A 53-year-old man who has a history of CSOM in the right ear was suffering for more than 3 months. He had visited various healthcare facilities and hospitals. He was on an antibacterial drug from the last 10 days but was not benefited. Finally, he visited the department of ENT and Head and Neck Surgery OPD of a tertiary-care hospital and underwent clinical investigation and treatment in March 2016.
During his visit to the Department of Otorhinolaryngology-Head and Neck Surgery (ENT-HNS), Tribhuvan University and Teaching Hospital (TUTH), a medical team observed his ears with an otoscope and immediately spotted the ear infection with fungus, i.e., otomycosis. Furthermore, the doctor performed microscopic suction clearance of fungal mass. Subsequently, the use of topical antibiotic was discontinued.
For clinical investigation, we aseptically collected four swab samples from the right ear discharge and conducted further downstream assays, primarily microbiological investigations, Gram stain, KOH mount, and bacterial and fungal culture of aural discharge. The patient was advised for follow-up after a week for further treatment.
The first aural swab was processed for the preparation of Gram staining and potassium hydroxide (KOH) mount preparation. The second swab was inoculated into Robertson's cooked meat (RCM) medium, while the third was aliquoted into the nutrient broth (NB) and then the aliquots were inoculated into chocolate agar (CHOC), 5% sheep blood agar (BAP), and MacConkey medium (MAC), respectively. The fourth swab was utilized for the culture in Sabouraud dextrose agar (SDA), and clonal selection and subculture were conducted in specific media, i.e., malt yeast agar (MYA) and corn meal agar (CMA). All the clinical investigation procedures have been performed in accordance with the guidelines of the Clinical Microbiology Procedures Handbook, Volume 2 by Lynne S. Garcia, and Textbook of Medical Mycology, the Third Edition by Jagdish Chander. All the experiments were performed in triplicate utilizing the relevant positive and negative controls.
The Gram staining of the aural discharge revealed the presence of plenty of pus cells with fungal spores (conidia); however, no bacteria were detected (Figure 1).
The KOH mount exhibited the septate fungal hyphae with dichotomous branching (a typical feature of Aspergillus spp.) (Figure 2). For further microbial characterization and investigation, the aliquot of the culture was incubated for 18-24 hours in all three media plates, i.e., chocolate agar, blood agar, and MacConkey agar. The results indicated that there was no bacterial growth; however, fungal growth was observed in all three media plates (Figure 3).
To rule out bacterial a growth, Gram stain was performed with the colonies/growth from all three media plates. For the precise phenotypic characterization of the fungal growth, LPCB preparation was assessed and observed.
After 48 hours of incubation in RCM, the growth was further subcultivated into blood agar with disks of metronidazole plus gentamicin and cultured anaerobically for 48 hours. Pseudomonas aeruginosa (previously isolated in the department) was used as a control strain for aerobic culture and Clostridium sporogenes (previously isolated in the department) was used as a control for anaerobic culture in this study.
In the blood agar medium, no bacterial growth was observed; however, the light growth of the fungus was spotted which was conformed through Gram staining and LPCB mount preparation.
After 48 hours of incubation in an SDA medium, the fungal growth (Figure 4) was further inoculated into a corn meal agar (CMA) and cultured for 72 hours. The fungal colonies from SDA and CMA media were proceeded into the lactophenol cotton blue (LPCB) tease mount. The microscopy results from the LPCB mount revealed the presence of multiple conidia, conidiophores, vesicles, phialides, and metulae resembling Aspergillus spp. (Figure 5). Furthermore, we have also conducted the clonal selection and utilized Malt yeast agar (MYA) and CMA-specific culture media for the growth of Aspergillus spp. and successfully isolated and confirmed Aspergillus niger. From all microbial observations and analysis, the finding of the vital characterization of the specific biseriate structure of phialides and the black-coloured conidia phenotypically confirmed Aspergillus niger as the sole etiological agent for this CSOM.