Electronic equipment and appliances in special wards of hospitals as a source of azole-resistant Aspergillus fumigatus: a multi-centre study from Iran.

BACKGROUND: Azole-resistant Aspergillus fumigatus (ARAf), reported as a global public health concern, has been unexpectedly observed in different countries. AIM: To identify ARAf and detect azole resistance related to the CYP51A mutation in different hospital environmental samples. METHODS: In this multi-centre study from Iran, surfaces of electronic equipment and appliances from different hospitals in Iran were sampled using cotton swabs. All samples were cultured using azole-containing agar plates (ACAPs). Recovered Aspergillus isolates were identified at the species level using partial DNA sequencing of the ß-tubulin gene. The azole susceptibility testing of A. fumigatus isolates was performed using the Clinical and Laboratory Standards Institute M38-A3 guideline. The sequencing of the CYP51A gene was also performed to detect mutations related to resistance. FINDINGS: Out of the 693 collected samples, 89 (12.8%) Aspergillus species were recovered from ACAPs. Aspergillus fumigatus (41.6%) was the most prevalent, followed by A. tubingensis (23.6%) and A. niger (15.6%). Among 37 isolates of A. fumigatus, 19 (51.3%) showed high minimum inhibitory concentration (MIC) values to at least one of the three azoles, voriconazole, itraconazole, and posaconazole. CYP51A polymorphisms were detected in all 19 isolates, of which 52.6% showed the TR34/L98H mutation. Other detected mutations were G432C, G448S, G54E/G138C, F46Y, and Y121F/M220I/D255E. T289F and G432C were the first reported mutations in ARAf. CONCLUSION: There was a considerable level of azole resistance in hospital environmental samples, a serious warning for patients vulnerable to aspergillosis. Our findings have also revealed a different mutation pattern in the CYP51A gene.

Aspergillus fumigatus and aspergillosis: From basics to clinics.

The airborne fungus Aspergillus fumigatus poses a serious health threat to humans by causing numerous invasive infections and a notable mortality in humans, especially in immunocompromised patients. Mould-active azoles are the frontline therapeutics employed to treat aspergillosis. The global emergence of azole-resistant A. fumigatus isolates in clinic and environment, however, notoriously limits the therapeutic options of mould-active antifungals and potentially can be attributed to a mortality rate reaching up to 100 %. Although specific mutations in CYP 51A are the main cause of azole resistance, there is a new wave of azole-resistant isolates with wild-type CYP 51A genotype challenging the efficacy of the current diagnostic tools. Therefore, applications of whole-genome sequencing are increasingly gaining popularity to overcome such challenges. Prominent echinocandin tolerance, as well as liver and kidney toxicity posed by amphotericin B, necessitate a continuous quest for novel antifungal drugs to combat emerging azole-resistant A. fumigatus isolates. Animal models and the tools used for genetic engineering require further refinement to facilitate a better understanding about the resistance mechanisms, virulence, and immune reactions orchestrated against A. fumigatus. This review paper comprehensively discusses the current clinical challenges caused by A. fumigatus and provides insights on how to address them.

Prevalence of allergic bronchopulmonary aspergillosis in cystic fibrosis patients using two different diagnostic criteria.

Summary: Objective.There are different diagnostic criteria for the diagnosis of Allergic bronchopulmonary aspergillosis (ABPA) in CF patients. In this present study we evaluated the prevalence of ABPA in Iranian CF patients by two more usual diagnostic criteria as ISHAM working criteria (A) and CF Foundation Consensus Conference criteria (B). Methods.Eighty-six CF patients were included in the study. All CF patients underwent for Aspergillus skin prick test (AST), Aspergillus-specific IgE (sIgEAf) and Aspergillus-specific IgG (sIgGAf), total IgE. The ABPA prevalence was estimated by two diagnostic criteria, (A) and (B) and compared. Results. The frequency of positive AST, total IgE, sIgEAf and sIgGAf were 47 (54.6%), 9 (10.5%), 42 (48.8%) and 67 (77.9%), respectively. The obtained rate of ABPA prevalence (10.5%) was identical in two diagnostic criteria A and B (kappa value of 1.000). Conclusions.The applied diagnostic criteria had no significant effect on the reported rate of ABPA prevalence.

