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In immunocompetent individuals, Fusarium spp. stands out as the causative agent of onychomycosis, among the non-dermatophyte molds. Despite evidence indicating that Fusarium oxysporum organizes itself in the form of a biofilm causing onychomycosis, there is little literature on the etiopathogenesis of the biofilm on the nail, specifically the signaling molecules present, known as quorum sensing (QS). Thus, this study detected the presence of a molecule related to QS from the ex vivo biofilm of F. oxysporum on human nail and investigated its effect on preformed biofilm in vitro. The detection and physicochemical characterization of a QS molecule, from the extracellular matrix (ECM), was carried out by Fourier transform infrared (FTIR) spectroscopy with an attenuated total reflectance (ATR) accessory and by headspace gas chromatography coupled to mass spectrometry (GC-MS) analyses. Determination of viable cells, cell activity, total biomass, ECM components and scanning electron microscopy (SEM) were performed to evaluate the influence of the QS molecule on the in vitro biofilm of F. oxysporum. The beginning, in the ex vivo biofilm of F. oxysporum on human nails, the volatile organic compound 2-ethyl-1-hexanol (2EH) was detected as a component of QS. Thereafter in vitro analyses, synthetic 2EH was able to modulate the biofilm by stimulating its filament, increasing total biomass and ECM production in terms of total carbohydrates, but with a reduction in total proteins and nucleic acids. We thus evidence, for the first time, the presence of 2EH in the biofilm of F. oxysporum, developed on the human nail, and the in vitro action of this compound as a QS molecule.

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INTRODUCTION: The Trichophyton rubrum complex comprises the majority of dermatophyte fungi (DM) responsible for chronic cases of onychomycosis, which is treated with oral or topical antifungals. However, owing to antifungal resistance, alternative therapies, such as photodynamic therapy (PDT), are needed. This study investigated the frequency of the T. rubrum species complex in onychomycosis cases in the northwestern region of Paraná state, Brazil, and evaluated the efficacy of (PDT) using P123-encapsulated hypericin (Hyp-P123) on clinical isolates of T. rubrum in the planktonic cell and biofilm forms. MATERIAL AND METHODS: The frequency of the T. rubrum complex in onychomycosis cases from 2017 to 2021 was evaluated through a data survey of records from the Laboratory of Medical Mycology (LEPAC) of the State University of Maringa (UEM). To determine the effect of PDT-Hyp-P123 on planktonic cells of T. rubrum isolates, 1 × 105 conidia/mL were treated with ten different concentrations of Hyp-P123 and then irradiated with 37.8 J/cm2. Antibiofilm activity of PDT-Hyp-P123 was tested against T. rubrum biofilm in the adhesion phase (3 h), evaluated 72 h after irradiation (37.8 J/cm2), and the mature biofilm (72 h), evaluated immediately after irradiation. In this context, three different parameters were evaluated: cell viability, metabolic activity and total biomass. RESULTS: The T. rubrum species complex was the most frequently isolated DM in onychomycosis cases (approximately 80 %). A significant reduction in fungal growth was observed for 75 % of the clinical isolates tested with a concentration from 0.19 µmol/L Hyp-P123, and 56.25 % had complete inhibition of fungal growth (fungicidal action); while all isolates were azole-resistant. The biofilm of T. rubrum isolates (TR0022 and TR0870) was inactivated in both the adhesion phase and the mature biofilm. CONCLUSION: PDT-Hyp-P123 had antifungal and antibiofilm activity on T. rubrum, which is an important dermatophyte responsible for onychomycosis cases.

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BACKGROUND: Our previous proteomics data obtained from Candida albicans recovered after serial passage in a murine model of systemic infection revealed that Orf19.36.1 expression correlates with the virulence of the fungus. Therefore, the impact of ORF19.36.1 upon virulence was tested in this study. MATERIALS & METHODS: CRISPR-Cas9 technology was used to construct homozygous C. albicans orf19.36.1 null mutants and the phenotypes of these mutants examined in vitro (filamentation, invasion, adhesion, biofilm formation, hydrolase activities) and in vivo assays. RESULTS: The deletion of ORF19.36.1 did not significantly impact the phenotypes examined or the virulence of C. albicans in two infection models. CONCLUSION: These results suggest that, although Orf19.36.1 expression correlates with virulence, this protein is not essential for C. albicans pathobiology.

