Isolates Of Aspergillus Species Research Articles

First report of Aspergillus species in green pistachio of Bronte

Aspergillus contamination of pistachios causes significant product losses and potential presence of mycotoxins, particularly aflatoxin B1 (AFB1), and ochratoxin A (OTA). These toxins, which threaten human health, are strictly monitored by most nations. Italian pistachios produced in Bronte, Sicily, have high nutritional value and unique organoleptic properties, but the extent to which they contain these contaminants is unknown. Aspergillus spp. isolated from Bronte pistachios (cultivar Napoletana) were assessed for their ability to synthesize OTA or AFB1. Aspergillus occurrence in pistachio samples was measured at 1137 cfu g-1 for in shell pistachios and 770 cfu g-1 for kernels. The predominant isolated Aspergillus species was A. niger representing 74% of section Nigri (black isolates) and 47% of all Aspergillus isolates. Within section Flavi, A. flavus comprised 83% of green isolates. Only one black isolate (identified as A. carbonarius) had high OTA production, but all the A. flavus isolates had potential to produce AFG1 and AFB1, with AFB1 produced amount ranging from 0.1 to 8498 ng mL-1 of culture filtrate.

Antifungal Susceptibility Profile of Clinical and Environmental Isolates of Aspergillus Species From a Tertiary Care Center in North India.

Aspergillus species are ubiquitously found in the environment worldwide and are important causative agents for infection. Drug resistance among Aspergillus species is emerging, hencethe present study was undertaken to look for antifungal susceptibility profiles of clinical and environmental isolates of Aspergillus species. During the period from January 2018 to June 2019, a total of 102 Aspergillus isolates (40 clinical, 40 hospital, and 22 community environment) were tested for antifungal susceptibility testing for determination of minimum inhibitory concentration (MIC)/minimum effective concentration (MEC) as per Clinical and Laboratory Standards Institute(CLSI) M38-A3 method for itraconazole, voriconazole, amphotericin B, and caspofungin. Out of these 102 Aspergillus isolates, A. flavus was the most common species present. Aspergillus species were found to have low MIC values to azoles such as itraconazole and voriconazole except for one clinical isolate, which showed a MIC value of 2 μg/ml to voriconazole. Two isolates were non-wild-type for amphotericin B, but all isolates were wild-type for caspofungin. Antifungal susceptibility testing among clinical Aspergillus isolates and environmental surveillance studies in view of emerging drug resistance should be undertaken at a larger scale.

PCR-RFLP metoda u kombinaciji sa elektroforezom na čipu kao alat za određivanje varijabilnosti između važnih vrsta Aspergillus

Aspergillus species are among the most significant producers of aflatoxins, which can contaminate a wide range of agricultural and food products at any stage of production. The aim of this research was to utilize molecular methods to determine and characterize the variability between isolates of standard Aspergillus species. Genomic DNA was isolated from the mycelium of all tested Aspergillus isolates. PCR amplifications were performed using gene-specific primers. The PCR method successfully amplified the ITS1-5.8S rDNA-ITS2 region and portions of the β-tubulin and calmodulin genes of all tested Aspergillus isolates. PCR products obtained after amplification with primer pairs (ITS1/ITS4 and Bt2a/Bt2b), followed by digestion with restriction enzymes HhaI, MwoI, and AlwI in RFLP analysis, facilitated the identification of variability among the studied Aspergillus species. The results of PCR-RFLP analysis on the tested isolates were consistent with those previously obtained through morphological examinations, indicating the effectiveness of this molecular method for identification and determination of variability among important Aspergillus species. The presented molecular method based on PCR-RFLP analysis, due to its advantages such as reproducibility, speed, and high sensitivity, represents a valuable tool for monitoring and controlling contamination by Aspergillus species in the food supply chain. The method described in this study can be successfully used for rapid identification and determination of variability between isolates of Aspergillus species, contributing to improved food safety control and public health.

Azole-Containing Agar Plates and Antifungal Susceptibility Testing for the Detection of Azole-Resistant Aspergillus Species in Hospital Environmental Samples.

