Aspergillosis

Background: Allergic bronchopulmonary aspergillosis (ABPA), which is predominantly a disease of asthmatic subjects, is caused by hypersensitivity to Aspergillus antigens. Screening for Aspergillus sensitization in asthmatic subjects could identify those who are at risk for ABPA. Few studies have shown that fungal sensitization could be an important risk factor for asthma severity. We sought to determine the frequency of sensitization to Aspergillus antigens in asthmatic subjects and its effect on disease severity. We also determined the occurrence of ABPA in these subjects.

Design: Prospective study of consecutive patients with asthma.

Setting: Tertiary university referral hospital, outpatient department.

Patients and methods: One hundred five asthmatic subjects and 26 volunteers underwent skin testing with aeroallergens, including Aspergillus, serum precipitins against Aspergillus antigens, and specific IgG against Aspergillus fumigatus, total serum IgE levels, and routine blood and radiologic investigations. ABPA was diagnosed when all eight major criteria were fulfilled. Results: Thirty patients (28.5%) had a positive skin reactivity to Aspergillus antigens. Eleven patients (10.4%) had positive specific reactions to IgG, and 8 patients (7.6%) demonstrated positive reactions to serum precipitins. Eight of these 30 patients (26.6%) received diagnoses of ABPA, which was 7.6% of the total. None of the control subjects were sensitized to Aspergillus antigens. The patients were classified into the following four groups: negative skin test results; positive reactions to aeroallergens other than Aspergillus; positive reactions to aeroallergens including Aspergillus antigens; and patients with ABPA. Based on clinical and serologic parameters, patients with Aspergillus-sensitive asthma and ABPA had a significantly more severe form of the disease.

Conclusions: Sensitization to the mold Aspergillus increases the severity of asthma. ABPA should be excluded in all patients with Aspergillus-sensitive asthma.

Key words: allergic bronchopulmonary aspergillosis; Aspergillus; asthma; sensitization; severity

Abbreviations: ABPA = allergic bronchopulmonary aspergillosis; AEC = absolute eosinophil count; ELISA = enzyme-linked immunosorbent assay. TLC = total leukocyte count

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Aspergillus species are ubiquitous, occur worldwide, and are known to cause four distinct clinically recognizable forms of hypersensitivity respiratory disorders (ie, allergic bronchopulmonary aspergillosis [ABPA], allergic Aspergillus sinusitis, IgE-mediated asthma, and hypersensitivity pneumonitis). (1) ABPA, the most frequently recognized manifestation of allergic aspergillosis, is an indolent disease with a protracted course, occurs worldwide, and is now seen as an important emerging disease in India. (2)

The prevalence of ABPA is speculative as the few earlier studies (3-7) that were performed adopted widely different diagnostic criteria. In order to determine the incidence of ABPA, which is a disease predominantly of asthmatic subjects, it may be appropriate to study the frequency of sensitization to Aspergillus antigens in asthmatic subjects. Sensitization to Aspergillus conidia occurs in asthmatic subjects when the thick secretions, which are usually present in the airways, trap the fungal spores. This generally develops in atopic subjects and is sustained by the continuous inhalation of Aspergillus antigens, resulting in acute asthma. (8) The reported frequency of skin test reactivity to Aspergillus antigens in patients with asthma has varied from 16 to 38% in different parts of the world. (4,9-13) Similarly, the prevalence of ABPA also varies from 1 to 11% in patients with asthma, (4-7,14-16) and from 25 to 28% in Aspergillus skin test-positive asthmatic subjects. (6,7) These variable prevalence rates probably reflect the lack of a single diagnostic criterion with a standardized test. (17) Data from the subcontinent in this regard are lacking.

