Aspergillus Fumigatus Isolates Research Articles

Comparison of ISSR indicators in distinguishing aspergillus fumigatus isolates from different sources

Comparison of ISSR indicators in distinguishing aspergillus fumigatus isolates from different sources

Pan-azole resistance in clinical Aspergillus fumigatus isolates carrying TR34/L98H from birds and mammals in Belgium

Aspergillosis causes significant health risks to both birds and mammals. The outcome of these infections is often poor due to delayed diagnosis and treatment failure. We investigated 152 cases of aspergillosis from birds and mammals in Belgium. Most samples originated from the taxonomic orders Artiodactyla (40.1 %) and Columbiformes (19.7 %). Five isolates (3.3 %) showed phenotypical resistance against at least one medical azole. Three of these isolates were pan-azole resistant bearing the TR34/L98H mutation. The predominance of this resistance mutation supports an environmental route for exposure and resistance selection, highlighting the importance of the One Health concept.

Antifungal Susceptibility and Genotypic Analysis of cyp51A Mutations in Aspergillus fumigatus Isolates in Malaysia.

Azole resistance in Aspergillus fumigatus poses a significant challenge in the management of invasive aspergillosis. This study aimed to investigate the antifungal susceptibility and cyp51A mutation profiles of A. fumigatus isolates in Malaysia. Sixty clinical A. fumigatus isolates were collected and subjected to antifungal susceptibility testing (AFST) and molecular analysis. The antifungal susceptibility testing was performed according to CLSI M38 guideline. The geometric mean (GM) minimum inhibitory concentration (MIC), MIC50/MIC90 for voriconazole, itraconazole, posaconazole, amphotericin B, and isavuconazole against A. fumigatus in non-invasive cases and invasive cases were calculated. In addition, the presence of cyp51A mutations was also identified. The present study revealed an overall resistance rate of 6.7% among the isolates. In non-invasive cases, isavuconazole and posaconazole demonstrated the lowest GM MIC of 0.08 µg/mL. Following them were itraconazole, voriconazole, and amphotericin B with concentrations of 0.15µg/mL, 0.16µg/mL and 0.90µg/mL, respectively. Similarly, in invasive cases, isavuconazole and posaconazole exhibited the lowest GM MIC of 0.09µg/mL. Following them were itraconazole, voriconazole, and amphotericin B with concentrations of 0.14µg/mL, 0.17µg/mL and 0.80µg/mL, respectively. Genotypic analysis revealed various cyp51A mutations, including F46Y, M172V, N248K, R34L, V244A, V244S, and E427K. However, not all mutations corresponded to antifungal resistance. The majority of clinical Aspergillus fumigatus isolates demonstrated susceptibility to the antifungal agents tested, with isavuconazole and posaconazole demonstrating the lowest MIC values. However, cyp51A mutations were discovered without a consistent correlation to antifungal resistance, emphasising the need for additional research.

First detection of triazole-resistant aspergillus fumigatus harbouring the TR34/L98H Cyp51A mutation in Burkina Faso.

Triazole-resistant Aspergillus fumigatus (TRAF) isolates are a growing public health problem with worldwide distribution. Epidemiological data on TRAF is limited in Africa, particularly in West Africa. This study aimed to screen for the environmental presence of TRAF isolates in the indoor air of two hospitals in Burkina Faso. Air samples were collected in wards housing patients at risk for invasive aspergillosis, namely infectious diseases ward, internal medicine ward, nephrology ward, pulmonology ward, medical emergency ward and paediatric ward. Sabouraud Dextrose Agar supplemented with triazoles was used to screen the suspected TRAF isolates and EUCAST method to confirm the resistance of suspected isolates. Sequencing of cyp51A gene was used to identify the resistance mechanism of confirmed TRAF isolates. Of the 198 samples collected and analysed, 67 showed growth of A. fumigatus isolates. The prevalence of TRAF isolates was 3.23% (4/124). One TRAF isolate exhibited a pan-triazole resistance. Sequencing of cyp51A gene identified the TR34/L98H mutation for this pan-triazole resistant isolate. This study showed for the first time the circulation of the pan-azole resistant isolate harbouring the TR34/L98H mutation in Burkina Faso. These findings emphasise the need to map these TRAF isolates in all parts of Burkina Faso and to establish local and national continuous surveillance of environmental and clinical TRAF isolates in this country.

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.

