Diagnosis of dermatophytoses: conventional methods and recent molecular biological methods]

Dermatophytoses such as tinea pedis and tinea unguium are very common diseases in the field of dermatology. The diagnosis of dermatophytoses is usually performed by direct microscopy and culture. The identification of species is based on morphological features of giant culture and slide culture. However, in some cases, it is difficult to identify the species clearly because the culture shows an atypical appearance or is false negative. Therefore, several molecular biological methods have been developed for precise identification of a species. The analysis of patterns of random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) of mitochondrial DNA is useful for identifying isolates which are not clearly identifiable by conventional biological techniques. The phylogenetic analysis of dermatophytes was made by using DNA direct sequencing of nuclear ribosomal internal transcribed spacer 1 (ITS1). Sequence analysis of chitin synthase 1 (CHS 1) is a rapid tool for species level identification. We attempted the identification and viability assessment of dermatophytes based on the quantitative measurement of dermatophyte actin (ACT) mRNA. An internal fragment of the ACT, 725 to 762 bp, was isolated by PCR from the genomic DNA of dermatophytes and sequenced. ACT intron based primers were dermatophyte species-specific and primer pairs crossing the intron were dermatophyte genus-specific. The results indicated that quantification of dermatophyte ACT mRNA correlated with the results of culture and KOH examination. It is important that the identification of dermatophyte be done by combining conventional methods with molecular biological methods. In some cases results of the two methods do not correspond, and is those the fungal species needs to be re-examined.     

Chitin synthase (CHS) gene analysis of dermatophytes]

About 620-bp genomic DNA fragments of CHS1 genes were amplified from 13 species of dermatophytes by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of CHS1 gene fragments of these dermatophyte species revealed that 3 genera of Epidermophyton, Microsporum and Trichophyton were genetically different from each other. The molecular analysis of CHS1 genes will provide useful information for the identification of dermatophytes. The species-specific primers were designed from the nucleotide sequences of CHS1 gene in 3 teleomorphs of T. mentagrophytes. Using these primers the PCR analysis identified the clinical isolates of T. mentagrophytes from rabbit as A. benhamiae. By PCR analysis with the dermatophyte specific primer pair, dermatophyte DNA could be diagnosed directly and rapidly in clinical skin samples. The full length of CHS1 and CHS2 genes of Arthroderma benhamiae (one of the teleomorphs of T. mentagrophytes) was sequenced by 5'-RACE and 3'-RACE methods using cDNA as a template. The full length cDNA sequences of CHS1 gene (3158bp) and CHS2 gene (3392bp) were proved to encode 890 and 419 amino acids, respectively. The amino acid sequences of A. benhamiae CHS1 and CHS2 in the conserved regions shared, respectively, about 70% and 80% sequence similarity with those of the other filamentous ascomycetes registered in the data base of the GeneBank. RT-PCR analysis suggested that chitin synthase inhibitors (nikkomycin Z and polyoxin D) might stimulate the expression of CHS1 mRNA in A. benhamiae, and not the expression of CHS2 mRNA.     

Clinical benefit of recovering dermatophytes from skin swabs sent for bacterial culture

We incubated Sabouraud dextrose agar plates to recover dermatophytes from skin swabs sent for bacterial culture. Dermatophytes were recovered from 66 (0.3%) of 22,613 cultures. Twenty-one patients received specific antifungal treatment when their dermatophyte was reported. Most clinicians thought recovering and reporting the dermatophyte contributed to patient management.     

