Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.     

The Pathogenesis of Aspergillus fumigatus,Host Defense Mechanisms,and the Development of AFMP4 Antigen as a Vaccine

Aspergillus fumigatus is one of the ubiquitous fungi with airborne conidia, which accounts for most aspergillosis cases. In immunocompetent hosts, the inhaled conidia are rapidly eliminated. However, immunocompromised or immunodeficient hosts are particularly vulnerable to most Aspergillus infections and invasive aspergillosis (IA), with mortality from 50% to 95%. Despite the improvement of antifungal drugs over the last few decades, the therapeutic effect for IA patients is still limited and does not provide significant survival benefits. The drawbacks of antifungal drugs such as side effects, antifungal drug resistance, and the high cost of antifungal drugs highlight the importance of finding novel therapeutic and preventive approaches to fight against IA. In this article, we systemically addressed the pathogenic mechanisms, defense mechanisms against A. fumigatus, the immune response, molecular aspects of host evasion, and vaccines’ current development against aspergillosis, particularly those based on AFMP4 protein, which might be a promising antigen for the development of anti-A. fumigatus vaccines.     

<Emphasis Type="Italic">Aspergillus</Emphasis> Species in Bronchiectasis: Challenges in the Cystic Fibrosis and Non-cystic Fibrosis Airways

Bronchiectasis is a chronic irreversible airway abnormality associated with infectious agents that either cause or superinfect the airways. While the role of bacteria is well studied, much remains to be determined about fungi in both cystic fibrosis- and non-cystic fibrosis-related bronchiectasis. The airway is constantly exposed to inhaled ambient moulds of which Aspergillus represent the most ubiquitous. In a normal healthy host, this situation is of little consequence. The presence of anatomical or immunological abnormalities such as those in bronchiectasis leads to a range of fungal-related pathologies from asymptomatic airway colonization to fungal sensitization, allergic bronchopulmonary aspergillosis or chronic pulmonary aspergillosis. These entities are difficult to recognize, diagnose and treat due in part to a lack of validated biomarkers. Our true understanding of the complex relationships that regulate fungal–host interactions is still in its infancy and, several questions remain. This includes if fungal epidemiology in bronchiectasis is uniform across countries, and to what extent immunopathological mechanisms—related to fungal airway infections—occurs in different disease states. Specific triggers to allergic or infectious responses to Aspergillus require further exploration. How transition occurs between allergic and invasive phenotypes and their respective biomarkers is also important. Whether anti-fungal treatment is warranted in all cases and what the optimal management strategy is, particularly when treatment should commence and its expected duration remains unclear. Further research is clearly necessary and should be prioritized to better understand the clinical effects and impact of Aspergillus in the setting of bronchiectasis.     

Developmental regulators in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

The filamentous fungus Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing severe and usually fatal invasive aspergillosis in immunocompromised patients. This fungus produces a large number of small hydrophobic asexual spores called conidia as the primary means of reproduction, cell survival, propagation, and infectivity. The initiation, progression, and completion of asexual development (conidiation) is controlled by various regulators that govern expression of thousands of genes associated with formation of the asexual developmental structure conidiophore, and biogenesis of conidia. In this review, we summarize key regulators that directly or indirectly govern conidiation in this important pathogenic fungus. Better understanding these developmental regulators may provide insights into the improvement in controlling both beneficial and detrimental aspects of various Aspergillus species.     

Allergic Aspects of Aspergillosis

Aspergillus species and principally A. fumigatus cause a spectrum of pulmonary diseases depending on underlying host genetic risk factors. These include invasive aspergillosis, hypersensitivity lung disease, allergic asthma, allergic bronchopulmonary aspergillosis, and mycetomas. These disorders, pathogenesis and host risk factors will be discussed.     

Pathogenesis of Invasive Pulmonary Aspergillosis in Transplant Recipients

Purpose of Review

Transplant patients are at high risk for invasive pulmonary aspergillosis, and the associated mortality is high. The purpose of this study is to review the pathogenesis of invasive pulmonary aspergillosis (IPA) in transplant patients.

Recent Findings

The pathogenesis of aspergillosis is multifactorial and results from a complex interplay between the pathogen and host. It is well recognized that Aspergillus causes IPA in immunocompromised patients. Recent studies have shown that Aspergillus might also cause diseases likely attributed to an unmodulated immune response in certain transplant recipients such as bronchopulmonary aspergillosis or bronchiolitis obliterans syndrome in lung transplant recipients.

Summary

This review focuses on two crucial axes of the damage response framework applicable to aspergillosis: (1) Aspergillus virulence attributes that enable it to survive and proliferate in the host (thermotolerance, stress and hypoxic response, secretion of secondary metabolites) and (2) host response with specific focus on innate immunity and angiogenesis.

