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Invasive aspergillosis (IA), caused predominantly by Aspergillus fumigatus, is the most common opportunistic mold infection in immunocompromised patients. Resistance of A. fumigatus to triazoles has been increasingly reported, leading to poor outcomes of IA to the front-line azoles. Triazole resistance is in part driven by exposure to agricultural azoles through mechanisms that are poorly understood beyond mutations in ergosterol biosynthetic genes. Priming is defined as a process in which prior exposures to sublethal stressful stimuli, such as antimicrobial drugs, can enhance the ability of pathogens to withstand reexposure to the same or other stressors. Here, we describe, for the first time, triazole priming, where exposure of conidia of three A. fumigatus strains to subinhibitory concentrations of either agricultural (tebuconazole difenoconazole, epoxiconazole) or medical triazoles (voriconazole) increases germination and growth during subsequent reexposure to subinhibitory triazole challenge. We demonstrate that priming in A. fumigatus is class specific to triazoles, is not confined to a particular isolate, and is retained for extended periods in primed dormant conidia, but is not transferred to subsequent generations. Furthermore, azole priming at subinhibitory triazole concentrations increased the frequency of development of stable resistance development at inhibitory triazole exposures. Triazole priming could have far-reaching clinical implications in generating resistance due to the widespread use of agricultural triazoles or breakthrough IA in patients with subtherapeutic serum levels of azoles.

Pseudomonas aeruginosa (PA) and Aspergillus fumigatus (AF) chronically colonize the airways of patients with cystic fibrosis or chronic immunosuppression and mutually affect each other's pathogenesis. Here, we evaluated IncuCyte time-lapse imaging and NeuroTrack TM (NT) analysis (Wurster et al., 2019, mBio) as a toolbox to study mycelial expansion and morphogenesis of AF during interaction with PA. Co-incubation of AF with supernatant filtrates of wild-type (WT) PA strains strongly inhibited hyphal growth and branching. Consonant with prior metabolic studies, pyoverdine-deficient PA mutants had significantly attenuated inhibitory capacity. Accordingly, purified PA products pyoverdine and pyocyanin suppressed mycelial expansion of AF in a concentration-dependent way. Using fluorescence-guided tracking of GFP-AF293 mycelia during co-culture with live WT PA cells, we found significant inoculum-dependent mycelial growth inhibition and robust precision of the NT algorithm. Collectively, our experiments position IncuCyte NT as an efficient platform for longitudinal analysis of fungal growth and morphogenesis during bacterial co-infection.

Cutaneous fungal infection is a challenging condition to treat that primarily afflicts immunocompromised patients. Local antifungal therapy may permit the delivery of high concentrations of antifungals directly to wounds while minimizing systemic toxicities. However, the field currently lacks suitable in vivo models. Therefore, a large cutaneous wound was created in immunosuppressed mice and inoculated with Aspergillus fumigatus. We fabricated biodegradable polymer microparticles (MPs) that were capable of locally delivering antifungal and characterized in vitro release kinetics. We compared wound bed size, fungal burden, and histological presence of fungi in mice treated with antifungal-loaded MPs. Mice with a cutaneous defect but no infection, mice with infected cutaneous defect but no treatment, and infected mice treated with blank MPs were used as controls. Infection of large wounds inhibited healing and resulted in tissue invasion in an inoculum-dependent manner. MPs were capable of releasing antifungals at concentrations above A. fumigatus Minimum Inhibitory Concentration (MIC) for at least 6 days. Wounds treated with MPs had significantly decreased size compared with no treatment (64.2% vs. 19.4% wound reduction, p = 0.002) and were not significantly different from uninfected controls (64.2% vs. 58.1%, p = 0.497). This murine model may serve to better understand cutaneous fungal infection and evaluate local biomaterials-based therapies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1867-1874, 2019., (© 2019 Wiley Periodicals, Inc.)

Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus , even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR 34 (a 34-bp tandem repeat in the promoter region), TR 46 , G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A -associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A -associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications., (Copyright © 2019 American Society for Microbiology.)

Breakthrough mucormycosis in patients receiving isavuconazole prophylaxis or therapy has been reported. We compared the impact of isavuconazole and voriconazole exposure on the virulence of clinical isolates of Aspergillus fumigatus and different Mucorales species in a Drosophila melanogaster infection model. In contrast to A. fumigatus , a hypervirulent phenotype was found in all tested Mucorales upon preexposure to either voriconazole or isavuconazole. These findings may contribute to the explanation of breakthrough mucormycosis in isavuconazole-treated patients., (Copyright © 2019 American Society for Microbiology.)

LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway regulated by Rubicon, with an emerging role in immune homeostasis and antifungal host defence. Aspergillus cell wall melanin protects conidia (spores) from killing by phagocytes and promotes pathogenicity through blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of LAP. However, the signalling regulating LAP upstream of Rubicon and the mechanism of melanin-induced inhibition of this pathway remain incompletely understood. Herein, we identify a Ca 2+ signalling pathway that depends on intracellular Ca 2+ sources from endoplasmic reticulum, endoplasmic reticulum-phagosome communication, Ca 2+ release from phagosome lumen and calmodulin (CaM) recruitment, as a master regulator of Rubicon, the phagocyte NADPH oxidase NOX2 and other molecular components of LAP. Furthermore, we provide genetic evidence for the physiological importance of Ca 2+ -CaM signalling in aspergillosis. Finally, we demonstrate that Ca 2+ sequestration by Aspergillus melanin inside the phagosome abrogates activation of Ca 2+ -CaM signalling to inhibit LAP. These findings reveal the important role of Ca 2+ -CaM signalling in antifungal immunity and identify an immunological function of Ca 2+ binding by melanin pigments with broad physiological implications beyond fungal disease pathogenesis.

