Wiener Medizinische Wochenschrift Volume 163 Issue 1-2 2013 [Doi 10.1007%2Fs10354-012-0139-3]...

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Onychomycosis: modern diagnostic and treatment approaches 1 1 3

Onychomykose: Moderne Diagnostik undBehandlungsansätze

Zusammenfassung Der medizinische erminus Ony-chomykose steht für eine chronische Infektion desNagelapparates durch einen Pilz. Zu den häufigsten verursachenden Erregern zählen Dermatophyten sowieCandida-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 häufigstenisolierte Dermatophyt ist Trichophyton (T .) rubrum, wei-tere relevante Spezies für eine Onychomykose sind T. in-terdigitale (früher T. mentagrophytes), Epidermophyton

floccosum und T. tonsurans. Die wichtigsten, eine Ony-chomykose verursachenden Hefepilze sind Candida al-bicans und Candida parapsilosis. Zu den disponierendenFaktoren, die eine Onychomykose begünstigen, zäh-len vor allem Stoffwechselerkrankungen, wie Diabetes

mellitus, aber auch Gefäßerkrankungen, wie peripherearterielle Verschlusskrankheit, chronisch-venöse Insuf-

Wien Med Wochenschr (2013) 163:1–12

DOI 10.1007/s10354-012-0139-3

Onychomycosis: modern diagnosticand 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. chernev () · . anevaPolyclinic for Dermatology and Venerology, University HospitalLozenetz, Academic Educational Hospital of the Saint KlimentOhridski University, Medical Faculty, Koziak Street 1, 1407 Sofia,Bulgariae-mail: [email protected]

P. K. Penev, MDDepartment of Dermatology and Venerology, rakia University,Medical faculty, Armeiska Street 11, 6000 Stara Zagora, Bulgariae-mail: [email protected]

Prof. Dr. med. P. Nenoff Haut- und Laborarzt/Allergologie, Andrologie, Labor fürmedizinische Mikrobiologie, Straße des Friedens 8, 04579 Mölbis,Germany e-mail: [email protected]

Assoc. Prof. L. G. Zisova, MD, PhwD · R. Hristova, MDD. Nocheva, MDDepartment of Dermatology and Venerology, Medical UniversityPlovdiv, Vasil Aprilov 15A Street, Plovdiv, Bulgariae-mail: [email protected]

R. Hristova, MDe-mail: [email protected]

D. Nocheva, MDe-mail: [email protected]

Received: 29 April 2012 / Accepted: 2 August 2012 / Published online: 30 September 2012

© Springer-Verlag Wien 2012

J. C. CardosoDermatology and Venerology Department, University Hospitalof Coimbra, Praceta Mota Pinto, 3000-075 Coimbra, Portugale-mail: [email protected]

Assoc. Prof. G. Ginter-Hanselmayer, MDDepartment of Dermatology and Venerology, Medical Universityof Graz, Auenbruggerplatz 8, 8036 Graz, Austriae-mail: [email protected]; [email protected]

J. Ananiev · Assoc. Prof. M. Gulubova, MD, PhDDepartment of General and Clinical Pathology, Medical Faculty,rakia University, Armeiska Street 11, 6000 Stara Zagora, Bulgariae-mail: [email protected]

Assoc. Prof. M. Gulubova, MD, PhDe-mail: [email protected]

Claudio Guarneri, MDDepartment of Social erritorial Medicine, Section of Dermatology,

University of Messina, c/o A.O.U. “G. Martino"—via Consolare Valeria, Gazzi, 98125 Messina, Italy e-mail: [email protected]

Nobuo Kanazawa, MD, PhDDepartment of Dermatology, Wakayama Medical University

e-mail: [email protected]

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1 32 Onychomycosis : modern diagnostic and treatment approaches

fizienz, Polyneuropathien unterschiedlicher Ätiologieund 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 Vorschubleisten, sind lokale raumen bei Profi- oder Leistungss-portlern, oft vergesellschaftet mit starker Hyperhidrose.In dermatologischen Kliniken und Praxen kommen verschiedene diagnostischen Methoden zur Anwend-ung Ein einheitlicher diagnostischer Algorithmus wäre wünschenswert, nach wie vor ist jedoch die persönlicheErfahrung des Untersuchers entscheidend für die einge-setzten Methoden. Entscheidend ist, dass der gewähltetherapeutische Ansatz im Wesentlichen vom nachgewi-esenen Erreger abhängt. In dieser Übersicht wird diekonventionelle Diagnostik von Onychomykosen darg-estellt. Außerdem wird auf moderne und neu entwick-

elte labordiagnostische Methoden, die zum direktenNachweis und zur Identifizierung der nachgewiesenenErreger der Onychomykose Einzug in die Dermatolo-gie und Mikrobiologie gefunden haben, eingegangen.Darüber hinaus wird auf die Auswahl der erfolgver-sprechendsten lokalen und systemischen Terapiefor-men erläutert, abhängig davon, ob Dermatophyten,Hefepilze oder Schimmelpilze nachweisbar waren. Die verschiedenen Schemata der Onychomykosetherapiefür bestimmte Patientenkollektive werden ausführlichdargestellt.

