Aspergillus Section Fumigati Research Articles

Evaluation of the Activity of Triazoles Against Non-fumigatus Aspergillus and Cryptic Aspergillus Species Causing Invasive Infections Tested in the SENTRY Program.

The activity of isavuconazole and other triazoles against non-fumigatus (non-AFM) Aspergillus causing invasive aspergillosis was evaluated. A total of 390 non-AFM isolates were collected (1/patient) in 2017-2021 from 41 hospitals. Isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and/or internal spacer region/β-tubulin sequencing and tested by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. CLSI epidemiological cutoff values were applied, where available. Isavuconazole showed activity against Aspergillus sections Flavi (n = 122; minimum inhibitory concentration [MIC]50/90, 0.5/1 mg/L), Terrei (n = 57; MIC50/90, 0.5/0.5 mg/L), Nidulantes (n = 34; MIC50/90, 0.12/0.25 mg/L), Versicolores (n = 7; MIC50, 1 mg/L), and Circumdati (n = 2; MIC range, 0.12-2 mg/L). Similar activity was displayed by other triazoles against those Aspergillus sections. Most of the isolates from Aspergillus sections Fumigati (n = 9), Nigri (n = 146), and Usti (n = 12) exhibited elevated MIC values to isavuconazole (MIC50/90, 2/-, 2/4, and 2/8 mg/L), voriconazole (MIC50/90, 2/-, 1/2, and 4/8 mg/L), itraconazole (MIC50/90, 2/-, 2/4, and 8/>8 mg/L), and posaconazole (MIC50/90, 0.5/-, 0.5/1, and >8/>8 mg/L), respectively. Isavuconazole was active (MIC values, ≤1 mg/L) against Aspergillus parasiticus, Aspergillus tamarii, Aspergillus nomius, Aspergillus nidulans, Aspergillus unguis, Aspergillus terreus, Aspergillus alabamensis, and Aspergillus hortai, while isavuconazole MIC values between 2 and 8 mg/L were observed against cryptic isolates from Aspergillus section Fumigati. Isavuconazole inhibited 96.1% of Aspergillus niger and 80.0% of Aspergillus tubingensis at ≤4 mg/L, the CLSI wild-type cutoff value for A niger. Voriconazole, itraconazole, and posaconazole showed similar activity to isavuconazole against most cryptic species. Isavuconazole exhibited potent in vitro activity against non-AFM; however, the activity of triazoles varies among and within cryptic species.

Poultry farms as a One health priority for Aspergillus section Fumigati surveillance

Abstract In poultry production environments, particulate matter (PM) is considered one of the harmful air pollutants [2]. Considering potential biological pathogens, the genus Aspergillus in particular, Aspergillus fumigatus, is a widely recognized human and animal pathogen [3]. Hot-humid farm environments could contribute to the proliferation of these fungi [4]. Therefore, poultry production workers, besides animals [4], may be at a higher risk of inhaling fungi spores [4,5]. This study aimed to determine particulate matter (PM) contamination and analyze the prevalence of Aspergillus section Fumigati in air samples from poultry pavilions. Indoor and outdoor air samples were taken in the 1st, 2nd and 3rd weeks of birds growth cycle (n = 58). Air was collected through the impaction method (MAS-100) onto polycarbonate filters, set at 100 L/min, for 5 min. Followed by DNA extraction, and real-time PCR detection of Aspergillus section Fumigati. For PM evaluation, portable direct-reading equipment (Lighthouse, model 3016 IAQ) was used. Particle concentrations were measured (n = 67) at five distinct fractions during 5 min. Considering particles fractions, PM10 and PM5 where prevalent in 1st (58% PM10; 28% PM5), 2nd (63% PM10; 26% PM5) and 3rd (64% PM10; 25% PM5) weeks. These results evidence that coarse particles (between 2.5 and 10 µm) are the most relevant PM present in this setting. Regarding fungal detection, Aspergillus section Fumigati was widespread inside poultry pavilions (62%, 26 out of 42), highlighting this environment has a potential reservoir of microbial pathogens [7]. Furthermore, PM can act as a host of biological fragments [6]. Overall, in addition to molecular analysis, future studies should consider using culture-based methods to assess the viability of pathogenic microorganisms and their infection potential [3]. From a One Health perspective, quantitative and qualitative research is required to fully understand this environment. Key messages • This study evidence that coarse particles (between 2.5 and 10 µm) are the most relevant PM present and Aspergillus section Fumigati is widespread in poultry pavilions. • A One health intervention should be considered in future studies.

Emerging Aspergillus lentulus infections in Taiwan: clinical and environmental surveillance.

