This cross-sectional, prospective study was carried out in the Department of Microbiology and the Department of ENT, Vydehi Institute of Medical Sciences and Research Centre, Bengaluru, Karnataka, India, from January 2015 to June 2016 (18 months). Ethical clearance was sought from the institutes ethics committee bearing no: VIMS & RC/IEC/016/2014-15. Patients of all age groups and either sex with clinical presentation of otomycosis, who gave their consent to be a part of the study, were included and those with other ear ailments (auricular cellulitis, chronic suppurative otitis media, and ear infections due to bacteria only) were excluded from the study. In cases of mixed infection (bacterial as well as fungal), only the fungal isolate was processed further.

Ear examination was done using an autoscope, the ear canal was wiped with 70% alcohol and allowed to air dry before sample collection. Patient details were recorded on pre-determined proforma. Any discharge or debris was collected on three swabs which were labeled and transported to the laboratory within 2 hours for further processing.

Swab 1 was used for Gram staining [13] and wet mount preparation using 10% potassium hydroxide (KOH) [14], swab 2 was inoculated on Blood agar and MacConkey agar and the third swab was inoculated onto two sets of Sabouraud’s Dextrose agar (SDA) which were incubated at 37°C and 25°C for 3-4 weeks. All the media was procured from Hi-Media, Mumbai, India. Any bacterial growth on Blood agar and MacConkey agar was not subjected to further processing. On SDA the following were observed: morphology of the colony, rate of growth and pigment production.

Any yeast like colony was subjected to Gram staining [13] germ tube test [13] inoculation on to Cornmeal agar (CMA), [13] and carbohydrate fermentation test [14] to identify and speciate the fungus. Lactophenol Cotton Blue (LPCB) mount [15] was made for fungal colonies on SDA resembling filamentous fungi. These were subsequently taken up for agar block slide culture and cavity side culture [12] Statistical analysis was done using one-way ANOVA (analysis of variance) method and a p-value of <0.05 was taken as significant.

A total of 120 cases that fulfilled the inclusion criteria were included in the study. Higher incidence of otomycosis (61.7%) was seen in rainy season (from August to December); (n=74; 61.7%) with maximum number of patients being in the age group of 31-40 years (n=39; 32.5%). The disease was more prevalent in females (n=79; 65.8%) with a 1:3 male to female ratio. Unilateral disease was the usual presentation (n=114; 95%) with the left side being affected more (n=79; 65.8%).

Majority of the patients (n=80; 66.7%) gave a history of habitual use of unsterile wooden sticks/metal wax pickers, whereas 48.3% (n=58) of patients used oil drops. Prior antibiotic and/or steroid use was seen in 25% (n=30) of cases. Tympanic membrane perforation was seen in 15% (n=18) of the patients. The most common otoscopic finding was the presence of moist brown green mouldy material with epithelial debris (n=60; 50%). Itching was the most common symptom (n=81; 67.5%) followed by otalgia (n=79; 65.8%) and ear discharge (n=37; 30.8%). CSOM was seen in 12.5% (n=15) of the cases whereas 9.2% (n=11) patients had ASOM associated with otomycosis.

Species of Aspergillus were the most common (n=97; 80.8%) whereas the Candida species (n=10; 8.3%) were the least common fungi isolated in patients with otomycosis [Table/Fig-1]. Isolation of Aspergillus species was statistically significant (p<0.05). Comparison of agar block method and cavity slide technique for the identification of fungi and proper appreciation of morphological characteristics is shown in [Table/Fig-2]. The amount of media used as well as the processing time and risk of contamination was less for cavity slide culture method. Most importantly the morphological features of fungi were intact and better appreciated in this method than the conventional agar block technique as shown in [Table/Fig-3,4,5 and 6].

[Table/Fig-3]:

Appearance of A. flavus on agar block (left) and cavity slide culture (right).

a) Day 3: Broken conidiphore, scattered conidial heads and vesicle not visible; b) Day 2: Intact conidiophore, clear biserate philaides covering ¾ of vesicle with hyline conidia

[Table/Fig-4]:

Appearance of A. fumigatus on agar block and cavity slide culture.

a) Day 3: Dbroken conidiophore, haphazard morphology.

b) Day 2: Clear vesicle, hyaline conidia covering 2/3 of vesicle, undisturbed uniseriate phialides

[Table/Fig-5]:

Appearance of A. niger on agar block and cavity slide culture.

a) Scattered Conidia’s disturbed conidial heads.

b) Clear large biseriate vesicle and irregular black conidia’s radiating from entire conidial head

[Table/Fig-6]:

