We employed an endpoint genotyping method to update the prevalence rate of positivity for the TR34/L98H mutation (a 34-bp tandem repeat mutation in the promoter region of the cyp51A gene in ...combination with a substitution at codon L98) and the TR46/Y121F/T289A mutation (a 46-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with substitutions at codons Y121 and T289) among clinical Aspergillus fumigatus isolates obtained from different regions of Iran over a recent 5-year period (2010 to 2014). The antifungal activities of itraconazole, voriconazole, and posaconazole against 172 clinical A. fumigatus isolates were investigated using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. For the isolates with an azole resistance phenotype, the cyp51A gene and its promoter were amplified and sequenced. In addition, using a LightCycler 480 real-time PCR system, a novel endpoint genotyping analysis method targeting single-nucleotide polymorphisms was evaluated to detect the L98H and Y121F mutations in the cyp51A gene of all isolates. Of the 172 A. fumigatus isolates tested, the MIC values of itraconazole (≥16 mg/liter) and voriconazole (>4 mg/liter) were high for 6 (3.5%). Quantitative analysis of single-nucleotide polymorphisms showed the TR34/L98H mutation in the cyp51A genes of six isolates. No isolates harboring the TR46/Y121F/T289A mutation were detected. DNA sequencing of the cyp51A gene confirmed the results of the novel endpoint genotyping method. By microsatellite typing, all of the azole-resistant isolates had genotypes different from those previously recovered from Iran and from the Dutch TR34/L98H controls. In conclusion, there was not a significant increase in the prevalence of azole-resistant A. fumigatus isolates harboring the TR34/L98H resistance mechanism among isolates recovered over a recent 5-year period (2010 to 2014) in Iran. A quantitative assay detecting a single-nucleotide polymorphism in the cyp51A gene of A. fumigatus is a reliable tool for the rapid screening and monitoring of TR34/L98H- and TR46/Y121F/T289A-positive isolates and can easily be incorporated into clinical mycology algorithms.
In this study, 15
Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of ...the Diagnostic Fragment 3 (DF3) of
Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these
Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of
Acanthamoeba through animal faeces in Iran.
Overall, the widespread distribution of pathogenic
Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.
Introducing the effect of RNAi in fungi to downregulate essential genes has made it a powerful tool to investigate gene function, with potential strategies for novel disease treatments. Thus, this ...study is an endeavor to delve into the silencing potentials of siRNA on cyp51A and MDR1 in voriconazole-resistant Aspergillus flavus as the target genes.
In this study, we designed three cyp51A-specific siRNAs and three MDR1-specific siRNAs and after the co-transfection of siRNA into Aspergillus flavus, using lipofectamine, we investigated the effect of different siRNA concentrations (5, 15, 25, 50nM) on cyp51A and MDR1 expressions by qRT-PCR. Finally, the Minimum Inhibitory Concentrations (MICs) of voriconazole for isolates were determined by broth dilution method.
Cyp51A siRNA induced 9, 22, 33, 40-fold reductions in cyp51A mRNA expres-sion in a voriconazole-resistant strain following the treatment of the cells with concentrations of 5, 15, 25, 50nM siRNA, respectively. Identically, the same procedure was applied to MDR1, even though it induced 2, 3, 4, 10-fold reductions. The results demonstrated a MIC for voriconazole in the untreated group (4µg per ml), when compared to the group treated with cyp51A-specific siRNA and MDR1-specific siRNA, both at concentrations of 25 and 50nM, yielding 2µg per ml and 1µg per ml when 25 nM was applied and 2µg per ml and 0.5µg per ml when the concentration doubled to 50 nM.
In this study, we suggested that siRNA-mediated specific inhibition of cyp51A and MDR1 genes play roles in voriconazole-resistant A.flavus strain and these could be apt target genes for inactivation. The current study promises a bright prospect for the treatment of invasive aspergillosis through the effective deployment of RNAi and gene therapy.
Aflatoxin is known as one of the most important mycotoxins threatening human life. This toxin is produced by
species, which is the common cause of agricultural product contamination. The use of ...organic compounds has been always considered for the inhibition of fungal growth and toxin production. Regarding this, the aim of the present study was to investigate the effect of vitamin C on the rate of fungal growth,
gene expression, and toxin production.
For the purpose of the study, first,
ATCC15517 was cultured in Sabouraud dextrose agar medium containing vitamin C at concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.1 mg/ml and temperature of 28°C for 72 h. Then, the amount of aflatoxin produced in the presence of vitamin C was measured through high performance liquid chromatography. Finally, by extracting the DNA of the cultured samples, the
gene expression level was evaluated by means of real-time polymerase chain reaction at different concentrations of vitamin C.
