Correction to: Molecular identification of aflatoxigenic Aspergillus species in dried nuts and grains collected from Tehran, Iran.

[This corrects the article DOI: 10.1007/s40201-021-00734-6.].

Molecular identification of aflatoxigenic Aspergillus species in dried nuts and grains collected from Tehran, Iran.

Introduction: Agricultural commodities contaminated by molds and mycotoxins can be considered as public health problems in less developed countries, particularly in Iran. Hence the main purpose of this study was to identify mold fungi and molecular analysis of the most important species of aflatoxin-B1-producing Aspergillus species in some dried nuts and grains in local markets in Tehran. Materials and methods: Two hundred fifty samples of wheat, rice, corn, pistachios, and peanuts were collected from the five different locations of Tehran between January 2018 and January 2019. The samples were analyzed by using direct seed inoculation method and grain crushing method. Fungal strains were identified as Aspergillus spp. on the basis of morphological characters and further confirmed by using of ß-tubulin gene sequencing. To differentiate between aflatoxigenic and non-aflatoxigenic Aspergillus spp., the isolates were screened for the presence of aflatoxigenic genes (nor-1, ver-1, omtA, and aflR). Results: One-handed forty-eight aflatoxigenic Aspergillus isolates (144 A. flavus and 4 A. parasiticus) were identified and aflR gene was the most frequent gene in these species. Five isolates (4 A. flavus, 1 A. parasiticus) had quadruplet pattern, 64 isolates (63 A. flavus, 1 A. parasiticus) had more than 1 gene and 39 isolates (38 A. flavus,1 A. parasiticus) did not have any genes. Conclusion: According to the contamination of dried nuts and grains by some aflatoxigenic fungi, an extensive surveillance is necessary to provide a wider view on these products. Moreover, effective and efficient aflatoxin control program requires identifying and managing key elements that are effective in reducing mycotoxin production at farm level or in storage conditions.

Synthesis, Cytotoxicity Evaluation, and Antifungal Activity of Novel Nitroglycerin Derivatives against Clinical Candida albicans Isolates.

BACKGROUND: Candida albicans remains the main cause of candidiasis in most clinical settings. Available drugs for candidiasis treatment have many side effects. In this work, novel nitroglycerin derivatives were synthesized and their cytotoxic and antifungal effects evaluated against fluconazole susceptible and resistant clinical C. albicans isolates. METHODS: This experimental study was performed in Tehran University of Medical Sciences and Baqiatallah University of Medical Sciences, Tehran, Iran between Feb to Dec 2019. The in vitro activities of two novel nitroglycerin derivatives (1b and 2b) against 25 clinical fluconazole-susceptible and resistant C. albicans isolates and four standard C. albicans strains were determined according to CLSI reference M27-A3 documents. The cytotoxicity of chemical compounds was investigated near the SNL76/7 cells by colorimetric assay. Real-time PCRs were performed to evaluate the alterations in the regulation of ERG11 and CDR1 genes under nitroglycerin derivatives-treated and untreated conditions. RESULTS: The derivatives 1b and 2b exhibited potent antifungal activity against C. albicans isolates; MICs and MFCs varied from 18 µg/ml to 72 µg/ml and 36 µg/ml to 144 µg/ml, respectively. The cell viability evaluation demonstrated that both chemical compounds are safe within 24h. The nitroglycerin derivatives were able to reduce the transcription level of CDR1 and ERG11 genes in all susceptible and resistant C. albicans isolates. CONCLUSION: Considering the potential and efficacy of these compounds against clinical C. albicans isolates, the complementary in vivo and clinical trials should be investigated.

First molecular report of causative agent of otomycosis due to Aspergillus luchuensis.

Otomycosis is a fungal infection of the external auditory canal caused mainly by the genus Aspergillus. Aspergillus luchuensis, an industrially important fungus, is a member of Aspergillus section Nigri. In this report, we present a case of otomycosis due to Aspergillus luchuensis in a 43-year-old female patient. We performed a partial PCR-sequencing of ß-tubulin and calmodulin genes to identify the isolate to the species level. Further, we determined the in vitro susceptibility of the isolate to nystatin, clotrimazole and itraconazole according to the Clinical & Laboratory Standards Institute (CLSI) M38-A2 protocol. Accordingly, the minimum inhibitory concentrations of clotrimazole, nystatin and itraconazole were 0.25µg/mL, 0.5µg/mL and 1µg/mL, respectively. This is the first report of clinically relevant isolation of Aspergillus luchuensis identified by a molecular technique as a causative agent of otomycosis.

