Abstract
Introduction
Psoriasis is a chronic inflammatory disease that affects over 125 million people worldwide. Many studies have shown the importance of the microbiome for psoriasis exacerbation.
Aim
Explore the fungal load and species composition of cultivable yeasts on the skin of psoriatic patients (PP) and healthy volunteers living in a tropical area and evaluate the susceptibility to antifungals.
Methodology
A cross-sectional study with 61 participants (35 patients and 26 healthy controls) was performed during August 2018 and May 2019. Clinical data were collected from patient interviewing and/or medical records review. Samples were collected by swabbing in up to five anatomic sites. Suggestive yeast colonies were counted and further identified by phenotypical tests, PCR-REA, and/or MALDI-TOF. Susceptibility of Malassezia spp. and Candida spp. to azoles, terbinafine, and amphotericin B was evaluated by broth microdilution.
Results
Nearly 50% of the patients had moderate to severe psoriasis, and plaque-type psoriasis was the most common clinical form. Yeast colonies count was significantly more abundant among PP than healthy controls. Malassezia and Candida were the most abundant genus detected in all participants. Higher MIC values for ketoconazole and terbinafine were observed in Malassezia strains obtained from PP. Approximately 42% of Candida isolates from PP showed resistance to itraconazole in contrast to 12.5% of isolates from healthy controls. MIC values for fluconazole and amphotericin B were significantly different among Candida isolates from PP and healthy individuals.
Conclusion
This study showed that Malassezia and Candida strains from PP presented higher MIC values to widespread antifungal drugs than healthy individuals.
Supplementary Information
The online version contains supplementary material available at 10.1007/s42770-022-00883-2.
Keywords: Psoriasis, Yeast, Mycobiota, Antifungal susceptibility
Introduction
Psoriasis is a chronic inflammatory disease that affects over 125 million people worldwide—approximately 2 to 3% of the global population [1, 2]. Scientific evidences attest that psoriasis is a T-cell mediated disease in which the interaction between T lymphocytes and keratinocytes triggers innate and adaptative responses, with up regulation of proinflammatory cytokines [1, 3]. Genetic and immunological factors of the host are well related to the pathobiology of the disease. In addition, environmental conditions and allergens from skin microbiota exacerbate the disease: Streptococcus pyogenes, Staphylococcus aureus, and Malassezia have been repeatedly related to psoriasis [3–5].
Malassezia are members of the skin mycobiota of humans and warm-blooded animals. Since the first description of M. furfur in 1889, the taxonomy of the genus has evolved and advances in molecular biology techniques have allowed the recognition of 16 species, six of them found exclusively in animals [6]. Lipid-dependent Malassezia species are found in the infundibulum of sebaceous glands where they can escape local immune response and coexist in a commensal/symbiotic status [6, 7]. However, under conditions that suppress this equilibrium state, Malassezia can act as a true pathogen causing many diseases, such as pityriasis versicolor, dandruff, folliculitis, seborrheic dermatitidis, onychomycosis, otitis, or even exacerbating previous diseases, such as atopic dermatitis and psoriasis [7–9].
The purpose of this study was to explore the fungal load and species composition of cultivable yeast mycobiota on the skin of patients with psoriasis and healthy volunteers living in a tropical area. Yeasts recovered from both patients and controls seem to present different antifungal susceptibility responses.
Material and methods
Subjects
The study was conducted with patients with psoriasis and healthy individuals attended at the Dermatology Service of Walter Cantídio Teaching Hospital/Federal University of Ceará, Brazil (HUWC / UFC) during August 2018 and May 2019. Patients under 18 years old, with clinically known infectious diseases, chronic inflammatory diseases, other autoimmune diseases, and/or immunosuppression were excluded from the study. This cross-sectional study included 61 participants categorized into three groups: I patients with active psoriasis, but who have not started treatment or underwent a wash out period of at least 4 weeks for topic medications or 12 weeks for systemic medications (n = 9); II patients with active psoriasis despite current topical or systemic treatment (n = 26); III healthy individuals included patients that attended dermatology clinics for other reasons (n = 26). The study was approved by the Ethics Committee of the Federal University of Ceará, Brazil (90,710,218.5.0000.5054).
