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Purpose: The increasing resistance to onychomycosis treatment globally is a major concern as it results in treatment failures. This research aimed to investigate the effectiveness of incorporating nanostructured lipid carriers (NLCs) in a gel formulation for treating mild to moderate Candida-associated onychomycosis using Zataria multiflora (Zt) essential oils. Methods: The characteristics of Zataria multiflora-loaded nanostructured lipid carriers (Zt-NLCs) were validated using a carbopol gel (1 % w/w). In vitro tests were conducted using CLSI M60 standards to evaluate the susceptibility of ten commonly encountered dermatophyte species to antifungal agents. A clinical study was conducted involving 40 volunteers randomly assigned to two groups: one receiving the Zt-NLCs gel and the other receiving a placebo, using a double-blind, placebo-controlled design. The objective was to assess clinical manifestations and mycological findings after topical application for 2 and 4 weeks. The causative agents' species were identified using a PCR-RFLP method. Results: The preparation of Zt-NLCs gel resulted in a uniform suspension of spherical nanoparticles with favourable characteristics and no cytotoxic effects. Zt-NLCs demonstrated significant inhibitory effects on fungal growth and effectively improved clinical and mycological criteria in comparison to the placebo group (p

Candida auris, an emerging non-albicans multidrug-resistant yeast, has become a significant cause of invasive candidiasis in healthcare settings. So far, data on the metabolites of C. auris in different clades are minimal, and no studies have focused on clade V metabolites. Therefore, Gas chromatography–mass spectrometry (GC-MS) was used for the metabolomic profiling of clade I C. auris compared with fluconazole-resistant and susceptible C. auris in clade V strains. GC-MS chromatography revealed 28, 22, and 30 compounds in methanolic extracts of the fluconazole-susceptible and fluconazole-resistant C. auris clade V and C. auris clade I strain, respectively. Some compounds, such as acetamide and metaraminol, were found in fluconazole-susceptible and resistant C. auris clade V and clade I. N-methyl-ethanamine and bis(2-ethylhexyl) phthalate metabolites were found in both fluconazole -susceptible and resistant C. auris clade V, as well as 3-methyl-4-isopropylphenol, 3,5-bis(1,1-dimethyl)-1,2-benzenediol, and diisostyl phthalate metabolites in both fluconazole resistant C. auris clade V and I. Identifying these metabolites contributes to understanding the morphogenesis and pathogenesis of C. auris, highlighting their potential role in antifungal drug resistance and the control of fungal growth. However, further experiments are warranted to fully comprehend the identified metabolites’ regulatory responses, and there may be potential challenges in translating these findings into clinical applications.

Background: Recently, niosomes are becoming popular in drug delivery. The current work aimed to investigate the characteristics, cellular safety, and antifungal activity of ketoconazole-loaded niosome (ketosome). Methods: Ultrasonic approach was employed to prepare ketosome including cholesterol, nonionic surfactant and ketoconazole. The size characteristics and morphological features of ketosome and physicochemical properties of ketoconazole in ketosomes were evaluated using dynamic light scattering (DLS), differential scanning calorimetry (DSC), powder x-ray diffractometer (PXRD), scanning electron microscopy (SEM), and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Also, the dissolution rate, cellular safety test and antimycotic properties of ketosome were examined. Results: According to the results, the particle size of the ketosome decreased from 491.400±10.622 to 121.300±7.274 nm by the increment of cholesterol. According to further research, changes in the cholesterol:surfactants ratio can modulate the zeta potential from -27.866±1.069 to -12.500±1.153 mV. The highest entrapment of ketoconazole was about 87% when the cholesterol concentration in the ketosome was high. Ketosome with the maximum cholesterol:surfactants ratio showed the fastest drug release. Furthermore, the cell viability assay revealed that the ketosome had lower cytotoxicity in comparison with pure drug. The cell viability of the ketosome was estimated to be about 90% (HGF cell line). The ketosome had a lower MIC than the pure drug when tested against Candida albicans. Conclusion: The results of this study revealed that the optimized ketoconazole-loaded niosome could be used as a possible nanovesicle for ketoconazole drug delivery, potentially opening up new ways for the management of cutaneous candidiasis complaints.

Background and Purpose: Given the high mortality rate of invasive candidiasis inhospitalized pediatric patients, it is crucial to establish a predictive system to achieveearly diagnosis and treatment of patients who are likely to benefit from early antifungal treatment. This study aimed to assess the Candida colonization index, species distribution, and antifungal susceptibility pattern of Candida strains isolated frompediatric patients with high Candida colonization index (CI)Materials and Methods: This study was carried out at the Children’s Medical Center inTehran-Iran. In total, 661 samples were collected from 83 patients. The Candida CI wascalculated according to the descriptions of previous studies. The isolates were identified using polymerase chain reaction-based techniques. The Clinical and Laboratory Standard Institute protocol M60 was used to conduct the antifungal susceptibility test.Results: A colonization index greater than 0.5 was confirmed in 29 cases (58% ofpositive samples) with two children developing candidemia. Candida albicans (n=53,49.5%) was the most common Candida species in patients with CI > 0.5. Except foracute lymphoblastic leukemia, no risk factors were linked to a high index in colonizedchildren (P > 0.05). Twelve isolates (7.01%) were multi-azole resistant with high MICsagainst both isavuconazole and ravuconazole and seven strains (4.09%) wereechinocandins resistant.Conclusion: In pediatric intensive care units, patients are at risk of fungal infection,particularly candidemia. In this study, more than half of the children with positive yeastcultures had CI > 0.5, and 6.8% developed candidemia.