Objectives: Over the last 30 years, the number of invasive fungal infections among immunosuppressed patients has increased significantly, while the number of effective systemic antifungal drugs remains low. The aim of this study was to identify and characterize antifungal compounds that inhibit fungus-specific metabolic pathways not conserved in humans., Methods: We screened a diverse compound library for antifungal activity in the pathogenic mould Aspergillus fumigatus . We determined the in vitro activity of bromoquinol by MIC determination against a panel of fungi, bacteria and cell lines. The mode of action of bromoquinol was determined by screening an Aspergillus nidulans overexpression genomic library for resistance-conferring genes and by RNAseq analysis in A. fumigatus . In vivo efficacy was tested in Galleria mellonella and murine models of A. fumigatus infection., Results: Screening of a diverse chemical library identified three compounds interfering with fungal iron utilization. The most potent, bromoquinol, shows potent wide-spectrum antifungal activity that was blocked in the presence of exogenous iron. Mode-of-action analysis revealed that overexpression of the dba secondary metabolite cluster gene dbaD , encoding a metabolite transporter, confers bromoquinol resistance in A. nidulans , possibly by efflux. RNAseq analysis and subsequent experimental validation revealed that bromoquinol induces oxidative stress and apoptosis in A. fumigatus . Bromoquinol significantly reduced mortality rates of G. mellonella infected with A. fumigatus , but was ineffective in a murine model of infection., Conclusions: Bromoquinol is a promising antifungal candidate with a unique mode of action. Its activity is potentiated by iron starvation, as occurs during in vivo growth., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation.

Concealing pathogen-associated molecular patterns (PAMPs) is a principal strategy used by fungi to avoid immune recognition. Surface exposure of PAMPs during germination can leave the pathogen vulnerable. Accordingly, β-glucan surface exposure during Aspergillus fumigatus germination activates an Atg5-dependent autophagy pathway termed LC3-associated phagocytosis (LAP), which promotes fungal killing. We found that LAP activation also requires the genetic, biochemical or biological (germination) removal of A. fumigatus cell wall melanin. The attenuated virulence of melanin-deficient A. fumigatus is restored in Atg5-deficient macrophages and in mice upon conditional inactivation of Atg5 in hematopoietic cells. Mechanistically, Aspergillus melanin inhibits NADPH oxidase-dependent activation of LAP by excluding the p22phox subunit from the phagosome. Thus, two events that occur concomitantly during germination of airborne fungi, surface exposure of PAMPs and melanin removal, are necessary for LAP activation and fungal killing. LAP blockade is a general property of melanin pigments, a finding with broad physiological implications., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Filamentous fungi are renowned for the production of bioactive secondary metabolites. Typically, one distinct metabolite is generated from a specific secondary metabolite cluster. Here, we characterize the newly described trypacidin (tpc) cluster in the opportunistic human pathogen Aspergillus fumigatus. We find that this cluster as well as the previously characterized endocrocin (enc) cluster both contribute to the production of the spore metabolite endocrocin. Whereas trypacidin is eliminated when only tpc cluster genes are deleted, endocrocin production is only eliminated when both the tpc and enc non-reducing polyketide synthase-encoding genes, tpcC and encA, respectively, are deleted. EncC, an anthrone oxidase, converts the product released from EncA to endocrocin as a final product. In contrast, endocrocin synthesis by the tpc cluster likely results from incomplete catalysis by TpcK (a putative decarboxylase), as its deletion results in a nearly 10-fold increase in endocrocin production. We suggest endocrocin is likely a shunt product in all related non-reducing polyketide synthase clusters containing homologues of TpcK and TpcL (a putative anthrone oxidase), e.g. geodin and monodictyphenone. This finding represents an unusual example of two physically discrete secondary metabolite clusters generating the same natural product in one fungal species by distinct routes., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus fumigatus that conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the β-glucan catalytic subunit of glucan synthase, FKS1, increased macrophage NF-κB/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis in C. albicans or when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z.

We studied the effect of noninvasive radiofrequency-induced hyperthermia on the viability of Aspergillus fumigatus hyphae in vitro. Radiofrequency-induced hyperthermia resulted in significant (>70%, P < 0.0001) hyphal damage in a time and thermal dose-dependent fashion as assessed by XTT [(sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl] (1)-2H-tetrazolium inner salt)], DiBAC [bis-(1,3-dibutylbarbituric acid) trimethine oxonol] staining, and transmission electron microscopy. For comparison, water bath hyperthermia was used over the range of 45 to 55°C to study hyphal damage. Radiofrequency-induced hyperthermia resulted in severe damage to the outer fibrillar layer of hyphae at a shorter treatment time compared to water bath hyperthermia. Our preliminary data suggest that radiofrequency-induced hyperthermia might be an additional therapeutic approach to use in the management of mold infections.

Aspergillus fumigatus is the predominant airborne fungal pathogen in immunocompromised patients. Genetic defects in NADPH oxidase (chronic granulomatous disease [CGD]) and corticosteroid-induced immunosupression lead to impaired killing of A. fumigatus and unique susceptibility to invasive aspergillosis via incompletely characterized mechanisms. Recent studies link TLR activation with phagosome maturation via the engagement of autophagy proteins. In this study, we found that infection of human monocytes with A. fumigatus spores triggered selective recruitment of the autophagy protein LC3 II in phagosomes upon fungal cell wall swelling. This response was induced by surface exposure of immunostimulatory β-glucans and was mediated by activation of the Dectin-1 receptor. LC3 II recruitment in A. fumigatus phagosomes required spleen tyrosine kinase (Syk) kinase-dependent production of reactive oxygen species and was nearly absent in monocytes of patients with CGD. This pathway was important for control of intracellular fungal growth, as silencing of Atg5 resulted in impaired phagosome maturation and killing of A. fumigatus. In vivo and ex vivo administration of corticosteroids blocked LC3 II recruitment in A. fumigatus phagosomes via rapid inhibition of phosphorylation of Src and Syk kinases and downstream production of reactive oxygen species. Our studies link Dectin-1/Syk kinase signaling with autophagy-dependent maturation of A. fumigatus phagosomes and uncover a potential mechanism for development of invasive aspergillosis in the setting of CGD and corticosteroid-induced immunosupression.

