Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

Formononetin protects against Aspergillus fumigatus Keratitis: Targeting inflammation and fungal load.

PURPOSE: To investigate the potential treatment of formononetin (FMN) on Aspergillus fumigatus (A. fumigatus) keratitis with anti-inflammatory and antifungal activity. METHODS: The effects of FMN on mice with A. fumigatus keratitis were evaluated through keratitis clinical scores, hematoxylin-eosin (HE) staining, and plate counts. The expression of pro-inflammatory factors was measured using RT-PCR, ELISA, or Western blot. The distribution of macrophages and neutrophils was explored by immunofluorescence staining. The antifungal properties of FMN were assessed through minimum inhibitory concentration (MIC), propidium iodide (PI) staining, fungal spore adhesion, and biofilm formation assay. RESULTS: In A. fumigatus keratitis mice, FMN decreased the keratitis clinical scores, macrophages and neutrophils migration, and the expression of TNF-α, IL-6, and IL-1ß. In A. fumigatus-stimulated human corneal epithelial cells (HCECs), FMN reduced the expression of IL-6, TNF-α, IL-1ß, and NLRP3. FMN also decreased the expression of thymic stromal lymphopoietin (TSLP) and thymic stromal lymphopoietin receptor (TSLPR). Moreover, FMN reduced the levels of reactive oxygen species (ROS) induced by A. fumigatus in HCECs. Furthermore, FMN inhibited A. fumigatus growth, prevented spore adhesion and disrupted fungal biofilm formation in vitro. In vivo, FMN treatment reduced the fungal load in mice cornea at 3 days post infection (p.i.). CONCLUSION: FMN demonstrated anti-inflammatory and antifungal properties, and exhibited a protective effect on mouse A. fumigatus keratitis.

Aspergillus fumigatus secretes a protease(s) that displays in silico binding affinity towards the SARS-CoV-2 spike protein and mediates SARS-CoV-2 pseudovirion entry into HEK-293T cells.

BACKGROUND: The novel coronavirus disease of 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Data from the COVID-19 clinical control case studies showed that this disease could also manifest in patients with underlying microbial infections such as aspergillosis. The current study aimed to determine if the Aspergillus (A.) fumigatus culture media (i.e., supernatant) possessed protease activity that was sufficient to activate the SARS-CoV-2 spike protein. METHODS: The supernatant was first analysed for protease activity. Thereafter, it was assessed to determine if it possessed proteolytic activity to cleave a fluorogenic mimetic peptide of the SARS-CoV-2 spike protein that contained the S1/S2 site and a full-length spike protein contained in a SARS-CoV-2 pseudovirion. To complement this, a computer-based tool, HADDOCK, was used to predict if A. fumigatus alkaline protease 1 could bind to the SARS-CoV-2 spike protein. RESULTS: We show that the supernatant possessed proteolytic activity, and analyses of the molecular docking parameters revealed that A. fumigatus alkaline protease 1 could bind to the spike protein. To confirm the in silico data, it was imperative to provide experimental evidence for enzymatic activity. Here, it was noted that the A. fumigatus supernatant cleaved the mimetic peptide as well as transduced the HEK-293T cells with SARS-CoV-2 pseudovirions. CONCLUSION: These results suggest that A. fumigatus secretes a protease(s) that activates the SARS-CoV-2 spike protein. Importantly, should these two infectious agents co-occur, there is the potential for A. fumigatus to activate the SARS-CoV-2 spike protein, thus aggravating COVID-19 development.

Trends in the activity of mold-active azole agents against Aspergillus fumigatus clinical isolates with and without cyp51 alterations from Europe and North America (2017-2021).

