Abstract

The colonization of airways by filamentous fungi and the development of respiratory infections require some predisposing factors as encountered in patients with cystic fibrosis (CF). Indeed, the defective mucociliary clearance which characterizes the disease is associated with local immunological disorders. In addition, the prolonged therapy with antibiotics and the use of corticosteroid treatments also facilitate fungal growth. An important fungal biota has been described in respiratory secretions of patients suffering from CF. Aspergillus fumigatus, Scedosporium apiospermum and Aspergillus terreus for filamentous fungi and Candida albicans for yeasts are the main fungal species associated with CF. Although less common, several fungal species including Aspergillus flavus and Aspergillus nidulans may be isolated transiently from CF respiratory secretions, while others such as Exophiala dermatitidis and Scedosporium prolificans may chronically colonize the airways. Moreover, some of them like Penicillium emersonii and Acrophialophora fusispora are encountered in humans almost exclusively in the context of CF. As fungal complications in CF patients are essentially caused by filamentous fungi the present review will not include works related to yeasts. In CF patients, fungi may sometimes be responsible for deterioration of lung function, as occurs in allergic broncho-pulmonary aspergillosis (ABPA) which is the most common fungal disease in this context. Additionally, although the clinical relevance of the fungal airway colonization is still a matter of debate, filamentous fungi may contribute to the local inflammatory response, and therefore to the progressive deterioration of the lung function.

Introduction

Cystic fibrosis (CF) is the major genetic disease in the European Caucasian population and, by the number of patients (about 6,500), the third orphan disease in frequency in France 1. It results from mutations in the gene CFTR (Cystic Fibrosis Transmembrane conductance Regulator), which is located on chromosome 7 and encodes a chloride channel involved in electrolytic exchanges through the plasma membrane of numerous epithelial cell types. While several organs are involved, morbidity and mortality in CF patients is mainly dependent on the severity of lesions of the respiratory tract 2. Mutations in CFTR gene result in a defective mucociliary clearance and, as a consequence, production of thick and sticky bronchial mucus facilitates the entrapment of the airborne bacteria and fungal spores and provides a suitable environment for the growth of these microorganisms.

Bacteria, particularly Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia, are typical CF pathogens that cause recurrent exacerbations of the pulmonary disease and often determine the vital prognosis of patients 3. However, the respiratory tract of the patients may also be colonized by filamentous fungi. Most of the epidemiological studies that have been conducted in CF populations deal with Aspergillus fumigatus, but the fungal biota is obviously more complex. Among clinically significant fungi, Aspergillus spp., Scedosporium apiospermum and Exophiala dermatitidis are the most frequent molds recovered from respiratory secretions of CF patients, but their isolation rate greatly varies according to the studies 4, 5. Moreover, Nagano et al. 4 reported an increasing prevalence of fungi, from 6% in 1995 to 13% in 2005, being isolated from sputum samples of CF patients in the USA. In addition, over the past decade, some cases of infection or chronic colonization have been associated with species that have only rarely or never been previously reported in human cases, such as Penicillium emersonii and Acrophialophora fusispora. Such changes in the epidemiology of fungal species involved in CF are probably related to the improvement in global disease management, which has led to a marked increase in life expectancy of the patients 1. Nevertheless, they may also result from the improvement of the procedures used for examination of sputum samples in clinical laboratories. In this review, the various filamentous fungi associated with CF will be presented, with a particular emphasis on Scedosporium species.

Aspergillus spp.

Aspergillus fumigatus

Aspergillus fumigatus is by far the major agent involved in the colonization of the airways of CF patients. As illustrated in Table 1, mycological examination of respiratory secretions from CF patients in previous studies revealed a prevalence rate ranging from 16 to 56.7% 6–21.

