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Keywords:

  • atopic eczema dermatitis syndrome;
  • Malassezia furfur;
  • Candida albicans;
  • mannan;
  • IgE;
  • skin prick test

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References

Background:  Elevated and correlative Malassezia furfur (M. furfur) and Candida albicans (C. albicans) mannan-specific IgE have been demonstrated in atopic eczema dermatitis syndrome (AEDS) of the head, neck and shoulder (HNS) region of the skin. The significance of these antibodies in vivo has not been demonstrated.

Methods:  Sixty-five AEDS patients with HNS distribution were included. Serum total IgE (S-IgE) and yeast antigen-specific (Cetavlon-purified mannan and whole extract antigens of M. furfur and C. albicans) IgE were measured and skin prick tests (SPT) were performed with the yeast antigens.

Results:  Mannan-specific IgE and SPT were positive in 51 and 48% of patients with M. furfur and in 42 and 22% with C. albicans, respectively. Whole extract-specific IgE and SPT were positive in 85 and 95% of patients with M. furfur and in 91 and 57% with C. albicans, respectively. The highest correlation between specific IgE and SPT was seen with M. furfur mannan (r = 0.60; P < 0.0001). Both M. furfur mannan-specific IgE (r = 0.76; P < 0.0001) and SPT (r = 0.44; P = 0.0005) correlated with S-IgE.

Conclusions:  Mannan-induced immediate hypersensitivity in vivo was demonstrated in SPT. The significant correlation between M. furfur mannan-specific IgE and SPT suggests that mannan is an important allergen in yeast hypersensitive AEDS in vivo.

Atopic eczema dermatitis syndrome (AEDS) is a chronic inflammatory skin disease characterized by elevated specific serum IgE levels and positive skin prick tests (SPT) to a variety of allergens. Immediate hypersensitivity to saprophytic Malassezia yeasts is typical of AEDS involving the seborrhoeic, i.e. head, neck and shoulder (HNS), region of the skin (1–4). Specific IgE antibodies have been detected against a wide range of protein (9–96 kDa) and recombinant allergens (rMal s1, rMal s5, rMal s6) of Malassezia (4–6). In addition, immediate hypersensitivity reactions (specific IgE) to Candida albicans (C. albicans) protein antigens (27, 37, 43 and 46 kDa) are frequent among AEDS patients (7–9). In few existing studies with simultaneous measurements of specific IgE and SPT reactions to crude or protein yeast antigens, 45–100% of AEDS patients showed specific IgE antibodies and 40–60% were SPT positive to Malassezia (6, 10–13) whereas specific IgE and positive SPT reaction to C. albicans were found in 41–60 and 27–52% of AEDS patients, respectively (8, 9, 11).

Parallel positive SPT reactions to several saprophytic and airborne yeasts have been proposed to indicate allergenic cross-reactivity (14). Malassezia furfur (M. furfur) and C. albicans have been shown to cross-react at the IgE level with mannan, a polysaccharide, being an important cross-reactive allergen in yeast hypersensitive AEDS patients (15–17).

Until now, type I immediate hypersensitivity to M. furfur or C. albicans mannan has not been studied in vivo with SPT. In this study, we compared an in vitro (specific IgE) and an in vivo (SPT) method in measuring immediate hypersensitivity to both carbohydrate and whole extract (protein-rich) antigens of yeasts. We examined whether specific IgE and SPT results with mannan and whole extract antigens of M. furfur and C. albicans correlated and whether these results matched with serum total IgE (S-IgE), clinical severity of eczema (SCORAD) (18) or therapeutic outcome of antifungal treatment. Our main goal was to demonstrate that mannan is capable of inducing immediate hypersensitivity reaction in vivo.

Patients

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References

The patients (n = 65) participating the study were adult voluntary outpatients from the Department of Dermatology of the University Central Hospital of Turku (Finland). All of them gave a written informed consent. They also attended the previously published antifungal treatment study (19). The included patients had specific IgE antibodies and positive SPT reaction to M. furfur mannan (n = 24 and n = 31, respectively), M. furfur whole extract (n = 55 and n = 62, respectively), C. albicans mannan (n = 19 and n = 14, respectively) or C. albicans whole extract antigen (n = 59 and n = 37, respectively). AEDS was established by a specialist in dermatology according to the criteria of Hanifin and Rajka (20) and EAACI position statement (21) with eczema typically located in the HNS region. The clinical severity of eczema was evaluated by calculating the SCORAD score as earlier described (18, 19).

Preparation of antigens

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References

Malassezia furfur (CBS 7854) was grown on agar plates on a medium containing reduced-fat cream supplement as previously described (5, 22). The crude cytoplasmic extract was prepared after disruption of the yeast cells in X-Press chamber (AB Biox, Gothenburg, Sweden) as previously reported (5). Candida albicans whole extract was the In House Reference (IHR) preparation described earlier (23). Malassezia furfur and C. albicans mannan antigens were purified according to the Cetavlon method (24, 25).

