Dermatological Specimens Research Articles

Rapid clearing and imaging of Mohs and melanoma surgical margins using a low-cost tissue processor.

Tissue clearing methods render biological tissues transparent while maintaining tissue structure, enabling visualization of entire tissues. Recent developments in tissue clearing have predominantly emphasized preserving intrinsic fluorescent proteins or aqueous-based tissue clearing and so typically involve complex procedures and long processing times. The utilization of tissue clearing protocols in standard of care histology settings has been less well explored, and protocols for rapid clearing of human tissue specimens are limited. This study presents a novel rapid clearing protocol and demonstrates a low-cost tissue processor for high volume rapid tissue clearing that can be intergraded into standard histology workflow. We demonstrate rapid clearing in dermatological specimens, including both nonmelanoma and melanoma excisions.

Diagnostic Performance of Ex Vivo Fluorescence Confocal Microscopy in the Assessment of Diagnostic Biopsies of the Prostate

Simple SummaryFluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. In this study, we compare intraoperative diagnoses from the real-time application of FCM in pre-therapeutic prostate biopsies with the final diagnoses from conventional histology. We found FCM to be an effective tool for the timely assessment of prostate biopsies enabling reliable real-time diagnosis of prostate cancer in patients requiring therapy.Background: Fluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. The method has been well established for the diagnosis of tumors in dermatological specimens. Methods: We compare intraoperative diagnoses of the real-time application of FCM in pre-therapeutic prostate biopsies (35 patients, total number of biopsy specimens: n = 438) with the findings of conventional histology. Results: Prostate carcinoma was reliably diagnosed in all patients. Depending on scan quality and experience of the examiner, smaller lesions of well differentiated carcinoma (ISUP1) could not be consistently differentiated from reactive changes. Furthermore, in some cases there was difficulty to distinguish ISUP grade 2 from ISUP grade 1 tumors. ISUP grades 3–5 were reliably detected in FCM. Conclusions: Despite some limitations, FCM seems to be an effective tool for the timely assessment of prostate biopsies enabling reliable diagnosis of prostate cancer in patients requiring therapy.

Feasibility study for ex vivo fluorescence confocal microscopy (FCM) on diagnostic prostate biopsies.

Fluorescence confocal microscopy (FCM) is a novel micro-imaging technique providing optical sections of examined tissue. The method has been well established for the diagnosis of tumours in dermatological specimens. Preliminary results found good feasibility when this technique was used to examine prostate cancer (PCa) specimens. We report on the application of FCM in magnet resonance imaging (MRI)-fused prostate biopsies (10 patients, total number of biopsy specimens: n=121) and compare the results to conventional histology. Specific structures of the prostatic tissue were very well represented in the FCM images comparable to conventional histology. Prostate carcinoma was diagnosed with good sensitivity (79/68%) and high specificity (100%) by two pathologists with substantial/almost perfect levels of agreement with the results of conventional histology (kappa 0.79/0.86). Depending on the quality of the scans, malignant lesions of 1.8 mm and more in diameter were reliably diagnosed. Smaller lesions were rated as suspect for malignancy, but could not be consistently differentiated from reactive changes. Optimal image qualities were achieved in focus depths of up to 50 µm, whereas deeper scans led to insufficient representation of cytological features. Pre-treatment with acridine orange (AO) did not alter immunoreactivity of the tissue or its feasibility for fluorescence in situ hybridization (FISH) analyses and adequate amounts of DNA could be extracted for further polymerase chain reaction (PCR)-based examinations. FCM seems to be a promising tool for the timely diagnosis in cases of PCa in patients requiring therapy. In particular, this technique is a material-sparing method that conserves the biopsies as unfixed material for further analysis such as molecular tumour companion diagnosis.

Mixed Borrelia burgdorferi and Helicobacter pylori Biofilms in Morgellons Disease Dermatological Specimens.

