Marine Bioactive Compounds against Aspergillus fumigatus: Challenges and Future Prospects

With the mortality rate of invasive aspergillosis caused by Aspergillus fumigatus reaching almost 100% among some groups of patients, and with the rapidly increasing resistance of A. fumigatus to available antifungal drugs, new antifungal agents have never been more desirable than now. Numerous bioactive compounds were isolated and characterized from marine resources. However, only a few exhibited a potent activity against A. fumigatus when compared to the multitude that did against some other pathogens. Here, we review the marine bioactive compounds that display a bioactivity against A. fumigatus. The challenges hampering the discovery of antifungal agents from this rich habitat are also critically analyzed. Further, we propose strategies that could speed up an efficient discovery and broaden the dimensions of screening in order to obtain promising in vivo antifungal agents with new modes of action.

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In vitro and in vivo characterization of two nonsporulating Aspergillus fumigatus clinical isolates from immunocompetent patients

Medical Mycology ◽

10.1093/mmy/myz076 ◽

2019 ◽

Vol 58 (4) ◽

pp. 543-551

Author(s):

Zheng Zhang ◽

Yuan Jiang ◽

Jun Chen ◽

Peiying Chen ◽

Qingtao Kong ◽

...

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Pathogenic Fungus ◽

Antifungal Drugs ◽

Causative Role ◽

Immunocompromised Patients ◽

Pulmonary Aspergillosis ◽

Brla Gene

Abstract Aspergillus fumigatus is a pathogenic fungus responsible for invasive aspergillosis (IA). Typically, it can produce abundant conidia to survive and spread. The infection by A. fumigatus usually occurs in immunocompromised patients due to failed clearance of inhaled conidia. However, the incidence of aspergillosis in immunocompetent hosts has been increasing, the pathogenesis of which is still unknown. Our team previously obtained two clinical nonsporulating A. fumigatus isolates from non-immunocompromised patients, which only have the form of hyphae. This present study demonstrated the in vitro and in vivo characteristics of the two nonsporulating A. fumigatus isolates and verified that their conidiation defects are associated to abolished expression of the sporulation-related central regulatory pathway brlA gene. In addition, we confirmed the mutation site of brlA gene (c.657_660delTCCT) contributes to the nonsporulating phenotype in one clinical isolate. Plate assay showed that the two nonsporulating isolates have a similar resistance to antifungal drugs, cell wall disturbing substances, and oxidative stress compared with the wild-type reference Af293. Most important of all, we employed an immunocompetent mouse model to mimic the pathogenesis of pulmonary aspergillosis in non-immunocompromised patients. It revealed that the hyphae of two nonsporulating isolates and Af293 have similar virulence in immunocompetent hosts. Interestingly, the hyphae fragments of Af293 but not conidia are able to induce invasive aspergillosis in immunocompetent mice. In conclusion, our study indicate that the form of hyphae may play a dominant causative role in pulmonary aspergillosis of immunocompetent hosts rather than conidia.

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Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis

Applied and Environmental Microbiology ◽

10.1128/aem.01288-08 ◽

2008 ◽

Vol 74 (22) ◽

pp. 7023-7035 ◽

Cited By ~ 68

Author(s):

Matthias Brock ◽

Grégory Jouvion ◽

Sabrina Droin-Bergère ◽

Olivier Dussurget ◽

Marie-Anne Nicola ◽

...

