Wiener Medizinische Wochenschrift
Wien Med Wochenschr
Onychomycosis: modern diagnostic and
Georgi Tchernev, Plamen Kolev Penev,
Pietro Nenoff, Liliya Georgieva Zisova,
José Carlos Cardoso, Teodora Taneva,
Gabriele Ginter-Hanselmayer, et al.
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Onychomycosis: modern diagnostic and treatment approaches 1
Onychomykose: Moderne Diagnostik und
Zusammenfassung Der medizinische Terminus Ony-
chomykose steht für eine chronische Infektion des
Nagelapparates durch einen Pilz. Zu den häugsten
verursachenden Erregern zählen Dermatophyten sowie
Candida-Arten. Zahlenmäßig weniger bedeutsam sind
bestimmte Schimmelpilze (nicht- Dermatophyten-
Schimmelpilze oder engl. non-dermatophyte moulds).
In etwa 60–80 % der Fälle wird die Onychmoykose jedo-
ch durch Dermatophyten verursacht. Der am häugsten
isolierte Dermatophyt ist Trichophyton (T.) rubrum, wei-
tere relevante Spezies für eine Onychomykose sind T. in-
terdigitale (früher T. mentagrophytes), Epidermophyton
occosum 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 Stowechselerkrankungen, wie Diabetes
mellitus, aber auch Gefäßerkrankungen, wie periphere
arterielle Verschlusskrankheit, chronisch-venöse Insuf-
Wien Med Wochenschr
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
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 Soa,
P. K . Penev,MD
Department of Dermatology and Venerology, Trakia University,
Medical faculty, Armeiska Street 11, 6000 Stara Zagora, Bulgaria
Prof. Dr. med.P.Neno
Haut- und Laborarzt/Allergologie, Andrologie, Labor für
medizinische Mikrobiologie, Straße des Friedens 8, 04579 Mölbis,
Assoc. Prof. L. G. Zisova,MD, PhwD· R.Hristova,MD
Department of Dermatology and Venerology, Medical University
Plovdiv, Vasil Aprilov 15A Street, Plovdiv, Bulgaria
Received: 29 April 2012 / Accepted: 2 August 2012
© Springer-Verlag Wien 2012
J. C. Cardoso
Dermatology and Venerology Department, University Hospital
of Coimbra, Praceta Mota Pinto, 3000-075 Coimbra, Portugal
Department of Dermatology and Venerology, Medical University
of Graz, Auenbruggerplatz 8, 8036 Graz, Austria
e-mail: firstname.lastname@example.org; gabriele.ginter@
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
Assoc. Prof. M.Gulubova,MD, PhD
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
Nobuo Kanazawa, MD, PhD
Department of Dermatology, Wakayama Medical University
Author's personal copy
2 Onychomycosis: modern diagnostic and treatment approaches
zienz, 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 Pro- 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 Identizierung 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 erapiefor-
men erläutert, abhängig davon, ob Dermatophyten,
Hefepilze oder Schimmelpilze nachweisbar waren. Die
verschiedenen Schemata der Onychomykosetherapie
für bestimmte Patientenkollektive werden ausführlich
rubrum, MALDI-TOF Massenspektroskopie, Uniplex-
PCR-ELISA-Test, Antimykotische erapie, Terbinan,
Fluconazol, Itraconazol, Laserbehandlung
Summary e medical term onychomycosis should be
understood as chronic infection of the nails caused by
a fungus. e most common causative agents are the
dermatophytes and Candida species. e 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
occosum, and T. tonsurans. e most signicant 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-
suciency, 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. e diagnostic meth-
ods that are often applied in dierent dermatologic de-
partments and ambulatory units are also dierent. is
precludes the creation of a unied diagnostic algorithm
that could be used everywhere as a possible standard.
In most of the cases, the method of choice depends on
the specialist’s individual experience. e therapeutic
approach depends mostly on the fungal organism iden-
tied by the dermatologist or mycologist. is review
hereby includes the conventional as well as the newest
and most reliable and modern methods used for the
identication 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 identied 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,
Terbinane, Itraconazole, Laser treatment
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, conrmed the
following results: 6.5%  and 14% , respectively. e
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% .
