BACKGROUND:The resistance of Aspergillus flavus to the azole antifungal drugs is an emerging problem. Mutations in the molecular targets of the azole antifungals - CYP 51 A, B and C - are possible mechanisms of resistance, but data to confirm this hypothesis are scarce. In addition, the behaviour of resistant strains in vitro and in vivo is not yet understood. OBJECTIVES:This study had 3 objectives. The first was to compare the sequences of CYP51 A, B and C in resistant and susceptible strains of A. flavus. The second was to look for the existence of a fitness cost associated with resistance. The third was to evaluate the activity of voriconazole and posaconazole on resistant strains in the Galleria mellonella model. METHODS:The CYP51 A, B and C sequences of seven resistant strains with those of four susceptible strains are compared. Fitness costs were assessed by growing the strains in RPMI medium and testing their virulence in G. mellonella larvae. In addition, G. mellonella larvae infected with strains of A. flavus were treated with voriconazole and posaconazole. RESULTS:In the CYP51A sequences, we found the A91T, C708T and A1296T nucleotide substitutions only in the resistant strains. The resistant strains showed a fitness cost with reduced in vitro growth and reduced virulence in G. mellonella. In vivo resistance to posaconazole is confirmed in a strain with the highest MIC for this antifungal agent. CONCLUSIONS:These results allow to conclude that some substitutions in CYP51 genes, in particular CYP51A, contribute to resistance to azole drugs in A. flavus. The study of the relationship between drug dosage and treatment duration with resistance and the reduction of fitness costs in resistant strains is a major perspective of this study. This work could help to establish recommendations for the treatment of infections with resistant strains of A. flavus.

(1) Background: Aspergillus flavus is a cosmopolitan mold with medical, veterinary, and agronomic concerns. Its morphological similarity to other cryptic species of the Flavi section requires molecular identification techniques that are not routinely performed. For clinical isolates of Aspergillus section Flavi, we present the molecular identification, susceptibility to six antifungal agents, and clinical context of source patients. (2) Methods: One hundred forty fungal clinical isolates were included in the study. These isolates, recovered over a 15-year period (2001–2015), were identified based on their morphological characteristics as belonging to section Flavi. After the subculture, sequencing of a part of the β-tubulin and calmodulin genes was performed, and resistance to azole antifungals was screened on agar plates containing itraconazole and voriconazole. Minimum inhibitory concentrations were determined for 120 isolates by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. (3) Results: Partial β-tubulin and calmodulin sequences analysis showed that 138/140 isolates were A. flavus sensu stricto, 1 isolate was A. parasiticus/sojae, and 1 was A. nomiae. Many of the isolates came from samples collected in the context of respiratory tract colonization. Among probable or proven aspergillosis, respiratory infections were the most frequent, followed by ENT infections. Antifungal susceptibility testing was available for isolates (n = 120, all A. flavus ss) from one hospital. The MIC range (geometric mean MIC) in mg/L was 0.5–8 (0.77), 0.5–8 (1.03), 0.125–2 (0.25), 0.03–2 (0.22), 0.25–8 (1.91), and 0.03–0.125 (0.061) for voriconazole, isavuconazole, itraconazole, posaconazole, amphotericin B, and caspofungin, respectively. Two (1.67%) isolates showed resistance to isavuconazole according to current EUCAST breakpoints with MICs at 8 mg/L for isavuconazole and voriconazole. One of these two isolates was also resistant to itraconazole with MIC at 2 mg/L. (4) Conclusions: The present characterization of a large collection of Aspergillus belonging to the Flavi section confirmed that A. flavus ss is the predominant species. It is mainly implicated in respiratory and ENT infections. The emergence of resistance highlights the need to perform susceptibility tests on section Flavi isolates.

