Evaluation of antifungal activity of natural compounds on growth and aflatoxin B1 production of Aspergillus parasiticus and Aspergillus flavus.

BACKGROUND: Aspergillus species cause broad spectrum infections especially invasive lethal infections in immunocompromised patients. This study aimed to assess the antifungal activity of plants and compounds including Aloe vera, Thyme, carvacrol, and nano-encapsulation of carvacrol on the growth and production of aflatoxin B1 production by Aspergillus parasiticus and Aspergillus flavus. METHODS AND RESULTS: Minimum inhibitory concentrations of extracts Aloe vera, Thyme, carvacrol, and nanocarvacrol, and fluconazole as a control were determined according to Clinical and Laboratory Standards Institute by serial microdilution protocol. Then, the effect of inhibitory concentrations of these compounds on the aflatoxin B1 production level was evaluated by real-time PCR and high-performance liquid chromatography. Our results indicate that the Aspergillus parasiticus and Aspergillus flavusare sensitive to selected plants and compounds. CONCLUSION: Our findings showed that the compounds are appropriate alternative candidates against growth and production of aflatoxin of Aspergillus spp.

Biomaterials with antifungal strategies to fight oral infections.

Oral fungal infections pose a threat to human health and increase the economic burden of oral diseases by prolonging and complicating treatment. A cost-effective strategy is to try to prevent these infections from happening in the first place. With this purpose, biomaterials with antifungal properties are a crucial element to overcome fungal infections in the oral cavity. In this review, we go through different kinds of biomaterials and coatings that can be used to functionalize them. We also review their potential as a therapeutic approach in addition to prophylaxis, by going through traditional and alternative antifungal compounds, e.g., essential oils, that could be incorporated in them, to enhance their efficacy against fungal pathogens. We aim to highlight the potential of these technologies and propose questions that need to be addressed in prospective research. Finally, we intend to concatenate the key aspects and technologies on the use of biomaterials in oral health, to create an easy to find summary of the current state-of-the-art for researchers in the field.

Combination of Farnesol with Common Antifungal Drugs: Inhibitory Effect against Candida Species Isolated from Women with RVVC.

Background and Objectives: Vulvovaginal candidiasis (VVC) is a mucous membrane infection, with an increased rate of antifungal resistance of Candida species. In this study, the in vitro efficacy of farnesol alone or in combination with traditional antifungals was assessed against resistant Candida strains recovered from women with VVC. Materials and Methods: Eighty Candida isolates were identified by multiplex polymerase chain reaction (PCR), and the antifungal susceptibility to amphotericin B (AMB), fluconazole (FLU), itraconazole (ITZ), voriconazole (VOR), clotrimazole (CTZ), and farnesol was tested by the standard microdilution method. The combinations of farnesol with each antifungal were calculated based on the fractional inhibitory concentration index (FICI). Result: Candida glabrata was the predominant species (48.75%) isolated from vaginal discharges, followed by C. albicans (43.75%), C. parapsilosis (3.75%), a mixed infection of C. albicans and C. glabrata (2.5%) and C. albicans and C. parapsilosis (1%). C. albicans and C. glabrata isolates had lower susceptibility to FLU (31.4% and 23.0%, respectively) and CTZ (37.1% and 33.3%, respectively). Importantly, there was "synergism" between farnesol-FLU and farnesol-ITZ against C. albicans and C. parapsilosis (FICI = 0.5 and 0.35, respectively), reverting the original azole-resistant profile. Conclusion: These findings indicate that farnesol can revert the resistance profile of azole by enhancing the activity of FLU and ITZ in resistant Candida isolates, which is a clinically promising result.

Biofilm formation in clinically relevant filamentous fungi: a therapeutic challenge.

Biofilms are highly-organized microbial communities attached to a biotic or an abiotic surface, surrounded by an extracellular matrix secreted by the biofilm-forming cells. The majority of fungal pathogens contribute to biofilm formation within tissues or biomedical devices, leading to serious and persistent infections. The clinical significance of biofilms relies on the increased resistance to conventional antifungal therapies and suppression of the host immune system, which leads to invasive and recurrent fungal infections. While different features of yeast biofilms are well-described in the literature, the structural and molecular basis of biofilm formation of clinically related filamentous fungi has not been fully addressed. This review aimed to address biofilm formation in clinically relevant filamentous fungi.