The indoor environment of hospitals should be considered as an important reservoir of azole resistant Aspergillus species. In this study, we evaluated azole-containing agar plates (ACAPs) and antifungal susceptibility testing (AFST) for the detection of azole-resistant Aspergillus species in hospital environmental samples. Between September 2021 and January 2022, environmental samples (108 instruments and 12 air) were collected from different wards of 4 educational hospitals in Mazandaran province, Iran. All samples were cultured using ACAPs. Recovered Aspergillus isolates were molecularly identified at species level using partial DNA sequencing of beta-tubulin gene. AFST of Aspergillus species was performed using the Clinical and Laboratory Standards Institute M38-A3 guideline. Screening for cyp51A mutations was also done. Overall, 18 (15.0%) isolates of Aspergillus species were recovered from ACAPs, of which Aspergillus tubingensis (50%) and Aspergillus fumigatus (38.9%) were the commonest species. No isolate of Aspergillus species grew on posaconazole (PCZ)-containing agar plates. Among the 18 Aspergillus isolated species from ACAPs, 83.3% were related to samples from instruments. Of the nine isolates of A. tubingensis, 22.2% and 44.4% isolates showed minimum inhibitory concentration (MIC) = 2 μg/mL against voriconazole (VCZ) and itraconazole, respectively; and 44.4% isolates showed MIC = 1 μg/mL against PCZ. Of the seven isolates of A. fumigatus, one (14.3%) was resistant to VCZ. This isolate showed F46Y, G54E, G138C, M172V, M220I, D255E, T289F, G432C, and G448S mutation in cyp51A. Our finding showed the emergence of high MICs in cryptic and non-fumigatus species of Aspergillus such as A. tubingensis and VCZ resistance in A. fumigatus in indoor environment of hospitals.

Pulmonary Aspergillosis in Critically Ill COVID-19 Patients Admitted to the Intensive Care Unit: A Retrospective Cohort Study.

COVID-19-associated pulmonary aspergillosis (CAPA) is a life-threatening fungal infection that mainly affects critically ill patients. The aim of this study was to assess the incidence and clinical outcomes of putative CAPA in critically ill COVID-19 patients. This retrospective observational cohort study included 181 cases from 5 ICUs at Vienna General Hospital between January 2020 and April 2022. Patients were diagnosed with putative CAPA according to the AspICU classification, which included a positive Aspergillus culture in a bronchoalveolar lavage sample, compatible signs and symptoms, and abnormal medical imaging. The primary outcome was adjusted 60-day all-cause mortality from ICU admission in patients with vs. without putative CAPA. Secondary outcomes included time from ICU admission to CAPA diagnosis and pathogen prevalence and distribution. Putative CAPA was identified in 35 (19.3%) of 181 COVID-19 patients. The mean time to diagnosis was 9 days. Death at 60 days occurred in 18 of 35 (51.4%) patients with CAPA and in 43 of 146 (29.5%) patients without CAPA (adjusted HR (95%CI) = 2.15 (1.20-3.86, p = 0.002). The most frequently isolated Aspergillus species was Aspergillus fumigatus. The prevalence of putative pulmonary aspergillosis in critically ill COVID-19 patients was high and was associated with significantly higher mortality.

Epidemiology and antifungal susceptibilities of clinically isolated Aspergillus species in South China.

Aspergillosis is a rising concern worldwide; however, its prevalence is not well documented in China. This retrospective study determined Aspergillus's epidemiology and antifungal susceptibilities at Meizhou People's Hospital, South China. From 2017 to 2022, the demographic, clinical, and laboratory data about aspergillosis were collected from the hospital's records and analysed using descriptive statistics, chi-square test, and ANOVA. Of 474 aspergillosis cases, A. fumigatus (75.32%) was the most common, followed by A. niger (9.92%), A. flavus (8.86%), and A. terreus (5.91%). A 5.94-fold increase in aspergillosis occurred during the study duration, with the highest cases reported from the intensive care unit (52.74%) - chronic pulmonary aspergillosis (79.1%) and isolated from sputum (62.93%). Only 38 (8.02%) patients used immunosuppressant drugs, while gastroenteritis (5.7%), haematologic malignancy (4.22%), and cardiovascular disease (4.22%) were the most prevalent underlying illnesses. In A. fumigatus, the wild-type (WT) isolates against amphotericin B (99.1%) were higher than triazoles (97-98%), whereas, in non-fumigatus Aspergillus species, the triazole (95-100%) WT proportion was greater than amphotericin B (91-95%). Additionally, there were significantly fewer WT A. fumigatus isolates for itraconazole and posaconazole in outpatients than inpatients. These findings may aid in better understanding and management of aspergillosis in the region.

