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Onychomycosis: Modern diagnostic and treatment approaches

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DOI: 10.1007/s10354-012-0139-3 · Source: PubMed

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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

review

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
In etwa 60–80 % der Fälle wird die Onychmoykose jedo-
ch durch Dermatophyten verursacht. Der am häufigsten
Onychomykose: Moderne Diagnostik und isolierte Dermatophyt ist Trichophyton (T.) rubrum, wei-
Behandlungsansätze tere relevante Spezies für eine Onychomykose sind T. in-
terdigitale (früher T. mentagrophytes), Epidermophyton
Zusammenfassung  Der medizinische Terminus Ony- floccosum und T. tonsurans. Die wichtigsten, eine Ony-
chomykose steht für eine chronische Infektion des chomykose verursachenden Hefepilze sind Candida al-
Nagelapparates durch einen Pilz. Zu den häufigsten bicans und Candida parapsilosis. Zu den disponierenden
verursachenden Erregern zählen Dermatophyten sowie Faktoren, die eine Onychomykose begünstigen, zäh-
Candida-Arten. Zahlenmäßig weniger bedeutsam sind len vor allem Stoffwechselerkrankungen, wie Diabetes
bestimmte Schimmelpilze (nicht- Dermatophyten- mellitus, aber auch Gefäßerkrankungen, wie periphere
Schimmelpilze oder engl. non-dermatophyte moulds). arterielle Verschlusskrankheit, chronisch-venöse Insuf-

Assoc. Prof. G. Tchernev () · T. Taneva J. C. Cardoso

Polyclinic for Dermatology and Venerology, University Hospital Dermatology and Venerology Department, University Hospital
Lozenetz, Academic Educational Hospital of the Saint Kliment of Coimbra, Praceta Mota Pinto, 3000-075 Coimbra, Portugal
Ohridski University, Medical Faculty, Koziak Street 1, 1407 Sofia, e-mail: [email protected]
Bulgaria
e-mail: [email protected] Assoc. Prof. G. Ginter-Hanselmayer, MD
Department of Dermatology and Venerology, Medical University
P. K. Penev, MD of Graz, Auenbruggerplatz 8, 8036 Graz, Austria
Department of Dermatology and Venerology, Trakia University, e-mail: [email protected]; gabriele.ginter@
Medical faculty, Armeiska Street 11, 6000 Stara Zagora, Bulgaria meduni-graz.at
e-mail: [email protected] J. Ananiev · Assoc. Prof.  M. Gulubova, MD, PhD
Prof. Dr. med. P. Nenoff Department of General and Clinical Pathology, Medical Faculty,
Haut- und Laborarzt/Allergologie, Andrologie, Labor für Trakia University, Armeiska Street 11, 6000 Stara Zagora, Bulgaria
medizinische Mikrobiologie, Straße des Friedens 8, 04579 Mölbis, e-mail: [email protected]
Germany Assoc. Prof. M. Gulubova, MD, PhD
e-mail: [email protected] e-mail: [email protected]
Assoc. Prof.  L. G. Zisova, MD, PhwD · R. Hristova, MD Claudio Guarneri, MD
D. Nocheva, MD Department of Social Territorial Medicine, Section of Dermatology,
Department of Dermatology and Venerology, Medical University University of Messina, c/o A.O.U. “G. Martino"—via Consolare
Plovdiv, Vasil Aprilov 15A Street, Plovdiv, Bulgaria Valeria, Gazzi, 98125 Messina, Italy
e-mail: [email protected] e-mail: [email protected]
R. Hristova, MD Nobuo Kanazawa, MD, PhD
e-mail: [email protected] Department of Dermatology, Wakayama Medical University
D. Nocheva, MD e-mail: [email protected]
e-mail: [email protected]

