Comparison of diagnostic methods in onychomycosis

Abstract

Aim: Onychomycosis is a chronic fungal infection of the nail bed, plate, or matrix. This study aimed to compare the sensitivity of three diagnostic methods in the diagnosis of onychomycosis. Material and Method: This study included 39 patients with a clinical diagnosis of onychomycosis of the toenails, who presented to Medipol Mega University Hospital between May 2019 and August 2022. Using the nail samples taken from the patients, the results of the direct microscopic examination with standard potassium hydroxide (KOH), histopathological examination performed with periodic acid-Schiff (PAS) staining, and fungal agents that grew in fungal culture were noted. Results: Eleven (28.2%) patients were female, and 28 (71.8%) were male, with the mean age being 43.1±13.9 years. Of the patients, 53.8% had distal subungual onychomycosis and 46.2% had total subungual onychomycosis. The mean disease duration was 38.8±24.5 (12-120) months. Fungal infection was detected on direct microscopic examination with standard KOH in 66.7% of the patients, culture growth in 38.5%, and PAS staining on histopathological examination in 71.8%, and the sensitivities of these methods were determined as 74.3%, 49.2%, and 80%, respectively, with the negative predictive values being 30.8%, 16.7%, and 36.4%, respectively. Conclusion: Among the investigated methods, histopathological examination with PAS staining was found to have the highest sensitivity and negative predictive value in the diagnosis of onychomycosis.

Full text

HEALTH SCIENCES
MEDICINE
Original Article
is work is licensed under a Creative Commons Attribution 4.0 International License.
J Health Sci Med 2023; 6(2): 353-358
DOI: 10.32322/jhsm.1204419
Received: 15.11.2022 Accepted: 03.02.2023Corresponding Author: Ece Altun, altunece@hotmail.com
Comparison of diagnostic methods in onychomycosis
Ece Altun1, Elif Kuzucular2, Ayşe İstanbullu Tosun3
Department of Dermatology and Venereology, School of Medicine, İstanbul Medipol University, İstanbul, Turkey
Department of Medical Pathology, School of Medicine, İstanbul Medipol University, İstanbul, Tu rkey
Department of Medical Microbiology, School of Medicine, İstanbul Medipol University, İstanbul, Turk ey
Cite this article as: Altun E, Kuzucular E, İstanbullu Tosun A. Comparison of diagnostic methods in onychomycosis. J Health Sci Med
2023; 6(2): 353-358.
ABSTRACT
Aim: Onychomycosis is a chronic fungal infection of the nail bed, plate, or matrix. is study aimed to compare the sensitivity
of three diagnostic methods in the diagnosis of onychomycosis.
Material and Method: is study included 39 patients with a clinical diagnosis of onychomycosis of the toenails, who presented
to Medipol Mega University Hospital between May 2019 and August 2022. Using the nail samples taken from the patients,
the results of the direct microscopic examination with standard potassium hydroxide (KOH), histopathological examination
performed with periodic acid-Schi (PAS) staining, and fungal agents that grew in fungal culture were noted.
Results: Eleven (28.2%) patients were female, and 28 (71.8%) were male, with the mean age being 43.1±13.9 years. Of the
patients, 53.8% had distal subungual onychomycosis and 46.2% had total subungual onychomycosis. e mean disease
duration was 38.8±24.5 (12-120) months. Fungal infection was detected on direct microscopic examination with standard
KOH in 66.7% of the patients, culture growth in 38.5%, and PAS staining on histopathological examination in 71.8%, and the
sensitivities of these methods were determined as 74.3%, 49.2%, and 80%, respectively, with the negative predictive values
being 30.8%, 16.7%, and 36.4%, respectively.
Conclusion: Among the investigated methods, histopathological examination with PAS staining was found to have the highest
sensitivity and negative predictive value in the diagnosis of onychomycosis.
