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 Universityof Toronto, 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 of Toronto, Toronto, Ontario, Canada; }Sahlgrenska UniversityHospital, 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], 28 with psoriasis[PS], 28with seborrheicdermatitis [SD] and23 with pityriasisversicolor [PV]). Contact platesŽ lledwith specialculture medium were used to obtain a quantitativeculture from Žvebody 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 identiŽcation to specieslevel usingmicroscopic, physiological and molecular characteristics. The mean cfu counts observedamong patients with AD,PSandSD wassigniŽ cantly 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 signiŽ cantly lower than innon-lesional sites (P <0 05).Furthermore, the meancfu counts from lesionalsites in patients ¢ with ADandPS weresigniŽ 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 ADandPV 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. sloofŽae were recoveredmore frequently from the upper body (scalpand forehead) than from the lowerbody. Among normal individuals and for patientswith ADandPV, M.sympodialis wassigniŽ cantly 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 Simmonsand 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. sloofŽae, 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 classiŽcation system, however, the speciŽc 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 signiŽcance [5]. colony location. These plates were Žlled 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 amodiŽed 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 signiŽcant 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 inammation 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 deŽciency 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 sufŽciently 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 Whitney test, to compare and analyze the data at two Downloaded from https://academic.oup.com/mmy/article/39/3/243/946776 by guest on 27 September 2021 To obtain asample fromthe scalp, individuals were different levels: (i)within and between different derma- requested topart hairs and expose an area at the vertex toses and healthy controls, and (ii)within and between of the scalp. Acontact plate was then pressed Žrmly lesional and non-lesional skin of individuals with against the exposed surface for 20 s. Similarly, for each different dermatoses. These tests were performed using subject four additional contact plates were pressed in density data, i.e., data recorded as the number of succession against at areas of the forehead, arm, trunk Malassezia cfu obtained within the sample categories and leg for 20 seach. For each individual all the samples (see Results, Tables 2and 3). Two other tests for the were collected in one sitting. Immediately after the microhabitat (body site) speciŽcity of individual Malas- collection of samples, the plates were placed at 32 oC in sezia species were carried out using frequency data, i.e., the dark for 7days and the number of cfu obtained from data based on the number of contact plates yielding at different body sites was then recorded. Fromeach least one isolate of the species in question (see Results, contact plate showing multiple colonies, three to Žve Table 4). To determine if the various patient categories colonies were picked fromdifferent locations on the differed in the extent to which an individual Malassezia plate; when only one or two colonies grew they were species was associated with different anatomic sites, the likewise subcultured. Cochrane nonparametric statistic was used. For agiven dermatosis and any particular Malassezia species, the Identi®cation of Malassezia species McNemartest was used to determine if the species could be recovered more frequently than by chance alone from Malassezia species were identiŽed on the basis of agiven anatomic site. microscopicobservation of cells and physiological properties such as presence of catalase and the ability Results to utilize Tween compounds as alipid source in asimple medium [16]. For some ambiguous cases molecular Recoveryrate of Malassezia fromdifferent body sites of criteria for identiŽcation were used [17]; these were individuals used mainly to differentiate M.sympodialis from M. In at least 67% of individuals fromeach skin condition, a furfur and M. sloofŽae. positive culture was obtained fromone or more body sites. The frequency at which contact plates yielded a Physiological tests culture in each patient category, recovery of apositive Catalasereaction culture fromlesional and non-lesional sites, the number Presence of catalase was determined by application of a of colonies analyzed for identiŽcation and the number of drop of hydrogen peroxide (3%, 80% solution) on to a species recovered in each dermatoses category is culture smear on aglass slide. Production of gas bubbles presented in Table 1. was considered apositive reaction. Among all seven species of Malassezia,only the recently described Occurrenceof Malassezia onindividuals with and M.restricta lacks catalase activity [1]. without dermatoses The mean cfu counts for individuals with dermatoses and Utilizationof Tweencompounds the healthy controls were more variable than would be This test was modiŽed fromthe one reported by Guillot expected by chance (chi square, 21 0; degrees of freedom ¢ et al. [18]. Yeast suspensions (at least 10 7 cfu ml–1) were (df), 4; P <0 001) (Table 2). The mean cfu counts from ¢ made insterilized normal saline (0 85%) and inoculated patients with AD,PSand SDwere signiŽcantly lower ¢ on Sabouraud peptone-glucose agar. The inoculum was than those fromnormal individuals ( P < 0 05). On the ¢ spread evenly. Each plate was divided into four sections other hand, the mean cfu counts in patients with PV and awell was made in the center of each section. Four were similar to those in normal individuals. In apair wise ã 2001 ISHAM, Medical Mycology , 39, 243±251 246 Gupta et al.

