[Molecular biological detection of dermatophytes in clinical samples when onychomycosis or tinea pedis is suspected. A prospective study comparing conventional dermatomycological diagnostics and polymerase chain reaction]. / Molekularbiologischer Direktnachweis von Dermatophyten im klinischen Material bei Verdacht auf Onychomykose und Tinea pedis. Eine prospektive Studie zum Vergleich konventioneller dermatomykologischer Diagnostik und der Polymerasekettenreaktion.

BACKGROUND: The prevalence of onychomycosis is rising worldwide. Before starting antifungal treatment, an exact mycological diagnosis should be obtained. The current laboratory diagnosis of dermatomycoses is based on the detection of the causative agent by microscopy and culture. These conventional diagnostic methods for fungal infections often are not the best solution because they are time-consuming, cultures are false-negative and direct examination identifies non-vital structures which cannot be used for speciation. PATIENTS AND METHODS: A total of 218 patients presenting in a surgical practice over 3 months with clinical signs of tinea pedis and/or onychomycosis were involved in the prospective study. All patients had predisposing factors for tinea pedis and tinea unguium, such as vascular insufficiency, diabetes mellitus, and leg ulcers. Nail specimens and skin scrapings were investigated for fungi using Blancophor® preparation, and cultured. In addition to conventional diagnostics, PCR (polymerase chain reaction) for detection of dermatophyte DNA was employed. This PCR-Elisa assay is based on the use of specific primers which target the topoisomerase II gene. This allows the highly specific molecular identification of Trichophyton (T.) rubrum, T. interdigitale, and Epidermophyton floccosum directly in clinical samples. RESULTS: 23.9 % of patients were culture-positive for dermatophytes (either T. rubrum, or T. interdigitale). With PCR, dermatophyte DNA either of T. rubrum or T. interdigitale could be detected in nail samples and skin scrapings from at least 29.9 % of all patients. Epidermophyton floccosum was not found in this study, neither by cultivation nor by PCR. The diagnostic sensitivity of the PCR-Elisa assay was calculated as 79.0% ; the diagnostic specificity as 85.5 %. CONCLUSION: PCR-Elisa evaluation makes possible a rapid, specific and sensitive diagnosis of dermatophytosis of the nails and skin within 24 (maximal 48) hours with identification of the involved species.

Development of a novel polymerase chain reaction-enzyme-linked immunosorbent assay for the diagnosis of Trichophyton rubrum onychomycosis.

BACKGROUND: The prevalence of onychomycosis has increased steadily in the past decade. An accurate diagnosis at the outset is important for successful and cost-effective treatment of patients. However, current diagnostic tests for onychomycosis are not rapid, sensitive or specific. OBJECTIVES: To develop a microsatellite-based polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (MS-ELISA) for the detection of Trichophyton rubrum, which is the most common aetiological agent of onychomycosis. METHODS: An archival set of 434 nail and skin specimens from 217 patients was included as the test sample in this study. We also compared MS-ELISA with an earlier published topoisomerase PCR-ELISA (TI-ELISA) using template DNA extracted by another method. RESULTS: The MS-ELISA detected the highest number of positive samples (69%) followed by direct microscopy (56%), TI-ELISA (44%) and fungal culture (30%). When an identical DNA extraction method was applied to 120 specimens, the MS-ELISA proved to be twice as sensitive as the TI-ELISA. CONCLUSIONS: We have optimized a target gene and DNA extraction method for rapid detection of T. rubrum onychomycosis.

Detection of cutaneous herpes simplex virus infections by immunofluorescence vs. PCR.

BACKGROUND: Detection of cutaneous infections with herpes simplex virus (HSV) has proven difficult, as serum antibody tests sometimes are not sensitive and specific enough for that purpose. OBJECTIVE: This study was conducted to compare the sensitivity for detection of HSV of an immunofluorescence method (Syva Microtrak) and an internally controlled PCR. METHODS: Cutaneous swabs from skin lesions were analysed by immunofluorescence separately for HSV types 1 and 2 and by competitive PCR. Detection of PCR products was done by ELISA, if positive additionally by agarose gel electrophoresis. RESULTS: Of 79 samples 34 were PCR-positive by ELISA (34 = 100%), of which 23 (68%) were also positive on the agarose gel. Eleven samples (32%) were positive by immunofluorescence. No sample was positive by immunofluorescence and negative by PCR. CONCLUSIONS: These results demonstrate that immunofluorescence using Syva Microtrak is not suitable for exclusion of herpes simplex virus infection as sensitivity was only 32%. However, as immunofluorescence is cheaper and faster than PCR, first screening can be done with immunofluorescence, and negative samples can be investigated by PCR to finally prove or exclude the presence of HSV DNA.