Candida auris: A Quick Review on Identification, Current Treatments, and Challenges.

Candida auris is a novel and major fungal pathogen that has triggered several outbreaks in the last decade. The few drugs available to treat fungal diseases, the fact that this yeast has a high rate of multidrug resistance and the occurrence of misleading identifications, and the ability of forming biofilms (naturally more resistant to drugs) has made treatments of C. auris infections highly difficult. This review intends to quickly illustrate the main issues in C. auris identification, available treatments and the associated mechanisms of resistance, and the novel and alternative treatment and drugs (natural and synthetic) that have been recently reported.

The effect of Candida cell wall beta-glucan on treatment-resistant LL/2 cancer cell line: in vitro evaluation.

Candida albicans (C. albicans) cell wall beta-glucan has been considered as a potential agent in the treatment of cancers due to its anti-tumor properties. Therefore, in the present study, we investigated the anti-cancer effects of Candida cell wall beta-glucan on Lewis lung carcinoma cell line (LL/2) cells. Beta-glucan of C. albicans cell wall was extracted. LL/2 cell line was cultured, then sphere cells and parental cells were exposed to the different concentrations of beta-glucan extracted from C. albicans (10-6000 µg/ml), for 24, 48 and 72 h. Cytotoxicity of beta-glucan was assayed by MTT test, then RNA extracted from cells population (treated and untreated cells), cDNA synthetized and expression level of Sox2, Oct4, C-myc, Nanog genes were also investigated using Real-time methods. At optimal concentrations of 800 and 1000 µg/ml, the extracted beta-glucan showed a significant cytotoxic effect on both parental and sphere cell populations (p < 0.05). Real-time PCR analysis revealed a decreased expression of Oct4 and Sox2 genes in treatment of cells with beta-glucan compared with control group. Since the extracted beta-glucan showed an inhibitory effect on the expression of Oct4 and Sox2 genes involved in LL/2 metastasis, therefore, beta-glucan can be considered as an anti-tumor agent because of its anti-metastatic properties, however, more in vitro and in vivo studies are needed to provide further evidence on this topic in the future.

Low Level of Antifungal Resistance in Iranian Isolates of Candida glabrata Recovered from Blood Samples in a Multicenter Study from 2015 to 2018 and Potential Prognostic Values of Genotyping and Sequencing of PDR1.

Establishing an effective empirical antifungal therapy requires that national surveillance studies be conducted. Herein, we report the clinical outcome of infections with and the microbiological features of Iranian isolates of Candidaglabrata derived from patients suffering from candidemia. C. glabrata isolates were retrospectively collected from four major cities in Iran; identified by a 21-plex PCR, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and large subunit of ribosomal DNA sequencing; and genotyped by amplified fragment length polymorphism (AFLP). Mutations in PDR1, ERG11, and hot spot 1 (HS1) of FKS1 and FKS2 were investigated, and antifungal susceptibility testing (AFST) was performed (by the CLSI M27-A3 and M27-S4 methods). Seventy isolates of C. glabrata were collected from 65 patients with a median age of 58 years. Fluconazole was the most widely used (29.23%) and least effective antifungal agent. The overall crude mortality rate was 35.4%. Only one strain was resistant to fluconazole, and 57.7% and 37.5% of the isolates were non-wild type (non-WT) for susceptibility to caspofungin and voriconazole, respectively. All isolates showed the WT phenotype for amphotericin B, posaconazole, and itraconazole. HS1 of FKS1 and FKS2 did not harbor any mutations, while numerous missense mutations were observed in PDR1 and ERG11 AFLP clustered our isolates into nine genotypes; among them, genotypes 1 and 2 were significantly associated with a higher mortality rate (P = 0.034 and P = 0.022, α < 0.05). Moreover, 83.3% of patients infected with strains harboring a single new mutation in PDR1, T745A, died despite treatment with fluconazole or caspofungin. Overall, Iranian isolates of C. glabrata were susceptible to the major antifungal drugs. Application of genotyping techniques and sequencing of a specific gene (PDR1) might have prognostic implications.