All subjects gave their written informed consent; details regarding the demographic characteristics of all individuals were collected using a questionnaire. Clinical data were collected from patient interviewing and/or medical records review. Severity of psoriasis was evaluated using psoriasis area and severity index (PASI) [9].
Sample collection
Samples were collected based on Jagielski et al. [10], with adaptations. For each participant, five samples from each different anatomic body site were collected. Patients with more than 20 lesions and/or moderate to severe disease had samples collected at five anatomic sites. Patients with less than 20 lesions and/or mild disease, as well as healthy individuals, had samples collected in at least three anatomic sites. Samples were chosen from the following anatomic sites: head (scalp), intertriginous skin (armpit, gluteus, navel, inframammary skin), upper limbs (arm, forearm, elbow, back, hand, fingernails), and lower limbs (knee, leg, foot). For some patients with psoriasis, samples were also collected from non-affected areas adjacent to psoriatic lesions.
Skin areas were previously cleaned with sterile gauze embedded with sterile saline. For each anatomic site, a sterile cotton swab soaked with sterile saline was rubbed against a 2 cm2 skin surface for 15 s and immediately spread onto modified Sabouraud agar (mSAB) supplemented with 2% olive oil and 0,5% Tween 80 pH 5.6. The plates were incubated for up to 4 weeks at 35 °C.
Culture conditions and yeast isolation
Cream-colored, smooth, and pasty colonies, larger than 1 mm in diameter, suggestive of yeast growth on mSAB were counted using a stereomicroscope. For each anatomic site, one colony is suggestive of Malassezia spp., Candida spp., Trichosporon spp., and/or Rhodotorula spp. was selected for further identification. Phenotypic identification was carried out by growth on chromogenic medium, Dalmau slide culture on cornmeal-Tween 80 agar, carbohydrate assimilation, and urease test [11]. Confirmation of phenotypic identification of Malassezia was performed by 26S rDNA-RFLP reaction with CfoI (Thermo Fisher Scientific, EUA) and BstF51 (Thermo Fisher Scientific, EUA), as suggested by Mirhendi et al. [12]. For the accurate identification, proteomic analysis by MALDI-TOF MS (Autoflex III, Bruker Daltonics Inc., USA/Germany) of all yeast strains were performed. The obtained spectra were compared with the spectra of the BiotyperTM version 3.1 database (Bruker Daltonics, Germany/USA) for yeast identification, considering the identification with score values ≥ 2 [13].
Antifungal susceptibility of Malassezia spp. and Candida spp. strains
Susceptibility of Malassezia spp. and Candida spp. strains was evaluated by broth microdilution technique according to document M27-A3 [14]. Assays were conducted in RPMI 1640 medium buffered with 0.165 M morpholinopropanesulfonic acid (MOPS; Sigma Chemical Co., USA), at pH 7.0 for Candida spp. or modified RRPI medium (mRPMI; 2% glucose, 1% peptone, 1% ox bile, 3% olive oil, 0.2% glycerol, 0.5% malt extract, 0.5% yeast extract) for Malassezia spp., as suggested by Rojas et al. [15, 16], with adaptations. The drugs tested were miconazole (MCZ; Sigma Chemical Co., USA), ketoconazole (KTZ; Sigma Chemical Co., USA), fluconazole (FCZ; Merck KGaA, Germany), itraconazole (ITZ; Janssen Pharmaceutica, Belgium), voriconazole (VCZ; Pfizer, USA), terbinafine (TRB; Sigma Chemical Co., USA), and amphotericin B (AMB; Sigma Chemical Co., USA). The concentrations tested ranged from 0.03 to 16 µg/mL for all drugs, except for FCZ, which ranged from 0.125 to 64 μg/mL.
