Voriconazole resistance genes in Aspergillus flavus clinical isolates.

OBJECTIVE: The present study was designed to discover novel biomarkers involved in voriconazole resistance in clinical isolates of Aspergillus flavus. MATERIALS AND METHODS: Two voriconazole non-wild-type and two voriconazole-wild-type A. flavus clinical isolates were selected to evaluate possible molecular mechanism involved in A. flavus resistance to voriconazole using the mutation assessment, Quantitative real- time PCR of cyp51A and cyp51C genes and complementary DNA- amplified fragment length polymorphism technique. RESULTS: No mutations were seen in the cyp51A and cyp51C genes in voriconazole non-wild-type isolates compared to wild- type and reference strains. Regarding to mRNA expression results, no changes were observed in expression fold of cyp51A and cyp51C mRNA expression level in first non- wild- type isolate compared to wild-type isolate. For second isolate cyp51C mRNA expression level was down regulated (5.6 fold). The set of genes including ABC fatty acid transporter XM- 002375835 and aldehydereductase XM- 002376518 and three unknown functional genes were identified. Based on results, the over-expression of AKR1 and ABC fatty acid transporter in the voriconazole non- wild- type isolates suggests these genes could represent a novel molecular marker linked to the voriconazole resistance in A. flavus. CONCLUSION: The results obtained in this study showed a novel finding as the authors identified AKR1 and ABC fatty acid transporter genes as possible voriconazole target genes in Iranian clinical isolates of A. flavus.

The antagonistic effects of Candida parapsilosis on the growth of Fusarium species and fumonisin production.

BACKGROUND AND PURPOSE: Fusarium species are avid producers of secondary toxic and carcinogenic metabolites such as fumonisin. Contamination of food and feed products with fumonisin can be hazardous to the health of humans and animals and may lead to agricultural loss. Accordingly, in this study, we aimed to evaluate the effects of Candida parapsilosis on the growth and fumonisin production of Fusarium species. MATERIALS AND METHODS: Mycelial growth rate of 26 Fusarium isolates, including F. verticillioides (n=6), F. proliferatum (n=18), F. solani (n=1), and F. oxysporum (n=1), in the presence of 42 C. parapsilosis strains was investigated by pour-plate method. The decline in fumonisin production was measured in co-cultured fungi in coarsely ground maize after four weeks of incubation in the dark at 22°C, using ELISA technique. For data analysis, paired t-test was performed, using SPSS version 20. RESULTS: The mycelial growth and fumonisin production of Fusarium isolates significantly decreased in the presence of C. parapsilosis in comparison with the control cultures (P<0.05). The percentage of mycelial growth inhibition ranged from 56.36% to 74.54%. The minimum and maximum decline in total fumonisin production was 12% and 78%, respectively. F. oxysporum and F. solani were found to be minor fumonisin producers among the studied Fusarium species. On the other hand, a decline was reported in the growth of Fusarium species and fumonisin production in the presence of C. parapsilosis. CONCLUSION: C. parapsilosis showed notable inhibitory activities against Fusarium isolates. Therefore, this fungal species could be considered as a biocontrol agent against the growth and fumonisin production of toxigenic Fusarium species in the future.

Candida infection in the intensive care unit: A study of antifungal susceptibility pattern of Candida species in Milad hospital, Tehran, Iran.

OBJECTIVE: The occurrence of Candida infections has improved during the past two decades as a result of increase in the number of immunocompromised patients. In this study the antifungal susceptibility patterns of Candida species isolated from sterile body sites of patients admitted in Milad Intensive Care Unit (ICU) during 6 months were determined. METHODS: Candidal isolates were obtained from 50 patients admitted in Milad ICUs from April to September 2013. Identification of the isolates was performed by using morphological and polymerase chain reaction assay. Resistance to the antifungal agents containing caspofungin, posoconazole, voriconazole and amphotericin B was determined using E-test method. RESULTS: Out of 67 Candida isolates 47.8% were Candida glabrata, 28.3% were C. albicans, 7.5% were C. tropicalis, 7% were C. guilliermondii, 3% were C. krusei and 2% were C. dubliniensis. C. glabrata was the least susceptible species, with 9.4% of the isolates resistant to amphotericin B and 6.3% resistant to posoconazole and voriconazole. No resistance to caspofungin was observed among C. glabrata isolates. One of the C. krusei isolates was resistant to amphotericin B while no resistance to voriconazole, caspofungin and posoconazole was detected among C. krusei strains. Increase in the prevalence of antifungal-resistant non-C. albicans species in recent years has become a problematic event amongst clinicians caring for ICU patients. C. glabrata as the most common species isolated from ICU patients in this study indicated higher levels of antifungal resistance in comparison with other species. This observation accentuates the importance of managing preventive treatments to avoid development of resistance to the current antifungal drugs.

