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Onychomycosis: Modern diagnostic and treatment approaches

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Onychomycosis: modern diagnostic and
treatment approaches

Georgi Tchernev, Plamen Kolev Penev,

Pietro Nenoff, Liliya Georgieva Zisova,
José Carlos Cardoso, Teodora Taneva,
Gabriele Ginter-Hanselmayer, et al.
Wiener Medizinische Wochenschrift

ISSN 0043-5341

Wien Med Wochenschr

DOI 10.1007/s10354-012-0139-3

1 23
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1 23
 Author's personal copy
Wien Med Wochenschr
DOI 10.1007/s10354-012-0139-3
Onychomycosis: modern diagnostic
and treatment approaches
Georgi Tchernev, Plamen Kolev Penev, Pietro Nenoff, Liliya Georgieva Zisova, José Carlos Cardoso,
Teodora Taneva, Gabriele Ginter-Hanselmayer, Julian Ananiev, Maya Gulubova, Reni Hristova,
Desislava Nocheva, Claudio Guarneri, Nobuo Kanazawa
Received: 29 April 2012 / Accepted: 2 August 2012
© Springer-Verlag Wien 2012
Onychomykose: Moderne Diagnostik und
Behandlungsansätze
Zusammenfassung  Der medizinische Terminus Ony-
chomykose steht für eine chronische Infektion des
Nagelapparates durch einen Pilz. Zu den häufigsten
verursachenden Erregern zählen Dermatophyten sowie
Candida-Arten. Zahlenmäßig weniger bedeutsam sind
bestimmte Schimmelpilze (nicht- Dermatophyten-
Schimmelpilze oder engl. non-dermatophyte moulds).
Assoc. Prof. G. Tchernev () · T. Taneva
Polyclinic for Dermatology and Venerology, University Hospital
Lozenetz, Academic Educational Hospital of the Saint Kliment
Ohridski University, Medical Faculty, Koziak Street 1, 1407 Sofia,
Bulgaria
e-mail: georgi_tchernev@yahoo.de
P. K. Penev, MD
Department of Dermatology and Venerology, Trakia University,
Medical faculty, Armeiska Street 11, 6000 Stara Zagora, Bulgaria
e-mail: pacypenev@gmail.com
Prof. Dr. med. P. Nenoff
Haut- und Laborarzt/Allergologie, Andrologie, Labor für
medizinische Mikrobiologie, Straße des Friedens 8, 04579 Mölbis,
Germany
e-mail: nenoff@mykologie-experten.de
Assoc. Prof.  L. G. Zisova, MD, PhwD · R. Hristova, MD
D. Nocheva, MD
Department of Dermatology and Venerology, Medical University
Plovdiv, Vasil Aprilov 15A Street, Plovdiv, Bulgaria
e-mail: lzisova@abv.bg
R. Hristova, MD
e-mail: reni_hristova@abv.bg
D. Nocheva, MD
e-mail: desi_nocheva@abv.bg
13
review
In etwa 60–80 % der Fälle wird die Onychmoykose jedo-
ch durch Dermatophyten verursacht. Der am häufigsten
isolierte Dermatophyt ist Trichophyton (T.) rubrum, wei-
tere relevante Spezies für eine Onychomykose sind T. in-
terdigitale (früher T. mentagrophytes), Epidermophyton
floccosum und T. tonsurans. Die wichtigsten, eine Ony-
chomykose verursachenden Hefepilze sind Candida al-
bicans und Candida parapsilosis. Zu den disponierenden
Faktoren, die eine Onychomykose begünstigen, zäh-
len vor allem Stoffwechselerkrankungen, wie Diabetes
mellitus, aber auch Gefäßerkrankungen, wie periphere
arterielle Verschlusskrankheit, chronisch-venöse Insuf-
J. C. Cardoso
Dermatology and Venerology Department, University Hospital
of Coimbra, Praceta Mota Pinto, 3000-075 Coimbra, Portugal
e-mail: ze_carlos_cardoso@yahoo.com.br
Assoc. Prof. G. Ginter-Hanselmayer, MD
Department of Dermatology and Venerology, Medical University
of Graz, Auenbruggerplatz 8, 8036 Graz, Austria
e-mail: gabriele.ginter@klinikum-graz.at; gabriele.ginter@
meduni-graz.at
J. Ananiev · Assoc. Prof.  M. Gulubova, MD, PhD
Department of General and Clinical Pathology, Medical Faculty,
Trakia University, Armeiska Street 11, 6000 Stara Zagora, Bulgaria
e-mail: operation@abv.bg
Assoc. Prof. M. Gulubova, MD, PhD
e-mail: mgulubova@hotmail.com
Claudio Guarneri, MD
Department of Social Territorial Medicine, Section of Dermatology,
University of Messina, c/o A.O.U. “G. Martino"—via Consolare
Valeria, Gazzi, 98125 Messina, Italy
e-mail: cguarneri@unime.it
Nobuo Kanazawa, MD, PhD
Department of Dermatology, Wakayama Medical University
e-mail: nkanazaw@wakayama-med.ac.jp
Onychomycosis: modern diagnostic and treatment approaches   1
 Author's personal copy
review
fizienz, Polyneuropathien unterschiedlicher Ätiologie
und immunsupprimierende Krankheiten, z. B. myelo-
proliferative Neoplasien (wie z. B. Lymphome und Para-
proteinämien), HIV/AIDS, etc. Weitere Faktoren, die der
Entstehung einer mykotischen Nagelinfektion Vorschub
leisten, sind lokale Traumen bei Profi- oder Leistungss-
portlern, oft vergesellschaftet mit starker Hyperhidrose.
In dermatologischen Kliniken und Praxen kommen
verschiedene diagnostischen Methoden zur Anwend-
ung Ein einheitlicher diagnostischer Algorithmus wäre
wünschenswert, nach wie vor ist jedoch die persönliche
Erfahrung des Untersuchers entscheidend für die einge-
setzten Methoden. Entscheidend ist, dass der gewählte
therapeutische Ansatz im Wesentlichen vom nachgewi-
esenen Erreger abhängt. In dieser Übersicht wird die
konventionelle Diagnostik von Onychomykosen darg-
estellt. Außerdem wird auf moderne und neu entwick-
elte labordiagnostische Methoden, die zum direkten
Nachweis und zur Identifizierung der nachgewiesenen
Erreger der Onychomykose Einzug in die Dermatolo-
gie und Mikrobiologie gefunden haben, eingegangen.
Darüber hinaus wird auf die Auswahl der erfolgver-
sprechendsten lokalen und systemischen Therapiefor-