Azole resistance in Aspergillus fumigatus (AFM) is increasing and often associated with cyp51 alterations. We evaluated the activity of isavuconazole and other mold-active azoles against 731 AFM isolates causing invasive aspergillosis collected in Europe (EU; n = 449) and North America (NA; n = 282). Isolates were submitted to CLSI susceptibility testing and epidemiological cutoff value (ECV) criteria. A posaconazole ECV of 0.5 mg/L was used as no CLSI ECV was determined. Azole non-wild-type (NWT) isolates were submitted for cyp51 sequencing by whole genome sequencing. Overall, isavuconazole activity (92.7%/94.0% WT in EU/NA) was comparable to other azoles (WT rate range, 90.9%-96.4%/91.8%-98.6%, respectively), regardless of the region. A total of 79 (10.8%) azole NWT isolates were detected, and similar rates of these isolates were noted in EU (10.7%) and NA (11.0%). Although most AFM were WT to azoles, increasing azole NWT rates were observed in NA (from 6.0% in 2017 to 29.3% in 2021). Azole NWT rates varied from 4.9% (2019) to 20.6% (2018) in EU without an observed trend. cyp51 alterations occurred in 56.3%/54.8% of azole NWT from EU/NA, respectively. The cyp51A TR34/L98H alteration was observed only in EU isolates (72.0% of EU isolates), while cyp51A I242V occurred only in NA isolates (58.3%). Isavuconazole remained active (MIC, ≤1 mg/L) against 18.5/47.1% of azole NWT AFM exhibiting cyp51 alterations in EU/NA, along with voriconazole (29.6/82.4%; MIC, ≤1 mg/L) and posaconazole (48.1/88.2%; MIC, ≤0.5 mg/L). Fourteen different cyp51 alterations were detected in 44 of 79 NWT isolates. The in vitro activity of the azoles varied in AFM that displayed cyp51 alterations. IMPORTANCE A few microbiology laboratories perform antifungal susceptibility testing locally for systemically active antifungal agents. The identification of emerging azole-resistant Aspergillus fumigatus is worrisome. As such, there is a critical role for antifungal surveillance in tracking emerging resistance among both common and uncommon opportunistic fungi. Differences in the regional prevalence and antifungal resistance of these fungi render local epidemiological knowledge essential for the care of patients with a suspected invasive fungal infection.

Synergistic effects of putative Ca2+-binding sites of calmodulin in fungal development, temperature stress and virulence of Aspergillus fumigatus.

In pathogenic fungi, calcium-calmodulin-dependent serine-threonine-specific phosphatase calcineurin is involved in morphogenesis and virulence. Therefore, calcineurin and its tightly related protein complexes are attractive antifungal drug targets. However, there is limited knowledge available on the relationship between in vivo Ca2+-binding sites of calmodulin (CaM) and its functions in regulating stress responses, morphogenesis, and pathogenesis. In the current study, we demonstrated that calmodulin is required for hyphal growth, conidiation, and virulence in the human fungal pathogen, Aspergillus fumigatus. Site-directed mutations of calmodulin revealed that a single Ca2+-binding site mutation had no significant effect on A. fumigatus hyphal development, but multiple Ca2+-binding site mutations exhibited synergistic effects, especially when cultured at 42 °C, indicating that calmodulin function in response to temperature stress depends on its Ca2+-binding sites. Western blotting implied that mutations in Ca2+-binding sites caused highly degraded calmodulin fragments, suggesting that the loss of Ca2+-binding sites results in reduced protein stability. Moreover, normal intracellular calcium homeostasis and the nuclear translocation of the transcriptional factor CrzA are dependent on Ca2+-binding sites of AfCaM, demonstrating that Ca2+-binding sites of calmodulin are required for calcium signalling and its major transcription factor CrzA. Importantly, in situ mutations for four Ca2+-binding sites of calmodulin resulted in an almost complete loss of virulence in the Galleria mellonella wax moth model. This study shed more light on the functional characterization of putative calcium-binding sites of calmodulin in the morphogenesis and virulence of A. fumigatus, which enhances our understanding of calmodulin biological functions in cells of opportunistic fungal pathogens.

Protective role of CFTR during fungal infection of cystic fibrosis bronchial epithelial cells with Aspergillus fumigatus.

