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Abstract Dermatophytes show a wide geographic distribution and are the main causative agents of skin fungal infections in many regions of the world. Recently, their resistance to antifungal drugs has led to an obstacle to effective treatment. To address the lack of dermatophytosis data in Iraq, this study was designed to investigate the distribution and prevalence of dermatophytes in the human population and single point mutations in squalene epoxidase gene (SQLE) of terbinafine resistant isolates. The identification of 102 dermatophytes isolated from clinical human dermatophytosis was performed through morphological and microscopic characteristics followed by molecular analysis based on ITS and TEF-1α sequencing. Phylogeny was achieved through RAxML analysis. CLSI M38-A2 protocol was used to assess antifungal susceptibility of the isolates to four major antifungal drugs. Additionally, the presence of point mutations in SQLE gene, which are responsible for terbinafine resistance was investigated. Tinea corporis was the most prevalent clinical manifestation accounting for 37.24% of examined cases of dermatophytosis. Based on ITS, T. indotineae (50.98%), T. mentagrophytes (19.61%), and M. canis (29.41%) was identified as an etiologic species. T. indotineae and T. mentagrophytes strains were identified as T. interdigitale based on TEF-1α. Terbinafine showed the highest efficacy among the tested antifungal drugs. T. indotineae and T. mentagrophytes showed the highest resistance to antifungal drugs with MICs of 2–4 and 4 μg/mL, while M. canis was the most susceptible species. Three of T. indotineae isolates showed mutations in SQLE gene Phe397Leu substitution. A non-previously described point mutation, Phe311Leu was identified in T. indotineae and mutations Lys276Asn, Phe397Leu and Leu419Phe were diagnosed in T. mentagrophytes XVII. The results of mutation analysis showed that Phe397Leu was a destabilizing mutation; protein stability has decreased with variations in pH, and point mutations affected the interatomic interaction, resulting in bond disruption. These results could help to control the progression of disease effectively and make decisions regarding the selection of appropriate drugs for dermatophyte infections.

Background and Purpose: The current study aimed to report a multiplex polymerase chain reaction (PCR) assay as a monitoring technique to differentiate aflatoxigenic from non-aflatoxigenic strains of Aspergillus flavus isolated from pistachio orchards soil.Materials and Methods: In total, 25 A. flavus strains were isolated from soil samples of pistachio orchards. To test the strains for Aflatoxin B1(AFB1)-producing ability, thin-layer chromatography (TLC) was used and the amounts of AFB1 were measured by high performance liquid chromatography (HPLC). Multiplex PCR was used as a genome-based method to detect genes responsible for AFB1 production by A. flavus and the results were analyzed in terms of speed and specificity of detection. A set of four primers was designed specifically for the omtA, omtB, ver-1, and aflR genes which are commonly present in aflatoxin biosynthetic pathways.Results: The AFB1 production by the A. flavus strains ranged from 0 to 321 ρg/μl. Four band patterns of the primer sets were observed only in AFB1-producing A. flavus strains. Moreover, 18 out of the 25 strains showed all four bands belonging to omtA, omtB, ver-1, and aflR, whereas 7 strains did not display omtA, or aflR-related bands, in non-toxigenic and low toxin-producing A. flavus.Conclusion: The multiplex PCR is a supplementary strategy to current conventional mycotoxin analytical techniques, such as TLC and HPLC. It could be used as an efficient method to differentiate aflatoxigenic from non-aflatoxigenic strains of A. flavus. This achievement is crucial to minimize fungal contamination of food, feed, and agricultural commodities, thereby reducing the risk of subsequent aflatoxin consumption.

Background: Understanding the microbiota of disease vectors can help for developing new strategies to prevent the transmis­sion of vector pathogens. Ixodes ricinus is one of the most notorious tick vectors with increasing importance in Iran and other parts of the world while there is limited data on its microbiota. This study aimed to use metagenomics for identifying the I. ricinus tick’s microbiota of Iran. Methods: A total of 39 adult ticks were collected from Mazandaran (21 females), Gilan (17 females), and Golestan (1 male). Five tick pools prepared from 39 adults of I. ricinus were subjected to metagenomics analysis. The data were analyzed by targeting the V6 region of the 16S rRNA gene by Illumina 4000 Hiseq sequencing. Results: Among hundreds of intestinal microbiota identified by metagenomics, various pathogenic microorganisms distributed in 30 genera and species including those responsible for tick-borne diseases resided in the genera Coxiella, Rickettsia, and Burkholderia were found. Conclusion: Our results indicated that metagenomics identifies bacteria genera and species which cannot be easily rec­ognized by routine methods. The presence of such pathogenic bacteria indicates the importance of possible zoonotic diseases in this region which could affect public health. These results further substantiate the importance of advanced metagenomics analyses to identify neglected tick-borne pathogens which enable researchers to provide efficient mapping roads for the management of emerging and re-emerging infectious diseases.