Unveiling the structure of GPI-anchored protein of Malassezia globosa and its pathogenic role in pityriasis versicolor.

Glycosylphosphatidylinositols (GPI)-anchored proteins (GpiPs) are related to the cell wall biogenesis, adhesion, interactions, protease activity, mating, etc. These proteins have been identified in many organisms, including fungi such as Neurospora crassa, Candida albicans, Saccharomyces cerevisiae, and Fusarium graminearum. MGL-3153 gene of Malassezia globosa (M. globosa) encodes a protein which is homologous of the M. restricta, M. sympodialis, M. Pachydermatis, and U. maydis GpiPs. Real-time PCR assay showed that the expression of MGL_3153 gene was significantly up-regulated among M. globosa isolated from patients with pityriasis versicolor (PV) compared to a healthy individual, suggesting the contribution of this gene in the virulence of M. globosa. Accordingly, the sequence of this protein was analyzed by bioinformatics tools to evaluate the structure of that. The conservation analysis of MGL-3153 protein showed that the C-terminal region of this protein, which is responsible for GPI-anchor ligation, was highly conserved during evolution while the N-terminal region just conserved in Malassezia species. Moreover, the predicted tertiary structure of this protein by homology modeling showed that this protein almost has alpha helix structure and represented a stable structure during 150 ns of molecular dynamic simulation. Our results revealed that this protein potentially belongs to GPI-anchored proteins and may contribute to the virulence of M. globosa which warrants further investigations in this area.

Virulence Factors and Azole-Resistant Mechanism of Candida Tropicalis Isolated from Candidemia.

BACKGROUND: Limited knowledge exists on the virulence factors of Candida tropicalis and the mechanisms of azole resistance that lead to an intensified pathogenicity and treatment failure. We aimed to evaluate the virulence factors and molecular mechanisms of azole resistance among C. tropicalis isolated from patients with candidemia. MATERIALS AND METHODS: Several virulence factors, including extracellular enzymatic activities, cell surface hydrophobicity (CSH), and biofilm formation, were evaluated. Antifungal susceptibility pattern and expression level of ERG11, UPC2, MDR1, and CDR1 genes of eight (4 fluconazole resistance and 4 fluconazole susceptible) clinical C. tropicalis isolates were assessed. The correlation between the virulence factors and antifungal susceptibility patterns was analyzed. RESULTS: During a 4 year study, forty-five C. tropicalis isolates were recovered from candidemia patients. The isolates expressed different frequencies of virulence determinants as follows: coagulase 4 (8.9%), phospholipase 5 (11.1%), proteinase 31 (68.9%), esterase 43 (95.6%), hemolysin 44 (97.8%), biofilm formation 45 (100%) and CSH 45(100%). All the isolates were susceptible to amphotericin B and showed the highest resistance to voriconazole. There was a significant positive correlation between micafungin minimum inhibitory concentrations (MICs) and hemolysin production (rs = 0.316). However, we found a negative correlation between fluconazole MICs and esterase production (rs = -0.383). We observed the high expression of ERG11 and UPC2 genes in fluconazole-resistant C. tropicalis isolates. CONCLUSION: C. tropicalis isolated from candidemia patients extensively displayed capacities for biofilm formation, hemolysis, esterase activity, and hydrophobicity. In addition, the overexpression of ERG11 and UPC2 genes was considered one of the possible mechanisms of azole resistance.

Dysregulation of Key Proteinases in Aspergillus fumigatus Induced by Blood Platelets.