In invasive pulmonary aspergillosis, direct invasion and occlusion of pulmonary vasculature by Aspergillus hyphae causes tissue hypoxia, which is enhanced by secreted fungal metabolites that downregulate compensatory angiogenic signaling pathways. We assessed the effects of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on survival rates, fungal burden, and in situ angiogenesis in a murine invasive pulmonary aspergillosis model. bFGF and VEGF monotherapy significantly increased survival rates and potentiated the activity of amphotericin B. bFGF-containing regimens were associated with reduced tissue fungal burdens. bFGF and VEGF reversed the antiangiogenic activity of Aspergillus fumigatus; however, VEGF induced the formation of immature neovessels, providing an explanation for its lesser efficacy. Treatment with bFGF plus amphotericin B was associated with neutrophil influx into Aspergillus-infected pulmonary tissue, suggesting that this combination limits fungal growth through neutrophil trafficking. Vasculogenic pathways are unexplored targets for the treatment of invasive pulmonary aspergillosis and may potentiate both innate immunity and antifungal drug activity against A. fumigatus.

The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new "function-omic" avenues downstream of the metabolomics, identification, and purification phases.

Mucormycosis is an uncommon fungal infection that has been increasingly reported in severely immunocompromised patients receiving Aspergillus-active antifungals. Although clinical studies and pre-clinical animal models have suggested a unique predisposition for breakthrough mucormycoses in patients receiving voriconazole, no study has specifically evaluated the selection dynamics of various Aspergillus -active antifungal classes in vivo. We utilized an Aspergillus fumigatus:Rhizopus oryzae (10:1) model of mixed fungal pneumonia in corticosteroid-immunosuppressed mice to compare the selection dynamics of daily liposomal-amphotericin B (L-AMB), micafungin (MCFG) and voriconazole (VRC) therapy. A. fumigatus and R. oryzae lung fungal burden were serially monitored in parallel using non-cross-amplifying quantitative real-time PCR assays for each fungal genus. Additionally, experiments were performed where the R. oryzae component of the mixed inoculum was serially-passed on VRC-containing agar before animal infection. We found prior exposure to voriconazole in vitro, consistently resulted in a 1.5-2 log 10 increase in R. oryzae fungal burden by day +5 in vivo relative to animals infected with the non-VRC preexposed inoculum, irrespective of the antifungal-treatment administered in mice (P ≤ 0.02 all treatment groups). Mice infected with the VRC-preexposed inoculum and subsequently treated with saline or VRC had the highest mortality rates (82-86%), followed by MCFG (55%) then L-AMB (39%, P = 0.04 vs. control). However, in vivo treatment alone with voriconazole alone did not consistently increase the virulence of non- voriconazole preexposed R. oryzae versus controls. We conclude that exposure of R. oryzae sporangiospores to voriconazole in vitro modulates the subsequent growth rate and/or virulence of the fungus in vivo, which reduces effectiveness of Mucorales-active antifungals. The mechanisms underlying this phenotypic change are unknown.

Background: Echinocandin resistance in Aspergillus species is rare. We examined if mutations in FKS1 would result in a complete loss of echinocandin activity in vivo in an experimental model of aspergillosis., Methods: Neutropenic mice were infected with either an echinocandin-susceptible Aspergillus fumigatus (AF 293) or an echinocandin-resistant A. fumigatus laboratory strain harbouring 'hot-spot' substitution in Fks1p (AF Ser678Pro). Mice then received daily treatment with either anidulafungin or caspofungin at varying dosages (0.25-16 mg/kg/day) for 5 days and Aspergillus lung fungal burden was assessed by quantitative real-time PCR., Results: Both strains produced histological evidence of progressive invasive pulmonary aspergillosis, but AF Ser678Pro was less virulent than AF 293, as evidenced by lower lung fungal burden and longer median survival time. At > 0.5 mg/kg/day, both anidulafungin and caspofungin reduced the lung fungal burden in neutropenic animals infected with AF 293, but had mixed efficacy against the resistant AF Ser678Pro strain. For caspofungin, the fungal burden was reduced only at doses <1 mg/kg/day. Anidulafungin also modestly reduced AF Ser678Pro lung fungal burden, but only at > 4 mg/kg/day., Conclusions: Despite a lack of appreciable antifungal activity in vitro, both anidulafungin and caspofungin were still modestly effective in vivo against a laboratory-generated A. fumigatus mutant harbouring the Ser678Pro mutation in Fks1p. This persistent activity, combined with impaired fitness of the isolate in vivo, could partially explain why microbiologically documented echinocandin-resistance in Aspergillus species remains a rare clinical occurrence.

Drug combinations are increasingly being explored as a means of improving outcomes in cases of invasive aspergillosis. However, the optimal methods for assessing in vitro drug combinations are unclear. We investigated whether echinocandin drug combinations have potentially useful interactions against Aspergillus fumigatus. We explored the in vitro interactions of three echinocandins (caspofungin [CSP], anidulafingin [ANF], and micafungin [MCF]) by three methods, i.e., (i) checkerboard assay, (ii) disk diffusion assay, and (iii) E-test/agar dilution. The checkerboard experiments revealed different interactions between echinocandin pairs depending on the drug combination tested. Specifically, the combination of CSF and either ANF or MCF yielded a fractional inhibitory concentration (FIC) index range of 0.15-2.0. Whereas the combination of MCF and ANF yielded an FIC index range of 0.19-0.31, consistent with synergistic interaction. In contrast, all echinocandin pairs appeared indifferent by the disk diffusion method. Moreover, the combination of MCF and ANF appeared antagonistic when the two drugs were tested using the E-test/agar dilution method with both the FKS1 wild-type and echinocandin-resistant fks1 mutant strains. Our results highlight methodological problems inherent in in vitro antifungal combination testing. We did not find compelling evidence of inter-echinocandin synergy that could serve as a basis for further in vivo experimentation.

