Aspergillus in the Indoor Air of Critical Areas of a Tertiary Hospital in Brazil.

Airborne Aspergillus spp. are critical pathogens that cause nosocomial infections in hospitals. Despite their importance, little is known about the distribution of Aspergillus species in the indoor air of hospitals in Brazil. We investigated Aspergillus spp. in the indoor air of critical areas in a tertiary hospital in Brazil. Air samples (n = 238) were collected from the intensive care unit (ICU), medical clinic unit (MCU), and urgency and emergency unit (UEU) using an air sampler (100 L/min). Of the 324 Aspergillus isolates, 322 were identified using phenotypic methods, and 37 were identified using DNA sequencing. Aspergillus spp. was grouped into five sections: Fumigati (29.3%), Nidulantes (27.8%), Nigri (27.5%), Flavi (11.7%), and Terrei (3.1%). The predominant species identified via sequencing were Aspergillus sydowii (n = 9), Aspergillus flavus (n = 7), and Aspergilus fumigatus (n = 6). The number of Aspergillus spp. and their sections varied according to the collection day. A. fumigatus was isolated more frequently during winter and in the ICU. This study is the first to demonstrate the diversity of airborne Aspergillus (saprophytic, allergenic, toxigenic, and potentially pathogenic) strains in a hospital located in the Midwest region of Brazil. It contributes to the knowledge of the diversity of cryptic species in the hospital environment.

Hereditary Severe Insulin-resistance Syndrome and Acanthosis Nigricans Caused by Novel Mutations in the INSR Gene.

Most cases associated with Hereditary Severe Insulin Resistance Syndrome (H-SIRS) are linked to mutations in the insulin receptor (INSR) gene. Patients with H-SIRS typically manifest symptoms of hyperinsulinemia, insulin resistance, and diabetes mellitus. Other symptoms include impaired glucose regulation, hyperandrogenism, and the presence of acanthosis nigricans (AN). In this report, we present two cases of H-SIRS in female children exhibiting various symptoms, such as hyperinsulinemia, fasting hypoglycemia, postprandial hyperglycemia, overweight, fatty liver, hyperandrogenism, and varying degrees of AN. One patient also presented with mental retardation. Gene sequencing identified specific mutations in the INSR gene for both patients: c.2663A > G (p.Tyr888Cys) and c.38_61del (p.Pro13_Ala20del). These mutations have the potential to disrupt the interaction between INSR and insulin, leading to abnormal insulin signaling, insulin resistance, and various clinical manifestations.

Aspergillus hubkae, a Novel Species Isolated from a Patient with Probable Invasive Pulmonary Aspergillosis.

A 50-year-old man, previously diagnosed with pulmonary tuberculosis and lung cavities, presented with symptoms including fever, shortness of breath, and cough. A pulmonary CT scan revealed multiple cavities, consolidation and tree-in-bud in the upper lungs. Further investigation through direct examination of bronchoalveolar lavage fluid showed septate hyphae with dichotomous acute branching. Subsequent isolation and morphological analysis identified the fungus as belonging to Aspergillus section Nigri. The patient was diagnosed with probable invasive pulmonary aspergillosis and successfully treated with a three-month oral voriconazole therapy. Phylogenetic analysis based on partial ß-tubulin, calmodulin and RNA polymerase second largest subunit sequences revealed that the isolate represents a putative new species related to Aspergillus brasiliensis, and is named Aspergillus hubkae here. Antifungal susceptibility testing demonstrated that the isolate is resistant to itraconazole but susceptible to voriconazole. This phenotypic and genetic characterization of A. hubkae, along with the associated case report, will serve as a valuable resource for future diagnoses of infections caused by this species. It will also contribute to more precise and effective patient management strategies in similar clinical scenarios.

Description and management of Aspergillus section Nigri causing post-harvest bulbs rot of onion.

