INTRODUCTION

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Aspergillus fumigatus is a saprophytic fungus that plays an essential role in recycling environmental carbon and nitrogen (235, 506, 676). Its natural ecological niche is the soil, wherein it survives and grows on organic debris. Although this species is not the most prevalent fungus in the world, it is one of the most ubiquitous of those with airborne conidia (443, 444, 466). It sporulates abundantly, with every conidial head producing thousands of conidia. The conidia released into the atmosphere have a diameter small enough (2 to 3 µm) to reach the lung alveoli (518, 577). A. fumigatus does not have an elaborate mechanism for releasing its conidia into the air; dissemination simply relies on disturbances of the environment and strong air currents. Once the conidia are in the air, their small size makes them buoyant, tending to keep them airborne both indoors and outdoors. Environmental surveys indicate that all humans will inhale at least several hundred A. fumigatus conidia per day (99, 222, 271). For most patients, therefore, disease occurs predominantly in the lungs, although dissemination to virtually any organ occurs in the most severely predisposed.

Inhalation of conidia by immunocompetent individuals rarely has any adverse effect, since the conidia are eliminated relatively efficiently by innate immune mechanisms. Thus, until recent years, A. fumigatus was viewed as a weak pathogen responsible for allergic forms of the disease, such as farmer's lung, a clinical condition observed among individuals exposed repeatedly to conidia, or aspergilloma, an overgrowth of the fungus on the surface of preexisting cavities in the lungs of patients treated successfully for tuberculosis (169, 341, 500). Because of the increase in the number of immunosuppressed patients, however, and the degree of severity of modern immunosuppressive therapies, the situation has changed dramatically in recent years (114, 556, 572). Over the past 10 years, A. fumigatus has become the most prevalent airborne fungal pathogen, causing severe and usually fatal invasive infections in immunocompromised hosts in developed countries (13, 43, 61, 142, 170, 231). A fourfold increase in invasive aspergillosis (IA) has been observed in the last 12 years. In 1992, IA was responsible for approximately 30% of fungal infections in patients dying of cancer, and it is estimated that IA occurs in 10 to 25% of all leukemia patients, in whom the mortality rate is 80 to 90%, even when treated (59, 140, 141, 231, 682). IA is now a major cause of death at leukemia treatment centers and bone marrow transplantation (BMT) and solid-organ transplantation units (119, 159, 489, 575).

Although A. fumigatus is the most common etiologic agent, being responsible for approximately 90% of human infections (61, 159, 169, 334, 350, 587, 676), it is not the only pathogen in this genus. A. flavus, A. terreus, A. niger, and A. nidulans can also cause human infections. Since A. fumigatus is the most common, however, this review is devoted exclusively to it. Fundamental and clinical aspects of the pathobiology of A. fumigatus infections are presented, with special emphasis on IA. The topics discussed include (i) taxonomic characterization of the species, (ii) clinical and laboratory diagnosis of the disease, (iii) host immune response to the fungus and putative fungal virulence factors, and (iv) antifungal drugs used in treatment.

TAXONOMY OF A. FUMIGATUS

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Species Identification

Culture and morphological characteristics. Identification of A. fumigatus is based predominantly upon the morphology of the conidia and conidiophores. The organism is characterized by green echinulate conidia, 2.5 to 3 µm in diameter, produced in chains basipetally from greenish phialides, 6 to 8 by 2 to 3 µm in size. A few isolates of A. fumigatus are pigmentless and produce white conidia (582). The chains of conidia are borne directly on broadly clavate vesicles (20 to 30 µm in diameter) in the absence of metulae (Fig. 1). No sexual stage is known for this species. A. fumigatus is a fast grower; the colony size can reach 4 ± 1 cm within a week when grown on Czapek-Dox agar at 25°C (518). A. fumigatus is a thermophilic species, with growth occurring at temperatures as high as 55°C and survival maintained at temperatures up to 70°C (235, 341, 518, 577).

View larger version (84K): [in this window] [in a new window]   FIG. 1.   Light microscopy of typical A. fumigatus sporulating structures.

Biochemical and molecular characterizations used in species determination. The need for a better taxonomic definition of the species A. fumigatus and the possible misidentification of a teleomorph stage of A. fumigatus among the Neosartorya species have led to the study of selected biochemical and molecular criteria, in addition to morphological data, as adjuncts to species determination. Biochemical characterizations which have been studied include the detection and identification of secondary metabolites (200), the identification the ubiquinone system (400), and the examination of isoenzyme patterns (369, 400, 543, 554). Molecular data have been obtained on total DNA (105, 218, 501), mitochondrial DNA (mtDNA) (127, 543) or ribosomal DNA (rDNA) (105, 127, 210, 543, 618) by using various methodological approaches, mainly restriction fragment length polymorphisms (RFLP) visualized with or without hybridization to specific probes and sequencing of characteristic DNA regions. Criteria which have been suggested as useful in the identification of A. fumigatus are summarized in Table 1.

Aside from its fundamental interest, intraspecific characterization of this species has potent epidemiological and clinical implications. Since strain typing requires methods that are highly discriminative, reproducible, and independent of growth conditions, phenotypic analysis based on protein patterns detected by antibodies or enzymatic substrates should be discouraged (84, 637). At best, protein patterns can be used to rank strains at a subspecies level. In contrast, genotypic methods are independent of the external milieu. Some of the earlier molecular methods, however, are helpful only for analysis at the subspecies level. RFLP following digestion of total genomic DNA by XbaI, SalI, or XhoI shows a limited degree of discrimination among strains. The complex banding patterns with large numbers of faint bands displayed in ethidium bromide-stained gels are difficult to interpret, and only major bands can be used to designate subspecific clusters (82, 144). Similarly, heterogeneity in the IGS region can be used to group strains of A. fumigatus only at a subspecific level (514, 618).