Abstract Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectives Aspergillus flavus and closely related species could be pathogenic for humans, animals, and plants and could also produce mycotoxins. The members of the Flavi section are morphologically quite similar making precise identification to the species level difficult. In this study, we present the antifungal susceptibility profiles of French clinical isolates belonging to the Flavi section. Isolates have been characterized by molecular methods and the potential fitness-cost associated with azole-resistance has been determined. Methods A total of 120 isolates phenotypically identified as A. flavus were included in the study. These clinical isolates were recovered over a 15-year period (2001-2015). For all isolates, specific identification was confirmed by sequencing a part of the β-tubulin and calmodulin genes. The isolates were first screened for their susceptibility to azoles antifungal agents by using 3-sectors agar plates containing itraconazole, voriconazole, and a drug-free control. Susceptibility to six antifungal drugs was further determined by using the EUCAST reference microdilution broth technique. Fitness cost was evaluated by growth curve kinetics in RPMI and by evaluation of virulence in a Galleria mellonella invertebrate animal model. Results Out of 120 isolates, molecular analysis of the partial β-tubulin and calmodulin sequences showed that 117 isolates were A. flavus sensu stricto and the three remaining corresponded to A. parasiticus, A. nomius, and A. tamarii. Two isolates were azole-resistant by the screening test. For the A. flavus sensu stricto isolates, the geometric mean MIC values (range) of amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole, and caspofungin were 1.84 (0.25-16), 0.29 (0.125-2), 0.82 (0.5-8), 0.27 (0.06-2), 1.15 (0.25-8), and 0.061 (0.03-0.125) μg/ml, respectively. For A. parasiticus, A. nomius, and A. tamarii, MICs were in the same range. Two A. flavus sensu stricto isolates (AfR1 and AfR2) had voriconazole and isavuconazole MICs at 8 μg/ml. Compared to susceptible isolates, these two azole-resistant isolates had a delayed growth in RPMI liquid medium. In the G. mellonella model, the mortality was 100% for susceptible isolates. In contrast, the Galleria infected by AfR1 and AfR2 showed a significantly lower mortality rate. Conclusion Antifungal susceptibility to six drugs was determined on a large collection of clinical isolates belonging to Aspergillus Flavi section. Most of the isolates were identified as A. flavus sensu stricto and most of them were susceptible to antifungal drugs. Nevertheless, the occurrence of two resistant isolates highlights the need for susceptibility testing for A. flavus. It seems that azole-resistance is associated with a fitness-cost including a lower growth rate and a lower virulence.

Mucormycosis is a life-threatening invasive fungal disease that affects a variety of patient groups. Although Mucorales are mostly opportunistic pathogens originating from soil or decaying vegetation, there are currently few data on prevalence of this group of fungi in the environment. The aim of the present study was to assess the prevalence and diversity of species of Mucorales from soil samples collected in France. Two grams of soil were homogenized in sterile saline and plated on Sabouraud dextrose agar and RPMI agar supplemented with itraconazole or voriconazole. Both media contained chloramphenicol and gentamicin. The plates were incubated at 35 ± 2 °C and checked daily for fungal growth for a maximum of 7 d. Mucorales were subcultured for purity. Each isolate was identified phenotypically and molecular identification was performed by ITS sequencing. A total of 170 soil samples were analyzed. Forty-one isolates of Mucorales were retrieved from 38 culture-positive samples. Among the recovered isolates, 27 Rhizopus arrhizus, 11 Mucor circinelloides, one Lichtheimia corymbifera, one Rhizopus microsporus and one Cunninghamella bertholletiae were found. Positive soil samples came from cultivated fields but also from other types of soil such as flower beds. Mucorales were retrieved from samples obtained in different geographical regions of France. Voriconazole-containing medium improved the recovery of Mucorales compared with other media. The present study showed that pathogenic Mucorales are frequently recovered from soil samples in France. Species diversity should be further analyzed on a larger number of soil samples from different geographic areas in France and in other countries.

In vitro interaction of voriconazole with micafungin was evaluated against 33 clinical Aspergillus fumigatus isolates, including azole-resistant (n=31) and -susceptible (n=2) isolates. Interaction was synergistic for only 1 resistant isolate carrying the TR34/L98H mutation. No antagonistic effects were observed for 96.8% of azole-resistant isolates.

Mucormycoses are life-threatening fungal diseases that affect a variety of patients including those with diabetes mellitus or hematological malignancies. The responsible agents, the Mucorales, are opportunistic pathogens originating from the environment such as soil or decaying organic matter. The aim of the present study was to assess the prevalence and diversity of human-pathogenic species of Mucorales in commercially available foodstuffs in France. All food samples were purchased from January 2014 to May 2015 in France. A total of 159 dried food samples including spices and herbs (n = 68), herbal tea (n = 19), cereals (n = 19), vegetables (n = 14), and other foodstuffs (n = 39) were analyzed. Each strain of Mucorales was identified phenotypically, and molecular identification was performed by ITS sequencing. From the 28 (17.6%) samples that were culture-positive for Mucorales, 30 isolates were recovered. Among the isolates, 13 were identified as Rhizopus arrhizus var. arrhizus, 10 R. arrhizus var. delemar, two Rhizopus microsporus, one Lichtheimia corymbifera, three Lichtheimia ramosa, and one Syncephalastrum racemosum. Culture-positive samples originated from different countries (Europe, Asia) and brands. The samples most frequently contaminated by Mucorales were spices and herbs (19/68, 27.9%), followed by herbal tea (2/19, 10.5%), cereals (2/19, 10.5%), other food products (5/39, 12.8%). The present study showed that human-pathogenic Mucorales were frequently recovered from commercially available foodstuffs in France with a large diversity of species. The potential danger represented by Mucorales present in food for immunocompromised patients should be further analyzed.

