Diagnosis of Dermatophytosis

Dermatophytoses are infections of the skin, hair or nails caused by dermatophytes. Dermatophytes can induce typical diagnostic clinical lesions (tinea), but can also mimic other dermatoses. Therefore, physicians need to be familiar with the whole spectrum of tinea and must constantly be mindful of possible dermatophytosis. An examination with Wood’s light can be helpful. In superficial lesions, the demonstration of dermatophytes may be achieved by dermatoscopy or laser scanning. An essential step of the following diagnostic procedure is the skilful collection of samples for the proof of fungi. Microscopy of KOH mounts or equivalent preparations produced from infected material and histology are approved methods. The necessary identification of dermatophytes on species level can finally be accomplished by conventional methods based on cultures or by new techniques based on molecular differentiation or MALDI-TOF analysis. These modern methods are clearly on the increase and can considerably improve and accelerate dermatophyte identification.     

Molecular Markers Useful for Intraspecies Subtyping and Strain Differentiation of Dermatophytes

Dermatophytosis is a very common skin disorder and the most frequent infection encountered by practicing dermatologists. The identification, pathogenicity, biology, and epidemiology of dermatophytes, the causative agents of dermatophytosis, are of interest for both dermatologists and medical mycologists. Recent advances in molecular methods have provided new techniques for identifying dermatophytes, including intraspecies variations. Intraspecies subtyping and strain differentiation have made possible the tracking of infections, the identification of common sources of infections, recurrence or reinfection after treatment, and analysis of strain virulence and drug resistance. This review describes molecular methods of intraspecies subtyping and strain differentiation, including analyses of mitochondrial DNA and non-transcribed spacer regions of ribosomal RNA genes, random amplification of polymorphic DNA, and microsatellite markers, along with their advantages and limitations.     

Contribution to the study of dermatomycosis in Catalonia

We report the results of a study which aim was the mycological identification of specimens coming from patients included in a clinical trial. A total of 445 specimens from patients with clinical diagnosis of dermatomicosis were processed during 8 months (138 pityriasis versicolor, 28 cutaneous candidosis and 279 dermatophytosis). A 48% of pityriasis versicolor cultures were positive for Malassezia furfur, 50% of candidosis cultures were positive for yeasts and 67% of dermatophytosis cultures were positive for dermatophytes. According to our results Candida albicans was the principal causative agent for cutaneous candidosis and Trichophyton mentagrophytes and Trichophyton rubrum were the most frequent isolated species causing dermatophytosis.     

Update on the diagnosis of dermatomycosis

Dermatomycosis are mycotic diseases of skin caused by a few mycetes: dermatophytes, and some opportunistic fungi as Malassezia, Candida (not C. albicans), Trichosporon, Rhodutorula, Cryptococcus or Aspergillus, Geotrichum, Alternaria, etc. Dermatophytes are a group of closely related filamentous fungi that invade keratinized tissue (skin, hair, nails) of humans and other animals and produce infection called dermatophytosis or ringworm or "tinea". The etiological agents of dermatophytosis are classified in three genera: Microsporum, Trichophyton and Epidermophyton (Deuteromycetes). On the basis of their primary habitat dermatophytes are divided in Anthropophilic dermatophytes (parasitic organisms that infect humans), Zoophilic dermatophytes (parasitic organisms that infect animals, but also humans: agents of zoonosis) and Geophilic dermatophytes (saprobic fungi associated with keratinous materials in soil). In the soil there are also structure associated with contagion, ("spore", "arthroconidium", or "clamydospore") of anthropophilic and zoophilic dermatophytes that may persist for years, in the environment, in hair or skin scales. Since on the skin of animals there are many saprobic organisms (Malassezia) and many fungi may infect the fur, it is important to make an accurate diagnosis. Dermatophytosis are communicable diseases acquired from infected animals or from fomites. Infections caused by dermatophytes is a ringworm. These infections may range from mild and superficial, almost subclinical, to a few areas of scaling to a highly inflammatory reaction with extensive areas of scarring and alopecia. Granuloma formations (mycetoma-like) may occur especially in cats. Dermatophytes, as filamentous fungi, undergo radial fungi: collection of skin material is best made by collecting the scales near the edges of the rings. Hairs are best sampled by plucking; a scalpel may be used to scrape scales; brushes have also been used. Sample materials are best transported in dry packet. The Wood's light may be used to identify infected fluorescent hairs. Direct microscopy, although false negative up to 50% of cases, is a highly efficient screening technique. Scraping and hairs should mixed to 10-15% KOH. Culture is a valuable adjunct to direct microscopy and is essential to identify more dermatophytes. A medium selective against most nondermatophytic moulds and bacteria is used as a primary isolation medium. Many typical isolates of common dermatophytes can be identified directly from primary isolation media. Identification characters include: colony pigmentation, texture, morphological structure (macroconidia, microconidia, spirals, pectinate branches, etc). Nutritional requiment, growth in special media, "in vitro" perforation, mating studies are procedures used to identify atypical isolates. Serological approaches have revealed difficulties. Many kinds of molecular biologic techniques have been made available for clinical diagnosis recently; almost all of these techniques involve the polymerase chain reaction (PCR).     

