Journal of Dermatology 2015; 42: 232–235
doi: 10.1111/1346-8138.12813
REVIEW ARTICLE
Molecular markers useful for epidemiology of dermatophytoses Takashi MOCHIZUKI,1,2 Kiminobu TAKEDA,1 Kazushi ANZAWA1,2 1
Department of Dermatology, and 2Division of Dermatomycology, Research Institute of Medical Science, Kanazawa Medical University, Uchinada, Ishikawa, Japan
ABSTRACT Dermatophytosis is a very common skin disorder and the most prevalent infectious disease treated by dermatologists. Recent developments in molecular techniques have markedly changed methods of identifying dermatophytes, with these methods showing intraspecies polymorphisms in some molecular markers. Intraspecies subtyping and strain differentiation have made possible the tracking of infections, the identification of common sources of infections and recurrence or reinfection after treatment. This review describes methods of intraspecies differentiation using mitochondrial DNA, random amplification of polymorphic DNA, non-transcribed spacer regions of ribosomal RNA genes and microsatellite markers, as well as their usefulness and limitations.
Key words: dermatophyte, dermatophytosis, epidemiology, microsatellite marker, molecular technique, ribosomal RNA gene.
INTRODUCTION Dermatophytosis is a very common skin disorder and the most prevalent infectious disease treated by dermatologists. For example, a randomized epidemiological survey of outpatients who visited Japanese dermatologists in May 2006, revealed 3848 cases (49.4%) of fungal infections on the feet among 7783 patients.1 The dermatophyte species isolated from tinea lesions have been identified by their morphological characteristics, with/or without mating behavior. Although culture-based methods are still useful for species level identification, these methods are not well suited to reveal intraspecies polymorphisms. Methods were therefore needed to distinguish among strains belonging to particular species.2 Progress in molecular techniques has markedly altered methods of identifying dermatophytes. Methods including dermatophyte DNA–DNA hybridization,3 restriction fragment polymorphisms (RFLP) of mitochondrial DNA4,5 and random amplification of polymorphic DNA (RAPD) using arbitrary primers6,7 have been reported useful for species level identification of dermatophytes. In addition, molecular methods targeting particular genes including the internal transcribed spacer region (ITS) of ribosomal RNA genes (rDNA),8,9 chitin synthase I gene10 and DNA topoisomerase II gene11 have been found useful for identifying dermatophyte species, as well as for understanding their phylogenetic relationships, and the relationships between dermatophyte species and other related fungi. Some of these molecular markers were found to demonstrate some degree of intraspecies polymorphisms, however, the discrimination power of subtypes in each species was
limited. Comprehensive review articles have described such molecular approaches to identify dermatophytes in detail.2,12,13 Subtyping based on intraspecies polymorphisms may be helpful in evaluating the maintenance and disappearance of strains within a community, and further strain level differentiation may make the tracking of infections, common sources of infections and recurrence or reinfection after treatment.2 In addition, genotyping may contribute to understand their virulence and drug resistance. This review describes methods for intraspecies subtyping and strain differentiation, which may enable determination of the molecular epidemiology of dermatophytoses.
INTRASPECIES SUBTYPING AND STRAIN DIFFERENTIATION IN PRACTICE Mitochondrial DNA Mitochondrial DNA analysis using restriction enzymes has been shown to be a powerful tool for species level identification. Some of the earliest studies investigated methods of distinguishing dermatophyte species, for example, differences between Arthroderma (Nanizzia) otae and Microsporum canis4 and between two subtypes of Trichophyton rubrum.5 Although the method of choice for species level identification, this method is much less sensitive for subspecies level discrimination. A recent sequencing study revealed that cob-nad3 intergenic sequences of different dermatophyte species have diverged considerably, suggesting that these intergenic regions may serve as potential genetic markers for strain identification.14
Correspondence: Takashi Mochizuki, M.D., Ph.D., Department of Dermatology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan. Email: [email protected] Received 5 January 2015; accepted 7 January 2015.