Over the past few years, attention has been focused on the role of sensitization to fungi in asthmatic subjects, as this is an important risk factor for the increasing severity of the disease. (18,19) A European Community respiratory health survey (18) in 30 centers demonstrated that the frequency of sensitization to Alternaria alternata and/or Cladosporium herbarum increased significantly with increasing asthma severity. No association between severity of asthma and sensitization to pollens or eats was found in this cross-sectional population-based study. Previous studies have shown that sensitization or exposure to fungi increases the risk of death from asthma (20) and also of acute attacks of asthma requiring ICU admission. (21) However, in none of these studies was sensitivity to Aspergillus antigens was assessed. The only study to link Aspergillus sensitization with severity of asthma was conducted simultaneously in Cleveland and London. (11) The investigators recorded that a positive Aspergillus skin test result in patients with asthma was related to the severity of airway obstruction and stated that this was an unexpected finding.

In light of this situation, we attempted to ascertain the frequency of sensitization to Aspergillus antigens in patients with asthma and its effect on the severity of the disease. The study, which was conducted in a large tertiary center, also sought to determine the occurrence of ABPA in asthmatic subjects in India.

MATERIALS AND METHODS

Study Group

A total of 115 consecutive patients who had asthma with or without rhinitis were included in the study. Patients were included if they were [greater than or equal to] 15 years of age, had never smoked, and if their asthma had been confirmed by demonstrating a reversibility of at least 12% and an increase of 200 mL in FE[V.sub.1] after inhaling 200 [micro]g of salbutamol. (22) Pregnant and lactating women, and patients with other disorders were excluded. Of the 115 patients, 105 (59 men and 46 women) completed the study protocol (mean age, 27.4 years; age range, 15 to 56 years).

Control Group

The control group comprised 26 healthy volunteers (17 men and 9 women) with no history of atopy (mean age, 27.2 years; age range, 25 to 35 years). None of them had ever smoked or were on any kind of medication.

Methodology

Both the study and control subjects underwent an identical workup. All the subjects were asked to respond to a questionnaire on asthma. A thorough clinical examination with special attention to the respiratory system was performed. A history of the subject's oral corticosteroid use and antitubereulous therapy was recorded.

Pulmonary function testing and skin tests were performed on different days in both groups. It was ensured that neither the patient nor the control group had received antihistamines prior to undergoing the skin tests. Intradermal skin testing was performed with the usual set of 63 local aeroallergens including Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus tamari. Antigens were obtained from the Institute of Genomics and Integrative Biology, Delhi, India. Intradermal testing was performed with 0.01 mL of the allergens (1:500 weight/volume) along with negative control (buffer saline solution) and positive control (histamine, 100 [micro]g/mL). The results of the skin test were interpreted as per the method described by Schwartz and Greenberger, (6) which is as follows: negative (-), wheal and erythema different from those in control subjects; 1+, 3 to 6 mm wheal and 11 to 20 mm erythema; 2+, 6 to 9 mm wheal and 21 to 30 mm erythema; 3+, 9 to 13 mm wheal and 31 to 40 mm erythema; and 4+, > 15 mm wheal with pseudopods and > 40 mm erythema. Patients with [greater than or equal to] 2+ were considered to be positive for type I hypersensitivity. None of the subjects had ever undergone a skin allergy testing. Blood was drawn for total leukocyte count (TLC) and differential leukocyte count, absolute eosinophil count (AEC), serum precipitins against Aspergillus species, specific IgG antibodies against A fumigatus, and serum IgE levels.

The double diffusion-in-gel method, as described by Ouchterlony, (23) was used to detect serum precipitins against Aspergillus species. The four species tested were A fumigatus, A flavus, A niger, and A tamari. Neat serum samples were used. The antigens employed were prepared from the cultural filtrate of 4 weeks growth of the representative strains of Aspergillus species by Seitz filtration and dialysis, and were concentrated 10-fold to 15-fold. (24) Specific IgG antibodies against A fumigatus were determined by the enzyme-linked immunosorbent assay (ELISA) method. The cutoff for the specific IgG ELISA was the mean absorbance value obtained for 20 healthy individuals plus 3 SDs (0.135). (25) The total serum IgE levels in patients and control subjects were determined by the Sandwich-ELISA method (Human IgE ELISA quantification kit; Bethyl Laboratories; Montgomery, TX). The cutoff for the total IgE ELISA was the mean absorbance value obtained for 26 healthy individuals in our study plus 3 SDs (0.210 [equivalent to 226.29 [U/mL]).