Evaluation of Aflatoxin-Producing Fungi in Indoor Air of Warehouses and Houses of Farmers in Giwa, Kaduna State Nigeria

The health risks associated with ingesting food contaminated with mycotoxins, particularly aflatoxin-contaminated staple foods like maize and other cereals, have been widely studied. However, there is little knowledge about the role of inhalation of pathogenic fungi as bioaerosols in contaminated air from handling crops as an occupational health risk. This paper presents a study aimed at determining the level of airborne aflatoxin-producing fungi in the indoor air of grain stores in the Giwa community of Kaduna State. Indoor air was sampled using the settling plate technique from grain stores, warehouses and living rooms. Metrological data of the studied area were collected from the Institute of Agricultural Research, ABU, Zaria. Airborne mycofloral concentrations were determined, and colonies of Aspergillus flavus were identified. The isolates were screened for aflatoxin production on Neutral Red Desiccated Coconut Agar (NRDCA). Selected aflatoxin-producing fungal isolates were screened for the presence of aflD (nor-1), aflM (ver-1) and AflR genes by PCR. Sampling was done once every month from October to December 2020. Mycofloral concentrations were in the range of 2.77x103−4.05x103 and 1.55x103−2.17x103CFUm-3for grain stores and living rooms respectively. A total of twelve (12) strains of A. flavus were isolated from the indoor air of the grain stores and warehouses while none was obtained from the living room. Eleven (11) isolates were confirmed to be aflatoxigenic on NRDCA, presenting 30 CFUm-3 of the indoor air mycofloral composition. The aflD, aflM and aflR were amplified with aflD being the most detected gene from all the aflatoxin-producing mould isolates of Aspergillus species. The mycofloral concentrations in the grain stores were higher than those in the living room and, in all the sampling sites, exceeded the limit of the total mycofloral concentration of 500 CFUm-3 for agricultural and industrial environments. There were significant differences (p<0.05) in the indoor mycofloral concentrations between the grain stores/warehouses and the living room. The presence of aflatoxigenic strains of A. flavus in the stores indicates that grain handlers and traders are at risk of occupational exposure to aflatoxigenic fungi and aflatoxins. Hence, they should wear protective materials for their safety while working in such stores.

Trends in the Prevalence of Amphotericin B-Resistance (AmBR) among Clinical Isolates of Aspergillus Species

The challenges of the invasive infections caused by the resistant Aspergillus species include the limited access to antifungals for treatment and high mortality. This study aimed to provide a global perspective of the prevalence of amphotericin B resistance (AmBR), geographic distribution, and the trend of AmBR from 2010 to 2020. To analyze the prevalence of in vitro AmBR in clinical Aspergillus species, we reviewed the literature and identified a total of 72 articles. AmBR was observed in 1128 out of 3061 Aspergillus terreus (36.8%), 538 out of 3663 Aspergillus flavus (14.9%), 141 out of 2691 Aspergillus niger (5.2%), and 353 out of 17,494 Aspergillus fumigatus isolates (2.01%). An increasing trend in AmB-resistant isolates of A. fumigatus and a decreasing trend in AmB-resistant A. terreus and A. flavus isolates were observed between 2016 and 2020. AmB-resistant A. terreus and A. niger isolates, accounting for 40.4% and 20.9%, respectively, were the common AmB-resistant Aspergillus species in Asian studies. However, common AmB-resistant Aspergillus species reported by European and American studies were A. terreus and A. flavus isolates, accounting for 40.1% and 14.3% in 31 studies from Europe and 25.1% and 11.7% in 14 studies from America, respectively. The prevalence of AmB-resistant A. niger in Asian isolates was higher than in American and European. We found a low prevalence of A. terreus in American isolates (25.1%) compared to Asian (40.4%) and European (40.1%). Future studies should focus on analyzing the trend of AmBR on a regional basis and using the same methodologies.

In Vitro Susceptibilities of Worldwide Isolates of Intrapulmonary Aspergillus Species and Important Candida Species in Sterile Body Sites against Important Antifungals: Data from the Antimicrobial Testing Leadership and Surveillance Program, 2017-2020.

