ARTICLE IN PRESS Respiratory Medicine (2007) 101, 2352–2357
Allergic bronchopulmonary aspergillosis’ diagnosis remains a challenge Edilamara de Oliveiraa, Pedro Giavina-Bianchia,, Luiz Augusto Marcondes Fonsecaa, Alfeu Tavares Franc-ab, Jorge Kalila a
Division of Clinical Immunology and Allergy, University of Sa˜o Paulo, R. Prof. Artur Ramos 178 ap.211A, 01454-904, Sa˜o Paulo, SP, Brazil b Division of Clinical Immunology and Allergy, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Received 22 March 2007; accepted 15 June 2007 Available online 3 August 2007
KEYWORDS Allergic bronchopulmonar aspergillosis; Aspergillus fumigatus; Difficult to control asthma; Recombinant allergen; Specific IgE
Summary Introduction: Allergic bronchopulmonary aspergillosis (ABPA) is a complex disease, triggered by a hypersensitivity reaction to the allergens of Aspergillus fumigatus, a fungus that opportunistically colonizes the lungs of patients with asthma. The diagnosis of ABPA is difficult. A major problem is the lack of standardized allergens used in the determination of specific IgE, but the use of recombinant allergens has been proposed to overcome this. The aim of the present study is to evaluate whether serological tests for IgE specific to recombinant allergens of A. fumigatus (rAsp) can aid in the detection of sensitization to this fungus and in the diagnosis of ABPA. Methods: This was an observational, cross-sectional study. The diagnosis of ABPA, using classical criteria, was searched in 65 asthmatics patients with immediate cutaneous reactivity to A. fumigatus. After that, serum titers of IgE against rAsp f 1, rAsp f 2, rAsp f 3, rAsp f 4 and rAsp f 6 were determined. In order to compare the differences between patients with confirmed and excluded diagnosis of ABPA, the two-tailed Fisher’s exact test was used. Results: Although 19 of 65 patients had IgE against at least one recombinant, the disease was diagnosed in only six patients by classical criteria. One of them had IgE against all recombinant allergens tested and another one had antibody against Asp f 3. Discussion: The determination of serum IgE against recombinant A. fumigatus allergens in this group was not helpful to make the diagnosis of ABPA, neither to detect sensitization to fungus. & 2007 Published by Elsevier Ltd.
Corresponding author. Tel.: +5511 30320562; fax: +5511 30713189.
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[email protected] (P. Giavina-Bianchi). 0954-6111/$ - see front matter & 2007 Published by Elsevier Ltd. doi:10.1016/j.rmed.2007.06.018
ARTICLE IN PRESS ABPA’ diagnosis remains a challenge
Introduction Allergic bronchopulmonary aspergillosis (ABPA) is a complex disease, triggered by a hypersensitivity reaction to the allergens of Aspergillus fumigatus (Asp f).1 The clinical evolution of the disease consists of periods of remission and exacerbation, and may include the appearance of central bronchiectasis and progress to terminal stages with the presence of pulmonary fibrosis.2 The disease may be divided into two categories: ABPA-S (serological) that is the initial phase of the disease, when no bronchiectasis is observable by high-resolution tomography but other serological criteria of the disease are present, and ABPA-CB (central bronchiectasis), when bronchiectasis occurs.3 In Brazil, a study carried out in Rio de Janeiro in 1997 found 20% prevalence of ABPA among asthmatic patients sensitive to the fungus A. fumigatus.4 A form of diagnosis based on clinical, laboratory, radiological, and immunological aspects should be sought, since the early recognition and treatment of ABPA can prevent the progression of the disease to pulmonary fibrosis. The clinical criteria most widely used are those proposed by Rosenberg and coworkers,5 modified by Greenberger and Patterson.6 The criteria are non specific for ABPA and it is very difficult for all of them to be present at once, since, during the natural evolution of the disease and during treatment with anti-inflammatory drugs, there are fluctuations in the levels of antibodies and in the presence of eosinophilia and pulmonary infiltrates. Years are often necessary before the diagnosis is confirmed, by which time irreversible pulmonary lesions are already present. The difficulty in obtaining satisfactory amounts of purified, standardized Asp f extract for performing reliable and reproducible immunological tests, along with the need for fulfilling multiple criteria, render ABPA a difficult and complex disease to diagnose. In order to overcome these difficulties and seeking to simplify and standardize ABPA diagnosis, great efforts have been invested in techniques for the production of recombinant allergens.7 Some works have demonstrated that, depending on which antibodies against these allergens are present, one can distinguish patients with ABPA from asthmatic patients only sensitized by the mold.8–10 Asp f antigens belong to two functional categories: secreted proteins and cytoplasmic proteins; allergens such as Asp f 1, Asp f 3, and Asp f 5 belonging to the first category and Asp f 6 and, possibly, Asp f 4 and Asp f 2, to the second. Cytoplasmic proteins are unlikely to be found in a free form as aeroallergens, which partly explains the lack of IgE specific for these allergens in asthmatic patients sensitized to Asp f. By contrast, patients with ABPA show growth of the fungus in the lungs, and its interaction with the local cellular defense system leads to the exposure of cytoplasmic proteins, eliciting a specific immune response.11 Therefore, a new diagnostic tool was proposed, which uses a panel of recombinant antigens (rAsp f) capable of detecting only sensitization to fungus (rAsp f 1, rAsp f 3 and rAsp f 5) or ABPA (rAsp f 2, rAsp f 4 and rAsp f 6). However, this tool requires further evaluation before it is universally accepted.
