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 Approaches in the Diagnosis of Dermatophytosis

Dermatophytosis is one of the most common infectious diseases in the world and can be caused by several dermatophyte species. These species are closely related in genetic structure in spite of different phenotypic and ecological features. The morphological similarity, variability, and polymorphism of dermatophytes have meant that species identification for dermatophytes is time consuming and requires a significant degree of knowledge and technological expertise. Molecular biology-based techniques have solved problems concerning the morphology-based identification of dermatophytes and have improved our knowledge on the epidemiology of dermatophytosis. Further development of molecular diagnosis of dermatophytosis requires the investigation of additional molecular markers for diagnostic tools targeting multiple loci as well as the improvement of techniques.     

Diagnosis of Dermatophytosis Using Molecular Biology

Identification of fungi in dermatological samples using PCR is reliable and provides significantly improved results in comparison with cultures. It is possible to identify the infectious agent when negative results are obtained from cultures. In addition, identification of the infectious agent can be obtained in 1 day. Conventional and real-time PCR methods used for direct fungus identification in collected samples vary by DNA extraction methods, targeted DNA and primers, and the way of analysing the PCR products. The choice of a unique method in a laboratory is complicated because the results expected from skin and hair sample analysis are different from those expected in cases of onychomycosis. In skin and hair samples, one dermatophyte among about a dozen possible species has to be identified. In onychomycosis, the infectious agents are mainly Trichophyton rubrum and, to a lesser extent, Trichophyton interdigitale, but also moulds insensitive to oral treatments used for dermatophytes, which renders fungal identification mandatory. The benefits obtained with the use of PCR methods for routine analysis of dermatological samples have to be put in balance with the relative importance of getting a result in a short time, the price of molecular biology reagents and equipment, and especially the time spent conducting laboratory manipulations.     

Bedside Diagnosis for Disseminated Deep Dermatophytosis: a Case Series Study

Mycopathologia - Deep cutaneous fungal infections including deep dermatophytosis are responsible for significant morbidity and mortality, especially in immunocompromised patients. Variable and...     

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.     

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.     

Pathogenesis of Dermatophytosis

Despite the superficial localization of most dermatophytosis, host-fungus relationship in these infections is complex and still poorly elucidated. Though many efforts have been accomplished to characterize secreted dermatophytic proteases at the molecular level, only punctual insights have been afforded into other aspects of the pathogenesis of dermatophytosis, such as fungal adhesion, regulation of gene expression during the infection process, and immunomodulation by fungal factors. However, new genetic tools were recently developed, allowing a more rapid and high-throughput functional investigation of dermatophyte genes and the identification of new putative virulence factors. In addition, sophisticated in vitro infection models are now used and will open the way to a more comprehensive view of the interactions between these fungi and host epidermal cells, especially keratinocytes.     

Use of Endo-Ovarian Tissue Biopsy and Pelvic Aspirated Fluid for the Diagnosis of Female Genital Tuberculosis by Conventional versus Molecular Methods

Background

Til date, none of the diagnostic techniques available for the detection of female genital tuberculosis (FGTB) are 100% accurate. We therefore, proposed to use the endometrial tissue biopsies (ETBs), ovarian tissue biopsies (OTBs) and pelvic aspirated fluids (PAFs) for the diagnosis of FGTB among infertile women by conventional versus molecular methods.

Methodology/Principal Findings

A total of 302 specimens were collected both from 202 infertile women highly suspected of having FGTB on laparoscopy examination and 100 control women of reproductive age. Out of 302 specimens, 150 (49.67%) were ETBs, 95 (31.46%) were OTBs and 57 (18.87%) were PAFs. All specimens were tested by conventional techniques, later compared with multi-gene PCR for the detection of Mycobacterium tuberculosis (MTB) and correlated with laparoscopic findings. The presence of MTB DNA was observed in 49.5% of ETBs, 33.17% of OTBs and 5.44% of PAF specimens collected from highly suspected FGTB patients. All women of control group were confirmed as negative for tuberculosis. The conventional methods showed 99% to 100% specificity with a low sensitivity, ranging from 21.78% to 42.08% while hematoxylin and eosin staining showed a sensitivity of 51.48%. Multi-gene PCR was found to have much higher sensitivity of 70.29% with MTB64 gene, 86.63% with 19 kDa antigen gene at species and TRC4 element at regional MTB complex and 88.12% with 32 kDa protein gene at genus level. The specificity of multi-gene PCR was 100%. Compared with culturing and Ziehl-Neelsen''s staining, multi-gene PCR demonstrated improvement in the detection of FGTB (χ² = 214.612, 1 df, McNemar''s test value <0.0001).

Conclusions Significance

We suggest site specific sampling, irrespective of sample type and amplification of the 19 kDa antigen gene in combination with TRC4 element as a successful multi-gene PCR for the diagnosis of FGTB and differentiation of mycobacterial infection among endo-ovarian tissue biopsies and PAFs taken from infertile women.     