Aspergillus fumigatus is an opportunistic filamentous fungus that is currently the most frequent cause of invasive fungal disease in immunosuppressed individuals. Recent advances in our understanding of the pathogenesis of invasive aspergillosis have highlighted the multifactorial nature of A. fumigatus virulence and the complex interplay between host and microbial factors. In this review, we outline current concepts of immune recognition and evasion, angioinvasion and angiogenesis, secondary metabolism and the fungal stress response, and their respective roles in this often lethal infection.

The pathogenicity of six mutants of Aspergillus fumigatus that had been previously characterized in mice was assessed in Toll-deficient Drosophila melanogaster flies. Four out of six mutants of A. fumigatus, which displayed attenuated virulence in mice due to defects in siderophore biosynthesis (DeltasidA, DeltasidD), PABA metabolism (H515), and starvation stress response (DeltacpcA), also had attenuated virulence in the fly model. In addition, similarly to previous findings in the mouse model, DeltasidG mutant that is defective in extracellular siderophore biosynthesis retained full virulence in Toll-deficient flies. Overall, our studies reveal a high level of concordance between fly and murine models of invasive aspergillosis.

Cutaneous models have proven useful in studies of the pathogenesis and treatment of Gram-positive bacterial infections. Because cutaneous invasive aspergillosis (IA) occurs in the clinical setting, we sought to develop a nonlethal murine cutaneous model of IA. We induced cutaneous IA in cyclophosphamide-treated nude BALB/c mice by subcutaneous injection of Aspergillus fumigatus conidia. Skin lesion areas correlated well with tissue fungal burdens, allowing dynamic visual monitoring of cutaneous infections. The cutaneous model accurately reflected alterations in A. fumigatus pathogenicity resulting from deletions of recognized virulence genes (pabaA, sidA, and pksP). Moreover, analysis of the roles of conidial and mycelial catalases revealed that the former is required for the initiation of cutaneous aspergillosis, whereas the latter contributes to its propagation. Finally, posaconazole treatment reduced skin lesion areas relative to those of untreated and fluconazole-treated controls. This novel cutaneous model system should be applicable to comparative studies of the pathogenesis, treatment, and tissue specificity of IA.

The hypomethylating agent 5-azacytidine (5AC) is widely used in patients at risk of invasive mycoses. We sought to determine whether 5AC affects the developmental competence and virulence of Aspergillus fumigatus. Incubation of A. fumigatus strain 293 with 5AC induced high-frequency conversion to a fluffy-variant (Af293 (FL) ). The conidiation defect was bypassed by exposing Af293 (FL) to light during the initial 18 hours of growth on solid media. Transcriptional profiling revealed differential expression of multiple genes involved in G-protein signaling, including a putative G-protein coupled photoreceptor (opsin), suggesting that impaired signaling through a light-responsive pathway upstream of brlA is responsible for this phenotype. Af293 (FL) was fully virulent in fruit fly and murine models of invasive aspergillosis. Moreover, Af293 (FL) overexpressed aspergillopepsin F, had increased elastase activity and was more angioinvasive than the parental wild-type strain. The 5AC-induced A. fumigatus fluffy variant illustrates the potential effects of chemotherapeutic agents on the developmental and pathobiologic characteristics of opportunistic fungi.

Despite progress over the last decade, opportunistic mold infections continue to be associated with high rates of morbidity and mortality in immunocompromised patients. Given the propensity of molds to invade blood vessels, vasculopathy may be a barrier to effective delivery of antifungal drugs to infected tissue. In a recent study (Ben-Ami R et al. Blood, 2009), we found that A. fumigatus suppresses endothelial cell migration, differentiation and capillary tube formation both in vitro and in an animal model system. This effect is mediated by secreted secondary metabolites such as gliotoxin. Herein, I discuss the potential implications of how invasive molds modulate host angiogenesis in experimental and clinical mold infections. Strategies that employ reversal of vasculopathy, neutralization of metabolites that inhibit endothelial function, exploration of pro-angiogenic factors as diagnostic or prognostic markers affected patients will likely be the focus of future studies. This complex, yet emerging field might add another level of knowledge and therapeutic choices in the management of these devastated infections.

Members of the genus Aspergillus are opportunistic fungal pathogens characterized by their genomic diversity. However, whether variations among Aspergillus strains and species at the genome level translate into significant differences in virulence is unclear. Therefore, we studied the interstrain and interspecies variations in virulence for a collection of Aspergillus fumigatus and Aspergillus terreus isolates using a previously described model of invasive aspergillosis in Toll-deficient fruit flies. We then looked for associations between survival in the fly model and strain relatedness as defined by repetitive-sequence polymerase chain reaction (rep-PCR). We observed no significant differences in the survival of flies infected with A. fumigatus vs. A. terreus or flies infected with colonizing vs. invasive isolates of either species. However, in both Aspergillus species we observed significant interstrain variability in fly survival (P<0.001 by the log-rank test). Using rep-PCR, we identified two dominant A. fumigatus clades that were associated with significantly different survival rates in Toll-deficient flies (P=0.007). We conclude that the fly model of invasive aspergillosis enables high-throughput screening of Aspergillus species for variations in virulence and may uncover distinct A. fumigatus clades that differ in their pathogenicity.

In 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.

Aspergillus fumigatus, an important human fungal pathogen, encounters high levels of reactive oxygen species following its ingestion by phagocytes. Reactive oxygen species are important mediators of the fungicidal activities of phagocytes. In yeasts, YAP1 encodes for transcriptional factors that contribute to their oxidative stress response and given the importance of the stress response, we hypothesized that the YAP1 homologue in A. fumigatus plays a similar role in this fungus. In this study, we found that Afyap1, the Yap1 homologue of A. fumigatus, confers protection against oxidative stress. Replacement of Afyap1 with the marker gene pyrG (DeltaAfyap1) resulted in hypersensitivity of A. fumigatus to oxidants such as H(2)O(2) and menadione. In contrast, an A. fumigatus strain harboring multiple-copy Afyap1 was resistant to these two oxidants as well as the oxidant diamide. However, DeltaAfyap1 and strain harboring multiple-copy Afyap1 were comparable in their virulence to a wild-type A. fumigatus strain in a murine model of invasive pulmonary aspergillosis. Taken together, these results demonstrate that Afyap1 is involved in oxidative stress response but is not an essential virulence factor for A. fumigatus.