What are the advantages of living in a community? A microbial biofilm perspective!

Biofilm formation is the preferred mode of growth lifestyle for many microorganisms, including bacterial and fungal human pathogens. Biofilm is a strong and dynamic structure that confers a broad range of advantages to its members, such as adhesion/cohesion capabilities, mechanical properties, nutritional sources, metabolite exchange platform, cellular communication, protection and resistance to drugs (e.g., antimicrobials, antiseptics, and disinfectants), environmental stresses (e.g., dehydration and ultraviolet light), host immune attacks (e.g., antibodies, complement system, antimicrobial peptides, and phagocytes), and shear forces. Microbial biofilms cause problems in the hospital environment, generating high healthcare costs and prolonged patient stay, which can result in further secondary microbial infections and various health complications. Consequently, both public and private investments must be made to ensure better patient management, as well as to find novel therapeutic strategies to circumvent the resistance and resilience profiles arising from biofilm-associated microbial infections. In this work, we present a general overview of microbial biofilm formation and its relevance within the biomedical context.

Lung cancer and ß-glucans: review of potential therapeutic applications.

The potential of natural substances with immunotherapeutic properties has long been studied. ß-glucans, a cell wall component of certain bacteria and fungi, potentiate the immune system against microbes and toxic substances. Moreover, ß-glucans are known to exhibit direct anticancer effects and can suppress cancer proliferation through immunomodulatory pathways. Mortality of lung cancer has been alarmingly increasingly worldwide; therefore, treatment of lung cancer is an urgent necessity. Numerous researchers are now dedicated to using ß-glucans as a therapy for lung cancer. In the present attempt, we have reviewed the studies addressing therapeutic effects of ß-glucans in primary and metastatic lung cancer published in the time period of 1991-2016.

Antifungal effect of Echinophora platyloba on expression of CDR1 and CDR2 genes in fluconazole-resistant Candida albicans.

BACKGROUND: Several studies examined the effect of the Echinophora platyloba extract in treatment of azole-resistant Candida albicans clinical isolates. OBJECTIVE: We investigated the effect of E. platyloba extract on expression of CDR1 and CDR2 genes in fluconazole-resistant clinical isolates of C. albicans using real-time PCR. MATERIALS AND METHODS: The crude extract of E. platyloba was obtained using percolation method. Using serial dilution method, different concentrations of extract were achieved. Two hundred microlitres of fungal suspension (10(6) CFU/ml) was added to the media and cultured with different concentrations and then incubated at 37 °C for 48 h. The concentration of extract in the first tube, which inhibited the growth of C. albicans, was recorded as the Minimal Inhibitory Concentration (MIC). In order to analyse the expression of CDR1 and CDR2 genes, RNA was extracted from C. albicans isolates before and after treatment with MIC of E. platyloba using glass beads and the denaturing buffer agents in an RNase-free environment and then the cDNA was synthesised and used for real-time PCR assay. RESULTS: Twenty of total of 148 isolates were resistant to fluconazole. The MIC and MFC for the alcoholic extract of E. Platyloba were 64 mg/ml and 128 mg/ml, respectively. Real-time PCR results revealed that the mRNA levels of CDR1 and CDR2 genes significantly declined after incubation with E. Platyloba (both p values < 0.001). CONCLUSION: E. Platyloba is effective in reducing CDR1 and CDR2 expression which in turn plays an important role in fluconazole resistance in Candida species.

The First Case of Total Dystrophic Onychomycosis Caused by Aspergillus clavatus Resistant to Antifungal Drugs.

Onychomycosis is a common fungal infection of nails which is mainly caused by dermatophyte species and less often by yeasts and non-dermatophyte molds. We present a case of onychomycosis due to Aspergillus clavatus for the first time worldwide. The patient was an immunocompetent 32-year-old woman who identified with Psoriasis of the nail. The presence of A. clavatus in a nail sample was confirmed using microscopic and culture analysis followed by PCR of the ß-tubulin gene. After antifungal susceptibility test, it is revealed that the isolate was resistant to the majority of common antifungal drugs, but finally the patient was treated with itraconazole 200 mg daily. A. clavatus and drug-resistant A. clavatus have not previously been reported from onychomycosis.