Validation of PCR–reverse line blot, a method for rapid detection and identification of nine dermatophyte species in nail, skin and hair samples

A dermatophyte-specific PCR–reverse line blot (PCR-RLB) assay based on internal transcribed sequences was developed. This assay allows the rapid detection and identification of nine clinically relevant species within the three dermatophyte genera Trichophyton , Microsporum and Epidermophyton in nail, skin and hair samples within 1 day. Analysis of 819 clinical samples (596 nail, 203 skin and 20 hair) revealed a positive PCR-RLB result in 93.6% of 172 culture-positive and microscopy-positive samples. PCR-RLB was superior to culture and direct microscopy, in both detection and species identification. Comparison of identification results of 208 PCR-positive and culture-positive clinical samples showed five discrepancies (2.4%) between PCR-RLB identification and classical microscopic/biochemical identification of isolates. Comparison of PCR-RLB identification and classical identification of 98 other isolates (dermatophytes and non-dermatophytes) revealed 13 discrepancies (13.3%) and five incomplete identifications of Trichophyton spp. Sequence analysis of ITS1 regions of 23 samples with discrepant or incomplete identification results (four Centraalbureau voor Schimmelcultures dermatophyte strains, four clinical samples and 15 clinical isolates) confirmed identification results of PCR-RLB in 21 of the 23 analyzed samples. PCR-RLB proved to be extremely suitable for routine detection and identification of dermatophytes directly in nail, skin and hair samples because it is rapid, sensitive, specific and accurate.     

PCR identification of dermatophyte fungi Trichophyton rubrum, T. soudanense and T. gourvilii

Diagnosis of dermatophytosis employing conventional laboratory procedures has been complicated by the slow growth and varied morphological features shown by dermatophytes. After analysis of the nucleotide base sequences of a 1.2-kb fragment amplified from a dermatophyte fungus Trichophyton rubrum by arbitrarily primed PCR with random primer OPD18, a pair of primers (TRIF and TR1R) was designed and evaluated for specific identification of T. rubrum. The sensitivity of the primers TR1F and TR1R was high, as a specific PCR band of c. 600 bp was detected from as little as 7 pg of T. rubrum DNA. By examining 92 dermatophyte strains and clinical isolates, it was found that this pair of primers reacted in PCR with T. rubrum, T. soudanense and T. gourvilii through formation of the specific fragment of 600 bp, but not with any other of the dermatophyte species or varieties, fungi, yeasts or bacteria tested. As T rubrum is one of the most frequently isolated dermatophyte fungi, and T. soudanense and T. gourvilii are relatively uncommon in many parts of the world, these primers can be used for rapid, sensitive and specific identification and differentiation of T. rubrum from other fungi and micro-organisms.     

Successful identification of clinical dermatophyte and Neoscytalidium species by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Dermatophytes are keratinolytic fungi responsible for a wide variety of diseases of glabrous skin, nails, and hair. Their identification, currently based on morphological criteria, is hindered by intraspecies morphological variability and the atypical morphology of some clinical isolates. The aim of this study was to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a routine tool for identifying dermatophyte and Neoscytalidium species, both of which cause dermatomycoses. We first developed a spectral database of 12 different species of common and unusual dermatophytes and two molds responsible for dermatomycoses (Neoscytalidium dimidiatum and N. dimidiatum var. hyalinum). We then prospectively tested the performance of the database on 381 clinical dermatophyte and Neoscytalidium isolates. Correct identification of the species was obtained for 331/360 dermatophytes (91.9%) and 18/21 Neoscytalidium isolates (85.7%). The results of MALDI-TOF MS and standard identification disagreed for only 2 isolates. These results suggest that MALDI-TOF MS could be a useful tool for routine and fast identification of dermatophytes and Neoscytalidium spp. in clinical mycology laboratories.     

Application of PCR to the identification of dermatophyte fungi

Infection of the keratinised tissues (skin, hair and nails) in man and animals by keratinophilic fungi (dermatophytes) results in dermatophytosis (also known as tinea or ringworm). As conventional laboratory procedures for the identification of dermatophytes are either slow or lack specificity, improved diagnostic methods are required. The application of nucleic acid amplification technology has made rapid and precise identification of dermatophytes possible. Recent studies have shown that when one of the four random primers (OPAA11, OPD18, OPAA17 and OPU15) was used in arbitrarily primed PCR (AP-PCR), up to 20 of the 25 dermatophyte species or subspecies under investigation could be distinguished on the basis of characteristic band patterns detected in agarose gel electrophoresis. A combination of two random primers (OPD18 and OPAA17) used in separate reaction tubes identified 23 of the 25 dermatophyte species or subspecies examined. AP-PCR provides a rapid and practical tool for identification of dermatophyte isolates that is independent of morphological and biochemical characteristics and thus enhances laboratory diagnosis of dermatophytosis.     