Keywords: Onychomycosis, potassium hydroxide examination, periodic acid-Schi staining, fungal culture
INTRODUCTION
Onychomycosis is a fungal infection of the nail that causes
the thickening and discoloration of the aected nail plate
(1). According to recently published studies, the global
prevalence of onychomycosis approximately 5.5% in the
general population (2,3). Onychomycosis accounts for
50% of all nail diseases and is the most common disorder
aecting the nail unit (1). Predisposing factors for this fungal
infection include diabetes, human immunodeciency virus,
immunosuppression, diabetes, obesity, smoking, trauma,
tinea pedis, psoriasis, and older age (4). Onychomycosis
most commonly involves the toenails, usually aecting
the rst (great) toenail. It typically presents as the white or
yellow-brown discoloration of the nail and oen causes the
hyperkeratosis of the nail bed, which results in varying degrees
of onycholysis (1,4). Organisms that cause onychomycosis
include dermatophytes, non-dermatophyte molds (NDMs),
and yeasts. Dermatophytes, particularly
Trichophyton
mentagrophytes
and
Trichophyton rubrum
, are responsible
for approximately 90% of toenail onychomycosis cases,
and the remaining dermatophyte infections are caused
by
Epidermophyton occosum
,
Microsporum
species,
Trichophyton verrucosum
,
Trichophyton tonsurans
,
Trichophyton violaceum
,
Trichophyton soudanense
,
Trichophyton krajdenii
,
Trichophyton equinum
,
and Arthroderma species (1,5,6). e most common
NDM organisms associated with onychomycosis are
Aspergillus
spp.,
Scopulariopsis brevicaulis
,
Fusarium
spp.,
Acremonium
spp.,
Neoscytalidium
spp., and
Syncephalastrum spp. (7,8). Yeast-induced onychomycosis
is rare. Candida albicans accounts for approximately 70%
of yeast-induced onychomycosis cases (9). ere is a need
for eective and sensitive diagnostic tests that can conrm
the diagnosis of onychomycosis before initiating systemic
antifungal therapy. Currently, the main diagnostic methods
for onychomycosis are direct microscopic examination,
histological examination, and culture analysis (10). However,
direct microscopic examination with potassium hydroxide
(KOH) and histological examination with periodic acid-
Schi (PAS) staining cannot identify fungal species.
erefore, despite the disadvantage of being the slowest

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Altun et al . Diagnostic methods in onychomycosis J Health Sci Med 2023; 6(2): 353-358
method, culture analysis has the benet of identifying the
species causing onychomycosis (11). e current study
aimed to compare the diagnostic value of direct microscopic
examination, histopathological examination, and fungal
culture analysis in the clinical diagnosis of onychomycosis.
MATERIAL AND METHOD
e study was carried out with the permission of İstanbul
Medipol University Clinical Researches Ethics Committee
(Date: 26/08/2022, Decision No: E-10840098-772.02-4808).
All procedures were carried out in accordance with the
ethical rules and the principles of the Declaration of Helsinki.
is study included 39 patients with onychomycosis who
presented to the dermatology outpatient clinic of Medipol
Mega University Hospital between May 2019 and August
2022. e patients’ age, gender, comorbidities, disease
duration, and examination ndings were recorded from
their les. e results of the direct microscopic examination
with KOH, histopathological examination with PAS
staining, and fungal agents that grew in culture were noted.
Direct Microscopic Examination
For this examination, a 10-20% KOH solution was utilized
as the most commonly used material (10). e sample taken
from the suspicious nail was placed on a slide, one or two
drops of this solution were dropped onto the slide, which
was then covered with a coverslip. e slide was le in a
petri dish with moist blotting paper for 30-60 minutes and
examined under a light microscope.
Histopathological Examination
Sections of 3-m thickness were taken from paran blocks,
placed on positively charged slides, and kept in an oven at 60
°C for 30 minutes. Histochemical staining was automatically
performed with the Ventana Benchmark® Special Stain
device (Ventana, Roche, USA) using the PAS staining kit,
BSS deparanization, BSS liquid cover slip, and BSS wash
solutions. is device has a two-stage operating system, in
which the deparanization process is performed in the
rst stage and in the second. PAS, background staining
(hematoxylin), and bluing were performed. Aer the
staining was completed, the sections were passed through
an increasing alcohol series (80%, 90%, and 96%) and le in
xylene for two minutes to remove chemicals. e samples
were covered with a lm using an automatic closure device
(Tissue Single Film, Sakura, Japan). e preparations were
analyzed under a light microscope (Eclipse Ni, Nikon,
Japan).
Fungal Culture
Specimens were inoculated to Sabouraud agar (Becton
Dickenson, USA) and dermatophyte agar (Becton
Dickenson, USA) and incubated at 25 °C for 28 days
(12). Media were monitored every 24 hours in terms of
colony growth. When visible colonies were formed, the
microscopic examination was performed. Identication
was made based on the presence and shape of microconidia
and macroconidia, as well as the shape of the colonies.
