Establishing Minimal Conditions Sufficient for the Development of Titan-like Cells in Cryptococcus neoformans/gattii Species Complex.

Opportunistic pathogens of the anamorphic genus Cryptococcus are unique considering their virulence factors that in the context of pathogenesis allowed them to achieve evolutionary success. Morphological transformation into giant (Titan) cells is one of the factors contributing to cryptococcosis. Recently established in vitro protocols demonstrate that 5 or 10% fetal bovine serum (FBS) combined with 5% CO2, 37 °C, and sufficiently low cell density, triggers cellular enlargement (Serum protocols). However, the FBS components that promote this morphological transition remain incompletely characterized. In search of minimal conditions necessary for stimulating the formation of Titan cells, we performed a study where we eliminated serum from the protocol (Serum-free protocol) and instead systematically adjusted the amount of glucose, source of nitrogen (ammonium sulfate), and the pH. We found that exposing cells to PBS with adjusted pH to 7.3, and supplemented with 0.05% glucose, 0.025% ammonium sulfate, 0.004% K2HPO4, 0.0035% MgSO4, in the presence of 5% CO2 at 37 °C triggers Titan-like cell formation to the same degree as the previously established protocol that utilized 10% FBS as the sole nutrient source. Titan-like cells obtained according to this Serum-free protocol were characterized by cell body size over ten microns, a single enlarged vacuole, thick cell wall, extensive polysaccharide capsule, and changes in the level of cell ploidy, all currently known hallmarks of Titan cells found in vivo. Strikingly, we found that in both, Serum and Serum-free protocols, an optimal pH for Titan-like cell development is ~7.3 whereas relatively acidic pH (5.5) prevents this morphological transition and promotes robust proliferation, while alkaline pH (~8.0) has a profound growth inhibitory effect. Our study demonstrates a critical role of pH response to the formation of Titan cells and indicates that conditions that allow restricted proliferation in the presence of 5% CO2 are sufficient for this morphological transition to form enlarged cells in Cryptococcus neoformans and Cryptococcus gattii species complex.

In Vitro Activity of Ebselen and Diphenyl Diselenide Alone and in Combination with Drugs against Trichophyton mentagrophytes Strains.

BACKGROUND: Dermatophytoses are one of the most prevalent infectious diseases in the world for which the pace of developing new drugs has not kept pace with the observed therapeutic problems. Thus, searching for new antifungals with an alternative and novel mechanism of action is necessary. OBJECTIVE: This study aimed to evaluate the antifungal activity of ebselen and diphenyl diselenide against Trichophyton mentagrophytes clinical isolates. METHODS: In vitro antifungal susceptibility was assessed for organoselenium compounds used alone or in combination with allylamines and azoles according to the 3rd edition of the CLSI M38 protocol. RESULTS: Ebselen demonstrated high antifungal activity with MICGM equal to 0.442 µg/mL and 0.518 µg/mL in the case of human and animal origin strains, respectively. The values of MICGM of diphenyl diselenide were higher: 17.36 µg/mL and 13.45 µg/mL for the human and animal isolates, respectively. Synergistic or additive effects between terbinafine and ebselen or diphenyl diselenide were observed in the case of 12% and 20% strains, respectively. In turn, the combination of itraconazole with diphenyl diselenide showed a synergistic effect only in the case of 6% of the tested strains, whereas no synergism was shown in the combination with ebselen. CONCLUSIONS: The results highlight the promising activity of organoselenium compounds against Trichophyton mentagrophytes. However, their use in combinational therapy with antifungal drugs seems to be unjustified due to the weak synergistic effect observed.

Laboratory Diagnosis and In Vitro Antifungal Susceptibility of Trichophyton quinckeanum from Human Zoonoses and Cats.

