1. Introduction

Otomycosis or fungal otitis externa has typically been described as a fungal infection of the external auditory canal, auricle, and less frequently the middle ear [1]. It is estimated that approximately 10% of the world’s population will develop otitis externa at some point in their life [2,3]. A vast array of pathogens can lead to otitis externa amongst which are most commonly bacteria, fungi, and other pathogens. Fungal otitis externa (otomycosis) remains a prominent cause of otitis externa with less classical treatment options as the more common bacterial infections. Globally, the prevalence of otomycosis varies greatly by geographical region, sex, and age amongst other factors with a current global prevalence ranging between about 9-25% [4]. Although this disease is rarely life-threatening, it can present a challenging and frustrating situation for the otologist and patients due to long-term treatment and high rate of recurrence with some studies reporting recurrence rates up to about 7.3% [5]. Common complications include hearing loss, tympanic membrane perforations, and invasive temporal bone infection.

Several predisposing factors have been described, including humid climate, presence of cerumen, instrumentation of the ear, increased use of topical antibiotics/steroids preparations, immunocompromised host, patients who have undergone open cavity mastoidectomy, and those wearing hearing aids with occlusive ear molds [6]. In Sub-Saharan Africa, the prevalence of otomycosis is even higher at 39.6%, as described by several studies, and is favored by several factors such as the increase in number of immunocompromised patients, the humid climate and increased antibiotic and steroid abuse [7]. Few studies however have been carried out in Cameroon to date on this subject. In 1996, Lohdoue et al described a 6.09% prevalence in Yaoundé [8] with very little updates carried out on this subject in the country. We set an objective to describe the mycological profile and antifungal susceptibility testing in patients with otomycosis in Yaounde.

2. Methodology

We carried out a cross-sectional study from November 2020 to August 2021.  The study sites included Yaounde University Teaching Hospital (CHUY) and Yaounde Central Hospital (HCY) where samples were collected and analyzed.  The study population was a pool of samples at the study sites. Samples with incomplete information such as identification were excluded. One hundred samples were obtained through convenient sampling.

2.1 Study materials

2.1.1 Specimen collection material

1. Sterile cotton swab
2. Sterile distilled water

2.1.2 Materials for mycologic analysis

1. Sabouraud + chloramphenicol culture medium
2. Petri dishes, cotton blue, and other Laboratory reagents, equipment, and personnel
3. Optic microscope
4. Antifungal disks

2.2 Procedure

2.2.1 Specimen collection

-               Ear specimens were collected from the external auditory canal using sterile cotton after signing an informed consent form at the ENT (Ear, Nose, and Throat) services of HCY and CHUY

-               Specimens were collected in duplicate for each ear.

-               The specimens were immediately transported to the bacteriology laboratory of  CHUY  for analysis within two hours. The macroscopic exam was done to know the ordour, colour and consistency.

-               Afterwards, the specimens were inoculated on Sabouraud-chloramphenicol

2.2.2 Inoculation and incubation

-               Specimen from the two ear was inoculated on the culture medium Sabouraud-chloramphenicol which had initially undergone sterility checks.

-               Inoculation was done by gentle streaks on the surface of the sabouraud dextrose agar.

-               After inoculation, the inoculated culture media were incubated at 37° for 1 to 5 days.

-               After incubation collect for the wet mount

2.3 Wet mount

The specimen collected with the cotton swab was mounted on a new glass slide containing a drop of sterile normal saline. The mounted slide was examined with low magnification. The morphologic characteristics of the fungus, if present are recorded; presence of hyphae, and conidia distribution amongst others.

2.3.1 Observation of culture growth

-               Each day, the incubated media will be analyzed macroscopically for growth

-               Specimens that produce growth were described (color, texture, borders)

-               Specimens that fail to produce growth within 5 days were considered negative and discarded

2.3.2 Microscopy of growth colonies

-               Two drops of Lactophenol Cotton Blue were placed on the slide.

-               Two colonies of fungal culture were added to the drops of Lactophenol Cotton Blue.

-               The fungal colony and lactophenol cotton blue were mixed using forceps. A coverslip was placed on top of the preparation. The mixture was observed under the microscope at objective 40X. When yeasts were observed the test of filamentation was done, when the filamentous fungus was observed, its image was correlated with those in the reference atlas for identification.

2.3.3 Germ tube tests for identification of yeasts

-               The Germ tube test was used to identify yeast isolates. One colony of the yeast isolated from the culture was incubated in 5ml animal serum for 4 hours at 37°C.

-               After incubation, microscopy of a drop of the incubated mixture was done at low magnification

-               Microscopy that reveals germ tubes were considered positive for C. albicans. A germ tube is an appendage that is half the width and 3-4 times the length of the yeast cell that produced it. There is no point of constriction at the origin of the germ tube from the yeast cell.

