Immunodiagnosis of Aspergillosis · IMMUNODIAGNOSIS OF ASPERGILLOSIS 441 allergic rhinitis is basedonclinical history andIgE-mediated skin reactivity to appropriate aeroallergens

CLINICAL MICROBIOLOGY REVIEWS, Oct. 1991, p. 439-4560893-8512/91/040439-18$02.00/0Copyright C 1991, American Society for Microbiology

Immunodiagnosis of AspergillosisVISWANATH P. KURUP* AND ANOOPA KUMAR

Allergy-Immunology Division, Department of Medicine, Medical College of Wisconsin, and Research Service,Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295-1000

INTRODUCTION ............................................................. 439

CLASSIFICATION OF DISEASES CAUSED BY ASPERGILLUS SPECIES .....................................439

CHARACTERISTIC FEATURES OF ASPERGILLUS-INDUCED DISEASES....................................440

Saprophytic Colonization ............................................................. 440

Bronchopulmonary colonization ............................................................. 440

Aspergilloma ............................................................. 440

Allergic Diseases ............................................................. 440

Allergic asthma............................................................. 440

Allergic rhinitis............................................................. 440

Allergic sinusitis............................................................. 441

HP............................................................. 441

ABPA ............................................................. 441

(i) Clinical presentation of ABPA............................................................. 441

(ii) Diagnostic criteria of ABPA ............................................................. 441

Systemic Disease ............................................................. 442

IA ............................................................. 442

IMMUNOLOGY OF ASPERGILLOSIS ............................................................. 443

Humoral Immune Response............................................................. 443

Cellular Immune Response ............................................................. 443

ETIOLOGY AND EPIDEMIOLOGY ............................................................. 443

Etiology of Aspergillosis............................................................. 443

Epidemiology of Aspergillosis............................................................. 444

IMMUNODIAGNOSIS ............................................................. 444

Antigens ............................................................. 444

Antigen preparation............................................................. 444

Fractionation and purification ............................................................. 445

Serological Tests ............................................................. 447

ID............................................................. 447

Immunogold assay ............................................................. 447

RIA............................................................. 448

ELISA and BALISA ............................................................. 448

Immunoblotting ............................................................. 448

Skin Tests............................................................. 449

Detection of Circulating Antigens for Diagnosis of IA ............................................................. 449

CONCLUSION............................................................. 450

ACKNOWLEDGMENTS..............................................................450

REFERENCES ............................................................. 450

INTRODUCTION

Members of the genus Aspergillus are fungi ubiquitouslydistributed from the Arctic region to the tropics sparing no

materials or objects. The term aspergillus was first describedby Micheli in 1729 (149), and the first description of a humandisease caused by aspergilli was made in 1847 (212). Theaspergilli have become increasingly important since then andhave been implicated as the causative agents of a number ofdiseases in humans and other animals. The aspergilli are

considered opportunistic pathogens, and a variety of under-lying conditions including impaired immune status contrib-ute to disease production. In addition, however, the durationand quantity of exposure to the organism are importantdeterminants of host response, and selected forms of as-

* Corresponding author.

pergilloses have been detected in apparently healthy sub-jects. The number of Aspergillus conidia in the air and onvarious substrates apparently influences the development ofvarious clinical forms of aspergillosis.

CLASSIFICATION OF DISEASES CAUSED BYASPERGILLUS SPECIES

Aspergillus species have been associated with a widespectrum of diseases in humans and other animals. Coloni-zation and exposure to antigens in normal hosts can lead to

allergic diseases ranging from asthma to allergic broncho-pulmonary aspergillosis (ABPA). Aspergillomas may de-

velop in paranasal sinuses or in preexisting pulmonarycavities. Invasive aspergillosis (IA) is primarily an infection

of immunocompromised patients, but severe infection can

also occur in patients with chronic diseases such as diabetes,alcoholism, and cancer. IA may also develop in patients as a

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440 KURUP AND KUMAR

postoperative or posttraumatic complication or as a super-infection following antibiotic therapy or the insertion ofprosthetic devices or indwelling catheters. Although neutro-penia is the most common predisposing factor, infectionshave been reported in completely normal individuals, insome instances, following exposure to exceptionally highconcentrations of Aspergillus conidia (1, 93, 151).

In most patients, the clinical entities are distinct; in a fewpatients, however, two or more entities may coexist, orthere may be progression from one to the other. Hence, nosingle classification of Aspergillus-induced diseases is en-

tirely satisfactory. However, these diseases can be broadlyclassified according to the following clinical conditions:saprophytic colonization, including saprophytic bronchopul-monary aspergillosis and pulmonary aspergilloma; allergicdiseases, including allergic asthma, allergic rhinitis, allergicsinusitis, hypersensitivity pneumonitis (HP), and ABPA,and systemic disease, i.e., IA.

CHARACTERISTIC FEATURES OF ASPERGILLUS-INDUCED DISEASES

Saprophytic Colonization

Bronchopulmonary colonization. Bronchopulmonary colo-nization refers to organism infestation of airways withoutevidence of direct tissue damage. The most important fac-tors predisposing to colonization of the respiratory tract withaspergilli or other fungi include chronic destructive pulmo-nary disease, prolonged use of broad-spectrum antibiotics,malnutrition, and debilitation. A high incidence of sapro-

phytic colonization has also been demonstrated in patientswith bronchial asthma, bronchiectasis, chronic bronchitis,cystic fibrosis, and primary ciliary dyskinesia syndrome (19,20). Some of these patients mount an immunologic responsemanifested by serum precipitins, immediate skin reactivity,and an Arthus skin reaction to Aspergillus antigen. Althoughaspergilli have been isolated from the respiratory tracts ofsuch patients, no direct correlation with underlying pulmo-nary disease could be made. Thus, saprophytic colonizationin its usual and uncomplicated form is not an infection in thestrict sense. However, its presence could adversely affectthe immune responsiveness in a host and may interfere withthe diagnosis of various disease entities (21).

Aspergilloma. Aspergilloma, also known as mycetoma or

fungus ball, is a saprophytic manifestation of a fungusgrowing in a preformed and poorly drained lung space. In1938, Deve coined the term aspergilloma to describe themycetomalike lesion caused by Aspergillus species (48). Anintrathoracic cavity, whether bronchial, parenchymatous, or

pleural, can become colonized by Aspergillus species if itcommunicates with the bronchial tree. Aspergillus speciesmay grow in the preformed spaces in the lung for months or

years without invading the wall or other tissues. Manypulmonary diseases favor the development of aspergillomas.The most common antecedent diseases are pulmonary tuber-culosis with healed cavities and chronic pulmonary sarcoi-dosis with cystic spaces (31, 90). Fungal balls may alsodevelop in the presence of active pulmonary tuberculosis or

in ectatic bronchi with accompanying ABPA (187, 232).Other antecedent diseases include ankylosing spondylitis,histoplasmosis, lung abscess, bronchiectasis, lung tumors,radiation fibrosis, pneumoconiosis, and pulmonary infarc-tion. Diagnosis usually occurs in the fifth or sixth decade oflife. The most common symptom associated with as-

pergilloma is recurrent hemoptysis. The cause is uncertain,

but hemoptysis has been ascribed to intracavitary growth,mechanical friction due to mycetomal movement, and atrypsinlike proteolytic enzyme produced by aspergilli (39,232). A chronic cough with the production of purulentsputum and generalized symptoms of fever, weight loss, andmalaise are common in patients who are atopic. Patientswith aspergilloma and concomitant ABPA may also demon-strate wheezing and tightness of chest.Pulmonary aspergillomas can often be diagnosed by their

characteristic appearance in roentgenographs (167). Thecharacteristic sign is an opacity occupying some or most ofa round or ovoid cavity that is partially surrounded by acrescent-shaped patch of air. Aspergillomas most often arelocated in the upper lobes or the superior segment of lowerlobes. Although they tend to be solitary, bilateral andmultiple aspergillomas have been described (35, 165). In theearly stage of the disease, the presence of pleural thickeningoverlying the involved pulmonary parenchyma may suggestthe development of Aspergillus superinfection. Additionalradiographic findings may include proximal lobar shrinkage,bronchiectasis, and extensive patchy infiltrate contiguous tothe cavity or enlargement of the cavity by the fungus ball.

Allergic Diseases

Allergic asthma. Asthma is a chronic disease characterizedby increased responsiveness of the tracheobronchial tree tovarious stimuli, particularly following exposure to specificallergens. Fungal spore-induced asthma may cause chronicand even severe respiratory symptoms such that patientsrequire inhaled or alternate-day oral corticosteroids. Mold-associated asthma may be more prevalent in children than inadults in some geographical areas. A number of fungi,including Aspergillus species, have been implicated in aller-gic asthma. Exposure to fungal conidia results in nasalsymptoms like those of a cold followed by coughing andwheezing. The development of asthma is slow and persistentin mold-sensitive patients. The symptoms due to moldasthma can be differentiated from those attributable to otherallergen-induced asthmas by a history of exposure, skin testreactivity, or in vitro demonstration of specific immunoglob-ulin (immunoglobulin E [IgE]) antibodies in patient sera.Many patients with allergic asthma exhibit a biphasic re-sponse to inhalation challenge with appropriate antigens(166). However, the lack of sufficiently pure and reliableallergenic extracts poses a major problem in the develop-ment of in vivo and in vitro tests for diagnosis (256).