Depending on its origin, onychomycosis can be divi-
ded into primary and secondary. In primary onycho-
mycosis, fungal invasion aects an intact nail, whereas
secondary onychomycosis occurs in an already abnor-
mal nail aected by various diseases or traumas . It
should be noted, however, that when strictly dened,
primary onychomycosis is a rare occurrence.
Nowadays, an increasing prevalence of onychomy-
cosis has been noted . e 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.
ere 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 deciencies [6, 7].
A seriously disturbing fact is the gradually increasing
frequency of onychomycosis in children . Some aut-
hors estimate onychomycosis prevalence in children at
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Onychomycosis: modern diagnostic and treatment approaches 3
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 dierential diagnostic procedures are essential to
exclude the presence of coexisting fungal infection .
Previously conducted studies report a prevalence rate
of onychomycosis in patients with psoriasis vulgaris vary-
ing in wide ranges from 4.6  to 47.6% , 56% ,
and 43–62% . 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 specically the Candida spp., are more often isola-
ted from ngernails. A correlation between psoriatic nail
change—Nail Psoriasis Area Severity Index—and posi-
tive mycology is observed.
e huge number of psoriatic patients diagnosed with
onychomycosis requires mycological tests to be perfor-
med in all patients with psoriasis .
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 .
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 aected.
Genetic predisposition in patients with onychomyco-
sis should not be neglected . 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 .
e reasons why children and adults with Down syn-
drome are more often aected by onychomycosis and
tinea pedis have not been determined yet .
In the cases of leading clinical manifestation, the
accurate identication of the causative fungal pathogen
is highly advised. e accurate identication of fungal
causative pathogens is obligatory in patients with sig-
nicant 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 .
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 identication. 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-
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 , and involvement of the surrounding
skin is a common event. However, spreading of the infec-
tion to aect other sites of the body is a rare event. Never-
theless, it is important to note that without treatment,
patients can suer from low self-esteem, shame and fear,
and often avoid participating in community activities .
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]. e 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. e sample from the aected area is obtai-
ned through sharp scissors, nail buer, 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 sucient. Some authors consider the best method for
obtaining material for the conrmation of fungal invasion
to be the one in which the diagnostician takes the most
proximal part of the diseased nail as sample .
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
e easiest and quickest method for the identication of
nail fungal infection is a KOH preparation. However, it is
characterized by low diagnostic sensitivity .
e nail fragments are placed on a slide adding 1–2
drops of KOH solution (10–30%) . 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
Author's personal copy
4 Onychomycosis: modern diagnostic and treatment approaches
be observed under the microscope . Some clinicians
heat the slides to accelerate the process, or add color
stains to make hyphae easier to identify.
Another frequently used method for the diagnosis of ony-
chomycosis is fungal cultures. e 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).
e 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-
A typical fungal culture requires 2–5 weeks at a cons-
tant temperature around 37 °C to grow. After that a
macroscopic and microscopic identication of the cau-
sative pathogen should be done (Figs. 1 and 2). Some
additional substances are recommended to be used in
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 identication 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 identication of the causative pathogens
of onychomycosis, especially regarding dermatophytes
and yeasts, Sabouraud’s dextrose agar+ Actidion (cyclohe-
ximide 400mg/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-
mide . Another alternative is the modied agar for
dermatophytes (SIFIN, Berlin)—2% Sabouraud’s dextrose
agar with additional cycloheximide and chloramphenicol.
e identication of yeasts and dermatophytes is prece-
ded by macroscopic, microscopic, and molecular biolo-
gical observation of the infected people. Dermatophytes
grow for about 2–4 weeks at a temperature of 26–32°C.
Yeasts, especially that of the genus Candida, grow
within 2–4 days at 26–32°C, or up to 37°C. For Candida
albicans and nonalbicans species, culture on the so-cal-
led CHROM agar or biochemical tests (e.g., API 20°C, ID
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
with AIDS. e cultivation of Malassezia spp. is performed
onto lipid-enriched Dixon’s agar medium, and the Tween
test is used to identify the dierent Malassezia species.
e growth of moulds begins during the rst days
after the cultivation on Sabouraud agar. ey can be
both causative pathogens or laboratory contaminants.