A new medium for the presumptive identification of dermatophytes.

A new medium, Dermatophyte Identification Medium (DIM) (trade mark pending), was specifically developed to eliminate problems of false-positive results associated with commercially marketed media, such as dermatophyte test medium (DTM). Previous investigations had demonstrated that DTM only partially suppressed growth of nondermatophytes and that several of these nondermatophytic fungi that were morphologically similar to dermatophytes caused false-positive results. Presumptive identification of an unknown isolate as a dermatophyte required only the transfer of a portion of the suspected colony recovered from the specimen to DIM. Positive results, evidenced by a change in the color of the medium, were observed within 24 to 48 h. In studies of over 500 isolates of dermatophytes and common nondermatophyte molds, as well as close to 600 yeast isolates, false-positive results were always associated with bacterial contamination of the mold isolates while false negatives were only observed with occasional isolates of Trichophyton verrucosum. DIM culture was an inexpensive, rapid, and accurate method for the presumptive identification of dermatophytes in the clinical mycology laboratory.     

Evaluation of a single-tube real-time PCR for detection and identification of 11 dermatophyte species in clinical material

We developed a dermatophyte-specific single-tube real-time PCR assay based on internal transcribed sequences. This assay allows the rapid detection and identification of 11 clinically relevant species within the three dermatophyte genera Trichophyton, Microsporum and Epidermophyton in nail, skin and hair samples within a few hours. Analysis of 145 clinical samples (107 nail, 36 skin scale, and two hair) by both real-time PCR and a PCR--reverse line blot (PCR-RLB) assay described earlier revealed that 133 of the 145 samples had concordant real-time PCR and PCR-RLB detection results (83 -positive, 49 negative, and one inhibited). Six samples were -positive by real-time PCR and negative by PCR-RLB, and two were negative by real-time PCR and -positive by PCR-RLB. Four samples demonstrated inhibition in one of the two PCR assays. Only one of 83 -positive samples had discordant identification results between both assays (Trichophyton verrucosum and Trichophyton erinacei by real-time PCR and Trichophyton erinacei by PCR-RLB). Dermatophytes present in seven -positive samples that were incompletely identified as Trichophyton sp. by PCR-RLB were identified to the species level by real-time PCR as Trichophyton interdigitale and Trichophyton rubrum in six cases and one case, respectively. One hundred and twenty of 145 samples were also analysed by conventional dermatophyte culture and by direct microscopy. Our single-tube real-time PCR assay proved to be suitable for direct detection and identification of dermatophytes in nail, skin and hair samples with minimal total assay time (4 h after overnight lysis) and hands-on time, without the need for post-PCR analysis, and with good sensitivity and specificity.     

Advances in molecular biology of dermatophytes]

During the 44th meeting of The Japanese Society for Medical Mycology in Nagasaki, 2000, a forum was held entitled Advances in Molecular Biology of Dermatophytes. Based on the subject, target molecules and kind of approach, we selected seven presentations from over 100 of the poster abstracts. Six of them concerned identification and one concerned viability. Summaries of the 7 presentations are given in this article. Of presentations on the identification methods, 5 demonstrated their usefulness: 1) A sequence analysis of ITS 1 region in ribosomal DNA of several Microsporum species showed ITS 1 genospecies Arthroderma otae to be composed of A. otae, M. canis, M. equinum and M. audouinii. 2) RAPD may be useful for identifying isolates which are not clearly identifiable by conventional biological techniques. 3) Sequence analysis of CHS 1 was shown to be a rapid tool for species level identification of M. gypseum. 4) PCR-SSCP analysis was also useful for discrimination of dermatophytes with high reproducibility and sensitivity. 5) Strain identification of A. benhamiae isolates may be possible using RFLP analysis of NTS regions in ribosomal DNA. The other presentation concerning identification pointed out some important problems: RFLP of mitochondrial DNA and ITS sequencing of A. benhamiae showed that the results are sometimes in conflict with those obtained from biological techniques, or in some cases, between other molecular techniques. This implies that our concept of fungal species needs to be re-examined and perhaps amended. The presentation on viability introduced quantitative analysis of mRNA of ACT gene, a new application of a molecular technique. Since the mRNA expresses only in living cells, the method is highly useful as an indicator of fungal viability.     