The sputum of all patients was tested for pathogenic fungi by direct microscopy (10 to 20% KOH), and culture was performed on Sabouraud dextrose agar containing chloramphenicol (0.05 mg/mL), and on Sabouraud dextrose agar containing chloramphenicol and cycloheximide (0.5 mg/L). Cultures were identified by a detailed study of their colonial and microscopic features.

Roentgenograms of the chest and paranasal sinuses were also carried out in all subjects. CT scans of the thorax and the paranasal sinuses were performed in patients when required. This study had the approval of the institutional ethics committee.

The statistical analysis was performed using a statistical software package (SPSS, version 11.0; SPSS; Chicago, IL). The data were represented as range, mean, and SD. The unpaired t test, [chi square] test, and analysis of variance were applied. When two or more sets of variables were compared, and the p value was < 0.05, it was considered to be significant.

RESULTS

A total of 105 patients (59 men and 46 women) completed the study protocol. Symptoms of rhinitis were present in 89 patients (84.7%), and a family history of atopy was present in 44 patients (42%). The TLC ranged from 3,500 to 19,300 cells/[micro]L (mean [[+ or -] SD], 8,400 [+ or -] 2,200 cells/[micro]L), and the AEC ranged from 110 to 3,720 cells/[micro]L (mean, 1,550 [+ or -] 380 cells/[micro]L). FE[V.sub.1] ranged from 0.56 to 4.6 L (mean, 2.4 [+ or -] 0.86 L), and FVC from 1.5 to 5.0 L (mean, 3.3 [+ or -] 0.85 L). Mild airflow obstruction was seen in 60 patients (57%), moderate airflow obstruction was seen in 27 patients (25%), and severe airflow obstruction was seen in 18 patients (17.2%). The total serum IgE level ranged from 17.27 to 2,489.11 IU/mL (mean, 1,340 [+ or -] 524.35 IU/mL). In control subjects, the level ranged from 17.27 to 155.50 IU/mL (mean, 72.55 [+ or -] 21.38 IU/mL). Sensitization to local aeroallergens was observed in 79 patients (75%). Of the fungal aeroallergens other than Aspergillus that were tested, sensitization to Trichoderma was present in 18 patients (17.1%), Cladosporium was present in 17 patients (16.1%), Alternaria was present in 13 patients (12.3%), and Candida was present in 10 patients (9.5%). In the 26 control subjects, skin testing was performed with the 17 most common aeroallergens, which were identified in 79 patients. Eight control subjects (30.7%) were sensitized to one or more aeroallergens.

Skin Testing With Aspergillus Antigens

Thirty patients (28.5%) showed immediate hypersensitivity to one or more Aspergillus antigens. Of these patients, 24 (80%) were sensitized to more than one Aspergillus antigen. Twenty-nine patients (97%) were sensitive to A fumigatus, 21 patients (70%) were each sensitive to A flavus and A tamari, and 19 patients (64%) were sensitive to A niger. Late cutaneous reactions were seen in 19 patients (64%), while 1 patient (1.2%) was exclusively positive to Aspergillus antigens. All control subjects were negative for Aspergillus antigens.

Serum Precipitins Against Aspergillus Antigens

Eight patients (7.6%) demonstrated the presence of positive serum precipitins to Aspergillus species. Four patients (3.8%) showed the presence of serum precipitins against A fumigatus only, two patients (1.9%) against A flavus only, and two patients (1.9%) against both. Serum precipitins were not detected in any of the control subjects.

Specific IgG Antibodies Against A fumigatus

Specific IgG antibodies were positive in 11 patients (10.4%). This was not detected in any of the control subjects.

Chest Radiology

Abnormalities in chest radiographs were seen in 13 of 105 patients (12.3%). Transient pulmonary infiltrates were present in eight patients (7.6%), ring opacities were present in seven patients (6.6%), bilateral hyperinflated lung was present in three patients (2.8%), air-fluid levels were present in one patient (0.95%), and calcified opacities were present in one patient (0.95%).