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Molecular epidemiology
Random amplification of polymorphic DNA Random amplification of polymorphic DNA analysis is a method that uses one or two short primers of arbitrary sequence to generate polymerase chain reaction (PCR) products from template DNA. The amplified DNA fragments are separated by electrophoresis and their fingerprint-like profiles have been used as molecular markers.15 For example, amplification using the primer R28 (50 -ATGGATCCGC-30 ) showed a slight degree of intraspecies variation between isolates of Trichophyton mentagrophytes var. interdigitale,6 and amplification of animal isolates of T. mentagrophytes using the primer OPAO-15 (50 -GAAGGCTCCC-30 ) showed diverse band patterns, in contrast to the uniform band patterns of human isolates.7 In addition, amplification of 67 clinical isolates of T. rubrum using the primers 1 (50 -GGTGCGGGAA-30 ) and 6 (50 -CCCGTCAGCA-30 ) yielded 12 and 11 profiles, respectively.16 The method is suitable for obtaining results promptly and from several isolates at the same time. In addition, it can be used even in the absence of prior genetic information. However, RAPD profiles are highly dependent on PCR conditions, including primer and template concentrations, annealing temperatures, the concentration of magnesium ion in reaction solutions and the quality of template DNA; and on electrophoresis conditions.6 The poor reproducibility of the profiles obtained has reduced interest in this method.
Non-transcribed spacer regions of rDNA Non-transcribed spacer (NTS) regions of rDNA accumulate high degrees of sequence variations, and detection of these variations may be the most frequent method used to identify dermatophytes. First, strain typing of T. rubrum was performed by Southern blot hybridization based RFLP analysis,17 the method used in the most recent study which found the strain switching in patients with onychomycosis.18 Then, species-specific PCR primers targeting tandemly repetitive subelements in NTS (TRS-1, TRS-2) were synthesized to detect interspecies variations. Assays of TRS-1 and TRS-2 polymorphisms in 101 clinical isolates identified 23 separate PCR types,19 with these markers later used to identify species and subspecies of T. rubrum.17,20 Similar findings were observed when this method was used to assess T. mentagrophytes-related species. Because T. mentagrophytes is a complex of at least three teleomorphic species, Arthroderma vanbreuseghemii, Arthroderma benhamiae and Arthroderma simii, and several anamorphic taxa,8 species level identification is required prior to applying the NTS method. T. interdigitale (T. mentagrophytes var. interdigitale) is the most predominant dermatophyte species following T. rubrum. Southern blot hybridization-based RFLP analysis of 60 strains identified by RFLP profiles of ITS of rDNA identified 23 molecular types.21 PCR primers targeting three individual parts of the NTS were synthesized and used to analyze combinations of RFLP of three amplicons, identifying 19 molecular types among 42 strains.22 Application of this reproducible and discriminatory method to 65 clinical strains isolated at one regional hospital in Japan identified 15 molecular types.23
© 2015 Japanese Dermatological Association
A. benhamiae, a pathogen mainly carried by small pet animals, was first isolated in 1996 in Japan, with Southern blot hybridization identifying molecular polymorphisms in Japanese isolates.24 This method was able to identify a patient with a laboratory-based A. benhamiae infection.25 Sequence analysis was then used to generate NTS specific primers and identify an RFLP site, enabling molecular typing (Fig. 1).26 This NTS targeted PCR–RFLP analysis revealed 11 molecular types among 46 A. benhamiae strains, including four types among 22 Japanese strains, with three of the latter also detected outside Japan.26 Outbreaks of Trichophyton tonsurans have led to interest in the molecular epidemiology of the isolates, enhancing understanding of the transmission of fungus and its spread inside and outside the community. Molecular polymorphisms were first identified by RFLP of variable internal repeat (VIR) regions insertions and deletions, combined with single nucleotide polymorphisms (SNP) detected by restriction enzyme analysis targeting NTS of rDNA.27 Using this method, 94 isolates from the USA could be divided into 12 molecular types. Later, primers used for NTS analysis of T. rubrum19 were found to amplify the NTS region of T. tonsurans.28 This method, combined with digestion with restriction enzymes Hae III and Dde I, identified four molecular types among 19 isolates cultured in Brazil, Italy and China.28 RFLP analysis of the VIR region of T. tonsurans showed homogeneity of 101 strains isolated from Japanese judo athletes.29 RFLP analysis of a similar NTS region using Mva I and Ava I identified six molecular types among 232 strains isolated from judo athletes, wrestlers, sumo wrestlers and several sporadically infected individuals in Japan.30 Of the six types, NTS I was predominant, being present in 160 of 164 isolates from judo athletes, and suggesting a clonal lineage.