Background: Morgellons disease (MD) is a dermopathy that is associated with tick-borne illness. It is characterized by spontaneously developing skin lesions containing embedded or projecting filaments, and patients may also experience symptoms resembling those of Lyme disease (LD) including musculoskeletal, neurological and cardiovascular manifestations. Various species of Borrelia and co-infecting pathogens have been detected in body fluids and tissue specimens from MD patients. We sought to investigate the coexistence of Borrelia burgdorferi (Bb) and Helicobacter pylori (Hp) in skin specimens from MD subjects, and to characterize their association with mixed amyloid biofilm development. Methods: Testing for Bb and Hp was performed on dermatological specimens from 14 MD patients using tissue culture, immunohistochemical (IHC) staining, polymerase chain reaction (PCR) testing, fluorescent in situ hybridization (FISH) and confocal microscopy. Markers for amyloid and biofilm formation were investigated using histochemical and IHC staining. Results: Bb and Hp were detected in dermatological tissue taken from MD lesions. Bb and Hp tended to co-localize in foci within the epithelial tissue. Skin sections exhibiting foci of co-infecting Bb and Hp contained amyloid markers including β-amyloid protein, thioflavin and phosphorylated tau. The biofilm marker alginate was also found in the sections. Conclusions: Mixed Bb and Hp biofilms containing β-amyloid and phosphorylated tau may play a role in the evolution of MD.

Dermatophytes Isolated From Dogs and Cats Suspected Dermatophytoses in Istanbul, Turkey Within A 15-Year-Period: An Updated Report

The present research was aimed to determine the prevalence of dermatophytes isolated from symptomatic dogs and cats, within a 15-year-period, in the city of Istanbul, Turkey. Dermatological specimens were collected from 1504 dogs and 846 cats, which were presented clinical signs of ringworm. Direct microscopy and mycological cultures were performed. The fungal growth rate was detected at 8.2% and 22.8% from dogs and cats, respectively. Microsporum canis was the most frequently isolated species followed by Trichophyton spp., M. gypseum , T. mentagrophytes , M. nanum , other Microsporum spp. moreover T. tonsurans . The cats less than two-year age and more than ten-year age showed a statistically significant higher isolation rate of infection ( p < 0.05). There were no statistically significant differences between the age of the dogs and the dermatophyte isolation rate and between the gender of the dogs and cats and the dermatophyte isolation rate. As a conclusion, the data suggest an updated report on local epidemiology and define potential etiologic agents.

Detection of tick-borne infection in Morgellons disease patients by serological and molecular techniques.

BackgroundMorgellons disease (MD) is a skin condition associated with Lyme disease (LD) and tick-borne illness. Patients with this skin disorder experience ulcerative lesions that contain multicolored filamentous collagen and keratin inclusions. Infection with various species of Borrelia and other tick-borne pathogens has been detected in tissue and body fluid specimens from MD patients. We sought to explore this association further in a cohort of MD patients.Patients and methodsSera from 30 patients with MD were tested for antibody reactivity to antigens from the Borrelia burgdorferi (Bb) group and the relapsing fever Borrelia (RFB) group of spirochetes. Tissue and/or body fluid specimens from these patients were also tested for the presence of Bb and RFB infection using PCR technology. In addition, tissue and body fluid specimens were tested for the presence of Bartonella henselae using PCR, and formalin-fixed skin sections from a subset of patients were tested using fluorescent in situ hybridization (FISH) with B. henselae-specific DNA probes.ResultsSeroreactivity to Bb, RFB or both was detected in 63% of the cohort, while positive PCR testing for Bb, RFB or both was detected in 53% of the cohort. Overall, 90% of patients tested positive for exposure and/or infection with Borrelia spirochetes. B. henselae infection was detected by PCR in skin sections or body fluids from 20% of the subjects, and B. henselae FISH testing was positive in 30% of the dermatological specimens submitted for study.ConclusionThe study demonstrates an association between MD and positive tests for both Bb and RFB spirochetes. In conjunction with previous studies, our study provides corroborative evidence linking MD to Borrelia infection and tick-borne illness.

Trichophyton verrucosum infection in livestock in the Chitral district of Pakistan.