For Malassezia spp., inoculum suspensions were prepared in sterile saline with 0.5% Tween 80 from 5-day cultures grown on mSAB at 32 °C. Suspensions were vigorously vortexed for dispersing cell clumps, then inoculum was adjusted spectrophotometrically at 530 nm to 2 McFarland standard and further validated by colony counts on mSAB medium (106 UFC/mL). Inoculum suspensions were diluted 1:100 in mRPMI to reach cell density of approximately 0.5 to 2.5 × 103 UFC/mL. Each microdilution well was inoculated with 100 μl of the diluted inoculum suspension and the final volume in each well was 200 μl. Plates were read visually after incubation at 35 °C during 24 h or 32 °C during 72 h, for Candida spp. [14] or Malassezia spp. [15, 16], respectively. Minimum inhibitory concentrations (MIC) of azoles and TRB were defined as the lowest concentrations capable of inhibiting fungal growth by 50% when compared to drug-free growth control; MIC for AMB was defined as the lowest concentration able to inhibit fungal growth completely. All tests were performed in duplicate and Candida parapsilosis ATCC 22,019 and C. krusei ATCC 6258 were included as quality control for each assay [14].
Statistical analysis
Differences in the frequencies of fungal isolation among patients and healthy individuals were analyzed by Qui-square and exact Fisher test (IBM SPSS, version 20, EUA). The unpaired t-test was used to analyze antifungal sensitivity. P value < 0.05 was considered significant. Geometric mean and standard deviation for continuous variables and absolute and relative frequencies of categorical variables of clinical and epidemiological data were also calculated.
Results
Clinical and epidemiological data
Regarding patients with psoriasis, 40% were female (n = 14) and 60% were male (n = 21); patients from group I were significantly younger (36.7 years ± 14.4) than those from group II (47.7 years ± 15.2). The most common clinical presentation was plaque-type psoriasis (85.71%), followed by pustular disease (5.71%). Flexural, guttate psoriasis, and erythroderma were each represented by one patient. Nearly half of the patients with psoriasis (48.58%) had moderate to severe disease (PASI > 10). In terms of treatment, 13 (50%) of patients in group II received phototherapy plus topical treatment (keratolytic agents and/or topical corticosteroids); remaining 13 (50%) received systemic therapy (corticosteroids and/or immunobiologics). In the present study, a total of 22 patients showed early-onset psoriasis (group I, n = 8; group II, n = 14); whereas 13 showed late-onset disease (group I, n = 2; group II, n = 11). Clinical and epidemiological information concerning to each group of participants is shown in Table 1. Details regarding each participant are shown in the supplementary material (Tables S1 and S2).
Table 1.
Main clinical characteristics of patients investigated in this study
| Groups(n) | Ageδ | Gender n (%) | Clinical forms n (%) | Severity* n (%) | Treatment n (%) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Female | Male | Plaque-type | Erythroderma | Pustular | Inverse | Guttate | Mild | Moderate to severe | W/T | PH + TP | SYS | ||
|
I (9) |
36.7 ± 14.4 |
4 (44.4) |
5 (55.6) |
7 (77.8) |
0 |
2 (22.2) |
0 | 0 |
3 (42.8) |
4 (57.2) |
9 (100) |
0 | 0 |
|
II (26) |
47.7 ± 15.2 |
10 (38.5) |
16 (61.5) |
23 (88.5) |
1 (3.8) |
0 |
1 (3.8) |
1 (3.8) |
13 (50) |
13 (50) |
0 |
13 (50) |
13 (50) |
|
III (26) |
54.3 ± 14.4 |
12 (46.2) |
14 (53.8) |
n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a |
Mild psoriasis: limited disease with PASI ≤ 10; moderate to severe psoriasis: PASI > 10 (Naldi & Gambini, 2007)
Group I: untreated patients; Group II: treated patients; Group III: healthy individuals
n/a, not applicable; PASI, psoriasis area and severity index; BSA, body surface area index; W/T, without treatment; PH, phototherapy; TP, topic treatment; SYS, systemic treatment
δ Age (years): mean ± standard deviation; * measured by PASI
Of the 35 patients, 97.4% reported aggravating factors for psoriasis. Most patients from both patient groups reported stress as an aggravating factor (80%). Other aggravating factors and comorbidities including smoking, alcohol abuse, positive family history of psoriasis, and diabetes were less common (Table 2).