In vitro antifungal activities of Euphorbia macroclada and fluconazole against pathogenic Candida species.

BACKGROUND AND PURPOSE: Candida species constitute an important group of opportunistic fungi, which cause various clinical diseases. Considering the resistance of some Candida species to conventional antifungal agents, treatment of such cases may be challenging and complicated. The purpose of this study was to evaluate and compare the antifungal activities of Euphorbia macroclada latex and fluconazole against different Candida species. MATERIALS AND METHODS: A total of 150 Candida isolates including C. albicans (n=77), C. glabrata (n=28), C. parapsilosis (n=23), C. tropicalis (n=15), C. krusei (n=4), C. famata (n=1), C. kefyr (n=1) and C. inconspicua (n=1) were included in this study. In vitro antifungal activities of Euphorbia macroclada latex and fluconazole against these Candida species were evaluated, according to M27-A2 protocol on broth macrodilution method by the Clinical and Laboratory Standards Institute (CLSI). RESULTS: Among 150 Candida isolates, 98 isolates (65.33%), i.e., C. albicans (n=41), C. glabrata (n=23), C. tropicalis (n=12) and C. parapsilosis (n=22) with minimal inhibitory concentration (MIC) ≤ 8 µg/ml were susceptible to fluconazole. Resistance to fluconazole was noted in 15 isolates, i.e., C. albicans (n=10), C. glabrata (n=2), C. krusei (n=1), C. kefyr (n=1), and C. inconspicua (n=1), with MICs of 64 µg/ml. The remaining isolates (n=37) including C. albicans (n=26), C. glabrata (n=3), C. tropicalis (n=3), C. parapsilosis (n=1), C. krusei (n=3) and C. famata (n=1) with MIC= 16-32 µg/ml showed dose-dependent susceptibility. The latex of Euphorbia macroclada was able to inhibit the growth of 30 out of 150 tested Candida isolates with MIC range of 128-512 µg/ml. These isolates were as follows: C. albicans (n=2), C. glabrata (n=4), C. parapsilosis (n=19), C. krusei (n=2) and C. tropicalis (n=3). Compared to other isolates, higher MIC values were noted for C. albicans and C. glabrata (512 µg/ml), respectively. CONCLUSION: The latex of Euphorbia macroclada showed notable antifungal activities against some pathogenic Candida species. Therefore, it can be potentially used as an alternative antifungal agent in future. However, further research is required to identify its active components.

Sporotrichosis in Iran: A mini review of reported cases in patients suspected to cutaneous leishmaniasis.

Sporotrichosis is a chronic subcutaneous fungal infection with global distribution. It is a rare fungal infection with nine reported cases in Iran, including eight humans and one animal, within the past 30 years. Among the human cases, seven were of the fixed cutaneous type of sporotrichosis and one had sporotrichoid lymphocutaneous. The reported patients were within the age range of 23-60 years, and six of them were female. The most frequent sites of infection were forearms and hands, as well as the face and legs. In addition, the majority of the cases had previously been suspected of leishmaniasis and received treatment. Sporotrichosis is not a well-known condition in Iran and is often misdiagnosed and erroneously treated for other cutaneous parasitic or bacterial infections with similar clinical manifestations. Therefore, sporotrichosis should be taken into account in the differential diagnosis of nodular-ulcerative skin lesions.

A survey of the etiological agents of scalp and nail dermatophytosis in Yazd, Iran in 2014-2015.

BACKGROUND AND PURPOSE: Tinea capitis and tinea unguium are regarded as global public health concerns. The purpose of the present study was to identify the etiological agents of tinea capitis and tinea unguium in patients, referring to the Central Laboratory of Yazd University of Medical Sciences, Yazd, Iran. MATERIALS AND METHODS: This study was conducted during 2014-2015. Skin scraping, scalp hair, and nail clipping specimens were collected from 134 patients (80 males and 54 females) with clinical features suggesting fungal involvement. Direct microscopic examinations were carried out, using potassium hydroxide 10%, while culture studies were performed on Sabouraud dextrose agar, containing chloramphenicol and cycloheximide at 28°C for four weeks. Fungal colonies were identified based on their macroscopic and microscopic characteristics, as well as supplementary diagnostic tests. RESULTS: Among 134 patients, 12 cases showed positive results on direct examination and culture studies. The frequency of infections was equal among male and female subjects. Among 12 affected cases, the frequency of tinea capitis and tinea unguium was 91.6% and 8.4%, respectively. Microsporum canis (50%) was the most prevalent species, followed by Trichophyton verrucosum (25%) and Trichophyton mentagrophytes (25%). Also, tinea unguium, caused by T. mentagrophytes, was found in a female patient. CONCLUSION: The etiological agents of scalp and nail dermatophytosis have changed in Yazd over the past 13 years. In the present study, replacement of anthropophilic dermatophytes by zoophilic species was noteworthy, highlighting the necessity of efficient surveillance for the management and prevention of infections.