Table 1 Recoveryrate for Malassezia fromdifferent sample categories and number of Malassezia speciesrecovered among the cultures analyzed for identiŽ cation

Characteristics AD PS SD PV Healthy

Sample size 31 28 28 23 20 No.of patients positive 22 19 23 21 19 for Malassezia Recoveryrate (%) 70 9 67 8 82 1 91 3 95 ¢ ¢ ¢ ¢ No.of contact plate 155 (49) 140 (44) 140(55) 115 (61) 100 (62) sampled(No. yielding Malassezia) Downloaded from https://academic.oup.com/mmy/article/39/3/243/946776 by guest on 27 September 2021 Lesionalsites* 93 (35) 86 (27) 58 (23) 36 (23) – Non-lesionalsites 62 (14) 54 (17) 82 (33) 79(38) 100 (62) y Numberof patients 17 11 17 19 19 analyzedfor culture identiŽcation Numberof cultures 69 50 80 113 83 analyzed Numberof species 6 3 4 5 5 recovered

*,Numbersin parenthesesrepresent the number of lesionalsites sampled that grew Malassezia onthe contact plates; ,numbersin parentheses represent the number of non-lesionalsites sampled that grew Malassezia onthe contact plates. y comparison of cfu counts between various skin condi- the mean cfu counts on the scalp, forehead and trunk, tions, the mean cfu counts in patients with PSwas were signiŽcantly higher than on the armsand legs. signiŽcantly lower than the corresponding value for SD. However, in patients with ADthere was no signiŽcant difference in the mean cfu counts on the scalp, forehead, trunk and legs. In PS, the mean cfu counts on the scalp, cfuCounts fromlesional and non-lesional sites forehead, and trunk were signiŽcantly higher than the combined counts on the armsand legs. In SD, the mean cfu counts For each dermatosis, the total number of cfu was were not signiŽcantly different on the scalp, forehead, recorded at Žve different anatomic sites, namely the trunk and arms.The counts on the legs were signiŽcantly scalp, forehead, arm, trunk and leg (Table 2). In general, lower than those on the scalp, forehead, armsand trunk.

Table 2 Distributionof cfu bybody site and dermatosis

Distributionof cfu bybody site Average (lesionaland non-lesional sites combined) Number age Total Dermatosis ofpatients (years) Sex (M/ F)Scalp ForeheadArm Trunk Leg cfu

AD 31 41 3 17/14 ¢ Total cfu* 265(1) 367 (2) 21 49 134 (1) 836 Mean cfu 8 5 11 8 0 7 1 6 4 3 27 0 ¢ ¢ ¢ ¢ ¢ ¢ y PS 28 48 9 12/16 ¢ Total cfu 120 106 9 155 2 392 Mean cfu 4 3 3 8 0 3 5 5 0 07 14 ¢ ¢ ¢ ¢ ¢ SD 28 57 8 23/5 ¢ Total cfu 328 (1) 130 160 663 (3) 16 1285 Mean cfu 11 7 4 6 5 7 23 7 0 6 45 9 ¢ ¢ ¢ ¢ ¢ ¢ PV 23 32 5 9/14 ¢ Total cfu 449(2) 952 (2) 129 576 (2) 85 2191 Mean cfu 19 5 41 4 5 6 25 3 7 95 3 ¢ ¢ ¢ ¢ ¢ Healthyindividuals 20 38 3 6/14 ¢ Total cfu 794(2) 388 (1) 77 88 9 1356 Mean cfu 39 7 19 4 3 9 4 4 0 5 67 8 ¢ ¢ ¢ ¢ ¢ ¢