ABPA

Of the 105 patients with asthma, ABPA was diagnosed in 8 (7.6%) [Table 1] as they fulfilled all eight major criteria for diagnosis, including the presence of specific IgE against A fumigatus, which was specifically tested for in these patients. (26,27) CT scans of the thorax also were performed in only these eight patients, all of whom had central bronchi with normal tapering of the distal bronchi. None of these patients had been previously recognized as having ABPA.

Clinical Categorization

Based on these results, the following five groups emerged: group A, control subjects (n = 26); group B, asthmatic subjects with negative skin test results (n = 26); group C, asthmatic subjects with positive skin test results for common aeroallergens, but negative results for Aspergillus antigens (n = 49); group D, patients with skin test results that were positive for common aeroallergens including Aspergillus antigens (n = 22); and group E, patients with ABPA (n = 8). The clinical profile of these five groups is summarized in Table 2.

Comparison of the Five Groups

Duration of Illness: The total duration of illness was significantly longer in patients with ABPA (group E) than in those in all other groups (p = 0.003). Patients in group D also had a significantly longer duration of illness compared to those in groups B (p < 0.0001) and C (p < 0.0002). Patients in group C also had a significantly longer duration of illness than those in group B (p = 0.04). Similarly, the mean duration of asthma was significantly longer in group E than in all other groups (p < 0.001). Patients in group D also had asthma for a significantly longer duration than those in groups B (p < 0.0001) and C (p < 0.0001). The mean duration of rhinitis was also significantly longer in group E than in all other groups (p < 0.001). Here too, the duration of rhinitis was significantly longer in group D than in groups B (p < 0.0012) and C (p < 0.0001) [Table 3].

Age at Onset of Illness: The mean age at onset of illness was significantly lower in group E than in groups B (p < 0.0001), C (p = 0.02), and D (p < 0.0001). However, this did not differ significantly among groups B, C, and D. The mean age at onset of asthma was significantly earlier in group E than groups B (p = 0.02), C (p = 0.01), and D (p < 0.0001). This too did not differ significantly among groups B, C, and D. The mean age at onset of rhinitis was significantly lower in group E than groups B (p < 0.0001), C (p < 0.0001), and D (p < 0.0001). Patients in group D also had earlier onset of rhinitis when compared with those in groups B (p = 0.04) and C (p = 0.05) [Table 3].

Symptomatology: The number of patients with a history of hemoptysis (four patients; 50%), fever (four patients; 50%), chest pain (four patients; 50%), and expectoration of sputum plugs (three patients; 37.5%) was significantly higher in group E compared to the other groups (p < 0.03).

Therapy: A history of frequent prescriptions for short courses of oral corticosteroids was elicited from 10 patients (20.4%) in group B, 4 patients (15.3%) in group C, 10 patients (45.4%) in group D, and 6 patients (75%) in group E. The number was significantly greater in group D than in group C (p = 0.004). Group E also had significantly more prescriptions than groups B (p = 0.05) and C (p = 0.03). Antituberculous therapy had been prescribed in one patient (13%) in group C, one patient (4.5%) in group D, and six patients (75%) in group E. This was significantly more in group E than in groups C (p < 0.0001) and D (p = 0.0136) [Table 3].

TLC: The mean TLC in group E was significantly higher than those in groups B (p = 0.005), C (p = 0.002), and D (p = 0.0168). This did not differ among other the groups (Table 4).

AEC: The mean AEC in group E was significantly higher than those in groups B (p < 0.0001), C (p < 0.0001), and D (p = 0.0024). Similarly, group D had a mean AEC that was significantly higher than those in groups B (p < 0.0001) and C (p < 0.0001). This did not differ between groups B and C (Table 4).