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Light Emission ◽

Screening Method ◽

Growth Conditions ◽

Antifungal Drugs ◽

In Vivo Studies ◽

Drug Efficiency

ABSTRACT Aspergillus fumigatus is the main cause of invasive aspergillosis in immunocompromised patients, and only a limited number of drugs for treatment are available. A screening method for new antifungal compounds is urgently required, preferably an approach suitable for in vitro and in vivo studies. Bioluminescence imaging is a powerful tool to study the temporal and spatial resolutions of the infection and the effectiveness of antifungal drugs. Here, we describe the construction of a bioluminescent A. fumigatus strain by fusing the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene from A. fumigatus with the luciferase gene from Photinus pyralis to control the expression of the bioluminescent reporter. A. fumigatus transformed with this construct revealed high bioluminescence under all tested growth conditions. Furthermore, light emission correlated with the number of conidia used for inoculation and with the biomass formed after different incubation times. The bioluminescent strains were suitable to study the effectiveness of antifungals in vitro by several independent methods, including the determination of light emission with a microplate reader and the direct visualization of light emission with an IVIS 100 system. Moreover, when glucocorticoid-treated immunosuppressed mice were infected with a bioluminescent strain, light emission was detected from infected lungs, allowing the visualization of the progression of invasive aspergillosis. Therefore, this new bioluminescence tool is suitable to study the in vitro effectiveness of drugs and the disease development, localization, and burden of fungi within tissues and may also provide a powerful tool to study the effectiveness of antifungals in vivo.

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In Vivo Efficacy of Liposomal Amphotericin B against Wild-Type and Azole-Resistant Aspergillus fumigatus Isolates in Two Different Immunosuppression Models of Invasive Aspergillosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02479-16 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 3

Author(s):

Seyedmojtaba Seyedmousavi ◽

Johan W. Mouton ◽

Willem J. G. Melchers ◽

Paul E. Verweij

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Amphotericin B ◽

Liposomal Amphotericin ◽

Treated Group ◽

Liposomal Amphotericin B ◽

Wild Type ◽

Resistance Mutations ◽

Content Type

ABSTRACT Using an immunocompetent murine model of invasive aspergillosis (IA), we previously reported that the efficacy of liposomal amphotericin B (L-AmB) (Ambisome) is not hampered by the presence of azole resistance mutations in Aspergillus fumigatus (S. Seyedmousavi, W. J. G. Melchers, J. W. Mouton, and P. E. Verweij, Antimicrob Agents Chemother 57:1866–1871, 2013, https://doi.org/10.1128/AAC.02226-12 ). We here investigated the role of immune suppression, i.e., neutropenia and steroid treatment, in L-AmB efficacy in mice infected with wild-type (WT) A. fumigatus and with azole-resistant A. fumigatus harboring a TR34/L98H mutation in the cyp-51A gene. Survival of treated animals at day 14 in both immunosuppressed models was significantly better than that of nontreated controls. A dose-response relationship was observed that was independent of the azole-resistant mechanism and the immunosuppression method used. In the neutropenic model, 100% survival was reached at an L-AmB dose of 16 mg/kg of body weight for the WT strain and the TR34/L98H isolate. In the steroid-treated group, 90.9% survival and 100% survival were achieved for the WT isolate and the TR34/L98H isolate with an L-AmB dose of 16 mg/kg, respectively. The 50% effective dose (ED50) was 1.40 mg/kg (95% confidence interval [CI], 0.66 to 3.00 mg/kg) for the WT isolate and 1.92 mg/kg (95% CI, 0.60 to 6.17 mg/kg) for the TR34/L98H isolate in the neutropenic model and was 2.40 mg/kg (95% CI, 1.93 to 2.97 mg/kg) for the WT isolate and 2.56 mg/kg (95% CI, 1.43 to 4.56 mg/kg) for the TR34/L98H isolate in the steroid-treated group. Overall, there were no significant differences between the two different immunosuppressed conditions in the efficacy of L-AmB against the wild-type and azole-resistant isolates (P > 0.9). However, the required L-AmB exposure was significantly higher than that seen in the immunocompetent model.

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Efficient Clearance of Aspergillus fumigatus in Murine Lungs by an Ultrashort Antimicrobial Lipopeptide, Palmitoyl-Lys-Ala-dAla-Lys

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00526-08 ◽

2008 ◽

Vol 52 (9) ◽

pp. 3118-3126 ◽

Cited By ~ 29

Author(s):

Alexandra Vallon-Eberhard ◽

Arik Makovitzki ◽

Anne Beauvais ◽

Jean-Paul Latgé ◽

Steffen Jung ◽

...