Animal dermatophytosis. Recent advances

The proportion of positive samples in relation to the number of samples examined from cases of dog and cat dermatophytosis varies considerably from one investigation to another. In dogs, it ranges between 4% and 10% and few studies show higher prevalences. On the other hand, the percentages of positive cultures cited in the reviewed literature from dogs with or without suspected dermatophytosis are quite similar. In dogs with suspected lesions of dermatophytosis, with few exceptions, Microsporum canis is the most common species isolated. Trichophyton mentagrophytes and Microsporum gypseum are less frequently isolated. In cats the prevalence of dermatophytes is usually higher than in dogs, and it is usually higher than 20%. However the frequency of positive findings is higher in cats with suspected dermatophytosis than in cats without visible lesions, with the exception of asymptomatic infected cats and transient carrier cats. Cats are accepted as the principal reservoir for M. canis. Griseofulvin is the drug of choice in canine and feline dermatophytosis.     

Reappraisal of Conventional Diagnosis for Dermatophytes

Dermatophytoses include a wide variety of diseases involving glabrous skin, nails and hair. These superficial infections are a common cause of consultation in dermatology. In many cases, their diagnosis is not clinically obvious, and mycological analysis therefore is required. Direct microscopic examination of the samples using clearing agents provides a quick response to the clinician and is usually combined with cultures on specific media, which must be used to overcome the growth of contaminating moulds that may hamper the recovery of dermatophytes. Accurate identification of the causative agent (i.e. at the species level), currently based on morphological criteria, is necessary not only to initiate an appropriate treatment but also for setting prophylactic measures. However, conventional methods often lack sensitivity and species identification may require up to 4 weeks if subcultures are needed. Histological analysis, which is considered the “gold standard” for the diagnosis of onychomycoses, is seldom performed, and as direct examination, it does not allow precise identification of the pathogen. Nevertheless, a particular attention to the quality of clinical specimens is warranted. Moreover, the sensitivity of direct examination may be greatly enhanced by the use of fluorochromes such as calcofluor white. Likewise, sensitivity of the cultures could be enhanced by the use of culture media containing antifungal deactivators. With the generalization of molecular identification by gene sequencing or MALDI-TOF mass spectrometry, the contribution of historical biochemical or physiological tests to species identification of atypical isolates is now limited. Nevertheless, despite the recent availability of several PCR-based kits and an extensive literature on molecular methods allowing the detection of fungal DNA or both detection and direct identification of the main dermatophyte species, the biological diagnosis of dermatophytosis in 2016 still relies on both direct examination and cultures of appropriate clinical specimens.     