Only three methods can be used to genotypically type A. fumigatus strains. Two of these methods use PCR, each with different primers and amplification protocols (microsatellite and random amplified polymorphic DNAs [RAPD]), whereas the third uses RFLP visualized after hybridization with a repeated DNA sequence.

RAPD is the method most commonly used to type strains of A. fumigatus (11, 23, 81, 354, 369, 385, 422, 670). To date, the decamer primer R108 (GTATTGCCCT) generated the best strain differentiation (23). However, RAPD patterns are difficult to repeat or interpret due to the low annealing temperature (44, 386, 687). Moreover, the distance of migration scanned is only a few centimeters, and the variability in banding pattern is too limited to make the comparison of a large number of strains feasible. The second PCR-based method involves microsatellites. This method, which has been used to construct the physical map of the human genome, has been successfully applied recently to A. fumigatus (34). The method is rapid and highly reproducible and, in contrast to RAPD, uses unique primers and specific sequences flanking the microsatellite. Four CA repeats have been identified to date: (CA)₉(GA)₂₅, (CA)₂C(CA)₂₃, (CA)₈, and (CA)₂₁.

Hybridization of restriction enzyme fragments with repeated DNA sequences, a method successfully used to type other fungal pathogens, has also been used to type A. fumigatus strains. Screening of a phage library resulted in the isolation of a phage (3.9) which contains a species-specific repeat sequence. Use of this phage as a probe provides unique and highly discriminative Southern blot hybridization patterns for each strain tested (133, 217). The repeat sequence AFUT1, inserted into phage 3.9 and used for strain fingerprinting, is a defective retrotransposon element of 6.9 kb bounded by two long terminal repeats (LTR) of 282 bp (459) (Fig. 2). The 5' and 3' LTRs are not totally homologous, since they have only 90% identity. Moreover, the 5' LTR of another copy of AFUT1, isolated from a different phage (4.11), which cross-hybridizes with 3.9, is 86.5% identical to the 5'LTR of the retrotransposon isolated from the 3.9 phage. A 5-bp duplication site was found at the border of AFUT1. AFUT1 encodes amino acid sequences homologous to the reverse transcriptase, RNase H, and endonuclease encoded by the pol gene of retroviruses.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Schematic representation of the 3.9 probe of A. fumigatus used for molecular studies and typical hybridization patterns obtained with EcoRI-digested total DNA probed with the entire SalI-SalI fragment (A) and EcoRI fragments of the repeated sequence (B to E). The repeated element Afut1 (squares) is an inactive retroelement of 6.9 kb bounded by two LTRs () and with sequences homologous to reverse transcriptase (RT), RNase H, and endonuclease (endo) encoded by the pol genes of retrotransposons.

Comparison of AFUT1 with other fungal and nonfungal LTR retrotransposons showed that AFUT1 has a sequence and organization characteristic of the gypsy family of Drosophila (459). At least 10 copies of the retrotransposon element are found in the genome of A. fumigatus. However, AFUT1 is a defective element; the putative coding domains contain multiple stop codons due exclusively to transitions from C · G to T · A. Such a pattern of nucleotide variation is reminiscent of the repeated-induced point mutation (RIP) in Neurospora repeated sequences. However, no sexual reproduction is known in A. fumigatus, and no methylation of cytosine, an event typically associated with mutations in sequences affected by RIP, was detected (459). This result would suggest that AFUT1 was subjected to RIP at a time when A. fumigatus possessed a functional sexual cycle and an active DNA methylation process. The copies of this repeated sequence found today could be relics of RIP consecutive to and fixed at a time where A. fumigatus had lost its sexual stage.

Although most researchers have used the PCR- and RFLP-based typing methods separately, a study is under way to compare their discriminatory potential and to evaluate if combination of data obtained by more than one typing method will lead to better strain discrimination (369). To date, strain typing has been most successful by using microsatellite polymorphism or analysis of Southern hybridization patterns obtained with repeated DNA sequences.

For most patients, the main portal of entry and site of infection for A. fumigatus is the respiratory tract. Although other sites of infections have been described in the normal or immunocompromised host, such as the skin, peritoneum, kidneys, bones, eyes, and gastrointestinal tract, nonrespiratory infections are infrequent and are not discussed here (142, 169, 341, 383, 511). Pulmonary diseases caused by A. fumigatus can be classified according to the site of the disease within the respiratory tract and the extent of mycelial colonization or invasion, both of which are influenced by the immunological status of the host (61, 169, 341). Allergic diseases, including asthma, allergic sinusitis, and alveolitis, are not covered in this review. They occur following repeated exposure to conidia or antigens of Aspergillus in the absence of mycelial colonization, and in most cases, removal of the patient from the environmental source results in clinical improvement. In contrast, allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and IA, syndromes involving mycelial growth of A. fumigatus in the body, usually require therapeutic intervention. The primary symptoms and diagnostic features of these three forms of aspergillosis are described in the following section.