BACKGROUND: Aspergillus fumigatus is the most common species causing invasive aspergillosis (IA), a life-threatening infection with more than 80% mortality. Interactions between A. fumigatus and human blood platelets lead to intravascular thrombosis and localized infarcts. To better understand A. fumigatus pathogenesis, we aimed to analyze the genetic basis of interactions between the pathogen and blood platelets. METHODS: A bioinformatic pipeline on microarray gene expression dataset, including analysis of differentially expressed genes (DEGs) using Limma R package and their molecular function, as well as biological pathways identification, was conducted to find the effective genes involved in IA. In the wet phase, the gene expression patterns following fungal exposure to blood platelets at 15, 30, 60, and 180 min were evaluated by quantitative reverse transcriptase-PCR analysis. RESULTS: Three genes encoding aspartic endopeptidases including (Pep1), (Asp f 13), and (ß-glucanase) were the standing candidates. The invasion-promoting fungal proteinase-encoding genes were down-regulated after 30 min of hyphal incubation with blood platelets, and then up-regulated at 60 and 180 min, although only Pep1 was greater than the control at the 60and 180 min time points. Also, the same genes were downregulated in more the clinical isolates relative to the standard strain CBS 144.89. CONCLUSION: Our findings delineate the possible induction of fungal-encoded proteinases by blood platelets. This provides a new research line into A. fumigatus' molecular pathogenesis. Such insight into IA pathogenesis might also guide researchers toward novel platelet-based therapies that involve molecular interventions, especially in IA patients.

Eco-friendly control of licorice aqueous extract to increase quality and resistance to postharvest decay in apple and tangerine fruits.

BACKGROUND: Postharvest diseases in fruits and vegetables are one of the major problems in storing them as a fresh agri-product. This study aimed to investigate the antifungal activity of licorice (Glycyrrhiza glabra) aqueous extract against the Penicillium expansum and the Penicillium digitatum in apple and tangerine fruits as well as their postharvest decay during storage time. METHODS: The minimum inhibitory concentration (MIC) of the molds, and the decay inhibition percentage (%DI) with the P.expansum for apple and P.digitatum for tangerine after treatment with licorice aqueous extract were measured. Additionally, the lesion diameter, titratable acidity (TA), total soluble solids (TSS), pH, and organoleptic properties were determined. RESULTS: The growth of molds was almost inhibited at the concentration of 62.5 mg/mL. The ability of licorice aqueous extract to significantly control and reduce the growth of P. expansum in apple by 60 and 20 % after 7 days and 21 days of storage time was proved, respectively. Furthermore, significant differences in pH and TSS (p < 0.05) were observed in apples. Also, the growth of P. digitatum in the tangerine reduced by 33.3 % after 7 days, while there was no significant difference between the control and treatment groups in pH and TSS for apples, and similarly, there was no significant difference in TA for tangerine samples. CONCLUSIONS: Therefore, the licorice aqueous extract treatment could postpone the blue mold decay in apple fruits and green mold decay in tangerine without any significant effect on fruit quality characteristics. It can be considered as a new eco-friendly control in fruit preservation, while it did not result in any significant adverse effect on  the quality.

Molecular identification and clinical features of fungal rhinosinusitis: A 3-year experience with 108 patients.

This study aimed to determine the prevalence, the causative agents, clinical features, and the risk factors associated with the fungal rhinosinusitis in a tertiary health center with a view to providing valid grounds that may guide healthcare professionals to effectively prevent, control, and treat fungal infections. All patients were subjected to diagnostic nasal endoscopy and CT scan of paranasal sinuses and FRS were confirmed by routine and complementary mycological and molecular methods. The inclusion criteria for invasive FRS were: confirmed diagnosis of IFRS according to the guidelines of the EORTC/MSG criteria (i.e., clinical, microbiological, and histological evidence of invasive fungal infection). From a total of 512 suspected patients, FRS was confirmed in 108 cases (21.1%). Our results showed FB (38/108; 35.2%) is the most common form of FRS followed by AIFRS (33/108; 30.6%), AFS (32/108; 29.6%), and CIFRS (5/108; 4.6%). A. flavus and Rhizopus oryzae were the most common causes of infection in AFS, FB, CIFRS, and AIFRS, respectively. Univariate analysis of variables predictive of AIFRS revealed 3 variables significantly associated with AIFRS. These included mucosal abnormalities of the middle turbinate and septum, and specifically, necrosis of the middle turbinate (P < .0001). Microbiological cultures, although useful for mycological speciation, are less sensitive. Furthermore, we used molecular methods to confirm the identity of some isolates that were not detectable using routine methods. Our data showed that the molecular methods and histologic diagnosis in all patients were more sensitive than the unenhanced sinus CT scan, and conventional microbiological methods.