Exposure to resistant fungi across working environments and time

Antifungal resistance has emerged as a significant health concern with increasing reports of resistant variants in previously susceptible species. At present, little is known about occupational exposure to antifungal-resistant fungi. This study aimed to investigate Danish workers' occupational exposure to airborne fungi resistant to first-line treatment drugs. A retrospective study was performed on a unique collection of personal exposure samples gathered over a twenty-year period from Danish working environments, in sectors including agriculture, animal handling, waste management, and healthcare. A total of 669 samples were cultivated at 37 °C and fungal colonies were identified using MALDI-TOF MS. Subsequently, identification was confirmed by amplicon sequencing the genes of calmodulin and beta-tubulin to unveil potential cryptic species.Infectious fungi (495 isolates from 23 species) were tested for resistance against Itraconazole, Voriconazole, Posaconazole, and Amphotericin B. Working environments were highly variable in the overall fungal exposure, and showed vastly different species compositions. Resistance was found in 30 isolates of the species Aspergillus fumigatus (4 of 251 isolates), A. nidulans (2 of 13), A. niger complex (19 of 131), A. versicolor (3 of 18), and A. lentulus (2 of 2). Sequence analysis revealed several cryptic species within the A. niger complex including A. tubingensis, A. luchuensis, and A. phoenicis. Among the resistant A. fumigatus isolates, two contained the well-described TR34/L98H mutation in the cyp51A gene and promoter region, while the remainder harbored silent mutations.The results indicate that the working environment significantly contributes to exposure to resistant fungi, with particularly biofuel plant workers experiencing high exposure. Differences in the prevalence of resistance across working environments may be linked to the underlying species composition.

Impact of Aspergillus fumigatus inoculation on the composting of wood shaving bedding for horses.

Equine farming generates a significant amount of waste, prompting the need for effective management. Composting enhanced by filamentous fungi holds promise for this purpose. This study focused on inoculating Aspergillus fumigatus isolates in composting horse bedding made with wood shavings (Pinus elliottii). The experiment lasted 90 days, with two treatment groups, control and inoculated, analyzing temperature, pH, electrical conductivity, total organic carbon and nitrogen content, and cellulose, hemicellulose, and lignin contents. Both treatments entered the thermophilic phase by the fourth day, reaching temperatures above 55°C and mesophilic maturation at 35 days (41±0.2°C). The inoculated treatment exhibited higher electrical conductivity after 30 days and a more pronounced reduction in the total carbon content (42.85%vs. 38.29%) compared to the control. While there was no significant nitrogen difference, the inoculated treatment had a sharper reduction in carbon/nitrogen ratio, and cellulose and hemicellulose contents. Both treatments showed low coliform counts, no Salmonella sp., and reduced Strongyloides sp. larvae. Inoculating A. fumigatus in saturated horse bedding made from wood shavings improved compost quality, providing a possibility for sustainable equine farming waste treatment.

Variability in competitive fitness among environmental and clinical azole-resistant Aspergillus fumigatus isolates.

Azole resistance in the human fungal pathogen Aspergillus fumigatus presents a global public health challenge. Understanding the epidemic trends and evolutionary patterns of azole resistance is critical to prevent and control the spread of azole-resistant isolates. The primary cause is the mutation of the drug target 14α-sterol-demethylase Cyp51A, yet its impact on competitive ability remains uncertain. Our competition assays revealed a diverse range of fitness outcomes for environmental and clinical cyp51A-mutated isolates. We have shown that this fitness cost is not reliant on cyp51A mutations but might be linked to unknown mutations induced by stress conditions. Among these isolates, the majority displayed fitness costs, while a few displayed enhanced competitive ability, which may have a potential risk of spread and the need to closely monitor these isolates. Our observation reveals the variation in fitness costs among azole-resistant isolates of A. fumigatus, highlighting the significant role of fitness cost in the spread of resistant strains.

In vivo pharmacodynamic characterization of a next-generation polyene, SF001, in the invasive pulmonary aspergillosis mouse model.