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Microscopic Analysis ◽

Current Standard ◽

New Family ◽

Therapeutic Properties ◽

Survival Assay ◽

Opportunistic Fungal Pathogen

ABSTRACT Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The inefficiency of antifungal agents and high mortality rate resulting from invasive aspergillosis remain major clinical concerns. Recently, we reported on a new family of ultrashort cationic lipopeptides active in vitro against fungi. Mode of action studies supported a membranolytic or a detergent-like effect. Here, we screened several lipopeptides in vitro for their anti-A. fumigatus activity. To investigate the therapeutic properties of the selected peptides in vivo, we challenged immunosuppressed C57BL/6 wild-type mice intranasally with DsRed-labeled A. fumigatus conidia and subsequently treated the animals locally with the lipopeptides. Confocal microscopic analysis revealed the degradation of DsRed-labeled hyphal forms and residual conidia in the lungs of the mice. The most efficient peptide was tested further using a survival assay and was found to significantly prolong the life of the treated animals, whereas no mice survived with the current standard antifungal treatment with amphotericin B. Moreover, as opposed to the drug-treated lungs, the peptide-treated lungs did not display any toxicity of the peptide. Our results highlight the potential of this family of lipopeptides for the treatment of pulmonary invasive aspergillosis.

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Aspergillus fumigatus inhibits angiogenesis through the production of gliotoxin and other secondary metabolites

Blood ◽

10.1182/blood-2009-07-231209 ◽

2009 ◽

Vol 114 (26) ◽

pp. 5393-5399 ◽

Cited By ~ 78

Author(s):

Ronen Ben-Ami ◽

Russell E. Lewis ◽

Konstantinos Leventakos ◽

Dimitrios P. Kontoyiannis

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Capillary Tube ◽

Umbilical Vein ◽

Tube Formation ◽

Dependent Manner ◽

Antiangiogenic Effect ◽

Umbilical Vein Endothelial Cells

AbstractIn susceptible hosts, angioinvasion by Aspergillus fumigatus triggers thrombosis, hypoxia, and proinflammatory cytokine release, all of which are stimuli for angiogenesis. We sought to determine whether A fumigatus directly modulates angiogenesis. A fumigatus culture filtrates profoundly inhibited the differentiation, migration, and capillary tube formation of human umbilical vein endothelial cells in vitro. To measure angiogenesis at the site of infection, we devised an in vivo Matrigel assay in cyclophosphamide-treated BALB/c mice with cutaneous invasive aspergillosis. Angiogenesis was significantly suppressed in Matrigel plugs implanted in A fumigatus–infected mice compared with plugs from uninfected control mice. The antiangiogenic effect of A fumigatus was completely abolished by deletion of the global regulator of secondary metabolism, laeA, and to a lesser extent by deletion of gliP, which controls gliotoxin production. Moreover, pure gliotoxin potently inhibited angiogenesis in vitro in a dose-dependent manner. Finally, overexpression of multiple angiogenesis mediator–encoding genes was observed in the lungs of cortisone-treated mice during early invasive aspergillosis, whereas gene expression returned rapidly to baseline levels in cyclophosphamide/cortisone-treated mice. Taken together, these results indicate that suppression of angiogenesis by A fumigatus both in vitro and in a neutropenic mouse model is mediated through secondary metabolite production.

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In VitroInteraction between Alginate Lyase and Amphotericin B against Aspergillus fumigatus Biofilm Determined by Different Methods

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01875-12 ◽

2012 ◽

Vol 57 (3) ◽

pp. 1275-1282 ◽

Cited By ~ 32

Author(s):

Francesca Bugli ◽

Brunella Posteraro ◽

Massimiliano Papi ◽

Riccardo Torelli ◽

Alessandro Maiorana ◽

...

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Amphotericin B ◽

Extracellular Polymeric Substances ◽

Alginate Lyase ◽

Antifungal Drugs ◽

Antibiofilm Activity ◽

Content Type ◽

Time Kill

ABSTRACTAspergillus fumigatusbiofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treatAspergillus-related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study,in vitrointeractions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, againstA. fumigatusbiofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that whenA. fumigatusbiofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed inA. fumigatusbiofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination of AlgL and a polyene antifungal may prove to be a new therapeutic strategy for invasive aspergillosis, while reinforcing the EPS as a valuable antibiofilm drug target.

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Antifungal effect of all-trans retinoic acid against Aspergillus fumigatus in vitro and in a pulmonary aspergillosis in vivo model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01874-20 ◽

2020 ◽

Author(s):

Elena Campione ◽

Roberta Gaziano ◽

Elena Doldo ◽

Daniele Marino ◽

Mattia Falconi ◽

...