Figure 1. Structures of non-transcribed spacer (NTS) region of the ribosomal DNA of Arthroderma benhamiae. Detectable length variation of the NTS are derived from a variable number of repetitive units comprising relatively similar sequences from 205 to 233 bp in length at the 50 -end side of the NTS, whereas the sequences of the 30 -end are very similar. (Reproduced from Takeda et al.,26 with permission.)
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Isolates from wrestlers fell into two major molecular types, NTS I and NTS II. Later, two additional types were identified based on this method,31 but NTS typing of the isolates obtained from 2006 to 2010 showed a prevalence similar to that observed in isolates obtained before 2005.32 In addition, all strains isolated from sumo wrestlers were classified as NTS I, suggesting that the epidemic among sumo wrestlers originated from the epidemic among judo athletes.32 Evaluation of genetic variations in the international population using a mixed marker strategy found 27 variations using 13 gene loci, including variations in length of the VIR region and SNP in NTS; and sequence variations in enzyme coding regions such as those encoding alkaline proteinase-1 and subtilisin-like proteinases 2, 3 and 5.33 Even using the typing method, Japanese isolates showed no strain variations, suggesting that Japanese outbreaks arose from strains introduced from other global regions.33
Microsatellite DNA Microsatellite DNA sequences are short, tandem-repeating DNA sequences comprised of 1–6 bp per repeating unit. These sequences are polymorphic in populations due to the propensity for insertion/deletion mutations of multiples of the repeating units to be introduced during replication.34 Variations in the number of repeating units at a genetic locus can be determined by PCR amplification of alleles using unique primers flanking the repeating sequence, followed by resolution of the PCR products on denaturing gels.34 This multilocus microsatellite typing (MLMT) was able to detect variations among strains of T. rubrum35 and was later applied to additional isolates to better understand the pathogenesis of T. rubrum.36 Using 22 microsatellite markers, 55 genotypes were recognized among 233 isolates, suggesting that populations of T. rubrum are geographically separated, in conjugation with their predilection on human hosts.36 However, no diagnostic correlation was observed between the genotypes and any of the phenotypical characteristics of the isolates.36 The MLMT method was also used to assess variations in Microsporum persicolor, a geophilic keratinophilic fungus,37 and Microsporum canis, a zoophilic dermatophyte that is not only the most frequent fungal species isolated from dogs but a major pathogen in patients with tinea capitis.38 The imbalance in the prevalence of MLMT genotypes among human and animal isolates of M. canis suggested that population differentiation was due to the emergence of a virulent genotype with a high potential to infect human hosts.38 MLMT was observed on silverstained polyacrylamide gels after electrophoresis of PCR products. However, when primers labeled with fluorescent dyes were used and the PCR products loaded onto a genetic analyzer, the results were observed as colored peaks, of sizes calculated by alignment with initial size standards.34 This more advanced method was used for several additional MLMT studies on M. canis. Using eight of 38 microsatellite markers, 22 genotypes were found among 26 M. canis strains isolated from 13 countries.34 Using two microsatellite markers, 102 strains isolated in Brazil, including 19 from human patients, yielded a total of 14 genotypes, which could be sorted into six large populations.39 Taken together, these findings
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indicated that MLMT is a reliable method for the differentiation of M. canis strains.34,39
CONCLUSIONS AND FUTURE PROSPECTS The ideal marker for molecular epidemiology must be sufficiently sensitive and easy to use on a number of strains at once, as well as yielding reproducible results. Species level identification of isolates from patients with dermatophytoses are now easily performed using reliable molecular methods such as sequence analysis of ITS, because the dermatophytes have been shown to constitute a homogenous species with low genetic diversity. However, this makes the detection of intraspecies polymorphisms more difficult. PCR primers that amplify VIR of NTS or microsatellite sequences are needed for each species, and the usefulness of methods and primers must be checked empirically by each investigator. Rapidly developing whole genome comparative analysis13,40 in the same species may be a robust method of identifying highly variable regions. If different sequences are found among strains in the same species, they are considered unimportant, because they do not encode characteristics associated with species pathogenicity or morphology. However, they may be useful as markers suitable to detect intraspecies polymorphisms.