Abstract In the present study, metal and metal oxide nanoparticles were successfully synthesized using Aspergillus kambarensis. UV–Vis spectroscopy showed maximum absorbance of 417 nm for silver (AgNPs), 542 nm for gold (AuNPs), 582 nm for copper (CuNPs) and 367 nm for zinc oxide (ZnONPs) nanoparticles. Fourier transform infrared spectroscopy indicated the presence of various mycochemicals with diverse functional groups in the fungal cell‐free filtrate. Transmission electron microscopy revealed mono and poly dispersed particles with an estimate size of 50 nm and different shapes for synthesized manufacture metallic nanoparticles (MNPs. Dynamic light scattering confirmed that MNPs were dispersed in the size range less than 50 nm. Zeta potential analysis showed values of −41.32 mV (AgNPs), −41.26 mV (AuNPs), −34.74 mV (CuNPs) and 33.72 mV (ZnONPs). X‐ray diffraction analysis demonstrated crystalline nature for MNPs. All the synthesized MNPs except AuNPs showed strong antifungal and antibacterial activity in disc diffusion assay with growth inhibition zones of 13.1–44.2 mm as well as anticancer activity against HepG‐2 cancer cell line with IC50 in the range of 62.01–77.03 µg/ml. Taken together, the results show that biologically active MNPs synthesized by A. kambarensis for the first time could be considered as promising antimicrobial and anticancer agents for biomedical applications.

Background: Ticks are vectors of many pathogens that involve various important diseases in humans and animals, they have several diverse hosts consequently can retain a diverse group of indigenous microbes, from bacteria to fungi. Little is known about the prevalence and diversity of tick microflora colonizing the midgut and their effects on ticks and their interaction. This information is important for development of vector control strategies. Methods: This study was carried out in northern Iran during autumn 2019. Ticks, Ixodes ricinus caught alive on the bodies of domestic animals in the fall. The tick homogenate was prepared. The identification of fungal isolates was car­ried out according to a combination of macro and microscopic morphology and molecular sequencing. Pathogenic bac­teria of the family Borreliaceae, Francisella tularensis, Borrelia burgdorferi and Coxiella burnetii were tested by real-time PCR. Results: A total of 133 mature I. ricinus ticks were collected from domestic animals, including 71.5% cattle and 28.5% sheep. The tick frequency rates were 87.21% for Mazandaran, 8.28% for Golestan and 4.51% for Gilan Provinces. Total prevalence of fungal tick contamination was 53.4% (75/133) of which Trichoderma harzianum (57%) was the most prevalent species followed by Aspergillus spp. (42%), Mortierella alpine (19%) and Penicillium polonicum (14%). All tick samples were negative for three pathogenic bacteria including Francisella tularensis, Coxiella burnetii, and Borre­lia burgdorferi by real-time PCR analysis. Conclusion: These results show a first picture of the microbial diversity of ticks and highlight the importance of micro­biota and their role in host-pathogen interaction.

Background and Purpose: This study aimed to assess the effect of Allium cepa ethanolic extract (ACE) loaded polyacrylonitrile (PAN) and polyvinyl pyrrolidone (PVP) nanofibers on Candida albicans (C. albicans) CDR1 and CDR2 genes expression.Materials and Methods: The minimum inhibitory concentrations (MICs) of ACE against C. albicans ATCC 10231 and clinical fluconazole (FLC)-resistant C. albicans PFCC 93-902 were determined using the Clinical and Laboratory Standards Institute (CLSI) protocol (M27-Ed4) at a concentration range of 45.3-5800 µg/ml. The nanofibers containing ACE (60 wt%) were fabricated using the electrospinning technique. The expression of the CDR1 and CDR2 genes was studied in the fungus exposed to ACE loaded nanofibers and 0.5×MIC concentration of FLC using the real-time polymerase chain reaction.Results: MIC50 and MIC90 of ACE against FLC-resistant C. albicans were 725 and 1450/mL, respectively. The expression of CDR1 (4.5-fold) and CDR2 (6.3-fold) were down-regulated after the exposure of FLC-resistant C. albicans to ACE-loaded nanofibers (P