Lung infection with the fungus Aspergillus fumigatus (Af) is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function. We established a fungal epithelial co-culture model to examine the impact of Af infection on CF bronchial epithelial barrier function using Af strains 10AF and AF293-GFP, and the CFBE41o- cell line homozygous for the F508del mutation with (CF+CFTR) and without (CF) normal CFTR expression. Following exposure of the epithelial surface to Af conidia, formation of germlings (early stages of fungal growth) was detected after 9-12 hours and hyphae (mature fungal growth) after 12-24 hours. During fungal morphogenesis, bronchial epithelial cells showed signs of damage including rounding, and partial detachment after 24 hours. Fluorescently labeled conidia were internalized after 6 hours and more internalized conidia were observed in CF compared to CF+CFTR cells. Infection of the apical surface with 10AF conidia, germlings, or hyphae was performed to determine growth stage-specific effects on tight junction protein zona occludens protein 1 (ZO-1) expression and transepithelial electrical resistance (TER). In response to infection with conidia or germlings, epithelial barrier function degraded time-dependently (based on ZO-1 immunofluorescence and TER) with a delayed onset in CF+CFTR cell monolayers and required viable fungi and apical application. Infection with hyphae caused an earlier onset and faster rate of decline in TER compared to conidia and germlings. Gliotoxin, a major Af virulence factor, caused a rapid decline in TER and induced a transient chloride secretory response in CF+CFTR but not CF cells. Our findings suggest growth and internalization of Af result in deleterious effects on bronchial epithelial barrier function that occurred more rapidly in the absence of CFTR. Bronchial epithelial barrier breakdown was time-dependent and morphotype-specific and mimicked by acute administration of gliotoxin. Our study also suggests a protective role for CFTR by turning on CFTR-dependent chloride transport in response to gliotoxin, a mechanism that will support mucociliary clearance, and could delay the loss of epithelial integrity during fungal development in vivo.

Comparative proteome analysis identifies species-specific signature proteins in Aspergillus pathogens.

Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests. KEY POINTS: • CSPs and exoproteins could differentiate A. flavus and A. fumigatus. • A. fumigatus conidial surface harbored more antigenic proteins than A. flavus. • Identified species-specific signature proteins of A. flavus and A. fumigatus.

Inhibition of cGAS aggravated the host inflammatory response to Aspergillus fumigatus.

Backgroud: Aspergillus fumigatus (A. fumigatus) is a clinically important fungal pathogen. Invasive pulmonary aspergillosis (IPA) is the main fungal infection with increased morbidity and mortality in immunocompromised populations, although treatments are available. An innate DNA sensor known as cyclic GMP-AMP Synthase (cGAS) has recently been discovered that senses invading pathogens and has a significant impact on innate immunity. It can activate the cGAS-STING signaling pathway to stimulate downstream signals. But it is still unclear what role it plays in IPA's pathogenesis.Methods: An investigation into the infection of A. fumigatus was conducted by inhibiting cGAS activity in vivo and in vitro using siRNA and RU.521(an inhibitor of cGAS).Results: We discovered that suppressing cGAS increased the host's susceptibility to A. fumigatus and harmed those with infections by enhancing pulmonary tissue damage and edema, as well as decreasing fungal clearance. Furthermore, our findings show that inhibiting or silencing cGAS can exacerbate the inflammatory response in IPA mouse models and human bronchi epithelial cells (HBECs) treated with A. fumigatus by upregulating the production of inflammatory genes with non-type 1 interferon.Conclusion: Based on our analysis, we conclude that activating cGAS might increase host resistance to A. fumigatus, protect against pulmonary illnesses brought on by A. fumigatus and that exploring the cGAS-STING signaling pathway is beneficial not only for the immunological investigation of IPA but also may be a potential therapeutic objective.

The therapeutic role and mechanism of 4-Methoxycinnamic acid in fungal keratitis.

Fungal keratitis is an infectious vision-threatening disease that has a poor prognosis, and the clinical therapeutic drugs have multiple limitations, such as epithelial toxicity and low bioavailability. Therefore, new antifungal treatment strategies must be developed. 4-Methoxycinnamic acid (MCA) is a widely occurring natural phenolic acid that has been proven to have multiple effects, such as antibacterial, antifungal, anti-inflammatory, neuroprotective, and inhibiting cancer. In this research, we explored the effects and underlying mechanisms of MCA on A. fumigatus keratitis and the antifungal effects of the combination of MCA and natamycin (NATA) on A. fumigatus. We found that MCA exerts antifungal effects by inhibiting the synthesis of the fungal cell wall, changing the permeability of fungal cell membranes. Moreover, the MCA-NATA combination exhibited synergy for A. fumigatus. In addition, MCA exerted an anti-inflammatory effect by downregulating the inflammatory factors (IL-1ß, TNF-α, IL-6, and iNOS) in C57BL/6 mice and RAW264.7 cells. The anti-inflammatory mechanism of MCA was associated with the Mincle signal pathway. In summary, MCA acts as a potential therapeutic drug for fungal keratitis and a potential antifungal sensitizer for natamycin. MCA inhibits fungal cell wall synthesis, destroys the permeability of fungal cell membranes, and mediates the anti-inflammatory, immune response of the host.