Statistical Analysis
SPSS 15.0 for Windows (SPSS Inc., Chicago, Illinois, USA)
was used for statistical analyses. As descriptive statistics,
numbers and percentages were used for categorical variables,
and mean, standard deviation, minimum, and maximum
values for numerical variables. Dierences between
the screening tests were examined with the McNemar
test. Taking any test positivity as the gold standard, the
eectiveness of each screening test was evaluated based on
sensitivity dened as the test’s ability to produce a positive
result in individuals that truly had the disease, selectivity
as the negative test rate among the individuals without the
disease, positive predictive value as the probability that the
individuals with a positive test truly have the disease, negative
predictive value as the probability that the individuals with
a negative test do not have the disease, accuracy as the rate
of correct identication of the individuals with and without
the disease, and negative likelihood ratio as the ratio of the
probability of an individual with the disease testing negative
to the probability of an individual without the disease
testing negative. e statistical alpha signicance level was
accepted as p < 0.05.
RESULTS
e study included a total of 39 patients with a diagnosis
of onychomycosis, 11 (28.2%) female and 28 (71.8%) male,
with a mean age of 43.1±13.9 years. Distal subungual
onychomycosis was present in 53.8% of the patients and
total dystrophic onychomycosis in 46.2%. e mean
disease duration was 38.8±24.5 months, with a minimum
value of 12 and a maximum value of 120 months. Tab le 1
summarizes the characteristics of the patients participating
in the study.
Table 1. General characteristics of the patients
Age, mean±SD (min-max/median) 43.1±13.9 (19-75/41)
Gender, n (%)
Female 11 (28.2%)
Male 28 (71.8%)
Distal subungual onychomycosis, n (%) 21 (53.8%)
Total dystrophic onychomycosis, n (%) 18 (46.2%)
Disease duration (month), mean±SD
(min-max/median) 38.8±24.5 (12-120/36)
SD: Standard deviation
Of the patients, 66.7% tested positive in the direct
microscopic examination with KOH, 71.8% in the
histopathological examination with PAS staining, and
38.5% in the culture analysis. e rate of positivity detected

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Altun et al. Diagnostic methods in onychomycosis
J Health Sci Med 2023; 6(2): 353-358
in the culture analysis was lower compared to the remaining
diagnostic methods. e rates negative test results were
33.3%, 28.2%, and 61.5% for KOH examination, PAS
staining, and culture analysis, respectively. Ta bl e 2 presents
the rates of positive and negative test results of the cases
according to the diagnostic methods.
Table 2. Positivity and negativity rates of the diagnostic methods
Onychomycosis
KOH
examination Culture PAS staining
N % n % n %
Positive 26 66.7 15 38.5 28 71.8
Negative 13 33.3 24 61.5 11 28.2
KOH vs. culture, p = 0.02; KOH vs PAS, p = 0.774; culture vs PAS, p = 0.011, KOH:
Potassium hydroxide; PAS: Periodic acid-Schi
e histopathological examination with PAS staining was
the method with the highest sensitivity (80%) and negative
predictive value (36.4%). e sensitivity of the direct
microscopic examination with KOH was 74.3%, and that
of the culture analysis was 49.2%. e negative predictive
values of the KOH examination and culture analysis were
found to be 30.8% and 16.7%, respectively (Tabl e 3).
e distribution of the fungal agents that grew in culture
was as follows: Trichophyton spp. in nine cases,
Aspergillus
spp. in three, Penicillium spp. in one, and Candida spp. in
one. e study data are summarized in Table 4 .
