Received: 13 December 2019 | Revised: 10 January 2020
DOI: 10.1002/jobm.201900675
REVIEW
Recent advances in the diagnosis of dermatophytosis
Jubeda Begum1 | Nasir A. Mir2
4 2
Bidyarani Buyamayum
1
Department of Veterinary Microbiology,
College of Veterinary and Animal
Sciences, GBPUAT, Pantnagar, India
2
AN & FT Division, ICAR‐Central Avian
Research Institute, Bareilly, India
3
Division of Pharmacology & Toxicology,
ICAR‐Indian Veterinary Research
Institute, Bareilly, India
4
Department of Microbiology, Jawaharlal
Nehru Institute of Medical Science,
Porompat, Manipur, India
Correspondence
Jubeda Begum, Department of Veterinary
Microbiology, College of Veterinary and
Animal Sciences, GBPUAT, Pantnagar
263145, India.
Email: jubedavet@gmail.com
1 | INTRODUCTION
Dermatophytosis is a disease of global significance
caused by pathogenic keratinolytic fungi called
dermatophytes in both animals and humans. Earlier
all pathogenic dermatophytes were classified into
three genera, namely Microsporum, Trichophyton and
Epidermophyton. However, with the advancement
J Basic Microbiol. 2020;1–11.
| Accepted: 18 January 2020
| Madhu C. Lingaraju3 |
| Kapil Dev
Abstract
Dermatophytosis is a disease of global significance caused by pathogenic
keratinolytic fungi called dermatophytes in both animals and humans.
The recent taxonomy of dermatophytes classifies them into six pathogenic
genera, namely Microsporum, Trichophyton, Epidermophyton, Nannizzia,
Lophophyton and Arthroderma. It is because of the delayed diagnostic
nature and low accuracy of dermatophyte detection by conventional
methods that paved the path for the evolution of molecular diagnostic
techniques, which provide the accurate and rapid diagnosis of dermato-
phytosis for an appropriate, timely antifungal therapy that prevents the
nonspecific over‐the‐counter self‐medication. This review focuses on the
importance of rapid and accurate diagnosis of dermatophytosis, limita-
tions of conventional methods, selection of targets in diagnosis, and fac-
tors affecting sensitivity and specificity of various molecular diagnostic
technologies in the diagnosis of dermatophytosis. Generally, all the
molecular techniques have a significant edge over the conventional
methods of culture and microscopy in the dermatophytosis diagnosis.
However, in mycology laboratory, the suitability of any molecular diag-
nostic technique in the diagnosis of dermatophytosis is driven by the
requirement of time, economy, complexity, the range of species spectrum
detected and the scale of diagnostic output required. Thus, various choices
involved in the pursuit of a diagnosis of dermatophytosis are determined
by the available conditions and the facilities in the laboratory.
KEYWORDS
dermatophytes, molecular techniques, sensitivity, specificity, target sequences
of diagnostic techniques, these dermatophytes
were classified into six genera, with Nannizzia,
Lophophyton and Arthroderma as new three genera
[1]. Globally, approximately 20–25% of the population
is affected by superficial mycosis with a pre-
ponderance of dermatophytosis [2]. However, over the
past two decades, there has been a dramatic increase
in the incidence of dermatophytosis in humans as a
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2 |
result of socioeconomic problems, large‐scale inter-
national travels, immigration from tropical countries,
and contact with animals, particularly pets. The
increasing age and the use of immunosuppressive
drugs are the predisposing factors for the rise of
dermatophytosis‐induced morbidity in humans [3].
Due to the zoonotic nature of some dermatophytosis,
an interdisciplinary approach, involving dermatolo-
gists, paediatricians, primary healthcare physicians
and veterinarians, is warranted to curtail its
spread [4].
The dermatophytosis is generally chronic in nature,
and its control requires proper identification of aetiological
agents and its prevalence for the prescription of specific
treatment [5]. The rapid identification of aetiological
agents from the clinical samples of dermatophytosis and
route of infection is vital for specific antifungal therapy
and for the prevention of further dissemination to human
and/or other animals [6]. The conventional diagnosis of
dermatophytosis involves the easily available techniques
of culture and morphological identification of the fungus or
the detection of fungal elements by the direct microscopy
of clinical specimens. Although the diagnosis by conven-
tional culture and microscopy is suitable for fresh isolates,
it is difficult to maintain and reproduce the reference
strains for standardisation due to rapid degeneration on
cultures [1]. Furthermore, such conventional diagnostic
techniques are highly time‐consuming to arrive at a diag-
nosis, and the accuracy of the results depends on the
expertise of the personnel. Thus, the need for polymerase
chain reaction (PCR)‐based techniques was felt to increase
the sensitivity, specificity and the speed of diagnosis. The
rapid diagnosis reduces the inconvenience caused to the
patients due to delayed diagnosis and prolonged treatment
course [7].
