Onychomycosis-Sampling , Diagnosing as E ffi ciant Part of Hospital Pharmacology

Vesna A. Ignjatović1, Milica T. Stevanović1, Vera S. Djurdjević2, Mirjana M. Petrović3, Milica P. Stanucević4, Aleksandar M. Džamić5, Ivana V. Čolović Čalovski5 A 1 Esthetic center ̋Dr VAIS ̋, Belgrade, Serbia 2 Esthetic center ̋Dr VAIS ̋, Niš, Serbia 3 Institute for endocrinology Clinical Center of Serbia, Belgrade, Serbia 4 Department of Radiology, Clinical center „Zvezdara“, Belgrade, Serbia 5 Institute for microbiology and immunology, Medical Faculty, University of Belgrade, Belgrade, Serbia


INTRODUCTION
Th e term onychomycosis comes from a Greek word "νύχι" (nail), "μύκης" (mushroom) and it represents a fungal infection of one or more nails.Based on various studies prevalence of onychomycosis is aproximately 2% to 13% of general population [1,2].Predisposing factors of onychomycosis are: age, sex, occupation, climate, previous onychomycosis, inadequate hygiene of legs, sweating, inadequate footwear, sports and trauma, contact with soil and animals.Th e prevalence was signifi cantly higher in patients with immunodefi ciency, compromised peripheral circulation, diabetes, psoriasis as well as the patients on long-term antibiotic therapy [3][4][5][6][7].

Causes of onychomycosis
Onychomycosis is caused by dermatophytes, yeasts and non-dermatophyte molds, but certainly the most common cause are the fungi of the dermatophyte group.Dermatophytes are strictly pathogenic fungi which have keratinophilic and keratinolytic features and aff ect the skin, hair and nails.Dermatophytes include three genera: Trichophyton, Microsporum and Epidermophyton, which are divided by ecology into anthropophilic, geophilic and zoophilic types [8].Th ese diseases are highly contagious and can be transmitted by direct and indirect contact [8].
Onychomycosis is most frequently caused by T. rubrum, T. interdigitale and E. fl occosum from the group of dermatophytes causing about 90% of onychomycosis on the feet and 50% of onychomycosis on the hands [9].Th e most common cause is the anthropophilic species T. rubrum.Various causes can oft en give an identical clinical presentation, especially in advanced cases of onychomycosis.

Virulence factors and immune response
Th e main eff ect of dermatophytes is through their endoproteases which can be classifi ed into two large protein families: serin proteases and metalo-proteases [10].A humoral and cell-mediated immunity occurs as a defensive response of the host.Th e immune response to Trichophyton species is particularly unusual because it can cause immediate hypersensitivity and delayed hypersensitivity reaction in diff erent individuals [11].

Clinical forms
Depending on the location of the primary infection and its spread, Tinea unguium (onychomycosis) can be divided in four subtypes:

Diagnostic methods
Th e fi rst important step in diagnosing onychomycosisis is sampling.Many studies have shown that proper sampling of the nail is very important for the correct diagnosis.Methods for collecting the infected nails are: cutting the free edge of the nail, scratching hyperkeratotic subungual material and scraping the surface of the nail with a scalpel [16].
Aft er proper sampling the diagnosis of onychomysosis usually involves making a direct microscopic preparations with 10-30% potassiumhydroxide (KOH) in dimethylsulfoxide (DMP) which allows identifi cation of fungal elements but cannot make the identifi cation to the level of species.Cultivation remains the gold standard in the diagnosis of onychomycosis based up on which it is possible to identify the level of genus or species in most routine laboratories.It is performed by seeding the material on Sabouraud Dextrose Agar (SDA) and Dermatophyte Test Medium (DTM).Further more, the metod of molecular diagnostics can also be used, most frequently the Polymerase Chain Reaction (PCR) [14,15].
European Mycologycal Association suggested in the year of 2013 the methodological laboratory procedures for the diagnosis of onychomycosis which will be included in the European standard protocols.Th e protocol recommends PCR as a fi rst method of choice for onychomycosis of the feet, and PCR and conventional mycologycal methods for onychomycosis of the hands.