Table 1

Prevalence rates for Aspergillus fumigatus and ABPA in cystic fibrosis

References Country (total no. of patients) A. fumigatus positive patients (%) ABPA positive patients (%) 
Nelson et al. 6 USA (46) 21/37 (56.7) 5 (10.8) 
Laufer et al. 7 USA (100) 5/55 (9) 10 (10) 
Bauernfeind et al. 8 Germany (102) 6 (5.9) Not specified 
Simmonds et al. 9 UK (137) Not specified 8 (5.8) 
Marchant et al. 10 UK (160) Not specified 11 (6.8) 
Mroueh and Spock 11 USA (236) 60 (25.4) 15 (6.4) 
Becker et al. 12 USA (49) 8 (16) 1 (1.9) 
Milla et al. 13 USA (212) 45 (21.2) Not specified 
Hutcheson et al. 14 USA (118) Not specified 6 (5.1) 
Cimon et al. 15 France (210) 45 (21.4) 2 (0.95) 
Cimon et al. 16 France (128) 59 (46.1) 5 (3.9) 
Skov et al. 17 Denmark (238) 61 (25.6) 26 (10.9) 
Bakare et al. 18 Germany (94) 43 (45.7) Not specified 
Taccetti et al. 19 Italy (3089) Not specified 191 (6.18) 
Skov et al. 20 Australia (270) 22 in 1998 (7.4) 13 (4.7) 
  52 in 2002 (18.8)  
Valenza et al. 21 Germany (60) 35 (58.3) Not specified 
References Country (total no. of patients) A. fumigatus positive patients (%) ABPA positive patients (%) 
Nelson et al. 6 USA (46) 21/37 (56.7) 5 (10.8) 
Laufer et al. 7 USA (100) 5/55 (9) 10 (10) 
Bauernfeind et al. 8 Germany (102) 6 (5.9) Not specified 
Simmonds et al. 9 UK (137) Not specified 8 (5.8) 
Marchant et al. 10 UK (160) Not specified 11 (6.8) 
Mroueh and Spock 11 USA (236) 60 (25.4) 15 (6.4) 
Becker et al. 12 USA (49) 8 (16) 1 (1.9) 
Milla et al. 13 USA (212) 45 (21.2) Not specified 
Hutcheson et al. 14 USA (118) Not specified 6 (5.1) 
Cimon et al. 15 France (210) 45 (21.4) 2 (0.95) 
Cimon et al. 16 France (128) 59 (46.1) 5 (3.9) 
Skov et al. 17 Denmark (238) 61 (25.6) 26 (10.9) 
Bakare et al. 18 Germany (94) 43 (45.7) Not specified 
Taccetti et al. 19 Italy (3089) Not specified 191 (6.18) 
Skov et al. 20 Australia (270) 22 in 1998 (7.4) 13 (4.7) 
  52 in 2002 (18.8)  
Valenza et al. 21 Germany (60) 35 (58.3) Not specified 

Interestingly, colonization of the airways by A. fumigatus is uncommon in young children and usually follows bacterial respiratory infections. In an epidemiological study conducted in collaboration between our two CF centers (Angers and Giens), mean age of the patients at date of first isolation of A. fumigatus and Scedosporium apiospermum from respiratory secretions was about 12.3 and 14.1 years, respectively. In contrast, the first isolation occurred at 5.4 and 8.1 years for S. aureus and P. aeruginosa, respectively 15. Bronchopulmonary epithelial tissue damage caused by the bacterial proteases or the leukocyte elastase released during the inflammatory reaction were therefore suggested to be required for adherence of the airborne fungal spores to the host tissues. The resulting exposition of the subepithelial extracellular matrix proteins laminin and fibronectin or the fibrinogen/fibrin deposits formed at the surface of wounded epithelia would mediate the adherence process 22. In addition to laminin and fibronectin, receptors have been identified at the surface of A. fumigatus conidia 23, 24. Furthermore, several studies showed that filamentous fungi may also be responsible for direct damage of the respiratory mucosa that is similar to those produced by bacteria 15. Indeed, A. fumigatus produces some secondary metabolites and proteolytic enzymes that are able to impair the mucociliary clearance or to inhibit phagocytosis. For example, it has been shown that A. fumigatus alkaline protease may cause the detachment of A549 cells, a cell line with characteristics of pneumocytes type II 25.