2 | IMPORTANCE OF RAPID AND
S E N S I T I V E DE R M A T O P H Y T E
DIAGNOSIS
Dermatophytosis is one of the most common commu-
nicable human infectious diseases, often chronic in nat-
ure, causing considerable irreversible destruction to skin,
hair and nails, if not detected and treated timely [3]. The
problem with the zoophilic dermatophytes is that the
clinical lesions it produces in humans are more severe
than those by the typical anthropophilic ones that
are always transmitted by direct or indirect contact from
person to person [8]. The clinical presentations of
dermatophytosis such as multifocal alopecia, scaling and
circular lesions are often similar to those of other
BEGUM ET AL.
skin diseases [9], which make the treatment course
highly uncertain. The isolation and identification of
dermatophytes involved in infections are essential to
prescribe species‐specific treatment regimens. For
example, Trichophyton tonsurans requires shorter treat-
ment duration than that of Microsporum canis involved
in tinea capitis, and nondermatophyte infections may not
respond to the standard therapy used for dermatophy-
tosis [10]. A number of nondermatophyte fungi such as
Fusarium, Acremonium and Aspergillus have been
recovered as unique infectious agents in many cases of
onychomycosis that did not respond to multiple systemic
treatments with terbinafine and itraconazole [11].
Although an oral treatment with terbinafine/azoles is
well established against dermatophytes in onychomy-
cosis, the cases like above may make one falsely arrive at
the conclusion of resistant dermatophyte strains. The
isolation and identification of the aetiological agent in
such nonresponding cases are necessary to prescribe an
alternative therapy. For example, in case of non-
dermatophyte onychomycosis [12] or Fusarium onycho-
mycosis [13], the topical amphotericin B or
photodynamic therapy, respectively, were found to be
efficient, safe and easy to apply. Thus, the accurate
identification of dermatophytes at species level de-
termines the choice of the most appropriate antifungal
treatment and helps in tracking sources of infection,
thereby helping avoid reinfection, investigate and control
epidemics [14].
The isolation of fungi from clinical specimens is pri-
marily done on Sabouraud's dextrose agar or potato
dextrose agar supplemented with antibiotics and cyclo-
heximide [10]. The direct microscopic examination of
clinical samples and the recovery of the causative agent
in culture were the classical approaches used for the la-
boratory diagnosis of the dermatophytosis [15]. However,
the isolation of these fungi in culture requires several
weeks (even up to 6 weeks) and is often complicated by
the growth of contaminant fungi normally present in skin
and hair samples from animals, such as Alternaria,
Cladosporium, Mucor, Penicillium, Rhizopus, and so on.
However, the growth of these contaminant species can be
avoided by adding antibiotics and cycloheximide in the
agar used for the culture. As most of the superficial
fungal infections are caused by dermatophytes, their
rapid and sensitive detection is of great importance [16].
The molecular diagnostic methods offer a significantly
faster diagnosis of dermatophytosis, and in most of the
cases, they provide the direct species identification for a
targeted treatment to avoid unnecessary antifungal
treatment and toxicity, and also for epidemiological
purposes [17–20]. There is an argument that the
BEGUM ET AL.
implementation of new molecular diagnostic approaches
is associated with an added cost, and the employment of
an expensive rapid diagnostic approach for dermatophy-
tosis, a nonlife‐threatening chronic infection, may not be
appropriate [7]. However, it is noteworthy to consider
that certain dermatophytosis cases, particularly nail in-
fections (15%), maybe culture negative at the first sam-
pling [18]. This may lead to additional cost and
inconvenience to the patient associated with a second
visit to the physician [7]. Furthermore, over‐the‐counter
self‐medication can significantly reduce the sensitivity of
culture but not of the molecular approaches [7].
3 | L IM ITA TI O N S O F
CONVENTIONAL METHODS
The isolation followed by identification of dermatophytes
from the clinical samples was earlier considered as a gold
standard method for diagnosis purpose, even though it
took a long time to grow in culture and sporulate. The
outcome of the conventional culture isolation and iden-
tification in the diagnosis of dermatophytosis is largely
limited by the quality of the sample collected. For the
successful isolation of the aetiological agent, the lesion
site needs to be cleaned with 70% alcohol before sample
collection to remove contaminants. Also, the sample
collection needs to be done before initiating the systemic
treatment; however, a withdrawal period of 15 days or
3 months is required if a topical or oral antifungal
treatment is already going on, respectively, before sample
collection [21]. The samples from the skin need to be
collected from the advancing edge of the lesions, the nail
samples should be collected according to the type of
onychomycosis, and hair samples need to be collected
along with their follicles by plucking, not clipping, for
better results [22].