2.3.4 Antifungal Susceptibility Testing

The antifungal susceptibility was done by the disc diffusion method for yeasts following the CASFM 2021 method. The macro-dilution method in liquid milieu was used for other species of filamentous fungi following the procedure described [44]. The antifungal susceptibility was determined by the disc diffusion method on an agar medium. The fungal inoculum was prepared from 24-hour culture and the turbidity was adjusted to 0.5McFarland standard which corresponds to a suspension of approximately 1 to 2×108 CFU/ml. Sabouraud chloramphenicol agar plates were then inoculated with a swab. Fifteen minutes later, the antifungal discs were deposited on the surface of the agar. The whole was left at room temperature for 15 minutes for pre-diffusion and then incubated at 37°C for 24 to 72 hours. Inhibition zones were measured using calipers.

As regards the determination of MIC, 1ml of Sabouraud broth + chloramphenicol was introduced into six test tubes, afterward a stock solution of 139.2ug/ml of antifungal was prepared, and a series of double dilution was done in the culture medium until obtention of a series of concentration varying from 69.6ug/ml to 2.17ug/ml. Next, 1ml of inoculum prepared at 1 to 2×108 CFU/ml was introduced into each tube until obtention of a series of concentrations of antifungal ranging from 34.8ug/ml to 1.08ug/ml. Simultaneously, one case-control tube containing Sabouraud and the inoculum and a negative case-control containing only the culture media were prepared under the same conditions. The tubes were incubated for 24-72 hours at 37°C. The Minimum Inhibitory Concentration (MIC) corresponds to the least concentration of antifungal capable of inhibiting all visible growth was determined after adding almar blue. Alamar Blue reduction may signify an impairment of cellular metabolism and is not necessarily specific to the interruption of electron transport and mitochondrial dysfunction. This change from oxidized to reduced state allows flexibility of detection where measurements can be quantitative as colorimetric and/or fluorometric readings (the latter being more sensitive) or qualitative as a visible change in color indicating the presence or absence of viable cells [45].

2.4 Data entry and statistical analysis

Data was collected in Microsoft Excel 2016 and analyzed using SPSS version 23. Continuous variables were described with measures of central tendency (mean, median, mode) and dispersion measures (variance and standard deviation) stating their 95% confidence intervals. Qualitative variables were described with frequencies and proportions and differences between proportions, verified using the independent Chi-square test. P-values of <0.05 were considered statistically significant.

2.4.1 Ethical considerations

The research proposal was submitted to the Institutional Review Board of the Faculty of Medicine and Biomedical Sciences, for an ethical clearance. Research authorization was obtained from the directorates of the institutions.

3. Results

Samples were taken from 100 participants, of whom 50 samples showed positive cultures for fungi. Of the 50 patients with otomycosis, 58.0% (29/50) were female,

Figure 1: Frequency of participants with positive cultures according to sex

The distribution of patients according to marital status showed that 42% of patients were married and 58% were single, Figure 2.

Figure 2: Frequency of participants according to marital status

The distribution of patients with otomycosis according to level of education (Figure 3), showed that patients with high school education were the most infected (46%) followed by patients with secondary education (42%) and patients with primary education (10%). Only 2% were unschooled.

Figure 3: Distribution of patients with otomycosis according to level of education

Table I represents the proportion of patients who had clinical features versus age.  Pruritus was the most common symptom, followed by otalgia. There was predominance of clinical features of patients between 15 and 19 years of age.

Table 1: Frequency of symptoms by age (years)

Figure 4 shows the proportion of otomycosis by age. Patients aged between 15 and 49 years represented over half (58%) followed by 49-65 age group.

Figure 4: Proportion of otomycosis with respect to age

3.1 Mycologic profile in participant with otomycosis

Figure 5 shows the mycologic profile of participants in the study. Candida albicans was the most predominant fungi isolated (38%).

Figure 5: Frequency of fungi type by species

3.2 Antifungal susceptibility profiles of fungi isolated from patients with otomycosis

The antifungal susceptibility profile was done on all the specimens to ascertain their clinical categorization. Figure 6 represents the antifungal susceptibility of Candida albicans to the various antifungals tested.

Figure 6: Susceptibility profile of Candida albicans to antifungals

Figure 7: Susceptibility profile of Candida spp to antifungals

Figure 7 shows the susceptibility of candida species to the antifungals tested. Candida spp most sensitive to nystatin.

Figure 8: Susceptibility profile of Aspergillus niger to antifungals

Figure 8 shows the antifungal susceptibility profile of Aspergillus niger to the various antifungals tested. Aspergillus was 100% sensitive to Amphotericin B.

Figure 9: Susceptibility profile of Aspergillus fumigatus to antifungals

Figure 9 shows the antifungal susceptibility profile of Aspergillus fumigatus to the antifungals tested.