Introduction: This study was proposed to assess the potential role of efflux transporters in reversing fluconazole resistance in Candida glabrata isolates treated with fluconazole loaded nanostructured lipid carriers (FLZ-NLCs). Methods: The ultrasound technique was used to synthesize the FLZ-NLCs. Four fluconazole-resistant, as well as one susceptible standard C. glabrata isolates, were applied and exposed to FLZ/ FLZ-NLCs for 20 h at 37°C. Real-time PCRs were done to estimate the likely changes in ATP-binding cassette transporter genes. Results: Similar to the FLZ-exposed-susceptible standard strain which showed no alteration, the genes were not up-regulated significantly under the FLZ-NLCs treated condition. While they were over-expressed when the yeasts were treated with fluconazole. Conclusion: It is highly suggested that due to the nature of the NLCs which shields the whole conformation of the drug, FLZ is not recognized by the efflux transporter subunits and consequently the translocation would not happen.

Development of point-of-care (POC) diagnostic tools is an emerging area with significant potential for disease surveillance, monitoring, and diagnosis, especially for underdeveloped or developing countries. Our current research focuses on rapid, POC technologies for DNA or RNA detection that can be deployed to significantly decrease the turnaround time when encountering demands for massive quantities of tests, e.g. during a pandemic. Hairpin-like DNA or molecular beacon (MB) probes can be used as bioreceptors to specifically bind to a pathogen DNA or RNA. In the presence of complementary DNA, the immobilized MBs undergo a conformational change, and the fluorescent signal of 5’-FAM is restored from the internally quenched fluorophore. Here we studyinvestigating 3D polymer brush (PB) structures with antifouling surface properties, functionalized with a particular MB-DNA probe. Patterns of polymer brushes were created on foils of poly(ethylene-co-tetrafluoroethylene) (ETFE) activated through a metal mask using extreme ultraviolet (EUV) radiation, yielding patterns of initiators for the subsequent graft-copolymerization of vinylpyrrolidone (VP) and glycidyl methacrylate (GMA). The successful copolymerization of VP and GMA on the EUV-exposed areas was proved based on characteristic peaks of VP and GMA in ATR-IR spectra. Structure heights in the range of micrometers were achieved, which enables binding of considerably higher densities of probe molecules compared to monolayer systems. The grown polymer brush structures provide both hydrophilicity, beneficial to minimize bio-fouling, and epoxide functional groups for further functionalization. These were biotinylated and functionalized with streptavidin and 3′-biotinylated MBs, resulting in a promising platform for fluorescence-based DNA detection as demonstrated by significant fluorescence increase upon addition of target DNA down to nM concentrations. Finally, embedding of optimized MB/PB structures into a microfluidic channel and coupling to a mobile-phone-based fluorescence microscope for signal detection was demonstrated.

Background and Purpose: This study aimed to evaluate the species distribution and susceptibility pattern of the strains isolated from Candida colonization in pediatric patients staying at pediatric intensive care unit (ICU) and infant ICU of Children’s Medical Center in Tehran, Iran.Materials and Methods: This study was conducted in the Children’s Medical Center in Tehran, Iran. In total, 440 samples from 56 patients with oral cavity, skin surrounded catheters, and ear, throat, nasal, and urine cultures were collected. All patients were evaluated in terms of Candida colonization on the admission day as well as the days 7, 14, and 28 according to the previous studies. CHROMagar Candida medium was applied for primary/multiple species identification and the isolates were identified by using polymerase chain reaction-based methods to the species-specific complex level. The antifungal susceptibility test was performed according to the Clinical and Laboratory Standards protocol published as M27-A3 and M60 documents.Results: In total, 136 yeast samples from 26 individuals (30.9%) out of 440 samples were considered colonization. The most prevalent species in IICU was C. albicans (27%,n=20) followed by C. krusei (24 %, n=18) and C. parapsilosis (16%, n=12). In PICU, the predominant species was C. krusei (40%, n=24) followed by C. parapsilosis (18%, n=11) and C. dubliniensis (16%, n=10). Among the 40 tested isolates from both units, fluconazole-resistant isolates (n=11, 8.15%) were determined according to the new breakpoints. In the case of echinocandins, 2 isolates, including C. albicans (n=1) and C. krusei (n=1) were resistant against both caspofungin and anidulafungin (totally 1.48%).Conclusion: In the present study, since C. krusei is intrinsically-resistance against fluconazole, emphasizing the importance of species-level identification of Candida isolates is outstanding. However, according to the antifungal susceptibility testingresults, only 7.2% of the strains were resistant to fluconazole. It would be beneficial tomonitor the ICU patients who are at high risk of invasive Candida infection.

Background and Purpose: Invasive mucormycosis is a rare mycosis that affects most cases of uncontrolled diabetes and has a high mortality rate. Patients with COVID-19 are at high risk of developing invasive mucormycosis due to the consumption of anti inflammatory drugs such as corticosteroids and dexamethasone. Rhizopus species followed by Rhizomucor spp. and Mucor spp. are the main common etiological agents of rhino-orbital mucormycosis. Therefore, this study aimed to present a case of mucormycosis due to Syncephalastrum racemosum in a diabetic patient with COVID-19 for the first time in Iran.Case report: A 73-year-old diabetic female was referred to Ayatollah Rouhani Hospital in Babol, Iran, with a confirmed COVID-19 diagnosis, based on positive RT-PCR and computed tomography of the lungs. She has received methylprednisolone due to severe lung complications. Nasal involvement and left orbital swelling were observed 20 days after the hospitalization. By sinus endoscopic surgery, debridement was done and histopathology indicated wide hyphae (without septa). The sequenced PCR products displayed Syncephalastrum racemosum. In the antifungal susceptibility test, amphotericin B showed good activity against S. racemosum and the patient survived with timely treatment.Conclusion: This is the first case report of rhino-orbital mucormycosis due to S. racemosum in COVID-19 patient; therefore, S. racemosum can be considered one of the etiological factors of rhino-orbital mucormycosis in COVID-19 cases.

