Susceptibility of Candida spp. clinical isolates to antimycotics and disinfectants

The incidence of candidiasis among immunocompromised patients and emergence of antimycotics resistant strains has increased significantly. The aims of this study were: to examine the in vitro activity of antimycotics and biocides against Candida clinical isolates; to detect cross-resistance of fungi to these preparations and to estimate whether disinfectants applied in hospital areas are active against clinical Candida isolates. In vitro susceptibility of 102 Candida isolates to eight antimycotics was examined by Etest and ATB Fungus. Sensitivity of these strains to four disinfectants and an antiseptic agent was tested according to EN 1275:2005. Amphotericin B, caspofungin and 5-fluorocytosine were the most effective antimycotics against all Candida isolates. Resistance to itraconazole and fluconazole was observed among C. krusei and C. glabrata. The MICs (Minimal Inhibitory Concentrations) for ketoconazole, voriconazole and posaconazole against Candida albicans ranged: 0.003 - >32 μg/ml and one strain was resistant to three agents tested. All analysed Candida strains were sensitive to biocides containing either chlorine, aldehyde, alcohol mixtures, glucoprotamin or chlorhexidine gluconate with isopropanol. Sensitivity to these agents was observed at concentrations lower than those concentrations recommended by manufacturers to achieve proper biocidal activity to those preparations. Our data suggest that these disinfectants can be effectively applied in clinical wards to prevent nosocomial Candida infections.     

Trapped in keratin; a comparison of dermatophyte detection in nail, skin and hair samples directly from clinical samples using culture and real-time PCR

Traditionally, laboratory detection and identification of dermatophytes consists of culture and microscopy which yields results within approximately 2-6 weeks. In 2007 our medical microbiological diagnostic laboratory implemented a molecular method for the detection of dermatophytes. A real-time PCR assay was developed which simultaneously detects and identifies the most prevalent dermatophytes directly in nail, skin and hair samples and has a turnaround time of less than two days. For 1437 clinical samples, received by our diagnostic laboratory, we compared the results obtained from both culture and real-time PCR. This study showed that real-time PCR significantly increased the detection rate of dermatophytes compared to culture. Furthermore, excellent concordance between culture and real-time PCR identification was achieved.     

Antimicrobials and in vitro systems: antibiotics and antimycotics alter the proteome of MCF-7 cells in culture

Cell culture is widely used to study gene or protein changes in response to experimental conditions. The value of such experiments depends on stringent control and understanding of the in vitro environment. Despite well-documented evidence describing toxic effects in the clinical setting, antibiotics and antimycotics are routinely used in cell culture without regard for their potential toxicity. We cultured MCF-7 breast cancer cells in the presence/absence of antibiotics (penicillin/streptomycin) and/or the antimycotic amphotericin B. Differential protein expression was assessed using 2D-DIGE and MALDI-MS/MS. Antibiotics caused 8/488 spots (1.3% of the protein) to be generally down-regulated. The affected proteins were principally chaperones and cytoskeletal. In marked contrast, amphotericin B induced a more dramatic response, with 33/488 spots (9.5% of the total protein) generally up-regulated. The proteins were mostly involved in chaperoning and protein turnover. Combining antibiotics and amphotericin B had little overall effect, with only one (unidentified) protein being up-regulated. As this study identifies differential protein expression attributable to antibiotics/antimycotics, we urge caution when comparing and interpreting proteomic results from different laboratories where antibiotics/antimycotics have been used. We conclude that as antibiotics and antimycotics alter the proteome of cultured cells in markedly different ways their use should be avoided where possible.     

Molecular differentiation of Keratinomyces (Trichophyton) species

The taxonomy of the form-genus Keratinomyces (Trichophyton) within the group of the dermatophytes is based on morphological features which remain insufficient for the distinction of these anamorphic species. The three species included in the genus Keratinomyces, namely K. ajelloi, K. ceretanicus and K. longifusus were examined by means of their mitochondrial-like DNA diversity and compared to few other dermatophytes. The analysis of the mtDNA restriction fragments confirmed that the three species are different and well separate from the other dermatophytes.     