Onychomycosis therapy
Proper diagnosis and laboratory confi rmation of onychomycosis are needed for the use of antimycotic therapy and the most eff ective hospital pharmacology results.
Th erapy can be local/topycal, systemic/oral or combined.Topycal treatment consists of removing of the infected nail material and persistent application of topycal anti-fungal drugs over several weeks/months [16].Systemic antimycotic therapy is required if more than 50% of the nail is aff ected, but it is expensive, has a variety of adverse eff ects such as: disorders of various liver enzymes, interactions with other drugs and many others [17].

THE AIM
By sampling determination the most common clinical type of onychomycosis, localization and involvement of the nail plate, and monitoring the effi cacy of methods/tests in the diagnosis of nail onychomycosis.

MATERIAL AND METHODS
Th is paper is a part of the prospective academic (non-commercial) phase IV study, carried out in the Institute for microbiology and immunology, Medical Faculty, University of Belgrade, and Esthetic center ˝Dr VAIS˝.Study has been approved by the Esthetic center ˝Dr VAIS˝ Ethics Board and conducted in compliance with the EU clinical trials directives [18].Informed consent was obtained from all patients.
Patients with clinical symptoms suspicious of the onychomycosis, confi rmed by a dermatology specialist, were tested on onychomycosis till collecting 30 patients with nails onychomycosis confi rmed.Samples were collected from October 1 st 2013 to December 31 st 2013, from two medical centers: Esthetic center ˝Dr VAIS˝ in Belgrade and Niš.

Diagnosing procedure included:
Sampling the nail -Depending on the type of onychomycosis part of the nail plate was sampled in several ways: by cutting the distal free edge of the nail, scratching the and scraping the surface of the nail with a scalpel (by which we received the hyperkeratotic material from deeper layers) [16].All samples were collected in sterile containers and labeled properly.Laboratory methods -Each sample of the nail was cultivated on fi ve mediumes: • SDA at 28°C and 37°C • D-SDA at 28°C and 37°C • DTM at 28°C Period of incubation was completed in three weeks at the indicated temperatures and samples were controlled once a week to detect fungal growth.Th e samples with no fungal growth aft er four weeks were consid-ered negative.
Identifi cation of isolated fungi to the level of genus/species has been based on macroscopic and microscopic characteristics.We used KOH and Blancophor fl uorescent dye for making preparations for microscopy.Preparations were observed by light microscope and fl uorescent microscope at magnifi cation 10x and 40x for detecting and identifying fungal elements.
For the identifi cation of dermatophytes we used Dermatophytes Multiplex PCR (Statens Serum Institute, Denmark) that detects specifi c T. rubrum and pan-dermatophytic multiplex PCR product.
PCR method involved the following steps as instructed by the manufacturer: • Preparing the nail samples for each patient • Preparing Master Mix mixture • PCR amplifi cation in the Th ermal Power Device Termojet (Eurogentec, Searing Belgium) Th e mixtures were exposed to a temperature of 94°C for fi ve minutes, for denaturation of DNA; followed by a 45 temperature cycles consisted of: 1. Denaturation of DNA at the temperature of 94°C lasting for 30 seconds; 2. Reproduction at thetemperature of 60°C lasting for 30 seconds; 3. Binding of the primers to the matrix (Ta annealing) at the temperature of 72°C lasting for 30 seconds and 4. Elongationat the temperature of 72°C lasting for 3 minutes.
• Electrophoresis and visualization oft he polymerse chain reaction products For visualization of the products of PCR we performed electrophoresis in 1.5% agarose gel and brightening of the gel with ultraviolet light.Th e size of the resulting DNA products was compared to the size of the DNA control fragment from the PCR kit for T. rubrumand pan-dermatophytic marker.

RESULTS
Th irty patients with positive results were included in the study [Table 1], 21 (70%) female average age 54 years and 9 (30%) male average age 38 years.In clinical fi ndings DLSO prevailed (86.6%), followed by PSO (10%) and WSO (3.4%).In 83% of patients onychomycosis was found on the feet, while 17% of patients had changes on their hands.In most cases, thumbs were aff ected both on the hands (57%) and the feet (49.3%), including percentage involvement of each fi nger on the right and the left foot [Table 1, Figure 1].Th e size of involvement of the nail plates was diff erent in diff erent patients [Table 1].In 66.6% of patients the nail plate involvement was 1/3-2/3, while the percentage of nail involvement of <1/3 and >2/3 was the same (16.7%)[Picture 1].
Aft er cultivating on three diff erent cultures (SDA, D-SDA and DTM) at diff erent temperatures [Picture 1], we obtained 15 positive results.As the most common isolate in 60% of cases T. rubrum was isolated [Figure 2].For 15 samples (50%) where there was no growth found, we performed PCR.T. rubrum was detected in 11 patients (73.3%), pandermatophyte marker (PD) was found in 2 samples (13.3%), which shows that the cause was other than T. rubrum and two samples were negative (13.3%).Th e results are shown in Figure 1.