     

Papel futuro de la micafungina en el tratamiento de las micosis invasoras por hongos filamentosos

BackgroundMicafungin is a echinocandin. It inhibits β-1,3-D-glucan synthesis, thus achieving fungicidal activity against virtually all Candida spp., including those resistant to fluconazole, and fungistatic activity against Aspergillus spp., as well as several but not all pathogenic molds. Results from in vitro studies, animal models, small clinical trials, hint at possible future indications such as invasive aspergillosis and empirical viantifungal therapy, although currently there is little information published.AimsTo describe published data of micafungin as treatment against invasive mold infections, specially analysing its role in the inmunodepressed host and critical care setting.MethodsA sistematic review of literature using the principal medical search engines was performed. Terms such as micafungin, aspergillosis, zygomycosis, invasive fungal infections, emerging fungal infections, antifungal treatment or therapy, antifungal prophylaxis, empiric or pre-emptive therapy were crossed. Febrile neutropenia patients were excluded.ResultsSeveral studies in these setting were identified and were described in this review. Although there were no blinded randomized clinical trials published, treatment or prophylaxis of invasive aspergillosis and other invasive mould infections with micafungin described in open clinical studies were analyzed.ConclusionsMicafungin could play a future important role as a primary or rescue therapy, alone or in combination, in the treatment or prophylaxis of invasive fungal infections caused by moulds. New randomized clinical trials are needed to confirm their efficacy.     

Bronchoalveolar lavage in an immunosuppressed rabbit model of invasive pulmonary aspergillosis

A rabbit model of invasive aspergillosis has been used to investigate the pathogenesis of Aspergillus infection in the immunosuppressed host. The animals received hydrocortisone daily and a single dose of cyclophosphamide 2 days prior to intratracheal instillation of conidia from Aspergillus fumigatus. Bronchoalveolar lavage (BAL) was performed in 3 infected and 2 control saline treated animals sacrificed on days 1, 2, 4, 7 and 10 following inoculation. Infective load within the lung was quantified using an assay for chitin which is an important component of fungal cell walls (in particular the hyphal cell wall) and is not present in vertebrate tissue. The total BAL white cell count did not discriminate between infected and saline treated animals and Aspergillus was cultured from one lavage specimen only. Infected animals developed a marked neutrophil alveolitis by day 2 in contrast to a near total absence of neutrophils in the lavages of the control animals. Phagocytosis of conidia by alveolar macrophages was prominent but did not prevent progressive infection as confirmed by measurement of lung chitin. This pattern of cellular response within the alveolar airspace reflects the complex nature of the response to Aspergillus infection in the immunosuppressed host.     

Monitoring Environmental Aspergillus spp. Contamination and Meteorological Factors in a Haematological Unit

The opportunistic pathogens belonging to the Aspergillus genus are present in almost all seasons of the year, and their concentration is related to meteorological conditions. The high density of Aspergillus spp. conidia in a haematological hospital ward may be a significant risk factor for developing invasive fungal diseases in immunocompromised patients. Aim of the present study was to evaluate the variability of airborne Aspergillus spp. conidia contamination in a Haematological Unit (HU) within a period of 16 months in relation with some meteorological parameters. An environmental Aspergillus surveillance was conducted in the HU in four rooms and their bathrooms, in the corridor and in three external sites using an agar impact sampler. During each sampling, temperature and relative humidity at each site were recorded and current wind speed and rainfall events were taken from the official weather service. Aspergillus spp. conidia concentration differed significantly across the sampling sites. Internal Aspergillus spp. loads were significantly dependent on temperature, internal relative humidity and rain. External conidia concentrations were significantly influenced by outdoor temperature and relative humidity. A suitable indicator was introduced to evaluate the seasonal distribution of Aspergillus spp. conidia in the sampling sites, and a significant dependence on this indicator was observed inside the HU. Seventeen different fungal species belonging to the Aspergillus genus were detected during the sampling period. Aspergillus fumigatus was the most frequently isolated species and its distribution depended significantly on the seasonal indicator both inside and outside the hospital ward.     

Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella

The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen-associated molecular patterns) may cause self-damage that results in increased mortality of larvae infected with the color mutants. The current study underscores the limitations of using this insect model for inferring the pathogenic potential of A. fumigatus strains in mammals, but also points to the importance of understanding the innate immunity of the insect host in providing insights into the pathogenicity level of different fungal strains in this model. Additionally, our observations that melanization defective color mutants demonstrate increased virulence in the insect wax moth, suggest the potential of using melanization defective mutants of native insect fungal pathogens in the biological control of insect populations.     