The conventional methods, culture and direct micro-
scopy, of dermatophyte diagnosis, are plagued by the fact
that the rate of isolation of fungi from the clinical samples is
generally lower (even by 30%) than the positive rates ob-
tained by the direct microscopic examination, particularly
in case of nail samples [23], and this has been attributed
to various factors such as inadequate sample quantity,
presence of dead fungal hyphae, precollection antifungal
treatment and so on [10,14,24]. Furthermore, the limitation
of direct microscopy is that being relatively nonspecific, it
can detect the presence of hyphae but cannot differentiate
between the dermatophyte species or dermatophytes from
nondermatophyte fungi [22,25]. Moreover, it has been
found to be falsely negative in considerable number of cases
(5–15%) [23,25]. Also, the successful culture isolation of
dermatophytes from samples is hampered by the difficulty
| 3
in identification due to morphological similarities among
different species or polymorphism shown by dermato-
phytes, which necessitates the molecular techniques for
diagnosis [26]. The factors like morphological similarities
among fungal species and limited sporulation or lack of
sporulation render most of the dermatophytes unidentified
by the classical conventional methods [10]. Another dis-
advantage of conventional culture isolation is the prolonged
(up to 6 weeks) turnaround time in case of atypical isolates
or slow‐growing species like Trichophyton verrucosum,
which delays the treatment and may result in overgrowth
of more rapidly growing contaminating moulds [27].
Although the modern‐day PCR assays have demonstrated
much higher sensitivity and specificity in the diagnosis
of dermatophytosis than conventional methods, the
researchers have now become severely sceptic to the gold
standard status of conventional methods [7,28]. Rapid
diagnosis and accurate identification of dermatophytes help
in the successful management of dermatophytosis [29].
Besides the nucleic acid‐based methods for detection of
dermatophytes directly from the clinical specimen [30],
matrix‐assisted laser desorption/ionisation time‐of‐flight
mass spectrometry [31,32] and nanoelectrospray ionisation
spectrometry [20] were employed for more accurate
identification of dermatophytes, thereby overcoming
the limitations of phenotypic identification. Even after the
application of the topical antifungal agents, the PCR has the
ability to identify the non‐viable cells with intact nucleic
acids [33].
4 | THE RISE OF ADVANCED
TECHNIQUES
The culture‐based dermatophyte identification is limited by
a high false‐negative rate with longer incubation time [34],
which has paved the path for the emergence of numerous
sensitive and specific molecular tests [7,17–19,25]. The
molecular diagnostic methods are becoming widely acces-
sible with superior sensitivity, specificity, and shorter
turnaround times than the conventional methods. The ad-
vanced molecular techniques employed in the diagnosis of
dermatophytosis are conventional PCR or real‐time PCR
(RT‐PCR) for DNA extracted from the clinical specimens
[7]. The extraction of DNA from the clinical specimens can
be done by traditional phenol–chloroform method or by
using more efficient and convenient commercially available
DNA extraction kits [35]. However, the DNA extraction is
hampered by the presence of keratin in the clinical samples,
which is disrupted mechanically or enzymatically by using
keratin digesting proteinase K or nonenzymatically by
keratin digesting Na2S solution [36]. The DNA extraction
followed by the application of different methods of
4 |
utilisation for the diagnostic purpose has undergone
significant evolution over the past 15 years.
The molecular diagnosis of dermatophytosis requires
extraction of fungal DNA, followed by its amplification
using PCR. However, the initial study designs allowed for
the rapid detection of the dermatophyte presence in
clinical samples by targeting conserved ribosomal DNA
sequences without species identification, whereas, the
successive study designs were aimed at more specific
DNA sequences allowing the identification at the genus
and species level [7]. The application of other molecular
methods, such as restriction fragment length poly-
morphism (RFLP) analysis of mitochondrial DNA [37],
sequencing of the internal transcribed spacer (ITS) re-
gions of the ribosomal DNA [38], sequencing of protein‐
encoding genes, arbitrarily primed PCR [29], post‐PCR
electrophoresis or hybridisation [7], pan‐dermatophyte
nested PCR, and PCR fingerprinting [39], considerably
advanced the diagnosis of dermatophytosis to species and
strain level. Although all the molecular methods of der-
matophyte diagnosis are sensitive by far over the con-
ventional methods, they show considerable differences in
sensitivity and specificity among themselves. Thus, there
is the need of study designs that not only compare mo-
lecular assays with conventional diagnostics but also
make a comparison between different molecular diag-
nostics to develop an ideal molecular approach that is
rapid, economical, and involves less technical efforts for a
reliable diagnosis of dermatophytosis [35]. The higher
degree of phylogenetic relationship among the dermato-
phytes from the clinical samples warrants the use of
highly variable target sequences such as ITSs, a micro-
satellite region, topoisomerase II gene and so on, in a
quantitative RT‐PCR and other post‐PCR techniques in
the diagnosis of dermatophytes [23,35,40].