Background: We investigated whether caspofungin and other echinocandins have immune-enhancing properties that influence human polymorphonuclear neutrophil (PMN)-mediated mold hyphal damage., Materials and Methods: Using aniline blue staining, we compared patterns of beta-glucan exposure in Aspergillus fumigatus, Aspergillus terreus, Rhizopus oryzae, Fusarium solani, Fusarium oxysporum, Scedosporium prolificans, and Scedosporium apiospermum hyphae after caspofungin exposure. We also determined PMN-mediated hyphal damage occurring with or without preexposure to caspofungin or with preexposure to the combination of caspofungin and anti-beta-glucan monoclonal antibody, using 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-sH-tetrazolium hydroxide (XTT) assay., Results: Preincubation with caspofungin (32 microg/mL for R. oryzae; 0.0625 microg/mL for other isolates) increased exposure to beta-glucan. PMN-induced damage increased after caspofungin exposure and was further augmented by the addition of anti-beta-glucan antibody. Preincubation with micafungin or anidulafungin had similar effects on PMN-induced damage of A. fumigatus hyphae. Finally, preexposure of A. fumigatus, but not S. prolificans, to caspofungin induced expression of Dectin-1 by PMN., Conclusions: The results of the present study suggest inducement of beta-glucan unmasking by echinocandins and enhancement of PMN activity against mold hyphae, thereby supporting the immunopharmacologic mode of action of echinocandins.

We demonstrated that intranasal granulocyte-macrophage colony-stimulating factor given to immunosuppressed mice infected with pulmonary aspergillosis resulted in a sixfold reduction in the lung fungal burden compared to the result for saline-treated mice (P = 0.045). These data suggest that lung-targeted immunotherapy may be complementary to antifungal agents and may improve patient responses.

Background: Gliotoxin is a epipolythiodioxopiperazine toxin that is made by the filamentous fungus Aspergillus fumigatus. Gliotoxin has a wide range of effects on metazoan cells in culture, including induction of apoptosis through inhibition of Nf-kappaB, and inhibition of superoxide production by phagocytes. These activities have led to the proposal that gliotoxin contributes to pathogenesis during invasive aspergillosis. We tested this hypothesis by creating isogenic strains of gliotoxin-producing and nonproducing strains., Methods: We deleted gliP, the gene that encodes the nonribosomal peptide synthetase GliP. GliP catalyzes the first biosynthetic step in the synthesis of gliotoxin. We then tested for gliotoxin production and virulence in different animal models., Results: Deletion of gliP resulted in strains that were wild type for growth, but they did not synthesize gliotoxin. Transformation of gliP deletion mutants with a full copy of gliP restored gliotoxin production. The gliP deletion strain had attenuated virulence in nonneutropenic mice immunosuppressed with corticosteroids, but had normal virulence in neutropenic mice. It also had reduced virulence in a Drosophila melanogaster model., Conclusions: Gliotoxin only contributes to the virulence of A. fumigatus in nonneutropenic mice and in fruit flies with functional phagocytes. These results suggest that the principal targets of gliotoxin are neutrophils or other phagocytes.

Invasive aspergillosis is a leading cause of infectious death in immunosuppressed patients. Here, we adapted a phage display library-based selection to screen and identify binding peptides to the surface of Aspergillus fumigatus conidia and hyphae. We identified a peptide (sequence CGGRLGPFC) that reliably binds to the surface of Aspergillus fumigatus hyphae. Binding was not Aspergillus strain specific, as it was also observed in hyphae of other Aspergillus clinical isolates. Furthermore, CGGRLGPFC-displaying phage targets Aspergillus fumigatus hyphae on formalin-fixed paraffin-embedded histopathology sections of lung tissue recovered from mice with invasive pulmonary aspergillosis. This approach may yield reagents such as peptidomimetics for novel diagnostic and therapeutic interventions in invasive aspergillosis.

Aspergillus fumigatus (AF) is a ubiquitous mold and is the most common cause of invasive aspergillosis, an important source of morbidity and mortality in immunocompromised hosts. Using cytokine flow cytometry, we assessed the magnitude of functional CD4+ and CD8+ T-cell responses following stimulation with Aspergillus antigens. Relative to those seen with cytomegalovirus (CMV) or superantigen stimulation, responses to Aspergillus antigens were near background levels. Subsequently, we confirmed that gliotoxin, the most abundant mycotoxin produced by AF, was able to suppress functional T-cell responses following CMV or staphylococcal enterotoxin B (SEB) stimulation. Additional studies demonstrated that crude AF filtrates and purified gliotoxin inhibited antigen-presenting cell function and induced the preferential death of monocytes, leading to a marked decrease in the monocyte-lymphocyte ratio. Analysis of caspase-3 activation confirmed that gliotoxin preferentially induced apoptosis of monocytes; similar effects were observed in CD83+ monocyte-derived dendritic cells. Importantly, the physiologic effects of gliotoxin in vitro were observed below concentrations recently observed in the serum of patients with invasive aspergillosis. These studies suggest that the production of gliotoxin by AF may constitute an important immunoevasive mechanism that is mediated by direct effects on antigen-presenting cells and both direct and indirect effects on T cells.