Successful control of exacerbation of Allergic Bronchopulmonary Aspergillosis due to Aspergillus terreus in a cystic fibrosis patient with short-term adjunctive therapy with voriconazole: A case report.

A 12-year-old boy with cystic fibrosis (CF) and a history of glucocorticoid-dependent allergic bronchopulmonary aspergillosis (ABPA) was referred to our hospital. The ABPA was diagnosed when he was 8 years old and he had been treated with several course of oral glucocorticoids for recurrent exacerbations. He was readmitted when aged 12 with a history of worsening shortness of breath and chest tightness. A recurrence of ABPA was diagnosed based on eosinophilia and elevation of Aspergillusspecific IgE and IgG, and total IgE. Thoracic high-resolution computed tomography (HRCT) showed central bronchiectasis with parenchymal infiltrates. The treatment started with itraconazole and oral corticosteroid. After 2 months of treatment, he was re-admitted to the hospital due to a progressive worsening of respiratory symptoms. Chest HRCT revealed the a sub segmental atelectasis in the left lung. Microscopic examination of sputum and BAL samples demonstrated septate hyphae consistent with Aspergillus species. Sputum and BAL culture yielded Aspergillus ochraceus and Aspergillus terreus, which were both sensitive to itraconazole and voriconazole. The treatment was switched to voriconazole and the patient showed significant clinical, serological and mycological improvement after three months. This case shows that voriconazole may be used as an alternative for treatment of ABPA due to Aspergillus terreus.

Molecular identification and antifungal susceptibility testing of Candida species isolated from dental plaques.

OBJECTIVE: The aim of the present study was to provide insight into the prevalence and susceptibility profiles of Candida species isolated from the dental plaque of Iranian immunocompetent patients. As a biofilm, Candida species are responsible for several disorders common to the oral cavity including gingivitis, dental caries, periodontitis, and the less common severe systemic infections specifically in immunosuppressed individuals. METHOD: PCR-RFLP was performed to identify yeasts isolated from the dental plaques of 40 immunocompetent patients. Moreover, antifungal susceptibility testing was performed in according to CLSI guidelines (M27-A3). RESULTS: Among 40 yeasts isolated from the dental plaques of immunocompetent patients, Candida albicans was the most common species (92.5%), followed by P. kudriavzevii (7.5%). It is the first isolation of P. kudriavzevii from dental plaques and the first evaluation of antifungal effect of the new imidazole, luliconazole and echinocandins against these samples worldwide. Luliconazole, voriconazole, amphotericin B and anidulafungin showed the best activity with the lowest geometric mean (GM) 0.03, 0.06, 0.08 and 0.09µg/ml, respectively, followed by miconazole (0.14µg/mL), caspofungin (0.24µg/mL) fluconazole (0.38µg/mL) and itraconazole (0.5µg/mL). CONCLUSION: The current study demonstrated luliconazole and echinocandins displayed excellent activity against all Candida isolates from dental plaques, presenting promising and potent alternative for all oral Candidiasis.

Glabridin triggers over-expression of MCA1 and NUC1 genes in Candida glabrata: Is it an apoptosis inducer?

The growing trends of emergence of antifungal-resistant Candida strains has recently been inspired the researchers to design new antifungal agents with novel mechanisms of action. Glabridin is an originally natural substrate with multiple biological activities which propose it as a novel anticancer, antimicrobial and antifungal agent. In the present study, the antifungal effect of glabridin against Candida glabrata isolates and its possible mechanism of action were investigated. The minimum inhibitory concentrations (MIC) for glabridin against fluconazole-resistant and fluconazole-SDD strains of C. glabrata were investigated using the Clinical and laboratory standards institute document M27-A3 and M27-S4 as a guideline. Possible alternations in the expression of two critical genes involved in yeast apoptosis, MCA1 and NUC1, were assayed by real-time PCR. DNA damage and chromatin condensation was investigated using DAPI staining. Although glabridin led to a significant decrease in MICs against fluconazole-resistant C. glabrata (MIC50: 8µg/mL), no significant decreased was shown for fluconazole-SDD strains. Therefore, a distinct azole-independent mechanism could be responsible for the inhibitory activity of glabridin. Overexpression of MCA1 and NUC1 genes in addition to DNA damage and chromatin condensation suggesting the involvement of apoptosis signaling in C. glabrata stains exposed to glabridin. This study suggests that glabridin might be considered as a novel naturally originated agent to fight against fluconazole-resistance C. glabrata strains.