Understanding Aspergillus fumigatus galactosaminogalactan biosynthesis: A few questions remain.

Half a century after their discovery, polymers of N-acetylgalactosamine produced by the Aspergilli have garnered new interest as mediators of fungal virulence. Recent work has focused on the Aspergillus fumigatus secreted and cell wall-associated heteropolymer, galactosaminogalactan (GAG). This polymer, composed of galactose (Gal) and partially deacetylated N-acetylgalactosamine (GalNAc), plays a role in a variety of pathogenic processes including biofilm formation, immune modulation and evasion, and resistance to antifungals. Given its many potential contributions to fungal pathogenesis, GAG is a promising therapeutic target for novel antifungal strategies. As such, several studies have sought to elucidate the biosynthetic pathways required for GAG production and secretion. Herein we review the progress made in the understanding of the molecular mechanisms underlying GAG synthesis and identify several gaps in our understanding of this process.

Thymol Ameliorates Aspergillus fumigatus Keratitis by Downregulating the TLR4/ MyD88/ NF-kB/ IL-1ß Signal Expression and Reducing Necroptosis and Pyroptosis.

Fungal keratitis is a refractory kind of keratopathy. We attempted to investigate the anti-inflammatory role of thymol on Aspergillus fumigatus (A. fumigatus) keratitis. Wound healing and fluorescein staining of the cornea were applied to verify thymol's safety. Mice models of A. fumigatus keratitis underwent subconjunctival injection of thymol. The anti-inflammatory roles of thymol were verified by hematoxylin-eosin (HE) staining, slit lamp observation, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. In contrast with the DMSO group, more transparent corneas and less inflammatory cells infiltration were detected in mice treated with 50 µg/ml thymol. Thymol downregulated the synthesis of TLR4, MyD88, NF-kB, IL-1ß, NLRP3, caspase 1, caspase 8, GSDMD, RIPK3 and MLKL. In summary, we proved that thymol played a protective part in A. fumigatus keratitis by cutting down inflammatory cells aggregation, downregulating the TLR4/ MyD88/ NF-kB/ IL-1ß signal expression and reducing necroptosis and pyroptosis.

Caspase-8 is involved in pyroptosis, necroptosis and the maturation and release of IL-1ß in Aspergillus fumigatus keratitis.

PURPOSE: To explore the role of caspase-8 in mediating the transition between different death modes in fungal keratitis. METHODS: The expression of caspase-8 in Aspergillus fumigatus (A. fumigatus) keratitis was detected using western blotting and immunofluorescence. After subconjunctival injection of Z-IETD-FMK (caspase-8 inhibitor) or VX765 (caspase-1 inhibitor), the mice corneas of A. fumigatus keratitis were observed and scored under a slit lamp. Colony plate count, immunofluorescence staining, western blotting and qRT-PCR experiments were used to detect fungal load, inflammatory cells, and the production of related mRNAs and proteins. In vitro experiments, the LDH release test, Cell Count Kit-8(CCK-8) assay, ELISA, qRT-PCR and western blotting were used to detect cell viability, related mRNAs and proteins. RESULTS: The caspase-8 protein was upregulated following fungal infection. Compared with the A. fumigatus keratitis group, the mice treated with Z-IETD-FMK had heavier corneal turbidity, higher clinical scores, more fungal load and fewer inflammatory cells. The expression of NLRP3, cleaved-caspase-1, N-GSDMD, and IL-1ß in the fungal infection group after Z-IETD-FMK pretreatment were downregulated, while RIPK3 and p-MLKL were upregulated. In the fungal infection group after VX765 pretreatment, the expression of cleaved-caspase-8 was up-regulated, while N-GSDMD was downregulated. CONCLUSIONS: Caspase-8 is involved in the early immune defense response of A. fumigatus keratitis. It is essential for the recruitment of inflammatory cells and the clearance of the fungus. In A. fumigatus keratitis, activated caspase-8 promoted the caspase-1/GSDMD signaling pathway to participate in pyroptosis, inhibited RIPK3/MLKL signaling pathway-mediated necroptosis, and promoted IL-1ß maturation and release by activating the NLRP3 inflammasomes.

