Keywords
otomycosis - yeast - aspergillus - antifungal agents - candida - dermatophytes
Introduction
Otomycosis is a common condition, frequently encountered in otolaryngology outpatient
clinics. It is defined as a fungal infection affecting the external ear canal in the
majority of cases. Less commonly, it can involve the middle ear, if the drum is perforated,
and the mastoid cavity following an open mastoidectomy. Common associated symptoms
include itching, pain, aural fullness, aural discharge, hearing impairment and tinnitus.[1]
[2]
[3]
Various types of fungal species were encountered in otomycosis. However, the genus
Aspergillus seemed to be the most common causative agent, particularly Aspergillus niger. Other fungal agents include species from the genera Penicillium, Fusarium, Mucoraceae, Scopulariopsis, Alternaria, Malassezia, and Candida, as well as various dermatophytes.[4]
[5]
[6] Common risk factors for otomycosis are poor hygienic conditions; minor trauma; inflammation
or physical injury; use of swimming pools; being exposed to hot humid atmospheres,
as in tropical and subtropical areas; prolonged use of antibiotics, and use of steroid
ear drops in those with impaired immune system.[4]
[7]
[8]
[9]
Many antifungal agents were prescribed for otomycosis eradication, including azole
group antifungals, amphotericin B, boric acid, mercurochrome (1%–2% solution) and
phenylmercuric acetate (0.02%) in sterile water, urea-acetic acid solution, or aluminum
acetate solution (5%).[10]
[11]
[12]
[13] In the present study, we focused on the causative fungi for otomycosis in our region,
as well as on their susceptibility to the commonly used antifungal agents. In addition,
we tried to clarify the reason for newly emerged antifungal resistance.
Methods
This study fulfills the requirements of the regional and institutional ethical guidelines
on studies involving human participants as well as those of the Helsinki declaration.
An informed consent was obtained from all patients enrolled in this work.
Patients' selection: an experimental descriptive study was performed on patients clinically diagnosed
with otomycosis who attended our otolaryngology outpatient clinic for 1 year (April
2016 to April 2017). Those patients presented with different complaints, including
aural pain, itching, otorrhea, with or without hearing loss, and their examination
revealed erythema, fungal debris and creamy or blackish aural discharge. Those who
had recent history of antifungal topical medication were excluded from our study.
Specimen preparation & processing: After the clinical diagnosis was established, specimens from the external ear canal
were collected from all patients by means of sterile cotton swabs under aseptic conditions.
Each sample was divided into two parts for fungal analysis. One part was clarified
with a 10% potassium hydroxide solution on a glass slide for direct microscopic examination.
The second part of the specimen was mounted on the surface of two Sabouraud's dextrose
agar (SDA) plates supplemented with 0.05 mg/mL of chloramphenicol (AppliChem GmbH,
Darmstadt, Germany); one was incubated at 37°C and the other was incubated at 27°C.
Incubation of both plates continued for at least 4 weeks with daily examination for
the first week, then twice weekly for the next 3 weeks until the colonies appeared
or revealed no growth.
The growing fungi were kept for further mycological testing in SDA slants and sterile
Eppendorf tubes containing sterile glycerol in distilled water with a concentration
of 20% for yeasts, and a concentration of 10% for filamentous fungi. In addition,
Vitek-2 (bioMérieux, Marcy-L'Etoile, France) was used for complete identification
of Candida species.[14]
Antifungal susceptibility testing: the antifungal susceptibility testing was performed using the disc diffusion method.[15] The antifungal agents tested were: polyenes; (amphotericin B 100 units and nystatin
100 units), azoles; (fluconazole 25 µg, ketoconazole 10 µg, clotrimazole 10 µg, voriconazole
1 µg and itraconazole 10 µg) and terbinafine 25 µg. All the antifungal discs were
obtained from HiMedia, India, except for terbinafine (25 µg) and itraconazole (10µg),
which were locally prepared. The isolated yeast and filamentous fungi were tested
for extracellular enzyme (urease, lipase and protease) production, according to previously
described methods.[16]
[17]
Statistical analysis; Data were analyzed using the STATA intercooled version 12.1 (Stata Corp. LP, College
Station, TX). Quantitative data were expressed as mean, standard deviation, median
and range. Qualitative data were presented as numbers and percentages.
Results
Our study included 112 patients clinically diagnosed with otomycosis. Of these patients,
102 revealed fungal pathogens. Fifty-nine of the patients were male (57.84%). Their
ages ranged between 9 months and 71 years, with the highest proportion among the 21–30
years age group, median 25.7 years (27%).
The most common presenting symptom was pruritis, in 94 cases (92.16%), followed by
otalgia, in 52 cases (50.98%), otorrhea, in 21cases (20.59%), hearing loss, in 22
cases (23.53%) and tinnitus, in 10 cases (9.8%).
