Medical Mycology February 2004, 42, 35 /42

Prevalence of Malassezia species on various body sites in clinically healthy subjects representing different age groups
ADITYA K. GUPTA*,$ & YATIKA KOHLI$,% *Division of Dermatology, Department of Medicine, Sunnybrook and Womens College Health Science Center (Sunnybrook site) and the University of Toronto, Toronto, $Mediprobe Laboratories, London, Ontario and %The Hospital for Sick Children, Toronto, Ontario, Canada

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To investigate the distribution of Malassezia species on four body sites (scalp, forehead, chest and back), we employed contact plates filled with Leeming Notman agar to sample 245 clinically healthy subjects, representing six age groups (AG) (AG I, 0 3 years; AG II, 4 14 years; AG III, 15 25 years; AG IV, 26 40 years; AG V, 41 60 years; AG VI, over 60 years). The number of colony forming units was recorded for every plate positive for Malassezia species, and the species were identified. Younger individuals ( B 14 years) yielded a culture positive for Malassezia significantly less frequently than did older individuals ( ] 15 years). M. globosa was cultured at significantly elevated frequency on younger subjects. M. sympodialis was present at low frequency on younger subjects, but was found in higher amounts on the skin of adolescents and adults. The amount and kind of Malassezia species that can be recovered from human skin varies with age and body site.
/ / / / / / / /

Keywords

age, contact plates, Malassezia , skin, yeasts

Introduction
Yeasts of the genus Malassezia are a normal part of the flora of human skin. In susceptible individuals, however, these yeasts appear to play a causative role in various superficial dermatoses, including pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, and some variants of psoriasis and atopic dermatitis [1]. The yeasts have been found on upwards of 90% of normal adults and are also commonly isolated from lesional skin [2]. While Malassezia species tend to colonize lipid-rich body sites such as the chest, back, face and scalp [3], the relationship between colonization patterns and age group is less clear. A number of studies indicate that there is a difference in the colonization pattern of Malassezia species in different

Received 8 November 2002; Accepted 2 July 2003 Correspondence: Dr Aditya K. Gupta, Suite 6, 490 Wonderland Road South, London, Ontario, Canada, N6K 1L6. Tel.: 519 657 4222; E-mail: agupta@execulink.com

age groups, with an increase in colonization appearing to occur at puberty when quantities of skin lipids increase [4,5]. In addition, when colonization does occur in children, it appears to more likely to happen on the head than on the trunk [6]. With the recent reclassification of the genus Malassezia , there has been a resurgence of interest in these yeasts and in the possibility of variation in species distribution in different body sites and dermatoses. This study investigates whether there is a difference in the amount and kinds of Malassezia species in different age groups. Previous studies investigating the relationship between age and colonization with Malassezia species were based on an older classification in which the yeasts were classified as the genus Pityrosporum [4,7]. In the previous nomenclature, the two lipophilic variants were known as P. ovale and P. orbiculare ; however, there was controversy over whether these variants represented different species, or simply different variants of a single species. The relative prevalence of these yeasts in human hosts has been found to vary with age, body site and geographical location [2,4,8].
DOI: 10.1080/13693780310001610056

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Age has also been implicated as a factor in the amount of Malassezia isolated from the skin. In general, colony counts are higher in adults than in children, though the amount of yeast obtained decreases again in elderly individuals [9]. The yeasts are less frequently isolated from infants and children, though Bergbrant and Broberg found P. ovale to be quite common in children [7]. The taxonomy of Malassezia is now quite different than that used in these studies. Despite the controversy over the relationship between P. orbiculare and P. ovale , most investigators believed that the two forms represented a single species [10]. However, there was also controversy over whether the genus should be named Pityrosporum or Malassezia . Some investigators even used both designations, describing the yeasts cultivated from healthy skin as P. orbiculare or P. ovale and reserving the name Malassezia furfur to indicate the pathogenic mycelial form of the organism cultured from lesions of pityriasis versicolor. The currently accepted generic name is Malassezia [11]. In 1996, the genus Malassezia was reclassified and seven species were delineated [12,13]. M. pachydermatis remains the sole recognized species that does not require exogenous lipids in order to grow. The remaining six lipophilic species are M. furfur, M. sympodialis , M. restricta , M. slooffiae , M. obtusa and M. globosa . All of these species have been isolated from human skin, though with varying frequencies. Since this reclassification, several studies have begun to determine the differences in prevalence of the six lipophilic Malassezia species. Some progress has been made in discovering the geographical variation in species distribution; in addition, there are indications that the dominant species may vary at different body sites and in different skin conditions [3,14]. To our knowledge, however, this is the first study to investigate differences in the type and amount of Malassezia species in different age groups. Because the amount and composition of skin surface lipids varies with age [15,16]. we hypothesized that alterations in the habitat provided by human skin will affect the amount and species of Malassezia yeasts present on the skin.