Keyword(s):

Retinoic Acid ◽

Aspergillus Fumigatus ◽

Rat Model ◽

Fungal Infections ◽

Invasive Pulmonary Aspergillosis ◽

Antifungal Drugs ◽

Pulmonary Aspergillosis ◽

Fungistatic Activity

AIM: Aspergillus fumigatus is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. Fungistatic activity of all-trans retinoic acid (ATRA) has been recently described in vitro. We evaluated the efficacy of ATRA in vivo and its potential synergistic interaction with other antifungal drugs. MATERIALS AND METHODS: A rat model of IPA and in vitro experiments were performed to assess the efficacy of ATRA against Aspergillus in association with classical antifungal drugs and in silico studies used to clarify its mechanism of action. RESULTS: ATRA (0.5 and 1 mM) displayed a strong fungistatic activity in Aspergillus cultures, while at lower concentrations, synergistically potentiated fungistatic efficacy of sub-inhibitory concentration of Amphotericin B (AmB) and Posaconazole (POS). ATRA also enhanced macrophagic phagocytosis of conidia. In a rat model of IPA, ATRA reduced mortality similarly to Posaconazole. CONCLUSION: Fungistatic efficacy of ATRA alone and synergistically with other antifungal drugs was documented in vitro, likely by inhibiting fungal Hsp90 expression and Hsp90-related genes. ATRA reduced mortality in a model of IPA in vivo. Those findings suggest ATRA as suitable fungistatic agent, also to reduce dosage and adverse reaction of classical antifungal drugs, and new therapeutic strategies against IPA and systemic fungal infections.

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Identification of Essential Genes in the Human Fungal Pathogen Aspergillus fumigatus by Transposon Mutagenesis

Eukaryotic Cell ◽

10.1128/ec.2.2.247-255.2003 ◽

2003 ◽

Vol 2 (2) ◽

pp. 247-255 ◽

Cited By ~ 48

Author(s):

Arnaud Firon ◽

François Villalba ◽

Roland Beffa ◽

Christophe d'Enfert

Keyword(s):

Aspergillus Fumigatus ◽

Transposable Element ◽

Drug Targets ◽

Fungal Infections ◽

Fungal Growth ◽

Developed Countries ◽

Transposon Mutagenesis ◽

Essential Genes ◽

Antifungal Drugs

ABSTRACT The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.

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Vaccinations with Recombinant Variants of Aspergillus fumigatus Allergen Asp f 3 Protect Mice against Invasive Aspergillosis

Infection and Immunity ◽

10.1128/iai.00815-06 ◽

2006 ◽

Vol 74 (9) ◽

pp. 5075-5084 ◽

Cited By ~ 57

Author(s):

James I. Ito ◽

Joseph M. Lyons ◽

Teresa B. Hong ◽

Daniel Tamae ◽

Yi-Kuang Liu ◽

...

Keyword(s):