Pulmonary Function Testing: Mild obstruction was observed in 18 patients (69.2%) in group B, 28 patients (57.1%) in group C, 11 patients (50%) in group D, and 3 patients (37.5%) in group E. Moderate obstruction was seen in 6 patients (23%) in group B, 12 patients (24.4%) in group C, 8 patients (36.3%) in group D, and 1 patient (12.5%) in group E. Severe obstruction was observed in two patients (7.69%) in group B, nine patients in group C (18.3%), three patients (13.7%) in group D, and four patients (50%) in group E. The total number of patients with severe obstruction was significantly higher in group E when compared with other groups (p < 0.04). The mean FE[V.sub.1], FVC, and peak expiratory flow rate did not differ significantly among groups B, C, and D (Table 4).

Serum Total IgE: The mean total IgE level was significantly higher in group E than in groups B (p = 0.0002), C (p < 0.0001), and D (p < 0.0011). The mean total IgE level in group D was also significantly higher than that in groups B (p = 0.0033) and C (p = 0.0009) [Table 4].

DISCUSSION

The close link between the mold Aspergillus and asthma makes it imperative to establish the frequency of Aspergillus sensitization in asthmatic subjects in each geographic region. Our study, which carried out to document the frequency of sensitization to Aspergillus antigens in asthmatic subjects in Delhi, found that 28.5% of our patients had a positive skin test reaction to Aspergillus antigens and that 7.6% of patients had test results that were positive for the presence of serum precipitins. Incidentally, ABPA was later diagnosed in the patients with serum precipitins. However, it has been well documented that test results in asthmatic subjects without ABPA can be positive for the presence of serum precipitins. (4,9,11,13) Furthermore, 10.4% of our patients had test results that were positive for specific IgG. Our study corroborates the results of earlier studies from the West. Longbottom and Pepys (9) in 1964 demonstrated that 38% of their 238 asthmatic subjects had a positive skin test result for Aspergillus antigens, while 9% had positive test results for the presence of serum precipitins. This was further confirmed by Henderson et al (4) when they found that 24% of their 39 asthmatic patients had skin test results that was positive for Aspergillus antigens and that 13% of asthmatic patients had skin test results that were positive for the presence of serum precipitins to Aspergillus antigens. Another large study (10) from the United Kingdom reported that 16% of their 656 asthmatic patients had a skin test that was positive for A fumigatus. In a comparative study (11) of the prevalence of sensitization to Aspergillus antigens among asthmatic subjects in Cleveland and London, it was observed that 28% of asthmatic subjects from Cleveland and 23% of those from London had immediate skin reactivity to Aspergillus antigens. Furthermore, 7.5% of the patients from Cleveland and 10.5% from London had Aspergillus precipitins in their serum. A Canadian study (12) found that 21.5% of 200 asthmatic subjects reacted to Aspergillus antigens, while another report from South Africa (13) observed a skin test positivity of 22%, while 5% were positive for serum precipitins. Overall, Aspergillus skin test reactivity has been recorded in 16 to 38% of asthmatic patients from different parts of the world, (4,9-13) whereas 5 to 13% of the asthmatic subjects were positive for serum precipitins. (4,9,11,13) It appears that the levels of sensitization to Aspergillus antigens in Delhi are similar to those from previous studies performed in the West.

Among our 30 patients with skin reactivity to Aspergillus antigens, 8 (26.6%) fulfilled all of the major criteria for the diagnosis of ABPA. This was 7.6% of our patients with asthma. In 1968, Henderson et al (4) reported that, of their 46 asthmatic patients, 11% had definite allergic aspergillosis and 22% had probable or definite allergic aspergillosis. An Australian group (14) diagnosed ABPA in 10% of their 250 patients with asthma. Greenberger and Patterson (5) evaluated 531 patients with asthma and observed ABPA in 32 patients (6%), 19 of whom (3.6%) had central bronchiectasis and 13 of whom (2.4%) had positive serology findings only. Donnelly et al (15) found the period prevalence of allergic bronchopulmonary mycosis (ABPM) due to Aspergillus and Candida to be just > 1%. Schwartz and Greenberger (6) determined that 28% of Aspergillus skin test-positive asthmatic subjects had ABPA, 10 of whom (35.7%) had ABPA-central bronchiectasis and 18 of whom (64.3%) had positive serology findings. Subsequently, Eaton et al (7) demonstrated the presence of ABPA in 25% of Aspergillus skin test-positive patients and supported the use of skin testing as a screening tool for ABPA in asthmatic subjects. A report from Saudi Arabia (16) suggested the period prevalence of ABPM to be 2.7% in 264 consecutive patients with asthma. Overall, the prevalence of ABPA varied from 1 to 11% in patients with asthma, (4-7,14-16) and from 25 to 28% in Aspergillus skin test-positive asthmatic subjects. (6,7)