Aspergillus-specific antibodies – Targets and applications

Aspergillus is a fungal genus comprising several hundred species, many of which can damage the health of plants, animals and humans by direct infection and/or due to the production of toxic secondary metabolites known as mycotoxins. Aspergillus-specific antibodies have been generated against polypeptides, polysaccharides and secondary metabolites found in the cell wall or secretions, and these can be used to detect and monitor infections or to quantify mycotoxin contamination in food and feed. However, most Aspergillus-specific antibodies are generated against heterogeneous antigen preparations and the specific target remains unknown. Target identification is important because this can help to characterize fungal morphology, confirm host penetration by opportunistic pathogens, detect specific disease-related biomarkers, identify new candidate targets for antifungal drug design, and qualify antibodies for diagnostic and therapeutic applications. In this review, we discuss how antibodies are raised against heterogeneous Aspergillus antigen preparations and how they can be characterized, focusing on strategies to identify their specific antigens and epitopes. We also discuss the therapeutic, diagnostic and biotechnological applications of Aspergillus-specific antibodies.     

Aspergillus felis sp. nov., an Emerging Agent of Invasive Aspergillosis in Humans,Cats, and Dogs

We describe a novel heterothallic species in Aspergillus section Fumigati, namely A. felis (neosartorya-morph) isolated from three host species with invasive aspergillosis including a human patient with chronic invasive pulmonary aspergillosis, domestic cats with invasive fungal rhinosinusitis and a dog with disseminated invasive aspergillosis. Disease in all host species was often refractory to aggressive antifungal therapeutic regimens. Four other human isolates previously reported as A. viridinutans were identified as A. felis on comparative sequence analysis of the partial β-tubulin and/or calmodulin genes. A. felis is a heterothallic mold with a fully functioning reproductive cycle, as confirmed by mating-type analysis, induction of teleomorphs within 7 to 10 days in vitro and ascospore germination. Phenotypic analyses show that A. felis can be distinguished from the related species A. viridinutans by its ability to grow at 45°C and from A. fumigatus by its inability to grow at 50°C. Itraconazole and voriconazole cross-resistance was common in vitro.     

Fungi in healthy and diseased sea fans (<Emphasis Type="Italic">Gorgonia ventalina)</Emphasis>: is <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> always the pathogen?

Caribbean corals, including sea fans (Gorgonia spp.), are being affected by severe and apparently new diseases. In the case of sea fans, the pathogen is reported to be the fungus Aspergillus sydowii, and the disease is named aspergillosis. In order to understand coral diseases and pathogens, knowledge of the microbes associated with healthy corals is also necessary. In this study the fungal community of healthy Gorgonia ventalina colonies was contrasted with that of diseased colonies. In addition, the fungal community of healthy and diseased tissue within colonies with aspergillosis was contrasted. Fungi were isolated from healthy and diseased fans from 15 reefs around Puerto Rico, and identified by sequencing the nuclear ribosomal ITS region and by morphology. Thirty fungal species belonging to 15 genera were isolated from 203 G. ventalina colonies. Penicillum and Aspergillus were the most common genera isolated from both healthy and diseased fans. However, the fungal community of healthy fans was distinct and more diverse than that of diseased ones. Within diseased fans, fungal communities from diseased tissues were distinct and more diverse than from healthy tissue. The reduction of fungi in diseased colonies may occur prior to infection due to environmental changes affecting the host, or after infection due to increase in dominance of the pathogen, or because of host responses to infection. Data also indicate that the fungal community of an entire sea fan colony is affected even when only a small portion of the colony suffers from aspergillosis. An unexpected result was that A. sydowii was found in healthy sea fans but never in diseased ones. This result suggests that A. sydowii is not the pathogen causing aspergillosis in the studied colonies, and suggests several fungi common to healthy and diseased colonies as opportunistic pathogens. Given that it is not clear that Aspergillus is the sole pathogen, calling this disease aspergillosis is an oversimplification at best. Communicated by Biology Editor Dr Michael Lesser     

Production of Extracellular Traps against Aspergillus fumigatus In Vitro and in Infected Lung Tissue Is Dependent on Invading Neutrophils and Influenced by Hydrophobin RodA

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading.     

Aspergillus fumigatus: contours of an opportunistic human pathogen

Aspergillus fumigatus is currently the major air‐borne fungal pathogen. It is able to cause several forms of disease in humans of which invasive aspergillosis is the most severe. The high mortality rate of this disease prompts increased efforts to disclose the basic principles of A. fumigatus pathogenicity. According to our current knowledge, A. fumigatus lacks sophisticated virulence traits; it is nevertheless able to establish infection due to its robustness and ability to adapt to a wide range of environmental conditions. This review focuses on two crucial aspects of invasive aspergillosis: (i) properties of A. fumigatus that are relevant during infection and may distinguish it from non‐pathogenic Aspergillus species and (ii) interactions of the pathogen with the innate and adaptive immune systems.     