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.

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.

SummaryAn increasing number of reports have described the emergence of acquired resistance of Aspergillus fumigatus to azole compounds. The primary mechanism of resistance in clinical isolates is the mutation of the azole drug target enzyme, which is encoded by the cyp51A gene. The aim of this study was to evaluate the impact of silencing the cyp51A gene in azole‐resistant A. fumigatus isolates. A 21‐nucleotide small‐interfering RNA (siRNA) was designed based on the cDNA sequence of the A. fumigatus cyp51A gene. After silencing the cyp51A gene in germinated conidia (15, 20, 25 and 50 nM), azole‐resistant A. fumigatus was cultured on broth media and gene expression was analysed by measuring the cyp51A mRNA level using RT‐PCR assay. Hyphae were successfully transfected by siRNA and expression of the cyp51A gene was significantly reduced by siRNA at the concentration of 50 nM (P ≤ 0.05). In addition, at this siRNA concentration, the minimum inhibitory concentration of itraconazole for the treated cells was decreased, compared with that for untreated control cells, from 16 to 4 μg/ml.

Triazole antifungal drugs such as itraconazole, posaconazole and voriconazole are the mainstay of therapy in the management and prophylaxis of aspergillosis. However, in recent years, triazole-resistant clinical isolates of Aspergillus fumigatus have emerged in Europe (the Netherlands, Denmark, Spain, UK, Belgium, Germany and France) and Asia (China, India) resulting in several cases of therapeutic failure caused by triazole-resistant A. fumigatus. Azole resistance may develop in patients who are treated with long-term azole therapy or may develop in the environment through the exposure of the fungus to the azole fungicides used in agriculture. In the present study, we investigated the presence of azole resistance in environmental and clinical A. fumigatus isolated both from hospital surroundings area and infected patients. In vitro antifungal susceptibility testing has shown high MICs of itraconazole (> 16 mg/L) and voriconazole (> 8 mg/L). The TR34/L98H mutation was the only resistance mechanism with no mutation in G54, M220, G138C, T289A, Y121F and G432C. Azole-resistant A. fumigatus isolates had the same TR34/L98H STRAf genotype with Netherlands and India. It is emphasized that routine resistance surveillance studies focusing on environmental and clinical samples are warranted to yield the true prevalence of azole resistance in A. fumigatus in Iran. The widespread application of triazole fungicides in the environment in Iran could have contributed to the emergence of environmental azole-resistant A. fumigatus. Given the emergence of azole resistance in environmental strains, continued surveillance of resistance in clinical A. fumigatus strains in Iran is desirable for successful therapy of aspergillosis. Furthermore, the need for enhanced understanding of the evolution of azole resistance and measures to prevent the emergence of multiple-azole-resistant A. fumigatus strains in countries using fungicides can hardly be over-emphasized.

Background and purpose: Aspergillus is a ubiquitous fungus, which causes a wide spectrum of infections including invasive pulmonary aspergillosis (IPA), depending on the patient's immune status and underlying lung disease. Among the Aspergillus spp, A. fumigatus remains the predominant agent of IPA. In patients with a preexisting lung cavity from a variety of causes, such as pulmonary tuberculosis (TB) Aspergillus can colonize and grow into the cavity to form a pulmonary aspergilloma. In this present study we assessed TB patients for aspergilloma based on culture and non-culture based methods Materials and methods: During one year, we studied 124 patients with TB at Massih Daneshvari hospital from Tehran, Iran. Sputum specimens were analyzed by direct microscopic examination (DME) with 20% potassium hydroxide. These samples were also processed for fungal culture. The clinical and radiological features or CT-scan report of all patients were recorded. All patients were screened for serum specific IgG against A. fumigatus, using Aspergillus IgG ELISA Kit (Genesis Diagnostics Ltd, Cambridgeshire, UK). The results are expressed in U/mL. IgG 12 U/mL was considered as positive result based on kit manufacturer instruction. Results: Out of 124 patients with tuberculosis (66 male, 58 female, age range: 10-91 years), 54 had abnormal chest radiographic findings. Chest X-ray findings showed that 48 patients (38.7%) exhibited residual cavities (31 cases in right lobe, 10 in left lobe and 7 in both lung). Round shaped mass lesion was detected only in 6 patients (6.8%). DME of sputum was positive in 10 patients for septate fungal hyphae. A. fumigatus was grown from 14 samples of TB patients. Out of 124 TB patients, 55(44.3%) cases were positive for specific serum IgG against A. fumigatus. There was a significant relationship between positive culture, DME and serum IgG profile level (P< 0.05). Totally, three patients (2.4%) met criteria for aspergilloma Conclusion: Colonization with Aspergillus in preexisting lung cavity produced by TB should be considered as a risk factor for aspergilloma

