Corneofungimetry: A bioassay for dermato-mycology.

Corneofungimetry is a bioassay in dermatomycology evaluating the antifungal effect of drugs. It is based on the method of culture of fungi on human stratum corneum harvested by cyanoacrylate skin surface strippings. Using corneofungimetry, it is possible to establish a classification of topical antifungals according to their spectrum of activity and their fungitoxicity. Similarly, the potency of oral antifungals can be evaluated at the level of the stratum corneum.     

Antifungal Susceptibility Testing of Filamentous Fungi

Methods developed for testing filamentous fungi (molds) include standardized broth microdilution (Clinical and Laboratory Standards Institute [CLSI] and European Committee for Antimicrobial Susceptibility Testing [AFST-EUCAST]) methods and disk diffusion (CLSI) methods. Quality control limits also are available from CLSI for MIC (minimal inhibitory concentration), MEC (minimal effective concentration), and zone diameters. Although clinical breakpoints based on correlations of in vitro results with clinical outcome have not been established, epidemiologic cutoff values have been defined for six Aspergillus species and the triazoles, caspofungin, and amphotericin B. The link between resistance molecular mechanisms, elevated MICs, and clinical treatment failure has also been documented, especially for Aspergillus and the triazoles. Other insights into the potential clinical value of high MICs have also been reported. Various commercial methods (e.g., YeastOne, Etest, and Neo-Sensitabs) have been evaluated in comparison with reference methods. This review summarizes and discusses these developments.     

A Case of Relapsed Chromoblastomycosis Due to Fonsecaea monophora: Antifungal Susceptibility and Phylogenetic Analysis

Chromoblastomycosis is a chronic cutaneous and subcutaneous mycosis. The management of this infection continues to be challenging because there is no consensus on the therapeutic regimen. We report here a case of a 69-year-old male patient with cauliflower-like lesions on his left leg and foot. He had already been treated with itraconazole at a dose of 200 mg/day for 5 months, with mycological cure for all the affected areas. However, the lesions relapsed at both sites, and treatment with itraconazole was resumed at the dose previously used. Initially, direct mycological examination, cultural, and microculture slide observation were performed. Afterward, sequencing of the ITS1-5.8S rDNA-ITS2 region of the fungal DNA and evaluation of its susceptibility to antifungal agents alone and in combination were performed. In direct mycological examination, the presence of sclerotic cells was verified, and the fungus was identified as Fonsecaea based on cultural and microscopic examinations. Identification as Fonsecaea monophora was confirmed after sequencing of the ITS region and phylogenetic analysis. The isolate was susceptible to itraconazole and terbinafine. The combinations of amphotericin B and terbinafine and terbinafine and voriconazole were synergistic. The use of drugs for which the causative agent is susceptible to singly or in combination may be an alternative for the treatment of mycosis. Furthermore, the identification of the agent by molecular techniques is important for epidemiological purposes. To the best of our knowledge, this is the first case of relapsed chromoblastomycosis caused by F. monophora in Brazil.     

Occurrence of Dermatomycoses and In-Vitro Therapeutic Efficacy of Three Antifungal Drugs on the Growth of Epidermophyton Floccosum

The occurrence of dermatomycoses and the in-vitro therapeutic efficacy of some antifungal agents on dermatomycotic organisms were investigated. Of the 550 primary school children screened, the incidence was one hundred (18%), 70 were males (representing 20% of the males screened) and 30 females (15% of the females sampled). The differences between male and female prevalence were insignificant. Three species of dermatophytes were isolated and identified. These were Microsporum canis, Trichophyton tonsurans and Epidermophyton floccosum. The antifungal agents tested on E. floccosum were griseofulvin, terbinafine and ketoconazole. They produced different sized zones of inhibition against the growth of E. floccosum. Griseofulvin exhibited a 50% inhibition of growth on E. floccosum at 63.00 mg/L. Terbinafine on the other hand exhibited varying levels of inhibition of growth at varying concentrations, at 0.07 mg/L, terbinafine achieved 46% inhibition of growth on E. floccosum. The drug achieved 100% inhibition of growth on the isolate at 61.81 mg/L. In the case of ketoconazole, 50% inhibition of growth was achieved at 100 mg/L while 100% inhibition of growth was achieved at 200 mg/L. The antifungal effects of the three drugs were confirmed by broth dilution tests where terbinafine was found to be fungistatic on the growth of E. floccosum at concentrations ranging from 0.013-1.700 mg/L and was fungicidal at concentrations ranging from 0.027-1.700 mg/L. Ketoconazole was found to inhibit the growth of E. floccosum at 0.003-1.700 mg/L and was fungicidal at concentrations ranging from 0.027-1.700 mg/L. It however did not succeed in killing the isolate under the same range of concentrations. Griseofulvin exhibited fungistatic effects on the growth of E. floccusum at 0.013-1.700 mg/L. In conclusion, ketoconazole and griseofulvin were found to be fungistatic and not fungicidal while terbinafine was both fungistatic and fungicidal on the pathogen. Terbinafine was found to be the most effective drug in inhibiting E. floccosum.     