The New Species Concept in Dermatophytes—a Polyphasic Approach

The dermatophytes are among the most frequently observed organisms in biomedicine, yet there has never been stability in the taxonomy, identification and naming of the approximately 25 pathogenic species involved. Since the identification of these species is often epidemiologically and ethically important, the difficulties in dermatophyte identification are a fruitful topic for modern molecular biological investigation, done in tandem with renewed investigation of phenotypic characters. Molecular phylogenetic analyses such as multilocus sequence typing have had to be tailored to accommodate differing the mechanisms of speciation that have produced the dermatophytes that are commonly seen today. Even so, some biotypes that were unambiguously considered species in the past, based on profound differences in morphology and pattern of infection, appear consistently not to be distinct species in modern molecular analyses. Most notable among these are the cosmopolitan bane of nails and feet, Trichophyton rubrum, and the endemic African agent of childhood tinea capitis, Trichophyton soudanense, which are effectively inseparable in all analyses. The molecular data require some reinterpretation of results seen in conventional phenotypic tests, but in most cases, phylogenetic insight is readily integrated with current laboratory testing procedures.     

Use of arbitrarily primed polymerase chain reaction to differentiate Trichophyton dermatophytes

Abstract Dermatophytes such as Trichophyton species are common human pathogens, the infection of which results in dermatophytosis (also known as ringworm). Several laboratory tests are used routinely for the diagnosis of dermatophytosis, but they are either slow or lacking specificity. Through examination of genomic DNA from Trichophyton dermatophytes and other fungi in arbitrarily primed PCR, it was shown that a random primer 5'-ACCCGACCTG-3' produced bands of 4.3 kb, 1.9 kb, 1.7 kb and 0.7 kb in T. rubrum DNA, bands of 2.5 kb, 1.9 kb and 0.8 kb in T. mentagrophytes var. interdigitale and T. mentagrophytes var. mentagrophytes DNA, and bands of 2.5 kb, 1.9 kb, 1.5 kb and 0.9 kb in T. tonsurans DNA. This primer amplified bands of different sizes in other fungal DNA. Therefore, based on the distinct band patterns observed in arbitrarily primed PCR using this primer, T. rubrum , T. mentagrophytes and T. tonsurans dermatophytes could be rapidly differentiated.     

Dermatophyte and non dermatophyte fungi in Riyadh City,Saudi Arabia

BackgroundDermatophytes are a scientific label for a group of three genera (Microsporum, Epidermophyton and Trichophyton) of fungus that causes skin disease in animals and humans. Conventional methods for identification of these fungi are rapid and simple but are not accurate comparing to molecular methods.ObjectiveThis study aimed to isolate human pathogenic dermatophytes which cause dermatophytosis in Riyadh City, Saudi Arabia and to identify these fungi by using conventional and molecular methods.MethodsThe study was conducted in Medical Complex, Riyadh and King Saud University. Samples of infected skin, hairs and nails were collected from 112 patients. Diagnosis of skin infections, direct microscopic test, isolation and identification of dermatophytes by conventional and molecular methods were carried out.ResultsThe results indicated that the tinea capitis infection had the highest prevalence among the patients (22.3%) while Tinea barbae had the lowest. In this study the identified dermatophyte isolates belong to nine species as Trichophyton violaceum, Trichophyton verrucosum, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton schoenleinii, Trichophyton concentricum, Microsporum canis, Microsporum audouinii and Epidermophyton floccosum which cause skin infections were isolated during this study. Non dermatophyte isolates included 5 isolates from Aspergillus spp. 4 isolates from Acremonium potronii and 15 isolates from Candida spp. M. canis were the most common species (25% of isolated dermatophytes). Out of the 52 dermatophyte isolates identified by conventional methods, there were 45 isolates identified by the molecular method.ConclusionsThe results concluded that approximately M. canis caused a quarter of dermatophyte cases, tinea capitis infection was prevalent and the molecular method was more accurate than conventional methods.     

Unusual Species of Dermatophytes: Rarely Identified or New?

Dermatophytes are causing superficial mycosis in animals and humans. Depending on the geophilic, zoophilic or anthropophilic origin of the fungus but also on the immunological status of the patient, symptomatology can widely differ. Nevertheless, each species is currently associated with typical clinical manifestations, even if atypical localizations and/or clinical pictures are sometimes also reported. Diagnostic tools applied to species identification have been changing since the last two decades with the more frequent use of molecular methods currently considered nowadays as reference methods for species identification. It becomes obvious that the algorithm used for the distinction of closely related species needs to combine phenotypic and genomic methods. All these different points are discussed, and the most recent novel species causing or involved in human dermatophytosis are reported.     