Azole resistance in Aspergillus is emerging in European and Asian countries. As azoles are mainstay of therapy in the management of aspergillosis, azole resistance has serious implications in patient management. We report the emergence of resistance to triazoles in environmental Aspergillus fumigatus isolates in Iran. The TR34/L98H mutation was the only resistance mechanism. Overall 3.3% of the A. fumigatus isolates from hospital surroundings in Sari and Tehran had the same TR34/L98H STRAf genotype and were related to some resistant clinical and environmental TR34/L98H isolates from the Netherlands and India. It is emphasised that routine resistance surveillance studies focusing on environmental and clinical samples are warranted to yield the true prevalence of azole resistance in A. fumigatus in Iran.

Invasive Aspergillosis (IA) is an important cause of mortality and morbidity in the immunocompromised host such as, neutropenic individuals, chronic granulomatous disorder, leukemia, those undergoing solid organ transplantation, patients using broad spectrum antibiotics and steroids, patients with severe underlying diseases and patients with chronic pulmonary obstructive disease are among the main risk groups. Successful management in the treatment of IA depends on early diagnosis and treatment, the adequate choice of therapy, and antifungal resistance. The diagnosis of IA remains difficult and significant proportions of cases of IA remain undetected, thus in case of IA treatment should be considered as early as possible and carried out until the improvements. The treatment is usually based on surgery, antifungal therapy and reduction of immunosuppression. Azole-resistant Aspergillus fumigatus was first observed in Netherlands in 1999. Full mechanism of evolution of azole resistance is not completely known, however, increasing evidence indicates a role for azole fungicide used in agriculture. Due to the presence of A. fumigatus as an agent of IA in our environment and risk for patients, understanding the evolution of the increasing azole resistance in A. fumigatus is crucially recommended. Therefore, induction of azole resistance or its spread can be possibly prevented to allow future treatment of IA due to A. fumigatus.

Inter- and intraspecific genomic variability of 18 isolates of Veronaea botryosa originating from clinical and environmental sources was studied using amplified fragment length polymorphism (AFLP). The species was originally described from the environment, but several severe cases of disseminated infection in apparently healthy individuals have been reported worldwide. All tested strains of V. botryosa, identified on the basis of sequencing and phenotypic and physiological criteria prior to our study, were confirmed by AFLP analysis, yielding a clear separation of V. botryosa as a rather homogeneous group from related species. In vitro antifungal susceptibility testing resulted in MIC90s across all strains in increasing order posaconazole (0.25 μg/ml), itraconazole (1 μg/ml), voriconazole (4 μg/ml), terbinafine (4 μg/ml), caspofungin (8 μg/ml), anidulafungin (8 μg/ml), isavuconazole (16 μg/ml), amphotericin B (16 μg/ml), and fluconazole (32 μg/ml). Overall, the isolates showed a uniform pattern of low MICs of itraconazole and posaconazole, but high MICs for remaining agents. The echinocandins (caspofungin and anidulafungin) had no activity against V. botryosa. There was no statistically significant difference between susceptibilities of environmental (n = 11) and clinical (n = 7) isolates of V. botryosa (P > 0.05).

We report two cases of chromoblastomycosis due to Fonsecaea pedrosoi and F. monophora in otherwise healthy Cuban males. Direct microscopic examination of biopsies revealed muriform cells, the hallmark of chromoblastomycosis. The suspected agents were recovered in culture, identified on the basis of morphological criteria and confirmed by sequencing of the internal transcribed spacer regions of rDNA. Final treatment consisted of surgical excision. The patients were successfully cured since there was no relapse after a follow-up of more than a year. In vitro antifungal susceptibility testing of both isolates showed that itraconazole and posaconazole had potent activity. High MICs of amphotericin B (2 μg/ml), fluconazole (>64 μg/ml), anidulafungin (8 μg/ml) and caspofungin (8 μg/ml) were found.