The relationship between biofilm formation and mortality in patients with Candida tropicalis candidemia.

BACKGROUND: Biofilm formation by Candida species is an influential virulence factor in candidemia pathogenesis. We investigated the relationship between biofilm formation of Candida tropicalis isolates with the clinical characteristics and mortality outcomes in patients with candidemia. MATERIALS AND METHODS: Thirty-nine C. tropicalis isolates were recovered from patients with candidemia admitted to two university hospitals in Tehran, Iran. Biofilm mass and metabolic activity of C. tropicalis biofilms were assessed in vitro with two colorimetric methods. The sessile minimum inhibitory concentrations (SMICs) were evaluated in vitro by treating preformed biofilms with diluted concentrations of azoles according to CLSI-M27 A3/S4 protocol, followed by metabolic activity quantification. The expressions of ERG11, UPC2, MDR1, and CDR1 genes were also evaluated. RESULTS: All C. tropicalis isolates produced biofilm. Respectively, higher <7-day and ≥7-day mortality rates were found among cases with high metabolic activity (46.7% vs. 13%, P = 0.03) and high biofilm mass (31.8% vs. 0, P = 0.029). Sessile cells had high resistance to fluconazole, voriconazole, and itraconazole. The azole minimum inhibitory concentrations (MICs) of C. tropicalis sessile were significantly greater than the planktonic minimum inhibitory concentrations (PMICs). In fluconazole-treated biofilms, the expression of ERG11 and UPC2 genes was increased. CONCLUSION: Our findings highlight the importance of C. tropicalis biofilm formation as an important factor in candidemia pathogenesis and the clinical outcome of patients with candidemia.

The double-edged sword of systemic corticosteroid therapy in viral pneumonia: A case report and comparative review of influenza-associated mucormycosis versus COVID-19 associated mucormycosis.

Acute respiratory distress syndrome is a common complication of severe viral pneumonia, such as influenza and COVID-19, that requires critical care including ventilatory support, use of corticosteroids and other adjunctive therapies to arrest the attendant massive airways inflammation. Although recommended for the treatment of viral pneumonia, steroid therapy appears to be a double-edged sword, predisposing patients to secondary bacterial and invasive fungal infections (IFIs) whereby impacting morbidity and mortality. Mucormycosis is a fungal emergency with a highly aggressive tendency for contiguous spread, associated with a poor prognosis if not promptly diagnosed and managed. Classically, uncontrolled diabetes mellitus (DM) and other immunosuppressive conditions including corticosteroid therapy are known risk factors for mucormycosis. Upon the background lung pathology, immune dysfunction and corticosteroid therapy, patients with severe viral pneumonia are likely to develop IFIs like aspergillosis and mucormycosis. Notably, the combination of steroid therapy and DM can augment immunosuppression and hyperglycaemia, increasing the risk of mucormycosis in a susceptible individual. Here, we report a case of sinonasal mucormycosis in a 44-year-old woman with hyperglycaemia secondary to poorly controlled diabetes following dexamethasone therapy on a background of influenza pneumonia and review 15 available literatures on reported cases of influenza and COVID-19 associated mucormycosis.

Evaluation of phenotypes and genotypes of airborne Fungi during middle eastern dust storms.