Background and Purpose: This study aimed to investigate the effects of Allium cepa ethanolic extract (EAC) on Cryptococcus neoformans biological activities and LAC1 gene expression. Materials and Methods: The minimum inhibitory concentration (MIC) of EAC was determined based on the Clinical and Laboratory Standards Institute M27-A4 method at a concentration range of 125- 4000 µg/ml. The EAC synergism activity was determined in combination with fluconazole (FCZ) as an antifungal azole. Laccase activity, melanin production, and cell membrane ergosterol content of C. neoformans were assessed at the 0.5× MIC concentration of EAC (1000 μg/ml) and FCZ (64 μg/ml) by approved methods. The expression of the LAC1 gene was studied in the fungus exposed to 0.5× MIC concentration of EAC and FCZ using the real-time polymerase chain reaction. Results: Based on obtained results, MIC of EAC and FCZ were 2000 and 128 μg/ml,respectively. A combinatory effect was reported for FCZ and EAC by a fractional inhibitory concentration index of 0.25. The cell membrane ergosterol content was inhibited in EAC- and FCZ-treated C. neoformans by 58.25% and 49.85%, respectively.The laccase activity and melanin production were reduced in EAC-treated C. neoformans by 45.37% and 51.57%, and in FCZ-treated fungus by 54.64% and 53.68%, respectively.The expression of fungal LAC1 at messenger RNA (mRNA) level was measured 0.46 and 0.58 folds and significantly decreased in both EAC- and FCZ-treated C. neoformans at the 0.5×MIC concentration, respectively (p

Background and Purpose: Soil bacteria have extreme population diversity among natural sources and are able to produce a wide array of antifungal metabolites. This study aimed to isolate and identify the bioactive metabolite-producing bacteria from forest soils and evaluate their antimicrobial potent against some pathogenic organisms. Materials and Methods: In this study, soil samples were screened for antifungal activity against Aspergillus fumigatus on glucose-yeast extract (GY) agar using a visual agar plate assay method. All growing bacteria were examined for antifungal activity, and antagonistic bacteria were identified based on 16S ribosomal RNA sequence analysis. For optimization of the production of antifungal bioactive metabolites, inhibitory bacteria were cultured on different culture conditions, including media, pH, temperature, and incubation time. Results: In total, 110 bacterial strains were isolated from the forest soils and four species with high antifungal activity were identified as Streptomyces libani, Streptomyces angustmyceticus, Bacillus subtilis, and Sphingopyxis spp. on the basis of 16s ribosomal RNA sequencing. Dichloromethane extract of the starch casein broth culture filtrate of the S. libani (incubated at 30° C for five days) showed strong antifungal activity against A. fumigatus, Aspergillus niger, and Aspergillus flavus. Conclusion: Based on the results, forest soils contain organisms with antifungal activity and could be considered as a good source for novel antifungal metabolites as effective and safe therapeutics.

Background: Zoonotic diseases as health concerns worldwide account for more than half of the emerging infectious diseases. Arachnids are powerful vectors to transmit several diseases to humans. Additionally, these emerging zoonotic diseases have been a considerable health threat in the Eastern Mediterranean Region of the WHO (EMRO) due to the large population living close to farms and international trade with nearby countries. Methods: This review study is based on the reported three tick-borne diseases, Lyme disease, Tularemia, and Q fever, from Iran and other EMRO countries. To this end, we searched PubMed central, ISI web of Science, and Google with the related keywords in English at any time. The reported data are then sorted by countries for each disease. Results: According to the published data, 15 countries in the region have one/more emerging infectious diseases. Q fever has been the most frequent infection in EMRO countries, while Lyme was less recorded. Furthermore, Iran is among the countries with documented history of all three investigated diseases. Conclusion: Tick-borne disease is popular among EMRO countries, indicating that they have natural conditions for infections in animals and humans. It appears necessary to develop a disease management strategy and control programs against tick-borne diseases (TBDs). Moreover, the disease-resistant animal could be bred instead of susceptible livestock. Therefore, research studies to control TBDs should be regarded as a top priority plan.