Background and objectives: The incidence of candiduria caused by Candida spp. has increased in recent years, particularly in hospitalized patients. Candiduria is most commonly caused by Candida albicans; however, an increase in the prevalence of non-albicans species has been observed during last decades. This study aimed at molecular identification of Candida species isolated from candiduria in hospitalized patients. Methods: This cross-sectional study was conducted on 530 hospitalized patients in two hospitals in the Mazandaran Province, Iran. Midstream urine specimens were collected and then cultured on CHROMagar Candida medium. Molecular identification of common Candida species was carried out using the polymerase chain reaction-restriction fragment length polymorphism method after enzymatic digestion with MspI. C. albicans and Candida parapsilosis species complexes were identified by amplification of the HWP1 and intein-containing vacuolar ATPase precursor genes, respectively. Results: The frequency of candiduria was estimated at 14% among hospitalized patients. Of 74 samples positive for candiduria, 65 (87.8%) were isolated from females. The most common predisposing factor to candiduria was diabetes (n=36; 48.6%). The most frequent isolates were C. albicans complex (n=44; 59.4%), followed by Candida glabrata (n= 16; 21.6%), Candida tropicalis (n= 10; 13.5%), Candida Krusei (n= 3; 4%) and C. parapsilosis (n= 1; 1.3%). Conclusion: Based on the results, the conventional and molecular methods produced similar results for identification of Candida species. However, accurate identification of Candida spp. requires the use of molecular techniques such as PCR-RFLP, HWP1, and intein-containing vacuolar ATPase precursor genes. Nevertheless, chromogenic methods such as CHROMagar Candida can be used for diagnosis of Candida spp. in laboratories with limited resources.

Background: Recently, nanoparticles were widely used in drug delivery. Fluconazole (FLZ) is a lipid-soluble antifungal, which is utilized in treating fungal infections. The current work aimed to investigate the characteristics and antifungal activity of FLZ nanosuspension. Methods: FLZ nanosuspensions were prepared by Ultrasonication (simple and containing polymer). Surfactants in various concentrations were dissolved in the deionized water, and the drug powder was dispersed in the surfactant solution by a high-speed homogenizer to achieve nanosuspension. The polymer was added to the selected formula. FLZ nanosuspension characteristics, including polydispersity, mean particle size, entrapment efficacy, and zeta potential, were assessed. The release profile via dialysis membrane, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (ATR-FTIR), and TEM were performed for nanosuspension evaluation. Antifungal activity against resistant strains of C. albicans was defined according to the CLSI document guideline. To analyze the results, one-way ANOVA was used, followed by Tukey test. Results: The results showed that increased sonication time and hydrophilic-lipophilic balance (HLB) significantly affected particle size reduction. Moreover, modification in the formulation components had a significant effect on the drug release process, furthermore affecting the properties of the nanoparticles. ATR-FTIR showed no chemical interaction between FLZ and formulation components. Compare to FLZ, a significant reduction (p < 0.05) was detected in the MIC values of both FLZ-resistant and FLZ-susceptible strains of C. albicans against FLZ nanosuspension. Conclusion: It can be concluded that the ratio and amount of surfactants, the HLB, and the sonication process have effects on the properties of the nanoparticle’s characteristics, and selected nanoparticles show suitable antifungal effect against resistant strains of C. albicans.

Background and purpose: Removable prostheses made of polymethyl methacrylate are susceptible to Candida-dependent denture stomatitis and Candida albicans is considered to be the first common cause. The aim of the present study was to investigate the antifungal effect of acrylic resin modified by zinc oxide nanoparticles. Materials and methods: In this laboratory study, 72 disks (10*4 mm) made of polymethyl methacrylate (Acropars, Marlic, Iran) were divided into three groups (n= 24). Concentrations of zinc oxide nanoparticles (ZnO NPs) were 1 (group A), 0.5 (group B), and 0 (group C, control) mg/ml. Samples were studied in laboratory using Candida albicans strains (ATCC 10231) according to CLSI M27-A3 and M27-S4 documents. For data analysis, one-way ANOVA and Scheffe's post-test were performed using SPSS V24. Results: There were significant differences in mean numbers of fungal colonies in study groups. The amount of fungal colonies in group A was lower than those in group B and in group B was lower than those in group C (P

Background and purpose: Invasive candidiasis in infants has high mortality rate and is often associated with colonization on medical devices such as catheters. Candida colonization is considered as a predisposing factor for Candida related infection. The aim of this study was to evaluate the rate of catheters infectivity by Candida species. Materials and methods: In this descriptive cross-sectional study, sampling was done from catheters of hospitalized children (0-18 years old) in pediatric intensive care unit (PICU) in Mashhad Dr. Sheikh Hospital, June-November 2018. All catheters were evaluated to determine the extent of contamination and early identification of isolates was done by morphological and molecular methods. Results: The types of catheter infectivity for 114 samples included colonization or infective (n=25 isolates, 21.92%), contamination (n=4, 3.5%), and no growth (n=85 isolates, 56.74%). The most isolated species was C. albicans (n=18, 13.39%), followed by C. glabrata (n=9, 19.56%), C. krusei and C. parapsilosis (n=7, 15.21%, each isolate), C. tropicalis (n=3, 6.25%), and C. lusitaniae and C. kefyr (n=1, 2.17% each isolate). Conclusion: Isolation of Candida species in 25% of catheters raised the possibility of colonization, considering the catheters as an exogenous potential source of candidiasis which is very important in PICU.