A study on etiologic agents and clinical manifestations of dermatophytosis in Yazd, Iran.

BACKGROUND AND PURPOSE: Dermatophytosis is one of the most common infections of skin, hair, and nails, caused by a group of keratinophilic fungi known as dermatophytes. Species identification of these fungi is of great significance from epidemiological and therapeutic points of view. The objective of the present study was to investigate dermatophytosis and its causative agents in patients, referring to the Central Mycology Laboratory of Yazd University of Medical Sciences, Yazd, Iran. MATERIALS AND METHODS: In total, 139 clinically suspected cases of dermatophytosis were examined during 12 months from February 2014 to February 2015. Skin scrapings were assessed through direct microscopic examinations and culture studies. Dermatophyte isolates were identified based on colony morphology on potato dextrose agar and dermatophyte test medium, nutritional requirements, urease and hair perforation tests, and microscopic characteristics on slide cultures. RESULTS: Dermatophytosis was mycologically confirmed in 26 (18.70%) out of 139 cases. Although there was a statistically insignificant difference between male and female subjects, men were dominantly affected. Infection was significantly common in the age group of ≤ 29 years (P<0.043). The most common clinical manifestation of dermatophytosis was tinea corporis (69.2%), followed by tinea cruris (15.4%), tinea manuum (11.5%), and tinea pedis (3.8%). Trichophyton mentagrophytes complex was the main etiologic agent (38.5%), followed by T. rubrum (23%), T. violaceum (15.5%), T. verrucosum (11.5%), Microsporum canis (7.7%), and Epidermophyton floccosum (3.8%). CONCLUSION: In comparison with previous research, epidemiology of dermatophytosis has changed in Yazd over the past decades. Therefore, periodical investigations on the epidemiological aspects of this infection are required for efficient control and prevention of this cutaneous dermatophytic disease.

Use of Single-enzyme PCR-restriction Digestion Barcode Targeting the Internal Transcribed Spacers (ITS rDNA) to Identify Dermatophyte 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.

Identification of Prototheca zopfii from Bovine Mastitis.

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.

Candida parapsilosis as a Potent Biocontrol Agent against Growth and Aflatoxin Production by Aspergillus Species.

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.

Aflatoxins in iran: nature, hazards and carcinogenicity.

Many studies have shown that mycotoxin contamination of agricultural products is a challenge for individual's health especially in developing countries. Improper production and storage of foods, prepare conditions for aflatoxin production in crops, especially rice, wheat, pistachio, walnut, almond, etc which are the main sources of foods for people. Feeding livestock by contaminated bread is another way of human exposure to mycotoxins, especially aflatoxin and because of expensive methods for detecting and analyzing aflatoxin in laboratory; it is not measured in foods. This manuscript is a review of some Iranian and nonIranian reports about aflatoxin, its exposure ways, its adverse effect on human health and nutrition, as well as methods for reducing its exposure. Based on studies on foods, aflatoxin exposure is high in Iran. Since livestock feeding by contaminated bread is one of the potential ways for milk contamination, we should control and reduce aflatoxin contamination by improving production process, storage condition and livestock feeding as soon as possible. Pistachio is one of the most important exporting products of Iran and to maintain Iran's position in exporting of this product, specific regulations on lowering its contamination with aflatoxin should be considered seriously. Finally, effective controlling of all food and feedstuffs which are vulnerable to aflatoxin contamination is necessary to prevent its effects.

Aflatoxin Detoxification in Rice using Citric Acid.

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 B(1) and aflatoxin B(2) were detected in 211(76.72% of total) samples. Aflatoxin B(1) was detected in 203(73.82% of total) samples with a mean and standard deviation of 2.3±10.21ppb. Aflatoxin B(2) together with aflatoxin B(1) were detected in only 8(2.91% of total) samples with a mean and standard deviation of 1.38±2.7ppb of aflatoxin B(2) and 2.99±1.56 of aflatoxin B(1) respectively. Aflatoxin B(1) level in 5 samples (1.82%) was above the maximum tolerated level of aflatoxin B(1) 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 <30 and <4 ppb in the rice samples were completely degraded, but 97.22% degradation occurred in rice contaminated with ≥30 and ≥4ppb when treated with 1N citric acid. These results revealed the efficacy of 1N citric acid in reducing aflatoxins levels in rice.