2353 The aims of the present study are (1) to establish early serological diagnosis of ABPA in a group of patients sensitized to Asp f; (2) to evaluate clinical, laboratory, and radiological differences between asthmatic patients with ABPA and those only sensitized; (3) to evaluate whether serological tests for IgE specific for recombinant Asp f allergens can aid in the detection of sensitization to this fungus and in the diagnosis of ABPA.
Methods An observational, cross-sectional study approved by our Institutional Review Board was done in the outpatient facility of Clinical Immunology and Allergy Division of Medical School of Sa ˜o Paulo University. All the patients were informed and gave their consents. In the study 65 patients with prior diagnosis of asthma and positive immediate-reading skin test for A. fumigatus were included, between March 2004 and October 2005. The percutaneous immediate-reading test was performed by prick test, using antigenic extracts of Asp f at 5% (Alk-Abello `s).12 If subjects did not react to the prick test, the Asp f antigenic extract was re-administered, intradermal, at 1% (Alk-Abello `s).13
Clinical history Patients were first submitted to judicious clinical anamnesis. Asthma diagnoses were confirmed and the disease was classified into one of its severity categories, according to the GINA criteria.14 Clinical data relevant to the study were: age at onset of asthma, personal and family history of the disease, use and dosage of systemic and inhaled corticosteroids, and frequency and intensity of asthma symptoms in the last 12 months.
Laboratory tests On the same day as the anamnesis was performed, blood samples were drawn for eosinophil count, measurement of serum IgE, and serological studies. The presence of 350 cells or more per mm3 of blood was considered as eosinophilia.15 Total IgE was measured by the UniCAPs method, using the UniCAP 100 apparatus (Pharmacia-diagnostics), and results were calculated in ng/ml. In addition to blood tests, patients underwent parasitological stool tests, lung function tests and radiological studies (thorax X-rays and highresolution computerized tomography (HRCT)). Lung function tests were analyzed according to national and international consensus criteria.16,17 Serological tests Precipitin detection was carried out by gel diffusion.18 Three Asp f antigens were used, two of which were purchased from the Greer Laboratory (USA) and the other was provided by the Clementino Fraga Filho University Hospital, RJ, Brazil. Sera were concentrated three times by lyophilization prior to use.19 Evaluation of serum specific IgE for A. fumigatus (IgEm3) and for recombinant A. fumigatus antigens (rAsp f 1-IgERm218, rAsp f 2-IgERm219, rAsp f 3-IgERm220, rAsp f
ARTICLE IN PRESS 2354 4-IgERm221 and rAsp f 6-IgERm222) were evaluated by the UniCAPs method. Results were given in kUA/L, where A represents allergen-specific antibodies (1 kUA/L ¼ 2.4 ng/ml). Classes (0–IV) were attributed to predefined level ranges. Specific IgG for A. fumigatus (IgGm3) were evaluated by the UniCAPs method. Results were given in mgA/l, where A represents antigen-specific antibodies. For a 1:100 sample dilution, the dosage range is 2–200 mgA/l.