Table 3. Sensitivity percentages obtained as a result of analyses
performed with the diagnostic methods
Sensitivity
%
Specicity
%
Positive
predictive
value %
Negative
predictive
value %
Accuracy
%
KOH 74.3 100% 100% 30.8 76.9
Culture 49.2 100% 100% 16.7 48.7
PAS 80.0 100% 100% 36.4 82.1
KOH: Potassium hydroxide; PAS: Periodic acid-Schi
Table 4. Summary of the study data
Patient number Age Gender DSO TDO Disease duration (month) KOH PAS Culture
1 70 M + - 24 + + + Trichophyton
2 61 M - + 60 - + - -
3 29 M + - 24 - - +
Aspergillus
4 31 M - + 60 + + - -
5 48 M - + 12 + + - -
6 40 M - + 48 + + - -
7 44 M - + 48 + + - -
8 26 M + - 12 + + + Trichophyton
9 39 F + - 36 + + - -
10 34 F - + 24 + + - -
11 40 M - + 24 + + + Trichophyton
12 19 M + - 36 + + - -
13 64 F - + 24 + + - -
14 31 M + - 24 - + - -
15 46 M + - 60 + + - -
16 59 M + - 72 + - - -
17 28 M + - 12 - - + Candida
18 55 F + - 60 - + - -
19 41 M - + 36 + + - -
20 30 F - + 48 - - - -
21 43 F - + 12 - - - -
22 72 M + - 60 + + - -
23 27 F + - 72 - - - -
24 51 F + - 60 + + - -
25 35 M + - 24 + + - -
26 25 M + - 24 + - - -
27 47 F + - 24 - + +
Aspergillus
28 75 M - + 36 + + + Penicillium
29 22 F - + 12 - + + Trichophyton
30 40 M - + 60 + + - -
31 31 F + - 60 + - + Trichophyton
32 50 M - + 12 + + + Trichophyton
33 45 M + - 24 + - + Trichophyton
34 47 M - + 12 - - - -
35 57 M + - 12 - + +
Aspergillus
36 46 M - + 60 - + - -
37 51 M + - 72 + - + Trichophyton
38 41 M - + 120 + + + Trichophyton
39 40 M + - 12 + + + Penicillium
KOH: Potassium hydroxide; PAS: Periodic acid-Schi; F, Female; M, Male; +: Positive result; -: Negative result; DSO: Distal subungual
onychomycosis; TDO: Total dystrophic onychomycosis

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Altun et al . Diagnostic methods in onychomycosis J Health Sci Med 2023; 6(2): 353-358
e microscopic images of fungal hyphae and yeast
visualized using the three diagnostic techniques are
summarized in Figure 1.
Figure 1. Microscopic images. A: Histochemical examination of
fungal hyphae with periodic acid-Schi staining (x200); B:
Candida
yeast cells stained with methylene blue (x40); C: Direct microscopic
examination of fungal hyphae with potassium hydroxide
DISCUSSION
Onychomycosis is one of the most common fungal
diseases. Direct microscopic examination with KOH
is a fast and inexpensive diagnostic method for
onychomycosis. However, when using this method, false
negative results may be obtained due to the examination
of the infected nail not containing any fungal hyphae, the
poor quality of the KOH solution, the presence of a history
of topical and systemic treatments, and the insucient
experience of the clinician ( 13-16). Furthermore,
secondary contamination and air bubbles mimicking
fungal structures can produce false positive results. In the
literature, the positivity rates of the direct microscopic
examination with KOH in onychomycosis vary between
32 and 96% (17-22). In many studies, the positivity rate of
this diagnostic method was found to be lower compared
to the histopathological examination with PAS staining
and higher compared to the culture analysis (17,18,23-
25); however, there are also researchers reporting that
the direct microscopic examination with KOH had the
lowest positivity rate (19,20). In contrast, in two studies
conducted in Turkey, Aydıngöz et al. (21) and Ceren et
al. (22) determined the KOH method to have the highest
positivity at 96% and 85%, respectively. In onychomycosis,
the sensitivity of this test varies in a wide range from 44
to 92% (17-20,22,26-29). It was found to be the most
sensitive method in the diagnosis of onychomycosis
by Ceren et al. (22) (92%) and Hsiao et al. (27) (87%).
However, Wilsmann-eis et al. (26) determined that
the KOH method had the lowest sensitivity with a rate
of 48%. In the current study, the positivity and sensitivity
rates of the direct microscopic examination with KOH
were 66.7% and 74.3%, respectively. ese rates were
lower than the histopathological examination with PAS
staining and higher than the culture analysis.
