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Evaluation of candidal colonization and specific humoral responses against Candida albicans in patients with psoriasis Mehdi Taheri Sarvtin1,2,3, PhD, Tahereh Shokohi3,4, PhD, Zohreh Hajheydari4,5, MD, Jamshid Yazdani6, PhD, and Mohammad T. Hedayati3,4, PhD
1 Department of Medical Mycology and Parasitology, School of Medicine, Jiroft University of Medical Sciences, Jiroft, Iran, 2 Department of Medical Mycology and Parasitology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran, 3Invasive Fungi Research Center, Mazandaran University of Medical Sciences, Sari, Iran, 4Department of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, 5Department of Dermatology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, and 6Department of Statistics, School of Health, Mazandaran University of Medical Science, Sari, Iran
Correspondence Mohammad T. Hedayati, PhD Department of Medical Mycology and Parasitology School of Medicine, Mazandaran University of Medical Sciences PO Box 48175-1665 Km18 Khazarabad Road, Sari 48175, Iran E-mail: [email protected]
Abstract Background Psoriasis is an inflammatory skin disease that can considerably affect a patient’s quality of life. Environmental and genetic factors, as well as superantigens and toxins from Candida species, may play various roles in the exacerbation and persistence of psoriasis. In the present study, we evaluated candidal colonization and specific humoral responses against Candida albicans in patients with psoriasis. Methods A total of 100 patients with psoriasis vulgaris and 50 healthy control individuals were enrolled in the study. Skin and oral specimens from all participants were cultured on CHROMagar Candida medium. Isolated yeast-like fungi were identified using the sequence of the D1/D2 domain of the 26S rRNA gene. Enzyme-linked immunosorbent assays (ELISAs) were used to detect immunoglobulin M (IgM), IgA, and IgG antibodies against C. albicans in sera of patients and healthy individuals. Results Candida species were isolated from the skin of 15% of patients and 4% of controls and from oral specimens of 60% of patients and 20% of controls. There was a significant difference in candidal colonization between patients and controls (P < 0.05). Serum IgM, IgA, and IgG levels against C. albicans were significantly lower in patients with psoriasis than in controls (P < 0.05). There was no significant association between serum levels of specific antibodies against C. albicans or the frequency of candidal colonization with the clinical severity of the disease (P > 0.05). Conclusions The results of the present study show a higher rate of candidal colonization in patients with psoriasis in comparison with controls and a reduction in humoral immune responses in patients.
Funding: This study was supported by Mazandaran University of Medical Sciences. Conflicts of interest: None.
Introduction Psoriasis is an immune-mediated, chronic, and non-contagious skin disease characterized by lesions covered with silvery scales and the infiltration of leukocytes into both the dermis and the epidermis.1,2 It affects 1–3% of the world’s population, especially those in the two age groups of 16–22 and 57–60 years.3,4 Most psoriasis patients have a diminished quality of life in comparison with healthy individuals, particularly with reference to sexual dysfunction, anxiety, depression, self-esteem, and nutritional condition.5 ª 2014 The International Society of Dermatology
The exact etiology of psoriasis remains unknown, but it is believed that a combination of several factors such as genetic predisposition and environmental and immunological factors contribute to the development of this disease.6 Among environmental factors, various microorganisms such as viruses (retroviruses, papillomaviruses, and endogenous retroviruses), bacteria (Staphylococcus aureus, Streptococcus pyogenes), and fungi (Candida spp., Malassezia spp.) are known to be associated with the provocation and exacerbation of psoriasis via the production of either toxins or their metabolites.7 Candida spp. represent some of the most important fungal colonizers International Journal of Dermatology 2014, 53, e555–e560
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on the skin and mucosal surfaces of the body, such as in the genitourinary tract, oral cavity, and gastrointestinal tract.8,9 Candida spp. can cause a wide range of disorders, such as vulvovaginitis, oral thrush, and skin and diaper rash, as well as life-threatening diseases in immunocompromised patients.8–11 So far, over 200 species of Candida have been identified, among which Candida albicans, Candida glabrata, Candida tropicalis, and Candida parapsilosis are responsible for the majority of candidal infections.8,10 Depending on the species, Candida is capable of secreting different toxins and superantigens, which can provoke psoriasis.12 Several previous studies have aimed to investigate the prevalence of Candida colonization in different body sites of patients with psoriasis but have varied in their results.13– 16 Recent studies have considered psoriasis as an immunological skin disease that involves substantial changes in humoral immune response.17 In addition, it has been hypothesized that an abnormal response of the humoral immune system in patients with psoriasis attributable to microorganisms can alter the course of the disease.18 However, few studies have evaluated the humoral response against Candida spp. in patients with psoriasis. Accordingly, we assessed the colonization of Candida spp. of the skin and oral cavity and the specific humoral response against C. albicans in patients with psoriasis. Materials and methods Patients A total of 100 patients with psoriasis vulgaris and 50 healthy control subjects were enrolled in this study between January 2011 and March 2012. All patients enrolled in the study were either attending our facility for the first time or had discontinued medication for at least five months. Patients and control subjects completed a consent form giving their agreement to their participation in the present research. The study was approved by the ethical committee of Mazandaran University of Medical Sciences, Sari, Iran. Most of the patients were diagnosed on the basis of the findings of a clinical examination conducted by a dermatologist. Histopathology was used in cases of suspected disease. Control subjects were selected from among persons referred for cosmetic problems. People with diabetes, individuals who had used broad-spectrum antibiotics or steroids, and pregnant patients were excluded from the study. In order to assess the clinical severity of the disease, the Psoriasis Area and Severity Index (PASI) was calculated as elucidated by Fredriksson and Pettersson in 1978.19 Based on this definition, the clinical severity of psoriasis was categorized as mild (PASI scores of < 11), moderate (PASI scores of 11–49), or severe (PASI scores of > 50). International Journal of Dermatology 2014, 53, e555–e560
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Mycological investigation Samples were collected from the oral cavity and skin (lesions and normal skin adjacent to lesions) by swab and scalpel, respectively. Samples were obtained from all areas with lesions (flexures, trunk, extremities) in all patients. All of the samples were cultured on CHROMagar Candida medium (CHROMagar Microbiology, Paris, France). Isolated species of Candida were subcultured on Sabouraud’s dextrose agar containing chloramphenicol (SC) and incubated at 27 °C for four days. Molecular investigation