When onions are improperly stored, a post-harvest disease known as black mold of onion bulbs can result in considerable economic losses. Aspergillus section Nigri, one of many species, has been implicated in the development of black mold. In the present study, rot onion bulbs were collected from markets in Qena, Egypt. Thirteen Aspergillus section Nigri isolates were obtained and identified by morphological and molecular characterization. The ochratoxins potential of isolated A. section Nigri was tested, and three isolates were producers at the range of 1.5-15 ppm. For the presence of pks gene, no amplification product was detected. Using the fungal growth inhibition test, the isolates of A. niger were inhibited by eco-friendly materials Cement and Zeolite. Cement exhibited maximum percentage growth inhibition against the tested isolates at 74.7-86.7%. The pathogenicity activity of the A. niger isolates was tested by inoculation of healthy onion bulbs, other onion bulbs covered with Cement and Zeolite before inoculation by A. niger was used. The two treatments significantly reduced bulbs rot disease of onion than untreated bulbs. Seven and nine isolates showed 0% rot on covered bulbs by Cement and Zeolite, respectively as compared with inoculated onions, which exhibited rot ranging from 55 to 80%. Using eco-friendly materials with efficiency against post-harvest bulbs rot of onion was evaluated in this study.

Molecular Analysis for Potential Hospital-Acquired Infection Caused by Aspergillus Tubingensis Through the Environment.

The identification of Aspergillus species has been performed mainly by morphological classification. In recent years, however, the revelation of the existence of cryptic species has required genetic analysis for accurate identification. The purpose of this study was to investigate five Aspergillus section Nigri strains isolated from a patient and the environment in a university hospital. Species identification by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identified all five black Aspergillus strains as Aspergillus niger. However, calmodulin gene sequence analysis revealed that all five strains were cryptic species, four of which, including the clinical strain, were Aspergillus tubingensis. Hospital-acquired infection of the patient with the A. tubingensis strain introduced from the environment was suspected, but sequencing of six genes from four A. tubingensis strains revealed no environmental strain that completely matched the patient strain. The amount of in vitro biofilm formation of the four examples of the A. tubingensis strain was comparable to that of Aspergillus fumigatus. An extracellular matrix was observed by electron microscopy of the biofilm of the clinical strain. This study suggests that various types of biofilm-forming A. tubingensis exist in the hospital environment and that appropriate environmental management is required.

Ochratoxin A Contamination of California Pistachios and Identification of Causal Agents.

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium spp., which contaminates many crops, including pistachios. Pistachios contaminated with OTA may be subjected to border rejections resulting in significant economic losses to the United States agricultural revenues. The current study examined prevalence of OTA in California-grown pistachios and identified its causal agents. OTA was detected in 20% of samples from 2018 to 2021 (n = 809), with 18% of samples exceeding the European Union regulatory limit of 5 µg/kg. Fungi potentially responsible for OTA contamination were isolated from leaves, nuts, and soil collected from 14 pistachio orchards across California. A total of 1,882 isolates of Aspergillus section Nigri and 85 isolates of section Circumdati were recovered. Within section Nigri, 216 (11.5%) isolates were identified as potential OTA producers using a boscalid-resistance assay. Phylogenetic analyses of partial gene sequences for ß-tubulin and calmodulin genes resolved section Circumdati into four species: A. ochraceus (33%), A. melleus (28%), A. bridgeri (21%), and A. westerdijkiae (19%). A. westerdijkiae produced the highest levels of OTA in inoculated pistachios (47 µg/g), followed by A. ochraceus (9.6 µg/g) and A. melleus (3.3 µg/g). A. bridgeri did not produce OTA. OTA production by section Circumdati was optimal from 20 to 30°C. All 216 boscalid-resistant isolates from section Nigri were identified as A. tubingensis, and representative isolates (n = 130) produced 3.8 µg/kg OTA in inoculated pistachios. This is the first detailed report on OTA contamination and causal fungi in California pistachios and will be helpful in devising effective management strategies.

α-Tomatine degradation to tomatidine by food-related Aspergillus species belonging to the section Nigri.