The "One Health" concept increasingly demonstrates the global spread of pathogenic (also eukaryotic) microorganisms and their zoonotic potential. Dermatophytes can cause superficial mycoses in humans and animals. Furthermore, the number of transmissions from asymptomatic carriers to humans has been on the rise over the last few years. This study was focused on the detailed characterisation of clinical isolates of Trichophyton quinckeanum with epidemiological analyses and characterisation of their in vitro antifungal susceptibility patterns. The isolated dermatophytes were identified with a combination of conventional and molecular methods. In turn, their susceptibility in vitro was tested according to the Clinical and Laboratory Standards Institute (CLSI) M38 ed.3 protocol. A total of 36 strains were isolated, with 21 cases of T. quinckeanum zoonoses resulting from direct contact with symptomatic cats (58.3%). The other 15 strains (41.7%) were isolated simultaneously from healthy cats and their owners. All strains showed high susceptibility to allylamine, pyridinone, and phenyl morpholine derivatives but were resistant to fluconazole and ketoconazole. In conclusion, our study shows the frequency of zoonoses contracted from asymptomatic cats. Moreover, the antifungal susceptibility profiles indicate the serious risk posed to animal owners by resistant strains of T. quinckeanum, which are often responsible for recalcitrant-to-treatment cases.

Airborne dermatophyte propagules concentration in cowsheds as an underestimated reservoir of potential zoonoses.

AIMS: Indoor air quality in stables, cowsheds or henhouses has recently become in interest due to the potential risks of zoonotic infections. Cowsheds are commonly known to have high fungal loads, particularly if insufficient attention is paid to the monitoring and control of the indoor microclimate around three elements, that is heating, ventilation and lighting. The aim of this study was to determine the concentrations and spectrum of dermatophyte propagules in the indoor air of cowsheds. METHODS AND RESULTS: Air samples were collected on five farms, and the dermatophyte species were identified using MALDI-TOF MS analysis. The quantitative analysis of the fungal pollutants showed an average of 0.084 dermatophyte propagules (CFU) per m3 of flowing air in spring and 0.0239 CFU/m3 in the summer. Dermatophyte species were identified in case of 64.6% of the obtained colonies. Trichophyton verrucosum as dominant species was isolated on all five farms. In turn, Nannizzia gypsea was isolated on four farms and Trichophyton mentagrophytes as well as Paraphyton cookei were isolated on two farms. CONCLUSIONS: This study demonstrated that indoor aerosol appears to be one of the underestimated risks of dermatophyte infections. Moreover, the risk of zoonotic infections is posed by airborne zoophilic dermatophytes, especially T. verrucosum, whose prevalence of infections has been increasing in recent years. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of dermatophytes to infect animals and humans is thought to be a consequence of not only their adaptation to new ecological niches but also occurring as an aerosol component, which we demonstrate for the first time in this study. The microclimate of the cowshed may be an underestimated reservoir of zoophilic dermatophytes, which pose a zoonotic threat to farmers, animal breeders and veterinarians.

Update on Stachybotrys chartarum-Black Mold Perceived as Toxigenic and Potentially Pathogenic to Humans.

In nature, there are many species of fungi known to produce various mycotoxins, allergens and volatile organic compounds (VOCs), as well as the commonly known etiological agents of various types of mycoses. So far, none of them have provoked so much emotion among homeowners, builders, conservators, mycologists and clinicians as Stachybotrys chartarum. This species compared to fungi of the genera Fusarium and Aspergillus is not as frequently described to be a micromycete that is toxigenic and hazardous to human and animal health, but interest in it has been growing consistently for three decades. Depending on the authors of any given review article, attention is focused either on the clinical aspects alongside the role of this fungus in deterioration of biomaterials, or aspects related to its biology, ecology and taxonomic position. On the one hand, it is well established that inhalation of conidia, containing the highest concentrations of toxic metabolites, may cause serious damage to the mammalian lung, particularly with repeated exposure. On the other hand, we can find articles in which authors demonstrate that S. chartarum conidia can germinate and form hyphae in lungs but are not able to establish an effective infection. Finally, we can find case reports that suggest that S. chartarum infection is linked with acute pulmonary hemorrhage, based on fungal structures recovered from patient lung tissue. New scientific reports have verified the current state of knowledge and note that clinical significance of this fungus is exceedingly controversial. For these reasons, understanding S. chartarum requires reviewing the well-known toxigenic features and harmful factors associated with this fungus, by gathering the newest ones into a coherent whole. The research problem related to this fungus seems to be not overly publicized, and there is still a demand to truthfully define the real threats of S. chartarum and phylogenetically related species. The most important problem, which should be fully elucidated as soon as possible, remains the clarification of the pathogenicity of S. chartarum and related species. Maybe it is urgent time to ask a critical question, namely what exactly do we know 28 years after the outbreak of pulmonary hemorrhage in infants in Cleveland, Ohio, USA most likely caused by S. chartarum?

