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Acquired azole resistance in opportunistic fungi causes severe clinical problems in immunosuppressed individuals. This study investigated the molecular mechanisms of azole resistance in clinical isolates of Candida glabrata. Six unmatched strains were obtained from an epidemiological survey of candidiasis in immunocompromised hosts that included azole and amphotericin B susceptible and azole resistant clinical isolates. Candida glabrata CBS 138 was used as reference strain. Antifungal susceptibility testing of clinical isolates was evaluated using Clinical and Laboratory Standards Institute (CLSI) methods. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) technology, semi-quantitative RT-PCR, and sequencing were employed for identification of potential genes involved in azole resistance. Candida glabrata Candida drug resistance 1 (CgCDR1) and Candida glabrata Candida drug resistance 2 (CgCDR2) genes, which encode for multidrug transporters, were found to be upregulated in azole-resistant isolates (≥2-fold). Fatty acid activator 1 (FAA1) gene, belonging to Acyl-CoA synthetases, showed expression in resistant isolates ≥2-fold that of the susceptible isolates and the reference strain. This study revealed overexpression of the CgCDR1, CgCDR2, and FAA1 genes affecting biological pathways, small hydrophobic compounds transport, and lipid metabolism in the resistant clinical C.glabrata isolates.

Background: Aflatoxin contamination of food and feed stuff is a serious health problem and significant economic concerns. In the present study, the inhibitory effect of Candida parapsilosis IP1698 on mycelial growth and aflatoxin production in aflatoxigenic strains of Aspergillus species was investigated. Methods: Mycelial growth inhibitions of nine strains of aflatoxigenic and non-aflatoxigenic Aspergillus species in the presence of C. parapsilosis investigated by pour plate technique at different pH, temperature and time of incubation. Reduction of aflatoxin was evaluated in co-cultured fungi in yeast extract sucrose broth after seven days of incubation using HPLC method. The data were analyzed by SPSS 11.5. Results: The presence of the C. parapsilosis at different pH did not affect significantly the growth rate of Aspergillus isolates. On the other hand, temperature and time of incubation showed to be significantly effective when compared to controls without C. parapsilosis (P≤0.05). In aflatoxigenic strains, minimum percentage of reductions in total aflatoxin and B1, B2, G1, G2 fractions were 92.98, 92.54, 77.48, 54.54 and 72.22 and maximum percentage of reductions were 99.59, not detectable, 94.42, and not detectable in both G1 and G2, respectively. Conclusion: C. parapsilosis might employ as a good biocontrol agent against growth and aflatoxin production by aflatoxigenic Aspergillus species

Background: Dermatophytes are the most common causative agents of superficial mycoses. Species identification of these fungi is important from therapeutic and epidemiological point of wive. Traditional approaches for identification of dermatophytes at the species level, relying on macroscopic and microscopic features of the colonies, usually are time-consuming and unreliable in many circumstances. Recently a broad varieties of rapid and accurate DNA-based techniques were successfuly utilized for species delineation of dermatophytes. Methods: The ITS1-5.8S-ITS2 region of rDNA from various reference strains of dermatophyte species were amplified using the universal fungal primers ITS1 and ITS4.The PCR products were digested by a single restriction enzyme, MvaI. The enzyme was evaluated in both in silico and practical PCR-RFLP assay to find the exact differentiating restriction profiles for each species. To validate the standardized PCR-RFLP system, all tested strains were subjected to sequencing and sequence analysis. Results: The obtained RFLP patterns were specific for many species including T. interdigitale, T. rubrum, T. violaceum, M. persicolor, M. audouinii, M. nanum (A. obtusum) and E. floccosum but were similar for some closely related species such as M. canis / M. ferrugineum. Sequencing of the ITS1-5.8S-ITS2 fragment from all type strains affirmed the RFLP findings. Conclusion: It was practically revealed that the ITS-PCR followed by MvaI-RFLP is a useful and reliable schema for identification and differentiation of several pathogenic species and can be used for rapid screening of even closely related species of dermatophytes in clinical and epidemiological settings.