5 | THE CONVENTIONAL PCR
The most widely used conventional PCR is a simple and
inexpensive molecular technique for rapid detection of
aetiological agents accurately in clinical cases pertaining
to dermatophytosis by employing specific primers, fol-
lowed by interpretation of the results based on the am-
plicon size in agarose gel [7,41]. The species‐specific
primers, pan dermatophyte primers, or panfungal pri-
mers that usually target ITS, 28S region of ribosomal
DNA, or the sequences coding for topoisomerase II or
chitin synthase I (CSH1) have been used for the identi-
fication of aetiological agents involved in the dermato-
phytosis [30,42] (Table 1). However, the sample type,
sample preparation and laboratory conditions have a
direct bearing on the PCR results in dermatophyte
BEGUM ET AL.
diagnosis. In a study of 107 clinical samples by Gordon
et al. [3], the conventional PCR could not only detect all
the dermatophytes from all the culture‐positive samples,
but it also detected the CHS1 gene from seven samples
that grew other nondermatophyte fungi or yeast.
This suggests that PCR has the potential to detect the
pathogens in mixed infections or contamination with
environmental fungi. They also detected the target gene
in 10 of the 66 clinical samples from which a fungi was
not isolated, suggesting the superiority of PCR in terms of
sensitivity over conventional culture and microscopy
methods. A number of other studies have reported the
use of PCR kits in the diagnosis of dermatophytosis
increased rate of detection by 10–19.5% compared with
culture method [14,18,25,44,52]. However, the outcome
of the PCR assays may vary, based on the source of the
clinical sample and the selection of the target sequence.
For example, a one‐step PCR assay with specificity of
94.4% and sensitivity of 62.5% was found to be moderately
accurate for cat and highly accurate for dog samples be-
cause of the small amounts of fungal DNA in samples
from cats, which has been related to self‐grooming habit
in cats [26]. However, a PCR assay, using ITS primers,
tested on 58 known isolates could identify 42 isolates
including 35/35 Trichophyton isolates of different species,
3/3 M. canis, and 4/4 Microsporum audouinii isolates.
However, 2 Epidermophyton floccosum, 11 Microsporum
gypseum, and 3 Microsporum persicolor could not be
identified [18]. Although conventional PCR benefits from
simplicity and lower cost, its inability to determine the
appropriate quantity of sample to assure a positive result
and to quantify fungal load [52] makes way for the
RT‐PCR and other post‐PCR techniques for the derma-
tophyte diagnosis.
6 | REAL ‐ T I M E P OL Y M E R A S E
C H A I N RE AC T I O N
The RT‐PCR is a rapid and sensitive approach to detect
one or several dermatophyte species directly from clinical
samples using different species‐specific probes, and it is
conducted in a closed tube system without post‐PCR
analysis, which limits the risk of contamination [7,41]. In
dermatophyte diagnosis, RT‐PCR is characterised by
short handling time, quantification of fungal load, mon-
itoring of therapy success, and determination of the ex-
tent of inhibitions in clinical samples along with the high
incurring cost. However, in high‐throughput diagnostic
laboratories, this high incurring cost can be compensated
by a lower cost per sample [35]. The feature of quantifi-
cation of fungal load by RT‐PCR would help us differ-
entiate between the infection and the contamination of
BEGUM ET AL. | 5

TABLE 1 Summary of PCR techniques available for diagnosis of dermatophytes

PCR technique Target sequences Target species Developer Reference

Real‐time PCR 18S, 5.8S rRNA Pan dermatophyte Lab developed tool [17]
ITS1, ITS2 Trichophyton tonsurans Lab developed tool [43]
Trichophyton
mentagrophytes
Trichophyton violaceum
Microsporum canis
Trichophyton rubrum
Microsporum audouinii
Multiplex PCR CSH, ITS Pan dermatophyte Lab developed tool [25]
Trichophyton rubrum
ITS2, CSH1 Pan dermatophyte Statens Serum Institut, [44]
Trichophyton rubrum Copenhagen, Denmark
PCR‐ELISA ITS Trichophyton interdigitale Lab developed tool [45]
(OnychoDiag)
Nested PCR CHS1 Pan dermatophyte Lab developed tool [46]
ITS1 Trichophyton rubrum Amersham Bioscience [47]
Trichophyton interdigitale UK Ltd.