European Hedgehogs (Erinaceus europaeus L.) as a Reservoir of Dermatophytes in Poland.

The European hedgehog (Erinaceus europaeus Linnaeus) frequently colonises areas located close to human life in cities, as these are more suitable nest sites offering an abundance of food and allowing avoidance of predators. However, urbanisation has a significant impact on the epidemiology of infectious diseases, including dermatophytoses, the primary source of which are wild animals. In this study, we determined the spectrum of dermatophytes isolated from the European hedgehog and assessed their susceptibility profile to antifungal drugs. Symptomatic and asymptomatic dermatophyte infections were observed in 7.7% and 8% of the 182 examined free-living hedgehogs, respectively. In the pool of the isolated dermatophyte strains, Trichophyton erinacei was dominant (29.9%), followed by Trichophyton mentagrophytes (17.9%), Trichophyton benhamiae (13.4%), Nannizzia gypsea (11.9%), Microsporum canis (10.4%), Nannizzia nana (7.5%), Paraphyton cookei (6.0%), and Nannizzia fulva (3.0%). Susceptibility tests revealed the highest activity of luliconazole and the lowest of activity fluconazole among the azole drugs applied. Although terbinafine generally exhibited high efficacy, two Trichophyton mentagrophytes isolates showed resistance to this drug (MIC = 2 µg/ml) resulting from missense mutations in the SQLE gene corresponding to the amino acid substitution Leu393Phe. Summarising, our study has also revealed that such wildlife animals as hedgehogs can be a reservoir of pathogenic human dermatophytes, including harmful strains resistant to commonly used antifungal drugs.

New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions.

Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be used in the identification of dermatophytes is of great importance for the development of this branch of diagnostics. In this article, the suitability of six candidate reference genes (TUBB, ACTB, ADPRF, RPL2, SDHA, and EEF1A1) was investigated for gene expression analysis in the dermatophyte Trichophyton verrucosum, which was cultured in various mycological media that are commonly used in a diagnostic laboratory, i.e., Sabouraud, potato dextrose, and keratin-supplemented MM-Cove. The different culture conditions are extremely important factors for the growth and physiology of dermatophytes. Gene expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Regarding the stability of expression, SDHA was the most stable housekeeping gene; hence, this gene is recommended for future qRT-PCR studies on T. verrucosum strains. These results allow us to conclude that the SDHA gene can be an additional good candidate as an identification target in the qRT-PCR technique.

Cold atmospheric pressure plasma (CAPP) as a new alternative treatment method for onychomycosis caused by Trichophyton verrucosum: in vitro studies.

PURPOSE: Anthropophilic dermatophytes as etiological factors of onychomycoses are more common than zoophilic fungi. In the case of the latter, reverse zoonoses are possible, which poses a threat to the persistence of dermatophytes in the environment. Nevertheless, without treatment, both types of tinea unguium may lead to complete nail plate destruction and secondary mixed infections with fungi and bacteria. One of the zoophilic dermatophytes that cause onychomycosis is Trichophyton verrucosum, whose prevalence has been increasing in recent years. Such infections are usually treated with allylamines and/or azoles, but such a conventional treatment of infections caused by T. verrucosum often fails or is discontinued by patients. METHODS: Herein, we reveal the results of our in vitro studies related to direct application of cold atmospheric pressure plasma (CAPP) on Trichophyton verrucosum growth, germination and adherence to nail as a new alternative treatment method of such types of dermatomycoses. RESULTS: Our in vitro studies showed that, while exposure to CAPP for 10 min delays germination of conidia and clearly impairs the fitness of the fungal structures, 15 min is enough to kill all fungal elements exposed to plasma. Moreover, the SEM images revealed that T. verrucosum cultures exposed to CAPP for 10 and 15 min were not able to invade the nail fragments. CONCLUSION: The results revealed that single exposure to CAPP was able to inhibit T. verrucosum growth and infection capacity. Hence, cold atmospheric pressure plasma should be considered as a promising alternative treatment of onychomycoses.