DNA extraction A part of fresh pure yeast colony obtained from three days of culture on SC was transferred to a microtube and suspended in 300 ll of lysis buffer (10 mM Tris [hydroxymethyl] aminomethane [Thermo Fisher Scientific, Inc., Waltham, MA, USA], 1 mM EDTA, 1% SDS, 100 mM NaCl [Merck KGaA, Darmstadt, Germany], 2% Triton X-100 [Sigma-Aldrich Corp., St Louis, MO, USA]), 300 ll of phenol : chloroform (1 : 1) solution and 0.3 g of 0.5-mm diameter glass beads, vortexed for five minutes, and centrifuged at 19,000 g for 10 minutes. Then, the supernatant was transferred to a new tube, mixed with the same volume of chloroform, and centrifuged at 19,000 g for 10 minutes, after which the supernatant was separated and transferred to a new microtube. For alcohol precipitation, 2.5 ml volumes of cold 96% ethanol were added and kept at 20 °C for three hours. After this period, the tube was centrifuged at 19,000 g for 10 minutes and the supernatant discarded. The precipitate was washed by adding 300 ll of cold 70% ethanol and centrifuged at 19,000 g for 10 minutes. The pellet was air-dried and resuspended in 50 ll of TE buffer (10 mM Tris, 1 mM EDTA) and stored at 4 °C until use.20
Polymerase chain reaction amplification, sequencing, and yeast identification Yeasts were identified to species level using sequence analysis of the D1/D2 domain of the 26S ribosomal RNA gene. For amplification of the D1/D2 domain, the external primers NL-1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and NL-4 (5′-GGT CCG TGT TTC AAG ACG G-3′)21 were used. The reactions were performed in an automatic thermal cycler (C1000 thermal cycler; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with initial denaturation at 95 °C for three minutes, followed by 35 cycles at 95 °C for 30 seconds, 56 °C for 30 seconds and 72 °C for 30 seconds, and a final extension at 72 °C for seven minutes. The quality of polymerase chain reaction (PCR) products was determined by electrophoresis in 1% (w/v) agarose gel in 19 TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA) at 80 V for two hours. GenRuler DNA ladder mix was used as a marker. Sequencing of the purified PCR products was performed using the primer NL-1. Sequences
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were aligned to the 26S rRNA gene sequences obtained from the National Center for Biotechnology Information (NBCI)
statistical significance. Cronbach’s alpha was used to calculate the reliability of the PASI instrument. The correlations between
GenBank database (http://blast.ncbi.nlm.gov/Blast.cgi), and
carriage of Candida spp. and levels of antibodies against
yeast species were identified by searches using the
C. albicans and the severity of disease (PASI score) were
BLAST
algorithm.
examined using the chi-squared test and Pearson correlations, respectively.
Detection of anti-C. albicans antibodies Serum immunoglobulin G (IgG), IgM, and IgA levels were measured with enzyme-linked immunosorbent assay (ELISA) test kits (Genesis Diagnostics Ltd, Littleport, UK) according to the manufacturer’s instructions. Briefly, serum samples were diluted to 1 : 200 in sample diluent. Quantities of 100 ll of each standard, positive control and the diluted patient samples were dispensed into appropriate microplate wells coated with a purified C. albicans extract. After 30 minutes, the well contents were decanted and washed three times by an automatic ELISA washer (Washer MPW1; SCO Tech GmbH, Diagnostic, Germany). Then, 100 ll of conjugate was added to each well and incubated for 30 minutes at room temperature. The microplate wells were washed four times. A quantity of 100 ll of TMB (3,3′,5,5′-tetramethylbenzidine) substrate was dispensed into each well and incubated for 10 minutes at room temperature. The reaction was stopped by dispensing 100 ll of stop solution into each well. The optical density (OD) of each well was measured at 450 nm in a microplate reader (Bio-Rad 680; Bio-Rad Laboratories, Inc.).
Results In this study, 100 patients (44 male and 56 female patients; mean standard deviation [SD] age: 40.47 11.03 years [range: 12–72 years]) and 50 control subjects (22 male and 28 female subjects; mean SD age: 39.90 11.45 years [range: 13–63 years]) were examined. A Cronbach's alpha value of 0.7 indicated the reliability of the PASI tool for measuring the severity of psoriasis. PASI scores of < 11, 11–50, and > 50 were observed in 76%, 9%, and 15%, respectively, of the psoriasis patients. Overall, 63% of psoriasis patients and 24% of control subjects demonstrated colonization by yeast species (v2 = 20.28, P < 0.01). In all, 41% of patients and 22% of control subjects were colonized by only one yeast species, 14% of patients and 2% of control subjects were colonized by two different yeast species, and 8% of patients were colonized by three different yeast species (v2 = 25.04, P < 0.01). Isolated yeast species are listed in Table 1. Candida spp. were isolated from the oral cavity of 60 (60%) patients and 10 (20%) control subjects (v2 = 21.49, P < 0.01). Candida spp. were isolated from the skin of the trunk only in 15 (15%) patients and two (4%) control subjects (v2 = 4.01, P = 0.045). Samples from the scalp, flexures, and extremities were negative for Candida growth in culture. Candida parapsilosis was isolated from normal skin adjacent to lesions in one (1%) patient (v2 = 4.08, P = 0.047). A total of 45 (45%)
Statistical analysis Numbers of individuals with yeast growth were compared using the chi-squared test. Independent t-tests were used to compare the results of IgG and IgM assays in the two groups. P-values of < 0.05 were considered to indicate statistical significance. The results of IgA assays were analyzed using the Kruskal– Wallis test. Paired comparisons were made using the Mann– Whitney U-test. P-values of < 0.015 were considered to indicate
Table 1 Yeast species isolated from psoriasis patients and control subjects Oral specimens
Skin specimens
Yeast species
Patients, n (%)
Controls, n (%)
Patients, n (%)
Controls, n (%)
Candida albicans Candida glabrata Debaryomyces hansenii Pichia guilliermondii Candida tropicalis Candida dubliniensis Saccharomyces cerevisiae Candida parapsilosis Yarrowia lipolytica Dipodascus capitatus Issatchenkia orientalis Aureobasidum pullulans Total
45 10 10 6 4 4 4 3 2 2 2 2 94
3 4 0 0 1 0 0 3 0 0 0 0 11
9 2 2 2 0 0 0 0 0 0 2 0 17
2 (100%) 0 0 0 0 0 0 0 0 0 0 0 2 (100%)
(47.9%) (10.6%) (10.6%) (6.4%) (4.3%) (4.3%) (4.3%) (3.2%) (2.1%) (2.1%) (2.1%) (2.1%) (100%)
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(27.3%) (36.4%)
(9.1%)
(27.3%)
(100%)
(52.8%) (11.8%) (11.8%) (11.8%)
(11.8%) (100%)
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35 30
31 32 26
25
22
20 15
12
10
0
0–10.0
Patients without Candida species colonization Patients with Candida species colonization Control without Candida species colonization Control with Candida species colonization
30.1+
IgM level, U/mL Figure 1 Distribution of serum levels of immunoglobulin M (IgM) specific against Candida albicans in psoriasis patients and control subjects. Mean standard deviation IgM levels: patients, 9.96 6.85 U/ml; controls, 14.48 12.00 U/ml International Journal of Dermatology 2014, 53, e555–e560
0 0. –1 0
10 0. 0+
2 11
0
0
0
6 2
.1
20.1–30.0
0 0
6
80
10.1–20.0
0
4
30
0–10.0
2
88
0 30
3
45
14
11
0.