Allergic rhinitis. The classical forms of nasal allergy, hayfever and allergic rhinitis, may develop in susceptible indi-viduals on exposure to mold spores and mycelia (34). Aller-gic rhinitis develops as a result of interaction of allergens andIgE fixed to specific mast cells in the epithelial surface of thenasal mucosa. This results in the release of a number ofchemical mediators, including histamine, eosinophil andother chemotactic factors, leukotrienes, and prostaglandins,which contribute to the pathogenesis of the disease (53, 65,94, 210). Allergic rhinitis can occur as a seasonal or anonseasonal disease. In addition to Aspergillus species, avariety of other fungi can also act as causative agents ofallergic rhinitis. The symptoms and signs of fungus-inducedallergic rhinitis are usually indistinguishable from thosecaused by the inhalation of pollen, dust, danders, and insectallergens. The clinical symptoms may include persistent orintermittent nasal obstruction, nasal discharge, pruritis, andeye irritation. On subsequent exposures to fungal spores,sneezing and nocturnal cough may develop. The diagnosis of

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allergic rhinitis is based on clinical history and IgE-mediatedskin reactivity to appropriate aeroallergens. In vitro testssuch as the radioallergosorbent test and paper radioimmu-nosorbent test are useful in the diagnosis (34, 53, 65, 94, 210,218, 256).

Allergic sinusitis. Sinus disease due to fungi includesseveral clinicopathological syndromes, including fungal ballproduction and allergic fungal sinusitis (32, 94, 137). Thepathophysiology of allergic sinusitis differs considerablyfrom that of fungal mycetoma. Allergic fungal sinusitis,which occurs in atopic individuals, is chronic and affectsmultiple sinuses without tissue invasion. Several fungi havebeen implicated in allergic fungal sinusitis. The most impor-tant ones are Aspergillus, Curvularia, Alternaria, and Bipo-laris species (32, 63, 94, 137). Most affected individuals areyoung and atopic, and nasal polyposis, asthma, and rhinitisare the frequently noted symptoms (63). Mucoid impaction("allergic mucin"), resulting in plugs containing fragmentsof the fungi similar to those seen in the sputum plugs ofpatients with ABPA, is a consistently noticed feature inindividuals with allergic sinusitis (94). The sinus exudatemay contain eosinophils, Charcot-Leyden crystals, epithe-lial cells, cellular debris, and abundant fungal hyphae. Ra-diological examination may show progressive thickening andclouding of several or all paranasal sinuses. The patientsmay show immediate and late cutaneous reactivity, precip-itating antibodies to an appropriate fungal antigen, andmarkedly elevated levels of total and specific serum IgG andIgE antibodies (24, 32).HP. HP, or extrinsic allergic alveolitis, is caused by the

exposure of susceptible individuals to a number of organ-isms. Included in this disease group are farmer's lung,bagassosis, and mushroom worker's lung, which resultfrom occupational exposure to thermophilic actinomycetespresent in hay, bagasse, and mushroom compost, respec-tively (94). Contaminated heating or air-conditioning sys-tems in residential and commercial buildings may result inventilation system-induced pneumonitis (111). These dis-eases result mostly from the inhalation of actinomycetesbelonging to the genera Faenia and Thermoactinomyces.Among the fungi, Aspergillus and Penicillium species arecommonly associated with HP (111). The mode of onset ofHP may be acute or insidious and probably is dependent onthe intensity and duration of exposure to the responsibleantigen. The acute form of HP may result from intermittentexposure to an antigenic source. Sensitization occurs follow-ing inhalation of antigens, and on subsequent exposures thepatient develops characteristic symptoms usually 4 to 8 hafter inhalation. The symptoms persist for up to 12 h andrecovery is spontaneous. The recurrence occurs each timethe patient is exposed to the antigens, and there are asymp-tomatic periods between exposures. In the untreated chronicform of HP, irreversible lung damage frequently occurs.Continuous exposure to the antigens may result in intenseimmunological inflammatory reaction in the lung. Lungbiopsies often reveal interstitial fibrosis with granulomaformation and alveolar wall thickening due to infiltration oflymphocytes, plasma cells, and eosinophils. The most char-acteristic feature of HP is the universal presence of serumantibodies against offending antigens (94, 111). The complex-ity and cross-reactivity of antigens and the variable immu-nological responses of patients to these antigens are themajor limitations in the diagnosis of HP.ABPA. Early reports of Aspergillus disorders, possibly

ABPA and characterized by severe bronchitis accompaniedby asthmatic symptoms, were given by Popoff in 1887 (175)

and Hamman in 1927 (73). The first definitive case of ABPAwas reported by Hinson et al. from the United Kingdom in1952 (84). Originally considered a rarity, ABPA is currentlyrecognized with much greater frequency. Since the report ofHinson et al., many cases have been reported from all overthe world, and considerable information on the clinicopatho-logic features has been obtained. ABPA with varied clinicalpresentations has been reported to occur in 20% of asthmaticpatients admitted to hospitals and in 5% of all rhinitis cases,while the incidence in patients with cystic fibrosis may varyfrom 10 to 25% (21, 208). Nevertheless, the onset of ABPAcan be traced to early childhood or even infancy, and thedisease may remain undiagnosed for years or decades (67).Thus, the actual incidence of ABPA seems to be muchhigher than is indicated from the reported number of cases.ABPA is the result of hypersensitivity to Aspergillus

antigens in patients with long-standing atopic asthma. ABPAusually presents as an acute, easily reversible asthmaticsyndrome that progresses to a more intractable asthmaticstate with transient pulmonary infiltrates and a strikingsensitivity to Aspergillus antigen. Subsequently, a chronicstage with admixture of reversible and irreversible obstruc-tive lung diseases emerges (138).

(i) Clinical presentation of ABPA. With few exceptions,patients with ABPA are atopic and have a history of bron-chial asthma (26, 209, 226). Although there is no special ageor sex distribution for ABPA, the disease tends to affectyounger people, with most cases occurring before the age of40. The disease has been reported even in children of <2years of age (89). The clinical symptoms of an acute episodeof ABPA should be differentiated from those of more chronicforms. The acute onset is characterized by severe asthmaand wheezing, fever, malaise, weightloss, chest pain, andproductive cough with blood-streaked sputum. As the dis-ease becomes chronic, the patient develops bronchiectasiswith production of copious purulent sputum. Patients maycough up green, brown, or often blood-tinged mucous plugscontaining eosinophils, Charcot-Leyden crystals, and fungalelements.The radiological findings are quite varied. The commonest

finding is homogeneous or nonhomogeneous consolidation,with or without atelectasis, involving a segment or a lobe.Generally, the upper lobes are involved (184). The upper-lobe infiltrates shift from one place to another as the diseaseprogresses. Repeated episodes of ABPA result in edema ofbronchial walls, their dilation, and their eventual destruc-tion. Radiographically, these changes are manifested asthickening of the walls and saccular dilation. With multipleepisodes, bronchiectasis, cavitation, atelectasis, and subse-quent fibrosis may also develop. Because the chest X-rayand symptoms may not exclude the possibility of ABPA insome patients, and some patients with ABPA show noremarkable symptoms, procedures such as tomography orcomputerized tomography scanning may be essential fordemonstrating bronchiectasis. These procedures usually re-veal cylindrical or saccular bronchiectasis with distal spar-ing, which suggests a pattern of localized obstruction andinflammation resulting from mucus and mycelial plugs in thelarger air passages (53, 152). The administration of oralcorticosteroids clears the mucoid impactions which areresponsible for much of the radiological involvement inpatients with ABPA.

(ii) Diagnostic criteria of ABPA. ABPA has been describedas an immunological disease that ranges from asthma to fataldestructive lung disease with defined clinical, serological,radiological, and pathological features (64, 66, 156, 157, 159,

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TABLE 1. Criteria for diagnosis of ABPA'

Occurrence ofsymptom

Symptom CbCRb CRI

present absent

Asthma + +Peripheral blood eosinophilia (>1,000/mm3) + +Immediate cutaneous reactivity to A. fumigatus + +Serum precipitins to A. fumigatus + +Elevated level of total serum IgE (>1,000 ng/ml) + +Proximal bronchiectasis + +Elevated level of serum IgE and IgG to + +A. fumigatusca Adapted from Greenberger (65).b CRI, chest roentgenographic infiltrate.c Compared with serum from patients with asthma and immediate cutane-

ous reactivity to A. fumigatus but in whom ABPA has been ruled out. Thisassay is not widely used and is not absolutely necessary in the diagnosis ofclassic cases. However, it is valuable for early diagnosis when all of thefeatures of ABPA are not present.