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Aim: This study evaluated the antifungal efficacy of gentian violet (GV) in an experimental vulvovaginal candidiasis (VVC) model. Materials & methods: In vitro susceptibility and cytotoxicity assays were performed to validate the antifungal potential and safety of GV. The antifungal efficacy was then evaluated in vivo through comparative analysis of the fungal burden following treatment with GV or nystatin, as well as assessment of the vaginal tissue by histology and electron microscopy. Results: GV demonstrated a safe antifungal profile against C. albicans, with a significant decrease in fungal burden and an improvement in the inflammatory process evaluated histologically. Conclusion: The results of this study motivate further assessment of GV as a promising alternative for VVC therapy.

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Infections caused by biofilm-forming agents have important implications for world health. Mixed infections, caused by more than one etiological agent, are also an emerging problem, especially regarding the standardization of effective diagnosis and treatment methods. Cases of mixed onychomycosis (OM) have been reported; however, studies on the microbial interactions between the different fungi in biofilms formed on nails are still scarce. We describe a case of mixed OM caused by the dermatophyte Trichophyton rubrum and the black yeast-like fungus Rhinocladiella similis. Identical growths of both fungi were observed in more than 50 cultures from different nail samples. Additionally, both species were able to form organized single and mixed biofilms, reinforcing the participation of both fungi in the etiology of this OM case. R. similis seemed to grow faster during the process, suggesting that T. rubrum benefits from biofilm development when in combination. Moreover, the biofilm of the Rhinocladiella isolate exhibited exacerbated production of the extracellular matrix, which was not observed with that of a Rhinocladiella reference strain, suggesting that the isolate had natural abilities that were possibly perfected during development in the nail of the patient.

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BACKGROUND: Sporothrix brasiliensis is a pathogenic dimorphic fungus that affects humans and animals causing sporotrichosis. The treatment of this disease with conventional antifungals commonly results in therapeutic failures and resistance. Therefore, this study aimed to evaluate the in vitro effect of curcumin (CUR) mediated by photodynamic therapy (PDT) in its pure state and incorporated into pharmaceutical formulation in gel form, on the filamentous and yeast forms of S. brasiliensis. METHODS: Cells from both forms of the fungus were treated with pure curcumin (PDT-CUR). For this, CUR concentrations ranging from 0.09 to 50 µM were incubated for 15 min and then irradiated with blue LED at 15 J/cm². Similarly, it was performed with PDT-CUR-gel, at lower concentration with fungistatic action. After, a qualitative and quantitative (colony forming units (CFU)) analysis of the results was performed. Additionally, reactive oxygen species (ROS) were detected by flow cytometry. Results PDT with 0.78 µM of CUR caused a significant reduction (p < 0.05) in cells of the filamentous and yeast form, 1.38 log10 and 1.18 log10, respectively, in comparison with the control. From the concentration of 1.56 µM of CUR, there was a total reduction in the number of CFU (≥ 3 log10). The PDT-CUR-gel, in relation to its base without CUR, presented a significant reduction (p < 0.05) of 0.83 log10 for the filamentous form and for the yeast form, 0.72 log10. ROS release was detected after the PDT-CUR assay, showing that this may be an important pathway of death caused by photoinactivation. Conclusion PDT-CUR has an important in vitro antifungal action against S. brasiliensis strains in both morphologies.

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Onychomycosis is a chronic fungal nail infection caused by several filamentous and yeast-like fungi, such as the genus Candida spp., of great clinical importance. Black yeasts, such as Exophiala dermatitidis, a closely related Candida spp. species, also act as opportunistic pathogens. Fungi infectious diseases are affected by organisms organized in biofilm in onychomycosis, making treatment even more difficult. This study aimed to evaluate the in vitro susceptibility profile to propolis extract and the ability to form a simple and mixed biofilm of two yeasts isolated from the same onychomycosis infection. The yeasts isolated from a patient with onychomycosis were identified as Candida parapsilosis sensu stricto and Exophiala dermatitidis. Both yeasts were able to form simple and mixed (in combination) biofilms. Notably, C. parapsilosis prevailed when presented in combination. The susceptibility profile of propolis extract showed action against E. dermatitidis and C. parapsilosis in planktonic form, but when the yeasts were in mixed biofilm, we only observed action against E. dermatitidis, until total eradication.