ey cannot be cultivated on media containing Actidion
(cycloheximide). If a medium with Actidion (cyclohexi-
mide) is routinely used, mould identication as a causa-
tive pathogen can sometimes be impossible. In order for
the moulds to be identied as causative pathogens rather
than laboratory contaminants, they should be isolated in
several consecutive microbiological cultures. e exact
type of causative pathogen is determined by microbiolo-
gical culture .
is method gains higher sensitivity, when compared
with the direct microscopic examination using KOH pre-
paration. However, UV light (UVA 365nm, special lter)
and a uorescence microscope are needed. A special
uorescent substance is added to the KOH (Blankophor,
Fig. 1 Infection due to T. rubrum, 32-year-old swimmer
Fig. 2 Diagnostics of T. rubrum by culture
Author's personal copy
Onychomycosis: modern diagnostic and treatment approaches 5
Calcouor, or acridinium orange). It binds to fungal
chitin, and marks hyphae and arthrospores appear as
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-schi (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 . e 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 identication of the exact species, although
dermatophytes or yeasts can be suspected [25, 26]. Com-
pared with direct microscopy, histological diagnosis is
much more reliable .
e 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 .
Molecular biological methods as diagnostic tool
Dermatophytes are considered to be one of the most
signicant 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. occosum, M. gypseum, and rarely M. canis .
For their diagnosis, conventional methods can be used—
direct microscopy with KOH preparation, uorescence
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 identied only through
molecular biological methods [29, 30]. e conven-
tional classic (macromorphology and miscroscopic
examination) and biochemical methods cannot provide
identication of Trichophyton species of Arthroderma
Benhamiae . e 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 specicity and long
duration—around 6 weeks . In addition, for the cor-
rect identication of the results a mycologist with a good
knowledge of the morphological features of dermatophy-
tes is needed.
A precise diagnosis and exact identication 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 ight mass spectro-
metry). e last one is used to identify dermatophytes in
fungal material that has been isolated from culture .
Polymerase chain reaction (PCR)
PCR is a process of in vitro amplication of a DNA mole-
cule, as a result of which within a few hours millions of
copies of a particular molecule can be generated .
Hence, it is a very sensitive and specic method. ere
are dierent primers available to detect dierent species,
including T. rubrum, T. interdigitale, M. gypseum, M.
canis, T. tonsurans, T. violaceum, and E. occosum.
In 1999, the rst special gene probe was used for the
detection of T. rubrum in nail material (Fig. 3) .
Polymerase chain reaction-enzyme-linked
immunosorbent assay (PCR-ELISA) for direct
detection of dermatophyte DNA
is new established method comprises an ampli-
cation and hybridization technique, which is used to
detect sequences within the PCR products of ampli-
ed DNA of dermatophytes. e 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.
e rst step of the amplication process follows those
of the PCR—denaturation, annealing of the primers to
the single-stranded DNA template and elongation. e
ready copies of DNA sequences are used in the second
step—ELISA—in which specic probes (primers) labeled
with biotin, are used to bind to amplied DNA. If derma-
Fig. 3 Onychomycosis, 57-year-old man with diabetes
Author's personal copy
6 Onychomycosis: modern diagnostic and treatment approaches
tophyte DNA is available in the sample, the biotin-labe-
led probe will be xed to streptavidin that is xed 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 conrmed and the identi-
cation can be performed.
Uniplex-PCR-ELISA-Test includes T. rubrum, T. inter-
digitale, E. occosum, 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 .
is molecular–biological method allows a conside-
rably quick identication of the causative agent directly
from nail material within 24hours . A multiplex-PCR
for T. rubrum-DNS identication as well as for other cli-
nically relevant dermatophytes (Pan-Dermatophyten-
primer) allows identication within 5hours .
e morphological dierentiation between anthro-
pophilic and zoophilic T. interdigitale strains by clas-
sical microscopical and biochemical methods is often
problematic. In particular, it is impossible to dierent-
iate between the zoophilic strains of T. interdigitale, T.
mentagrophytes, and the Trichophyton anamorph of A.
benhamiae. In these cases, molecular identication met-
hods may be applied to answer epidemiological, taxono-
mical, and therapeutical questions .
Matrix-assisted laser desorption ionization
MALDI-TOF MS is a routine technique for the identica-
tion of certain bacteria and it is nowadays gaining increa-
sing popularity . At present, the use of the method is
restricted to the identication of a causative agent that
has been cultivated in a microbiological culture [35, 36].
