Identification of ptototheca zopfii from bovine mastitis

The aim of this study was identification of the epidemiology of Prototbeca zopfu species from the milk samples of dairy cattle in Isfahan, central Iran. Milk samples were obtained from 230 daily cattle, 130 with and 100 without mastitis, in Isfahan. The samples were cultured in Prototbeca Isolation Medium [PIM] and Sabouraud's dextrose agar. All P. zopfu isolates were identified by morphological and biochemical methods. Then, as a confirmatory test they wrere examined by genotype-specific PCR. Four P. zopfu strains [3.07° o] 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. It seems that P. zopfu 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

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. 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. The presence of the C. parapsilosis at different pH did not affect significantly the growth rate of Aspergillm 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 BI, 62, Gi, 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 GI and G2, respectively. C parapsilosis might employ as a good biocontrol agent against growth and aflatoxin production by aflatoxigenic Aspergillus species

Use of single-enzyme PCR-restriction digestion barcode targeting the internal transcribed spacers [ITs rDNA] to identify dermatophyte species

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. 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. 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. 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

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

Upregulation of the ERG11 gene in Candida krusei by azoles

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 ERG 11 in C. krusei which had been identified to be resistance to azoles. 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. 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 ERG 11 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 Even though such a polymorphism creates a new Earl 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

Aflatoxin detoxification in rice using citric acid

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. 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. 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. 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

Molecular characterization of cyclophilin protein gene in skin normal microflora: malassezia furfur

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. 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. 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. 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

Fungal nail infections in Tehran, Iran

Onychomycosis results from invasion of the nail plate by dermatophytes, yeasts or mould species of fungi. The objective was to determine the etiological agents of onychomycosis. A total of 549 patients clinically suspected of onychomycosis were examined for causative fungal agents. Both direct microscopy and the cultures of the nail material were performed to identify the causative agents between 2004-2005 in Tehran, Iran. Out of 549 cases examined, 263 [47.9%] were mycologically proven cases of onychomycosis [139 finger, 124 toe nails], among those 33 [6.09%] were only positive in direct microscopic examination. From an etiological point of view, 21.85% of nail infections were caused by yeasts, 10.55% were infected by dermatophytes and 15.5% by non-dermatopyte moulds.Candida albicans was the common yeast causative agent [16.73%] followed by A. flavus [11.78%], T. mentagrophytes [10.26%], [2.66%], Aspergillus spp [1.90%], each of Rhizopus spp and Cladosporium spp [1.52%], C.giulliermondii [1.14%], Scopolariopsis spp. [1.14%], each of C. famata, C.glabrata, C. krusei, S. lusitania, Acremonium spp. [0.76%] and C. homicola [0.38%], T. rubrum [4.94%]. Candida species were most common responsible agent for onychomycosis in female hands [74.1%] followed by 17.26% non-dermatophyte moulds. Dermatophytes caused tinea unguim of hand [8.63%] and peduum [37.1%] in males. The yeasts of the Genus Candida and non-dermatophyte moulds are dominant cause of female finger nail onychomycosis and dermathophytes are principal causes of both finger and toe nails in males in Tehran

Inhibitory effect of farnesol on biofilm formation by Candida tropicalis

Candidiasis associated with indwelling medical devices is especially problematic since they can act as substrates for biofilm growth which are highly resistant to antifungal drugs. Farnesol is a quorum-sensing molecule that inhibits filamentation and biofilm formation in Candida albicans. Since in recent years Candida tropicalis have been reported as an important and common non-albicans Candida species with high drug resistance pattern, the inhibitory effect of farnesol on biofilm formation by Candida tropicalis was evaluated. Five Candida tropicalis strains were treated with different concentration of farnesol [0, 30 and 300 micro M] after 0, 1 and 4 hrs of adherence and then they were maintained under biofilm formation condition in polystyrene, 96-well microtiter plates at 37°C for 48 hrs. Biofilm formation was measured by a semiquantitative colorimetric technique based on reduction assay of 2,3- bis -2H-tetrazolium- 5- carboxanilide [XTT]. The results indicated that the initial adherence time had no effect on biofilm formation and low concentration of farnesol [30 micro M] could not inhibit biofilm formation. However the presence of non-adherent cells increased biofilm formation significantly and the high concentration of farnesol [300 micro M] could inhibit biofilm formation. Results of this study showed that the high concentration of farnesol could inhibit biofilm formation and may be used as an adjuvant in prevention and in therapeutic strategies with antifungal drugs

enzymatic activity and molecular characterization of a secreted subtilisin-like protease in Microsporum gypseum and Trichophyton vanbreuseghemii