Immunomodulatory Effect of ß-Glucan on Peritoneal Macrophages of Bab1/c Mice.

We assessed the effect of ß-Glucan on macrophages by Griess reagent and viability by MTT assay and cytotoxicity. Assay of macrophages culture supernatants were carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay were done. NO release was increased at the dose of 10 µg/ml (P = 0.001) of ß-Glucan while the viability of macrophages in all concentrations was the same. In TNF-α bioassay, the supernatant of macrophages stimulated with ß-Glucan had a significant cytotoxic effect on WEHI-164 cells (P = 0.023). ß-Glucan had a positive effect on increasing tumoricidal activity of macrophages which may help in anti-cancer immune responses.

Exploring fungal diversity in Vietnam: A citizen science initiative.

Invasive fungal diseases are increasing globally, causing a large burden of disease in vulnerable populations. At the same time, antifungal resistance is rapidly emerging. Affordable nationwide and regional surveillance of fungal pathogens is needed. We have adapted a citizen-science methodology developed by a United Kingdom research group to study six key fungi in Vietnam, where there is no existing formal surveillance. These pathogens were ranked as high or critical in the World Health Organization fungal priority pathogens list and recognized as major disease-causing agents in Vietnam. Secondary school students (n = 90) in Hanoi were our citizen scientists, collecting soil (n = 90) and air (n = 90) samples for fungal identification and characterisation of drug-susceptibility in the laboratory. Pilot studies confirmed the effectiveness of our revised isolation procedure, which used selective culture media to improve the isolation of target fungi. Through active school and student involvement, optimized protocols, and our cost-effective sampling, the study could be scaled across Vietnam. We demonstrate an approach to fungal surveillance which also enhances science education, and awareness of fungal diseases. It addresses critical healthcare and education challenges in Vietnam while combating the growing issues of invasive fungal diseases and antifungal resistance.

Hospitalized COVID-19 Patients with Urinary Tract Infection in Iran: Candida Species Distribution and Antifungal Susceptibility Patterns.

Candida species, typically part of the human skin and mucous membrane flora, can cause opportunistic fungal infections, notably urinary tract infections (UTIs), which are on the rise among hospitalized COVID-19 patients. The lack of understanding of UTIs in this population, coupled with the emergence of multidrug-resistant strains, poses significant challenges for effective treatment and further investigations. In this study, urine samples were collected from 70 COVID-19 patients with UTIs in sterile containers for microbiology examination. After microscopic observation, the isolates were identified both by phenotypic and molecular techniques such as multiplex PCR. Antifungal susceptibility testing (AFST) against fluconazole (Flu), itraconazole (Itr), and amphotericin B (AMB) was performed according to CLSI M27/S4 standard methods, with the frequency of isolates including Candida albicans (n = 20, 51.3%), Candida tropicalis (n = 15, 38.4%), Nakaseomyces glabrata (previously Candida glabrata) (n = 2, 5.1%), Pichia kudriavzevii (previously Candida krusei), and Candida parapsilosis (n = 1, 2.5%). All isolates of C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis were sensitive to amphotericin B, while C. kruzei was resistant to AMB. Around 70% of C. albicans isolates were sensitive to Flu; 20% of C. tropicalis were resistant to itraconazole, while 33% were resistant to fluconazole. C. albicans and C. tropicalis were the main causes of candiduria in infected cases and both Flu and AMB showed good results in AFST in these species. Performing drug susceptibility testing for clinical isolates of Candida spp. provided guidance for appropriate management and control, and timely antifungal treatment.

Thymoquinone Antifungal Activity against Candida glabrata Oral Isolates from Patients in Intensive Care Units-An In Vitro Study.