In vitro antitumor activity of patulin on cervical and colorectal cancer cell lines.

BACKGROUND AND PURPOSE: Patulin is a mycotoxin produced by some molds, especially Aspergillus and Penicilium, and is responsible for mycotoxicosis in animals and humans.There is still not very detailed data about the anti-cancer potency of patulin, but some reports demonstrated that it induces cellular apoptosis and toxicity. MATERIALS AND METHODS: To determine the efficacy of patulin as a therapeutic strategy for cervical and colorectal cancers, we investigated its effects on HeLa,SW-48, and MRC-5 cell lines. Cell lines were exposed to various concentrations of patulin (i.e., 0.5, 1, 2, and 4 µM), then using methyl thiazolyl tetrazolium (MTT) and bromo-2'-deoxyuridine (BrdU) assays, the rates of apoptosis and cell viability were determined. RESULTS: The obtained results showed a significant reduction in cell viability and apoptosis induction in a dose-dependent manner. Among all the cell lines, the highest growth inhibition rate was obtained at the 4 µM concentration of patulin. CONCLUSION: Our results suggested that patulin could significantly decrease tumor growth in human cervical and colorectal cancer models.

Hyphal wall protein 1 gene: A potential marker for the identification of different Candida species and phylogenetic analysis.

BACKGROUND AND PURPOSE: Hyphal wall protein 1 (HWP1) is an important adhesin which usually is expressed on the germ tube and hyphal surface produced by different Candida species. The hyphal wall protein-coding gene (HWP1) was evaluated as a novel identification and phylogenetic marker in Candida tropicalis, C. orthopsilosis, C. parapsilosis and C. glabrata. MATERIALS AND METHODS: Initially, four specific primer pairs were designed, and the target was amplified and finally sequenced. A total of 77 Candida isolates from four different species were included in the study. Consensus sequences were used for the evaluation of phylogenetic tree using the CLC Genome Workbench, GENEIOUS, and MEGA softwares and the levels of nucleotide and amino acid polymorphism were assessed. RESULTS: According to the results, the specific amplified fragments of HWP1 gene were useful for the differentiation of four species. Intra-species variation was observed only in C. tropicalis with two DNA types. The phylogenetic tree of Candida species based on the HWP1 gene showed consistency in topology with those inferred from other gene sequences. CONCLUSION: We found that HWP1 gene was an excellent marker for the identification of non-albicansCandida species as well as the phylogenetic analysis of the most clinically significant Candida species.

Aspergillus species in indoor environments and their possible occupational and public health hazards.

The genus Aspergillus, which consists of a few hundred opportunistic mold species found in various climatic conditions, causes diseases including localized infections, fatal diseases, allergic responses, and inhaled conidia in humans. Herein, we present an overview of the most common diseases and allergic infections caused by Aspergillus species and their associated health hazards in various indoor environments worldwide.

Prevalence of chronic pulmonary aspergillosis in patients with tuberculosis from Iran.

In patients with preexisting lung disease, especially a cavity, Aspergillus can infect the surface of the cavity, causing chronic cavitary pulmonary aspergillosis (CCPA), and may form an aspergilloma, collectively called chronic pulmonary aspergillosis (CPA). In the present study, we assessed tuberculosis (TB) patients for CPA based on culture and serological methods. During a period of 1 year (from March 2013 to March 2014), we studied 124 patients with TB (94 with current TB and 30 with previous TB) at Masih Daneshvari Hospital in Tehran, Iran. Sputum specimens were analyzed by direct microscopic examination (DME) and fungal culture. The clinical and radiological features of all patients were recorded, to categorize the patients into CCPA and aspergilloma. All patients were screened for serum-specific IgG against A. fumigatus, by enzyme-linked immunosorbent assay (ELISA). Out of 124 patients with TB (66 male, age range: 10-91 years), 48 patients (38.7 %) exhibited residual cavities. Eighteen (14.5 %) patients had cavities with pleural thickening. A round-shaped mass lesion was detected in six patients (6.8 %). DME was positive in ten patients for septate fungal hyphae. A. fumigatus was grown from 14 samples. Fifty-five (44.3 %) cases were positive for serum-specific IgG against A. fumigatus. Of 124 patients with TB, 3 (2.4 %) met criteria for aspergilloma and 14 (11.3 %) for CCPA. CPA is a common clinical presentation in individuals with healed TB in Iran, as reported by previous studies from other countries.

