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

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,

Fungus as an Exacerbating Factor

species may be related to the severity of AD.

**2.2 Control of fungi in AD patients** 

double-blind, placebo-controlled study.

**3. Fungal infection in animals with AD** 

inflammation in AD patients was reported (Klein et al., 1999).

administration is expected to be highly effective in treating AD patients.

of Atopic Dermatitis, and Control of Fungi for the Remission of the Disease 143

other AD patients (mild and moderate) and healthy subjects. Since the species-specific DNA of *M. globosa* and *M. restricta* were frequently and massively detected, the two *Malassezia*

Besides the *Malassezia* species, *Candida* species and dermatophytes are also involved in the pathogenesis of AD, and especially *C. albicans* may play a role in the alimentary canal of AD patients, becuase *Candida* species have been cultured more frequently from the gastrointestinal tract in AD patients than healthy subjects (Arzumanyan et al., 2000; Savolainen et al., 2003). Moreover, the possible involvement of dermatophytes, especially *Trichophyton rubrum*, in the

Ketoconazole and itraconazole, azole antimycotics, have been the most frequently studied therapeutic agents for AD. The antimycotics showed strong antifungal activities against *Malassezia* species isolated from AD patients *in vitro* (Sugita et al. 2005). In clinical studies, ketoconazole and itraconazole have shown a significant therapeutic effect on AD patients. Bäck et al. (1995) assessed the efficacy of oral ketoconazole treatment on 20 AD patients using a positive radioallergosorbent test. The AD patients were treated with ketoconazole 200 mg daily for 2 months and 200 mg twice a week for another 3 months. Of the 20 patients, 18 completed the ketoconazole treatment regimen for 5 months and most patients showed a good to moderate response for ketoconazole 200 mg daily during the 2 months but no further improvement after the administration of ketoconazole 200 mg twice a week for another 3 months. Svejgaard et al. (2004) evaluated the efficacy of oral itraconazole in the treatment of AD patients with head and neck dermatitis in a randomized, double-blind, placebo-controlled study. The AD patients were treated daily with itraconazole 200 mg, 400 mg or placebo for 7 days. The treatment with 200 mg and 400 mg of itraconazole exerted a remarkable therapeutic effect on AD patients. Therefore, the systemic antimycotic

Meanwhile, the application of topical antimycotics could decrease *Malassezia* colonization and the severity of eczematous lesions in AD patients. For instance, as reported by Broberg et al. (1995), the treatment of AD patients who had head and neck dermatitis with twicedaily miconazole-hydrocortisone cream and twice weekly ketoconazole shampoo for 4 weeks resulted in decreased *Malassezia* colonization although clinical scores were not greatly improved. In addition, they confirmed the effect of ciclopiroxolamine on AD patients with moderate to severe head and neck dermatitis, which is often difficult to be treated, in a

Fungal infection in animals with AD has been reported mainly in canines and felines. For instance, Morris et al. (2002) reported that cell-mediated and humoral reactivities to *M. pachydermatis* contribute to the pathogenesis of AD in dogs but are not directly correlated. They investigated whether the potential cell-mediated immune response of atopic dogs to the yeast *M. pachydermatis* is correlated with the type-1 hypersensitivity (humoral) response of the same population of dogs. Atopic dogs with cytologic evidence of *Malassezia* dermatitis had an increased lymphocyte blastogenic response to crude *M. pachydermatis* extract, compared with clinically normal dogs and dogs with *Malassezia* otitis. The blastogenic responses in atopic

are used in AD patients with signs of a fungal infection (Sugita et al., 2005; Bäck et al., 1995). Antifungal therapy may remit the severity of AD by controlling these *Malassezia* yeasts.

#### **2.1 Related pathogenic fungi**

The yeasts of the genus *Malassezia* are members of the normal cutaneous flora. However, *Malassezia* colonization on the skin of AD patients shows a different pattern from that on healthy skin (Faergemann, 2002; Gupta et al., 2001; Nakabayashi et al., 2000; Sandström et al., 2005; Sugita et al., 2004, 2006) and may aggravate AD due to an allergic reaction, especially on the head and neck area in adults (Brehler & Luger, 2001; Broberg et al., 1992; Faergemann, 1999; Huang et al., 1995; Jensen-Jarolim et al., 1992; Lintu et al., 1997; Rokugo et al., 1990; Schmidt et al., 1997; Nakabayashi et al., 2000; Savolainen et al., 2001; Scalabrin et al., 1999). Scalabrin et al. (1999) measured total IgE and specific IgE to *Malassezia furfur* in 73 AD patients. In the AD patients, specific IgE to *M. furfur* was observed more frequently in adults than children. The reaction of specific IgE to *M. furfur* was 132 times higher than that in healthy subjects. This result suggests that *Malassezia* yeast is associated with IgE-mediated skin inflammation in AD.

Culture-dependent methods have been used for the detection of *Malassezia* species from AD patients (Nakabayashi et al., 2000; Sandström et al., 2005). However, in recent years, many researchers have attempted the detection of *Malassezia* species from AD patients by means of a molecular-based culture-independent method that is not affected by the isolation medium, sampling method, or incubation period. Table 1 summarizes the three major studies applying molecular based PCR assay to detect *Malassezia* species from AD patients and healthy subjects, indicating that the number of detected *Malassetia* species was similar to AD patients and healthy subjects (Sugita et al., 2001; Tajima et al., 2008; Kaga et al., 2009).


\* Number of cases. \*\* Percentage of the number of patients. AD, atopic detmatitis; HS, healthy subjects. -, not detected.

Table 1. Comparison of published research on *Malassezia* colonization in AD patients and healthy subjects.

In both AD patients and healthy subjects, the predominant species were *M. globosa* and *M. restricta*. However, the study by Kaga et al. (2009), who applied real-time PCR to determine the number of rDNA copies of *M. globosa* and *M. restricta*, revealed that *Malassezia* colonization in severe AD patients was approximately two to five times higher than that in other AD patients (mild and moderate) and healthy subjects. Since the species-specific DNA of *M. globosa* and *M. restricta* were frequently and massively detected, the two *Malassezia* species may be related to the severity of AD.

Besides the *Malassezia* species, *Candida* species and dermatophytes are also involved in the pathogenesis of AD, and especially *C. albicans* may play a role in the alimentary canal of AD patients, becuase *Candida* species have been cultured more frequently from the gastrointestinal tract in AD patients than healthy subjects (Arzumanyan et al., 2000; Savolainen et al., 2003). Moreover, the possible involvement of dermatophytes, especially *Trichophyton rubrum*, in the inflammation in AD patients was reported (Klein et al., 1999).
