Efficacy of antifungal agents against fungal spores: An in vitro study using microplate laser nephelometry and an artificially infected 3D skin model.

Dermal fungal infections seem to have increased over recent years. There is further a shift from anthropophilic dermatophytes to a growing prevalence of zoophilic species and the emergence of resistant strains. New antifungals are needed to combat these fungi and their resting spores. This study aimed to investigate the sporicidal effects of sertaconazole nitrate using microplate laser nephelometry against the microconidia of Trichophyton, chlamydospores of Epidermophyton, blastospores of Candida, and conidia of the mold Scopulariopsis brevicaulis. The results obtained were compared with those from ciclopirox olamine and terbinafine. The sporicidal activity was further determined using infected three-dimensional full skin models to determine the antifungal effects in the presence of human cells. Sertaconazole nitrate inhibited the growth of dermatophytes, molds, and yeasts. Ciclopirox olamine also had good antifungal activity, although higher concentrations were needed compared to sertaconazole nitrate. Terbinafine was highly effective against most dermatophytes, but higher concentrations were required to kill the resistant strain Trichophyton indotineae. Sertaconazole nitrate, ciclopirox olamine, and terbinafine had no negative effects on full skin models. Sertaconazole nitrate reduced the growth of fungal and yeast spores over 72 h. Ciclopirox olamine and terbinafine also inhibited the growth of dermatophytes and molds but had significantly lower effects on the yeast. Sertaconazole nitrate might have advantages over the commonly used antifungals ciclopirox olamine and terbinafine in combating resting spores, which persist in the tissues, and thus in the therapy of recurring dermatomycoses.

Current knowledge on the etiology and epidemiology of Scopulariopsis infections.

Scopulariopsis is a common fungus in the environment, characterized by its intrinsic resistance to the available antifungal drugs. Around 70 cases of infection by this fungus have been described in the literature. Pulmonary and disseminated infections are the most common and their treatment is difficult; therefore, very diverse approaches have been taken, with varied results. A successful outcome has been reported in only a few cases, generally attributed to a multitreatment strategy combining medical and surgical procedures that ultimately led to the resection of the infected tissue if possible, identification of the mould, and an aggressive long-term antifungal therapy. Although most of the infections are caused by Scopulariopsis brevicaulis, a few other species have also been linked to these cases, although molecular evidence has not been proven for all of them. On this basis, more knowledge on the epidemiology, presentation, diagnosis, treatment, and prognosis of these unusual infections would improve their management. This review aims to compile the current data on Scopulariopsis infections.

In vitro photodynamic inactivation (PDI) of pathogenic germs inducing onychomycosis.

Onychomycosis is a fungal nail infection caused primarily by the dermatophytes Trichophyton rubrum and Trichophyton interdigitale or, less frequently, by molds like Aspergillus spp. and Scopulariopsis brevicaulis. Photodynamic treatment of onychomycosis is considered a promising future therapy to overcome the frequent failure of currently used antifungals. In this study, we tested the potential of three photosensitizers for photodynamic inactivation of the onychomycosis causing pathogens T. rubrum, T. interdigitale and S. brevicaulis. Photosensitizers used are 10,15,20-Tetrakis(1-methylpyridinium-4-yl) porphyrintetra(p-toluenesulfonate) (TMPyP), 5,10,15-tris-(1-methylpyridinium-2-yl)corrolato-(trans-dihydroxo)phosphorus(V) (PCor+) and 2',4',5',7'-tetrabromo-3',6'-dihydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one (Eosin Y). The phototoxic effects caused by the cationic photosensitizers (PCor+ and TMPyP) were tested on suspension cultures of spores as well as on fungi during growth on surfaces where both photosensitizers cause high phototoxicity. The anionic Eosin Y was tested on surface-growing fungi only and induces remarkable phototoxic effects on dermatophytes and molds. In all cases, no spore regrowth was detected after PDI. This study is considered a first step towards successful and cost efficient treatment of onychomycosis.

