Airborne Aspergillus fumigatus conidia: a risk factor for aspergillosis

Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis, a usually fatal infection. The disease has risen in prominence in recent years due to the increasing numbers of severely immunocompromised patients becoming infected. The fungus is ubiquitous in the environment, producing large numbers of conidia that are dispersed in the air. Humans inhale numerous conidia everyday, but infections are not seen in healthy individuals. As inhalation of conidia is the main route of infection, considerable efforts are required to prevent infection in susceptible patients. This review summarises the current knowledge on airborne concentrations of A. fumigatus conidia, their background levels in outdoor air and seasonal distribution patterns. New and established methods of air sampling for airborne A. fumigatus conidia are discussed. Common environmental sources of the fungus are reviewed, including its presence in compost heaps. Finally, the lack of stringent guidelines on the monitoring and control of airborne A. fumigatus concentrations in hospitals is discussed.     

Inhibition of quantitative PCR analysis of fungal conidia associated with indoor air particulate matter

Environmental samples analyzed by quantitative PCR (qPCR) are subject to interference by inhibitors present in the environment being sampled. A controlled determination of the effect of inhibitors associated with sampling indoor air and the ability of internal standard controls to detect inhibition was carried out by filter collection of air samples followed by spiking of the filters with green fluorescent protein-expressing Aspergillus fumigatus conidia. Microscopic conidial counts were compared with qPCR results and correlated with levels of particulate matter and viable airborne microorganisms. Our data showed that PCR can be inhibited by masses of particulate matter as low as 50 μg and that the amount of inhibition was positively correlated with the mass of particulate (r = 0.75) and the number of non-filamentous organisms (r = 0.73). The use of internal standard DNA identified the presence of inhibitors and indicated the need for additional sample processing or qualification of sample results.     

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.     

Phagocytosis of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> conidia by primary nasal epithelial cells <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Invasive aspergillosis, which is mainly caused by the fungus Aspergillus fumigatus, is an increasing problem in immunocompromised patients. Infection occurs by inhalation of airborne conidia, which are first encountered by airway epithelial cells. Internalization of these conidia into the epithelial cells could serve as a portal of entry for this pathogenic fungus.     

Contamination of air systems: 1987–88 assessment and prospects of the Grenoble intervention group

Summary In order to evaluate bacterial and fungal contamination, the authors used a qualitative and quantitative procedure and investigated 47 air conditioning and humidifying units. Air samples were studied using Biotest RCS biocollector (160×6 liters on 6 different selective media), whilst dust samples were analysed after spreading 10 mg onto fungal media. Selective research ofLegionella and fungi were performed using water filtrates and specific media. Even though, the most frequently identified species were quite common environmental fungi, mainlyPenicillium, Alternaria andAspergillus fumigatus (36 out of 47 collections), the health effects of intense exposure to these common moulds are not really known.Staphylococcus aureus was identified 6 times and thermophilic actinomyces species (A. candidus) once. From the quantitative analysis a significant relationship was found between some risk factors and airborne contamination. Indeed bacteria and fungi number depends on the humidifying system (steam or washer), efficiency of secondary filters, relative humidity percentage (< or >60%), installation maintenance, industrial activity and complaints of people at risk. The presence of air recycling and biocide use seems only to have an influence on fungal flora. Having determined a global risk score for each air conditioning unit, the authors underline the fact that bacterial and fungal airborne contamination increases with score. Moreover, for a global, metrological, medical and technical evaluation, a multidisciplinary approach has proved itself to be indispensable.     

Effects of Conidia of Various Aspergillus Species on Apoptosis of Human Pneumocytes and Bronchial Epithelial Cells

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species. F. Féménia and D. Huet made an equal contribution to this work.     

Isolation and toxigenicity of Aspergillus fumigatus from moldy silage

Thirty-nine silage samples were collected from various siloson Terceira Island in the Azores. Samples were examined for the presence of total fungi, and isolates of Aspergillus fumigatus were analyzed for their ability to produce fumitremorgens B and C, fumigaclavines B and C, and gliotoxin. Thirty-four silage samples (87%) were contaminated with fungi, and A. fumigatus was isolated from 27 samples (69%). Samples that were taken from the surface of silos had significantly higher populations of both total fungi and A. fumigatus than did samples taken from the middle of silos. Analysis of 27 A. fumigatus isolates (one representing each positive sample) showed that 59.3% produced fumitremorgen B; 33.3% produced fumitremorgen C; 29.6% produced fumigaclavine B; 7.4% produced fumigaclavine C; and 11.1% produced gliotoxin. Fifty-two percent of the isolates produced multiple toxins, and 25.9% did not produce any of these toxins. Gliotoxin and fumigaclavine C were always produced in combination with other toxins. Because of the demonstrated potential of these A. fumigatus isolates to producemycotoxins, it is important to properly construct and manage silos to prevent their contamination with A. fumigatus.This revised version was published online in October 2005 with corrections to the Cover Date.     