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Introduction: In vitro 3D equivalent tissues can be used for studies of fungal infections. Objectives: To develop 3D electrospun nanofibers using polycaprolactone (PCL) colonized by HeLa cells as a possible in vitro model for the investigation of fungal infection. Materials & methods: A PCL solution was synthesized and electrospun. HeLa cells were cultured on the nanostructured PCL scaffolds, forming a 3D structure. Physicochemical, biological and Candida albicans infection assays were performed in this model. Results: The nanostructured PCL scaffolds showed favorable physicochemical characteristics and allowed the colonization of HeLa cells, which showed indications of extracellular matrix production. Conclusions: Fungal infection was evidenced in the 3D nanostructured PCL scaffolds, being viable, economical and compatible to study fungal infections in vitro.

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Infections by Trichosporon spp. are increasing worldwide and its treatment remains a challenge. Colonization of medical devices has been considered as a predisposing factor for trichosporonosis, which is related to fungal biofilm production. Thus, this study aimed to evaluate the ability of six hospital T. asahii isolates to form biofilm on abiotic surface, as well as to investigate the impact of three classic antifungals on both planktonic and biofilm forms. The fungal identification was based on macro and micromorphological characteristics, biochemical tests and confirmation by mass spectrometry assisted by the flight time desorption/ionization matrix (MALDI-TOF MS). Antifungal susceptibility assay of planktonic cells showed inhibitory and fungicidal concentrations ranging from 2.5 to 10 µg/mL for voriconazole, 2 to 8 µg/mL for fluconazole, and 1 to 4 µg/mL for amphotericin B. All T. asahii strains were able to form biofilms on the polystyrene microplates surface within 24 h, showing a simple architecture when compared with Candida spp. biofilm. On the other hand, the same antifungals did not show action in neither the inhibition of biofilm formation nor on the formed biofilm. Concluding, the present study reinforced the relevance of the MALDI-TOF MS methodology for a safe identification of T. asahii. Classic antifungals were active on the planktonic form, but not on the biofilms. All isolates formed biofilms on the polystyrene microplates and showed a simple architecture.

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Considering the multifaceted and increasing application of photodynamic therapy (PDT), in recent years the antimicrobial employment of this therapy has been highlighted, because of the antiviral, antibacterial, antiparasitic, and antifungal activities that have already been demonstrated. In this context, research focussed on antimycological action, especially for treatment of superficial infections, presents promising growth due to the characteristics of these infections that facilitate PDT application as new therapeutic options are needed in the field of medical mycology. Among the more than one hundred classes of photosensitizers the antifungal action of hypericin (Hyp) stands out due to its ability to permeate the lipid membrane and accumulate in different cytoplasmic organelles of eukaryotic cells. In this review, we aim to provide a complete overview of the origin, physicochemical characteristics, and optimal alternative drug deliveries that promote the photodynamic action of Hyp (Hyp-PDT) against fungi. Furthermore, considering the lack of a methodological consensus, we intend to compile the best strategies to guide researchers in the antifungal application of Hyp-PDT. Overall, this review provides a future perspective of new studies and clinical possibilities for the advances of such a technique in the treatment of mycoses in humans.

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This article describes Neocosmospora keratoplastica as an etiological onychomycosis agent. Ex vivo studies were initially performed to demonstrate the ability of this species to grow and form a well-organized characteristic biofilm on sterilized healthy nails. Based on the history of excellent results, both for antifungal and antibiofilm, of propolis resin gum, we evaluated its activity using artificially formed biofilm. In vitro, the minimal biofilm eradication concentration of the propolis extract (PE) was 375 µg of total polyphenol content (TPC) per mL, while for the propolis gel (PG) it was 450 µg of TPC per mL. In biofilm exposed to the propolis products, a decrease in hyphae and conidia was evident, accompanied by a disorganization of the extracellular matrix. Additionally, this low concentration of PE was able to significantly reduce the number of colony-forming units and the metabolic activity. Furthermore, the treatment of a 15-year nail infection due to N. keratoplastica was carried out exclusively using a topical treatment with a gel containing propolis (30%) with a daily dosage. This treatment achieved complete remission of the onychomycosis in 12 months. It is important to point out that some inconveniences previously reported by other patients treated with propolis extract were eliminated, increasing adherence to treatment.