men erläutert, abhängig davon, ob Dermatophyten,
Hefepilze oder Schimmelpilze nachweisbar waren. Die
verschiedenen Schemata der Onychomykosetherapie
für bestimmte Patientenkollektive werden ausführlich
dargestellt.
Schlüsselwörter:­  Onychomykose, Trichophyton
rubrum, MALDI-TOF Massenspektroskopie, Uniplex-
PCR-ELISA-Test, Antimykotische Therapie, Terbinafin,
Fluconazol, Itraconazol, Laserbehandlung
Summary  The medical term onychomycosis should be
understood as chronic infection of the nails caused by
a fungus. The most common causative agents are the
dermatophytes and Candida species. The less common
are certain types of moulds (nondermatophyte moulds
or NDMs). In approximately 60–80  % of the cases, ony-
chomycosis is due to dermatophytes. Among dermato-
phytes, the most often isolated causative pathogen is Tri-
chophyton (T.) rubrum. Other common species are T. in-
terdigitale (formerly T. mentagrophytes), Epidermophyton
floccosum, and T. tonsurans. The most significant yeasts
causing onychomycosis are Candida albicans and Can-
dida parapsilosis. Predisposing factors for onychomy-
cosis include mainly diseases such as diabetes mellitus,
peripheral vascular arterial disease, chronic venous in-
sufficiency, polyneuropathies of diverse etiologies, and
immunosuppression, e.g., myeloproliferative diseases
(such as lymphoma and paraproteinemia), HIV/AIDS,
etc. Other factors facilitating the fungal infection are fre-
quent trauma in professional sportsmen, often accom-
panied by excessive perspiration. The diagnostic meth-
ods that are often applied in different dermatologic de-
partments and ambulatory units are also different. This
precludes the creation of a unified diagnostic algorithm
that could be used everywhere as a possible standard.
2   Onychomycosis: modern diagnostic and treatment approaches
In most of the cases, the method of choice depends on
the specialist’s individual experience. The therapeutic
approach depends mostly on the fungal organism iden-
tified by the dermatologist or mycologist. This review
hereby includes the conventional as well as the newest
and most reliable and modern methods used for the
identification of the pathogens causing onychomycosis.
Moreover, detailed information is suggested, about the
choice of therapeutic scheme in case whether dermato-
phytes, moulds, or yeasts have been identified as causa-
tive agents. A thorough discussion of the schemes and
duration of the antifungal therapy in certain groups of
patients have been included.
Keywords: Onychomycosis, Trichophytonrubrum, ­MALDI-
TOF MS, Uniplex-PCR-ELISA-Test, Antifungal therapy,
Terbinafine, Itraconazole, Laser treatment
Introduction
Onychomycosis is а fungal infection of the nail plate,
caused by dermatophytes, yeasts, and moulds [1, 2].
Onychomycosis is the most common disease of the nails
worldwide and constitutes about a half of all nail abnor-
malities [2, 3].
Recent studies, concerning onychomycosis prevalence
among population in the USA and Canada, confirmed the
following results: 6.5 % [2] and 14 % [1], respectively. The
AHIL survey on the other hand, the largest survey under-
taken in 20 European countries on patients with onycho-
mycosis, estimated its prevalence in 29.6 % [4].
Depending on its origin, onychomycosis can be divi-
ded into primary and secondary. In primary onycho-
mycosis, fungal invasion affects an intact nail, whereas
secondary onychomycosis occurs in an already abnor-
mal nail affected by various diseases or traumas [5]. It
should be noted, however, that when strictly defined,
primary onychomycosis is a rare occurrence.
Nowadays, an increasing prevalence of onychomy-
cosis has been noted [1]. The most common reasons for
the increasing number of patients with onychomyco-
sis are the increased lifespan of the general population,
the wide use of antibiotics and corticosteroids, the pre-
valence of synthetic clothes over cotton ones, commu-
nity swimming pools, gyms, saunas and spa procedures
becoming a part of everyday life, as well as wearing tight
shoes and sneakers.
There are numerous factors that can cause or act like
catalysts for the clinical manifestation of onychomycosis.
Among them are diabetes mellitus, smoking, peripheral
vascular arterial disease, varicose syndrome, as well as
some systemic diseases such as paraproteinemias, lympho-
mas, congenital or acquired immune deficiencies [6, 7].
A seriously disturbing fact is the gradually increasing
frequency of onychomycosis in children [8]. Some aut-
hors estimate onychomycosis prevalence in children at
0–2.6 % [8].
13
 Author's personal copy
Another problem also exists, derived from the fact that
often nail psoriasis is misdiagnosed as onychomycosis.
Many psoriatic patients have nail changes which morpho-
logically resemble onychomycosis, and in such patients
further differential diagnostic procedures are essential to
exclude the presence of coexisting fungal infection [9].
Previously conducted studies report a prevalence rate
of onychomycosis in patients with psoriasis vulgaris vary-
ing in wide ranges from 4.6 [10] to 47.6 % [10], 56 % [11],
and 43–62 % [9]. In such cases, secondary fungal invasion
is most probable. Dystrophic nail changes in psoriasis
vulgaris are a predisposing condition to fungal infections.