This study aimed to investigate the prevalence and characteristics of Aspergillus lentulus clinical and environmental isolates in Taiwan. Aspergillus isolates obtained from patients at three hospitals and from 530 soil samples across Taiwan were screened. A. lentulus, confirmed by calmodulin sequencing, was subjected to antifungal susceptibility testing and cyp51A analyses. Soil samples yielding A. lentulus were analysed for residues of 25 azole fungicides. Nine A. lentulus isolates were identified, which included seven (1.2%, 7/601) isolates from three antifungal-naïve patients out of 601 Aspergillus section Fumigati clinical isolates and two (0.3%, 2/659) isolates out of 659 Aspergillus soil isolates. All isolates developed white colonies and failed to grow at 48°C. They were susceptible to anidulafungin but showed reduced susceptibility to amphotericin B (AmB), voriconazole and azole fungicides. One heart transplant recipient with proven invasive pulmonary aspergillosis (IPA) initially showed suboptimal response to voriconazole monotherapy but was cured with a combination of voriconazole-caspofungin, liposomal AmB (LAmB)-caspofungin, along with surgery, followed by voriconazole maintenance therapy. Among two critically ill patients with probable IPA, one survived with micafungin, while the other died of aspergillosis despite sequential isavuconazole and LAmB monotherapy. Clinical and environmental isolates sharing identical Cyp51A sequence are identified, matching the Cyp51A sequence of A. lentulus NIID0096. Flusilazole (0.0009 mg/kg) was detected in one soil sample. This study raises concerns about health threat posed by human pathogenic A. lentulus originating from natural environments and underscores the need for increased clinical and laboratory vigilance regarding A. lentulus infections.

Microbial Assessment in A Rare Norwegian Book Collection: A One Health Approach to Cultural Heritage.

Microbial contamination poses a threat to both the preservation of library and archival collections and the health of staff and users. This study investigated the microbial communities and potential health risks associated with the UNESCO-classified Norwegian Sea Trade Archive (NST Archive) collection exhibiting visible microbial colonization and staff health concerns. Dust samples from book surfaces and the storage environment were analysed using culturing methods, qPCR, Next Generation Sequencing, and mycotoxin, cytotoxicity, and azole resistance assays. Penicillium sp., Aspergillus sp., and Cladosporium sp. were the most common fungi identified, with some potentially toxic species like Stachybotrys sp., Toxicladosporium sp., and Aspergillus section Fumigati. Fungal resistance to azoles was not detected. Only one mycotoxin, sterigmatocystin, was found in a heavily contaminated book. Dust extracts from books exhibited moderate to high cytotoxicity on human lung cells, suggesting a potential respiratory risk. The collection had higher contamination levels compared to the storage environment, likely due to improved storage conditions. Even though overall low contamination levels were obtained, these might be underestimated due to the presence of salt (from cod preservation) that could have interfered with the analyses. This study underlines the importance of monitoring microbial communities and implementing proper storage measures to safeguard cultural heritage and staff well-being.

Assessment of Portuguese fitness centers: Bridging the knowledge gap on harmful microbial contamination with focus on fungi

The lack of knowledge regarding the extent of microbial contamination in Portuguese fitness centers (FC) puts attendees and athletes at risk for bioaerosol exposure. This study intends to characterize microbial contamination in Portuguese FC by passive sampling methods: electrostatic dust collectors (EDC) (N = 39), settled dust (N = 8), vacuum filters (N = 8), and used cleaning mops (N = 12). The obtained extracts were plated in selective culture media for fungi and bacteria. Filters, EDC, and mop samples' extracts were also screened for antifungal resistance and used for the molecular detection of the selected Aspergillus sections. The detection of mycotoxins was conducted using a high-performance liquid chromatograph (HPLC) system and to determine the cytotoxicity of microbial contaminants recovered by passive sampling, HepG2 (human liver carcinoma) and A549 (human alveolar epithelial) cells were employed. The results reinforce the use of passive sampling methods to identify the most critical areas and identify environmental factors that influence microbial contamination, namely having a swimming pool. The cardio fitness area presented the highest median value of total bacteria (TSA: 9.69 × 102 CFU m−2.day−1) and Gram-negative bacteria (VRBA: 1.23 CFU m−2.day−1), while for fungi it was the open space area, with 1.86 × 101 CFU m−2.day−1. Aspergillus sp. was present in EDC and in filters used to collect settled dust. Reduced azole susceptibility was observed in filters and EDC (on ICZ and VCZ), and in mops (on ICZ). Fumonisin B2 was the only mycotoxin detected and it was present in all sampling matrixes except settled dust. High and moderate cytotoxicity was obtained, suggesting that A549 cells were more sensitive to samples’ contaminants. The observed widespread of critical toxigenic fungal species with clinical relevance, such as Aspergillus section Fumigati, as well as Fumonisin B2 emphasizes the importance of frequent and effective cleaning procedures while using shared mops appeared as a vehicle of cross-contamination.

Highly effective decontamination in a hospital environment: An easy-to-operate, low-cost prototype.