Invasive Aspergillosis ◽

Aspergillus Fumigatus ◽

Mononuclear Cells ◽

Protein Vaccine ◽

Novel Approach ◽

In Vivo Testing ◽

Asp F 1 ◽

Fungal Allergen ◽

Subcutaneous Immunization

ABSTRACT A vaccine that effectively protects immunocompromised patients against invasive aspergillosis is a novel approach to a universally fatal disease. Here we present a rationale for selection and in vivo testing of potential protein vaccine candidates, based on the modification of an immunodominant fungal allergen for which we demonstrate immunoprotective properties. Pulmonary exposure to viable Aspergillus fumigatus conidia as well as vaccination with crude hyphal extracts protects corticosteroid-immunosuppressed mice against invasive aspergillosis (J. I. Ito and J. M. Lyons, J. Infect. Dis. 186:869-871, 2002). Sera from the latter animals contain antibodies with numerous and diverse antigen specificities, whereas sera from conidium-exposed mice contain antibodies predominantly against allergen Asp f 3 (and some against Asp f 1), as identified by mass spectrometry. Subcutaneous immunization with recombinant Asp f 3 (rAsp f 3) but not with Asp f 1 was protective. The lungs of Asp f 3-vaccinated survivors were free of hyphae and showed only a patchy low-density infiltrate of mononuclear cells. In contrast, the nonimmunized animals died with invasive hyphal elements and a compact peribronchial infiltrate of predominately polymorphonuclear leukocytes. Three truncated versions of rAsp f 3, spanning amino acid residues 15 to 168 [rAsp f 3(15-168)], 1 to 142, and 15 to 142 and lacking the known bipartite sequence required for IgE binding, were also shown to be protective. Remarkably, vaccination with either rAsp f 3(1-142) or rAsp f 3(15-168) drastically diminished the production of antigen-specific antibodies compared to vaccination with the full-length rAsp f 3(1-168) or the double-truncated rAsp f 3(15-142) version. Our findings point to a possible mechanism in which Asp f 3 vaccination induces a cellular immune response that upon infection results in the activation of lymphocytes that in turn enhances and/or restores the function of corticosteroid-suppressed macrophages to clear fungal elements in the lungs.

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Ten Years of Experience with Invasive Aspergillosis: Has Anything Changed?.

Blood ◽

10.1182/blood.v114.22.3687.3687 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3687-3687

Author(s):

Gloria Mattiuzzi ◽

Jorge Cortes ◽

Jennifer Ho ◽

Hagop M. Kantarjian

Keyword(s):

Mortality Rate ◽

Invasive Aspergillosis ◽

Hematologic Malignancies ◽

Mortality Rates ◽

Antifungal Drugs ◽

Lymphocytic Leukemia ◽

Myelogenous Leukemia ◽

Myelocytic Leukemia ◽

Incidence And Mortality ◽

The Moment

Abstract Abstract 3687 Poster Board III-623 Invasive Aspergillosis (IA) has represented a threat for patients and physicians for many years. This is especially true for patients with hematologic malignancies. Several new antifungal agents with broad antifungal activity became available in 2000 ushering in a new millennium with hopes that with this new armamentarium of antifungal drugs will decrease mortality due to IA and perhaps even prevent them better. Mortality rates due to IA have been described in recent reports as acceptable and even in some cancer centers significantly lower than before. The objective of the present study was to evaluate IA in patients with hematologic malignancies in the leukemia department at MDACC during the past 10 years (1998 to 2007) and compare the incidence and mortality rates between these two times periods. Methods retrospective review of all patients that presented at the leukemia department at MDACC from January 1998 to December 2007. Proven or probable IA was defined as per EORTC criteria. Results From 1998 to 2007, 12,845 patients were presented to our department, including 4,243 pts with acute myelogenous leukemia (AML); 940 with acute lymphocytic leukemia (ALL); 3,642 with chronic lymphocytic leukemia (CLL); and 4,276 with other type of leukemia (chronic myelocytic leukemia, low risk-myelodysplastic syndrome and miscellaneous). Overall median age was 58 (range 17-83); 60% were male, 83% did not have history of diabetes mellitus; 71% were neutropenic (ANC < 500) at the moment of the infection; and 11% had a previous bone marrow transplantation. The overall incidence of IA has significantly decreased from 4 % to 2% over the 10 study years (p= 0.005, IRR=0.45), however, the overall mortality rate (60%) has not decreased significantly. Comparison between the incidence of IA and mortality in the different subsets of patients between 1998-1999 and 2000-2007 is shown in Table 1 The incidence of IA decreased significantly between the two time periods only in patients with AML and in patients with ALL (p<0.001 and 0.004, respectively). However, although there has been a reduction in the mortality rate in all the subsets of patients in the two periods, this difference is not significant. Overall, A. fumigatus was the pathogen more frequently isolated (30%), followed by A. flavus (21%), A. terreus (18%) and A. niger (11%). In 10% of the episodes more than one Aspergillus species was isolated. In conclusion, our data suggests that the incidence of IA in patients with leukemia traditionally at high risk for IA has significantly decreased since 2000. Despite the availability of new antifungal drugs for the treatment of IA, the mortality rate in patients with hematological malignancies has not significantly decreased. Disclosures: Mattiuzzi: Merck: Research Funding.

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