Detection of galactomannan in bronchoalveolar lavage of the intensive care unit patients at risk for invasive aspergillosis.

BACKGROUND AND PURPOSE: Invasive aspergillosis (IA) is one of the most common life-threatening fungal infections among the critically ill patients including intensive care unit (ICU) patients. Delayed diagnosis and therapy may lead to poor outcomes. Diagnosis may be facilitated by a test for molecular biomarkers, i.e. detection of galactomannan (GM) antigen based on enzyme immunoassay, which is of increasing interest in the clinical settings for the diagnosis of IA. In the present study, we assessed GM testing of bronchoalveolar lavage (BAL) fluid as a tool for early diagnosis of IA among ICU patients who were at risk for developing IA. MATERIAL AND METHODS: A prospective study was performed in ICU patients with underlying predisposing conditions for IA between August 2010 and September 2011. BAL samples for direct microscopic examination, culture, and GM detection were obtained once or twice weekly. GM in BAL levels was measured using the Platellia Aspergillus EIA test kit. According to modified European Organization for the Research and Treatment of Cancer/ Mycoses Study Group (EORTC/MSG) criteria, patients were classified as having probable or possible IA. RESULTS: Out of 43 suspected patients to IA, 13 (30.2%) cases showed IA. According to the criteria presented by EORTC/MSG, they were categorized as: 4 cases (30.8%) of possible IA and 9 (69.2%) of probable IA. Out of 21 BAL samples from patients with IA, 11 (52.4%) had at least one positive BAL GM index. Using a cutoff index of 0.5, the sensitivity and specificity, positive and negative predictive values of GM detection in BAL fluid were 100%, 85.7%, 65.7% and 96%, respectively. The sensitivity and specificity was 73% and 92.7% at cutoff ≥1.0, respectively. In 6 of 13 IA cases, BAL culture or direct microscopic examination remained negative, whereas GM in BAL was positive. CONCLUSION: Our data have revealed that the sensitivity of GM detection in BAL was better than that of conventional tests. It seems that GM detection in BAL is beneficial to establish or exclude the early diagnosis of IA in ICU patients.

A real time PCR assay on blood for diagnosis of invasive candidiasis in immunocompromised patient.

BACKGROUND AND PURPOSE: Invasive candidiasis (IC) is a significant cause of morbidity and mortality in patients with hematologic disorders and bone marrow transplant recipients. Rapid, specific and sensitive test for the timely accuracy in immunocompromised patients to reduce mortality rates and prevent IC progress is necessary. We established a real-time PCR assay on blood for the diagnosis and differentiation of the causative Candida species. MATERIALS AND METHODS: Whole blood samples were collected twice, from 72 patients for Real Time PCR and blood culture assays. The primers and hybridization probes were designed to potentiate the specific sequence of 18S rRNA genes using Light Cycler system and Fluorescence Resonance Energy Transfer (FERT). The patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC based on the revised European Organization for Research and Treatment of Cancer/ Mycoses Study Group (EORTC/MSG) criteria. RESULTS: From 2009 to 2011, 72 patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC. The female to male ratio was 27:45; the mean age was 32.1 years. The most common malignancy in this patient was acute myeloid leukemia (AML) (27.8%) and acute lymphoblastic leukemia (ALL) (26.4%). Out of 72 patients, 11 patients (15.3%) had positive real time PCR /probe results. Based on the melting temperature (Tm) analysis, 5 (45.4%) C. krusei, 3 (27.2%) C. tropicalis, 2 (18.1%) C. parapsilosis and 1 C. albicans (9%) were identified. According to the revised EORTC / MSG, 1 patient (9%) and 10 patients (91%) were defined as proven and possible groups of IC, respectively. The mortality rate in proven and possible IC patient was found 54.5%. CONCLUSION: The established Real-time PCR/FRET probe assay is an appropriate diagnostic tool for the detection of Candida species DNA and the management of patients suffering from hematologic malignancies and bone marrow recipient are at risk for IC.