160, 163, 164). Hinson et al. originally described ABPA as anallergic response to Aspergillus fumigatus in bronchial se-cretions. However, they could not demonstrate skin testreactivity to an Aspergillus extract in their patients (84).Since then, a number of additional features of ABPA havebeen recognized (49). The diagnostic criteria have beenrevised several times and elaborated upon since their initialdescription (21, 65, 145, 158, 185). The diagnosis of ABPAdepends on central bronchiectasis and the presence orabsence of chest roentgenographic infiltrate. The criteria arelisted in Table 1. Patients in whom proximal bronchiectasisis not detected by radiological procedures are designatedABPA-S (for seropositive), and those with central bron-chiectasis are designated ABPA-CB. These designationscombined with currently available serodiagnostic methodsaid in the early diagnosis of ABPA (65).According to Greenberger and Patterson, ABPA should be

ruled out in all patients with chronic asthma (67). Althoughthe classic patients meet the criteria for ABPA, other sus-pected patients should be evaluated over a period of time(65, 72, 125, 181). After being tested with A. fumigatusextracts, patients usually manifest dual cutaneous reactivitythat consists of a wheal and erythema reaction within 10 to20 min and erythema and induration (without a wheal) in 4 to8 h. A dual response following bronchial provocation with A.fumigatus and production of sputum plugs containing hy-phae are the characteristic features of a classic patient withABPA (146). A different set of diagnostic criteria was usedby Sandhu et al. in their study (190). They consideredasthma, dual cutaneous reactions to A. fumigatus, recurrentroentgenographic infiltrates, elevated peripheral blood eo-sinophilia, positive sputum culture, and precipitins to A.fumigatus as diagnostic. These criteria would identify onlythe classic cases and miss the early cases of ABPA and casesof ABPA in remission.ABPA should be ruled out in patients with chronic asthma

exhibiting immediate reactivity to A. fumigatus or otherAspergillus antigens. Most of these patients demonstrate a3+ to 4+ skin prick test response and will not require anintracutaneous injection. Although an elevated level of totalserum IgE is suggestive, it is not specific for ABPA. Theroentgenographic consolidation resulting from bacterialpneumonia can be differentiated from that of pulmonaryinfiltrates of ABPA (66). Posterior segments of upper lobes

are often affected, simulating tuberculosis, but middle andlower lobes have also been involved. The possibility ofABPA should also be considered in patients with a history ofsurgical resection for bronchiectasis (66). There are alsoreports of ABPA-like syndromes caused by Curvularia,Candida, and Helminthosporium species and other fungi (5,65, 71, 83). Skin tests and tests for precipitins to A. fumiga-tus may be negative in these cases, whereas the culture ofsputum or the presence of precipitins to other fungi may aidin specific diagnosis.

Systemic disease

IA. Although IA has been considered an infection exclu-sively of immunocompromised patients, sporadic cases havebeen reported even in normal hosts (1, 93, 151). Infection iscommon in patients with leukemia and other hematologicalmalignancies, in patients undergoing organ transplantation,and in chronic granulomatous disease of childhood (17, 29,148, 176, 177). The association of IA with AIDS is stillcontroversial in spite of the few reported cases (43, 62). Anincreased prevalence of IA has been noted in bone marrowtransplant recipients (42, 168, 251). Predisposing factors forthe development of IA in immunocompromised patients aremanifold. They include neutropenia, granulocytopenia, un-derlying chronic diseases, and treatments such as those withcorticosteroids, broad-spectrum antibiotics, and cytotoxicdrugs (3, 54, 59, 70). The IA detected in apparently healthyindividuals may be due to unrecognized subtle immunologi-cal defects, tissue damage, viral infections, or antibiotictherapy for a bacterial infection (30). Several recent noso-comial outbreaks of IA that occurred among leukemic pa-tients and renal transplant recipients have been attributed toa variety of environmental sources. The potential sources ofAspergillus conidia from contaminated environments in-cluded hospital renovations, road constructions, and fire-proofing materials. A. fumigatus conidia from contaminatedair conditioners have been implicated in a number of cases ofIA (4, 11, 126, 191).The pathogenesis of IA is complex. The lung serves as the

portal of entry for the fungus, and colonization of therespiratory tract is followed by endobronchial proliferationand bronchial ulceration. The fungus may spread to the lungparenchyma, resulting in necrotizing bronchopneumonia,lobar pneumonia, or hemorrhagic infarction. An alternativeroute of colonization is through necrotic lung tissue follow-ing pulmonary emboli or bacterial infection or from thecavitary growth. As a result, single or multiple abscessesmay be formed, in which case the diagnosis is made only atautopsy. Hematogenous spread from sites such as the gas-trointestinal tract or intravenous catheters results in multiplemiliary microabscesses throughout all organs (154, 155, 253).The symptoms of IA are mostly nonspecific. They include

cough, wheezing, dyspnea, and fever. In Aspergillus-in-duced bronchitis, the chest radiograph may be normal, butsingle or multifocal nodules can be seen in establishedinvasive disease. These nodules may undergo infarction orcavitation and appear often as bilateral consolidations (155).The widespread radiological infiltrates give an indication ofhow extensively the disease has spread. The differentialdiagnosis of IA includes infections such as those due toPneumocystis caring or Nocardia, Candida, Mycobacte-rium, or Mycoplasma species as well as some of the commonbacterial infections.


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IMMUNOLOGY OF ASPERGILLOSIS

Because of the universal distribution of aspergilli, allindividuals are exposed to these organisms, but the normaldefense mechanisms act as a barrier to infection frominhaled Aspergillus conidia. The aerosolized conidia areeasily inhaled, and they encounter alveolar macrophages asthe first line of defense. Studies in mice have shown thatalveolar macrophages are efficient scavengers of inhaledAspergillus conidia (51). Macrophages do not readily kill theconidia, but they prevent their germination (107). The mu-cociliary cells efficiently clear conidia from the lung into thegastrointestinal tract during swallowing (44). The differentclinical manifestations found in humans are frequently due toimmunological responses of the host to A. fumigatus anti-gens or to other environmental factors. Only a limitednumber of studies on the cellular immune mechanisms inaspergillosis have been done. Although a number of reportshave emphasized the role of humoral immune mechanisms inaspergillosis, particularly in patients with ABPA or as-pergilloma, very little is known about the cellular immuneresponses in these diseases.

Humoral Immune Response

Humoral immune responses in patients with ABPA andaspergilloma have been well studied. Sera from patients withABPA consistently demonstrate high levels of A. fumigatus-specific circulating antibodies of the IgG and IgE isotypes(130, 185). Specific antibodies belonging to other immuno-globulin classes have also been reported in these patients.According to Brummund et al., in addition to elevated levelsof IgE and IgG antibodies, there was evidence of polyclonalelevation of levels of all immunoglobulin isotypes in patientswith Aspergillus-induced diseases (33). In a recent study,Kurup et al. showed that, along with all immunoglobulinisotypes, levels of subclasses of IgG, particularly IgG1 andIgG2, are also elevated in ABPA patients (120). Similarly, A.fumigatus-specific antibodies belonging to the IgG class ofimmunoglobulins have been reported to be the predominantantibody isotype in patients with aspergilloma (240). Re-cently, we have demonstrated that A. fumigatus-specificIgGl is the predominant subclass of IgG in patients withaspergilloma (120).

Specific humoral responses differ considerably in patientswith different clinical forms of aspergillosis because of theparticipation of diverse antigens of A. fumigatus (21, 65,240). Serum antibodies in patients with ABPA frequentlyreact with protein antigens of A. fumigatus. Identical levelsof A. fumigatus-specific antibodies belonging to the IgG1and IgG2 subclasses are usually found in the sera of thesepatients (120). Although aspergilloma patients frequentlyreact to carbohydrate and glycoprotein antigens of A. fumi-gatus, their sera show more IgG1 than IgG2 subclasses. Thecharacteristic expression of A. fumigatus-specific antibodiesin the sera of patients with ABPA and aspergilloma suggestsa difference in the fundamental immunoregulatory responseto Aspergillus antigens in these disorders.

Cellular Immune Response

Lymphocytic infiltration and granuloma formation in thelung clearly demonstrate the presence of a cell-mediatedimmune response in ABPA patients (208). In some studies ofA. fumigatus antigen-induced lymphocyte proliferation inABPA patients, no correlation with disease activity was

detected. In addition, proliferation was not significantlyhigher in ABPA patients than in patients with a positiveimmediate skin test reaction to A.fumigatus (77, 211). Thesefindings suggest that T-lymphocyte sensitization is not animportant feature of ABPA. The lack of lymphocyte stimu-lation reported in these studies may be due to the use of lessrelevant antigens or to the treatment of the patients withprednisone. In contrast, other reports indicate that antigen-induced lymphocyte transformation can be demonstrated inpatients with ABPA (57, 104, 234, 235). An antigen-inducedproliferative response of lymphocytes from these ABPApatients has been correlated with disease activity. During theexacerbation of disease, the proliferative response of periph-eral blood lymphocytes was undetectable. Analysis of pe-ripheral blood by lymphocyte phenotyping demonstrated asignificant increase in T-helper or inducer cells, whereas thesuppressor cell population was normal (104). Antibody-dependent, cell-mediated cytotoxicity has also been re-ported in both ABPA and aspergilloma (117).The pathogenesis of ABPA is not completely understood.