SF001 is a next-generation polyene antifungal drug in development, designed to have increased specificity to fungal ergosterol, which is absent in humans, and decreased binding to cholesterol. SF001 demonstrates long-acting, potent, broad-spectrum fungicidal activity. The goal of the current study was to determine the pharmacodynamic index and target of SF001 in an immunocompromised mouse model of invasive pulmonary aspergillosis against six Aspergillus fumigatus isolates. Minimum inhibitory concentration (MIC) values ranged from 0.5 to 2.0 mg/L. Plasma and epithelial lining fluid (ELF) pharmacokinetics were performed following single intraperitoneal doses of 1, 4, 16, and 64 mg/kg. Treatment efficacy was assessed with each of the six fungal isolates using daily doses of SF001 ranging from 0.25 to 64 mg/kg/day over a 96-h treatment duration. Efficacy was assessed by A. fumigatus quantitative PCR of conidial equivalents from lung homogenates. Nonlinear regression analysis using the Hill equation demonstrated that the 24-h exposure-response relationships for both plasma and ELF area under the concentration/MIC and Cmax/MIC ratios were strong and relatively similar [coefficient of determination (R2) = 0.74-0.75). Exposure-response relationships included a median plasma 24-h Cmax/MIC target for stasis and 1-log kill endpoint of 0.5 and 0.6, respectively. The present studies demonstrated in vitro and in vivo SF001 potency against A. fumigatus. These results have potential relevance for SF001 clinical dose selection and evaluation of susceptibility breakpoints.

Epidemiology and Azole Resistance of Clinical Isolates of Aspergillus fumigatus from a Large Tertiary Hospital in Ningxia, China.

The objective of this study was to determine the clinical distribution, in vitro antifungal susceptibility and underlying resistance mechanisms of Aspergillus fumigatus (A. fumigatus) isolates from the General Hospital of Ningxia Medical University between November 2021 and May 2023. Antifungal susceptibility testing was performed using the Sensititre YeastOne YO10, and isolates with high minimal inhibitory concentrations (MICs) were further confirmed using the standard broth microdilution assays established by the Clinical and Laboratory Standards Institute (CLSI) M38-third edition. Whole-Genome Resequencing and RT-qPCR in azole-resistant A. fumigatus strains were performed to investigate the underlying resistance mechanisms. Overall, a total of 276 A. fumigatus isolates were identified from various clinical departments, showing an increasing trend in the number of isolates over the past 3 years. Two azole-resistant A. fumigatus strains (0.72%) were observed, one of which showed overexpression of cyp51A, cyp51B, cdr1B, MDR1/2, artR, srbA, erg24A, and erg4B, but no cyp51A mutation. However, the other strain harbored two alterations in the cyp51A sequences (L98H/S297T). Therefore, we first described two azole-resistant clinical A. fumigatus strains in Ningxia, China, and reported one azole-resistant strain that has the L98H/S297T mutations in the cyp51A gene without any tandem repeat (TR) sequences in the promoter region. This study emphasizes the importance of enhancing attention and surveillance of azole-resistant A. fumigatus, particularly those with non-TR point mutations of cyp51A or non-cyp51A mutations, in order to gain a better understanding of their prevalence and spread in the region.

Most Azole-Susceptible Isolates of Aspergillus fumigatus in Hokkaido, Japan are Clustered with Worldwide Strains That Do Not Have Tandem Repeats in Cyp51A Promoter.

In this study, we analyzed Aspergillus fumigatus short tandem repeat patterns of 106 strains isolated from the outdoor air, clinical specimens, and king penguins (Aptenodytes patagonicus) with aspergillosis in Japan, and compared them with those of 668 strains from AfumID (including six isolates from Japan). The results showed that the isolates were classified into three major groups. Group II contained most of the azole-resistant strains with 34- and 46-bp tandem repeats in cyp51A promoter. As in our previous study, OKH50 and Env1 strains were classified in Group II. Most of the azole-susceptible strains obtained in Japan were classified in Group III.

Aspergillus Outbreak in an Intensive Care Unit: Source Analysis with Whole Genome Sequencing and Short Tandem Repeats.

Whole genome sequencing (WGS) is widely used for outbreak analysis of bacteriology and virology but is scarcely used in mycology. Here, we used WGS for genotyping Aspergillus fumigatus isolates from a potential Aspergillus outbreak in an intensive care unit (ICU) during construction work. After detecting the outbreak, fungal cultures were performed on all surveillance and/or patient respiratory samples. Environmental samples were obtained throughout the ICU. WGS was performed on 30 isolates, of which six patient samples and four environmental samples were related to the outbreak, and twenty samples were unrelated, using the Illumina NextSeq 550. A SNP-based phylogenetic tree was created from outbreak samples and unrelated samples. Comparative analysis (WGS and short tandem repeats (STRs), microsatellite loci analysis) showed that none of the strains were related to each other. The lack of genetic similarity suggests the accumulation of Aspergillus spores in the hospital environment, rather than a single source that supported growth and reproduction of Aspergillus fumigatus. This supports the hypothesis that the Aspergillus outbreak was likely caused by release of Aspergillus fumigatus spores during construction work. Indeed, no new Aspergillus cases were observed in the ICU after cessation of construction. This study demonstrates that WGS is a suitable technique for examining inter-strain relatedness of Aspergillus fumigatus in the setting of an outbreak investigation.

