Journal of Microbiological Methods 100 (2014) 32–41
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Identification of dermatophyte species using genomic in situ hybridization (GISH) Mariusz Worek a, Aleksandra Kwiatkowska b, Anita Ciesielska c, Adam Jaworski d, Jakub Kaplan a, Beata Miedziak a, Anna Deregowska a, Anna Lewinska e, Maciej Wnuk a,⁎ a
Department of Genetics, University of Rzeszow, Kolbuszowa, Poland Department of Botany and Biotechnology of Economic Plants, University of Rzeszow, Kolbuszowa, Poland Department of Microbial Genetics, University of Lodz, Lodz, Poland d University of Social Sciences, Lodz, Poland e Department of Biochemistry and Cell Biology, University of Rzeszow, Rzeszow, Poland b c
a r t i c l e
i n f o
Article history: Received 30 January 2014 Received in revised form 22 February 2014 Accepted 22 February 2014 Available online 28 February 2014
a b s t r a c t A genomic in situ hybridization (GISH)-based method for dermatophyte identification has been developed. Using specific GISH probes, discrimination between Trichophyton interdigitale, Trichophyton rubrum and Microsporum canis has been conducted. Moreover, GISH has been found particularly helpful when proper dermatophyte identification was difficult due to ambiguous PCR–RFLP patterns. © 2014 Elsevier B.V. All rights reserved.
Keywords: Dermatophytes Trichophyton Microsporum PCR–RFLP GISH
1. Introduction Dermatophytes (Arthrodermataceae family) are a group of filamentous and keratinophilic fungi causing dermatophytoses (tinea infections) — superficial infections of keratinized tissues, such as skin, hair and nails (e.g. athlete's foot, ringworm, jock itch and onychomycosis) (Achterman and White, 2012; Cafarchia et al., 2013; Degreef, 2008; Weitzman and Summerbell, 1995). Dermatophyte-mediated host colonization is assisted by the release of keratinases and other proteolytic enzymes, thus dermatophyte infections are limited to keratinized surfaces without invading deeper localized tissues (Monod, 2008; Vermout et al., 2008). Dermatophyte species can be divided into three environmental groups, namely anthropophilic (human-type dermatophytes), zoophilic (animal-type dermatophytes) and geophilic (soil-type dermatophytes) (Achterman and White, 2012; Cafarchia et al., 2013; Weitzman and Summerbell, 1995). In general, anthropophilic dermatophytes are associated with human infections. Nevertheless, dermatophytes belonging to all three groups may promote fungal infections, e.g. cats and other animals may be considered as infection vectors
⁎ Corresponding author at: Department of Genetics, University of Rzeszow, Rejtana 16C, PL 35-959 Rzeszow, Poland. Tel.: +48 178723711. E-mail address:
[email protected] (M. Wnuk).
http://dx.doi.org/10.1016/j.mimet.2014.02.012 0167-7012/© 2014 Elsevier B.V. All rights reserved.
(Seebacher et al., 2008). Taxonomically, dermatophytes comprise three closely related genera: Epidermophyton, Microsporum and Trichophyton (Weitzman and Summerbell, 1995). Microsporum and Trichophyton are represented by multiple species, e.g. Trichophyton genus is composed of more than fifteen species and a common Trichophyton interdigitale species is characterized by several different variants (Weitzman and Summerbell, 1995). Since clinical manifestation is not necessary dermatophyte speciesspecific, accurate dermatophyte identification is absolutely essential in order to facilitate antifungal therapy and to prevent the spread of tinea infections (Gupta and Tu, 2006). It has been repeatedly reported that dermatophyte sensitivity to antifungal treatment may vary (Dragos and Lunder, 1997; Fleece et al., 2004). Epidermophyton spp. and Trichophyton spp. were found susceptible to terbinafine treatment, whilst Microsporum spp. were less sensitive (Dragos and Lunder, 1997; Fleece et al., 2004). Until recently, dermatophyte identification was based on morphological markers, such as presence, appearance and arrangement of macroconidia and microconidia, and other unique mycelial structures (e.g. racquet hyphae, favic chandeliers, spiral hyphae, nodular organs or pectinate body) (Robert and Pihet, 2008; Weitzman and Padhye, 1996; Weitzman and Summerbell, 1995). Other conventional methods may be also used, e.g. methods based on biochemical tests: nutritional requirement analysis, in vitro hair penetration test or urease activity