ABPA diagnosis According to the essential criteria for the diagnosis of ABPA proposed in the literature,1,2,5,6,15,20 the following findings were valued in our group of patients: (1) positive precipitins; (2) total IgEX1000 ng/ml; (3) IgEm3Xclass II; (4) IgGm3 above 70 mg/ml and (5) central bronchiectasis. The presence of eosinophilia in the leucogram and of pulmonary infiltrates in the radiological tests were not considered as essential criteria, but reinforced the diagnosis when present. The following diagnoses were considered: (1) certain serological diagnosis: presence of the first four criteria; (2) certain serological and radiological diagnosis: presence of the second and fifth criteria along with at least one of the other three criteria; (3) excluded diagnosis: absence of the five criteria and (4) possible diagnosis: presence of any of the five criteria. Due to the great difficult in making ABPA diagnosis, we decided to use these less strict criteria.
Statistical analysis In order to compare the differences between patients with confirmed and excluded diagnosis of ABPA, the two-tailed Fisher’s exact test was used. Statistical significance was established at 5%.
Results General characteristics Our study sample comprised 65 patients, 40 women and 25 men, with diagnosed asthma and sensitized to Asp f as determined by immediate-reading skin tests. Age ranged from 12 to 81 years (mean 44.7 years; s.d. 15.83 years). According severity classification, 72% of the patients presented severe persistent asthma, 23% moderate persistent asthma, 2% mild persistent asthma and 3% intermittent asthma. Twelve percent patients did not use corticosteroids, 66% used only inhaled corticosteroids, and 22% used inhaled and oral corticosteroids simultaneously. None of the patients presented atopic dermatitis.
Sensitization to Asp f Of the 65 patients showing reactivity to the immediatereading skin test, 58.46% were positive to the percutaneous test and did not undergo to the intradermal test. The others 41.54% were negative to the percutaneous test and positive to the intradermal test. Serum IgE specific for Asp f (IgEm3)
E. de Oliveira et al. was detected in 40% patients, with different levels of antibodies for each patient. This antibody was detected in 47.37% percutaneous test-positive patients and in 29.63% intradermal test-positive patients. It was equal or greater than class II in 31.58% percutaneous test-positive patients and in 14.81% intradermal test-positive patients.
ABPA diagnosis Of the patients studied, 17 (26.15%) did not meet any of the criteria for ABPA diagnosis, with the exception of positive skin tests to Asp f. The disease was excluded in these cases. Twenty-six patients met only one criterion, 15 met two criteria, and a single patient met three criteria. ABPA diagnosis could not be confirmed for any of these 42 cases (64.60%), which correspond to the group of possible disease. None of the patients met the criteria for serological diagnosis (ABPA-S). Diagnosis was confirmed in six patients who showed central bronchiectasis and the minimal essential criteria. Therefore, 9.20% of sensitized patients to A. fumigatus presented ABPA. Major laboratory findings in the subgroup of patients with confirmed ABPA diagnosis are shown (Table 1).
Comparison between patients with diagnosed ABPA and patients sensitized to Asp f A comparison of the clinical data of patients with (certain diagnosed) and without (excluded) ABPA, including age, gender distribution, age at onset of asthma, associated atopic disease symptoms, severity of asthma, use of oral corticosteroids, and absence from work, showed no statistically significant differences between the two subgroups. Concerning the immediate-reading skin tests for Asp f, patients with confirmed ABPA diagnosis showed a trend towards greater positivity to the percutaneous test, whereas the group with excluded diagnosis showed a trend towards greater positivity to the intradermal test. Most patients did not show eosinophilia (66% in the confirmed ABPA diagnosis subgroup and 53% in the excluded diagnosis subgroup). An analysis of each of the essential diagnostic criteria in the sample showed that 63% of patients showed total serum IgE above 1000 ng/ml. Sixty-six percent of ABPA patients showed a predominance of severe obstruction, a higher prevalence than in patients without ABPA. However, this difference was not significant. Table 1 Essential criteria in patients with ABPA diagnosis confirmed. Patients
Total IgE (ng/ml)
IgE m3 (kUA/l)
IgG m3 (mgA/l)
Pp
Bc
Case1 Case 2 Case 3 Case 4 Case 5 Case 6
6400 1061 2820 1380 1608 2520
43.2 0.61 2.81 0 1.36 1.25
96.3 25.6 13 8.42 9.05 14.8
+ + + +
+ + + + + +
(+) (+) (+) (+) (+) (+)
(+) () (+) () (+) (+)
Pp, precipitin; Bc, bronchiectasis.