Schlüsselwörter: Onychomykose, Trichophyton

rubrum, MALDI-OF Massenspektroskopie, Uniplex-PCR-ELISA-est, Antimykotische Terapie, erbinafin,Fluconazol, Itraconazol, Laserbehandlung

Summary Te medical term onychomycosis should beunderstood as chronic infection of the nails caused bya fungus. Te most common causative agents are thedermatophytes and Candida species. Te less commonare certain types of moulds (nondermatophyte mouldsor NDMs). In approximately 60–80 % of the cases, ony-chomycosis is due to dermatophytes. Among dermato-phytes, the most often isolated causative pathogen is Tri-chophyton (T.) rubrum. Other common species are T. in-

terdigitale (formerly T. mentagrophytes), Epidermophyton floccosum, and T. tonsurans. Te most significant yeastscausing onychomycosis are Candida albicans and Can-dida parapsilosis. Predisposing factors for onychomy-cosis include mainly diseases such as diabetes mellitus,peripheral vascular arterial disease, chronic venous in-sufficiency, polyneuropathies of diverse etiologies, andimmunosuppression, 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. Te diagnostic meth-ods that are often applied in different dermatologic de-

partments and ambulatory units are also different. Tisprecludes the creation of a unified diagnostic algorithmthat could be used everywhere as a possible standard.

In most of the cases, the method of choice depends onthe specialist’s individual experience. Te therapeuticapproach depends mostly on the fungal organism iden-tified by the dermatologist or mycologist. Tis review

hereby includes the conventional as well as the newestand most reliable and modern methods used for theidentification of the pathogens causing onychomycosis.Moreover, detailed information is suggested, about thechoice of therapeutic scheme in case whether dermato-phytes, moulds, or yeasts have been identified as causa-tive agents. A thorough discussion of the schemes andduration of the antifungal therapy in certain groups ofpatients have been included.

Keywords: Onychomycosis, Trichophytonrubrum, MALDI-OF MS, Uniplex-PCR-ELISA-est, Antifungal therapy,erbinafine, Itraconazole, Laser treatment

Introduction

Onychomycosis is а fungal infection of the nail plate,caused by dermatophytes, yeasts, and moulds [1, 2].Onychomycosis is the most common disease of the nails worldwide and constitutes about a half of all nail abnor-malities [2, 3].

Recent studies, concerning onychomycosis prevalenceamong population in the USA and Canada, confirmed thefollowing results: 6.5 % [2] and 14 % [1], respectively. Te AHIL survey on the other hand, the largest survey under-

taken in 20 European countries on patients with onycho-mycosis, estimated its prevalence in 29.6 % [4].

Depending on its origin, onychomycosis can be divi-ded into primary and secondary. In primary onycho-mycosis, fungal invasion affects an intact nail, whereassecondary onychomycosis occurs in an already abnor-mal nail affected by various diseases or traumas [5]. Itshould be noted, however, that when strictly defined,primary onychomycosis is a rare occurrence.

Nowadays, an increasing prevalence of onychomy-cosis has been noted [1]. Te most common reasons forthe increasing number of patients with onychomyco-sis are the increased lifespan of the general population,

the wide use of antibiotics and corticosteroids, the prevalence of synthetic clothes over cotton ones, commu-nity swimming pools, gyms, saunas and spa proceduresbecoming a part of everyday life, as well as wearing tightshoes and sneakers.

Tere are numerous factors that can cause or act likecatalysts for the clinical manifestation of onychomycosis. Among them are diabetes mellitus, smoking, peripheral vascular arterial disease, varicose syndrome, as well assome systemic diseases such as paraproteinemias, lympho-mas, congenital or acquired immune deficiencies [6, 7].

A seriously disturbing fact is the gradually increasingfrequency of onychomycosis in children [8]. Some aut-

hors estimate onychomycosis prevalence in children at0–2.6 % [8].

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Another problem also exists, derived from the fact thatoften nail psoriasis is misdiagnosed as onychomycosis.Many psoriatic patients have nail changes which morpho-logically resemble onychomycosis, and in such patients

further differential diagnostic procedures are essential toexclude the presence of coexisting fungal infection [9].

Previously conducted studies report a prevalence rateof onychomycosis in patients with psoriasis vulgaris vary-ing in wide ranges from 4.6 [10] to 47.6 % [10], 56 % [11],and 43–62 % [9]. In such cases, secondary fungal invasionis most probable. Dystrophic nail changes in psoriasis vulgaris are a predisposing condition to fungal infections.