ABPA is currently the most severe allergic pulmonary complication caused by Aspergillus species. It occurs in patients suffering from atopic asthma or cystic fibrosis. ABPA occurs in approximately 1 to 2% of asthmatic patients (15% of asthmatic patients sensitized to A. fumigatus) and 7 to 35% of cystic fibrosis patients (38, 311, 312, 318, 352, 464). It follows the same course as classic asthma, with a unique cellular immune response and pathophysiologic findings caused by the response of T-cell products (447, 492). Its effects range from asthma to fatal destruction of the lungs with defined clinical, serological, radiological, and pathological features (493, 568).

Clinically, ABPA manifests as a bronchial asthma with transient pulmonary infiltrates that may proceed to proximal bronchiectasis and lung fibrosis (228, 708). It is a very difficult syndrome to diagnose. The criteria classically listed for a definitive diagnosis are the following: asthma, peripheral blood eosinophilia (>1,000 mm³), immediate skin reactivity to A. fumigatus antigenic extracts within 15 ± 5 min, precipitating (immunoglobulin G [IgG] and IgM) and IgE antibodies against A. fumigatus, elevated levels of total IgE in serum (>1 µg/ml), a history of pulmonary infiltrates, and central bronchiectasis. Less importantly, isolation of A. fumigatus from sputum, expectoration of brown plugs containing eosinophils and Charcot-Leyden crystals, and a skin reaction occurring 6 ± 2h after the application of antigen are also used diagnostically (40, 130, 227, 229, 311, 352, 568, 608, 609, 704).

All the above criteria are rarely fulfilled for each patient with ABPA (226, 227, 601, 746). Moreover, most diagnostic features are not specific and, as a consequence of the intermittent course of the disease, not all criteria are fulfilled at the same time (226, 229, 360). Central bronchiectasis is, for example, detected only in the late stages of the disease (492, 493), and the predictive value of several of these criteria, such as radiographic findings, eosinophilia, or observation of precipitins, may depend on the group (cystic fibrosis patients or asthmatic patients without cystic fibrosis) and age of patients studied (275, 276, 285, 438, 569). The ability to diagnose ABPA would be greatly improved by the use of standardized antigens. Efforts in this direction are being pursued (123, 256). The limitations of the diagnosis of ABPA have led to the concept of "silent" ABPA (598). In some cystic fibrosis patients, for example, damage to the respiratory mucosa in response to exposure to Aspergillus conidia occurs even though all of the diagnostic criteria are not met. In untreated patients, ABPA eventually progresses to pulmonary fibrosis and respiratory failure, although some patients have remissions. Obviously, there is a need for improved diagnosis of ABPA and ABPA-related syndromes.

Aspergilloma, commonly referred to as "fungus ball," occurs in preexisting pulmonary cavities that were caused by tuberculosis, sarcoidosis, or other bullous lung disorders and in chronically obstructed paranasal sinuses (280, 307, 341, 731). Historically, in the early 1950s, this syndrome was the classical form of aspergillosis. It still occurs today in 10 to 15% of patients with cavitating lung diseases (3). Aspergilloma consists of a spheroid mass of hyphae embedded in a proteinaceous matrix with sporulating structures at the periphery, all of which are found external to the lining of the cavity, i.e., in the airway. A common symptom of aspergilloma is hemoptysis. Hemoptysis results from the disruption of blood vessels in the wall of the cavity occupied by the fungus or in the bronchial artery supply, centimeters away from the aspergilloma (169). Most frequently, internal bleeding occurs, but hemoptysis may be massive and even fatal (3, 101, 128, 190). Aspergillomas appear on chest radiographs as spherical masses usually surrounded by a radiolucent crescent (40, 75). Marked pleural thickening characteristically occurs. High antibody titers (precipitins) are detected in patients with aspergillomas (137, 158, 245, 334, 653). Patients are usually asymptomatic, and aspergillomas are most often detected on chest radiographs obtained for the evaluation of another pulmonary or allergic disease. Today, an increasing number of aspergillomas occur when a solid lesion of IA erodes to the surface of the lung in an immunocompromised host (169). As patients recover from granulocytopenia, cavitation ensues without pleural thickening, and a concomitant increase in anti-A. fumigatus antibody occurs. Lesions of this type are best demonstrated by computed tomography (CT) scans of the chest. Their existence should be taken into consideration when the underlying disease relapses or worsens, thereby requiring renewed immunosuppressive therapy (474, 535, 551, 558).

IA has become a leading cause of death, mainly among hematology patients. The average incidence of IA is estimated to be 5 to 25% in patients with acute leukemia, 5 to 10% after allogeneic BMT, and 0.5 to 5% after cytotoxic treatment of blood diseases or autologous BMT and solid-organ transplantation. IA which follows solid-organ transplantation is most common in heart-lung transplant patients (19 to 26%) and is found, in decreasing order, in liver, heart, lung, and kidney recipients (1 to 10%) (119, 221, 489, 533, 682, 718). Although IA is recognized today as the main fungal infection in cancer patients, its true incidence is probably underestimated because of the low sensitivity of diagnostic tests (59, 231, 296, 693). IA also occurs in patients with nonhematogenous underlying conditions; it is increasingly reported in AIDS patients (1 to 12%) (80, 145, 305, 384, 451, 454, 605, 700) and is also a common infectious complication of chronic granulomatous disease (CGD) (25 to 40%) (142, 208). In contrast, it is rarely found in immunocompetent hosts (300).

