Prevalence of antibodies to aspergilli in bronchial asthmatics.AA Kumar, RC Sahu, KK Subbannayyar, Jyothirlata, PV Rau, PG Shivananda
Sputum and bleed samples were analysed from 330 cases of bronchial asthmatics and 50 normal persons without features of bronchial asthma. The prevalence of aspergilli and antibodies to different Aspergillus species were detected using cultural methods and serological technique like agar gel double diffusion (DD), counter-immuno-electrophoresis (CIEP) and enzyme-linked immunosorbent assay (ELISA). Out of total 6.06% yielded Aspergillus species in sputum specimens repeatedly. Precipitins against Aspergillus species were detected in 7.88% of cases by DD and CIEP. ELISA test showed 20% of cases to be having antibodies to A. fumigatus. Allergic broncho-pulmonary aspergillosis can be detected in the early course of the disease in bronchial asthmatics using highly sensitive technique like ELISA.
Keywords: Antibodies, analysis,Aspergillosis, Allergic Bronchopulmonary, diagnosis,Aspergillus, immunology,Asthma, blood,immunology,Human, Sputum, analysis,immunology,
The members of the genus Aspergillus form an important group of pathogens of the broncho-pulmonary system. The spectrum of diseases caused by them encompasses aspergilloma, chronic necrotising pulmonary aspergillosis, invasive pulmonary aspergillosis, allergic broncho-pulmonary aspergillosis (ABPA), bronchial asthma and allergic alveolitis. The association between asthma and aspergilli was noted as early as in 1925. Investigations have confirmed that Aspergillus spores are more common in the sputum of patients with asthma than with other lung disorders.,
The present study was carried out to find out the prevalence of aspergilli and antibodies to Aspergillus species in bronchial asthmatics using cultural methods and serological techniques like agar gel double diffusion, counter-immuno-electrophoresis and enzyme-linked immunosorbest assay.
Sputum and blood samples were collected from 330 cases of bronchial asthma. These cases were selected from patients with various lung disorders attending the Pulmonary Tuberculosis and Chest Diseases Department of Kasturba Medical College Hospital, Manipal. The proven ABPA cases were excluded out of the study. The cases were subjected to routine blood examination, pulmonary function tests, absolute eosinophil count, immunoglobulin profile and bronchogram. Fifty normal persons without any features of bronchial asthma or any other lung complaints were included as controls.
Early morning sputum samples were collected from these cases on 3 consecutive days. After gross examination, the sputum samples were subjected to direct microscopy using KOH mounts, Gram's and Ziehl-Neelsen staining techniques. The smears were examined for fungal elements. The sputum samples were then inoculated into Sabouraud's dextrose agar slants with chloramphenicol (0.05 mg/ml) in duplicate. The tubes were incubated at room temperature and 37°C. Examination of the tubes was done regularly for upto 10 days of incubation and the aspergilli grown if any were subcultured onto Czapek-Dox synthetic agar medium. The macro and microscopic characters of fungal colonies were studied as per Raper and Fennell.
Serum samples collected from the patients were preserved at -70°C until further testing.
Antigens were prepared from A. fumigatus, A. niger and A. flavus species isolated from the sputum of patients. The preparation of antigens was done according to Kurup et al., The protein and carbohydrate content of the antigens were determined using standard methods.,
Agar gel double diffusion (D) :
The antigen-antibody interactions were carried out using DD technique according to the modified method of Wadsworth. The antigens were used at a protein concentration of 10 mg/ml.
Counter-immunoelectrophoresis (CIEP) :
The CIEP test was performed as per the method of Warnock using antigens at a protein concentration of 5 mg/ml.
Enzyme-linked immunosorbent assay (ELISA ) :
The culture filtrate antigen of A. fumigatus only was used as antigen in ELISA test. The antigen containing 40-50 µg protein/ml, in carbonate buffer (0.05 M, pH 9.6), was used to coat the wells of the micrititer plates (Dynatech MicroELISA systems, Germany). The concentration of the antigen and conjugate were determined by means of checkerboard titrations using positive and negative sera.