ACKNOWLEDGMENTS: This study was supported in part by Grants-in-Aid for Emerging and Reemerging Infectious Diseases (H25-shinko-ippan 006) from the Ministry of Health, Labor and Welfare of Japan. CONFLICT OF INTEREST: The authors declare that there are no conflicts of interest including industrial links and affiliations. REFERENCES 1 Watanabe S, Harada T, Hiruma M et al. Epidemiological survey of foot diseases in Japan: results of 30 000 foot checks by dermatologists. J Dermatol 2010; 37: 397–406. 2 Abdel-Rahman SM. Strain differentiation of dermatophytes. Mycopathologia 2008; 166: 319–333. 3 Davison FD, Macenzie DWR. DNA homology studies in the taxonomy of dermatophtytes. Sabouraudia 1984; 22: 117–123. 4 Kawasaki M, Aoki M, Ishizaki H, Watanabe S. Phylogeny of Nannizia incurvata, N. gypsea, N. fulva and N. otae by restriction enzyme analysis of mitochondrial DNA analysis. Mycopathologia 1990; 112: 173–177. 5 Nishio K, Kawasaki M, Ishizaki H. Phylogeny of the genera Trichophyton using mitochondrial DNA analysis. Mycopathologia 1992; 117: 127–132. 6 Mochizuki T, Sugie N, Uehara M. Random amplification of polymorphic DNA is useful for the differentiation of several anthropophilic dermatophytes. Mycoses 1997; 40: 405–409. 7 Kim JA, Takahashi Y, Tanaka R, Fukushima K, Nishimura K, Miyaji M. Identification and subtyping of Trichophyton mentagrophytes by random amplification of polymorphic DNA. Mycoses 2001; 44: 157– 165. 8 Makimura K, Mochizuki T, Hasegawa A et al. Phylogenetic classification of Trichophyton mentagrophytes complex strains based on
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DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol 1998; 36: 2629–2633. Makimura K, Tamura Y, Mochizuki T et al. Phylogenetic classification and species identification of dermatophyte strains based on DNA sequence of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol 1999; 37: 920–924. Kano R, Nakamura Y, Watari T et al. Species-specific primers of chitin synthase 1 gene for the differentiation of the Trichophyton mentagrophytes complex. Mycoses 1999; 42: 71–74. Kanbe T, Suzuki Y, Kamiya A et al. Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes. J Dermatol Sci 2003; 33: 41–54. Kanbe T. Molecular approaches in the diagnosis of dermatophytosis. Mycopathologia 2008; 166: 307–317. €ser Y, Otranto D. Molecular Cafarchia C, Iatta R, Latrofa MS, Gra epidemiology, phylogeny and evolution of dermatophytes. Infect Genet Evol 2013; 20: 336–351. Wu Y, Yang J, Yang F et al. Recent dermatophyte divergence revealed by comparative and phylogenetic analysis of mitochondrial genomes. BMC Genom 2009; 10: 238. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res 1990; 18: 6531–6535. Baeza LC, Matsumoto MT, Almeida AMF, Mendes-Giannini MJS. Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer. J Med Microbiol 2006; 55: 429–436. Jackson CJ, Barton RC, Evans EGV. Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal DNA spacer regions. J Clin Microbiol 1999; 37: 931–936. Gupta AK, Nakrieko KA. Trichophyton rubrum DNA strain switching increases in patients with onychomycosis failing antifungal treatments. Br J Dermatol 2014; 172: 74–80 Jackson CJ, Barton RC, Kelly SL, Evans EGV. Strain identification of Trichophyton rubrum by specific amplification of subrepeat elements in the ribosomal DNA nontranscribed spacer. J Clin Microbiol 2000; 38: 4527–4534. Yazdanparast A, Jackson CJ, Barton RC, Evans EVG. Molecular typing of Trichophyton rubrum indicates multiple strain involvement in onychomycosis. Br J Dermatol 2003; 148: 51–54. Mochizuki T, Ishizaki H, Barton RC et al. Restriction fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful for differentiating strains of Trichophyton mentagrophytes. J Clin Microbiol 2003; 41: 4583–4588. Jackson