Dermatophytes are the most common causativepathogens of onychomycosis of toenails, while yeasts,more specifically the Candida spp., are more often isola-ted from fingernails. A correlation between psoriatic nailchange—Nail Psoriasis Area Severity Index—and posi-

tive mycology is observed.Te huge number of psoriatic patients diagnosed with

onychomycosis requires mycological tests to be perfor-med in all patients with psoriasis [9].

An interesting fact is that in most patients onychomy-cosis is triggered by a long persisting interdigital mycosisand vice versa—in at least one-third of the patients withonychomycosis, a coexisting tinea pedis is observed [12].

Microtraumata, especially in people practicing sportsare considered to be a predisposing condition of greatimportance. An interesting fact is that football playersturn out to be the most affected.

Genetic predisposition in patients with onychomyco-

sis should not be neglected [13]. It had been described byseveral authors that the autosomal-dominant inheritanceis of utmost importance in the clinical manifestation of dis-tal subungual onychomycosis, caused by T. rubrum [14].

Te reasons why children and adults with Down syn-drome are more often affected by onychomycosis andtinea pedis have not been determined yet [15].

In the cases of leading clinical manifestation, theaccurate identification of the causative fungal pathogenis highly advised. Te accurate identification of fungalcausative pathogens is obligatory in patients with sig-nificant comorbidities and polymedication as well as inimmunosuppressed patients. In such cases, high risk of

frequent fungal infections exists. Systemic candidosisand sepsis are possible complications due to a blood orlymphatic spread of fungal infection.

In addition, fungal infections predispose to relapsingerysipelas, which can be followed by lymphedema [16].

It is assumed that topical antifungal treatment shouldbe prescribed only after positive microscopic examina-tion for fungal elements and systemic treatment—afterfungal culture, followed by identification. Despite esta-blished rules, not every dermatologist and only a lowpercentage of general practitioners in Europe initiateantifungal therapy after testing for fungal infection. Aninteresting fact is that according to some European stu-

dies, barely around 50 % of the dermatologists conductdiagnostic procedures before initiating systemic antifun-gal therapy. Without systemic therapy there is a conside-

rable risk for the fungal infection to spread to other areasof the body, most commonly via autoinoculation [17–19].

reatment should be considered in every patient withonychomycosis; but decision to treat should be made on

individual grounds taking into account several factorsincluding the degree of nail involvement and the patient’sgeneral status, comorbidities, and concomitant medicati-ons. Fungal infection may advance to complete destructionof the nail plate [20], and involvement of the surroundingskin is a common event. However, spreading of the infec-tion to affect other sites of the body is a rare event. Never-theless, it is important to note that without treatment,patients can suffer from low self-esteem, shame and fear,and often avoid participating in community activities [20].

Ambulatory and clinical diagnosis

According to some European studies, low percentage ofdermatologists and general practitioners conduct dia-gnostic procedures before initiating topical or systemicantifungal therapy [21, 22]. Te samples for fungal micro-scopy should be taken after at least 4–6 days without topi-cal antifungal therapy; otherwise false negative results canbe obtained. Immediately before the sample is taken, thenail plate should be cleaned with 70 % alcohol, thus dimi-nishing the possibility of contaminating the samples withmoulds or bacteria. Disinfection procedure is not requi-red if selective agars which contain cycloheximide (activeagainst moulds) or chloramphenicol (active against bac-

teria) are used. Te sample from the affected area is obtai-ned through sharp scissors, nail buffer, or scalpel, and atleast 15–20 nail scrapings are needed. A special electricalgrinding machine can be used if the obtained material isnot sufficient. Some authors consider the best method forobtaining material for the confirmation of fungal invasionto be the one in which the diagnostician takes the mostproximal part of the diseased nail as sample [23].

Conventional methods for laboratorydiagnosis 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’sdextrose agar.

Fungal microscopy

Te easiest and quickest method for the identification ofnail fungal infection is a KOH preparation. However, it ischaracterized by low diagnostic sensitivity [10].

Te nail fragments are placed on a slide adding 1–2drops of KOH solution (10–30 %) [24]. After a cover slip

is placed, the specimen is put into a humid environmentfor at least 2 hours or more (best overnight). Immedia-tely after that typical morphology of fungal hyphae can



4 Onychomycosis : modern diagnostic and treatment approaches

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be observed under the microscope [24]. Some cliniciansheat the slides to accelerate the process, or add colorstains to make hyphae easier to identify.

Fungal cultures

Another frequently used method for the diagnosis of ony-chomycosis is fungal cultures. Te specimen is put intoa Petri dish, containing agar—usually Sabouraud’s dex-trose agar (a selective medium that is formulated to allowthe growth of fungi and inhibit the growth of bacteria).

Te 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 anexperienced clinician, who will be able to distinguish themicroscopic 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 amacroscopic and microscopic identification of the cau-sative pathogen should be done (Figs. 1 and 2). Someadditional substances are recommended to be used inorder for the bacterial growth in fungal cultures to beinhibited. Sabouraud’s dextrose agar allows considerablyfaster fungal growth in comparison with Kimmig agar.