α-Tomatine is a steroidal glycoalkaloid in tomato plants and degrades with ripening. The aglycone form, tomatidine, is reported to have beneficial effects. In this study, the ability of food-related microorganisms to produce tomatidine from α-tomatine was evaluated. A total of 11 strains of Aspergillus species belonging to the section Nigri exhibited tomatinase activity, and Aspergillus luchuensis JCM 22302 was selected for optimization due to its high activity in its mycelia, conidia, and non-mycotoxin-producing property. Next, using A. luchuensis JCM22302 conidia, the highest yield was obtained in a 24-h reaction with 50 m m of acetic acid-sodium acetate buffer (pH 5.5) at 37 °C. Similar to the tomato pathogen Fusarium oxysporum f. lyceopersici, the time course analysis suggested that A. luchuensis JCM 22302 removed the entire sugar moiety in a single step. Future research will focus on utilizing conidia for large-scale tomatidine production because of their high tolerance and manageability.

Combined Toxicity of the Most Common Indoor Aspergilli.

The most common Aspergilli isolated from indoor air samples from occupied buildings and a grain mill were extracted and analyzed for their combined (Flavi + Nigri, Versicolores + Nigri) cytotoxic, genotoxic and pro-inflammatory properties on human adenocarcinoma cells (A549) and monocytic leukemia cells induced in macrophages (THP-1 macrophages). Metabolite mixtures from the Aspergilli series Nigri increase the cytotoxic and genotoxic potency of Flavi extracts in A549 cells suggesting additive and/or synergistic effects, while antagonizing the cytotoxic potency of Versicolores extracts in THP-1 macrophages and genotoxicity in A549 cells. All tested combinations significantly decreased IL-5 and IL-17, while IL-1ß, TNF-α and IL-6 relative concentrations were increased. Exploring the toxicity of extracted Aspergilli deepens the understanding of intersections and interspecies differences in events of chronic exposure to their inhalable mycoparticles.

Otomycosis: The foremost aetiological agent causing otitis externa and the antifungal susceptibility pattern in North-Western Iran.

BACKGROUND: Otomycosis is considered a recurring fungal ear infection. The external auditory canal provides an appropriate and optimal situation for fungal growth. OBJECTIVES: The study aimed to identify the causative agents of otomycosis and determine corresponding antifungal drug susceptibility patterns in north-western Iran. METHODS: From October 2020 until November 2021, 200 patients attended an otolaryngology referral centre with otitis externa, and their ear discharge and debris were examined and cultured. The identification of the fungal agents was implemented by polymerase chain reaction-restriction fragment length polymorphism and sequencing. In vitro antifungal susceptibility testing of the isolates was conducted in accordance with the CLSI broth microdilution protocols. RESULTS: The prevalence of otomycosis was measured 50.5% (n = 101/200). The majority of patients were in their forties (n = 35, 34.6%) and female (n = 57, 56.4%), and the most prevalent symptom was otalgia (56.4%). The most underlying factor was remarked manipulation employing a cotton swab (65.3%). Regarding fungus, Aspergillus section Nigri (58.57%) was the foremost isolate, followed by Aspergillus section Flavi (19.23%) and Candida parapsilosis (14.96%). The predominance of Aspergillus isolates had minimal in vitro sensitivity to tioconazole and nystatin. Candida species represented higher geometric mean minimum inhibitory concentrations (MIC) against nystatin. The MIC of three Aspergillus species isolates shown above the epidemiologic cut-off values (ECV) against itraconazole. CONCLUSIONS: Otomycosis incidence surpassed in comparison with the previous study as the most common cause of otitis externa. The MIC distribution of Aspergillus species isolates against triazole antifungals is close to the defined ECVs and likely outrun it over time.

Poly-(lactic-co-glycolic) Acid Nanoparticles Entrapping Pterostilbene for Targeting Aspergillus Section Nigri.