Drug resistance is one of the major challenges to skin fungal infections, especially in tropical and subtropical infections caused by dermatophytes. This study aimed to determine the antifungal susceptibility of clinically dermatophytes and evaluate point mutations in terbinafine-resistant isolates. A total number of 123 clinical dermatophyte isolates in eight species were evaluated in terms of sensitivity to seven major antifungals. Furthermore, the point mutation in squalene epoxidase (SQLE) gene responsible for terbinafine resistance was studied. The dermatophytes species were identified by morphological characteristics and confirmed by the ITS sequencing. Also, the phylogenetic tree was drawn using the RAxML analyses for 123 dermatophytes isolates. A new XXIX genotype was also found in 4 Trichophyton mentagrophytes isolates. Based on the results obtained, terbinafine was the most effective antifungal drug followed by itraconazole and voriconazole. Trichophyton rubrum and Trichophyton tonsurans were the most susceptible species (MIC50 = 0.01, 0.09 μg/ml), and T. mentagrophytes was the most resistant species (MIC50 = 0.125 μg/ml) to terbinafine. Of the 123 dermatophytes isolates, six isolates showed reduced susceptibility to terbinafine, and only Trichophyton indotineae had a mutation in SQLE gene as a Phe397Leu substitution. Overall, the antifungal susceptibility test is necessary for managing dermatophytosis. These results help physicians to control the course of the disease and provide further insights to select effective drugs for patients with dermatophytosis, especially in tropical and subtropical regions of the world, where dermatophytosis is still a public health problem.

Background and Purpose: The present study was conducted to investigate the inhibitory effects of Carum carvi essential oil (EO) against ERG6 gene expression in relation to fungal growth and some important virulence factors in Candida albicans. Materials and Methods: The minimum inhibitory concentration (MIC) of C. carvi EO against C. albicans was determined by the Clinical and Laboratory Standards Institute M27-A4 method at a concentration range of 20-1280 μg/ml. Furthermore, the expression of ERG6 gene was studied at the 0.5× MIC concentration of C. carvi EO using real-time polymerase chain reaction. The proteinase and phospholipase activities, cell surface hydrophobicity (CSH), and cell membrane ergosterol (CME) content of C. albicans were also assessed at the 0.5× MIC concentration of the plant EO using the approved methods. In addition, fluconazole (FLC) was used as a control antifungal drug. Results: The results indicated that the MIC and minimum fungicidal concentration of C. carvi EO for C. albicans growth were 320 and 640 μg/ml, respectively. The expression of fungal ERG6 at an mRNA level and ergosterol content of yeast cells were significantly decreased by both C. carvi EO (640 μg/ml) and FLC (2 μg/ml). The proteinase and phospholipase activities were also reduced in C. carvi EO by 49.82% and 53.26%, respectively, while they were inhibited in FLC-treated cultures by 27.72% and 34.67%, respectively. Furthermore, the CSH was inhibited in EO- and FLC-treated cultures by 12.75% and 20.80%, respectively. Conclusion: Our findings revealed that C. carvi EO can be considered a potential natural compound in the development of an efficient antifungal agent against C. albicans.

Objective Precise identification of dermatophyte species significantly improves treatment and controls measures of dermatophytosis in human and animals. This study was designed to evaluate molecular tools effectiveness of the gene sequencing and DNA-based fragment polymorphism analysis for accurate identification and differentiation of closely- related dermatophyte species isolated from clinical cases of dermatophytosis and their antifungal susceptibility to the current antifungal agents. Materials And Methods In this experimental study, a total of 95 skin samples were inoculated into mycobiotic agar for two weeks at 28˚C. Morphological characteristics of the isolated dermatophytes were evaluated. DNA was extracted from the fungal culture for amplification of topoisomerase II gene fragments and polymerase chain reaction (PCR) products were digested by Hinf I enzyme. Internal transcribed spacer (ITS) rDNA and TEF-1α regions of the all isolates were amplified using the primers of ITS1/4 and EF-DermF/EF-DermR, respectively. Results Based on the morphological criteria, 24, 24, 24 and 23 isolates were identified as T. rubrum, T. interdigitale, T. tonsurans and E. floccosum, respectively. PCR-restriction fragment length polymorphism (RFLP) results provided identification pattern of the isolates for T. rubrum (19 isolates), T. tonsurans (28 isolates), T. interdigitale (26 isolates) and E. floccosum (22 isolates). Concatenated dataset results were similar in PCR-RFLP, except six T. interdigitale isolates belonging to T. mentagrophytes. Conclusion Our results clearly indicated that conventional morphology and PCR-RFLP were not able to precisely identify all dermatophyte species and differentiation of closely related species like T. interdigitale and T. mentagrophytes, while ITS rDNA and TEF-1α gene sequence analyses provided accurate identification of all isolates at the genus and species level.