Singlet oxygen luminescence kinetics under PDI relevant conditions of pathogenic dermatophytes and molds.

A treatment of onychomycosis using the photodynamic effect would be a favorable alternative to currently used antimycotic drugs. This study should be considered as a first step towards development and control of an efficient photodynamic inactivation of onychomycosis causative pathogens. Here, we evaluate the usage of time-resolved 2D singlet oxygen luminescence detection in combination with 2D fluorescence scanning as a tool to understand the behavior of the photosensitizer when applied to fungi on Petri dishes. To investigate the interaction of photosensitizer with fungi in various concentrations and in different stages of live, a photodynamic inactivation was avoided by keeping the samples in darkness. Scans of singlet oxygen luminescence and photosensitizer fluorescence were performed over a period of 24days. Two different photosensitizer, a cationic porphyrin and cationic corrole and two fungi strains, the dermatophyte Trichophyton rubrum and the mold Scopulariopsis brevicaulis, were investigated in this study. The two-dimensional correlation of photosensitizer fluorescence and singlet oxygen luminescence revealed differences in the diffusion of both photosensitizer. Even though the singlet oxygen luminescence was quenched with increasing growth of fungi, it was found that the kinetics of singlet oxygen luminescence could be detected on Petri dishes for both photosensitizers and both fungi strains for up to seven days.

Deprotonation and protonation of humic acids as a strategy for the technological development of pH-responsive nanoparticles with fungicidal potential.

Humic acids (HAs) are macromolecules of undefined compositions that vary with origin, the process by which they are obtained and functional groups present in their structure, such as quinones, phenols, and carboxylic acids. In addition to agriculture, there is an increased interest in HAs due to their important pharmacological effects. However, HAs are not readily soluble in water at physiological pH, which may limit their bioavailability. Although primary aggregation forms non-uniform pseudo-micelles, the presence of ionisable groups in the HA molecule makes pH an environmental stimulus for controlled aggregation and precipitation. The aim of this work was to induce HA deprotonation and protonation, without compromising their colloidal dispersion, by means of pH changes as a strategy to produce nanoparticles. Deprotonation and protonation were achieved by treating HAs with sodium hydroxide and acetic acid, respectively, at various concentrations. Non pH-treated HAs at the same concentrations were used as control. The evolution of the treatments was monitored by pH changes in bulk solutions as a function of time. At equilibrium, the conformation of the colloidal structures was characterised by the predominant mean diameter, polydispersity index and absorbance of the solutions. The zeta potential was also measured in protonation assays. Moreover, the fungicidal activity of the nanoparticles was evaluated on the mycelial growth of three fungal genera. The results showed the pH decrease or increment as a function of the balance between hydroxyl and carboxyl groups and of the diffusion rate inside the structures. Deprotonation followed by protonation produced nanosized (100-200nm), electrostatically stable (-30mV) and pH-responsive particles with a polydispersity index <0.5. The protonated nanoparticles significantly inhibited (P≤0.05) the mycelial growth of Candida albicans in vitro, when compared with control, and the fungicidal activity was dose-dependent. No activity was observed for the deprotonated HAs nanoparticles. These results show that deprotonation followed by protonation is an easy and useful strategy for the controlled production of HA nanoparticles, which exhibit a tendency to elicit fungicidal effects, with potential to develop new classes of cosmetics and pharmaceuticals.

Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species.

Bioremediation and biological-control by fungi have made tremendous strides in numerous biotechnology applications. The aim of this study was to test Byssochlamys nivea and Scopulariopsis brumptii in sensitivity and degradation to pentachlorophenol (PCP) and in biological-control of Phytophthora cinnamomi and Phytophthora cambivora. B. nivea and S. brumptii were tested in PCP sensitivity and degradation in microbiological media while the experiments of biological-control were carried out in microbiological media and soil. The fungal strains showed low PCP sensitivity at 12.5 and 25 mg PCP L(-1) although the hyphal size, fungal mat, patulin, and spore production decreased with increasing PCP concentrations. B. nivea and S. brumptii depleted completely 12.5 and 25 mg PCP L(-1) in liquid culture after 28 d of incubation at 28 °C. Electrolyte leakage assays showed that both fungi have low sensitivity to 25 mg PCP L(-1) and produced no toxic compounds for the plant. B. nivea and S. brumptii were able to inhibit the growth of the two plant pathogens in laboratory studies and reduce the mortality of chestnut plants caused by two Phytophthorae in greenhouse experiments. The two fungal strains did not produce volatile organic compounds able to reduce the growth of two plant pathogens tested.