Serum immunoglobulin E and immunoglobulin G reactivity to Agaricus bisporus proteins in mushroom cultivation workers.

BACKGROUND AND PURPOSE: Although molds are regarded as the main fungal allergen sources, evidence indicates that spores of Basidiomycota including Agaricus bisporus (A. bisporus) can be also found at high concentrations in the environment and may cause as many respiratory allergies as molds. The aim of the present study was to evaluate specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibodies against A. bisporus via immunoblotting technique in individuals working at mushroom cultivation centers. MATERIALS AND METHODS: In this study, 72 workers involved in the cultivation and harvest of button mushrooms were enrolled. For the analysis of serum IgE and IgG, A. bisporus grown in Sabouraud dextrose broth was harvested and ruptured by liquid nitrogen and glass beads. The obtained sample was centrifuged and the supernatant was collected as "crude extract" (CE). CE was separated via Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The separated proteins were transferred to a nitrocellulose filter and the bands responsive to IgE and IgG were identified by anti-human conjugated antibodies. All participants were screened in terms of total IgE level. RESULTS: Among 72 workers, 18 (25%) had a total IgE level higher than 188 IU/mL. In SDS-PAGE, the CE of A. bisporus showed 23 different protein bands with a molecular weight range of 13-80 kDa. The sera of 23.6% and 55.5% of participants showed positive response, with specific IgE and IgG antibodies against A. bisporus in the blot, respectively. The bands with molecular weights of 62 and 68 kDa were the most reactive protein components of A. bisporus to specific IgE antibodies. Moreover, bands with molecular weights of 57 and 62 kDa showed the highest reactivity to IgG, respectively. Also, 62 and 68 kDa components were the most reactive bands with both specific IgG and IgE antibodies. CONCLUSION: The obtained findings revealed that A. bisporus has different allergens and antigens, which contribute to its potential as an aeroallergen in hypersensitivity-related reactions of the lungs.

Study on fungi in archives of offices, with a particular focus on Stachybotrys chartarum.

OBJECTIVE: This study aimed at evaluating fungi in archives of different offices in Sari city the capital of Mazandaran, a northern province of Iran, with a particular focus on Stachybotrys chartarum. MATERIALS AND METHODS: The samples were collected from twenty archives of offices and controls (n=7) using a SKC single-stage impactor which draws air at 20L/min (100L) and impacts the sampled material onto Petri dishes containing malt extract agar (MEA) (n=22) and also cellulose agar (CA) (n=22). Surface samples were also collected by pressing a sterile cotton swab on different areas of archives and cultured on MEA and CA. The grown fungi were identified by standard mycological techniques. The counted fungal colonies were converted to CFUs per cubic meter. RESULTS: In indoor air of archives, Cladosporium spp (25.1%), Aspergillus spp (22.9%) and Penicillium spp (22.9%) had the most frequencies. Stachybotrys chartarum (7.9%) was the fourth most common fungus isolated from the surface samples. Cladosporium spp had the highest total CFU concentration in indoor air of archive samples (1227/m(3)). Stachybotrys chartarum was recovered from surface collected samples of 4 archives of offices on CA. Out of the 22 rooms of archives, 45.4%, 45.4% and 9.1% had concentration level < 170 CFU/m(3), > 170 < 560 CFU/m(3) and > 560 < 1000 CFU/m(3), respectively. CONCLUSION: The results of our study have shown the high concentration levels of airborne fungi in some archives of offices that might put the workers at risk from respiratory diseases.

Clinical risk factors and bronchoscopic features of invasive aspergillosis in intensive care unit patients.