Background and Purpose: Because of the growing incidence of Aspergillus infection,typing methods of Aspergillus species are increasingly being used. Accordingly,studying the spread and population dynamics of strains isolating from clinical and environment, from a single host to large scale ecosystems is definitely needed. In the current study, we carried out a genetic analysis of nine microsatellite loci in isolates from different regions of Iran to compare and explore the genetic diversity between environmental and clinical A. fumigatus strains. Materials and Methods: Sixty-six clinical (n=43) and environmental (n= 23) isolates of A. fumigatus, have collected from six cities of Iran. All A. fumigatus isolates identified based on macroscopic and microscopic characters, the ability to grow at above 45°C, and confirmed using DNA sequencing of the partial b-tubulin gene.Sixty-six A. fumigatus isolates were subjected by microsatellite typing using three separate multiplex PCRs with a panel of nine short tandem repeats (STR) to evaluate the genetic relatedness. Results: The STR typing of 66 A. fumigatus isolates revealed 38 distinct genotypes distributed among environmental and clinical isolates. We identified 12 clones including 40 different isolates representing 60% of all isolates tested, which each clone included 2–7 isolates. Conclusion: The STR typing is considered as a valuable tool with excellent discriminatory power to study the molecular epidemiology and genotypic diversity of A.fumigatus isolates. These findings show that the high genetic diversity observed of Iranian A. fumigatus isolates with those outside Iran and formed a separate cluster.

Background and Purpose: The present study aimed to evaluate the effect of cyproconazole, the most used fungicide in Iranian wheat farms, on the induction of voriconazole resistance in Aspergillus fumigatus isolates. Materials and Methods: A collection of 20 clinical and environmental isolates were selected for investigation of the in vitro activity of fungicides. The minimum inhibitory concentrations (MICs) were determined by the documented broth microdilution method M38-A2 (CLSI, 2008). Induction experiments were performed and the possibly induced isolate(s) were subjected to antifungal susceptibility testing, sequencing of the CYP51A promoter, and full coding gene. Furthermore, CYP51-protein homology modeling and docking modes were evaluated using SWISS-MODEL (https://swissmodel.expasy.org/) and SEESAR software (version 9.1). Results: Among 10 susceptible isolates, only one strain showed a high MIC value against voriconazole (MIC=4μg/ml) after 25 passages. Nevertheless, sequencing of the CYP51A promoter and full coding gene did not reveal any mutations. Cyproconazole, which has three nitrogen atoms in the aromatic ring, coordinated to the iron atom of heme through a hydrogen bond contact to residue Lys147 present in the active site of the A. fumigates Cyp51 homology model. Conclusion: Cyproconazole is being applied extensively in wheat farms in Iran. According to the results, cyproconazole may not play a key role in the induction of azole resistance in the isolates through the environmental route. However, the potential ability of the fungicide to induce medically triazole-resistant strains over a long period of application should not be neglected.

Objectives: The rate of resistance of Candida parapsilosis to echinocandins remains unexplored in Iran. The main aims of this study were to investigate the susceptibility patterns and possible mechanisms of echinocandin resistance in echinocandin-resistant clinical C. parapsilosis isolates in Iran. Methods: A total of 105 isolates of C. parapsilosis sensu stricto underwent antifungal susceptibility testing to echinocandins by the broth microdilution reference method. Sequences of the CpERG3 and CpFKS1 genes were analysed using MEGA6 software, and alterations in CHS3, FKS1 and Rho gene expression were evaluated by quantitative reverse transcription (RT-qPCR). REST® software was used to analyse the results. Results: The rate of echinocandin cross-resistance was 2.9% (3/105). No substitutions were detected in Fks1p except for the naturally occurring P660A amino acid substitution observed in isolates both with high and low minimum inhibitory concentrations (MICs). Moreover, the G111R amino acid substitution was not found in Erg3p. Following echinocandin exposure, expression of Rho and FKS1 genes was significantly increased in resistant isolates, whilst the CHS3 gene showed no change. Conclusion: Alterations in the expression of some key genes may be responsible for echinocandin resistance among C. parapsilosis isolates. Understanding the mechanisms responsible for drug resistance in C. parapsilosis is not only crucial for the development of new antifungals but is also important in choosing appropriate antifungals for patient treatment at the earliest stage.

Background and Purpose: Encapsulation can lead to improved efficacy and safety of antifungal compounds. The attention of scientists has recently turned to biocompatible lipids as the carriers for the delivery of antifungal drugs, such as fluconazole. Although several research reports have already been published on fluconazole loaded solid lipid nanoparticles (FLZ-SLNs) and fluconazole loaded nanostructured lipid carriers (FLZ-NLCs), the possible advantages of NLCs over SLNs have not yet been fully established. Studies performed so far have given several contradictory results. Materials and Methods: Both formulations of fluconazole were synthesized using probe ultrasonication method and the characteristics were analyzed. Antifungal susceptibility testing (AFST) was performed with FLZ, FLZ-SLNs, and FLZ-NLCs using CLSI document M60 against some common fluconazole-resistant Candida species. Results: A significant decrease was observed in minimum inhibitory concentration values when both formulations were applied. Nonetheless, FLZ-NLCs were significantly more effective (P Conclusion: Based on the obtained results, FLZ-NLCs could reverse the azole-resistance phenomenon in the most common Candida species more effectively, as compared to FLZ-SLNs.