The host immunological response presumably is exaggeratedin terms of specific antibody isotype, total serum IgE pro-duction, sensitized lymphocytes, cellular hyperreactivity,and complement activation in the bronchoalveolar compart-ment. Studies of cell-mediated immunity in Aspergillus-induced diseases are conflicting. The limited knowledge andconflicting results of studies on cell-mediated immune re-sponses in patients with ABPA may be due partly to the lackof pure, dependable, and reproducible antigens. Studies withpurified antigens would be of value in understanding bothhumoral and cell-mediated arms of immune response inaspergillosis. In contrast, the lymphokines and the cell-mediated mechanisms by which T cells participate in theregulation of IgE synthesis and eosinophil activation anddifferentiation in certain other allergic diseases have beenwell documented (99, 231). Similar mechanisms may play amajor role in the pathogenesis of ABPA.

ETIOLOGY AND EPIDEMIOLOGY

Etiology of AspergillosisA. fumigatus is the species of Aspergillus most frequently

isolated from human beings with saprophytic, allergic, orinvasive disease manifestations. It is also associated withfood spoilage and with diseases of plants and animals (2, 12,28, 37, 92, 182). Although A. fumigatus is the principaletiological agent of ABPA, other species such as A. terreus,A. flavus, A. nidulans, A. oryzae, and A. niger have beenassociated with the disease (6, 124, 190). Most aspergillomasare caused by A. fumigatus. Other species, such as A. niger,have also been incriminated as causative agents (207). Thepredominant species involved in IA is A. fumigatus, butother species including A. flavus, A. glaucus, A. niger, A.nidulans, and A. terreus have also been implicated (253).

Opinions vary on the importance of the number of As-pergillus conidia in the immediate environment of patients.ABPA has resulted from smoking moldly marijuana andfrom exposure to A. fumigatus-contaminated dump sites andbird droppings (106, 119, 128, 145). In addition, closedenvironments, pets, house plants, and carpets often contrib-ute to the excessive concentration of A. fumigatus conidiainside houses or other buildings (213, 214, 230, 233). Beau-mont et al. measured airborne concentrations of Aspergillusconidia in and around the dwellings of two patients withABPA (24). They found heavy spore contamination in the

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environment of one patient, with lower spore concentrationsin the other. Both patients had increased bronchial obstruc-tion during periods when the Aspergillus spore content in theoutside air was high. The results of the study suggest thatavoiding an Aspergillus spore-contaminated environmentand limiting overall exposure may play a major role inpreventing exacerbation of the disease. Such aerobiologicalstudies in the microenvironments of patients with ABPAmay aid in detecting a particular mold source and in moni-toring the intensity of exposure. These parameters may bevital in immunosuppressed patients at risk of contracting IA.

Epidemiology of AspergillosisBetter epidemiological tools for the characterization of

aspergilli associated with IA in immunosuppressed patientsare needed. Although a detailed epidemiological investiga-tion may not be necessary when the causative agent isreadily identifiable from a particular source, it may be ofutmost importance in hospital-acquired infections (87). Iden-tification of aspergilli is based mainly on morphologicalcharacteristics, but variants within a species are known tooccur, and strain recognition is then a problem. In a study onthe morphological variation of a series of A. fumigatusisolates from aspergilloma patients, Leslie and co-workersfound strains exhibiting progressively more abnormal char-acteristics during the course of the infection (127).

Recently, typing schemes for the identification of as-pergilli have been explored. A biotyping scheme known askiller typing, based on the toxins released from the fungus,has been reported to be an effective epidemiological markerin the differentiation of A. fumigatus strains (174). Burnieand associates used immunoblot fingerprinting for the exam-ination of strains of A. fumigatus (36). They found 11immunoblot types among 21 isolates obtained from eightaspergilloma patients. However, it is not known whether thisdelineation of types was due to quantitative or qualitativedifferences in proteins. Restriction enzyme analysis of mito-chondrial DNA has been used as an aid in determining thetaxonomy of the genus Aspergillus (105). A recent study hasshown that restriction fragment length polymorphism analy-sis of total cellular DNA can be used to distinguish or matchdiverse or related A.fumigatus isolates (46). The analysis of31 epidemiologically characterized isolates from three con-tinents revealed 24 patterns (DNA types). Among these,three DNA types were represented by three isolates eachand one DNA type was represented by two isolates. Twentytypes were unique. This kind of a typing system might helpanswer questions concerning the genetics, pathogenicity,epidemiology, and ecology of A. fumigatus. An explorationof the clonality of A. fumigatus by using this typing methodmight throw light on the epidemiological aspects of as-pergillosis.

IMMUNODIAGNOSIS

AntigensThe diagnosis of ABPA is dependent on clinical and

immunological findings. Circulating antibodies to specificantigens have been detected in patients with most of theAspergillus-induced diseases. Extracts from the wholemycelium or metabolic products secreted into the mediumduring the growth of A. fumigatus have been used asantigens to detect antibodies in the sera of patients. Inaddition, a few semipurified antigens with more predictable

reactivity with patient sera have been isolated in recentyears (80, 82, 115, 132, 172, 194, 197, 249). However,standardized antigens are still not available for dependableuse in the serological diagnosis of ABPA and other Aspergil-lus-induced diseases. Pure antigens of known physicochem-ical properties would be invaluable in defining the differentimmunological mechanisms involved in these diseases.Demonstration of circulating antibodies is considered an

important criterion in the diagnosis of Aspergillus-induceddiseases. However, currently available antigen and antibodydetection methods are not altogether satisfactory and needto be improved for dependable results. The major reasonsfor the nonavailability of standardized antigens are as fol-lows. (i) Antigen preparations are highly variable. Culturefiltrate and mycelial extracts show considerable differencesin their antigenic components. The variation is further ag-gravated by the use of different strains of A. fumigatus anddifferent cultural conditions and extraction procedures (114).(ii) Antigens show cross-reactivity. Significant cross-reactiv-ity exists between A. fumigatus strains and other aspergillias well as between A. fumigatus and other fungi and bacteria(19, 22). This cross-reactivity may be due to epitope sharingbetween the antigens or to the universal presence of bacte-rial and fungal antibodies in the sera of normal subjects. Inaddition to immunological cross-reactivity due to sharedantigens, some Aspergillus antigens frequently show reac-tivity to C-reactive protein in the sera of patients with avariety of infectious, neoplastic, and autoimmune diseases.The cross-reactivity of epitopes in most cases is due to thelimited number of shared epitopes, mostly between polysac-charides from different fungi. (iii) Dependable serologicalmethods are lacking. Not all available serological assays aresensitive and specific when crude antigens are used. Sensi-tive methods such as the enzyme-linked immunosorbentassay (ELISA) and radioimmunoassay (RIA) need purifiedantigens for reliable and reproducible results (22, 98, 122,123). (iv) Toxins and enzymes may be present. Antigenpreparations frequently contain toxins such as endotoxins,hemolytic toxins, and, rarely, aflatoxins (121). In addition,several proteolytic enzymes from A. fumigatus antigens maycause interference and adversely affect the reproducibility oftest results.

In light of these problems, purified or semipurified andstandardized antigens are essential for the development ofreliable and sensitive immunoassays. For rapid progress inthe standardization of antigens for laboratory and clinicaluse, identification of relevant components of crude antigenicextracts is of primary importance. Once such componentsare identified, it is possible to obtain fractions with fewer orhomogeneous components. Such preparations can be stan-dardized with less difficulty and can be successfully used inimmunodiagnosis as well as in understanding the immuno-pathogenesis of the disease.The chemical nature of antigens of Aspergillus species

may be polysaccharide, protein, or glycoprotein. In manyearly studies, carbohydrate antigens (14-16, 188, 189, 216)were used in immunoassays, but the cross-reactivities ofthese antigens with those of other fungi was a major draw-back in interpreting test results. Most later investigatorsused the protein and glycoprotein antigens to detect antibod-ies in patient serum (74, 115, 122, 197, 248, 250). On theother hand, in IA, galactomannan and glycoproteins are themajor circulating antigens (25, 50, 179).