Aspergillus fumigatus is a ubiquitous fungus that is a frequent opportunistic pathogen in immunosuppressed patients. Because of its role as a pathogen, it is of considerable experimental interest. A set of auxotrophic isogenic strains in the A. fumigatus genome reference strain AF293 has been developed. Using molecular genetic methods, arginine and lysine auxotrophs were made by deletion of argB and lysB, respectively. Transformation of these auxotrophic strains with plasmids carrying argB or lysB, respectively, results in efficient integration at these loci. Finally, these strains are able to form stable diploids, which should further facilitate analysis of gene functions in this fungus. Furthermore, the development of this isogenic set of auxotrophic strains in the AF293 background will enable investigators to study this important opportunistic human pathogen with greater facility.

Background: A paucity of data exists regarding the pharmacodynamics of caspofungin (CAS) during invasive pulmonary aspergillosis (IPA). We conducted a dosage-fractionation study to characterize the in vivo pharmacodynamics of CAS activity during IPA, using immunosuppressed mice inoculated intranasally with Aspergillus fumigatus., Methods: After single intraperitoneal doses (0.25, 1.0, and 4.0 mg/kg), plasma CAS concentrations were assayed by high-performance liquid chromatography. The pharmacokinetic data were analyzed by nonparametric population pharmacokinetic analysis. Three dosage groups (0.25, 1.0, and 4.0 mg/kg) fractionated into 3 different dosing intervals (q6, q24, or q48 h) were then used to evaluate the pharmacokinetic/pharmacodynamic effects (percentage of time greater than the minimum effective concentration [MEC], 96-h area under the plasma concentration curve:MEC ratio, and peak concentration in plasma [Cmax]:MEC ratio) at clinically achievable exposures. Mice were treated for 96 h and were then euthanized, and their lungs were harvested for analysis of pulmonary fungal burden by real-time quantitative polymerase chain reaction., Results: A concentration-dependent reduction in mean pulmonary fungal burden was evident in mice in the 1 mg/kg dosage-fractionation group, with significantly lower mean pulmonary fungal burden in mice dosed q48 h versus q6 h (P < .01). A paradoxical increase in pulmonary fungal burden was observed in the highest dosage-fractionation group., Conclusions: CAS demonstrates concentration-dependent pharmacodynamics in the treatment of IPA. The Cmax : MEC ratio appears to be the parameter most closely associated with the reduction of pulmonary fungal burden., (Copyright 2004 Infectious Diseases Society of America)

Objective: To clone Aspergillus fumigatus squalene epoxidase gene and to further investigate its role in terbinafine resistance., Methods: The A.fumigatus genomic DNA library was transformed into pyrG-A. fumigatus strain protoplasts with polyethylene glycol-mediated transformation protocol. TRB-resistant pyrG+ transformants were then isolated by being plated on MM-U with TRB (0.625 mg/L) plates. After confirmation of terbinafine-resistance by using both disk diffusion and NCCLS M38-A microdilution antifungal susceptibility testing, the gene conferring terbinafine-resistance was identified. Finally, the gene was cloned and retransformed into pyrG-A. fumigatus strain., Results: From a total of 5x10(4) transformants, one TRB-resistant pyrG+ transformant was isolated, which showed the terbinafine-specific resistance without cross-resistance to any other antifungals. A. fumigatus squalene epoxidase gene was further identified to confer this terbinafine-resistance. As a result, the complete A. fumigatus squalene epoxidase gene was firstly cloned. Finally, the transformants with extra copies of A. fumigatus squalene epoxidase gene, again, showed the specific resistance to terbinafine., Conclusion: Extra copies of A. fumigatus squalene epoxidase gene, which was cloned for the first time in this study, could result in A. fumigatus resistance to terbinafine. This is a novel mechanism of terbinafine-resistance that needs further investigation for its clinical significance.

With the increasing use of antifungals such as amphotericin B, itraconazole, voriconazole, caspofungin, and terbinafine (TRB) in patients at high risk for invasive aspergillosis, resistance of Aspergillus fumigatus to these agents will ultimately emerge. Due to the limited availability of molecular genetics for A. fumigatus, few studies have addressed its mechanisms of resistance to antifungals. We transformed A. fumigatus protoplasts with a pyrG-based A. fumigatus genomic DNA library (constructed in the multicopy nonintegrating vector pRG3-AMA1-NotI, which also has the pyr-4 gene for selection). We obtained one pyrG(+) transformant that grew in medium containing a fungicidal concentration (0.625 microg/ml) of TRB. To determine whether TRB resistance in that transformant was plasmid dependent, we evicted the plasmid and found concomitant loss of uracil prototrophy and TRB resistance. DNA sequence analysis identified the gene responsible for TRB resistance as the A. fumigatus squalene epoxidase gene (ERG1), which encodes the target enzyme of TRB. Authentic A. fumigatus ERG1, amplified from the genome and cloned into pRG3-AMA1-NotI, also conferred TRB-specific resistance. This molecular approach has the potential to enhance our knowledge of the mechanisms of A. fumigatus resistance to modern antifungals.

Fluconazole (FLC), a triazole with limited activity against Aspergillus species, is frequently used as prophylaxis in leukemia patients and bone marrow transplant recipients. Prior FLC use has been associated with an increasing incidence of invasive aspergillosis in these patients. We hypothesized that prior exposure of Aspergillus fumigatus to FLC could result in altered in vitro susceptibility of this fungus to other, more active triazoles. Thus, we performed serial passages of conidia of 10 clinical isolates of A. fumigatus (all itraconazole [ITC] susceptible) on FLC-containing yeast agar glucose plates. The MICs and minimal fungicidal concentrations (MFCs) of amphotericin B, FLC, ITC, and voriconazole (VRC) for A. fumigatus conidia were measured following four passages on FLC-containing medium according to the National Committee for Clinical Laboratory Standards microdilution method. Serial passages on FLC-containing plates resulted in a fourfold increase in the MFCs (but not the MICs) of ITC for nine isolates. The attenuated ITC fungicidal activity against A. fumigatus following FLC preexposure was medium independent and was also observed against FLC-preexposed A. fumigatus hyphae with the viability staining FUN-1 dye. Moreover, FLC preexposure of A. fumigatus conidia resulted in an analogous increase in the MFCs (but not the MICs) of VRC. Our findings suggest that preexposure of A. fumigatus to FLC attenuates the in vitro fungicidal activity of subsequent ITC use against it. This phenotypic adaptation is not captured by a routine MIC determination but requires MFC measurement. The in vivo significance of this in vitro phenomenon requires further investigation.