7 6
16
14
.0
10
5 0
21
14 6
52
25
20
12 11
10
30
0–
15
40
–8
20
20
30.1+
60
.1
25
0 0
Our results showed a significantly higher prevalence of Candida colonization in the oral cavity of patients with psoriasis compared with the control group. This result is
60
26
20.1–30.0
3 2
Figure 2 Distribution of serum levels of immunoglobulin A (IgA) specific against Candida albicans in psoriasis patients and control subjects. Mean standard deviation IgA levels: patients, 9.81 9.33 U/ml; controls, 13.18 4.74 U/ml
0
26
10.1–20.0
2
IgA level, U/mL
Subjects, %
30
Patients without Candida species colonization Patients with Candida species colonization Control without Candida species colonization Control with Candida species colonization
5 4
3
5
50
33 34
8
6
40 35
Patients without Candida species colonization Patients with Candida species colonization Control without Candida species colonization Control with Candida species colonization
40
0.
In this study, we investigated the associations between specific IgG, IgM, and IgA levels and the frequency of colonization with Candida spp. and clinical severity in patients with psoriasis in order to establish which factors are correlated with clinical severity.
44
45
–6
Discussion
50
.1
patients and three (6%) controls were colonized by C. albicans (v2 = 23.3, P < 0.01). Ten (10%) patients were colonized by only Debaryomyces hansenii (Candida famata) (v2 = 5.35, P = 0.02). Of patients with mild, moderate, and severe disease, respectively, 47%, 15%, and 1% showed colonization with yeast species (P = 0403). The mean level of IgM against C. albicans in patients and controls was 9.96 and 14.68 U/ml, respectively (P = 0.00). The mean level of IgG against C. albicans in patients and controls was 68.83 and 90.03 U/ml, respectively (P = 0.00). Mean levels of IgA against C. albicans in patients and control subjects were 9.81 and 13.18 U/ml, respectively (Z = 3.337, P= 0.001). Pearson correlation statistics for the association between levels of IgM, IgG, IgA, and disease severity were 0.049, 0.710, and 0.022, respectively (P > 0.05). Among participants with candidal colonization, 33% of patients and 12% of control subjects showed an IgM, level of < 10 U/ml (Fig. 1), 31% of patients and 6% of control subjects showed an IgA level of < 10 U/ml (Fig. 2), and 5% of patients and 4% of control subjects showed an IgG level of < 30 U/ml (Fig. 3).
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IgG level, U/mL Figure 3 Distribution of serum levels of immunoglobulin G (IgG) specific against Candida albicans in psoriasis patients and control subjects. Mean standard deviation IgG levels: patients, 68.83 22.59 U/ml; controls, 90.03 40.21 U/ml ª 2014 The International Society of Dermatology
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incompatible with those of Leibovici et al.13 but is consistent with the findings of Waldman et al.14 Our results also showed a significantly higher prevalence of Candida spp. in the psoriatic lesions of patients with psoriasis vulgaris compared with adjacent normal skin in these patients and skin of the control group. Psoriasis is an immunological skin disease which reflects a humoral immune disorder, low cellular immune function, and a disturbance in the normal structure of the skin.17 These conditions can increase the carriage of Candida spp. in patients with psoriasis. It is suggested that superantigens and toxins released from Candida spp. activate the secretion of cytokines from T cells, keratinocytes, and inflammatory cells that contribute to the provocation and exacerbation of psoriasis.12 Moreover, Candida spp. can provoke psoriasis via the activation of complement system and neutrophil recruitment.22 By contrast with previous studies,13,14 we applied molecular methods in the identification of yeast species and thus identified a greater diversity of yeast species than in previous studies. In the present study, C. albicans was the most common species of Candida, both in the oral cavity and in the skin of patients with psoriasis, a finding that concords with those of other studies. Among the Candida spp., C. albicans is the most prevalent and is able to cause different diseases under predisposing conditions which include the humoral and cellular immune disorder8 seen in psoriasis.17 Thus, colonization by this species in patients with psoriasis should be considered to represent a risk factor for the exacerbation of psoriatic signs in skin. Debaryomyces hansenii (C. famata) is a hemiascomycetous yeast commonly found in dairy products and the environment, which rarely causes disease in humans.23 In the present study, D. hansenii was isolated from the oral cavity and skin in participants within the patient group. This species of Candida can adapt itself to differences in environmental pH and salinity by changing the sterol to phospholipid ratio of its plasma membranes.24 Thus it would seem that patients with psoriasis are predisposed to colonization by D. hansenii by the abnormal condition of their skin and mucous. Debaryomyces hansenii is capable of secreting antigens and different metabolites, including protease B, arginyl aminopeptidase, and toxin,25 which may provoke psoriasis. Therefore, further studies are required to show the possible roles of these metabolites in psoriasis. In the present study, IgM and IgA levels against C. albicans were significantly lower in patients than in control subjects. This result concords with those reported by Liang et al.,18 who also reported a higher level of IgG against C. albicans in patients with psoriasis than in control subjects, which differs from the equivalent finding in our study. In the present study, we found ª 2014 The International Society of Dermatology