基因表达常规分析方法概述

在基因表达的分析中,RNA转录的稳态水平是检测细胞株和组织的基因表达活性的最方便的参数之一.本概述了用于检测mRNA丰度的常规分析方法,即Northern印迹、RNase保护试验和实时定量PCR技术,特别是为对数量不多的基因进行表达情况的测定时提供了较为实用的方法.     

Immunoprophylaxis of Dermatophytosis in Animals

Dermatophytosis is a relatively common disease in many countries occurring endemically both in companion and food animals. Fungi belonging to the genera Trichophyton and Microsporum are most often isolated from clinical cases. Measures to control and prevent dermatophytosis include sanitation, hygienic measures and treatment. In some countries, successful control and eradication have been achieved by mass vaccination of cattle and fur-bearing animals. Vaccines containing live attenuated cells of the fungus stimulate a cell-mediated immune response conferring long-lasting protection against subsequent challenge by the homologous fungus. A delayed type hypersensitivity (DTH) skin test using appropriate dermatophyte antigens is suitable to assess the response. Inactivated dermatophyte vaccines are available for use in cattle, horse, dog, and cat in some countries. However, the scientific literature is scarce making it difficult to conclude on efficacy and appropriate use. Current vaccines are all first generation vaccines. Attempts have been made to prepare subunit vaccines based on new knowledge about virulence factors like the keratinases, so far with limited success. Candidate antigens must be able to stimulate a strong T helper 1 cell response and future research should focus on identification of major T-cell epitopes that specifically elicit a DTH reaction. Dermatophytosis is a zoonotic disease. In Norway and a few other countries, systematic vaccination against cattle ringworm has almost eliminated the disease, and ringworm in man caused by T. verrucosum is almost nonexistent. A similar benefit could be expected if a safe and efficacious vaccine was available for Microsporum canis infection in cats and dogs.     

Methods for Accelerating the Fluorescent-Antibody Test for Rabies Diagnosis

The time required to perform the fluorescent-antibody test for rabies was reduced by eliminating acetone fixation of the brain impressions and by incubating the conjugate-impression reaction at room temperature for only 10 min. Elimination of the preliminary acetone fixation had no effect on the diagnosis of impression smears from 246 mammalian brains by immunofluorescence. Staining at 37 C for 30 min and staining at room temperature for 10 min were found to be equally effective in the examination of impression smears from 161 brain samples. The procedure, as modified, shortens the time required for the diagnosis of rabies by immunofluorescence from about 5.5 hr to approximately 45 min.     

New Rapid Methods for Tissue Diagnosis

Two new methods applicable to the staining of fixed and fresh frozen tissue sections are presented herein. In addition certain improvements are suggested for the technic reported by Geschickter, Walker, Hjort and Moulton (1931). In brief the procedures are as follows:

The thionin eosinate method of Geschickter et al (1931). This procedure has been modified as follows:

A mixture of diethylene glycol, 40 parts, ethylene glycol, 40 parts, and grain alcohol, 20 parts is superior to ethylene glycol, 80 parts, and ethyl alcohol, 20 parts, as a solvent for the compound stain in that the staining is intensified.

Ethylene glycol monobutyl ether supplants diethylene glycol monobutyl ether because of its lower viscosity.

Ethyl phthalate replaces butyl phthalate on account of a more satisfactory viscosity.

The methyl green eosinate procedure is the same as the modified thionin eosinate method except for the following variations:

The staining time is increased to one minute.

Decolorization and washing are reduced to about 15 seconds.

The hematoxylin-eosin method. After cutting, the tissue sections are carried thru the following steps:

Unfold in water; transfer to formalin (4 to 40%) for at least 30 seconds; stain in hematoxylin (Harris) for 30 to 60 seconds; wash in water, 5 seconds; decolorize in 0.1% HC1 or saturated aqueous picric acid, 5 seconds; wash in water, S seconds; float in 0.5% ammonia, 5 to 10 seconds; wash in water, 5 seconds; stain in 5% aqueous eosin, 15 seconds¹; wash in water, 5 to 10 seconds; dehydrate in a mixture of diethylene glycol, 30 parts, and ethyl alcohol, 70 parts, 5 to 10 seconds; dehydrate in ethylene glycol monobutyl ether, 5 to 10 seconds; clear in ethyl phthalate, 5 to 10 seconds; float on a glass slide, blot with photographic lintless blotter, place a drop of neutral gum damar on the section, and cover with glass cover slip.     

