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Candida parapsilosis is one of the main emerging non-Candida albicans species leading to superficial and systemic fungal infections in humans. Candida has the ability to produce biofilms associated with pathogenesis. The aim of the study was to estimate biofilm-producing ability of clinical isolates of C. parapsilosis sp. complex.Clinical samples of C. parapsilosis complex have been analyzed. Crystal violet (CV) staining and tetrazolium reduction assay (MTT) have been used to analyze the clinical isolates ability to produce biofilms. The biofilm's structural characteristics have been assessed by using scanning electron microscopy.All 65 isolates were able to form biofilm. In addition, no significant difference was found between biofilm quantification based on two assays at different time intervals (24h, 48h, 72h, 96h) (P0.05), with the exception of Candida orthopsilosis, which exhibited higher metabolic activity at 24h time point (P0.05). Moreover, metabolic activity and production of biofilm biomass demonstrated statistically significant correlation (r=0.685, P0.01). According to microscopic observations, the investigated clinical strains formed the similar surface topography with the slight differences in morphology; in addition, there was no statistically significant difference between efficiency of two assays to quantify biofilm.It was shown that, similar to C. parapsilosissensu stricto, two cryptic identified species (C. orthopsilosis and Candida metapsilosis) obtained from different clinical samples, were biofilm producers, while C. parapsilosissensu stricto exhibited the highest biofilm production.

Background and Purpose: Aspergillus flavus 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. Materials and Methods: For the purpose of the study, 10 strains of A. flavus 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 mdr1 and cyp51C genes were studied in the strains showing drug resistance. Results: 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 mdr1 and cyp51c genes in these strains showed a decrease and increase in adjacency with diazinon and benomyl, respectively. Conclusion: 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 A. flavus by altering the expression of genes involved in drug resistance.

Background and purpose Aflatoxin is known as one of the most important mycotoxins threatening human life. This toxin is produced by Aspergillus 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, aflR gene expression, and toxin production. Materials and methods For the purpose of the study, first, Aspergillus parasiticus 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 aflR gene expression level was evaluated by means of real-time polymerase chain reaction at different concentrations of vitamin C. Results 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 B1, B2, G1, and G2, 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 aflR 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. Conclusion This study showed that vitamin C, as a human-compatible compound, could be considered a good agent to protect agricultural products against fungal aflatoxin.

A total of 105 independent Candida albicans strains isolated from patients in Iran were investigated. According to CLSI documents M27-A3 and M27-S4, the 24 h geometric mean MICs of caspofungin, itraconazole, and fluconazole were 0.27, 3.19, and 11.91 μg/ml, respectively. Microsatellites analysis of CEF3, CAIII, LOC4 Loci identified 93 different allelic genotypes clustered apart into six different clades. Antifungal susceptibility was not linked with the source of isolation and the corresponding genotype of C. albicans strains.

Purpose: 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. Methods: 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. Results: Cyp51A siRNA induced 9, 22, 33, 40-fold reductions in cyp51A mRNA expres­sion 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. Conclusion: 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.

Objective Aspergillus sections Terrei and Nidulantes are the less common causes of invasive aspergillosis and pulmonary aspergillosis (PA) in immunocompromised patients when compared to A. fumigatus and A. flavus. Identifying these fungi as the infectious agent is crucial because of the resistance to amphotericin B (AMB) and increased lethality. The aim of this study was to identify the molecular status, evaluate the genetic diversity and examine the antifungal susceptibility profile of the uncommon Aspergillus species. Forty-five uncommon Aspergillus species were identified based on the microscopic and macroscopic criteria. Then, the molecular identification was performed using the sequencing beta tubulin (benA) gene. In vitro antifungal susceptibility to amphotericin B (AMB), itraconazole (ITC), ravuconazole (RAV), voriconazole (VRC), caspofungin (CFG) isavuconazole (ISA) and posaconazole (POS) test was performed according to the CLSI M38-A2 guidelines. Results A. terreus was the most species detected, followed by A. nidulans, A. latus, A. ochraceus, and A. citrinoterreus, respectively. The analysis of the benA gene showed the presence of 12 distinct genotypes among the A. terreus isolates. The other species did not show any intraspecies variation. CFG exhibited the lowest MEC50/MIC50 (0.007 μg/mL), followed by POS (0.125 μg/mL), VRC, ITC, ISA (0.25 μg/mL), RAV (0.5 μg/mL), and AMB (8 μg/mL). Among all the isolates, only 15.5% (7/45) were susceptible to AMB. Conclusion Antifungal susceptibility pattern of the uncommon Aspergillus species is useful to improve patient management and increase knowledge concerning the local epidemiology. Moreover, this information is necessary when an outbreak dealing with drug-resistant infections occurs.