Analysis of the dermatophyte species isolated in the British Isles between 1980 and 2005 and review of worldwide dermatophyte trends over the last three decades.

Infections of the skin, hair and nails by dermatophyte fungi are common in developed and developing countries alike. However, the species involved and the resulting clinical entities vary both geographically and with time. We have surveyed 15,333 dermatophytes obtained from primary isolations at the Mycology Reference Laboratory, Bristol, UK from 1980 through 2005. Several striking trends in dermatophyte prevalence were apparent over this period. The relative frequencies of isolations of Microsporum canis (cat and dog ringworm), Trichophyton verrucosum (cattle ringworm), T. mentagrophytes var. mentagrophytes (rodent ringworm) and Epidermophyton floccosum (a cause of human groin and foot infections) all decreased by 90%. Conversely, the contributions of T. tonsurans and T. violaceum (two anthropophilic scalp-infecting species) to total dermatophyte isolations increased by 1000% over the same period. Finally, T. rubrum and T. mentagrophytes var. interdigitale, the two common causes of foot infection comprised 80% of all dermatophytes isolated in 1980 and 90% of isolations in 2005. Similar trends in dermatophyte prevalence were evidenced throughout the British Isles, based on the voluntary reporting of isolations from a large number of British laboratories at 5-yearly intervals over the same period. The implications of these changing patterns of dermatophyte species, and the clinical entities they produce are discussed in the context of a review of worldwide dermatophyte isolations over the last three decades, with emphasis on the causal agents of tinea capitis.     

Five-hour diagnosis of dermatophyte nail infections with specific detection of Trichophyton rubrum

A rapid two-step DNA extraction method and a multiplex PCR for the detection of dermatophytes in general and Trichophyton rubrum specifically were developed and evaluated with DNA extracted from pure cultures and from clinically diseased nails. DNA from the following dermatophytes was used: Epidermophyton floccosum, Microsporum audouinii, Microsporum canis, Microsporum gypseum, Microsporum nanum, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton soudanense, Trichophyton terrestre, Trichophyton tonsurans, Trichophyton verrucosum, and Trichophyton violaceum. Human DNA and DNA from the following nondermatophyte fungi were included as controls: Alternaria, Aspergillus niger, Candida albicans, Candida glabrata, Candida krusei, Malassezia furfur, Saccharomyces cerevisiae, and Scopulariopsis brevicaulis. A total of 118 nail samples received for routine microscopy and culture for dermatophytes were subsequently tested by the two PCRs separately and in a multiplex format. Using DNA extracted from pure cultures and the pan-dermatophyte PCR, the T. rubrum-specific PCR sequentially and in a multiplex format correctly detected all dermatophytes and additionally correctly identified T. rubrum. Comparison of the traditional diagnostic evaluation (microscopy and culture) of nail samples with PCR on DNA directly extracted from the nails showed excellent agreement between PCR and microscopy, but the number of samples with dermatophyte species identification was increased considerably from 22.9% to 41.5%, mainly due to the identification of T. rubrum by PCR in microscopy-positive but culture-negative samples. In conclusion, this 5-hour diagnostic test was shown to increase not only the speed but also the sensitivity of investigation for nail dermatophytosis.     