INTRODUCTION: Invasive aspergillosis (IA) is an important cause of morbidity and mortality in immunocompromised patients. During recent years, a rising incidence of IA in Intensive Care Unit (ICU) patients has been reported. The patterns of IA related infection may differ according to the type of underlying disease. Unfortunately little is known about the characteristics of IA in ICU patients. In the present study we assessed IA related clinical and bronchoscopy findings in ICU patients. MATERIALS AND METHODS: This study was performed at the ICU units in Sari and Babul, Mazandaran from August 2009 through September 2010. We analysed 43 ICU patients with underlying predisposing conditions for IA. Bronchoalveolar lavage (BAL) samples were collected by bronchoscope twice a weekly. The samples were analyzed by direct microscopic examination, culture and non-culture based diagnostic methods. Patients were assigned a probable or possible diagnosis of IA according to the consensus definition of the EORTC/MSG. RESULTS: Out of 43 suspected patients to IA, 13 (36.1%) cases showed IA. According to criteria presented by EORTC/MSG, they were categorized as: 4 cases (30.8%) of possible IA and 9 (69.2%) of probable IA. The observed mortality was 69.2%. The main underlying predisposing conditions were neutropenia, hematologic malignancy, and COPD. The macroscopic finding in bronchoscopy included of Prulent secretion (46.6%), Mucosal bleeding (30.7%), Mucosal erythema (23%), Trachobronchomalasia (15.3%). CONCLUSION: The diagnosis of IA in patients with critical illness in ICU is even more difficult. The clinical diagnostic process is often dependent on indirect circumstantial data enhancing the probability of IA. Bronchoscopy with inspection of the tracheobronchial tree, sampling of deep airway secretions and BAL can be helpful.

Exophiala oligosperma involved in a refractory chronic rhinosinusitis.

Fungal rhinosinusitis refers to a wide variety of conditions caused by fungal infections of the paranasal sinuses. Allergic fungal rhinosinusitis and sinus fungus balls are mostly noted in healthy individuals. Aspergillus species are supposed to be the most common etiologic agents of the disorder, but melanized fungi also occur, and these potentially are able to lead to fatal dissemination into brain parenchyma. We report on a case of fungus ball in a 20-year-old female with refractory chronic rhinosinusitis (RCRS) and bronchial asthma due to the black yeast Exophiala oligosperma which was confirmed by mycological and molecular (sequences of ITS rDNA) investigations. Exophiala oligosperma has previously not been reported to cause fungus balls or invasive fungal rhinosinusitis. Patient underwent functional endoscopic sinusitis surgery and the hypertrophic mucosa was removed completely. Without antifungal therapy, successful cure was achieved after spray therapy with corticosteroids for 1 month, without any relapse after a 6 month-follow up.

Study on fungal flora of tap water as a potential reservoir of fungi in hospitals in Sari city, Iran.

OBJECTIVE: The aim of the present investigation was to evaluate the fungal flora of tap water from university hospitals of Sari city, Iran. MATERIALS AND METHODS: During a 1-year period, 240 water samples were collected from four university hospitals. All water samples were collected in sterile polystyrene bottles. A volume of 100ml of the samples passed through sterile 0.45-micrometer filters. The filters were placed directly on malt extract agar and incubated at 27°C for 3 to 7 days. Routine mycological techniques were applied to identification of grown fungi. RESULTS: Out of 240 plates, 77.5% were positive for fungal growth. Twelve different genera were identified. Aspergillus (29.7%), Cladosporium (26.7%) and Penicillium (23.9%) were the most common isolated. Among Aspergillus species, A. flavus had the highest frequency. Highest colony counts were found in autumn. Aspergillus predominated in autumn, Cladosporium in winter and spring and Penicillium in summer. CONCLUSION: The results of our study showed that hospital water should be considered as a potential reservoir of fungi particularly Aspergillus.

Identification of Candida species using PCR-RFLP in cancer patients in Iran.

UNLABELLED: Opportunistic infections caused by Non-Candida albicans. have been increasing. Traditional methods that are used to identify clinical isolates of Candida species are time-consuming and not appropriate for rapid, accurate and reliable identification. PURPOSE: To identify Candida spp isolated from cancer patients using PCR-restriction enzyme. MATERIALS AND METHODS: Using universal primers, ITS1 and ITS4, in this study, we could amplify ITS1-5.8S-ITS2 rDNA regions at both 80 clinical isolates and 3 standard strains. The PCR products were digested with two restriction enzymes MspI and BlnI separately. RESULT: We successfully identified all isolated species using two restriction enzymes (MspI, BlnI). Candida albicans was the most common species (77.5%), followed by C. glabrata (15%), C. tropicalis (5%), C. krusei (2.5%). Although the primers and enzyme had the ability to identify C. parapsilosis, C. guilliermondii, C. dubliniensis, present isolates did not include these among identified ones. CONCLUSION: RFLP-PCR using ITSI and ITS4 primers and restriction enzyme is a rapid, easy, reliable and also applicable method in clinical laboratory for identification of medically important Candida spp.