Quantification of indoleamine 2,3-dioxygenase gene induction in atopic and non-atopic monocytes after ligation of the high-affinity receptor for IgE, Fc(epsilon)RI and interferon-gamma stimulation.

Antigen-presenting cells (APCs) are crucial in regulating the outcome of T cell responses. Certain APCs are able to down-regulate T cell proliferation in vitro by inducing the enzyme indoleamine 2,3-dioxygenase (IDO) upon interferon-gamma (IFN-gamma) stimulation. IDO is the rate-limiting enzyme in the catabolism of the essential amino acid tryptophan. A lack of extracellular tryptophan creates environments in which cells become starved for this amino acid. The high-affinity receptor for IgE, Fc(epsilon)RI, is the principal receptor for the binding of specific IgE in type I-mediated allergies. We demonstrated recently that IDO is overexpressed in Fc(epsilon)RI-stimulated monocytes. In the present study, we performed quantification of IDO gene induction after treatment of atopic (Fc(epsilon)RI(high)) and non-atopic (Fc(epsilon)RI(low/-)) monocytes with IgE/anti-IgE and IFN-gamma. By quantitative PCR ELISA, we found IDO molecule induction in atopic monocytes was enhanced about 50-fold over non-atopic monocytes after ligation of Fc(epsilon)RI. Stimulation with IFN-gamma at a concentration of 100 U/ml in culture medium caused an increase in IDO gene copy numbers in atopics of about fourfold over that of non-atopics. This comparative quantification study demonstrates clearly the regulation of IDO gene expression by Fc(epsilon)RI and discloses differences thereof in atopic and non-atopic cells upon inflammatory stimuli.

Varicella zoster viraemia during herpes zoster is not associated with neoplasia.

BACKGROUND: Shingles are caused by an endogenous or exogenous reinfection with varicella zoster virus (VZV). Up to 50% of individuals with Hodgkin's disease develop herpes zoster; however, no association could be shown between the occurrence of herpes zoster and underlying subclinical malignancies. OBJECTIVE: This study was conducted to investigate whether VZV DNA could be detected by polymerase chain reaction (PCR) in the blood of herpes zoster patients and whether there was an association between VZV viraemia and previous or concurrent neoplasias. METHODS: At least five blood samples from 28 patients with herpes zoster were investigated by internally controlled PCR enzyme-linked immunosorbent assay prior to and during therapy with aciclovir. RESULTS: None of 13 patients, two with a history of neoplasia and two with a neoplasia at the time of the study, showed any signs of viraemia with VZV, and 14 patients had inconsistent viraemia, one with a history of neoplasia and two with neoplasia at the time of the study. In one patient VZV DNA was detected in the blood for 6 days. This patient died soon after from metastatic malignant melanoma. CONCLUSIONS: VZV viraemia may occur during herpes zoster episodes, even in patients without evidence of immunosuppression; however, this viraemia is, in most cases, inconsistent and does not provide any specific information concerning underlying unrecognized malignancies.

Competitive polymerase chain reaction used to diagnose cutaneous leishmaniasis in German soldiers infected during military exercises in French Guiana.

A competitive polymerase chain reaction (PCR) followed by enzyme-linked-immunoassay-based verification of PCR products has been developed, which facilitated the diagnosis of leishmaniasis in two German soldiers who underwent survival training in the jungle of French Guiana and returned with therapy-resistant pyoderma-like lesions. After treatment with liposomal amphotericin B, the skin manifestations disappeared, and leishmania DNA could no longer be detected by PCR. In the context of growing military involvement in areas where leishmaniasis is prevalent, this assay may help detect or, due to its internal controls, exclude cases of infection with this parasite.

[Allergic contact dermatitis in beauty parlor clients]. / Allergische Kontaktdermatitis bei Friseurkunden.