Both the agars contain antibiotics (Chloramphenicol50 mg/L, Penicillin, Streptomycin 40,000 IE/L), whichinhibit 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 pathogensof onychomycosis, especially regarding dermatophytesand 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-mide [25]. Another alternative is the modified agar fordermatophytes (SIFIN, Berlin)—2 % Sabouraud’s dextroseagar with additional cycloheximide and chloramphenicol.Te identification of yeasts and dermatophytes is prece-

ded by macroscopic, microscopic, and molecular biolo-gical observation of the infected people. Dermatophytesgrow 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 Candidaalbicans and nonalbicans species, culture on the so-cal-led CHROM agar or biochemical tests (e.g., API 20 °C, ID32 °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. Te cultivation of Malassezia spp. is performedonto lipid-enriched Dixon’s agar medium, and the weentest is used to identify the different Malassezia species.

Te growth of moulds begins during the first daysafter the cultivation on Sabouraud agar. Tey can beboth causative pathogens or laboratory contaminants.

Tey cannot be cultivated on media containing Actidion(cycloheximide). If a medium with Actidion (cyclohexi-

mide) is routinely used, mould identification as a causa-tive pathogen can sometimes be impossible. In order forthe moulds to be identified as causative pathogens ratherthan laboratory contaminants, they should be isolated inseveral consecutive microbiological cultures. Te exacttype of causative pathogen is determined by microbiolo-gical culture [25].

Fluorescence microscopy

Tis method gains higher sensitivity, when compared with the direct microscopic examination using KOH pre-

paration. However, UV light (UVA 365 nm, special filter)and a fluorescence microscope are needed. A specialfluorescent 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

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Calcofluor, or acridinium orange). It binds to fungalchitin, and marks hyphae and arthrospores appear asbrightening structures.

Histopathology

Histological examination of nail material is rarely used,but it is a highly informative method. One of the following stainings is used—periodic acid-schiff (PAS), orGroccot–Gomori silver staining. It should be conside-red that although there is a fungal invasion of the tis-sues, histopathology is not always positive. Accordingto some retrospective researches undertaken in famousEuropean University Hospitals, the diagnostic sensitivity of the fungal culture and microscopy is not always very high. In some cases, they are positive in just about

50–70 % of patients with onychomycosis [25]. Te dia-gnostic sensitivity can be considerably increased by con-ducting biopsy. However, biopsy is not always advisableor possible to conduct, e.g., in diabetics. An additionalproblem regarding histological diagnosis is that it doesnot provide identification of the exact species, althoughdermatophytes or yeasts can be suspected [25, 26]. Com-pared with direct microscopy, histological diagnosis ismuch more reliable [27].

Te histological diagnosis of fungal elements in tissuesis a highly sensitive method in comparison with the directmicroscopy with KOH preparation and fungal culture.However, patients often reject this diagnostic procedure [24].

Molecular biological methods as diagnostic tool

Dermatophytes are considered to be one of the mostsignificant causative pathogens of onychomycosis worldwide. Dermatоphytes belong to three genera offungi—Trichophyton, Epidermophyton, and Microspo-rum. T. rubrum is the most common causative agent ofdermatophytosis, followed by T. interdigitale , T. tonsu-rans, E. floccosum, M. gypseum, and rarely M. canis [28].For their diagnosis, conventional methods can be used—direct microscopy with KOH preparation, fluorescence

staining, or cultivation on Sabouraud’s dextrose agar.Certain dermatophytes such as Trichophyton spe-

cies of Arthroderma benhamie (genus Trichophyton)—azoophilic dermatophyte can be identified only throughmolecular biological methods [29, 30]. Te conven-tional classic (macromorphology and miscroscopicexamination) and biochemical methods cannot provideidentification of Trichophyton species of ArthrodermaBenhamiae [26]. Te direct microscopy with KOH prepa-ration, cultivation on Sabouraud’s dextrose agar followedby specifying of microconidia and macroconidia mor-phology, presence of chlamydospores, urease activityare classical methods, showing low specificity and long

duration—around 6 weeks [31]. In addition, for the cor-rect identification of the results a mycologist with a good

knowledge of the morphological features of dermatophy-tes is needed.

A precise diagnosis and exact identification of thecausative agent could be performed through some newmolecular biological methods—such as polymerasechain reaction (PCR) and matrix-assisted laser desorptionionization (MALDI-OF MS—time of flight mass spectro-metry). Te last one is used to identify dermatophytes infungal material that has been isolated from culture [24].

Polymerase chain reaction (PCR)

PCR is a process of in vitro amplification of a DNA mole-cule, as a result of which within a few hours millions ofcopies of a particular molecule can be generated [24].Hence, it is a very sensitive and specific method. Tereare different primers available to detect different species,including T. rubrum, T. interdigitale , M. gypseum, M.canis, T. tonsurans, T. violaceum, and E. floccosum.