*,Thenumbers in parenthesesrepresent the number of patientsfor whom >100 cfu wererecovered from the sampled body site(s); ,thesum of meanswithin apatientcategory corresponds to themean number of coloniesisolated per patient at allŽ vebodysites combined. y ã 2001 ISHAM, Medical Mycology , 39, 243±251 Malassezia speciesin patientswith cutaneousdermatoses 247

In PV,the cfu counts were not signiŽcantly different on Association of Malassezia specieswith different the scalp, forehead and trunk, and were signiŽcantly dermatoses higher than those obtained fromsites on the armsand All six known lipophilic Malassezia species were the legs. In normal individuals, the cfu counts were recovered (Table 4). In many individuals, more than signiŽcantly higher on the scalp, forehead, armsand one Malassezia species was recovered froma given trunk, than on the legs ( P < 0 05). ¢ anatomic site. The mostcommon species were M.sympodialis and M. globosa.The remaining species, cfuComparisons oflesional versusnon-lesional sites M. sloofŽae, M. furfur, M.restricta and M. obtusa, were

Mean values for the cfu counts fromlesional and non- less frequently isolated. Downloaded from https://academic.oup.com/mmy/article/39/3/243/946776 by guest on 27 September 2021 lesional sites in all Žve patient categories are shown in The distributions of M.sympodialis and M. globosa Table 3. For all dermatoses considered together, the cfu were signiŽcantly different among body sites in ADand counts in lesional sites were signiŽcantly lower than the PVpatients, as well as in healthy individuals (Table 4). corresponding factor for non-lesional sites (chi-squared In atopic dermatitis, M.sympodialis was recovered test, 4 1; df, 1; P <0 05). This trend appeared to derive ¢ ¢ signiŽcantly more frequently on the forehead than on mainly froma strong tendency of SDpatients to have the armor leg. In PVand healthy individuals, higher colony counts on non-lesional than on lesional M.sympodialis was observed signiŽcantly more skin. Asimilar but less pronounced trend was also seen frequently on the forehead and trunk than on the legs. in ADpatients (sample numbers were not high enough For M. globosa,in healthy individuals the forehead was to test whether the latter apparent trend was signiŽcant colonized signiŽcantly more frequently than were the independently). PSand PVpatients actually had slightly armsand legs. For the remaining Malassezia species, the higher mean counts on lesional than on non-lesional number of observed occurrences at the different skin, but the difference was not statistically signiŽcant anatomic sites within patient categories was too small (Table 3). to permit statistics to be performed. In overview, however, combining patient categories, cfuComparisons oflesional sites among different the four less common Malassezia species appeared to fall patient categories into two groups on the basis of their distribution on the The cfu counts fromlesional sites in patients with AD upper body (scalp and forehead) versus lower body (arm, and PSwere signiŽcantly lower than in patients with PV trunk and leg). The numbers obtained suggested that (Table 3). Noother signiŽcant differences were found. additional investigation of these relatively rare organ- ismsmight also reveal signiŽcant trends in body site cfuComparisons ofnon-lesional sites in differentpatient preferences. M. sloofŽae and M.restricta were frequently categories isolated fromscalp and forehead. M. sloofŽae was observed in 10 of 16 records (62 5%) and M.restricta The cfu counts fromnon-lesional sites of patients with ¢ was observed in Žve of six records (83 3%) fromthese ADand PSwere signiŽcantly lower than those of healthy ¢ upper body sites. M. furfur and M. obtusa,on the other control subjects and of the remaining two patient groups hand, were recorded in12 of 17 positive samples (70 6%) (P < 0 05) (Table 3). No signiŽcant difference in cfu ¢ ¢ and Žve of six positive samples (83 3%), respectively, on counts was observed among non-lesional skin samples ¢ fromPV and SDpatients, and normal individuals. the lower body (trunk, arms,legs). There were also some signiŽcant differences in Malassezia species counts among patient categories Table 3 Meancfu countsfor lesional and non-lesionsal sites in when results fromall body sites were amalgamated. variousdisease states M.sympodialis was the mostfrequently recorded species in contact plate samples fromAD patients (found in Meancfu fromall Meancfu fromall 51 3% of samples) and PVpatients (62 7% of samples). Diseasestate lesionalsites non-lesionalsites ¢ ¢ M. globosa,by contrast, was the mostfrequently AD 3 67 7 62 recorded species in patients with PS(57 7% of samples) ¢ ¢ ¢ PS 3 09 2 8 and SD (45 0% of samples). ¢ ¢ SD 4 3 16 05 ¢ ¢ ¢ There was one 17-year-old female PVpatient in whom PV 19 28 18 48 ¢ ¢ four different species were isolated. The 13 isolates Healthypatients N/ A 13 56 ¢ identiŽed fromthis patient included Žve M.sympodialis N/A,Not applicable. isolates fromthe forehead, two M. obtusa fromthe arm, ã 2001 ISHAM, Medical Mycology , 39, 243±251 248 Gupta et al.

