Samia A. Girgis*, Nehal M. Zu El -Fakkar**, Hala Badr*, Omnia A. Shaker*, Fatma E. Metwally* and Hadia H. Bassim*

Egyptian Dermatology Online Journal 2 (2): 2, December  2006.

Abstract:

Background: Dermatophytosis accounts for the majority of fungal infection all over the world. The conventional laboratory methods for identification of dermatophytes are slow and lack specificity. Genetic amplification has made rapid and precise identification of dermatophytes possible. With the increasing variety of drugs available for the treatment of dermatophytosis and with the lack of effective and safe antifungal, the need for a reference method for testing the antifungal susceptibilities of dermatophytes has become apparent.

Aim of the study: The current study was conducted to compare the rapid diagnostic molecular technique arbitrarily primed polymerase chain reaction (AP-PCR) with the conventional culture method for identification of the dermatophyte fungal infections of hair, nail and skin. Also to determine the antifungal susceptibility pattern of different dermatophyte isolates to Terbinafine, Griseofulvin, Itraconazole and Ketoconazole as the routinely used antifungal agents.

 Patients and Methods: The present study included 115 patients with dermatomycosis of the hair, skin and nail. Their age ranged from 3 to 50 years (mean 19.8±12.5 SD). Specimens from the infected sites were collected and subjected to conventional examination by direct (potassium hydroxide) KOH microscopic examination, culture on primary and selective media. Dermatophyte isolates were identified by their characteristic morphology, physiological tests and AP-PCR. Antifungal susceptibility was tested for all isolates according to the National Committee for Clinical Laboratory Standards NCCLS microdilution method M38-A for filamentous fungi with modifications in temperature and incubation period.

 Results: Out of the 115 cases with ringworm infection, dermatophytes were isolated in culture from 46.1% of specimens and nondermatophytes from 18%. Trichophyton (T) rubrum (32.1%) was the most commonly isolated dermatophyte from all types of skin fungal infection except tinea capitis (P<0.001). T. mentagrophytes and T. violaceum were the main causes of tinea capitis. By genotypic identification (AP-PCR) of dermatophytes, all isolates formed distinct DNA band patterns on gel electrophoresis which was in agreement with the conventional methods in 86.8% of isolates. Out of the eleven phenotypically identified T. mentagrophytes; two only were diagnosed to the strain level, two strains were genotypically identified as T. rubrum and one as T. tonsurans. Also two isolates of T. violaceum were diagnosed by PCR as T. schoenleinii, one T. rubrum was diagnosed as T. ajelloi and one T. soudanense as T. violaceum. The direct KOH examination had sensitivity of 88% and specificity of 74%. The antifungal susceptibility pattern of the isolated dermatophytes were for terbinafine 0.06-0.5 (0.121) µg/ml, itraconazole 0.06-4 (0.62) µg/ml, ketoconazole ranged from 0.06-4 (0.857) µg/ml and griseofulvin from 0.5-8 (2.151) µg/ml. Terbinafine was the most powerful antimycotic and T. rubrum had the highest ( minimal inhibitory concentration) MIC values for the four antifungal agents.

Conclusion: The genotypic differentiation by AP-PCR provides a rapid and practical tool for identification of dermatophyte isolates to the species and strain level within one day that is independent of the culture variations. The standard NCCLS M38-A broth microdilution method with the modifications in temperature and incubation period is convenient for antifungal susceptibility testing of dermatophytes.

Introduction

Dermatophytes infections of the skin affects a large proportion of population and have emerged as important causes of morbidity especially in aging population and in immunocompromised patients (Marie et al., 2001 [46] and Osborne at al., 2003[59]). These dermatophytes are of the genera Microsporum, Trichophyton and Epidermophyton. The genus Epidermophyton is represented by a single pathogenic species (E. floccosum), the genera Microsporum and Trichophyton are complex and made of multiple species (Koichi et al., 1999)[38].

For many years they were accustomed to diagnose dermatophytes on the basis of morphological and biochemical characteristics by using direct microscopic examination and in vitro culture. Although they are economic, these procedures suffer from the drawbacks of being either slow or non specific showing false negative results (Howell et al., 1999)[33]. Also the application of chemotherapy has contributed to the occasional modification and alteration of the morphological characteristics of dermatophytes cultures and complicating laboratory identification procedures based on phenotypic features (Faggi et al., 2001)[22].