In the context of CF, A. fumigatus may be responsible for various diseases including asthma, bronchitis and aspergilloma. Moreover, this fungus may also cause invasive pulmonary infections after lung transplantation 26, 27. However, the main clinical form caused by A. fumigatus in patients with CF remains allergic broncho-pulmonary aspergillosis (ABPA) 28, for which the prevalence rate varies from 0.95 to 10.9% depending on the studies (Table 1). These variations are due in large part to differences in the definition of ABPA since some of the criteria initially proposed for its diagnosis were not always considered in these studies. Therefore, it may be useful to consider revisions of the definitions which have remained unchanged since an international conference held in Bethesda, USA, in June 2001 29. The established diagnosis included acute or subacute clinical deterioration not attributable to another etiology, associated with (i) an elevated total serum IgE titer, (ii) an immediate cutaneous reactivity to A. fumigatus or the presence of specific serum IgE, and (iii) the presence of specific serum IgG, or recent signs on chest radiography or tomodensitometry of infiltrates or bronchiectasis that are not cured by antibiotics.

Additionally, recent genetic or immunological studies of the host provided new insights into the pathogenesis of ABPA. It has been demonstrated that some HLA-DR molecules. i.e., DR2, DR5, and possibly DR4 or DR7 contribute to susceptibility, while HLA-DQ2 contributes to resistance, and that a combination of these genetic events determines the outcome of ABPA in patients with CF 30. Moreover, the presence of single-strand polymorphisms in the interleukin 4 receptor α chain, in particular the substitution I75V, appears to be a genetic risk for the development of ABPA 31. Likewise, it has been suggested that polymorphisms in the promoter region of the interleukin 10 gene may influence the CF host response to A. fumigatus32. In this study of a large number of CF patients, a significant relationship was seen between the GG genotype at position –1082 in the promoter region of this gene and both ABPA and colonization of the airways by A. fumigatus.

But genotype studies of the isolates collected from patients with CF have also provided interesting information about the colonization of the airways. Indeed, genotype study of multiple and sequential isolates recovered from patients recently colonized by A. fumigatus, performed by sequence-specific DNA primer and by random amplification of polymorphic DNA (RAPD) using the primers NS3 and NS7, revealed a huge diversity of genotypes even in the same sputum sample 33. However, as colonization continues, a genotype common to the chronically colonized patients tended to become dominant. Moreover, this genotype was associated with the presence of anti-catalase antibodies which have been suggested as markers of an altered lung function 34. Interestingly, similar observations were reported by Neuvéglise et al. 35 using restriction fragment length polymorphism (RFLP) followed by hybridization with the repetitive probe Afut1, a retrotransposon-like element from A. fumigatus. A great variety of genotypes were detected in sputum samples collected at the beginning of the colonization of the airways, whereas a dominant genotype was observed in later samples. More recently, 256 isolates recovered from 89 respiratory samples, obtained from eight CF patients from Belgium and France, were genotyped using a highly discriminant microsatellite-based typing method 36. Although different genotypes were identified in 88.3% of the patients, recurrent genotypes for seven out of the eight CF patients were isolated on several occasions more than five months apart. These results indicate that some genotypes are capable of chronically colonizing the airways. Thus, it seems that some particular genotypes are selected with prolonged colonization, but the mechanisms of this genotype selection have not been investigated.

Other Aspergillus species

Other Aspergillus species may be encountered in the context of CF. For instance, Aspergillus flavus, Aspergillus niger and Aspergillus nidulans are commonly reported, but are usually found transiently. Conversely, Aspergillus terreus is usually responsible for the chronic colonization of the airways of CF patients. In our experience, this fungus ranked third among filamentous fungi colonizing the airways of CF patients, with a prevalence rate ranging from 1.9 to 6.2% 15, 16. Moreover, A. terreus was responsible in a few cases for clinical signs, including ABPA and IA which are similar to those reported for A. fumigatus.

