Analysis of Malassezia microbiota in healthy superficial human skin and in psoriatic lesions by multiplex real-time PCR

Abstract

Yeasts from the genus Malassezia are members of the normal biota of human skin, and may play a role in dermatopathology. Our previous study of the fungal microbiota from healthy subjects and from patients with psoriasis using clone library analysis revealed the presence of five Malassezia species and four uncharacterized phylotypes. We now compared the Malassezia microbiota from six healthy body locations and two psoriatic lesions, and evaluated its stability over time using multiplex real-time PCR. Samples from each body location were obtained monthly, for 4 months. Dual-labeled probes were designed to recognize four Malassezia sp. and two uncharacterized groups, and a genus-specific probe was also developed. A good correspondence was obtained between real-time PCR data and clone library analyses. Malassezia restricta was the most abundant species in the majority of samples, and high amounts of Malassezia globosa were also detected. The uncharacterized phylotype 1 was usually detected in lower proportions, nevertheless it was present in most samples. The microbiota was host-specific and relatively stable over time. In accordance with our previous observations, no significant dichotomy between samples from healthy skin and from psoriatic lesions was found; the samples clustered according to the subject, rather than health status.

Malassezia, psoriasis, real-time PCR, skin

Introduction

Yeasts from the genus Malassezia are receiving increasing attention, because some species have been considered as emerging pathogens (Jahagirdar & Morrison, 2002; Batra et al., 2005). Malassezia species are members of the normal cutaneous microbiota from humans and other warm-blooded animals, and also have been associated with skin diseases such as pityriasis versicolor, seborrheic dermatitis, dandruff, atopic dermatitis, folliculitis, and psoriasis (Gueho et al., 1998; Crespo Erchiga & Delgado Florencio, 2002; Baroni et al., 2004; Gupta et al., 2004a; Batra et al., 2005), as well as with systemic infections in immunocompromised patients (Ashbee & Evans, 2002; Devlin, 2006).

The genus Malassezia currently includes 13 species, with six described in the past 5 years (Simmons & Gueho, 1990; Gueho et al., 1996; Sugita et al., 2002, 2003b, 2004b; Hirai et al., 2004; Cabañes et al., 2007). All species are lipid-dependent; except for Malassezia pachydermatis (Gueho et al., 1996). The distribution of Malassezia species in healthy body locations and in skin diseases has been investigated using culture-based methods (Aspiroz et al., 1999; Nakabayashi et al., 2000; Gupta et al., 2001; Prohic, 2003; Rendic et al., 2003; Gupta & Kohli, 2004; Sandstrom Falk et al., 2005; Lee et al., 2006); however, difficulties in cultivating Malassezia organisms may limit the analyses and bias the observations (Batra et al., 2005). Thus, molecular approaches, particularly analyses of ribosomal genes and internal transcribed regions, have been used for detection, identification, and characterization of Malassezia species, and efforts have been made for the development of new systems and for the improvement and optimization of methodologies (Sugita et al., 2001, 2003a, 2004a; Gaitanis et al., 2002, 2006a; Gemmer et al., 2002; Gupta et al., 2004b; Morishita et al., 2006). The complete genome of Malassezia globosa and partial genome of Malassezia restricta described recently (Xu et al., 2007) reflect the increasing interest in this genus, and open new perspectives on the study of Malassezia species and for better understanding of their role in the development of skin diseases.

Previously, using clone library analyses, we studied the bacterial microbiota from flexor forearms from healthy subjects (Gao et al., 2007), as well as the fungal microbiota in 25 skin samples from healthy subjects and from patients with psoriasis (Paulino et al., 2006). Broad-range 18S rRNA gene primers revealed that most of the organisms belonged to the genus Malassezia. Subsequently, using 5.8S rRNA gene/internal transcribed spacer 2 (ITS2) Malassezia-specific primers, five Malassezia species and four unknown phylotypes were identified (Paulino et al., 2006). Because this methodology is laborious, and does not allow accurate quantitation of the organisms, we aimed to develop an alternative system to detect and quantify Malassezia sp. The present study compared the Malassezia microbiota from six healthy body locations and two psoriatic lesions, and evaluated its stability over time using multiplex real-time PCR.

