CULTURE TO CLADE: DEMOGRAPHIC AND TEMPORAL INSIGHTS INTO OTOMYCOSIS USING CLASSICAL, MOLECULAR AND PHYLOGENETIC SEQUENCE ANALYSES

Abstract

Background: Otomycosis is a common superficial fungal infection of the external auditory canal, particularly in tropical and subtropical regions. However, long-term data combining epidemiological, conventional mycological, and molecular findings remain limited in Nigeria. This study assessed the distribution of recorded otomycosis cases over seven years and evaluated selected fungal isolates using conventional and molecular identification methods. Methods: A descriptive cross-sectional study incorporating retrospective review of fungal isolation records from January 2018 to December 2024 was conducted at the National Ear Care Centre, Kaduna, Nigeria. Demographic and microbiological data were summarized using descriptive statistics, while associations between categorical variables were assessed using the chi-square test. Selected stored fungal isolates available for laboratory investigation between April 2022 and September 2025 were identified by microscopy and culture, followed by PCR amplification of the internal transcribed spacer (ITS) region using ITS1 and ITS4 primers. Successful amplicons were sequenced and subjected to BLASTN analysis for molecular identification. Results: A total of 217 recorded otomycosis cases were reviewed. Females accounted for 148 (68.2%) cases and males for 69 (31.8%). The largest proportion of cases occurred among individuals aged 0–9 years (53; 24.4%), followed by those aged 10–19 years (50; 23.0%), with fewer cases recorded in older age groups. The number of recorded cases was highest in 2024 (62) and lowest in 2020 (6). Aspergillus flavus was the most frequently identified fungal species, followed by A. niger and Candida albicans. A significant association was observed between fungal diagnosis and gender (χ² = 13.93, p = 0.0075). Of five selected isolates subjected to molecular investigation, four produced PCR amplicons. Sequencing of isolate 612 produced a 523-bp sequence showing 99.05% identity, 100% query coverage and an E-value of 0.0 with A. flavus, confirming the conventional identification. Isolate 775, initially identified phenotypically as Candida tropicalis, produced a 537-bp sequence showing 99.07% identity, 100% coverage and an E-value of 0.0 with Meyerozyma caribbica. Isolate 574 yielded an approximately 45- bp sequence that was insufficient for reliable molecular identification, while isolate 641 was PCR-negative and isolate 670 was not sequenced. Conclusion: The study provides seven-year descriptive data on otomycosis in a Nigerian tertiary ear-care setting and demonstrates a predominance of cases among females and younger age groups. A. flavus was the leading fungal diagnosis. Molecular analysis complemented conventional identification and revealed a phenotypic–molecular identification discrepancy in one isolate, highlighting the value of molecular confirmation while also demonstrating limitations associated with PCR amplification and short sequence reads. These findings support the integration of conventional mycology with appropriately interpreted molecular methods for improved fungal identification and surveillance