Antigen preparation. A. fumigatus strains vary consider-ably in their antigenic makeup. Kurup et al. studied 11strains of A. fumigatus from different sources and tested


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their culture filtrate antigens against sera from 33 patientswith aspergillosis (114). Individual antigens reacted differ-ently with these sera, and the detection of specific antibodiesin these 33 sera varied from 40 to 76% (114). Similar resultshave been reported by other investigators (102, 220, 221,223, 236). Variation in the antigenic components was alsonoted with different batches of antigens prepared from thesame strain. On the other hand, Odds et al. reported quali-tative similarity of all batches of antigens from A. fumigatusand considered that the variability was only quantitative innature (153). Our studies as well as the results reported byother investigators indicate that use of synthetic medium andincubation of A. fumigatus at 370C resulted in more consis-tently reactive antigens (101, 114, 228, 229). From theavailable reports it can be concluded that the most reliablecell-associated antigens for immunoassay can be obtainedfrom 4-day-old growth of A. fumigatus in aerated cultures.Reliable culture filtrate antigen for immunoassays can beobtained from 2- to 4-week-old stationary cultures of theorganism. The number of antigenic components is higherduring the first 4 days but decreases as time goes on until thecell components are liberated into the medium after 2 to 4weeks (101, 114, 228, 229). Medium with an initial pH ofabout 5 consistently gave good results. Glucose-asparagineor a mixture of Czapek-AOAC broth (Difco) is the mostwidely accepted medium for the best growth of A. fumigatus(101, 114). Stationary cultures for culture filtrate and shakecultures for mycelial antigens have been reported to be ideal.

Fractionation and purification. In recent years severalinvestigators have attempted to characterize and purifyrelevant antigens of A. fumigatus (Table 2). Most of theearlier attempts involved harsh physical and chemical treat-ments, which had an adverse effect on antigenicity. Bothculture filtrate and mycelial extracts contain a number ofantigenic components capable of reacting with patient sera.These antigens include heterogeneous proteins, carbohy-drates, and glycoproteins (38, 60, 78, 79, 81, 96, 103, 115,171, 200, 247). Kim et al. demonstrated up to 28 bands bypolyacrylamide gel electrophoresis (103), while Piechura etal. showed about 200 demonstrable components by two-dimensional electrophoresis (171). Immunoblots from one-and two-dimensional electrophoreses have shown that anumber of these components react with sera from patientswith aspergillosis. Crossed immunoelectrophoresis of As-pergillus culture filtrate antigen against sera from patientswith ABPA demonstrated approximately 35 precipitin arcs,of which 8 to 10 showed specific reactivity with IgE antibod-ies (118, 133). Using crossed isoelectric focusing, Kim andassociates demonstrated 52 precipitin arcs (103). Thesefindings indicate that the A. fumigatus antigens constitute avery heterogeneous group.

Aspergillus antigens demonstrating a variety of enzymeactivities have been reported by several investigators. Byusing the enzyme staining method of Uriel et al., Tran vanKy et al. detected 18 enzymatically active components fromAspergillus species (222-225). Of the 18 antigens showingenzyme activity, 13 reacted specifically with sera frompatients with aspergillosis. Biguet et al. demonstrated anti-gen fractions having chymotryptic activity that specificallyreacted with sera from patients with aspergilloma but notwith sera from patients with ABPA (27). A fraction having amolecular weight of 250,000, probably a tetramer demon-strating catalase activity, has been reported by Schonheyderet al. (200). This fraction had sugar subunits with affinity toconcanavalin A (ConA) and was detected in 13 of 14 clinicalisolates of A. fumigatus. However, this component with

catalase activity was also shared by other Aspergillus spe-cies and has not been clinically evaluated. Antibodies tocatalase antigen have been found in patients with cysticfibrosis having ABPA (199). Fractions with protease activityhave been reported from both pathogenic and nonpathogenicspecies of Aspergillus. Schonheyder and Andersen partiallypurified a 25- to 50-kDa protease fraction having reactivitywith patient sera (197). We have isolated two acid proteasesfrom membrane vesicles of A. fumigatus. The molecularweights of these fractions were 73,000 and 43,000. Bothfractions bound to ConA and showed reactivity with serafrom patients with ABPA in a biotin-avidin-linked immuno-sorbent assay (BALISA) (172).

Polysaccharide fractions from either cell wall or cyto-plasm show reactivity with specific antibodies. However,these antigens frequently demonstrate cross-reactivity withother fungal antigens. Galactomannan, with a molecularweight of 50,000 or more, has been purified by Azuma et al.It showed both skin test reactivity and a precipitin reaction(13-16). These authors also characterized and purified agalactomannan peptide complex by using a Sephadex G-200column. This galactomannan has been identified as thesignificant circulating antigen in patients with IA (25, 50,179).

Several investigators identified ConA-binding componentsof A. fumigatus as the major antigens consistently showingreactivity with patient sera (74, 76, 123, 196, 248). Theseantigens have been isolated from both culture filtrate andmycelial extracts of A. fumigatus (100, 204, 246, 249, 256).Wilson and Hearn fractionated mycelial extracts by prepar-ative isoelectric focusing and compared the reactivity of thethree major fractions with that of crude water-soluble ex-tracts (248). They found that all fractions reacted with serafrom patients with aspergillosis but also showed cross-reactivity with antibodies against other fungi. By usingpreparative isoelectric focusing and affinity chromatogra-phy, we have isolated two fractions, one a glycoprotein andthe other a protein (123). In an ELISA, both fractionsdemonstrated reactivity against patient sera. We have alsoisolated another fraction which focused at pH 6.5 andshowed 20-, 40-, and 80-kDa components in two-dimensionalelectrophoresis. This fraction also showed good reactivitywith both IgE and IgG antibodies (116). Longbottom andassociates characterized four antigens (Ag 3, Ag 5, Ag 7, andAg 13) by using gel filtration, isoelectric focusing, andaffinity chromatography (74-76, 132, 134, 135, 219). Two ofthese fractions, Ag 7 and Ag 13 with molecular masses of150,000 to 200,000 and 70,000, respectively, showed ConAbinding. Both of these antigens reacted in an ELISA withsera from patients with ABPA and aspergilloma. Two otherfractions, Ag 5 and Ag 3, which were thermolabile peptideswith molecular masses of 35 and 18 kDa (24 kDa by gelfiltration), respectively, were also isolated from A. fumiga-tus and found to be useful in detecting antibodies in patientswith ABPA.Schonheyder and associates identified several fractions of

A. fumigatus antigens with molecular masses of 25,000 to>470,000 Da (196, 200). Some of these fractions wereevaluated and found to be very specific in the diagnosis ofaspergilloma. Using hydrophobic interaction chromatogra-phy, Schonheyder and Andersen obtained a componenthaving a molecular mass distribution of 25 to 50 kDa (196). Inpolyacrylamide gel electrophoresis this fraction showed 14components, of which 3 had ConA-binding ability. None ofthese components, however, were isolated in pure form. Byusing hydrophobic interaction and affinity chromatography,

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TABLE 2. Recent reports on fractionation and evaluation of Aspergillus antigens

Antigen extract Disease category Antibody assay Antibodies Results and conclusion Reence-CF ABPA, aspergilloma RIST, RAST, XIEF IgE, precip- Individual components and reactivity with 95

itins sera varied

ABPA, aspergilloma,allergic asthma, asper-gilloma with ABPA

ABPA, aspergilloma

ABPA, aspergilloma, IA,other mycoses

ID, EAST, RAST,RIEF

2-DIEF, ID, PAGE

ELISA, IIF, ID

IgE, IgG

Precipitins

IgE binding components found in early loga-rithmic phase; precipitating antigenic com-ponents low in early phase, increased duringlate phase

Concentrated ABPA sera showed precipitinssimilar to aspergilloma

Precipitins Aspergillosis patients reacted with wide rangeof antigens of A. fumigatus; some antigenscross-reacted with sera from other mycoticdiseases

194

82

45

ABPA, aspergilloma,bronchial asthma

ID, ELISA

ABPA RAST, self-XRIEF,ELISA, PRIST

ABPA ELISA, XIEF,XRIEF, fusedRIEF

ABPA, A. fumigatus STP ELISA, XIEF

ABPA, aspergilloma,farmer's lung, A.fumigatus STP

ABPA, aspergilloma

ABPA, aspergilloma

ABPA, aspergilloma

ELISA, XIEF, fused IgE, IgGRIEF

ELISA, XIEF, ID

BALISA

FAST, BALISA,Western blot

IgE, precip- Polysaccharides cross-reacted with otheritins Aspergillus species and fungi

IgE

IgE

Two components: major antigens with strongIgE binding and poor precipitating activityand minor antigens with strong precipitatingactivity but weak IgE binding

Ag 3, low molecular wt heat labile, non-ConAbinding, reacted with 75% of ABPA sera

IgG, IgG1, Elevated IgG to Ag 7 detected in 97% ofIgG4 ABPA sera

Ag 13, heat labile, ConA binding, with chymo-tryptic activity, poor binding to IgE

Glycoprotein antigen reacted with aspergillosisIgE, IgG

IgE, IgG Fractions 18, 19, 20 were ConA binding,reacted with ABPA and aspergilloma

IgG, IgE Low-molecular-wt, non-ConA-binding antigensreacted with ABPA

204

131

132

75

76

123

116

115

ABPA, aspergilloma,EAA, persistent lunginfiltration, COPD

ABPA, aspergilloma

ELISA

ID, CIEP

IgG 70,000-Da antigen was reactive

Precipitins Triton X fraction superior to water-solublefraction; cross-reactivity between otheraspergilli