Modulation of ERG gene expression in fluconazole-resistant human and animal isolates of Trichophyton verrucosum.

Dermatophytes are a group of eukaryotic microorganisms characterized by high capacity to colonize keratinized structures such as the skin, hair, and nails. Over the past years, the incidence of infections caused by zoophilic species, e.g., Trichophyton verrucosum, has been increasing in some parts of the world, especially in Europe. Moreover, the emergence of recalcitrant dermatophytoses and in vitro resistant dermatophytes has become a cause of concern worldwide. Here, we analyzed the mechanisms underlying resistance to fluconazole among clinical isolates of T. verrucosum. Quantitative RT-PCR was carried out to determine the relative expression levels of mRNA transcripts of ERG3, ERG6, and ERG11 genes in the fungal samples using the housekeeping gene GAPDH as a reference. Our results showed that the upregulation of the ERG gene expression is a possible mechanism of resistance to fluconazole in this species. Furthermore, ERG11 is the most statistically significantly overexpressed gene in the pool of fluconazole-resistant T. verrucosum isolates. Additionally, we have demonstrated that exposure to fluconazole increases the levels of expression of ERG genes in fluconazole-resistant isolates of T. verrucosum. In conclusion, this study has shown one of the possible mechanisms of resistance to fluconazole among zoophilic dermatophytes, which involves the maintenance of high levels of expression of ERG genes after drug exposure.

In vitro evaluation of photodynamic activity of methylene blue against Trichophyton verrucosum azole-susceptible and -resistant strains.

The intense search for the "Holy Grail" of antifungal therapy can be observed today. The searches are not limited only to discovery of potential antifungal drugs, but also new therapeutic strategies involving the use of chemosensitizers to achieve synergistic effect or physicochemical factors inducing stress conditions in fungal cells. In this study was examined in vitro effectiveness of photodynamic antifungal strategy with methylene blue using a light beam with a wavelength equal to 635 nm toward the Trichophyton verrucosum susceptible and itraconazole- and/or fluconazole-resistant strains. Methylene blue used at concentration equal to 5 µg/mL and in the presence of 40 J/cm2 of light energy showed fungicidal effect toward the susceptible strains. However, for azole-resistant isolates, only the energy dose equal to 60 J/cm2 at 5 µg/mL of methylene blue allowed to kill the pathogen. This study confirms that methylene blue induced by red light has a definite inhibitory effect on zoophilic dermatophytes.

Therapeutic efficacy of topically used luliconazole vs. terbinafine 1% creams.

OBJECTIVES: Dermatomycoses of zoophilic origin, especially those caused by Trichophyton mentagrophytes, often pose considerable therapeutic problems. This is reflected in the growing number of strains of this species with resistance to terbinafine caused by a mutation in the squalene epoxidase (SQLE) gene. Therefore, it is reasonable to look for alternative therapies to the commonly used terbinafine. The aim of the present study was to assess the in vivo effectiveness of topical therapy with luliconazole or terbinafine 1% cream. METHODS: Therapeutic efficacy was assessed using direct examination in KOH with DMSO, qPCR analysis with pan-dermatophyte primers and culturing. Moreover, in vitro susceptibility tests for luliconazole and terbinafine were performed. RESULTS: The results demonstrated significantly higher antifungal activity of luliconazole than terbinafine against dermatomycoses caused by T. mentagrophytes. The geometric mean of the MIC value for luliconazole against all T. mentagrophytes strains was 0.002 µg/ml, while this value for terbinafine was 0.004 µg/ml. In all studied cases, 28-day local therapy with luliconazole contributed to complete eradication of the aetiological agent of infection. CONCLUSIONS: Given the increasingly frequent reports of difficult-to-treat dermatophytoses caused by zoophilic terbinafine-resistant strains, the 1% luliconazole cream can be alternative solution in topical therapy.