Healthcare-associated infections (HAI) are illnesses acquired during healthcare and are often the most important adverse event during healthcare. With the aim of increasing the effectiveness of disinfection/decontamination processes in the health service with safe and not promote microbial resistance, we propose the development of portable equipment associated with type C ultraviolet light (UVC). The efficiency of the irradiance emitted by the equipment (at dosages 3.5, 5.0, and 60 mJ/cm2) was determined by the action exerted after exposure against four different bacterial (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) and three different fungi (Candida albicans, C. parapsilosis, and Aspergillus section Fumigati). It was possible to observe that all treatments were capable of inactivating the bacterial species evaluated (p < 0.05), causing the irreversible death of these microorganisms. The most effective elimination of fungal agents was at a dose of 60 mJ/cm2 of UVC radiation, with a decrease in the fungal inoculum varying between 94% and 100% in relation to the control without exposure. Thus, our study showed that the application of the portable prototype with UVC light (254 nm) at a distance of 48 mm, allowed an average irradiance of 3.5 mW/cm2, with doses of 3.5 ≈ 60 mJ/cm2 (from 1 to 60 s of exposure), which can promote the total reduction of the bacteria evaluated and significantly reduce fungal growth. Therefore, this prototype could be used safely and effectively in the hospital environment, considerably reducing contamination and contributing to the reduction of healthcare-associated infection risk.

Prevalence of Azole-Resistant Aspergillus Section Fumigati Strains Isolated from Romanian Vineyard Soil Samples.

A total of 265 soil samples from various Romanian vineyards were screened for fungi resistant to azoles. Aspergillus section Fumigati isolates exhibited significant resistance to itraconazole and voriconazole, but no azole-resistant Aspergillus fumigatus strains were detected. Six percent of the samples were positive for Aspergillus section Fumigati strains, all of which were azole-resistant. The strains were mainly Aspergillus udagawae (93.75%) and Aspergillus lentulus (6.25%). The predominant azole-resistant Aspergillus species were Aspergillus section Nigri strains, which were found in 75 soil samples. This study highlights the importance of understanding fungal resistance in vineyard soils for both the agricultural and clinical sectors. The presence of resistant strains may affect vine health and wine production while also constituting a challenge in the selection of effective treatments against severe and potentially fatal fungal infections in humans, stressing the importance of species-specific antifungal resistance knowledge.

Antifungal susceptibility of Aspergillusgenus determined by the Etest® method: eleven years of experience at the Instituto Médico La Floresta. Caracas, Venezuela.

Abstract. This research aimed to determine the susceptibility of Aspergil-lus spp. to four antifungal agents using the Etest® method in several clinical samples (respiratory samples, soft tissue, otic tissue, and ocular tissue, among others) from a private health center in Venezuela. Thirty-three strains were evaluated: 11 Aspergillus section Flavi, eight Aspergillus section Fumigati, six Aspergillus section Nigri, four Aspergillus section Terrei, and four Aspergillus spp. A 0.5 McFarland standard suspension of a 5-day culture of each Aspergillusstrain was prepared on Potato Dextrose agar and then inoculated on Sabouraud agar plates with 2% glucose. Voriconazole (VCZ), amphotericin B (AMB), caspo-fungin (CAS), and posaconazole (PCZ) were tested. Minimal inhibitory concen-trations (MIC) in μg/mL were determined after 24 and 48 hours of incubation at 35 °C and th range (R), geometric mean (GM), MIC50, and MIC90 were calcu-lated. The results for the 33 Aspergillus spp. tested after 24 h were the follow-ing: VCZ (R = 0.031- 16; GM = 0.145; MIC50 = 0.125 and MIC90 = 0.5), AMB (R = 0.031-16; GM = 0.644; MIC50 = 0.5 and MIC90 = 8), CAS (R = 0.031-16; GM = 0.1076; MIC50 = 0.063 and MIC90 = 1), PCZ (R =0.031 - 0.5; GM = 0.0755; MIC50 = 0.063 and MIC90 = 0.25). This investigation allowed assessing the an-tifungal susceptibility profiles of Aspergillus spp. isolated from clinical samples by the Etest® method, which is practical, reproducible and easy to perform in microbiology laboratories.

Surveillance of Amphotericin B and Azole Resistance in Aspergillus Isolated from Patients in a Tertiary Teaching Hospital.