To understand the changes of resistance in clinically commonly encountered fungi, we used the Antimicrobial Testing Leadership and Surveillance (ATLAS) database to explore in vitro antifungal susceptibilities against clinically important isolates of Aspergillus and Candida species (collected from intrapulmonary and sterile body areas, respectively). We applied the CLSI antifungal 2020 and the EUCAST antifungal 2020 guidelines. From 2017 to 2020, isolates of intrapulmonary Aspergillus fumigatus (n = 660), Aspergillus niger (n = 107), Aspergillus flavus (n = 96), Aspergillus terreus (n = 40), and Aspergillus nidulans species complex (n = 26) and sterile site-originated isolates of Candida albicans (n = 1,810), Candida glabrata (n = 894), Candida krusei (n = 120), Candida dubliniensis (n = 107), Candida lusitaniae (n = 82), Candida guilliermondii (n = 28), and Candida auris (n = 7) were enrolled in this study. Using the EUCAST 2020 breakpoints, it was demonstrated that amphotericin B and posaconazole displayed poor in vitro susceptibility rates against A. fumigatus isolates (<50% and 18.9%, respectively). In contrast, isavuconazole and itraconazole showed high in vitro potency against most Aspergillus isolates (>92%). Most intrapulmonary Aspergillus isolates exhibited MICs of ≤0.06 μg/mL to anidulafungin. Furthermore, intrapulmonary A. fumigatus isolates collected from Italy and the United Kingdom exhibited lower in vitro susceptibility to isavuconazole (72.2% and 69%, respectively) than those in the remaining ATLAS participant countries (>85%). Higher isavuconazole MIC90s against C. auris and C. guilliermondii (1 and 4 μg/mL, respectively) were observed compared to the other five Candida species. Despite the aforementioned MICs and susceptibilities against fungi, research needs to consider the pharmacokinetic (PK) profiles, pharmacodynamic (PD) parameters, and clinical treatment experience with antifungals against specific Aspergillus species. IMPORTANCE In addition to monitoring the antifungal susceptibilities of clinically important fungi, reviewing the PK/PD indices and the clinical therapy experience of antifungals under evaluation are important to guide an appropriate antifungal prescription. The efficacies of liposomal amphotericin B complex and anidulafungin for the treatment of pulmonary aspergillosis caused by different Aspergillus species need to be periodically evaluated in the future.

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

Otomycosis is considered a recurring fungal ear infection. The external auditory canal provides an appropriate and optimal situation for fungal growth. The study aimed to identify the causative agents of otomycosis and determine corresponding antifungal drug susceptibility patterns in north-western Iran. 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. 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. 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.

P398 COVID-19-associated pulmonary aspergillosis: Species distribution and susceptibility profiles

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesThis study aimed to investigate the species distribution and susceptibility profiles of Aspergillus species isolated from patients admitted to the intensive care unit with severe COVID-19 in Isfahan, Iran, between April 2021 and March 2022.MethodsThis retrospective study included intubated patients with COVID-19 in three referral COVID-19 hospitals. Tracheal aspirate (TA) samples were taken from 267 patients to investigate pulmonary co-infections. COVID-19-associated aspergillosis (CAPA) was defined according to the 2020 European Confederation of Medical Mycology/International Society of Human and Animal Mycosis consensus criteria. Aspergillus species obtained from samples were characterized based on conventional and molecular assays. In vitro antifungal susceptibility testing was performed on the obtained isolates according to the guidelines from the Clinical and Laboratory Standards Institute.ResultsThe mean age of the patients was 61.73 ± 12.69 years. The mean length of hospitalization and admission in ICU were 18.77 ± 12.94 and 13.51 ± 9.83 days, respectively. A total of 61 (22.9%) patients presented with a single cavity lesion. Pulmonary artery pseudoaneurysm was seen in seven patients and post-COVID-19 changes were seen in all patients. Based on the conventional and molecular techniques, 72 isolates of Aspergillus species (26.9%), including A. flavus (10.1%%), A. fumigatus (8.6%%), A. niger (3.3%), A. tubingensis (2.9%), A. terreus (1.1%), A. luchuensis (0.37%) A. quadrillineatus, and (0.37%), were obtained from 267 patients. MIC results showed that all Aspergillus species were susceptible to all tested antifungal drugs.ConclusionAccess to priority clinical groups, improving the care of patients with simultaneous pulmonary aspergillosis with COVID-19, and identifying Aspergillus species are essential steps in the care cascade to manage those affected by them.