28S rRNA Trichophyton rubrum Amersham Bioscience [48]
Trichophyton interdigitale UK Ltd.
CHS1 Trichophyton Lab developed tool [16]
mentagrophytes
PCR‐RFLP Metalloproteinase‐1 (MEP1) using Trichophyton rubrum Lab developed tool [49]
BsrD1 restriction enzyme Microsporum canis
Microsporum gypseum
ITS2 using BstUI restriction enzyme Trichophyton spp. Lab developed tool [50]
Microsporium spp.
Microsporum audouinii
Athroderma spp.
ITS rDNA using MvaI restriction Trichophyton interdigitale Lab developed tool [51]
enzyme Epidermophyton floccosum
Trichophyton rubrum
Microsporum canis
Trichophyton erinacei
Trichophyton violaceum
Trichophyton tonsurans
Microsporum gypseum

Abbreviations: ELISA, enzyme‐linked immunosorbent assay; ITS, internal transcribed spacer; PCR, polymerase chain reaction; RFLP, restriction fragment
length polymorphism.

clinical samples by establishing the threshold of the in-
fection. A number of studies have suggested that RT‐PCR
can be applied successfully for the diagnosis of derma-
tophytosis with much higher precision compared with
the classical diagnostic methods. In a study, comparing
RT‐PCR and classical methods, targeting the diagnosis of
Trichophyton rubrum by using species‐specific primers
revealed that considerable misdiagnosis by classical
methods was overcome by applying RT‐PCR technique
[53]. The concurrence between the results of culture and
the RT‐PCR in the identification of the dermatophytes to
the species and genus level has been reported to be 94.3%
and 97.4%, respectively [54]. The RT‐PCR could not only
detect and correctly identify different dermatophyte
species, using ITS primers, from culture‐positive clinical
specimens but it also identified a dermatophyte species
from seven specimens that were negative on microscopy
and culture [43].
In an RT‐PCR assay, based on the ITS1 primers,
Bergmans et al. [17] and Alexander et al. [53] differ-
entiated 11 dermatophyte species belonging to Tricho-
phyton, Microsporum and Epidermophyton genera from
6 |
nail, skin and hair samples with melt curve analysis
within a time period of 72 hr. They also could differ-
entiate dermatophytes from nondermatophytes with a
sensitivity of 97%, compared with culture identification.
In the same year, another study, targeting the same ITS1
sequence, also reported a significant increase in the de-
tection rate of dermatophytes in clinical samples with a
sensitivity of 97%, compared with culture [19]. Generally,
most of the PCR assays have been designed for the si-
multaneous detection of several dermatophyte species
without considering nondermatophytes as a possible in-
fectious agent. However, while studying the onychomy-
cosis, an RT‐PCR assay was developed by Miyajima et al.
[55] using two species‐specific Trichophyton primers (T.
rubrum and Trichophyton mentagrophytes) and a pan-
fungal primer to detect any other possible dermatophyte
or nondermatophyte aetiological agent. They not only
identified the T. rubrum and T. mentagrophytes but also
detected the mould species of Fusarium, Acremonium,
Aspergillus and Scopulariopsis as the infectious agents,
not transient contaminants, of onychomycosiswithout
species identification and without differentiation with
nontarget pathogenic dermatophyte species.
7 | NESTED PCR
Various post‐PCR steps have been performed to increase
the sensitivity of dermatophytes detection and identifi-
cation from clinical samples using panfungal primers
[41]. A nested PCR assay is the modification of a con-
ventional PCR approach in which the first amplification
is followed by another amplification of a smaller region
inside the initial amplified fragment [30]. The nested
PCR consists of primary and secondary amplification to
increase the specificity and sensitivity of a PCR [56] in
which the primary PCR product is used as a template for
secondary PCR to avoid primer dimer artefacts. Thus, it
eliminates false‐positive and false‐negative results as well
as discriminates between the specific and nonspecific
DNA bands or, in other words, nested PCR tends to re-
duce the nonspecific binding in products due to the
amplification of unexpected primer binding sites. A pan‐
dermatophyte nested PCR performed for the diagnosis of
onychomycosis targeting CSH1 gene showed a sensitivity
of 83.8%, which is far higher than the KOH wet mount
microscopy [39]. In 2009, the same protocol directed
against CSH1 for the direct detection and identification of
dermatophytes in skin and hair showed the same sensi-
tivity of 83.8% [16]. In the same year, the nested PCR
directed against ITS1 also successfully detected T. rubrum
and T. mentagrophytes in nail and skin samples [47],
whereas, earlier in 2005 a Trichophyton‐specific primer
BEGUM ET AL.