Materials and methods

Subjects and sample collection

Two subjects, a female (age 33) with normal skin and a male (age 58) with mild psoriasis localized on extensor surfaces were enrolled in this study. The patient had never received therapy for psoriasis. Both subjects provided written informed consent, approved by the NYU Institutional Review Board (Paulino et al., 2006; Gao et al., 2007). Healthy skin samples were obtained from left and right forearms, forehead, scalp, upper back and lower back from both subjects. Elbow and finger lesions also were analyzed from the patient with psoriasis. Samples were obtained from each body location, each month, for 4 months. The skin was rubbed using sterile cotton swabs, as described (Paulino et al., 2006). To detect possible contamination, negative controls were prepared using cotton swabs without any contact with skin, and then subjected to the same procedures.

PCR amplification

Total genomic DNA was extracted from the skin swabs, as described (Paulino et al., 2006), and an c. 300-bp fragment containing 5.8S rRNA gene and ITS2 was amplified using Malassezia-specific PCR primers (Table 1). PCR was performed using 3.5 mM MgCl₂, 0.4 μg μL⁻¹ bovine serum albumin, 0.25 mM of each deoxynucleoside triphosphate, 20 pmol of each primer, 2.5 U Taq DNA polymerase (Qiagen, Valencia, CA), and 5 μL of extracted DNA in a final 50 μL volume. PCR conditions were 5 min at 94 °C, 40 cycles of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C, followed by 10 min at 72 °C. PCR products were analyzed by electrophoresis on 1% (w/v) Tris–acetate–EDTA (TAE)-agarose gels containing ethidium bromide, and visualized under UV. After excision from TAE-agarose gels, PCR products were purified using the QIAquick gel extraction kit (Qiagen), following the manufacturer's instructions.

1

Primers and probes used in this study

Designation	Primer/probe	Target	Fluorescent reporter/ quencher designation	Multiplex group	Sequence (5′→3′)
Mal1F	Primer	Genus Malassezia	–	–	TCTTTGAACGCACCTTGC
Mal1R	Primer	Genus Malassezia	–	–	AHAGCAAATGACGTATCATG
Mal	Probe	Genus Malassezia	5′ 6FAM/3′ MGBNFQ	1	ATGCCTGTTTGWGTGC
glob	Probe	M. globosa	5′ VIC/3′ MGBNFQ	2	ATAACTGCTTTTCTTCTCT
restr	Probe	M. restricta	5′ VIC/3′ MGBNFQ	3	CGCCTCCTCCCAAAC
symp	Probe	M. sympodialis	5′ CAL Fluor Red 610/3′ BHQ-2	3	TGCATAAGCGCCAGGA
pachyd	Probe	M. pachydermatis	5′ 6FAM/3′ MGBNFQ	3	CCTCGCCTGACTGTTT
phy1(1)	Probe	Phy1 (subgroup 1)	5′ 6FAM/3′ MGBNFQ	4	TGCCAAACCTTAGCCC
phy1(2)	Probe	Phy1 (subgroup 2)	5′ VIC/3′ MGBNFQ	4	CCTGTGCTTACCTTTT

*

6FAM and VIC, Applied Biosystems (Foster City, CA); CAL Fluor Red 610, Biosearch Technologies Inc. (Novato, CA).

†

Probes indicated as being in the same multiplex group were analyzed in the same multiplex reaction. Probes included in groups 1 and 2 were analyzed in single reactions.

‡

H, T/C/A; W, A/T.

Quantitative PCR (qPCR)