Silencing of mitochondrial alternative oxidase gene of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.     

烟曲霉黑色素的研究进展

烟曲霉(Aspergillus fumigatus)是一种分布于世界各地的腐生真菌,属于人类临床常见的三大机会性致病真菌之一,是侵袭性曲霉菌病的主要病原菌。烟曲霉可以产生DHN-黑色素(dihydroxynaphthalene melanin)和脓黑素(pyomelanin)这2种类型黑色素。本综述介绍烟曲霉黑色素产生的遗传代谢途径、功能以及与宿主免疫系统相互作用的最新认识,有助于更好地理解烟曲霉的病理生理特征,为烟曲霉感染快速诊断技术和新型抗真菌药物的研发提供理论依据。     

Verruculogen associated with <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> hyphae and conidia modifies the electrophysiological properties of human nasal epithelial cells

Background  

The role of Aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. In a previous study, we found that filtrates of A. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells (HNEC) compared to those of non pathogenic moulds.     

MonitoringAspergillus fumigatus aerosols from a composting facility

The large, outdoor Islip Yard Waste Composting Facility on Long Island, New York was investigated as a source of airborne fungus spores. The Burkard-Hirst volumetric spore trap was used for the first extensive sampling of small mold spores for this application. Samplers were operated continuously from 21 August to 30 November 1992 in the facility and in a suburban community about 540 m from the facility. A control site approximately 10 000 m from the facility was also sampled to establish background levels of fungus spores. The facility site had higher average readings ofAspergillus fumigatus spores than did the community and both were higher than the control.A. fumigatus was the only fungus among 30 categories tracked that differed significantly between the facility and control sites. It was also isolated repeatedly from the compost. Higher average levels ofA. fumigatus were measured in the community when winds blew from the facility, and also during times when the compost was moved or mixed at the facility. No correlation was found between wind direction or work times andA. fumigatus conidia at the control site. The study shows that this compost facility can produce a measurable increase in the number of airborneA. fumigatus conidia both at the edge of the facility and at 540 m downwind. It also demonstrates that the Burkard spore trap can be used for monitoring small, airborne mold spores, but it is a difficult and labor intensive task.     

Aspergillosis in Larus cachinnans micaellis: Survey of Eight Cases

Avian aspergillosis is reported in several avian species, with Aspergillus fumigatus as the main aetiological agent. Predisposing factors such as starvation, thermal stress, migratory stress, primary infectious disease or toxicosis may play a role. Eight cases of disseminated aspergillosis in free ranging seagulls sheltered at C.R.U.M.A. (Centro Recupero Uccelli Marini e Acquatici, Livorno, Italy) with different clinical histories are presented. The infection was demonstrated by cultural and histological methods from lesions of all birds, and the presence of airborne A. fumigatus viable elements ranging from 450 to 525 CFU/m³ inside and outside the shelter by means of a surface air sampler (SAS) Super-90 was also assessed. The role of this fungal species as an opportunistic factor in the captivity of seagulls is considered and some control measures, such as a clean and stress free environment and the use of antifungal drugs are suggested.     

Development and Use of Flow Cytometry for Detection of Airborne Fungi

Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of Aspergillus fumigatus and Penicillium brevicompactum conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.     