In the literature, the positivity and sensitivity rates of
the culture analysis are generally found to be lower
compared to the histopathological examination with
PAS staining and direct microscopic examination
with KOH. e culture positivity rate as reported to
be low (19%) by Ceren et al. (22), higher (52%) by
Gianni et al. (30) and vary between 19 and 52% in
other studies (17,21,23-25,28). However, the absence
of growth in culture does not exclude the diagnosis
of onychomycosis. Among the reasons for negative
results are insucient analysis material, the incorrect
placement of samples in the culture medium, material
being kept in the culture medium longer than required,
contamination with or growth of secondary pathogens,
removal of nail material from the distal portion that
does not contain live fungi, and the use of topical or
systemic antifungals. erefore, positivity increases
in repeat culture analyses. In a study by Gupta (31),
when the culture analysis was performed once, the
positivity rate was 44.5%, but when it was performed
four times, the positivity rate increased to 63.7%. Test
results are aected by dierences in the sampling and
handling of clinical specimens in centers, skill levels,
or clinical samples (11). In the literature, the sensitivity
of the culture analysis varies between 20 and 70% (17-
20,22,26-29). In a study on onychomycosis, Jeelani et
al. (19) found the sensitivity of the culture analysis
to be as high as 70%; however, this rate was lower
compared to other diagnostic methods. Consistent
with the literature, in the current study, the positivity
and sensitivity rates of the culture analysis were 38.5%
and 49.2%, respectively, but it had the lowest sensitivity
among the three diagnostic methods. Although this
analysis allows for the fungal agent to be classied as
a dermatophyte, non-dermatophyte mold, or yeast, it
does not provide information on whether the growing
agent is a true pathogen or there is any contamination
(21,31). Grover et al. (23) detected fungi in 44% of 120
cases, and 70.2% of these positive cases were identied
to have Trichophyton spp. Hajar et al. (32) identied
Trichophyton spp. in 80% of positive cultures. In
another study, Trichophyton spp. were also shown to be
the most isolated organisms (33). In the current study,
Trichophyton spp. grew in 60% of the positive cultures.
In the literature, it has been shown that the most
sensitive method in the diagnosis of onychomycosis is
the histopathological examination with PAS staining. In
previous studies, the positivity of this test varied between
47 and 90%, and its sensitivity ranged from 80 to 92%
(13,19-26,28-30,33). e PAS method was reported to
have a high sensitivity rate of 80% by Karimzadegan-Nia
et al. (29), 82% by Wilsmann-eis et al. (26), 90% by
Shenoy et al. (18), 91.6% by Jeelani et al. (19), and 92% by
Weinberg et al. (20). However, in other studies, despite
the high sensitivity rates of this test (80, 81, and 90%), this
method still had lower sensitivity values compared to the
direct microscopic examination with KOH (21,22,27).
In the current study, the histopathological examination
with PAS staining had the highest positivity (71.8%) and
sensitivity (80%).

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Altun et al. Diagnostic methods in onychomycosis
J Health Sci Med 2023; 6(2): 353-358
e rates of negative predictive values of the direct
microscopic examination with KOH, histopathological
examination with PAS staining, and culture analysis were
previously reported as 53%, 42%, and 10%, respectively
by Ceren et al. (22), 58%, 77%, and 43%, respectively by
Weinberg et al. (20), and 50%, 40%, and 28%, respectively
by Hsiao et al. (27). In the current study, the negative
predictive value was 30.8% for the KOH method, 36.4%
for the PAS method, and 16.7% for the culture analysis.
e histopathological examination with PAS staining
had the highest negative predictive value.
CONCLUSION
is study investigated the sensitivity of the direct
microscopic examination with KOH, histopathological
examination with PAS staining, and culture analysis in
the diagnosis of onychomycosis. e histopathological
examination with PAS was found to be superior
to the remaining two methods in the diagnosis of
onychomycosis, with a high negative predictive value and
sensitivity. e culture analysis had the lowest sensitivity
and negative predictive value.
ETHICAL DECLARATIONS
Ethics Committee Approval: e study was carried
out with the permission of İstanbul Medipol University
Clinical Researches Ethics Committee (Date: 26/08/2022,
Decision No: E-10840098-772.02-4808).
Informed Consent: Written informed consent was
obtained from all participants who participated in this
study.
Referee Evaluation Process: Externally peer-reviewed.
Conict of Interest Statement: e authors have no
conicts of interest to declare.
Financial Disclosure: e authors declared that this
study has received no nancial support.
Author Contributions: All of the authors declare that
they have all participated in the design, execution, and
analysis of the paper, and that they have approved the
nal version.
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