Table 4 Distributionof Malassezia speciesby bodysite in variousdermatoses

Dermatosisfrequency of isolation* (frequencyof isolation at lesionalsite )[%frequencywithin disease category ] Overall y z Malassezia Anatomic frequencyand species site AD (n = 31) PS (n = 28) SD (n = 28) PV (n =23)Healthy ( n =20)(% frequency)

M.sympodialis Scalp 5 (2) 2 (2) 2 7 4 20 Forehead7 (5) 3 (2) 3 (3) 8 10 31 Arm 1 (1) 1 (1) 2 5 (1) 6 15 Trunk 6 (6) 1 5 (2) 10 (10) 5 27 Leg 1 (1) 1 (1) 3 2 0 7 Downloaded from https://academic.oup.com/mmy/article/39/3/243/946776 by guest on 27 September 2021 Species 20 (15) [51 3%] 8 (6) [30 8%]15 (5)[37 5%]32 (11)[62 7%]25 (N/ A)[47 2%] 100 (47 8%) ¢ ¢ ¢ ¢ ¢ ¢ msubtotal M. globosa Scalp 1 4 (3) 4 (2) 5 5 19 Forehead4 (3) 4 (1) 5 (4) 3 6 22 Arm 0 2 4 (1) 0 0 6 Trunk 2 (2) 4 (4) 5 (4) 1 (1) 4 16 Leg 0 1 0 0 0 1 Species 7 (5) [17 9%]15 (8) [57 7%]18 (11)[45 0%] 9 (1) [17 6%]15 (N/ A)[28 3%] 64 (30 6%) ¢ ¢ ¢ ¢ ¢ ¢ msubtotal M. furfur Scalp 1 (1) 0 1 0 1 3 Forehead1 (1) 0 0 0 1 2 Arm 0 0 0 1 1 2 Trunk 1 (1) 0 2 2 (2) 1 6 Leg 1 (1) 0 0 1 2 4 Species 4 (4) [10 3%] 3 (0) [7 5%] 4 (2) [7 8%]6 (N/A)[11 3%] 17 (8 1%) ¢ ¢ ¢ ¢ ¢ msubtotal M. sloofŽae Scalp 1 (1) 2 (1) 2 1 1 7 Forehead 0 0 0 1 2 3 Arm 0 0 0 0 0 0 Trunk 0 1 2 (2) 1 (1) 0 4 Leg 0 0 0 1 1 2 Species 1 (1) [2 6%] 3 (1) [11 5%] 4 (2) [10 0%] 4 (1) [7 8%]4 (N/A)[7 5%] 16 (7 7%) ¢ ¢ ¢ ¢ ¢ ¢ msubtotal M. obtusa Scalp 0 0 0 0 0 0 Forehead 0 0 0 1 0 1 Arm 1 (1) 0 0 1 (1) 0 2 Trunk 1 (1) 0 0 0 0 1 Leg 2 (2) 0 0 0 0 2 Species 4 (4) [10 3%] 0 0 2 (1) [3 9%] 0 (N/A) 6 (2 9%) ¢ ¢ ¢ msubtotal M.restricta Scalp 1 0 0 0 2 3 Forehead2 (1) 0 0 0 0 2 Arm 0 0 0 0 0 0 Trunk 0 0 0 0 0 0 Leg 0 0 0 0 1 1 Species 3 (1) [7 7%] 0 0 0 3 (N/A) [5 7%] 6 (2 9%) ¢ ¢ ¢ msubtotal Totals 39 26 40 51 53 209