CJ, Mochizuki T, Barton RC. PCR fingerprinting of Trichophyton mentagrophytes var. interdigitale using polymorphic subrepeat loci in the rDNA nontranscribed spacer. J Med Microbiol 2006; 55: 1349–1355. Wakasa A, Anzawa K, Kawasaki M, Mochizuki T. Molecular typing of Trichophyton mentagrophytes var. interdigitale isolated in a university hospital in Japan based on the non-transcribed spacer region of the ribosomal RNA gene. J Dermatol 2010; 37: 431– 440. Mochizuki T, Kawasaki M, Ishizaki H et al. Molecular epidemiology of Arthroderma benhamiae, an emerging pathogen of dermatophytoses
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in Japan, by polymorphisms of the non-transcribed spacer region of the ribosomal DNA. J Dermatol Sci 2001; 27: 14–20. Mochizuki T, Watanabe S, Kawasaki M, Tanabe H, Ishizaki H. A Japanese case of tinea corporis caused by Arthroderma benhamiae. J Dermatol 2002; 29: 221–225. Takeda K, Nishibu A, Anzawa K, Mochizuki T. Molecular epidemiology of a major subgroup of Arthroderma benhamiae isolated in Japan by restriction fragment length polymorphism analysis of the non-transcribed spacer region of ribosomal RNA gene. Jpn J Infect Dis 2012; 65: 233–239. Gaedigk A, Gaedigk R, Adbel-Rahman SM. Genetic heterogeneity in the rRNA gene locus of Trichophyton tonsurans. J Clin Microbiol 2003; 41: 5478–5487. Abliz P, Takizawa K, Nishimura K et al. Molecular typing of Trichophyton tonsurans by PCR-RFLP of the ribosomal DNA nontranscribed spacer region. J Dermatol Sci 2004; 36: 125–127. Sugita T, Shiraki Y, Hiruma M. Genotype analysis of the variable internal repeat region in the rRNA gene of Trichophyton tonsurans isolated from Japanese judo practitioners. Microbiol Immunol 2006; 50: 57–60. Mochizuki T, Kawasaki M, Tanabe H et al. Molecular epidemiology of Trichophyton tonsurans isolated in Japan using RFLP analysis of non-transcribed spacer regions of ribosomal RNA genes. Jpn J Infect Dis 2007; 60: 188–192. Mochizuki T, Kawasaki M, Fujita J et al. Epidemiology of sporadic (non-epidemic) cases of Trichophyton tonsurans infection in Japan based on RFLP analysis of non-transcribed spacer region of ribosomal RNA Gene. Jpn J Infect Dis 2008; 61: 219–222. Anzawa K, Mochizuki T, Nishibu A et al. Molecular epidemiology of Trichophyton tonsurans isolated in Japan between 2006 and 2010 and their susceptibility to oral antimycotics. Jpn J Infect Dis 2011; 64: 458–462. Adbel-Rahman SM, Sugita T, Gonzalez GM et al. Divergence among an international population of Trichophyton tonsurans isolates. Mycopathologia 2010;169:1–13. Pasquetti M, Peano A, Soglia D et al. Development and varidation of microsatellite marker-based method for tracing infections by Microsporum canis. J Dermatol Sci 2013; 70: 123–129. €ser Y. Origins of Ohst T, de Hoog GS, Presber W, Stavrakieva V, Gra microsatellite diversity in the Trichophyton rubrum-T. violaceum clade (Dermatophytes). J Clin Microbiol 2004; 42: 4444–4448. €ser Y, Fro € hlich J, Presber W, de Hoog GS. Microsatellite markGra ers reveal geographic population differentiation in Trichophyton rubrum. J Med Microbiol 2007; 56: 1058–1065. €ser Y. Molecular detection of Sharma R, Presber W, Rajak RC, Gra Microsporum persicolor in soil suggesting widespread dispersal in central India. Med Mycol 2008; 46: 67–73. €ser Y. A virulent genotype Sharma R, de Hoog GS, Presber W, Gra of Microsporum canis is responsible for the majority of human infections. J Med Microbiol 2007; 56: 1377–1385. da Costa FV, Farias MR, Bier D et al. Genetic variability in Microsporum canis isolated from cats, dogs and humans in Brazil. Mycoses 2013; 56: 582–588. €ser Y et al. Comparative genome analyMartinez DA, Oliver BG, Gra sis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection. MBio 2012; 3: e00259–12.