Table 2.
Self-reported aggravating factors and/or comorbidities for psoriasis
| Groups (n) | Factors and comorbidities n (%) | ||||
|---|---|---|---|---|---|
| Emotional | Stress | Diabetes | Smoking/alcohol | Family history | |
|
I (9) |
7 (87.5) | 7 (87.5) | 2 (22.2) | 1 (11.1) | 3 (33.3) |
|
II (26) |
22 (88) | 21 (87.5) | 4 (15.4) | 3 (11.5) | 7 (26.9) |
Group I: untreated patients; Group II: treated patients; Group III: healthy individuals
Mycological studies
A total of 3944 yeast colonies were obtained from clinical samples, being significantly more abundant among patients (n = 2,644 cfu) than in healthy controls (n = 1300) (p < 0.05). In patients with psoriasis, higher yeast counts were observed in the upper limbs (47.04%; 1244/2644 cfu); lower counts were seen in the lower limbs (4.72%; 125/2644 cfu). There was no difference in the detection rates between lesional and non-lesional skin areas in this group. With regard to healthy individuals, the areas with the highest yeast counts were intertriginous skin and upper limbs (42.15%; 548/1300 cfu for both sites); the areas with the lowest counts were the lower limbs (2.46%; 32/1300 cfu). Details regarding the number and percentages of positive sites, as well as yeast counting are shown in Table 3.
Table 3.
Fungal load from psoriatic patients and controls
| Groups(n) | Investigated areas | |||||||
|---|---|---|---|---|---|---|---|---|
| Head | Intertriginous | Upper limbs | Lower limbs | |||||
| na (%) | CFU | na (%) | CFU | na (%) | CFU | na (%) | CFU | |
|
I + II (35) |
23/34 (67.6) |
501 |
17/26 (65.4) |
774 |
16/32 (50) |
1244 |
6/10 (60) |
125 |
|
III (26) |
15/26 (57.7) |
172 |
18/24 (75) |
548 |
14/22 (63.6) |
548 |
3/7 (42.9) |
32 |
Group I: untreated patients; Group II: treated patients; Group III: healthy individuals
na number of positive sites/total of investigated sites
CFU counting forming units
Malassezia was isolated from 62.9% (22/35) of patients with psoriasis and from 61.5% (16/26) of healthy individuals. On the other hand, Candida was isolated from 45.71% (16/35) of psoriasis cases and from 76.92% (20/26) of healthy controls (p < 0.05) (Table 4). Based on conventional, molecular e proteomic approaches, a total of 23 yeast isolates were thoroughly identified as follows: Malassezia furfur (n = 3), Candida orthopsilosis (n = 1), C. parapsilosis (n = 16), Candida haemulonii (n = 2), Trichosporon coremiforme (n = 1). Inconsistencies arising from the polyphasic approach allowed confirmation of some isolates only to the genus level: Malassezia spp. (n = 47); Candida spp. (n = 18); Trichosporon spp. (n = 15); Rhodotorula spp. (n = 6). Table 5 details the identification of each fungal isolate, presenting their origin per anatomical sites and population groups investigated.
Table 4.
Positivity in psoriatic patients and controls. For each anatomic site, one colony is suggestive of Malassezia spp., Candida spp., Trichosporon spp. and/or Rhodotorula spp. was counted
| Groups (n) | Positive individuals n (%) | |||
|---|---|---|---|---|
| Malassezia | Candida | Rhodotorula | Trichosporon | |
|
I + II (35) |
22 (62.9) | 16 (45.7) | 1 (2.9) | 6 (17.1) |
|
III (26) |
16 (61.5) | 20 (76.9)* | 5 (19.2) | 10 (38.5) |
Group I: untreated patients; Group II: treated patients; Group III: healthy individuals
*p < 0.05
Table 5.