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. 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. 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. 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

Detection of Aflr gene and toxigenicity of Aspergillus flavus group isolated from patients with fungal sinusitis

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. 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. 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. 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

Performance of five phenotypical methods for identification of Candida isolates from clinical materials

Although Candida albicans is the most common etiologic agent of candidiasis, C. dubliniensis, has been emerged, as another pathogen resembles C. albicans in many phenotypic aspects and noted for its in vitro potential for fluconazole resistance. Since there was no evidence of any report about detection of this organism in Iran, this study was designed to use of five different tests for identification of Candida species with special reference to C. dubliniensis among 313 suspected Candida isolates in Tehran, capital of Iran. Overall, 199 [63.6%] C. albicans and 114 [36.6%] Candida spp. were identified. All 199 C. albicans isolates were found germ tube and chlamydospore positive. Different shades of green color colonies were yielded on CHROMagar Candida of which 23 [11.6%] showed dark green color indicative of C. dubliniensis. All but four C. albicans isolates grew well at 45 °C. These 4 isolates beyond to 23 dark green colony producers were suspected of being C.dubliniensis, later examined by API 20C AUX system. The results indicated that all 27 isolates were able to assimilate both xylose and alpha-methyl-D-glucoside, therefore these isolates were identified as C. albicans. Overall, C. dubliniensis had not been found in present study. It must be concluded that no single phenotypic test has proven to be highly effective, and the use of several tests may be necessary of these two closely related Candida species for definitive identification

Fungi as Causative agent of nasal polyps

Nasal polyposis is an inflammatory condition of unknown etiology that involves nasal and sinus mucous membrane. These polyps can impair a person's quality of life by nasal obstruction, recurrent sinusitis, persistent postnasal drainage, hyposmia, anosmia, changes in sense of taste and even bony destruction. It has been shown that chronic inflammation causes a reactive hyperplasia of the intranasal mucosal membrane which results in the formation of polyps. Recently, fungal elements were suspected to be the causative agent of chronic rhinosinusitis and a fungal etiology has been proposed to underlie severe nasal polyposis. The present study was undertaken to determine the role of fungi in development of nasal polyps. In this study resected polyps from 100 patients were examined by mycological and pathological methods for the presence of fungi. Fungal elements were shown in 9 samples by mycological methods and isolated fungi were Aspergillus flavus, Aspergillus fumigatus and Rhizopus sp. Tissue invasion by fungi also was seen by histopathological examination in 6 patients. Therefore, fungi could be considered as the causative factor in the development of nasal polyposis in those patients and since medical treatment of nasal polyps have become increasingly recognized in recent years, the present study also implying the benefits of topical antifungal therapy in such cases

Isolation of keratinophilic fungi from soil samples of forests and farm yards

Soil is well known to support the transient or ongoing existence of kerathophilic fungi and potential sources of infection for humans and animals. Fifty soil samples were collected from various areas of forests and farmyards at Golestan Province in the north part of Iran to determine the prevalence of keratinophilic fungi and dominant species. A total of 357 fungal colonies including 13 genera with 11 species were isolated as follows: Anixiopsis stercoraria [16.24%], Arthroderma cuniculi [12.04%], Reniospora flavissima [9.24%], Fusarium oxysporum [9.24%], Aspergillus flavus [8.68%], Chrysosporium keratinophilum [8.40%], Trichophyton vanbreuseghemii [7.84%], and other fungi [37.56%]. McNemar's test showed that non-keratinolytic fungi were dominant in this investigation [P<0.05]. Anixiopsis stercoraria [16.24%] was the most prevalent and dominant keratinophilc fungus [P<0.05]. It can be concluded that soils from forest and farmyards of Golestan Province are rich in keratiophilic fungi including dermatophytes

Study on the sources of nosocomial fungal infections at intensive care unit and transplant wards at a teaching hospital in Tehran