Dermatophytes are the most common causative
pathogens of onychomycosis of toenails, while yeasts,
more specifically the Candida spp., are more often isola-
ted from fingernails. A correlation between psoriatic nail
change—Nail Psoriasis Area Severity Index—and posi-
tive mycology is observed.
The huge number of psoriatic patients diagnosed with
onychomycosis requires mycological tests to be perfor-
med in all patients with psoriasis [9].
An interesting fact is that in most patients onychomy-
cosis is triggered by a long persisting interdigital mycosis
and vice versa—in at least one-third of the patients with
onychomycosis, a coexisting tinea pedis is observed [12].
Microtraumata, especially in people practicing sports
are considered to be a predisposing condition of great
importance. An interesting fact is that football players
turn out to be the most affected.
Genetic predisposition in patients with onychomyco-
sis should not be neglected [13]. It had been described by
several authors that the autosomal-dominant inheritance
is of utmost importance in the clinical manifestation of dis-
tal subungual onychomycosis, caused by T. rubrum [14].
The reasons why children and adults with Down syn-
drome are more often affected by onychomycosis and
tinea pedis have not been determined yet [15].
In the cases of leading clinical manifestation, the
accurate identification of the causative fungal pathogen
is highly advised. The accurate identification of fungal
causative pathogens is obligatory in patients with sig-
nificant comorbidities and polymedication as well as in
immunosuppressed patients. In such cases, high risk of
frequent fungal infections exists. Systemic candidosis
and sepsis are possible complications due to a blood or
lymphatic spread of fungal infection.
In addition, fungal infections predispose to relapsing
erysipelas, which can be followed by lymphedema [16].
It is assumed that topical antifungal treatment should
be prescribed only after positive microscopic examina-
tion for fungal elements and systemic treatment—after
fungal culture, followed by identification. Despite esta-
blished rules, not every dermatologist and only a low
percentage of general practitioners in Europe initiate
antifungal therapy after testing for fungal infection. An
interesting fact is that according to some European stu-
dies, barely around 50  % of the dermatologists conduct
diagnostic procedures before initiating systemic antifun-
gal therapy. Without systemic therapy there is a conside-
13
review
rable risk for the fungal infection to spread to other areas
of the body, most commonly via autoinoculation [17–19].
Treatment should be considered in every patient with
onychomycosis; but decision to treat should be made on
individual grounds taking into account several factors
including the degree of nail involvement and the patient’s
general status, comorbidities, and concomitant medicati-
ons. Fungal infection may advance to complete destruction
of the nail plate [20], and involvement of the surrounding
skin is a common event. However, spreading of the infec-
tion to affect other sites of the body is a rare event. Never-
theless, it is important to note that without treatment,
patients can suffer from low self-esteem, shame and fear,
and often avoid participating in community activities [20].
Ambulatory and clinical diagnosis
According to some European studies, low percentage of
dermatologists and general practitioners conduct dia-
gnostic procedures before initiating topical or systemic
antifungal therapy [21, 22]. The samples for fungal micro-
scopy should be taken after at least 4–6 days without topi-
cal antifungal therapy; otherwise false negative results can
be obtained. Immediately before the sample is taken, the
nail plate should be cleaned with 70 % alcohol, thus dimi-
nishing the possibility of contaminating the samples with
moulds or bacteria. Disinfection procedure is not requi-
red if selective agars which contain cycloheximide (active
against moulds) or chloramphenicol (active against bac-
teria) are used. The sample from the affected area is obtai-
ned through sharp scissors, nail buffer, or scalpel, and at
least 15–20 nail scrapings are needed. A special electrical
grinding machine can be used if the obtained material is
not sufficient. Some authors consider the best method for
obtaining material for the confirmation of fungal invasion
to be the one in which the diagnostician takes the most
proximal part of the diseased nail as sample [23].
Conventional methods for laboratory
diagnosis of onychomycosis
Conventional methods used for the diagnosis of onycho-
mycosis have been potassium hydroxide (KOH) prepara-
tion and fungal culture of nail samples on Sabouraud’s
dextrose agar.
Fungal microscopy
The easiest and quickest method for the identification of
nail fungal infection is a KOH preparation. However, it is
characterized by low diagnostic sensitivity [10].
The nail fragments are placed on a slide adding 1–2
drops of KOH solution (10–30 %) [24]. After a cover slip
is placed, the specimen is put into a humid environment
for at least 2  hours or more (best overnight). Immedia-
tely after that typical morphology of fungal hyphae can
Onychomycosis: modern diagnostic and treatment approaches   3
 Author's personal copy

