**4. Treatment with antifungal agents**

#### **4.1 Anti-***Malassezia* **IgE in the serum of AD patients**

Skin prick tests positive for *Malassezia* antigen and specific IgE antibodies have been demonstrated in head and neck AD (HANAD) patients. A delayed-type hypersensitivity to *Malassezia* antigen also seems to play a role. Of 33 HANAD patients, 79% were prick-test positive for *Malassezia* antigen, but only 44% of 22 AD patients without head and neck involvement were prick-test positive (Kieffer *et al*. 1990). Rokugo *et al*. (1990) found that 71% of 35 AD patients who were prick-test positive for *Malassezia* antigen also demonstrated delayed hypersensitivity to *Malassezia* antigen in 64% of 118 AD patients. The presence of anti-*Malassezia* IgE antibody has been demonstrated in several studies (Table 3). The frequency of *Malassezia* specific IgE antibody in serum was higher in AD patients with head and neck dermatitis than without. For example, Bayrou *et al*. (2005) found IgE antibodies against *Malassezia* antigen in 100% of 106 HANAD patients, but in only 28% of 25 AD patients without head and neck involvement. Total IgE levels were also significantly higher in the AD group with head and neck dermatitis (mean, 2,823 kU/L) than without (546 kU/L).

full-length cDNA encoding MGp42 has been cloned and sequenced by the rapid amplification of cDNA ends method. MGp42 exhibits properties similar to those of heat shock protein (hsp) family members, and evidence indicates that MGp42 may be a cleavage product of intact HSP70. However, no IgE cross-reactivity has been observed between MGp42 and recombinant human HSP70, suggesting that the epitopes of MGp42 recognized by serum IgE of AD patients are masked by steric hindrance in the presence of intact HSP70 and become exposed

**Allergens Function Species Reference** 

Mala s1 Unknown *M. sympodialis* Schmidt et al. 1997 Mala f2 peroxisomal protein *M. furfur* Yasueda et al. 1998 Mala f3 peroxisomal protein *M. furfur* Yasueda et al. 1998 Mala f4 Malate dehydrogenase *M. furfur* Onishi et al. 1999 Mala s5 peroxisomal protein *M. sympodialis* Hemmann et al. 1997 Mala s6 Cyclophilin *M. sympodialis* Hemmann et al. 1997 Mala s7 Unknown *M. sympodialis* Weichel et al. 2002 Mala s8 Unknown *M. sympodialis* Weichel et al. 2002 Mala s9 Unknown *M. sympodialis* Weichel et al. 2002 Mala s10 Heat shock protein *M. sympodialis* Lindborg et al. 1999 Mala s11 MnSOD *M. sympodialis* Lindborg et al. 1999 Mala s12 GMC oxidoreductase *M. sympodialis* Rasool et al. 2000 Mala s13 Thioredoxin *M. sympodialis* Limacher et al. 2007 MGp42 Heat shock protein *M. globosa* Ishibashi et al. 2009

Skin prick tests positive for *Malassezia* antigen and specific IgE antibodies have been demonstrated in head and neck AD (HANAD) patients. A delayed-type hypersensitivity to *Malassezia* antigen also seems to play a role. Of 33 HANAD patients, 79% were prick-test positive for *Malassezia* antigen, but only 44% of 22 AD patients without head and neck involvement were prick-test positive (Kieffer *et al*. 1990). Rokugo *et al*. (1990) found that 71% of 35 AD patients who were prick-test positive for *Malassezia* antigen also demonstrated delayed hypersensitivity to *Malassezia* antigen in 64% of 118 AD patients. The presence of anti-*Malassezia* IgE antibody has been demonstrated in several studies (Table 3). The frequency of *Malassezia* specific IgE antibody in serum was higher in AD patients with head and neck dermatitis than without. For example, Bayrou *et al*. (2005) found IgE antibodies against *Malassezia* antigen in 100% of 106 HANAD patients, but in only 28% of 25 AD patients without head and neck involvement. Total IgE levels were also significantly higher in the AD group

with head and neck dermatitis (mean, 2,823 kU/L) than without (546 kU/L).

as a result of conformational changes during HSP70 cleavage.

Table 2. *Malassezia* allergens

**4. Treatment with antifungal agents** 

**4.1 Anti-***Malassezia* **IgE in the serum of AD patients** 


Table 3. *Malassezia* IgE antibodies in sera of patient with atopic dermatitis

There was also a significant correlation between the level of *Malassezia* specific IgE antibody and clinical severity criteria, as reflected by the SCORAD index (*p* < 0.0001, *r2* = 0.55), whereas total IgE showed only a slight correlation with severity criteria (*p* < 0.001, *r2*=0.29). No correlation was found between age or gender, and specific or total IgE. Based on pricktest results and specific IgE antibody levels, treatment of HANAD patients with antifungal agents has been recommended for the previous two decades.