The causative agents for otomycosis in our study were described in [Table 1]. Aspergillus niger was the most common fungus, having been found in 49 cases (48.04%).
Table 1
Distribution of patients with otomycosis according to isolated fungal pathogens
|
Microorganisms
|
Number (%)
|
|
Aspergillus niger
|
49 (48.04%)
|
|
Aspergillus flavus
|
31 (30.39%)
|
|
Candida famata
|
3 (2.94%)
|
|
Aspergillus terreus
|
2 (1.96%)
|
|
Candida parapsilosis
|
2 (1.96%)
|
|
Candida utiliz
|
2 (1.96%)
|
|
Rhizopus stolonifer
|
2 (1.96%)
|
|
Candida guilliermondii
|
1 (0.98%)
|
|
Candida krusei
|
1 (0.98%)
|
|
Cryptococcus laurentii
|
1 (0.98%)
|
|
Penicillium duclauxi
|
1 (0.98%)
|
|
A. flavus + C. utiliz
|
2 (1.96%)
|
|
A. flavus + C. famata
|
1 (0.98%)
|
|
A. niger + C. guillerimondii
|
1 (0.98%)
|
|
A. niger + C. krusei
|
1 (0.98%)
|
|
A. niger + C. utiliz
|
1 (0.98%)
|
|
A. terreus + Cryptococccus laurentii
|
1 (0.98%)
|
|
Total
|
102 (100%)
|
Antifungal Susceptibility Testing (AST) of Isolated Filamentous Fungi
Antifungal susceptibility testing of 92 mold isolates was performed using the disc
diffusion method ([Table 2]). The highest percentage of sensitivity among mold isolates was to voriconazole
(93.48%), followed by terbinafine (75%). Surprisingly, all isolates were resistant
to fluconazole.
Table 2
Antifungal susceptibility pattern of mold isolates against various antifungal agents
|
Name
|
No
|
Drug sensitivity
|
AP
|
NS
|
FU
|
IT
|
VOR
|
KT
|
CLO
|
TF
|
|
N (%)
|
N (%)
|
N (%)
|
N (%)
|
N (%)
|
N (%)
|
N (%)
|
N (%)
|
|
A. niger
|
52
|
S
|
48 (92.3)
|
29 (55.77)
|
0
|
29 (55.77
|
51 (98.08)
|
0
|
0
|
37 (71.15)
|
|
I
|
2 (3.85)
|
22 (42.31)
|
0
|
12 (23.08)
|
0
|
23 (44.23)
|
36 (69.23)
|
11 (21.15)
|
|
R
|
2 (3.85)
|
1 (1.92)
|
52 (100)
|
11 (21.15)
|
1 (1.92)
|
29 (55.77)
|
16 (30.77)
|
4 (7.69)
|
|
A. flavus
|
34
|
S
|
1 (2.94)
|
18 (52.94)
|
0
|
17 (50)
|
32 (94.12)
|
13 (38.24)
|
5 (14.71)
|
30 (88.24)
|
|
I
|
2 (5.88)
|
13 (38.24)
|
0
|
7 (20.59)
|
2 (5.88)
|
21 (61.76)
|
29 (85.29)
|
1 (2.94)
|
|
R
|
31 (91.18)
|
3 (8.82)
|
34 (100)
|
10 (29.41)
|
0
|
0
|
0
|
3 (8.82)
|
|
A. terreus
|
3
|
S
|
0
|
0
|
0
|
0
|
3 (100)
|
0
|
2 (66.67)
|
1 (33.33)
|
|
I
|
0
|
3 (100)
|
0
|
2 (66.67)
|
0
|
3 (100)
|
1 (33.33)
|
2 (66.67)
|
|
R
|
3 (100)
|
0
|
3 (100)
|
1 (33.33)
|
0
|
0
|
0
|
0
|
|
Rhizopus stolonifer
|
2
|
S
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
|
I
|
1 (50)
|
2 (100)
|
0
|
0
|
0
|
0
|
0
|
0
|
|
R
|
1 (50)
|
0 0
|
2 (100)
|
2 (100)
|
2 (100)
|
2 (100)
|
2 (100)
|
2 (100)
|
|
Penicillium duclauxi
|
1
|
S
|
0
|
1 (100)
|
0
|
0
|
0
|
0
|
0
|
1 100)
|
|
I
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
|
R
|
1 (100)
|
0
|
1 (100)
|
1 (100)
|
1 (100)
|
1 (100)
|
1 (100)
|
0
|
|
Total
|
92
|
S
|
49 (53.26)
|
48 (52.17)
|
0
|
46 (50)
|
86 (93.48)
|
13 (14.13)
|
7 (7.61)
|
69 (75.00)
|
|
I
|
5 (5.43)
|
40 (43.48)
|
0
|
21 (22.83)
|
2 (2.17)
|
47 (51.09)
|
66 (71.74)
|
14 (15.22)
|
|
R
|
38 (41.3)
|
4 (4.35)
|
92 (100)
|
25 (27.17)
|
4 (4.35)
|
32 (34.78)
|
19 (20.65)
|
9 (9.78)
|
Abbreviations: AP, amphotericin; CLO, clotrimazole; FU, fluconazole; I, intermediate;
IT, itraconazole; KT, ketoconazole; NS, nystatin; R, resistant; S, sensitive; TF,
terbinafine; VOR, voriconazole.