Trichophyton verrucosum belongs to the dermatophyte fungi, closely related organisms that cause skin infections in animals and humans. T. verrucosum infection has been reported in livestock and people in different countries from all continents. Human cases have been reported in different areas of Pakistan, but there is little information about the animal source of the fungus. Dermatological specimens collected in the Chitral district of Pakistan for a study on mange in livestock were retrospectively analyzed for the presence of T. verrucosum. In total, 5,873 animals (1,087 cows, 2,033 goats, and 2,753 sheep) were screened for evidence of dermatological lesions during two surveys performed in the summer and winter seasons. Skin scrapings collected from animals with lesions were analyzed by direct microscopic examination after digestion in sodium hydroxide and a real-time polymerase chain reaction (PCR) targeting pathogenic Trichophyton species. At microscopy, samples from 18 cows (1.6%), 3 sheep (0.1%), and 4 goats (0.2%) were positive for fungal elements consistent with T. verrucosum. PCR confirmed the microscopy results. The prevalence was lower than that reported in other countries in intensive breeding farms. Results agree with the literature regarding factors affecting T. verrucosum diffusion, i.e., infection was more prevalent in cattle, especially in younger animals during the winter season. This study reports, for the first time, the presence of T. verrucosum in livestock in Pakistan. A better knowledge of the animal role in the spread of this fungus may allow the adoption of more efficient control measures and prophylaxis.

Novel Use of Preoperative Epidermal Coloring of Very Small Dermatological Specimens-Protocol for Reduction of Lost Specimens.

Small tissue biopsies are often difficult to visualize and can be easily lost or mishandled. The authors hypothesized that full epidermal surface coloration of small skin lesions with a sterile skin marker (gentian violet ink) before performing shave biopsy would make small gross specimens easier to identify without impacting microscopic appearance. Live evaluation of 4 inked and 4 noninked gross (2-3 mm) specimens in covered and uncovered formalin-containing jars by 50 consecutive health care personnel demonstrated that inked specimens were significantly (P < 0.001) easier to visualize than noninked specimens. Additionally, a blinded dermatopathologist evaluated 25 inked and 25 noninked specimens microscopically. Utilization of this inking process did not interfere with histopathologic assessment or impede diagnosis. This pilot study describes an easily implementable quality improvement measure that may decrease the rate of loss and mishandling of specimens.

Canine Filamentous Dermatitis Associated with Borrelia Infection

Canine Filamentous Dermatitis Associated with Borrelia Infection Background: Although canine clinical manifestations of Lyme disease vary widely, cutaneous manifestations are not well documented in dogs. In contrast, a variety of cutaneous manifestations are reported in human Lyme disease caused by the spirochete Borrelia burgdorferi. A recently recognized dermopathy associated with tickborne illness known as Morgellons disease is characterized by brightly-colored filamentous inclusions and projections detected in ulcerative lesions and under unbroken skin. Recent studies have demonstrated that the dermal filaments are collagen and keratin biofibers produced by epithelial cells in response to spirochetal infection. We now describe a similar filamentous dermatitis in canine Lyme disease. Methods and Results: Nine dogs were found to have cutaneous ulcerative lesions containing embedded or projecting dermal filaments. Spirochetes characterized as Borrelia spp. were detected in skin tissue by culture, histology, immunohistochemistry, polymerase chain reaction (PCR) and gene sequencing performed at five independent laboratories. Borrelia DNA was detected either directly from skin specimens or from cultures inoculated with skin specimens taken from the nine canine study subjects. Amplicon sequences from two canine samples matched gene sequences for Borrelia burgdorferi sensu stricto. PCR amplification failed to detect spirochetes in dermatological specimens from four healthy asymptomatic dogs. Conclusions: Our study provides evidence that a filamentous dermatitis analogous to Morgellons disease may be a manifestation of Lyme disease in domestic dogs.

Exploring the association between Morgellons disease and Lyme disease: identification of Borrelia burgdorferi in Morgellons disease patients.