3.3 Potential factors associated with otomycosis infection

Table II shows a statistically significant association between swimming, excessive cleaning of ears, and otomycosis. This means that people who engage in swimming and excessive cleaning of the ears are more likely to develop otomycosis than people who do not.

Table 2: Study of the association between risk factors and otomycosis

4. Discussion

4.1 Mycological profile

The main fungi isolated in these centres were Candida albicans 38%, Aspergillus niger 26%, Candida spp 22%, and Aspergillus fumigatus 14%.  The findings were different from those reported in several other countries in terms of predominant fungi causing otomycosis. In Germany, Khaled et al reported the most common species to be Aspergilus niger (41%), Aspergilus flavus (30%) and Candida albicans only made up about 3% [46]. Still in Europe, Turkey, Ozcan et al reported in one study the most common fungi to be Aspergilus niger (44.8%) [47]. Studies in south America, Asia are similar to those in Europe. They showed Aspergilus fumigatus and Aspergilus niger as predominating organisms causing ear infections [23,48,49]. In Nigeria, Fasunla et al in their study showed that the predominant organisms were Aspergilus niger and Aspergilus fumigatus at 48.36% and 33.96% respectively [50]. As shown in these studies, the results vary from country to country. The difference in results between countries could be explained by the differences in the ecological system, hence the importance to always do an antifungal sensitivity test. Also, in one recent Brazilian study, the most frequently isolated species were C. albicans (30%), C. parapsilosis (20%), A. niger (20%), A. flavus (10%), A. fumigatus (5%), C. tropicalis (5%), Trichosporon asahii (5%) and Scedosporium apiospermum (5%) [51]. This is similar to the results   of the present study.

4.2 Antifungal susceptibility testing

This study showed that nystatin was highly sensitive to most microorganisms tested with 86.6% of microorganisms being sensitive, followed by 46%, 36%, 36% for ketoconazole, amphotericin B and fluconazole respectively. Meanwhile, high resistance was noticed with voriconazole and amphotericin B at 58% and 56% respectively. In a 2020 study done in Iran, the most sensitive antifungal was terbinafine which has great activity whereas Fluconazole has low activity and 77.8% of candida were resistant to Caspofungin [52]. These antifungi were not routinely tested in our study. In a study in Germany by Ali et al, molds isolates had highest susceptibility to voriconazole whereas yeast highest sensitivity to Nystatin (88.24%) followed by Amphotericin B (82.35%) [46]. The results of sensibility to nystatin are similar to the present study. In this same study yeast was 100% resistant to terbinafine and 94.12% resistant to itraconazole [46]. The resistances are different from those described in this present study. The difference could be explained by local antifungal policies and local ecology.

4.3 Clinical presentation and associated factors

In the index study, otomycosis was more common between 15-49 years followed by 49 - 65 years than 0 - 15 years with prevalence of 58%, 22% and 12% respectively. While some series have reported otomycosis to be more common in children, in this study otomycosis was more common between adults compared to children. The results in this study are similar to those described in Iran by Prasad et al. [49]. This could be explained by the fact that adults spent more time outside and, in the farm, hence more likely to get in contact with these air-containing pathogens. In this series, these ear infections had varied clinical presentations with the most common presentations being pruritus (80%), otalgia (26%), tinnitus (26%), aural fullness (24%), and otorrhea (16%). The findings are similar to those published by Prasad et al [49] in Iran and Musa et al in Nigeria [53]. In a univariate analysis, factors associated to otomycosis included swimming and excessive ear cleanings. These factors were similar to those described by Prasad et al [49] in Iran. This could be explained by the fact that obsessive manipulation of the external ear with any hard objects such as wooden sticks or earphones to clean the ear of wax and vigorous rubbing of the ears for relief from itching (in case of otalgia due to eustachian catarrh, serous otitis, or acute otitis media) is a common practice. These practices may cause trauma (usually minor hence unnoticed) to the skin of external auditory canal and deposition of fungal conidia in the wound leading to fungal infection. Further, earphones are usually shared amongst populations hence increasing the risk of contamination.

5. Conclusion

The diagnosis of otomycosis is based on a high index of suspicion. Culture is an indispensable arm for confirmatory diagnosis and specific treatment options. Candida albicans was the most predominant followed by Aspergillus fumigatus and candida species. Nystatin was highly sensitive to most microorganisms tested, followed by ketoconazole, amphotericin B, and fluconazole respectively. Among predisposing factors were excessive cleaning of ears, and swimming.

Study Limitations

This study may not be completely representative of Cameroon, though the study area, Yaounde, receives patients coming from diverse zones. Hence, a larger study is recommended to get a broader representation. We recommend further studies be done with more advanced techniques that are more precise and accurate.

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