The number of Candida spp. infections and drug resistance are dramatically increasing worldwide, particularly among immunosuppressed patients, and it is urgent to find novel compounds with antifungal activity. In this work, the antifungal and antibiofilm activity of thymoquinone (TQ), a key bioactive constituent of black cumin seed Nigella sativa L., was evaluated against Candida glabrata, a WHO 'high-priority' pathogen. Then, its effect on the expression of C. glabrata EPA6 and EPA7 genes (related to biofilm adhesion and development, respectively) were analyzed. Swab samples were taken from the oral cavity of 90 hospitalized patients in ICU wards, transferred to sterile falcon tubes, and cultured on Sabouraud Dextrose Agar (SDA) and Chromagar Candida for presumptive identification. Next, a 21-plex PCR was carried out for the confirmation of species level. C. glabrata isolates underwent antifungal drug susceptibility testing against fluconazole (FLZ), itraconazole (ITZ), amphotericin B (AMB), and TQ according to the CLSI microdilution method (M27, A3/S4). Biofilm formation was measured by an MTT assay. EPA6 and EPA7 gene expression was assessed by real-time PCR. From the 90 swab samples, 40 isolates were identified as C. glabrata with the 21-plex PCR. Most isolates were resistant to FLZ (n = 29, 72.5%), whereas 12.5% and 5% were ITZ and AMB resistant, respectively. The minimum inhibitory concentration (MIC50) of TQ against C. glabrata was 50 µg/mL. Importantly, TQ significantly inhibited the biofilm formation of C. glabrata isolates, and EPA6 gene expression was reduced significantly at MIC50 concentration of TQ. TQ seems to have some antifungal, antibiofilm (adhesion) effect on C. glabrata isolates, showing that this plant secondary metabolite is a promising agent to overcome Candida infections, especially oral candidiasis.

Oral candidiasis in patients with kidney transplantation in Iran: prevalence and antifungal susceptibility pattern.

Aim: This study aimed to identify Candida species recovered from the oral cavity of patients with kidney transplantation. Materials & methods: Two swabs were taken from the oral cavities of 40 patients before and after transplantation, cultured on Sabouraud dextrose agar, and yeasts identified. Antifungal drug susceptibility testing was performed with fluconazole and itraconazole. Results: Candida glabrata was the most frequently isolated species in patients, followed by Candida albicans and Rhodotorula. C. glabrata isolates from patients before transplantation were resistant to fluconazole, whereas C. albicans was fluconazole-resistant both before and after transplantation. Conclusion: The importance of non-albicans Candida species in the oral cavity of patients sheds light on performing antifungal tests for achieving the best outcome to prevent therapeutic failure.

Antifungal activity of green-synthesized curcumin-coated silver nanoparticles alone and in combination with fluconazole and itraconazole against Candida and Aspergillus species.

Background and Purpose: Regarding the wide-spectrum antimicrobial effects of curcumin and silver, this study aimed to evaluate the antifungal activity of green-synthesized curcumin-coated silver nanoparticles (Cur-Ag NPs) against a set of Candida and Aspergillus species. Materials and Methods: Cur-Ag NPs were synthesized by mixing 200 µL of curcumin solution (40 mM) and 15 mL of deionized water. The mixture was stirred for 3-5 min, followed by the addition of 2.5 mL of silver nitrate solution (2.5 mM). The resulting solution was incubated for 3 days. Antifungal susceptibility of 30 fungal isolates of Aspergillus and Candida to fluconazole and itraconazole, as well as the activity of Cur-Ag NPs against the isolates, were determined, both alone and in combination, using broth microdilution according to the Clinical and Laboratory Standards Institute guidelines. Results: Cur-Ag NPs demonstrated promising antifungal activity, particularly against Candida species. The geometric mean value of the minimum inhibitory concentration of Cur-Ag NPs was significantly lower than that of fluconazole for all the studied fungi. Similarly, it was lower than those of itraconazole in C. albicans and A. fumigatus. The minimum fungicidal concentrations of Cur-Ag NPs were markedly better than those of fluconazole but still inferior to those of itraconazole. Conclusion: Cur-Ag NPs demonstrated indisputable antifungal activity and great potential that can be harnessed to combat fungal infections, particularly those caused by azole-resistant strains of Aspergillus and Candida.

Overview on the Infections Related to Rare Candida Species.