Influence of the soil on certain dermatophytes and their evolutional trend

Conclusions From our experiments it could be seen that a quite definite relationship exists between various dermatophytes and the soil which is expressed on the one side by antagonistic action of the microflora in the soil inhibiting the growth of dermatophytes and on the other, by ability of some dermatophytes to persist in the soil or to use the soil as habitat in their saprophytic life. This condition appears to be the reflection of the evolutive tendency of dermatophytes to develop from its primary habitat, the soil, to the parasitic life on man and animal. It is persumed that this evolution of dermatophytes is based on mutations of gens as one of the fundamental phenomenons in nature. It is obvious that this evolution of dermatophytes is not completed. Our experiments regardingT. mentagrophytes indicate that this species is at present still to a certain extent in a transitional evolutive stage with retained but limited ability to the life in the soil and with an evident tendency for adaption on small rodents and human beings. In this course of development on a lower level isK. ajelloi andM. gypseum with full ability of saprophytic life in the soil and with slowly but increasing pathogenicity for men and animals. E. I. Grin, Prof. of Dermatology, Med. Faculty, Univ. Sarajevo, Director Institute of Dermatovenerology. L. Oegovi, Prof. of Intern. Dis., Veter. Faculty, Univ. Sarajevo, Chief of Dept. of Mycology Inst. of Dermatovenerology.     

实验动物皮肤病原真菌检测方法的改良

目的对实验动物皮肤病原真菌2种培养方法进行了比较。方法将采集到的3只皮肤真菌感染病兔样品经由沙氏平皿法和沙氏试管斜面培养法分别进行培养。结果在3只真菌感染病兔中应用试管斜面法我们只检测到1例皮肤病原真菌阳性,而采用沙氏平皿法3例阳性全部检出。结论结合临床检测经验,我们认为本研究的沙氏平皿法优于沙氏试管斜面法,在实验动物皮肤病原真菌常规检测中具有推广应用价值。     

In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes

To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis‐causing non‐dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non‐dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non‐azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis.     

Effectiveness of five antidandruff cosmetic formulations against planktonic cells and biofilms of dermatophytes

This study aimed to investigate the in vitro antifungal effectiveness of five different formulations against dandruff and ringworm dermatophytes. Candida albicans was also included in our assays. Fungal susceptibility tests were performed with planktonic cells and biofilms of reference strains. Microbiological and physicochemical quality parameters were assessed for all formulations. Our data indicated that the formulations were effective against the dermatophytes strains, and to our knowledge, the effectiveness of cosmetic formulations against fungal biofilms is shown for the first time. The formulations were considered effective against the explored dermatophytes and were considered safe given the adequate microbiological and physicochemical characteristics shown in the proposed assays.     

In vitro susceptibility to 9 antifungal agents of 14 strains of Zygomycetes isolated from clinical specimens

Fourteen clinical isolates of Zygomycetes were tested for their in vitro susceptibility to nine antifungal agents. Susceptibility assessment was performed using a microtiter broth dilution method. Synthetic broth with YNB and glucose was used for 5-fluorocytosine and BHI broth for all the other antimycotics. Amphotericin B exhibited the strongest activity against all isolates tested. MIC values of other two polyenes — nystatin and pimaricin — ranged within the susceptibility limits, with a little pronounced higher activity of pimaricin. The isolates of the genusAbsidia andSyncephalastrum were well sensitive to all antimycotics with the exception of 5-fluorocytosine and naftifine. A very weak or zero growth inhibitory effect against all members of the generaMucor andRhizopus was found in azoles, 5-fluorocytosine and naftifine.     