Background and Purpose: The present study was targeted toward investigating the effects of eugenol on Cryptococcus neoformans biological activity and Cxt1p gene expression. Materials and Methods: For the purpose of the study, the growth, urease, synergism activity, and disk diffusion of C. neoformans were assessed in eugenol-treated culture. The minimum inhibitory concentration (MIC) was determined by the Clinical and Laboratory Standards Institute M27-A3 method at a concentration range of 0.062-2 mg/mL. Subsequently, the expression of Cxt1p genes was studied at the MIC50 concentration of eugenol using real-time polymerase chain reaction. Results: The obtained results showed that eugenol at the concentrations of 125 and 500 μg/mL resulted in 50% and 100% growth inhibition in C. neoformans, respectively. In terms of urease activity, the results showed that the addition of MIC50 of eugenol and fluconazole to urea medium reduced urease activity in C. neoformans. In the culture treated with eugenol, the inhibition zone of antifungal drugs, namely amphotericin B, itraconazole, and fluconazole, was increased to 36±0.002, 22±0.001, and 12±0.002 mm, respectively. The expression levels of Cxt1p in the eugenol-treated, fluconazole-treated, and non-treated samples were estimated at 46%, 58%, and 100%, respectively. Conclusion: The findings of the current study revealed that eugenol could cause C. neoformans growth inhibition and reduce Cxt1p expression in this species. As the results indicated, the susceptibility of C. neoformans to fluconazole was increased when combined with eugenol.

Introduction: This study aimed to evaluate antifungal activity and cytotoxicity of two new nitroheteroaryl-1,3,4-thiadiazole derivatives containing N-methoxyethyl (9) and N-benzyl (10) moiety against Aspergillus fumigatus with a special focus on their mechanism of action at cellular level. Materials and Methods: The fungal growth rate was evaluated by microbioassay technique. Ergosterol content of the cell membrane was determined, and morphological changes of fungal compartments were assessed by electron microscopy. Cytotoxicity against Vero and Hep2 cell lines was determined by cell culture technique. Results: Based on the data obtained, compound 9 showed 31.90-100% inhibition at concentrations of 25-800 μM, while compound 10 presented 24.26-100% inhibition at higher concentrations (25-3200 μM). Compounds 9 and 10 showed minimum fungicidal concentration of 500 and 8000 μM and IC50 values of 43.39 and 1008.67 μM, respectively. Ergosterol content was not meaningfully affected by both compounds. Electron microscopy showed deformation of A. fumigatus hyphae, depletion of hyphae contents, and destruction of cell membrane and membranous organelles in the fungus exposed to both compounds. Compounds 9 and 10 had no obvious cytotoxicity against Vero and Hep2 cell lines in vitro. Conclusion: Taken together, our results showed that both compounds, especially compound 9, could be considered as potential candidates for developing antifungal drugs against A. fumigatus as the main etiologic agent of life-threatening invasive aspergillosis.

Due to the increasing rate of invasive fungal infections and emerging antifungal resistance, development of novel antifungal drugs has been an urgent necessity. Antifungal peptides (AFPs) have recently attracted attention due to their unique ability to evade drug-resistant fungal pathogens. In this study, a novel AFP, Cc-AFP1, with a molecular weight of ~3.759 kDa, was isolated from Carum carvi L., purified by ammonium sulfate precipitation and reversed-phase HPLC and finally identified by sequence analysis using Edman degradation. Peptide sequence analysis revealed a fragment of 36 amino acid residues as RVCFRPVAPYLGVGVSGAVRDQIGVKLGSVYKGPRG for Cc-AFP1 with a net charge of +5 and a hydrophobicity ratio of 38%. The antifungal activity of Cc-AFP1 was confirmed against Aspergillus species with MIC values in the range of 8–16 µg/ml. Cc-AFP1 had less than 5% hemolytic activity at 8–16 µg/ml on human red blood cells with no obvious cytotoxicity against the HEK293 cell line. Stability analysis showed that the activity of Cc-AFP1 was maintained at different temperatures (20°C to 80°C) and pH (8 to 10). The results of a propidium iodide uptake and transmission electron microscopy showed that the antifungal activity of Cc-AFP1 could be attributed to alteration in the fungal cell membrane permeability. Taken together, these results indicate that Cc-AFP1 may be an attractive molecule to develop as a novel antifungal agent combating fungal infections cause by Aspergillus species.