13 Onychomycosis: modern diagnostic and treatment approaches   1

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fizienz, Polyneuropathien unterschiedlicher Ätiologie In most of the cases, the method of choice depends on
und immunsupprimierende Krankheiten, z. B. myelo- the specialist’s individual experience. The therapeutic
proliferative Neoplasien (wie z. B. Lymphome und Para- approach depends mostly on the fungal organism iden-
proteinämien), HIV/AIDS, etc. Weitere Faktoren, die der tified by the dermatologist or mycologist. This review
Entstehung einer mykotischen Nagelinfektion Vorschub hereby includes the conventional as well as the newest
leisten, sind lokale Traumen bei Profi- oder Leistungss- and most reliable and modern methods used for the
portlern, oft vergesellschaftet mit starker Hyperhidrose. identification of the pathogens causing onychomycosis.
In dermatologischen Kliniken und Praxen kommen Moreover, detailed information is suggested, about the
verschiedene diagnostischen Methoden zur Anwend- choice of therapeutic scheme in case whether dermato-
ung Ein einheitlicher diagnostischer Algorithmus wäre phytes, moulds, or yeasts have been identified as causa-
wünschenswert, nach wie vor ist jedoch die persönliche tive agents. A thorough discussion of the schemes and
Erfahrung des Untersuchers entscheidend für die einge- duration of the antifungal therapy in certain groups of
setzten Methoden. Entscheidend ist, dass der gewählte patients have been included.
therapeutische Ansatz im Wesentlichen vom nachgewi-
esenen Erreger abhängt. In dieser Übersicht wird die Keywords: Onychomycosis, Trichophytonrubrum, ­MALDI-
konventionelle Diagnostik von Onychomykosen darg- TOF MS, Uniplex-PCR-ELISA-Test, Antifungal therapy,
estellt. Außerdem wird auf moderne und neu entwick- Terbinafine, Itraconazole, Laser treatment
elte labordiagnostische Methoden, die zum direkten
Nachweis und zur Identifizierung der nachgewiesenen
Erreger der Onychomykose Einzug in die Dermatolo- Introduction
gie und Mikrobiologie gefunden haben, eingegangen.
Darüber hinaus wird auf die Auswahl der erfolgver- Onychomycosis is а fungal infection of the nail plate,
sprechendsten lokalen und systemischen Therapiefor- caused by dermatophytes, yeasts, and moulds [1, 2].
men erläutert, abhängig davon, ob Dermatophyten, Onychomycosis is the most common disease of the nails
Hefepilze oder Schimmelpilze nachweisbar waren. Die worldwide and constitutes about a half of all nail abnor-
verschiedenen Schemata der Onychomykosetherapie malities [2, 3].
für bestimmte Patientenkollektive werden ausführlich Recent studies, concerning onychomycosis prevalence
dargestellt. among population in the USA and Canada, confirmed the
following results: 6.5 % [2] and 14 % [1], respectively. The
Schlüsselwörter:­  Onychomykose, Trichophyton AHIL survey on the other hand, the largest survey under-
rubrum, MALDI-TOF Massenspektroskopie, Uniplex- taken in 20 European countries on patients with onycho-
PCR-ELISA-Test, Antimykotische Therapie, Terbinafin, mycosis, estimated its prevalence in 29.6 % [4].
Fluconazol, Itraconazol, Laserbehandlung Depending on its origin, onychomycosis can be divi-
ded into primary and secondary. In primary onycho-
Summary  The medical term onychomycosis should be mycosis, fungal invasion affects an intact nail, whereas
understood as chronic infection of the nails caused by secondary onychomycosis occurs in an already abnor-
a fungus. The most common causative agents are the mal nail affected by various diseases or traumas [5]. It
dermatophytes and Candida species. The less common should be noted, however, that when strictly defined,
are certain types of moulds (nondermatophyte moulds primary onychomycosis is a rare occurrence.
or NDMs). In approximately 60–80  % of the cases, ony- Nowadays, an increasing prevalence of onychomy-
chomycosis is due to dermatophytes. Among dermato- cosis has been noted [1]. The most common reasons for
phytes, the most often isolated causative pathogen is Tri- the increasing number of patients with onychomyco-
chophyton (T.) rubrum. Other common species are T. in- sis are the increased lifespan of the general population,
terdigitale (formerly T. mentagrophytes), Epidermophyton the wide use of antibiotics and corticosteroids, the pre-
floccosum, and T. tonsurans. The most significant yeasts valence of synthetic clothes over cotton ones, commu-
causing onychomycosis are Candida albicans and Can- nity swimming pools, gyms, saunas and spa procedures
dida parapsilosis. Predisposing factors for onychomy- becoming a part of everyday life, as well as wearing tight
cosis include mainly diseases such as diabetes mellitus, shoes and sneakers.
peripheral vascular arterial disease, chronic venous in- There are numerous factors that can cause or act like
sufficiency, polyneuropathies of diverse etiologies, and catalysts for the clinical manifestation of onychomycosis.
immunosuppression, e.g., myeloproliferative diseases Among them are diabetes mellitus, smoking, peripheral
(such as lymphoma and paraproteinemia), HIV/AIDS, vascular arterial disease, varicose syndrome, as well as
etc. Other factors facilitating the fungal infection are fre- some systemic diseases such as paraproteinemias, lympho-
quent trauma in professional sportsmen, often accom- mas, congenital or acquired immune deficiencies [6, 7].
panied by excessive perspiration. The diagnostic meth- A seriously disturbing fact is the gradually increasing
ods that are often applied in different dermatologic de- frequency of onychomycosis in children [8]. Some aut-
partments and ambulatory units are also different. This hors estimate onychomycosis prevalence in children at
precludes the creation of a unified diagnostic algorithm 0–2.6 % [8].
that could be used everywhere as a possible standard.