Upregulation of the ERG11 gene in Candida krusei by azoles.

BACKGROUND AND THE PURPOSE OF THE STUDY: Candida species are the agents of local and systemic opportunistic infections and have become a major cause of morbidity and mortality in the last few decades. Azole resistance in Candida krusei (C. krusei) species appears to be the result of gene alterations in relation to the ergosterol biosynthesis pathway, as well as efflux pumps. The main objective of this study was to examine the RNA expression of ERG11 in C. krusei which had been identified to be resistance to azoles. METHODS: The ERG11 mRNA expression was investigated in four Iranian clinical isolates of C. krusei, which were resistant to fluconazole and itraconazole by a semiquantitative RT-PCR. RESULTS: The mRNA expression levels were observed in all four isolates by this technique. Furthermore, it was found that ERG11 expression levels vary among four representative isolates of C. krusei. Although DNA sequencing revealed no significant genetic alteration in the ERG11 gene, one heterozygous polymorphism was observed in two isolates, but not in others. This polymorphism was found in the third base of codon 313 for Thr (ACT>ACC). MAJOR CONCLUSION: Even though such a polymorphism creates a new Ear1 restriction site, no significant effect was found on the resistance of C. krusei to azoles. RESULTS of this investigation are consistent with previous studies and may provide further evidence for the genetic heterogeneity and complexity of the ergosterol biosynthetic pathway or efflux pumps.

The antigenic composition and protein profiles of eumycetoma agents.

The protein profiles of different eumycetoma agents were compared by SDS gel electrophoresis. Dendrograms confirmed the homogeneity of isolates of Pseudallescheria boydii but amongst Madurella species, particularly isolates identified as M. grisea, there were substantial differences in protein composition. However using Western blotting reference isolates of the different species showed distinct antigen patterns in response to immune rabbit sera. In particular there was little evidence of cross reactivity between M. mycetomatis and M. grisea. However this specificity was not apparent when human sera from patients with different eumycetoma infections were compared in an ELISA system using the same antigens. It is possible that the formation of a mycetoma grain may limit a patient's exposure to antigens which confer specificity, an explanation which may also account for the variability in antibody responses seen.

Antifungal drugs affecting the chemotaxis of polymorphonuclear neutrophils.

Investigations on the chemotaxis of polymorphonuclear neutrophils (PMNs) towards a cytoplasmic extract of Trichophyton rubrum in the presence and absence of antifungal drugs are described. It is shown that with griseofulvin, clotrimazole, econazole, ketoconazole, miconazole and natamycin at 1 mg l(-1), the number of PMNs migrating was significantly reduced. After 3 h of exposure to 10 mg l(-1), not one of the drugs tested had any discernable effect on the viability of the PMNs, or the complement. The anti-inflammatory activity of the drugs is discussed and whilst the chemosuppression of PMN chemotaxis may be an undesirable feature in a drug used to treat systemic mycoses, it is unlikely to have any adverse effect in the therapy of the dermatophytoses.

Enzymic activities of Trichophyton rubrum and the chemotaxis of polymorphonuclear leucocytes.

The enzymic activity of Trichophyton rubrum has been investigated in relation to the plasma-dependent chemotaxis of polymorphonuclear leucocytes (PMNs). In Boyden-type experiments use of a cytoplasmic extract of T. rubrum (CETr) produces neutrophil chemotactic factor (NCF) from plasma. CETr was shown to have activity for eight enzymes: heat treatment of CETr led to a partial loss of activity for seven enzymes and a significant reduction in the number of PMNs migrating. Addition of CETr to plasma and incubation for 18 h at 37 degrees C before use led to complete loss of chemotactic activity. The similar incubation of plasma with trypsin led to a complete loss of chemotactic activity. CETr has greater activity than trypsin in the production of NCF from plasma. The results are discussed in relation to reports on the importance of serine esterases in PMN chemotaxis. The failure of PMNs to migrate into keratinized tissue infected with T. rubrum is noted and it is suggested that the high enzymic activities necessary for the colonization of keratinized tissue effect a breakdown of NCF.

Trichophyton rubrum and the chemotaxis of polymorphonuclear leucocytes.

In experiments with a Boyden-type chamber, both the cell wall and a cytoplasmic extract of Trichophyton rubrum acted as chemotaxigens for polymorphonuclear neutrophils in the production of neutrophil chemotactic factor from plasma. Activation of complement by the cytoplasmic extract to produce neutrophil chemotactic factor is shown to be predominantly through the alternative pathway and the cytoplasmic extract mimics the activity of the complementary system.