Objective Dermatophytes are a group of keratinophilic fungi that invade and infect the keratinized tissues and cause dermatophytosis. We investigated effectiveness of novel triazole (luliconazole and lanaconazole) in comparison with available antifungal agents against dermatophyte species isolated from patients with tinea pedis. Material and methods A total of 60 dermatophytes species were isolated from the patients with tinea pedis. Identification of species was done by DNA sequencing of the ITS1-5.8S rDNA-ITS2 rDNA region. In vitro antifungal susceptibility testing with luliconazole and lanaconazole and available antifungal agent was done in accordance with the Clinical and Laboratory Standards Institute, M38-A2 document. Results In all investigated isolates, luliconazole had the lowest minimum inhibitory concentration (MIC) (MIC range = 0.0005–0.004 μg/mL), while fluconazole (MIC range = 0.4–64 μg/mL) had the highest MICs. Geometric mean MIC was the lowest for luliconazole (0.0008 μg/mL), followed by lanoconazole (0.003 μg/mL), terbinafine (0.019 μg/mL), itraconazole (0.085 μg/mL), ketoconazole (0.089 μg/mL), econazole (0.097 μg/mL), griseofulvin (0.351 μg/mL), voriconazole (0.583 μg/mL) and fluconazole (11.58 μg/mL). Conclusion The novel triazoles showed potent activity against dermatophytes and promising candidates for the treatment of tinea pedis caused by Trichophyton and Epidermophyton species. However, further studies are warranted to determine the clinical implications of these investigations.

Background and Purpose: Formation of pseudohyphae is considered a virulence factor in Candida species. Generally, Candida glabrata grows as budding yeast cells; however, reports illustrated that C. glabrata could form pseudohyphal cells in response to some stimuli. In this study, we provided insight into the ability of C. glabrata in forming pseudohyphal cells under different levels of carbon dioxide (CO2). Materials and Methods: Candida glabrata reference strain (ATCC 90030) was used in this study. Yeast samples were cultured on Sabouraud dextrose broth (SDB) medium and incubated under 3%, 5%, and 10% CO2 levels for 24, 48 and72h.Control cultures were prepared without CO2 pressure for three days. The possibility of pseudohyphae and mycelium formation in C. glabrata was investigated. Results: The results of this study revealed that the most branching filament-like cells were obtained at high CO2 pressure (10%) after 72 h. After three days of low CO2 pressure (3%), only yeast and budding cells were observed without any pseudohyphae formation. Conclusion: CO2 could act as a stimulus and induced formation of pseudohyphae in Candida glabrata yeast cells. &nbsp

Aspergillus flavus is the second leading cause of invasive and non-invasive aspergillosis, as well as the most common cause of fungal sinusitis, cutaneous infections, and endophthalmitis in tropical countries. Since resistance to antifungal agents has been observed in patients, susceptibility testing is helpful in defining the activity spectrum of antifungals and determining the appropriate drug for treatment. A collection of 199 clinical and environmental strains of Aspergillus flavus consisted of clinical (n=171) and environmental (n=28) were verified by DNA sequencing of the partial b-tubulin gene. MICs of amphotericin B, itraconazole, voriconazole, posaconazole, and MEC of caspofungin were determined in accordance with the Clinical and Laboratory Standards Institute M38-A2 document. Caspofungin, followed by posaconazole, exhibited the lowest minimum inhibitory concentrations (MIC). All isolates had caspofungin MEC90 (0.063μg/ml) lower than the epidemiologic cutoff values, and 3.5% of the isolates had amphotericin B MIC higher than the epidemiologic cutoff values. However, their clinical effectiveness in the treatment of A. flavus infection remains to be determined.