New Rapid Methods for Tissue Diagnosis

Two new methods applicable to the staining of fixed and fresh frozen tissue sections are presented herein. In addition certain improvements are suggested for the technic reported by Geschickter, Walker, Hjort and Moulton (1931). In brief the procedures are as follows:

1. The thionin eosinate method of Geschickter et al (1931). This procedure has been modified as follows:
   1. A mixture of diethylene glycol, 40 parts, ethylene glycol, 40 parts, and grain alcohol, 20 parts is superior to ethylene glycol, 80 parts, and ethyl alcohol, 20 parts, as a solvent for the compound stain in that the staining is intensified.
   2. Ethylene glycol monobutyl ether supplants diethylene glycol monobutyl ether because of its lower viscosity.
   3. Ethyl phthalate replaces butyl phthalate on account of a more satisfactory viscosity.
2. The methyl green eosinate procedure is the same as the modified thionin eosinate method except for the following variations:
   1. The staining time is increased to one minute.
   2. Decolorization and washing are reduced to about 15 seconds.
3. The hematoxylin-eosin method. After cutting, the tissue sections are carried thru the following steps:

Unfold in water; transfer to formalin (4 to 40%) for at least 30 seconds; stain in hematoxylin (Harris) for 30 to 60 seconds; wash in water, 5 seconds; decolorize in 0.1% HC1 or saturated aqueous picric acid, 5 seconds; wash in water, S seconds; float in 0.5% ammonia, 5 to 10 seconds; wash in water, 5 seconds; stain in 5% aqueous eosin, 15 seconds¹; wash in water, 5 to 10 seconds; dehydrate in a mixture of diethylene glycol, 30 parts, and ethyl alcohol, 70 parts, 5 to 10 seconds; dehydrate in ethylene glycol monobutyl ether, 5 to 10 seconds; clear in ethyl phthalate, 5 to 10 seconds; float on a glass slide, blot with photographic lintless blotter, place a drop of neutral gum damar on the section, and cover with glass cover slip.     

Accuracy of Screening Methods for the Diagnosis of Breast Disease

Clinical examination, thermography, and 70-mm. mammography were performed in 891 patients—414 presented to hospital with symptoms of breast disease and 477 were asymptomatic. Comparison of the diagnostic accuracy of these methods showed that neither thermography nor 70-mm. mammography has a useful place as an isolated screening procedure for breast cancer. In fact, we consider such a policy dangerous.     

Comparative Evaluation of Five Serological Methods for the Diagnosis of Sporotrichosis

The diagnosis of sporotrichosis can be time consuming. Serological procedures could facilitate the rapid and accurate diagnosis of this disease. A slide latex agglutination (SLA) test for sporotrichosis was developed and compared with the tube agglutination (TA), complement fixation (CF), and immunodiffusion (ID) tests in the serological study of 80 proven human cases of sporotrichosis representing the cutaneous, subcutaneous, and extracutaneous forms of the disease. In addition, the indirect fluorescent antibody (IFA) technique was applied to 61 case sera. In the SLA test, latex particles sensitized with culture filtrate antigens from the yeast form of Sporothrix schenckii (B 959) detected 94% of the cases, as compared to 96% of the cases detected by the TA test, 68% by the CF test, and 56% by the ID test. The IFA test detected 90% of the 61 cases. The SLA and ID tests were specific, showing no reactions with sera from 86 persons with no disease or with diseases other than sporotrichosis. Because of its sensitivity, specificity, ease of performance, and ability to provide results in 5 min, the SLA test is highly recommended for routine use in the clinical laboratory.     

Update in Antifungal Therapy of Dermatophytosis

Treatment of dermatophyte infection involves primarily oral and/or topical formulations of azoles or allylamines, particularly itraconazole and terbinafine. Topical medications applied once or twice daily are the primary treatment indicated for tinea corporis/cruris, and tinea pedis/manuum. Use of oral antifungals may be practical where the tinea involvement is extensive or chronic, or where application of a topical is not feasible. For tinea unguium (onychomycosis) and tinea capitis, oral therapies are the primary treatments provided. Recently, topical amorolfine and ciclopirox formulations have been approved for use in milder onychomycosis cases, and their role in the treatment of the different clinical forms of onychomycosis is currently being defined. Relapse of infection remains a problem, particularly with tinea pedis/unguium. Appropriate follow-up duration and education of patients on proper foot hygiene are also important components in providing effective therapy.     

Dermatophytosis of pigs by Trichophyton mentagrophytes

We describe the isolation of Trichophyton mentagrophytes in two family-operated farms where the animals were suffering skin ailments characterized by a swelling and a reddening of the back and flanks.This condition affected 2 and 5% respectively of the animals on the farms, the younger ones being more frequently affected.     

奶牛结核病诊断方法的比较研究

旨在比较目前实验室用于检测牛结核方法及PPD皮内变态反应检测牛结核的敏感性和特异性.对145头进行了PPD结核菌素实验的牛进行了IFN-γ体外释放试验,ELISA方法检测ESAT-6,CFP-10,MPB70,MPB83四个蛋白的抗体,胶体金检测两种相似蛋白MPB70和MPB83.结果表明,IFN-γ体外释放试验的敏感性和特异性最高,与PPD皮内变态反应有较高的符合性.在抗体检测方面,iELISA的敏感性高于胶体金方法.虽然抗体检测(iELISA和胶体金方法)比细胞介导的免疫方法(IFN-γ和PPD)敏感性要低,前者更适合于检测处于结核病程发展后期的样本,并且可以有效降低假阳性反应.