review

be observed under the microscope [24]. Some clinicians

heat the slides to accelerate the process, or add color
stains to make hyphae easier to identify.

Fungal cultures

Another frequently used method for the diagnosis of ony-

chomycosis is fungal cultures. The specimen is put into
a Petri dish, containing agar—usually Sabouraud’s dex-
trose agar (a selective medium that is formulated to allow
the growth of fungi and inhibit the growth of bacteria).
The usage of the Kimmig agar is similar. It is a non-
selective agar, which allows the growth of yeasts, derma-
tophytes, and moulds. Its disadvantage is the need of an
experienced clinician, who will be able to distinguish the
microscopic and macroscopic morphology of the causa-
tive pathogen.
A typical fungal culture requires 2–5 weeks at a cons-
tant temperature around 37  °C to grow. After that a
macroscopic and microscopic identification of the cau-
sative pathogen should be done (Figs. 1 and 2). Some
additional substances are recommended to be used in Fig. 1  Infection due to T. rubrum, 32-year-old swimmer
order for the bacterial growth in fungal cultures to be
inhibited. Sabouraud’s dextrose agar allows considerably
faster fungal growth in comparison with Kimmig agar.
Both the agars contain antibiotics (Chloramphenicol
50  mg/L, Penicillin, Streptomycin 40,000 IE/L), which
inhibit bacterial growth. In routine diagnostic procedu-
res for the accurate identification of the fungal causative
pathogen, Sabouraud’s 2 or 4 %-dextrose agar are predo-
minantly used; Kimmig agar can be used as an alternative.
For selective identification of the causative pathogens
of onychomycosis, especially regarding dermatophytes
and yeasts, Sabouraud’s dextrose agar+ Actidion (cyclohe-
ximide 400 mg/L) is recommended. Cycloheximide inhi-
bits the growth of moulds, bacteria, and certain yeasts.
A possible alternative is Mycosel agar (Becton Dickin-
son, Heidelberg, Germany), which contains cyclohexi-
Fig. 2  Diagnostics of T. rubrum by culture
mide [25]. Another alternative is the modified agar for
dermatophytes (SIFIN, Berlin)—2 % Sabouraud’s dextrose
agar with additional cycloheximide and chloramphenicol. They cannot be cultivated on media containing Actidion
The identification of yeasts and dermatophytes is prece- (cycloheximide). If a medium with Actidion (cyclohexi-
ded by macroscopic, microscopic, and molecular biolo- mide) is routinely used, mould identification as a causa-
gical observation of the infected people. Dermatophytes tive pathogen can sometimes be impossible. In order for
grow for about 2–4 weeks at a temperature of 26–32 °C. the moulds to be identified as causative pathogens rather
Yeasts, especially that of the genus Candida, grow than laboratory contaminants, they should be isolated in
within 2–4 days at 26–32 °C, or up to 37 °C. For Candida several consecutive microbiological cultures. The exact
albicans and nonalbicans species, culture on the so-cal- type of causative pathogen is determined by microbiolo-
led CHROM agar or biochemical tests (e.g., API 20 °C, ID gical culture [25].
32  °C, bioMérieux SA, France) is used. Rarely, onycho-
mycosis can be caused by yeasts from the genus Malas-
sezia—especially in immunosuppressed patients or those Fluorescence microscopy
with AIDS. The cultivation of Malassezia spp. is performed
onto lipid-enriched Dixon’s agar medium, and the Tween This method gains higher sensitivity, when compared
test is used to identify the different Malassezia species. with the direct microscopic examination using KOH pre-
The growth of moulds begins during the first days paration. However, UV light (UVA 365 nm, special filter)
after the cultivation on Sabouraud agar. They can be and a fluorescence microscope are needed. A special
both causative pathogens or laboratory contaminants. fluorescent substance is added to the KOH (Blankophor,