#### **4.2 Susceptibility of** *Malassezia* **to drug treatment**

Compared with the plethora of antibacterial agents, only a small number of antifungal agents are available, which limits the treatment options for HANAD. Ketoconazole and itraconazole are highly effective *in vitro* (Sugita *et al*. 2005, Miranda *et al*. 2007, Sancak *et al*. 2005, Velegraki *et al*. 2004). In a large-scale study using 125 strains of 11 *Malassezia* species, all of the *Malassezia* species were highly susceptible to both itraconazole and ketoconazole, with minimum inhibitory concentrations (MICs) ranging from 0.016 to 0.25 mg/ml; approximately 80% of the strains had a MIC of ≤0.03 mg/ml (Sugita *et al*. (2005). This efficacy is not specific to these species, but applies to all members of the genus *Malassezia*. To our knowledge, no resistant strain has been detected. Ketoconazole and itraconazole are chemically classified as azole compounds, but other azole agents, fluconazole, voriconazole, and terbinafine, cannot inhibit the growth of *Malassezia*.

A calcineurin inhibitor, topical tacrolimus, is widely used to treat AD. This compound had antifungal activity against half of the known *Malassezia* strains, with MICs of 16–32 mg/mL (Sugita *et al*. 2005). The immunosuppressive drugs cyclosporine and tacrolimus target calcineurin and are also toxic to *Candida albicans* and *Cryptococcus neoformans* (Cruz *et al*. 2001). A combination of either ketoconazole or itraconazole and tacrolimus had a synergistic effect against *Malassezia* strains, based on a fractional inhibitory index of 0.245 to 0.378. These observations follow earlier reports on the effectiveness of a combination of tacrolimus and fluconazole against *C. albicans* and *C. neoformans* strains. The combination of topical tacrolimus and an azole agent can simultaneously treat AD and reduce the number of exacerbating *Malassezia* cells colonizing the skin surface. Although the synergistic mechanism of this combination is not known, Maesaki *et al*. (1998) demonstrated that tacrolimus increases the intracellular concentration of the azole agent in *C. albicans*. This observation may provide the basis for future clinical trials of these agents aimed at reducing the number of *Malassezia* cells colonizing the skin of AD patients (more details are provided in the following section).

Atopic Dermatitis and Skin Fungal Microorganisms 135

improved clinical scores and reduced the levels of *Malassezia* specific IgE, particularly in the head and neck area (Bäck *et al*. 1995). However, in a double-blind study with 53 HANAD patients, no difference in the clinical score was detected between those treated with miconazole-hydrocortisone cream and ketoconazole shampoo and those treated with hydrocortisone cream and placebo shampoo, although the ketoconazole group showed decreased *Malassezia* colonization (Broberg and Faergemann 1995). In another randomized double-blind placebo-controlled study comparing treatment with 200 mg ketoconazole daily *versus* placebo for 3 months in 29 HANAD patients with specific IgE antibodies to *Malassezia*, the clinical score decreased in both groups, and the improvement was correlated with the use of topical steroids in the control group, but not in the ketoconazole group (Bäck

A number of studies have also been conducted with itraconazole. In one study, 53 HANAD patients were divided into three groups that received 200 mg itraconazole, 400 mg itraconazole, or placebo daily (Svejgaard *et al*. 2004). The study included a 7-day treatment period and a follow-up period of 105 days. At days 7 and 14, a significant improvement was observed in the SCORAD of the head and neck area in the groups given 400 and 200 mg itraconazole daily. At day 14, a comparison among all three groups showed a significant improvement in the SCORAD of the head and neck area in the 200 mg itraconazole group compared with the placebo group. A randomized double-blind crossover study was also conducted (Ikezawa *et al*. 2004). One group was treated with a combination of itraconazole (100 mg daily) plus a conventional *Lactobacillus* preparation for 8 weeks, followed by the *Lactobacillus* preparation alone for 8 weeks. The other group received the *Lactobacillus* preparation alone for 8 weeks, followed by itraconazole (100 mg daily) plus *Lactobacillus* for 8 weeks. In both groups, a decrease in the dose or strength of concomitant topical steroids was observed at the end of the treatment course with itraconazole, and improvements in the eosinophil count, serum IgE, and fungi-specific IgE were found after