The genus Aspergillus harbors human infection-causing pathogens and is involved in the complex one-health challenge of antifungal resistance. Here, a 6-year retrospective study was conducted with Aspergillus spp. isolated from patients with invasive, chronic, and clinically suspected aspergillosis in a tertiary teaching hospital. A total of 64 Aspergillus spp. clinical isolates were investigated regarding molecular identification, biofilm, virulence in Galleria mellonella, antifungal susceptibility, and resistance to amphotericin B and azoles. Aspergillus section Fumigati (A. fumigatus sensu stricto, 62.5%) and section Flavi (A. flavus, 20.3%; A. parasiticus, 14%; and A. tamarii, 3.1%) have been identified. Aspergillus section Flavi clinical isolates were more virulent than section Fumigati clinical isolates. Furthermore, scant evidence supports a link between biofilm formation and virulence. The susceptibility of the Aspergillus spp. clinical isolates to itraconazole, posaconazole, voriconazole, and amphotericin B was evaluated. Most Aspergillus spp. clinical isolates (67.2%) had an AMB MIC value equal to or above 2 µg/mL, warning of a higher probability of therapeutic failure in the region under study. In general, the triazoles presented MIC values above the epidemiological cutoff value. The high triazole MIC values of A. fumigatus s.s. clinical isolates were investigated by sequencing the promoter region and cyp51A locus. The Cyp51A amino acid substitutions F46Y, M172V, N248T, N248K, D255E, and E427K were globally detected in 47.5% of A. fumigatus s.s. clinical isolates, and most of them are associated with high triazole MICs. Even so, the findings support voriconazole or itraconazole as the first therapeutic choice for treating Aspergillus infections. This study emphasizes the significance of continued surveillance of Aspergillus spp. infections to help overcome the gap in knowledge of the global fungal burden of infections and antifungal resistance, supporting public health initiatives.

86 Fungi and Mycotoxins Occupational Exposure – Unveiling the Contamination in Different Settings from Portugal

Abstract This study intends to present an overview from the assessments of fungal contamination and mycotoxins performed in three different occupational environments (Firefighters headquarters - FFH; Waste collection trucks – WCT and; Cemeteries - CM). Active sampling (impaction and impingement methods) and several passive sampling methods (swabs, settled dust, settled dust filters, electrostatic dust cloths – EDC, cleaning materials,…) were employed. Azole resistance screening, the molecular detection of Aspergillus sections, as well as mycotoxin analysis were also conducted. In FFH microbial contamination didn´t comply with Portuguese IAQ legal requirements in most of the FFH, the widespread of Aspergillus section Fumigati in all the FFH was observed, as well as a trend of multidrug resistance with focus on Fumigati isolates. Several mycotoxins (fumonisin B2, nivalenol, mycophenolic acid and sterigmatocystin) were detected. Concerning WCT Aspergillus sp. (4.18 %) was one of the most prevalent species. Aspergillus section Fumigati was detected in 5 samples. Mycotoxins were detected in filters (N=1) and in settled dust samples (N=16). The mycotoxin detected in the filter was fumonisin B1 and the most detected in settled dust was mycophenolic acid. In what concerns CM, Aspergillus sp. presented the highest counts in DG18 (18.38%) and it wasn´t observed in azole-supplemented SDA media. Mycophenolic acid was detected in one settled dust sample.Overall, our study reveals that a comprehensive sampling approach and combined analytic methods is an important asset in microbial exposure assessments. Furthermore, in all the three occupational environments mycotoxins were detected unveiling this occupational health threat.

Aspergillus Species Causing Invasive Fungal Disease in Queensland, Australia

BackgroundAspergillus species are important causes of invasive fungal disease, particularly among those with an impaired immune system. Increasing reports have revealed a rising incidence of antifungal drug resistance among Aspergillus spp., particularly among cryptic species. Understanding local antifungal susceptibility patterns is paramount to delivering optimal clinical care.MethodsAspergillus spp. recovered from clinical specimens between 2000 and 2021 from Pathology Queensland were collected. Aspergillus spp. were identified routinely morphologically, and where there was ambiguity or a lack of sporulation, by sequencing of the internal transcribed spacer (ITS) region. All Aspergillus spp. that underwent antifungal susceptibility testing according to the CLSI M38-A3 method and were recorded and included in the study. Amphotericin B, voriconazole, posaconazole, isavuconazole, micafungin, caspofungin, and anidulafungin were tested. Pathology Queensland services all public healthcare facilities in Queensland, Australia.Results236 Aspergillus spp. were identified from clinical specimens during the study period. The most frequent species identified were Aspergillus section Fumigati (n = 119), Aspergillus section Flavi (n = 35), Aspergillus terreus (n = 32) and Aspergillus niger (n = 29). Overall, MIC50/90 values for voriconazole, posaconazole, itraconazole, and isavuconazole were 0.25/1, 0.25/0.5, 0.25/0.5, and 0.5/2 mg/L respectively. Echinocandins demonstrated low MIC values overall with micafungin and anidulafungin both having an MIC50/90 of 0.015/0.03 mg/L. A total of 15 cryptic species were identified; high triazole MIC values were observed with a voriconazole MIC50/90 of 2/8 mg/L. From 2017 to 2021 we observed an increase in incidence of isolates with high voriconazole MIC values. There was no difference in voriconazole MIC values between Aspergillus spp. acquired in North Queensland when compared to Southeast Queensland, Australia.ConclusionIncreasing reports of antifungal resistance among Aspergillus spp. is concerning and warrants further investigation both locally and worldwide. Active surveillance of both the emergence of different Aspergillus spp. and changes in antifungal susceptibility patterns over time is crucial to informing clinicians and treatment guidelines.