Sample and sampling procedure

We sampled 245 individuals from six different age groups (AG). These six age groups were: AG I, 0 /3 years (n 0/33); AG II, 4 /14 years (n 0/55); AG III, 15 / 25 years (n 0/43); AG IV, 26 /40 years (n 0/40); AG V, 41 /60 years (n 0/44); AG VI, over 60 years (n 0/30). All of the individuals sampled were clinically healthy individuals (i.e. with no Malassezia -related skin conditions) and informed consent (on the protocol approved by an independent Review Board) was obtained from the volunteers or their guardians. All of the individuals were being seen for conditions other than a dermatosis (i.e. individuals with psoriasis, atopic dermatitis, seborrheic dermatitis, etc., were excluded). For each individual, four body sites (scalp, forehead, chest and back) were sampled by pressing the contact plates filled with Leeming /Notman agar on the skin for 20 s. The flattest available surface of the body site was sampled in order to maximize the recovery of yeast in each sample. The sampling procedure, incubation of contact plates, counting the colony forming units (c.f.u.), and identification of Malassezia species among selected colonies was performed as described previously [3]. Contact plates were incubated at 358C for 7 days. Prior to starting the experiment we had done a pilot study to determine the appropriate incubation period. Our pilot data indicated that all six lipophilic species could be grown adequately and form colonies in this time frame. Moreover, with longer incubation times (i.e. 10 /14 days) the medium in Rodac plates ( /9 /9.5 ml medium) usually dries out at 358C. Up to five isolated single colonies with varied macromorphology were picked from the contact plates and subcultured on Leeming /Notman agar slants before processing them for species identification. Malassezia species were identified on the basis of their micromorphology (shape and size of cells), physiological features (catalase reaction and ability to grow on Tween 20, 40, 60 and 80, as described by Guillot et al. [17]), and molecular characteristics (based on PCR-restriction enzyme analysis [REA] of the nuclear ribosomal internal transcribed spacer [ITS] and large subunit [LSU] regions as described previously [18]).

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Statistics

Materials and methods

Preparation of contact plates
BBL RODAC (Beckton Dickinson, MD, USA) contact plates (4.5 cm diameter) filled with Leeming / Notman agar were used for sampling, as described previously [3].

Statistical tests were performed as follows: (i) Samples from individuals in different age groups and from different body sites that yielded a positive culture for Malassezia species were compared using a chi-squared test, with standardized residuals computed for each cell. Phi-coefficients were computed to estimate the strength of relationship between variables. (ii) A uni 2004 ISHAM, Medical Mycology, 42, 35 /42

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variate analysis of variance was conducted to compare the mean c.f.u. and adjusted c.f.u. from each age group. (iii) A univariate analysis of variance was conducted to evaluate the effect of age and sex on total c.f.u., followed by multivariate analysis to evaluate the effects of age and sex on the c.f.u. counts from each of the four body sites. (iv) Chi-square tests, with standardized residuals for each cell, were calculated to analyze the differential likelihood of Malassezia species, if any, to be recovered across different age groups and from different body regions. Phi coefficients were computed to estimate the strength of relationship between variables. Tests mentioned in points (i) and (ii) were performed using density data (i.e. data recorded as the number of Malassezia c.f.u. obtained within the sample categories (Tables 1 and 2). Statistical tests based on the specificity of individual Malassezia species as mentioned in point (iii) were carried out using frequency data (data based on the number of contact plates yielding at least one isolate of the species in question; Tables 3 and 4).

individuals sampled in each age category. This was further adjusted (adjusted c.f.u.) by limiting the maximum counts from each contact plate to 100. Overall, the anova indicated a significant difference among the age groups for both the mean c.f.u. (F [5,238] 0/7.714; P B/0.0001) and the adjusted c.f.u. (F [5,239] 0/9.997; P B/0.0001). The mean c.f.u. in AG-1 and AG-II was significantly lower than in AG-III and AG-VI. Similarly, the adjusted c.f.u. in AG-I and AG-II was significantly lower than in AG-III, IV, V and VI. (Table 1).