BackgroundMorgellons disease (MD) is a complex skin disorder characterized by ulcerating lesions that have protruding or embedded filaments. Many clinicians refer to this condition as delusional parasitosis or delusional infestation and consider the filaments to be introduced textile fibers. In contrast, recent studies indicate that MD is a true somatic illness associated with tickborne infection, that the filaments are keratin and collagen in composition and that they result from proliferation and activation of keratinocytes and fibroblasts in the skin. Previously, spirochetes have been detected in the dermatological specimens from four MD patients, thus providing evidence of an infectious process.Methods & ResultsBased on culture, histology, immunohistochemistry, electron microscopy and molecular testing, we present corroborating evidence of spirochetal infection in a larger group of 25 MD patients. Irrespective of Lyme serological reactivity, all patients in our study group demonstrated histological evidence of epithelial spirochetal infection. Strength of evidence based on other testing varied among patients. Spirochetes identified as Borrelia strains by polymerase chain reaction (PCR) and/or in-situ DNA hybridization were detected in 24/25 of our study patients. Skin cultures containing Borrelia spirochetes were obtained from four patients, thus demonstrating that the organisms present in dermatological specimens were viable. Spirochetes identified by PCR as Borrelia burgdorferi were cultured from blood in seven patients and from vaginal secretions in three patients, demonstrating systemic infection. Based on these observations, a clinical classification system for MD is proposed.ConclusionsOur study using multiple detection methods confirms that MD is a true somatic illness associated with Borrelia spirochetes that cause Lyme disease. Further studies are needed to determine the optimal treatment for this spirochete-associated dermopathy.

Advances in Microbiology, Infectious Diseases and Public Health: Fungal Occurrence in the Hair and Skin of Symptomatic Pets in Turin, Italy.

Companion animals, often asymptomatic reservoir of fungi, can be important sources of infection in humans, due to the close contact with their owners. The present study was aimed to assess the occurrence of dermatophytes and other fungi isolated from pet dermatological lesions in Turin, Italy. Dermatological specimens were examined for fungal elements by direct microscopy and cultured to detect dermatophytes, other filamentous fungi and yeasts: 247 pets (118 cats, 111 dogs and 18 dwarf rabbits) were positive for fungal detection in culture. Microsporum canis was the most frequent dermatophyte in cats and dogs, whereas Trichophyton mentagrophytes was the most common in rabbits. Among the other fungi, for all examined pets, dematiaceous fungi were the most isolated, followed by Mucorales, penicilli, yeasts and yeast-like fungi, and aspergilli. No gender predisposition was detected for dermatophyte growth; on the contrary, for the other fungi male cats were more susceptible than female. The highest fungal occurrence was recorded in <1-year-old cats for dermatophytes, and in <5-year-old cats and dogs for the other fungi. Autumn was the period associated with a relevant incidence of fungal infection. Finally, fungi were more frequent in non pure-breed cats and in pure-breed dogs. These data underline the importance to timely inform pet owners about the potential health risk of infection caused not only by dermatophytes but also by non-dermatophyte fungi, routinely considered to be contaminants or harmless colonizers, since their role as source of zoonotic infections is not to be excluded.

Processing dermatological biopsies.

Dermatological biopsies can be obtained from many anatomic areas and fulfill a variety of diagnostic roles. Due to this variety, the handling and processing of dermatological specimens pose unique challenges. The small size, disparate shapes, and anatomic variation seen on skin biopsies require specialized techniques to enable sufficient sampling, proper orientation, and accurate diagnoses. This chapter addresses issues related specifically to the processing of small specimens, evaluation for biopsies for alopecia, nail specimens, frozen sections, and specimens requiring direct immunofluorescence.