Appearance of Penicillium spp on agar block and cavity slide culture.

a) Day 4: Condiophores not well defined, few visible condial phialides not appreciated clearly

b) Day 2: Claerly defined conidiophore, smooth phialides proper conidia’s in chains (brush broder appearance)

Otomycosis is a fairly common ear infection encountered in clinical practice. Owing to the varied microbiological flora (both bacterial and fungal) known to cause this clinical entity early diagnosis is of paramount importance to guide proper treatment. In the present study; majority of the patients were in the age group of 31 to 40 years and females were affected more than males. Similar findings have been reported by many authors [4,16,17]. Unilateral presentation and a higher incidence of otomycosis in rainy season as has been reported previously in many studies [16,18-20] was seen in our study as well. Habitual use of wooden sticks and metal wax pickers were found to be the predisposing factors causing damage to the ear canal. Likewise prior topical antibiotic use that alters defense mechanism facilitating fungal growth was also seen in many patients in our study. Findings similar to ours have been reported by other authors as well [16,18-21].

The commonest presentation of cases in this study was itching followed by otalgia and ear discharge. A study by Bhat KS et al., has reported otalgia followed by ear discharge to be the most common complaints in patients of otomycosis [22]. Agarwal P et al., in their study found that the most common presenting complaint was a blocking sensation in the affected ear followed by itching, pain, discharge and tinnitus [6]. Likewise, Gupta S et al., and Sangavi AKB et al., in their respective studies found itching to be a common presenting symptom [20,21].

Aspergillus species followed by Penicillium and Candida were isolated from all the cases of otomycosis. Among the Aspergillus species, A. flavus was the most common isolate followed by A. niger and A. fumigatus. C. glabrata was the most common species of Candida isolated from cases of otomycosis followed by C. albicans. Similar results have been reported previously by Agarwal P et al., where the authors found Aspergillus spp. (n=302; 87.3%) to be the predominant fungi isolated from the cases followed by Candida spp. (n=35; 10%) and Penicillium (n=2; 0.6%) [6]. Gupta S et al., in their study found A. niger (n=20; 51.3%), A. flavus (n=7; 17.9%), A. fumigatus and Candida spp (n=6; 15.4% respectively) to be the most common fungal isolates recovered from clinically suspect cases of otomycosis [20]. Likewise Sangavi AKB et al., have reported A. niger (n=15; 46.9%) followed by A. flavus (n=6; 18.8%) and Candida spp. (n=10; 31.3%) to be the most common fungal isolates recovered from 32 samples [21]. Penicillium species are generally considered as contaminants except Penicillium marneffei which is a pathogen in patients with AIDS or those receiving immunotherapy.

In the present study, on examination of Aspergillus and Penicillium species on the agar block culture, conidial arrangement was disturbed and very scanty whereas in comparison it was undisturbed and well preserved on the cavity slide culture. Earlier visualisation of fungal structures under both 10x and 40x objective was possible with cavity slide culture technique which was stained with LPCB using a micropipette without fully removing the coverslip. The mycelia growth occurs in a single plane along the undersurface of the cover slip in cavity slide culture thus providing clear visualisation of the fungi. With the agar block identification of fungal elements under the 40x objective was difficult due to its thickness. Also, growth and visualisation of fungi was appreciated on the third day on cavity slide culture whereas it was appreciated on the fourth day on agar block culture. Advantages of cavity slide culture include flexibility and relative ease of handling, quick turnaround time and lesser amount of media required (15 μL for cavity slide culture as compared to 12 ml for agar block culture).

Wijedsa MH et al., in their study found that using the cavity slide culture technique, earlier visualisation of structures that are required for the identification of fungi is possible [12]. In the agar block the cover slip has to be removed for staining with LPCB which invariably distorts the fungal structures and their arrangement. Also, repeated visualisation of the slide to observe different growth stages of the fungi is not possible. Furthermore, growth in the agar block takes place in various planes which can’t be better appreciated due to the thickness of the block.

Less number of cases (n=120) was a drawback of the present study. Also, the fungal isolates recovered from the cases of otomycosis were not very diverse; hence a generalisation regarding the advantages of cavity slide culture technique over agar block slide culture cannot be made. A study that tests the cavity slide culture technique for the identification different fungal groups can make us more wiser as to the utility of this method.

The present study highlights the increased prevalence of otomycosis in females as compared to males with majority of the cases occurring in the rainy season. Aspergillus species was the most common fungi isolated. Also, an increased incidence of non-albicans Candida was seen. Cavity slide culture technique gave a better and early identification of the fungi as compared to agar block slide culture and should be done routinely.