all areas of the body, qualitative 65 15 mm-style dish is specially designed to allow a £ culture of these organisms has no diagnostic value. raised convex surface of culture medium. The 10 mm However, aquantitative assessment in certain circum- grid on the bottom of the plate facilitates counting and stances may correlate with clinical signicance [5]. colony location. These plates were lled with alipid-rich Additionally, such culturing may also be useful in glucose peptone yeast extract medium described by monitoring the effect of antimycotic treatments in SD Leeming &Notman [10]. The composition of Leeming– patients. Quantitative cultures of M. furfur complex can Notman agar (LNA)is as follows: 1lglass distilled H 2O; be made with the scrub technique [6], atape method [7] 10 gbacteriological peptone (DIFCO,Detroit, MI, or contact plates [5,8,9]. Bergbrant et al. [9] reported the USA); 5gglucose; 0 1gyeast extract; 4gOx-Bile ¢ use of contact plates containing amodied medium (desiccated; DIFCO);12 gagar; with lipid supplements described by Leeming &Notman [10] for quantitative as: 1mlglycerol; 0 5gglycerol monostearate; 0 5 ml ¢ ¢ culturing of M. furfur. Tween 60, 10 mlhomogenized milk (3 25% fat), and the ¢ 1 Several studies on quantitative culturing of Malassezia antimicrobial supplements chloramphenicol (50 mgml ¡ ) using different techniques have reported contradictory 1mland cycloheximide (2%) 9 9ml.The pHof the ¢ results. McGinley et al. [11], in astudy of quantitative media was 6 2. microbiology of the scalp, showed that for patients with ¢ dandruff, the population densities of M. furfur were Sample twice as high on nonaffected scalp. Faergemann [5] used contact plates for quantitative culture of Pityrosporum Twenty clinically healthy individuals and 110 individuals orbiculare (M. furfur complex pro parte)and reported a with the following skin conditions: atopic dermatitis muchhigher number of cfu frompatients with SDthan (AD;31 individuals), psoriasis (PS; 28 individuals), SD clinically healthy individuals. Bergbrant &Faergemann (28 individuals) and PV(23 individuals), were sampled [12], on the other hand, reported no signicant difference for quantitative culturing. Healthy control subjects were in the number of P. ovale for patients with SDthan individuals with normal appearing skin and without any control subjects. They reasoned that an abnormal evidence of adermatosis. We included only those reaction in the skin to P. ovale may cause the patients who had not used any topical or oral treatment inammation and that the number of P. ovale could be for their dermatoses for the previous 2and 4weeks, of lesser importance. In addition, SDmay be unrelated respectively. There were no known human immuno- to the sebum excretion rate and not necessarily deciency virus (HIV)-positive individuals in the study associated with seborrhea [13,14]. Ingham &Cunning- populations. Five body sites, namely scalp, forehead, ham [15], in astudy on pityriasis versicolor (PV)lesions, arm, trunk and leg were sampled for each subject. For reported that population densities of the yeast cells were patients with skin conditions, aseparate record was up to 105 cells cm–2 in lesions which is two log stages made for affected and unaffected sites. Because body higher than on unaffected skin. Although some of these sites with lesions often have no expanse of unaffected studies successfully used contact plates for quantitative skin sufciently large to allow exposure of acontact culturing, there have been no studies to our knowledge plate, and because inoculum fromlesions could easily where quantitative cultures have been taken with the cross-contaminate adjacent non-lesional areas, patient ã 2001 ISHAM, Medical Mycology , 39, 243±251 Malassezia speciesin patientswith cutaneousdermatoses 245 body sites were sampled on lesional or non-lesional drops of aTween compound, i.e. Tween 20, 40, 60 and areas, but not both. Lesional skin was sampled whenever 80, were applied in each separate well. These plates were possible. The protocol was reviewed and approved by an incubated at 32 oCfor 7days and the growth in each Independent Review Board (IRB)an institutional section was recorded. service in Aurora, Ontario. Each participant provided written, informed consent after the nature of the study Statistics had been explained to them. Nonparametric statistical analysis was conducted, using either the Kruskal-Wallis chi-square test or Mann- Sampling procedure
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