Microbial species such as bacteria and fungi can be transported by dust storms over long distances, and may change the mycobiota in downwind. This study aimed to evaluate phenotypes and genotypes of airborne fungi during the Middle Eastern dust (MED) events and normal days in Khorramabad, Iran. The samples were collected regularly every six days at three locations during April 2018-March 2019, with additional samplings during MED days. For phenotypic analyses, the Petri dishes were incubated at 25 °C for 72-120 h. Molecular identification of fungi was carried out using polymerase chain reaction (PCR). The average (±SD) of total fungal concentration was 460.9 (±493.2) CFU/m3. The fungi with the highest average concentrations included Cladosporium cladosporioides, Penicillium brevicompactum, and Cladosporium iridis, respectively. The average concentration of fungi during dust days (967.65 CFU/m3) was 3.6 times higher than those in normal days (267.10 CFU/m3). During normal and dust days, 61 and 45 species were detected, respectively. Aspergillus and Cladosporium spp. were relatively more dominant during normal and dust days, respectively. Eight fungal species were only observed during MED days, including Talaromyces albobiverticillius that was detected for the first time in Iran. Despite air temperature, relative humidity and wind speed were associated to the fungal concentrations. Dust events lead to the changes in the air pollutants composition and mycobiota, identification of new fungi, and elevated fungal concentrations that may extremely affect the public health.

In vitro synergy of echinocandins with triazoles against fluconazole-resistant Candida parapsilosis complex isolates.

INTRODUCTION: Candida parapsilosis (C. parapsilosis) is a common non-albicans Candida species ranked as the second common cause of bloodstream infections. Azole resistance and elevated echinocandin MICs have been reported for these fungi. This study was conducted to determine the interactions between azoles and echinocandins against C. parapsilosis species complex. MATERIALS AND METHODS: Fifteen fluconazole-resistant clinical isolates of C. parapsilosis complex were included: C. parapsilosis sensu stricto (n = 7), C. orthopsilosis (n = 5) and C. metapsilosis (n = 3). The activity of azoles (fluconazole, itraconazole) and echinocandins (anidulafungin, micafungin) alone and in combination was determined using checkerboard broth microdilution. The results were determined based on the fractional inhibitory concentration index (FICI). RESULTS: In vitro combination of fluconazole with anidulafungin was found to be synergistic (FICI 0.07-0.37) and decreased the MIC range from 4-64 µg/mL to 0.5-16 µg/mL for fluconazole and from 2-8 µg/mL to 0.125-1 µg/mL for anidulafungin. Similarly, interactions of fluconazole with micafungin (FICI 0.25-0.5), itraconazole with anidulafungin (FICI 0.15-0.37) and itraconazole with micafungin (FICI 0.09-0.37) were synergistic. CONCLUSION: The combination of fluconazole and itraconazole with either anidulafungin or micafungin demonstrated synergistic interactions against C. parapsilosis species complex, especially against isolates with elevated MIC values. However, the use of these combinations in clinical practice and the clinical relevance of in vitro combination results remain unclear.

Investigation of cgrA and cyp51A gene alternations in Aspergillus fumigatus strains exposed to kombucha fermented tea.

BACKGROUND AND PURPOSE: Aspergillus fumigatus is one of the most common opportunistic fungus, which causes infection in immunocompromised and neutropenic patients. The current guidelines recommend voriconazole as the initial therapeutic and prophylactic agent for almost all cases, especially in patients with organ transplants, which leads to increased medication resistance in A. fumigatus. The aim of the present study was to evaluate the antifungal activity and effect of kombucha as a natural compound on A. fumigatus growth, as well as on the expression of cgrA and cyp51A genes. MATERIALS AND METHODS: A panel of 15 A. fumigatus strains with two quality controls of CM237 and CM2627 as susceptible and resistant strains were obtained from Tehran Medical Mycology Laboratory, Tehran,Iran(TMML).Antifungal susceptibility testing assay was performed according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. Moreover, the mycelial dry weight of the fungus was calculated before and after being treated with kombucha. In addition, the quantitative changes in the expression of cgrA and cyp51A genes were analyzed by real-time polymerase chain reaction (real-time PCR) technique. RESULTS: In the present study, the minimum inhibitory concentration ranges of kombucha were measured at 6,170 and 12,300 µg/mL for ten A. fumigatus azole-susceptible strains and 24,700 µg/mL for five A. fumigatus resistant strains. Moreover, changes in mycelial dry weight under kombucha treatment conditions underwent a significant reduction (P≤0.05). A coordinate down-regulation of expression in cgrA and cyp51A genes was observed in all azole-susceptible and -resistant A. fumigatus strains, after treating the fungus with different concentrations of kombucha (P≤0.05). CONCLUSION: According to the obtained results, kombucha as a natural antioxidant , can exert inhibitory effects against the growth and expression of some genes in A. fumigatusstrains.