The incidence of nosocomial fungal infections has increased dramatically during the past two decades as the consequence of continuous increase in the number of severely immunocompromised patients. This study was done to determine the presumptive sources of nosocomial fungal infections at the intensive care unit and transplant wards [in a university- based teaching hospital in Tehran] during a 10-month period. Totally 583 samples were obtained from the air, surfaces, health care workers and also from the patients at those wards. Mycological culture of the samples yielded growth of 25 different genus and species of fungi and the most common isolated fungi were Candida albicans, Penicillium spp., Aspergillus niger, and Cladosporium spp., respectively. It was noted that health care workers were carrying fungi on their hands [50%], nasal mu-cosa [57.6%], in oral cavity [38.6%] and also by their shoes [92.3%] and uniforms [92.7%]. Environmental fungal contamination was shown and it was more prominent at the intensive care unit. Hospitalization also had more significant effect on colonization of fungi in the patients at the latter ward. Therefore, the highly susceptible patients in present study were at the greatest risk of developing fungal infections and preventive measures were critical for prevention and control of these life-threatening fatal infections

Anti-Cryptococcal-Globulin-Latex production for rapid detection of cryptococcus neoformans polysaccharide antigen in cryptococcosis

Cryptococcosis has become the fourth leading life-threatening opportunistic infection in patients with AIDS, but also occurs in non-AIDS patients. In view of the increasing numbers of infection during last decade in Iran, use of rapid, sensitive and specific test for diagnosis of cryptococcal disease has become important than ever. We aimed to produce the reagents for latex cryptococcal antigen test. The antigen was prepared from NCPF 3168 strain of Cryptococcus neoformans. Anticapsular antiserum of C. neoformans raised in rabbits and latex carboxylate- modified beads were coated with antiserum. The maximally- reactive globulin dilution was obtained at dilution of 1:400. For evaluation of efficacy of reagents, challenged 38 BALB/C mice and other 38 mice were used as controls. The mice were bled and autopsied. Brain, heart and lung were checked by direct, histopathological and cultural examination for cryptococcosis. The sera from case and control mice were tested with Immunomycologic [Immy] kit and also our produced reagents [OPR] for detection of cryptococcal antigen. Moreover, 15 cerebrospinal fluid and 15 serum samples from patients with cryptococcal meningitis, 30 with aspergillosis, 30 with suspected other fungal infections, and 30 from healthy individuals were tested as well. The results showed that the sensitivity [97.3%] and specificity [100%] of OPR was quite comparable with those of Immy kit. Therefore, it could be regarded as a substitute for commercial kits

Fungal Involvement in Patients with Paranasal Sinusitis

Fungal involvement of the paranasal sinuses is frequently observed in the immunocompromised host and it can become lifethreatening if it is not diagnosed. Definitive diagnosis is made by tissue biopsy and culture. In this study biopsy materials of maxillary, ethmoidal and frontal sinuses of 60 patients with clinical manifestation of sinusitis and no response to medical therapy were assessed by mycological and pathological methods for the presence of fungi. Invasive fungal sinusitis was diagnosed in 3 patients and etiologic agents were C and ida albicans, Rhizopus sp. and Aspergillus fumigatus. Predisposing factors in these patients were leukemia, diabetes mellitus and previous sinus and polyp surgery, respectively. Allergic fungal sinusitis also was seen in one patient and Alternaria sp. isolated from the biopsy material. Only the patient with allergic form of disease survived but all the patients with invasive form of fungal infection were expired. This clearly underscores the need of early recognition of fungal sinusitis in at risk population in order to start urgent treatment. In this study Nocardia asteroids also was isolated from the biopsy sample in a patient with sinunasal adenocarcinoma

relationship between blood group isoantigens and dermatophytosis

The relationship between ABO blood groups and dermatophytosis was studied. Blood grouping was done for 308 dermatophyte-infected patients. Eighty six patients belonged to blood group A, 68 to blood group B, 34 to blood group AB and 120 to blood group O. The patients with blood group 0 and A were the most dermatophyte infected persons. Chronicity of the disease was more frequent in those with blood groups A and O. Our study suggests that blood group A subjects might be more susceptible to Trichophton mentagrophytes [TM], Trichophton rubrum [TR] and Epidemophyton floccosum [EF] than others

Enzyme activities in trichophyton, rubrum, trichophyton mentagrophytes and trichophyton verrucosum

Since most of the studies on chemical compounds of dermatophytes have shown the existence of a relationship between their pathogenisity and proteolytec enzymes. Activities of 19 different enzymes in viable mycelia and cytoplasmic extracts of T.rubrum [CETr], T.mentagrophytes [CETm] and T.verrucosum [CETv] were investigated by the API-Zym System. The results showed that Viable mycelia of T.rubrm and T.mentagrophytes had valine arylamidase and cystine arylamidase activity where as no such activity was observed in CETr and CETm or in the viable mycelia and cytoplasmic extracts of T.verrucosum. Also the viable mycelia of T.rubrum showed