Four types of IA have been described (142, 682): (i) acute or chronic pulmonary aspergillosis, the most common form of IA; (ii) tracheobronchitis and obstructive bronchial disease with various degrees of invasion of the mucosa and cartilage as well as pseudomembrane formation, seen predominantly in AIDS patients (145, 305, 454); (iii) acute invasive rhinosinusitis (173, 431, 584, 691, 714); and (iv) disseminated disease commonly involving the brain (10 to 40% in BMT patients) and other organs (for example, the skin, kidneys, heart, and eyes) (59, 481, 532, 729). Clinical features of the different types of IA depend on the organ localization listed above and the underlying disease. These features have been reviewed recently (142, 682) and are not detailed here. However, the diagnostic procedures currently available for IA and their associated problems are discussed. IA remains difficult to diagnose even today, particularly when it is in the early stages. Consensus has not been reached regarding the most appropriate diagnostic criteria for IA. In fact, to prove IA, one must provide histopathological evidence of mycelial growth in tissue. Unfortunately, this is most often demonstrated only at autopsy (64, 231, 682). Moreover, since the hyphae of other filamentous fungi such as Fusarium or Pseudollescheria spp. may resemble Aspergillus spp., definitive identification may require immunohistochemical staining or in situ hybridization techniques (292, 303, 429, 487). Since there is no consensus regarding the criteria used to establish a diagnosis of IA, the terms "highly probable," "probable," "possible," or "suspected" are often used to define IA cases, and definitions vary from study to study.

Features currently considered in the diagnosis of IA include (i) a positive CT scan, (ii) culture and/or microscopic evidence of disease, and (iii) detection of Aspergillus antigen(s) in serum. Clinical symptoms are usually too nonspecific to be helpful in narrowing the focus to IA.

As with other forms of aspergillosis, the general symptoms of IA, primarily fever, chest pain, cough, malaise, weight loss, and dyspnea, are variable and nonspecific. The presence of a fever of >38.5°C that is unresponsive to antibacterial therapy, previously recognized as the hallmark for initiating antifungal treatment, is no longer applicable, since corticosteroid-treated patients with IA frequently do not have elevated temperatures (532, 583).

A positive CT scan may be the first definitive suggestion of IA. CT scanning is more sensitive than radiography and shows the extent and number of lesions (75). In the early stages of the infection, CT scans may reveal specific signs of an infection, such as the typical "halo" resulting from hemorrhagic necrosis surrounding the fungal lesion or pleura-based lesions (86, 142, 214, 273, 319, 373-375, 682). Radiographic appearances of pulmonary IA are very heterogenous and can vary from single or multifocal nodules, with and without cavitation, to widespread and large infiltrates which are often bilateral. In nonpulmonary forms of the disease, e.g., rhinosinusitis or cerebral aspergillosis, a CT scan can indicate the extent of the disease and whether bone invasion has occurred. CT scanning can be used in conjunction with brain magnetic resonance imaging in patients with cerebral aspergillosis (532).

The use of culture or microscopic examination of respiratory tract specimens has been criticized because of the presence of airborne conidia of Aspergillus and the possibility that a positive culture from such specimens results from accidental contamination (80, 166, 192, 295, 480). The presence of A. fumigatus in clinical samples from patients at risk for IA is, however, highly suggestive of an infection, a conclusion which is supported by a careful statistical reassessment of published data (99, 230, 453, 657, 743, 745). In patients with leukemia and BMT for example, microscopic examinations and/or cultures are positive in 50 to 100% of bronchoalveolar lavage fluid (BAL) samples from patients who have definitive or probable aspergillosis (4) and the positive predictive value of a sputum culture in neutropenic or BMT patients has been reported to be >70% (270). The results obtained with BAL samples and sputum samples vary from study to study, however, and some patients have a positive sputum culture and a negative BAL culture or vice versa (270, 453, 743). In some, but not all studies, cultures from nasal swabs of patients were positive repeatedly for Aspergillus spp. (4, 6, 399). Bronchoscopy may also provide a suitable specimen for culture, since there is a trend to accept a positive culture from normally sterile sites as a definitive diagnosis for IA (142, 367). Percutaneous lung biopsy specimens or aspirated specimens obtained with radiological or ultrasound guidance, as well as BAL samples, are the specimens of choice. However, since the patient is often quite debilitated, invasive procedures in neutropenic patients demand careful consideration and cannot be repeated.

The recent development of a capture enzyme-linked immunosorbent assay (ELISA) which measures the presence of serum antigens is both sensitive and specific for the diagnosis of IA (345). More information on this topic is given in the next section.

