AspergillusIgG: Preliminary Prevalence Studies in Pulmonary Diseases.
Yuri Lebedin, Svetlana Vlasenko, Andrey Kuleshov.
The fungi of Aspergillus species are important human pathogens and established causative agents of systemic and local infections as well as allergic diseases. The isotypic pattern of antibody response to Aspergillus is variable and depends on the form of the disease. Low titre IgG-antibody response reflects anamnestic infection and has little or no clinical significance. However, in allergic broncho-pulmonary aspergillosis (ABPA) the level of Aspergillus-IgG is significantly elevated and is used as one of diagnostic criteria of this nosological form. Higher levels of Aspergillus-IgG can be detected in aspergilloma and invasive aspergillosis. The latter form of aspergillosis is a life-threatening disease mostly affecting immunosuppressed patients. The elevation of Aspergillus-specific IgG antibody titres in above mentioned diseases is very dramatic and these antibodies can be detected by immunoprecipitation (double immunodiffusion, DID).
However, our studies showed that in pulmonary diseases, e.g. chronic bronchitis, bronchial asthma, pulmonary fibrosis etc. the titres of Aspergillus-IgG are significantly elevated, but do not reach the sensitivity threshold of immunoprecipitation (DID). This milder form of aspergillosis (so called ‘fungal bronchitis’) is underdiagnosed and the specific anti-fungal treatment is not applicated.
Materials and Methods
1. Aspergillus-IgG EIA
Aspergillus-IgG EIA manufactured by Xema Co. Ltd., Moscow, Russia, was used in this study. This test for was designed for the detection of moderately elevated concentrations of specific antibody to Aspergillus specific antigens. Xema Aspergillus IgG EIA is a two-stage solid phase enzyme-linked immunosorbent assay. The test specimen (serum or plasma) is added to the Aspergillus antigen purified from culture filtrate, immobilized on polystyrene microwells (solid phase), and incubated in the Incubation Buffer. Specific IgG antibodies, if present in the specimen, bind to the antigen on the microwell surface. The microwells are then washed by detergent solution to remove unbound material, and mouse monoclonal gamma-chain specific antibody-enzyme (horseradish peroxidase) conjugate reagent is added. During the second incubation, the conjugate reagent binds immunologically to the IgG on the microwell surface, forming a complex labelled with peroxidase. The subsequent washing removes unbound conjugate. A solution of 3,3¢,5,5¢-Tetramethylbenzidine (TMB) is then added and incubated for 15-30 minutes, resulting in the development of a blue colour. The colour development is stopped by addition of 5% H2SO4 and the resulting yellow colour is measured spectrophotometrically at 450 nm. The concentration of Aspergillus-IgG is directly proportional to the OD value of the test sample and is calculated from the standard curve. The normal range was determined by testing serum specimens of 120 healthy blood donors. The distribution obtained was assymmetrical; the upper limit of the normal range was determined based on cumulative distribution percentage. All donor sera gave values less than 21 A.U. (arbitrary units) while 97.5% of them gave values less than 16 A.U. (Fig. 1). The latter value was chosen as the upper limit of the normal range.
The sensitivity of the method was ca. 10 times greater comparing to double immunodiffusion method (DID) -see Table 1.
Table 1. A comparative sensitivity of anti-A.f.-IgG EIA and DID in different patient groups.
2. Patient groups
The following patient groups were investigated in this study:
- 65 patients with bronchial asthma and suggested bronchopulmonary mycosis (age - 19-82 years, 38 females and 27 males);
- 50 patients with pulmonary or mixed pulmonary-intestinal types of cystic fibrosis ( 30 children of age 2-15 years, 17 males and 18 females, and 15 adults of 16-36 years, 6 males and 9 females);
- 15 patients with primary or secondary inborn anatomical defects of lung tissue (16-74 years, 8 males and 7 females);
- 30 patients with interstitial lung diseases (sarcoidosis, idiopathic fibrotic alveolitis, exogeneous allergic alveolitis - 12 patients, other interstitial lung disseminations of unknown ethiology - 18 patients).
Results and Discussion
1. Bronchial asthma
The preliminary suggestion of possible participation of Aspergillus infection in asthma course was made by physicians by clinical manifestations (anamnestic exposition to fungi, inadequate reactions to antibiotics, yellowish inclusions in sputum) or indirect laboratory indications (high peripheral or sputal eosinophilia, high total IgE, positive specific IgE to Aspergillus). The data below do not reflect serologic prevalence in random asthmatic patients.