Background and Purpose: Candida parapsilosis isolates usually have a low minimum inhibitory concentration (MIC) against azoles. Although Candida parapsilosis isolates usually have low MICs against azoles, recent studies candida invasive infections due to azole resistant-C. parapsilosis isolates . Regarding this, the main aim of this study was to determine the susceptibility pattern of Iranian clinical C. parapsilosis against available azole antifungal drugs. Materials and Methods: This study was conducted on 105 previously-identified isolates of C. parapsilosis sensu stricto. For the purpose of the study, the isolates were subjected to antifungal susceptibility testing against fluconazole (FLZ), itraconazole (ITZ), voriconazole (VRZ), and two new azole drugs, namely luliconazole (LUZU) and lanoconazole (LZN). The broth microdilution reference method adopted in this study was according to the Clinical & Laboratory Standards Institute M27-A3 and M27-S4 documents. Results: According to the results, 89% (n=94) of C. parapsilosis isolates showed a MIC of ≥ 1 μg/ml, indicating resistance against ITZ. Multi-azole resistance was observed in 3.8% of the isolates. In addition, LUZU and LZN demonstrated the highest efficacy with the MIC50 values of 0.5 and 1 μg/ml, respectively. Conclusion: The majority of the isolates showed high MIC values against ITZ. This may have been associated with the long-term ITZ prophylaxis/therapy in patients infected with candidiasis. Hence, the adoption of an appropriate antifungal agent is a crucial step for starting the treatment. Background and Purpose: Although Candida parapsilosis isolates have usually low MICs against azoles but recent study confirmed Candida –related invasive infections due to azoles resistance C. parapsilosis isolates. The main aim of this study was to determine the susceptibility pattern of Iranian clinical C. parapsilosis against available azolesantifungal drugs. .Material and Methods: One hundred and five previously-identified isolates of C. parapsilosis sensu stricto were subjected to antifungal susceptibility testing against fluconazole, itraconazole, voriconazole and two new azole drugs, loliconazole and lanoconazole using the broth microdilution reference method according to CLSI M27-A3 and M27-S4 document.Results: Eighty nine percent (n=94) of C. parapsilosis isolates showed MIC ≥ 1µg/ml which indicated resistance against itraconazole. Multi-azoles resistances were observed in 3.8% of the isolates. Loliconazole and lanoconazole demonstrated the highest efficacy with MIC50 values of 0.5 and 1µg/ml, respectively.Conclusion: The majority of the isolates showed high MIC values against Itraconazole. It may associated with the long term Itraconazole prophylaxis/therapy in patients Infected with candidasis. Hence, choosing the appropriate antifungal is the crucial step for starting treatment.

Background and Purpose: Although the mechanism of action for echinocandins is known, the physiological mechanisms by which these antifungal agents cause cell death via the classical apoptotic pathways are not well-defined yet. Regarding this, the present study aimed to evaluate the mechanisms of caspofungin-induced Candida glabrata cell death. Materials and Methods: For the purpose of the study, the minimum inhibitory concentration (MIC) of caspofungin against C. glabrata (ATCC 90030) was determined using the broth microdilution reference method (CLSI M27-A2 and M27-S4). The annexin V and propidium iodide staining was performed to determine the way through which caspofungin acts against C. glabrata (i.e., through the induction of apoptosis and/or necrosis). Additionally, the possible effect of caspofungin on inducing the expression of two apoptotic genes, namely MCA1 and NUC, was studied using the real-time polymerase chain reaction assay. Results: According to the obtained MIC value (0.5 μg/mL), C. glabrata, exposed to 0.25, 0.5, and 1 μg/mL of caspofungin, exhibited the features of late apoptosis/necrosis after 18 h of incubation. Furthermore, the use of 0.25, 0.5, and 1 μg/ml caspofungin induced apoptosis (early/late) in 14.67%, 17.04%, and 15.89% of the cells, respectively. The results showed a significant difference between the percentages of early-apoptotic cells at the three concentrations (p Conclusion: As the findings of the present study indicated, caspofungin promoted both necrosis and apoptosis of C. glabrata cells at concentrations higher than or equal to the MIC value.

Background: Recent studies from multiple countries have shown a high prevalence of coronavirus disease 2019 (COVID-19)-associated pulmonary aspergillosis (CAPA) among severely ill patients. Despite providing valuable insight into the clinical management of CAPA, large-scale prospective studies are limited. Here, we report on one of the largest multicenter epidemiological studies to explore the clinical features and prevalence of COVID-19-associated pulmonary mold infections (CAPMIs) among mechanically ventilated patients.Methods: Bronchoalveolar lavage (BAL) and serum samples were collected for culture, galactomannan (GM), and β-D-glucan (BDG) testing. Patients were classified as probable CAPMI based on the presence of host factors, radiological findings, and mycological criteria.Results: During the study period, 302 COVID-19 patients were admitted to intensive care units (ICUs), among whom 105 were mechanically ventilated for ≥4 days. Probable CAPMI was observed among 38% of patients (40/105), among whom BAL culture of 29 patients turned positive for molds, while galactomannan testing on BAL (GM index ≥1) and serum (GM index >0.5) samples were positive for 60% (24/40) and 37.5% (15/39) of patients, respectively. Aspergillus (22/29; 75.8%) and Fusarium (6/29; 20.6%) constituted 96.5% of the molds isolated. Diaporthe foeniculina was isolated from a COVID-19 patient. None of the patients who presented with CAPMI were treated with antifungal drugs.Conclusion: Despite being prevalent, the absence of appropriate antifungal treatment highlights that CAPMI is a neglected complication among mechanically ventilated COVID-19 patients admitted to ICUs. CAPMI can be caused by species other than Aspergillus.