ABPA, aspergilloma, lungdisorders with As-pergillus colonization

ABPA, aspergilloma,asthma, cystic fibrosiswith A. fumigatus pre-cipitins

ABPA, aspergilloma

ID, CIEP, ELISA,XIEF

BALISA

ELISA, XIEF, ID

Precipitins ConA-bound fraction superior to non-ConA-bound fraction

IgE, IgG ConA-binding glycoprotein reacted with IgGand IgE compared with non-ConA-bindingantigen

IgG 470,000-Da fraction reacted with normal con-trols and patients; 250,00-Da antigen hadcatalase activity

246

118

196

Mycelial and ABPA, aspergilloma,CF bronchial asthma, bron-

chiectasis, tuberculosis

ELISA, ID IgG and IgM Cytoplasmic extracts bound more weakly toIgG than mycelial fractions

Continued on following page

CF

CF

CF

CF

CF

CF

CF

CF

CF

CF

CF

Mycelial

Mycelial

Mycelial

Mycelial

Mycelial andCF

194

80

247


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TABLE 2-Continued

Antigen extract Disease category Antibody assay Antibodies Results and conclusion Reenfce-

Mycelial and ABPA, aspergilloma, ID, PAGE, XRIEF Precipitins CS2 reacted with 75% of ABPA and as- 38CF other fungal dis- pergilloma

eases

Mycelial and ABPA, aspergilloma, ID, IEF, PAGE, IgG, IgA, IgM CS2 fraction reacted with aspergillosis sera 171CF other fungal dis- XIEF, ELISA

eases

Mycelial, CF, ABPA, aspergilloma, ELISA, ID, RAST, IgE, IgG Spore cytoplasm is similar to CF and mycelial 96spore extract Aspergillus asthma EAST antigen, but showed low ELISA titers;

spore surface antigen had low immunologi-cal activity

CF, germinated ABPA IF, XIEF, XRIEF, IgE, IgG Ag 5, heat labile, non-ConA binding, reacted 219conidia, co- RIEF, fused with IgG and IgEnidial surface RIEF, ELISAantigens

Membrane ves- ABPA, aspergilloma ID, XIEF, BALISA IgE, IgG Two fractions with protease activity reacted 172icles with ABPA and aspergilloma

a CF, culture filtrate; EAA, extrinsic allergic alveolitis; STP, skin test positive; COPD, chronic obstructive pulmonary disease; IEF, immunoelectrophoresis;RIST, radioimmunosorbent test; RAST, radioallergosorbent test; XIEF, crossed immunoelectrophoresis; EAST, enzyme allergosorbent test; RIEF, rocketimmunoelectrophoresis; 2-DIEF, two-dimensional immunoelectrophoresis; PAGE, polyacrylamide gel electrophoresis; self-XRIEF, self-crossed radioimmuno-electrophoresis; PRIST, paper radioimmunosorbent test; FAST, fluorescent allergosorbent test; CIEP, counterimmunoelectrophoresis.

we have isolated a fraction with two components of 35 and65 kDa. This fraction consistently showed binding to IgGand IgE antibodies in sera from patients with ABPA (118).Using gel filtration, ConA affinity chromatography, iso-

electric focusing, and trichloroacetic acid precipitation,Kauffman et al. obtained several antigen fractions from A.fumigatus (97). They observed that the low-molecular-weight (5,000 to 28,000) fraction usually demonstrated weaklinkage to the polystyrene surface used in ELISA and gaveweak precipitins. In contrast, high-molecular-weight compo-nents reacted strongly in the ELISA and precipitin tests.Longbottom found that low-molecular-weight peptidesshowed strong IgE-binding capacity and were more specificin the diagnosis of allergic aspergillosis (131).Monoclonal antibodies produced against Aspergillus spe-

cies will be of value in purifying the relevant Aspergillusantigens (55, 109, 110, 215). Using several such monoclonalantibodies, we purified antigens by employing monoclonalantibody affinity chromatography. Some of these antigensshowed both IgE- and IgG-binding activities and will be ofuse in immunodiagnosis (109, 110). Thus, it can be seen thata number of homogeneous fractions that are reactive withpatient sera can be purified. However, such antigens are notyet available commercially or for general use.

Serological Tests

Serological tests serve as important aids in the effectivediagnosis of various types of aspergillosis. Circulating anti-bodies against A. fumigatus or other species of Aspergilluscan be readily detected in immunocomponent patients.However, a different approach may be necessary in immu-nosuppressed patients with no demonstrable antibodiesagainst the invading fungus. In these instances antigendetection systems may provide information helpful in diag-nosing the disease. A variety of tests with diverse sensitiv-ities have been developed to detect circulating antibodies toaspergilli.

ID. Because of its simplicity and ease of performance,immunodiffusion (ID) is still the most widely used techniquefor the serodiagnosis of aspergillosis. Several investigatorshave highlighted its usefulness in demonstrating precipitinarcs in agar gels as a diagnostic tool for aspergillosis (18, 19,40, 113, 122, 123, 136, 140, 170, 239). Antigens used in IDmay be extracts from mycelia or culture filtrates, whichshould be free of C-reactive substance (136). Concentratingthe serum specimens before testing may yield better resultswithout reduction in specificity (137). However, the ID testlacks sensitivity and gives no quantitative information onantibody concentrations. A micromodification of Wads-worth's agar gel diffusion technique has been used in ourlaboratory for testing antibodies in the sera of patientsagainst a panel of antigens (52). This method yielded repro-ducible results and has correlated well with other diagnosticmethods, although the sensitivity is much lower than that ofELISA or RIA.Immunogold assay. An immunogold assay was developed

by Gugnani et al. for the detection of A. fumigatus-specificIgG and IgE antibodies (69). This method is similar toindirect immunofluorescence (IIF) but more sensitive anddoes not need any specialized equipment. Slide cultures ofA. fumigatus, fixed in methanol, are treated with differentdilutions of patient sera, then with goat anti-human IgG orIgE, and finally with rabbit anti-goat IgG conjugated withcolloidal gold. After being dehydrated with alcohol andxylol, the slides are mounted and observed under light andphase-contrast microscopes. A positive reaction is indicatedby a brownish color on the surface of hyphae caused bybinding of gold particles. Gugnani et al. found a positivecorrelation between the degree of reactivity detected by theimmunogold assay, the BALISA titers, and the precipitinsdetected by agar gel diffusion. To make the sensitivity of theimmunogold assay comparable to that of the BALISA orRIA, silver enhancement can be used (141). Recent studiesusing immunogold electron microscopy with thin sections ofAspergillus hyphae and conidia have shown that both spores

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and hyphae react identically with IgG and IgE antibodies ofserum from aspergillosis patients (178). Sera from patientswith ABPA and aspergilloma reacted with cell envelopeantigens, whereas sera from patients with IA also bound tocell sap.RIA. A RIA for the measurement of Aspergillus antibody

was first described by Bardana et al. (22). The antigen was

prepared by ultrasonic disruption of A. fumigatus myceliafollowed by ammonium sulfate precipitation. The radiola-beled trichloroacetic acid-soluble antigen fraction was usedto detect antibodies to A. fumigatus. Several investigatorshave developed and utilized RIA as a reliable tool fordetecting antibody in patients with aspergillosis (142, 161,162). A radio allergosorbent test developed by Arbesman etal. used the culture filtrate antigen covalently bound tocellulose disks for the detection of IgE antibodies to A.fumigatus (10). RIA has several disadvantages includinglimited storage life of the radioisotopes, length of timerequired to complete the assay, need for expensive radioac-tivity detectors, and potential hazards and difficulties con-nected with exposure to and safe disposal of radioactivewastes.ELISA and BALISA. Because of its high sensitivity,

reliability, and versatility, the ELISA is being widely used inthe serodiagnosis of various forms of aspergillosis. Althoughthe sensitivity of the ELISA system depends on many

variables, the nature and type of antigens used and theirabilities to bind to solid surfaces are of prime importance.The complexity of A. fumigatus antigens used in the tech-niques and the inherent variations in antigen preparationsmay lead to major discrepancies in results from differentlaboratories. Both mycelial and culture filtrate antigens of A.fumigatus have been used in ELISA (100, 204, 249). Wilsonand Hearn used partially purified mycelial and culture filtrateantigens of A. fumigatus, rich in protein and carbohydrate,to compare the efficacy of these antigens in ELISA (249).They found that fractionation of mycelial extract yieldedcomponents that proved to be more sensitive in detectingspecific antibodies in patient sera. However, they observedbatch-to-batch variation in the antigen preparations. Khan etal. used a fraction from a culture filtrate of A. fumigatus todetect specific IgG in patients with allergic aspergillosis(100). They noticed rapid binding of IgG antibodies to bothwhole and fractionated antigens. Mohan et al. compareddifferent A. fumigatus antigens and found the metabolicantigen prepared from 24-h-old culture to be specificallyreactive against patient sera (150). Schonheyder andAndersen studied the reactivities of three antigen fractionsof molecular weights 470,000, 250,000, and 25,000 to 50,000(197). The 470,000-molecular-weight fraction reacted withmost sera from normal controls and patients with nonfungallung diseases. The antibody response in aspergillosis pa-

tients was much stronger against this fraction than againstcontrol sera. When the 250,000-molecular-weight antigenwas used in ELISA to test the sera from patients with

aspergilloma, 13 of 14 patients gave positive results, but onlyone of nine serum samples from patients with ABPA showedantibodies to this antigen. Schonheyder and Andersen con-

cluded that these three fractions may all be of value in the

immunodiagnosis of patients with A. fumigatus-induceddiseases, particularly when other methods fail to demon-

strate specific antibodies.We have compared antigens from three strains of A.

fumigatus by using the serological methods ID, IIF, and

ELISA, all with patient sera (122). The results showed wide

differences in the reactivity of antigens by different methods.