Colony forming units from different body sites and variation among age groups
Table 2 summarizes the data for mean c.f.u. from each age group and different body sites. We conducted a multivariate anova, using c.f.u. scores (both adjusted and unadjusted) from scalp, forehead, chest and back as dependent variables. A significant difference was found between age groups when all body parts were considered as an optimally weighted composite for both adjusted c.f.u. (F [20,952] 0/2.486; P B/0.0001) and unadjusted c.f.u. (F [20,952] 0/2.522; P B/0.0001). Based on a pairwise comparison of means from different age groups, the mean c.f.u. on the scalp, back, and chest of individuals in AG-III and VI were significantly higher than the counts for individuals in AG-I and II (Table 2).

Results
Recovery of Malassezia species from individuals of different age groups
Overall, a positive culture was obtained from 70% of individuals sampled in all age groups from one or more body sites. Occurrence of a positive culture was highest (93.3%) in AG VI (over 60 years) and lowest (36.3%) in AG-I and II (0 /14 years) (Table 1). Based on a calculation of standardized residual scores, significantly fewer than expected AG-I and AG-II individuals yielded a positive culture.

Interaction between age and sex

There was no significant difference in the number of men and women sampled for each of the age groups (Table 1; x2 [5] 0/3.5; P 0/0.6). While the main effect of sex was not significant, the interaction between age and sex was significant with women in AG-V and VI having significantly lower mean c.f.u. than men. (F [5,233] 0/ 2.554; P B/0.05). The same trend was noted for the forehead (P 0/0.02) and the chest (P 0/0.006).

Mean and adjusted c.f.u.

The mean c.f.u. for each age group was calculated by dividing the total c.f.u. count by the number of

Table 1 Malassezia colony forming units (c.f.u.) from contact plate samples of individuals from different age groups Age group Age (years) No. individuals sampled (M/F) 33 55 43 40 44 30 (15/18) (27/28) (18/25) (14/26) (17/27) (16/14) No. positive for Malassezia (%) No. negative for Malassezia (%) Total c.f.u. count 228 466 2000 1427 1502 2233 Mean c.f.u. (standard error) 6.9 8.5 47.6 35.7 34.1 74.4 (4.0) (3.3) (8.5) (6.0) (20.7) (113.2) Adjusted c.f.u. (standard error) 6.1 8.2 38.3 33.8 27.3 45.7 (3.3) (3.1) (5.8) (5.2) (5.4) (7.4) Tukeys HSD**

I II III IV V VI

0 /3 4 /14 15 /25 26 /40 41 /60 Over 60

12 20 39 36 37 28

(36.3) (36.3) (90.7) (90) (84.1) (93.3)

21 35 4 4 7 2

(63.6)* (63.6)* (9.3) (10) (15.9) (6.7)

I B/III, IV, V, VI II B/III, IV, V, VI III !/I, II IV !/I, II V !/I, II VI !/I, II

*Represent values of statistical significance where significantly less growth was observed than would be expected due to chance alone. **Tukeys HSD was computed, in the post hoc testing of the mean scores to do a pairwise comparison of adjusted means from different age groups. M/F: Males/Females.

