Serial systemic candidiasis alters Candida albicans macromorphology associated with enhancement of virulence attributes.

Candida albicans is one of the major pathogens found in superficial and invasive infections. This fungus expresses several virulence factors and fitness attributes that are essential to the pathogenesis. In our previous study using a murine model of serial systemic candidiasis, virulence of the recovered C. albicans was enhanced and several virulence factors were also modified after five successive passages through mice (P1-P5). In this study, we aimed to correlate the different fungal morphologies, as well as the filamentation, invasion, and stress resistance abilities, of the cells recovered after passing through this model of infection with our previous findings regarding virulence. We obtained two colony morphology types from the recovered cells, differing in their peripheral filamentation. The morphotype 1, which presented zero to five filaments in the colony edge, was higher in P2, while morphotype 2, which presented more than five filaments in the colony edge, was predominant from P3 to P5. In general, morphotype 1 showed similar levels regarding filamentation in serum, invasion of agar and cells, and resistance to osmotic, oxidative, and thermal stress in all passages analyzed. The morphotype 2, however, exhibited an enhancement in these abilities over the passages. We observed an accordance with the increased virulence over the passages obtained in our previous study and the increased adaptability profile of morphotype 2. Therefore, we suggest that the behavior observed previously in the pathogenesis and virulence could be attributed, at least in part, to the greater presence and ability of morphotype 2.

Repurposing approach identifies new treatment options for invasive fungal disease.

Drug repositioning is the process of discovery, validation and marketing of previously approved drugs for new indications. Our aim was drug repositioning, using ligand-based and structure-based computational methods, of compounds that are similar to two hit compounds previously selected by our group that show promising antifungal activity. Through the ligand-based method, 100 compounds from each of three databases (MDDR, DrugBank and TargetMol) were selected by the Tanimoto coefficient, as similar to LMM5 or LMM11. These compounds were analyzed by the scaffold trees, and up to 10 compounds from each database were selected. The structure-based method (molecular docking) using thioredoxin reductase as the target drug was performed as a complementary approach, resulting in six compounds that were tested in an in vitro assay. All compounds, particularly raltegravir, showed antifungal activity against the genus Paracoccidioides. Raltegravir, an antiviral drug, showed promising antifungal activity against the experimental murine paracoccidioidomycosis, with significant reduction of the fungal burden and decreased alterations in the lung structure of mice treated with 1 mg/kg of raltegravir. In conclusion, the combination of two in silico methods for drug repositioning was able to select an antiviral drug with promising antifungal activity for treatment of paracoccidioidomycosis.

In vitro interaction of Candida tropicalis biofilm formed on catheter with human cells.

Candida tropicalis has emerged as one of the major Candida non-C. albicans species, in terms of epidemiology and virulence. Despite its virulence, C. tropicalis pathogenic mechanism has yet not been fully defined. The current study aimed to demonstrate the interaction of mature C. tropicalis ATCC 750 biofilm formed on catheter with different human cell lines. In vitro mature (72 h) C. tropicalis biofilms were produced on small catheter fragments (SCF) and were mainly composed by blastoconidia. Then, migration of yeast cells from mature biofilm to human cell surfaces (HeLa and HUVEC) was investigated. After contact with both cell lines, the surface of SCF, containing mature C. tropicalis biofilm, exhibited predominantly the filamentous form. Meanwhile, fresh biofilm formed on human cell surfaces also revealed mainly of blastoconidia involved by extracellular matrix. Total biomass and metabolic activity from the remaining biofilm on SCF surface, after direct contact with human cells, exhibited a significant reduction. Mature C. tropicalis biofilm modified its extracellular matrix components, after contact with human cells. Thus, we described for the first time an easy and simple in vitro model with catheter, which could be a powerful tool for future studies that desires to elucidate the mechanisms involved in C. tropicalis biofilm.

Healthcare workers' hands as a vehicle for the transmission of virulent strains of Candida spp.: A virulence factor approach.