BackgroundDermatophytes are a homogeneous group of species with low genetic diversity, and there are still many uncertainties about the boundaries among species.ObjectivesAiming at clarifying the relationships among species in the genus and introducing suitable genes for multilocus sequence typing (MLST), a new MLST scheme approach was developed to characterize the major pathogenic dermatophytes.MethodsWe performed maximum parsimony (MP), MrBayes, RAxML, and eBURST analyses, based on the MLST scheme to scrutinize the evolution within 95 clinical isolates and four reference strains belonging to the four major dermatophytes species. Then, the discriminatory power, pairwise genetic distances, ratio dN/dS, and sequence types (STs) of these isolates were determined. Also, to study taxonomy, sequences of the internal transcribed spacer (ITS), Beta-tubulin (BT2), and translation elongation factor 1-α (TEF-1α) genes of other dermatophytes species available in the GenBank were analyzed.ResultsFindings of the present study indicated that three genes: BT2, ITS, and TEF−1α, which showed the greatest diversity among dermatophyte species, were suitable for MLST. The most prevalent STs were seen among the species of Trichophyton interdigitale. Also, two new genotypes, i.e., XXVII and XXVIII, were introduced for T. interdigitale and Trichophyton mentagrophytes. The least informative sites were found in Epidermophyton floccosum, Trichophyton rubrum, and T. mentagrophytes, while the most informative sites were observed in T. interdigitale. Furthermore, the most informative locus was TEF-1α. The phylogenetic tree, constructed by the combination of the three genes, shows a new topological pattern that confirms the derivation of the anthropophilic and zoophilic genera from the geophilic genus. Also, the phylogenetic analyses and pairwise distances of the combination of the three loci showed that Trichophyton tonsurans and Trichophyton equinum were a species complex, where T. equinum is derived from T. tonsurans.ConclusionsResults of this study showed that MLST is very effective in determining the boundaries between species and taxonomy. Considering that there is no database for MLST dermatophytes, further studies are needed to determine the suitable genes for MLST. Also, the determination of STs in epidemiological studies and raising epidemiological information are helpful. This study was a new starting point to determine the ST and a foundation for a dermatophyte MLST database.

Fungal species resistant to current antifungal agents are considered as a serious threat to human health, the dilemma that has dragged attentions toward other sources of antifungals such as antimicrobial peptides (AMPs). In order to improve biological activity of a recently described antifungal peptide MCh-AMP1 from Matricaria chamomilla flowers, MCh-AMP1dimer (DiMCh-AMP1), containing 61 amino acid residues connected by flexible linker (GPDGSGPDESGPDES), was designed and expressed in Escherichia coli, and its structure was analyzed using bioinformatics tools. DiMCh-AMP1 synthetic gene was cloned into pET-28a expression vector, which was then used to transform E. coli BL21 (DE3) strain. His-tag purification was achieved using metal-chelate affinity chromatography. Because there is no methionine residue in the DiMCh-AMP1 sequence, cyanogen bromide was successfully used to separate the target product from the tag. Reverse-phase high-performance liquid chromatography was used as the final step of purification. Results showed that recombinant peptide was produced in considerable amounts (0.9 mg/L) with improved antifungal activity toward both yeasts and molds compared to its monomeric counterpart. The minimum inhibition concentration and minimum fungicidal concentration values of DiMCh-AMP1 against Candida and Aspergillus species were reported in the range of 1.67–6.66 μM and 3.33–26.64 μM, respectively. Our results showed that while antifungal activity of dimerized peptide was improved considerably, its cytotoxicity was decreased, implying that DiMCh-AMP1 could be a potential candidate to design an effective antifungal agent against pathogenic yeasts and molds.

Over the past decades, the incidence of life-threatening fungal infections has increased dramatically in particular among patients with hampered immune function. Fungal infections cause around 1.5 million deaths annually, superior to malaria and tuberculosis. With respect to high toxicity, narrow spectrum of activity and drug resistance to current antifungals, there is an urgent need to discover novel leads from molecules of natural origin especially those derived from plants and microorganisms for antifungal drug discovery. Among antifungal drugs introduced into the clinic, those affecting ergosterol biosynthesis are still superior to other classes and the vital role of ergosterol in fungal growth and development. This review highlights current knowledge about available antifungal agents and further issues on antifungal drug discovery from compounds of natural origin which affect ergosterol biosynthesis. Special attention is made to the fungal sterol C24-methyltransferase (SMT), a crucial enzyme in ergosterol biosynthesis pathway as a novel target for rational drug design.