(+) () () () () ()
ARTICLE IN PRESS ABPA’ diagnosis remains a challenge
2355
In the HRCT of the thorax, the most common finding in the large airways in both subgroups was thickening of the bronchial wall, which was present in 100% of patients with confirmed ABPA diagnosis and in 72% of patients with excluded diagnosis. All patients with ABPA showed central bronchiectasis. Of the 17 patients without ABPA, only one showed no alterations in the computerized tomography. The only case of presence of signs of fibrosis came from the ABPA subgroup. Regarding the small airways, bronchiolar dilatation was present only in patients with diagnosed ABPA. Centrolobular nodules were found in 60% of patients with ABPA and in only 12% of sensitized patients, but this difference was not significant. Central bronchiectasis was not seen in the thorax X-rays of three (50%) patients in the confirmed ABPA subgroup. This confirms that chest radiographs are insensitive compared to chest CT. Of the alterations detected by X-ray, pulmonary hyperinflation and thickening of the bronchial walls were common findings in both groups. Irregular nodules and opacities were found only in patients with ABPA.
Serological tests Only eight patients (12%) showed positive results in the precipitin assay, four of them from the ABPA group. Nineteen patients (29%) showed detectable levels of IgE specific for at least one of the recombinant allergens. Analyzed individually, IgE against rAsp f 2 and rAsp f 3 were those most frequently found, and in some cases were the only antibodies detected. The majority of patients with detectable levels of IgE specific for the recombinant allergens also showed IgE reactive to the natural antigens of Asp f (IgEm3). Of the six patients in the diagnosed ABPA subgroup, only two showed serum IgE specific for the recombinant allergens (Table 2). One of the patients showed different levels (classes I–IV) of these antibodies against the five allergens tested, whereas one showed antibodies only against Asp f 3 (class I). In the group of 17 patients for which ABPA was excluded, no specific serum IgE against any of the recombinant allergens was detected.
Discussion Difficulties in diagnosing ABPA, as those frequently reported in the literature, were faced at each of the stages of the present work. An older Brazilian study had already showed low rate of asthma and rhinitis patients with positive Table 2
Specific IgE against recombinant allergens in patients with ABPA diagnosis confirmed.
Serum IgE
Patients Case 1
IgERm IgERm IgERm IgERm IgERm
218 219 220 221 222
percutaneous test to Asp f (4.44%).21 When analyzing patient sensitization, there were great discrepancies between skin test results and the presence of serum IgE specific for Asp f (IgEm3); of the 65 patients with positive percutaneous or intradermal tests, only 26 (40%) also had detectable IgEm3. Most intradermal test-positive patients had IgEm3 less than class II (85.19%). Similar discrepancies have also been reported in the literature.4 Some factors must be considered, such as 1) the use of extracts from different sources for in vivo and in vitro tests; 2) the use of fungal extracts that are not yet standardized; 3) false positive skin tests, mainly the intradermal test; 4) the patients may be sensitized to an Asp f allergen other than those tested; 5) the patients may be sensitized to a fungal which shows cross-reactivity to Asp f; 6) low titers of circulating IgE against the allergenic components, which are present in low concentrations in the allergenic extract. This last reason could be due to a strong suppression of the IgE immune response by corticoteroids. ABPA diagnosis was excluded in 17 (26%) and confirmed in six (9.20%) of the patients sensitized to Asp f. Even in these six patients, ABPA diagnosis could be questionable, because we used less strict criteria. Studies of the prevalence of this disease among A. fumigatus sensitized asthmatic patients show results that range from 6% to 28%,4,15,22 which may be due to the lack of a single test to establish ABPA diagnosis. There were no statistically significant differences with respect to clinical, laboratory, or radiological findings between patients with confirmed ABPA and those with excluded diagnosis, probably due to the small number of patients in the confirmed diagnosis subgroup. Asthma was severe in all patients with ABPA and in 76% of patients without the disease. A purely serological diagnosis (ABPA-S) was not able to be established. The six patients diagnosed with ABPA showed central bronchiectasis. Only one of these patients fulfilled all serological criteria, another patient fulfilled 3 of the criteria and the other four patients fulfilled two criteria. Diagnosis could not be confirmed but remained suspected in 42 patients (64.60%), with greater probability in three patients showing central bronchiectasis alone, in two patients with bronchiectasis associated with one of the serological criteria, and in a single patient who fulfilled three serological criteria but showed none of the radiological criteria. There is a well-known advantage of the early diagnosis and adequate treatment of ABPA to the clinical evolution of
(r (r (r (r (r
Asp Asp Asp Asp Asp
f f f f f
1) 2) 3) 4) 6)
D, detectable; U, undetectable. Serum level—kU/l.