Canine and feline dermatophytosis due to Microsporum gypseum: A retrospective study of clinical data and therapy outcome with griseofulvin

Microsporum gypseum is a common inhabitant of the soil, occasionally responsible for human and animal ringworm. Few reports describe the treatment of dermatologic diseases due to M. gypseum. The objective of this study was to evaluate retrospectively cases of M. gypseum infection in dogs and cats. The occurrence of infection by this dermatophyte was retrospectively evaluated in dermatological specimens from 15,684 dogs and cats dermatologically diseased from Italy. Clinical outcome after treatment with griseofulvin combined with topical enilconazole was evaluated in 41 dogs and, out of label, 10 cats. Furthermore, in vitro susceptibility to griseofulvin and enilconazole was evaluated on 31 clinical isolates of M. gypseum. One hundred and eighty-five specimens out of 15,684 (1.1%) scored positive for M. gypseum. The treatment failed to achieve both mycological and clinical cure in 16 dogs (39%) and four cats (40%), as well as fungal isolates demonstrated a very poor in vitro sensitivity when tested versus griseofulvin: the MIC value was 150 μg/mL. The ED50 value was calculated at 66 μg/mL. Blind treatments with griseofulvin in ringworm due to M. gypseum should be avoided.

Characterization and evolution of dermal filaments from patients with Morgellons disease.

Morgellons disease is an emerging skin disease characterized by formation of dermal filaments associated with multisystemic symptoms and tick-borne illness. Some clinicians hypothesize that these often colorful dermal filaments are textile fibers, either self-implanted by patients or accidentally adhering to lesions, and conclude that patients with this disease have delusions of infestation. We present histological observations and electron microscopic imaging from representative Morgellons disease samples revealing that dermal filaments in these cases are keratin and collagen in composition and result from proliferation and activation of keratinocytes and fibroblasts in the epidermis. Spirochetes were detected in the dermatological specimens from our study patients, providing evidence that Morgellons disease is associated with an infectious process.

Black yeast‐like fungi in skin and nail: it probably matters

Black yeast-like fungi are rarely reported from superficial infections. We noticed a consistent prevalence of these organisms as single isolations from mycological routine specimens. To investigate the prevalence of black yeast-like fungi in skin, hair and nail specimens and to discuss the probability of these species to be involved in disease. Slow-growing black yeast-like fungi in routine specimens were prospectively collected and identified. A questionnaire regarding patient information was sent to physicians regarding black yeast-like fungus positive patients. A total of 20,746 dermatological specimens were examined by culture. Black yeast-like fungi accounted for 2.2% (n=108) of the positive cultures. Only 31.0% of the samples, culture positive for black yeast-like fungi were direct microscopy positive when compared with overall 68.8% of the culture positive specimens. The most prevalent species were Phialophora europaea (n=29), Coniosporium epidermidis (n=12), Ochroconis cf. humicola (n=6) and Cladophialophora boppii (n=4). These are not common saprobes and thus less likely to be coincidental colonizers. In 10/30 cases, discolouration of nail/skin had been noticed. A limited number of black yeast-like fungi were repeatedly isolated from routine specimens suggesting that they may play a role in superficial infections or as colonizers.

Developing best practice for fungal specimen management: audit of UK microbiology laboratories

This study represents an audit of microbiology laboratories in the UK to ascertain whether they are aware of, or follow, the Health Protection Agency (HPA) National Standard Methods Standard Operating Procedure (NSM SOP) for the investigation of dermatological specimens for superficial mycoses, or use a locally adapted version. A questionnaire audit was distributed to 179 NHS microbiology laboratories throughout England, Wales, Scotland and Northern Ireland. The NSM SOP was followed by 92% of laboratories for the microscopy of dermatological samples; light microscopy/ KOH digestion was used by 63% and fluorescence microscopy/KOH digestion by 29% of laboratories. Preliminary reports post-microscopy were issued by 98% of laboratories, with 93% issuing reports within 48 hours. Adherence to the NSM SOP guidelines for culture was low; only 34% of laboratories incubated microscopy-negative specimens for the recommended 14 days, while approximately 60% incubated microscopy-positive specimens for 21 days. The culture medium recommended by the NSM SOP was used in 82% of laboratories. Comments were added to culture reports by 51% of laboratories; most were added manually and comments varied between laboratories. Nail samples were the most common sample received from primary care, followed by skin and hair. These results show no significant difference in the rate of microscopy positives versus culture positives. Microscopy and culture are the easiest and cheapest methods available to UK laboratories for the investigation of suspected superficial fungal infections. Although most laboratories included in this audit claimed to follow the NSM SOP for microscopy and culture, these results show that the techniques used vary throughout the UK. To maximise the service provided to primary care, UK laboratories should use standardise methods based on the NSM SOP.