Complementary effect of mechanism of multidrug resistance in Trichophyton mentagrophytes isolated from human dermatophytoses of animal origin.

BACKGROUND: Dermatophytoses have gained interest worldwide due to the increased resistance to terbinafine and azoles and difficulty in management of these refractory diseases. OBJECTIVES: In this study, we identified and analysed Trichophyton mentagrophytes clinical isolates obtained from humans with infections of animal origin. METHODS: We used quantitative real-time PCR (qRT-PCR) to examine the transcriptional modulation of three MDR genes (PDR1, MDR2 and MDR4) and analysed squalene epoxidase (SQLE) gene sequences from multidrug-resistant Trichophyton mentagrophytes isolates. RESULTS: The expression profile revealed a 2- to 12-fold increase in mRNA accumulation in the presence of any of the antifungals, compared to cells incubated without drugs. A statistically significant relationship between the isolates exposed to itraconazole and increased expression of the tested genes was revealed. Substantially lower transcription levels were noted for cells exposed to luliconazole, that is, a third-generation azole. Additionally, in the case of 50% of terbinafine-resistant strains, Leu397Phe substitution in the SQLE gene was detected. Furthermore, the reduced susceptibility to itraconazole and voriconazole was overcome by milbemycin oxime. CONCLUSIONS: In conclusion, our study shed more light on the role of the ABC transporter family in T. mentagrophytes, which, if overexpressed, can confer resistance to single azole drugs and even cross-resistance. Finally, milbemycin oxime could be an interesting compound supporting treatment with azole drugs in the case of refractory dermatomycoses.

A case of anti- pityriasis versicolor therapy that preserves healthy mycobiome.

BACKGROUND: The impact of Malassezia yeasts on skin mycobiome and health has received considerable attention recently. Pityriasis versicolor (PV), a common dermatosis caused by Malassezia genus worldwide, is a manifestation of dysbiosis. PV can be associated with hyper- and/or hypopigmented skin lesions. This disease entity is characterized by high percentage of relapses, which demands a proper antifungal therapy that is based on unambiguous species identification and drug susceptibility testing. CASE PRESENTATION: Comprehensive analysis of PV case in man presenting simultaneously hyper- and hypopigmented skin lesions was performed. Conventional and molecular diagnostic procedures revealed Malassezia furfur and Malassezia sympodialis, respectively as etiological agents of skin lesions observed. Susceptibility tests showed significantly lowered sensitivity of M. furfur cells to fluconazole. Based on susceptibility profiles local antifungal therapy with drugs characterized by entirely different mechanism of action was included. CONCLUSIONS: Our study indicates that cases of PV represented by two types of skin lesions in one patient may be associated with distinct Malassezia species. Moreover, as observed in this case, each of the isolated etiological agents of PV may differ significantly in susceptibility to antifungals. This can significantly complicate the treatment of dermatosis, which by definition is associated with a significant percentage of relapses. In the presented case localized topical treatment was sufficient and successful while allowing maintaining the physiological mycobiome.

Intrinsic resistance to terbinafine among human and animal isolates of Trichophyton mentagrophytes related to amino acid substitution in the squalene epoxidase.