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(Robert and Pihet, 2008). For morphological identification, dermatophytes have to be cultured for up to four weeks (Bosshard, 2011). However, phenotypic variations and pleomorphism may account for inappropriate dermatophyte identification (Weitzman and Padhye, 1996; Weitzman and Summerbell, 1995). To improve dermatophyte identification, polymerase chain reaction (PCR)-based methods have been introduced, such as conventional PCR (Harmsen et al., 1999; Kanbe et al., 2003a), PCR fingerprinting (Faggi et al., 2002), random amplification of polymorphic DNA (Kano et al., 1998), PCR and restriction fragment length polymorphism analysis (PCR–RFLP) (Dobrowolska et al., 2006; Kanbe et al., 2003b), arbitrarily primed PCR (Liu et al., 2000a), multiplex PCR (Brillowska-Dabrowska et al., 2010; Kim et al., 2011) or real-time PCR (Miyajima et al., 2012). The main targets (genes or DNA fragments) were the following: the ribosomal DNA region, DNA topoisomerase II genes, and the chitin synthase gene (Kanbe et al., 2003a; Kano et al., 2003). In the present study, 47 dermatophyte samples were analyzed using morphological and molecular (PCR–RFLP analysis) markers. Since we were unable to identify two clinical isolates based on PCR–RFLP patterns, a complementary molecular tool for dermatophyte identification
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has been developed. Biotin-labeled dermatophyte species-specific genomic probes have been constructed and genomic in situ hybridization (GISH) has been performed. The usefulness of GISH-based dermatophyte identification is discussed. 2. Materials and methods 2.1. Ethics statement This study was approved by the Ethics Committee of the Faculty of Medicine, University of Rzeszow, Poland. All samples were analyzed anonymously. The dermatophyte samples were obtained from the clinical microbiology laboratory (Department of Diagnostic Medicine, Provincial Medical Specialist Unit, Rzeszow, Poland). 2.2. Clinical specimens and reference strains A total of 47 clinical samples used in this study are listed in Table 1. Clinical isolates were originated from human skin, nails and hair. The following three reference dermatophyte strains were used: T. interdigitale
Table 1 Clinical dermatophyte isolates used in this study. Isolate
Morphological characteristics
Patient sex/age
Source
PCR–RFLP patterns
340 343 412 449 451 459 482 489 498 503 510 554 587 648 697 698 701 711 712 720 736 782 820 826 912 976 985 1001 1016 1050 1070 1074 1098 1109 1189 1202 1203 1223 1286 1290 1347 1348 1395 1413 1479 1495 1536
T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. rubrum T. interdigitale T. interdigitale T. interdigitale T. rubrum T. interdigitale T. rubrum T. interdigitale T. rubrum T. rubrum T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. rubrum T. interdigitale T. interdigitale T. interdigitale T. rubrum T. interdigitale T. interdigitale T. rubrum M. canis T. rubrum T. rubrum T. rubrum M. canis M. canis T. interdigitale T. interdigitale T. interdigitale
M/19 F/46 F/26 M/67 F/5 F/81 F/37 M/24 M/63 M/9 M/47 F/30 F/61 M/40 F/22 M/25 F/66 F/66 F/26 F/48 M/6 F/37 F/67 F/63 F/39 M/8 M/14 F/51 F/42 M/58 F/32 M/22 M/65 F/25 M/31 M/14 M/3 M/64 F/6 F/63 F/12 F/78 F/23 M/7 M/9 F/57 M/4
Onychomycosis Onychomycosis Onychomycosis Onychomycosis Tinea corporis Onychomycosis Tinea pedis Onychomycosis Onychomycosis Onychomycosis Onychomycosis Onychomycosis Onychomycosis Tinea pedis Tinea corporis Tinea corporis Onychomycosis Onychomycosis Onychomycosis Onychomycosis Tinea faciei Onychomycosis Tinea pedis Onychomycosis Tinea pedis Tinea corporis Tinea corporis Onychomycosis Tinea pedis Onychomycosis Onychomycosis Tinea capitis Onychomycosis Tinea pedis Tinea pedis Tinea corporis Tinea faciei Onychomycosis Tinea capitis Onychomycosis Tinea corporis Tinea corporis Tinea corporis Tinea capitis Tinea pedis Onychomycosis Tinea corporis
T. rubruma T. interdigitale T. interdigitale T. interdigitale T. rubruma T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. interdigitalea T. interdigitale T. interdigitale T. interdigitale T. interdigitalea T. interdigitale T. interdigitalea T. rubruma T. rubrum T. interdigitalea T. interdigitale ?b T. interdigitale T. interdigitale T. interdigitale T. interdigitale T. rubrum T. interdigitale T. interdigitale T. interdigitale T. rubrum T. interdigitale T. interdigitale T. rubrum M. canis T. rubrum T. rubrum ?b M. canis M. canis T. rubruma T. interdigitale T. interdigitale
a b
Discrepancies in the dermatophyte identification based on morphological examination and molecular markers (PCR–RFLP analysis) are marked bold. Question mark indicates a problem with proper molecular identification due to ambiguous PCR–RFLP patterns.