2   Onychomycosis: modern diagnostic and treatment approaches 13

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Another problem also exists, derived from the fact that rable risk for the fungal infection to spread to other areas
often nail psoriasis is misdiagnosed as onychomycosis. of the body, most commonly via autoinoculation [17–19].
Many psoriatic patients have nail changes which morpho- Treatment should be considered in every patient with
logically resemble onychomycosis, and in such patients onychomycosis; but decision to treat should be made on
further differential diagnostic procedures are essential to individual grounds taking into account several factors
exclude the presence of coexisting fungal infection [9]. including the degree of nail involvement and the patient’s
Previously conducted studies report a prevalence rate general status, comorbidities, and concomitant medicati-
of onychomycosis in patients with psoriasis vulgaris vary- ons. Fungal infection may advance to complete destruction
ing in wide ranges from 4.6 [10] to 47.6 % [10], 56 % [11], of the nail plate [20], and involvement of the surrounding
and 43–62 % [9]. In such cases, secondary fungal invasion skin is a common event. However, spreading of the infec-
is most probable. Dystrophic nail changes in psoriasis tion to affect other sites of the body is a rare event. Never-
vulgaris are a predisposing condition to fungal infections. theless, it is important to note that without treatment,
Dermatophytes are the most common causative patients can suffer from low self-esteem, shame and fear,
pathogens of onychomycosis of toenails, while yeasts, and often avoid participating in community activities [20].
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- Ambulatory and clinical diagnosis
tive mycology is observed.
The huge number of psoriatic patients diagnosed with According to some European studies, low percentage of
onychomycosis requires mycological tests to be perfor- dermatologists and general practitioners conduct dia-
med in all patients with psoriasis [9]. gnostic procedures before initiating topical or systemic
An interesting fact is that in most patients onychomy- antifungal therapy [21, 22]. The samples for fungal micro-
cosis is triggered by a long persisting interdigital mycosis scopy should be taken after at least 4–6 days without topi-
and vice versa—in at least one-third of the patients with cal antifungal therapy; otherwise false negative results can
onychomycosis, a coexisting tinea pedis is observed [12]. be obtained. Immediately before the sample is taken, the
Microtraumata, especially in people practicing sports nail plate should be cleaned with 70 % alcohol, thus dimi-
are considered to be a predisposing condition of great nishing the possibility of contaminating the samples with
importance. An interesting fact is that football players moulds or bacteria. Disinfection procedure is not requi-
turn out to be the most affected. red if selective agars which contain cycloheximide (active
Genetic predisposition in patients with onychomyco- against moulds) or chloramphenicol (active against bac-
sis should not be neglected [13]. It had been described by teria) are used. The sample from the affected area is obtai-
several authors that the autosomal-dominant inheritance ned through sharp scissors, nail buffer, or scalpel, and at
is of utmost importance in the clinical manifestation of dis- least 15–20 nail scrapings are needed. A special electrical
tal subungual onychomycosis, caused by T. rubrum [14]. grinding machine can be used if the obtained material is
The reasons why children and adults with Down syn- not sufficient. Some authors consider the best method for
drome are more often affected by onychomycosis and obtaining material for the confirmation of fungal invasion
tinea pedis have not been determined yet [15]. to be the one in which the diagnostician takes the most
In the cases of leading clinical manifestation, the proximal part of the diseased nail as sample [23].
accurate identification of the causative fungal pathogen
is highly advised. The accurate identification of fungal
causative pathogens is obligatory in patients with sig- Conventional methods for laboratory
nificant comorbidities and polymedication as well as in diagnosis of onychomycosis
immunosuppressed patients. In such cases, high risk of
frequent fungal infections exists. Systemic candidosis Conventional methods used for the diagnosis of onycho-
and sepsis are possible complications due to a blood or mycosis have been potassium hydroxide (KOH) prepara-
lymphatic spread of fungal infection. tion and fungal culture of nail samples on Sabouraud’s
In addition, fungal infections predispose to relapsing dextrose agar.
erysipelas, which can be followed by lymphedema [16].
It is assumed that topical antifungal treatment should
be prescribed only after positive microscopic examina- Fungal microscopy
tion for fungal elements and systemic treatment—after
fungal culture, followed by identification. Despite esta- The easiest and quickest method for the identification of
blished rules, not every dermatologist and only a low nail fungal infection is a KOH preparation. However, it is
percentage of general practitioners in Europe initiate characterized by low diagnostic sensitivity [10].
antifungal therapy after testing for fungal infection. An The nail fragments are placed on a slide adding 1–2
interesting fact is that according to some European stu- drops of KOH solution (10–30 %) [24]. After a cover slip
dies, barely around 50  % of the dermatologists conduct is placed, the specimen is put into a humid environment
diagnostic procedures before initiating systemic antifun- for at least 2  hours or more (best overnight). Immedia-
gal therapy. Without systemic therapy there is a conside- tely after that typical morphology of fungal hyphae can