We investigated the in vitro activity of sequential itraconazole and caspofungin against 10 isolates of Aspergillus fumigatus. Previous exposure of A. fumigatus to itraconazole resulted in dose-dependent enhanced effects of caspofungin and vice versa. Our finding suggests a preferential role for azole-caspofungin sequential combinations and merits further in vivo investigation.

Invasive aspergillosis (IA) caused by inherently more antifungal-resistant non-fumigatus Aspergillus species has become an important life-threatening complication in severely immunocompromised patients with cancer. The purpose of this study was to compare the relative incidence of, risk factors for, and in vitro correlation of amphotericin B and itraconazole with the clinical outcome of IA caused by Aspergillus fumigatus with those of IA caused by non-fumigatus Aspergillus spp. in patients with cancer. A retrospective search of our tertiary care cancer center's microbiology laboratory reports from 1998-2001 revealed 40 patients with cancer and IA. A non-fumigatus Aspergillus species caused IA in 28 (70%) of those patients. A. fumigatus was the predominant cause of late-onset IA after bone marrow transplantation (p = 0.05), whereas IA due to non-fumigatus Aspergillus spp. was more common in patients with neutropenia (p = 0.01). The minimum inhibitory concentration (50/90) and minimum fungicidal concentration (50/90) for amphotericin B were higher in the non-fumigatus Aspergillus spp. group than in the A. fumigatus one. The Aspergillus species distribution in IA cases in our institution shows a predominance of the more antifungal-resistant or -tolerant non-fumigatus Aspergillus spp.

Objectives: Over the last 30 years, the number of invasive fungal infections among immunosuppressed patients has increased significantly, while the number of effective systemic antifungal drugs remains low. The aim of this study was to identify and characterize antifungal compounds that inhibit fungus-specific metabolic pathways not conserved in humans.Methods: We screened a diverse compound library for antifungal activity in the pathogenic mould Aspergillus fumigatus . We determined the in vitro activity of bromoquinol by MIC determination against a panel of fungi, bacteria and cell lines. The mode of action of bromoquinol was determined by screening an Aspergillus nidulans overexpression genomic library for resistance-conferring genes and by RNAseq analysis in A. fumigatus . In vivo efficacy was tested in Galleria mellonella and murine models of A. fumigatus infection.Results: Screening of a diverse chemical library identified three compounds interfering with fungal iron utilization. The most potent, bromoquinol, shows potent wide-spectrum antifungal activity that was blocked in the presence of exogenous iron. Mode-of-action analysis revealed that overexpression of the dba secondary metabolite cluster gene dbaD , encoding a metabolite transporter, confers bromoquinol resistance in A. nidulans , possibly by efflux. RNAseq analysis and subsequent experimental validation revealed that bromoquinol induces oxidative stress and apoptosis in A. fumigatus . Bromoquinol significantly reduced mortality rates of G. mellonella infected with A. fumigatus , but was ineffective in a murine model of infection.Conclusions: Bromoquinol is a promising antifungal candidate with a unique mode of action. Its activity is potentiated by iron starvation, as occurs during in vivo growth. [ABSTRACT FROM AUTHOR]

We examined the qualitative polymorphonuclear neutrophil (PMN)-associated immune impairment in patients with chronic lymphocytic leukemia (CLL) by characterizing phagocytic killing of key non-opsonized bacterial ( Staphylococcus aureus and Pseudomonas aeruginosa) and fungal ( Candida albicans and Aspergillus fumigatus) pathogens. Neutrophils were collected from 47 non-neutropenic patients with CLL (PMN count > 1000/mm3) and age-matched and young healthy controls (five each). A subset of patients (13%) had prior or subsequent infections. We found that the patients with CLL had diminished PMN microbicidal response against bacteria but not against fungi compared with the controls. Compared to patients with effective PMN responses, we did not identify differences of basal PMN pathogen-associated molecular pattern receptor gene expression, soluble pathogen-associated molecular pattern gene expression or inflammatory cytokine signatures in patients with impaired PMN responses when PMNs were analyzed in multiplex real-time polymerase chain reaction assays. However, differences in PMN microbicidal response against A. fumigatus in patients with CLL were associated with the degree of hypogammaglobulinemia. [ABSTRACT FROM AUTHOR]

Drug combinations are increasingly being explored as a means of improving outcomes in cases of invasive aspergillosis. However, the optimal methods for assessing in vitro drug combinations are unclear. We investigated whether echinocandin drug combinations have potentially useful interactions against Aspergillus fumigatus . We explored the in vitro interactions of three echinocandins (caspofungin [CSP], anidulafingin [ANF], and micafungin [MCF]) by three methods, i.e., (i) checkerboard assay, (ii) disk diffusion assay, and (iii) E-test/agar dilution. The checkerboard experiments revealed different interactions between echinocandin pairs depending on the drug combination tested. Specifically, the combination of CSF and either ANF or MCF yielded a fractional inhibitory concentration (FIC) index range of 0.15 – 2.0. Whereas the combination of MCF and ANF yielded an FIC index range of 0.19 – 0.31, consistent with synergistic interaction. In contrast, all echinocandin pairs appeared indifferent by the disk diffusion method. Moreover, the combination of MCF and ANF appeared antagonistic when the two drugs were tested using the E-test/agar dilution method with both the FKS1 wild-type and echinocandin-resistant fks1 mutant strains. Our results highlight methodological problems inherent in in vitro antifungal combination testing. We did not find compelling evidence of inter-echinocandin synergy that could serve as a basis for further in vivo experimentation. [ABSTRACT FROM AUTHOR]