MALDI-TOF MS allows identication of the causative
agents by the molecular weight of their specic protein
e principle of the method—the proteins are added
to crystals of UV-absorbing proteins (matrix). Laser ash
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. e dierences in
the time required for the ions to reach the detector show
dierences in analyzed spectrum, thus the causative
agent is identied. e spectrum for every microorga-
nism is individual, so it can be used for identication of
fungal species and subspecies from nail and skin sam-
ples. is method can be used not only for onychomyco-
sis diagnosis, but also in dermatomycosis diagnosis.
is method is specic, sensitive, quick to process,
and has a relatively low cost, being able to detect not
only fungi but also dierent types of bacterial species in
a sample [37–39].
According to other sources in the literature, MALDI-
TOF MS, used with special software (SARAMIS), is capa-
ble of identifying the dierent phenotypic variations of
Aspergillus species, even in their specic growth phases
. is technique can be applied and completed wit-
hin a few hours directly from Aspergillus mycelium, and
within a few days if spores are present .
MALDI-TOF MS can be used as an additional test
for conrming the results of fungal cultures, especially
when accurate identication of the causative agent is
dicult—Trichophyton species of Arthoderma banha-
miae, for example, an often ignored and misclassied
zoophilic dermatophyte, which causes tinea corporis
and tinea capitis. is method allows successful mor-
phological dierentiation between Arthroderma ben-
hamiae and M. canis (causative agent of tinea capitis).
Such dierentiation turns out to be impossible in above
75% of the cases in which classical diagnostic methods
have been used.
With MALDI-TOF MS dierent species of Candida can
also be dierentiated (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 identication of fungal
species in nail material . However, this direct assess-
ment of fungi in clinical samples has to be proved in furt-
Restriction fragment length polymorphism (RFLP)
e accurate identication 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 .
Therapeutic schemes, choice of treatment
e therapeutic scheme is chosen depending on the
microbiological culture, the results of PCR, and/or MAL-
DI-TOF MS for accurate identication of the causative
agent (see Table1).
Topical antifungal therapy
Topical antifungal therapy is used only in supercial ony-
chomycosis, which aects up to one-third of the nail plate.
e most commonly used medications are Ciclopirox
(Polinail nail lacquer, Batrafen nail lacquer), Naftine
(Exoderil solution), and others.
An antifungal nail lacquer can be used in onychomy-
cosis which aect 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 aection should not exceed 50 % of the nail sur-
face. However, some nail lacquers are approved for topi-
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Onychomycosis: modern diagnostic and treatment approaches 7
cal treatment of an onychomycosis up to 80% of the nail
Systemic antifungal therapy
Itraconazole exhibits ecacy against fungal infections
caused by dermatophytes, yeasts, and moulds.
A so-called pulse therapy with itraconazole 200mg
twice daily for 1 week is recommended. After a thera-
py-free period of 3 weeks, a second pulse therapy with
itraconazole pulse 200mg twice daily should follow .
e 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 .
If dermatophytosis of the nails is conrmed then oral
terbinane therapy is recommended. Various therapeu-
tic schemes have been proposed.
In practice, terbinane is usually prescribed as fol-
lows—one oral tablet of 250mg once a day with die-
rent continuity of the therapy course: at least for about 6
weeks in onychomycosis of the ngernails, 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 .
One important therapeutic scheme is the following:
from the 1st to the 14th day—terbinane 250mg once a
day, followed by 250mg terbinane once a week; treat-
ment duration is until recovery and can achieve up to 1
Another intermittent therapeutic scheme is repor-
ted by Gupta et al. . Terbinane 250mg once daily is
administered for 4 weeks, followed by a 4-weeks break.
According to treatment success, a second and third pulse
may follow .
Fluconazole as well as itraconazole are eective against
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 .
Onychomycosis due to moulds
Terbinane is the drug of choice with highest evidence
for treatment of onychomycosis due to Scopulariopsis
brevicaulis and Aspergillus spp. Topical drugs may be
eective , in particular ciclopirox-containing nail
lacquer in infections due to Scopulariopsis brevicaulis
and Acremonium spp., best in combination with chemi-
cal keratolysis of the nails using 40% urea preparations.