In 1999, the first special gene probe was used for thedetection of T. rubrum in nail material (Fig. 3) [27].

Polymerase chain reaction-enzyme-linked

immunosorbent assay (PCR-ELISA) for directdetection of dermatophyte DNA

Tis new established method comprises an amplifi-cation and hybridization technique, which is used todetect sequences within the PCR products of ampli-fied DNA of dermatophytes. Te topoisomerase II geneof the dermatophytes is used as target for the primers(one of them is labeled by digoxigenin). DNA isolationis carried out using the Qiagen QIAamp DNA Mini Kit.Te first step of the amplification process follows thoseof the PCR—denaturation, annealing of the primers tothe single-stranded DNA template and elongation. Te

ready copies of DNA sequences are used in the secondstep—ELISA—in which specific probes (primers) labeled with biotin, are used to bind to amplified DNA. If derma-

Fig. 3 Onychomycosis, 57-year-old man with diabetes

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tophyte DNA is available in the sample, the biotin-labe-led probe will be fixed to streptavidin that is fixed to thebottom of the microtiter plate. After an antibody–antigenreaction 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 inthe examined sample can be confirmed and the identifi-cation can be performed.

Uniplex-PCR-ELISA-est includes T. rubrum, T. inter-digitale , E. floccosum, T. tonsurans, M. canis, T. violaceum,and richophyton species of Arthoderma benhamiae separately. According to some researches, the diagnostic value of the selective culture media for Dematophytes isevaluated to be around 82.1 %, and that of PCR-ELISA-est 85.8 %, respectively [24].

Tis molecular–biological method allows a conside-rably quick identification of the causative agent directly

from nail material within 24 hours [32]. A multiplex-PCRfor T. rubrum-DNS identification as well as for other cli-nically relevant dermatophytes (Pan-Dermatophyten-primer) allows identification within 5 hours [33].

Te morphological differentiation between anthro-pophilic and zoophilic T. interdigitale strains by clas-sical microscopical and biochemical methods is oftenproblematic. In particular, it is impossible to different-iate between the zoophilic strains of T. interdigitale , T.mentagrophytes, and the Trichophyton anamorph of A.benhamiae . In these cases, molecular identification met-hods may be applied to answer epidemiological, taxono-mical, and therapeutical questions [34].

Matrix-assisted laser desorption ionization(MALDI-TOF MS)

MALDI-OF MS is a routine technique for the identifica-tion of certain bacteria and it is nowadays gaining increa-sing popularity [27]. At present, the use of the method isrestricted to the identification of a causative agent thathas been cultivated in a microbiological culture [35, 36].

MALDI-OF MS allows identification of the causativeagents by the molecular weight of their specific proteinfragments.

Te principle of the method—the proteins are addedto crystals of UV-absorbing proteins (matrix). Laser flashionizes the matrix molecules and as a result, positive ionsare formed, which are captured by a detector. Small ionsreach the detector before large ions. Te differences inthe time required for the ions to reach the detector showdifferences in analyzed spectrum, thus the causativeagent is identified. Te spectrum for every microorga-nism is individual, so it can be used for identification offungal species and subspecies from nail and skin sam-ples. Tis method can be used not only for onychomyco-sis diagnosis, but also in dermatomycosis diagnosis.

Tis method is specific, sensitive, quick to process,

and has a relatively low cost, being able to detect notonly fungi but also different types of bacterial species ina sample [37–39].

According to other sources in the literature, MALDI-OF MS, used with special software (SARAMIS), is capa-ble of identifying the different phenotypic variations of

Aspergillus species, even in their specific growth phases

[40]. Tis technique can be applied and completed wit-hin a few hours directly from Aspergillus mycelium, and within a few days if spores are present [41].

MALDI-OF MS can be used as an additional testfor confirming the results of fungal cultures, especially when accurate identification of the causative agent isdifficult—Trichophyton species of Arthoderma banha-miae , for example, an often ignored and misclassifiedzoophilic dermatophyte, which causes tinea corporisand tinea capitis. Tis method allows successful mor-phological differentiation between Arthroderma ben-hamiae and M. canis (causative agent of tinea capitis).Such differentiation turns out to be impossible in above

75 % of the cases in which classical diagnostic methodshave been used.

With MALDI-OF MS different species of Candida canalso be differentiated (such as C. albicans, C. parapsilo-sis, C. magnoliae , C. dubliniensis, C. lusitaniae , C. krusei ,C. glabrata, C. tropicalis, and C. guilliermondii ).

According to other sources in the literature, MALDI-OF MS can be used for direct identification of fungalspecies in nail material [41]. However, this direct assess-ment of fungi in clinical samples has to be proved in furt-her studies.

Restriction fragment length polymorphism (RFLP)

Te accurate identification of moulds such as Fusarium, Acremonium, and Aspergillus, is performed in speciali-zed laboratories. Firstly, PCR is used for the extraction ofribosomal RNA followed by RFLP [40].