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(formerly Trichophyton mentagrophytes var. interdigitale) (CBS 120357), Trichophyton rubrum (CBS 120358) and Microsporum canis (CBS 113480). The reference strains were obtained from Centraalbureau voor Schimmelcultures (CBS), Fungal Biodiversity Center, Utrecht, Netherlands. 2.3. Culture conditions Clinical isolates were cultured on Mycoline medium supplemented with cycloheximide (bioMerieux) at 25 °C until visible fungal growth occurred and then dermatophyte cells were passaged on Sabouraud Glucose Agar (SGA) medium, pH 5.6 (ARGENTA). For morphological and molecular (PCR–RFLP, GISH) identification, dermatophyte cells were cultured at 25 °C for a maximum of 4 weeks. The reference strains were cultured on SGA medium for a maximum of 4 weeks. 2.4. Morphological characteristics Strain-specific morphological macroscopic and microscopic features were monitored and especially the analysis of reproductive structures by slide culture method (Haley and Stonerod, 1954) was conducted. The dermatophyte microcultures were stained with lactophenol cotton blue and inspected under a light microscope. 2.5. Genomic DNA extraction Extraction of clinical isolate and reference strain genomic DNA was performed by rapid mini-preparation of fungal DNA for PCR method (Liu et al., 2000b). DNA samples were inspected and quantified using NanoDrop2000 Spectrophotometer (Thermo Scientific) and agarose electrophoresis. 2.6. PCR–RFLP For polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), the following primers were used: ITS1 primer (5′-TCCGTAGGTGAACCTGCGG) and ITS4 primer (5′-TCCTCCGCTTATTGATATGC) (Sigma). Taq PCR Master Mix Kit (Qiagen) was used according to the manufacturer's instructions. The PCR reaction conditions were the following: initial denaturation at 95 °C for 5 min, 30 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, elongation at 72 °C for 1 min, and final extension step at 72 °C for 10 min. Subsequently, the PCR products (~ 700 bp) were subjected to restriction analysis (restriction fragment length polymorphism, RFLP) by digestion with HinfI (Fermentas), MvaI (Fermentas) and HhaI (Fermentas) endonucleases at 37 °C for 3 h. Digested DNA were electrophoresed on 8% polyacrylamide gel (8% acrylamide/ bisacrylamide, 90 mM boric acid, 90 mM Tris–HCl, 2 mM EDTA, 6.51 mM TEMED, 0.07% APS) using 0.5 × TBE running buffer (45 mM boric acid, 45 mM Tris–HCl, 1 mM EDTA), stained with ethidium bromide, and inspected under UV light using G:BOX gel imaging system (Syngene, Cambridge, UK). PCR–RFLP patterns of clinical samples were compared with reference strain PCR–RFLP patterns. 2.7. Genomic in situ hybridization (GISH) For GISH assay, a small portion of hyphae was washed with 1× PBS buffer and centrifuged. Pellets were resuspended in 1 × PBS supplemented with 3.7% formaldehyde, incubated at 30 °C for 1 h with shaking, and then centrifuged (12,000 g, 5 min). Fixed cells were washed three times with sterile 1× PBS, pH 7.4, resuspended in 1× PBS containing 1.2 M sorbitol, 25 μl of 5 mg/ml zymolyase 100 T and 16 μl of 1.42 M β-mercaptoethanol in a total volume of 1 ml and incubated for 1 h. Then, cells were subjected to 24-h treatment with a mixture of glacial acetic acid:methanol (1:3) at −20 °C. Next, the cells were centrifuged (12,000 g, 5 min), placed on microscopic slide and dried at 50 °C for
10 min. Subsequently, slides were washed twice for 3 min in 4 × SSC buffer with 0.05% Tween 20 and once in 2× SSC buffer at room temperature. The slides with fixed cells were incubated with 100 μg/ml RNase in 2 × SSC buffer, pH 7.2, at 37 °C in a humidified chamber. The slides were washed three times for 3 min in 2 × SSC buffer, pH 7.2, at room temperature and then treated with 1% pepsin in 10 mM HCl for 2 min at room temperature. Slides were washed twice for 5 min with 1 × PBS, pH 7.4, and once with 1× PBS, pH 7.4, supplemented with 50 mM MgCl2 at room temperature and air-dried. Next, the slides were passed through a grade series of ethanol solutions (70%, 80% and 95%) at room temperature for 3 min and air-dried. A biotinylated genomic probe specific to T. interdigitale, T. rubrum or M. canis DNA (biotin-labeled dermatophyte-specific DNA) (Polish Patent Office, registration number P.404901) was added to the slide (Wnuk et al., 2013). The production of genomic probes fairly specific to T. interdigitale, T. rubrum or M. canis DNA is described comprehensively in the application number P.404901 (Polish Patent Office). Briefly, dermatophyte DNA was extracted as previously described (Section 2.5), dried with SpeedVac Concentrator (Savant) and suspended in 50 μl of ultra pure water (UPW). Then, dermatophyte DNA was labeled using Biotin-High Prime DNA labeling Kit with biotin-16-dUTP using random oligonucleotides as primers (Roche) according to standard procedure provided by manufacturer. To precipitate biotin-labeled DNA, 100 μg/ml yeast tRNA, 5 μl of 10 M ammonium acetate and 100 μl of ice-cold absolute ethanol were added. After 30-min centrifugation (14,000 g, 4 °C), supernatant was discarded and pellet containing labeled DNA was dried using SpeedVac Concentrator. Biotin-labeled dermatophyte-specific DNA with yeast tRNA (100 μg/ml) were added to hybridization buffer (Kreatech). To dissolve genomic dermatophyte probes, biotin-labeled dermatophytespecific DNA was incubated at 4 °C for 48 h. Genomic dermatophyte probes are stable at 4 °C for 6 months. Co-denaturation of the dermatophyte sample and genomic probe on a microscope slide was carried out on a hot plate at 80 °C for 7 min. Hybridization was performed in a humidified chamber at 37 °C for 48 h. The slides were washed with 1× Wash Buffer I (0.4× SSC, pH 7.2, containing 0.3% Igepal CA-630, Sigma) at 70 °C for 2 min and next washed with 1 × Wash Buffer II (2 × SSC containing 0.1% Igepal CA-630) at room temperature for 1 min. To detect biotinylated probe, Star*FISH© Biotin Painting Kit — FITC Label (Cambio, UK) was used. For DNA visualization, the slides were counterstained with a drop of mounting medium with 4′,6′-diamino-2-phenylindole (DAPI) (Cambio, UK) and were analyzed using an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software. FITC (GISHpositive nuclear signals) and DAPI fluorescent signals were collected using FITC and DAPI filters (λex = 495 nm, λem = 519 nm and λex = 345 nm, λem = 455), respectively. All images were collected at 1024 × 1024 pixel resolution. 3. Results and discussion A total of 47 clinical dermatophyte samples were isolated from patients in southeastern Poland (Subcarpathia province) and subjected to conventional morphological characterization and molecular identification using both PCR–RFLP analysis and genomic in situ hybridization (GISH). According to morphological marker analysis, T. interdigitale, T. rubrum and M. canis were established as major dermatophyte species causing of about 70%, 24%, and 6% superficial fungal infections (skin, hair, nails), respectively (Fig. 1 and Table 1). Our morphological data are in agreement with previously published results on pathogenic dermatophyte infections (dermatophytoses) throughout the world: T. interdigitale, T. rubrum and M. canis are considered main causative agents (Ameen, 2010). It is widely accepted that morphological dermatophyte identification and other conventional methods may be insufficient and errorprone (Robert and Pihet, 2008), especially discrimination between T. interdigitale and T. rubrum may be difficult due to dermatophyte
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Fig. 1. The dermatophyte identification based on morphological markers. Clinical isolates (n = 47) were cultured using slide culture method and microcultures were stained with lactophenol cotton blue. Representative features of Trichophyton interdigitale (top panel), Trichophyton rubrum (middle panel) and Microsporum canis (bottom panel) are shown.