Drug-Resistant Aspergillus spp.: A Literature Review of Its Resistance Mechanisms and Its Prevalence in Europe.

Infections due to the Aspergillus species constitute an important challenge for human health. Invasive aspergillosis represents a life-threatening disease, mostly in patients with immune defects. Drugs used for fungal infections comprise amphotericin B, triazoles, and echinocandins. However, in the last decade, an increased emergence of azole-resistant Aspergillus strains has been reported, principally belonging to Aspergillus fumigatus species. Therefore, both the early diagnosis of aspergillosis and its epidemiological surveillance are very important to establish the correct antifungal therapy and to ensure a successful patient outcome. In this paper, a literature review is performed to analyze the prevalence of Aspergillus antifungal resistance in European countries. Amphotericin B resistance is observed in 2.6% and 10.8% of Aspergillus fumigatus isolates in Denmark and Greece, respectively. A prevalence of 84% of amphotericin B-resistant Aspergillus flavus isolates is reported in France, followed by 49.4%, 35.1%, 21.7%, and 20% in Spain, Portugal, Greece, and amphotericin B resistance of Aspergillus niger isolates is observed in Greece and Belgium with a prevalence of 75% and 12.8%, respectively. The prevalence of triazole resistance of Aspergillus fumigatus isolates, the most studied mold obtained from the included studies, is 0.3% in Austria, 1% in Greece, 1.2% in Switzerland, 2.1% in France, 3.9% in Portugal, 4.9% in Italy, 5.3% in Germany, 6.1% in Denmark, 7.4% in Spain, 8.3% in Belgium, 11% in the Netherlands, and 13.2% in the United Kingdom. The mechanism of resistance is mainly driven by the TR34/L98H mutation. In Europe, no in vivo resistance is reported for echinocandins. Future studies are needed to implement the knowledge on the spread of drug-resistant Aspergillus spp. with the aim of defining optimal treatment strategies.

Optimization of the EUCAST reference broth microdilution method for echinocandin susceptibility testing of Aspergillus fumigatus.

Because of the high inoculum (105 cfu/mL) used in the EUCAST susceptibility testing of Aspergillus spp., determination of the minimal effective concentration (MEC) of echinocandins is challenging as the morphological differences are subtle. The MECs of 10 WT and 4 non-WT Aspergillus fumigatus isolates were determined with the EUCAST E.Def 9.4. Plates were inoculated with increasing inocula (102-105 cfu/mL) and after 24 and 48 h of incubation, MECs were determined macroscopically (magnifying mirror) and microscopically (inverted microscope) by two observers, spectrophotometrically (OD at 405 nm) and colorimetrically (absorbance at 450/630 nm after 2 h incubation with 400 mg/L XTT/6.25 μM menadione). The interobserver (between observers)/intermethod (compared with the microscopic method) essential agreement (EA, ±1 2-fold dilution) and categorical agreement (CA) were determined for each inoculum. Echinocandin-induced microscopic hyphal alterations or macroscopic changes in turbidity were subtle with a 105 cfu/mL inoculum compared with the lower inocula of 103 and 102 cfu/mL, where more distinct changes in turbidity and formation of characteristic rosettes were obvious at the MEC after 48 h. A 105 cfu/mL inoculum resulted in wider MEC distributions (3-6 dilutions) and lower interobserver EA (69%), macroscopic-microscopic EA (26%) and CA (71%) compared with a 103 cfu/mL inoculum (2-3 dilutions, 100%, 100% and 100%, respectively). Spectrophotometric readings using a 103 cfu/mL inoculum showed good EA (57-93%) and excellent CA (86%-100%), while the XTT assay demonstrated excellent EA (93%) and CA (100%). A 48 h incubation using a 103 cfu/mL inoculum improved echinocandin MEC determination for A. fumigatus with the EUCAST method, while the colorimetric assay could allow automation.

Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.

Work in nursing homes and occupational exposure to endotoxin and bacterial and fungal species.