"nBackground: Malassezia are dimorphic, lipid-dependent yeasts, which are responsible for causing several cutaneous and sys­temic conditions. Although cyclophilins (CyPs) are highly conserved cytosolic proteins that catalyze the peptidyl-prolyl cis-trans isomerazation reaction before protein folding process, it has been suggestive of an allergen in a few numbers of fungi such as Aspergillus fumigatus and Malassezia species. Allergenic cyclophilins are IgE-binding components, which have been characterized in other species of Malassezia; and are considered as Mala s 6 in Malassezia sympodialis. In the pre­sent study we tried to identify the molecular characterization of cyclophilin gene in M. furfur."nMethods: Pairs of oligonucleotide primers were designed from highly conserved regions of the gene counterparts in other fungi. The primers were then applied to amplify the primer-specific DNA fragment. Afterward, PCR product fragments were sequenced to be used in further analysis."nResults: About 573 nucleotides, encoding a polypeptide of 190 amino acids, have been sequenced. Sequence comparison was performed in Gene Bank, both for the nucleotides and their deduced amino acid sequence. It revealed a significant homol­ogy with cyclophilin genes and proteins of other eukaryotic cells. The amino acid sequence of the encoded protein was about 86% identical to the sequence of cyclophilin protein from other fungi."nConclusion: The molecular characterization of cyclophilin gene may open the way to disclosure of the functional char­acteris­tics of cyclophilin and is a fundamental step for understanding the molecular basis of its pathogenesis in AEDS dis­ease.

"nBackground: Acute fulminant fungal sinusitis is a rapidly progressive disease with high mortality (50-80%) and occurring with increasing frequency in patients with acute leukemia. The purpose of the present study was early diagnosis of this le­thal infection."nMethods: In a cross-sectional study, 142 patients with hematological malignancies were studied immediately by initiation of chemotherapy to determine early clinical and radiological findings of invasive fungal sinusitis. This infection was con­firmed by pathological and mycological methods."nResults: Acute fulminant fungal sinusitis was diagnosed in 8 patients with acute leukemia. The most common isolated fungi was Aspergillus flavus (n= 5) followed by Aspergillus fumigatus (n= 2) and Rhizopus sp. (n= 1). Despite prompt surgical and medical therapy, the disease in our patients was very aggressive with a rapid clinical course and high mortality."nConclusion: The present report shows the poor prognosis of invasive fungal sinusitis in neutropenic patients; the necessity to take intensive preventive measures and the application of new diagnostic methods for early detection of fungal infection in these high risk patients.

In the framework of a survey on dermatophytoses, 14,619 clinical specimens taken from outpatients with symptoms suggestive of tinea and referred to a Medical Mycology laboratory in Tehran, Iran, were analyzed by direct microscopy and culture. In total, 777 dermatophyte strains recovered in culture were randomly identified by a formerly established RFLP analysis method based on the rDNA ITS regions. For confirmation of species identification, 160 isolates representing the likely entire species spectrum were subjected to ITS-sequencing. Infection was confirmed in 5,175 collected samples (35.4%) by direct microscopy and/or culture. Tinea pedis was the most prevalent type of infection (43.4%), followed by tinea unguium (21.3%), tinea cruris (20.7%), tinea corporis (9.4%), tinea manuum (4.2%), tinea capitis (0.8%) and tinea faciei (0.2%). Trichophyton interdigitale was the most common isolate (40.5%) followed by T. rubrum (34.75%), Epidermophyton floccosum (15.6%), Microsporum canis (3.9%), T. tonsurans (3.5 %) and M. gypseum (0.5%). Other species included M. ferrugineum, T. erinacei, T. violaceum, T. schoenleinii, and a very rare species T. eriotrephon (each one 0.25%). The two strains of T. eriotrephon isolated from tinea manuum and tinea faciei are the second and third reported cases worldwide. Application of DNA-based methods is an important aid in monitoring trends in dermatophytosis in the community.

"nBackground: Subtilisin -like proteases are the group of proteases including keratinases found in dermatophytes which de­graded keratin. Determination of the proteases activity of Trichophyton vanbreuseghemii isolates which were obtained from soil and clinical and soil isolates of Microsporum gypseum in Iran and characterization of their genome were aim of present study."nMethods: Ezymatic activity was determined by use of chromogenic substrates. The genes, which coded subtilisin-like pro­teases in above-mentioned dermatophytes, was identified and amplified by using specific primers in PCR."nResults: The highest yield of enzyme production was observed in only one isolate of T. vanbreuseghemii Ir-84 whereas low enzyme activity was observed in M. gypseum isolates. Homology study of obtained nucleotide as well as amino acid sequences indicated different rates of homology with other subtilisin-like proteases genes in other pathogenic dermato­phytes."nConclusion: Intra-strain differences were observed in production of serine proteinases and molecular characterization of genes encoding such enzymes could be of great interest for studies on pathogenicity and other purposes. &nbsp

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.

