dermatophytes comprise a highly specialized group of pathogenic fungi that infect keratinized tissues ( skin , hair , and nails ) of humans and animals , resulting in dermatophytoses , also referred to as tinea infections .
taxonomically , dermatophytes are classified into three genera : epidermophyton , microsporum , and trichophyton , with the latter being the most complex , containing more than 15 species and several different variants within the species t. mentagrophytes . the most frequently observed dermatophyte species worldwide is t. rubrum , whose infections usually manifest as tinea pedis and tinea unguium ( onychomycosis ) .
the prevalence of t. rubrum as a causative agent of onychomycosis is particularly high and exceeds 90% in europe .
the identification of t. rubrum by means of conventional laboratory methods may not always be easy or straightforward , since t. rubrum exhibits substantial phenotypic variability .
contrastingly , a high degree of homogeneity of the t. rubrum genome , as revealed by using several anonymous molecular markers , significantly impedes discrimination at the strain level [ 3 , 4 ] . yet , as new molecular typing methods are becoming increasingly available , species determination and strain typing are still being improved .
the aim of this study was to apply some of the recently devised dna fingerprinting methods to the identification and strain differentiation of t. rubrum .
the study included 57 isolates of t. rubrum recovered from as many dermatological patients from lower silesia , poland ( 40 ) , krakw , poland ( 8) , and tbingen , germany ( 9 ) .
clinically , onychomycosis showed the highest number of cases ( 40 patients ; 70.2% of all patients ) , followed by tinea pedis ( 12 ; 21% ) , tinea corporis ( 2 ; 3.5% ) , tinea cruris ( 2 ; 3.5% ) , and tinea manuum ( 1 ; 1.8% ) .
the isolates were primarily identified as t. rubrum on the basis of their phenotypic characteristics , such as colony surface texture , reverse pigmentation , the ability of micro- and macroconidia formation , and urease activity , assessed by standard mycological procedures .
the identification of t. rubrum was achieved by restriction fragment length polymorphism ( rflp ) analysis of the internal transcribed spacer ( its ) region of rdna , as previously described .
the amplified products were digested with mvni , hinfi , and , where necessary , with mvai restriction endonucleases ( roche ) , at a 2-h incubation at 37c .
the resulting restriction fragments were separated electrophoretically on 8% polyacrylamide gels and visualized under uv light after ethidium bromide ( et - br ) staining .
two primers , designated 1 and 6 , as devised by baeza and mendes - giannini , were used in two separate randomly amplified polymorphic dna ( rapd ) assays .
amplification products were resolved by electrophoresis using 1.5% agarose gels and were photographed under uv light after et - br staining .
the gel images were analyzed by the gel doc system and quantity one ( bio - rad , usa ) software .
a dendrogram of the rapd profiles obtained with primer 1 was constructed using dice s coefficient of similarity and the unweighted pair - group arithmetic averaging ( upgma ) clustering method .
of the 57 clinical isolates assessed in this study and recognized as t. rubrum by conventional identification methods , 55 ( 96.5% ) were further confirmed as t. rubrum by means of pcr - rflp analysis .
using mvni , all of the isolates except for one ( 841/05 ) yielded restriction patterns consistent with that of trichophyton taxon . using hinfi , 55 ( 96.5% )
one isolate ( 841/05 ) could not be assigned to any of the dermatophyte species , distinguishable by their unique rflp profiles obtained with mvni , hinfi , or mvai enzymes . among the 55
t. rubrum isolates , a total of 40 distinct patterns ( a  an ) were obtained by rapd with primer 1 .
the four most prevalent patterns , designated as t , aj , ae , and an , contained five , four , three , and three isolates , respectively .
four patterns ( c , af , ag , and al ) contained two isolates each , and the remaining 34 patterns were represented by single isolates .
the rapd with primer 6 generated five different profiles in t. rubrum isolates ( a  e ) and two additional profiles in non - t .
rubrum isolates ( designated as  f  and  g  for isolate nos .
b  , found in 23 isolates , followed by profiles designated as  a  and  c  , represented by 19 and 11 isolates , respectively .
patterns designated as  d  and  e  were each identified for one isolate .
the combination of the profiles obtained with both primers resulted in 47 different genotypes for the t. rubrum isolates .
six of those genotypes were common to three ( type t - a and type aj - a ) or two ( types c - a , ae - b , af - b , and an - a ) isolates , whereas the remaining 41 genotypes were unique , which are represented by single isolates only .