Virulence and Resistance to Antifungal Therapies of Scopulariopsis Species.

Scopulariopsisis an emerging opportunistic fungus characterized by its high resistance to antifungal therapies. We have developed a murine model of disseminated infection in immunosuppressed animals by intravenous inoculation ofScopulariopsis brevicaulisandScopulariopsis brumptii, the most clinically relevant species, in order to evaluate their virulence and their responses to conventional antifungal treatments. Survival and tissue burden studies showed thatS. brumptiiwas more virulent thanS. brevicaulis The three drugs tested, liposomal amphotericin B, posaconazole, and voriconazole, prolonged the survival of mice infected withS. brumptii, but none showed efficacy againstS. brevicaulis The different therapies were only able to modestly reduce the fungal burden of infected tissue; however, in general, despite the high serum levels reached, they showed poor efficacy in the treatment of the infection. Unfortunately, the most effective therapy forScopulariopsisinfections remains unresolved.

Scopulariopsis sp. and Fusarium sp. in the Documentary Heritage: Evaluation of Their Biodeterioration Ability and Antifungal Effect of Two Essential Oils.

Fungi produce pigments and acids, generating particular local conditions which modify the physicochemical properties of materials. The aims of this work are (i) to investigate bioadhesion, foxing production and biofilm formation by Scopulariopsis sp. and Fusarium sp. isolated from document collections under laboratory conditions; (ii) to verify attack on cellulose fibres and (iii) to study the possibility of reducing fungal growth using natural products. Biofilm formation and extracellular polymeric substance (EPS) production by fungi were demonstrated in laboratory assays and by scanning electron microscopy (SEM) observations. The biocidal activity of two essential oils of Origanum vulgare L. and Thymus vulgaris L. was evaluated using the microatmosphere method. SEM observations showed that these strains were able to attach to paper and form biofilms, causing damage on them, which demonstrates the biodeterioration ability of these microorganisms. Scopulariopsis sp. and Fusarium sp. isolated from paper books showed the formation of fox-like reddish-brown colour spots, attack to the paper structure and pigment production on aged paper samples. The strains tested produced a decrease in the pH of one unit. This would substantiate the effect of the strains in paper biodeterioration. The microatmosphere method showed that volatile compounds of the essential oils have antifungal activity.

In vitro activities of a wide panel of antifungal drugs against various Scopulariopsis and Microascus species.

The in vitro activities of 11 antifungal drugs against 68 Scopulariopsis and Microascus strains were investigated. Amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, ketoconazole, miconazole, posaconazole, voriconazole, and ciclopirox showed no or poor antifungal effect. The best activities were exhibited by terbinafine and caspofungin, where the MIC and MEC (minimal effective concentration) ranges were 0.0313 to >16 µg/ml and 0.125 to 16 µg/ml, respectively. The MIC and MEC modes were both 1 µg/ml for terbinafine and caspofungin; the MIC50 and MEC50 were 1 µg/ml for both drugs, whereas the MIC90 and MEC90 were 4 µg/ml and 16 µg/ml, respectively.

In Vitro Triple Combination of Antifungal Drugs against Clinical Scopulariopsis and Microascus Species.

Broth microdilution checkerboard techniques based on the methodology of the Clinical and Laboratory Standards Institute (CLSI) were employed to study the triple antifungal combination of caspofungin, posaconazole, and terbinafine against 27 clinical isolates of Scopulariopsis and Microascus species. Synergy was observed for 26 isolates, whereas antagonism was observed for Scopulariopsis candida in this study.