Background: Candida parapsilosis is an emergent agent of invasive fungal infections. This yeast is one of the five most widespread yeasts concerned in invasive candidiasis. C. parapsilosis stands out as the second most common yeast species isolated from patients with bloodstream infections especially in neonates with catheter. Recently several reports suggested that its reduced susceptibility to azoles and polyene might become a cause for clinical concern, although C. parapsilosis is not believed to be intensely prone to the development of antifungal resistance. Methods: In the present report, One hundred and twenty clinical isolates of C. parapsilosis complex were identified and differentiated by using PCR-RFLP analysis. The isolates were then analyzed to determine their susceptibility profile to fluconazole (FLU), itraconazole (ITC) and amphotericin B. The minimum inhibitory concentration (MIC) results were analyzed according to the standard CLSI guide. Results: All of isolates were identified as C. parapsilosis. No C. metapsilosis and C. orthopsilosis strains were found. Evaluation of the antifungal susceptibility profile showed that only three (2.5%) C. parapsilosis were resistant to fluconazole, three (2.5%) C. parapsilosis were resistant to itraconazole and two (1.7%) C. parapsilosis were amphotericin B resistant. Conclusion: Profiles in clinical isolates of C. parapsilosis can provide important information for the control of antifungal resistance as well as distribution and susceptibility profiles in populations.

Background: Aflatoxins cause health hazards to human and animals and has also economical problems. Therefore, the detoxification effect of citric acid was investigated in rice as the main food of Iranian people.Methods: Initially 275 samples of rice were examined for aflatoxins by HPLC. The aflatoxins contaminated samples were later treated by aqueous citric acid and detoxification of aflatoxins were quantified using HPLC.Results: Among the 275 samples analyzed, aflatoxin B1 and aflatoxin B2 were detected in 211(76.72% of total) samples. Afla­toxin B1 was detected in 203(73.82% of total) samples with a mean and standard deviation of 2.3±10.21ppb. Aflatoxin B2 together with aflatoxin B1 were detected in only 8(2.91% of total) samples with a mean and standard deviation of 1.38±2.7ppb of aflatoxin B2 and 2.99±1.56 of aflatoxin B1 respectively. Aflatoxin B1 level in 5 samples (1.82%) was above the maximum tolerated level of aflatoxin B1 in Iran (5ppb). However considering the Iranian maximum tolerated level for aflatox­ins in rice (30ppb), only 3(1.09%) samples were above the 30ppb and also in regard to the European maximum tolerated level for aflatoxins in rice (4ppb), only 9(3.27%) samples were considered as higher than 4ppb.Conclusion: The HPLC assay showed that although aflatoxins with a concentration of

"nBackground: Aspergillus flavus is the second most important Aspergillus species causing human infections particu­larly fun­gal sinusitis. Since little is known about aflatoxin producing ability of clinical isolates, this study was under­taken to de­tect the aflatoxigenic isolates amongst these isolates."nMethods: A total of 23 isolates of A. spp. which were recovered from patients proved to have fungal sinusitis by morpho­logi­cal and histological methods and also 5 additional aflatoxigenic and non-aflatoxigenic reference of A. fla­vus group strains were studied. The isolates were identified morphologically using Czapek Yeast Agar and A. flavus and parasiticus Agar (AFPA). Aflatoxin producing ability of the isolates was confirmed by Thin Layer Chromatogra­phy. Existing of aflR gene the regulatory gene in aflatoxin biosynthesis, were studied in all isolates by PCR method."nResults: All twenty three Aspergillus isolates confirmed as A. flavus group by their macroscopic and microscopic fea­tures. One clinical isolate confirmed as A. oryzae by mycological methods. A. oryzae as well as A. flavus JCM2061 and NCPF2008 and 3 clinical isolates were not able to produce orange pigment on AFPA. From total of 23 iso­lates 4 (17.4%) con­firmed to be aflatoxigenic by TLC method. A banding pattern which matched to aflR primers was amplified with approxi­mate size of 800 bp in all 23 clinical A. flavus isolates. A larger banding pattern 1050 bp was revealed in clinical iso­late; strain no.20 as well."nConclusion: Some clinical sinus isolates are able to produce aflatoxin and all of studied isolates including; A. oryzae, A. parasiti­cus and A. sojae were able to amplify aflR gene under our laboratory conditions. &nbsp

Background: The aim of this study was identification of the epidemiology of Prototheca zopfii species from the milk samples of dairy cattle in Isfahan, central Iran. Methods: Milk samples were obtained from 230 dairy cattle, 130 with and 100 without mastitis, in Isfahan. The samples were cultured in Prototheca Isolation Medium (PIM) and Sabouraud's dextrose agar. All P. zopfii isolates were identified by morphological and biochemical methods. Then, as a confirmatory test they were examined by genotype-specific PCR. Results: Four P. zopfii strains (3.07%) were isolated from the 130 samples of dairy cattle with clinical mastitis and there was no isolation from totally 100 samples of healthy bovines without mastitis. Specific PCR product (about 946 bp) was detected in four isolates. Conclusion: It seems that P. zopfii genotype II plays a key role in affecting bovine mastitis that confirmed other previous studies. Our study was the first, which identified the Prototheca species by traditional and molecular methods in Iran and Middle East as well.