Background and Purpose: The incidence of invasive fungal infections has been increased in recent years. The growing use of azole drugs for prophylactic and therapeutic purposes has resulted in the gradual emergence of azole-resistant species. Accordingly, the introduction of a new strategy to improve the management of Candida infections is an urgent need. Regarding this, the present study was performed to evaluate the antifungal activities of crocin (Cro) alone and in combination with fluconazole. Materials and Methods: This study was conducted on 50 clinical isolates of four different Candida species. The identity of the isolates was confirmed using the internal transcribed spacer identification system. The interactions of Cro with fluconazole were investigated using a microdilution checkerboard method based on the Clinical and Laboratory Standards Institute reference technique with 96-well microtiter plates. Furthermore, the assessment of the interaction of drug combinations was accomplished using the fractional inhibitory concentration index (FICI) based on the Loewe additivity theory. Results: According to the results, Cro alone showed a relatively high MIC50 value (1 g/ml) against Candida species. Our results demonstrated indifferent interactions between Cro and fluconazole with a FICI range of 0.5-4 against Candida strains. Conclusion: The high MIC value for Cro against Candida species indicated its failure to show appropriate antifungal activity against this species. The MIC of this agent was not significantly reduced even by the addition of fluconazole. Therefore, other mechanisms which are not related to the mechanism of azole drugs are involved at high concentration of Cro.

Objective: Aflatoxin is known as one of the most important mycotoxins that threatens of human life. The toxin is produced by Aspergillus species which are common cause of contamination of agricultural products. For this reason, the use of organic compounds has always been considered in order to inhibit the growth of fungi and production of toxin. The aim of this study was to investigate the effect of vitamin C on the growth rate of fungi and the level of aflR gene expression (gene responsible for aflatoxin production). Material and method: At first, Aspergillus parasiticus ATCC15517 was cultured in SDA medium containing vitamin C with concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.1 mg / ml at 28 ° C for 72 hours. Then, the amount of aflatoxin produced in the presence of vitamin C was measured by HPLC method. Finally, by extracting the DNA of cultured samples, the aflR gene expression level was evaluated by real-time PCR at different concentrations of vitamin C. Result: The results showed that the deformation of mycelium was started in medium with 50 mg / ml of vitamin C and only fungal spores were observed at higher concentrations. The results of measurement of toxin showed that the level of total aflatoxin and the subset of B 1, B 2, G 1 and G 2 were 5.9, 1.9, 0.2, 3.5 and 0.3 ppm in the presence of vitamin, respectively. While without the presence of vitamin C, these values were 207.5, 73.6, 4.5, 123.4, 6 ppm, respectively. Measuring the expression level of aflR genes, showed that at a concentration of 25 mg / ml of vitamin C, the level of gene expression is down 68%, and at the concentration of 50 mg / ml, the level of gene expression is decreased up to 81%. Conclusion: This study showed that vitamin C, as a human-compatible compound, could be considered as a good way to keep agricultural products from fungal aflatoxin.

Background and Purpose: Candida albicans is a prevalent human fungal pathogen that can cause a wide spectrum of diseases, from superficial mucosal infections to systemic disorders, in patients with impaired immunity. Glabridin is a pyranoisoflavan originally extracted from root extract of Glycyrrhiza glabra. Glabridin can also mediate apoptosis in yeast cells by changing the mitochondrial membrane potential, activation of caspase-like proteases, and DNA cleavage. The aim of this study was to investigate the mechanism of action of glabridin in C. albicans. Materials and Methods: Candida albicans ATCC14053 was applied as the standard strain. Total RNA was extracted from the isolate under glabridin-treated and untreated conditions. To evaluate the alternations in the apoptosis inducing factor (AIF) gene expression, real-time polymerase chain reaction (real-time -PCR) was performed, and the obtained data were analyzed using REST software. Results: Expression of the AIF gene was represented as the ratio of expression relative to the reference gene. According to the REST® output, the expression of the AIF gene increased significantly (P

Nosocomial fungal infections are amongst the main causes of mortality in patients admitted to healthcare settings, especially in immunocompromised populations. The predominant pathogens include Candida spp., Aspergillus spp., Mucorales spp., and Fusarium spp. Nosocomial fungal infections are increasing due to the underlying factors in decades ahead. One of the predisposing factors includes immune system suppressing modalities due to the extensive use of invasive treatments such as stem cell transplantation, organ transplantation, chemotherapy, and immunosuppressive drugs. Infection control methods recommended, can avoid catheter-related candidiasis and also minimize exposure to airborne Aspergillus spores in immunocompromised patients in hospital settings. Many of these infections can be prevented without advanced equipment and high costs by training healthcare workers in using medical equipment. Treatment for these infections is costly due to increased length of stay in health settings. Antifungal prophylaxis should be considered in patients at risk of invasive fungal infections during the periods of severe immunosuppression. In this study the epidemiology, diagnosis, treatment, and prevention strategies of nosocomial fungal infections have been fully reviewed.

Background and Purpose: Candida parapsilosis is a predominant species found in nosocomial infection, particularly in hospitalized patients. The molecular epidemiology of the clinical strains of this species has not been well studied. The present study was performed with the aim of investigating the microsatellite genotyping of Candida parapsilosis among the Iranian clinical isolates. Materials and Methods: This study was conducted on 81 independent clinical C. parapsilosis isolates that were genotyped by using a panel of six microsatellite markers. Results: The short tandem repeat (STR) typing of clinical C. parapsilosis isolates demonstrated 68 separate genotypes, among which 57 genotypes were observed once and the remaining 11 cases were identified for multiple times. The Simpson’s diversity index for the panel of combined six markers yielded a diversity index of 0.9951. The heterogeneity was observed among the Iranian and the Netherlands clinical C. parapsilosis isolates. Conclusion: As the findings indicated, the clinical C. parapsilosis isolates from Iran showed a high genetic diversity. It can be concluded that molecular epidemiology could be useful for screening during outbreak investigation where C. parapsilosis is involved.