In vitro antifungal susceptibility of Scopulariopsis brevicaulis isolates.

In humans, Scopulariopsis is mainly associated with onychomycoses, rarely with cutaneous infections or with invasive mycoses. However, during the last two decades, deep infections caused by members of this genus have been increasing. Scopulariopsis brevicaulis is the most common species described as an etiologic agent of human disease. Previous antifungal susceptibility studies indicate that this species is resistant in vitro to the broad-spectrum antifungal agents that are available today. Here, we describe the antifungal activity of amphotericin B, terbinafine, ciclopirox, itraconazole, ketoconazole, and voriconazole against 40 S. brevicaulis isolates. Antifungal susceptibility tests were performed using a modified Clinical and Laboratory Standards Institute M38-A2 procedure. The results showed that itraconazole had the highest minimal inhibitory concentration (MIC) of >16 mg/l; amphotericin B, voriconazole, and ketoconazole MICs were ranging from 4 to >16 mg/l, 8 to >16 mg/l, and 8 to >16 mg/l, respectively; and the best activity was found with terbinafine and ciclopirox with MICs ranging from 0.5 to 16 mg/l and 1 to 8 mg/l, respectively.

[Antifungal susceptibility of clinical isolates of Scopulariopsis species]. / Sensibilidad a antifúngicos de especies de Scopulariopsis de origen clínico.

INTRODUCTION: Scopulariopsis is a common soil saprophyte. In the last years the infections caused by Scopulariopsis species have increased, included superficial and invasive mycoses. This fungi has been reported resistant in vitro to some antifungal agents, although there is little information about this. The aim of the study was to establish in vitro antifungal susceptibility of clinical isolates of Scopulariopsis species against to broad-spectrum antifungal agents. METHODS: A total of 28 Scopulariopsis strains (10 S. brevicaulis, 7 S. koningii, 3 S. acremonium, 3 S. candida, 3 S. flava, 1 S. brumptii and 1 S. fusca) were tested using Sensititre Yeast One and broth microdilution methods to determine the minimum inhibitory concentrations (MICs) to amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole and 5-fluorocytosine, and minimun effective concentration (MECs) to anidulafungin, caspofungin and micafungin. RESULTS: Our data confirm the high in vitro resistance of Scopulariopsis to antifungal agents. Anidulafungin, caspofungin, micafungin (MICs ≥ 8 mg/L), 5-fluorocytosine (MICs ≥ 64 mg/L), and fluconazole (MICs ≥ 128 mg/L) were inactive in vitro in all species. MICs of amphotericin B (range 2 to ≥ 8 mg/L) and itraconazole (0.5 to ≥ 16 mg/L) were high. The best antifungal activity was observed for posaconazole and voriconazole (0.5 to ≥ 8 mg/L). With Sensititre Yeast One method MICs obtained slightly lower. Scopulariopsis candida, S. flava and S. fusca were the most resistant species, while S. acremonium and S. brevicaulis showed the lowest MICs. CONCLUSIONS: MICs of all tested antifungal agents for Scopulariopsis were very high. Infections caused by Scopulariopsis species may not respond to antifungal treatment. Voriconazole is the drug of choice for treatment. We consider it appropriate to add amphotericin B in serious infections.

Scopulariopsis, a poorly known opportunistic fungus: spectrum of species in clinical samples and in vitro responses to antifungal drugs.

Ninety-nine isolates of clinical origin, tentatively identified as Scopulariopsis or Microascus, were morphologically and molecularly characterized by a combined analysis of the D1/D2 domains of the 28S rRNA gene and a fragment of the elongation factor 1-α gene (EF1-α) sequences. The most prevalent species was Scopulariopsis brevicaulis (49.4%), followed by Scopulariopsis gracilis (14.4%), Scopulariopsis brumptii (7.2%), Microascus cinereus (5.2%), the Scopulariopsis candida species complex (3.1%), and Microascus cirrosus (2.1%). The most common anatomic sites of isolation were the respiratory tract (61.6%), superficial tissue (19.2%), and deep tissue or fluid samples (19.2%). The antifungal susceptibilities of the isolates to eight drugs were tested in vitro, with all the drugs generally showing poor activity.

