P434 Anti fungal susceptibility of Malassezia to azoles by broth microdilution method and their phylogenetic relationship based on multi-locus sequence analysis

POSTER SESSION 3, SEPTEMBER 23, 2022, 12:30 PM - 1:30 PM: OBJECTIVES: To analyze the epidemiological pattern of disease with regards to age, sex, and site of predilection of skin infection. To identify the species of Malassezia causing infection by phenotypic methods. To conclude with the most effective anti-fungal drugs (azoles) to treat infections caused by the Malassezia by obtaining the MIC (Minimum inhibitory concentration) values in terms of MIC 50 and MIC 90. Molecular identification of the species of Malassezia by Sangers Sequencing. Comparison between phenotypic and genotypic methods of Identification. To do phylogenetic analysis to know the strain relatedness among Malassezia species isolated globally using Bio-informatics tools. METHOD: Part 1—Epidemiological pattern, phenotypic Identification, and anti-fungal susceptibility pattern by broth microdilution method: Type of study—cross-sectional study. Period of study—August 2016-October2018. Sample size—151 samples. After obtaining Institutional Ethical Clearance, skin scrappings were collected from patients and microscopic examination was done with 10% KOH. Samples were cultured and Phenotypic Identification was done based on colony growth characteristics, gram staining, urease test, and catalase test, bile esculin with tween 60 hydrolysis and tween assimilation. AFST was performed by the broth microdilution method, according to the Clinical and Laboratory Standard Institute (CLSI) guidelines M27-A3. Systemic antifungals used—fluconazole, itraconazole, and voriconazole. Topical antifungals used—clotrimazole, miconazole, sertaconazole, and luliconazole. Part 2-Molecular Identification, Sangers Sequencing, Phylogenetic Analysis and Dendogram Molecular Identification: 1. Extraction of DNA by in-house Phenol: Chloroform method. 2. Amplification of extracted DNA—using Internal Transcribed Spacer (ITS) region and Malassezia -specific nested primers. 3. Agarose Gel Electrophoresis. 4. Nucleic acid sequencing (Sanger's Sequencing)—The results were obtained as trimmed FASTA files and Chromatogram files. 5. Species Identification—The sequenced product was blasted in NCBI (National Centre for Biotechnology Information, USA) blast. 6. Phylogenetic analysis—. 7. DNA sequences were aligned using ClustalW in MEGA11 software. 8. The obtained sequences were concatenated using bioEdit software. 9. Phylogenetic analysis was done using maximum likelihood ratio and maximum parsimony analysis. 10. Phylogenetic Tree/Dendrogram drawn using the bootstrap method. RESULTS: Figure 1 Epidemiological pattern, phenotypic Identification, and AFST by broth microdilution Figure 2 Molecular Identification, Sangers Sequencing, Phylogenetic Analysis, and Dendrogram CONCLUSIONS: The most effective systemic anti-fungal with the least MIC values were itraconazole and voriconazole. Fluconazole shows resistant pattern with high MIC values. The most effective topical antifungals were clotrimazole with low MIC values compared with miconazole, sertaconazole, and lulliconazole. Malassezia sympodialis responded to antifungals better than other four species isolated. Speciation of Malassezia is important because of variation in resistant pattern to antifungals among different species. Phenotypic speciation showed M. sympodialis as the predominant species. Genotypic speciation showed M. furfur as the predominant species. Genotypic speciation is more confirmatory than phenotypic method of speciation in case of Malassezia. Among the primers used, NCBI blasting results were better with Pan fungal ITS primers than the Malassezia specific primers. Dendogram showed very good strain relatedness among other strains obtained from India placing them in same clade.