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a significant reduction in the level of IgG against C. albicans in patients with psoriasis in comparison with control subjects. Levels of antibodies against microbial agents are associated with a variety of factors, including the type of infectious agent and duration of infection.26 For this reason, we did not find any significant relationship between candidal colonization and levels of specific antibodies against C. albicans in patients or control subjects. However, the inability of the immune system to produce sufficient antibodies against Candida spp. in patients with psoriasis can help to increase candidal colonization in these subjects. Psoriasis is a chronic disease, and most of the patients included in the present study did not remember the exact date of onset of the disease. For this reason, we were unable to investigate the relationship between duration of disease, carriage of Candida spp., and antibody levels in patients with psoriasis. Most of the patients included in this study had a mild form of disease, and therefore we did not observe any significant association between PASI score, carriage of Candida spp., and antibody levels in patients with psoriasis. Further studies are required to investigate the correlations between duration of disease, PASI score, carriage of Candida spp., and antibody levels in patients with psoriasis. However, the results of our study showed a higher rate of candidal colonization in patients with psoriasis in comparison with control subjects but a reduction in humoral immune responses in the patient group. Moreover, our results demonstrate that Candida spp. in patients with psoriasis are more diverse than in healthy subjects. Hence, the periodic determination of candidal colonization, the early screening of patients with hypercolonization of Candida spp., and the treatment of such patients with antifungal agents are recommended. Acknowledgment The authors would like to thank all patients for their kind cooperation in the completion of the study. References 1 Bijlmakers MJ, Kanneganti SK, Barker JN, et al. Functional analysis of the RNF114 psoriasis susceptibility gene implicates innate immune responses to double-stranded RNA in disease pathogenesis. Hum Mol Genet 2011; 15: 3129–3137. 2 van Beelen AJ, Teunissen MB, Kapsenberg ML, et al. Interleukin-17 in inflammatory skin disorders. Curr Opin Allergy Clin Immunol 2007; 7: 374–381. 3 Fraga NA, de F atima Paim M, Follador I, et al. Refractory erythrodermic psoriasis in a child with an
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excellent outcome by using etanercept. An Bras Dermatol 2011; 86: 144–147. Henseler T, Christophers E. Psoriasis of early and late onset: characterization of two types of psoriasis vulgaris. J Am Acad Dermatol 1985; 13: 450–456. Heller MM, Wong JW, Nguyen TV, et al. Quality-of-life instruments: evaluation of the impact of psoriasis on patients. Dermatol Clin 2012; 30: 281–291. Bataille V, Lens M, Spector TD. The use of the twin model to investigate the genetics and epigenetics of skin diseases with genomic, transcriptomic and methylation data. J Eur Acad Dermatol Venereol 2012; 26: 1067–1073. Fry L, Baker BS. Triggering psoriasis: the role of infections and medications. Clin Dermatol 2007; 25: 606–615. Mohandas V, Ballal M. Distribution of Candida species in different clinical samples and their virulence: biofilm formation, proteinase and phospholipase production: a study on hospitalized patients in southern India. J Glob Infect Dis 2011; 3: 4–8. Ben-Aryeh H, Blumfield E, Szargel R, et al. Oral Candida carriage and blood group antigen secretor status. Mycoses 1995; 38: 355–358. Scully EP, Baden LR, Katz JT. Fungal brain infections. Curr Opin Neurol 2008; 21: 347–352. Feja KN, Wu F, Roberts K, et al. Risk factors for candidemia in critically ill infants: a matched case–control study. Pediatr 2005; 147: 156–161. Macias ES, Pereira FA, Rietkerk W, et al. Superantigens in dermatology. Am Acad Dermatol 2011; 64: 455–472. Leibovici V, Alkalay R, Hershko K, et al. Prevalence of Candida on the tongue and intertriginous areas of psoriatic and atopic dermatitis patients. Mycoses 2008; 51: 63–66. Waldman A, Gilhar A, Duek L, et al. Incidence of Candida in psoriasis – a study on the fungal flora of psoriatic patients. Mycoses 2001; 44: 77–81. Flytstrom I, Bergbrant IM, Brared J, et al. Microorganisms in intertriginous psoriasis: no evidence of Candida. Acta Derm Venereol 2003; 83: 121–123.
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16 Leibovici V, Hershko K, Ingber A, et al. Increased prevalence of onychomycosis among psoriatic patients in Israel. Acta Derm Venereol 2008; 88: 31–33. 17 Lima HC, Kimball AB. Targeting IL-23: insights into the pathogenesis and the treatment of psoriasis. Indian J Dermatol 2010; 55: 171–175. 18 Liang YS, Wen HQ, Xiao R. [Serum levels of antibodies for IgG, IgA, and IgM against the fungi antigen in psoriasis vulgaris.]. Hunan Yi Ke Da Xue Xue Bao 2003; 28: 638–640. 19 Fredriksson T, Pettersson U. Severe psoriasis – oral therapy with a new retinoid. Dermatologica 1978; 157: 238–244. 20 Yamada Y, Makimura K, Merhendi H, et al. Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts. Jpn J Infect Dis 2002; 55: 122–125. 21 Kurtzman CP, Robnett CJ. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 1998; 73: 331–371. 22 Rosenberg EW, Noah PW, Skinner RB Jr. Psoriasis is a visible manifestation of the skins defense against micro-organisms. J Dermatol 1994; 21: 375–381. 23 Desnos-Ollivier M, Ragon M, Robert V, et al. Debaryomyces hansenii (Candida famata), a rare human fungal pathogen often misidentified as Pichia guilliermondii (Candida guilliermondii). J Clin Microbiol 2008; 46: 3237–3242. 24 Turk M, Montiel V, Zigon D, et al. Plasma membrane composition of Debaryomyces hansenii adapts to changes in pH and external salinity. Microbiology 2007; 153: 3586–3592. 25 Breuer U, Harms H. Debaryomyces hansenii – an extremophilic yeast with biotechnological potential. Yeast 2006; 23: 415–437. 26 van Tiel FH, Harmsen T, Wagenaar M, et al. Rapid determination of neutralizing antibodies to Semliki Forest virus in serum by enzyme immunoassay in cell culture with virus-specific monoclonal antibodies. J Clin Microbiol 1986; 24: 665–668.