Distribution of yeasts isolated in untreated patients (Group I), treated patients (Group II), and healthy individuals (Group III) and their occurrence regarding the anatomical site. Numbers in parentheses indicate the quantity of positive individuals for each fungal species
| Investigated areas | Group I | Group II | Group III |
|---|---|---|---|
| Head |
Malassezia spp. (1) C. orthopsilosis (1) C. parapsilosis (1) |
Malassezia spp. (4) C. parapsilosis (2) C. haemulonii (1) Candida spp. (1) Trichosporon spp. (1) |
Malassezia spp. (4) M. furfur (1) C. parapsilosis (2) Candida spp. (1) Rhodotorula spp. (1) Trichosporon spp. (2) |
| Intertriginous | Malassezia spp. (4) |
Malassezia spp. (4) Candida spp. (4) C. parapsilosis (1) Rhodotorula spp. (1) Trichosporon spp. (1) |
Malassezia spp. (8) Candida spp. (7) M. furfur (1) C. parapsilosis (4) T. coremiforme (1) Rhodotorula spp. (2) Trichosporon spp. (3) |
| Upper limbs |
Malassezia spp. (2) C. parapsilosis (3) Trichosporon spp. (2) |
Malassezia spp. (9) C. parapsilosis (1) Trichosporon spp. (1) |
Candida spp. (5) Malassezia spp. (4) M. furfur (1) C. haemuloni (1) Rhodotorula spp. (2) Trichosporon spp. (4) |
| Lower limbs |
C. parapsilosis (1) Trichosporon spp. (1) |
Malassezia spp. (4) C. parapsilosis (1) |
Malassezia spp. (3) |
Modified RPMI medium allowed abundant Malassezia growth after 72 h of incubation. Variations in MIC values of all antifungals were observed in Malassezia strains from both patients and control groups. KTC and TRB MIC values were higher in Malassezia strains obtained from patients than in healthy controls; poor susceptibility to FCZ, ITZ, and VCZ was observed in 80%, 20%, and 65%, respectively of Malassezia strains obtained from psoriatic patients (p < 0.05). All Malassezia strains were able to grow in the presence of AMB > 1 µg/mL. Table 6 shows in absolute frequency the number of isolated Malassezia strains according to the MIC value obtained.
Table 6.
Minimum Inhibitory Concentration (MIC) of antifungals against Malassezia spp. from patients with psoriasis and control subjects. Information in parentheses indicates the number of positive individuals for each fungal species. Results are shown in μg/mL
| Antifungal | Psoriatic patients (groups I and II) | Healthy individuals (group III) | ||
|---|---|---|---|---|
| MIC (n) | Geometric mean ± SD | MIC (n) | Geometric mean ± SD | |
| Miconazole (MCZ) |
0.5 (3) 2.0 (2) 8.0 (5) 16.0 (9) > 16.0 (1) |
6.20 ± 6.42 |
0.25 (1) 0.5 (2) 1.0 (1) 2.0 (1) 4.0 (4) 16 (6) |
3.82 ± 7.09 |
| Ketoconazole (KTZ) |
0.06 (1) 0.125 (10) 0.25 (1) 0.5 (4) 2.0 (4) |
0.29 ± 0.74* |
0.03 (6) 0.125 (6) 0.5 (2) 1.0 (1) |
0.097 ± 0.27 |
| Fluconazole (FCZ) |
4.0 (4) 16.0 (6) 32.0 (8) 64.0 (2) |
18.38 ± 17.30 |
4.0 (5) 8.0 (4) 16.0 (1) 32.0 (1) 64.0 (4) |
12.70 ± 26.12 |
| Itraconazole (ITZ) |
0.03 (8) 0.06 (7) 0.125 (1) 0.5 (2) 1.0 (1) 2.0 (1) |
0.22 ± 0.47 |
0.03 (9) 0.06 (6) |
0.03 ± 0.01 |
| Voriconazole (VCZ) |