could detect T. rubrum from paraffin‐embedded material
without resorting to culture [57]. A nested PCR assay,
using multiple primers ITS+ (ITS1 + 5.8S rRNA gene +
ITS2) for the detection of dermatophytes in samples from
cats and dogs, was highly accurate with higher specificity
(94.4%) and sensitivity (94.9%) for both animal species
[26]. In this study, one‐step PCR could successfully
identify M. canis directly, but it could not discriminate
Trichophyton interdigitale from M. gypseumand Tricho-
phyton terrestre, which was satisfactorily achieved by the
nested PCR assay. Thus, owing to its higher sensitivity
compared with KOH microscopy, the pan dermatophyte
nested PCR was advocated to be considered as the gold
standard for the diagnosis of dermatomycosis under such
settings [16,39]. However, this recommendation was
contested by Petinataud et al. [58] because of the higher
chances of contamination due to the two successive
amplification steps involved in the nested PCR. Although
it increases the sensitivity of fungal detection, nested PCR
has the disadvantages of more manipulations, the risk of
contamination, and the prolonged time to get a diag-
nostic response [56,59].
8 | MULTIPLEX P CR
The application of PCR in any diagnostic laboratory is
constrained by the cost incurred and occasionally by
the sample volume, which can be overcome by using
multiple target sequences in a single PCR reaction. The
use of multiple targets in a single reaction constitutes
the multiplex PCR, which has a considerable potential
to save time and efforts within the laboratory without
compromising the test utility [60]. However, the de-
signed primers should be checked for dimerisation,
which may lead to unspecific amplification. The mul-
tiplex PCR increases the efficiency of detection and
reduces the chances of false‐negative results. A duplex
RT‐PCR was developed by Arabatzis et al. [43] for the
direct detection of dermatophytes in nail and skin
clinical samples by using ITS1 and ITS2 target regions.
The Statens Serum Institute in Denmark developed a
commercial duplex PCR kit for the diagnosis of der-
matophytes in general and T. rubrum in particular
from nail samples within 5 hr by targeting CSH1 and
ITS2 regions, respectively [25], and later the same
group applied the multiplex PCR technique in the de-
tection of dermatophytes from tinea unguium cases
[18]. In the same year, duplex PCR was evaluated by
targeting CSH1 and ITS2 regions, and a positivity rate
of 44% for dermatophytes compared with 34% by cul-
ture was reported [44]. They also reported that the
positive predictive value, negative predictive value,
BEGUM ET AL.
specificity, and sensitivity of the duplex PCR were 93%, 87%,
94% and 85%, respectively, when confirmed by either po-
sitive culture or microscopy or both. The multiplex PCR
targeting CHS1 and ITS regions for identification of T. ru-
brum and T. mentagrophytes in nail samples showed a
sensitivity of 97%, compared with 81.1% in the conventional
methods [30]. The duplex PCR is mostly used for the de-
tection of T. rubrum from onychomycosis and tinea pedis
cases in many countries [17,23].