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.

Improvement of saccharification of native grass, Pennisetum sp. using cellulase from isolated Aspergillus fumigatus for bioethanol production: an insight into in silico molecular modelling, docking and dynamics studies

Lignocellulosic grass biomass is potential substrate for economical and sustainable bioethanol production. However, the processing cost of bioethanol that majorly includes the hydrolysis of cellulose by cellulases is still a major concern for its industrial production. Thus, knowledge on the sequence to the structural study of cellulase enzyme with consideration of its catalytic region can give important information for effective enzyme engineering and consequently towards enhanced bioethanol production from Pennisetum sp. Therefore, in this study, sequence conservativeness of different cellulosic site among a group of endoglucanase family of cellulase from previously isolated Aspergillus species has been determined. Furthermore, comparative molecular modeling of the endoglucanase from eight different Aspergillus species including Aspergillus fumigatus was conducted and the obtained structures revealed a high degree of difference in their conformational folds. Analysis from InterProScan revealed that the modeled endoglucanase has similar types of domains and share homology with protein family, such as glycoside hydrolase family-61 and fungal cellulose binding domain. Furthermore, molecular docking and interaction studies demonstrated the presence of residues in the endoglucanase of A. fumigatus viz. His20, His88, Asp96, Ala99, Ser100, Ser101, His102, His169, Glu170, Arg173, Glu178, and Tyr218 that are responsible in forming the substrate interaction. An interesting molecular phenomenon, i.e., catalytic promiscuity has been noted for all the substrate bound complexes of A. fumigatus endoglucanase which also depicts the degree of ligand binding efficacy of the studied enzyme. The molecular interaction study, binding energy analysis and molecular dynamics simulation, demonstrated that heteromeric substrate XylGlc3 is more strongly interacting with the receptor enzyme. Overall, the present findings revealed that important amino acid residues can help in increasing the specificity of endoglucanase from A. fumigatus towards hydrolysis of Pennisetum sp. and other biomass that has an adequate amount of XylGlc3, for possible industrial applications.

Evaluation of catalase activity of clinical and environmental isolates of Aspergillus species.

Background and Objectives:Catalases are a good scavenger of H2O2 which degrades hydrogen peroxide into water and oxygen. They are considered as a virulence factor that are present in both spores and hypha of fungi. There is limited data regarding catalase activity in Aspergillus species. This study aimed to assess the mycelial catalase activity of clinical and environmental isolates of Aspergillus niger, A. tubingensis, A. flavus, A. luchuensis, A. piperis and A. terreus.Materials and Methods:Briefly, clinical and environmental Aspergillus species were used in the current study. Catalase activity was assessed for both groups of isolates including 13 A. flavus (12 clinical, 1 environmental), 13 A. terreus (8 clinical, 5 environmental), 26 A. tubingensis (13 clinical, 13 environmental), and 44 A. niger (25 environmental, 19 clinical) species. Fungal balls of mycelia were separated from the liquid culture and were crushed using homogenizer. The supernatants were collected and used for a catalase activity assay.Results:Totally, in our study 98 Aspergillus including 45 environmental and 53 clinical isolates were assessed for catalase activity. High catalase activity was detected among environmental Aspergillus species (Mean= 1.62 mU/ml) and the mean of mycelial catalase activity among clinical A. terreus isolates was higher than environmental strains.Conclusion:In summary, mycelial catalase activity varied among species and environmental isolates demonstrated higher catalase activity. Totally a significant difference was found between clinical and environmental Aspergillus isolates.