phenotypic variability (different shapes of microconidia, low occurrence rate of macroconidia) and pleomorphism (the spontaneous appearance of white fluffy tufts of aerial mycelium on the surface of colonies, which results in the loss of characteristic pigmentation and conidiation) (Abdel-Rahman, 2008; Graser et al., 2000; Weitzman and Summerbell, 1995). Therefore, in order to make identification of dermatophytes more accurate, advanced genetic-based techniques have been introduced, such as conventional polymerase chain reaction (PCR), realtime PCR and post-PCR techniques (Jensen and Arendrup, 2012; Liu et al., 2000a; Miyajima et al., 2012). In the present study, a comprehensive restriction analysis (PCR–RFLP) involving three endonucleases, namely HinfI, HhaI and MvaI was performed (Fig. 2, Table 1).
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With three reference dermatophyte strains: T. interdigitale (CBS 120357), T. rubrum (CBS 120358) and M. canis (CBS 113480), and HinfI, HhaI and MvaI digestion, species-specific PCR–RFLP patterns were revealed (Fig. 2). Similar restriction profiles have been previously published (Dobrowolska et al., 2006; Mochizuki et al., 2003a,b). In 79% of cases examined, PCR–RFLP-based dermatophyte identification was identical with identification based on morphological markers (Table 1). However, some discrepancies between morphological versus molecular identification were observed (Table 1). Incorrect identification was recorded in 8 clinical isolates (17% of all cases examined) and involved Trichophyton genus (e.g. isolate 826 identified as T. rubrum based on morphological markers and finally as T. interdigitale based on PCR–RFLP analysis using HinfI, HhaI and MvaI digestion) (Table 1, Fig. 2). Since macroconidia of M. canis are characteristic and relatively easy to identify, improper M. canis species identification was not observed (Table 1). Our findings are consistent with previously published results on M. canis identification (Dobrowolska et al., 2006; Liu et al., 2001; Nardoni et al., 2007). Surprisingly, we were unable to identify two clinical isolates based on PCR–RFLP analysis (Table 1, Fig. 2). PCR–RFLP patterns of isolate 976 (identified as T. interdigitale based on morphological markers) and isolate 1348 (identified as T. rubrum based on morphological markers) were different from PCR–RFLP patterns of three reference strains used (Fig. 2). We have conducted a combined restriction analysis involving three different endonucleases, namely HinfI, HhaI and MvaI (Fig. 2). PCR–RFLP profile (HhaI digestion) of isolate 976 was similar to PCR– RFLP profile (HhaI digestion) of reference strain T. rubrum (Fig. 2), whilst other PCR–RFLP profiles (HinfI and MvaI digestions) of isolate 976 were totally different when compared to PCR–RFLP profiles (HinfI and MvaI digestions) of reference strains used (Fig. 2). Moreover, PCR–RFLP profiles of isolate 1348 were distinct from PCR–RFLP profiles of reference strains used (Fig. 2). Some minor similarities between PCR– RFLP patterns of isolate 1348 (HhaI digestion) and PCR–RFLP patterns of reference strain T. rubrum (HhaI digestion) have been observed (Fig. 2). Nevertheless, one restriction fragment (above 100 bp) of PCR–RFLP profile of isolate 1348 (HhaI digestion) was missing compared to two digestion products of similar size (above 100 bp) of PCR–RFLP profile of reference strain T. rubrum (HhaI digestion) (Fig. 2). Products of similar size may migrate together, which may result in a single band product on a gel (Dobrowolska et al., 2006). It is widely accepted that PCR-based systems, sensitive, reliable and rapid methods for dermatophyte identification, are particularly useful for large-scale epidemiological studies and for clinical trials of antifungal therapy (Brillowska-Dabrowska et al., 2010; Jensen and Arendrup, 2012; Kim et al., 2011; Miyajima et al., 2012). More recently, matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has also been adopted for dermatophyte identification (de Respinis et al., 2013; Nenoff et al., 2013). Nevertheless, some of the proposed advanced techniques are quite costly and require well-qualified staff and well-equipped laboratories, thus it would be a challenge to validate their applications in routine laboratory practice for dermatophyte identification. It is also worthwhile to remember that PCR-based procedures have some limitations/disadvantages, which may lead to false-positive or false-negative results. Some genotyping failures may be due to the use of Taq DNA polymerase, which is sensitive to phenol or ethanol (common contaminations after genomic DNA extraction). Contamination-mediated inhibition of Taq DNA polymerase may result in false-negative signals. Clinical samples may be also contaminated with other biological materials, analyzed samples may be heterogeneous or the quality of DNA template may be poor. Moreover, it is crucial to use the same reagents and lab equipment from defined suppliers, which is particularly important for random amplified polymorphic DNA (RAPD)-PCR. Successful PCR-based identification may be also limited due to the sensitivity of DNA restriction enzymes to a plethora of inhibitors, Taq DNA polymerase-associated errors during DNA amplification or the unavailability of sufficient amount of DNA material,