13 Onychomycosis: modern diagnostic and treatment approaches   3

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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,

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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 Fig. 3  Onychomycosis, 57-year-old man with diabetes
50–70  % of patients with onychomycosis [25]. The dia-
gnostic sensitivity can be considerably increased by con- knowledge of the morphological features of dermatophy-
ducting biopsy. However, biopsy is not always advisable tes is needed.
or possible to conduct, e.g., in diabetics. An additional A precise diagnosis and exact identification of the
problem regarding histological diagnosis is that it does causative agent could be performed through some new
not provide identification of the exact species, although molecular biological methods—such as polymerase
dermatophytes or yeasts can be suspected [25, 26]. Com- chain reaction (PCR) and matrix-assisted laser desorption
pared with direct microscopy, histological diagnosis is ionization (MALDI-TOF MS—time of flight mass spectro-
much more reliable [27]. metry). The last one is used to identify dermatophytes in
The histological diagnosis of fungal elements in tissues fungal material that has been isolated from culture [24].
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]. Polymerase chain reaction (PCR)

PCR is a process of in vitro amplification of a DNA mole-

Molecular biological methods as diagnostic tool cule, as a result of which within a few hours millions of
copies of a particular molecule can be generated [24].
Dermatophytes are considered to be one of the most Hence, it is a very sensitive and specific method. There
significant causative pathogens of onychomycosis are different primers available to detect different species,
worldwide. Dermatоphytes belong to three genera of including T. rubrum, T. interdigitale, M. gypseum, M.
fungi—Trichophyton, Epidermophyton, and Microspo- canis, T. tonsurans, T. violaceum, and E. floccosum.
rum. T. rubrum is the most common causative agent of In 1999, the first special gene probe was used for the
dermatophytosis, followed by T. interdigitale, T. tonsu- detection of T. rubrum in nail material (Fig. 3) [27].
rans, E. floccosum, M. gypseum, and rarely M. canis [28].
For their diagnosis, conventional methods can be used—
direct microscopy with KOH preparation, fluorescence Polymerase chain reaction-enzyme-linked
staining, or cultivation on Sabouraud’s dextrose agar. immunosorbent assay (PCR-ELISA) for direct
Certain dermatophytes such as Trichophyton spe- detection of dermatophyte DNA
cies of Arthroderma benhamie (genus Trichophyton)—a
zoophilic dermatophyte can be identified only through This new established method comprises an amplifi-
molecular biological methods [29, 30]. The conven- cation and hybridization technique, which is used to
tional classic (macromorphology and miscroscopic detect sequences within the PCR products of ampli-
examination) and biochemical methods cannot provide fied DNA of dermatophytes. The topoisomerase II gene
identification of Trichophyton species of Arthroderma of the dermatophytes is used as target for the primers
Benhamiae [26]. The direct microscopy with KOH prepa- (one of them is labeled by digoxigenin). DNA isolation
ration, cultivation on Sabouraud’s dextrose agar followed is carried out using the Qiagen QIAamp DNA Mini Kit.
by specifying of microconidia and macroconidia mor- The first step of the amplification process follows those
phology, presence of chlamydospores, urease activity of the PCR—denaturation, annealing of the primers to
are classical methods, showing low specificity and long the single-stranded DNA template and elongation. The
duration—around 6 weeks [31]. In addition, for the cor- ready copies of DNA sequences are used in the second
rect identification of the results a mycologist with a good step—ELISA—in which specific probes (primers) labeled
with biotin, are used to bind to amplified DNA. If derma-

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tophyte DNA is available in the sample, the biotin-labe- According to other sources in the literature, MALDI-
led probe will be fixed to streptavidin that is fixed to the TOF MS, used with special software (SARAMIS), is capa-
bottom of the microtiter plate. After an antibody–antigen ble of identifying the different phenotypic variations of
reaction the enzymatic change of the substrate produces Aspergillus species, even in their specific growth phases
a color change in the microtube that is considered posi- [40]. This technique can be applied and completed wit-
tive. In this way, the presence of dermatophyte DNA in hin a few hours directly from Aspergillus mycelium, and
the examined sample can be confirmed and the identifi- within a few days if spores are present [41].
cation can be performed. MALDI-TOF MS can be used as an additional test
Uniplex-PCR-ELISA-Test includes T. rubrum, T. inter- for confirming the results of fungal cultures, especially
digitale, E. floccosum, T. tonsurans, M. canis, T. violaceum, when accurate identification of the causative agent is
and Trichophyton species of Arthoderma benhamiae difficult—Trichophyton species of Arthoderma banha-
separately. According to some researches, the diagnostic miae, for example, an often ignored and misclassified
value of the selective culture media for Dematophytes is zoophilic dermatophyte, which causes tinea corporis
evaluated to be around 82.1  %, and that of PCR-ELISA- and tinea capitis. This method allows successful mor-
Test 85.8 %, respectively [24]. phological differentiation between Arthroderma ben-
This molecular–biological method allows a conside- hamiae and M. canis (causative agent of tinea capitis).
rably quick identification of the causative agent directly Such differentiation turns out to be impossible in above
from nail material within 24 hours [32]. A multiplex-PCR 75 % of the cases in which classical diagnostic methods
for T. rubrum-DNS identification as well as for other cli- have been used.
nically relevant dermatophytes (Pan-Dermatophyten- With MALDI-TOF MS different species of Candida can
primer) allows identification within 5 hours [33]. also be differentiated (such as C. albicans, C. parapsilo-
The morphological differentiation between anthro- sis, C. magnoliae, C. dubliniensis, C. lusitaniae, C. krusei,
pophilic and zoophilic T. interdigitale strains by clas- C. glabrata, C. tropicalis, and C. guilliermondii).
sical microscopical and biochemical methods is often According to other sources in the literature, MALDI-
problematic. In particular, it is impossible to different- TOF MS can be used for direct identification of fungal
iate between the zoophilic strains of T. interdigitale, T. species in nail material [41]. However, this direct assess-
mentagrophytes, and the Trichophyton anamorph of A. ment of fungi in clinical samples has to be proved in furt-
benhamiae. In these cases, molecular identification met- her studies.
hods may be applied to answer epidemiological, taxono-
mical, and therapeutical questions [34].
Restriction fragment length polymorphism (RFLP)

