Title: -

A study on Mycological Profile, Phenotype Identification and Antifungal Susceptibility

Pattern of Fungus Causing Otomycosis with Emphasis on their Enzymatic Activity.

Introduction: -

Otomycosis is a superficial fungal infection affecting external auditory canal, mostly caused by

perforation of tympanic membrane. Mostly occur at tropical and subtropical climate with hot and

humid regions and dusty area. Common symptoms are itching, otalgia, hearing loss, tinnitus, and

aural discharge. Hearing loss and tinnitus are mostly due to obstruction of ear canal by aural

discharge or by fungal hyphae. It is a common medical disease and health hazard in India.

Several predisposing factors are present, such as profuse and random use of topical antibiotics,

unhygienic cleaning of ear, use of hot oil/water in the ear, use of hearing aid, swimming in defile

or polluted water, immunosuppressive diseases, etc. [1][8].

Wide range of fungi causes otomycosis but Aspergillus and Candida species are most common

type. Aspergillus species accounts for 75% of cases with A. niger (most common) followed by A.

flavus and then A. fumigates. Less frequently involving fungi are Penicillium, Mucor, Rhizopus,

Scopulariopsis, Alternaria, Malassezia, Fusarium. [2]

Fungi are known to describe extracellular enzymes based on the substrate they use for growth.

Production and secretion of hydrolytic enzymes are important factors for virulence. It is

sometimes very difficult to manage in terms of long-term care and follow up, it also has high

recurrence rate [3]. However detailed information on otomycosis is still limited in hospitals in

Indian population. Hence this study aimed to explain detailed mycological profile, phenotype,

identification, and antifungal susceptibility pattern with emphasis on their enzymatic activity.
Objectives: -

1.To isolate and identify fungal species causing otomycosis in suspected patients attending

otolaryngology OPD at Hi tech medical colleges and hospitals.

2.To perform antifungal susceptibility testing of fungal isolates.

3.To detect the production of different enzymes produced by the isolated fungus by phenotypic

methods.

Review of literature: -

Anusheela Howlader et al. reported out of 126 specimens collected, 92 (73.02%) were positive

for fungal growth by culture. The most common fungal isolates belonged to the species of

Aspergillus [3] .

Yenisehirli and Szigeti et al. reported resistance to fluconazole in all the Aspergillus strains

recovered from otomycosis with a high MIC value (≥256 and >64 µg/ml) [4].

Diekema et al. found that all their Aspergillus species were sensitive to itraconazole,

Miconazole, bifonazole, and econazole had a low MIC range against A. niger complex, A.

terreus complex, and A. nidulans complex strains. However, the MIC range was slightly higher

for A. flavus complex isolates [5].

Mohammed S. Alhussaini et al. reported out of 33 isolates which were chosen randomly to test

their ability to produce lipase enzyme, 25 (75.75%) were able to produce lipase but with variable

capabilities. High lipase production was exhibited by 8 isolates (32%) which were mainly

belonging to genus Aspergillus. Three isolates (12%) which include A. brasiliensis (AUMC

9396), A. flavus var. columnaris (AUMC 9393) and P. aurantiogriseum (AUMC9398) were
moderately able to produce lipase. Fourteen isolates (56%) which were belonging to Aspergillus,

Candida, Penicillium and Scopulariopsis genera exhibited low activity of lipase. Many

investigators have emphasized the ability of several Aspergillus strains belonging to A. niger, A.

flavus, A. parasiticus and A. terreus to produce extracellular lipases 43-46 found that

extracellular lipases play a role during microbial infections and suggested their role is to digest

lipids for nutrient acquisition by pathogenic microbe and that these enzymes help the microbe

(bacteria or fungi) to grow in environments where lipids are the sole carbon source [1].

Material and methods:

Study design: - Cross sectional study

Study settings: - Department of Microbiology and Department of ENT of Hi-tech Medical

College and Hospitals.

Study period: - March 2024 to September 2025

Study participants: - All patients attending otolaryngology OPD and admitted in ENT

ward in Hi-tech Medical Collages and Hospitals.

Study tool: - 10% KOH solution, SDA media, lactophenol cotton blue stain, Antifungal

susceptibility test using disc diffusion methods, proteolytic activity using test tube containing

modified casein hydrolysis medium, lipolytic activity using Ullman and blasins media.

Sample size: - 250.

Sampling: - randomized sampling method.

Selection criteria: -
 Inclusion criteria-

1) Patients with symptoms of itching of the ear, otalgia, scaling, inflammation, hole in

tympanic membrane, aural discharge, hearing impairment and tinnitus.

2) Patients who give their consents for study.

3) Patients age group above 10 yrs.

Exclusion criteria-

1) Patients with perforated tympanic membrane under treatment.

2) Patients with Chronic otitis media under treatment.

3) Those who refuses to give their consent for the study.

4) Children below 10 yrs. are excluded.

