Envelope Protein in Differential Serodiagnosis of Dengue, Zika, and Chikungunya Viruses: A Systematic Review.

OBJECTIVES: This systematic review was conducted to evaluate the applicability of the envelope (E) protein in the diagnosis of arboviruses. METHODS: This review was performed in accordance with the PRISMA statement. Five databases were explored (PubMed, Web of Science, Scopus, EMBASE, and IEDB). The inclusion and exclusion criteria were applied to study eligibility. After data extraction, the risk of bias and evidence certainty were evaluated according to QUADAS and GRADE assessments, respectively. RESULTS: Eleven studies were included. A total of 11 studies were included in the review. ELISA was the most frequently utilized technique, with two studies employing it for antigen detection and nine for antibodies. The E protein was used as a whole protein, heterologous protein, and peptides. The diagnostic metrics were enhanced by optimizations on techniques, such as antibody capture, competitors, and nanosensors. Monoclonal antibodies showed improved specificity, including in coinfected samples. Seven studies demonstrated a minimal risk of bias, and the evidence certainty was considered moderate for dengue diagnosis. CONCLUSIONS: The E protein was successfully employed in different immunological assays with large-scale strategies, enhancing the applicability potential for differential arboviruses' diagnosis. Furthermore, both the antigen design and the implementation of innovative methodologies will have a substantial impact on the quality of the new tests. The PROSPERO protocol related to this work: CRD42021265243.

"Antibacterial effect and possible mechanism of action of 1,3,4-oxadiazole in Staphylococcus aureus".

Staphylococcus aureus is one of the main etiological agents causing foodborne diseases, and the development of new antibacterial agents is urgent. This study evaluated the antibacterial activity and the possible mechanism of action of the 1,3,4-oxadiazole LMM6 against S. aureus. The minimum inhibitory concentration (MIC) of LMM6 ranged from 1.95 to 7.81 µg ml-1. The time-kill assay showed that 48-h treatment at 1× to 8× MIC reduced S. aureus by 4 log colony forming unit (CFU), indicating a bacteriostatic effect. Regarding the possible mechanism of action of LMM6, there was accumulation of reactive oxygen species (ROS) and an increase in the absorption of crystal violet (∼50%) by the cells treated with LMM6 at 1× and 2× MIC for 6-12 h. In addition, there was increased propidium iodide uptake (∼84%) after exposure to LMM6 for 12 h at 2× MIC. After 48 h of treatment, 100% of bacteria had been injured. Scanning electron microscopy observations demonstrated that LMM6-treated cells were smaller compared with the untreated group. LMM6 exhibited bacteriostatic activity and its mechanism of action involves increase of intracellular ROS and disturbance of the cell membrane, which can be considered a key target for controlling the growth of S. aureus.

Photodynamic inactivation by hypericin-P123 on azole-resistant isolates of the Trichophyton rubrum complex as planktonic cells and biofilm.

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.

Successful treatment of experimental murine vulvovaginal candidiasis with gentian violet.

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.

Cell wall associated proteins involved in filamentation with impact on the virulence of Candida albicans.

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.

New approach to the use of propolis against dermatomycosis.

In recent years, propolis extract (PE) has demonstrated antimicrobial and anti-inflammatory properties. The aim of this study was to evaluate the antifungal activity of a bioadhesive thermoresponsive system containing 16% propolis (BTSP 16%) against Microsporum canis, Nannizzia gypsea, Trichophyton mentagrophytes and T. rubrum. We also evaluated PE alone against the same strains. The results showed that both PE and BTSP 16% significantly reduced the fungal viability of all evaluated strains. In addition, they interacted with the biofilm of these species in different stages of biofilm formation. We observed that the bioadhesive and thermoresponsive properties of BTSP 16% prolonged propolis presence at infection sites, leading to positive results against planktonic fungal cells and mature biofilms. These characteristics make this formulation a valuable alternative treatment for dermatomycosis.

Anti-mycotoxigenic and antifungal activity of ginger, turmeric, thyme and rosemary essential oils in deoxynivalenol (DON) and zearalenone (ZEA) producing Fusarium graminearum.

This study aimed to evaluate the antimycotoxigenic effect of essential oils (EOs) obtained from four different aromatic plants on the production of deoxynivalenol (DON) and zearalenone (ZEA) by Fusarium graminearum. The EOs from ginger (GEO), turmeric (TEO), thyme (ThEO) and rosemary (REO) were obtained by hydrodistillation and identified by gas chromatography/mass spectrometry (GC/MS). The major compounds found were mostly monoterpenes and sesquiterpenes. The minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were 11.25, 364, 366 and 11,580 µg mL-1 for ThEO, GEO, REO and TEO, respectively. The results evidenced that the assessed EOs inhibited DON and partially ZEA production by F. graminearum. ThEO and GEO were the EOs with most potent antimycotoxigenic action for DON and ZEA, respectively. These EOs have shown promising results in vitro regarding inhibition of mycotoxin production and might be used in the future as substitutes for synthetic fungicides.

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.

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.

Different expression levels of ALS and SAP genes contribute to recurrent vulvovaginal candidiasis by Candida albicans.

