PCR Identification of Trichophyton Mentagrophytes Var. Interdigitale And T. Mentagrophytes var. Mentagrophytes Dermatophytes with a Random Primer

Liu, D.; Coloe, S.; Baird, Robert W.; Pedersen, J.

doi:10.1099/00222615-46-12-1043

Cited by 30 publications

(27 citation statements)

References 8 publications

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“…For example, based on ribosomal small subunit rRNA gene sequences, a primer system specific for the detection of dermatophytic DNA has been described, though this system is not designed to distinguish individual dermatophyte species (5). Restriction fragment length polymorphism analysis (35), PCR fingerprinting (12), arbitrarily primed PCR (29), random amplified polymorphic DNA analysis (30,34), or sequencing of the ITS region (13,14,23) has been stated to be useful for the identification of species in dermatophytes and other fungi. Some of these methods, however, lack reproducibility; in particular, few of the random amplified polymorphic DNA techniques proposed for fungi in the 1990s are in use today.…”

Section: Discussionmentioning

confidence: 99%

Forty-Eight-Hour Diagnosis of Onychomycosis with Subtyping ofTrichophyton rubrumStrains

et al. 2006

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A novel strategy for the molecular identification of fungal agents of onychomycosis (including Trichophyton rubrum) has been designed based on the use of species-specific and universal primers in conjunction with a commercial kit that allows the extraction of DNA directly from the nail specimens. The microsatellite marker T1, which is based on a (GT)n repeat, was applied for the species-specific identification of Trichophyton rubrum. To evaluate how often Scopulariopsis spp. are detected in nail specimens, a second primer pair was designed to amplify specifically a 336-bp DNA fragment of the 28S region of the nuclear rRNA gene of S. brevicaulis and closely related species. Other fungal species were identified using amplification of the internal transcribed spacer (ITS) region of the rRNA gene, followed by restriction fragment length polymorphism analysis or sequencing. In addition, polyacrylamide gel separation of the T1-PCR product allowed subtyping of T. rubrum strains. We studied 195 nail specimens (the "nail sample") and 66 previously collected etiologic strains (the "strain sample") from 261 onychomycosis patients from Bulgaria and Greece. Of the etiologic agents obtained from both samples, T. rubrum was the most common organism, confirmed to be present in 76% of all cases and serving as the sole or (rarely) mixed etiologic agent in 199 of 218 cases (91%) where the identity of the causal organism(s) was confirmed. Other agents seen included molds (6% of cases with identified etiologic agents; mainly S. brevicaulis) and other dermatophyte species (4%; most frequently Trichophyton interdigitale). Simultaneous infections with two fungal species were confirmed in a small percentage of cases (below 1%). The proportion of morphologically identified cultures revealed by molecular study to have been misidentified was 6%. Subtyping revealed that all but five T. rubrum isolates were of the common type B that is prevalent in Europe. In comparison to microscopy and culture, the molecular approach was superior. The PCR was more sensitive (84%) than culture (22%) in the nail sample and was more frequently correct in specifically identifying etiologic agents (100%) than microscopy plus routine culture in either the nail or the strain samples (correct culture identifications in 96% and 94% of cases, respectively). Using the molecular approach, the time for diagnosing the identity of fungi causing onychomycosis could be reduced to 48 h, whereas culture techniques generally require 2 to 4 weeks. The early detection and identification of the infecting species in nails will facilitate prompt and appropriate treatment and may be an aid for the development of new antifungal agents.

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Section: Discussionmentioning

confidence: 99%

Forty-Eight-Hour Diagnosis of Onychomycosis with Subtyping ofTrichophyton rubrumStrains

et al. 2006

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“…The species included in this anthropo-zoophilic complex, which were first defined on the basis of morphological features and mating-type studies, have more recently been investigated using molecular methods, revealing an organization more complex than expected. These molecular approaches have been based primarily on the G+C content of chromosomal DNA (Davison et al, 1980), total DNA homology (Davison & Mackenzie, 1984), restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) (Mochizuki et al, 1990(Mochizuki et al, , 1996, arbitrarily primed PCR (AP-PCR) (Liu et al, 2000), random amplification of polymorphic DNA (RAPD) analysis (Kac et al, 1999;Liu et al, 1997;Mochizuki et al, 1997) and PCR fingerprinting (Faggi et al, 2001;Graser et al, 1999b). More recently, sequence analysis of the rDNA regions (Graser et al, 1999a;Harmsen et al, 1995;Makimura et al, 1998Makimura et al, , 1999Mochizuki et al, 1999Mochizuki et al, , 2003 and, in particular, analysis of internal transcribed spacer (ITS) regions (Graser et al, 1999a;Makimura et al, 1998Makimura et al, , 1999Mochizuki et al, 1999), which appeared more suitable than the gene coding for the small-subunit rRNA (18S rRNA) (Harmsen et al, 1995), were used successfully to evaluate the phylogenetic relationships within the T. mentagrophytes complex.…”