TaqMan-based real-time qPCR was used to detect and to quantify the particular Malassezia species that had been previously identified in skin samples by clone library analyses (Paulino et al., 2006). Dual-labeled probes were designed based on an alignment between 5.8S rRNA gene/ITS2 sequences from Malassezia organisms (Paulino et al., 2006) using primers Mal1F/Mal1R (Table 1), as well as sequences available in public databases. The alignment was performed using clustalw v. 1.83 (http://sbcr.bii.a-star.edu.sg/clustalw/), including at least two sequences from each species. Species-specific probes were selected to detect four Malassezia species (M. restricta, M. globosa, Malassezia sympodialis, and M. pachydermatis) and two uncharacterized organisms (designated phylotype 1, subgroups 1 and 2), as well as a Malassezia genus-specific probe. Three fluorescent reporter dyes with minimum spectral overlap were used, allowing the simultaneous detection of the probes in multiplex reactions. qPCR assays were performed using the probes in combination with Malassezia-specific primers (Table 1). One microliter of PCR products or extracted DNA was used as template. The PCR reactions were performed as described above, except that 0.2 μM of each probe was added, in a final 25 μL volume. PCR conditions were 5 min at 94 °C, 50 cycles of 30 s at 94 °C, 30 s at 50 °C [probes phy1(1), and phy1(2)] or 60 °C (probes mal, restr, glob, symp, and pachyd) and 30 s at 72 °C, followed by 10 min at 72 °C. The assays were done using a Rotor-Gene 6000 system (Corbett Life Science, Sydney, Australia). Standard curves were built using serial dilutions of cloned PCR products corresponding to each tested species, or a mix including equal amounts of cloned PCR products from all species, in the case of the genus-specific probe. Each sample was tested at least four times in two independent experiments, and the results were analyzed using software rotor-gene 3000 v. 6.0.22.

Sensitivity and specificity of the qPCR assays

To determine the sensitivity of the assays, serial dilutions of cloned PCR products from each species were used, ranging from 10⁹ to 10¹ amplicon copies per reaction. The assay specificity was verified using 10⁹ copies of cloned PCR products from five of the Malassezia species and four phylotypes previously identified in skin samples (Paulino et al., 2006). The level of detection of each species and phylotypes by the genus-specific probe was tested using 10⁸ copies of cloned PCR products per reaction as templates. One nanogram of human genomic DNA also was used to assess the presence of background contamination.

Comparison between qPCR and clone library analyses

The distribution of Malassezia organisms in two skin samples from healthy subjects previously studied by clone library analyses (Paulino et al., 2006) also was analyzed by qPCR. 5.8S rRNA gene/ITS2 PCR products previously obtained using Malassezia-specific primers (Table 1) were used as templates in qPCR assays to detect and quantify six Malassezia species or phylotypes, following the procedures described above.

Principal coordinate analysis and cluster analysis

Principal coordinate analysis (multidimensional scaling) was used to explore similarities in the data. The sample dissimilarity matrix was calculated based on the proportions of the organisms in the samples, and their overall prevalence. To facilitate the visualization of the similarities, the first two orthogonal principal axes were obtained and plotted to show the distribution of samples in a two-dimensional space. Unsupervised hierarchical clustering techniques also were used to evaluate the similarities among samples based on the sample dissimilarity matrix. The results are represented by hierarchical trees, where samples are clustered according to similarities observed in their microbiota in both proportion of species and the overall prevalence of the taxa. To assess the robustness of the clustering, bootstrap resampling was done with 1000 simulations. Based on the dissimilarity measure, the total quadratic entropy can be defined and decomposed into within- and between-samples entropies (Rao, 1982). This allowed the use of a ‘pseudo F’ statistic (the ratio of within-cluster entropy and between-cluster entropy) to examine the possible clustering phenomena, and significance was evaluated by permutation tests (Eckburg et al., 2005). The analyses were performed using r statistical software (http://www.r-project.org/).

Results

Standard curves, sensitivity and specificity of the qPCR assays

One Malassezia genus-specific and six species-specific dual-labeled probes were used in combination with genus-specific primers, to detect and to quantify Malassezia organisms from skin samples. The number of amplicon copies was calculated using 10-fold dilution samples of cloned PCR products from each species as standards, or a mix of equal amounts of cloned PCR products for standardizing the genus-specific probe. Standard curves were constructed by measuring the threshold cycle (C_t) from each standard, defined as the PCR cycle in which the fluorescence exceeds the determined baseline. At least four values were considered for building the curves. The correlation coefficient (R²) obtained from the standard curves ranged between 0.991 and 0.998, indicating the excellent accuracy of the linear regressions.

The sensitivity of the assays was assessed using 10-fold dilutions of the same templates used for the standard curves, corresponding to 10⁹ through 10¹ amplicon copies per reaction. To distinguish the actual DNA amplification from inherent fluorescence variation, values lower than the water blank or near to the threshold fluorescence baseline were excluded. The genus-specific probe for Malassezia sp. allowed the detection of 10² amplicon copies per reaction, and the limit of detection for the species-specific probes ranged from 10³ to 10⁵ amplicon copies per reaction (supplementary Fig. S1). The mean percentage of PCR efficiency (E) was 70 ± 10%, calculated according to the formula E=10^(−1/m)−1, where m is the curve slope.