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Evaluation of candidal colonization and specific humoral responses against Candida albicans in patients with psoriasis Mehdi Taheri Sarvtin1,2,3, PhD, Tahereh Shokohi3,4, PhD, Zohreh Hajheydari4,5, MD, Jamshid Yazdani6, PhD, and Mohammad T. Hedayati3,4, PhD
1 Department of Medical Mycology and Parasitology, School of Medicine, Jiroft University of Medical Sciences, Jiroft, Iran, 2 Department of Medical Mycology and Parasitology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran, 3Invasive Fungi Research Center, Mazandaran University of Medical Sciences, Sari, Iran, 4Department of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, 5Department of Dermatology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, and 6Department of Statistics, School of Health, Mazandaran University of Medical Science, Sari, Iran
Correspondence Mohammad T. Hedayati, PhD Department of Medical Mycology and Parasitology School of Medicine, Mazandaran University of Medical Sciences PO Box 48175-1665 Km18 Khazarabad Road, Sari 48175, Iran E-mail: [email protected]
Abstract Background Psoriasis is an inflammatory skin disease that can considerably affect a patient’s quality of life. Environmental and genetic factors, as well as superantigens and toxins from Candida species, may play various roles in the exacerbation and persistence of psoriasis. In the present study, we evaluated candidal colonization and specific humoral responses against Candida albicans in patients with psoriasis. Methods A total of 100 patients with psoriasis vulgaris and 50 healthy control individuals were enrolled in the study. Skin and oral specimens from all participants were cultured on CHROMagar Candida medium. Isolated yeast-like fungi were identified using the sequence of the D1/D2 domain of the 26S rRNA gene. Enzyme-linked immunosorbent assays (ELISAs) were used to detect immunoglobulin M (IgM), IgA, and IgG antibodies against C. albicans in sera of patients and healthy individuals. Results Candida species were isolated from the skin of 15% of patients and 4% of controls and from oral specimens of 60% of patients and 20% of controls. There was a significant difference in candidal colonization between patients and controls (P < 0.05). Serum IgM, IgA, and IgG levels against C. albicans were significantly lower in patients with psoriasis than in controls (P < 0.05). There was no significant association between serum levels of specific antibodies against C. albicans or the frequency of candidal colonization with the clinical severity of the disease (P > 0.05). Conclusions The results of the present study show a higher rate of candidal colonization in patients with psoriasis in comparison with controls and a reduction in humoral immune responses in patients.
Funding: This study was supported by Mazandaran University of Medical Sciences. Conflicts of interest: None.
Introduction Psoriasis is an immune-mediated, chronic, and non-contagious skin disease characterized by lesions covered with silvery scales and the infiltration of leukocytes into both the dermis and the epidermis.1,2 It affects 1–3% of the world’s population, especially those in the two age groups of 16–22 and 57–60 years.3,4 Most psoriasis patients have a diminished quality of life in comparison with healthy individuals, particularly with reference to sexual dysfunction, anxiety, depression, self-esteem, and nutritional condition.5 ª 2014 The International Society of Dermatology
The exact etiology of psoriasis remains unknown, but it is believed that a combination of several factors such as genetic predisposition and environmental and immunological factors contribute to the development of this disease.6 Among environmental factors, various microorganisms such as viruses (retroviruses, papillomaviruses, and endogenous retroviruses), bacteria (Staphylococcus aureus, Streptococcus pyogenes), and fungi (Candida spp., Malassezia spp.) are known to be associated with the provocation and exacerbation of psoriasis via the production of either toxins or their metabolites.7 Candida spp. represent some of the most important fungal colonizers International Journal of Dermatology 2014, 53, e555–e560
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Candida species in psoriasis
on the skin and mucosal surfaces of the body, such as in the genitourinary tract, oral cavity, and gastrointestinal tract.8,9 Candida spp. can cause a wide range of disorders, such as vulvovaginitis, oral thrush, and skin and diaper rash, as well as life-threatening diseases in immunocompromised patients.8–11 So far, over 200 species of Candida have been identified, among which Candida albicans, Candida glabrata, Candida tropicalis, and Candida parapsilosis are responsible for the majority of candidal infections.8,10 Depending on the species, Candida is capable of secreting different toxins and superantigens, which can provoke psoriasis.12 Several previous studies have aimed to investigate the prevalence of Candida colonization in different body sites of patients with psoriasis but have varied in their results.13– 16 Recent studies have considered psoriasis as an immunological skin disease that involves substantial changes in humoral immune response.17 In addition, it has been hypothesized that an abnormal response of the humoral immune system in patients with psoriasis attributable to microorganisms can alter the course of the disease.18 However, few studies have evaluated the humoral response against Candida spp. in patients with psoriasis. Accordingly, we assessed the colonization of Candida spp. of the skin and oral cavity and the specific humoral response against C. albicans in patients with psoriasis. Materials and methods Patients A total of 100 patients with psoriasis vulgaris and 50 healthy control subjects were enrolled in this study between January 2011 and March 2012. All patients enrolled in the study were either attending our facility for the first time or had discontinued medication for at least five months. Patients and control subjects completed a consent form giving their agreement to their participation in the present research. The study was approved by the ethical committee of Mazandaran University of Medical Sciences, Sari, Iran. Most of the patients were diagnosed on the basis of the findings of a clinical examination conducted by a dermatologist. Histopathology was used in cases of suspected disease. Control subjects were selected from among persons referred for cosmetic problems. People with diabetes, individuals who had used broad-spectrum antibiotics or steroids, and pregnant patients were excluded from the study. In order to assess the clinical severity of the disease, the Psoriasis Area and Severity Index (PASI) was calculated as elucidated by Fredriksson and Pettersson in 1978.19 Based on this definition, the clinical severity of psoriasis was categorized as mild (PASI scores of < 11), moderate (PASI scores of 11–49), or severe (PASI scores of > 50). International Journal of Dermatology 2014, 53, e555–e560
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Mycological investigation Samples were collected from the oral cavity and skin (lesions and normal skin adjacent to lesions) by swab and scalpel, respectively. Samples were obtained from all areas with lesions (flexures, trunk, extremities) in all patients. All of the samples were cultured on CHROMagar Candida medium (CHROMagar Microbiology, Paris, France). Isolated species of Candida were subcultured on Sabouraud’s dextrose agar containing chloramphenicol (SC) and incubated at 27 °C for four days. Molecular investigation