Cryptococcus neoformans: The Model Organism for Yeast Antifungal Drug Susceptibility Testing

We describe an approach to antifungal susceptibility testing of the yeast Cryptococcus neoformans that shows promise for predicting the mycological response in patients to treatment. Quantitative cultures of the cerebrospinal fluid provide a direct measure of the patient's mycological response to treatment and have been used in multiple studies to identify the most promising antifungal drugs for subsequent testing in larger clinical studies. Using these quantitative measures of response, a modified macrobroth dilution assay system shows the potential for predicting the response of an individual patient to treatment with amphotericin B, fluconazole, or the combination of amphotericin B plus flucytosine. We describe this modified macrobroth dilution assay method, the statistical approach for assessing susceptibility, and the clinical decisions that can be guided by this in vitro antifungal drug susceptibility testing.     

EUCAST and CLSI: Working Together Towards a Harmonized Method for Antifungal Susceptibility Testing

The U.S. Clinical and Laboratory Standards Institute (CLSI) and the European Committee of Antimicrobial Susceptibility Testing (AFST-EUCAST) have developed broth microdilution methodologies for testing yeasts and filamentous fungi (molds). The mission of these methodologies is to identify in vitro antifungal resistance, which is accomplished by the use of either clinical breakpoints (CBPs), or to a lesser degree, epidemiologic cutoff values (ECVs). The newly adjusted and species-specific CLSI CBPs for Candida spp. versus fluconazole and voriconazole have ameliorated some of the differences between the two methodologies. In the absence of CBPs for mold testing, CLSI ECVs are available for six Aspergillus species versus the triazoles, caspofungin and amphotericin B. Recently, breakpoints were developed by the EUCAST for certain Aspergillus spp. versus amphotercin B, itraconazole and posaconazole, which to some extent are comparable to ECVs. We summarize these latest accomplishments, which have made possible the harmonization of some susceptibility cutoffs, if not methodologies for some agent/species combinations.     

Biofilm Formation by and Antifungal Susceptibility of Candida Isolates from Urine

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC₅₀ of <1 μg/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.     

Significance of Molecular Identification and Antifungal Susceptibility of Clinically Significant Yeasts and Moulds in a Global Antifungal Surveillance Programme

The increasing diversity of opportunistic fungi causing serious invasive fungal infections (IFI) has been documented. Accurate identification (ID) is important in guiding therapy, determining prognosis for IFIs and in epidemiological surveys. We assessed the utility of PCR-based methods for the ID of yeasts and moulds that either were uncommon, failed conventional ID, or represented unusual biochemical or phenotypic profiles of common species. Among 1,790 viable fungal clinical isolates received during the SENTRY Program in 2010, 322 strains from 40 study sites had ID confirmed by molecular methods. Isolates were previously identified in participant institutions. Yeasts that were not confirmed by morphology on CHROMagar, growth at 45?°C (Candida albicans/dubliniensis), or assimilation of trehalose (C. glabrata) as well as non-Candida yeasts and all moulds were amplified and sequenced using primers amplifying one or more of the following genes: ITS, 28S, β-tubulin (Aspergillus spp.), TEF (Fusarium spp.), IGS (Trichosporon spp.). The isolates selected for molecular ID included 149 isolates of Candida species, 77 of Aspergillus species, 73 non-Candida yeasts, and 23 other moulds (a total of 41 different species). Overall, the ID determined by the submitting site was confirmed for 189 isolates (58.7?%): Aspergillus spp. (64.1?% correct); Candida spp. (60.1?% correct); non-Candida yeasts (58.9?% correct); non-Aspergillus moulds (30.4?% correct). Species with high levels of concordance between conventional and molecular ID included A. fumigatus (95.0 %), C. lusitaniae (100?%), C. dubliniensis (92.3?%), C. kefyr (100?%), and C. neoformans (90.2?%). Only 50.0?% of isolates of C. albicans and 59.1?% of C. glabrata selected due to unusual phenotypic or biochemical features were found to be correctly identified by the submitting site. Molecular methods for the identification of fungal pathogens are an important adjunct to the conventional identification of many less common clinically relevant yeasts and moulds including species of Candida with unusual or erroneous phenotypic or biochemical profiles. Molecular confirmation of fungal identification is essential in epidemiological surveys such as SENTRY.     