To verify the specificity of the species-specific probes, 10⁹ copies per reaction from the same templates were used, and the level of detection of each species and phylotype by the genus-specific probe was tested using 10⁸ copies per reaction. All Malassezia organisms were detected by the genus-specific probe, and the species-specific probes showed high specificity, because no significant cross-reaction was detected (supplementary Fig. S1). The M. restricta and M. sympodialis probes were able to detect phylotypes that are closely related to the species; however, in most cases the level of detection was more than 100-fold lower, and the fluorescence curves projected different slopes. Although phylotypes 2 and 4 presented significant cross-reactions with M. restricta and M. sympodialis probes, respectively, this may not be a major problem, because prior data involving clone libraries showed that unlike phylotype 1, they are not commonly encountered organisms; each was found in only one subject, corresponding to 0.6% and 0.4% of all analyzed clones (Paulino et al., 2006). No significant amplification signal from human DNA was observed using any probe (supplementary Fig. S1), indicating that the presence of human DNA in the skin samples did not interfere with the detection of Malassezia species.

To determine whether the probes could be used in multiplex reactions, the detection of cloned PCR products was compared in single and in multiplex formats. Similar values were obtained when M. restricta, M. sympodialis, and M. pachydermatis probes were tested in triplex compared with the single formats. In addition, the slope of the fluorescence curves did not show significant variation. Similar results also were obtained for phylotype 1 (subgroup 1), and phylotype 1 (subgroup 2) probes in single and triplex format (data not shown). In contrast, use of the genus-specific probe did not yield good results in multiplex reactions, possibly because more than one probe was competing for the same DNA template; therefore, this probe was analyzed in single reactions only. The single reaction format also was used for the M. globosa probe because use of only three different reporter dyes does not allow multiplexing more than three probes.

Templates for the qPCR assays

Although the technique is sensitive (supplementary Fig. S1), detection of most of the Malassezia organisms using genomic DNA obtained from skin samples as template for the qPCR assays was limited by the low levels of Malassezia DNA in the samples. Consequently, PCR products amplified using the same pair of primers, were used as an alternative template. To determine whether preamplified templates would modify the proportions between species in the samples, qPCR results obtained using both PCR products and genomic DNA as templates were compared. The Malassezia sp. and M. restricta probes are expected to detect high amounts of DNA in the samples, because our preliminary experiments have shown that M. restricta is abundant in many samples, and the Malassezia sp. probe detects the total amount of Malassezia DNA present in the samples. Therefore, these two probes were selected for the test, because their use will make possible detecting and quantifying genomic DNA used as template.

Malassezia organisms were quantified using both probes in 56 skin samples from the two subjects, and the results generated by both types of templates were compared (Fig. 1). For both probes, a high correspondence was found between results obtained using PCR products and genomic DNA as templates, indicating no strong bias due to PCR amplification. The correlation values calculated for Malassezia sp. and M. restricta, respectively, were 0.38 (P=0.006) and 0.66 (P<0.0001).

1

Number of amplicon copies representing the genus Malassezia and M. restricta detected in skin samples from a healthy subject (a) and from a subject with psoriasis (b), obtained at 1-month intervals. Six healthy skin sites were sampled from both subjects, and for the psoriasis patient, two lesions also were analyzed. PCR products (solid symbols), and genomic DNA (empty symbols) were used as templates for the qPCR assays.

Comparison between qPCR and clone library analyses

The distribution of Malassezia organisms in two skin samples from healthy subjects previously studied by clone library analyses (Paulino et al., 2006) was measured using qPCR, and the results from both techniques were compared (Table 2). Overall, there was good correspondence between results obtained using qPCR and clone library analyses; both methods revealed the presence of the same species, and the difference between proportion of species calculated by qPCR and by clone library analyses was lower than 20% in most cases. Higher differences were observed in the case of phylotype 1, in both subjects tested.