Identification of Malassezia species isolated from Iranian seborrhoeic dermatitis patients.

BACKGROUND AND OBJECTIVES: In recent years, the genus Malassezia has come to be considered important in the etiology of seborrhoeic dermatitis (SED). The aim of present study was identification of Malassezia species on the lesions of Iranian SED patients. METHODS: 100 patients with SED were enrolled in the study. The patients were evaluated both clinically for the severity of SED and microscopically for the presence of the yeast Malassezia. Diagnosis of Malassezia was made after the yeast Malassezia was microscopically observed on skin scales stained with methylene blue. All samples were also cultivated on Leeming and Notman and Sabouraud's dextrose agar culture media. The agar plates were incubated at 32 degrees C for 2 weeks and evaluated for the existence of growth every day for one week. Identification of isolated yeast was based on morphological and physiological characteristics. RESULTS: From 100 patients with SED, 60% were female. The age range was 12-65 years with median 27.3 years. The highest prevalence of SED was seen in 20-29 years age group. 59% and 41% of patients had local and generalized lesions, respectively. 58% of patients showed lesion on scalp. Microscopic examination of skin scales was positive in 100% of SED lesions. 96% of patients showed more than 1-3 yeasts in each microscopic field whereas only 4% patients showed 1-3 yeasts in whole slide. Totally, 77% of the specimens yielded Malassezia in culture. Malassezia globosa was the most commonly isolated Malassezia species (55.8%). Malassezia globosa had also most frequencies on scalp and face lesions. Malassezia furfur had most frequency on trunk lesions. CONCLUSION: The results of our study showed high recovery rate of Malassezia species on lesions of patients with SED. So it might be playing a causative role in the etiology of this disease.

Specific IgE against Alternaria alternata in atopic dermatitis and asthma patients.

Alternaria alternata (A. alternata) is one of the most common airborne fungi in outdoor and indoor environment. A. alternata has also considered as an aeroallergen. So, it could be responsible for an allergen release and may be responsible for allergic reactions in sensitive patients. The aim of this study was the analyzing of specific IgE against A. alternata in atopic dermatitis (AD) and asthma patients. A total of 50 AD patients (male 17 and female 33) and 50 asthma patients (male 20 and female 30) were entered in study. The range age vary from 4 months to 60 years. To analyzing of specific IgE, A. alternata was cultured in Sabouraud's dextrose agar. The grown fungi were harvested and ruptured by liquid nitrogen and glass beads. Samples were centrifuged at 3000rpm in 15 minutes and then at 15,500 rpm (4 degrees C) in 2 hours and then supernatant were collected as crude extract. The crude extract was separated by Sodium Dodecyl Sulfate-Polyacryl Amide Gel Electrophoresis (SDS-PAGE). The separated proteins transferred to nitrocellulose filter and then socked with atopic dermatitis and asthma patient's sera. The responsive bands to IgE were revealed by antihuman IgE antibodies conjugated with enzyme in chromogenic substrate. 16 (32%) and 19 (38%) of AD and asthma patients had specific IgE against A. alternata, respectively. Among the AD and asthma patients who were positive for specific IgE to A. alternata, 14 (87.5%) and 9 (47.4%) were women, respectively. Of the 16 AD patients for specific IgE positive, 9 (56.3%) were >12 years old. Of the 19 asthma patients for specific IgE positive, 10 (52.6%) were 20-39 years old. This study suggests that A. alternata is a major aeroallergen. Our previous studies as well as different studies from other countries have shown that A. alternata is one of the most common indoor and outdoor airborne fungi, so it could permanently present some allergens to susceptible individuals. Therefore, control of A. alternata growth in indoor areas and avoidance with A. alternata propagules could play an important role in reducing allergic reaction in susceptible individuals.