BACKGROUND: Although the majority of Candida infections are thought to come from endogenous sources, the healthcare workers' (HCWs) hands are being increasingly reported as vehicles for the transmission of pathogens. The aim of the present study was to evaluate the susceptibility of yeast isolated from the HCWs' hands and ICU (Intensive Care Unit) surfaces to antifungal agents and to determine the virulence potential and the genetic similarity between the same. METHODS: The susceptibility of yeasts from the HCWs' hands (n = 57) and ICU surfaces (n = 98) to conventional antifungals (fluconazole, voriconazole, amphotericin B and micafungin) was evaluated using the broth microdilution assay accordance with CLSI M27-A3. Additionally, some virulence factors such as adhesion and biofilm capacity on abiotic surfaces and on endothelial cells were evaluated, as well as germ tube formation. The similarity among yeast isolates were evaluated by the RAPD technique using the P4, OPA18 and OPE18 primers. RESULTS: Five species of Candida were found on the HCWs' hands (C. albicans, C. parapsilosis (sensu stricto), C. glabrata, C. tropicalis and C. krusei) and two on ICU surfaces (C. albicans and C. parapsilosis (sensu stricto)). The isolates from hands had higher resistance rates, with C. glabrata having the highest indices (100% FLU; 100% MFG). The similarity of C. albicans from HCWs and ICU surfaces was ≥80% according to the three primers analyzed. Candida spp. from hands had a greater potential for adhesion and biofilm formation on abiotic surfaces (p < 0.05). C. albicans from ICU surfaces had the greatest potential of adhesion on endothelial cells after 2 and 24 h, and presented high filamentation in SEM images and formed more and larger germ tubes (p < 0.05). CONCLUSION: the present study showed the significant virulence potential of yeasts transmitted in the hospital environment for the first time. Additionally, healthy people working in the ICU can carry these yeasts, which are capable of surviving in hospital surfaces, on their hands, offering a risk to patients, especially those who are immunocompromised.

Promising onychomycosis treatment with hypericin-mediated photodynamic therapy: Case reports.

BACKGROUND: Onychomycosis (OM) is a common nail plate disorder caused by dermatophyte molds, yeasts, and non-dermatophyte molds, which use keratin in the nail plate as an energy source. OM is characterized by dyschromia, increased nail thickness, subungual hyperkeratosis, and onychodystrophy, and is typically treated with conventional antifungals despite frequent reports of toxicity, fungal resistance, and OM recurrence. Photodynamic therapy (PDT) with hypericin (Hyp) as a photosensitizer (PS) stands out as a promising therapeutic modality. When excited by a specific wavelength of light and in the presence of oxygen, to lead to photochemical and photobiological reactions on the selected targets. METHODS: OM diagnosis was made in three suspected cases, and the causative agents were identified by classical and molecular methods, and confirmed by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Susceptibility of planktonic cells of the clinical isolates to conventional antifungals and PDT-Hyp was evaluated, and photoacoustic spectroscopy (PAS) of Hyp permeation in nail fragments ex vivo was analyzed. Furthermore, the patients opted to undergo PDT-Hyp treatment and were subsequently followed up. The protocol was approved by the human ethics committee (CAAE, number 14107419.4.0000.0104). RESULTS: The etiological agents of OM in patients ID 01 and ID 02 belonged to the Fusarium solani species complex, being identified as Fusarium keratoplasticum (CMRP 5514) and Fusarium solani (CMRP 5515), respectively. For patient ID 03, the OM agent was identified as Trichophyton rubrum (CMRP 5516). PDT-Hyp demonstrated a fungicidal effect in vitro, with reductions of p3 log10 (p < 0.0051 and p < 0.0001), and the PAS analyses indicated that Hyp could completely permeate through both healthy and OM-affected nails. After four sessions of PDT-Hyp, mycological cure was observed in all three cases, and after seven months, clinical cure was confirmed. CONCLUSION: PDT-Hyp showed satisfactory results in terms of efficacy and safety, and thus can be considered a promising therapy for the clinical treatment of OM.

Fungicidal Activity of a Safe 1,3,4-Oxadiazole Derivative Against Candida albicans.

Candida albicans is the most common species isolated from nosocomial bloodstream infections. Due to limited therapeutic arsenal and increase of drug resistance, there is an urgent need for new antifungals. Therefore, the antifungal activity against C. albicans and in vivo toxicity of a 1,3,4-oxadiazole compound (LMM6) was evaluated. This compound was selected by in silico approach based on chemical similarity. LMM6 was highly effective against several clinical C. albicans isolates, with minimum inhibitory concentration values ranging from 8 to 32 µg/mL. This compound also showed synergic effect with amphotericin B and caspofungin. In addition, quantitative assay showed that LMM6 exhibited a fungicidal profile and a promising anti-biofilm activity, pointing to its therapeutic potential. The evaluation of acute toxicity indicated that LMM6 is safe for preclinical trials. No mortality and no alterations in the investigated parameters were observed. In addition, no substantial alteration was found in Hippocratic screening, biochemical or hematological analyzes. LMM6 (5 mg/kg twice a day) was able to reduce both spleen and kidneys fungal burden and further, promoted the suppresses of inflammatory cytokines, resulting in infection control. These preclinical findings support future application of LMM6 as potential antifungal in the treatment of invasive candidiasis.