Section: Introductionmentioning

confidence: 99%

Phylogenetic analysis of Trichophyton mentagrophytes human and animal isolates based on MnSOD and ITS sequence comparison

et al. 2007

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Dermatophytes are keratinophilic fungi able to infect keratinized tissues of human or animal origin. Among them, Trichophyton mentagrophytes is known to be a species complex composed of several species or variants, which occur in both human and animals. Since the T. mentagrophytes complex includes both anthropophilic and zoophilic pathogens, accurate molecular identification is a critical issue for comprehensive understanding of the clinical and epidemiological implications of the genetic heterogeneity of this complex. Here, 41 T. mentagrophytes isolates from either human patients (14 isolates) or animals (27 isolates) with dermatophytosis were prospectively isolated by culture and identified on morphological bases at the University Hospital Centres of Lille and Poitiers, and the Veterinary School of Alfort, respectively. The isolates were differentiated by DNA sequencing of the variable internal transcribed spacer (ITS) regions flanking the 5.8S rDNA, and of the housekeeping gene encoding the manganese-containing superoxide dismutase (MnSOD), an enzyme which is involved in defence against oxidative stress and has previously provided interesting insight into both fungal taxonomy and phylogeny. ITS1-ITS2 regions and MnSOD sequences successfully differentiate between members of the T. mentagrophytes complex and the related species Trichophyton rubrum. Whatever the phylogenetic marker used, members of this complex were classified into two major clades exhibiting a similar topology, with a higher variability when the ITS marker was used. Relationships between ITS/MnSOD sequences and host origin, clinical pattern and phenotypic characteristics (macroscopic and microscopic morphologies) were analysed.

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“…Molecular methods, such as restriction fragment length polymorphism analysis of mitochondrial DNA (1,7,8), sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA (3,4), sequencing of protein-encoding genes (5,6), and PCR (random amplification of polymorphic DNA [RAPD] [15], arbitrarily primed PCR [AP-PCR] [10,11], and PCR fingerprinting [2]), have brought important progress in distinguishing between species and strains. However, most of these techniques (e.g., restriction fragment length polymorphism analysis, sequencing) are complex, laborious, time-consuming, and not easily employable for routine identification of dermatophytes; in contrast, PCR technology is simple, rapid, and, in the absence of specific nucleotide sequence information for the many dermatophyte species, able to generate species-specific or strain-specific DNA polymorphisms on the basis of characteristic band patterns detected by agarose gel electrophoresis (2,11).…”

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confidence: 99%

Application of PCR to Distinguish Common Species of Dermatophytes

et al. 2001

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This report describes the application of PCR fingerprinting for the identification of species and varieties of common dermatophytes and related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA) 4 . The primer was able to amplify all the strains, producing species-specific profiles for Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton ajelloi, and Epidermophyton floccosum. Intraspecific variability was not observed for these species. Instead, three different profiles were observed in the Trichophyton mentagrophytes group.Routine procedures for dermatophyte species identification rely on examination of the colony (pigmentation of the surface and reverse sides, topography, texture, and rate of growth) and microscopic morphology (size and shape of macroconidia and microconidia, spirals, nodular organs, and pectinate branches). Further identification characteristics include nutritional requirements (vitamins and amino acids) and temperature tolerance, as well as urease production, alkaline production of bromocresol purple medium, in vitro hair perforation, etc. (9, 16). Morphological and physiological characteristics can frequently vary; in fact, the phenotypic features can be easily influenced by outside factors such as temperature variation, medium, and chemotherapy (11) and therefore strain identification is often difficult.In the last few years genotypic approaches have proven to be useful for solving taxonomic problems regarding dermatophytes; in fact, genotypic differences are considered more stable and more precise than phenotypic characteristics (2, 11).Molecular methods, such as restriction fragment length polymorphism analysis of mitochondrial DNA (1,7,8) , have brought important progress in distinguishing between species and strains. However, most of these techniques (e.g., restriction fragment length polymorphism analysis, sequencing) are complex, laborious, time-consuming, and not easily employable for routine identification of dermatophytes; in contrast, PCR technology is simple, rapid, and, in the absence of specific nucleotide sequence information for the many dermatophyte species, able to generate species-specific or strain-specific DNA polymorphisms on the basis of characteristic band patterns detected by agarose gel electrophoresis (2, 11).This report describes the application of PCR fingerprinting for the identification of species and varieties of common dermatophytes and related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA) 4 previously used by Meyer and others to distinguish strains of Cryptococcus neoformans (12, 13) and species of the genus Candida (14 Furthermore, the study was conducted on various strains both from collections and from clinical isolation with the aim of finding the presence of an intraspecific variability.Strains. Out of a total of 140 strains selected for the study, 29 were obtained from the collection of the Institut Pasteur of Paris, France. One hundred eleven clinical isolates were re...

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PCR Identification of Trichophyton Mentagrophytes Var. Interdigitale And T. Mentagrophytes var. Mentagrophytes Dermatophytes with a Random Primer

Cited by 30 publications

References 8 publications

Forty-Eight-Hour Diagnosis of Onychomycosis with Subtyping ofTrichophyton rubrumStrains

Forty-Eight-Hour Diagnosis of Onychomycosis with Subtyping ofTrichophyton rubrumStrains

Phylogenetic analysis of Trichophyton mentagrophytes human and animal isolates based on MnSOD and ITS sequence comparison

Application of PCR to Distinguish Common Species of Dermatophytes

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