Background and Aims: Cryopreservation is one of the best long time preservation methods for fungi and using suitable cryoprotectants can increase the success of cryopreservation process. The aim of this study was to examine the effect of various cryoprotectants to protect of Candida glabrata in freezing conditions. Materials and Methods: In this study, 50 clinical isolates of C. glabrata were stored at low temperature within three common cryoprotectants including; glycerol 10% and 40%, glucose 4% and DMSO 10%. After completing the preservation period, samples recovery rate was evaluated after two years using descriptive statistics and chi-square test. Results: In this study, 4% glucose with the ability to keep 100% of the samples, was determined as the best cryoprotectant for C. glabrata. Also, 40% glycerol and 10% DMSO with 94% success in retrieving samples, were comparable to each other and had a fairly good protective characteristic. While the 10% glycerol showed the lowest protective effect on C. glabrata. Recovery of samples cultured in SDA slant at 4ºC with a total of 32 (64%) positive cultures, showed a significant difference compared to preservation in freezing conditions. Also, the sample recovery in SDA had priority over SDB and yielded better results. Conclusions: There was a significant difference between the cryoprotectants and by selecting a suitable cryoprotectant and appropriate concentration, survival of microorganisms can be increased in freezing conditions. Generally, assessment of a valid and reliable method for long-term preservation of a fungal strain should be associated with further studies on assessing changes in infectivity and pathogenicity, sporulation power and other features of microorganisms.

Pneumocystis jirovecii is an opportunistic fungus, which causes Pneumocystis pneumonia (PJP) in immunocompromised, COPD and TB positive patients with a high rate of colonization, morbidity and mortality. Dihydropteroate synthase (DHPS) gene mutations are well-reported in PJP. Although sulfa prophylaxis generally is associated with DHPS mutant infection, Multiple molecular techniques applied for detect sulfa resistance single-base mutation. Conformation sensitive gel electrophoresis (CSGE) is a rapid screening method for detection of DNA sequence variation, specifically single-base changes or small insertions and deletions. The current study is investigate on the DHPS sequence single-base dislocation among strains isolated from Iranian TB positive co-infected with PJP in association to increased levels of serum Lactate Dehydrogenase. Through high serum lactate dehydrogenase (LDH) levels have been associated with established Pneumocystis pneumonia. We investigated the DHPS mismatches in five P. jirovecii isolated of TB infected patients. For genetic identification of Pneumocystis isolates and detection of intraspecific variation, we developed a method for heteroduplex analysis. Our utilizing fragments was the DHPS gene regions, amplified by PCR method with specific primers. Serum LDH indicator was analysed for lung acute damages. In our results, at least 4 suspected isolates show more slowly migrating bands containing single heteroduplexes, reveal single-base mislocation in studied sequences. LDH level Peak was higher (p

Background and Objectives: Cutaneous candidiasis is a multipicture fungal infection caused by members of the genus Candida which is considered as a public health problem all over the world with urgency of effective treatment and control. This study was performed to analyze the clinical epidemiology and molecular aspects of cutaneous candidiasis in Tehran-Iran in relation to antifungal susceptibility and virulence factors of etiologic Candida species. Materials and Methods: Candida species were isolated from skin (27.3%) and nail scrapings (72.7%) of suspected patients and identified by ITS sequencing. Phylogeny of the isolates was evaluated using multilocus sequence typing (MLST) and antifungal susceptibility and virulence factors of the isolates were determined in relation to clinical presentation. Results: Candida albicans was the most prevalent species (39.8%), followed by C. parapsilosis (32.9%), C. orthopsilosis (10.4%), C. tropicalis (7.9%), C. glabrata and C. guilliermondii, each (4.5%). Molecular typing of 35 C. albicans isolates by MLST revealed 28 novel sequence types with 11 singletons with 80.0% new diploid sequence types (DSTs). Majority of the isolates were susceptible to amphotericin B (91.5%), followed by posaconazole (90.3%), fluconazole (84.3%), itraconazole (74.1%), caspofungin (53.6%), and voriconazole (26.8%). Biofilm formation, yeast-to-hyphae transformation and phospholipase activity were reported species-dependent. Conclusion: Our results demonstrated clinical epidemiology of various Candida species from cutaneous candidiasis distributed in new molecular types with increasing importance of drug resistant of non-albicans Candida species. Our results showed that drug susceptibility and genetic variability of Candida species may be attributed to their clinical features and source of isolation.