Little is known about the epidemiology of this fungus. Aspergillus terreus is seldom reported from environmental sources, contrasting with its occurrence in CF. Therefore, additional studies should be conducted to improve our knowledge on the habitat of the fungus and on the influence of human activities which might, in turn, contribute to prophylactic measures. In the framework of an epidemiological study performed at the CF clinic of the Hospital Renée Sabran (Giens, France), potential sources of fungal contamination were investigated by analysis of air and surface samples from patient rooms, and soil samples from the park adjacent to the hospital. Aspergillus terreus was detected exclusively from soil samples 37. Moreover, environmental isolates were typed by random amplification of polymorphic DNA (RAPD), using the two-primer set NS3 and NS7 according to Symoens et al. 38, and compared to clinical isolates from CF patients followed in this hospital during the study period 37. As expected, the three patients studied were colonized by a unique genotype, different from one patient to another, that persisted during the study period. The clonality of the colonization pattern suggests real colonization of CF patients by A. terreus, rather than a transient occurrence of airborne spores entrapped in the viscous mucus. However, no genotype was found to be common to clinical and environmental isolates. Considering the usual chronicity of the colonization of the airways by this fungus, a hospital-acquired infection seems to be unlikely and additional environmental studies should be conducted, particularly at the homes of the patients.

Scedosporium apiospermum

Usually a saprophyte, S. apiospermum (formerly proposed as the anamorph state of the Ascomycete Pseudallescheria boydii), may cause various mycoses as a result of traumatic or iatrogenic inoculation of its telluric conidia (for a review see reference 39). Respiratory diseases, likely due to the inhalation of airborne conidia, may also occur in receptive hosts. The diseases associated with this fungus are sinusitis, lung fungus-ball and necrotizing pneumonia, as well as disseminated infections in immunocompromised patients 39. However, all these clinical pictures are rare, contrasting with the relatively high occurrence of the fungus in CF patients.

Cystic fibrosis was first described as a risk factor for airway colonization by S. apiospermum, following the concomitant isolation of this fungus from three patients after the eradication of A. fumigatus by itraconazole treatment 40. This risk factor was further demonstrated in an epidemiological survey, conducted in collaboration between Angers and Giens hospitals (France). First, 210 patients with CF were investigated by means of mycological examination of a single sputum sample and by serological analysis 15. Then, a longitudinal study was performed on 128 patients who were followed for five years 41. Mycological results from the two studies were compared to clinical data, and to other biological data (including results from bacteriological analysis of sputum samples, blood eosinophil count, total IgE level and presence of specific anti-A. fumigatus IgE). In both studies, sputum analyses were conducted using the same procedures. The latter included the inoculation of a defined volume of the sample on two yeast extract-peptone-dextrose-agar (YPDA) plates, one of which was supplemented with cycloheximide. Plates were incubated at 37°C, and cultures were examined daily for seven days, even if some rapidly growing fungi were recovered. As shown in Table 2, S. apiospermum ranked the second most common filamentous fungus associated with CF, with a prevalence rate of 8.6% in our longitudinal survey 41. This high occurrence in CF cases was later confirmed in two distinct surveys performed in Australia and Austria, in which prevalence rates of 10% 42 and 6.5% 43, respectively were reported. The recovery of S. apiospermum from respiratory secretions was found to usually follow the colonization of the airways by A. fumigatus. Scedosporium apiospermum was found with A. fumigatus in ten out of the eleven positive patients, and emerged subsequently, after an average delay of 14 months in nine patients. Considering the frequency of this association, it is obvious that the use of a semi-selective culture medium such as YPDA +cycloheximide greatly facilitates the detection of S. apiospermum. However, this procedure might be improved, for example by the use of other semi-selective culture media such as dichloran-rose bengal-chloramphenicol agar supplemented with benomyl. Likewise, another semi-selective culture medium, called SceSel+, has been described recently 44. It has been shown to facilitate the isolation of S. apiospermum from environmental sources, and its potential value in mycological analyses of sputum specimens should be evaluated.

Table 2

The main filamentous fungi responsible for colonization of the airways in our experience

 Prevalence rate (%) 
Fungal species Transversal study [15] (210 patients) Longitudinal study [41] (128 patients) 
Aspergillus fumigatus 21.4 46.1 
Scedosporium apiospermum 3.3 8.6 
Aspergillus terreus 1.9 6.2 
 Prevalence rate (%) 
Fungal species Transversal study [15] (210 patients) Longitudinal study [41] (128 patients) 
Aspergillus fumigatus 21.4 46.1 
Scedosporium apiospermum 3.3 8.6 
Aspergillus terreus 1.9 6.2 