the distribution of the genotypes varied among the three geographical regions from which the isolates originated . of the 40 rapd genotypes produced with primer 1 , each
twenty - six genotypes occurred in isolates from lower silesia , eight genotypes were observed in isolates from krakw , and six in isolates from tbingen , germany . regarding the genotypes obtained by rapd with primer 6 , genotypes 
b  and  c  were found in isolates from all three regions studied , and genotype  a  was observed in the lower silesia and tbingen isolates , but not in the krakw isolates .
c  were the most prevalent in single regions , i.e. , in lower silesia ( 44.7% of isolates ) , krakw ( 62.5% ) , and tbingen ( 44.4% ) , respectively .
a dendrogram based on the rapd profiles with primer 1 allowed the separation of the t. rubrum isolates into genetic similarity groups ( clusters ) .
a total of 48 isolates could be allocated into six main clusters ( i  vi ) , with the similarity index between the isolates within the cluster being 80% or higher ( fig .  1 ) .
the largest cluster ( vi ) comprised 21 isolates , for which 11 different patterns were obtained .
clusters iv and v consisted of eight isolates each , being representative of either four ( cluster iv ) or eight ( cluster v ) distinct patterns .
the numbers of isolates ( and corresponding patterns ) belonging to the remaining clusters were 3 ( 2 patterns ) for cluster i , 5 ( 5 ) for cluster ii , and 3 ( 3 ) for cluster iii . 

1dendrogram showing genetic relationships among 55 trichophyton rubrum strains inferred from the randomly amplified polymorphic dna ( rapd ) patterns generated by using primer 1 dendrogram showing genetic relationships among 55 trichophyton rubrum strains inferred from the randomly amplified polymorphic dna ( rapd ) patterns generated by using primer 1
the traditional , culture - based methods of identifying dermatophytes are cumbersome , laborious , and often inconclusive , due to fungal phenotypic variability and pleomorphism [ 1 , 8 ] .
however , the advent of molecular biology tools has enabled the development of new molecular approaches to the diagnosis of dermatophyte infections .
, relies upon rflp analysis of pcr - amplified its regions of the rdna gene complex .
the only modification to the original procedure was the use of mvni and hinfi instead of mvai . whereas digestion with mvni allows discrimination between the three main dermatophyte genera ( trichophyton , microsporum , and epidermophyton ) , the hinfi digestion results in species - specific restriction profiles . by using those two restriction enzymes , we wanted to verify their usefulness in the molecular identification of dermatophyte species , and t. rubrum in particular .
the results from this study showed high concordance between conventional and molecular techniques for identifying t. rubrum .
rubrum isolates : one was identified as t. tonsurans ( upon restriction analysis with mvai ) and the other was concluded to be a non - dermatophyte fungus . a possible explanation for the latter may relate to the laboratory contamination of the specimen and/or culture .
it is also noteworthy that the application of the pcr - rflp analysis , as in this study , does not allow to distinguish between t. rubrum and t. soudanense .
the distinction between the two species is only possible with methods that target single nucleotide polymorphisms ( snps ) within the its regions of rdna .
given , however , that t. rubrum differs substantially from t. soudanense in terms of phenotypic properties and geographical distribution ( the former is cosmopolitan , while the latter is restricted mainly to the sub - saharan part of africa ) , the assignment to the t. rubrum species provided by pcr - rflp seems unambiguous .
differentiation of t. rubrum at the strain level has been attempted using a number of genotyping methods , though with unsatisfactory results [ 3 , 13 , 14 ] .
have recently reported on intraspecific variability within t. rubrum by pcr - amplifying two tandemly repetitive subelements ( trss ) , located in the non - transcribed spacer ( nts ) region of the rrna gene cluster .
more recently , the rapd analysis performed by baeza et al . with two decameric primers , designated 1 and 6 , has been shown to produce a high degree of interstrain polymorphism [ 7 , 16 ] .
the rapd analysis with primers 1 and 6 was also applied in the present study , and this choice was motivated by a high discriminatory potential of the method , higher than that of the trs typing system .
based on the combined rapd profiles , a total of 47 distinct genotypes were obtained .
hence , the overall genetic diversity rate ( gdr ) , calculated as the number of different genotypes divided by the number of isolates , was 85.4% .
it is noteworthy that most of the polymorphism was generated by rapd with primer 1 .