Comparison of in vitro antifungal activities of efinaconazole and currently available antifungal agents against a variety of pathogenic fungi associated with onychomycosis.

Onychomycosis is a common fungal nail infection in adults that is difficult to treat. The in vitro antifungal activity of efinaconazole, a novel triazole antifungal, was evaluated in recent clinical isolates of Trichophyton rubrum, Trichophyton mentagrophytes, and Candida albicans, common causative onychomycosis pathogens. In a comprehensive survey of 1,493 isolates, efinaconazole MICs against T. rubrum and T. mentagrophytes ranged from ≤ 0.002 to 0.06 µg/ml, with 90% of isolates inhibited (MIC90) at 0.008 and 0.015 µg/ml, respectively. Efinaconazole MICs against 105 C. albicans isolates ranged from ≤ 0.0005 to >0.25 µg/ml, with 50% of isolates inhibited (MIC50) by 0.001 and 0.004 µg/ml at 24 and 48 h, respectively. Efinaconazole potency against these organisms was similar to or greater than those of antifungal drugs currently used in onychomycosis, including amorolfine, ciclopirox, itraconazole, and terbinafine. In 13 T. rubrum toenail isolates from onychomycosis patients who were treated daily with topical efinaconazole for 48 weeks, there were no apparent increases in susceptibility, suggesting low potential for dermatophytes to develop resistance to efinaconazole. The activity of efinaconazole was further evaluated in another 8 dermatophyte, 15 nondermatophyte, and 10 yeast species (a total of 109 isolates from research repositories). Efinaconazole was active against Trichophyton, Microsporum, Epidermophyton, Acremonium, Fusarium, Paecilomyces, Pseudallescheria, Scopulariopsis, Aspergillus, Cryptococcus, Trichosporon, and Candida and compared favorably to other antifungal drugs. In conclusion, efinaconazole is a potent antifungal with a broad spectrum of activity that may have clinical applications in onychomycosis and other mycoses.

In vitro antifungal susceptibility testing of Scopulariopsis brevicaulis strains using agar diffusion method.

The genus Scopulariopsis is a common soil saprotroph and has been isolated from air, organic waste and also from plant, animal and human tissues. Scopulariopsis has mainly been associated in humans with superficial mycoses, but it has also been described as the cause of subcutaneous and invasive infections. The most common aetiological agent of infections in humans is Scopulariopsis brevicaulis. This species has been reported to be resistant in vitro to broad-spectrum antifungal agents available today. The aim of the study was to establish in vitro antifungal susceptibility of 35 S. brevicaulis strains against amphotericin B (AMB), flucytosine (FC), caspofungin (CAS), terbinafine (TER), ciclopirox (CIC), voriconazole (VOR), clotrimazole (CTR), miconazole (MCZ), econazole (ECO), ketoconazole (KET), itraconazole (ITR), and fluconazole (FLU). Antifungal susceptibility tests were evaluated by an agar diffusion method (Neo-Sensitabs, Rosco, Denmark). AMB, FC, CAS, ITR and FLU showed no antifungal activity against S. brevicaulis. TER, CIC, CTR, KET, VOR, ECO, and MCZ revealed inhibitory activity for S. brevicaulis, but it varied for each of the drugs. The best antifungal effect was observed for TER and CIC. All isolates had large inhibition zones for TER and CIC. CTR was also inhibitory for all tested S. brevicaulis isolates, but the diameters of inhibition zones were smaller than for TER and CIC. Nearly 89% isolates showed inhibition zones for KET and the mean diameter of the inhibition zone was comparable to CTR. The least antifungal activity exhibited VQR, ECO and MCZ. Because of the multiresistance of S. brevicaulis, infections due to this species may not respond to particular antifungal treatment and other therapeutic approaches should be considered, e.g., combined therapy and/or surgery.