Several questions are raised by the relatively high occurrence of S. apiospermum in CF patients. First, what is the origin of the fungus recovered from the patients. Indeed, S. apiospermum has been described mainly in highly polluted soils and water 45. Several studies showed the capacity of this fungus to use various aromatic compounds as a carbon source 46, 47. However, apart from these substrates, S. apiospermum has rarely been encountered in the environment. When Beguin and Nolard 48 investigated the air and surfaces in 130 dwellings in Brussels (Belgium) over a 10-year period, 52 fungal genera were recovered and among them, the genus Scedosporium ranked the 49th most common. Additionally, the fungus was not found on surfaces, and it was recovered from the air only once, representing only 1 colony forming unit (CFU) out of a total number of about 20,000 CFUs 48. An environmental study was therefore performed at the homes of six CF patients colonized by S. apiospermum, and followed in Angers University Hospital 49. Air and surfaces were sampled in the patient's bedroom, the bathroom and living room. In addition, soil from the garden, as well as from all potted plants present at home of the patients, were sampled. Thus, a total number of 164 samples were analyzed. Among the air and surface samples, only one from a radiator in the bedroom of one patient revealed the presence of S. apiospermum. In contrast, out of 55 potted plants that were sampled, S. apiospermum was isolated from 36 of these samples. The fungus was recovered at homes of each patient, and almost all positive samples were highly contaminated. These results, which are in agreement with those from Summerbell et al. 50 who reported several years ago the presence of the fungus in the soil of potted plants, clearly indicate that potted plants constitute a risk factor of airway colonization by S. apiospermum. Therefore, the presence of potted plants at home of CF patients should be avoided.

In addition, the discrepancy between the relatively high occurrence of S. apiospermum in CF and its paucity in the environment raises questions about the mechanisms by which it is acquired by patients. Obviously, this fungus expresses specific virulence factors which distinguish it from other more common airborne moulds. However, we have only limited knowledge about the pathogenic mechanisms of S. apiospermum. Regarding the adherence step, it was demonstrated that conidia of S. apiospermum attached to, and were internalized by HEp 2 cells through a lectin-mediated process involving a peptido-rhamnomannan of the fungal wall 51. Likewise, several extracellular peptidases have been identified in S. apiospermum, including a 28-kDa pepdidase which is fully inhibited by 1,10-phenanthroline (a potent zinc-metallopeptidase inhibitor). The pepdidase may cleave different protein substrates, such as extracellular matrix proteins and disialylated proteins 52, 53. Similarly, the release of siderophores, which are essential for iron uptake and therefore for growth of all microorganisms, has been suggested by the growth of the fungus in culture on casamino acids agar 54. However, none of these compounds has been characterized as yet, and only two putative virulence factors have been currently purified and fully characterized in S. apiospermum. These factors include an extracellular proteinase belonging to the subtilisin subfamily of serine-proteinase, and very close to the alkaline proteinase produced by A. fumigatus55, and a copper-zinc cytosolic superoxide dismutase 56.

One of the most important questions for physicians involved in the follow-up of CF patients remains the clinical significance of the isolation of this fungus from respiratory secretions. As demonstrated in a genotype study performed on multiple and sequential isolates recovered from long-term colonized patients, S. apiospermum is usually responsible for a chronic colonization of the airways. Each patient was colonized by a unique genotype different from one patient to another, and conserved over time despite antifungal treatments 57. Usually, this chronic colonization of the airways is not associated with clinical signs. However, Cimon et al. 41 showed that S. apiospermum may cause allergic broncho-pulmonary mycoses similar to the well known ABPA. Indeed, an allergic broncho-pulmonary mycosis was suspected for 6 out of the 128 patients followed up in our longitudinal study, and one of them presented episodes of bronchospasm associated with high counts of blood eosinophils and with high titers of total serum IgE. Specific anti-A. fumigatus IgE were not detected, and mycological examination of successive sputum samples revealed exclusive growth of S. apiospermum, suggesting its involvement in the clinical pattern. Moreover, S. apiospermum may be also responsible for cerebral or disseminated mycoses in severely immunocompromised patients, such as lung transplant recipients 39.