The results showed that mycelium deformation was started at the vitamin C concentration of 50 mg/ml, and that only fungal spores were observed at higher concentrations. The levels of total aflatoxin and its subsets, namely B
, B
, G
, and G
, in the presence of vitamin C were estimated as 5.9, 1.9, 0.2, 3.5, and 0.3 ppm, respectively. On the other hand, these values were respectively obtained as 207.5, 73.6, 4.5, 123.4, and 6 ppm in the absence of vitamin C. Measurement of the expression level of
genes showed that the level of gene expression decreased to 68% and up to 81% at the vitamin C concentrations of 25 and 50 mg/ml, respectively.
This study showed that vitamin C, as a human-compatible compound, could be considered a good agent to protect agricultural products against fungal aflatoxin.
is an important pathogen in immunodeficient patients. Due to the abundance of this fungus in nature, fungicides are commonly used to preserve and maintain agricultural products. Long-term exposure to ...these pesticides can lead to the induction of drug resistance in this fungus.
For the purpose of the study, 10 strains of
ATCC 204304 were cultured in benomyl and diazinon pesticides at the concentrations of 62.5, 125, 250.500, 750, 1000, 1500, 2000, and 2500 mg/L in nine steps. Morphological changes and resistance to voriconazole, itraconazole, and amphotericin B were evaluated at the end of each step. Subsequently, changes in the expression of
and
genes were studied in the strains showing drug resistance.
The results showed that during the nine stages of the adjacency of strains with benomyl and diazinon at different concentrations, resistance to voriconazole, itraconazole, and amphotericin B in these toxins increased by 30% and 10%, respectively. In addition, the microscopic examination of resistant strains revealed the absence of sporulation, and only mycelium was found. Macroscopically, the color of the colonies changed from green to white. Furthermore, the investigation of the expression of
and
genes in these strains showed a decrease and increase in adjacency with diazinon and benomyl, respectively.
As the findings indicated, exposure to agricultural pesticides can lead to the incidence of morphological changes and resistance to amphotericin B, itraconazole, and voriconazole in the sensitive species of
by altering the expression of genes involved in drug resistance.
Fascioliasis is a zoonotic disease caused by the liver fluke
. Drug resistance, high costs of treatment and economic losses in meat production have emerged the need of alternative control measures ...into consideration. The aim of this study was to evaluate the in vitro ovicidal activity of
fungus on
eggs.
isolated from the soil of natural environment was challenged on
eggs to observe the bio control effect of nematophagous fungi on trematode helminth eggs. The study was conducted in Tehran University of Medical Sciences, in 2015. Within 21 d of experiment, destructive effects exhibited on the eggshells were investigated using optical and Scanning Electron Microscopy.
The effective role of
on damaging the eggs of
was noticed.
This finding is promising for advantageous use of nematophagus fungi as a natural constituent in hyper endemic areas for certain helminthic infections like fascioliasis with diverse kinds of herbivores as egg passer hosts.
is the most common
species (sp.) isolated from fungal infections. Azole resistance in
species has been considerably increased in the last decades. Given the toxicity of the antimicrobial drugs, ...resistance to antifungal agents, and drug interactions, the identification of new antifungal agents seems essential. In this study, we assessed the antifungal effects of biogenic selenium nanoparticles on
and determined the expression of
and
genes.
Selenium nanoparticles were synthesized with
sp. MSH-1. The ultrastructure of selenium nanoparticles was evaluated with a transmission electron microscope. The antifungal susceptibility test was performed according to the modified Clinical and Laboratory Standards Institute M27-A3 standard protocol. The expression levels of the
and
genes were analyzed using the quantitative real-time polymerase chain reaction (PCR) assay.
The azole-resistant
and wild type
strains were inhibited by 100 and 70 µg/mL of selenium nanoparticle concentrations, respectively. The expression of
and
genes was significantly down-regulated in these selenium nanoparticle concentrations.
As the findings indicated, selenium nanoparticles had an appropriate antifungal activity against fluconazole-resistant and -susceptible
strains. Accordingly, these nanoparticles reduced the expression of
and
genes associated with azole resistance. Further studies are needed to investigate the synergistic effects of selenium nanoparticles using other antifungal drugs.
The
species are among the most important fungi in the medical, veterinary and agricultural fields.
In the present study, 172 strains of these fungi have been analyzed. The high molecular weight DNAs ...were extracted from 23 reference strains as well as from 149 isolated
species. Using the designed nucleotide primers from rDNA of
species, PCR analysis was performed for the amplification of ITS regions. Afterwards, the location of the effective endonuclease enzymes has been evaluated within approximately 930 bp of rDNA sequence.
Through the selected enzymes including;
I,
I,
I and
I, the mentioned
species have been divided into 33 groups. The first three enzymes were able to classify
species into 23 groups of which 19 groups included one member, one group included two members and three groups included three members of the
species. This study also revealed the possibility in the identification of
,
complex,
,
,
and
species by one unique enzyme. In addition, our study indicated the ability of the differentiation of
from
.
As Compared to previous studies with more endonuclease enzymes and with limited in identifications, the ITS-RFLP patterns reported here an attempted to evaluate most of the
species successfully.
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