0.03 (3) 0.06 (3) 0.125 (1) 0.25 (6) 0.5 (1) 1.0 (4) 2.0 (2) |
0.23 ± 0.61 |
0.03 (4) 0.06 (1) 0.125 (5) 0.25 (1) 1.0 (2) 2.0 (2) |
0.47 ± 0.69 |
| Terbinafine (TRB) |
2.0 (5) 4.0 (8) 8.0 (5) 16.0 (2) |
4.60 ± 4.16* |
0.5 (7) 1.0 (2) 2.0 (1) 4.0 (1) 8.0 (3) 16.0 (1) |
1.51 ± 4.58 |
| Amphotericin B (AMB) |
8.0 (4) 16.0 (4) > 16.0 (12) |
11.31 ± 4.27 |
2.0 (1) 4.0 (2) 8.0 (2) 16.0 (4) > 16.0 (6) |
8.0 ± 6.0 |
Group I: untreated patients; Group II: treated patients; Group III: healthy individuals
n number of strains, SD standard deviation
*p < 0.05
Most of Candida isolates from both psoriatic patients and healthy individuals showed susceptibility to antifungals, except for ITZ: 42.1% (8/19) of Candida isolates from psoriatic patients showed resistance to this drug, in contrast to 12.5% (1/8) of isolates from healthy controls. FCZ and AMB MICs were significantly different (p < 0.05) in Candida isolates from psoriatic patients (Table 7).
Table 7.
Minimum Inhibitory Concentration (MIC) of antifungals against Candida spp. from patients with psoriasis and control subjects. Information in parentheses indicates the number of positive individuals for each fungal species. Results are shown in μg/mL
| Antifungal | Psoriatic patients (groups I and II) | Healthy individuals (group III) | ||
|---|---|---|---|---|
| MIC (n) | Geometric mean ± SD | MIC (n) | Geometric mean ± SD | |
| Miconazole (MCZ) |
0.03 (1) 0.06 (6) 0.125 (8) 0.25 (2) 1.0 (1) 0.5 (1) |
0.18 ± 0.23 |
0.03 (2) 0.06 (2) 0.125 (3) 0.5 (1) |
0.13 ± 0.15 |
| Ketoconazole (KTZ) |
0.03 (16) 0.06 (2) 0.25 (1) |
0.04 ± 0.05 |
0.03 (6) 0.06 (2) |
0.04 ± 0.01 |
| Fluconazole (FLC) |
0.25 (7) 0.5 (3) 1.0 (6) 2.0 (3) |
0.80 ± 0.62* |
0.125 (1) 0.25 (5) 0.5 (2) |
0.30 ± 0.13 |
| Itraconazole (ITZ) |
0.03 (5) 0.125 (6) 0.25 (5) 0.5 (1) 0.06 (2) |
0.15 ± 0.12 |
0.03 (2) 0.06 (1) 0.125 (4) 0.25 (1) |
0.11 ± 0.07 |
| Voriconazole (VCZ) |
0.03 (13) 0.06 (5) 0.5 (1) |
0.06 ± 0.11 | 0.03 (8) | 0.03 ± 0.0 |
| Terbinafine (TRB) |
0.03 (3) 0.06 (5) 0.125 (6) 0.25 (3) 2.0 (1) 16.0 (1) |
1.05 ± 3.65 |
0.03 (1) 0.06 (3) 0.125 (2) 0.25 (2) |
0.12 ± 0.09 |
| Amphotericin B (AMB) |
0.06 (2) 0.125 (1) 0.25 (12) 0.5 (3) 2.0 (1) |
0.36 ± 0.42* |
0.125 (1) 0.25 (1) 0.5 (5) 1.0 (1) |
0.48 ± 0.25 |
Group I: untreated patients; Group II: treated patients; Group III: healthy individuals
Candida parapsilosis ATCC 22,019 MIC values: MCZ 0.125 µg/mL; KTZ 0.03 µg/mL; FLC 1 µg/mL; ITZ 0.25 µg/mL; VCZ 0.03 µg/mL; TRB 0.125 µg/mL; AMB 0.5 µg/mL
C. krusei ATCC 6258 MIC values: MCZ 0.25 µg/mL; KTZ 0.25 µg/mL; FLC 8 µg/mL; ITZ 0.25 µg/mL; VCZ 0.06 µg/mL; TRB 0.5 µg/mL; AMB 0.5 µg/mL
n number of strains, SD standard deviation
*p < 0.05
Discussion
The proportion of male (60%) and female (40%) psoriatic patients found in the present study was in agreement with previous researches [17–19]. However, it is important to note that some authors have described a lack of agreement about the incidence rates of psoriasis by sex [20] and clinical presentation [21].