Additionally, even in the absence of true respiratory infection, we can't disregard the chronic colonization of the airways by this fungus. Indeed, S. apiospermum undoubtedly contributes to the inflammatory reaction and the progressive deterioration of the lung function by secreting proteases and releasing polysaccharide and protein antigens. In addition, since the fungus is able to cause disseminated human infections and responds poorly to antifungal treatment, it would be advisable to critically evaluate in CF patients, especially those that have undergone lung transplantation 58, 59. This is highlighted by the clinical case recently reported by Symoens et al. 60, which occurred in 1999 in a 26-year old female CF patient followed in Brussels. In April of that year, the individual underwent a double lung transplant due to the major alteration of the lung function of the patient. The lungs had been colonized for several years by S. apiospermum. She was prophylactically treated with antibacterial and antifungal agents, the latter consisting of oral itraconazole associated with aerosols of amphotericin B. However, four weeks later, the patient presented a bilateral chorioretinitis and subcutaneous nodules. Direct examination of the clinical samples (vitreal fluid and biopsies of the subcutaneous lesions) revealed the presence of septate hyphae, and cultures yielded numerous colonies of S. apiospermum. An antifungal treatment was therefore initiated which consisted of daily doses of voriconazole, associated with intra-ocular injections of miconazole for two months which lead to a cure of the lesions. Miconazole was therefore stopped, but voriconazole was maintained for an additional four months. However, two days after total cessation of therapy, the patient developed a subacute meningitis and S. apiospermum was recovered from the cerebro-spinal fluid. Voriconazole therapy was started again, but the patient died three weeks later. Unfortunately, isolates from the cerebro-spinal fluid were not preserved. Available isolates were analysed by RAPD using the three-primer set described by Zouhair et al. 61. Genotyping revealed that the isolates from vitreal fluid were identical to those from sputum samples collected a few days after transplantation, as well as to isolates obtained from two sputum samples collected 18 months and the day before the transplantation, respectively.

Furthermore, probably as a result of local immunological disorders which characterize CF, dissemination of the fungus from the respiratory tract may also occur in those with severe immunodepression. For instance, a multifocal spondylitis was reported recently in a 28-year-old male CF patient. This patient was colonized by S. apiospermum since 1993 and presented with diabetes (a common complication of CF) diagnosed in 2003 as the sole risk factor for invasive fungal infection 62. In 2002, a left pleurisy was diagnosed, which was attributed to the fungus because of its isolation from sputum, and an antifungal therapy was started. However, in December 2004, the patient who was still on voriconazole treatment, began to complain of inflammatory back pain. Four months later, a spondylitis was diagnosed by X-ray of the back and discal magnetic resonance imaging. Three vertebral biopsies were carried out, but bacteriological and mycological examination was negative. An empiric antibiotic treatment was therefore started, but it was stopped one month later because of the progression of the disease. A surgical biopsy was then carried out, which permitted the recovery of S. apiospermum. Additionally, serum samples from the patient were analyzed retrospectively by counter-immunoelectrophoresis (CIE) which revealed 9–11 precipitin lines using a somatic extract (personal data).

Finally, recent studies performed by the group of Josep Guarro in Reus, Spain, have demonstrated that P. boydii is a complex of species. Sixty clinical or environmental isolates from different countries, including 16 isolates from CF patients from France, were studied by Gilgado et al. 63. On the basis of morphological and sequencing data (partial sequence of beta-tubulin and calmodulin genes, and sequence of the internal transcribed spacer regions), five clades were identified. The study resulted in the proposal of two of the clades as new species, i.e., Pseudallescheria minutispora (exclusively for environmental samples) and Scedosporium aurantiacum. More recently, on the basis of physiological tests such as resistance to cycloheximide and assimilation of various carbohydrate sources, the three other clades were proposed as new species, namely Scedosporium dehoogii, Scedosporium apiospermum sensu stricto, and Scedosporium boydii which corresponds to the anamorph of P. boydii64, 65. Interestingly, most of the isolates from CF patients were identified as S. apiospermum sensu stricto or as the new combination S. boydii, but two isolates from French patients were found to be S. aurantiacum. Further studies are therefore needed to define the respective prevalence of these new species in CF, and their clinical significance.