Ochratoxin A in airborne dust and fungal conidia

Farm workers are often exposed to high concentrations of airborne organic dust and fungal conidia, especially when working with plant materials. The purpose of this investigation was to study the possibility of exposure to the mycotoxin ochratoxin A (OTA) through inhalation of organic dust and conidia. Dust and aerosol samples were collected from three local cowsheds. Aerosol samples for determination of total conidia and dust concentrations were collected by stationary sampling on polycarbonate filters. Total dust was analysed by gravimetry, and conidia were counted using scanning electron microscopy. A method was developed for extraction and determination of OTA in small samples of settled dust. OTA was extracted with a mixture of methanol, chloroform, HCI, and water, purified on immunoaffinity column, and analysed by ion-pair HPLC with fluorescence detection. Recovery of OTA from spiked dust samples (0.9–1.0 μg/kg) was 74% (quantitation limit 0.150 μg/kg). OTA was found in 6 out of 14 settled dust samples (0.2–70 μg/kg). The total concentration of airborne conidia ranged from < 1.1 × 10⁴ to 3.9 × 15⁵ per m³, and the airborne dust concentration ranged from 0.08 to 0.21 mg/m³. Conidia collected from cultures of Penicillium verrucosum and Aspergillus ochraceus contained 0.4–0.7 and 0.02–0.06 pg OTA per conidium, respectively. Testing of conidial extracts from these fungi in a Bacillus subtilis bioassay indicated the presence of toxic compounds in addition to OTA. The results show that airborne dust and fungal conidia can be sources of OTA. Peak exposures to airborne OTA may be significant, e.g., in agricultural environments. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Inhibition and killing of fungi by the polyamine oxidase-polyamine system

Both components of the polyamine oxidase (PAO)-polyamine system are known to be present in phagocytes and have thus been postulated to contribute to the antimicrobial activity of these cells. Therefore, the effects of the PAO-polyamine system on three medically important opportunistic fungi were examined. Yeasts of Cryptococcus neoformans, but not Candida albicans blastoconidia or Aspergillus fumigatus conidia, were efficiently killed by the system. Two putative end products of the system, hydrogen peroxide and acrolein, both killed C. neoformans at concentrations attainable with the whole system. However, catalase failed to inhibit activity of the whole system, making hydrogen peroxide an unlikely mediator of killing. Although C. albicans blastoconidia and A. fumigatus conidia were not killed by the PAO-polyamine system, germ tube formation by the former, and hyphal growth by the latter, were markedly inhibited. These data establish that the PAO-polyamine system possesses antifungal activity.     

In vitro determination of phagocytosis and intracellular killing of Aspergillus species by mononuclear phagocytes

We investigated phagocytosis and intracellular killing of clinical and environmental isolates of Aspergillus spp. by human monocyte-derived macrophages (MDMs). Serial pathogens such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus were examined with a microbiological assay. Phagocytosis for resting conidia of Aspergillus spp. was similar for all isolates tested. During 30 min of incubation phagocytosis ranged from 49.9% to 85.5% for clinical isolates and from 40.3% to 87.1% for environmental isolates. MDMs killed A. fumigatus, A. flavus and A. terreus conidia after ingestion for 120 min, as shown by a decrease in colony forming units (cfu) count of intracellular fungi. The killing index for all isolates of Aspergillus spp., ranged from 12.1 ± 1.1% to 90.3 ± 10.4%; isolate-dependent (P < 0.01) differences against the fungicidal action of MDMs were observed. In conclusion, significant differences were noted for killing indices between several strains of Aspergillus spp. whereas phagocytosis was similar for all isolates tested in vitro. No differences were observed within environmental and clinical isolates.     

Deletion of the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> lysine biosynthesis gene <Emphasis Type="Italic">lysF</Emphasis> encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and -aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5×10³ conidia per mouse. In this murine model, the lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.     

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.     

Changes in atmospheric CO2 influence the allergenicity of Aspergillus fumigatus

Increased susceptibility to allergies has been documented in the Western world in recent decades. However, a comprehensive understanding of its causes is not yet available. It is therefore essential to understand trends and mechanisms of allergy‐inducing agents, such as fungal conidia. In this study, we investigated the hypothesis that environmental conditions linked to global atmospheric changes can affect the allergenicity of Aspergillus fumigatus, a common allergenic fungal species in indoor and outdoor environments and in airborne particulate matter. We show that fungi grown under present‐day CO₂ levels (392 ppm) exhibit 8.5 and 3.5 fold higher allergenicity compared to fungi grown at preindustrial (280 ppm) and double (560 ppm) CO₂ levels, respectively. A corresponding trend is observed in the expression of genes encoding for known allergenic proteins and in the major allergen Asp f1 concentrations, possibly due to physiological changes such as respiration rates and the nitrogen content of the fungus, influenced by the CO₂ concentrations. Increased carbon and nitrogen levels in the growth medium also lead to a significant increase in the allergenicity. We propose that climatic changes such as increasing atmospheric CO₂ levels and changes in the fungal growth medium may impact the ability of allergenic fungi such as A. fumigatus to induce allergies.