Background and Purpose :Candidiasis is a widespread fungal infection caused by different Candida species. Rapid identification of Candida species in clinical laboratory is becoming increasingly important since the identification and discrimination of ethological agents for early treatment. We aimed at molecular identification of commonly Candida species isolated from clinical samples by using both PCR-RFLP assay and amplification of hwp1 gene. Materials and Methods: Clinical samples comprising of vaginal specimens ,cutaneous, sputum, bronchoalveolar lavage (BAL,( and blood cultures were recovered from suspected patients. Candida isolates were initially identified phenotypically and confirmed by molecular approaches based on restriction fragment length polymorphism (PCR-RFLP (with MspI restriction enzyme. Amplification of hwp1 gene was performed for discrimination of C. albicans from C. dubliniensis and C. africana. Results: The most abundant species were C. albicans (n=67; 44.6 %), C. glabrata (n=10; 20 %), C. tropicalis (n=20; 13.3 %), C. krusei (n=12; 8 %), C. parapsilosis (n=11; 7.3 %). Out of 67 C. albicans species, 6 species identified as C. dubliniensis and 4 species identified as C. africana. Conclusion: High frequency of non-albicans Candida species and differences in levels of susceptibility to the antifungal agents are important issues in medicine .Therefore, to manage the Candida-related infections properly, molecular diagnostic methods would be fast, reliable and even cost-effective approaches for identification of Candida species.

Background and Purpose: Microorganism-based synthesis of nanostructures has recently been noted as a green method for the sustainable development of nanotechnology. Nowadays, there have been numerous studies on the emerging resistant pathogenic bacteria and fungal isolates, the probable inability of bacteria and fungi to develop resistance against silver nanoparticles’ (SNPs) antibacterial, antifungal, antiviral and, particularly antibacterial activities. In this study, we aim to use the yeast Saccharomyces cerevisiae model for synthesis of SNPs and to investigate its antifungal activity against some isolates of Candida albicans. Materials and Methods: A standard strain of S. cerevisiae was grown in liquid medium containing mineral salt then, it was exposed to 2 mM AgNO3. The reduction of Ag+ ions to metal nanoparticles was virtually investigated by tracing the color of the solution, which turned into reddish-brown after 72 hours. Further characterization of synthesized SNPs was performed afterwards. In addition, antifungal activity of synthesized SNPs was evaluated against fluconazolesusceptible and fluconazole-resistant isolates of Candida albicans. Results: The UV-vis spectra demonstrated a broad peak centering at 410 nm, which is associated with the particle sizes much less than 70 nm. The results of TEM demonstrated fairly uniform, spherical and small in size particles with almost 83.6% ranging between 5 and 20 nm. The zeta potential of SNPs was negative and equal to -25.0 (minus 25) mv suggesting that there was not much aggregation. Silver nanoparticles synthesized by S. cerevisiae, showed antifungal activity against fluconazole-susceptible and fluconazole-resistant Candida albicans isolates, and exhibited MIC90 values of 2 and 4 &mug/ml, respectively. Conclusion: The yeast S. cerevisiae model demonstrated the potential for extracellular synthesis of fairly monodisperse silver nanoparticles

Background and Purpose: Mycotoxins are secondary fungal metabolites with a very high diversity that are produced by some species of Aspergillus which frequently leads to contaminate food and agricultural products. Recently, elimination of aflatoxin contamination in food and feed has been considered by scientists worldwide. Although, the antibacterial and antifungal effects of vitamins as natural compounds have been proven, the mechanism of vitamins effect on Aspergillus parasiticus growth and aflatoxin production is not yet clear. In this study, the effect of thiamine (vitamin B1) was studied on Aspergillus parasiticus growth, aflatoxins production and the afIR gene expression. Materials and Methods: A standard strain of Aspergillus parasiticus was applied for performing antifungal susceptibility test in different concentrations of thiamine. Antifungal susceptibility test was performed according to CLSI M38-A2 document. The concentration of aflatoxin was determined by HPLC. Moreover, the quantitative changes in the aflR gene expression were analyzed by Real Time PCR method. Results: The minimum inhibitory concentration was yielded as > 500 mg/ml. However, HPLC analysis results showed that aflatoxin production reduced in samples treated with 500 mg/ml of thiamine. In addition, the level of afIR gene expression was significantly reduced after treating with 500 and 250 mg/ml of vitamin B1. Conclusion: Based on the obtained results, thiamine could not inhibit the fungal growth completely. However, the rate of afIR gene expression and aflatoxin production was significantly reduced after fungal treating with thiamine. Consequently, using natural compounds such as vitamins may be regarded as potential antitoxic agent in food industry and the industries related to agriculture.

Abstract Objective(s): The enzymatic activity of fungi has recently inspired the scientists with re-explore the fungi as potential biofactories rather than the causing agents of humans and plants infections. In very recent years, fungi are considered as worthy, applicable and available candidates for synthesis of smaller gold, silver and other nano-sized particles. Materials and Methods: A standard strain of Aspergillus parasiticus was grown on a liquid medium containing mineral salt. The cell-free filtrate of the culture was then obtained and subjected to synthesize SNPs while expose with 1mM of AgNO 3. Further characterization of synthesized SNPs was performed afterward. In addition, antifungal activity of synthesized SNPs was evaluated against a standard strain of Candida albicans. The reduction of Ag+ ions to metal nanoparticles was investigated virtually by tracing the color of the solution which turned into reddish-brown after 72h. Results: The UV-vis spectra demonstrated a broad peak centering at 400nm which corresponds to the particle size much less than 70nm. The results of TEM demonstrated that the particles were formed fairly uniform, spherical, and small in size with almost 90% in 5-30nm range. The zeta potential of silver nanoparticles was negative and equal to -15.0 which meets the quality and suggested that there was not much aggression. Silver nanoparticles synthesized by A. parasiticus showed antifungal activity against yeast strain tested an d exhibited MIC value of 4 μg/mL. Conclusion: The filamentous fungus, A. parasiticus has successfully demonstrated potential for extra cellular synthesis of fairly monodispersed, tiny silver nanoparticles.