Poly-(lactic-co-glycolic) acid (PLGA) is a biodegradable, biosafe, and biocompatible copolymer. The Aspergillus section Nigri causes otomycosis localized in the external auditory canal. In this research, Aspergillus brasiliensis, a species belonging to the Nigri section, was tested. Coumarin 6 and pterostilbene loaded in poly-(lactic-co-glycolic) acid nanoparticles (PLGA-coumarin6-NPs and PLGA-PTB-NPs) were tested for fungal cell uptake and antifungal ability against A. brasiliensis biofilm, respectively. Moreover, the activity of PLGA-PTB-NPs in inhibiting the A. brasiliensis infection was tested using Galleria mellonella larvae. The results showed a fluorescence signal, after 50 nm PLGA-coumarin6-NPs treatment, inside A. brasiliensis hyphae and along the entire thickness of the biofilm matrix, which was indicative of an efficient NP uptake. Regarding antifungal activity, a reduction in A. brasiliensis biofilm formation and mature biofilm with PLGA-PTB-NPs has been demonstrated. Moreover, in vivo experiments showed a significant reduction in mortality of infected larvae after injection of PLGA-PTB-NPs compared to free PTB at the same concentration. In conclusion, the PLGA-NPs system can increase the bioavailability of PTB in Aspergillus section Nigri biofilm by overcoming the biofilm matrix barrier and delivering PTB to fungal cells.

Discovery of Tolerance to Itraconazole in Japanese Isolates of Aspergillus Section Nigri, Aspergillus tubingensis and Aspergillus welwitschiae, by Microscopic Observation.

Aspergillus section Nigri, a group of black Aspergillus, has several cryptic species that were recently discovered to be intrinsically resistant to azole antifungals. In this study, susceptibility testing of 35 clinical isolates of Aspergillus tubingensis and Aspergillus welwitschiae in Japan was carried out using microdilution method. Strains tolerant to itraconazole in A. tubingensis (14/17 strains) and A. welwitschiae (6/18 strains) were discovered with hyphal growth and conidial germination above the minimal inhibitory concentration by microscopic observation, while no resistant strain was observed macroscopically. In contrast, no strain with reduced susceptibility to voriconazole, posaconazole, and amphotericin-B was found. Further examination may be required to determine the susceptibility of cryptic species in Aspergillus section Nigri to antifungals.

Enzyme activity of Aspergillus section Nigri strains isolated from the Korean fermentation starter, nuruk.

Aspergillus section Nigri is a fungus used industrially because of its ability to produce enzymes such as cellulolytic, amylolytic and proteolytic enzymes. In this study, we obtained twenty-eight strains of Aspergillus section Nigri from the traditional Korean fermentation starter, nuruk, which is known as a mixed culture of enzymatic filamentous fungi and yeasts. All strains were identified as Aspergillus section Nigri through combined phylogenetic analysis using partial ß-tubulin and calmodulin gene sequences. The cellulase, amylase and protease activities of Korean strains were measured and compared with ten reference strains of Aspergillus niger. Most Korean strains showed higher cellulolytic activity than reference strains, and Aspergillus neoniger KCN5 showed the highest ß-glucosidase activity. Two-thirds of the Korean strains showed similar levels of α- and glucoamylase activity as the reference strains. The protease activity of Aspergillus section Nigri strains was the highest at pH 3.0, and A. niger KSJ2 showed the highest acidic protease activity. By comparing ten reference strains and twenty-eight Korean strains, our results suggested useful Aspergillus section Nigri strains from nuruk with high enzyme activity, such as KCN5 and KSJ2, and their potential for industrial applications as enzyme producers.

Aspergillus tubingensis Endocarditis: A Case Report and Review of the Literature.