It has been concluded from the results that simultaneous use

of several different A. fumigatus antigen preparations andserological methods might help eliminate both false-positiveand -negative results, although such procedures are cumber-some and time-consuming.The studies described above reveal that ELISA results

vary widely depending on the type of antigen used fordetection of antibodies to A. fumigatus. The conventionalELISA system has been used successfully to detect A.fumigatus-specific IgG in patients with aspergillosis. Thisassay detected and quantitated antigen-specific IgG when itwas present in relatively high concentrations in the sera.However, the test was not sensitive enough to detect rela-tively low levels of antigen-specific IgE or subclasses of IgG.To overcome this disadvantage of the conventional ELISA(108), Brummund et al. have developed a modified tech-nique, the BALISA (33). This method has a sensitivitycomparable to that of RIA. The technique makes use ofhigh-affinity binding between biotin and avidin, which can becovalently coupled to other molecules such as proteins andpolysaccharides. We have been using the BALISA tech-nique routinely in our laboratory for the detection of A.fumigatus-specific antibodies and have obtained reproduc-ible results. BALISA correlates well with clinical features ofdisease and has sensitivity comparable to that of RIA.

Immunoblotting. Several studies have shown that glyco-protein antigens, a major constituent of both the myceliumand the culture filtrate of A. fumigatus, have high immuno-logical reactivity (23, 74, 115, 197, 246, 250). These antigenscan be characterized by their molecular size, isoelectricpoints, ability to bind to ConA or other lectins, and enzy-matic activity. The reactivity of specific components of A.fumigatus antigens to specific antibodies in the sera ofpatients can be studied by immunoblotting. This methodinvolves the separation of denatured antigens in polyacryl-amide gel followed by transfer onto nitrocellulose mem-branes. The reactivity of such antigens with antibody in thesera of patients can be visualized by the color reaction.Immunoblotting has been used with great success in detect-ing specific antibodies to Aspergillus species. The detailedmethods of immunoblotting for the demonstration of anti-A.fumigatus IgG and IgE in the sera of patients with aspergillo-sis have been reported (115).

Several other diagnostic procedures that have also beendeveloped for detecting aspergillosis include immunoelectro-phoresis, passive hemagglutination, IIF, latex agglutination,complement fixation, and enzyme-linked immunofiltrationassay (7, 22, 45, 58, 60, 61, 85, 87, 88, 95, 112, 129, 130, 139,147, 173, 193, 195, 198, 201-203, 237, 238, 252).

Several types of immunoelectrophoretic techniques havebeen used in diagnosing aspergillosis. Among these, coun-terimmunoelectrophoresis has been widely used, mainlybecause of the short time required to obtain results with thistest. By using counterimmunoelectrophoresis, Longbottomcould detect precipitins in 78% of patients with allergicaspergillosis; the ID test was positive in only 64% of thesame cases (129). However, compared with that for ID, theimproved efficacy of immunoelectrophoresis has not beendemonstrated because of the lack of specificity due to anincreased number of false-positive results (112, 139, 147).Crossed immunoelectrophoresis and crossed radioimmuno-electrophoresis have been reported to have added signifi-cance in the demonstration of A. fumigatus-specific antibod-ies. By using an intermediate gel and a reference serum, over40 specific precipitin arcs have been detected (95, 130). Withsuch an immunoelectrophoretic technique, enzymatically


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active antigens can also be detected (95, 200), but thesetechniques are not often used because of the lengthy andcumbersome procedures. The occasional failures of thesemethods have been related to either a shortcoming in thetechniques or the nonprecipitating nature of antigen-anti-body complexes (95, 241).Apart from the use of immunoelectrophoresis in the char-

acterization of antigens, the use of other tests is fairly limitedbecause of factors such as false positivity, instability ofantigen-treated erythrocytes, and the recent availability ofmore versatile and more sensitive detection methods. Whilethe ID test and ELISA are the most widely used serodiag-nostic methods in diagnosing aspergillosis, immunoblottinghas the advantage of revealing additional characteristics ofthe relevant and reactive antigens.

Skin Tests

Skin test reactivity to Aspergillus antigen extracts iswidely used to screen patients with suspected ABPA, inwhom at least two types of skin reactions can be observed.The type I (immediate) reaction consists of cutaneouswheall and flare" within 15 to 20 min of skin test challenge.A type III reaction, or the Arthus reaction, can be seen as aninduration usually developing by 4 to 10 h after skin testing.This reaction subsides after 24 h, but occasionally a delayedreaction, which might persist for 72 h or more, can also beseen. In patients with allergic asthma, only the immediatereaction is visible, whereas some aspergilloma patients hav-ing concomitant ABPA demonstrate both immediate and lateskin test reactivities. A delayed-type skin test reactivity mayalso be present in patients with HP.

Detection of Circulating Antigens for Diagnosis of IA

Aspergillosis is a major infection in patients with underly-ing diseases and other predisposing factors such as organtransplantation. The mortality of IA is very high and can besubstantially reduced if an early diagnosis is made. Such adiagnosis is often difficult because major clinical symptomsare nonspecific and blood cultures are usually negative.Bronchoscopy and biopsy of the lung and involved organsare essential for diagnosis, but these invasive procedureshave their own associated risks and high rates of false-negative results (91). Interest in developing rapid serologicalmethods for the diagnosis of this disease has thereforeincreased.The usual serological studies have not been useful in the

diagnosis of IA. Because of the unusual predisposition ofpatients with IA, namely, lack of a functionally normalimmune system because of malignancy or intentional immu-nosuppression as in organ transplantation, there is very littleor no antibody response in these patients. Even so, allprevious reports have dealt with the detection of Aspergil-lus-specific antibodies in the sera and body fluids of IApatients. Young and Bennett could not detect any signifi-cant antibodies against A. fumigatus in 15 patients tested byID, complement fixation, and IIF (252). All of their patientshad underlying hematological malignancies. Coleman andKaufman reported precipitins in the sera of 14 of 16 patientsstudied (41). Other investigators have also reported variousdegrees of success in detecting specific antibodies against A.fumigatus in the sera of patients with IA (60, 68, 86, 143, 144,148, 192). When success in demonstrating specific antibodiesagainst Aspergillus spp. has been reported, it has not re-ceived widespread recognition because of the lack of reli-

TABLE 3. Summary of recent published reports onantigen detection in IA

No.Assay Antigen Specimen positive/ Refer-

total encepatients

RIA Galactomannan Serum 3/3 205RIA, ELISA Galactomannan Serum 7/9 244Inhibition ELISA Carbohydrate Serum 11/19 186RIA Carbohydrate Serum 4/7 245ELISA Carbohydrate Serum 8/9 250RIA, ELISA Galactomannan Serum 2/12 56

Urine 7/13RIA Carbohydrate Serum 22/22 217Inhibition BALISA Glycoprotein Serum 4/5 91Inhibition BALISA Glycoprotein Serum 18/19 183

Urine 16/19Western blotting Galactomannan Urine 10/10 78

ability and sensitivity of the tests. Since patients develop theantibody only at the terminal stage of the disease, whentreatment is no longer effective, it is essential to look forrapid and more reliable assays for the early diagnosis of IA.Hence, in recent years considerable attention has been paidto the early detection of A. fumigatus antigens in the bloodand urine of patients suspected of having aspergillosis.