Genotyping and Antifungal Drug Susceptibility of Trichosporon asahii Isolated from Chinese Patients

As a major pathogenic agent of trichosporonosis, Trichosporon asahii can cause life-threatening infection in immunocompromised patients. In this study, we analyzed the genotypes of the intergenic spacer (IGS) 1 region of the rRNA gene and the antifungal drug susceptibility of eight T. asahii isolates obtained from Chinese patients. Five genotypes were identified from the eight isolates, including three novel genotypes, three genotype 1, and two genotype 4. The eight T. asahii isolates were resistant to amphotericin B, 5-flucytosine, and terbinafine, but were highly sensitive to fluconazole (FLC), itraconazole (ITC), and voriconazole (VRC). The mean minimum inhibitory concentrations (MICs) of FLC and VRC were significantly lower than those reported in most other countries, while that of ITC was slightly higher. Our results suggest that genotypes of the T. asahii isolated from China are different from those of other countries, and azole drugs appeared to be more effective on the Chinese isolates. These results provide new insights into the epidemiology and antifungal treatment for T. asahii.     

Virulence Factors and Antifungal Susceptibility in Candida Species Isolated from Dermatomycosis Patients

Mycopathologia - Dermatomycoses caused by Candida spp. are increasingly common, however there are few reports in the literature regarding their epidemiology, pathogenesis and antifungal...     

Antifungal Susceptibility, Exoenzyme Production and Cytotoxicity of Novel Oximes against Candida

Novel oximes were synthesized, their in vitro antifungal activity against Candida was evaluated and their cytotoxicity was determined. The procedure used for the synthesis of the oximes is aligned with the current green chemistry trend; water is employed as the solvent in this reaction. The minimum inhibitory and minimum fungicidal concentrations of the oximes were evaluated using the CLSI M27-A3 method. The influence of these compounds on the inhibition of the production of hydrolytic enzymes, phospholipase and proteinase by Candida was also investigated. The compounds showed a good ability to inhibit phospholipase, with a 50 % reduction in most cases. However, the tested compounds did not affect proteinase. The current results showed a substantial reduction in the phospholipase production, which suggests that compounds of this class may interfere with host infection and disease progression. The oximes examined showed lower fungicidal activities than fluconazole but interfered significantly with the expression of phospholipase. Some of the oximes included in this study could be a suitable matrix for the development of novel antifungal compounds.     

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.Download video file.^{(44M, mov)}     

Regional Differences in Antifungal Susceptibility of the Prevalent Dermatophyte Trichophyton rubrum

Mycopathologia - In vitro susceptibility testing for Trichophyton rubrum has shown resistance to terbinafine, azoles and amorolfine, locally, but epidemiological cutoffs are not available. In order...     

Investigation of the Physiological,Biochemical and Antifungal Susceptibility Properties of Candida auris

Background

Candida auris is an emerging pathogen associated with outbreaks in clinical settings. Isolates of the pathogen have been geographically clustered into four clades with high intra-clade clonality. Pathogenicity varies among the clades, highlighting the importance of understanding these differences.

Objectives

To examine the physiological and biochemical properties of each clade of C. auris to improve our understanding of the fungus.

Methods

Optimal growth temperatures of four strains from three clades, East Asia, South Asia and South Africa, were explored. Moreover, assimilation and antifungal susceptibility properties of 22 C. auris strains from the three clades were studied.

Results

The optimal growth temperatures of all strains were 35–37 °C. Assimilation testing demonstrated that the commercial API ID 32 C system can be used to reliably identify C. auris based on the biochemical properties of the yeast. Notably, C. auris can be uniquely differentiated from commonly clinical fungi by its ability to assimilate raffinose and inability to utilize D-xylose, suggesting a useful simple screening tool. The antifungal susceptibility results revealed that all strains are resistant against fluconazole (minimal inhibitory concentration (MIC) 4 to?>?64 µg/mL) and miconazole (MIC 8 to?>?16 µg/mL), with strains from the Japanese lineage showing relatively lower MIC values (1–4 µg/mL). Conversely, itraconazole, voriconazole, amphotericin B, micafungin and caspofungin were active against most of the tested strains. On the clade level, East Asian strains generally showed lower MICs against azoles comparing to the other clades, while they displayed MICs against flucytosine higher than those of strains from South Africa and South Asia clades.

Conclusion

Our data suggest a simple identification approach of C. auris based on its physiological and biochemical properties and highlight aspects of C. auris population from various clades.

     

Effects of Photodynamic Inactivation on the Growth and Antifungal Susceptibility of Rhizopus oryzae

Mycopathologia - Mucormycosis is an aggressive and high-mortality opportunistic fungal infection, especially in immunocompromised patients. Conventional antifungals or surgery showed a limited...