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Fig. 2. The dermatophyte molecular identification based on PCR–RFLP analysis. rDNA internal transcribed spacer (ITS) was amplified and PCR product (~700 bp) was digested using HinfI, MvaI and HhaI endonucleases followed by polyacrylamide gel electrophoresis. DNA was stained using ethidium bromide. Upper panel: Strain-specific PCR–RFLP patterns of three reference dermatophyte strains: Trichophyton interdigitale (CBS 120357) (lanes 1–3), Trichophyton rubrum (CBS 120358) (lanes 4–6) and Microsporum canis (CBS 113480) (lanes 7–9). A molecular marker (100–500 bp) is also shown (lane 10). Lower panel: PCR–RFLP patterns of three selected clinical isolates: 826 (lanes 1–3), 976 (lanes 4–6) and 1348 (lanes 7–9). Ambiguous PCR–RFLP patters are marked with a red frame. A molecular marker (100–500 bp) is also shown (lane 10).
which may be used as a template. Additionally, for primer design for PCR reaction (sequence-specific DNA amplification), the knowledge of DNA sequences is essential. Since we were unable to identify two isolates, namely isolate 976 and isolate 1348 based on PCR–RFLP analysis (Fig. 2), a complementary molecular tool for dermatophyte identification has been developed. Three dermatophyte species-specific genomic probes have been constructed (specific to T. interdigitale, T. rubrum or M. canis DNA). Briefly, DNA was extracted from reference strains used and was labeled using Biotin-High Prime DNA labeling Kit with biotin-16-dUTP using random oligonucleotides as primers according to standard procedure provided by manufacturer (Roche). The principles of a newly developed genomic
in situ hybridization (GISH) method using dermatophyte speciesspecific GISH probes are shown in a schematic, as shown in Fig. 3. To validate our system, genomic in situ hybridization (GISH) using dermatophyte species-specific and biotin-labeled DNA (GISH probes), and dermatophyte reference strain samples was performed (Figs. 3 and 4). Dermatophyte species-specific genomic probes (T. interdigitalespecific probe, T. rubrum-specific probe and M. canis-specific probe) were found to bind specifically to genomic DNA of T. interdigitale, T. rubrum and M. canis reference strains, respectively (Fig. 4). Some of age-related inherent autofluorescence was also revealed (Fig. 4). Green autofluorescence signals were localized within fungal cytosol
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Fig. 3. Schematic depicting the principles of genomic in situ hybridization (GISH) using dermatophyte species-specific DNA (GISH probes). After culture of dermatophyte reference strains (Trichophyton interdigitale, Trichophyton rubrum and Microsporum canis), dermatophyte DNA was isolated using phenol/chloroform extraction and labeled using Biotin-High Prime DNA labeling Kit with biotin-16-dUTP using random oligonucleotides as primers (Roche) according to standard procedure provided by manufacturer. For dermatophyte reference strain sample preparation, dermatophyte cultures were treated with formaldehyde, zymolyase 100 T and a mixture of glacial acetic acid:methanol (1:3). Fixed dermatophyte samples on microscopic slides were subjected to RNase, pepsin, MgCl2 and ethanol treatment. Then, dermatophyte samples and biotinylated genomic probes specific to T. interdigitale, T. rubrum or M. canis DNA (biotin-labeled dermatophyte-specific DNA) (Polish Patent Office, registration number P.404901) were co-denaturated using 7-min treatment at 80 °C. GISH was performed at 37 °C for 48 h. Two washing steps were included (at 70 °C and at room temperature). After FITC labeling (Star*FISH© Biotin Painting Kit — FITC Label, Cambio) and DAPI staining, FITC-positive nuclear signals (dermatophyte species-specific nuclear signals, white arrowhead) were analyzed using an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software. FITC (GISH-positive nuclear signals, green) and DAPI (DNA signals, blue) fluorescent signals were collected using FITC and DAPI filters (λex = 495 nm, λem = 519 nm and λex = 345 nm, λem = 455), respectively.
and did not interfere with nuclear dermatophyte species-specific FITC signals (Fig. 4). Moreover, nuclear dermatophyte species-specific FITC signals were verified with nuclear DAPI staining (DAPI/FITC colocalization) (Fig. 4). It has been previously published that fungi can emit a green autofluorescence (Baschien et al., 2001). The inherent autofluorescence emitted from the fungal strains was found to increase with the age of cultures, but was significantly decreased by chitinase treatment prior to in situ hybridization (Baschien et al., 2001). In our experimental system, cytosolic green autofluorescence did not limit the usage of green-labeled GISH probes to detect dermatophyte speciesspecific nuclear signals (Fig. 4). The signal specificity (FITC-positive nuclear signals, dermatophyte species-specific nuclear signals) was established to be in a range of 80 to 98% (Table 2).