Predisposing factors must be taken into account when assessing the risk of acquiring IA. Because of the difficulty in diagnosing IA and because of its rapid progression (1 to 2 weeks from onset to death) and severity, clinicians often treat the patient empirically rather than waiting for the diagnosis to be established. Moreover, waiting until the diagnosis is confirmed subjects the patient to a greater risk of untreatable IA, since the fungal burden might reach a level too high for antifungal therapy. The extent and duration of neutropenia correlate well with the risk of developing IA. Thus, profound (polymorphonuclear leukocytes [PMN] = 500 mm³ and especially 100 mm³) and prolonged (>12 to 15 days) neutropenia are associated with the greatest increased risk for pulmonary IA (140-142, 215). Cytomegalovirus infection is also a risk factor for IA in lung transplant recipients but not in BMT patients (85, 274, 532, 696). A major risk factor for all transplant patients is corticosteroid therapy, usually linked to graft-versus-host (GVH) disease and/or rejection in transplantation (696). However, the precise concentration of steroids, as either daily or cumulative doses, associated with the risk of acquisition of IA has not been identified (472, 483). Recently, Ribaud et al. (532) showed that GVH disease and a dose of prednisolone of >1 mg/kg/day for 4 weeks preceding a diagnosis of IA were poor prognostic indicators. A prednisolone dose of 1 mg/kg/day was also noted to be critical for kidney transplant patients to acquire IA (234). In summary, patients at greatest risk for developing IA include (i) allogeneic BMT recipients with prolonged neutropenia or under corticosteroid treatment for GVH disease, (ii) autologous BMT or solid-organ transplant recipients who have been neutropenic for >2 weeks, (iii) patients with acute leukemia and lymphomas undergoing intense chemotherapy, (iv) patients with aplastic anemias and prolonged neutropenia that is nonchemically induced, (v) patients with previously documented aspergillosis subjected to a new chemotherapy regimen or a BMT, (vi) patients with functional neutrophil deficits such as those seen in chronic granulomatous disease (CGD), and (vii) patients with advanced human immunodeficiency virus disease (119, 384, 415, 416, 532, 534, 572, 585, 694, 716, 723, 729).

The risk factors listed above and the severity of the infection illustrate the need for new and prospective methods to diagnose IA. The establishment of such a definition is made difficult by the limited knowledge of the natural history of the disease. For example, the median time to the development of IA is shorter in acute leukemia patients undergoing chemotherapy than in BMT patients, in whom IA occurs in 2 to 3 months after transplantation but often with a bimodal symptomatic distribution at 2 to 3 weeks and again 2 to 3 months after transplantation (403, 472, 532, 696). The occurrence of the disease at intervals between 1 week and 2 years after the start of immunosuppression suggests a different pathogenesis, which, in turn, may require the use of different diagnostic strategies. Comprehensive studies which show the relationships among the four criteria mentioned above (general symptoms, CT scanning, culture, and antigenemia) and the underlying disease and immunosuppressive treatment are urgently needed. Improvement in diagnosis should also lead to better management of IA. Of patients at risk for IA, 80% have fever, 40% have fever with pulmonary infiltrates, 25% are treated empirically, and only 6% are definitively diagnosed as having IA (602). A combination of diagnostic strategies is currently being evaluated. For example, when antigen is detected, the disease can be confirmed by performing a CT scan of the lungs and sinuses and radionuclide imaging with ¹¹¹In-labeled human IgG (602). Compared to the classical strategy for diagnosis (fever refractive to antibacterial agents and the presence of pulmonary infiltrates on chest radiograph), the alternative strategy mentioned above would significantly reduce the number of patients receiving empirical therapy. Finally, the establishment of accurate diagnostic criteria for early symptoms of IA would also lead to a better outcome, since several studies in the last 10 years have shown that reducing the time to obtain a definitive diagnosis was associated with a better prognosis (86, 684, 695).

ANTIGENS AND LABORATORY DIAGNOSIS

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Antigens

The antigenic properties of A. fumigatus extracts have been recognized for a long time and served as the basis for the early development of immunological assays used in the serological diagnosis of aspergillosis in the immunocompetent host (54, 656). Unfortunately, there are qualitative and quantitative differences in the composition of antigenic extracts prepared in various laboratories and even between batches in the same laboratory (244). In this section, the reasons for the antigenic variability and the most recent approaches to the production of pure antigens of A. fumigatus are discussed.

Variability in extracts of A. fumigatus does not appear to be related to strain or growth temperature, since the same antigenic pattern has been observed with multiple strains and at both 25 and 37°C (345), but a major source of variability clearly involves other conditions of culture. In particular, the incubation period, the conditions under which the cultures are held, and the composition of the culture medium are critical. There is no standard period of incubation; published periods of incubation have ranged from 1 or 2 days at 25°C with agitation to 5 weeks at 37°C under stationary conditions (244, 334, 376). Different antigenic patterns are produced when the organism is cultured in a defined medium such as Czapek-Dox medium and when it is cultured in a protein hydrolysate medium such as Sabouraud medium. Moreover, the presence of high concentrations of hexose in both media induces an acidic pH during growth and greatly influences the pattern of antigens produced (345, 351, 371, 436). The best complex antigenic preparations are obtained during active fungal growth (1 day at 37°C) in media without sugar but with a single protein substrate or a protein hydrolysate (345, 351). The composition of such a medium appears to be closer to the nutritional environment encountered by the fungus in the lungs, i.e., a protein-rich environment composed primarily of collagen and elastin with a pH close to 7.4.

Other factors that affect antigenic composition include the form of the fungus from which the antigenic mixture is extracted; the method of extraction, including the choice of reagents; and the subcellular source of the antigens (345, 351). With respect to fungal form, although conidial and mycelial (intracellular and extracellular) extracts contain a large number of identical immunologically reactive molecules, multiple qualitative and quantitative differences in their composition can be demonstrated (302, 512, 647). Procedurally, mild extractions, such as a short incubation of intact mycelium in a saline buffer in the presence or absence of a detergent, results in the extraction of loosely associated cell wall components (253, 727). In contrast, cell disruption techniques allow the recovery of all water-soluble mycelial glycoproteins, proteins, and polysaccharides (249, 351). Under these conditions, the choice of disruption buffer is critical as well, in that, e.g., a citrate buffer at pH 4 will solublize different antigens from an ammonium bicarbonate buffer at pH 8. Further, different antigen patterns appear from culture filtrates depending on how the antigenic components are concentrated (351).