Although psoriasis can appear at any age, it is more likely to occur between 15 and 30 years old [20, 21]. In the present study, the mean age of psoriatic patients varied accordingly to that described elsewhere [18, 19], even though larger populational studies have pointed that the disease is more common among older patients [22]. The age at onset seems to have great clinical-epidemiological importance and a bimodal pattern is recognized: early onset psoriasis (type I disease, associated with HLA class I antigens) which begins before 40 years old, with peak onset at 16–22 years old, and encompasses 70% of all cases; and late-onset psoriasis (type II disease, not associated with HLA antigens) that has onset at or after 40 years old, with a peak onset between 57 and 60 years old [21, 23].
As expected, most of the patients presented plaque-type or pustular psoriasis, regardless of treatment. Plaque psoriasis is the main form of the disease, affecting nearly 85 to 90% of patients [24, 25]; pustular psoriasis may occur in up to 12% of clinical cases [26], and erythrodermic psoriasis can affect up to 7% of patients [27].
Several comorbidities factors associated with psoriasis have been described and include inflammatory bowel disease, psychological, and psychiatric disorders as “classic factors”; metabolic syndrome, cardiovascular diseases; osteoporosis, among others as “emerging factors”; smoking habit, alcoholism, anxiety as “related to lifestyle”; and dyslipidemia, hypertension, and others, as “related to treatment” [28]. In the present study, more than 80% of patients reported the importance of emotional/stressful situations as aggravating factors for psoriasis; nearly one-third of them related that the disease had occurred among their family members. It is known that psychological stress can modulate immune responses and, probably, an abnormal neuroendocrine response to stress may contribute to the pathogenesis of chronic inflammatory diseases [24, 29].
Current studies suggest that Malassezia skin colonization has a secondary role in psoriasis, mainly as exacerbating factor [6–8]. It is believed that Malassezia can increase the inflammatory response in psoriasis by either of the following mechanisms: (a) damage of the epidermal barrier function by lipases and phospholipases; (b) increase in the local immune response by invading keratinocytes and modifying the cytokines synthesis, as keratinocytes can produce both pro-inflammatory (such as IL-6, IL-8, and TNF-α) and anti-inflammatory (IL-4, IL-10) cytokines after stimulation with Malassezia cells; and (c) sensitization to Malassezia antigens, such as pityriarubins and indirubin—ligands of the aryl hydrocarbon receptor in human cells [7].
Nevertheless, the role of Malassezia in psoriasis is difficult to prove. Divergent methodologies for selection of the study population and distinct Malassezia recovery tests (culture-based versus independent culture-based techniques) lead to ambivalent results: studies have pointed M. globosa, M. furfur, M. sympodialis, or M. restricta as the predominant species in psoriatic patients [3]. Additionally, divergences regarding abundance of Malassezia in psoriatic patients and healthy individuals have been reported [5, 30, 31].