Sequence-based genotyping HPV L1 DNA and RNA transcripts in clinical specimens

We developed a direct sequence-based genotyping method to detect single and multiple HPV L1 DNA and RNA types in genital and dermatological specimens. Our method couples PCR amplification of a highly conserved HPV L1 segment using a broad spectrum-generic primer cocktail mix with automated sequencing of amplified PCR products, followed by GenBank sorting of sequencing data. We genotyped 5 skin and 30 cervical HPV DNA-positive specimens using this method and established its first experimentally derived working cutoff value with the aid of commercial hybridization-based techniques. We suggest that sequence-based genotyping of appropriately amplified DNA and RNA products may serve as a primary HPV detection method in dermatological specimens. It can be applied as an all-purpose genotyping method for rare HPV types not detectable by commercial hybridization-based techniques and for sorting multiple HPV infections by order of prevalence.

Study of Mycological Examination Methods in Clinical Laboratories

We performed a comparative study of the effects of centrifugation, large amounts of inoculum and incubation temperature with regard to recovery of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus from fungal suspensions in order to identify optimal processing methods for mycological examination of clinical specimens. The number of fungal colonies, except for Candida spp., isolated from respiratory specimens, and the duration of incubation needed to isolate pathogenic fungi from clinical specimens were also analyzed retrospectively. There was a difference in the number of recovered colonies, with or without centrifugation, between inoculum sizes of 10 microl and 50 microl, but no differences were observed in the results obtained under two sets of centrifugation conditions: 2,000 x g for 15 minutes and 3,000 x g for 20 minutes. Candida albicans and Aspergillus fumigatus developed more rapidly at 35 degrees C than at 27 degrees C in the first 24 hours of incubation, while Cryptococcus neoformans formed a larger colony at 27 degrees C than at 35 degrees C. One to three colonies of Aspergillus spp. and Cryptococcus spp. were isolated from respiratory specimens in 73% and 50% of cases, respectively. The required incubation period was six days for isolation of 65 Aspergillus spp. strains from respiratory specimens, while 14 days was needed for isolation of 46 dermatophyte strains. Based on these results, we recommend a pretreatment of centrifugation and a large quantity of inoculum for respiratory specimen processing, as well as an incubation period of at least 7 days and 21 days for internal and dermatological specimens, respectively.

Application of polymerase chain reaction (PCR) and PCR based restriction fragment length polymorphism for detection and identification of dermatophytes from dermatological specimens.

Objective:To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes.Materials and Methods:Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods.Results:PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%.Conclusion:PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes.

Rapid identification and differentiation of fungal DNA in dermatological specimens by LightCycler PCR

The aim was to develop a LightCycler PCR method for the rapid detection and differentiation of fungal DNA in dermatological specimens such as skin scales and skin swabs. LightCycler PCR assays were established for seven primer sets specific for fungal DNA. For each primer set LightCycler melting points were defined by amplification of DNA from 21 fungi and sensitivity was determined by amplification of serial dilutions of fungal DNA. A protocol was established that allows detection and differentiation of mould and yeast DNA with one highly sensitive PCR reaction by assessment of LightCycler melting points. Two subsequent LightCycler PCR reactions and one RFLP reaction allowed the differentiation of dermatophytes and non-dermatophyte moulds and the subclassification of yeasts. Analysis of clinical samples from 38 patients with fungal skin diseases provided conclusive new diagnostic information in 9/38 cases (23.7 %) by this PCR protocol that was not equally provided by direct microscopy and mycological culture. Thus the LightCycler PCR protocol established here represents a rapid diagnostic tool that aids in the diagnosis of fungal skin disease in a substantial number of patients.