Background and Objectives: Dermatophytes possess a wide array of virulence factors and various antifungal susceptibility patterns which influence their pathogenesis in humans and animals. The aim of this study was to evaluate antifungal suscep- tibility and keratinase and proteinase activity of 49 dermatophyte strains from the genera Microsporum, Trichophyton and Epidermophyton which were isolated from human cases of dermatophytosis. Materials and Methods: Forty-nine dermatophyte strains isolated from clinical samples were cultured on general and spe- cific culture media. Keratinase and proteinase activity was screened on solid mineral media and confirmed in liquid cultures. Drug susceptibility toward azoles (fluconazole, ketoconazole and itraconazole), griseofulvin and terbinafine was evaluated using disk diffusion method on Mueller-Hinton agar and minimum inhibitory concentrations (MICs) were determined using microbroth dilution assay according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Our results indicated that clinically isolated dermatophytes from 7 major species produced keratinase and protein- ase at different extents. The mean keratinase and proteinase activity was reported as 6.69 ± 0.31 (U/ml) and 2.10 ± 0.22 (U/ ml) respectively. Disk diffusion and microbroth dilution (MIC) results of antifungal susceptibility testing showed that ke- toconazole was the most effective drug against Epidermophyton floccosum and Trichophyton mentagrophytes, itraconazole against T. rubrum and E. floccosum, and griseofulvin and terbinafine against Trichophyton verrucosum. Our results showed that all dermatophyte isolates were resistant to fluconazole. Overall, ketoconazole and itraconazole were the most effective drugs for all dermatophyte species tested. Conclusion: Our results showed that antifungal susceptibility testing is an urgent need to select drugs of choice for treatment of different types of dermatophytosis and further indicated the importance of keratinase and proteinase in pathogenesis of dermatophyte species.

Background and Objectives: The genus Malassezia contains an expanding list of lipophilic yeasts involve in the etiology of various superficial fungal infections. Pityriasis versicolor (PV) is the most prevalent Malassezia-related infection distributed worldwide. In the present study, clinical and epidemiological features of the genus Malassezia are discussed with special focus on PV in Iran. Materials and Methods: During June 2012 to April 2013, among 713 confirmed cases of fungal infections, 68 (9.5%) were diagnosed as PV by positive direct microscopy results in 20% potassium hydroxide (KOH) preparation of skin scrapings. All the specimens were cultured on modified Dixon agar and incubated at 32ºC for 10 days. Identification of the isolated yeasts was carried out based on macro- and microscopic morphology, catalase test, utilization of Tweens, polyethoxylated castor oil (EL slant), and hydrolysis of esculin and utilization of Tween-60 (TE slant). Results: Out of 68 skin scrapings, 55 (80.9%) yielded yeast colonies on mDixon’s agar which were finally identified as M. globosa (36.36%), M. pachydermatis (29.08%), M. furfur (23.65%), M. slooffiae (7.28%) and M. obtusa (3.64%). Conclusion: Results of the present study further indicate clinico-epidemiological importance of the genus Malassezia with growing importance of M. pachydermatis as a major species involve in the etiology of pityriasis versicolor. These findings are of major concern in management of Malassezia-related diseases.

Background and Objectives: Cattle dermatophytosis (syn. cattle ringworm), an important skin infection, has received major consideration not only for economical losses in the animal breeding industry but also in regards to its zoonotic transmission to humans. For effective control measures, it is important to determine the disease prevalence in cattle herds. Materials and Methods: To determine ringworm prevalence, a total number of 3,540 cattle in different age groups at three major farms of Mashhad including Kenebist (KB) in the east, Mazraeh Nemooneh (MN) in the south, and Moghoofat Malek (MM) in the north-east were examined. Skin scrapings were prepared from all animals clinically suspected to have dermato- phytosis. The samples were examined microscopically for fungal elements (hyphae and/or arthrospores) by adding potassium hydroxide (KOH) to samples. To isolate the etiologic dermatophytes, all samples were cultured on selective agar for patho- genic fungi medium for 4 weeks at room temperature. Results: Among 684 suspected cases (19.3%) selected from a total number of 3,540 cattle based on clinical signs, 604 cases (88.3%) were KOH positive in direct microscopy, while 490 cases (71.6%) were culture positive on selective agar for patho- genic fungi (SAPF) medium. The most frequent dermatophyte isolated was Trichophyton verrucosum (495 isolates accounting for 99% of total isolates) which was obtained from all culture positive cases except five cases (1.0%) infected with another dermatophyte named Trichophyton mentagrophytes. Conclusion: This work is the first comprehensive study on cattle ringworm in Iran. With respect to the high prevalence of cattle ringworm, particularly in young animals reported in the present study, effective management programs such as vaccina- tion and improved hygiene are necessary for disease control in the herds.