In Vitro Interaction of Geldanamycin with Triazoles and Echinocandins Against Common and Emerging Candida Species.

The aim of this study was to determine the in vitro interactions of geldanamycin (Hsp90-inhibitor) with triazoles and echinocandins against common and emerging Candida species. Twenty clinically important Candida strains comprising C. auris, C. albicans, C. parapsilosis, and C. glabrata (each five strains) were included. In vitro interactions of geldanamycin with fluconazole, itraconazole, caspofungin and anidulafungin were determined using a checkerboard method. The results were interpreted as synergistic, indifferent and antagonistic based on the fractional inhibitory concentration index (FICI). In vitro combination of fluconazole with geldanamycin resulted in synergistic effect against C. albicans (100%), C. glabrata (80%) and C. parapsilosis (80%) (FICI range 0.009-0.5), while indifferent interactions were obtained against C. auris (FICI range 1.5-2). The overall minimum inhibitory concentration (MIC) range of fluconazole against C. albicans, C. glabrata and C. parapsilosis reduced from 16-256 to 0.25-64 mg/L when combined with geldanamycin. Regarding the synergistic effect of geldanamycin with itraconazole against all strains of C. albicans, C. glabrata and C. parapsilosis (FICI range 0.009-0.375), the MIC range of this antifungal was reduced from 0.125-32 mg/L when tested alone, to 0.03-1 mg/L. Combinations of geldanamycin with fluconazole and itraconazole against C. auris, as well as combination of geldanamycin with caspofungin and anidulafungin against all studied Candida species, resulted in indifferent effects. No antagonism was observed. Simultaneous targeting of Hsp90 and lanosterol 14-α demethylase seems an effective approach against C. albicans, C. glabrata and C. parapsilosis. However, this combination is ineffective against the emerging pathogen C. auris.

Influence of postharvest application of chitosan combined with ethanolic extract of liquorice on shelflife of apple fruit.

BACKGROUND: Edible coatings are useful method that applied to preserve postharvest quality of production. The coatings can extend the shelf life of products and inhibit microbial growth. Chitosan based coatings are one of the best methods to prolong fruit and vegetable shelf life. The antimicrobial and other properties of chitosan are developed when it is combined with other functional ingredients. METHODS: The effectiveness of chitosan, ethanolic extract of liquorice (LE) and complex of chitosan-liquorice extract (CHLE) was evaluated for controlling blue mold and extending shelf life in apples. The fruits were coated with chitosan(1.0%), LE (62.5 mg/ml) and CHLE coating, and stored at 25 °C. Quality properties of fruit (such as weight loss, firmness, total soluble solid content(TSS), titrable acidity and pH) and decay incidence were assessed on 0,1,4,7 and 14 days of incubation, respectively. RESULTS: The results of experiments indicated that minimum of water loss(3.8%), TSS(14.53) and firmness(5.6 kg/cm2 ) were in CHLE coated apples. In addition, this coating significantly inhibited penicillium expansum during the storage and the lowest decay incidence was for apples coated with CHLE(29 mm). Chitosan and LE coating retarded undesirable changes during postharvest storage and inhibited decay incidence compared with uncoated samples. There was no significant difference (p ≤ 0/05) between treatments and control overtime in terms of titrable acidity and pH levels. CONCLUSIONS: The results reported here indicate importance and efficacy of CHLE coating in extending shelflife and reduction of postharvest losses of apple in storage time.

Corrigendum: Comparison of 21-Plex PCR and API 20C AUX, MALDI-TOF MS, and rDNA Sequencing for a Wide Range of Clinically Isolated Yeast Species: Improved Identification by Combining 21-Plex PCR and API 20C AUX as an Alternative Strategy for Developing Countries.

[This corrects the article DOI: 10.3389/fcimb.2019.00021.].

Investigation of Etiologic Agents and Clinical Presentations of Otomycosis at a Tertiary Referral Center in Tehran, Iran.