Efficacy of Ebselen Against Invasive Aspergillosis in a Murine Model.

Invasive aspergillosis is one of the major causes of morbidity and mortality among invasive fungal infections. The search for new antifungal drugs becomes imperative when existing drugs are not able to efficiently treat these infections. Ebselen, is an organoselenium compound, already successfully approved in clinical trials as a repositioned drug for the treatment of bipolar disorder and prevention of noise-induced hearing loss. In this study, we aimed to reposition ebselen for the treatment of invasive aspergillosis by showing ebselen effectiveness in a murine model. For this, BALB/c mice were immunosuppressed and infected systemically with Aspergillus fumigatus. Animals were divided and treated with ebselen, voriconazole, or drug-free control, for four days. The kidneys were used for CFU count and, histopathological and cytokine analysis. Ebselen was able to significantly reduce the fungal burden in the kidneys of infected mice with efficacy comparable with voriconazole treatment as both had reductions to the same extent. The absence of hyphae and intact kidney tissue structure observed in the histopathological sections analyzed from treated groups corroborate with the downregulation of IL-6 and TNF. In summary, this study brings for the first time in vivo evidence of ebselen efficacy against invasive aspergillosis. Despite these promising results, more animal studies are warranted to evaluate the potential role of ebselen as an alternative option for the management of invasive aspergillosis in humans.

Nanoencapsulated hypericin in P-123 associated with photodynamic therapy for the treatment of dermatophytosis.

The antifungal application of photodynamic therapy (PDT) has been widely explored. According to superficial nature of tinea capitis and the facility of application of light sources, the use of nanoencapsulated hypericin in P-123 associated with PDT (P123-Hy-PDT) has been a poweful tool to treat this pathology. Thus, the aim of this study was to evaluate the efficiency of P123-Hy-PDT against planktonic cells and in a murine model of dermatophytosis caused by Microsporum canis. In vitro antifungal susceptibility and in vivo efficiency tests were performed, including a skin toxicity assay, analysis of clinical signs by evaluating score, and photoacoustic spectroscopy. In addition, tissue analyses by histopathology and levels of pro-inflammatory cytokines, such as quantitative and qualitative antifungal assays, were employed. The in vitro assays demonstrated antifungal susceptibility with 6.25 and 12.5 µmol/L P123-Hy-PDI; these experiments are the first that have used this treatment of animals. P123-Hyp-mediated PDT showed neither skin nor biochemical alteration in vivo; it was safe for dermatophytosis treatment. Additionally, the treatment revealed rapid improvement in clinical signs at the site of infection after only three treatment sessions, with a clinical score confirmed by photoacoustic spectroscopy. The mycological reduction occurred after six treatment sessions, with a statistically significant decrease compared with untreated infected animals. These findings showed that P123-Hy-PDT restored tissue damage caused by infection, a phenomenon confirmed by histopathological analysis and proinflammatory cytokine levels. Our results reveal for the first time that P123-Hy-PDT is a promising treatment for tinea capitis and tinea corporis caused by M. canis, because it showed rapid clinical improvement and mycological reduction without causing toxicity.

Development and in vitro evaluation of buccal mucoadhesive films for photodynamic inactivation of Candida albicans.

Candidiasis is one of the most common diseases that occur in the oral cavity, caused mainly by the species Candida albicans. Methylene blue (MB) has a potential for microbial photoinactivation and can cause the destruction of fungi when applied in Photodynamic Therapy (PDT). Mucoadhesive films are increasingly being studied as a platform for drug application due to their advantages when compared to other pharmaceutical forms. The aim of this work was to develop mucoadhesive buccal film containing poloxamer 407 (P407), alcohol polyvinyl (PVA) and polyvinylpyrrolidone (PVP) for the release of MB aiming the photoinactivation of Candida albicans in buccal infections. Different amounts of P407 were added to the binary polymeric blends composed PVA and PVP. Formulations were characterized as morphology, thickness, density, bending strength, mechanical properties, water vapor transmission, disintegration time, mucoadhesion, DSC, ATR-FTIR, in vitro drug release profile and photodynamic inactivation. The films displayed physicochemical characteristics dependent of polymeric composition, mucoadhesive properties, fast MB release and were effective in photo inactivate the local growth of Candida albicans isolates. The formulation containing the lowest PVA and P407 amounts displayed the best performance. Therefore, data obtained from the film system show its potentially useful role as a platform for buccal MB delivery in photoinactivation of C. albicans, showing its potential for in vivo evaluation.