*,Speciesfrequency: number of contact platesyielding at least one isolate of theindicated species (including plates from both lesional and non-lesional sites); ,Numberof contact platesfrom lesional body site yielding species; y Percentageof contact platesamples within individualdisease category yielding the indicated species. z N/A,Not applicable. two M. furfur and two M. sloofŽae fromthe trunk and quantitative culturing of Malassezia yeasts fromhuman one M. sloofŽae and one M.sympodialis fromthe leg. skin. The colonies are easy to identify with high and reliable counts. Itis not known if all individual species Discussion grew in proportion to their true population density In this study we have demonstrated that the use of (a theoretical problem with all isolation studies); how- contact plates isa simple and effective method for ever, it is certain that they were all able to grow to a

ã 2001 ISHAM, Medical Mycology , 39, 243±251 Malassezia speciesin patientswith cutaneousdermatoses 249 signiŽcant extent. Although astudy with similar design Leeming–Notman medium [10]. At least one previous was reported by Faergemann [5] fromEurope using a study has also shown that lesional and non-lesional previous taxonomic system now considered insufŽciently skin of PVpatients yielded equivalent numbers of resolved, this is the ŽrstNorth American study of its Malassezia colonies, and that these numbers also kind. Arecent Spanish study of the distribution on skin differed insigniŽcantly fromthose obtained from of Malassezia species [19] identiŽed only three of the six control skin and fromlesional and non-lesional skin recognized lipophilic species and sampled only healthy of SDpatients [30]. volunteers. Another recent Spanish study identiŽed Žve Perhaps surprisingly, our study showed that lesional of the six recognized species fromnormal, SDand PV skin fromSD yielded signiŽcantly lower colony numbers patients sampled by means of ascraping inoculation than non-lesional skin. The existing literature in general Downloaded from https://academic.oup.com/mmy/article/39/3/243/946776 by guest on 27 September 2021 technique [20]. appears highly discrepant on the matter of whether or In the present study, recovery of Malassezia species not SDlesions are associated with excess numbers of fromskin was signiŽcantly lower in patients with the Malassezia cells, perhaps in part because of alack of inammatory dermatoses PSand ADthan in healthy standardization in the techniques used. Heng et al. [31], patients or in patients with the less inammatory using direct microscopy, found that numbers of yeast dermatosis PV.PSpatients in particular had the lowest cells present rose in proportion to the severity of the densities of Malassezia species on both lesional and lesions. At least two culturing studies have supported healthy skin. Non-lesional areas in patients with ADalso this Žnding [5,32], but three independent research groups had adensity of Malassezia species that was signiŽcantly using cultures, including one group that had earlier reduced compared to counts fromhealthy control associated high culture numbers with lesional SD, have subjects. The explanation for this phenomenon is not published contradictory results [12,30,33,34]. Ourstudy, clear. With regard to both lesional and non-lesional skin which used the mostcommonly employed culturing in these patient categories, it may be signiŽcant that technique yielding quantitative results (contact plates patients with certain skin conditions have been shown to with Leeming–Notman agar), is the Žrstto show a excrete skin lipids differing in composition fromthe lipid signiŽcantly lower recovery of cultures fromlesional mixtures characteristic of normal skin [21,22]. Malassezia than non-lesional skin in SD. species may be inuenced by differences among