Despite the introduction of new antifungal agents, resistances to antifungal therapy continue to increase and outcome of invasive fungal infections treatment is frequently suboptimal. A large amount of the recent effort in antifungal drug discovery has focused on a limited set of targets with functions known or expected to be important for fungal viability and virulence. A variety of techniques can be used to identify fungal genes of interest. Gene expression profiling, RNA mediated gene silencing and insertional mutagenesis are three main molecular genetics technologies used to identify and validate antifungal drug targets. The term RNA interference (RNAi) refers to a cellular process by which a sequence-specific double-stranded RNA (dsRNA) inhibits the expression of a gene. This mechanism is strongly conserved in eukaryotes and has been documented to be existed in different fungal species such as Candida albicans, Aspergillus nidulans and Penicillium marneffei. Many vital and virulence genes have been successfully knocked down using RNAi technology. RNAi can be regarded as a promising approach for discovery of new gene targets for the design of fungus-specific antifungal agents. Here we discuss about a novel approach and its application in designing new molecular antifungal targets.

Invasive aspergillosis (IA) are a major complication in immunocompromized patients where can be serious and rapidly fatal. Early diagnosis and early appropriate antifungal treatment is important in reducing mortality and morbidity. But despite many efforts to develop detection methods, the diagnosis of IA still remains challenging and current conventional methods are limited for adequate diagnosis. New rapid methods which can detect IA early in the course of disease with high sensitivity and specificity are needed since treating these infections at an early stage. Using molecular methods for Aspergillus species identification can be a cost-effective, rapid, discriminatory, and objective approach for delineating Aspergillus species in a clinical microbiology laboratory. PCR techniques for the diagnosis of IA have been studied for more than a decade and are still considered investigational, however until now PCR is not included in current EORTC/MSG diagnostic criteria.

The nanoparticles of Ti and TiO2 have attracted extensive research interest because of their diverse applications in, for instance, catalysis, energy conversion, pigment and cosmetic manufacturing and biomedical engineering. Through this project, a one-step bulk synthesis method of electromagnetic levitational gas condensation (ELGC) was utilized for the synthesis of monodispersed and crystalline Ti and TiO2 nanoparticles. Within the process, the Ti vapours ascending from the high temperature levitated droplet were condensed by an argon gas stream under atmospheric pressure. The TiO2 nanoparticles were produced by simultaneous injection of argon and oxygen into the reactor. The effects of flow rate of the condensing and oxidizing gases on the size and the size distribution of the nanoparticles were investigated. The particles were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and image analysis. The process parameters for the synthesis of the crystalline Ti and TiO2 nanoparticles were determined.

Efg1 transcription factor is believed to be the main regulator of hyphal formation under many different conditions. In addition, it is responsible for positive regulation of the expression of several hyphal-specific genes. SAP5, which encodes secreted aspartic proteinase, is one of the mentioned genes and is crucial for pathogenicity properties. In the present work we have established the experimental conditions for the use of siRNA in the diploid yeast Candida albicans in order to knock-down the EFG1 gene expression as well as the Efg1-dependent gene, SAP5. The 19-nucleotide siRNA was designed according to cDNA sequence of EFG1 gene in C. albicans and modified-PEG/LiAc method was applied for yeast transfection. To quantify the level of both EFG1 and SAP5 gene expression, the cognate mRNAs were measured in C. albicans by quantitative real-time RT-PCR and data was consequently analyzed by use of REST® software. Images taken by fluorescent microscopy method indicated the effectiveness of transfection. According to REST® software data analysis, expression of EFG1 gene decreased about 2.5-fold using 500 nM of siRNA. A 7-fold decrease in EFG1 gene expression was observed when applying 1 µM of siRNA (P

Due to the epidemiological alteration in distribution of Candida species as well as significant increasing trend of either intrinsic or acquired in resistance of some of these fungi, the precise identification of Candida species is necessary for effective antifungal therapy and also for prevention of nosocomial infections. PCR-RFLP method is indicated to be a reliable, rapid and simple technique which is able to differentiate the Candida species. In the present study, we applied this method to evaluate the distribution of Candida species in patients affected with cutaneous candidiasis in the Guilan province. 896 clinical cutaneous samples were collected from different parts of skin and nail of suspected patients referred to clinical centers all over the Guilan province during 24 months. Samples were examined directly with 15% KOH and cultured on fungal specific media. Genomic DNA was extracted and the restriction enzyme Msp1 was applied for polymorphism analysis. Totally, 47 yeast strains were successfully isolated from different clinical samples and identified by conventional as well as PCR-RFLP methods. The results indicated that Candida albicans (36.17%) was the most frequent species followed by C. parapsilosis (25.53%), C. tropicalis (19.14%), C. guilliermondii (14.89%), C. famata (2.12%) and C. krusei (2.12%). Female finger nails were the most common location to be affected by Candida species. In conclusion, PCR-RFLP method was successfully used for recognition of clinical Candida species within the Guilan province and obtained results revealed C. albicans as the predominant causative agent of cutaneous candidiasis. However, distribution of other Candida species did not completely consist with the reported distribution of Candida species in other parts of Iran with different climate to the Guilan province.