Using molecular methods for differentiation between the genotypic characteristics of the species of dermatophytes are more specific, precise, rapid and are less likely to be affected by external influences such as temperature variations and chemotherapy. These molecular methods, such as restriction fragment length polymorphism analysis of mitochondrial DNA (Kano et al., 2000)[36], sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA, sequencing of protein- encoding genes, and polymerase chain reaction (PCR); random amplification of polymorphic DNA [RAPD], arbitrarily primed PCR [AP-PCR] (Elisabetta et al., 2001)[18], and PCR fingerprinting, have brought important progress in distinguishing between species and strains. However, most of these techniques require additional manipulation such restriction endonuclease digestion, hybridization or sequencing after amplification, so they are complex, laborious, relatively time-consuming, and not easily employable for routine identification of dermatophytes (Graser et al., 1999)[29]. In contrast, AP-PCR technology is simple, rapid and in the absence of specific nucleotide sequence information for many dermatophyte species, AP-PCR is able to generate species- specific or strain specific DNA polymorphism on the basis of characteristic band patterns detected by agarose gel electrophoresis (Faggi et al., 2001[22]& Baeza and Giannini, 2004[7]).

Dermatophytes are eukaryotic and have machinery for protein and nucleic acid synthesis similar to that of higher animals. It is, therefore, very difficult to find out compounds that selectively inhibit fungal metabolism without exhibiting any toxicity to humans. Also there is evidence that dermatophytes have acquired resistance to certain antimycotic drugs. So, with the increasing variety of drugs available for the treatment of dermatophytoses and with the lack of effective and safe antifungal , the need for a reference method for testing of antifungal susceptibilities of dermatophytes has become apparent. Such standard method is not yet available. Establishment of a reference susceptibility testing method may allow the clinician to select the appropriate therapy for the treatment of infections caused by dermatophytes (Jessup et al., 2000 [35] and Augustine et al., 2005[5]).

Aim of the Study

The current study was conducted to compare the rapid diagnostic molecular technique AP-PCR with the conventional culture method for identification of the dermatophyte fungal infections of hair, nail and skin. In addition, to determine the antifungal susceptibility pattern of different dermatophyte isolates to Terbinafine, Griseofulvin, Itraconazole and Ketoconazole as the routinely used antifungal agents.

Patients and Methods

The present study was conducted on 115 patients attending the Dermatology Outpatient Clinic of Ain Shams University Hospitals. Their age ranged from 3 to 50 years (mean 19.8 ± 12.5 SD). They were 59 females and 56 males. The patients were clinically diagnosed as having tinea capitis (47), tinea corporis (29), tinea pedis (23), onychomycosis (9), tinea cruris (5) and tinea manuum (2). All patients were subjected to full history taking including age, sex and antifungal treatment.

Specimen collection and processing:

Clinical examination was done to differentiate between different types of ring worm infection. Specimens were collected from all patients after disinfection with 70% alcohol and were kept in dry sterile containers. The collected specimens were: 
 - Hair: suspiciously infected hairs were plucked with forceps.
 - Cutaneous skin scales: scrapings were taken from the definite edge of the lesions with sterile scalpel.
 - Nail: nail eclipses, scrapings or subangular curette.

The specimens were then subjected to the following (Milne, 2001)[50]:

 1. Direct microscopic examination using 10-20% KOH solution with methylene blue.
 2. Culture on:

 a) Sabouraud's dextrose agar medium with chloramphenicol (Oxoid, U.K.).
 b) Dermasel agar medium with chloramphenicol and cycloheximide (Oxoid, U.K.).

 c) Potato dextrose agar ( PDA )with chloramphenicol (Oxoid, U.K.).
 d) Oatmeal cereal agar for antifungal susceptibility (Sigma Chemicals Co., St., Louis, Mo., U.S.A.).
 e) Potato agar slants for storage of dermatophyte isolates at -80oC until time of use.

Identification of the fungal isolates was done through

1. Macroscopic examination of colonies on different media
2. Microscopic examination of cultures.
3. Physiological tests including urease and pigment production tests.
4. Arbitrarily primed polymerase chain reaction (AP-PCR).