4   Onychomycosis: modern diagnostic and treatment approaches 13

 Author's personal copy
Calcofluor, or acridinium orange). It binds to fungal
chitin, and marks hyphae and arthrospores appear as
brightening structures.
Histopathology
Histological examination of nail material is rarely used,
but it is a highly informative method. One of the follo-
wing stainings is used—periodic acid-schiff (PAS), or
Groccot–Gomori silver staining. It should be conside-
red that although there is a fungal invasion of the tis-
sues, histopathology is not always positive. According
to some retrospective researches undertaken in famous
European University Hospitals, the diagnostic sensiti-
vity of the fungal culture and microscopy is not always
very high. In some cases, they are positive in just about
50–70  % of patients with onychomycosis [25]. The dia-
gnostic sensitivity can be considerably increased by con-
ducting biopsy. However, biopsy is not always advisable
or possible to conduct, e.g., in diabetics. An additional
problem regarding histological diagnosis is that it does
not provide identification of the exact species, although
dermatophytes or yeasts can be suspected [25, 26]. Com-
pared with direct microscopy, histological diagnosis is
much more reliable [27].
The histological diagnosis of fungal elements in tissues
is a highly sensitive method in comparison with the direct
microscopy with KOH preparation and fungal culture.
However, patients often reject this diagnostic procedure [24].
Molecular biological methods as diagnostic tool
Dermatophytes are considered to be one of the most
significant causative pathogens of onychomycosis
worldwide. Dermatоphytes belong to three genera of
fungi—Trichophyton, Epidermophyton, and Microspo-
rum. T. rubrum is the most common causative agent of
dermatophytosis, followed by T. interdigitale, T. tonsu-
rans, E. floccosum, M. gypseum, and rarely M. canis [28].
For their diagnosis, conventional methods can be used—
direct microscopy with KOH preparation, fluorescence
staining, or cultivation on Sabouraud’s dextrose agar.
Certain dermatophytes such as Trichophyton spe-
cies of Arthroderma benhamie (genus Trichophyton)—a
zoophilic dermatophyte can be identified only through
molecular biological methods [29, 30]. The conven-
tional classic (macromorphology and miscroscopic
examination) and biochemical methods cannot provide
identification of Trichophyton species of Arthroderma
Benhamiae [26]. The direct microscopy with KOH prepa-
ration, cultivation on Sabouraud’s dextrose agar followed
by specifying of microconidia and macroconidia mor-
phology, presence of chlamydospores, urease activity
are classical methods, showing low specificity and long
duration—around 6 weeks [31]. In addition, for the cor-
rect identification of the results a mycologist with a good
13
review
Fig. 3  Onychomycosis, 57-year-old man with diabetes
knowledge of the morphological features of dermatophy-
tes is needed.
A precise diagnosis and exact identification of the
causative agent could be performed through some new
molecular biological methods—such as polymerase
chain reaction (PCR) and matrix-assisted laser desorption
ionization (MALDI-TOF MS—time of flight mass spectro-
metry). The last one is used to identify dermatophytes in
fungal material that has been isolated from culture [24].
Polymerase chain reaction (PCR)
PCR is a process of in vitro amplification of a DNA mole-
cule, as a result of which within a few hours millions of
copies of a particular molecule can be generated [24].
Hence, it is a very sensitive and specific method. There
are different primers available to detect different species,
including T. rubrum, T. interdigitale, M. gypseum, M.
canis, T. tonsurans, T. violaceum, and E. floccosum.
In 1999, the first special gene probe was used for the
detection of T. rubrum in nail material (Fig. 3) [27].
Polymerase chain reaction-enzyme-linked
immunosorbent assay (PCR-ELISA) for direct
detection of dermatophyte DNA
This new established method comprises an amplifi-
cation and hybridization technique, which is used to
detect sequences within the PCR products of ampli-
fied DNA of dermatophytes. The topoisomerase II gene
of the dermatophytes is used as target for the primers
(one of them is labeled by digoxigenin). DNA isolation
is carried out using the Qiagen QIAamp DNA Mini Kit.
The first step of the amplification process follows those
of the PCR—denaturation, annealing of the primers to
the single-stranded DNA template and elongation. The
ready copies of DNA sequences are used in the second
step—ELISA—in which specific probes (primers) labeled
with biotin, are used to bind to amplified DNA. If derma-
Onychomycosis: modern diagnostic and treatment approaches   5
 Author's personal copy
review
tophyte DNA is available in the sample, the biotin-labe-
led probe will be fixed to streptavidin that is fixed to the
bottom of the microtiter plate. After an antibody–antigen
reaction the enzymatic change of the substrate produces
a color change in the microtube that is considered posi-
tive. In this way, the presence of dermatophyte DNA in
the examined sample can be confirmed and the identifi-
cation can be performed.
Uniplex-PCR-ELISA-Test includes T. rubrum, T. inter-
digitale, E. floccosum, T. tonsurans, M. canis, T. violaceum,
and Trichophyton species of Arthoderma benhamiae
separately. According to some researches, the diagnostic
value of the selective culture media for Dematophytes is
evaluated to be around 82.1  %, and that of PCR-ELISA-
Test 85.8 %, respectively [24].
This molecular–biological method allows a conside-
rably quick identification of the causative agent directly
from nail material within 24 hours [32]. A multiplex-PCR
for T. rubrum-DNS identification as well as for other cli-
nically relevant dermatophytes (Pan-Dermatophyten-
primer) allows identification within 5 hours [33].
The morphological differentiation between anthro-
pophilic and zoophilic T. interdigitale strains by clas-
sical microscopical and biochemical methods is often
problematic. In particular, it is impossible to different-
iate between the zoophilic strains of T. interdigitale, T.
mentagrophytes, and the Trichophyton anamorph of A.
benhamiae. In these cases, molecular identification met-
hods may be applied to answer epidemiological, taxono-
mical, and therapeutical questions [34].
Matrix-assisted laser desorption ionization
(MALDI-TOF MS)
MALDI-TOF MS is a routine technique for the identifica-
tion of certain bacteria and it is nowadays gaining increa-
sing popularity [27]. At present, the use of the method is
restricted to the identification of a causative agent that
has been cultivated in a microbiological culture [35, 36].
MALDI-TOF MS allows identification of the causative
agents by the molecular weight of their specific protein