Dermatophytes isolated from patients in University Hospital,Kuala Lumpur,Malaysia

A total of 576 dermatophytes were isolated from patients with a variety of skin infections from January 1993 to May 2000. Ten species of dermatophytes were identified: Epidermophyton floccosum (0.7%), Microsporum audouinii (1.1%), M. canis (3.1%), M. gypseum (0.3%), Trichophyton concentricum (3.5%), T. equinum (0.2%), T. mentagrophytes (36.1%), T. rubrum (53.8%), T. verrucosum (0.2) and T. violaceum (1.0%). The body sites most frequently affected by dermatophytes were the buttocks, nails and trunk. Anthropophilic dermatophytes made up 60.1% of the isolates; the most common species was T. rubrum, T. mentagrophytes and M. canis were the two main zoophilic dermatophytes. T. mentagrophytes was isolated from all body sites except the scalp. M. canis was found to be associated with domestic dogs and was not isolated from ethnic Malays. The only geophilic dermatophyte was M. gypseum, an uncommon dermatophyte associated with tinea pedis.     

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).     

Susceptibility testing of dermatophytes

The dermatophytes are a specialized group of fungi that infect the keratinized tissues of humans (hair, nails, and skin) and cause superficial infections. Although several studies have been conducted to develop methods to determine the susceptibilities of yeast and filamentous fungi, similar studies for dermatophytes have only recently taken place. We review how susceptibility testing of dermatophytes was developed and how it has already been applied to clinical samples. We also review recent advances in the development of disk diffusion and colorimetric methodologies for determining the antifungal susceptibility of dermatophytes. With several agents now available for treating infections due to dermatophytes, susceptibility testing will serve as a valuable tool for clinicians as they choose the most appropriate treatment option. Studies are still needed to establish interpretive breakpoints for antifungal agents used in the treatment of superficial fungal infections.     

The effect of selected human steroid hormones upon the growth of dermatophytes with different adaptation to man

The inhibitory effect exerted by steroid hormones on thein vitro growth characteristics of dermatophytes is poorly understood. As a hypothesis this inhibition could result from fungal adaptation to the human host. Therefore, in this study the susceptibility of representative anthropophilic, zoophilic and geophilic dermatophytes to hormonal inhibition was compared. As a result, in agar dilution assaysprogesterone,testosterone, andestradiol proved to reduce fungal growth, whereashydrocortisone had no such effect. In general, anthropophilic dermatophytes were shown to be more responsive to steroid hormones than geophilic species, suggesting a correlation of steroid susceptibility with adaptation to human skin. However, since fungal response to hormones consisted of growth inhibition and occurred only at steroid concentrations much higher than present in human skin, it cannot be assumed to contribute to this adaptation.     

Cutaneous phaeohyphomycosis caused byPhialophora richardsiae and the effect of topical clotrimazole in its treatment

A 15 year-old girl and her 50-year-old mother from Jabalpur, India, were found to have phaeohyphomycotic skin lesions due toPhialophora richardsiae. Among the antimycotics testedin vitro, clotrimazole was found to be most active. Topical application of clotrimazole cream cured the lesions completely.     

Considerations sur la parabiose des dermatophytes dans les cultures cellulaires

Résumé Dans le but d'étudier l'action des dermatophytes sur les cultures de tissues, les auteurs ont utilisé 18 espèces et des cellules épithéliales humaines (à type de HeLa et Detroit 6) et animale (rein de lapin) et des fibroblastes de l'embryon humain, de poulet et de rat. L'ensemencement des dermatophytes choisis a été faite en des tubes contenant les cultures cellulaires, parfois additionnées de milieu liquide. L'effet protéolytique le plus rapide a été constaté pour les dermatophytes zoophyles, tels queTr. mentagrophytes etTr. quinckeanum et surtout vis-à-vis des cellules HeLa, tandis que les espèces d'origine anthropophyle ont montré une action cytotoxique un peu plus ralentie. Parmi les géodermatophytes, l'action exercée parTr. terrestre etKeratinomyces ajelloi s'approche de celle produite par les espèces d'origine animale, fait qui pourrait plaider, selon l'opinion des auteurs, en faveur de la prochaine adaptation à la vie parasitaire de ces deux derniers champignons. La lyse des cellules obtenue à la suite des produits metaboliques élaborés parTr. megninii fait incadrer ce dermatophyte dans le groupe des espèces d'origine humaine.