Other filamentous fungi

The respiratory tract of patients with CF may also be colonized transiently by other filamentous fungi such as Alternaria spp., Cladosporium spp., Penicillium spp. or Paecilomyces variotii4, 15. Scedosporium prolificans (formerly S. inflatum) and Exophiala dermatitidis (formerly Wangiella dermatitidis) have also been reported in the context of CF (Fig. 1), sometimes with a high frequency, but strikingly these species seem to be restricted to some geographic areas. Additionally, two thermophilic species, Penicillium emersonii (the anamorph state of Talaromyces emersonii) and Acrophialophora fusispora, have been isolated in human, almost exclusively in the context of CF.

Fig. 1

Two examples of mixed cultures from sputum samples of CF patients incubated at 37°C, showing the simultaneous growth of Candida albicans (green colonies), Candida glabrata (purple colonies), Aspergillus fumigatus (arrows) and Exophiala dermatitidis (black colonies) on CHROMagar Candida from Becton-Dickinson (A) and of Scedosporium apiospermum (pale grey velvety colonies) and Scedosporium prolificans (dark brown colonies) on in-house-prepared dichloran-rose bengal-chloramphenicol agar supplemented with benomyl (B). This figure is reproduced in colour in Medical Mycology online.

Fig. 1

Two examples of mixed cultures from sputum samples of CF patients incubated at 37°C, showing the simultaneous growth of Candida albicans (green colonies), Candida glabrata (purple colonies), Aspergillus fumigatus (arrows) and Exophiala dermatitidis (black colonies) on CHROMagar Candida from Becton-Dickinson (A) and of Scedosporium apiospermum (pale grey velvety colonies) and Scedosporium prolificans (dark brown colonies) on in-house-prepared dichloran-rose bengal-chloramphenicol agar supplemented with benomyl (B). This figure is reproduced in colour in Medical Mycology online.

Scedosporium prolificans

Conversely to S. apiospermum, S. prolificans is known principally as a causative agent of disseminated infections in patients with hematological malignancies 66. Only a few reports, mainly from Australia, Spain and the USA, deal with its isolation from respiratory secretions of CF patients 42, 67–71.

Moreover, its pathogenic role in this context remains questionable, since only one case of endophtalmitis after lung transplantation has been reported today in CF 72.

Exophiala dermatitidis

Although distributed worldwide, E. dermatitidis which grows as a black yeast at 37°C, and as a filamentous fungus at room temperature, has been described in the context of CF mainly from German patients 73–77. Its prevalence rate in Germany and Belgium, which varies from 4.8–15.7% 73–78, contrasts with its very low occurrence in other countries like France or North America. As it has been suggested for the colonization of the airways by P. aeruginosa79, genetic factors may account for this discrepancy. However, it may also result from differences in the way of life between the two countries, for example the use of sauna facilities since this fungus can survive for many months in hot and moist conditions 80. Nevertheless, this discrepancy may also reflect the lack of standardization of mycological examination of respiratory secretions from CF patients, since detection of this fungus may be difficult with routine isolation procedures. A prolonged incubation time (up to 4 weeks) seems to be needed for isolation of this usually slow growing fungus 73–75. Moreover, due to its frequent association with yeasts and other filamentous fungi as illustrated in Fig. 1, as well as with bacteria such as P. aeruginosa, the use of a selective medium such as erythritol-chloramphenicol agar (ECA) is highly recommended for its isolation 75. Besides, P. aeruginosa which produces toxic compounds, such as pyocyanin and 1-hydroxyphenazine capable of inhibiting the fungal growth and therefore to hamper the recovery of filamentous fungi 81, 82, was not detected on ECA 75.

In the context of CF, E. dermatitidis may be responsible for a transient or chronic colonization of the airways, but usually without any clinical signs. However, this fungus may also cause true respiratory infections, or invasive fungal infections 73, 76. Additionally, E. dermatitidis, as well as species of the genus Aspergillus and Scedosporium, isolated from respiratory secretions of patients with bronchiectasis, should be considered as a biomarker of a CFTR-related disease. For example, the regular isolation of E. dermatitidis from sputum samples of a 60-year-old female patient, who presented since several years bronchiectasis with recurrent episodes of bronchial infections, led us to search for an atypical form of CF. This was confirmed by sequencing of the gene CFTR, which revealed minor point mutations for both alleles. Moreover, CIE performed with a somatic antigenic extract of the fungus showed a serological response, with several precipitin lines (Bouchara et al., unpublished data).