BACKGROUND: Otomycosis is a superficial infection of the ear caused by a spectrum of various fungal agents and its epidemiology depends on geographical region and climatic condition. The aim of this study was to investigate the causal agents and clinical manifestations of otomycosis at a tertiary referral center in Tehran, Iran. METHODS: From Apr 2016 to Jan 2017 a set of 412 subjects with suspicion of external otitis were included. Clinical examination and specimen collection were performed by an otorhinolaryngologist. Subsequently, direct examination and culture were performed on specimens and isolated molds were identified morphologically. Yeast isolates were identified using CHROMagar Candida medium and PCR-RFLP of ribosomal DNA whenever needed. Data were analyzed using SPSS. RESULTS: Otomycosis was confirmed in 117 cases (28.39%) including 64 (54.7%) males and 53 (45.3%) females. Patients were within the age range of 10-75 yr and the highest prevalence was found in the age group of 46-55 yr (30.77%). Pruritus (89.74%) and auditory manipulation and trauma (83.76%) were the predominant symptom and predisposing factor, respectively. Among 133 isolates from 117 patients, Aspergillus niger (n=50, 37.59%) was the most common etiologic agent and Candida glabrata (n=25, 18.8%) was the predominantly isolated yeast. Furthermore, 16 cases of mixed infection were identified and coinfection due to A. niger and C. glabrata (seven cases) was the predominant pattern. CONCLUSION: Our results revealed the high prevalence of C. glabrata and mixed infections in otomycosis patients. Therefore, mycological examinations should be considered for proper treatment.

A simple and low cost tetra-primer ARMS-PCR method for detection triazole-resistant Aspergillus fumigatus.

The mutation at codon L98 accompanied by a tandem repeat of 34 base pairs (TR34/L98H) in the 5´upstream region of cyp51A is the principal mechanism of triazole resistance of Aspergillus fumigatus. We aimed to evaluate a simple and low-cost tetra-primer amplification refractory mutation system (ARMS)-PCR technique for detection of TR34/L98H mutations in the cyp51A gene of azole-resistant A. fumigatus. The tetra-primer ARMS-PCR assay optimized by four primers in one reaction consists of external primers for detection of tandem repeats in the promoter region and internal primers for detection of a point mutation in codon 98 (L98H) in the cyp51A gene of azole-resistant A. fumigatus. The specificity of TR34/L98H mutation detection was assessed by testing 36 clinical and environmental A. fumigatus strains. The tetra-primer ARMS-PCR assay from A. fumigatus, containing wild-type sequence (T allele) and L98H mutation at cyp51A (A allele), yielded two DNA fragments of 908 bp and 740 bp and two of 942 bp and 212 bp, respectively. None of the A. fumigatus isolates without the TR34/L98H mutation yielded false-positive results. The ARMS-PCR assay was 100% concordant with DNA sequencing results. Prevalence and screening of the TR34/L98H mutation in the cyp51A gene in A. fumigatus isolates may now be determined by a fast, low-cost, and simple method in resource-poor settings.

Optimization and Comparison of Different Methods and Factors for Efficient Transformation of Brucella abortus RB51strain.

BACKGROUND: The development of protective vaccines for Brucella spp. has been hampered by the difficulty in transformation of Brucella cells with foreign DNA for genetic manipulation. It seems that the formation of Brucella spheroplasts would increase the efficiency of transformation. The aim of this study was to devise an efficient method for the transformation of Brucella spp. MATERIALS AND METHODS: At first, spheroplast of Brucella was prepared by glycine and ampicillin induction and transformed using optimized protocols of CaCl2, electroporation, and lipofection methods. Then, the efficacy of transformation was compared between the three-mentioned methods. RESULTS: Ampicillin-induced spheroplasts from early-log phase culture of brucella when incubated in a medium-containing 0.2 M sucrose during cell recovery had higher transformation efficiency in three different methods. Comparison of the transformation efficiency of Brucella abortus RB51 using the CaCl2, lipofection, and electroporation methods revealed that the transformation efficiency with the lipofection method was significantly higher than with other two methods (P < 0.05). CONCLUSIONS: Lipofection method by lipofectamine 2000 on ampicillin-induced spheroplasts can be a suitable approach for Brucella transformation.