BACKGROUND: Dermatomycoses are the most common fungal infections in the world affecting a significant part of the human and animal population. The majority of zoophilic infections in humans are caused by Trichophyton mentagrophytes. Currently, the first-line drug for both oral and topical therapy is terbinafine. However, an increasing number of cases that are difficult to be cured with this drug have been noted in Europe and Asia. Resistance to terbinafine and other allylamines is very rare and usually correlated with point mutations in the squalene epoxidase gene resulting in single amino acid substitutions in the enzyme, which is crucial in the ergosterol synthesis pathway. PURPOSE: Here, we report terbinafine-resistant T. mentagrophytes isolates among which one was an etiological factor of tinea capitis in a man and three were obtained from asymptomatic foxes in Poland. METHODS: We used the CLSI protocol to determine antifungal susceptibility profiles of naftifine, amphotericin B, griseofulvin, ketoconazole, miconazole, itraconazole, voriconazole, and ciclopirox. Moreover, the squalene epoxidase gene of the terbinafine-resistant strains was sequenced and analysed. RESULTS: In the genomes of all four resistant strains exhibiting elevated MICs to terbinafine (16 to 32 µg/ml), single-point mutations leading to Leu393Phe substitution in the squalene epoxidase enzyme were revealed. Among the other tested substances, a MIC50 value of 1 µg/ml was shown only for griseofulvin. CONCLUSION: Finally, our study revealed that the terbinafine resistance phenomenon might not be acquired by exposure to the drug but can be intrinsic. This is evidenced by the description of the terbinafine-resistant strains isolated from the asymptomatic animals.

Titan cell formation is unique to Cryptococcus species complex.

Members of the Cryptococcus species complex stand out by unique virulence factors that allowed evolutionary transition to pathogenesis. Among the factors contributing to cryptococcosis is a morphological transformation into giant (Titan) cells. It remains unclear whether species outside of the C. neoformans/C. gattii species complex are capable of titanization. We utilized two recently developed protocols that allow obtaining Titan cells in vitro to test if titanization occurs in non-C. neoformans/C. gattii species. We find that none of the tested strains, representing 10 species of basidiomycetous yeasts and the ascomycetous yeast Saccharomyces cerevisiae, undergo significant titanization under conditions that promote robust Titan cell formation in C. neoformans/C. gattii species complex. C. terreus formed occasional enlarged cells through a mechanism potentially similar to that of titanization. Our findings suggest that titanization is a rare phenomenon among basidiomycetous yeasts that occurs mostly in members of the C. neoformans/C. gattii species complex.

Identification of emerging trends in the prevalence of dermatophytoses in alpacas (Vicugna pacos) farmed in Poland.

The increasing number of dermatophytoses among animals observed recently in developed countries may be connected with relocation of many exotic species outside their natural living environment. Moreover, an impact on this situation may also be exerted by relapses related to limited compliance with antifungal treatment regimes. Many exotic animals, including camelids, imported to European countries are connected with cases of tuberculosis or zoophilic dermatophytoses in humans. In the present study, we identified and comprehensively analysed dermatophyte infections in alpacas from breeding farms in Poland. As part of this study, we determined the prevalence of dermatophyte infections in alpacas. The conventional and molecular mycological diagnostic procedures applied led to unambiguous identification of the aetiological factors of symptomatic dermatophytoses and asymptomatic animals, that is Trichophyton benhamiae and T. verrucosum. Furthermore, the susceptibility tests allowed choosing the best therapeutic option and revealed superior activity of allylamine drugs against all strains. Finally, in the case of strains isolated from symptomatic dermatophytoses, our study revealed a significantly higher virulence level expressed by high activity of chosen enzymes, especially related to keratinolytic and haemolytic activity. In conclusion, this report indicates that farmed alpacas can be a reservoir or vector for contagious zoophilic dermatophyte infection. For this reason, imported animals should be subjected to meticulous monitoring to detect not only symptomatic infections but also asymptomatic animals.

Unusual dermatomycoses caused by Nannizzia nana: the geophilic origin of human infections.