these Ourresults, as well as those of other studies, may lipid compositions. With particular regard to lesional reect artifactual patient factors rather than differences skin, both PSand ADpatients have been shown in sampling techniques. Individuals with dermatoses are repeatedly to respond to Malassezia species antigens more likely than other persons to use home remedies, with strong immunological reactions that are not seen in synthetic detergents or shampoos to control their skin normal control subjects or SDpatients [23–28]. In AD,it condition. Useof these agents might be associated with has been proposed that P.orbiculare (an older name for reduced colony counts. Detergent soaps and shampoos round-celled elements of the lipophilic Malassezia with additives including climbazole, ketoconazole, ciclo- species complex, ergo aname suggestive of the modern pirox olamine, zincpyrithione, selenium sulŽde, or other M. globosa)may play arole in maintaining immuno- sulfur-containing substances are known to be quite globulin (Ig)E-mediated skin inammation [29]. The effective in patients with chronic disease [35], and it is present study suggests that if such aprocess does occur, not known how long such treatments mustbe discon- the inammation may be partially successful in control- tinued before the Malassezia populations fully recover. ling the Malassezia populations. Itis clearly not Itis possible that the various non-speciŽc hygienic impossible that such sensitization may ultimately affect practices and self-treatments used by our SDpopulation Malassezia populations on healthy as well as lesional were relatively effective in killing or removing Malas- skin, especially as particular skin areas may change in sezia cells fromlesional skin. disease status over the course of time. The results of our study are mostcomparable to that There was no signiŽcant difference in this study in of arecent study by Nakabayashi et al.[36] who used the the density of Malassezia species between patients with tape and swab method to isolate and identify Malassezia PVand normal individuals. Norwas there asigniŽcant species fromJapanese patients with SD,AD, PVand difference in the number of culturable colonies on normal subjects. Although lower numbers of positive lesional and non-lesional skin in PVpatients; cfu cultures were recovered fromlesional skin than non- counts were relatively high fromboth skin categories. lesional skin of patients with AD,no signiŽcant In aprevious study [17], we found that 56 2% of PV differences in the isolation frequency of lesional versus ¢ lesions sampled by conventional diagnostic scraping non-lesional skin of patients with SDwas found. More- failed to yield aculture even on the highly efŽcacious over, M. globosa was the mostpredominant species to be ã 2001 ISHAM, Medical Mycology , 39, 243±251 250 Gupta et al. isolated fromevery disease state as well as fromthe M.sympodialis was equally likely tobe recovered from normal subjects. In our study we have found M. globosa the scalp and forehead or the trunk and upper and lower to be frequently isolated frompatients with PSand SD. extremities, whereas Aspiroz et al. [19] found a M.sympodialis has by far been the mostpredominant signiŽcant association with the back, in contrast to the species to be associated with PV,ADas well as fromthe chest and scalp (forehead and leg were not studied). In normal subjects in our studies. The differences in the our study, M. furfur and M. obtusa,although not results of our study and that of Nakabayashi et al. [36] commonenough to yield deŽnitive results, appeared may also reect the differences in the patient population more likely to be associated with the lower body; again, that was sampled, i.e., North American versus Japanese. these species were not recorded by Aspiroz et al. [19].