fragments.
The principle of the method—the proteins are added
to crystals of UV-absorbing proteins (matrix). Laser flash
ionizes the matrix molecules and as a result, positive ions
are formed, which are captured by a detector. Small ions
reach the detector before large ions. The differences in
the time required for the ions to reach the detector show
differences in analyzed spectrum, thus the causative
agent is identified. The spectrum for every microorga-
nism is individual, so it can be used for identification of
fungal species and subspecies from nail and skin sam-
ples. This method can be used not only for onychomyco-
sis diagnosis, but also in dermatomycosis diagnosis.
This method is specific, sensitive, quick to process,
and has a relatively low cost, being able to detect not
only fungi but also different types of bacterial species in
a sample [37–39].
6   Onychomycosis: modern diagnostic and treatment approaches
According to other sources in the literature, MALDI-
TOF MS, used with special software (SARAMIS), is capa-
ble of identifying the different phenotypic variations of
Aspergillus species, even in their specific growth phases
[40]. This technique can be applied and completed wit-
hin a few hours directly from Aspergillus mycelium, and
within a few days if spores are present [41].
MALDI-TOF MS can be used as an additional test
for confirming the results of fungal cultures, especially
when accurate identification of the causative agent is
difficult—Trichophyton species of Arthoderma banha-
miae, for example, an often ignored and misclassified
zoophilic dermatophyte, which causes tinea corporis
and tinea capitis. This method allows successful mor-
phological differentiation between Arthroderma ben-
hamiae and M. canis (causative agent of tinea capitis).
Such differentiation turns out to be impossible in above
75 % of the cases in which classical diagnostic methods
have been used.
With MALDI-TOF MS different species of Candida can
also be differentiated (such as C. albicans, C. parapsilo-
sis, C. magnoliae, C. dubliniensis, C. lusitaniae, C. krusei,
C. glabrata, C. tropicalis, and C. guilliermondii).
According to other sources in the literature, MALDI-
TOF MS can be used for direct identification of fungal
species in nail material [41]. However, this direct assess-
ment of fungi in clinical samples has to be proved in furt-
her studies.
Restriction fragment length polymorphism (RFLP)
The accurate identification of moulds such as Fusarium,
Acremonium, and Aspergillus, is performed in speciali-
zed laboratories. Firstly, PCR is used for the extraction of
ribosomal RNA followed by RFLP [40].
Therapeutic schemes, choice of treatment
The therapeutic scheme is chosen depending on the
microbiological culture, the results of PCR, and/or MAL-
DI-TOF MS for accurate identification of the causative
agent (see Table 1).
Topical antifungal therapy
Topical antifungal therapy is used only in superficial ony-
chomycosis, which affects up to one-third of the nail plate.
The most commonly used medications are Ciclopirox
(Polinail nail lacquer, Batrafen nail lacquer), Naftifine
(Exoderil solution), and others.
An antifungal nail lacquer can be used in onychomy-
cosis which affect up to 40  % of the nail surface or not
more than three out of ten nails. According to the inter-
national consensus conference of onychomycosis the
fungal affection should not exceed 50 % of the nail sur-
face. However, some nail lacquers are approved for topi-
13
 Author's personal copy
Table 1.  Some of the most commonly used topical and systemic therapeutic options for onychomycosis
Modality Drug Dose
Systemic Terbinafine 250 mg per day
250 mg
250 mg per day
Itraconazol 200 mg twice a day for 1 week
Fluconazol 150–300 (450) mg once a week
Topical Ciclopirox Once a day
Amorolfine Once a week
cal treatment of an onychomycosis up to 80 % of the nail
surface [42].
Systemic antifungal therapy
Itraconazole  Fluconazole as well as itraconazole are effective against
Itraconazole exhibits efficacy against fungal infections
caused by dermatophytes, yeasts, and moulds.
A so-called pulse therapy with itraconazole 200  mg
twice daily for 1 week is recommended. After a thera-
py-free period of 3 weeks, a second pulse therapy with
itraconazole pulse 200 mg twice daily should follow [43].
The total number of pulses in onychomycosis is usually 3,
with a maximum 4.
During systemic therapy, an additional topical the-
rapy with Ciclopirox (Polinail nail lacquer, Batrafen nail
lacquer) or Bifonazole cream is recommended [43].
Terbinafine  lacquer in infections due to Scopulariopsis brevicaulis
If dermatophytosis of the nails is confirmed then oral
terbinafine therapy is recommended. Various therapeu-
tic schemes have been proposed.
In practice, terbinafine is usually prescribed as fol-
lows—one oral tablet of 250  mg once a day with diffe-
rent continuity of the therapy course: at least for about 6
weeks in onychomycosis of the fingernails, which is most
often of the distal subungual type. According to the clini-
cal response, therapy can be continued after 6 weeks. In
onychomycosis of toenails, the therapeutic course conti-
nues up to 12 weeks. In cases of slow nail growth, therapy
should be even more prolonged [43].
One important therapeutic scheme is the following:
from the 1st to the 14th day—terbinafine 250 mg once a
day, followed by 250 mg terbinafine once a week; treat-
ment duration is until recovery and can achieve up to 1
year.
Another intermittent therapeutic scheme is repor-
ted by Gupta et al. [44]. Terbinafine 250 mg once daily is
administered for 4 weeks, followed by a 4-weeks break.
13
review
Duration/therapeutic scheme
6 weeks—fingernails
12 weeks—toenails
Once a day for 2 weeks
Then once per week, up to 1 year
4-weeks course followed by 4-weeks break
Repeat second or third course according to clinical response
Two courses with 3-weeks intervals—fingernails
Three to four courses with 3-weeks intervals—toenails
Up to 9 months or until cure is achieved
Until cure is achieved
Until cure is achieved
According to treatment success, a second and third pulse
may follow [43].
Fluconazole 
infections caused by dermatophytes, and in particular
by yeasts (with the exception of Candida glabrata and
Candida krusei), and in case of itraconazole also moulds.
Fluconazole can be successfully administrated as
pulse therapy at a dosage of 150 up to 300 (450) mg once
a week for up to 9 months or until cure is achieved [45].
Onychomycosis due to moulds 
Terbinafine is the drug of choice with highest evidence
for treatment of onychomycosis due to Scopulariopsis
brevicaulis and Aspergillus spp. Topical drugs may be
effective [46], in particular ciclopirox-containing nail
and Acremonium spp., best in combination with chemi-