Aim: To study the behavior of Candida albicans in women with vulvovaginal candidiasis (VVC), recurrent VVC (RVVC) and asymptomatic (AS), regarding adhesion on HeLa cells and their ability to express secreted aspartic proteinases (SAP) genes, agglutinin-like sequence (ALS) genes and HWP1. Materials & methods: The adhesion of Candida albicans to HeLa cells was evaluated by colony-forming units, and the expressed genes were evaluated by qRT-PCR. Results: AS and VVC isolates showed greater ability to adhere HeLa cells when compared with RVVC isolate. Nevertheless, RVVC isolate exhibited upregulation of a large number of genes of ALS and SAP gene families and HWP1 gene. Conclusion: The results demonstrated that RVVC isolate expressed significantly important genes for invasion and yeast-host interactions.

One Century of Study: What We Learned about Paracoccidioides and How This Pathogen Contributed to Advances in Antifungal Therapy.

Paracoccidioidomycosis (PCM) is a notable fungal infection restricted to Latin America. Since the first description of the disease by Lutz up to the present day, Brazilian researchers have contributed to the understanding of the life cycle of this pathogen and provided the possibility of new targets for antifungal therapy based on the structural and functional genomics of Paracoccidioides. In this context, in silico approaches have selected molecules that act on specific targets, such as the thioredoxin system, with promising antifungal activity against Paracoccidioides. Some of these are already in advanced development stages. In addition, the application of nanostructured systems has addressed issues related to the high toxicity of conventional PCM therapy. Thus, the contribution of molecular biology and biotechnology to the advances achieved is unquestionable. However, it is still necessary to transcend the boundaries of synthetic chemistry, pharmaco-technics, and pharmacodynamics, aiming to turn promising molecules into newly available drugs for the treatment of fungal diseases.

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.

Impact of serial systemic infection on Candida albicans virulence factors.

Aim: To evaluate changes in virulence and pathogenicity approaches from Candida albicans after successive passages in a murine model of systemic candidiasis. Materials & methods: Phenotypic assays were performed using colonies recovered from animals infected serially, totalizing five passages. Results: A progressive infection was observed along the passages, with increased fungal burden and the presence of greater inflammatory areas in the histopathological findings. Recovered strains exhibited increased filamentation and biofilm abilities, along with modulation of phospholipase and proteinase activities. Conclusion: Repeated contact between yeast and host increased the expression of virulence factors. Furthermore, a correspondence between phenotypic profile and proteomic data obtained previously was observed.

Vulvovaginal candidiasis in a murine model of diabetes emphasizing the invasive ability of etiological agents.

Aim: To compare the pathogenesis of vulvovaginal candidiasis by three Candida species in diabetic mice. Materials & methods: Estrogenized and diabetic mice were challenged with C. albicans, C. tropicalis and C. glabrata. Results: Diabetic animals infected with C. albicans and C. tropicalis maintained the highest fungal burden, despite of high levels of proinflammatory cytokines (IL-6 and TNF-α), respectively. For C. glabrata, the results were similar in diabetic and nondiabetic groups. Conclusion:C. tropicalis was as invasive as C. albicans, and both were more effective than C. glabrata. This ability was attributed to filamentation, which may be stimulated by glucose levels from vaginal fluid. In addition, the high burden may be attributed to the apparent immunological inefficiency of the diabetic host.

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.

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.

Copolymeric micelles as efficient inert nanocarrier for hypericin in the photodynamic inactivation of Candida species.

Aim: To evaluate the efficacy of photodynamic inactivation (PDI) mediated by hypericin encapsulated in P-123 copolymeric micelles (P123-Hyp) alone and in combination with fluconazole (FLU) against planktonic cells and biofilm formation of Candida species Materials & methods: PDI was performed using P123-Hyp and an LED device with irradiance of 3.0 mW/cm2 . Results: Most of isolates (70%) were completely inhibited with concentrations up to 2.0 µmol/l of HYP and light fluence of 16.2 J/cm2. FLU-resistant strains had synergic effect with P123-HYP-PDI and FLU. The biofilm formation was inhibited in all species, in additional the changes in Candida morphology observed by scanning electron microscopy. Conclusion: P123-Hyp-PDI is a promising option to treat fungal infections and medical devices to prevent biofilm formation and fungal spread.

Epidemiological investigation and molecular typing of dermatophytosis caused by Microsporum canis in dogs and cats.

The objective of the present study was investigate the prevalence of dermatophytes in dogs, cats and environment floor through molecular epidemiology tools to identify the genetic profile of these infectious agents. This was an observational study with cross-sectional surveys design. Sample were collected from the hair and skin of 52 dogs and cats with the clinical suspicion of dermatophytosis, over a period of one year in Maringá, in the state of Paraná, Brazil. Household samples (carpets and floor), were collected from animals that were positive for dermatophytosis by morphological colonies characteristics, and samples of dogs or cats living in the same household as with the positive animals were also collected. After mycological confirmation, molecular typing was performed by random amplified polymorphic DNA (RAPD). Microsporum canis was the unic dermatophyto isolated whose prevalence was 26.9% (14/52) in animals with the clinical suspicion of dermatophytosis and four other animals that lived with positive animals. As some animals had more than one lesion site, there were 22 total positive cultures from samples from animals and another ten from abiotic sources. The majority of the animals that provided positive cultures for M. canis were aged up to five months (77.8%) and were female (66.7%). Molecular typing using the P1 primer revealed genetically distinct profiles in the symptomatic, asymptomatic and environmental animal samples, or the same animal, furthermore, showed that M. canis could have microevolution.