Inhibition of dermatophytes by the antimicrobial peptides human β-defensin-2, ribonuclease 7 and psoriasin

Previous studies have described some antibacterial effects of antimicrobial peptides (AMPs) expressed in human skin, but little is known about their possible activity against dermatophytes. Therefore we have tested the effects of human β-defensin 2 (hBD-2), ribonuclease 7 (RNase 7) and psoriasin on the in vitro growth of four dermatophyte species. Germinating conidia of Trichophyton rubrum, T. mentagrophytes, Microsporum canis and Epidermophyton floccosum were exposed in vitro to hBD-2, RNase 7, psoriasin and fluconazole. Subsequent fungal growth was measured photometrically over 168 hours. All AMPs significantly inhibited fungal growth, with the degree of inhibition dependent on the dermatophyte species and the specific AMP. E. floccosum was found to be the most susceptible species in that it was markedly suppressed by all AMPs, whereas M. canis was inhibited only by psoriasin. Overall, psoriasin was the most effective AMP and had even stronger inhibitory effects on some dermatophytes than fluconazole. Our findings show that AMPs expressed in human skin can, in principal, inhibit the growth of dermatophytes in vitro. Therefore the question whether AMPs are relevant for human protection against tineas is justified and should be addressed by investigating their role in vivo.     

Evaluation of new medium for identification of dermatophytes and primary dimorphic pathogens

Only 25 of 77 dermatophytic isolates caused dermatophyte identification medium (DIM) to turn purple after incubation at the recommended temperature (37 degrees C); the accuracy of the results was improved at 30 degrees C (71 of 77 isolates yielded positive results). Many dimorphic pathogenic fungi also tested positive at both incubation temperatures. Thus, DIM has limited usefulness for presumptive identification of dermatophytes.     

Dermatophyte identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry

The performance of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (MS) for the identification of dermatophytes from clinical cultures was compared to that of dermatophyte identification using 28S rRNA gene sequencing. The MALDI Biotyper library (MBL; version 3.0) was used alone and in combination with a supplemented library containing an additional 20 dermatophyte spectra (S-MBL). Acquired spectra were interpreted using both the manufacturer-recommended scores (genus, ≥1.7; species, ≥2.0) and adjusted cutoff values established by this study (genus, ≥1.5; species, ≥1.7); identifications required a minimum 10% difference in scores between the top two different organisms to be considered correct. One hundred well-characterized, archived dermatophyte isolates and 71 fresh dermatophyte cultures were evaluated using both libraries and both sets of cutoff criteria. Collectively, the S-MBL significantly outperformed the MBL at both the genus (93% versus 37.4%; P < 0,0001) and species (59.6% versus 20.5%; P < 0.0001) levels when using the adjusted score criteria. Importantly, application of the lowered cutoff values significantly improved genus (P = 0.005)- and species (P < 0.0001)-level identification for the S-MBL, without leading to an increase in misidentifications. MALDI-TOF MS is a cost-effective and rapid alternative to traditional or molecular methods for dermatophyte identification, provided that the reference library is supplemented to sufficiently encompass clinically relevant, intraspecies strain diversity.     

Use of commercially available cryogenic vials for long-term preservation of dermatophyte fungi

The use of commercially available cryogenic vials (Microbank vials) stored at -70 degrees C for the storage and preservation of dermatophyte fungi was investigated. None of the 200 strains of dermatophytes examined, representing 21 species, showed a loss of viability after they had been stored for periods ranging from 1 week to 2 years at -70 degrees C. All strains showed typical colonial and microscopic morphologies following revival.     

A specific PCR assay for the dermatophyte fungus Microsporum canis.

A DNA fragment of approximately 1.2 kb, generated from the common dermatophyte Microsporum canis by arbitrarily primed polymerase chain reaction (PCR) using random primer OPU13, was cloned and sequenced. Based on the resulting sequencing data, a forward primer (MC1F) and a reverse primer (MC1R) have been designed and assessed by PCR for their usefulness in the improved identification of M. canis. The results obtained suggest that these primers are specific for M. canis, as a band of 900 bp was amplified in PCR with genomic DNA from M. canis only, and not from any of the other dermatophyte species or varieties, other fungi or common bacteria examined. Combining this PCR technique with a rapid mini-preparation method for fungal DNA, a definitive diagnosis of M. canis can be achieved within a day from the primary cultures. Future refinement of a DNA purification protocol from clinical specimens would further enhance the potential of the PCR based test for improved detection and identification of M. canis.