We employed an endpoint genotyping method to update the prevalence rate of positivity for the TR 34 /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 TR 46 /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 TR 34 /L98H mutation in the cyp51A genes of six isolates. No isolates harboring the TR 46 /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 TR 34 /L98H controls. In conclusion, there was not a significant increase in the prevalence of azole-resistant A. fumigatus isolates harboring the TR 34 /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 TR 34 /L98H- and TR 46 /Y121F/T289A-positive isolates and can easily be incorporated into clinical mycology algorithms.

Objective Candida parapsilosis species complex, an important set of non-albicans Candida species, is known to cause candidaemia particularly in neonates and infants. However, the incidence has increased in recent years, owing to higher numbers of at individuals at risk for these infections. Our objective was to evaluate the in vitro susceptibility of clinical isolates of C. parapsilosis complex isolates from Iran to seven antifungal drugs. Material and methods One hundred-one clinical isolates of C. parapsilosis species complex cultured from humans were included. Species identification had been previously confirmed by combined phenotypic characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry-based assay and reconfirmed by DNA sequence analysis of the ITS rDNA region and D1/D2 gene. Minimum inhibitory concentrations (MICs) for amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, micafungin and anidulafungin were determined against well-characterized isolates by broth microdilution susceptibility testing according to the CLSI M27-A3 guideline. Results Species identifications were performed on 101 isolates, of which 88 (87.2%) C. parapsilosis sensu stricto and 13 (12.8%) C. orthopsilosis. Amphotericin B and posaconazole were the most active drugs with 100% of isolates being wild-type (WT). Voriconazole and micafungin, 99% of isolates were WT. The low activity was recorded for fluconazole and itraconazole with 93.1% and 89.1% of isolates being WT, respectively. At the species level, all Candida parapsilosis sensu stricto isolates were WT to amphotericin B and posaconazole and all Candida orthopsilosis isolates were WT to amphotericin B, voriconazole, posaconazole, anidulafungin and micafungin. In contrast, the highest rate of non-WT was observed in C. orthopsilosis to itraconazole (4 of 13, 30.8%). Conclusions Although almost all of the tested drugs demonstrated potent activity against C. parapsilosis species complex, it seems that more especially C. orthopsilosis isolates had decreased susceptibility to itraconazole. Further studies are needed to determine how these findings may switch into in vivo efficacy.

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.

Background and Purpose: The Fusarium species are among the most important fungi in the medical, veterinary and agricultural fields. Materials and Methods: 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 Fusarium species. Using the designed nucleotide primers from rDNA of Fusarium 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. Results: Through the selected enzymes including; HhaI, MspI, TaqI and FaqI, the mentioned Fusarium species have been divided into 33 groups. The first three enzymes were able to classify Fusarium species into 23 groups of which 19 groups included one member, one group included two members and three groups included three members of the Fusarium species. This study also revealed the possibility in the identification of F. semitectum, F. solani complex, F. pseudograminearum, F. nisikadoi, F. coeruleum and F. acuminatum species by one unique enzyme. In addition, our study indicated the ability of the differentiation of F. Compactum from F. equiseti. Conclusion: 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 Fusarium species successfully.

In this study we attempted to modify the PCR-RFLP method using restriction enzyme MwoI for the identification of medically important Aspergillus species. Our subjects included nine standard Aspergillus species and 205 Aspergillus isolates of approved hospital acquired infections and hospital indoor sources. First of all, Aspergillus isolates were identified in the level of species by using morphologic method. A twenty four hours culture was performed for each isolates to harvest Aspergillus mycelia and then genomic DNA was extracted using Phenol-Chloroform method. PCR-RFLP using single restriction enzyme MwoI was performed in ITS regions of rDNA gene. The electrophoresis data were analyzed and compared with those of morphologic identifications. Total of 205 Aspergillus isolates included 153 (75%) environmental and 52 (25%) clinical isolates. A. flavus was the most frequently isolate in our study (55%), followed by A. niger 65(31.7%), A. fumigatus 18(8.7%), A. nidulans and A. parasiticus 2(1% each). MwoI enabled us to discriminate eight medically important Aspergillus species including A. fumigatus, A. niger, A. flavus as the most common isolated species. PCR-RFLP method using the restriction enzyme MwoI is a rapid and reliable test for identification of at least the most medically important Aspergillus species.