Atypical Candida spp. infections are rising, mostly due to the increasing numbers of immunocompromised patients. The most common Candida spp. is still Candida albicans; however, in the last decades, there has been an increase in non-Candida albicans Candida species infections (e.g., Candida glabrata, Candida parapsilosis, and Candida tropicalis). Furthermore, in the last 10 years, the reports on uncommon yeasts, such as Candida lusitaniae, Candida intermedia, or Candida norvegensis, have also worryingly increased. This review summarizes the information, mostly related to the last decade, regarding the infections, diagnosis, treatment, and resistance of these uncommon Candida species. In general, there has been an increase in the number of articles associated with the incidence of these species. Additionally, in several cases, there was a suggestive antifungal resistance, particularly with azoles, which is troublesome for therapeutic success.

Prevalence, Molecular Identification, and Genotyping of Candida Species Recovered from Oral Cavity among Patients with Diabetes Mellitus from Tehran, Iran.

Background: Oral candidiasis (OC) has been noticed as a common mucous membrane infection in immunocompromised patients such as that diabetes. This study, focused on the genotyping of Candida albicans and enzymatic activities of Candida species recovered from oral mucosa among diabetes patients and healthy individuals. Materials and Methods: Specimens were obtained from oral mucosa of One-hundred and sixty patients with type 2 diabetic and 108 healthy individuals. All isolates were definitely identified by ribosomal DNA (rDNA) gene sequencinghHydrophobicity, hemolytic activities of Candida species and genotypes of C. albicans were determined through polymerase chain reaction (CA-INT). Results: , Eighty eight (55%) samples out of 160, were positive for Candida species in diabetic patients. Moreover, 79.5% (70/88) and 20.5% (18/88) isolates belonged to the C. albicans and non-albicans Candida species respectively. Three genotypes of C. albicans have recovered in diabetic patients: genotype A (71.42%), B (21.42%), and C (7.14%). In healthy individuals, 42.6% (46/102) Candida species recovered from oral cavity, with the highest prevalence of genotype A (76.6% of C. albicans). Additionally, hydrophobicity and hemolytic activities from Candida species were significantly greater in diabetes patients than healthy nondiabetic subjects. Conclusion: Collectively, C. albicans was the most causative agent isolated from diabetes patients and non-diabetes healthy individuals. Genotype A, as the most remarkable genotype, should be mentioned in both groups. Higher potential hydrophobicity and hemolytic activities of Candida species in diabetic patients compared to healthy cases suggest these features triggering pathogenicity of OC in diabetes patients.

Enhancing of Wound Healing in Burn Patients through Candida albicans ß-Glucan.

The mortality and disability-adjusted life years (DALYs) of burn patients are decreasing over time. However, finding novel effective treatment approaches using natural agents is highly considered to reduce the burden of burn injuries. One of the recent agents used in wound healing is ß-glucan, mainly extracted from fungi cell walls. This study aimed to evaluate the effect of 5% (m/m) of yeast ß-glucan ointment on burn wound healing and to assess the impact of ß-glucan on cytokines during the treatment. Thirty-three patients with second or third-degree burns were enrolled in this study. Two groups of twenty-three and ten patients used yeast 5% (m/m) ß-glucan ointment (study group) and Stratamed ointment (control), respectively, on a daily basis, for a maximum of four weeks. The size of the burn wounds was measured before and at the end of the treatment. Blood samples of 14 and 10 patients in the ß-glucan and control groups, respectively, were obtained before and after the treatment, and the enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum concentration of the IL-4, IL-17, and IFN-γ cytokines. The log-binomial model was used to assess the efficacy of the ß-glucan ointment on burn wound healing. ANOVA/ANCOVA was employed to assess the effects of ß-glucan on the serum concentration of cytokines. After adjusting for potential confounders/covariates, patients receiving ß-glucan had better wound healing (RR = 4.34; 95% CI: 0.73 to 25.67; p = 0.11). There was a significant difference in IL-4 secretion between the ß-glucan and control groups after adjusting for potential confounders/covariates (MD = 77.27; 95% CI: 44.73 to 109.82; Cohen's d = 2.21; 95% CI: 1.16 to 3.24; p = 0.0001). The results indicate that 5% (m/m) of ß-glucan has efficacy in burn wound healing, and a significant difference was found in the level of IL-4 after receiving ß-glucan. Further studies with a two-arm design and long-term use of ointment are needed to confirm the effect of ß-glucan on wound healing and cytokine secretion.