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Biofilms are important to the virulence of human pathogenic fungi, and some molecules have been found to play key roles in the growth and regulation of fungal biofilms. Farnesol, one of these molecules, is well-described for some microorganisms but is still scarcely known for Rhodotorula spp. This study aimed to evaluate the influence of farnesol on the biofilm of R. mucilaginosa. Initially, screening with 0.2 mM to 2.1 mM of farnesol was evaluated against planktonic forms. A concentration of this compound was then chosen and evaluated for its effect on biofilm in formation and on preformed biofilm after 24, 48 and 72 hours. The impact of farnesol was evaluated by colony-forming units (CFU) counts, determination of metabolic activity and quantification of total biomass. In the presence of 0.9 mM, farnesol was able to decrease the CFU number, at 48 hours, when the biofilm was in formation, although it did not affect the preformed biofilms. Thus, our results show that farnesol exerts a modulating activity during biofilm formation for R. mucilaginosa, with this compound reducing the metabolic activity and total biomass of the biofilms.

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A ocorrência das infecções do trato urinário (ITU) causadas por leveduras do gênero Candida estão aumentando consideravelmente nas últimas décadas, sendo a Candida albicans a mais comumente diagnosticada como causadora deste tipo de infecções. Contudo, outras espécies, como exemplo da Candida tropicalis, estão emergindo como preocupantes causadores da doença. Neste sentido, o objetivo do presente trabalho é revisar os aspectos relacionados com as ITU causadas por leveduras do gênero Candida. Foi realizada uma pesquisa na base de dados PubMed, buscando artigos sobre a epidemiologia, patogenia e tratamento das ITU causadas por leveduras do gênero Candida. As espécies de Candida são os fungos patogênicos oportunistas mais relevantes causadores de infecções nosocomiais e podem causar infecção no trato urinário, tanto inferior (ureteres, bexiga e uretra) quanto superior (rins), principalmente em pacientes imunocomprometidos. Existem alguns fatores predisponentes, como gênero feminino, idade avançada, diabetes mellitus, hospitalização prolongada, imunossupressão, gravidez, hipertensão, neutropenia, cálculos renais, infecções nosocomiais, terapia antibiótica e procedimentos, como a cateterização, que atuam como facilitadores das ITU por Candida spp. A doença pode ocorrer de forma assintomática, porém, pode evoluir para casos mais graves com comprometimento sistêmico em situações de candidemia que pode causar a morte do paciente, principalmente se tratando de indivíduos imunocomprometidos. Sendo assim, devido ao risco existente, a doença não pode ser negligenciada e um diagnóstico preciso e um tratamento adequado devem ser estabelecidos.

The occurrence of urinary tract infections (UTI) caused by yeasts of the genus Candida has increased considerably in recent decades, with Candida albicans being the most commonly diagnosed as causing this type of infections. However, other species, such as Candida tropicalis, are emerging as worrisome causes of the disease. In this sense, the objective of the present paper is to review the aspects related to the UTI caused by yeasts of the genus Candida. A search was carried out in the PubMed database, searching for articles on the epidemiology, pathogenesis and treatment of UTI caused by yeasts of the genus Candida. Candida species are the most relevant opportunistic pathogenic fungi that cause nosocomial infections and can cause both lower (ureters, bladder and urethra) and upper (kidneys) urinary tract infections, especially in immunocompromised patients. There are some predisposing factors, such as female gender, advanced age, diabetes mellitus, prolonged hospitalization, immunosuppression, pregnancy, hypertension, neutropenia, kidney stones, nosocomial infections, antibiotic therapy and procedures, such as catheterization, that act as facilitators of UTI by Candida spp. The disease can occur asymptomatically, however, it can progress to more severe cases with systemic involvement in situations of candidemia that can cause the death of the patient, especially in immunocompromised individuals. Therefore, due to the existing risk, the disease cannot be neglected and an accurate diagnosis and adequate treatment must be established.