Matrix-assisted laser desorption ionization The accurate identification of moulds such as Fusarium,
(MALDI-TOF MS) Acremonium, and Aspergillus, is performed in speciali-
zed laboratories. Firstly, PCR is used for the extraction of
MALDI-TOF MS is a routine technique for the identifica- ribosomal RNA followed by RFLP [40].
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 Therapeutic schemes, choice of treatment
has been cultivated in a microbiological culture [35, 36].
MALDI-TOF MS allows identification of the causative The therapeutic scheme is chosen depending on the
agents by the molecular weight of their specific protein microbiological culture, the results of PCR, and/or MAL-
fragments. DI-TOF MS for accurate identification of the causative
The principle of the method—the proteins are added agent (see Table 1).
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 Topical antifungal therapy
reach the detector before large ions. The differences in
the time required for the ions to reach the detector show Topical antifungal therapy is used only in superficial ony-
differences in analyzed spectrum, thus the causative chomycosis, which affects up to one-third of the nail plate.
agent is identified. The spectrum for every microorga- The most commonly used medications are Ciclopirox
nism is individual, so it can be used for identification of (Polinail nail lacquer, Batrafen nail lacquer), Naftifine
fungal species and subspecies from nail and skin sam- (Exoderil solution), and others.
ples. This method can be used not only for onychomyco- An antifungal nail lacquer can be used in onychomy-
sis diagnosis, but also in dermatomycosis diagnosis. cosis which affect up to 40  % of the nail surface or not
This method is specific, sensitive, quick to process, more than three out of ten nails. According to the inter-
and has a relatively low cost, being able to detect not national consensus conference of onychomycosis the
only fungi but also different types of bacterial species in fungal affection should not exceed 50 % of the nail sur-
a sample [37–39]. face. However, some nail lacquers are approved for topi-

6   Onychomycosis: modern diagnostic and treatment approaches 13

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Table 1.  Some of the most commonly used topical and systemic therapeutic options for onychomycosis
Modality Drug Dose Duration/therapeutic scheme
Systemic Terbinafine 250 mg per day 6 weeks—fingernails
12 weeks—toenails
250 mg Once a day for 2 weeks
Then once per week, up to 1 year
250 mg per day 4-weeks course followed by 4-weeks break
Repeat second or third course according to clinical response
Itraconazol 200 mg twice a day for 1 week Two courses with 3-weeks intervals—fingernails
Three to four courses with 3-weeks intervals—toenails
Fluconazol 150–300 (450) mg once a week Up to 9 months or until cure is achieved
Topical Ciclopirox Once a day Until cure is achieved
Amorolfine Once a week Until cure is achieved

cal treatment of an onychomycosis up to 80 % of the nail According to treatment success, a second and third pulse
surface [42]. may follow [43].