Modiﬁcation of treatment in certain groups
Individual modication of therapy is possible, someti-
mes even compulsory—in polymedicated patients with
signicant 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 . In patients with kidney diseases, thera-
py should also be carefully administrated. In patients
with immune deciency, 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
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
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
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8 Onychomycosis: modern diagnostic and treatment approaches
can have a negative inotropic eect 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 . In general, in these
patients azol antifungals should be avoided, in particular
Surgical treatment of onychomycosis
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 . Topical antifungal medications
are used at the same time and/or immediately after that,
aiming at elimination of the infected nail structures .
Surgical treatment could be accompanied by topical or
systemic therapy. Surgical nail plate removal could be
combined with topical antifungal therapy. is method
provides very good clinical results. Such treatment has
been applied in cases of Scopulariopsis brevicaulis and
Acremonium species infections . Surgical treatment
is also necessary in fungal infections resistant to sys-
temic or topical treatment . Besides avulsion (the
forcible tearing away of nail plate), the mechanical the-
rapy of onychomycosis includes abrasion (scraping o
the supercial layer) of the nail . 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
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
. It is applied as 0.65 ms pulsed Nd:Yag 1,064nm
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 eect in relation to clinic and microbiology,
as well as specifying the individual number of treatment
courses and optimal regimen . e 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 simplies the procedure. Adverse eects
and complications are not signicant. It is recommen-
ded for the nails not to be long for better results.
Laser therapy consists of radiation of 2mm area with
233J/sm energy without cooling spray, gel, or local anest-
hetic. Every nail is treated separately vertically and hori-
zontally, forming a cross over the surface of the nail. e
duration of treatment is 45seconds 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. e ecacy of therapy is
estimated after the secondary microbiological examina-
tion is performed .
Noveon is a laser with dual wavelength of 870nm and
930nm. Its parameters are close to those of the infrared
diods . ese machines are used for the treatment
of onychomycosis because of their unique photolethal
eect to infective agents . ese 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. e results from several clinical
researches show that these lasers are suitable for the tre-
atment of onychomycosis irrespective of the stage of nail
e femtosecond (f-sec) infrared titanium-sapphire
laser does not damage the surrounding tissues .
is laser achieves selective delivery of energy into deep
layers of the nail bed. In this kind of lasers, interaction
with environment is nonlinear . Besides, the deep
penetration of this laser contributes to the elimination
of deeply located dermatophytes without damaging the
surrounding tissues. Ecacy of treatment is evaluated
by subculture, while assessment of collateral damages is
done by scanning electron microscopic . Experience
shows that the f-sec laser inhibits fungus growth suc-
cessfully in examined specimens, while lower intensity
damages the nail plate .
CO2 laser also improves the condition of patients with
onychomycosis and gives good results [53, 54]. Other
authors have also reported an experimental method of
approaching the laser treatment of onychomycosis .
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
supercial nail infections. ere is a research concer-
ning the eciency of PDT in onychomycosis caused by
moulds—Acremonium sclerotigenum . PDT, combi-
ned with methyl-aminolevulinic acid, is administrated
in three sessions, with 15 days interval between each
procedure . Another study reveals the eect of 5-ami-
nolevulinic acid (ALA). e dermatophyte T. rubrum,
causative agent of onychomycosis, metabolizes ALA to
protoporphyrin IX (PP IX) in liquid culture. In optimal
conditions, a typical red uoroscence is seen. It is indu-
ced by PP IX and is estimated qualitatively with Wood’s
lamp or uoroscent microscope. Optimal concentration
of ALA is 1–10mmol/L. ALA causes signicant reduction
of dermatophyte growth and lack of PP IX uorescence,
if higher concentration of ALA is used. A combination
Author's personal copy
Onychomycosis: modern diagnostic and treatment approaches 9
between ALA and light clearly demonstrates the inhibi-
tory eect of PDT with ALA. is method is promising as
far as reduction of T. rubrum colonization in onychomy-
cosis is concerned .
One of the possible PDT schemes is: Damaged nail
surface is coated lavishly with 20% urea unguentum and
is covered with a folio for 10hours. e nails are subse-
quently treated with 20% solution of ALA methyl ester in
liquid cream for 5hours, but only after protection from
light with plaster and aluminium folio has been done.