it yielded 40 profiles , whereas rapd with primer 6 resulted in only five profiles ( gdrs of 72.7% and 9.1% , respectively ) . in the first study that used primers 1 and 6 , among ten clinical isolates of t. rubrum ,
five molecular patterns were observed for each primer . in a subsequent study including 67 t. rubrum isolates , a total of 12 and 11 individual patterns were obtained by rapd with primers 1 and 6 , respectively ( gdrs of 17.9% and 16.4% , respectively ) .
another study that investigated the intraspecific diversity of t. rubrum isolates originating from japan and china revealed a considerable tightness of the population structure .
all 150 isolates tested were split into only 19 fingerprinting genotypes , based on the combined rapd analyses with primers 1 and 6 ( gdr of 12.7% ) .
much higher genotypic variability with the same primers was shown in a recent study of santos et al . .
nineteen different molecular profiles were configured for 52 t. rubrum isolates when each of the primers was used independently , resulting in a gdr of 36.5% .
it was speculated by the authors that the greater genetic diversity revealed in their study might result from having used strains exclusively from patients with onychomycosis .
this condition , with its frequent chronicity , has been associated with mixed infections by multiple t. rubrum genotypes .
collectively , the results of the studies cited above differ in terms of genetic polymorphism achieved by rapd with both of the primers .
the polymorphism obtained in our study was exceptionally high , and this may relate to the specific , genetic structure of polish ( and german ) isolates , which is different from that of the so - far - analyzed t. rubrum populations .
the polymorphism obtained by rapd with primer 1 was further investigated by a dendrogram analysis derived from the similarity coefficients between the rapd patterns .
although the similarity coefficient value for the entire t. rubrum population studied was calculated to be 52% , a vast majority of isolates ( 87.3% ) were distributed into six clusters , within which all of the isolates shared at least 80% similarity .
this finding , considering that the similarity coefficient value of 0.80.99 is assumed to represent genetically related isolates , indicates that the clustered isolates might have originated from the same strain that had undergone microevolutionary changes .
thus , the high polymorphism resolved by rapd may relate to the reproducibility of the rapd technique itself .
indeed , rapd assays often suffer from poor reproducibility and variations in the amplification patterns between isolates may be caused by even the slightest changes in the pcr reaction conditions .
this explains the reluctance on the part of researchers to use the rapd method and their search for newer molecular tools with a better diagnostic performance .
one such promising alternative method is multilocus microsatellite typing ( mlmt ) , which has recently been developed by grser et al . .
finally , the fact that every genetic cluster identified in rapd with primer 1 was restricted to a single geographical locale , together with marked differences in the frequencies of the three most prevalent genotypes produced from rapd with primer 6 , may suggest that certain genotypes exhibit regional and/or geographical affinities .
however , since this study was carried out on a relatively small sample of isolates , the geographical specificity of the genotypes would need to be confirmed by further research involving a larger population . interestingly , the geographical differentiation of t. rubrum populations has , so far , been revealed only by using the mlmt methodology [ 4 , 21 ] . in conclusion , the results from this study demonstrated the usefulness of pcr - rflp in the rapid identification of t. rubrum , yet the insufficient suitability of the rapd analysis for t. rubrum strain differentiation , due to its poor reproducibility .
 trichophyton rubrum represents the most frequently isolated causative agent of superficial dermatophyte infections . 
 several genotyping methods have recently been introduced to improve the delineation between pathogenic fungi at both the species and the strain levels . 
 the purpose of this study was to apply selected dna fingerprinting methods to the identification and strain discrimination of t. rubrum clinical isolates . 
 fifty - seven isolates from as many tinea patients were subjected to species identification by polymerase chain reaction restriction fragment length polymorphism ( pcr - rflp ) analysis and strain differentiation using a randomly amplified polymorphic dna ( rapd ) method , with two primers designated 1 and 6 . using pcr - rflp , 55 of the isolates studied were confirmed to be t. rubrum . among those , 
 a total of 40 and five distinct profiles were obtained by rapd with primers 1 and 6 , respectively . 
 the combination of profiles from both rapd assays resulted in 47 genotypes and an overall genotypic diversity rate of 85.4% . a dendrogram analysis performed on the profiles generated by rapd with primer 1 showed most of the isolates ( 87.3% ) to be genetically related . 
 pcr - rflp serves as a rapid and reliable method for the identification of t. rubrum species , while the rapd analysis is rather a disadvantageous tool for t. rubrum strain typing . 
Introduction
Materials and methods
Results
Discussion