Fungal coexistence in the skin mycobiome: a study involving Malassezia, Candida , and Rhodotorula

**Background:** The skin microbiome includes commensal fungi such as Malassezia, Candida, and Rhodotorula, which can become pathogenic under dysbiosis. Fungal co-infections are rare but can complicate treatment, especially when multiple species coexist. This study reports a case of triple fungal coexistence in a patient with acne vulgaris (AV) and pityriasis versicolor (PV).

**Methods:** A 19-year-old healthy female presented with a one-year history of reddish hypopigmented asymptomatic lesions on the face and chest. Clinical examination revealed multiple erythematous papules and hypopigmented scaly macules. She was diagnosed with AV and PV and treated with fluconazole, minocycline, isotretinoin, clotrimazole, and other agents, but experienced relapses. Skin scrapings from the neck were collected and incubated on modified Dixon’s agar with chloramphenicol at 32°C. Three distinct colony types were purified and subjected to lactophenol cotton blue and DAPI staining, biochemical tests (catalase, urease, bile esculin, tween assimilation), PCR amplification of the ITS region using primers ITS3 and ITS4, RFLP with AluI, and Sanger sequencing of the ITS1 region. Reference strains included Malassezia furfur MTCC 1374 and a confirmed Candida parapsilosis isolate.

**Key Results:** Three fungal strains were isolated: Strain 1 (whitish), Strain 2 (pale yellow), and Strain 3 (pinkish). Biochemical tests showed all three were catalase-positive, urease-positive, and bile esculin-positive, but only Strain 2 showed tween assimilation. PCR produced bands of approximately 300 bp, 550 bp, and 350 bp. RFLP patterns matched Malassezia furfur (Strain 2) and Candida parapsilosis (Strain 1) compared to controls. Sequencing identified Strain 3 as Rhodotorula mucilaginosa (99% identity, 98% query cover, GenBank OR214976). Candida parapsilosis showed 99% identity (85% query cover, OR243746), and Malassezia furfur showed 97% identity (97% query cover, OR243747). The patient had no immunocompromising conditions or prior corticosteroid use.

**Clinical Implications:** This is likely the first report of Malassezia furfur, Candida parapsilosis, and Rhodotorula mucilaginosa coexisting in the same skin lesion. The finding underscores that fungal co-infections can occur in immunocompetent individuals and may contribute to treatment failure and relapse. Accurate species identification using molecular methods (PCR-RFLP and sequencing) is essential for selecting appropriate antifungals, as different species may have varying susceptibilities. Clinicians should consider the possibility of polymicrobial fungal involvement in recurrent or refractory skin infections.