Occupational contact dermatitis in hair dressers and beauticians has increased in importance in the past years. Type IV-allergies against glyceryl monothioglycate components of permanent waves are most common. Other occupational allergens include bleach components such as ammonium persulfate and hair dye ingredients such as p-phenylenediamine (PPD) and p-toluylene-diamine (PTD) base. Allergies to hair dyes in customers of hair dressers have rarely been observed. Two female patients developed allergic contact dermatitis of the scalp and face after repeated use of Polycolor intensivtönung schwarz and of Movida color. We also review the current literature on type IV-allergies to components of hair dressing products components.

Detection of cutaneous varicella zoster virus infections by immunofluorescence versus PCR.

Detection of localized, clinically atypical cutaneous infections with varicella zoster virus (VZV) has proven difficult, as serum antibody tests sometimes are not sensitive and specific enough for that purpose. Therefore immunofluorescence and an internally controlled PCR for VZV are compared for sensitivity. Detection of PCR products was done by ELISA, and if positive, additionally by agarose gel electrophoresis. Of 60 samples 44 were PCR-positive by ELISA (44 = 100%), of which 37 (84%) were also positive on the agarose gel. Thirty-four samples (77%) were positive by immunofluorescence. No sample was positive by immunofluorescence and negative by PCR. A combination of immunofluorescence and PCR with agarose gel analysis detected 42 samples out of 44 positive by PCR ELISA (95%). These results demonstrate that immunofluorescence is a suitable, fast and inexpensive method for routine diagnostics. Additional sensitivity can be achieved by screening immunofluorescence-negative samples by PCR, which is extremely sensitive but time-consuming and labor-intensive.

Hidden scabies: diagnosis by polymerase chain reaction.

Diagnosis of scabies infection can be difficult as in many cases only few mites are present on an infected person, and in some cases the skin manifestations can be subtle or atypical. We describe the use of polymerase chain reaction (PCR) to amplify Sarcoptes scabiei DNA in a patient presenting with clinically atypical eczema. Cutaneous scales were PCR positive for S. scabiei DNA before, and negative 2 weeks after, therapy. This method facilitates fast and very sensitive diagnosis of clinically atypical or inapparent scabies infection and therapy control in severely affected patients and may help to identify previously unrecognized scabies cases.

Coexpression patterns of EGFR, HER2, HER3 and HER4 in non-melanoma skin cancer.

The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR), HER2, HER3 and HER4 are involved in the pathogenesis of multiple human malignant neoplasias. However, their role in the carcinogenesis of basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) remains to be elucidated. In order to further define the role of these RTKs, 56 human skin tissue samples of normal skin, BCC and SCC were studied by conventional and differential and quantitative reverse transcriptase-polymerase chain reaction (rtPCR). EGFR and HER3 were predominantly expressed in the BCCs and SCCs, while HER2 was ubiquitously expressed. HER4 was not expressed in any sample. Since in vitro studies have provided compelling evidence that heterodimer formation of these receptors are associated with different signal transduction processes, coexpression patterns might be decisive for the induction and maintenance of a malignant phenotype. These results confirm this concept: isolated HER2 expression and EGFR/HER2 were predominantly found in normal skin, while HER2/HER3 and the triple expression of EGFR/HER2/HER3 were seen more frequently in the BCCs and SCCs compared with normal skin (50% and 40% compared with 26%, respectively). The activation of HER3, in addition to EGFR and HER2, might therefore be associated with the malignant phenotype. However, due to the small numbers in this study, further confirmation of the patterns is needed.

Quantitation of human herpes virus 8 DNA in paraffin-embedded biopsies of HIV-associated and classical Kaposi's sarcoma by PCR.

BACKGROUND: Kaposi's sarcoma occurs in patients seropositive and seronegative for the human immunodeficiency virus (HIV) and has been associated with human herpes virus 8 (HHV8). The purpose of this study was to determine and to compare the amount of HHV8 DNA in formalin-fixed tissue sections of Kaposi's sarcoma. METHODS: From 27 biopsies of Kaposi's sarcoma patients, tissue sections were taken and deparaffinized. Four patients were HIV seronegative and 13 were HIV seropositive. After extraction of DNA copy numbers of HHV8 and beta-globin were determined in every sample by quantitative PCR ELISA using an internal quantitation standard. Results were expressed as HHV8 per beta-globin. RESULTS: No significant differences were found between biopsies from HIV-positive and HIV-negative patients (14.8+/-19.6 HHV8 per 1000 beta-globin in HIV-positive versus 18.0+/-23.5 in HIV-negative patients). CONCLUSIONS: These data suggest that HHV8 viral load in Kaposi's sarcoma is relatively low and does not differ in HIV-positive and HIV-negative samples. The importance of viral load determination for prognosis or treatment monitoring remains to be elucidated.