Since to construct GISH probes, random primed labeling was applied and random oligonucleotides were used as primers, one cannot exclude that some of probe sequences may hybridize with the DNA of three dermatophyte reference strains applied, especially in the case of probe hybridization to repetitive sequences, such as rDNA. However, to prevent probe binding to repetitive sequences, tRNA was added to the reaction mixture. Additionally, post-hybridization step (washing step) is crucial for sequence-specific identification using GISH. During washing step, species-unspecific or partly specific hybridization is removed and finally species-specific signals are detected using random sequence-based GISH probes. In a case of no specific signal, one can speculate that fungal cell wall was inadequately digested (Table 2). Additionally, some weak unspecific signals were also observed (1 to 10% of all cases examined) and involved mainly Trichophyton genus samples and genomic probes,
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Fig. 4. The dermatophyte identification using the genomic in situ hybridization (GISH) using dermatophyte strain-specific GISH probes, namely Microsporum canis probe (left panel), Trichophyton rubrum probe (middle panel) and Trichophyton interdigitale probe (right panel). For the construction of biotin-labeled genomic probes fairly specific to M. canis, T. rubrum and T. interdigitale, respectively, see Materials and methods section. To validate GISH probes, three corresponding reference dermatophyte strains were used (from top to bottom: M. canis, T. interdigitale and T. rubrum). Merged fluorescence channels are shown: FITC channel (dermatophyte strain-specific nuclear signals, green) and DAPI channel (DNA signals, blue).
e.g. the use of T. rubrum-specific probe and subsequently incorrect detection of T. interdigitale (b 10% of all cases examined) (Table 2). GISH that resulted in incorrect identification of M. canis (weak unspecific nuclear signals) was estimated as a rare experimental event of less than 2% of all cases examined (Table 2). Taken together, constructed genomic probes were found fairly specific to three dermatophyte reference strains used (Table 2, Fig. 4).
Subsequently, we asked the question of whether isolate 976 and isolate 1348 may be correctly identified using GISH and dermatophyte species-specific probes (Figs. 5 and 6). As it has been previously stated, molecular identification of isolate 976 and isolate 1348 using PCR–RFLP analysis was unsuccessful (Fig. 2). Using T. interdigitale-specific probe, FITC-positive nuclear signals were revealed within nuclei of isolate 976 (Fig. 5). In contrast,
Table 2 The specificity of genomic probes used. Probe
FITC-positive nuclear signals in Trichophyton interdigitale
FITC-positive nuclear signals in Trichophyton rubrum
FITC-positive nuclear signals in Microsporum canis
Trichophyton interdigitale-specific probe Trichophyton rubrum-specific probe Microsporum canis-specific probe
96–98% b10% b2%
2–4% 82–94% b1%
b1% b1% 80–91%
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Fig. 5. Clinical isolate 976 identification using the genomic in situ hybridization (GISH) and Trichophyton interdigitale probe. A, E, I — T. interdigitale reference strain, B, F, J — Trichophyton rubrum reference strain, C, G, K — Microsporum canis reference strain, D, H, L — clinical isolate 976 identified as T. interdigitale. A–D: Merged fluorescence channels. E–H: FITC channel (T. interdigitale-specific nuclear signals, green). I–L — DAPI channel (DNA signals, blue). White arrowheads indicate T. interdigitale-specific nuclear signals.
after hybridization with T. rubrum-specific probe, FITC-positive nuclear signals were revealed within nuclei of isolate 1348 (Fig. 6). According to GISH results (Figs. 5 and 6), one can conclude that isolate 976 is indeed T. interdigitale and isolate 1348 is T. rubrum, which is also in agreement with morphological identification of isolates 976 and 1348 (Table 1). In situ hybridization (ISH) has been previously used to identify fungi (mainly Aspergillus genus) in histopathological specimens (Hanazawa et al., 2000; Hayden et al., 2001, 2002; Kobayashi et al., 1999; Montone and Litzky, 1995; Zimmerman et al., 2000). Moreover, ISH was also considered a valuable tool to identify medically important molds in formalin-fixed and paraffin-embedded tissue sections or cytological preparations (Shinozaki et al., 2009). PCR reaction and ISH were performed on formalin-fixed and paraffin-embedded tissue sections
identified as Aspergillus, Zygomycetes or Fusarium-positive samples and their specificities were compared (Shinozaki et al., 2009). Since inadequately amplified PCR products were revealed, ISH was considered a more accurate method to detect pathogenic fungi in formalin-fixed and paraffin-embedded tissue sections (Shinozaki et al., 2009). The authors speculated that ISH may have advantages over PCR reaction, e.g.: PCR results may be affected by formalin-mediated DNA fragmentation, contamination and improper DNA extraction due to rigid fungal cell wall (Shinozaki et al., 2009). In conclusion, we have shown for the first time that GISH and dermatophyte species-specific genomic probes may be successfully used for dermatophyte identification. Moreover, GISH was found particularly useful when PCR–RFLP yielded ambiguous results. GISH method may be considered a relatively cheap and easy to perform procedure, and more
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M. Worek et al. / Journal of Microbiological Methods 100 (2014) 32–41
Fig. 6. Clinical isolate 1348 identification using the genomic in situ hybridization (GISH) and Trichophyton rubrum (A, B) and Trichophyton interdigitale (C, D) probes. T. rubrum-specific nuclear signals are shown (A, B). In contrast, T. interdigitale-specific nuclear signals were not detected (C, D). Merged fluorescence channels (FITC channel and DAPI channel) (A, C) and FITC channel (B, D) are presented. FITC channel: T. rubrum-specific nuclear signals (green), DAPI channel: DNA signals (blue).