Aspergillus endocarditis is a rare infection that may affect immunocompetent patients following heart valve replacement or heart surgery. We report the case of a 39 year old woman with a history of intravenous drug use who developed endocarditis with direct examination of the resected valve and vegetation showing the presence of mycelia. Cultures were positive for an Aspergillus of section Nigri, which was subsequently identified as Aspergillus tubingensis by sequencing. The clinical course was favorable following surgery and prolonged antifungal therapy (8 months in total). Antifungal susceptibility testing showed good in vitro activity of amphotericin B, voriconazole and echinocandins against planktonic cells of this A. tubingensis isolate. However, only amphotericin B displayed significant activity against biofilms. In vitro combinations of voriconazole or amphotericin B with echinocandins did not meet the criteria of synergism. Our review of the literature identified 17 other cases of endocarditis attributed to Aspergillus of section Nigri with an overall mortality rate of 57% (100% in the absence of surgery). Endocarditis caused by Aspergillus niger and related cryptic species are rare events, for which surgical management appears to be crucial for outcome. While amphotericin B was the only antifungal drug displaying significant anti-biofilm activity, the type and duration of antifungal therapy remain to be determined.

Advances in understanding the enzymatic potential and production of ochratoxin A of filamentous fungi isolated from cocoa fermented beans.

In this work, we determined the degree of filamentous fungi contamination in cocoa fermented seeds and the fungal potential to produce enzymes that could contribute to the cocoa quality. Internal transcribed spacer amplicon sequencing (ITS) showed 11 different species with Byssochlamys spectabilis, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius, Circinella muscae and Penicillium citrinum as the most abundant species. Our results evidenced intra and inter-species differences in the enzymes production. Cellulases, ß-glucosidase, ornithine decarboxylase and phenylalanine decarboxylase were the most diffused enzymes expressed in the 53 strains here studied. Moreover, A. niger (6/12), A. carbonarius (2/3), and P. citrinum (3/3) showed high pectinolytic activity. Remarkable was the amino decarboxylase activity of P. citrinum, and A. flavus strains. For the first time we reported the presence of B. spectabilis in cocoa fermented beans, which could play an important role in the biogenic amines formation. In addition, we explored the capability of the Aspergillus section Nigri strains, to produce ochratoxin A (OTA) in a cacao model system (CPMS) and in malt extract medium (MEL). We observed that CPMS, but not MEL, stimulated the OTA production in 6 out 15 strains of Aspergillus section Nigri, reaching values ranging between 1.70 and 4995 µg OTA kg-1 dry mycelium.

Black aspergilli as causes of otomycosis in the era of molecular diagnostics, a mini-review.

Otomycosis refers to the fungal infection of the external auditory canal, and less commonly the middle ear. A wide range of fungi can cause this disease, however, the most common etiologies are species of Aspergillus and Candida. Until recent years, Aspergillus niger was thought to be the prevailing species of the genus Aspergillus that causes otomycosis. Using molecular methods, now, it is known that Aspergillus section Nigri comprises several morphologically similar species and all black Aspergillus isolates are not necessarily equivalent to Aspergillus niger. In this review, we focus on the species within the Aspergillus section Nigri and their role as the causative agents of otomycosis.

Aspergillus spp. Isolated from Lungs of Poultry (Gallus gallus) at the Mycology Laboratory, School of Veterinary Medicine, Universidad Nacional, Heredia, Costa Rica between 2008 and 2021 and Associated Factors.

Aspergillosis is a disease caused by some species of the fungus Aspergillus, occurring in both mammals (including humans) and birds, the latter being the most susceptible group. Aspergillus must be considered a public health concern as it affects the poultry industry economically and is an occupational risk to its workers. A retrospective study of fungal isolates from the lungs of chickens (Gallus gallus), analyzed between 2008 and 2021 at the Mycology Laboratory, School of Veterinary Medicine, Universidad Nacional, Heredia, Costa Rica was performed to report the prevalence of Aspergillus spp. in poultry farms in Costa Rica and their associated factors. A total of 1113 cases were received, of which 31% (n = 392; 95% CI: 28.3−33.7) were positive for fungal isolation. Aspergillus was the most frequently detected genus, and the most frequent sections were Fumigati (n = 197/392, 50.3%), Flavi (n = 90/392, 22.9%), and Nigri (n = 50/392, 12.7%). Significant effects (p < 0.05) related to the year, geographical origin, purpose, and age were identified in relation to the Aspergillus infection. The identified factors are explained by climatic variations in the tropics and the particularities of the birds. Future research including molecular characterization and antifungal susceptibility tests in animals, humans, and the environment, are needed to better understand the risks of the diseases caused by those fungi in this country.