9 | P OL Y M E R A S E C H A I N
REACTION ENZYME‐ LINKED
IM MUNOSORBENT ASSA Y
Polymerase chain reaction enzyme‐linked immunosorbent
assay (PCR‐ELISA) technique is a hybrid of PCR assay and
ELISA whose application is similar to that of ELISA with
the exception that it allows the direct detection of nucleic
acids amplified by PCR instead of the proteins on an
ELISA plate [61]. Initially, the PCR‐ELISA was developed
for direct analysis of labelled amplicons, which was
moderate in sensitivity and specificity. Thus, to improve
the sensitivity and specificity of the detection, a hy-
bridisation step with specific probes was included in the
technique. It involved the amplification of the gene of
interest in the presence of digoxigenin to make
digoxigenin‐labelled PCR product that binds with biotin
labelled oligonucleotide probe. The biotinylated PCR pro-
duct is immobilised on the microplate and detected using
anti‐digoxigenin peroxidase [62]. The other method of
PCR‐ELISA technique involves the hybridisation of PCR
product with fluorescein‐labelled oligonucleotide probe,
followed by detection using anti‐fluorescein antibodies
conjugated to horseradish peroxidase [63]. Although PCR‐
ELISA is more elaborate and time‐consuming, it is less
expensive compared to RT‐PCR, which makes it highly
suitable for low sample throughput laboratories [35]. It has
been reported that PCR‐ELISA could successfully detect
five dermatophyte species, namely T. rubrum, T. inter-
digitale, Trichophyton violaceum, M. canis, and E. flocco-
sum, directly from clinical samples within 24 h by the
amplification of topoisomerase II gene and detection by
hybridisation using digoxigenin‐labelled probes [23]. A
significantly better sensitivity of around 90% was observed,
compared to control, and no cross‐hybridisation was ob-
served, indicating a better specificity of this technique. The
selection of the target sequence for the PCR‐ELISA tech-
nique has a significant effect on the results. For example,
Kupsch et al. [35] compared three types of PCR‐ELISA
based on three targets used, namely Topoisomerase
II‐ELISA (TI‐ELISA), ITS‐ELISA, and Microsatellite‐ELISA
| 7
(MS‐ELISA). They observed that the detection rate of
T. rubrumwas highest by MS‐ELISA, followed by the
ITS‐ELISA, whereas the detection rate of T. interdigitale was
highest with ITS‐ELISA, followed by TI‐ELISA. However,
the sensitivity of these molecular assays determined by the
serial dilutions of reference fungal strain DNA could not be
observed when these assays were applied to clinical sam-
ples. Furthermore, the PCR‐ELISA technique was tested in
cases of onychomycosis, and the sensitivity of 79% and
specificity of 85.5% has been reported [30,64]. Although a
commercial kit called Onychodiag based on PCR‐ELISA
technique was launched in the market for the detection of
dermatophytes in onychomycosis [45], it was its require-
ment of elaborative manipulations and time‐consuming
feature that rendered it less interesting for routine use.
10 | POLY MERAS E C HAIN
RE ACTI ON‐ R E S T R I C T E D
FRAGMENT LENGTH
POLYM ORPHIS M
The polymerase chain reaction‐restricted fragment length
polymorphism (PCR‐RFLP) is a technique that amplifies
the specific fragments of nucleic acids containing
small‐scale genetic variations, followed by analysis with
appropriate restriction enzyme and identification by
electrophoretic resolvement of the fragments. Initially,
the PCR‐RFLP was performed to identify selected
infectious fungal species in dermatological samples such
as Trichophyton spp. and Scytalidium spp. [65,66] or
Trichophyton spp., Fusarium spp. and Aspergillus spp.
in onychomycosis [67,68]. The identification of two
different species in mixed nail infections was done by
interpreting band profiles on agarose gels or by amplicon
sequencing [56,67]. The PCR‐RFLP has been developed
to successfully detect T. rubrum, T. mentagrophytes and
E. floccosum directly from nails, skin or hair samples, but
no strain variations were detected among these species
[69]. It has been automated to a considerable extent for
the rapid diagnosis of fungal infections in clinical speci-
mens. The PCR terminal RFLP was found to detect fungi
in 74% of culture‐negative samples from onychomycosis
with a minimum amount of nail sample required and in
addition to Trichophyton spp., 12 nondermatophyte
species were also recovered [70]. The advantages of
PCR‐RFLP technique are inexpensiveness, easy design,
and no requirement of advanced instruments, whereas
the disadvantages include the requirement of restriction
enzymes, identification of desired genetic variations, and
time‐consuming nature [71]. However, it is its complex
and laborious nature that prevents its routine use.
8 |
11 | S E L E C T I O N O F TA R G E T FO R
D I A G N O S I S OF
D E R M A T O PH Y T O S I S
Since morphological identification of dermatophyte
species in cultures is often difficult and uncertain because
of variations from one isolate to another and overlapping
characters between species that requires considerable
expertise for correct identification, the DNA sequences are
very useful for accurate identification [41]. Furthermore,
generally, low quantity of fungal DNA is recovered from
clinical specimens, and the sensitivity of detection depends
on the selection of a target for amplification and the
molecular technique that could detect the specific ampli-
con [52]. The PCR technique adopted for the direct
identification of the fungus in collected samples varies
from laboratory to laboratory depending on available
facilities, DNA extraction methods, PCR primers, and the
way of analysing the PCR products. The primer used can
be species‐specific to detect only one dermatophyte
species, pan‐dermatophyte primer to detect any dermato-
phyte species or panfungal primer to detect any fungal
species [41]. Dermatophyte taxonomy has undergone sig-
nificant evolution over the recent years as a consequence
of characterisation of the dermatophyte genome [72,73].