As expected, our eight patients with ABPA had a more severe form of the disease. This was evidenced by the significantly longer duration of illness, the earlier age of onset of asthma as well as of rhinitis, and the higher mean TLC, AEC, and total serum IgE values. This group had significantly more patients with severe obstruction and had significantly more prescriptions for oral corticosteroids. When we evaluated our group of Aspergillus-sensitive asthmatic subjects, we had similar findings, suggesting that these patients also had a more severe form of the disease compared to patients with a skin test that was positive for antigens other than Aspergillus. In the study (11) conducted among asthmatic subjects simultaneously in Cleveland and London, a significant relationship was found between Aspergillus skin sensitivity and the severity of airway obstruction. Our group with Aspergillus-sensitive asthma had a greater number of patients with severe obstruction than the other two groups. In addition, more patients in this group had a history of rhinitis, eczema, and family history of atopy. However, this did not achieve statistical significance.

Some studies (18,19) have given recognition to the importance of fungal sensitization as a risk factor for the increasing severity of asthma. In a study (28) of 11 patients with a history of respiratory arrest due to asthma, 10 were sensitized to A alternata compared to 31 of the 99 asthmatic control subjects with no history of respiratory arrest. A retrospective study (20) from Chicago reported that mean mold spore levels, but not tree, grass, and ragweed pollen levels, were significantly higher for days on which asthma-related deaths occurred than for days on which no deaths occurred. They concluded that exposure to environmental molds may play a role in asthma-related mortality. A more recent study from New Zealand (21) reported that 20 of the 37 asthmatic subjects (54%) who had been admitted to the ICU had a skin test result that was positive for one or more fungal allergens compared with 15 of the 50 patients (30%) who had not been admitted to the ICU. This was further highlighted by the results of the multicenter European Community trial (18) in 1,132 adult asthmatic subjects, in which it was observed that sensitization to A alternata and/or C herbarum was a powerful risk factor for severe asthma. However, none of the above studies associated Aspergillus with severity of asthma.

Another aspect of considerable bearing was that a significant number of our patients had received antituberculous therapy because of their radiologic picture. The radiologic changes in ABPA are commonly seen in upper lobes and closely resemble those of pulmonary tuberculosis. This has serious clinical implications in countries with high tuberculosis prevalence, like India, as patients with ABPA often receive antituberculous therapy for a long time while lung damage continues to progress relentlessly. (29-32)

Our study brought forth the fact that sensitization to Aspergillus increases the severity of asthma. In view of this, it is crucial to screen asthmatic subjects for sensitization to Aspergillus antigens so as to identify those subject who are at risk. Furthermore, this study underscores the importance of excluding ABPA in all such subjects.

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* From the Departments of Respiratory Medicine (Drs. Maurya and Shah) and Medical Mycology (Dr. Gugnani), Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India; and the Institute of Genomics and Integrative Biology (Drs. Sarma and Madan), Delhi, India.

Presented in part at the Fifth Asia Pacific Congress of Allergology and Clinical Immunology and at the Seventh West Pacific Allergy Symposium, Seoul, South Korea, October 12-15, 2002.

Manuscript received April 21, 2004; revision accepted November 12, 2004.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Ashok Shah MD Department of Respiratory Medicine, Vallabhbhai Patel Chest Institute, University of Delhi, PO Box: 2101, Delhi 110 007, India; e-mail: ashokshah99@yahoo.com

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