2

Distribution of Malassezia organisms in left forearm skin samples from two healthy subjects obtained using qPCR (this study), in comparison to previous findings using clone library analyses Paulino et al., (2006)

Species/sample	3NL	5NL
% Species	% Species
qPCR	Clone analysis (n=74)	Difference	95% Confidence interval	qPCR	Clone analysis (n=65)	Difference	95% Confidence interval
M. restricta	61.7 ± 4.4	51.4 ± 5.8	10.3	(−4.0, 24.7)	8.0 ± 6.3	13.8 ± 4.3	−5.8	(−20.7, 9.1)
M. globosa	37.6 ± 4.4	14.9 ± 4.1	22.7	(10.9, 34.5)	14.8 ± 4.9	6.2 ± 3.0	8.6	(−2.7, 19.9)
M. sympodialis	0.6 ± 0.2	4.1 ± 2.3	−3.5	(−8.1, 1.0)	6.9 ± 2.0	15.4 ± 4.5	−8.5	(−18.1, 1.1)
M. pachydermatis	ND	ND	0	0	19.3 ± 5.9	40.0 ± 6.1	−20.7	(−37.3, −4.1)
Phylotype 1	0.1 ± 0.03	28.5 ± 5.2	−28.4	(−38.7, −18.1)	50.9 ± 9.2	24.6 ± 5.3	26.3	(5.4, 47.2)

*

The total is lower than 100% because 1.3% of the clones correspond to phylotype 3, not analyzed by qPCR.

†

Number of analyzed clones.

‡

Mean ± SD.

§

Not detected.

¶

Includes both subgroups 1 and 2.

Malassezia microbiota

Malassezia organisms were detected and quantified from six body locations with normal skin from a healthy subject (Fig. 2) and from a patient with psoriasis (Fig. 3a), as well as from two psoriatic lesions (Fig. 3b). Each location was sampled four times, at 1-month intervals.

2

Number of amplicon copies from Malassezia organisms detected by qPCR at six skin locations from a healthy subject. Samples were obtained at 1-month intervals. All data correspond to mean values of at least quadruplicate results obtained in two independent experiments.

3

Number of amplicon copies from Malassezia organisms detected by qPCR at six healthy skin locations (a) and from two lesions (b) from a subject with psoriasis. Samples were obtained at 1-month intervals. All data correspond to mean values of at least quadruplicate results obtained in two independent experiments. The left forearm was not sampled at months 0 and 1.

The total number of amplicon copies from Malassezia sp. was obtained using a Malassezia genus-specific probe. All Malassezia species we previously identified on skin samples in >0.6% of the clones (Paulino et al., 2006) are being assessed in the present study; therefore, the aggregate numbers of Malassezia obtained on the basis of the species-specific probes is expected to be similar to the value obtained by the genus-specific probe. We found that the difference between the value obtained by the genus-specific probe and the total generated by the species-specific probes was less than or equal to threefold for most samples, which is within the range of variation usually observed between experiments.

Malassezia restricta was the most abundant species in the majority of samples, accounting for 57–100% and 7–99% of all Malassezia organisms detected in samples from the healthy subject and the patient with psoriasis, respectively. High levels of M. globosa also were detected, exceeding the levels of M. restricta in many samples from the subject with psoriasis. Among samples from the healthy subject, M. sympodialis was found exclusively on the upper back, and it was detected consistently on all samples from this body location, although in low amounts (10²–10⁵-fold fewer amplicon copies compared with M. restricta). For the patient with psoriasis, in addition to the upper back, relatively high levels of M. sympodialis also were found in the lesions, the lower back, and one sample from scalp and right forearm.

Very low levels of M. pachydermatis were found on four body locations from the patient with psoriasis, including both lesions; and were present exclusively on samples obtained on the last month. Although not abundant, the uncharacterized phylotype 1 (subgroups 1 and 2) was detected in most samples from both subjects.

Controls for contamination

Samples prepared without contact with skin used as negative controls did not generate any PCR products detectable by gel electrophoresis. With analysis by qPCR, no fluorescent signal was detected from most samples; and when present, it was significantly lower than the signal from skin samples (data not shown).

Variability of the Malassezia microbiota

Cluster analysis and principal coordinate analysis were used to compare the Malassezia microbiota from samples obtained from each body location, different locations from the each subject, and between subjects, as well as samples from healthy skin and psoriatic lesions. The samples were grouped on the basis of similarities on the distribution of Malassezia species, adjusted by the species abundance. The majority of the samples from each individual subject clustered together (Fig. 4), suggesting that the Malassezia microbiota is host-specific. The same result was obtained by principal coordinate analysis (Fig. 5), and confirmed by statistical hypothesis testing (P<0.001).