In Vitro and In Vivo Antimicrobial Activity of Hypochlorous Acid against Drug-Resistant and Biofilm-Producing Strains.

The aims of this study were as follows. First, we determined the antimicrobial efficacy of hypochlorous acid (HClO) against bacterial, fungal, and yeast strains growing planktonically and growing in biofilms. Second, we sought to compare the activity of the combination of daptomycin and HClO versus those of the antimicrobial agents alone for the treatment of experimental catheter-related Staphylococcus epidermidis infection (CRI) using the antibiotic lock technique (ALT) in a rabbit model. HClO was generated through direct electric current (DC) shots at determined amperages and times. For planktonic susceptibility studies, 1 to 3 DC shots of 2, 5, and 10 mA from 0 to 300 s were applied. A DC shot of 20 mA from 0 to 20 min was applied to biofilm-producing strains. Central venous catheters were inserted into New Zealand White rabbits, inoculated with an S. epidermidis strain, and treated with saline solution or ALT using daptomycin (50 mg/mL), HClO (20 mA for 45 min), or daptomycin plus HClO. One hundred percent of the planktonic bacterial, fungal, and yeast strains were killed by applying one DC shot of 2, 5, and 10 mA, respectively. One DC shot of 20 mA for 20 min was sufficient to eradicate 100% of the tested biofilm-producing strains. Daptomycin plus HClO lock therapy showed the highest activity for experimental CRI with S. epidermidis. HClO could be an effective strategy for treating infections caused by extensively drug-resistant or multidrug-resistant and biofilm-producing strains in medical devices and chronic wounds. The results of the ALT using daptomycin plus HClO may be promising. IMPORTANCE Currently, drug-resistant infections are increasing and there are fewer antibiotics available to treat them. Therefore, there is an urgent need to find new antibiotics and nonantimicrobial strategies to treat these infections. We present a new nonantibiotic strategy based on hypochlorous acid generation to treat long-term catheter-related and chronic wounds infections.

Developing a new treatment for superficial fungal infection using antifungal Collagen-HSAF dressing.

Fungal pathogens are common causes of superficial clinical infection. Their increasing drug resistance gradually makes existing antifungal drugs ineffective. Heat stable antifungal factor (HSAF) is a novel antifungal natural product with a unique structure. However, the application of HSAF has been hampered by very low yield in the current microbial producers and from extremely poor solubility in water and common solvents. In this study, we developed an effective mode of treatment applying HSAF to superficial fungal infections. The marine-derived Lysobacter enzymogenes YC36 contains the HSAF biosynthetic gene cluster, which we activated by the interspecific signaling molecule indole. An efficient extraction strategy was used to significantly improve the purity to 95.3%. Scanning electron microscopy images revealed that the Type I collagen-based HSAF (Col-HSAF) has a transparent appearance and good physical properties, and the in vitro sustained-release effect of HSAF was maintained for more than 2 weeks. The effective therapeutic concentration of Col-HSAF against superficial fungal infection was explored, and Col-HSAF showed good biocompatibility, lower clinical scores, mild histological changes, and antifungal capabilities in animals with Aspergillus fumigatus keratitis and cutaneous candidiasis. In conclusion, Col-HSAF is an antifungal reagent with significant clinical value in the treatment of superficial fungal infections.

Genetic determinants of fungi-induced ROS production are associated with the risk of invasive pulmonary aspergillosis.

Reactive oxygen species (ROS) are an essential component of the host defense against fungal infections. However, little is known about how common genetic variation affects ROS-mediated antifungal host defense. In the present study, we investigated the genetic factors that regulate ROS production capacity in response to the two human fungal pathogens: Candida albicans and Aspergillus fumigatus. We investigated fungal-stimulated ROS production by immune cells isolated from a population-based cohort of approximately 200 healthy individuals (200FG cohort), and mapped ROS-quantitative trait loci (QTLs). We identified several genetic loci that regulate ROS levels (P < 9.99 × 10-6), with some of these loci being pathogen-specific, and others shared between the two fungi. These ROS-QTLs were investigated for their influence on the risk of invasive pulmonary aspergillosis (IPA) in a disease relevant context. We stratified hematopoietic stem-cell transplant (HSCT) recipients based on the donor's SNP genotype and tested their impact on the risk of IPA. We identified rs4685368 as a ROS-QTL locus that was significantly associated with an increased risk of IPA after controlling for patient age and sex, hematological malignancy, type of transplantation, conditioning regimen, acute graft-versus-host-disease grades III-IV, and antifungal prophylaxis. Collectively, this data provides evidence that common genetic variation can influence ROS production capacity, and, importantly, the risk of developing IPA among HSCT recipients. This evidence warrants further research for patient stratification based on the genetic profiling that would allow the identifications of patients at high-risk for an invasive fungal infection, and who would benefit the most from a preventive strategy.