Itraconazole appears to be a promising treatment option for HANAD patients who do not respond to conventional therapeutic approaches. To optimize the selection of patients most likely to respond to itraconazole treatment, the levels of *Malassezia* colonization of the skin

This study was supported in part by a research grant from the Japan Society for the Promotion of Science (TS), a research grant for "High-Tech Research Center Project" from

Albanesi C, Scarponi C, Giustizieri ML, Girolomoni G. 2005, Keratinocytes in inflammatory

Amaya M, Tajima M, Okubo Y, Sugita T, Nishikawa A, Tsuboi R. 2007, Molecular analysis of *Malassezia* microflora in the lesional skin of psoriasis patients. *J Dermatol.* 34, 619-624.

the Ministry of Education, Culture, Sports, Science, and Technology (TS).

skin diseases. *Curr Drug Targets Inflamm Allergy.* 4, 329-334.

Ashbee HR. 2007, Update on the genus Malassezia. *Med Mycol.* 45, 287-303.

and Bartosik 2001).

the administration of itraconazole.

**5. Acknowledgment** 

**6. References** 

and specific IgE antibody should be evaluated.


Table 4. Treatment for ketoconazole or itraconazole in HANAD patients

#### **4.3 Ketoconazole and itraconazole in AD treatment (Table 4)**

A relationship between *Malassezia* and AD was first suggested by Clemmensen and Hjorth (1983), who demonstrated that oral ketoconazole was efficacious in adult HANAD patients with positive prick tests for *Malassezia*. A study of 20 AD patients with positive radioallergosorbent test results for *Malassezia* showed that treatment with oral ketoconazole

20 AD patients with positive RAST to *Malassezia*

53 HANAD patients

29 HANAD patients with specific IgE antibodies to *Malassezia*

34 AD patients with positive RAST to *Malassezia*

53 HANAD patients

Group B: 400 mg itraconazole

A relationship between *Malassezia* and AD was first suggested by Clemmensen and Hjorth (1983), who demonstrated that oral ketoconazole was efficacious in adult HANAD patients with positive prick tests for *Malassezia*. A study of 20 AD patients with positive radioallergosorbent test results for *Malassezia* showed that treatment with oral ketoconazole

Group C: placebo

Table 4. Treatment for ketoconazole or itraconazole in HANAD patients

**4.3 Ketoconazole and itraconazole in AD treatment (Table 4)** 

**patients Dosage** 

weeks

weeks

200 mg daily for 2 months and 200 mg twice weekly for further 3 months.

Group B: hydrocortisone cream and placebo shampoo for 6

Group A: 200 mg ketoconazole

Group B: placebo for 3 months

Group A: 200 mg itraconazole

Group A: 100 mg daily of itraconazole and lactobacillus preparation for 8 weeks and lactobacillus preparation alone

daily for 3 months

for further 8 weeks

for 7 days

for 7 days

Group B: Lactobacillus preparation alone for 8 weeks and 100 mg daily of itraconazole and lactobacillus preparation for additionally 8 weeks

Group A: miconazolehydrocortisone cream and ketoconazole shampoo for 6

**Authors Drug Study design Number of** 

study

Randomized double-blind placebocontrolled study

Randomized double-blind placebocontrolled study

Randomized double-blind crossover study

Randomized double-blind placebocontrolled study

<sup>1995</sup> Ketoconazole Open-label

Ketoconazole shampoo

Ketoconazole

Bäck et al.

Broberg and Faergemann,

1995

Bäck and Bartosik, 2001

Ikezawa

Svejgaard

et al. 2004 Itraconazole

et al. 2005 Itraconazole

improved clinical scores and reduced the levels of *Malassezia* specific IgE, particularly in the head and neck area (Bäck *et al*. 1995). However, in a double-blind study with 53 HANAD patients, no difference in the clinical score was detected between those treated with miconazole-hydrocortisone cream and ketoconazole shampoo and those treated with hydrocortisone cream and placebo shampoo, although the ketoconazole group showed decreased *Malassezia* colonization (Broberg and Faergemann 1995). In another randomized double-blind placebo-controlled study comparing treatment with 200 mg ketoconazole daily *versus* placebo for 3 months in 29 HANAD patients with specific IgE antibodies to *Malassezia*, the clinical score decreased in both groups, and the improvement was correlated with the use of topical steroids in the control group, but not in the ketoconazole group (Bäck and Bartosik 2001).