Systemic antifungal therapy Fluconazole 

Itraconazole  Fluconazole as well as itraconazole are effective against

infections caused by dermatophytes, and in particular
Itraconazole exhibits efficacy against fungal infections by yeasts (with the exception of Candida glabrata and
caused by dermatophytes, yeasts, and moulds. Candida krusei), and in case of itraconazole also moulds.
A so-called pulse therapy with itraconazole 200  mg Fluconazole can be successfully administrated as
twice daily for 1 week is recommended. After a thera- pulse therapy at a dosage of 150 up to 300 (450) mg once
py-free period of 3 weeks, a second pulse therapy with a week for up to 9 months or until cure is achieved [45].
itraconazole pulse 200 mg twice daily should follow [43].
The total number of pulses in onychomycosis is usually 3,
with a maximum 4. Onychomycosis due to moulds 
During systemic therapy, an additional topical the-
rapy with Ciclopirox (Polinail nail lacquer, Batrafen nail Terbinafine is the drug of choice with highest evidence
lacquer) or Bifonazole cream is recommended [43]. for treatment of onychomycosis due to Scopulariopsis
brevicaulis and Aspergillus spp. Topical drugs may be
effective [46], in particular ciclopirox-containing nail
Terbinafine  lacquer in infections due to Scopulariopsis brevicaulis
and Acremonium spp., best in combination with chemi-
If dermatophytosis of the nails is confirmed then oral cal keratolysis of the nails using 40 % urea preparations.
terbinafine therapy is recommended. Various therapeu-
tic schemes have been proposed.
In practice, terbinafine is usually prescribed as fol- Modification of treatment in certain groups
lows—one oral tablet of 250  mg once a day with diffe- of patients 
rent continuity of the therapy course: at least for about 6
weeks in onychomycosis of the fingernails, which is most Individual modification of therapy is possible, someti-
often of the distal subungual type. According to the clini- mes even compulsory—in polymedicated patients with
cal response, therapy can be continued after 6 weeks. In significant comorbidities, including liver, kidney, or he-
onychomycosis of toenails, the therapeutic course conti- art failure, as well as immunosuppressed patients (Figs.
nues up to 12 weeks. In cases of slow nail growth, therapy 4 and 5). Antifungal therapy should be administrated ca-
should be even more prolonged [43]. refully in patients with chronic or active liver diseases.
One important therapeutic scheme is the following: Before antifungal therapy is initiated, an evaluation of
from the 1st to the 14th day—terbinafine 250 mg once a the possible presence or absence of liver disease should
day, followed by 250 mg terbinafine once a week; treat- be done [43]. In patients with kidney diseases, thera-
ment duration is until recovery and can achieve up to 1 py should also be carefully administrated. In patients
year. with immune deficiency, the full blood count should be
Another intermittent therapeutic scheme is repor- constantly observed especially when treatment conti-
ted by Gupta et al. [44]. Terbinafine 250 mg once daily is nues more than 6 weeks. In patients with heart failure,
administered for 4 weeks, followed by a 4-weeks break. itraconazol should be used with great caution, since it