IgE determinations

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References

S-IgE values were analyzed by Pharmacia IgE RIA (radioimmunoassay) (Uppsala, Sweden) and specific IgE levels against M. furfur and C. albicans crude antigens by Pharmacia CAP-RAST (radioimmunosorbent test). IgE antibodies to M. furfur and C. albicans mannan were analyzed by nitrocellulose RAST as earlier described (5, 25).

Skin prick testing

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References

The final SPT extracts were prepared by reconstituting the yeast antigens with ALK diluent (ALK, Hørsholm, Denmark) and 50% glycerol (Sigma, St Louis, MO, USA) and sterile-filtering with 0.8/0.2 μm Acrodisc® (Pall Gelman Laboratory, Ann Arbor, MI, USA). SPT was performed according to EAACI position paper (26). Antigen concentration of 1 mg/ml was used for testing. Histamine-HCl (10 mg/ml) served as a positive and ALK diluent as a negative control. Results were evaluated after 15 min and expressed as indices (mean wheal diameter/mean histamine wheal diameter) (19).

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References

Forty-two of the patients were females and 23 were males. Mean age (SD) was 31.9 (10.2) years, mean S-IgE (SD) was 2825 (2683) kU/l and mean SCORAD score (SD) was 44.1 (12.9). The percentages of M. furfur and C. albicans mannan and whole extract antigen-specific IgE and SPT positive AEDS patients are presented in Table 1. Positive results and higher levels in mannan-specific IgE and mannan and whole extract SPT were seen more often with M. furfur than C. albicans. In the whole extract-specific IgE there were no differences between M. furfur and C. albicans in frequencies of positive results. This was the only assay in which positive reactions to C. albicans were seen frequently.

Table 1.  Specific IgE and SPT values of AEDS patients (n = 65) and percentages of positive results
  1. * [Sample (cpm) − buffer (cpm)] × 102/[total activity (cpm) − buffer (cpm)].

  2. † Mean (SD).

  3. ‡ Mean wheal diameter/mean histamine wheal diameter.

Mannan-specific IgE (index)*% Positive (>0.32)
M. furfur2.3 (3.8)† (n = 45)51
C. albicans0.85 (1.5) (n = 45)42
Whole extract-specific IgE (kU/l)% Positive (>0.35 kU/l)
M. furfur19.3 (20.1)85
C. albicans15.2 (19.5)91
Mannan SPT (index)‡ % Positive (>0.50)
M. furfur0.41 (0.40)48
C. albicans0.17 (0.30)22
Whole extract SPT (index)% Positive (>0.50)
M. furfur0.98 (0.34)95
C. albicans0.47 (0.35)57

A correlation analysis was performed between specific IgE, SPT and S-IgE results. The specific IgE and SPT results of each antigen preparation had a significant or trend-like correlation (Table 2). Malassezia furfur mannan-specific IgE and SPT correlated better than those of C. albicans mannan. When whole extract antigen results were compared, C. albicans-specific IgE and SPT had higher correlation.

Table 2.  Correlation between yeast antigen-specific IgE and SPT results
Antigenr*Pn
  1. * Correlation coefficient.

  2. † Spearman's rank order correlation test.

Malassezia furfur
Whole extract0.36†0.004265
Mannan0.60<0.000145
Candida albicans
Whole extract0.59<0.000165
Mannan0.270.07545

All the specific IgE results correlated significantly (r = 0.52–0.73; P < 0.0005; n = 45) whereas in SPT comparison significant correlations were only noticed between the mannan antigens (r = 0.33; P = 0.0076; n = 65) and the mannan and crude antigen of the same yeast species (r = 0.39–0.48; P < 0.002; n = 65).

S-IgE was found to correlate significantly with all the specific IgE results, higher correlation seen with mannans. Of the SPT results, only mannans correlated significantly with S-IgE (Table 3).

Table 3.  Correlation between yeast antigen-specific IgE and SPT results and serum total IgE (S-IgE)
AntigenSpecific vs S-IgESPT vs S-IgE
r*PnrPn
  1. * Correlation coefficient.

  2. † Spearman's rank order correlation test.

Malassezia furfur
Whole extract0.74†<0.0001650.0900.4765
Mannan0.76<0.0001450.440.000565
Candida albicans
Whole extract0.64<0.0001650.210.08865
Mannan0.73<0.0001450.250.04865

Severity scoring of atopic dermatitis (SCORAD) or therapeutic outcome of antifungal treatment did not correlate with mannan-specific IgE and SPT results (data not shown).