Promising antifungal activity of new oxadiazole against Candida krusei.

Candida krusei is one of the most common agents of invasive candidiasis and candidemia worldwide, leading to high morbidity and mortality rates. This species has become a problem due to its intrinsic resistance and reduced susceptibility to azoles and polyenes. Moreover, the number of antifungal drugs available for candidiasis treatment is limited, demonstrating the urgent need for the discovery of novel alternative therapies. In this work, the in vivo and in vitro activities of a new oxadiazole (LMM11) were evaluated against C. krusei. The minimum inhibitory concentration ranged from 32 to 64 µg/mL with a significant reduction in the colony forming unit (CFU) count (~3 log10). LMM11 showed fungicidal effect, similar to amphotericin, reducing the viable cell number (>99.9%) in the time-kill curve. Yeast cells presented morphological alterations and inactive metabolism when treated with LMM11. This compound was also effective in decreasing C. krusei replication inside and outside macrophages. A synergistic effect between fluconazole and LMM11 was observed. In vivo treatment with the new oxadiazole led to a significant reduction in CFU (0.85 log10). Furthermore, histopathological analysis of the treated group exhibited a reduction in the inflammatory area. Taken together, these results indicate that LMM11 is a promising candidate for the development of a new antifungal agent for the treatment of infections caused by resistant Candida species such as C. krusei.

Preclinical approaches in vulvovaginal candidiasis treatment with mucoadhesive thermoresponsive systems containing propolis.

Vulvovaginal candidiasis (VVC) is a common vaginitis that affects women, especially in childbearing age, caused by Candida albicans in almost 80% of cases. Considering the limited drug arsenal available and the increasing fungal resistance profile, the search for new therapeutic sources with low toxicity and easy administration should be supported. Propolis has been used as a traditional medicine for multiple diseases, considering its particular composition and pharmaceutical properties that permits its wide applicability; it has also emerged as a potential antifungal agent. Thus, this study performed an in vitro and in vivo investigation into the efficacy of a new mucoadhesive thermoresponsive platform for propolis delivery (MTS-PRPe) in a preclinical murine model of VVC treatment caused by C. albicans. The methodologies involved chemical analysis, an assessment of the rheological and mucoadhesive properties of propolis formulations, in vitro and in vivo antifungal evaluations, histological evaluations and electron microscopy of the vaginal mucosa. The results demonstrated the antifungal activity of propolis extract and MTS-PRP against the standard strain and a fluconazole-resistant clinical isolate of C. albicans, in both in vitro and in vivo assays. These results were similar and even better, depending on the propolis concentration, when compared to nystatin. Thus, the formulation containing propolis exhibited good performance against C. albicans in a vulvovaginal candidiasis experimental model, representing a promising opportunity for the treatment of this infection.

Serial Systemic Candida albicans Infection Highlighted by Proteomics.

Candida albicans is the major pathogen isolated from nosocomial bloodstream infections, leading to higher mortality rates. Thus, due to its clinical relevance, studies aiming to understand host-pathogen interactions in C. albicans infection are necessary. Therefore, we performed proteomic analysis using a murine model of serial systemic infection by C. albicans to evaluate possible changes in the protein profile of the pathogen over time. Firstly, we observed a reduction in the median survival time of infected animals with increasing passage number, suggesting a higher pathogenicity acquired during repeated infections. By LC-MS/MS, it was possible to obtain protein profiles from the wild-type strain (WT) and compare them to proteins extracted from Candida cells recovered from infected tissues during passages one, three, and four (P1, P3, and P4). We obtained 56, 29, and 97 proteins in P1, P3, P4, respectively, all varying in abundance. Regarding biological processes, the majority of proteins were related to carbohydrate metabolism, stress responses and amino acid metabolism. The proteins were also categorized according to their potential role in virulence traits, such as biofilm production, yeast-to-hyphae transition, phenotypic switching, proteins related to stress responses, and uncharacterized proteins. Therefore, serial infection in combination with proteomic approach enabled us to deepen the existing knowledge about host-pathogen interactions.

Two New 1,3,4-Oxadiazoles With Effective Antifungal Activity Against Candida albicans.