cal keratolysis of the nails using 40 % urea preparations.
Modification of treatment in certain groups
of patients 
Individual modification of therapy is possible, someti-
mes even compulsory—in polymedicated patients with
significant comorbidities, including liver, kidney, or he-
art failure, as well as immunosuppressed patients (Figs.
4 and 5). Antifungal therapy should be administrated ca-
refully in patients with chronic or active liver diseases.
Before antifungal therapy is initiated, an evaluation of
the possible presence or absence of liver disease should
be done [43]. In patients with kidney diseases, thera-
py should also be carefully administrated. In patients
with immune deficiency, the full blood count should be
constantly observed especially when treatment conti-
nues more than 6 weeks. In patients with heart failure,
itraconazol should be used with great caution, since it
Onychomycosis: modern diagnostic and treatment approaches   7
 Author's personal copy
review
can have a negative inotropic effect with potential for
decompensation of the underlying cardiac disease.
Furthermore, in polymedicated patients, careful
choice of the systemic drug should be done according
to the potential for interactions [47]. In general, in these
patients azol antifungals should be avoided, in particular
itraconazol.
Surgical treatment of onychomycosis  930 nm. Its parameters are close to those of the infrared
It is considered that onychomycosys is one of the fungal
infections among population with the highest percen-
tage of unsuccessful treatment. Although rarely used
independently, surgical treatment is an alternative to
systemic therapy [48]. Topical antifungal medications
are used at the same time and/or immediately after that,
aiming at elimination of the infected nail structures [48].
Surgical treatment could be accompanied by topical or
systemic therapy. Surgical nail plate removal could be
combined with topical antifungal therapy. This method
provides very good clinical results. Such treatment has
been applied in cases of Scopulariopsis brevicaulis and
Acremonium species infections [49]. Surgical treatment
is also necessary in fungal infections resistant to sys-
temic or topical treatment [49]. Besides avulsion (the
forcible tearing away of nail plate), the mechanical the-
rapy of onychomycosis includes abrasion (scraping off
the superficial layer) of the nail [49]. Partial avulsion is
recommended in cases of distal lateral subungual ony-
chomycosis and partial subungual onychomycosis as an
adjuvant to local therapy.
Laser treatment of onychomycosis  authors have also reported an experimental method of
Because of the high morbidity rate of onychomycosis
and the low results of oral and topical therapy, adminis-
trated separately or at the same time, as well as common
relapses, modern and noninvasive treatment methods
have been investigated. One of them is laser treatment
[50]. It is applied as 0.65  ms pulsed Nd:Yag 1,064  nm
laser. Patients are treated 2–3 times with minimum
3-weeks interval between sessions. It is well tolerated.
According to the literature, in seven out of eight cases
(87.5 %) fungal cultures are negative after the second or
third procedure. Because of that, treatment with 1,064
Nd:YAG laser is to be carefully explored, concerning its
long-term effect in relation to clinic and microbiology,
as well as specifying the individual number of treatment
courses and optimal regimen [50]. The advantage of
Nd:YAG 1,064  nm laser compared with lasers with lon-
ger exposure to radiation is that there is no need of skin
cooling, which simplifies the procedure. Adverse effects
and complications are not significant. It is recommen-
ded for the nails not to be long for better results.
Laser therapy consists of radiation of 2 mm area with
233 J/sm energy without cooling spray, gel, or local anest-
8   Onychomycosis: modern diagnostic and treatment approaches
hetic. Every nail is treated separately vertically and hori-
zontally, forming a cross over the surface of the nail. The
duration of treatment is 45 seconds or less for every nail
plate. For prevention of reinfection, patients are secured
with daily use of antifungal cream. Revision is made 4
–6 months after the treatment. The efficacy of therapy is
estimated after the secondary microbiological examina-
tion is performed [50].
Noveon is a laser with dual wavelength of 870 nm and
diods [51]. These machines are used for the treatment
of onychomycosis because of their unique photolethal
effect to infective agents [51]. These lasers have no terato-
genic risks, unlike the photodynamic therapy (PDT) with
UV beam. Also, there is no toxic photoablation deriving
from the Nd:YAG lasers. The results from several clinical
researches show that these lasers are suitable for the tre-
atment of onychomycosis irrespective of the stage of nail
damage [51].
The femtosecond (f-sec) infrared titanium-sapphire
laser does not damage the surrounding tissues [52].
This laser achieves selective delivery of energy into deep
layers of the nail bed. In this kind of lasers, interaction
with environment is nonlinear [52]. Besides, the deep
penetration of this laser contributes to the elimination
of deeply located dermatophytes without damaging the
surrounding tissues. Efficacy of treatment is evaluated
by subculture, while assessment of collateral damages is
done by scanning electron microscopic [52]. Experience
shows that the f-sec laser inhibits fungus growth suc-
cessfully in examined specimens, while lower intensity
damages the nail plate [52].
CO2 laser also improves the condition of patients with
onychomycosis and gives good results [53, 54]. Other
approaching the laser treatment of onychomycosis [55].
Photodynamic therapy (PDT) 
Photodynamic therapy is based on the usage of photo-
sensitizing agents and light with exact wavelength. Sing-
let oxygen is generated, leading to cell death. It has been
examined whether PDT is appropriate for treatment of
superficial nail infections. There is a research concer-
ning the efficiency of PDT in onychomycosis caused by
moulds—Acremonium sclerotigenum [56]. PDT, combi-
ned with methyl-aminolevulinic acid, is administrated
in three sessions, with 15 days interval between each
procedure [56]. Another study reveals the effect of 5-ami-
nolevulinic acid (ALA). The dermatophyte T. rubrum,
causative agent of onychomycosis, metabolizes ALA to
protoporphyrin IX (PP IX) in liquid culture. In optimal
conditions, a typical red fluoroscence is seen. It is indu-
ced by PP IX and is estimated qualitatively with Wood’s
lamp or fluoroscent microscope. Optimal concentration
of ALA is 1–10 mmol/L. ALA causes significant reduction
of dermatophyte growth and lack of PP IX fluorescence,
if higher concentration of ALA is used. A combination
13
 Author's personal copy