It was done according to the standard microdilution method for filamentous fungi M38-A of the National Committee for Clinical Laboratory Standards (NCCLS, 2002) against four antifungal agents; Terbinafine, Griseofulvin, Itraconazole and Ketoconazole with some modifications according to Favre and colleagues (2003)[23].

I. Arbitrarily Primed Polymerase Chain Reaction (AP-PCR):

Principle:

Arbitrarily primed PCR technique relies on arbitrarily designed sequences of short primers (usually 10 nucleotides long) that anneal specifically to DNA templates in a target organism. If these primers anneal to target DNA sequences, the intervening segments that are proximal enough to these annealing sites will be amplified and will generate products of variable molecular weights. Such products can be resolved by agarose gel electrophoresis, which will display a band pattern for each strain. The primer sequences are determined empirically, since the target DNA are usually not known and the entire genome of the organism serves as the target for the strain comparison and differentiation (Liu et al., 2000a [43]& Rilley, 2004[60]).

Procedure:

The procedure was done according to Liu et al. (2000b)[44].

a-DNA extraction and precipitation:

Fungal isolates were subcultured in 100 ml of Sabouraud's broth (Oxoid, UK) and incubated with shaking for up to 7 days at 25 °C. Hyphal growth was harvested by filtration and washed twice with 100ml of sterile saline. Strains, which could not be processed immediately, were frozen at -80°C prior to extraction. Liquid nitrogen was added to 2-3g of frozen hyphae and the cells were ground finely. Approximately 200mg of frozen ground mycelium was placed in a 1.5 ml microcentrifuge tube. Fungal DNA was extracted from fungal cell suspension by using the Puregene DNA purification kit (supplied by Gentra system, U.S.A.).

b-cDNA amplification:

The primer pair used for Dermatophyte DNA amplification were OPAA 17 (5'-GAGCCCGACT-3') were prepared by ABI Applied Biosystem 394 DNA, RNA synthesizer, USA.

Six microliters of cDNA was added with 1.0μl of the primers sense and antisense (50pM/µl) to 50 l reaction mixture. The reaction mix included 5µl of 1 PCR buffer (50mM KCl, 10mM Tris-HCl, pH 8.3), 2.5mM MgCl2, 0.25μM dATP, 0.25 μM dCTP, 0.25 μM dGTP, 0.75 μM dUTP, 0.125 U of UNG, and 2U of Taq polymerase (0.4µl) (Promega, USA) and Sterile nuclease free water (38.6µl).

After initial denaturation at 95 C for 2 min, the GenAmp 9700 thermocycler was programmed for 30 cycles of amplification and a final extension period of 5 min at 72oC. Each cycle consists of:
* denaturation at 94°C for 30s,
* annealing at <40°C for 30s, and
* extension at 72°C for 30s.

The amplified product were then stored at -20°C .

Negative control of sterile nuclease-free water and positive control of Trichophyton (T). mentagrophytes var erinacei identified by conventional methods, were included in the procedure.

c-Detection and interpretation of the amplified products:

A100-bp DNA ladder (Pharmacia Biotech, U.S.A.) was used as a molecular size marker. Ten microliters of the PCR amplicon was mixed with 2μl of gel loading dye (Promega, U.S.A.) and the mixture was electrophoresed on 1.5% agarose gel in Tris-acetate EDTA buffer and stained with ethidium bromide (Amersco, U.S.A.). The gel was viewed under ultraviolet light and photographs were taken. The high intensity bands produced by AP-PCR for each isolate and the positive control were compared with the bands of the DNA ladder (figure 1).

Fig 1: Agarose gel electrophoresis of PCR products of the amplified Dermatophytes DNA Lane 1A&B: 100-bp DNA ladder Lane 2A: T. verrucosum; 500bp Lane 3A: M. canis; 700, 1200, 3000bp Lane 4A: M. canis; 700, 1200, 3000bp Lane 5A: T. rubrum; 400, 600, 2500, 3400bp Lane 6A: negative control Lane 2B: T. mentagrophytes var, erinacei; 400, 1500bp Lane 3B: T. ajelloi; 500, 800, 1900bp Lane3B: E. floccosum; 800bp

The identification and differentiation of the 53 isolates were done by comparing the molecular size of the DNA bands (bp) of the amplified arbitrarily primed PCR products with that performed by Liu et al. (2000b)[44] and with the culture results. Table (1) shows the examination of the DNA products from dermatophyte fungi by AP-PCR.