La aparición de infecciones del tracto urinario (ITU) causadas por levaduras del género Candida ha aumentado considerablemente en las últimas décadas. Candida albicans es la infección por levaduras más comúnmente diagnosticada. Sin embargo, otras especies, como la Candida tropicalis, están surgiendo como causa preocupante de la enfermedad. En este sentido, el objetivo del presente trabajo es revisar los aspectos relacionados con la ITU causada por levaduras del género Candida. Se realizó una búsqueda en la base de datos PubMed, buscando artículos sobre la epidemiología, la patogénesis y el tratamiento de la ITU causada por levaduras del género Candida. Las especies de Candida son los hongos patógenos oportunistas más relevantes que causan infecciones nosocomiales y pueden provocar infecciones del tracto urinario inferior (uréteres, vejiga y uretra) y superior (riñones), especialmente en pacientes inmunodeprimidos. Existen algunos factores predisponentes, como el sexo femenino, la edad avanzada, la diabetes mellitus, la hospitalización prolongada, la inmunosupresión, el embarazo, la hipertensión, la neutropenia, los cálculos renales, las infecciones nosocomiales, la terapia con antibióticos y los procedimientos como el cateterismo, que actúan como facilitadores de la ITU por Candida spp. La enfermedad puede presentarse de forma asintomática, pero puede evolucionar a casos más graves con afectación sistémica en situaciones de candidemia que pueden causar la muerte del paciente, especialmente en individuos inmunodeprimidos. Por lo tanto, debido al riesgo existente, no se puede descuidar la enfermedad y se debe establecer un diagnóstico preciso y un tratamiento adecuado.

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Most current protocols for the diagnosis of fungal infections are based on culture-dependent methods that allow the evaluation of fungal morphology and the identification of the etiologic agent of mycosis. Most current protocols for the diagnosis of fungal infections are based on culture-dependent methods that enable the examination of the fungi for further identification of the etiological agent of the mycosis. The isolation of fungi from pure cultures is typically recommended, as when more than one species is identified, the second agent is considered a contaminant. Fungi mostly survive in highly organized communities that provoke changes in phenotypic profile, increase resistance to antifungals and environmental stresses, and facilitate evasion from the immune system. Mixed fungal biofilms (MFB) harbor more than one fungal species, wherein exchange can occur that potentialize the effects of these virulence factors. However, little is known about MFB and their role in infectious processes, particularly in terms of how each species may synergistically contribute to the pathogenesis. Here, we review fungi present in MFB that are commensals of the human body, forming the mycobiota, and how their participation in MFB affects the maintenance of homeostasis. In addition, we discuss how MFB are formed on both biotic and abiotic surfaces, thus being a significant reservoir of microorganisms that have already been associated in infectious processes of high morbidity and mortality.

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Few antifungals available today are effective in treating biofilms. Thus, it is urgent to discover new compounds, such as natural products, that provide improvements to existing treatments or the development of new antifungal therapies. This study aimed to perform a comparative analysis between the green propolis extract (PE) and its by-product, a waste of propolis extract (WPE) through a screening with Candida sp., Fusarium sp. and Trichophyton sp. The antifungal property of PE and WPE was assessed by the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) determination in planktonic cells. The influence of both extracts on the inhibition of biofilm formation in these fungi was also tested. The WPE MIC and MFC values (68.75 to 275.0 µg/mL) were three to twelve times lower than the values obtained for PE (214.06 to 1712.5 µg/mL). PE was more efficient than WPE in inhibiting the biofilm initial phase, especially in C. albicans. Meanwhile, WPE had dose-dependent behavior for the three fungi, being more effective on filamentous ones. Both PE and WPE showed excellent antifungal activity on planktonic cells and demonstrated great efficacy for inhibiting biofilm formation in the three fungi evaluated.