This possibility should be explored further. SpeciŽcally in lesional sites, Crespo Erchiga et al. [20] Downloaded from https://academic.oup.com/mmy/article/39/3/243/946776 by guest on 27 September 2021 In the present study, differences were seen in species found M. globosa in more than twice the number of PV assemblages detected at the different anatomic sites in lesions as M.sympodialis , while M.restricta and healthy and diseased individuals. This Žnding has been M. globosa appeared to be strongly associated with SD observed previously [19,20,24,25]. However, our study is lesions. Ourcontact plate data on PVlesions, consistent the Žrstto characterize and report the frequency of each with our previous large, independent study on routine of the species of Malassezia in aNorth American clinical PVscrapings [17], showed that Ontario patients population. The report by Faergemann et al. [37] grew more M.sympodialis (11 samples) than M. globosa discusses the outmoded taxon P.orbiculare only and (one positive sample) fromlesions of this disease. In Bandhaya [38] limited the discussion to two species, contrast to Crespo Erchiga et al. [20], we only obtained M. furfur and M.pachydermatis ,the Žrstof which, as M.globosa, M.sympodialis and M. sloofŽae, in conceived at the time, is now known to be amulti-species descending order of frequency, fromlesional SD; complex. Aspiroz et al. [19], as mentioned above, M.restricta was mainly obtained fromhealthy control identiŽed only three of the six currently recognized subjects and ADpatients. Although this species is slow species. Only the recent study of Crespo Erchiga et al. growing and may be overgrown by other species in [20], conducted in Spain, has attempted to identify all densely crowded isolation plates, this phenomenon was recognized species; the data in that study, however, were anticipated in our study, and we did not observe the not subjected to statistical analyses. In the present study, dense overcrowding on contact plates that might cause a we observed that the Malassezia counts were generally sampling artefact of this nature. higher on the scalp, forehead and trunk than on the arms The signiŽcance of the trends seen here and elsewhere and legs of healthy controls as well as of subjects in each in Malassezia colonization are not clear at present, but of the patient categories. Patients with ADand PV,as may become clearer as future studies clarify the degree well as healthy control subjects, grew M.sympodialis in of causality of the various species in PV,and possibly approximately half or more of all samples but grew also in SDand other diseases. What is of interest here is M. globosa fromless than one-third of samples. By the clear preliminary conŽrmation that the Malassezia contrast, PSand SDpatients grew M.sympodialis in only species seen are by no means interchangeable in their about one-third of samples, but grew M. globosa in ecological relationships: they have varying degrees of approximately half of all samples. The Malassezia niche separation. Itispossible that the lipid composition species more likely to grow fromthe scalp and forehead in the stratum corneum plays arole in the growth of rather than the lower body were M.restricta and particular species of Malassezia in certain disease states M.sloofŽae. Aspiroz et al. [19], studying only healthy and at particular anatomic sites [39,40]. In addition, there individuals, also found M.restricta particularly isa very likely possibility that, as in other fungal associated with the scalp; they did not identify dermatoses, there may be signiŽcant geographic differ- M. sloofŽae.In another correspondence with our study, ences worldwide in species prevalences, both in etio- Aspiroz et al. [19] found M. globosa evenly distributed in logical and commensal Malassezia colonizations. all sites sampled. We also found that, even when the Even when results are discrepant fromstudy to study, more ecologically sensitive frequency data (number of however, the enhanced resolution seen when all isolates contact plates positive for species) were tested in are identiŽed tospecies often makes older studies based contrast to the density data (cfu)used by Aspiroz et al. on previous taxonomies difŽcult to integrate into current [19], our study populations as agroup showed no concepts. In general, the identiŽcation of individual signiŽcant difference in the association of M. globosa Malassezia species should greatly increase the accuracy with scalp, forehead and trunk. Only the armand leg, not and predictiveness of future studies attempting investigated by Aspiroz et al. [19], showed signiŽcantly to correlate Malassezia distributions with chemical, lower prevalences of this species. Ourstudy found that physical, immunological and biogeographic host factors.

ã 2001 ISHAM, Medical Mycology , 39, 243±251 Malassezia speciesin patientswith cutaneousdermatoses 251

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