DISCUSSION
Onychomycosisis widespread in general population.Most oft en itoccurs in adults and rarely in children [19].In this study, the average age of patients was 46 years, which matches the results of a large number of studies that show the average age of patients 40-50 years [20,21].Th e large number of patients included in our study were female (70%), matching the results of various studies where the uncomfortable shoes and household tasks were the main reasons for female morbidity [20,22,23].However, our re- Involvement of the nail sults do not match the studies where the main reasons for male morbidity are trauma and active sports [4].
Numerous studies have shown that the most common clinical typeis DLSO, which matches the result of our study, where DLSO was diagnosed in 86.6% [8].Other studies have shown that DLSO and TDO are representingmore than 90% cases of onychomycosis, which is also inaccordance with our results [24].Toenails are 25 times more infected than fi ngernails.Th e most frequently aff ected nails are the fi rst and second toenails because they suff er the mosttrauma and preasure from the shoes [9].Th e results of our study match earlier results where the feet thumb nails were aff ected in even 56.7%.
Cultivation is still considered to be the gold standard, although numerous studies have shown the sensitivity that ranges from 25-75% [5,7].According to the recommendations, the sample should be seeded on SDA and at least one more medium.In thisstudy we cultivated the samples on the SDA, D-SDA and DTM.SDA is a standard mycological medium which is used for isolation of the causes of onychomycosis and which sensitivity ranges from 50-70% [5].D-SDA has less glucose compared to standard SDA, which allows better sporulation of the fungi making the identifi cation much easier.DTM is a medium that is used for isolation of dermatophytes.Th eir growth becomes visible when the yellow color of the medium changes to red, due to the presence of alkaline metabolites as a result of dermatophyte growth [25].Th is study showed that dermatophyte isolates have the biggest sensitivity to the DTM (33.4%).In most studies the common cause of onychomycosis is T. rubrum.It is stated that T. rubrum and T. mentagrophytes causes nearly 90% ofonychomycosis followed by E. fl occosum [9,26].Our research shows the highest incidence of T. rubrum(60%).
PCR is a method that is increasingly used in the diagnosis of the onychomycosis.Recommendations are for PCR to be the metod of choice particulary for diagnosing feet onychomycosis.For onychomycosis ofh ands the conventional methods are recommened also.Th e sensitivity of PCR varies in diff erent studies, ranging from 40-95% [21,27,28].In this study, sensitivity was very high 86.7%,inwhich 73.3% identifi ed T. rubrum.Out of 30 patients included in this study with clinically suspected onychomycosis, using the methods of cultivation and PCR, onychomycosis was confi rmed in 28 patients (93.3%).Cultivatinggave negative results in 50% of cases, while the PCR was positive in 86.6%.In addition to the PCR results being signifi cantly faster than cultivation (expected in 2-3 days), where the results are issued aft er 3 weeks and longer, the main advantage of PCR methodis higher sensitivity compared to the other methods used in diagnosing onychomycosis.

CONCLUSIONS
Th e most common clinical type is DLSO.It was diagnosed in 86.6%.Th is study have shown that DLSO and TDO are representing more than 90% cases of onychomycosis.Our research shows the highest incidence of T. rubrum (60%).Th is study showed that dermatophyte isolates have the biggest sensitivity to the DTM (33.4%).PCR is a method that is increasingly used in the diagnosis of the onychomycosis.Its sensitivity was very high 86.7%, in which 73.3% identifi ed T. rubrum.In continuation of this study will be analyzed the choice and eff ectiveness of therapy.

Figure 1 .
Figure 1.The incidence of onychomycosis on certain nails of feet and hands