Therapeutic schemes, choice of treatment

Te therapeutic scheme is chosen depending on themicrobiological culture, the results of PCR, and/or MAL-DI-OF MS for accurate identification of the causative

agent (see able 1).

Topical antifungal therapy

opical antifungal therapy is used only in superficial ony-chomycosis, which affects up to one-third of the nail plate.Te most commonly used medications are Ciclopirox(Polinail nail lacquer, Batrafen nail lacquer), Naftifine(Exoderil solution), and others.

An antifungal nail lacquer can be used in onychomy-cosis which affect up to 40 % of the nail surface or notmore than three out of ten nails. According to the inter-

national consensus conference of onychomycosis thefungal affection should not exceed 50 % of the nail sur-face. However, some nail lacquers are approved for topi-

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cal treatment of an onychomycosis up to 80 % of the nail

surface [42].

Systemic antifungal therapy

Itraconazole

Itraconazole exhibits efficacy against fungal infectionscaused by dermatophytes, yeasts, and moulds.

A so-called pulse therapy with itraconazole 200 mgtwice daily for 1 week is recommended. After a thera-py-free period of 3 weeks, a second pulse therapy withitraconazole pulse 200 mg twice daily should follow [43].

Te 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 naillacquer) or Bifonazole cream is recommended [43].

Terbinafine

If dermatophytosis of the nails is confirmed then oralterbinafine therapy is recommended. Various therapeu-tic schemes have been proposed.

In practice, terbinafine is usually prescribed as fol-

lows—one oral tablet of 250 mg once a day with diffe-rent continuity of the therapy course: at least for about 6 weeks in onychomycosis of the fingernails, which is mostoften of the distal subungual type. According to the clini-cal response, therapy can be continued after 6 weeks. Inonychomycosis of toenails, the therapeutic course conti-nues up to 12 weeks. In cases of slow nail growth, therapyshould be even more prolonged [43].

One important therapeutic scheme is the following:from the 1st to the 14th day—terbinafine 250 mg once aday, followed by 250 mg terbinafine once a week; treat-ment duration is until recovery and can achieve up to 1 year.

Another intermittent therapeutic scheme is repor-ted by Gupta et al. [44]. erbinafine 250 mg once daily isadministered for 4 weeks, followed by a 4-weeks break.

According to treatment success, a second and third pulse

may follow [43].

Fluconazole

Fluconazole as well as itraconazole are effective againstinfections caused by dermatophytes, and in particularby yeasts (with the exception of Candida glabrata andCandida krusei ), and in case of itraconazole also moulds.

Fluconazole can be successfully administrated aspulse therapy at a dosage of 150 up to 300 (450) mg oncea week for up to 9 months or until cure is achieved [45].

Onychomycosis due to moulds

erbinafine is the drug of choice with highest evidencefor treatment of onychomycosis due to Scopulariopsisbrevicaulis and Aspergillus spp. opical drugs may beeffective [46], in particular ciclopirox-containing naillacquer 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

of patients

Individual modification of therapy is possible, someti-mes even compulsory—in polymedicated patients withsignificant 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 ofthe possible presence or absence of liver disease shouldbe done [43]. In patients with kidney diseases, thera-py should also be carefully administrated. In patients with immune deficiency, the full blood count should be

constantly observed especially when treatment conti-nues more than 6 weeks. In patients with heart failure,itraconazol should be used with great caution, since it

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—toenails250 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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can have a negative inotropic effect with potential fordecompensation of the underlying cardiac disease.

Furthermore, in polymedicated patients, carefulchoice of the systemic drug should be done according

to the potential for interactions [47]. In general, in thesepatients azol antifungals should be avoided, in particularitraconazol.

Surgical treatment of onychomycosis

It is considered that onychomycosys is one of the fungalinfections among population with the highest percen-tage of unsuccessful treatment. Although rarely usedindependently, surgical treatment is an alternative tosystemic therapy [48]. opical antifungal medicationsare used at the same time and/or immediately after that,

aiming at elimination of the infected nail structures [48].Surgical treatment could be accompanied by topical orsystemic therapy. Surgical nail plate removal could becombined with topical antifungal therapy. Tis methodprovides very good clinical results. Such treatment hasbeen applied in cases of Scopulariopsis brevicaulis and

Acremonium species infections [49]. Surgical treatmentis also necessary in fungal infections resistant to sys-temic or topical treatment [49]. Besides avulsion (theforcible tearing away of nail plate), the mechanical the-rapy of onychomycosis includes abrasion (scraping off the superficial layer) of the nail [49]. Partial avulsion isrecommended in cases of distal lateral subungual ony-

chomycosis and partial subungual onychomycosis as anadjuvant to local therapy.