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doi: 10.1111/1346-8138.12813
REVIEW ARTICLE
Molecular markers useful for epidemiology of dermatophytoses Takashi MOCHIZUKI,1,2 Kiminobu TAKEDA,1 Kazushi ANZAWA1,2 1
Department of Dermatology, and 2Division of Dermatomycology, Research Institute of Medical Science, Kanazawa Medical University, Uchinada, Ishikawa, Japan
ABSTRACT Dermatophytosis is a very common skin disorder and the most prevalent infectious disease treated by dermatologists. Recent developments in molecular techniques have markedly changed methods of identifying dermatophytes, with these methods showing intraspecies polymorphisms in some molecular markers. Intraspecies subtyping and strain differentiation have made possible the tracking of infections, the identification of common sources of infections and recurrence or reinfection after treatment. This review describes methods of intraspecies differentiation using mitochondrial DNA, random amplification of polymorphic DNA, non-transcribed spacer regions of ribosomal RNA genes and microsatellite markers, as well as their usefulness and limitations.
Key words: dermatophyte, dermatophytosis, epidemiology, microsatellite marker, molecular technique, ribosomal RNA gene.
INTRODUCTION Dermatophytosis is a very common skin disorder and the most prevalent infectious disease treated by dermatologists. For example, a randomized epidemiological survey of outpatients who visited Japanese dermatologists in May 2006, revealed 3848 cases (49.4%) of fungal infections on the feet among 7783 patients.1 The dermatophyte species isolated from tinea lesions have been identified by their morphological characteristics, with/or without mating behavior. Although culture-based methods are still useful for species level identification, these methods are not well suited to reveal intraspecies polymorphisms. Methods were therefore needed to distinguish among strains belonging to particular species.2 Progress in molecular techniques has markedly altered methods of identifying dermatophytes. Methods including dermatophyte DNA–DNA hybridization,3 restriction fragment polymorphisms (RFLP) of mitochondrial DNA4,5 and random amplification of polymorphic DNA (RAPD) using arbitrary primers6,7 have been reported useful for species level identification of dermatophytes. In addition, molecular methods targeting particular genes including the internal transcribed spacer region (ITS) of ribosomal RNA genes (rDNA),8,9 chitin synthase I gene10 and DNA topoisomerase II gene11 have been found useful for identifying dermatophyte species, as well as for understanding their phylogenetic relationships, and the relationships between dermatophyte species and other related fungi. Some of these molecular markers were found to demonstrate some degree of intraspecies polymorphisms, however, the discrimination power of subtypes in each species was
limited. Comprehensive review articles have described such molecular approaches to identify dermatophytes in detail.2,12,13 Subtyping based on intraspecies polymorphisms may be helpful in evaluating the maintenance and disappearance of strains within a community, and further strain level differentiation may make the tracking of infections, common sources of infections and recurrence or reinfection after treatment.2 In addition, genotyping may contribute to understand their virulence and drug resistance. This review describes methods for intraspecies subtyping and strain differentiation, which may enable determination of the molecular epidemiology of dermatophytoses.
INTRASPECIES SUBTYPING AND STRAIN DIFFERENTIATION IN PRACTICE Mitochondrial DNA Mitochondrial DNA analysis using restriction enzymes has been shown to be a powerful tool for species level identification. Some of the earliest studies investigated methods of distinguishing dermatophyte species, for example, differences between Arthroderma (Nanizzia) otae and Microsporum canis4 and between two subtypes of Trichophyton rubrum.5 Although the method of choice for species level identification, this method is much less sensitive for subspecies level discrimination. A recent sequencing study revealed that cob-nad3 intergenic sequences of different dermatophyte species have diverged considerably, suggesting that these intergenic regions may serve as potential genetic markers for strain identification.14
Correspondence: Takashi Mochizuki, M.D., Ph.D., Department of Dermatology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan. Email: [email protected] Received 5 January 2015; accepted 7 January 2015.