A number of studies have also been conducted with itraconazole. In one study, 53 HANAD patients were divided into three groups that received 200 mg itraconazole, 400 mg itraconazole, or placebo daily (Svejgaard *et al*. 2004). The study included a 7-day treatment period and a follow-up period of 105 days. At days 7 and 14, a significant improvement was observed in the SCORAD of the head and neck area in the groups given 400 and 200 mg itraconazole daily. At day 14, a comparison among all three groups showed a significant improvement in the SCORAD of the head and neck area in the 200 mg itraconazole group compared with the placebo group. A randomized double-blind crossover study was also conducted (Ikezawa *et al*. 2004). One group was treated with a combination of itraconazole (100 mg daily) plus a conventional *Lactobacillus* preparation for 8 weeks, followed by the *Lactobacillus* preparation alone for 8 weeks. The other group received the *Lactobacillus* preparation alone for 8 weeks, followed by itraconazole (100 mg daily) plus *Lactobacillus* for 8 weeks. In both groups, a decrease in the dose or strength of concomitant topical steroids was observed at the end of the treatment course with itraconazole, and improvements in the eosinophil count, serum IgE, and fungi-specific IgE were found after the administration of itraconazole.

Itraconazole appears to be a promising treatment option for HANAD patients who do not respond to conventional therapeutic approaches. To optimize the selection of patients most likely to respond to itraconazole treatment, the levels of *Malassezia* colonization of the skin and specific IgE antibody should be evaluated.

### **5. Acknowledgment**

This study was supported in part by a research grant from the Japan Society for the Promotion of Science (TS), a research grant for "High-Tech Research Center Project" from the Ministry of Education, Culture, Sports, Science, and Technology (TS).

### **6. References**

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**9** 

*Japan* 

**Fungus as an Exacerbating Factor** 

Takuji Nakashima and Yoshimi Niwano

*Kitasato University, Tohoku University* 

**of Atopic Dermatitis, and Control of** 

**Fungi for the Remission of the Disease** 

Atopic dermatitis (AD) is a common, chronic fluctuating skin disease with prevalence in children (Williams, 2000; Williams & Wüthrich, 2000). The disease is an inflammatory skin disorder characterized by itching, and chronically relapsing course. Moreover, it also produces vulnerablity to surface infections caused by pathogenic bacteria, fungi and viruses. The most common skin infections in AD patients are caused by *Staphylococcus aureus* and herpes simplex virus (Ong & Leung, 2010). *S. aureus* is frequently detected in AD patients (Abeck & Mempel, 1998; Katsarou & Armenaka, 2011) and becomes an aggravating factor. In addition, toxins, such as staphylococcal enterotoxins and toxic shock syndrome toxin-1 (McFadden et al., 1993; Bunikowski et al., 1999), generated from *S. aureus* may act as superantigens (Herz et al., 1999; Niebuhr et al., 2011; Yeung et al., 2011). In AD patients, viral infection is most often caused by herpes simplex virus (HSV) (Wollenberg et al., 2003). Eczema herpeticum is a potentially life-threatening disseminated HSV type 1 or type 2 infection that occurs in 10% to 20% of AD patients (Peng et al., 2007). However, not only bacteria and viruses but also fungi, such as *Malassezia* species and *Candida* species, may play an important role as aggravation factors in AD patients. It has been reported that antifungal therapy is beneficial in the treatment of some AD patients (Bäck et al. 1995; Svejgaard et al. 2004; Broberg et al. 1995; Mayser et al., 2006). In addition, several candidate *Malassezia* antigens have been implicated in the pathogenesis of AD. In this chapter, the involvement of

The genus *Malassezia* has recently been shown to consist of fifteen species based on the database of National Center for Biotechnology Information (2011), one lipid-independent species, *M. pachydermatis* and fourteen lipid-dependent species, *M. sympodialis*, *M. furfur*, *M. globosa*, *M. obtusa*, *M. restricta*, *M. slooffiae*, *M. caprae*, *M. equine*, *M. dermatis*, *M. equi*, *M. japonica*, *M. nana*, *M. yamatoensis* and *M. cuniculi*. *Malassezia* species have been recognized as members of the microbiological ora of human and animal skin. *M. globosa* and *M. restricta* are frequently isolated from the skin scales of human AD (Sugita et al., 2001; Tajima et al., 2008; Kaga et al., 2009) and *M. pachydermatis* and *M. nana* are isolated from some animals (Aizawa et al., 2001; Hirai et al., 2004). Antifungal drugs, e.g. ketoconazole and itraconazole,

**1. Introduction** 

fungi in the pathogenesis of AD is discussed.

**2. Fungi isolated from AD patients and treatment** 