D D D D D
(11.1) (22.9) (15.1) (0.42) (7.73)
Case 2
Case 3
Case 4
Case 5
Case 6
U U D (0.53) U U
U U U U U
U U U U U
U U U U U
U U U U U
ARTICLE IN PRESS 2356 the patient, allowing the prevention of installation of irreversible pulmonary lesions.23 The fact that no serological diagnosis could be made in the present study, and the large number of patients whose diagnoses remained uncertain reflects the difficulties in establishing an ABPA diagnosis. The analysis of the levels of Asp f-specific IgE (IgEm3) and IgG (IgGm3), serological criteria specific for Asp f, showed that 16 patients (25%) showed IgEm3X class II, and only two patients (3%) showed IgGm3X70 mgA/l. Generally speaking, these levels are low when compared to the results found by other groups.10,22 In the subgroup of patients diagnosed with ABPA, four out of six patients tested positive for precipitins. Positivity to this test among patients with ABPA ranges from 57.10% to 100% in different works in the literature.5,15,24 Precipitins not specific for ABPA were present in approximately 10% of patients with asthma and without ABPA.1 Gel immunodiffusion showed greater positivity when compared to the IgGm3 assay, perhaps due to the greater number of extracts used. The only two patients with IgGm3X70 mgA/l also showed precipitins; one of these patients was from the ABPA subgroup. The majority of patients with ABPA did not show eosinophilia. As is the case with serological exams, eosinophilia may be negative in periods of disease remission and during corticosteroid use. ABPA was confirmed in one of the 14 patients in the sample under continuous treatment with inhaled and oral corticosteroids. The majority of patients (64.30%) were placed in the suspected diagnosis group. The presence of central bronchiectasis established by HRCT, one of the essential diagnostic criteria established, was observed in 11 patients (17%) in the sample, of which six were confirmed as having ABPA. The individual analysis of each specific serum IgE (IgER) in the group of 65 patients showed that IgE anti-rAsp f 2 and anti-rAsp f 3 were detected in a greater number of patients, with a frequency of 8% and 26%, respectively. There are no studies comparing the positivity of skin tests performed with a natural extracts of Asp f and the levels of serum IgE specific for recombinant allergens derived from this fungus. The repeated analysis of each IgER in the group of 26 patients that, in addition to positive skin tests, also showed detectable levels of IgEm3, showed an increased positivity. IgE anti-rAsp f 2 was present in 19% of patients and IgE antirAsp f 3 was in 54%, maintaining their position as the most reactive antigens, whereas IgE anti-rAsp f 1 was detectable in only 12% of patients. IgE anti-rAsp f 2 and anti-rAsp f 3 were the only antibodies to appear alone. None of the 17 patients in the excluded diagnosis subgroup showed detectable IgER levels. The levels of specific serum IgE against recombinant allergens were below those described in the literature, and rAsp f 1 did not behave as a major allergen.11 In the subgroup of patients with ABPA, only one patient showed detectable levels of IgE against all recombinant allergens, while another patient showed only IgE against rAsp f 3. In the literature there is some evidence that recombinant Asp f allergens can be useful for elucidating the diagnosis of ABPA in cystic fibrosis patients, through their utilization in serum IgE detection25 or intracutaneous test.26 However, the data are not uniform and further studies with larger
E. de Oliveira et al. numbers of patients remain necessary.27–30 The evidence for the utility of these recombinant allergens in the diagnosis of ABPA in patients with asthma is scant.9,10 In the present study, the investigation of IgE specific for the recombinant allergens, that could constitute a tool for both the detection of sensitization to Asp f and for the diagnosis of ABPA, showed no advantage over the use of natural Asp f antigens. The lack of standardization of the allergens used in the diagnosis of ABPA, which affects the reproducibility of results, could not be overcome by using the recombinant allergens used in the present study. Asp f allergens, able to improve the detection of ABPA disease, making diagnosis more uniform across different healthcare facilities, remains a challenge.
Conflict of interest statement We do not have conflict of interest.
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