Molecular detection of Aspergilli from commercial chicken in selected areas of Bangladesh.

Objectives: This study was designed to isolate, identify, and determine the prevalence of Aspergilli in commercial chicken in selected areas of Bangladesh. Materials and Methods: A total of 50 lung samples from suspected dead chickens, comprising broilers (n = 32) and layers (n = 18), aged between 5 days and 45 weeks, were collected from poultry farms located in the Gazipur district in Bangladesh. Fungi were primarily identified based on the colony morphology using potato dextrose agar (PDA). DNA was extracted from the suspected colonies. Aspegillus spp. was detected by genus-specific ASAP-1 and ASAP-2. Aspergillus spp. were then screened by polymerase chain reaction targeting Aspergillus flavus (FLA-1 and FLA-2), Aspergillus fumigatus (ASPU and Af3r), and Aspergillus niger (ASPU and Nilr). Results: The overall prevalence of Aspergillus spp. was 44% (n = 22/50; p < 0.05). Among the Aspergilli, A. flavus was detected in 10% (n = 5/50) of the samples. Similarly, A. fumigatus and A. niger were detected at 26% (n = 13/50) and 8% (n = 4/50) respectively. Three samples were associated with more than one fungus; two fungi (A. flavus and A. niger) were in two samples, and three fungi (A. flavus, A. fumigatus, and A. niger) were in one sample. Conclusion: Isolation and prevalence of Aspergillus spp. in commercial chicken were studied for the first time in Bangladesh.

[Aspergillus mediastinitis following post-immunotherapy lobectomy for oligometastatic lung]. / Médiastinite à Aspergillus après lobectomie post immunothérapie pour un cancer du poumon oligométastatique.

The relationship between infectious disease and therapy with immune checkpoint inhibitors remains unknown. We report the case of a 50-year-old woman with metastatic lung adenocarcinoma who responded remarkably well to immunotherapy and underwent upper right lobectomy. Three weeks after hospital discharge, she was readmitted for severe dyspnea due to mainstem bronchus compression by mediastinal mass. Histological analysis of transbronchial needle aspiration revealed A. fumigatus. After six months of voriconazole regimen, her symptoms improved with the regression of bronchial compression. Postoperative progression of pseudo-tumoral mass in patients treated with long-term immunotherapy may be related to opportunistic infectious disease and requires investigation.

Therapeutic Potential of Green Synthesized Gold Nanoparticles Using Extract of Leptadenia hastata against Invasive Pulmonary Aspergillosis.

Gold nanoparticles are widely used in the biomedical field for the treatment of several diseases, including cancer, inflammatory diseases, and immune system disorders, due to their distinctive physicochemical characteristics. In this study, we investigated the therapeutic potential of green synthesized gold nanoparticles using ethanolic leaf extract of Leptadenia hastata (LH-AuNPs) against invasive pulmonary aspergillosis (IPA) in mice. UV/visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), and zeta potential were used to characterize the biofabricated LH-AuNPs. Antifungal activity of LH-AuNPs was determined by MTT assay, (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), time-kill assay, and radial growth inhibition. TEM and SEM were used to examine the mode of the antifungal action of LH-AuNPs. The in vivo activity of LH-AuNPs against IPA was studied using a well-established IPA mouse model. LH-AuNPs excreted antifungal activity against Aspergillus fumigatus with MIC 64 µg/mL and inhibited the radial growth of A. fumigatus by 30% compared to the control. LH-AuNPs caused distortion and collapse of fungal hyphae and deterioration of cell walls. Interestingly, LH-AuNPs did not display any cytotoxicity on cultured primary bone marrow stem cells (BMSCs) or A549 human lung cell line in vitro at MIC concentration. IPA mice treated with LH-AuNPs displayed significant lung tissue repair without any in vivo cytotoxicity. LH-AuNPs administration showed significant suppression of fungal burden and gliotoxin production in the lung. In addition, LH-AuNPs inhibited IPA-induced pro-inflammatory cytokines production, including interleukin-1 (IL-1), interleukin-17 (IL-17), and tumor necrosis factor-alpha (TNF-α), and reduced oxidative stress in lung. In conclusion, our data provide LH-AuNPs as a novel nanoparticle therapy for IPA.