Penicillium emersonii

Apart from Penicillium marneffei, species of the genus Penicillium are usually considered as contaminating moulds. However, some like Penicillium emersonii may chronically colonize the airways of CF patients. This thermophilic telluric fungus, which is characterized by its biverticillate asymmetrical penicilli and its cylindrical to ellipsoidal conidia, was first reported associated with humans in 1999. A case involving this fungus in a CF patient has been reported since this date 83. P. emersonii was isolated repeatedly for nearly 5 years, suggesting that it was continuously present in the airways during this period. In addition, a progressive sensitization of the patient to the fungus was revealed by Western-blotting. However, since colonization does not appear to contribute to the development of disease, it seems difficult to assign a pathogenic role to this fungus.

Acrophialophora fusispora

Acrophialophora fusispora is also a thermotolerant soil fungus which is rarely encountered in clinical samples. Only eight cases have been reported to date involving three cases of keratitis 84, 85, three pulmonary infections 86 in non-immunocompromised adults and one brain abscess in a child with acute lymphoblastic leukemia 87. In CF, a unique case was described, corresponding to a transient colonization in a Spanish patient aged 11 84. However, during the last few years, the fungus was detected in a 26-year-old patient followed in the South of France, as well as in three patients investigated at Necker University Hospital in Paris 88. In these observations, the detection of the fungus in respiratory secretions always followed the colonization of the airways by A. fumigatus. Two of the patients were chronically colonized by A. fusispora. However, for all patients, the clinical status remained unchanged, and particularly no clinical signs of bronchopulmonary infections were observed during colonization by this fungus.

The prevalence of A. fusispora in CF is therefore very low, but it may be underestimated due to the currently poor knowledge of this fungus in most of laboratories of medical mycology. For instance, misidentifications have been reported with other fungi, such as Scopulariopsis chartarum89 or Paecilomyces species 87, but also with S. prolificans90, 91. However, the morphological features of A. fusispora are different since it produces on YPDA plates buff colonies of 5–6 cm in diameter at day 7. Microscopically, the fungus presents basally inflated phialides with an elongated neck, arising mostly singly on pale brown vegetative hyphae. These conidiogenous cells produce long chains of pale brown limoniform or fusiform single-celled conidia with ornamentations arranged in spiral bands which may be visualized by interferential phase-contrast microscopy.

Conclusion

As illustrated in Fig. 2, a large variety of filamentous fungi may be encountered in CF patients. However, apart from A. fumigatus, the real prevalence of the different species associated with CF is certainly underestimated and their pathogenic role needs to be defined.

Fig. 2

Filamentous fungi associated with CF according to their frequency in this clinical context and to their capacity to chronically colonize the airways.

Fig. 2

Filamentous fungi associated with CF according to their frequency in this clinical context and to their capacity to chronically colonize the airways.

Large multicenter studies are therefore needed in order to determine the prevalence rates of the different filamentous fungi which may colonize the respiratory tract of these patients, the possible variations in their geographic distribution and their clinical significance. This implies the improvement of the biological diagnosis. Particularly, guidelines for mycological examination of sputum samples from patients with CF should be defined, including the number and type of culture media to be used, as well as the temperature and duration of incubation. Alternatively, molecular tools allowing both detection and direct identification of these fungi should be developed. Finally, environmental studies designed to identify the reservoirs of these fungi, and thus the origin of contamination of the patients, should be promoted. To reach these objectives, a working group on Fungal respiratory infections in cystic fibrosis was approved by the ISHAM council on October 2006.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Author notes

All authors are members of the ISHAM Working group on Fungal respiratory infections in cystic fibrosis, and MP, BC, DC, LD, FS and JPB are members of the ECMM/ISHAM Working group on Pseudallescheria/Scedosporium infections