Methods of Data Collection: -

Ear examination will be done by using an otoscope then samples from the external

ear canal will be collected with the help of sterile cotton swabs under aseptic

conditions. Each sample will divide into two parts for fungal analysis. One ear swab

will be used for direct microscopy (Gram’s staining, 10% Potassium hydroxide

(KOH) wet mount) to identify yeast-like fungi, and 10% KOH mount, will be used to

identify filamentous fungi [3].

The second part of the specimen will be inoculated on the surface of two Sabouraud's

dextrose agar (SDA) with antibiotic slants, which will incubated at 37°C and 25°C

for two to three weeks. If growth observed, it will be identified by Lacto Phenol

Cotton Blue (LPCB) mount. Candida isolates will be morphologically identified by

 gram stain, germ tube and chlamydospore formation and then it has to be inoculated

HI-Chrome differential agar for species identification [3].

Anti-fungal susceptibility test is done by disc diffusion assay performed according to

Clinical and Laboratory Standards Institute (CLSI) guidelines (M44-A2-method for

antifungal disk diffusion susceptibility testing of yeast) to determine the susceptibility

of Candida isolates, for the filamentous fungi CLSI guideline(M51-A-Method for

antifungal disk diffusion susceptibility testing of non-dermatophyte filamentous

fungi) [7].

Test tubes containing modified casein hydrolysis medium will be used to see

proteolytic activity. The isolated fungus will be inoculated and incubated at 25°C for

7 days. Degradation of milk protein will be measured as depth of clear zone (mm) [1].

The medium of Ullman and Blasins will be used to see lipolytic activity. Tween 80

(10 ml) will be autoclaved separately and added to the sterile and cooled basal

medium. The medium will be dispensed aseptically in test tubes (10 ml/tube)

followed by inoculation of fungal isolates. After incubation at 25°C for 7 days, the

lipolytic enzymatic ability will be observed as a visible precipitate due to the

formation of crystals of calcium salt of the oleic acid liberated by the enzymes. The

depth of precipitate will be measured. (mm) [1].

Statistical Analysis: -

Statistical analysis will be conducted using M.S. Excell, SPSS (version-21). For
 statistical significance test like Chi-square and student t-test will be applied.

Ethical Consideration: - IEC clearance will be taken before the start of study.

Implications: -

1) Identifying fungal agents and host factors

involved in otomycosis can improve patients’ outcomes.

2) Early diagnosis and antifungal susceptibility can prevent complications involved in

delayed treatments of otomycosis.

3) Misuse of antifungal drugs can be prevented by early diagnosis.

References: -

1) Mohammed S. Alhussaini*, Noha F. El-Tahtawi and Mohammed I. Alghonaim.

Phenotype and Genotype Identification of Fungal Isolates in Otomycosis Patients with

Emphasis on their Enzymatic Activity. J pure and applied microbiol. 2015; Vol. 9(Spl.

Edn. 1): p. 567-578.

2) Sampath Chandra Prasad,1 Subbannayya Kotigadde,2 Manisha Shekhar,3 Nikhil

Dinaker Thada,1 Prashanth Prabhu,1 Tina D’ Souza,1 and Kishore Chandra Prasad.

Primary Otomycosis in the Indian Subcontinent: Predisposing Factors, Microbiology,

and Classification. Int J Microbiol. 2014; article id 636493.

3) Anusheela Howlader, Prithiviraj Nagarajan, Latha Ragunathan. Mycological Profile in

Otomycosis Patients and their Drug Sensitivity: A Cross-sectional Study at Union

Territory of Puducherry, India. J Cli Diag Res. 2022; Vol-16: DC11-DC15.

4) Yenisehirli G, Bulut Y, Guven M, Gunday E. In vitro activities of fluconazole,

itraconazole and voriconazole against otomycotic fungal pathogens. J Laryngol

Otol. 2009; 123(9):978–81.

5) Diekema DJ, Messer SA, Hollis RJ, Jones RN, Pfaller MA. Activities of caspofungin,

itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448

recent clinical isolates of filamentous fungi. J Clin Microbiol. 2003; 41(8):3623–6.

6) Ashopa V, Verma U, Nareda P, Gupta E, Prakash P. Assessment of risk factors and

microbial profile of otomycosis in patients attending tertiary level hospital of Western

Rajasthan, India. J Clin Diag Res. 2020;14(2): DC01-DC04.

7) Alastruey-Izquierdo A, Melhem MS, Bonfietti LX, Rodriguez-Tudela JL. Susceptibility

test for fungi: Clinical and laboratorial correlations in medical mycology. Rev Inst Med

Trop Sao Paulo. 2015;57(Suppl 19):57-64.

8) Prakash S B, Leelatejaswini R M, Deekshita V. A Clinical and Microbial Study of

Otomycosis: An Original Study. J Evol Med Dental Sci. 2015;4(71):12376–12384.