Antifungal susceptibility profile of Aspergillus species from patients with increased risk for aspergillosis

Aspergillosis is the most common fungal infection caused by molds, especially in high-risk patients. The treatment of these diseases is based on the use of polyene and azole antifungal drugs. Resistance rates of Aspergillus species to antifungal drugs vary widely across medical centers around the world. Antifungal susceptibility testing of Aspergillus species to antifungal agents could provide useful information for clinicians to make decision regarding the patient therapy. The aim of the study was to evaluate the antifungal susceptibility profile of Aspergillus species towards amphotericin B, itraconazole, voriconazole and caspofungin, isolated from patients with increased risk for aspergillosis. During a 2-year period, clinical specimens from 125 patients divided into 4 groups according to clinical diagnosis and EORTC/MSG criteria, were analysed at the Institute of Microbiology and parasitology, Faculty of Medicine, Skopje, Republic of North Macedonia. These groups included patients with primary immune deficiency, critically ill patients treated in intensive care units, patients with chronic aspergillosis and cystic fibrosis. All specimens (from respiratory tract and blood culture) were investigated with conventional mycological methods, by inoculation of specimens on media for support of fungal growth. Identification of Aspergillus was performed with macroscopic analysis of mold colonies and additional microscopic analysis of their conidia with lactophenol blue method. E-test strips of voriconazole, itraconazole, amphotericin B and caspofungin (AB bioMerieux, France) were used for determination of the antifungal susceptibility profile of Aspergillus species. Seventy-one isolates of Aspergillus species were confirmed in our patients. Four isolates of A.fumigatus (5.6%) were confirmed in blood cultures, from patients with primary immune deficiencies, and 67 isolates (94.4%) originated from respiratory specimens from patients with different underlying diseases. A.flavus was identified in 11 patients and A.terreus in 3 patients. Resistance to amphotericin B was detected in 6 isolates (2 isolates of A.fumigatus, 1 isolate of A.flavus and 3 isolates of A.terreus). Only one isolate of A.fumigatus showed resistance to itraconazole. All isolates of Aspergillus species were sensitive to voriconazole and caspofungin. In vitro antifungal susceptibility testing with E-test demonstrated resistance to amphotericin B in 6 isolates of Aspergillus species. Only one isolate of A.fumigatus was resistant to itraconazole. All isolates of Aspergillus species showed sensitivity to voriconazole and caspofungin. Antifungal susceptibility testing of Aspergillus species with E-test provides useful information for clinicians for appropriate choice of antifungal agents for treatment of aspergillosis. Keywords: Aspergillus, mold, infection, susceptibility, E-test

A CASE OF CHRONIC PULMONARY ASPERGILLOSIS AS THE INITIAL PRESENTATION FOR SARCOIDOSIS

TOPIC: Chest Infections TYPE: Medical Student/Resident Case Reports INTRODUCTION: Subacute invasive pulmonary aspergillosis is a locally invasive subacute form of chronic pulmonary aspergillosis (CPA) which typically involves the upper lobes of individuals with pre-existing lung disease or immunodeficiency. Patients with chronic fibrocystic sarcoidosis are prone to CPA. We described a case of sarcoidosis complicated with CPA which was diagnosed concomitantly. CASE PRESENTATION: A 34 year old male with no past medical history presented with hemoptysis for 3 days, associated with chronic productive cough, exertional dyspnea and skin rashes over his arms and face for the past year. Physical examination revealed multiple violaceous plaque over bilateral upper arms and face; reduced breath sounds over bilateral upper zones. CT Chest revealed bilateral upper lobes cavitations, left upper lobe mass-like consolidation, approximately 2.9 cm with air crescent sign. Laboratory tests were notable for lymphopenia, ESR 43 mm/h, CD4+ count 244, Aspergillus Ag 0.6, Aspergillus IgG positive, serum ACE 178 U/L, fungal culture from bronchial alveolar lavage (BAL) isolated Aspergillus species. ANCA, Histoplasma Ag, Cryptococcal Ag, HIV Ag-Ab, Quantiferon TB, Sputum acid fast smear were negative. Endobronchial ultrasound with fine needle aspiration (FNA) of subcarinal lymph nodes showed non-caseating granuloma. Skin biopsies of upper extremities skin lesions showed non-caseating granulomas without microorganisms seen. The patient was started on Voriconazole for 3 months duration and scheduled for outpatient follow up. DISCUSSION: Patients with sarcoidosis are predisposed to CPA due to underlying pulmonary involvement especially with fibrocystic sarcoidosis in addition to the mildly immunocompromised state, as evidenced by low CD4 count. The diagnosis of CPA was made based on symptomatology (chronic productive cough, dyspnea and hemoptysis), bilateral upper lobe cavitations on CT Chest, positive Aspergillus IgG and positive BAL culture for Aspergillus species. It was a challenge in making concomitant diagnosis of Sarcoidosis as it is a diagnosis of exclusion. Diagnosis of sarcoidosis cannot be made based on the non-caseating granulomas resulted from the FNA of the lymph nodes as aspergillosis can present with similar picture. The skin rash which appeared to be plaque sarcoidosis ultimately leads us to the diagnosis of sarcoidosis after confirmation of non-caseating granuloma on skin biopsy. CONCLUSIONS: Patients with CPA typically present with background risk factors of underlying lung disease. It is essential to have a high clinical suspicion for underlying undiagnosed disease processes especially when individuals presented with CPA without any obvious risk factors. CPA is a serious complication of sarcoidosis and is associated with high mortality and morbidity. Unfortunately, there is no consensus currently on how to best treat patients diagnosed with sarcoidosis and CPA. REFERENCE #1: Denning DW, Cadranel J, Beigelman-Aubry C, et al. Chronic pulmonary aspergillosis: rationale and clinical guidelines for diagnosis and management. Eur Respir J. 2016;47(1):45-68. doi:10.1183/13993003.00583-2015 REFERENCE #2: Alastruey-Izquierdo A, Cadranel J, Flick H, et al. Treatment of Chronic Pulmonary Aspergillosis: Current Standards and Future Perspectives. Respiration. 2018;96(2):159-170. doi:10.1159/000489474 REFERENCE #3: Denning DW, Pleuvry A, Cole DC. Global burden of chronic pulmonary aspergillosis complicating sarcoidosis. Eur Respir J. 2013;41(3):621-626. doi:10.1183/09031936.00226911 DISCLOSURES: No relevant relationships by Farahnaz Anwar, source=Web Response No relevant relationships by James Choi, source=Web Response No relevant relationships by Si Yuan Khor, source=Web Response No relevant relationships by Jason Liu Liu, source=Web Response No relevant relationships by Akhil Sharma, source=Web Response No relevant relationships by Enhua Wang, source=Web Response