In a group of 65 patients with asthma and suggested bronchopulmonary mycosis specific Aspergillus-specific IgG (spIgG) was elevated in 23 (35.4%) of patients (Fig.2.). In 78.2% of them it was accompanied by specific IgE to Aspergillus. A diagnostic triad characteristic of allergic bronchopulmonary aspergillosis - elevated total IgE, positive spIgE and elevated spIgG) was found in 9 patients of the above 23. The other 9 patients shown elevated spIgG, positive spIgE and normal total IgE. The last subgroup of 5 patients with elevated spIgG were negative for spIgE; one of them shown total IgE over 1000 IU/ml. Among patients with asthma and normal spIgG only one female patient has shown positive spIgE to Aspergillus, normal total IgE and positive Aspergillus culture from sputum. This false-negative result may be attributed to the considerable variability of ABPA manifestations at different clinical stages of the disease.
2. Cystic fibrosis
In cystic fibrosis a probability of secondary bronchopulmonary mycoses is elevated as a result of chronic purulent inflammatory processes and continuous antibacterial treatment of patients. An early diagnosis of secondary mycosis is helpful to modify treatment and improve clinical state of a patient.
Elevated levels of spIgG was found in 6 of 35 chidren patients (17.1% - Fig.3.). In five of these six elevated spIgG was accompanied by elevated total IgE and positive spIgE. One female patient with elevated spIgG demonstrated neither elevated total IgE nor spIgE.
Among the adult patients, elevated levels of spIgG were found in 6 of 15 (40% - Fig.3.). In all these six cases this finding was accompanied by positive spIgE and in 3 of them - by elevated total IgE (more than 1000 IU/ml).
Therefore, laboratory findings characteristic of ABPA (elevated specific IgG and specific IgE to Aspergillus) were found in 14.3% of pediatric patients and 40% of adult patients with cystic fibrosis. This difference may reflect the fact that the probability of development of bronchopulmonary mycoses is elevated with age of patient.
3. Inborn or acquired anatomical defects of lung tissue
It is widely known that inborn or acquired anatomical defects of pulmonary tissue may promote development of secondary bronchopulmonary mycoses. We investigated 15 patients - 12 of them had secondary bronchoectases, 1 - disontogenetic bronchoectases, 1 - bullous pulmonary cyst and 1 - secondary bronchoectases with bullous emphysema.
In this patient group, elevated levels of spIgG were found in 2 patients (13.3%). No specific IgE to Aspergillus or elevated total IgE was found in any patient of this group. Thus, all the cases of mycosis revealed were suggested to be nonallergic and secondary by origin.
4. Interstitial lung diseases
The role of Aspergillus and other fungi in pathogenesis of interstitial lung diseases is not well established. There are some data concerning development of allergic alveolitis with interstitial pneumonitis following long-term inhalation of high doses of fungal allergens and development of secondary mycoses aggravating other interstitial lung diseases (sarcoidosis, idiopathic fibrotic alveolitis, etc.).
Long term and high dosage steroid therapy is considered to be one of the possible promoting agents in the development of secondary mycoses in severe interstitial lung diseases.
We investigated 30 patients with different forms of interstitial lung diseases, 18 of them - with unknown ethiology. Elevated serum levels of spIgG were found in 6 patients, all of them - with unknown ethiology. In 5 of these 6 patients the levels of spIgG were extremely high - 120-800 AU. None of 30 patients shown elevated total IgE. Nevertheless, in one female with normal total IgE and a moderate elevation of spIgG we found polyvalent sensibilization to fungal allergens by RAST.
All the patients with extremely high anti-A.f.-IgG were younger (the average age - 31 year) than the other patients in ILD group (average age - 53 years) and had an early onset of severe chronic lung diseases - in childhood or in early postpuberty. Interestingly, the dissemination in all these 5 patients was localised predominantly (3 cases) or solely (2 cases) in the upper lobes; however, in the rest of spIgG negative ILD patients most cases (22 of 25) has shown other localisations - either in medial or in lower lobes.
The results presented here give evidence that diseases caused by Aspergillus species are relatively widely spread and often underdiagnosed. They may be either primary or secondary but in both cases they need specific treatment to be cured. It seems critical to obtain the correct diagnosis as early as possible and this, in turn, needs more sensitive and specific diagnostic methods. A good example of such a method is Aspergillus IgG ELISA. It is simple, sensitive, specific and informative. We recommend to include this test as obligatory in the programm of laboratory investigation of patients with cystic fibrosis, defects of lung tissue, interstitial lung diseases and bronchial asthma with suggested fungal involvement.
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