Item does not contain fulltext Abstract Objective: The purpose of this study was to evaluate the effects of low-level laser irradiation (LLLI) on the in vitro growth characteristics and in vivo pathogenicity of Candida albicans in a murine model in the absence of a photosensitizer. Background data: C. albicans is an opportunistic commensal organism that causes a wide variety of diseases in human beings, ranging from superficial infections to life-threatening invasive candidiasis. The incidence of C. albicans infection is increasing, because of the greater frequency of acquired immunodeficiency conditions. A high recurrence rate has been reported for vulvovaginal and oral candidiasis, despite the best available treatments. Therefore, the search for new treatment modalities seems quite rational. Methods: Candida culture plates were exposed to common clinical energies of LLLI: 3, 5, 10, and 20 J at 685 nm (BTL Laser 5000, Medicinos Projektai, Czech Republic, Prague, max power output 50 mW) and 3, 5, 10, 30, and 50 J at 830 nm (BTL Laser 5000, Medicinos Projektai, Czech Republic, Prague, max power output 400 mW). Results: Following LLLI with energies >10 J at both 685 and 830 nm wavelengths, statistically significant effects were observed in vitro on the turbidimetric growth kinetics of C. albicans and in vivo on the survival rate of infected mice (p value

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.

Background: Voriconazole Resistance (VRC-R) in Aspergillus flavus isolates impacts the management of aspergillosis, since azoles are the first choice for prophylaxis and therapy. However, to the best of our knowledge, the mechanisms underlying voriconazole resistance are poorly understood. Objectives: The present study was designed to evaluate mRNA expression levels of cyp51A and mdr1 genes in voriconazole resistant A. flavus by a Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technique. Materials and Methods: Five A. flavus isolates with resistance to VRC were examined by a RT-PCR approach. Results: Four out of five isolates revealed cyp51A and mdr1 mRNA overexpression. Interestingly, the isolate, which was negative for cyp51A and mdr1 mRNA expression showed a high voriconazole Minimum Inhibitory Concentration (MIC). Furthermore, a computational-based analysis predicted that voriconazole resistance could be mediated through cooperation with a network protein interaction. Conclusions: Our experimental and in silico findings may provide new insight in the complex molecular pathways of drug resistance and also could assist design an efficient therapeutic strategy for aspergillosis treatment.

Summary Objective Among filamentous fungal pathogens, Aspergillus spp. and zygomycetes account for highest rates of morbidity and mortality among immunocompromised patients. Recently developed antifungal drugs offer the potential to improve management and therapeutic outcomes of fungal infections. The aim of this study was to analyse the in vitro activities of voriconazole, itraconazole, amphotericin B and caspofungin against clinical isolates of Aspergillus spp. and Rhizopus oryzae. Material and methods The in vitro antifungal susceptibility of 54 isolates belonging to different clinical isolates of Aspergillus spp. and R. oryzae was tested for four antifungal agents using a microdilution reference method (CLSI, M38-A2). All isolates were identified by typical colony and microscopic characteristics, and also characterized by molecular methods. Results Caspofungin (MEC range: 0.008–0.25 and MEC50: 0.0023 μg/mL) was the most active drug in vitro against Aspergillus spp., followed by voriconazole (MIC range: 0.031–8 and MIC50: 0.5 μg/mL), itraconazole (MIC range: 0.031–16 and MIC50: 0.25 μg/mL), and amphotericin B (MIC range: 0.125–4 and MIC50: 0.5 μg/mL), in order of decreasing activity. The caspofungin, voriconazole, and itraconazole demonstrated poor in vitro activity against R. oryzae isolates evaluated, followed by amphotericin B. Conclusion This study demonstrates that caspofungin had good antifungal activity and azole agents had better activity than amphotericin B against Aspergillus species. Although, azole drugs are considered ineffective against R. oryzae . This result is just from a small scale in vitro susceptibility study and we did not take other factors into consideration.