However, the identification process of dermatophyte
species based on the traditional NCBI database is highly
problematic because of the plethora of identical sequences
under different names or due to the presence of
misidentified sequences [74,75]. This problem can be
overcome by the use of publicly available new reliable,
up‐to‐date ITS sequence database at the Centraalbur-
eauvoorSchimmelcultures dermatophyte website (http://
www.cbs.knaw.nl/dermatophytes/DefaultPage.aspx) [72]
or by creating a database for the laboratory gradually.
However, the availability of sequences in NCBI database
varies from species to species. For example, in comparison
to Trichophyton species, there are fewer entries of
complete genome sequences of M. canis and Epidermo-
phyton floccosum to interrogate alignments [3].
The CHS1 gene has been the target for pan‐
dermatophyte assays, for several years, because it is a
highly conserved region across all the dermatophytes
[76,77]. However, because of high resolving power and
abundance of repetitive elements (multicopy genes),
which increase the sensitivity of the diagnostic tests, the
current molecular methods mostly target ribosomal DNA
region flanking 18S, ITS1, 5.8S, ITS2 and 26S sequences
with the exceptions of DNA topoisomerase II gene [23]
and microsatellite marker [14]. The polymorphism of the
ITS1 and ITS2 flanking the DNA sequence encoding the
5.8S rDNA is very sensitive and reliable for the identifi-
cation of different dermatophyte species [78]. Even the
BEGUM ET AL.
28S rDNA sequences were reported to be suitable for
dermatophyte diagnosis at the species level [79], but they
were, later on, declared less discriminating than other
rDNA sequences [80].
12 | FACTORS F OR SENSITIVITY
AND S PE CIFICITY OF
DIAGNOSTIC ASSAYS
There are a number of factors that could affect the sen-
sitivity and specificity of molecular diagnosis in the
identification of dermatophytes, such as methods of
preparation, handling, and processing of the clinical
samples for DNA extraction, choice of technique adopted,
presence of inhibitory substances, choice of target
sequence, selection of primers and probes, amplicon size,
the volume, concentration and age of template DNA [81].
Under clinical settings, the presence of the considerable
amount of human DNA or other contaminant DNA along
with the presence of inhibitory substances significantly
hampers the DNA extraction efficiency, which, in turn,
influences the performance of subsequent tests employed
[82]. Kupsch et al. [35] compared two DNA extraction
methods (QIAamp DNA Mini Kit and Illustra genomic
Prep Mini Spin Kit) to extract DNA from clinical samples
and observed that all the diagnostic techniques adopted
have shown a slight surplus of dermatophyte positives
when DNA extraction was performed with the QiaAmp
Mini DNA Kit.
The specificity and sensitivity of the molecular
techniques for the identification of dermatophytes are not
affected by the antifungal treatment of animals 10 days
before molecular testing [27]. It has been reported that
the culture‐negative samples from antifungal treated dogs
and cats were test‐positive in nested PCR, which suggests
that this assay could be used for monitoring of the
treatment efficacy, persistence of dermatophyte carriers,
and for the estimation of the posttreatment period
required for test results to become negative for both
culture and PCR [14,26]. Furthermore, the source of
DNA for dermatophyte identification purpose sig-
nificantly impacts the sensitivity of the molecular tech-
niques applied. The sensitivity of PCR applied to culture
was significantly higher than that of hair samples from
Arabian horses for the detection of T. mentagrophytes, M.
canis and M. equinum [81].
13 | CONCLUSIONS
The molecular diagnostic techniques in the diagnosis of
dermatophytosis have significantly evolved over the
BEGUM ET AL.
recent years, which provide the objective and
reproducible species identification for the specific treat-
ment, monitoring and control of dermatophytosis. All the
molecular techniques discussed in this review have a
significant edge in terms of sensitivity and specificity over
the conventional methods of culture and microscopy for
dermatophytes identification. However, it is equally
important to take into consideration the time required,
economics, complexity and range of species spectrum
detection while selecting an appropriate molecular
technique. The high acquisition cost RT‐PCR is quiet
suitable for high‐throughput diagnostic laboratories with
the advantages of short handling time, quantification of
the fungal load, monitoring of the therapy success, and
the determination of the extent of inhibitions in clinical
samples. Thus, the authors conclude that the agony of
choices involved in the diagnosis of dermatophytes is
multifactorial, which depends on the available laboratory
conditions and the facilities.
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
ORCID
Jubeda Begum http://orcid.org/0000-0002-3636-4792
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How to cite this article: Begum J, Mir NA,
Lingaraju MC, Buyamayum B, Dev K. Recent
advances in the diagnosis of dermatophytosis.
J Basic Microbiol. 2020;1–11.
https://doi.org/10.1002/jobm.201900675