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Table 2 Means and standard errors for c.f.u. by age group and body site Body site Mean adjusted c.f.u. (standard error) AG-I Scalp Forehead Back Chest 0.6 (0.3) 4.4 (3.1) 0.4 (0.2) 1.6 (1.0) AG-II 2.7 (1.4) 2.4 (1.6) 0.9 (1.5) 2.5 (1.7) AG-III 4.2 (1.7) 5.5 (2.1) 17.4 (3.7) 19.6 (5.7) AG-IV 3.1 (1.5) 5.0 (2.0) 15.3 (3.5) 12.4 (3.6) AG-V 3.8 (0.9) 2.7 (1.3) 14.4 (3.7) 13.2 (4.2) AG-VI 11.5 (5.2) 12.2 (7.1) 26.9 (5.5) 23.7 (8.3) anova F (P )* 2.67 (0.001) 1.3 (0.02) 4.004 (0.000) 7.24 (0.000) Tukeys HSD$ I, II B/III,VI I, II B/III,VI I, II B/III, VI I, II B/III, VI

*F -statistics for univariate anova calculations with unadjusted c.f.u. as a variable; P -value in parentheses represent the probability value of statistical significance. $Tukeys honestly significant difference (HSD) was computed in the post hoc testing of the mean scores to do a pairwise comparison of means from different age groups.

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Table 3 Distribution of Malassezia species in individuals from different age groups Characteristics AG-I AG-II 55 20 AG-III 43 39 AG-IV 40 36 AG-V 44 37 AG-VI 30 28 Total 245 172

No. individuals sampled 33 No. individuals that cultured 12 positive for Malassezia species No. colonies examined 22 No. individuals with positive 5 species identification for yeast colonies. No. species identified 3 Malassezia species M. furfur M. globosa M. obtusa M. restricta M. slooffiae M. sympodialis Total

57 13

161 33

143 27

155 29

123 27

661 134

No. colonies (% of total number of colonies for stated age group) [No. individuals from whom species isolated]* 8(36.4%) [1] 3(5.3%) [3] 5(3.1%) [4] 2(1.4%) [2] 11(7.1%) [7] 11(8.9%) [4] 40{6.1}$ 12(54.5%) [5] 34(59.7%) [11] 41(25.5%) [17] 42(29.4%) [3] 52(33.5%) [17] 29(23.6%) [13] 210{31.8%} 0 2(3.5%) [2] 4(2.5%) [3] 1(0.07%) [1] 2(1.3%) [1] 3(2.4%) [2] 12{1.8%} 0 0 2(1.2%) [2] 3(2.1%) [2] 0 0 5{0.7%} 2(9.1%) [1] 0 0 3(2.1%) [3] 3(1.9%) [3] 10(8.1%) [2] 18{2.7%} 0 18(31.6%) [6] 109(67.7%) [28] 92(64.3%) [26] 87(56.1%) [24] 70(56.9%) [19] 376{56.9%} 22 57 161 143 155 123 661

*In some individuals more than one colony was isolated. $Percent of total number of colonies (n 0/661).

Association of Malassezia species with different age groups and body sites
All six known lipophilic Malassezia species were recovered (Table 3). In many individuals, more than one Malassezia species was recovered from any given body site. Different Malassezia species were recovered amongst the different colonies picked from the same plate from a given body site. We did not find any evidence of mixed colonies containing more than one species. The most common species were M. sympodialis (56.9%) and M. globosa (31.8%) followed by M. furfur (6.1%), M. slooffiae (2.7%), M. obtusa (1.8%), and M. restricta (0.7%).

The distributions of M. sympodialis and M. globosa were significantly different among individuals from different age groups (Table 3). In AG-I, M. globosa and M. furfur were the predominant species accounting for 54.5% and 36.4%, respectively, of all the species recovered. M. furfur was recovered significantly more frequently from AG-I members than would be expected by chance (P B/0.0001). M. sympodialis on the other hand, was not cultured from individuals aged 0 /3 years. In AG-II, again M. globosa was the predominant species (59.7%) and occurred more frequently than expected by chance alone (P B/0.0001). In AGs III to VI, M. sympodialis was observed approximately twice as often as M. globosa (Table 3).
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Table 4 Prevalence of Malassezia species on body sites of members of different age groups Malassezia species M. furfur Scalp Forehead Chest Back Total M. globosa Scalp Forehead Chest Back Total M. obtusa Scalp Forehead Chest Back Total M. restricta Scalp Forehead Chest Back Total M. slooffiae Scalp Forehead Chest Back Total M. sympodialis Scalp Forehead Chest Back Total AG-I AG-II AG-III AG-IV AG-V AG-VI Total