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Fungal biofilms have been involved in the pathogenesis of onychomycosis, but the aspects contributing to this association need to be enlightened. This study aimed to investigate the ability of three different fungi to form biofilm on the nail. All evaluated fungi were able to grow on the nails, using them as the only nutritional source and formed a structure strongly suggestive of biofilms. However, their architecture and morphology were highly contrasting: Candida albicans showed dense growth, exhibited a well-structured community and a large amount of extracellular matrix (ECM), and FTIR-ATR spectroscopy reinforced these findings revealing components suggestive of the biofilm. For Fusarium oxysporum, these events were also observed, but in lower intensity. Furthermore, while Trichophyton rubrum presented a well-organized architecture, the ECM was not visualized. We hypothesize that these findings are related to the symptomatology of onychomycosis. When it is caused by a non-dermatophyte, it usually is accompanied with paronychia, pain, oedema, inflammation and few signals of keratolysis, while dermatophytes are more associated with intense onycholysis and absence of the inflammatory signals. Biofilm seems to be crucial for non-dermatophytes to cause onychomycosis, but not for T. rubrum.

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Onychomycosis (OM) is a fungal infection, responsible for about 50% of nail diseases. OM has been attributed to the ability of fungi to naturally organize themselves into biofilms on nail surfaces. However, little is known about the exact role of the biofilm in the etiopathogenesis of OM, as well as its influence in the permeation of a topical treatment. The objectives of this study were to review the literature for topical OM treatments in clinical trials, assess the efficiency of these treatments, and discuss factors that could affect the success of these treatments. First, a systematic search of articles published in the MEDLINE database (PubMed) between January 2010 and December 2019 was conducted, focusing on drugs under clinical trials for the topical treatment of OM. Of the publications selected, it was clear that none of them had considered the fungi organized in biofilm. Therefore, we reflected on some important variables involved in OM, such as the nail structure and the mechanism of fungal invasion. Some methods, such as histopathologic analysis and spectroscopy techniques, were found to be effective in the detection of nail biofilm, and could be used in future drug permeation studies. This review allowed us to conclude that novel antifungals for the topical treatment of OM must consider the drug to permeate through biofilm. Natural products, such as propolis, seem strong candidates in this respect.

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Candida albicans is a commensal microorganism of the human microbiota that can be associated with superficial to disseminated infections. This species possesses several attributes that contribute to pathogenesis and virulence, such as the ability to transition from yeast to hyphae forms. During this transition, several changes occur in the fungal cell wall, which is the first point of contact between the pathogen and the host. The cell wall is a bi-layered structure, containing chitin, glucan, and proteins, the latter of which play important roles in pathogenesis. Given the importance of this structure, particularly in filamentation, this review focuses on the studies of C. albicans mutants for genes that encode cell wall-associated proteins that have an important role in the virulence, and also have a role in hyphal morphogenesis. Thus, we highlight some proteins whose mutation is associated with attenuated virulence in vivo and have defective filamentation. We also provide examples of proteins whose inactivation does not impair the filamentation yet are still important for C. albicans virulence.

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Sporotrichosis is a worldwide zoonosis, prevalent in tropical and subtropical regions. In recent years, there has been a substantial increase in human and feline cases reported in Brazil. Despite this, the antifungal treatment for sporotrichosis is still limited, and thus, research into new therapeutic modalities must be encouraged. Recently, photodynamic therapy has been introduced as a treatment for sporotrichosis. This work presents an overview of both in vitro and in vivo studies that have used photodynamic therapy in the context of photoinactivation of Sporothrix species. Until now, as far as the authors are aware, this is the first scope review specifically on photodynamic therapy for the treatment of sporotrichosis. A systematic electronic search was conducted in two databases: Web of Science and PubMed. Seven original articles published from 2010 to July 2021 were selected, six of which met the proposed inclusion and exclusion criteria and were considered in this scoping review. Concerning the photoinactivation of Sporothrix spp. the results have been promising as studies, in both animals and humans, have reported significant clinical and mycological effects. The most used photosensitizers were methylene blue and its derivatives, and aminolevulinic acid and its methyl derivative, methyl aminolevulinic acid. In conclusion, photodynamic therapy has great potential in treatment of sporotrichosis, as its fungicidal effect both in vitro and in vivo has clearly been demonstrated. Photodynamic therapy could be used in conjunction with classic antifungal agents to optimize treatment outcomes.

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