In Vitro Antifungal Activity of Luliconazole, Efinaconazole, and Nine Comparators Against Aspergillus and Candida Strains Isolated from Otomycosis

Background: Aspergillus and Candida species are the most commonly identified fungal pathogens in otomycosis. However, we usually encounter some difficulties in its treatment because many patients show resistance to antifungal agents and present a high recurrence rate. Objectives: The current research was conducted to compare the in vitro activities of luliconazole (LUL), and efinaconazole (EFN) and the nine comparators on Aspergillus and Candida strains isolated from otomycosis. Methods: The in vitro activities of nine common antifungal drugs (amphotericin B (AMB), voriconazole (VRC), fluconazole (FLU), itraconazole (ITC), ketoconazole (KTO), clotrimazole (CLO), nystatin (NYS), terbinafine (TRB), and caspofungin (CAS)) and two novel new azoles (LUL and EFN) against 108 clinical isolates of Aspergillus and Candida species obtained from otomycosis were assessed according to the CLSI broth microdilution document. Results: The LUL and EFN had the geometric mean minimum inhibitory concentrations (GM MICs) of 0.098 and 0.109 μg/mL against all Aspergillus strains, respectively. Furthermore, the GM MICs of all Candida isolates for LUL, EFN, CAS, CLO, VRC, AMB, ITC, KTO, FLU, NYS, and TRB were calculated to be 0.133, 0.144, 0.194, 0.219, 0.475, 0.537, 0.655, 1.277, 4.905, 9.372, and 13.592 μg/mL, respectively. Additionally, 6 (35.29%), 2 (11.7%), and 1 (5.88%) Candida isolates were resistant to FLU, CAS, and VRC, respectively. Conclusions: As the findings indicated, LUL and EFN showed the lowest GM MIC values against the examined species. Accordingly, these novel imidazole and triazole antifungal agents can be regarded as proper candidates for the treatment of otomycosis caused by Aspergillus and Candida strains.