In addition to the complications surrounding the extraction of antigens when aspergilli are cultured in vitro under different conditions, there is some evidence that the antigens expressed in vivo during colonization of host tissues are different from those expressed in vitro (83, 156, 581). Since the quantification of antibodies directed specifically against antigens produced in the lung matrix would increase the predictive values of immunological tests, more work must be done in this area.

About 100 proteins or glycoproteins from A. fumigatus can bind human Ig, as determined by Western blotting techniques performed after one- or two-dimensional electrophoresis (19, 22, 39, 45, 50, 77, 84, 250, 251, 254, 349, 380, 381, 505, 578, 702). Initially, Western blotting was thought to be the answer to serodiagnostic problems in aspergillosis. Unfortunately, most if not all of these studies have added to the confusion surrounding the antigenic makeup of the fungus for the following reason. The antigenic molecules noted in most of these studies were characterized solely on the basis of molecular mass, which is insufficient to identify an antigen. Consideration must be given to the function of the protein and identification of the encoding gene.

Two examples are illustrative of the problems encountered when studying antigens based only on their molecular mass as identified in Western blots. Three antigens which cannot be separated easily by one-dimensional electrophoresis has been identified in the 90-kDa region, i.e., a catalase (90 kDa), a dipeptidyl peptidase (always present in vitro as a protein doublet of 87 and 88 kDa, each with different levels of glycosylation), and an 88-kDa heat shock protein (41, 83, 89). Biochemical and molecular characterization has been the only way to differentiate these proteins. The second example concerns antigen 7 of Harvey and Longbottom (239). A recent molecular characterization of a homologue of this protein in A. nidulans (87) and the use of monospecific antisera (346), as well as careful analysis of previous publications, suggest that the antigens identified as p60, p40, and p37 (380, 381), AspfII (26, 27), gp55 (648), 41 and 53 kDa of CS2 (90, 504), 58 kDa (197), 35 to 65 kDa (333, 338), and GP66 (372) are probably the same protein. Differences in molecular mass can be attributed to the extent of phosphorylation and glycosylation, both of which can alter the size as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behavior during chromatographic purification. Unfortunately, the isolation of these proteins from different laboratories, using antigenic preparations from A. fumigatus cultured and purified under different conditions, has confounded the identification of these proteins. Confirmation of identity of antigens with different molecular masses is possible only from protein sequence analysis. Because of this, considerable effort has been expended in the last few years to isolate and characterize to the molecular level polypeptide antigens responsible for specific antibody responses.

Two strategies have been used for molecular characterizations of antigens from A. fumigatus. The predominant strategy for antigens shown to be reactive by immunoblotting has been biochemical purification followed by cloning of the structural gene and sequencing (41, 89). A second strategy, however, involves the use of expression libraries to isolate clones which express antigens recognized by patient sera (20, 27, 123, 124, 324). A problem with the latter strategy, however, is that the antigen(s) identified will be highly dependent upon the culture conditions used, since the cDNA library constructed will reflect high-copy-numbers mRNAs expressed by the fungus under those particular culture conditions. By using this strategy, unexpected antigens have been identified, most of them with molecular masses below 40 kDa (123). Only about a dozen of the hundreds of A. fumigatus antigenic (glyco)proteins reported in the literature have been characterized at a molecular and biochemical level. They are summarized in Table 2. The most comprehensively characterized antigens, which include an RNase, a catalase, and a dipeptidylpeptidase and the galactomannan, are described in the following section.

The catalase of A. fumigatus that has been characterized extensively is a tetrameric protein with a monomeric subunit of 90 kDa (89, 252, 381, 382, 599). The oligomeric subunit contains an N-linked sugar moiety of 7 kDa which bears no antigen epitopes. The protein is remarkably stable, being relatively insensitive to high temperatures, as well as to reducing and denaturing agents. The structural gene for the protein, CAT1, has been cloned and sequenced (89). Analysis of the deduced amino acid sequence shows that CAT1 has both a signal peptide of 15 amino acids and a propeptide of 12 amino acids, with a pair of basic amino acids Arg26-Arg27 acting as a cleavage signal for a KEX2-like endopeptidase. Comparison of the CAT1 sequence with other catalase genes suggests conservation of the tripeptide His102, Ser141, and Asn175, which is involved in the binding of proteins to its heme prosthetic group.

The dipeptidylpeptidase V has also been characterized recently (41, 313). It releases mainly X-Ala dipeptides and also His-Ser and Ser-Tyr from the N terminus of polypeptides (41). It has a molecular mass of 79 kDa and a signal peptide of 18 amino acids. These data are in agreement with the biochemical data showing that the protein migrates as a doublet of 87 and 88 kDa and contains approximately 9 kDa of N-linked carbohydrate. The biochemical properties, as well as its exocellular localization, indicate that it is an enzyme belonging to a new class of dipeptidylpeptidases (DPPV). Comparison of the A. fumigatus DPPV sequence with those of other DPPs shows the presence of a Gly558-X-Ser560-X-Gly562 consensus motif of serine hydrolases with a putative catalytic triad of the DPP arranged as Ser560 Asp643 His675. This protein has no chymotrypsin activity, but it has been referred to as a chymotrypsin antigen (also known as Ag13 or AgC) on the basis of its reactivity resulting in the release of naphthol radicals from a precipitin band when placed in the presence of the chromogenic substrate N-acetyl-phenylalanine naphthyl ester (55, 240, 655).