4

Consensus tree of hierarchical clustering of skin samples from a healthy subject and a patient with psoriasis. Height corresponds to the dissimilarity between samples. The number at each node represents the bootstrap value, based on 1000 iterations. N, healthy subject; P, patient with psoriasis. 0 through 3 represent the months when the samples were obtained.

5

Scatterplot based on the sample dissimilarity matrix, representing samples from a healthy subject (gray circles) and a patient with psoriasis (empty circles). The sizes of the circles are proportional to the sample diversity, as determined by the Gini–Simpson diversity index. The diversity scale is shown in the lower right corner.

Samples from the same body location from each subject obtained monthly for 4 months were closely related (P=0.04), suggesting that the Malassezia microbiota is relatively stable over that time period. Although there are differences in absolute values, the proportions between species remain relatively constant between samples from the same body location in most cases.

No significant dichotomy between samples from healthy skin and from psoriatic lesions was found (P=0.28); the samples clustered according to the subject, rather than health status. There also was no evidence of relatedness between samples from the same body location between the two subjects (P=0.10).

Discussion

We have described the use of multiplex real-time PCR to detect and to quantify Malassezia organisms from human skin. Our previous study using clone library analyses revealed that the fungal microbiota in superficial dry skin from healthy subjects and from patients with psoriasis is mostly constituted by organisms from the genus Malassezia. Nine Malassezia organisms were detected, including five species and four uncharacterized phylotypes (Paulino et al., 2006). Although Malassezia sp. have been associated with psoriasis (Kanda et al., 2002; Prohic, 2003; Baroni et al., 2004; Narang et al., 2007), our prior data did not support such an association. Our next aim was to compare the Malassezia microbiota in different body locations, and to evaluate its stability over time; however, clone library analysis is expensive and time-consuming, limiting the number of samples that could be studied. Moreover, we considered that it would be valuable to obtain a more accurate quantitation of the organisms in the skin samples. As such, we developed a real-time qPCR technique as an alternative method. The Malassezia microbiota from 54 skin samples was characterized, including six healthy body locations and two psoriatic lesions. Using dual-labeled probes, four known species and two uncharacterized organisms were analyzed, and the total of the Malassezia sp. also was measured using a genus-specific probe. The method described here is entirely culture-independent. Data based on CFU's were not compared with qPCR results due to the difficulties in cultivating Malassezia organisms. The growth rate and the medium requirements substantially vary between species, which would bias analysis (Batra et al., 2005).

The skin swabbing used for sample collection allowed large areas to be sampled repetitively. The method is both simple and fast, and had been successfully used for studying both the bacterial and fungal microbiota from healthy skin and psoriatic lesions by clone library analyses (Paulino et al., 2006; Gao et al., 2007). The use of the multiplex format increased the efficiency of the method, as it requires less DNA, and simplifies the technical execution and data analyses, consequently allowing for more species and samples to be studied per unit time. Although there is no absolute gold standard, the Malassezia microbiota from two skin samples studied by both qPCR and analysis of clone libraries corresponded well, supporting the applicability of qPCR in the study of Malassezia communities.

The comparison between the skin samples was based on the distribution of Malassezia species, rather than absolute amounts of DNA detected in each sample. Therefore, the results are little influenced by possible differences in the amount of DNA recovered from the samples, in the yield of the PCR reactions, and in PCR product purification. Our data suggest that the use of PCR products as template for the qPCR assays do not significantly modify the proportions between species in the samples, and therefore can be used as alternatives for genomic DNA. This offers the possibility to study samples in which the DNA amounts are insufficient to be detected or to be accurately quantified. In fact, despite the high sensitivity of the technique, it was not possible to detect or to accurately quantify most of the Malassezia organisms from the skin samples studied using genomic DNA as template for the qPCR assays. As such, using PCR products as templates was the only practical option to analyze these organisms.