An Approach to Suspected Invasive Fungal Infection in Patients with Hematologic Malignancy and HCT Recipients with Persistent Neutropenic Fever Despite Mold-Active Prophylaxis

Purpose of Review

This review offers an approach to managing suspected invasive fungal infection (IFI) in a febrile neutropenic patient with hematologic malignancy or hematopoietic cell transplantation (HCT) while on mold-active prophylaxis. We take into consideration host characteristics, new diagnostic tools, and available therapeutics.

Recent Findings

Despite use of anti-Aspergillus prophylactic agents, invasive aspergillosis is the most commonly reported IFI breaking through common prophylactic agents including the newest azole, isavuconazole. While more fungal diagnostic modalities are available, how to best incorporate them in the work-up of IFI remains unclear, while sensitivity of any particular fungal biomarker or molecular test is low.

Summary

In a febrile neutropenic patient with hematologic malignancy or HCT and suspected IFI, consider particularly invasive aspergillosis, regardless of the mold-active prophylactic agent. Early diagnosis and intervention are especially important to a favorable outcome; treatment is directed based on the suspected IFI syndrome and suspected organism. Switching azoles, consideration of combination therapy, and reducing immunosuppression are proposed strategies for the management of breakthrough IFI, while surgical debridement remains crucial for Mucormycoses. More study is needed into the optimal antifungal approach in these clinical scenarios. Meanwhile, therapeutic drug monitoring and attention to drug-drug interactions are encouraged.

     

The role of adjuvant agents in treating fungal diseases

Invasive fungal infections (IFIs) remain a significant cause of morbidity and mortality despite the recent introduction of new antifungal medications. In this review, the available data on the use of adjuvant agents for the treatment of IFIs are discussed. Cytokines such as interferon-γ, colony-stimulating factors, granulocyte transfusions, and the monoclonal antibody efungumab may have in a role in the management of IFIs through augmentation of the host immune response, whereas pathogen-specific vaccines may help prevent infection. Pentraxin 3, an acute phase protein, may assist in the prevention and treatment of aspergillosis. Deferasirox, an iron chelator, is being investigated as an adjunctive therapy for the treatment of zygomycosis. Lactoferrin, an ironbinding protein, appears to have activity in Candida and Aspergillus infections, and omiganan may help prevent fungal catheter-related infections. Although none of these agents are currently approved for the treatment of IFIs, they may be involved in current and/or future treatment options when used in combination with antifungal drugs.     

NETs formed by human neutrophils inhibit growth of the pathogenic mold Aspergillus fumigatus

Neutrophil extracellular traps (NETs) represent a distinct mechanism to control and eliminate microbial infections. Our results show that conidia and germ tubes of the human pathogenic mold Aspergillus fumigatus are able to trigger the formation of NETs. Viable fungal cells are not essentially required for this host–pathogen interaction. Neutrophils engulf conidia and thereby inhibit their germination, a process that is independent of NETosis. In the experimental set-up used in this study neutrophils do not kill germ tubes, but reduce their polar growth and this inhibition depends on NETs as it can be overcome by the addition of DNase-1. The Zn²⁺ chelator calprotectin is associated with the Aspergillus-induced NETs and addition of Zn²⁺ abrogates the NET-mediated growth inhibition. In summary, our data provide evidence that NETs are not sufficient to kill A. fumigatus, but might be a valuable tool to confine infection.     

Review of Epidemiology, Diagnosis, and Treatment of Invasive Mould Infections in Allogeneic Hematopoietic Stem Cell Transplant Recipients

Invasive mould infections are a major cause of morbidity and mortality in hematopoietic stem cell transplant recipients (HSCT). Allogeneic HSCT recipients are at substantially higher risk than autologous HSCT recipients. Although neutropenia following the conditioning regimen remains an important risk factor for opportunistic fungal infections, most cases of invasive mould infection in allogeneic HSCT recipients occur after neutrophil recovery in the setting of potent immunosuppressive therapy for graft-versus-host disease. Invasive aspergillosis is the most common mould infection. However, there has been an increased incidence of less common non-Aspergillus moulds that include zygomycetes, Fusarium sp., and Scedosporium sp. Reflecting a key need, important advances have been made in the antifungal armamentarium. Voriconazole has become a new standard of care as primary therapy for invasive aspergillosis based on superiority over amphotericin B. There is significant interest in combination therapy for invasive aspergillosis pairing voriconazole or an amphotericin B formulation with an echinocandin. There have also been advances in novel diagnostic methods that facilitate early detection of invasive fungal infections that include galactomannan and beta-glucan antigen detection and PCR using fungal specific primers. We review the epidemiology, diagnosis, and management of invasive mould infection in HSCT, with a focus on allogeneic recipients. We also discuss options for prevention and early treatment of invasive mould infections.