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Summary The authors report their first observations on the cytotoxic activity of the dermatophytes inoculated in various tissue cultures. The 18 species of dermatophytes were isolated from different mycotic infections of human and animal origin; the tissue cultures were prepared from different strains of cells: HeLa, Detroit 6, rabbit kidney, human and animal embryos (rat, chicken). The contact of these cells with the dermatophytes induces a lysis which is earliest with the zoophylic species asTr. mentagrophytes andTr. quinckeanum. The geophylic dermatophytes had almost the same proteolytic activity as the human ones, fact that justifies their place in this group of fungi.Moreover the way in whichTr. terrestre andKeratinomyces ajelloi, usually saprophytes of the soil, acted on the tissue culture, as zoophylic species did, enables the authors to consider these fungi (and probably other geophylic dermatophytes), to be adapted to the parasitic life. By this same method of investigation, the problem of human or animal origin ofTr. megninii is studied and discussed.

     

The isolation of keratinophilic fungi from African soils

Summary Twenty-seven soil samples from Kenya and 9 from Egypt were investigated for the presence of keratinophilic and pathogenic fungi. No systemic pathogenic fungi was obtained, while a total of 25 dermatophytes belonging to four species was isolated. The majority of the Kenya isolates belonged toM. gypseum, while Egyptian isolates belonged toT. mentagrophytes. Shady sites frequented by humans or animals produced higher incidences of dermatophytes in Kenya than those of the unshady isolated areas.     

改良的KANE/FISHER系统对常见皮肤癣菌的鉴定

目的 以改良的KANE/FISHER系统对常见皮肤癣菌进行鉴定,探索建立一个临床实用的标准化皮肤癣菌鉴定系统.方法 选取KANE/FISHER鉴定系统中的6种主要培养基对皮肤癣菌ATCC株和经初步鉴定的临床株共63株进行培养鉴定.结果 改良的KANE/FISHER鉴定系统基本可以将实验菌株鉴定到种的水平.结论 改良的KANE/FISHER鉴定系统对常见皮肤癣菌能做到种的鉴定,可作为标准化鉴定方法在实验室中应用.     

实验动物皮肤病原真菌DNA快速少量提取法

PCR技术应用于实验动物皮肤病原真菌检测,方法简单、省时。但是,真菌的DNA提取较为困难。本文推荐一种既简单又经济快速的提取皮肤真菌DNA的方法,并能成功用于实验动物皮肤病原真菌质量检测研究。     

Dermatophytes isolated from patients in University Hospital,Kuala Lumpur,Malaysia

A total of 576 dermatophytes were isolated from patients with a variety of skin infections from January 1993 to May 2000. Ten species of dermatophytes were identified: Epidermophyton floccosum (0.7%), Microsporum audouinii (1.1%), M. canis (3.1%), M. gypseum (0.3%), Trichophyton concentricum(3.5%), T. equinum (0.2%), T. mentagrophytes (36.%), T . rubrum (53.8%), T. verrucosum (0.2) and T. violaceum (1.0%). The body sites most frequently affected by dermatophytes were the buttocks, nails and trunk. Anthropophilic dermatophytes made up 60.1% of the isolates; the most common species was T. rubrum, T. mentagrophytes and M. canis were the two main zoophilic dermatophytes. T. mentagrophytes was isolated from all body sites except the scalp. M. canis was found to be associated with domestic dogs and wasnot isolated from ethnic Malays. The only geophilic dermatophyte was M. gypseum, an uncommon dermatophyte associated with tinea pedis.This revised version was published online in October 2005 with corrections to the Cover Date.