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Molecular epidemiology
Random amplification of polymorphic DNA Random amplification of polymorphic DNA analysis is a method that uses one or two short primers of arbitrary sequence to generate polymerase chain reaction (PCR) products from template DNA. The amplified DNA fragments are separated by electrophoresis and their fingerprint-like profiles have been used as molecular markers.15 For example, amplification using the primer R28 (50 -ATGGATCCGC-30 ) showed a slight degree of intraspecies variation between isolates of Trichophyton mentagrophytes var. interdigitale,6 and amplification of animal isolates of T. mentagrophytes using the primer OPAO-15 (50 -GAAGGCTCCC-30 ) showed diverse band patterns, in contrast to the uniform band patterns of human isolates.7 In addition, amplification of 67 clinical isolates of T. rubrum using the primers 1 (50 -GGTGCGGGAA-30 ) and 6 (50 -CCCGTCAGCA-30 ) yielded 12 and 11 profiles, respectively.16 The method is suitable for obtaining results promptly and from several isolates at the same time. In addition, it can be used even in the absence of prior genetic information. However, RAPD profiles are highly dependent on PCR conditions, including primer and template concentrations, annealing temperatures, the concentration of magnesium ion in reaction solutions and the quality of template DNA; and on electrophoresis conditions.6 The poor reproducibility of the profiles obtained has reduced interest in this method.
Non-transcribed spacer regions of rDNA Non-transcribed spacer (NTS) regions of rDNA accumulate high degrees of sequence variations, and detection of these variations may be the most frequent method used to identify dermatophytes. First, strain typing of T. rubrum was performed by Southern blot hybridization based RFLP analysis,17 the method used in the most recent study which found the strain switching in patients with onychomycosis.18 Then, species-specific PCR primers targeting tandemly repetitive subelements in NTS (TRS-1, TRS-2) were synthesized to detect interspecies variations. Assays of TRS-1 and TRS-2 polymorphisms in 101 clinical isolates identified 23 separate PCR types,19 with these markers later used to identify species and subspecies of T. rubrum.17,20 Similar findings were observed when this method was used to assess T. mentagrophytes-related species. Because T. mentagrophytes is a complex of at least three teleomorphic species, Arthroderma vanbreuseghemii, Arthroderma benhamiae and Arthroderma simii, and several anamorphic taxa,8 species level identification is required prior to applying the NTS method. T. interdigitale (T. mentagrophytes var. interdigitale) is the most predominant dermatophyte species following T. rubrum. Southern blot hybridization-based RFLP analysis of 60 strains identified by RFLP profiles of ITS of rDNA identified 23 molecular types.21 PCR primers targeting three individual parts of the NTS were synthesized and used to analyze combinations of RFLP of three amplicons, identifying 19 molecular types among 42 strains.22 Application of this reproducible and discriminatory method to 65 clinical strains isolated at one regional hospital in Japan identified 15 molecular types.23
© 2015 Japanese Dermatological Association
A. benhamiae, a pathogen mainly carried by small pet animals, was first isolated in 1996 in Japan, with Southern blot hybridization identifying molecular polymorphisms in Japanese isolates.24 This method was able to identify a patient with a laboratory-based A. benhamiae infection.25 Sequence analysis was then used to generate NTS specific primers and identify an RFLP site, enabling molecular typing (Fig. 1).26 This NTS targeted PCR–RFLP analysis revealed 11 molecular types among 46 A. benhamiae strains, including four types among 22 Japanese strains, with three of the latter also detected outside Japan.26 Outbreaks of Trichophyton tonsurans have led to interest in the molecular epidemiology of the isolates, enhancing understanding of the transmission of fungus and its spread inside and outside the community. Molecular polymorphisms were first identified by RFLP of variable internal repeat (VIR) regions insertions and deletions, combined with single nucleotide polymorphisms (SNP) detected by restriction enzyme analysis targeting NTS of rDNA.27 Using this method, 94 isolates from the USA could be divided into 12 molecular types. Later, primers used for NTS analysis of T. rubrum19 were found to amplify the NTS region of T. tonsurans.28 This method, combined with digestion with restriction enzymes Hae III and Dde I, identified four molecular types among 19 isolates cultured in Brazil, Italy and China.28 RFLP analysis of the VIR region of T. tonsurans showed homogeneity of 101 strains isolated from Japanese judo athletes.29 RFLP analysis of a similar NTS region using Mva I and Ava I identified six molecular types among 232 strains isolated from judo athletes, wrestlers, sumo wrestlers and several sporadically infected individuals in Japan.30 Of the six types, NTS I was predominant, being present in 160 of 164 isolates from judo athletes, and suggesting a clonal lineage.