Background: The production and development of an effective fungicidal drug requires the identification of an essential fungal protein as a drug target. Aconitase (ACO) is a mitochondrial protein that plays a vital role in tricarboxylic acid (TCA) cycle and thus production of energy within the cell. Objectives: The current study aimed to sequence Candida krusei ACO gene and determine any amino acid residue differences between human and fungal aconitases to obtain selective inhibition. Materials and Methods: Candida krusei (ATCC: 6258) aconitase gene was determined by 5’Rapid Amplification of cDNA Ends (RACE) method and degenerate Polymerase Chain Reaction (PCR) and analyzed using bioinformatics softwares. Results: One thousand-four hundred-nineteen nucleotide of C. krusei aconitase gene were clarified and submitted in Genbank as a partial sequence and then taxonomic location of C. krusei was determined by nucleotide and amino acid sequences of this gene. The comparison of nucleotide and amino acid sequences of Candida species ACO genes showed that C. krusei possessed characteristic sequences. No significant differences were observed between C. krusei and human aconitases within the active site amino acid residues. Conclusions: Results of the current study indicated that aconitase was not a suitable target to design new anti-fungal drugs that selectively block this enzyme.

Cryptosporidium has been recognized as an emerging zoonotic agent of intestinal cryptosporidiosis leading to diarrhea, malabsorption syndrome, and weight loss in AIDS patients. In the present case, oocysts of zoonotic Cryptosporidium parvum were detected in the sputum and stool samples of an AIDS patient with a 3-month history of intestinal cryptosporidiosis. The oocysts were detected by modified Ziehl-Neelsen staining; confirmation was achieved by nested polymerase chain reaction (PCR), targeting the most polymorphic region of the 18S rRNA gene. Genotyping was done by restriction endonuclease digestion of the PCR product. The zoonotic C. parvum bovine genotype was identified in both intestinal and respiratory samples. Treatment with both azithromycin and paromomycin resulted in improvement of both intestinal and respiratory symptoms, as well as the elimination of the parasite. This is the first report of the identification of Cryptosporidium sp. oocysts in the respiratory samples obtained from an AIDS patient in Iran. Pulmonary cryptosporidiosis should be considered whenever an AIDS patient with intestinal cryptosporidiosis develops respiratory symptoms.

Acanthamoeba keratitis cases have emerged in the recent years in Iran. In this case, an amoebic keratitis due to a mixed infection with Acanthamoeba and Vahlkampfia species is reported. Corneal scrapes, contact lenses and contact lens cases obtained from the patient were analysed and were positive for cysts of Acanthamoeba and Vahlkampfia genera. Genus-specific PCR was carried out for both genera, confirming the microscopic observations. To our knowledge, this is the first reported case of a possible mixed amoebic infection due to Acanthamoeba and Vahlkampfia and raises awareness within contact lens wearers in Iran.

Toxoplasms gondii was detected in sera and urine of acutely infected mice. Animals were inoculated intraperitoneally with tachyzoites of T. gondii, RH strain. Ten animals were killed every day from day 1 up to day 7 post infection. Urine and sera of animals were collected and stored at -20 degree C until use. PCR performed by B1 gene amplification. Toxoplasma was detected in sera from 3 days post infection and in urine from 5 days post infection. No parasite was detected in control group.

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis , C. parvum , and C. meleagridis , were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.

Difficulty in disrupting cysts of Giardia intestinalis, a cosmopolitan protozoan parasite, decreases the yield of DNA extracted and reduces the effectiveness of the polymerase chain reaction (PCR). To improve the detection of the Giardia Glutamate Dehydrogenase (gdh) gene, we re-evaluated the effects of deoxyribonucleic acid (DNA) extraction methods. Purified and concentrated cysts from 33 fecal samples were disrupted using conventional methods, and DNA extraction was conducted using two protocols: the QIAamp Stool Mini Kit and phenol/chloroform/isoamyl alcohol (PCI). PCR amplification was successful for 12 extracted DNA samples (36%) using PCI following a glass bead and freeze/thaw pretreatment and for all 33 samples (100%) using the QIAamp Stool Mini Kit following the aforementioned pretreatment. Consequently, the pretreatment of cysts with glass beads and freeze/thaw cycles followed by extraction of DNA with the QIAamp Stool Mini kit was the more effective protocol.