Table (1): Examination of DNA products from dermatophyte fungi by AP-PCR obtained by OPAA17 primer (Liu et al., 2000b)[44]

II. Antifungal Susceptibility Testing of Dermatophyte isolates: (NCCLS, 2002):

A standard method for antifungal susceptibility testing of Dermatophytes is not yet available, mainly the NCCLS (M38-A) standard method for conidium forming filamentous fungi was followed with some modifications recommended by Favre et al., (2003)[23] such as changing temperature and incubation time.

a) Antifungal agents:

Terbinafine (Novartis Pharma, Basel, Switzerland), Griseofulvin (Pharco Pharmaceutical- Alexandria), Itraconazole and Ketoconazole (Janssen- Cilag Beerse, Belgium) were used in this study.

Stock solutions of Terbinafine, Itraconazole and Ketoconazole were prepared by dissolving 16µg of each antifungal in 10ml of 100% dimethyl sulfoxide (DMSO) (Sigma chemicals Co., St. Louis, Mo., U.S.A.) in separate tubes to get a concentration of 1600µg/ml. For terbinafine 5% Tween 80 was added to the 100% DSMO. While a stock solution of Griseofulvin was prepared by dissolving 32µg in 10ml of 100% DMSO to get a concentration of 3200µg/ml. Stock solutions were kept frozen in 1ml aliquots at -70oC.

A working solution of each antibiotic was prepared by diluting 100 µl of the stock solution in 900 µl of RPMI-1640 medium containing L-glutamine and 0.165M morpholine propane sulfonic acid (MOPS) without bicarbonate (GIBCO BRL, Life Technologies, Paisley, Scotland) to get a concentration of 32µg/ml for Terbinafine, Itraconazole and Ketoconazole and 64 µg/ml for Griseofulvin.

b) Quality Control Strains:

Candida parapsilosis ATCC 22019 (The American Type Culture Collection (ATCC) (Rockville, Md) was used as quality control strain to test for the used antifungal drugs. According to NCCLS M27-A standard method (2000) for antifungal susceptibility of yeast, the reference MIC range for C. parapsilosis is 0.06-0.5 µg/ml for both itraconazole and ketoconazole after 48h incubation (Jessup et al., 2000)[35]. Reference strain was grown in 10ml brain heart infusion broth (Difco) at 35oC overnight. The suspension was diluted two folds with brain heart infusion broth containing 20% glycerol (Sigma), dispensed in screw-capped tubes, sealed and stored at -70oC. The reference strain was tested with every batch of antifungal susceptibility of the isolated species (Gupta and Kohli, 2003)[30].

c) Preparation of the microdilution plates:

o Serial two fold dilution of the antifungal agents were prepared with RPMI 1640 medium. The final concentrations of the antifungal agents ranged from 64 to 0.125 for Griseofulvin and from 32 to 0.06µg/ml for Itraconazole, Terbinafine and Ketoconazole. Sterility control (negative control) and growth control (positive control) were included in each plate. With each batch of antifungal susceptibility, antifungal control using C. parapsilosis ATCC 22019 reference strain was inoculated to test for the validity of the four antifungal agents according to the NCCLS M27-A standard method (NCCLS, 1997).

o Uninoculated microtitration plates containing antifungal dilutions were kept covered for approximately 6 months at -70°C.

d) Preparation of the Dermatophyte inoculum:

Dermatophyte isolates were grown on oatmeal cereal agar slants for 7 days at 28°C; the best medium to support conidial growth (Jessup et al., 2000)[35]. Sterile normal saline (0.85%) was added to the slant culture and was gently swabbed with a cotton tip applicator to dislodge the conidia from the hyphal mat. The suspension was adjusted to 5 mL with sterile normal saline. The cell density was adjusted to give final inoculum concentration of 104CFU/ml. The suspension was counted on a hemocytometer and was diluted in RPMI 1640 to the desired concentration. 100µ1 of the organism suspension was transferred into all wells of the microdilution plates except for the negative control wells. Plates were incubated aerobically at 30°C, except for E. floccosum and M. canis at 35°C. All were incubated for 4-10 days according to the growth in the control wells.