accurate than PCR-based methods under certain circumstances. However, PCR may seem time-efficient and more rapid than GISH. GISH cannot be performed directly from the clinical specimens and GISH results may be affected by inadequate fungal cell wall digestion. We proposed that GISH may be used as a supplement to dermatophyte identification based on PCR reaction, which may improve the diagnosis of dermatophytoses and in turn may help to design the appropriate antifungal therapy. Acknowledgments The authors would like to thank Teresa Abramowicz and Jolanta Siuta-Borla (Department of Diagnostic Medicine, Provincial Medical Specialist Unit, Rzeszow, Poland) for their help in collecting the clinical specimens of dermatophytes. References Abdel-Rahman, S.M., 2008. Strain differentiation of dermatophytes. Mycopathologia 166, 319–333. Achterman, R.R., White, T.C., 2012. A foot in the door for dermatophyte research. PLoS Pathog. 8, e1002564. Ameen, M., 2010. Epidemiology of superficial fungal infections. Clin. Dermatol. 28, 197–201. Baschien, C., Manz, W., Neu, T.R., Szewzyk, U., 2001. Fluorescence in situ hybridization of freshwater fungi. Int. Rev. Hydrobiol. 86, 371–384. Bosshard, P.P., 2011. Incubation of fungal cultures: how long is long enough? Mycoses 54, e539–e545. Brillowska-Dabrowska, A., Nielsen, S.S., Nielsen, H.V., Arendrup, M.C., 2010. Optimized 5hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory. Med. Mycol. 48, 828–831. Cafarchia, C., Iatta, R., Latrofa, M.S., Graser, Y., Otranto, D., 2013. Molecular epidemiology, phylogeny and evolution of dermatophytes. Infect. Genet. Evol. 20, 336–351. de Respinis, S., Tonolla, M., Pranghofer, S., Petrini, L., Petrini, O., Bosshard, P.P., 2013. Identification of dermatophytes by matrix-assisted laser desorption/ionization time-offlight mass spectrometry. Med. Mycol. 51, 514–521. Degreef, H., 2008. Clinical forms of dermatophytosis (ringworm infection). Mycopathologia 166, 257–265. Dobrowolska, A., Staczek, P., Kaszuba, A., Kozlowska, M., 2006. PCR–RFLP analysis of the dermatophytes isolated from patients in Central Poland. J. Dermatol. Sci. 42, 71–74. Dragos, V., Lunder, M., 1997. Lack of efficacy of 6-week treatment with oral terbinafine for tinea capitis due to Microsporum canis in children. Pediatr. Dermatol. 14, 46–48. Faggi, E., Pini, G., Campisi, E., 2002. PCR fingerprinting for identification of common species of dermatophytes. J. Clin. Microbiol. 40, 4804–4805. Fleece, D., Gaughan, J.P., Aronoff, S.C., 2004. Griseofulvin versus terbinafine in the treatment of tinea capitis: a meta-analysis of randomized, clinical trials. Pediatrics 114, 1312–1315.
Graser, Y., Kuijpers, A.F., Presber, W., de Hoog, G.S., 2000. Molecular taxonomy of the Trichophyton rubrum complex. J. Clin. Microbiol. 38, 3329–3336. Gupta, A.K., Tu, L.Q., 2006. Dermatophytes: diagnosis and treatment. J. Am. Acad. Dermatol. 54, 1050–1055. Haley, L.D., Stonerod, M., 1954. The isolation and identification of dermatophytes. Am. J. Med. Technol. 20, 27–34. Hanazawa, R., Murayama, S.Y., Yamaguchi, H., 2000. In situ detection of Aspergillus fumigatus. J. Med. Microbiol. 49, 285–290. Harmsen, D., Schwinn, A., Brocker, E.B., Frosch, M., 1999. Molecular differentiation of dermatophyte fungi. Mycoses 42, 67–70. Hayden, R.T., Qian, X., Roberts, G.D., Lloyd, R.V., 2001. In situ hybridization for the identification of yeastlike organisms in tissue section. Diagn. Mol. Pathol. 10, 15–23. Hayden, R.T., Qian, X., Procop, G.W., Roberts, G.D., Lloyd, R.V., 2002. In situ hybridization for the identification of filamentous fungi in tissue section. Diagn. Mol. Pathol. 11, 119–126. Jensen, R.H., Arendrup, M.C., 2012. Molecular diagnosis of dermatophyte infections. Curr. Opin. Infect. Dis. 25, 126–134. Kanbe, T., Suzuki, Y., Kamiya, A., Mochizuki, T., Fujihiro, M., Kikuchi, A., 2003a. PCR-based identification of common dermatophyte species using primer sets specific for the DNA topoisomerase II genes. J. Dermatol. Sci. 32, 151–161. Kanbe, T., Suzuki, Y., Kamiya, A., Mochizuki, T., Kawasaki, M., Fujihiro, M., Kikuchi, A., 2003b. Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR–RFLP targeting of the DNA topoisomerase II genes. J. Dermatol. Sci. 33, 41–54. Kano, R., Nakamura, Y., Watanabe, S., Takahashi, H., Tsujimoto, H., Hasegawa, A., 1998. Differentiation of Microsporum species by random amplification of polymorphic DNA (RAPD) and southern hybridization analyses. Mycoses 41, 229–233. Kano, R., Hirai, A., Muramatsu, M., Watari, T., Hasegawa, A., 2003. Direct detection of dermatophytes in skin samples based on sequences of the chitin synthase 1 (CHS1) gene. J. Vet. Med. Sci. 65, 267–270. Kim, J.Y., Choe, Y.B., Ahn, K.J., Lee, Y.W., 2011. Identification of dermatophytes using multiplex polymerase chain reaction. Ann. Dermatol. 23, 304–312. Kobayashi, M., Sonobe, H., Ikezoe, T., Hakoda, E., Ohtsuki, Y., Taguchi, H., 1999. In situ detection of Aspergillus 18S ribosomal RNA in invasive pulmonary aspergillosis. Intern. Med. 38, 563–569. Liu, D., Coloe, S., Baird, R., Pedersen, J., 2000a. Application of PCR to the identification of dermatophyte fungi. J. Med. Microbiol. 49, 493–497. Liu, D., Coloe, S., Baird, R., Pederson, J., 2000b. Rapid mini-preparation of fungal DNA for PCR. J. Clin. Microbiol. 38, 471. Liu, D., Pearce, L., Lilley, G., Coloe, S., Baird, R., Pedersen, J., 2001. A specific PCR assay for the dermatophyte fungus Microsporum canis. Med. Mycol. 39, 215–219. Miyajima, Y., Satoh, K., Uchida, T., Yamada, T., Abe, M., Watanabe, S., Makimura, M., Makimura, K., 2012. Rapid real-time diagnostic PCR for Trichophyton rubrum and Trichophyton mentagrophytes in patients with tinea unguium and tinea pedis using specific fluorescent probes. J. Dermatol. Sci. 69, 229–235. Mochizuki, T., Ishizaki, H., Barton, R.C., Moore, M.K., Jackson, C.J., Kelly, S.L., Evans, E.G., 2003a. Restriction fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful for differentiating strains of Trichophyton mentagrophytes. J. Clin. Microbiol. 41, 4583–4588. Mochizuki, T., Tanabe, H., Kawasaki, M., Ishizaki, H., Jackson, C.J., 2003b. Rapid identification of Trichophyton tonsurans by PCR–RFLP analysis of ribosomal DNA regions. J. Dermatol. Sci. 32, 25–32.
M. Worek et al. / Journal of Microbiological Methods 100 (2014) 32–41 Monod, M., 2008. Secreted proteases from dermatophytes. Mycopathologia 166, 285–294. Montone, K.T., Litzky, L.A., 1995. Rapid method for detection of Aspergillus 5S ribosomal RNA using a genus-specific oligonucleotide probe. Am. J. Clin. Pathol. 103, 48–51. Nardoni, S., Franceschi, A., Mancianti, F., 2007. Identification of Microsporum canis from dermatophytic pseudomycetoma in paraffin-embedded veterinary specimens using a common PCR protocol. Mycoses 50, 215–217. Nenoff, P., Erhard, M., Simon, J.C., Muylowa, G.K., Herrmann, J., Rataj, W., Graser, Y., 2013. MALDI-TOF mass spectrometry — a rapid method for the identification of dermatophyte species. Med. Mycol. 51, 17–24. Robert, R., Pihet, M., 2008. Conventional methods for the diagnosis of dermatophytosis. Mycopathologia 166, 295–306. Seebacher, C., Bouchara, J.P., Mignon, B., 2008. Updates on the epidemiology of dermatophyte infections. Mycopathologia 166, 335–352. Shinozaki, M., Okubo, Y., Nakayama, H., Mitsuda, A., Ide, T., Yamagata Murayama, S., Shibuya, K., 2009. Application of in situ hybridization to tissue sections for identification of molds causing invasive fungal infection. Nihon Ishinkin Gakkai Zasshi 50, 75–83.
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Vermout, S., Tabart, J., Baldo, A., Mathy, A., Losson, B., Mignon, B., 2008. Pathogenesis of dermatophytosis. Mycopathologia 166, 267–275. Weitzman, I., Padhye, A.A., 1996. Dermatophytes: gross and microscopic. Dermatol. Clin. 14, 9–22. Weitzman, I., Summerbell, R.C., 1995. The dermatophytes. Clin. Microbiol. Rev. 8, 240–259. Wnuk, M., Worek, M., Kwiatkowska, A., Jaworski, A., Miedziak, B., Kaplan J., 2013. Genomowa sonda diagnostyczna typu in situ znakująca DNA Trichophyton mentagrophytes, sposób jej otrzymania oraz wykrywanie Trichophyton mentagrophytes na szkiełku mikroskopowym [in Polish]. Polish Patent Office, registration number P.404901. Zimmerman, R.L., Montone, K.T., Fogt, F., Norris, A.H., 2000. Ultra fast identification of Aspergillus species in pulmonary cytology specimens by in situ hybridization. Int. J. Mol. Med. 5, 427–429.