Comparison of Dermatophyte PCR Kit with Conventional Methods for Detection of Dermatophytes in Skin Specimens

The laboratory diagnosis of dermatophytosis is usually based on direct microscopic examination and culturing of clinical specimens. A commercial polymerase chain reaction kit (Dermatophyte PCR) has had favorable results when used for detection of dermatophytes and identification of Trichophyton rubrum in nail specimens. This study investigated the efficacy of the Dermatophyte PCR kit for detecting dermatophytosis in 191 hair or skin specimens from patients with suspected dermatophytosis. PCR was positive for 37 % of samples, whereas 31 and 39 % of the specimens were positive by culturing and direct microscopy, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for PCR analysis were 83, 84, 71, and 91 %, respectively. The sensitivity of the PCR test was higher in specimens obtained from skin (88 %) than in those obtained from hair (58 %), while the specificity remained almost the same (84 and 86 % for skin and hair, respectively). Our results show that the Dermatophyte PCR kit is a promising diagnostic tool for detection of dermatophytosis in skin samples, providing clinicians with a rapid diagnosis.     

Immunology of Dermatophytosis

The immune response to infection by dermatophytes ranges from a non-specific host mechanism to a humoral and cell-mediated immune response. The currently accepted view is that a cell-mediated immune response is responsible for the control of dermatophytosis. Indeed, some individuals develop a chronic or recurrent infection mediated by the suppression of a cell-mediated immune response. The immune response to Trichophyton is unusual in that this fungus can elicit both immediate hypersensitivity (IH) and delayed-type hypersensitivity (DTH) in different individuals when they are submitted to a skin test reaction. Understanding the nature and function of the immune response to dermatophytes is an exciting challenge that might lead to novel approaches in the treatment and immunological prophylaxis of dermatophytosis.     

Species Identification of Dermatophytes by MALDI-TOF MS

MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry (MS) is a new tool for the identification of microorganisms, inclusive of dermatophytes. The technique is faster, more straightforward, and powerful when compared to conventional dermatophyte identification methods (culture and microscopy). Accurate species identification in dermatophytes is not only essential to survey epidemiologic situations, but also for an appropriate medical treatment and to locate the source of infection (zoophilic or anthropophilic). Multiple platforms from a number of well-established commercial manufacturers (Andromas SAS, Bruker Daltonics, bioMérieux) have been used for dermatophyte identification with different success. Independent from the platform used, all of the studies reviewed here report on problems with the identification of phylogenetically closely related anthropophilic and zoophilic dermatophyte species. Thus, supplementation of the databases/libraries as well as standardized extraction protocols and cultivation methods are the precondition required for optimal species identification.     

Phenotypical and molecular characterization of Microsporum canis strains in north-east Brazil

AIMS: This study investigated the possible correlation between the phenotypical and genotypical characteristics of Microsporum canis isolated from cats and dogs in north-east Brazil. METHODS AND RESULTS: The mycological study was conducted by direct microscopic examination and by fungal culture. Polymerase chain reaction-restriction enzyme analysis and random amplification of polymorphic DNA techniques were used for the genotypical analysis. The morphological analysis showed a considerable diversity of colonies as well as different morphologies of conidia, despite the M. canis strains having been isolated under the same conditions. However, the molecular analysis showed that all analysed strains are genetically similar. CONCLUSIONS: This study, based on phenotypical and molecular analysis, evidences the wide spectrum of phenotypical variations in M. canis in contrast to the stable genotypes of such dermatophytes. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study indicate that M. canis isolated from cats and dogs with dermatophytosis in north-east Brazil may be clones, well adapted to the conditions of this region, despite M. canis showing different morphological features.     