Morphometric and Genomic Characterization of Toxigenic Aspergillus Isolates Associated with Poultry Feeds and Feed Ingredients from Five Agro-Ecological Zones of Nigeria

Introduction: Toxigenic aspergilli are storage contaminants of poultry feeds and feed ingredients which produce secondary metabolites known as mycotoxins in these products. This research is aimed at detecting and characterizing mycotoxin producing aspergillus species present in feeds given to poultry birds and in the ingredients used in the production of these feeds.&#x0D; Method: A total of 102 samples of feed (n=30) and feed ingredients (n=72) were collected across 5 agro-ecological zones of Nigeria and analyzed for toxigenic Aspergilli using morphometric and molecular genomic characterization techniques. Different mycological media was used for isolation, by deploying pour plate method. The Aspergilli isolates morphometric identity were characterized using macroscopic and microscopic observed features. The isolated Aspergillus species were further characterized molecularly using genomic characterization protocols.&#x0D; Results: The results of the morphometric characterization indicated that nine (9) Aspergillus species were detected in the feed and feed ingredients, with Aspergillus flavus having the highest rate of occurrence among the isolates. Furthermore, the genomic typing using internal transcribe spacer (ITS) as a barcode, as well as comparison of the fungal isolates with other Aspergillus species in the genebank showed 99-100% similarity. Also, toxigenic gene typing indicated that some of the A. flavus were toxigenic with the detection of the following genes Nor-1, ver-1, OmtB and AflR.&#x0D; Conclusion: The findings of this study showed that toxigenic Aspergilli isolates are present in both poultry feed and feed ingredients from 5 agro-ecological zones in Nigeria and poses great public health issues.

Antifungal Resistance in Isolates of Aspergillus from a Pig Farm

Antibiotic resistance in fungal isolates is increasing on a global scale. Despite knowledge that pig farmers are occupationally exposed to infectious species of fungi, such as Aspergillus spp., little is known regarding their potential exposure to antifungal-resistant Aspergillus spp. The aim of this study is to obtain knowledge regarding the antifungal resistance profiles of isolates of Aspergillus species taken from different source materials—including airborne dust, surface dust, faeces, and straw—within a pig farm. The EUCAST broth microdilution method was used for testing antifungal resistance from 43 isolates of Aspergillus sampled in 3 periods inside a Danish pig farm. Seven species of Aspergillus were obtained, including A. candidus (n = 5), A. fumigatus (n = 5), A. glaucus (n = 13), A. nidulans (n = 2), A. niger (n = 15), A. terreus (n = 1), and A. versicolor (n = 2). Overall, 27.9% of the Aspergillus isolates displayed resistance against at least one antifungal, and 11.6% of Aspergillus isolates displayed resistance against multiple antifungals. The most abundant group exhibiting antifungal resistance was affiliated with the species A. niger, with isolates exhibiting resistance to itraconazole, voriconazole, and caspofungin. One isolate of A. glaucus and two isolates of A. versicolor were resistant to amphotericin B (MIC ≥ 2 mg/L amphotericin B). Antibiotic-resistant fungi were found on all three sampling days.

Species identification and antifungal susceptibility testing of Aspergillus strains isolated from patients with otomycosis in northern China

There are limited studies on species distribution and susceptibility profiles of Aspergillus strains isolated from patients with otomycosis in China. A total of 69 confirmed Aspergillus species isolates were obtained from ear swabs of patients diagnosed with otomycosis from 2017 to 2018 in northern China. Identification of these Aspergillus isolates at the species level was performed using conventional morphological methods and MALDI-TOF MS in combination with molecular sequencing, and invitro susceptibility to nine antifungal agents was evaluated using the Sensititre YeastOne system. The Aspergillus section Nigri had the greatest distribution of Aspergillus isolates. A. welwitschiae (n=25) was the most predominant isolate in section Nigri, followed by A. tubingensis (n=12) and A. niger (n=11). Other Aspergillus species were also isolated, including A. terreus (n=11), A. flavus/A. oryzae (n=8), and A. fumigatus (n=2). Amphotericin B, posaconazole, and echinocandins were highly invitro active against all the isolates tested. 2.9% (2/69) of the isolates were resistant to azoles in our study, including one A. niger isolate with a high MIC value for itraconazole (ITR) (16mg/L) and one A. tubingensis isolate cross-resistant to both voriconazole (VOR) (MIC >8mg/L) and ITR (MIC >16mg/L). One A. welwitschiae and one A. niger isolate both had increased MIC values of 4mg/L against VOR. A. welwitschiae was the most prevalent Aspergillus species isolated from patients with otomycosis. Our findings also indicated that the azole-resistant Aspergillus section Nigri should be utilized to guide clinical medication for Otomycosis.