e) Reading and interpretation of the panel:

The minimal inhibitory concentration (MIC) endpoints were determined according to NCCLS M38-A standards as the point at which no visual turbidity where the organism was inhibited 80% when compared to the growth control. For the quality control Candida parapsilosis the MIC endpoint was determined as ≥80% inhibition of the positive growth control for Itraconazole and Ketoconazole (NCCLS M27-A).

Results

This study was carried out on 115 patients with ringworm infection. Their age ranged from 3-50 years. Forty seven patients were below age of ten years (41%), eleven were in the second decade (9.4%), thirty one patients were in the third (27%), twenty three patients were in the fourth (20%) and three patients were in the fifth decade (2.6%). The patients under study were 59 (51.3%) females and 56 (48.7%) males. Tinea capitis infection 47 (41%) was only prevalent below the age of ten years and double in females 31 (27%) than males 16 (13.9%). Also tinea pedis was higher in males 13 (11.3%) than females 10 (8.7%).

Table (2) shows the distribution of the Dermatophyte positive cultures among patients with ringworm infection. Tinea capitis represented 47 (41%) of cases followed by tinea corporis 29 (25.2%) and tinea pedis 23 (20%). Twenty three (43%) of dermatophyte isolates were separated from tinea capitis patients, followed by 14 (26.4%) and 12 (22.6%) from tinea corporis and tinea pedis respectively.
 

Table (2): Distribution of Dermatophyte Positive Cultures Among Patients with Skin fungal Infection

In spite that there was no statistically significant association between the isolation of dermatophytes from the infected sites and the sex of the patients (P>0.5), dermatophytes isolated from tinea capitis patients were higher in females 15 (28.3%) than males 8 (15.1%). Males show higher prevalence of tinea pedis than females.

Out of the 115 specimens of the patients under study, 53 (46.1%) yielded dermatophyte growth on culture and 21 (18%) specimens grew nondermatophytes; nine Candida, eight Aspergillus spp. (five A. fumigatus, three A. niger) and four cases showed Acremonium. Three (2.61%) of the patients showed both Dermatophytes and non dermatophytes growth. The later group grew mainly on Sabouraud's dextrose agar (SDA).

In the present study there was a highly significant association (P<0.001) between the results of direct microscopic examination by KOH and dermatophyte culture. Out of the 53 positive cases by culture, there were 38 positive cases (33%) by direct KOH examination and the remaining 15 cases (13%) were negative and out of 62 negative cases by culture, there were 16 (14%) cases positive by direct KOH examination. Out of those 16 dermatophyte culture negative specimens and KOH positive; five cases were on antimycotic therapy (two tinea cruris and three tinea capitis), five cases grew Aspergillus (three obtained from tinea capitis and two from onychomycosis), five cases Candida (two were onychomycosis, one tinea pedis, one tinea capitis and one tinea cruris) and one case of tinea capitis showed mixed growth of Aspergillus and Candida. The KOH method had a sensitivity of 88% and specificity of 74%. By direct KOH microscopic examination no definite identification was reached. However, hyphae ,arthrospores and chlamydospores were seen in some preparations. Acremonium species gave the fronded appearance and Aspergillus branched dichotomously at acute angles.

Out of the three cases that yielded growth of both dermatophytes and rapidly growing non Dermatophyte fungi, one was onychomycosis which showed the fruiting bodies of Aspergillus together with the hyphae of dermatophytes on KOH examination. The second two cases were tinea corporis which showed budding yeast cells of Candida species on direct microscopy together with the hyphae of dermatophytes.

Table (3) shows the dermatophyte species isolated from each type of fungal infection. The most common type was T. rubrum 17 (32.1%) which showed a highly significant association with almost all types of dermatophytosis except tinea capitis (P<0.001). There were a highly significant association between T. mentagrophytes 11 (20.8%), T. violaceum 10 (18.9%) with tinea capitis, corporis and pedis patients (P<0.001).