MALDI-TOF-Based Dermatophyte Identification

MALDI-TOF MS has become increasingly popular for microorganism identification in the routine laboratory. Compared with conventional morphology-based techniques, MALDI-TOF is relatively inexpensive (per-unit identification), involves a rapid result turnaround time and yields more accurate results without the need for highly qualified staff. However, this technology has been technically difficult to implement for filamentous fungi identification. Identification of dermatophytes, a type of filamentous fungi, remains particularly challenging, partly due to the lack of clear species definition for some taxa or within some species complexes. Review of the ten studies published between 2008 and 2015 shows that the accuracy of MALDI-TOF MS-based identification varied between 13.5 and 100 % for dermatophytes. This variability was partly due to inconsistencies concerning critical steps of the routine clinical laboratory process. Use of both a complete formic acid-acetonitrile protein extraction step and a manufacturer library supplemented with homemade reference spectra is essential for an accurate species identification. This technique is conversely unaffected by variations in other routine clinical laboratory conditions such as culture medium type, incubation time and type of mass spectrometry instrument. Provided that a reference spectra library is adequate for dermatophyte identification, MALDI-TOF MS identification is more economical and offers an accuracy comparable to that of DNA sequencing. The technique also represents an advantageous alternative to the protracted and labor-intensive dermatophyte identification via macroscopic and microscopic morphology in the routine clinical laboratory.     

Intérêt de la spectrométrie de masse de type MALDI-TOF pour l’identification des champignons filamenteux

Identification of filamentous fungi, molds and dermatophytes, is currently based on the morphological study of colonies and therefore the experience of the mycologist. These techniques are not sufficiently precise to distinguish between different species within the same section. Furthermore, identification can be delayed for several weeks due to subcultures on specific media. MALDI-TOF MS allows correct identification of filamentous fungi until the species level in more than 95% of cases in most studies. MALDI-TOF MS is a fast and precise identification technique for filamentous fungi; however most of the different databases need to be further evaluated in routine and completed to broaden the spectrum of species identified.     

Characterization of a cDNA clone, encoding a 70 kDa heat shock protein from the dermatophyte pathogen Trichophyton rubrum

Trichophyton rubrum is an anthropophilic fungus causing up to 90% of chronic cases of dermatophytosis. To characterize T. rubrum proteins at the molecular level, we established a cDNA library of this pathogen. Here we describe a recombinant cDNA clone identical to eukaryotic 70kDa heat-shock proteins (HSPs). Western blot analysis using an anti HSP70 monoclonal antibody detected a recombinant fusion protein in Escherichia coli transformed with the expression vector containing the cloned cDNA insert. Southern blot analysis of T. rubrum genomic DNA detected no other members of the HSP70 gene family. Further analysis revealed the presence of two introns within the ORF of the HSP70 gene. In Northern blot analysis, the cDNA clone was hybridized to a RNA species of about 3.5kb which was constitutively expressed by cells cultured at 27 degrees C and was strongly up-regulated after culture at 37 degrees C. In summary, we have cloned the first member of the HSP family of dermatophytes and characterized it as a member of the Dnak subfamily of 70kDa HSPs.     

Three years incidence of dermatophytes in a hospital in Porto (Portugal)

We evaluated the incidence of dermatophytes isolated at our hospital in the years of 1997 to 2000 and correlated it with anatomical site and age. Trichophyton rubrum was the predominant species in all anatomical sites, excluding scalp, followed by Microsporum canis, the leading agent of tinea capitis. All dermatophytosis, except tinea capitis by M. canis and Trichophyton schoenleinnii appeared mainly in adult patients. Our results revealed no substantial differences to other portuguese studies regarding the major agents. We found a relatively high incidence of T. schoenleinnii as second tinea capitis agent.     

Isolations of dermatophyte from clinically normal scalps inM. Canis infections using the hairbrush method

The scalp hair of patients with dermatophytosis due toM. canis but without scalp lesions, and that of their family members without dermatophytosis were examined using the hairbrush method. The dermatophyte was detected in 93.8% of the scalps of those who lived in homes where cats were kept, and in 25% of those without cats. After the source of infection had been treated, the dermatophyte showed a gradually decreasing presence, finally disappearing altogether, so that no case of the disease on the scalp hair could be found.We inferred from the above findings inM. canis infections that, since the dermatophytes are seen in a high proportion of cases without scalp lesions, the dermatophytes, in many cases, exist only as saprophytes on the hair.