The RNase of A. fumigatus is composed of 149 amino acids with a 27-amino-acid leader sequence and a putative active site composed of the six amino acids His49, Glu95, Phe96, Pro98, Arg120, and His136. This RNase cleaves a single phosphodiester bond in a highly conserved region and releases a 300- to 400-base fragment from the 3' end of the large rRNA (736). It is also known as ASPF1, Ag3, or restrictocin (erroneously named from "A. restrictus," since a taxonomical reexamination of the strains used in all recent studies has shown that they are indeed true A. fumigatus strains) (19, 343, 349, 377, 434, 735).

Galactomannan (GM) isolated from cell wall or culture filtrates by a variety of different purification methods has been analyzed (25, 28, 32, 33, 46, 248, 348, 418, 525, 671). It is the only polysaccharide antigen characterized from A. fumigatus. Although data differ slightly, a consensus structure has been established: the mannan core has a linear configuration containing (1-2)- and (1-6)-linked residues in a ratio of 3:1, and the antigenic, acid-labile side chains, branched on two (1-2)-linked mannose residues, are composed of (1-5) galactofuranosyl residues with an average degree of polymerization of 4 (Fig. 3). Numerous intra- and exocellular glycoproteins of A. fumigatus with molecular masses of >40 kDa have this galactofuran epitope as well (348). The type of glycosylation involved in the linkage of GM to proteins has not been studied.

View larger version (32K): [in this window] [in a new window]   FIG. 3.   Schematic representation of steps involved in the development of a diagnostic test for the detection of antigen in the biological fluids of patients with IA, using GM as an example.

Serological testing for the detection of antibodies to Aspergillus antigens can be very helpful in the diagnosis of aspergilloma or ABPA, the two forms of aspergillosis observed in immunocompetent individuals. Although growth of the fungus in association with tissue is limited in both of these syndromes, a strong humoral response to the organism frequently occurs (158, 334, 345). Of the more than 20 diagnostic procedures that have been developed to detect anti-Aspergillus antibodies, double immunodiffusion and counterimmunoelectrophoresis are the most commonly used in the clinical laboratory (245). These two methods are simple, cheap, easy to perform, and sufficiently insensitive to virtually eliminate false-positive results occurring as a result of the low levels of anti-Aspergillus antibodies present in most healthy individuals (246, 345). Historically, these procedures resulted in the discovery of the two major precipitins, the catalase and the dipeptidylpeptidase (the chymotrypsin), which are still used in the serodiagnosis of aspergillosis in immunocompetent hosts (54, 55, 655, 656). The primary disadvantages of the methods are an inability to quantitate the immune response, and lack of standardization due to the use of crude Aspergillus extracts (244).

Immunoassays with A. fumigatus antigens purified by biochemical procedures have only recently been reported (313, 335, 348, 435, 727). In addition to the difficulty in producing large quantities of pure antigens from in vitro cultures, a minor contamination of even <1% of the antigen of interest with another antigen of greater reactivity may lead to erroneous results (434). To avoid such problems, it is now possible to use molecular biological techniques to produce pure recombinant antigens. For example, proteins of A. fumigatus have been produced in Escherichia coli or Pichia pastoris (41, 88, 89, 434, 435, 607). The P. pastoris expression system can yield large quantities of secreted, glycosylated A. fumigatus proteins (0.1 to 0.2 mg/ml) (41, 89). Recombinant antigens of A. fumigatus reported in the literature (Table 2) are comparable in their antigenicity to the native molecules (41, 88, 89, 123, 126, 256, 433, 435). Such antigens serve as the basis for the development of ELISA methods which will allow the quantitation of the antibody response (245, 246, 313, 349, 727). Studies to select a single antigen or a mixture of antigens that will not only identify the type of aspergillosis but will also have prognostic significance are under way (168, 256, 434, 652). Quantitation of Ig isotypes as well as understanding of the kinetics of the antibody response over the course of the disease will be useful in this regard, since most healthy individuals already have anti-A. fumigatus antibodies as the direct result of continuous environmental exposure (278, 301, 309, 339, 349, 376, 660, 679). Since titers in healthy individuals are normally low, infection can be correlated with a rise in specific antibodies. However, selected individuals may have quite high titers owing to occupational exposure or to an underlying disease such as cystic fibrosis, making diagnosis of an infection difficult (123, 256).

Circulating antigens. In contrast to immunocompetent hosts, growth of A. fumigatus in the tissues of an immunosuppressed host is not correlated with an increase in anti-Aspergillus antibody titers. In fact, the presence of anti-Aspergillus antibody in immunocompromised individuals is more likely to represent antibody formed before the onset of immunosuppressive therapy rather than as a result of invasive infection. Contradictory data in this regard may be linked to the regimen of immunosuppression used in patient populations (30, 86, 155, 197, 250, 299, 402, 491, 652, 728, 741). An increase in antibody titer at the end of immunosuppression is indicative of recovery from IA, whereas absence of an antibody titer or declining antibody levels suggest a poor prognosis. Thus, antibody detection can be used prognostically but not diagnostically for IA. In fact, the serological diagnosis of IA is based on the detection of circulating antigens in biological fluids, e.g., serum, urine, and BAL fluid, obtained from patients (345). Although the presence of antigens in the serum of patients with IA was first reported in 1979, the number of different antigens identified in the serum or urine remains small (Table 3).