The data of M. furfur mannan SPT and IgE are presented in Fig. 1.

image

Figure 1. Correlations between Malassezia furfur mannan-specific IgE and skin prick test (SPT) and serum total IgE (S-IgE) results in atopic eczema dermatitis syndrome patients (Spearman's rank order correlation test). Regression lines are shown. Mannan-specific IgE and SPT indices are calculated as presented in Table 1.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References

This is the first report demonstrating in vivo immediate hypersensitivity reactions in SPT with polysaccharide allergen, mannan. To our knowledge this is also the first time that mannan SPT were compared with specific IgE results in AEDS patients, correlative specific IgE and SPT results have earlier been presented only with the crude antigens of M. furfur (r = 0.47) and C. albicans (r = 0.38) (9). It was shown that the specific IgE and SPT results of each yeast antigen preparation correlated, M. furfur mannan results better than those of whole extract antigen, suggesting specific IgE-binding. The in vivo allergenicity of mannan was further supported by the facts that M. furfur mannan SPT was positive in 48% of AEDS patients and that M. furfur and C. albicans mannan SPTs had a significant correlation with S-IgE and with each other. Previously, it was suggested that IgE antibodies directed against allergens containing cross-reactive carbohydrate determinants (CCD) had poor biological activity as these glycoallergens neither released histamine nor yielded positive SPT reactions thus resulting in false-positive specific IgE values (27, 28). However, since then, the biological activity and clinical relevance of CCD-specific IgE has been re-evaluated and anti-carbohydrate IgE containing sera have been shown to induce histamine release and lose that ability if the allergen was exposed to periodate oxidation prior to IgE binding and histamine release assay (29–31).

It was previously shown that M. furfur induces a higher IgE production than C. albicans which is a more potent IgG-inducer (32). This is in line with the present finding that specific IgE and SPT results of M. furfur mannan correlated better with themselves and with S-IgE than those of C. albicans. In addition, previously, the specific IgE level against C. albicans (protein-rich) whole extract antigen was found to be higher than against purified mannan polysaccharide in line with the good correlation between specific IgE and SPT results of whole extract antigen noticed in this study (33).

Malassezia furfur (r = 0.42 − 0.74) and C. albicans (r = 0.66 − 0.84) specific IgE have been shown to correlate with S-IgE (5, 34–37). In this study all the specific IgE results associated significantly with S-IgE and, more importantly, M. furfur and C. albicans mannan SPTs also correlated with S-IgE, which is a novel finding. To assess the possible interfering role of high S-IgE in specific IgE and SPT correlations (38), patients with S-IgE >10 000 kU/l (n = 2) were not included in the study and the rest of the patients were divided into three groups on the basis of S-IgE [S-IgE <500 (n = 17), 500–4000 (n = 27) and >4000 (n = 21) kU/l]. No significant differences in correlations between groups were detected (data not shown).

The merely moderate correlations between specific IgE and SPT results found in the present study may be a consequence of methodological, antigen- and/or patient-dependent factors. Assays for specific IgE, including RAST, are in vitro tests where the antigen is coupled on a paper or nitrocellulose disc or sponge and recognized by specific IgE in the serum, whereas SPT is an in vivo test in the skin where the mast-cells with FcεRI-bound IgE are the main effector cells in immediate hypersensitivity reaction. Yeast antigens possess several IgE binding epitopes and it might be possible that the epitopes recognized in vitro and in vivo could differ. In addition, with similar levels of specific IgE the amount of allergen required for a positive SPT result may differ 100-fold between patients (39). Therefore, the lack of threshold end-point dilution in skin prick testing may have affected especially the C. albicans mannan results of this study. On the contrary, gastrointestinal tract colonizing C. albicans could be better recognized in the mucosal barrier than in the skin where it is rarely found (8). The lack of correlation between SCORAD score or antifungal treatment outcome and mannan-specific IgE and SPT results might indicate that also non-IgE-mediated responses appear during the change of clinical course of mannan-sensitive AEDS.

Based on statistically significant correlations between yeast antigen-specific IgE and SPT results, it can be concluded that in our patients M. furfur, and especially its mannan, could be a clinically important yeast antigen aggravating AEDS and that C. albicans antigens could be for a large part cross-reactive. Previous ELISA- and RAST-inhibition studies where M. furfur allergens were the most potent fluid-phase inhibitors support this presumption (16, 17). Furthermore, there were no correlations between whole extract antigen SPT – unlike specific IgE – results or between mannan and whole extract SPTs of different yeast species suggesting SPT to be more specific than specific IgE in assessing immediate hypersensitivity reactions toward allergens used in this study. Therefore, both tests are recommended for optimal estimation of sensitization.

Malassezia furfur mannan SPT correlated with M. furfur mannan-specific IgE and S-IgE showing the first evidence of the histamine-releasing capacity of yeast mannan polysaccharide-specific IgE. These findings suggest that M. furfur mannan is an important allergen in yeast hypersensitive AEDS involving the HNS region of the skin.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References

This study was supported with grants from Finnish Allergy Research Foundation, Finnish Society of Allergology and Immunology, Väinö and Laina Kivi Foundation, Turku University Foundation, Research Foundation of Orion Corporation, Turku University Central Hospital Fund and National Graduate School of Clinical Investigation. We thank Leena Kavén-Honka for excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Patients
  5. Preparation of antigens
  6. IgE determinations
  7. Skin prick testing
  8. Statistical analysis
  9. Results
  10. Discussion
  11. Acknowledgments
  12. References
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