DNA extraction A part of fresh pure yeast colony obtained from three days of culture on SC was transferred to a microtube and suspended in 300 ll of lysis buffer (10 mM Tris [hydroxymethyl] aminomethane [Thermo Fisher Scientific, Inc., Waltham, MA, USA], 1 mM EDTA, 1% SDS, 100 mM NaCl [Merck KGaA, Darmstadt, Germany], 2% Triton X-100 [Sigma-Aldrich Corp., St Louis, MO, USA]), 300 ll of phenol : chloroform (1 : 1) solution and 0.3 g of 0.5-mm diameter glass beads, vortexed for five minutes, and centrifuged at 19,000 g for 10 minutes. Then, the supernatant was transferred to a new tube, mixed with the same volume of chloroform, and centrifuged at 19,000 g for 10 minutes, after which the supernatant was separated and transferred to a new microtube. For alcohol precipitation, 2.5 ml volumes of cold 96% ethanol were added and kept at 20 °C for three hours. After this period, the tube was centrifuged at 19,000 g for 10 minutes and the supernatant discarded. The precipitate was washed by adding 300 ll of cold 70% ethanol and centrifuged at 19,000 g for 10 minutes. The pellet was air-dried and resuspended in 50 ll of TE buffer (10 mM Tris, 1 mM EDTA) and stored at 4 °C until use.20
Polymerase chain reaction amplification, sequencing, and yeast identification Yeasts were identified to species level using sequence analysis of the D1/D2 domain of the 26S ribosomal RNA gene. For amplification of the D1/D2 domain, the external primers NL-1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and NL-4 (5′-GGT CCG TGT TTC AAG ACG G-3′)21 were used. The reactions were performed in an automatic thermal cycler (C1000 thermal cycler; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with initial denaturation at 95 °C for three minutes, followed by 35 cycles at 95 °C for 30 seconds, 56 °C for 30 seconds and 72 °C for 30 seconds, and a final extension at 72 °C for seven minutes. The quality of polymerase chain reaction (PCR) products was determined by electrophoresis in 1% (w/v) agarose gel in 19 TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA) at 80 V for two hours. GenRuler DNA ladder mix was used as a marker. Sequencing of the purified PCR products was performed using the primer NL-1. Sequences
ª 2014 The International Society of Dermatology
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were aligned to the 26S rRNA gene sequences obtained from the National Center for Biotechnology Information (NBCI)
statistical significance. Cronbach’s alpha was used to calculate the reliability of the PASI instrument. The correlations between
GenBank database (http://blast.ncbi.nlm.gov/Blast.cgi), and
carriage of Candida spp. and levels of antibodies against
yeast species were identified by searches using the
C. albicans and the severity of disease (PASI score) were
BLAST
algorithm.
examined using the chi-squared test and Pearson correlations, respectively.
Detection of anti-C. albicans antibodies Serum immunoglobulin G (IgG), IgM, and IgA levels were measured with enzyme-linked immunosorbent assay (ELISA) test kits (Genesis Diagnostics Ltd, Littleport, UK) according to the manufacturer’s instructions. Briefly, serum samples were diluted to 1 : 200 in sample diluent. Quantities of 100 ll of each standard, positive control and the diluted patient samples were dispensed into appropriate microplate wells coated with a purified C. albicans extract. After 30 minutes, the well contents were decanted and washed three times by an automatic ELISA washer (Washer MPW1; SCO Tech GmbH, Diagnostic, Germany). Then, 100 ll of conjugate was added to each well and incubated for 30 minutes at room temperature. The microplate wells were washed four times. A quantity of 100 ll of TMB (3,3′,5,5′-tetramethylbenzidine) substrate was dispensed into each well and incubated for 10 minutes at room temperature. The reaction was stopped by dispensing 100 ll of stop solution into each well. The optical density (OD) of each well was measured at 450 nm in a microplate reader (Bio-Rad 680; Bio-Rad Laboratories, Inc.).
Results In this study, 100 patients (44 male and 56 female patients; mean standard deviation [SD] age: 40.47 11.03 years [range: 12–72 years]) and 50 control subjects (22 male and 28 female subjects; mean SD age: 39.90 11.45 years [range: 13–63 years]) were examined. A Cronbach's alpha value of 0.7 indicated the reliability of the PASI tool for measuring the severity of psoriasis. PASI scores of < 11, 11–50, and > 50 were observed in 76%, 9%, and 15%, respectively, of the psoriasis patients. Overall, 63% of psoriasis patients and 24% of control subjects demonstrated colonization by yeast species (v2 = 20.28, P < 0.01). In all, 41% of patients and 22% of control subjects were colonized by only one yeast species, 14% of patients and 2% of control subjects were colonized by two different yeast species, and 8% of patients were colonized by three different yeast species (v2 = 25.04, P < 0.01). Isolated yeast species are listed in Table 1. Candida spp. were isolated from the oral cavity of 60 (60%) patients and 10 (20%) control subjects (v2 = 21.49, P < 0.01). Candida spp. were isolated from the skin of the trunk only in 15 (15%) patients and two (4%) control subjects (v2 = 4.01, P = 0.045). Samples from the scalp, flexures, and extremities were negative for Candida growth in culture. Candida parapsilosis was isolated from normal skin adjacent to lesions in one (1%) patient (v2 = 4.08, P = 0.047). A total of 45 (45%)
Statistical analysis Numbers of individuals with yeast growth were compared using the chi-squared test. Independent t-tests were used to compare the results of IgG and IgM assays in the two groups. P-values of < 0.05 were considered to indicate statistical significance. The results of IgA assays were analyzed using the Kruskal– Wallis test. Paired comparisons were made using the Mann– Whitney U-test. P-values of < 0.015 were considered to indicate
Table 1 Yeast species isolated from psoriasis patients and control subjects Oral specimens
Skin specimens
Yeast species
Patients, n (%)
Controls, n (%)
Patients, n (%)
Controls, n (%)
Candida albicans Candida glabrata Debaryomyces hansenii Pichia guilliermondii Candida tropicalis Candida dubliniensis Saccharomyces cerevisiae Candida parapsilosis Yarrowia lipolytica Dipodascus capitatus Issatchenkia orientalis Aureobasidum pullulans Total
45 10 10 6 4 4 4 3 2 2 2 2 94
3 4 0 0 1 0 0 3 0 0 0 0 11
9 2 2 2 0 0 0 0 0 0 2 0 17
2 (100%) 0 0 0 0 0 0 0 0 0 0 0 2 (100%)
(47.9%) (10.6%) (10.6%) (6.4%) (4.3%) (4.3%) (4.3%) (3.2%) (2.1%) (2.1%) (2.1%) (2.1%) (100%)
ª 2014 The International Society of Dermatology
(27.3%) (36.4%)
(9.1%)
(27.3%)
(100%)
(52.8%) (11.8%) (11.8%) (11.8%)
(11.8%) (100%)
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35 30
31 32 26
25
22
20 15
12
10
0
0–10.0
Patients without Candida species colonization Patients with Candida species colonization Control without Candida species colonization Control with Candida species colonization
30.1+
IgM level, U/mL Figure 1 Distribution of serum levels of immunoglobulin M (IgM) specific against Candida albicans in psoriasis patients and control subjects. Mean standard deviation IgM levels: patients, 9.96 6.85 U/ml; controls, 14.48 12.00 U/ml International Journal of Dermatology 2014, 53, e555–e560
0 0. –1 0
10 0. 0+
2 11
0
0
0
6 2
.1
20.1–30.0
0 0
6
80
10.1–20.0
0
4
30
0–10.0
2
88
0 30
3
45
14
11
0.