Microbial contamination in grocery stores from Portugal and Spain — The neglected indoor environment to be tackled in the scope of the One Health approach

Microbial contamination in grocery shops (GS) should be evaluated since food commodities are commonly handled by workers and customers increasing the risk of food contamination and disease transmission. The aim of this study was to evaluate the microbial contamination in Portuguese and Spanish GS with a multi-approach protocol using passive (electrostatic dust cloths and surface swabs) sampling methods. The molecular detection of Aspergillus sections, mycotoxin analysis, screening of azole resistance as well as cytotoxicity measurement were conducted to better estimate the potential health risks of exposure and to identify possible relations between the risk factors studied.Fruits/vegetables sampling location was the one identified has being the most contaminated (bacteria and fungi) area in GS from both countries. Aspergillus section Fumigati and Fusarium species were observed in samples from Portuguese groceries with reduced susceptibilities to azoles commonly used in the clinical treatment of fungal infections. Fumonisin B2 was detected in Portuguese GS possible unveiling this emergent threat concerning occupational exposure and food safety. Overall, the results obtained raise concerns regarding human health and food safety and must be surveilled applying a One Health approach.

Reactive Azlactone Intermediate Drives Fungal Secondary Metabolite Cross-Pathway Generation.

Pathogenic fungi of Aspergillus section Fumigati are known to produce various secondary metabolites. A reported isolation of a compound with an atypical carbon skeleton called fumimycin from A. fumisynnematus prompted us to examine a related fungus, A. lentulus, for production of similar products. Here we report the isolation of fumimycin and a related new racemic compound we named lentofuranine. Detailed analyses revealed that both compounds were assembled by a nonenzymatic condensation of a polyketide intermediate from the terrein biosynthetic pathway and a highly reactive azlactone intermediate produced by an unrelated nonribosomal peptide synthetase carrying a terminal condensation-like domain. While highly reactive azlactone is commonly used in chemical synthesis, its production by a conventional non-metalloenzyme and employment as a biosynthetic pathway intermediate is unprecedented. The observed unusual carbon skeleton formation is likely due to the reactivity of azlactone. Our finding provides another example of a chemical principle being aptly exploited by a biological system.

Tea contamination by mycotoxins and azole-resistant mycobiota – The need of a One Health approach to tackle exposures

Despite tea beneficial health effects, there is a substantial risk of tea contamination by harmful pathogens and mycotoxins. A total of 40 tea samples (17 green (raw) tea; 13 black (fermented) tea; 10 herbal infusions or white tea) were purchased from different markets located in Lisbon district during 2020. All products were directly available to consumers either in bulk (13) and or in individual packages (27). Bacterial analysis was performed by inoculating 150 μL of samples extracts in tryptic soy agar (TSA) supplemented with 0.2 % nystatin medium for mesophilic bacteria, and in Violet Red bile agar (VRBA) medium for coliforms (Gram-negative bacteria). Fungal research was performed by spreading 150 μL of samples in malt extract agar (MEA) supplemented with 0.05 % chloramphenicol and in dichloran-glycerol agar (DG18) media. The molecular detection of the Aspergillus sections Fumigati, Nidulantes, Circumdati and Flavi was carried out by Real Time PCR (qPCR). Detection of mycotoxins was performed using high performance liquid chromatograph (HPLC) with a mass spectrometry detector. Azole resistance screening was achieved following the EUCAST guidelines. The highest counts of total bacteria (TSA) were obtained in green raw tea (81.6 %), while for coliform counts (VRBA) were found in samples from black raw tea (96.2 %). The highest fungal counts were obtained in green raw tea (87.7 % MEA; 69.6 % DG18). Aspergillus sp. was the most prevalent genus in all samples on MEA (54.3 %) and on DG18 (56.2 %). In the raw tea 23 of the samples (57.5 %) presented contamination by one to five mycotoxins in the same sample. One Aspergillus section Fumigati isolate from green tea beverage recovered form itraconazole-Sabouraud dextrose agar (SDA) medium, presented itraconazole and posaconazole E-test MICs above MIC90 values. Our findings open further discussion regarding the One-Health approach and the necessary investment in researching biological hazards and azole-resistance associated with the production and consumption of tea (in particular green tea).

Diversity and Distribution of Aspergillus fumigatus and Its Related Species in Izu and Ogasawara Islands, Japan.