Laser treatment of onychomycosis

Because of the high morbidity rate of onychomycosisand the low results of oral and topical therapy, adminis-trated separately or at the same time, as well as commonrelapses, modern and noninvasive treatment methodshave been investigated. One of them is laser treatment[50]. It is applied as 0.65 ms pulsed Nd:Yag 1,064 nmlaser. 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 orthird procedure. Because of that, treatment with 1,064Nd:YAG laser is to be carefully explored, concerning itslong-term effect in relation to clinic and microbiology,as well as specifying the individual number of treatmentcourses and optimal regimen [50]. Te advantage ofNd:YAG 1,064 nm laser compared with lasers with lon-ger exposure to radiation is that there is no need of skincooling, which simplifies the procedure. Adverse effectsand complications are not significant. It is recommen-ded for the nails not to be long for better results.

Laser therapy consists of radiation of 2 mm area with233 J/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. Teduration of treatment is 45 seconds or less for every nailplate. For prevention of reinfection, patients are secured

with daily use of antifungal cream. Revision is made 4–6 months after the treatment. Te efficacy of therapy isestimated after the secondary microbiological examina-tion is performed [50].

Noveon is a laser with dual wavelength of 870 nm and930 nm. Its parameters are close to those of the infrareddiods [51]. Tese machines are used for the treatmentof onychomycosis because of their unique photolethaleffect to infective agents [51]. Tese lasers have no terato-genic risks, unlike the photodynamic therapy (PD) withUV beam. Also, there is no toxic photoablation derivingfrom the Nd:YAG lasers. Te results from several clinicalresearches show that these lasers are suitable for the tre-

atment of onychomycosis irrespective of the stage of naildamage [51].

Te femtosecond (f-sec) infrared titanium-sapphirelaser does not damage the surrounding tissues [52].Tis laser achieves selective delivery of energy into deeplayers of the nail bed. In this kind of lasers, interaction with environment is nonlinear [52]. Besides, the deeppenetration of this laser contributes to the eliminationof deeply located dermatophytes without damaging thesurrounding tissues. Efficacy of treatment is evaluatedby subculture, while assessment of collateral damages isdone by scanning electron microscopic [52]. Experienceshows that the f-sec laser inhibits fungus growth suc-

cessfully in examined specimens, while lower intensitydamages the nail plate [52].

CO2 laser also improves the condition of patients with

onychomycosis and gives good results [53, 54]. Otherauthors have also reported an experimental method ofapproaching the laser treatment of onychomycosis [55].

Photodynamic therapy (PDT)

Photodynamic therapy is based on the usage of photo-sensitizing agents and light with exact wavelength. Sing-let oxygen is generated, leading to cell death. It has been

examined whether PD is appropriate for treatment ofsuperficial nail infections. Tere is a research concer-ning the efficiency of PD in onychomycosis caused bymoulds—Acremonium sclerotigenum [56]. PD, combi-ned with methyl-aminolevulinic acid, is administratedin three sessions, with 15 days interval between eachprocedure [56]. Another study reveals the effect of 5-ami-nolevulinic acid (ALA). Te dermatophyte T. rubrum,causative agent of onychomycosis, metabolizes ALA toprotoporphyrin IX (PP IX) in liquid culture. In optimalconditions, a typical red fluoroscence is seen. It is indu-ced by PP IX and is estimated qualitatively with Wood’slamp or fluoroscent microscope. Optimal concentration

of ALA is 1–10 mmol/L. ALA causes significant reductionof dermatophyte growth and lack of PP IX fluorescence,if higher concentration of ALA is used. A combination

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between ALA and light clearly demonstrates the inhibi-tory effect of PD with ALA. Tis method is promising asfar as reduction of T. rubrum colonization in onychomy-cosis is concerned [57].

One of the possible PD schemes is: Damaged nailsurface is coated lavishly with 20 % urea unguentum andis covered with a folio for 10 hours. Te nails are subse-quently treated with 20 % solution of ALA methyl ester inliquid cream for 5 hours, but only after protection fromlight with plaster and aluminium folio has been done.Protoporphyrin fluorescence is confirmed with UV-beamand spectrophotometer before PD. It’s observed in nailbase and periphery of fungal lesions. Te nail (includingproximal and lateral nail borders) is exposed to radia-tion horizontally and vertically with pulsed laser 630 nmlight, 100 J/sm2 using excimer laser [58] PD with 5-ALA,applied once a week [59]. 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 occursafter 6–7 treatment courses (total dosage 600–700 J/sm2).Most frequently, dermatophytes are not found in post-treatment investigations—microscopy with KOH pre-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 torecent researches, PD with methylaminolevulinate isalso a successful treatment of refractory onychomycosiscaused by nondermatophytic moulds [58].

PD is suitable for treatment of distal and lateral

subungual onychomycosis caused by T. rubrum [60].Te advantages of PD are the lack of side effects, con-

cerning kidney and liver function, as well as the lack ofrisk to patients with systemic diseases and the absenceof interaction with other drugs [57, 59]. Old age is not acontraindication for treatment. On the other hand, oraltherapy with antifungal agents may not be effective and itmay be even contraindicated in case of intolerance. PDis a very good alternative in these cases [58, 60].