7 6
16
14
.0
10
5 0
21
14 6
52
25
20
12 11
10
30
0–
15
40
–8
20
20
30.1+
60
.1
25
0 0
Our results showed a significantly higher prevalence of Candida colonization in the oral cavity of patients with psoriasis compared with the control group. This result is
60
26
20.1–30.0
3 2
Figure 2 Distribution of serum levels of immunoglobulin A (IgA) specific against Candida albicans in psoriasis patients and control subjects. Mean standard deviation IgA levels: patients, 9.81 9.33 U/ml; controls, 13.18 4.74 U/ml
0
26
10.1–20.0
2
IgA level, U/mL
Subjects, %
30
Patients without Candida species colonization Patients with Candida species colonization Control without Candida species colonization Control with Candida species colonization
5 4
3
5
50
33 34
8
6
40 35
Patients without Candida species colonization Patients with Candida species colonization Control without Candida species colonization Control with Candida species colonization
40
0.
In this study, we investigated the associations between specific IgG, IgM, and IgA levels and the frequency of colonization with Candida spp. and clinical severity in patients with psoriasis in order to establish which factors are correlated with clinical severity.
44
45
–6
Discussion
50
.1
patients and three (6%) controls were colonized by C. albicans (v2 = 23.3, P < 0.01). Ten (10%) patients were colonized by only Debaryomyces hansenii (Candida famata) (v2 = 5.35, P = 0.02). Of patients with mild, moderate, and severe disease, respectively, 47%, 15%, and 1% showed colonization with yeast species (P = 0403). The mean level of IgM against C. albicans in patients and controls was 9.96 and 14.68 U/ml, respectively (P = 0.00). The mean level of IgG against C. albicans in patients and controls was 68.83 and 90.03 U/ml, respectively (P = 0.00). Mean levels of IgA against C. albicans in patients and control subjects were 9.81 and 13.18 U/ml, respectively (Z = 3.337, P= 0.001). Pearson correlation statistics for the association between levels of IgM, IgG, IgA, and disease severity were 0.049, 0.710, and 0.022, respectively (P > 0.05). Among participants with candidal colonization, 33% of patients and 12% of control subjects showed an IgM, level of < 10 U/ml (Fig. 1), 31% of patients and 6% of control subjects showed an IgA level of < 10 U/ml (Fig. 2), and 5% of patients and 4% of control subjects showed an IgG level of < 30 U/ml (Fig. 3).
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IgG level, U/mL Figure 3 Distribution of serum levels of immunoglobulin G (IgG) specific against Candida albicans in psoriasis patients and control subjects. Mean standard deviation IgG levels: patients, 68.83 22.59 U/ml; controls, 90.03 40.21 U/ml ª 2014 The International Society of Dermatology
Taheri Sarvtin et al.
incompatible with those of Leibovici et al.13 but is consistent with the findings of Waldman et al.14 Our results also showed a significantly higher prevalence of Candida spp. in the psoriatic lesions of patients with psoriasis vulgaris compared with adjacent normal skin in these patients and skin of the control group. Psoriasis is an immunological skin disease which reflects a humoral immune disorder, low cellular immune function, and a disturbance in the normal structure of the skin.17 These conditions can increase the carriage of Candida spp. in patients with psoriasis. It is suggested that superantigens and toxins released from Candida spp. activate the secretion of cytokines from T cells, keratinocytes, and inflammatory cells that contribute to the provocation and exacerbation of psoriasis.12 Moreover, Candida spp. can provoke psoriasis via the activation of complement system and neutrophil recruitment.22 By contrast with previous studies,13,14 we applied molecular methods in the identification of yeast species and thus identified a greater diversity of yeast species than in previous studies. In the present study, C. albicans was the most common species of Candida, both in the oral cavity and in the skin of patients with psoriasis, a finding that concords with those of other studies. Among the Candida spp., C. albicans is the most prevalent and is able to cause different diseases under predisposing conditions which include the humoral and cellular immune disorder8 seen in psoriasis.17 Thus, colonization by this species in patients with psoriasis should be considered to represent a risk factor for the exacerbation of psoriatic signs in skin. Debaryomyces hansenii (C. famata) is a hemiascomycetous yeast commonly found in dairy products and the environment, which rarely causes disease in humans.23 In the present study, D. hansenii was isolated from the oral cavity and skin in participants within the patient group. This species of Candida can adapt itself to differences in environmental pH and salinity by changing the sterol to phospholipid ratio of its plasma membranes.24 Thus it would seem that patients with psoriasis are predisposed to colonization by D. hansenii by the abnormal condition of their skin and mucous. Debaryomyces hansenii is capable of secreting antigens and different metabolites, including protease B, arginyl aminopeptidase, and toxin,25 which may provoke psoriasis. Therefore, further studies are required to show the possible roles of these metabolites in psoriasis. In the present study, IgM and IgA levels against C. albicans were significantly lower in patients than in control subjects. This result concords with those reported by Liang et al.,18 who also reported a higher level of IgG against C. albicans in patients with psoriasis than in control subjects, which differs from the equivalent finding in our study. In the present study, we found ª 2014 The International Society of Dermatology
Candida species in psoriasis