Indoor microbial exposure may cause negative health effects. Only little is known about the occupational microbial exposure in nursing homes and the factors that influence the exposure. The exposure in nursing homes may be increased due to close contact with elderly persons who may carry infectious or antimicrobial-resistant microorganisms and due to handling of laundry, such as used clothing and bed linen. We investigated the microbial exposure in 5 nursing homes in Denmark, by use of personal bioaerosol samples from different groups of staff members taken during a typical working day, stationary bioaerosol measurements taken during various work tasks, sedimented dust samples, environmental surface swabs, and swabs from staff members' hands. From the samples, we explored bacterial and fungal concentrations and species composition, endotoxin levels, and antimicrobial resistance in Aspergillus fumigatus isolates. Microbial concentrations from personal exposure samples differed among professions, and geometric means (GM) were 2,159 cfu/m3 (84 to 1.5 × 105) for bacteria incubated on nutrient agar, 1,745 cfu/m3 (82 to 2.0 × 104) for bacteria cultivated on a Staphylococcus selective agar, and 16 cfu/m3 air for potential pathogenic fungi incubated at 37 °C (below detection limit to 257). Bacterial exposures were elevated during bed making. On surfaces, the highest bacterial concentrations were found on bed railings. The majority of bacterial species found were related to the human skin microflora, such as different Staphylococcus and Corynebacterium species. Endotoxin levels ranged from 0.02 to 59.0 EU/m3, with a GM of 1.5 EU/m3. Of 40 tested A. fumigatus isolates, we found one multiresistant isolate, which was resistant towards both itraconazole and voriconazole, and one isolate resistant towards amphotericin B. In conclusion, we give an overview of the general microbial exposure in nursing homes and show that microbial exposures are higher for staff with more care and nursing tasks compared with administrative staff.

Quantitative and Qualitative Airborne Mycobiota Surveillance in High-Risk Hospital Environment.

(1) Background: The primary aim of the presented study was to assess the prevalence of fungi in the indoor air of selected hospital wards, and the additional goal was to evaluate the susceptibility of cultured isolates of Aspergillus fumigatus to triazoles. (2) Methods: Three hematology departments and a hospital for lung diseases were surveyed in 2015 and/or 2019. Air samples were taken with a MicroBio MB1 air sampler on Sabouraud agar. The susceptibility of Aspergillus fumigatus isolates to voriconazole, posaconazole and itraconazole was tested with a microdilution method, according to EUCAST. (3) Results: The amount of fungi cultured from rooms equipped with sterile air circulation, as well as flow devices for air disinfection, was significantly lower compared to that from unprotected rooms. The areas most contaminated with fungi were corridors and bathrooms. The dominant species were Cladosporium and Penicillium. A. fumigatus was rare in hematological departments (6/61, 9.8% examinations performed in 2014 and 2/40, 5% in 2019), whereas in the hospital for lung diseases an outbreak of A. fumigatus spores with up to 300 CFU/m3 was noted in March 2015. No triazole-resistant A. fumigatus isolate was detected. (4) Conclusions: Regular microbiological testing of the hospital environment can contribute to the detection of spore outbreaks, and thus enable the implementation of corrective procedures (e.g., additional disinfection, changing of HEPA filters).

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections.

In vitro and in vivo evaluation of antifungal combinations against azole-resistant Aspergillus fumigatus isolates.

Azole resistance in Aspergillus fumigatus (Af) has become a widespread threat and a major concern for optimal management of patients with invasive aspergillosis (IA). Combination of echinocandins with azoles is an attractive alternative option for the treatment of IA due to azole-resistant Af strains. The aim of this study was to evaluate the in vitro and in vivo combination of caspofungin (CAS) with either voriconazole (VRZ) or posaconazole (PSZ). In vitro interactions were assessed by two methods, and an animal model of IA in Galleria mellonella was used for in vivo evaluation. Assessment of efficacy was based on larvae mortality. Groups of 10 larvae were infected by 3 clinical strains of Af (azole susceptible, AfS; PSZ resistant, AfR1; VRZ and PSZ resistant strain, AfR2). In vitro, combination of CAS and azoles was indifferent against AfS, and AfR2, and a synergy was found for AfR1. When compared to VRZ monotherapy, the combination of VRZ at 4 µg/larva with CAS at 4 µg/larva improved survival of AfR2-infected larvae (p=0.0066). Combination of PSZ at 4µg/larva with CAS at 4 µg/larva improved survival of AfR1-infected larvae compared to CAS (p=0.0002) and PSZ (0.0024) monotherapy. Antagonism was never observed. In conclusion, the combination of caspofungin with azoles is a promising alternative for the treatment of azole resistant strains of Af.

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.