Discussion

Currently, the most frequently used diagnostic methodsfor fungal infection confirmation are the conventionalones—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 aresult, 30–50 % of fungal agents cannot be identified byconventional 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 invasivemanipulation [24, 25].

Fungal tests are very important in the differential dia-gnosis of nail diseases, since other condition such aslichen planus, psoriasis, and nail dystrophy of differentcauses may have features that can be confused with ony-

chomycosis. Having in mind that onychomycosis fre-quently occurs in cases of primary or genetically damagednails, this problem is not very easy to be solved [ 9, 24, 25].

Modern additional methods, which are molecular–biological—PCR, Uniplex-PCR-ELISA-est, and MALDI-OF MS—will probably play a very important role in thediagnosis of onychomycosis [24, 40].

PCR is an easy and rapid method. Extraction of DNAtakes up to 30 minutes, whereas elongation takes 5 hours.PCR results are ready within a day. Tis method is charac-terized by high sensitivity and diagnostic specificity thatexceed the classic diagnostic methods. Te specificity ofPCR analysis derives from the possibility of revealing the

exact causative agents in the presence of other microor-ganisms, viruses, and cells of macroorganisms [24].From this point of view, the ability of PCR can be defi-ned as unique. By using this method, it is easy to identifyand diagnose dermatophytes that are difficult to detect

Fig. 4 Tinea pedis, 57-year-old patient, caused by T. rubrum

Fig. 5 Onychomycosis, a 70-year-old man; causative patho-

gen—T. rubrum

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by classic methods. Te analysis is made with minimalamount of specimen and at the same time concomitantdiagnosis of a number of species in one clinical sampleis possible. By PCR, different biologic specimens can be

examined, including those directly taken from skin lesi-ons, nails, and hair (Figs. 4 and 5). Tese properties allow wide usage of the method by dermatologists and mycolo-gists [24]. In conclusion, PCR is a more reliable methodthan direct microscopy and culture. At the moment, thismethod is not available in all clinics and laboratories,although it is used as an additional diagnostic tool to theclassic methods. Tis method has considerably increasedthe percentage of positive results and has reduced thetime needed for achieving an accurate diagnosis. Te cul-ture gives results in 3–4 weeks, whereas PCR method wit-hin a maximum of 1–2 days. Tis method is also reliableas far was necessary investments and materials for main-

tenance are concerned. At present, the wide use of themethod is restricted only by labor intensity and the needof more staff. In recent years, it is considered that mole-cular biology methods will completely replace conventio-nal methods, which are currently considered as standardmethods. Te establishment of separate specialized cen-ters and laboratories aiming to focus their activity in thisfield could be significant to the exact identification of thecausative agent and administration of proper systemictherapy. Te choice of treatment and duration of antifun-gal therapy depends on the identification of the causativeagent—dermatophytes, moulds, and yeasts. Besides the well-known topical and systemic antifungal medicati-

ons, in recent years new treatment methods have gainedpopularity—PD and laser treatment have been success-fully applied.

Conclusion

Te most frequently used diagnostic methods for theconfirmation of fungal infection are still the conventionalones—direct microscopy with KOH preparation and cul-ture on Sabouraud’s dextrose agar. Only a restricted num-ber of laboratories perform modern diagnostic molecularbiology methods such as PCR and MALDI-OF MS. Tis

results in problems in creating a universally accepteddiagnostic algorithm. At present, treatment options foronychomycosis include topical and systemic antifungalmedications, as well as surgical treatment, PD, and lasertreatment for very particular situations.

Terapy with terbinafine is preferred in cases of ony-chomycosis caused by dermatophytes. It has a very goodeffect in infections caused by Trichophyton, Microspo-rum, and Epidermophyton species [61, 62]. Terapy withterbinafine has also good effect in infections with moulds[62, 63]. It is, however, less effective against yeasts, sinceit is primarily fungistatic against some species, for exam-ple, Candida albicans and Candida parapsilosis [62, 63].

Because of the powerful fungicid effect of terbinafineagainst dermatophytes and moulds, many clinicians pre-

fer systemic therapy with terbinafine instead of itracona-zole or fluconazole.

On the other hand, therapy using fluconazole or itra-conazole schemes (pulse treatment) is preferred when

yeasts are identified, but it is considered a second-linetreatment for dermatophyte infections.

Surgical treatment as well as more recently describedoptions such as PD or laser treatment should, in princi-ple, be reserved for cases that have not responded ade-quately and/or for patients with contraindications for themore conventional therapeutic options.

Conflict of interest

Te authors declare that there is no actual or potentialconflict of interest in relation to this article

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Wiener Medizinische Wochenschrift Volume 163 Issue 1-2 2013 [Doi 10.1007%2Fs10354-012-0139-3]...

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