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a significant reduction in the level of IgG against C. albicans in patients with psoriasis in comparison with control subjects. Levels of antibodies against microbial agents are associated with a variety of factors, including the type of infectious agent and duration of infection.26 For this reason, we did not find any significant relationship between candidal colonization and levels of specific antibodies against C. albicans in patients or control subjects. However, the inability of the immune system to produce sufficient antibodies against Candida spp. in patients with psoriasis can help to increase candidal colonization in these subjects. Psoriasis is a chronic disease, and most of the patients included in the present study did not remember the exact date of onset of the disease. For this reason, we were unable to investigate the relationship between duration of disease, carriage of Candida spp., and antibody levels in patients with psoriasis. Most of the patients included in this study had a mild form of disease, and therefore we did not observe any significant association between PASI score, carriage of Candida spp., and antibody levels in patients with psoriasis. Further studies are required to investigate the correlations between duration of disease, PASI score, carriage of Candida spp., and antibody levels in patients with psoriasis. However, the results of our study showed a higher rate of candidal colonization in patients with psoriasis in comparison with control subjects but a reduction in humoral immune responses in the patient group. Moreover, our results demonstrate that Candida spp. in patients with psoriasis are more diverse than in healthy subjects. Hence, the periodic determination of candidal colonization, the early screening of patients with hypercolonization of Candida spp., and the treatment of such patients with antifungal agents are recommended. Acknowledgment The authors would like to thank all patients for their kind cooperation in the completion of the study. References 1 Bijlmakers MJ, Kanneganti SK, Barker JN, et al. Functional analysis of the RNF114 psoriasis susceptibility gene implicates innate immune responses to double-stranded RNA in disease pathogenesis. Hum Mol Genet 2011; 15: 3129–3137. 2 van Beelen AJ, Teunissen MB, Kapsenberg ML, et al. Interleukin-17 in inflammatory skin disorders. Curr Opin Allergy Clin Immunol 2007; 7: 374–381. 3 Fraga NA, de F atima Paim M, Follador I, et al. Refractory erythrodermic psoriasis in a child with an
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excellent outcome by using etanercept. An Bras Dermatol 2011; 86: 144–147. Henseler T, Christophers E. Psoriasis of early and late onset: characterization of two types of psoriasis vulgaris. J Am Acad Dermatol 1985; 13: 450–456. Heller MM, Wong JW, Nguyen TV, et al. Quality-of-life instruments: evaluation of the impact of psoriasis on patients. Dermatol Clin 2012; 30: 281–291. Bataille V, Lens M, Spector TD. The use of the twin model to investigate the genetics and epigenetics of skin diseases with genomic, transcriptomic and methylation data. J Eur Acad Dermatol Venereol 2012; 26: 1067–1073. Fry L, Baker BS. Triggering psoriasis: the role of infections and medications. Clin Dermatol 2007; 25: 606–615. Mohandas V, Ballal M. Distribution of Candida species in different clinical samples and their virulence: biofilm formation, proteinase and phospholipase production: a study on hospitalized patients in southern India. J Glob Infect Dis 2011; 3: 4–8. Ben-Aryeh H, Blumfield E, Szargel R, et al. Oral Candida carriage and blood group antigen secretor status. Mycoses 1995; 38: 355–358. Scully EP, Baden LR, Katz JT. Fungal brain infections. Curr Opin Neurol 2008; 21: 347–352. Feja KN, Wu F, Roberts K, et al. Risk factors for candidemia in critically ill infants: a matched case–control study. Pediatr 2005; 147: 156–161. Macias ES, Pereira FA, Rietkerk W, et al. Superantigens in dermatology. Am Acad Dermatol 2011; 64: 455–472. Leibovici V, Alkalay R, Hershko K, et al. Prevalence of Candida on the tongue and intertriginous areas of psoriatic and atopic dermatitis patients. Mycoses 2008; 51: 63–66. Waldman A, Gilhar A, Duek L, et al. Incidence of Candida in psoriasis – a study on the fungal flora of psoriatic patients. Mycoses 2001; 44: 77–81. Flytstrom I, Bergbrant IM, Brared J, et al. Microorganisms in intertriginous psoriasis: no evidence of Candida. Acta Derm Venereol 2003; 83: 121–123.
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16 Leibovici V, Hershko K, Ingber A, et al. Increased prevalence of onychomycosis among psoriatic patients in Israel. Acta Derm Venereol 2008; 88: 31–33. 17 Lima HC, Kimball AB. Targeting IL-23: insights into the pathogenesis and the treatment of psoriasis. Indian J Dermatol 2010; 55: 171–175. 18 Liang YS, Wen HQ, Xiao R. [Serum levels of antibodies for IgG, IgA, and IgM against the fungi antigen in psoriasis vulgaris.]. Hunan Yi Ke Da Xue Xue Bao 2003; 28: 638–640. 19 Fredriksson T, Pettersson U. Severe psoriasis – oral therapy with a new retinoid. Dermatologica 1978; 157: 238–244. 20 Yamada Y, Makimura K, Merhendi H, et al. Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts. Jpn J Infect Dis 2002; 55: 122–125. 21 Kurtzman CP, Robnett CJ. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 1998; 73: 331–371. 22 Rosenberg EW, Noah PW, Skinner RB Jr. Psoriasis is a visible manifestation of the skins defense against micro-organisms. J Dermatol 1994; 21: 375–381. 23 Desnos-Ollivier M, Ragon M, Robert V, et al. Debaryomyces hansenii (Candida famata), a rare human fungal pathogen often misidentified as Pichia guilliermondii (Candida guilliermondii). J Clin Microbiol 2008; 46: 3237–3242. 24 Turk M, Montiel V, Zigon D, et al. Plasma membrane composition of Debaryomyces hansenii adapts to changes in pH and external salinity. Microbiology 2007; 153: 3586–3592. 25 Breuer U, Harms H. Debaryomyces hansenii – an extremophilic yeast with biotechnological potential. Yeast 2006; 23: 415–437. 26 van Tiel FH, Harmsen T, Wagenaar M, et al. Rapid determination of neutralizing antibodies to Semliki Forest virus in serum by enzyme immunoassay in cell culture with virus-specific monoclonal antibodies. J Clin Microbiol 1986; 24: 665–668.
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