/ 3 2 3 8* 4 4 3 1 12 / / / / 0 / / / / 0 / / / 2 2 / / / / 0$

/ 1 / 2 3 16 9 5 4 34* 1 / 1 / 2 / / / / 0 / / / / 0 2 3 7 6 18$

/ / 2 3 5 9 9 13 10 41 2 2 / / 4 / / 1 1 2 / / / / 0 14 13 44 38 109

/ 1 1 1 3$ 6 11 18 7 42 / / 1 / 1 1 1 / / 2 1 / / 2 3 4 6 38 44 92

3 1 3 4 11 16 16 7 11 50 / / 2 / 2 / / / / 0 / 1 1 1 3 8 7 28 44 87

3 / 4 3 10 8 3 6 8 25 2 / 1 / 3 / / / / 0 5 / / 4 9* 9 8 15 35 67

6 6 12 16

59* 52* 52 41$

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5 2 5 0$

1 1 1 1

6 1 1 9

37$ 37$ 132 167*

*Based on the standardized residual scores by computing z-test, a significantly greater number of cases than expected were observed. $Based on the standardized residual scores by computing z-test, a significantly lower number of cases than expected were observed.

All correlations of Malassezia species with body site were more prominent after combining the data across age groups (Table 4). For all age groups combined, M. globosa was recovered significantly more often from scalp and forehead and less frequently from the back (P B/0.0001). M. sympodialis , on the other hand, was recovered more frequently from the back and less frequently from the scalp and forehead (P B/0.0001).

Discussion
This is the first study that evaluates the type and amount of Malassezia species in the different age groups using the new taxonomy. The results of our study replicate and expand on the findings of some of
2004 ISHAM, Medical Mycology, 42, 35 /42

the earlier studies [9,14,19 /22]. While our results and those of investigators using the old taxonomy show similarities in the number of yeasts found in different age groups, caution should be used in any attempts to compare the species-level results generated using the two classification systems. Another factor that makes it difficult to generalize among studies is the different sampling procedures used. While we have used contact plates in this study, other studies have used skin scrapings [23] or tape samples [24], the detergent scrub technique [25] and even contact plates (with either Leeming /Notman [26] or a different medium) [27]. Aspiroz [20] used a cotton ball impregnated with 1 ml TritonX-100 and Nakabayashi et al . [14] used several methods: swab, tape and

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hairbrush (for scalp). Recently, Gemmer et al. [28] have described a new molecular technique named terminal fragment length polymorphism (tFLP), which allows the molecular detection and differentiation of Malassezia yeast species from dandruff. Whether this technique will be helpful in isolating and identifying Malassezia yeasts from samples other than dandruff remains to be investigated. The tFLP method, however, is not suitable for quantitative culturing of yeasts. While no studies have been performed comparing the contact plates with the detergent scrub technique, an animal study has shown a high correlation in yields provided by the two methods (P B/0.001) [29]. Contact plates were chosen for sampling because: (i) we [3] and others [22] have used contact plates successfully for quantitative culturing; (ii) the use of a contact plate minimizes the chances of losing a part or whole sample in the transit from the clinic to the laboratory; and (iii) in our experience, this sampling method was found to be best tolerated by children. In the various age groups that we have studied, there is a difference in both the amount as well as the kind of Malassezia species found on the skin. The two youngest age groups, representing individuals from 0 to 3 and 4 to 14 years of age, had significantly lower mean c.f.u. (6.12 and 8.20, respectively; mean adjusted c.f.u.) than groups III through VI. Our study shows some striking similarities and differences when contrasted with a study by Bergbrant and Broberg [7], who also used contact plates (containing peptone, ox bile, bacto agar, glucose and yeast extract supplemented with glycerol, glycerol monostearate, Tween 60 and cows milk) to investigate the prevalence of P. ovale in children between 2 months and 15 years of age. The colony counts in our two youngest age groups were similar to those of Bergbrant and Broberg [7]. They reported a mean c.f.u. of 10 for children between the ages of 2 and 7 years; in our study the mean c.f.u. were 6.9 and 8.5 for AG I and II, respectively. Our results however, differ from those of Bergbrant and Broberg [7] in the number of individuals who sampled positive for Malassezia in younger age groups (0 /14 years). In our study, while a positive culture was obtained from 70% of individuals sampled over all age groups, only 36.3% of individuals of younger ages (0 /14 years) were positive for Malassezia , which is in contrast with the 87% positive recovery rate observed by Bergbrant and Broberg [7]. Several other studies have reported varied rates of positive recovery from young children. Faergemann and Fredrickson [4] found no Malassezia (P. orbiculare/P.ovale ) yeasts on children younger than 5 years of age. Results of our study are closest to those of Silva et al . [19], who found Malassezia yeasts in 23.3% of