Background. We investigated whether caspofungin and other echinocandins have immune-enhancing properties that influence human polymorphonuclear neutrophil (PMN)-mediated mold hyphal damage. Materials and methods. Using aniline blue staining, we compared patterns of β-glucan exposure in Aspergillus fumigatus, Aspergillus terreus, Rhizopus oryzae, Fusarium solani, Fusarium oxysporum, Scedosporium prolificans, and Scedosporium apiospermum hyphae after caspofungin exposure. We also determined PMN-mediated hyphal damage occurring with or without preexposure to caspofungin or with preexposure to the combination of caspofungin and anti-β-glucan monoclonal antibody, using 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-sH-tetrazolium hydroxide (XTT) assay. Results. Preincubation with caspofungin (32 μg/mL for R. oryzae; 0.0625 μg/mL for other isolates) increased exposure toβ-glucan. PMN-induced damage increased after caspofungin exposure and was further augmented by the addition of anti-β-glucan antibody. Preincubation with micafungin or anidulafungin had similar effects on PMNinduced damage of A. fumigatus hyphae. Finally, preexposure of A. fumigatus, but not S. prolificans, to caspofungin induced expression of Dectin-1 by PMN. Conclusions. The results of the present study suggest inducement of β-glucan unmasking by echinocandins and enhancement of PMN activity against mold hyphae, thereby supporting the immunopharmacologic mode of action of echinocandins. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatusis a ubiquitous fungus that is a frequent opportunistic pathogen in immunosuppressed patients. Because of its role as a pathogen, it is of considerable experimental interest. A set of auxotrophic isogenic strains in theA. fumigatusgenome reference strain AF293 has been developed. Using molecular genetic methods, arginine and lysine auxotrophs were made by deletion ofargBandlysB, respectively. Transformation of these auxotrophic strains with plasmids carryingargBorlysB, respectively, results in efficient integration at these loci. Finally, these strains are able to form stable diploids, which should further facilitate analysis of gene functions in this fungus. Furthermore, the development of this isogenic set of auxotrophic strains in the AF293 background will enable investigators to study this important opportunistic human pathogen with greater facility. [ABSTRACT FROM AUTHOR]

Background IBR is a Bruton's tyrosine kinase inhibitor, and plays a key role in the treatment of CLL. In randomized clinical trials, <1% of IBR-treated CLL patients developed IFIs. However, several IFIs were reported with real-life use of IBR. Methods This is a retrospective observational study of all CLL patients (> 18 years) treated with IBR (2/2014–8/2018) at MD Anderson Cancer Center. We excluded patients with active IFI (proven and probable, EORTC/MSG criteria) at the start of IBR and patients with <6 months of follow-up. Results Of the 821 CLL IBR-treated patients, 24 developed probable or proven IFI (2.9%). Of these infections, 21 occurred within 30 days (d) of last IBR dose, while 3 IFIs occurred at 94, 135 and 221 d post IBR, respectively. The majority of patients with IFI were male (83%) with a median age of 66 years at IFI diagnosis. The median prior lines of therapy for CLL was 1 (range 0–7), with 29% receiving IBR as frontline treatment. Five patients had evidence of Richter's transformation at the time of IFI diagnosis, while two patients had prior stem cell transplant. The average time from start of IBR to diagnosis of IFI was 338 d, with only 7 cases of IFI within the first 3 months of IBR. The majority of IFIs were proven/probable aspergillosis (63%), including 9 cases of Aspergillus fumigatus. The remaining infections consisted of Cryptococcus neoformans (21%), Fusarium spp. (8%), with one case each of candidiasis, histoplasmosis, mucormycosis, and Pneumocystis jiroveci pneumonia. Three patients had evidence of poly-fungal IFI. The sites of infection were pulmonary (88%), blood (13%), CNS (13%), and sinus (8%). Five patients were diagnosed with disseminated IFI, including Cryptococcus spp. (2 cases), Rhizopus spp. Aspergillus spp. and Candida spp. The 42-day mortality rate post IFI diagnosis was 25%. Conclusion We report the largest single-center cohort of CLL patients on IBR to date. The IFI incidence of 2.9% (24/821) is consistent with most previous reports estimating a 0.5–4% incidence. In contrast to published reports, close to 1/3 of our patients with IFI received IBR as frontline therapy and most IFIs (71%) were diagnosed > 3 months after starting IBR. We are currently conducting a case–control comparison with IBR–treated CLL patients with no infection to uncover risk factors associated with IFIs in these patients. Disclosures Samuel L. Aitken, PharmD, Melinta Therapeutics: Grant/Research Support, Research Grant; Merck, Sharpe, and Dohme: Advisory Board; Shionogi: Advisory Board. [ABSTRACT FROM AUTHOR]

The present study was to determine the in vitro activity of posaconazole (POS) against 385 Candida and 268 Aspergillus clinical isolates from China. We found that POS was active against 85.5% Candida and 94.4% Aspergillus isolates. Non–wild-type (non-WT) phenotype was found in a subset of Candida albicans (15.4%), Candida tropicalis (11.9%), Aspergillus fumigatus (4.1%), and Aspergillus flavus (17.4%) isolates. Cross-resistance to POS and other triazoles was seen. Gene sequencing showed that 4 C. albicans , 1 C. tropicalis , and 9 A. fumigatus isolates with cross-resistance to POS and other triazoles had mutations in ERG11 or CYP51A. In conclusion, POS has potent in vitro activity against most of Candida and Aspergillus isolates from China. Non-WT phenotype and those with cross-resistance to POS and other triazoles exist, frequently driven by mutations of ERG11 in Candida spp. and CYP51A in Aspergillus spp. • The susceptibility to posaconazole in Candida spp. and Aspergillus spp. from China was determined for the first time. • Posaconazole was active against most of Candida and Aspergillus isolates in this study. • Non-WT phenotype for posaconazole and those with cross-resistance to posaconazole and other triazoles exist. • Cross-resistance may be caused by mutations of ERG11 in Candida spp. and CYP51A in Aspergillus spp. [ABSTRACT FROM AUTHOR]