Eugenol protects against Aspergillus fumigatus keratitis by inhibiting inflammatory response and reducing fungal load.

Fungal keratitis is a corneal infection, which severely impairs vision. The fungal pathogen provokes host immune response, but the excessive inflammatory response causes significant collateral damage to the cornea. Eugenol, the main component of clove oil, has been found to have a broad range of pharmacological activities including anti-microbial, antioxidation and anti-inflammation. However, the role of eugenol in Aspergillus fumigatus (A. fumigatus) keratitis is unknown. In this study, we demonstrated that eugenol reduced mice keratitis severity, inflammatory cells infiltration, pro-inflammatory cytokine expression, and the fungal load. Eugenol also decreased the expressions of pro-inflammatory cytokines in human corneal epithelial cells (HCECs). We confirmed that the anti-inflammatory effects of eugenol were related to activating nuclear factor erythroid 2-related factor 2/Heme Oxygenase-1 (Nrf2/HO-1) signaling pathway. Moreover, we demonstrated that eugenol could inhibit the A. fumigatus growth and adhesion to host cells, as well as damage the fungal biofilm. The antifungal mechanism seemed to be disrupting the integrity of the fungal membrane and reducing the biosynthesis of ergosterol. Taken together, our research suggested that eugenol exerted protective effects on mouse A. fumigatus keratitis, due to its anti-inflammatory and antifungal activity.

Evaluation of Rezafungin Provisional CLSI Clinical Breakpoints and Epidemiological Cutoff Values Tested against a Worldwide Collection of Contemporaneous Invasive Fungal Isolates (2019 to 2020).

Rezafungin is a new echinocandin under development for the treatment of candidemia and invasive candidiasis. CLSI recently approved provisional susceptible-only breakpoints and epidemiological cutoff values for Candida spp. and rezafungin. The activities of rezafungin and comparators against 2019 to 2020 invasive fungal isolates was evaluated by applying the new CLSI breakpoints. Rezafungin demonstrated potent activity against Candida albicans (MIC50/MIC90, 0.03/0.06 mg/L; 100.0% susceptible), Candida tropicalis (MIC50/MIC90, 0.03/0.06 mg/L; 100% susceptible), Candida glabrata (MIC50/MIC90, 0.06/0.06 mg/L; 98.3% susceptible), Candida krusei (MIC50/MIC90, 0.03/0.03 mg/L; 100% susceptible), and Candida dubliniensis (MIC50/MIC90, 0.06/0.12 mg/L; 100% susceptible) when tested by the CLSI broth microdilution method. Rezafungin inhibited 99.6% of Candida parapsilosis isolates (MIC50/MIC90, 1/2 mg/L) at the susceptible breakpoint of ≤2 mg/L. All C. albicans, C. tropicalis, and C. krusei isolates, as well as most C. glabrata (96.2% to 97.9%) and C. parapsilosis (86.2% to 100%) isolates, were susceptible to comparator echinocandins. Fluconazole resistance was detected among 0.5%, 4.5%, 10.5%, and 1.2% of C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis isolates, respectively. All echinocandins displayed limited activity against Cryptococcus neoformans. Rezafungin and other echinocandins were active against Aspergillus fumigatus (minimum effective concentration for 90% of isolates tested [MEC90] range, 0.015 to 0.06 mg/L) and Aspergillus section Flavi (MEC90 range, 0.015 to 0.03 mg/L). All but 16 (8.6%) A. fumigatus isolates were susceptible to voriconazole, and 100% of Aspergillus section Flavi isolates were WT to mold-active azoles. When applying the CLSI clinical breakpoints, rezafungin displayed high susceptibility rates (>98.0%) against Candida isolates from invasive fungal infections and showed potent activity against Aspergillus isolates.