BACKGROUND: Fungal infections of the skin, hair, and nails are the largest and most widespread group of all mycoses. Nannizzia nana is a relatively rare etiological factor of dermatomycosis in humans, as it usually affects animals, e.g. pigs and boars. In addition to the zoophilic nature, there are also reports of the geophilic reservoir of this dermatophyte species. OBJECTIVE: In this study, we present symptomatic infections with N. nana aetiology in humans reported recently in Poland. Interestingly, these cases had a non-specific clinical picture and occurred as skin lesions on the neck and foot as well as onychomycosis of the toenails. From the medical history, the patients had no contact with pigs. METHODS: Diagnostics of these infections was performed with a combination of classical phenotypic and molecular genomic methods. The genomic diversity of the isolates was determined using the MP-PCR method. In vitro antifungal susceptibility tests against itraconazole, ketoconazole, terbinafine and naftifine hydrochloride were also performed. RESULTS: Nannizzia nana has been identified as an etiological factor of dermatomycosis. Moreover, heterogeneity of the genomes was revealed for the obtained strains. In vitro activities of antifungal agents showed that isolates were susceptible to all tested drugs. The patients were treated with oral terbinafine and topical ketoconazole cream, which led to a complete recovery. CONCLUSIONS: In conclusion, the cases studied by us may indicate that the infrequency of N. nana infections may not necessarily be related to the low infectivity of this fungal agent, but they are rather associated with misdiagnosis. Furthermore, N. nana reservoirs should also be sought in soil.

Tinea corporis caused by Trichophyton equinum transmitted from asymptomatic dogs to two siblings.

Each year, millions of people worldwide are affected by superficial mycoses, which are frequently caused by dermatophytes having affinity to the scalp, nails, hair and the stratum corneum of the skin. The changing reservoirs of zoophilic dermatophytes, the numerous reports of animal carrier status, and the increasing number of pets make the diagnosis difficult, which is usually impossible based only on medical history. Herein we present a case report of tinea corporis caused by Trichophyton equinum in siblings who had no contact with horses. The routine laboratory diagnostic procedures for identification of isolates were based on phenotypic and genotypic characteristics, especially molecular techniques using rDNA internal transcribed spacer sequences. The results showed that both techniques proved to be insufficiently discriminatory to differentiate two closely related species, i.e. Trichophyton equinum and Trichophyton tonsurans. Introduction of a TEF1 sequence analysis to the diagnostic procedures revealed consistent differences between these two species and facilitated unambiguous identification. Interestingly, dogs that could leave the homestead freely were the source of the infection in children. In conclusion, Trichophyton equinum was considered in the past as a strict zoophilic dermatophyte associated with horses and rarely transmitted to humans. This study revealed that this species can have other reservoirs and live in the fur of asymptomatic animals.

3-Bromopyruvate as an Alternative Option for the Treatment of Protothecosis.

Protothecosis is an unusual infection of both humans and animals caused by opportunistically pathogenic microalgae of the genus Prototheca. Until now, no standardized treatment protocols exist for the protothecal disease, boosted by a remarkable resistance of Prototheca spp. to a wide array of antimicrobial agents currently available in clinical use. Consequently, there is an urgent need for new effective drugs against Prototheca algae. In this study, the anti-Prototheca activity of 3-bromopyruvate (3BP), either alone or in combination with amphotericin B (AMB) was assessed in vitro, as well as the cytotoxicity of 3BP toward the bovine mammary epithelial cells and murine skin fibroblasts. The mean minimum inhibitory concentrations (MIC) and minimum algaecidal concentrations (MAC) were 0.85 ± 0.21 and 2.25 ± 0.54 mM for Prototheca wickerhamii, 1.25 ± 0.47 and 4.8 ± 1.03 mM for Prototheca blaschkeae, and 1.55 ± 0.69 and 5.6 ± 1.3 mM for Prototheca zopfii gen. 2, respectively. For all Prototheca strains tested, a synergistic interaction between 3BP and AMB was observed, resulting in about 4-fold reduction of their individual MICs, when used together. The elevated content of intracellular glutathione (GSH) was associated with a decreased susceptibility to 3BP. Both epithelial and fibroblast cells retained high viability upon treatment with 3BP at concentrations equivalent to the highest MIC recorded (3 mM) and 10-fold higher (30 mM), with the mean cell viability exceeding 80%, essentially the same as for the untreated cells. The results from these in vitro studies emphasize the high activity of 3BP against the Prototheca algae, its synergistic effect when used in combination with AMB, and the safety of the drug toward the tested mammalian cells. Along with the advantageous physico-chemical and pharmacokinetic properties, 3BP may be considered an effective and safe novel agent against the protothecal disease.