Extra c-myc oncogene copies in high risk cutaneous malignant melanoma and melanoma metastases.

Amplification and overexpression of the c-myc gene have been associated with neoplastic transformation in a plethora of malignant tumours. We applied interphase fluorescence in situ hybridization (FISH) with a locus-specific probe for the c-myc gene (8q24) in combination with a corresponding chromosome 8 alpha-satellite probe to evaluate genetic alterations in 8 primary melanomas and 33 advanced melanomas and compared it to 12 melanocytic nevi, 7 safety margins and 2 cases of normal skin. Additionally, in metaphase spreads of 7 melanoma cell lines a whole chromosome 8 paint probe was used. We investigated the functionality of the c-myc gene by detecting c-myc RNA expression with RT-PCR and c-myc protein by immunohistochemistry. 4/8 primary melanomas and 11/33 melanoma metastases showed additional c-myc signals relative to the centromere of chromosome 8 copy number. None of the nevi, safety margins or normal skin samples demonstrated this gain. In 2/7 melanoma cell lines (C32 and WM 266-4) isochromosome 8q formation with a relative gain of c-myc copies and a loss of 8p was observed. The highest c-myc gene expression compared to GAPDH was found in melanoma metastases (17.5%). Nevi (6.6%) and primary melanomas (5.0%) expressed the c-myc gene on a lower level. 72.7% of the patients with c-myc extra copies had visceral melanoma metastases (UICC IV), patients without c-myc gain in 35.0% only. The collective with additional c-myc copies also expressed the gene on a significantly higher level. These results indicate that a c-myc gain in relation to the centromere 8 copy number might be associated with advanced cutaneous melanoma.

Accidental radiation exposure and azoospermia.

Seven Georgian male soldiers (19-25 years old) had accidentally been exposed to radiation by Cs-137 between April 1996 and May 1997. No information about the exact time and duration of exposure was available. All patients presented with the subacute stage of Cutaneous Radiation Syndrome with deep painful ulcers on different body sites, predominantly on the legs. Semen analyses showed complete azoospermia in 4 patients, with elevated follicle-stimulating hormone (FSH) in 3 and elevated luteinizing hormone (LH) in 2 of them. One patient had severe oligozoospermia of 7 million sperm per mL, with normal sperm motility and morphology; his FSH and LH levels were elevated. One patient had complete normozoospermia, and the seventh patient had polyzoospermia of 340 million per mL; both of these patients had normal serum hormone levels. Only the patient with oligozoospermia reported a history of delayed testicular descent; his physical examination showed relatively soft and small testicles and a varicocele with considerable reflux. The physical andrological examinations were normal in the other 6 patients. It is very likely that the azoospermia in the 4 patients can be attributed to the radiation accident. In conclusion, it is essential to perform andrological examinations in patients who have been exposed to radiation even if there are only cutaneous injuries detectable, as a high percentage of them can show azoospermia.

The radiation accident in Georgia: clinical appearance and diagnosis of cutaneous radiation syndrome.

BACKGROUND: Eleven male Georgian soldiers were accidentally exposed to radiation by cesium 137 during their training in a military exercise camp in Lilo, Georgia between November 1996 and May 1997. OBJECTIVE: The characteristic sequelae of accidental cutaneous irradiation and available diagnostic methods are described. METHODS: Magnetic resonance imaging (MRI) of radiation ulcers was performed in all patients; thermography was performed in 2. In 7 patients ulcers and white macules were examined with high-frequency 20 MHz sonography; histologic results were obtained from all patients. RESULTS: Predominant lesions were radiation ulcers in 11 patients and white hairless macules in 7. MRI showed ulcers down to the muscles and an increase of signal intensity in the musculature in 9 cases. The corresponding muscle histology demonstrated vasculitis in 7 patients and necrosis in 2. In 2 patients, MRI signal intensity of the musculature was normal. In 3 patients, 20 MHz sonography showed dermal defects; 1 patient had cutaneous fibrosis. Thermography demonstrated hypothermic zones with extended inflammatory zones adjacent to the radiation ulcers in both patients examined. CONCLUSION: High-frequency 20 MHz sonography, MRI, and thermography are useful noninvasive methods for diagnosis of the extent of cutaneous radiation syndrome and for therapy planning.