The taxon Aspergillus section Fumigati comprises several causative agents of aspergillosis. Here, the distribution of Aspergillus sect. Fumigati in outdoor environments of Izu and Ogasawara Islands was investigated. Different strains were isolated from soil samples collected from 68 sites on 9 islands (Izu-oshima, Toshima, Shikinejima, Kozushima, Miyakejima, Hachijojima, Mukojima, Chichijima, and Hahajima), including different landscapes, and identified using morphological characteristics and calmodulin (CaM) sequences. Seven Aspergillus sect. Fumigati species were identified. The occurrence frequency of Aspergillus fumigatus was higher in forest sites on the islands, except for Ogasawara Islands, whereas that of species other than A. fumigatus was higher in bare land and grassland sites on all islands. The occurrence frequency of A. fumigatus was more than 50% on islands between Izu-oshima and Toshima, decreased on islands between Shikinejima and Hachijojima, and was zero on Ogasawara Islands. Considering other Aspergillus species, Aspergillus felis showed high occurrence frequency on islands between Izu-oshima and Shikinejima, Aspergillus pseudoviridinutans on islands between Kozushima and Hachijojima, and Aspergillus udagawae on Ogasawara Islands. At two study sites (grassland and forest sites), the soil was sampled throughout the year to evaluate whether the occurrence frequency of each fungal species was affected by sampling season. At the grassland site, A. pseudoviridinutans was isolated at more than 90% frequency, regardless of the sampling season. A. fumigatus occurrence frequency at the forest site ranged from 0% to 60% and greatly varied among sampling seasons. Thus, differences in island location and landscape affected the distribution of Aspergillus sect. Fumigati.

Five-year surveillance study of clinical and environmental Triazole-Resistant Aspergillus fumigatus isolates in Iran.

Invasive aspergillosis is one of the most common fungal infections and azole resistance in Aspergillus fumigatus (ARAf) is a growing medical concern in high-risk patients. To our knowledge, there is no comprehensive epidemiological surveillance study on the prevalence and incidence of ARAf isolates available in Iran. The study aimed to report a five-year survey of triazole phenotypes and genotype patterns concerning the resistance in clinical and environmental A.fumigatus in Iran. During the study time frame (2016-2021), a total of 1208 clinical and environmental Aspergillus species were collected. Isolates were examined and characterised by in vitro antifungal susceptibility testing (CLSI M38 broth microdilution) and cyp51A sequencing. In total, 485 Aspergillus section Fumigati strains were recovered (clinical, n=23; 4.74% and environment, n=462; 95.26%). Of which A.fumigatus isolates were the most prevalent species (n=483; 99.59%). Amphotericin B and the echinocandins demonstrated good in vitro activity against the majority of isolates in comparison to triazole. Overall, 16.15% (n=78) of isolates were phenotypically resistant to at least one of the azoles. However, 9.73% of A.fumigatus isolates for voriconazole were classified as resistant, 89.03% were susceptible, and 1.24% were intermediate. While, for itraconazole and posaconazole, using the epidemiological cut-off value 16.15% and 6.83% of isolates were non-wild types, respectively. Remarkably, in 21.79% (n=17) phenotypically resistant isolates, no mutations were detected within the cyp51A gene. Although the incidence of ARAf varies from country to country, in Iran the rate has ranged from 3.3% to 18%, significantly increasing from 2013 to 2021. Strikingly, a quarter of the phenotypically resistant isolates harboured no mutations in the cyp51A gene. It seems that other mechanisms of resistance are importantly increasing. To fill a gap in our understanding of the mechanism for azole resistance in the non-cyp51A strains, we highly recommend further and more extensive monitoring of the soil with or without exposure to fungicides in agricultural and hospital areas.

P384 Characterization of the virulence potential of Aspergillus species of section Terrei in Galleria mellonella infection model

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesSpecies belonging to the genus of Aspergillus are among the most common causative agents of human and animal infections. Less than 40 species among all Aspergillus species are known to be associated with human infections, including allergic bronchopulmonary aspergillosis, chronic pulmonary aspergillosis, and invasive aspergillosis. And of these, Aspergillus section Fumigati is one of the major infectious causes of death, followed by members of sections Flavi, Nigri, and Terrei. Aspergillus species in section Terrei are categorized into three series: Ambigui, Nivei, and Terrei. A. terreus sensu stricto is the first species described and the most common species found worldwide in different ecological habitats.However, there are several other species within the section Terrei, stating cryptic species which are not distinguished by conventional morphological analysis, even though they are taxonomically accepted by forming a distinctive phylogenetic clade. Despite definitive species identification, there is still less known about the virulence potential of all species in this section, and it might be underestimated because of their lack of distinction by conventional diagnostic methods. In this ongoing study, the in vivo Galleria mellonella model has been utilized to examine the inter-and/or intraspecies virulence dependency of section Terrei.MethodsA total of 18 accepted Aspergillus species in section Terrei (n = 18) were tested, including A. terreus sensu stricto, A. citrinoterreus, A. hortae, A. pseudoterreus, A. alabamensis, A. aureoterreus, A. floccosus, A. iranicus, A. recifensis, A. carneus, A. microcysticus, A. niveus, A. bicephalus, A. neoindicus, A. neoafricanus, A. barbosae, A. ambiguus, and A. allahabadii. Species were identified by sequencing gene regions of b-tubulin, calmodulin, and RNA Polymerase II Subunit 2 (RPB2). Briefly, groups of larvae (n = 30) (0.3 to 0.4 g; SAGIP, Italy) were stored in wood shavings in the dark at 18°C for 24 h before the experiment. Three groups were included: larvae infected with 107 conidia/larva, larvae injected with 20 μL sterile insect physiological saline, and untouched larvae. The survival rate was monitored for up to 144 h at 37°C.ResultsMedian survival rates revealed a species-dependent virulence pattern. Larvae inoculated with A. aureoterreus, A. pseudoterreus (Serie Terrei) and A. niveus, A. carneus, and A. iranicus (Serie Nivei) exhibited high virulence potential by reflecting lower survival rates in comparison with other species. In contrast, species belonging to the series Ambigui showed low virulence potential.ConclusionIn conclusion, the virulence characteristics of section Terrei differ between species. Further studies are needed to unravel the species' invasiveness, such as histopathology and immune response of G. mellonella.