review

between ALA and light clearly demonstrates the inhibi- Fungal tests are very important in the differential dia-
tory effect of PDT with ALA. This method is promising as gnosis of nail diseases, since other condition such as
far as reduction of T. rubrum colonization in onychomy- lichen planus, psoriasis, and nail dystrophy of different
cosis is concerned [57]. causes may have features that can be confused with ony-
One of the possible PDT schemes is: Damaged nail chomycosis. Having in mind that onychomycosis fre-
surface is coated lavishly with 20 % urea unguentum and quently occurs in cases of primary or genetically damaged
is covered with a folio for 10 hours. The nails are subse- nails, this problem is not very easy to be solved [9, 24, 25].
quently treated with 20 % solution of ALA methyl ester in Modern additional methods, which are molecular–
liquid cream for 5 hours, but only after protection from biological—PCR, Uniplex-PCR-ELISA-Test, and MALDI-
light with plaster and aluminium folio has been done. TOF MS—will probably play a very important role in the
Protoporphyrin fluorescence is confirmed with UV-beam diagnosis of onychomycosis [24, 40].
and spectrophotometer before PDT. It’s observed in nail PCR is an easy and rapid method. Extraction of DNA
base and periphery of fungal lesions. The nail (including takes up to 30 minutes, whereas elongation takes 5 hours.
proximal and lateral nail borders) is exposed to radia- PCR results are ready within a day. This method is charac-
tion horizontally and vertically with pulsed laser 630 nm terized by high sensitivity and diagnostic specificity that
light, 100 J/sm2 using excimer laser [58] PDT with 5-ALA, exceed the classic diagnostic methods. The specificity of
applied once a week [59]. A bearable pain has been repor- PCR analysis derives from the possibility of revealing the
ted in patients during the procedure but it tends to disap- exact causative agents in the presence of other microor-
pear the next day. Improvement in the condition occurs ganisms, viruses, and cells of macroorganisms [24].
after 6–7 treatment courses (total dosage 600–700 J/sm2). From this point of view, the ability of PCR can be defi-
Most frequently, dermatophytes are not found in post- ned as unique. By using this method, it is easy to identify
treatment investigations—microscopy with KOH pre- and diagnose dermatophytes that are difficult to detect
paration and culture [57]. According to the literature,
there are no relapses of onychomycosis after 3–6 months.
In comparison, improvement is not observed after tre-
atment with ALA or radiation solely [59]. According to
recent researches, PDT with methylaminolevulinate is
also a successful treatment of refractory onychomycosis
caused by nondermatophytic moulds [58].
PDT is suitable for treatment of distal and lateral
subungual onychomycosis caused by T. rubrum [60].
The advantages of PDT are the lack of side effects, con-
cerning kidney and liver function, as well as the lack of
risk to patients with systemic diseases and the absence
of interaction with other drugs [57, 59]. Old age is not a
contraindication for treatment. On the other hand, oral
therapy with antifungal agents may not be effective and it
may be even contraindicated in case of intolerance. PDT
is a very good alternative in these cases [58, 60]. Fig. 4  Tinea pedis, 57-year-old patient, caused by T. rubrum

Discussion

Currently, the most frequently used diagnostic methods

for fungal infection confirmation are the conventional
ones—direct microscopy with KOH preparation and cul-
ture on Saboraud agar.
Culture identifies the exact causative agent and in this
way makes it easier to choose the most suitable medica-
tion [25].
Culture has a diagnostic sensitivity of 50–70  %. As a
result, 30–50 % of fungal agents cannot be identified by
conventional methods [25].
Fluorescence method and histology/PAS are less used.
A number of data reveals that PAS staining is a considera-
bly sensitive method in comparison with other methods
for diagnosis, but not all patients will accept this invasive
manipulation [24, 25]. Fig. 5  Onychomycosis, a 70-year-old man; causative patho-
gen—T. rubrum

13 Onychomycosis: modern diagnostic and treatment approaches   9

 Author's personal copy
review
by classic methods. The analysis is made with minimal
amount of specimen and at the same time concomitant
diagnosis of a number of species in one clinical sample
is possible. By PCR, different biologic specimens can be
examined, including those directly taken from skin lesi-
ons, nails, and hair (Figs. 4 and 5). These properties allow
wide usage of the method by dermatologists and mycolo-
gists [24]. In conclusion, PCR is a more reliable method
than direct microscopy and culture. At the moment, this
method is not available in all clinics and laboratories,
although it is used as an additional diagnostic tool to the
classic methods. This method has considerably increased
the percentage of positive results and has reduced the
time needed for achieving an accurate diagnosis. The cul-
ture gives results in 3–4 weeks, whereas PCR method wit-
hin a maximum of 1–2 days. This method is also reliable
as far was necessary investments and materials for main-
tenance are concerned. At present, the wide use of the
method is restricted only by labor intensity and the need
of more staff. In recent years, it is considered that mole-
cular biology methods will completely replace conventio-
nal methods, which are currently considered as standard
methods. The establishment of separate specialized cen-
ters and laboratories aiming to focus their activity in this
field could be significant to the exact identification of the
causative agent and administration of proper systemic
therapy. The choice of treatment and duration of antifun-
gal therapy depends on the identification of the causative
agent—dermatophytes, moulds, and yeasts. Besides the
well-known topical and systemic antifungal medicati-
ons, in recent years new treatment methods have gained
popularity—PDT and laser treatment have been success-
fully applied.
Conclusion
The most frequently used diagnostic methods for the
confirmation of fungal infection are still the conventional
ones—direct microscopy with KOH preparation and cul-
ture on Sabouraud’s dextrose agar. Only a restricted num-
ber of laboratories perform modern diagnostic molecular
biology methods such as PCR and MALDI-TOF MS. This
results in problems in creating a universally accepted
diagnostic algorithm. At present, treatment options for
onychomycosis include topical and systemic antifungal
medications, as well as surgical treatment, PDT, and laser
treatment for very particular situations.
Therapy with terbinafine is preferred in cases of ony-
chomycosis caused by dermatophytes. It has a very good
effect in infections caused by Trichophyton, Microspo-
rum, and Epidermophyton species [61, 62]. Therapy with
terbinafine has also good effect in infections with moulds
[62, 63]. It is, however, less effective against yeasts, since
it is primarily fungistatic against some species, for exam-
ple, Candida albicans and Candida parapsilosis [62, 63].
Because of the powerful fungicid effect of terbinafine
against dermatophytes and moulds, many clinicians pre-
10   Onychomycosis: modern diagnostic and treatment approaches
fer systemic therapy with terbinafine instead of itracona-
zole or fluconazole.
On the other hand, therapy using fluconazole or itra-
conazole schemes (pulse treatment) is preferred when
yeasts are identified, but it is considered a second-line
treatment for dermatophyte infections.
Surgical treatment as well as more recently described
options such as PDT or laser treatment should, in princi-
ple, be reserved for cases that have not responded ade-
quately and/or for patients with contraindications for the
more conventional therapeutic options.
Conflict of interest
The authors declare that there is no actual or potential
conflict of interest in relation to this article
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