Antifungal Susceptibility Testing of Isolated Yeasts
Antifungal susceptibility testing of 17 yeast isolates was performed using the disc
diffusion method. For the used antifungal agents, the highest percentage of sensitivity
among yeast isolates was to nystatin (88%), followed by amphotericin B (82%). All
yeast isolates were resistant to terbinafine (100%) and resistance to itraconazole
was observed in 94.12%.
Discussion
Otomycosis is a common clinical entity. Although not life threatening, it can be a
frustrating condition for both patient and physician due to the need for a long-term
therapy, regular follow-up and tendency for recurrence.[18]
In this study, the most common symptoms of otomycosis were pruritis (92.16%) and otalgia
(50.98%). Other common complaints were hearing loss (23.53%) and ear discharge (20.59%).
The mentioned complaints and their incidence were in accordance with the findings
of other studies.[19]
[20] However, some reports stated that otalgia was the most frequent symptom.[21] Accordingly, absence of pruritis does not exclude the possibility of otomycosis.
Previous reports have concluded that the diagnostic yield of direct microscopic examination
(DME) may range from 10 to more than 90%, depending on the fungal species.[22] In this study, only 10 samples were positive for fungal infection on DME (8.2%),
while on SDA culture, the percentage of samples positive for fungal infection was
83.61% (102 of total collected samples). Therefore, culture is the main diagnostic
tool.[22]
In our study, A. niger (50.98%) was the most common isolated organism, followed by Aspergillus flavus (33.33%), and then by Candida species (14.7%). This is in accordance with other studies.[23]
[24] However, the predominant fungal pathogens in otomycosis were different in various
literature reports, including A. flavus
[18]
[25] Aspergillus fumigates
[26] and Candida.[5]
[27] We owed this difference to the variability in geographic distribution and environmental
factors.
This study revealed that voriconazole and nystatin should have the upper hand in treatment
of otomycosis. Moreover, it was informative enough to explain one of the main reasons
for lack of response to commonly prescribed antifungals and high recurrence rate,
which was recently observed in our practice with prescription of fluconazole as a
first-line treatment for our cases.
Another reason is the urease activity, which was proven to play a crucial role for
fungal pathogens, particularly those that initiate infection via the lungs.[28] Stehr et al confirmed that Candida lack the urease enzyme, and urea is instead metabolized by a urea amidolyase.[29] In our study, 93.39% of isolated filamentous fungi and Cryptococcus were urease-positive. All Candida isolates were urease-negative. In addition, Yike mentioned the fact that the external
digestion of protein substrates by secreted proteases is an essential requirement
for survival and growth of both saprophytic and pathogenic fungal species.[30] In our study, 31.52% of mold isolates were high enzyme producers, 20.65% were intermediate
enzyme producers, 46.74% were low enzyme producers and only 1.09% were non-enzyme
producers. For isolated yeasts, 64.71% of isolates were low enzyme producers, 17.65%
of isolates were intermediate enzyme producers and 17.65% of isolates were non-enzyme
producers. These results might explain the high virulence and resistance of mold isolates
to the commonly prescribed antifungals as well as the high rate of recurrence. Moreover,
our results clarify how important the in vitro antifungal susceptibility testing is.
Filamentous fungi possess a high enzymatic ability, which makes them more virulent,
and the infections caused by these fungi are more aggressive. Our recommendation is
to avoid the empirical prescription of antifungal drugs. If in vitro antifungal susceptibility
testing cannot be routinely done, fluconazole should be discarded for treatment of
otomycosis for these types of patients.
Conclusion
This study revealed that Aspergillus and Candida species were the most common fungal pathogens causing otomycosis. Aspergillus niger is the predominant fungal isolate.
Mold isolates showed highest sensitivity to Voriconazole, while the highest resistance
was to Fluconazole. For yeast isolates, the highest sensitivity was to Nystatin, and
the highest resistance was to terbinafine. Filamentous fungi possess a high enzymatic
ability, which makes them more virulent, aggressive and resistant to treatment. Empirical
use of antifungals in otomycosis should not be a routine practice.