Candida infections have become a serious public health problem with high mortality rates, especially in immunocompromised patients, since Candida albicans is the major opportunistic pathogen responsible for systemic or invasive candidiasis. Commercially available antifungal agents are restricted and fungal resistance to such drugs has increased; therefore, the development of a more specific antifungal agent is necessary. Using assays for antifungal activity, here we report that two new compounds of 1,3,4-oxadiazoles class (LMM5 and LMM11), which were discovered by in silico methodologies as possible thioredoxin reductase inhibitors, were effective against C. albicans. Both compounds had in vitro antifungal activity with MIC 32 µg/ml. Cytotoxicity in vitro demonstrated that LMM5 and LMM11 were non-toxic in the cell lines evaluated. The kinetic of the time-kill curve suggested a fungistatic profile and showed an inhibitory effect of LMM5 and LMM11 in 12 h that remained for 24 and 36 h, which is better than fluconazole. In the murine systemic candidiasis model by C. albicans, the two compounds significantly reduced the renal and spleen fungal burden. According to the SEM and TEM images, we hypothesize that the mechanism of action of LMM5 and LMM11 is directly related to the inhibition of the enzyme thioredoxin reductase and internally affect the fungal cell. In view of all in vitro and in vivo results, LMM5 and LMM11 are effective therapeutic candidates for the development of new antifungal drugs addressing the treatment of human infections caused by C. albicans.

Antifungal activity of two oxadiazole compounds for the paracoccidioidomycosis treatment.

Paracoccidioidomycosis (PCM) is a neglected disease present in Latin America with difficulty in treatment and occurrence of serious sequelae. Thus, the development of alternative therapies is imperative. In the current work, two oxadiazole compounds (LMM5 and LMM11) presented fungicidal activity against Paracoccidioides spp. The minimum inhibitory and fungicidal concentration values ranged from 1 to 32 µg/mL, and a synergic effect was observed for both compounds when combined with Amphotericin B. LMM5 and LMM11 were able to reduce CFU counts (≥2 log10) on the 5th and 7th days of time-kill curve, respectively. The fungicide effect was confirmed by fluorescence microscopy (FUN-1/FUN-2). The hippocratic screening and biochemical analysis were performed in Balb/c male mice that received a high dose of each compound, and the compounds showed no in vivo toxicity. The treatment of experimental PCM with the new oxadiazoles led to significant reduction in CFU (≥1 log10). Histopathological analysis of the groups treated exhibited control of inflammation, as well as preserved lung areas. These findings suggest that LMM5 and LMM11 are promising hits structures, opening the door for implementing new PCM therapies.

Cell damage caused by vaginal Candida albicans isolates from women with different symptomatologies.

The present study aimed to characterize cell damage caused by vaginal Candida albicans isolates from women with different symptomatologies. It was evaluated 12 clinical isolates of C. albicans from vaginal samples: 4 from asymptomatic women (AS), 4 from women with a single episode of vulvovaginal candidiasis (VVC) and 4 from women with recurrent vulvovaginal candidiasis (RVVC). We evaluated the ability of C. albicans to adhere to human cervical cancer cells (SiHa), the yeast-SiHa cell interactions and cell damage. All of the clinical isolates presented a high adhesion capacity on SiHa cells. However, clinical isolates from symptomatic women (VVC and RVVC) had higher filamentation after contact (24 h) with SiHa cells and a greater capacity to cause cell damage (>80 %). Clinical isolates from symptomatic women had greater potential to invade SiHa cells, suggesting that they are more pathogenic than AS isolates.

Genetic Polymorphisms of IL17 and Chagas Disease in the South and Southeast of Brazil.

The aim of this study was to investigate possible associations between genetic polymorphisms of IL17A G197A (rs2275913) and IL17F T7488C (rs763780) with Chagas Disease (CD) and/or the severity of left ventricular systolic dysfunction (LVSD) in patients with chronic Chagas cardiomyopathy (CCC). The study with 260 patients and 150 controls was conducted in the South and Southeast regions of Brazil. The genotyping was performed by PCR-RFLP. The A allele and A/A genotype of IL17A were significantly increased in patients and their subgroups (patients with CCC; patients with CCC and LVSD; and patients with CCC and severe LVSD) when compared to the control group. The analysis according to the gender showed that the A/A genotype of IL17A was more frequent in female with LVSD and mild to moderate LVSD and also in male patients with LVSD. The frequency of IL17F T/C genotype was higher in male patients with CCC and severe LVSD and in female with mild to moderate LVSD. The results suggest the possible involvement of the polymorphisms of IL17A and IL17F in the susceptibility to chronic Chagas disease and in development and progression of cardiomyopathy.