In the present study, a higher fungal load was observed in psoriatic patients in comparison with healthy controls. Different results were obtained in the seminal study performed by Gupta et al. [32], after cfu analysis in 28 psoriatic patients and 20 healthy individuals, which revealed significantly higher counts on the scalp, forehead, and trunk. Possibly, differences in sampling procedures in both studies (swabbing versus contact plate) may account for these contradictory results.
Despite the differences in fungal load, patients and healthy individuals displayed similar frequencies of isolation of Malassezia (62.9% in patients versus 61.53% in controls). Analogous results were found by Gupta et al. [32] and Rudramurthy et al. [4], which reported similar isolation rates of Malassezia among psoriatic patients and controls. On the contrary, Gomez-Moyano et al. [31] found a significant difference in Malassezia frequency in samples taken from the scalp skin of psoriatic patients (85%) in comparison with samples from healthy subjects (50%). In face of all these controversies, it is accepted that the scientific available data do not attest differing levels of Malassezia in psoriatic patients.
Although C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis may be found in healthy skin, several studies have concluded that Candida species are not major components of human skin mycobiota [30, 33, 34]. By way of molecular studies, Malassezia species are pointed as the main colonizers of human healthy skin [30] and the abundance of Candida species on the skin seems to increase under conditions of primary immunodeficiencies [35]. Candida species are not frequently found on the skin of psoriatic patients as well [30, 36], although they are found abundantly in the oral mucosa of these individuals [37–39]. However, it is important to address that a common mycobiota pattern in psoriatic patients has not been identified yet. Differences in sampling methods and sequencing of diverse skin regions, besides interindividual variation have continuously generated different results in many studies [40]. In the present study, Candida species were less frequent in psoriatic patients (45.71%) than in healthy individuals (76.92%). Possibly, antimicrobial peptides/proteins of the human skin, such as calprotectin and dermcidin, which exhibit fungistatic activity and are highly expressed in psoriatic lesions [41], may suppress C. albicans density on the skin of psoriatic patients. The hypothesis that Malassezia density may not be reduced by these antimicrobial peptides/proteins has been suggested by other authors [4].
Although there is no available standardized protocol for susceptibility testing of lipid-dependent Malassezia species, some studies have been conducted with agar-diffusion or broth microdilution methodologies [15, 16, 42, 43]. However, variations regarding medium composition, inoculum size, incubation time, and MIC endpoints hinder comparisons among such studies. Overall, variations in susceptibility to azoles and TRB have been described; resistance to AMB is common in clinical Malassezia strains [3, 16, 42, 43], but the susceptibility pattern of Malassezia strains from psoriatic patients is unknown. Data obtained in our study showed that Malassezia strains from patients presented higher MIC values against KTZ and TRB than those obtained towards healthy controls; resistance to AMB was observed in all Malassezia isolates from both patients and controls. Previous studies have already described high variability of MIC for Malassezia against antifungals [42, 43].
In addition, our results showed that Candida strains from psoriasis patients presented higher MIC values to FCZ in comparison with isolates obtained from controls. Although the antifungal susceptibility pattern of Candida from psoriasis patients is unknown, we believe that this result might have some clinical relevance as FCZ has widespread prophylactic and therapeutic use, including on psoriatic patients, and acquired resistance to FCZ by Candida has already been reported [44].
The importance of mycobiota in the exacerbation of psoriasis has already been demonstrated. However, the presented data did not show a correlation between the severity of psoriasis and fungal density on the skin. Nevertheless, this study showed that Malassezia and Candida strains from psoriatic patients presented higher MIC values to widespread antifungals than healthy individuals. Further studies should evaluate the clinical significance of this finding.
Supplementary Information
Below is the link to the electronic supplementary material.
Funding
This work was supported by CNPq (National Council for Scientific and Technological Development, Brazil). Process number process 430193/2018–1).
Declarations
Conflict of interest
The authors declare no competing interests.
Footnotes
Responsible Editor: Luis Nero
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rossana de Aguiar Cordeiro and Ane Teles Reis contributed equally to this work.
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