13 Onychomycosis: modern diagnostic and treatment approaches   7

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review

can have a negative inotropic effect with potential for hetic. Every nail is treated separately vertically and hori-
decompensation of the underlying cardiac disease. zontally, forming a cross over the surface of the nail. The
Furthermore, in polymedicated patients, careful duration of treatment is 45 seconds or less for every nail
choice of the systemic drug should be done according plate. For prevention of reinfection, patients are secured
to the potential for interactions [47]. In general, in these with daily use of antifungal cream. Revision is made 4
patients azol antifungals should be avoided, in particular –6 months after the treatment. The efficacy of therapy is
itraconazol. estimated after the secondary microbiological examina-
tion is performed [50].
Noveon is a laser with dual wavelength of 870 nm and
Surgical treatment of onychomycosis  930 nm. Its parameters are close to those of the infrared
diods [51]. These machines are used for the treatment
It is considered that onychomycosys is one of the fungal of onychomycosis because of their unique photolethal
infections among population with the highest percen- effect to infective agents [51]. These lasers have no terato-
tage of unsuccessful treatment. Although rarely used genic risks, unlike the photodynamic therapy (PDT) with
independently, surgical treatment is an alternative to UV beam. Also, there is no toxic photoablation deriving
systemic therapy [48]. Topical antifungal medications from the Nd:YAG lasers. The results from several clinical
are used at the same time and/or immediately after that, researches show that these lasers are suitable for the tre-
aiming at elimination of the infected nail structures [48]. atment of onychomycosis irrespective of the stage of nail
Surgical treatment could be accompanied by topical or damage [51].
systemic therapy. Surgical nail plate removal could be The femtosecond (f-sec) infrared titanium-sapphire
combined with topical antifungal therapy. This method laser does not damage the surrounding tissues [52].
provides very good clinical results. Such treatment has This laser achieves selective delivery of energy into deep
been applied in cases of Scopulariopsis brevicaulis and layers of the nail bed. In this kind of lasers, interaction
Acremonium species infections [49]. Surgical treatment with environment is nonlinear [52]. Besides, the deep
is also necessary in fungal infections resistant to sys- penetration of this laser contributes to the elimination
temic or topical treatment [49]. Besides avulsion (the of deeply located dermatophytes without damaging the
forcible tearing away of nail plate), the mechanical the- surrounding tissues. Efficacy of treatment is evaluated
rapy of onychomycosis includes abrasion (scraping off by subculture, while assessment of collateral damages is
the superficial layer) of the nail [49]. Partial avulsion is done by scanning electron microscopic [52]. Experience
recommended in cases of distal lateral subungual ony- shows that the f-sec laser inhibits fungus growth suc-
chomycosis and partial subungual onychomycosis as an cessfully in examined specimens, while lower intensity
adjuvant to local therapy. damages the nail plate [52].
CO2 laser also improves the condition of patients with
onychomycosis and gives good results [53, 54]. Other
Laser treatment of onychomycosis  authors have also reported an experimental method of
approaching the laser treatment of onychomycosis [55].
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 Photodynamic therapy (PDT) 
relapses, modern and noninvasive treatment methods
have been investigated. One of them is laser treatment Photodynamic therapy is based on the usage of photo-