Based on epidemiological studies, Aspergillus candidus has been demonstrated as an emerging fungal agent of toenail onychomycosis. Here we report a case of a toenail infection caused by A. candidus in a healthy 60-year-old woman. Based on macroscopic and microscopic characteristics of the culture as well as nucleotide sequencing of 28S region, the causative agent was identified as A. candidus.

Background and Objective: Fungi are widely distributed in nature and they are usually present in attomospher but other sources such as water play an important role in their ecology. This study was done to evaluate mycoflora assessment in drinking tap water in Sari, North of Iran. The tap water collected form Sari water distribution system for fungi. Materials and Methods: In this descriptive study, a volume of 100 ml of tap drinking water samples (n=60) were collected in sterile bottles. All water samples passed through sterile 0.45 micrometer filters. The filters were placed directly on Malt extract agar and incubated at 27°C for 3-7 days. Routine mycological techniques were applied to identify the grown fungi. Results: Out of 468 grown fungal colonies, eight different fungal genera were identified. The total mean cfu per 100 ml for the positive samples were 8.4. Aspergillus (37.4%) and Penicillium (27.3%) were the most common isolated fungi. Rhizopus (0.6%) had the lowest frequency. Among Aspergillus species, A. flavus had the highest frequency. Conclusion: Our result showed that various fungi were present in the tap drinking water. We propose fungi should be considered as part of the microbiological analysis parameters in drinking tap water.

Volumetric Assessment of Airborne Indoor and Outdoor Fungi at Poultry and Cattle Houses in the Mazandaran Province, IranThe aim of this study was to assess the volume of airborne fungi in the indoor and outdoor environment of poultry and cattle houses in the Mazandaran Province in Iran. Indoor and outdoor air of twenty cattle houses and twenty-five poultry houses were sampled using a single-stage impactor, which draws air at 20 L min-1and impacts sampled material onto Petri plates containing malt extract agar. The plates were incubated at 30 °C for seven days, after which the resulting colonies were counted. The fungi were identified and counted microscopically and macroscopically. A total of 4,662 fungal colonies were isolated from 90 plates collected from indoor and outdoor air of cattle and poultry houses.Cladosporium(55.3 %), yeast (10.0 %), andAspergillus(9.4 %) were the most common findings. The concentration of airborne fungi in cattle and poultry houses ranged from 10 CFU m-3to 1700 CFU m-3in indoor and 10 CFU m-3to 2170 CFU m-3in outdoor environments.Cladosporiumhad the highest mean indoor (424.5 CFU m-3) and outdoor (449.7 CFU m-3) air concentration in the cattle houses. In the poultry houses, the highest mean concentrations were measured forCladosporium(551.0 CFU m-3) outdoors and yeast (440.7 CFU m-3) indoors. These levels might present an occupational risk, but threshold levels for these environments have yet to be established worldwide.

"n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Several studies have shown that propolis has antibacterial, antifungal, antiviral and antiparasitic activity. Furthermore propolis has been described to have medicinal usages in some fungal infections like Candidiasis. Our aim is to study the inhibitory effects of alcoholic extract of propolis on Candida spp. and Aspergillus spp. "n"n Methods: To determine inhibitory and fatality dose of propolis extract, we prepared serial dilution of the extract including 1/20, 1/40, 1/80, 1/160, 1/320 and 1/640 in 1 ml of liquid medium sabouraud broth. Given numbers of Candida yeasts in 1ml were added to above dilution tubes. Candida and Aspergillus cultures were incubated at 30&deg;C and 25&deg;C respectively for 24-72 hours."n"n Results: We obseved that the concentration of 0.25 g/dl of propolis extract showed an inhibitory and killing effect on more than 50% of the isolates. But there were no inhibitory and killing by the concentrations 0.0312 g/dl and 0.0625 g/dl on Candida isolates. Our findings showed that 0.0312 g/dl of the extract was partially active on Aspergillus fumigatus and dilution of 0.125 g/dl was active on Aspergillus. niger. In the agar dilution method, some changes were observed on morphological features (depends on the extract dilution) as well as quantitative effects of dilution of extract on the colonies. "n"nConclusion: We found that the alcoholic extract of propolis had a prominent antifungal activity and inhibitory effect on Candida and Aspergillus isolates.