Several investigators have detected A. fumigatus antigensin animals with experimentally induced IA (47, 50, 169, 179,180, 206, 217, 243). The methods used were ID, ELISA, andRIA. The sensitivity of these earlier methods was notsufficient to allow an early diagnosis. However, they wereinstrumental in the development of more refined assays fordemonstrating antigenemia in patients. Recently, Westernblotting (immunoblotting) has been used to detect circulatingantigens in the sera and urine of rats with experimental IA(254). Van Cutsem et al. used a commercially available latexagglutination test in experimental invasive aspergillosis inguinea pigs. The test had a 97.6% sensitivity in detectingcirculating galactomannan in the plasma samples from thoseanimals. The kit was tested with human sera and waspositive in 13 of 15 proven cases and 17 of 18 patients with ahigh probability of invasive aspergillosis (227).A number of investigators have evaluated methods for

detecting A. fumigatus antigens in the bronchoalveolar lav-age fluid, sera, and urine of patients with aspergillosis (9,50, 58, 78, 91, 186, 205, 244, 245, 250, 255). The recentreports on the detection of antigenemia are summarized inTable 3. Weiner and associates used competitive bindingRIA for detecting cell wall carbohydrates and found antigen-emia in 78% of the patients with aspergillosis and leukemia(244). Other investigators used inhibition ELISA to quanti-tate specific antigens in the sera of patients with aspergillosisand of those predisposed to aspergillosis (50, 56, 183, 186,250). The circulating antigen detected in these instances is amajor cell wall polysaccharide, galactomannan. The sensi-tivity is still not satisfactory or comparable to that of RIA.Sabetta et al. demonstrated antigenemia in 11 of 19 patients(186), while Dupont et al. could detect galactomannan inonly 2 of 12 patients studied (50). On the other hand, Wilsonet al., using a sonicated mycelial extract, were able to detectantigenemia in 78% of their patients (250). They consideredan antigen concentration of 100 ng or more diagnostic. Usinga BALISA method, Fujita and associates demonstratedantigenemia in 16 of 18 patients studied (56). Our study, inwhich we used a double-sandwich inhibition BALISA, dem-

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onstrated the presence of heat-stable A. fumigatus antigensin sera from all five bone marrow recipients with aspergillo-sis (91). In this assay, the antigens detected were mostlyheat-resistant glycoproteins. Using polyclonal antibodies,Haynes and associates detected antigens in the urine of all 10of their patients with aspergillosis and in 23 neutropenicpatients with no evidence of aspergillosis. However, no

reaction was found when monoclonal antibody against ga-lactomannan was used (78).The success in detecting antigenemia in a patient's serum

mainly depends on the frequent monitoring of samples (78,242, 244, 250). The importance of serial sampling and ade-quate storage conditions has been emphasized. When singleserum samples were tested, Wilson et al. found a frequencyof detection of only 3.5%, which was increased to 19.2%,however, when two or more serum samples were obtainedper patient (250). Weiner et al. reported a frequency of 70 to76% antigenemia in serum samples from proven cases ofinvasive disease (242, 244). The false-negative results oftenobtained when detecting antigenemia may be ascribed toantifungal drugs administered to the patient, rapid clearanceof circulating antigen, the presence of different antigeniccomponents in the serum or urine at different time periods.

Details of the three methods commonly used for detectingA. fumigatus antigenemia, RIA, inhibition BALISA, andimmunoblotting, may be found in the literature (8, 78, 91,108).

CONCLUSION

IA, with its wide spectrum of clinical manifestations, hasbecome a major fungal infection. Similarly, A. fumigatus-induced allergic diseases are increasingly being diagnosed.With the increasing incidence reported in recent years andthe paucity of appropriate treatment, there is a need for an

early diagnosis of these diseases for effective management.There has been significant progress towards the characteri-zation and purification of antigens of A. fumigatus, but thecommon antigen(s) that can be used universally for reliablediagnosis has not yet been identified. In this context, use ofmonoclonal antibodies in serodiagnostic methods may yieldimproved results, A. fumigatus-specific antigens can bepurified with monoclonal antibodies and standardized to givereliable results in immunodiagnosis. Rapid and reliable im-munodiagnosis may also be achieved by the use of molecularbiological methods to obtain purified antigens. Studies withthese reagents may then enhance our understanding of theimmune mechanisms of these diseases.

ACKNOWLEDGMENTS

This work was supported in part by a Public Health Service grant

from the National Institutes of Health (AI-23071) and by theDepartment of Veterans Affairs.

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Vol. 5, No. 2CLINICAL MICROBIOLOGY REVIEWS, April 1992, p. 2110893-8512/92/020211-01$02.00/0

Letter to the EditorAntigen Detection in Invasive Aspergillosis

In the article on immunodiagnosis of aspergillosis byKurup and Kumar (2) some of our work was incorrectlycited, and we wish to bring this to the attention of the readerswho may have been misled. We refer specifically to Table 3,page 449, and to text on pages 449 and 450.With regard to Table 3 and the information summarized

from our paper (3), we wish to point out that two inhibitionenzyme-linked immunosorbent assays (ELISAs), not the bi-otin-avidin-linked immunosorbent assay (BALISA), wereused; in one case a polyclonal antiserum derived from ahuman patient with aspergillosis was employed, and in theother, a monoclonal antibody to galactomannan was used.Because of the way our data were presented in that paper, itis somewhat difficult to ascertain values for serum versusurine specimens on a per patient basis. Therefore, we providethe following information. The sera of 17 of 19 patients and 16of 19 patients, all with invasive aspergillosis (IA), werepositive with the polyclonal antiserum and the monoclonalantibodies, respectively. Urine specimens from six of eightpatients and eight of eight patients were positive with thepolyclonal and monoclonal ELISAs, respectively.With regard to the information presented in Table 3 on

Western blotting of urine samples (1), again, two separateantibody preparations were employed for antigen detection.In the first instance, a polyclonal antiserum specific forunfractionated cell walls was used, and 10 of 10 IA patientstested were positive. Only three urine specimens wereavailable for blotting with the second antibody preparation,viz., the monoclonal antigalactomannan antibody, but allthree specimens were positive. The band patterns obtainedwith the two antibody preparations were completely differ-ent. The anti-cell wall antiserum revealed seven bands, themajor ones corresponding to molecular masses of 11, 18, and29 kDa, whereas the antigalactomannan monoclonal anti-body revealed bands corresponding to molecular massesgreater than 45 kDa plus a single band at 21 kDa. Weconcluded that none of the antigens detected by the poly-clonal antiserum were galactomannan.

In the text on page 450, again with respect to the work ofHaynes et al. (1), Kurup and Kumar state that ". . . antigens(were detected) in the urine of all 10 ... patients with as-pergillosis and in 23 neutropenic patients with no evidence ofaspergillosis." Technically, that is correct, but the informationis misleading. Urine samples from 23 neutropenic patientswithout fungal infection were pooled and tested with both apolyclonal anti-cell wall antiserum and the monoclonal antiga-lactomannan antibody. The polyclonal antiserum did react withthe pooled urine specimen, but the bands that appeared werediffuse, lightly stained, and of different molecular masses fromthe distinct bands of 11, 18, and 29 kDa detected in IA patients.No bands appeared when the pooled urine from neutropenicpatients was tested with the monoclonal antibody.We appreciate the opportunity to clarify these issues.

REFERENCES1. Haynes, K. A., J. P. Latge, and T. R. Rogers. 1990. Detection of

Aspergillus antigens associated with invasive infection. J. Clin.Microbiol. 28:2040-2044.

2. Kurup, V. P., and A. Kumar. 1991. Immunodiagnosis of as-

pergillosis. Clin. Microbiol. Rev. 4:439-456.3. Rogers, T. R., K. A. Haynes, and R. A. Barnes. 1990. Value of

antigen detection in predicting invasive aspergillosis. Lancet336:1210-1213.

Kenneth A. HaynesThomas R. RogersCharing Cross and Westminster Medical SchoolLondon SWIP 2AR, United KingdomJean Paul LatgeInstitute Pasteur75724 Paris Cedex 15, FranceRosemary A. BarnesUniversity of Wales College ofMedicineCardiff CF4 4XN, United Kingdom

Author's ReplyWe would like to thank Drs. Haynes, Rogers, Latge, and

Barnes for drawing our attention to some errors in Table 3 andin the text with regard to their work on antigen detection. Asthey indicated, it was difficult to ascertain data on a per patientbasis in one of their papers, and in an effort to be as concise aspossible, we might have misinterpreted some of the informa-tion. In our review we selectively incorporated results fromdifferent studies for comparison of similar procedures andmethods. Our objective was not to discuss any particular paperin detail. In addition, as we were examining Table 3 with thegoal of revising it, we noted a mistake with regard to reference56 in that a capture BALISA was used instead of an ELISAand 16 of 18 serum samples studied were positive. In an effortto correct any misconceptions these errors may have caused,we have revised Table 3 (see below). Our apologies to allconcemed.

TABLE 3. Summary of recently published reports in antigendetection in IA

Antigen Speci- No. of No. Ref-Assay detected men patients positive erence

RIA Galactomannan Serum 3 3 205RIA Galactomannan Serum 9 7 244Inhibition ELISA Carbohydrate Serum 19 11 186RIA Carbohydrate Serum 7 4 245ELISA Carbohydrate Serum 9 8 250Capture BALISA Galactomannan Serum 18 16 56RIA Carbohydrate Serum 22 16 217Inhibition BALISA Glycoprotein Serum 5 4 91Inhibition ELISA Unknown' Serum 19 17 183

Urine 8 6Galactomannan Serum 19 16

Urine 8 8Western blotting Cell wall derived' Urine 10 10 78

Galactomannan Urine 3 3

Polyclonal antibody from a human patient was employed.b A polyclonal antiserum raised againstAspergillus cell walls was used, and

it contained antibodies capable of reacting with compounds in a pool of urinespecimens taken from 23 neutropenic patients without fungal infection. Theantigens detected, however, had different molecular masses than the majorantigens of 11, 18, and 29 kDa detected in patients with IA.

V. KurupA. KumarMedical College of WisconsinMilwaukee, Wisconsin 53295-1000

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