[50]. It is applied as 0.65  ms pulsed Nd:Yag 1,064  nm sensitizing agents and light with exact wavelength. Sing-
laser. Patients are treated 2–3 times with minimum let oxygen is generated, leading to cell death. It has been
3-weeks interval between sessions. It is well tolerated. examined whether PDT is appropriate for treatment of
According to the literature, in seven out of eight cases superficial nail infections. There is a research concer-
(87.5 %) fungal cultures are negative after the second or ning the efficiency of PDT in onychomycosis caused by
third procedure. Because of that, treatment with 1,064 moulds—Acremonium sclerotigenum [56]. PDT, combi-
Nd:YAG laser is to be carefully explored, concerning its ned with methyl-aminolevulinic acid, is administrated
long-term effect in relation to clinic and microbiology, in three sessions, with 15 days interval between each
as well as specifying the individual number of treatment procedure [56]. Another study reveals the effect of 5-ami-
courses and optimal regimen [50]. The advantage of nolevulinic acid (ALA). The dermatophyte T. rubrum,
Nd:YAG 1,064  nm laser compared with lasers with lon- causative agent of onychomycosis, metabolizes ALA to
ger exposure to radiation is that there is no need of skin protoporphyrin IX (PP IX) in liquid culture. In optimal
cooling, which simplifies the procedure. Adverse effects conditions, a typical red fluoroscence is seen. It is indu-
and complications are not significant. It is recommen- ced by PP IX and is estimated qualitatively with Wood’s
ded for the nails not to be long for better results. lamp or fluoroscent microscope. Optimal concentration
of ALA is 1–10 mmol/L. ALA causes significant reduction
Laser therapy consists of radiation of 2 mm area with of dermatophyte growth and lack of PP IX fluorescence,
233 J/sm energy without cooling spray, gel, or local anest- if higher concentration of ALA is used. A combination

8   Onychomycosis: modern diagnostic and treatment approaches 13

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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

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by classic methods. The analysis is made with minimal fer systemic therapy with terbinafine instead of itracona-
amount of specimen and at the same time concomitant zole or fluconazole.
diagnosis of a number of species in one clinical sample On the other hand, therapy using fluconazole or itra-
is possible. By PCR, different biologic specimens can be conazole schemes (pulse treatment) is preferred when
examined, including those directly taken from skin lesi- yeasts are identified, but it is considered a second-line
ons, nails, and hair (Figs. 4 and 5). These properties allow treatment for dermatophyte infections.
wide usage of the method by dermatologists and mycolo- Surgical treatment as well as more recently described
gists [24]. In conclusion, PCR is a more reliable method options such as PDT or laser treatment should, in princi-
than direct microscopy and culture. At the moment, this ple, be reserved for cases that have not responded ade-
method is not available in all clinics and laboratories, quately and/or for patients with contraindications for the
although it is used as an additional diagnostic tool to the more conventional therapeutic options.
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- Conflict of interest
ture gives results in 3–4 weeks, whereas PCR method wit- The authors declare that there is no actual or potential
hin a maximum of 1–2 days. This method is also reliable conflict of interest in relation to this article
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 References
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12   Onychomycosis: modern diagnostic and treatment approaches 13

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