Figure 1. Structures of non-transcribed spacer (NTS) region of the ribosomal DNA of Arthroderma benhamiae. Detectable length variation of the NTS are derived from a variable number of repetitive units comprising relatively similar sequences from 205 to 233 bp in length at the 50 -end side of the NTS, whereas the sequences of the 30 -end are very similar. (Reproduced from Takeda et al.,26 with permission.)
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Isolates from wrestlers fell into two major molecular types, NTS I and NTS II. Later, two additional types were identified based on this method,31 but NTS typing of the isolates obtained from 2006 to 2010 showed a prevalence similar to that observed in isolates obtained before 2005.32 In addition, all strains isolated from sumo wrestlers were classified as NTS I, suggesting that the epidemic among sumo wrestlers originated from the epidemic among judo athletes.32 Evaluation of genetic variations in the international population using a mixed marker strategy found 27 variations using 13 gene loci, including variations in length of the VIR region and SNP in NTS; and sequence variations in enzyme coding regions such as those encoding alkaline proteinase-1 and subtilisin-like proteinases 2, 3 and 5.33 Even using the typing method, Japanese isolates showed no strain variations, suggesting that Japanese outbreaks arose from strains introduced from other global regions.33
Microsatellite DNA Microsatellite DNA sequences are short, tandem-repeating DNA sequences comprised of 1–6 bp per repeating unit. These sequences are polymorphic in populations due to the propensity for insertion/deletion mutations of multiples of the repeating units to be introduced during replication.34 Variations in the number of repeating units at a genetic locus can be determined by PCR amplification of alleles using unique primers flanking the repeating sequence, followed by resolution of the PCR products on denaturing gels.34 This multilocus microsatellite typing (MLMT) was able to detect variations among strains of T. rubrum35 and was later applied to additional isolates to better understand the pathogenesis of T. rubrum.36 Using 22 microsatellite markers, 55 genotypes were recognized among 233 isolates, suggesting that populations of T. rubrum are geographically separated, in conjugation with their predilection on human hosts.36 However, no diagnostic correlation was observed between the genotypes and any of the phenotypical characteristics of the isolates.36 The MLMT method was also used to assess variations in Microsporum persicolor, a geophilic keratinophilic fungus,37 and Microsporum canis, a zoophilic dermatophyte that is not only the most frequent fungal species isolated from dogs but a major pathogen in patients with tinea capitis.38 The imbalance in the prevalence of MLMT genotypes among human and animal isolates of M. canis suggested that population differentiation was due to the emergence of a virulent genotype with a high potential to infect human hosts.38 MLMT was observed on silverstained polyacrylamide gels after electrophoresis of PCR products. However, when primers labeled with fluorescent dyes were used and the PCR products loaded onto a genetic analyzer, the results were observed as colored peaks, of sizes calculated by alignment with initial size standards.34 This more advanced method was used for several additional MLMT studies on M. canis. Using eight of 38 microsatellite markers, 22 genotypes were found among 26 M. canis strains isolated from 13 countries.34 Using two microsatellite markers, 102 strains isolated in Brazil, including 19 from human patients, yielded a total of 14 genotypes, which could be sorted into six large populations.39 Taken together, these findings
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indicated that MLMT is a reliable method for the differentiation of M. canis strains.34,39
CONCLUSIONS AND FUTURE PROSPECTS The ideal marker for molecular epidemiology must be sufficiently sensitive and easy to use on a number of strains at once, as well as yielding reproducible results. Species level identification of isolates from patients with dermatophytoses are now easily performed using reliable molecular methods such as sequence analysis of ITS, because the dermatophytes have been shown to constitute a homogenous species with low genetic diversity. However, this makes the detection of intraspecies polymorphisms more difficult. PCR primers that amplify VIR of NTS or microsatellite sequences are needed for each species, and the usefulness of methods and primers must be checked empirically by each investigator. Rapidly developing whole genome comparative analysis13,40 in the same species may be a robust method of identifying highly variable regions. If different sequences are found among strains in the same species, they are considered unimportant, because they do not encode characteristics associated with species pathogenicity or morphology. However, they may be useful as markers suitable to detect intraspecies polymorphisms.
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