Traditionally, culture-dependent methods have been used to analyze the distribution of Malassezia organisms in different body locations from both healthy subjects and patients with skin diseases, from different populations. Malassezia globosa has been reported as the most frequently isolated species from healthy individuals (Aspiroz et al., 1999; Nakabayashi et al., 2000; Rendic et al., 2003; Tarazooie et al., 2004; Salah et al., 2005), as well as from patients with pityriasis versicolor (Crespo Erchiga et al., 2000; Nakabayashi et al., 2000; Aspiroz et al., 2002; Hernandez Hernandez et al., 2003; Tarazooie et al., 2004; Rincon et al., 2005; Salah et al., 2005; Gaitanis et al., 2006b; Prohic & Ozegovic, 2007) and psoriasis (Gupta et al., 2001; Prohic, 2003). Malassezia sympodialis also has been isolated in relatively high frequencies from healthy subjects (Nakabayashi et al., 2000; Gupta et al., 2001; Rendic et al., 2003; Tarazooie et al., 2004; Sandstrom Falk et al., 2005), and from patients with atopic dermatitis (Gupta et al., 2001; Sandstrom Falk et al., 2005); it was commonly found in nonlesional samples from trunk (Crespo Erchiga et al., 2000; Nakabayashi et al., 2000). Malassezia restricta has not been isolated frequently, except for its culture in relatively high proportions from forehead and scalp (Aspiroz et al., 1999; Gupta et al., 2001; Prohic, 2003; Lee et al., 2006).

Divergences are observed when such results are compared with data obtained using culture-independent techniques. For example, using nested PCR assays, M. restricta and M. globosa were the most prevalent species from both healthy subjects and from patients with psoriasis (Amaya et al., 2007), atopic dermatitis (Sugita et al., 2001; Takahata et al., 2007b), and pityriasis versicolor (Morishita et al., 2006). In addition, real-time PCR used to quantify M. restricta, M. globosa, and the total Malassezia DNA in lesions from patients with atopic dermatitis (Sugita et al., 2006; Takahata et al., 2007b), seborrheic dermatitis (Tajima et al., 2008) and in the scale of patients with psoriasis (Takahata et al., 2007a) revealed that M. restricta was the most abundant species. Similarly, in the present study, we found that M. restricta and M. globosa were the most abundant species in samples from both healthy skin and from psoriatic lesions. The discrepancies between molecular methods and culture-based techniques reflect the possible biases of cultivation, and reinforce the importance of developing molecular approaches for a better understanding of the role of Malassezia species as commensals and as pathogens.

Our study revealed that a phylotype distinct from the currently recognized species is prevalent, being detected in 74% of the skin samples. This organism, most closely related to M. restricta, also was detected in our previous study using analysis of clone libraries (Paulino et al., 2006), and should be further characterized. The high prevalence of an organism that potentially represents a new species emphasizes the importance of further studies for better understanding the microbiota of human skin, especially considering that other unidentified Malassezia organisms also may be present.

In conclusion, we have reported the use of multiplex real-time PCR to detect and to quantify six Malassezia organisms from skin samples. To our knowledge, this is the first time in which multiple Malassezia species were individually quantified using a qPCR-based system. The method is sensitive and specific, representing a faster and more cost-effective alternative to clone library analyses, especially in those cases in which preliminary information about the microbial community is available. We opted for including a small number of subjects because it allowed us to perform extensive analyses for optimizing the method and maximizing its use for this particular purpose, and although it is not nearly sufficient to understand the complexity of the Malassezia microbiota in human skin, the data reported here provide useful information required for further investigations. Future studies should increase the number of subjects analyzed, including individuals from different countries and ethnic backgrounds. For better comprehension of the association of Malassezia microbiota in skin with cutaneous diseases, the effects of antifungal treatments on the microbiota can be investigated as well. The development of culture-independent methods is particularly relevant for studying Malassezia organisms, considering the difficulties in cultivation, isolation, and identification of species by culture-based techniques (Gueho et al., 1996). The data reported here extend our previous findings (Paulino et al., 2006), providing further evidence that the distribution of Malassezia organisms is host-specific and relatively stable over the period of time analyzed, and not showing any consistent variation between psoriasis and healthy skin.

Acknowledgements

We thank Athena Kritharis and Alexander Browne for their technical assistance.

This work was supported in part by the Senior Scholar Award from the Ellison Medical Foundation, the Diane Belfer Program in Human Microbial Ecology in Health and Disease, and the Michael Saperstein Medical Scholars Research Fund.

Supplementary material

Fig. S1. Amplification profiles obtained using seven qPCR assays to detect Malassezia organisms.

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