P465 Aspergillosis in free-ranging Magellanic penguins

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMAspergillosis is an opportunistic fungal disease caused by the Aspergillus genus, mostly by Aspergillus section Fumigati. Captive Magellanic penguins are vulnerable to Aspergillus infection, being this mycosis a limiting factor during the process of rehabilitation with a mortality rate of around 50%.ObjectivesGiving the scarce data regarding the occurrence of aspergillosis in non-captive penguins, we aimed to evaluate the proportional mortality by aspergillosis in free-ranging Magellanic penguins during their migration and reproductive season.MethodsCarcasses of Magellanic penguins were collected from the Southern coast of Brazil between June 2017 and October 2019 between Barra do Chui beach (Southern RS, Brazil - 33°44'19.9‘S 53°21'56.3’W) and the isthmus with the Lagoa do Peixe (Southern RS, Brazil - 31°26' S, 51°10' W 31°14'S, 50°54'W). In addition, in January 2019, penguins found dead in the reproducing colony in four islands localized in Puerto Deseado City (Santa Cruz, Patagonian, Argentina - 47°45′00″S 65°55′00″W) were collected. All animals were necropsied, and macroscopic alterations were observed. Samples of macroscopic lesions and/or respiratory systems were collected for histopathology and mycological culture. Only proven aspergillosis cases, defined by suggestive lesions at necropsy, associated with hyaline, septate, and 45° branched hyphae in histopathological slides and isolation of Aspergillus sp. in the culture were computed. Fungal isolates were identified by molecular techniques.ResultsA total of 98 Magellanic penguins were included in our study, being 80 recovered on the Southern RS beach, and 18 from the Patagonian colony. Two penguins collected in Southern Brazil were diagnosed with aspergillosis, both juveniles, one showing nodules in the lung parenchyma, and the other nodules and fungal colonies at the lung and air sac, resulting in a proportionate mortality rate of 2.5%. Both isolates were identified as A. fumigatus sensu stricto. Regarding the carcasses collected from the reproductive colony (Patagonian islands), no penguin had anatomopathological or mycological evidence of aspergillosis.ConclusionGiven the already known importance of aspergillosis in seabirds undergoing rehabilitation, these data suggest that penguins may already arrive in these centers infected by Aspergillus spp. The absence of aspergillosis cases in the reproductive period could be attributed to the low number of carcasses included. Our study is an initial step to demonstrate aspergillosis as one of the causes of mortality also in free-living penguins, especially during the migration process, instigating more studies, in other routes of migration, as well as in reproductive colonies.