Protoporphyrin uorescence is conrmed with UV-beam
and spectrophotometer before PDT. It’s observed in nail
base and periphery of fungal lesions. e nail (including
proximal and lateral nail borders) is exposed to radia-
tion horizontally and vertically with pulsed laser 630nm
light, 100J/sm2 using excimer laser  PDT with 5-ALA,
applied once a week . A bearable pain has been repor-
ted in patients during the procedure but it tends to disap-
pear the next day. Improvement in the condition occurs
after 6–7 treatment courses (total dosage 600–700J/sm2).
Most frequently, dermatophytes are not found in post-
treatment investigations—microscopy with KOH pre-
paration and culture . 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 . According to
recent researches, PDT with methylaminolevulinate is
also a successful treatment of refractory onychomycosis
caused by nondermatophytic moulds .
PDT is suitable for treatment of distal and lateral
subungual onychomycosis caused by T. rubrum .
e advantages of PDT are the lack of side eects, 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 eective and it
may be even contraindicated in case of intolerance. PDT
is a very good alternative in these cases [58, 60].
Currently, the most frequently used diagnostic methods
for fungal infection conrmation are the conventional
ones—direct microscopy with KOH preparation and cul-
ture on Saboraud agar.
Culture identies the exact causative agent and in this
way makes it easier to choose the most suitable medica-
Culture has a diagnostic sensitivity of 50–70%. As a
result, 30–50 % of fungal agents cannot be identied by
conventional methods .
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].
Fungal tests are very important in the dierential dia-
gnosis of nail diseases, since other condition such as
lichen planus, psoriasis, and nail dystrophy of dierent
causes may have features that can be confused with ony-
chomycosis. Having in mind that onychomycosis fre-
quently occurs in cases of primary or genetically damaged
nails, this problem is not very easy to be solved [9, 24, 25].
Modern additional methods, which are molecular–
biological—PCR, Uniplex-PCR-ELISA-Test, and MALDI-
TOF MS—will probably play a very important role in the
diagnosis of onychomycosis [24, 40].
PCR is an easy and rapid method. Extraction of DNA
takes up to 30minutes, whereas elongation takes 5hours.
PCR results are ready within a day. is method is charac-
terized by high sensitivity and diagnostic specicity that
exceed the classic diagnostic methods. e specicity of
PCR analysis derives from the possibility of revealing the
exact causative agents in the presence of other microor-
ganisms, viruses, and cells of macroorganisms .
From this point of view, the ability of PCR can be de-
ned as unique. By using this method, it is easy to identify
and diagnose dermatophytes that are dicult to detect
Fig. 4 Tinea pedis, 57-year-old patient, caused by T. rubrum
Fig. 5 Onychomycosis, a 70-year-old man; causative patho-
Author's personal copy
10 Onychomycosis: modern diagnostic and treatment approaches
by classic methods. e 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, dierent biologic specimens can be
examined, including those directly taken from skin lesi-
ons, nails, and hair (Figs. 4 and 5). ese properties allow
wide usage of the method by dermatologists and mycolo-
gists . 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. is method has considerably increased
the percentage of positive results and has reduced the
time needed for achieving an accurate diagnosis. e cul-
ture gives results in 3–4 weeks, whereas PCR method wit-
hin a maximum of 1–2 days. is 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 sta. In recent years, it is considered that mole-
cular biology methods will completely replace conventio-
nal methods, which are currently considered as standard
methods. e establishment of separate specialized cen-
ters and laboratories aiming to focus their activity in this
eld could be signicant to the exact identication of the
causative agent and administration of proper systemic
therapy. e choice of treatment and duration of antifun-
gal therapy depends on the identication 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-
e most frequently used diagnostic methods for the
conrmation 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. is
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.
erapy with terbinane is preferred in cases of ony-
chomycosis caused by dermatophytes. It has a very good
eect in infections caused by Trichophyton, Microspo-
rum, and Epidermophyton species [61, 62]. erapy with
terbinane has also good eect in infections with moulds
[62, 63]. It is, however, less eective 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 eect of terbinane
against dermatophytes and moulds, many clinicians pre-
fer systemic therapy with terbinane instead of itracona-
zole or uconazole.
On the other hand, therapy using uconazole or itra-
conazole schemes (pulse treatment) is preferred when
yeasts are identied, 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.
Conict of interest
e authors declare that there is no actual or potential
conict of interest in relation to this article
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