children between the ages of 0 and 18 months and 26.7% of children between the ages of 11 and 15 years. Because of the different ways in which subjects were grouped according to age, comparison between these studies is difficult. With the exception of the study by Bergbrant and Broberg [7], in which the amount of Malassezia species recovered from the skin of children is quite high, other studies, including ours, show that amount of Malassezia inoculum recoverable from children is quite low. This may reflect the fact that compared to other age groups, children have very little sebum on their skin [16]. Both the amount and the composition [16] of skin lipids changes at puberty. With respect to the carriage of Malassezia species in adolescents and adults, our study found no significant difference between the four age groups (AG-III to AGVI). It is thought that the increase in skin lipid levels that occurs at puberty may be responsible for the increase in colonization of the skin by Malassezia species [4]. A previous study [9] reported that there was a decrease in Malassezia species colonization with increasing age, with 30-year-olds having significantly higher mean colony counts than persons aged 40, 50, 60, 70 or 80 years. By contrast, we found the highest mean colony count in our age group VI (93.3%), which contained individuals over 60 years of age, though, again, differences between the four oldest age groups were not significant. It is possible, however, that differences in the techniques used to recover Malassezia species from the skin, i.e., differences between detergent scrub [9] and contact plate (present study) results, may explain some of the observed differences. For the various age groups, there were differences among body sites in the mean numbers of c.f.u. found. The mean c.f.u. for AG-III and AG /IV were significantly higher than those for AG-I and AG-II for the back, chest, scalp and forehead. It is possible that these differences are again due to puberty-related change in skin lipid levels. We also examined the relationship between the six lipophilic Malassezia species, age group and body site. All six species were recovered; however, the prevalence rates were different. Overall, the most common species was M. sympodialis (56.8%); M. globosa was the second most common (31.7%). There were differences in the prevalence of these species among age groups: M. globosa was most common in AG-I and AG-II, while M. sympodialis was most common in the other four groups. The last result confirms an earlier report by our group [3], but differs from the findings of other studies. Aspiroz et al. [20] reported that M. globosa was most frequently observed overall in normal individuals; however, M. sympodialis was more prevalent
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on the back. Crespo Erchiga et al . [21], from Spain, found that M. sympodialis was the most common species isolated from normal skin. In Sweden, Sandstro m et al. [22] also reported that M. sympodialis was frequently recovered from normal volunteers. It is possible that climatic factors may play a role in the type of Malassezia species present in skin, with M. sympodialis more common in temperate climates and M. globosa occurring most frequently in warmer or tropical locations. The body sites sampled were combined into two groups: head (forehead and scalp) and trunk (chest and back). M. globosa was found in significantly higher proportions than expected (according to the statistical null hypothesis) on the head and in significantly lower than expected amounts on the trunk. For M. sympodialis , the reverse was true; this species was recovered less often from the head and more frequently from the trunk than would be expected by chance (data not shown). Taken together, our results indicate that M. sympodialis is overall the most common Malassezia species found on normal skin, and that it is most frequently recovered from the chest and back. In children, Malassezia colonization is more frequent on the scalp and forehead than on the trunk, and is carried out primarily by M. globosa . However, colonization by Malassezia in children up to the age of approximately 14 years is much less common than in adults. Our results indicate that the Malassezia species are not interchangeable in their ecological relationships; they have varying degrees of niche separation. Differences in the amount and composition of skin lipids at different ages and different body sites may account for part of the variability we found. In addition, hygienic practices change with age and with body site, possibly affecting the habitat of the yeasts. Following the completion of the study two other Malassezia species have been reported, one from humans and the other from the skin of horses [30,31]. As our study was carried out before these characterizations were published, we did not examine for the presence of these yeasts.

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