Detection of DNA in specimens. In addition to the detection in body fluids of polysaccharide or protein components of the fungus, it might be possible to develop ultrasensitive PCR-based techniques for the detection of A. fumigatus DNA. The data presented in Table 5 support this possibility. Initial studies focused on detection of DNA in BAL samples. Confirmed cases of IA were always associated with a positive PCR test (35, 73, 407, 617, 643). When using PCR, however, extreme care must be taken to avoid false-positive or false-negative results. False-negative results can be monitored by the use of competitive PCR. However, false-positive results are more difficult to control. Since conidia are often present in the air, false-positive results can be generated by the transient presence of aspergilli in the respiratory tract. In fact, up to 25% of BAL samples from healthy subjects are positive by PCR tests (35). In addition, PCR results and GM detection from BAL samples are not congruent (686). Moreover, the number of false-positive samples was higher for PCR assays with BAL samples than for ELISA (73, 628, 630, 688). Recently, very promising PCR results were obtained with serum or plasma (72, 184, 672, 733).

ARE THERE VIRULENCE FACTORS IN A. FUMIGATUS?

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Strategies

The ideal test for identifying a virulence factor is to compare the infectivity of the fungus in the absence or presence of the factor. Such comparisons have been performed in the past by using naturally occurring mutants or those obtained by UV or chemical mutagenesis (314, 315). The major drawback of these approaches in a fungal species without a sexual stage such as A. fumigatus is that the mutant strain may be deficient in more than just the factor being studied. The use of such mutants could lead to an erroneous conclusion about the putative role of the factor studied, as, for example, the proteases (see below).

Molecular biological techniques make it possible to avoid such problems by cloning and disrupting the gene encoding for the putative virulence factor studied. Moreover, the expression of the factor in a heterologous host makes it possible to study its effect in the absence of possible contaminants from the fungus itself, which can occur during any biochemical purification.

Several strategies are available to produce single or multiple mutants of A. fumigatus and are summarized in Fig. 4. The classic method involves the disruption of the gene of interest by the insertion of an antibiotic resistance gene. To date, only two genes, one conferring resistance to hygromycin and one conferring resistance to phleomycin, have been used (401, 513). They are placed under the control of either the GPD promotor or the TRP C terminator of A. nidulans or the promotor and terminator of the gene subjected to disruption (427, 486). Disruption is usually made in a nitrate reductase-deficient background to take into account the possibility of external contamination. However, these systems can lead to only two successive mutations (286). To compensate for this disadvantage, a PYRG blaster has been developed recently in our laboratory (138). This system is very similar to the URA blaster previously developed in Saccharomyces cerevisiae and Candida albicans (194). The system consists of the A. niger PYRG gene flanked by a direct repeat that encodes the neomycin phosphotransferase of Tn5. The PYRG cassette is inserted by gene replacement following transformation of a uridine/uracil-auxotrophic PYRG strain. Recombination is selected in the presence of 5-fluoroorotic acid, which results in the excision of the A. niger PYRG gene, producing A. fumigatus uridine/uracil auxotrophs which have retained their mutant phenotype because of the persistence of one of the two elements of the direct repeat at the site of insertion of the PYRG blaster. Selection for uridine/uracil prototrophy can be used again to disrupt another gene. Transformation can be performed with protoplasts or by electroporation (78, 486, 717).

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Strategies currently available to disrupt genes in A. fumigatus (A) and to create a bank of A. fumigatus mutants by using the restriction enzyme-mediated integration strategy (B). E1 and E2 are endonucleases favoring a single-copy integration of the marker in the A. fumigatus genome. wt, wild type; OMP, orotidine-5'-phosphate; 5FOA, 5-fluoroorotic acid.

Another possible approach to understanding virulence in A. fumigatus is the construction of libraries of mutants through random insertional mutagenesis (78, 79, 258, 717). Conditions for restriction enzyme-mediated integration (REMI) have been recently published; XhoI or KpnI digestion was used to obtain a single-copy integration of transforming DNA with the majority of the transformants (78, 266). The signature-tagged mutagenesis approach developed for bacteria has also been applied recently to A. fumigatus (79, 266). Mutations which will render strains avirulent will allow for the cloning of the virulence genes disrupted by the mutagenesis.

The second essential tool, in addition to gene disruption techniques, in the identification of a virulence factor is an appropriate animal model in which to test virulence in vivo. Invasive pulmonary aspergillosis has been established in mice, rabbits, rats, guinea pigs, chickens, cows, turkeys, ducks, and monkeys (96, 171, 289, 291, 293, 392, 507, 536, 674). Originally, the animal models were developed to study the efficacy of antifungal drugs in the treatment of aspergillosis (8, 14, 196, 224, 495, 596, 673) or to evaluate diagnostic methods (177, 178, 497, 540, 720). There is no consensus about the best model to use. Indeed, a survey of the literature reveals that there is variation among researchers not only with respect to the choice of an animal (strain, weight, and sex) and immunosuppressive regimen (dose, products, frequency of the injections) but also with respect to the challenge protocol (concentration of conidia and route of injection). In spite of the heterogeneity in the animal models used, however, several conclusions can be drawn.

(i) As with other fungal pathogens, there is a direct relationship between dosage of conidia and lethality. The weight of the animal is critical as well; for any given species, heavier animals require larger dosages of conidia to establish disease (116, 171).

(ii) Immunosuppressive treatments substantially increase the susceptibility of animals to infection,