Successfully treated bronchopulmonary oxalosis caused by Aspergillus tubingensis in a non-neutropenic patient: A case report and review of the literature.

Pulmonary oxalosis can be fatal, and Aspergillus tubingensis is commonly resistant to azoles in Japan. We report a case of bronchopulmonary oxalosis caused by A. tubingensis in a non-neutropenic patient who was successfully treated with voriconazole monotherapy. The susceptibility of the isolates to voriconazole and the effective elimination of contagious necrotic tissue by expectoration seemed to be two major factors contributing to the patient's survival. According to the literature review, pulmonary oxalosis is associated with a high mortality rate over a short term. An exploration of detailed information about the genomic characteristics and drug susceptibility of Aspergillus isolates is important for the development of treatment strategies for this life-threatening disease.

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.

Aspergillus niger LBM 134 isolated from rotten wood and its potential cellulolytic ability.

Aspergillus is a genus of filamentous and cosmopolitan fungi that includes important species for medical mycology, food, basic research and agro-industry areas. Aspergillus section Nigri are efficient producers of hydrolytic enzymes such as cellulases that are employed in the cellulose conversion. Hence, the search of new cellulolytic isolates and their correct identification is important for carrying out safe biotechnological processes. This study aimed to characterise the cellulolytic potential of Aspergillus sp. LBM 134, isolated from the Paranaense rainforest (Argentina) and to identify the isolate through a polyphasic approach. The fungus was identified as Aspergillus niger and its cellulolytic potential was evaluated by using Congo red technique and fluorescence plate assays for carboxymethyl cellulase, ß-glucosidase and cellobiohydrolase, respectively. All three cellulase activities were positive; this bio-prospective positioned A. niger LBM 134 as a promising alternative for industries that require organisms capable of carrying out cellulosic biomass processing.

Ochratoxin A and related fungi in Brazilian black pepper (Piper nigrum L.).

Ochratoxin A (OTA) is a mycotoxin with nephrotoxic, genotoxic, teratogenic and carcinogenic properties, produced by several species of Aspergillus, mainly those belonging to the A. section Circumdati and A. section Nigri. Although this toxin has been detected in spices and condiments, in black pepper (Piper nigrum L.) few studies have investigated the mycobiota (based on a molecular approach) and the presence of OTA in this food. The aim of this study was to investigate the presence of potentially ochratoxigenic species and ochratoxin A in black pepper marketed in Brazil, one of the largest producers in the world. A total of 60 samples of black pepper (29 in powder and 31 in grain) were collected in markets. The presence of OTA was investigated in black pepper samples using High-Performance Liquid Chromatography (HPLC), OTA was detected in 55% of the samples, with levels ranging from 0.05 to 13.15 µg/kg, all of which were below the Brazilian legal tolerances. A. section Nigri and A. section Circumdati were found in 80% of the samples, but the species of A. section Nigri were significantly more frequent than those of A. section Circumdati. The potential for OTA production by fungal isolates was tested using the agar plug technique and confirmed by HPLC. Among the isolates belonging to A. section Nigri (n = 1,083) and A. section Circumdati (n = 129), 3.7% and 3.8%, respectively, were able to produce OTA in Yeast Extract Sucrose Agar (YESA). A total of 25 strains from A. section Circumdati and 64 from A. section Nigri were identified using molecular data. The following potentially ochratoxigenic species were found in black pepper: A. niger, A. welwitschiae, A. carbonarius, A. westerdijkiae and A. ochraceus. The occurrence of these species denotes the need for continuous monitoring of black pepper by regulatory bodies in order to safeguard consumers' health.