One hundred µl of the antigen was added to each well of the micro-ELISA plate and was incubated at 4°C for 18 hours. The wells were washed 3 times with phosphate buffered saline (PBS, 0.01M, pH 7.2) containing 0.05% Tween-20 (PBS-Tween). Serum dilutions ranging from 1:50 to 1:6400 were done in PBS-Tween, added to each well and the plate was incubated for 2 hours at 37°C. Then the plate was washed 3 times with PBS-Tween. One hundred µl of 1:1000 dilution of goat antihuman gammaglobulin conjugated with horseradishperoxidase (Sigma Chemical Co., U.S.A.) was added to each well. The plate was incubated at 37°C for 2 hours and was washed 3 times with PBS-Tween. One hundred µl of substate and chromogen (0.01% H202, 0.05% 0-Phenylenediamine) in citrate phosphate buffer (pH 5.0) were added to each well. The plate was incubated in the dark at room temperature for 30 minutes. The reaction was stopped by the addition of 100 0 of 0.5N H2S04. The results were read visually as well as photometrically at 490 nm using a Dynatech MicroELISA reader. The test was considered positive with optical density (O.D.) of 0.30 or above. This cut-off value was obtained by taking 2 standard deviations (S.D. 2 x 0.041) above the mean value (0.223) in healthy normal subjects.
Results of the cultural and serological studies are shown in[Table - 1]. Out of the 330 cases studied, 20 cases (6.06%) yielded Aspergillus species in sputum samples repeatedly. Among the Aspergillus isolates, 12 were Aspergillus fumigatus (3.64%), 5 were A. niger (1.5%) and 3 were A. flavus (0.91%). Precipitins to Aspergillus species were detected in 26 cases (7.88%) both by DD and CIEP. ELISA test showed comparatively higher number of persons to be having antibodies to A. fumigatus (20%). Of the normal persons, sputum sample of only one case (2%) yielded A. fumigatus repeatedly. The serum sample of the same patient showed positive serology to A. fumigatus by DD, CIEP and ELISA test and all other serum samples were negative for antibodies to Aspergilli.
The genus Aspergillus is one of the most ubiquitous of all micro-organisms. Its approximately 150 species are common to soil, water and decaying vegetation throughout the world. Studies have shown that aspergilli have a significant potential to act as powerful allergens in the human respiratory tract., Depending on an individual's proclivity to develop allergy, and the setting, intensity and duration of a given aspergillus exposure, a variety of diverse, yet intimately related hypersensitivity syndromes may develop.
ABPA has been defined as a condition which usually presents as an acute, easily reversible asthmatic syndrome in a very atopic individual. Depending on the type of antigen used, the prevalence of precipitin antibodies to Aspergilli have been found to vary from 2.5% to 13.5% of bronchial asthmatics. In our study, the precipitins have been found in 7.88% of the cases. These findings agree with some of the earlier studies., In the present series of asthmatics, proven by history, clinical examination and pulmonary function studies. routine chest X-ray revealed no abnormalities or features suggestive of hyperinflation. As the presence of asthma is the usual feature of ABPA, some of the asthmatics may become potential candidates for ABPA after the onset of asthma and hence require long term follow up. With the employment of a highly sensitive technique like ELISA, antibodies to Aspergilli may be detected in more number of cases in this specific group of patients. This fact has been substantiated by our findings, where ELISA has shown antibodies to A. fumigatus in 20% of asthmatics whereas DD and CIEP detected precipitins in only 7.88% of cases. Therefore, bronchial asthmatics should be subjected to serological diagnosis of ABPA using a highly sensitive technique like ELISA. This serves as a useful aid in the diagnosis of the possibility of ABPA in the early cause of bronchial asthma.
[Table - 1]