Molecular Diagnosis and Characterization of Malassezia species Isolated from Patients with Pityriasis versicolor and Healthy Individuals in Sudan

Abstract

Pityriasis versicolor is a chronic superficial fungal disease that is characterized by the appearance of round to oval lesions, most commonly found on the trunk and upper aspects of the arms. The objectives of this study were to isolate and characterize the cutaneous Malassezia in patients with pityriasis versicolor and healthy individuals, as well as to study the antifungal activity of common antifungal agents against the isolated Malassezia species. A total of 370 scale samples were collected (140 by moist cotton swabs and 230 by OpsiteTM transparent dressing to lesional and non-lesional sites) from patients suspected of having Pityriasis versicolor and healthy individuals in Khartoum and Gedarif States. Swabs samples (n=140) were cultured on Sabouraud’s Dextrose agar with Olive oil and the isolated colonies were identified on the base of colonial morphology, microscopic examination and biochemical tests. DNA was extracted from the yielded colonies and directly from the Opsite tapes, then amplified by PCR, sequenced and analyzed by real- time PCR using a TaqMan probe. Antifungal susceptibility of the isolates to nine antifungal including azoles and other antifungal agents (amphotericin B, micafungin, anidulafungin, fluconazole, itraconazole, 5-flucytosine, voriconazole, posaconazole and caspofungin) was tested via the Sensititre Yeast One system. Obtained data were analysed using SPSS programme. Twenty eight isolates (20%) were identified according to colonial morphology, microscopic examination and biochemical tests as Malassezia species while other 112 (80%) showed negative growth. After PCR amplification and DNA sequencing 18 (64.3%) out of 28 isolates were found to be Malassezia furfur, and 10 (35.7%) ruled out of Malassezia which were found as Pichia mandshurica, Cryptococcus albidus and Clavispora lusitaniae. DNAs of Malassezia globosa and Malassezia restricta were detected in 71.7% and 21.8%, respectively in P.V patients. The distribution of M. globosa and M. restricta in P.V patients were higher (60.9%) in 20-30 age group and lower (1.1%) in 61-70 age group. On the other hand, the distribution of M. globosa and M. restricta in healthy subjects, males and females were higher (38.6% and 56.2%) in 20-30 age, and lower (8.6% and 6.8%) in 61-70 age. The overall level of colonization by Malassezia at the lesional sites was higher than that at the non-lesional sites for all body sites, including the face, neck, cheeks, and trunk (2.7- to 6.0-fold increase) (P< 0.05). Posaconazole (POS), Voriconazole (VOR) and Itraconazole (ITZ) were highly active against isolates of M. furfur since all isolates were inhibited by 0.06-0.12 μg/mL, 0.015-0.03 μg/mL and 0.25 μg/mL respectively. Amphotericin B and caspofungin have high MICs and low in vitro activities against M. furfur isolates, since most isolates were inhibited by 8μg/ml of amphotericin B, 0.5μg /ml of caspofungin and 1 μg/mL of fluconazole. This study indicates that M. furfur strains were susceptible to ITZ, VOR and POS. The Sensititre Yeast One seems to be a suitable commercial tool for the antifungal susceptibility of Malassezia. Moreover, the study confirmed that both DNAs of M. globosa and M. restricta can be detected in healthy subjects, although M. restricta was predominant in both sexes with slight difference in colonization level between males and females. M. furfur was susccefully isolated from scale samples of both patients and healthy subjects while other species, M. globosa and M. restricta were not isolated on cultures and found only after molecular characterization by real- time PCR directly from the specimen. M. globosa was predominant in Sudanese patients with pityriasis versicolor. Further studies are required in different part to establish a solid base for research on the basic molecular biology of Malassezia fungus in Sudan.