P454 Massive parallel fungal sequencing on formalin-fixed tissues: development and contribution in integrated histomolecular diagnosis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesHistopathology is the gold standard for distinguishing between colonization and fungal infection, but it does not provide a precise diagnosis of genera/species. However, if the culture is negative or if no specimen is sent to the Mycology laboratory, only the specimen sent to the Pathology department is available. Formalin fixation and paraffin embedding (FFPE) cause DNA damage, making it difficult to perform molecular techniques.The objective was to develop and evaluate the contribution of massive parallel sequencing (MPS) to FFPE tissues.MethodsHistopathological review of all cases was performed. Then, DNA extraction from FFPE tissues was optimized by: (1) macrodissecting the fungal-rich area on the paraffin block; (2) comparing the efficiency of two DNA extraction kits (QIAamp DNAmicro-kit, QIAGEN; Maxwell 16 LEVRNA FFPE Purification kit, Promega, optimized for RNA and DNA extraction), by comparison of Aspergillus fumigatus and Mucorale specific PCR results for 30 cases. For 124 other cases, the sensitivity of two primer pairs (ITS3/4 and MITS2A/2B) was tested for identification by Sanger sequencing and then MPS. Finally, a histomolecular comparison was performed. This work was funded by the Société de Pathologie Infectieuse de Langue Française (SPILF).ResultsTo optimize extraction, DNA was extracted by both kits from samples of 16 mucormycoses and 14 A. fumigatus infections. PCR sensitivity was better with the QIAGEN extraction kit [100% (30/30)] compared to the Promega kit [86.7% (26/30)].PCR amplification of fungal DNA from an additional 124 FFPE samples was performed. The primer pairs ITS3/4 and MITS2A/2B, allowed: (1) identification by Sanger sequencing-histopathological analysis in 38.7% (48/124) of the cases in total, and more specifically 33% (41/124) of cases with the ITS3/4 primers and 32.3% (40/124) of cases with the MITS2A/B primers; and (2) identification by integrated SMP-histopathological analysis in 75% (93/124) of all cases (primers ITS3/4 and MITS2A/2B), and more specifically 66.1% (82/124) for ITS3/4 and 62.1% (77/124) for MITS2A/B (both primer pairs did not detect/amplify the same fungal genera/species). The combination of all results from Sanger sequencing and MPS led to fungal identification in 75.8% (94/124) of cases. In total, the addition of NGS to Sanger sequencing increased the diagnostic proportion by 36.3% (45/124; P <.0001). Example of integrated histomolecular diagnosis (Fig. 1): patient with a pseudotumor presentation of pulmonary invasive aspergillosis (A: thoracic CT scan; B: macroscopic examination of lesion after formalin fixation; C, D, E: Hematoxylin-eosin-safran, x20 and x400: observation of a necrotic mass caused by hyalohyphomycetes; F: Grocott, x400) with no culture or molecular identification available on fresh tissue. In contrast, identification by MPS on FFPE tissue was compatible with morphological analysis: Aspergillus section Fumigati, leading to the integrated histomolecular diagnosis of pulmonary invasive aspergillosis.ConclusionThe development of the fungal MPS on FFPE tissues is innovative and unprecedented for the achievement of an integrated histomolecular diagnosis in fungal pathology. It increases significantly the diagnostic proportion by 36.3%. This strategy can be used in hospitals and could improve patient management, especially when no sample is sent to the Mycology laboratory or when the culture is negative.

P270 Otomycosis: Main challenging microbial agent causing otitis externa in northern Iran

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesOtitis externa is one of the most common ear diseases, caused by bacterial and fungal agents. Accumulation of epithelial cells, cellular detritus, waxy substances, and microorganisms in the ear canal provide a proper environment for fungal growth, leading to annoying congestion. In this study, we aimed to identify the fungal causative agents and determine their susceptibility to the antifungal drugs were examined.MethodsAll patients with suspected otitis externa, referred to Amir-al Momenin Referral Centre from October 2020 to November 2021 entered to study. A total of 200 patients’ ear discharge and debris collected samples were examined by direct examination and cultured on SDA supplemented with chloramphenicol. Definitive identification of grown fungi was made by PCR-RFLP and sequencing of ribosomal DNA. In vitro susceptibility testing of the fungal isolates against eleven different antifungal agents was analyzed using the CLSI broth microdilution methods.ResultsA total of 101 (50.5%) cases were confirmed with otomycosis. Most patients were in their fifth decade of life (n = 35, 34.6%), female (n = 57, 56.4%), and had unilateral ear involvement (n = 78, 77.2%). The most common symptoms were earache (56.4%) and itching (51.4%). Most underlying predisposing factors are overaggressive using cotton swabs (65.3%) and history of topical antibiotics (48.5%). Among the 117 fungal isolates, Aspergillus section Nigri was the most common agent 58 (49.57%), followed by Aspergillus section Flavi 19 (16.23%), C. parapsilosis 14(11.96%), Aspergillus section Fumigati 12 (10.25%), C. orthopsilosis 6 (5.12%), C. albicans complex 5 (4.27%), Mucor spp. 2 (1.71%), and Syncephalastrum spp. 1 (0.85%). Mainly, all tested antifungals were active against the most isolates of Aspergillus, aside from tioconazole, nystatin, and terbinafine, which showed low in vitro effects. Also, nystatin and itraconazole showed higher GM MICs in against all Candida species isolates. Conversely, amphotericin B (GM = 0.07129) in Aspergillus and voriconazole (GM = 0.03686) in Candida showed the highest antifungal activity. Regarding ECV values represented by ECOFFs, one of each A. niger (MIC 8 μg/mL), A. flavus (MIC 2 μg/ml), and A. fumigatus (MIC 2 μg/ml) isolates were resistant to itraconazole.ConclusionIn this study, inconsistent with previous ones, fungal agents have overcome bacterial ones as the etiology of otitis externa. This result may relate to misuse or improper use of topical steroids, antibiotics, and inappropriate control of infection. In addition, our sample collection site was an ENT referral center and many patients have been admitted to this center due to treatment failure and suffered from recurrence. Aspergillus section Nigri isolation domination was seen, in agreement with other results. The MICs distribution of Aspergillus species isolates against triazole antifungals are close to ECVs defined by the CLSI and likely outrun it over time. We recommend that physicians request drug susceptibility testing before antibiotic therapy, to prevent the development of resistance.