Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay

An immunogold assay (IGA) was developed to detect IgG and IgE antibodies to Aspergillus fumigatus. Sixteen sera from patients with allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and normal controls were studied. All sera were also evaluated for antibodies against A. fumigatus by biotin-avidin linked enzyme immunosorbent assay (BALISA) and by agar gel double diffusion method. A. fumigatus specific IgG and IgE antibodies could be detected by IGA in all the patients' sera but not in the sera of normal controls. Both IgG and IgE antibodies to A. fumigatus could be demonstrated in all the sera by BALISA and normal controls showed only low levels of these antibodies. There was a positive correlation between the degree of reactivity detected by IGA, the BALISA titer and the precipitins by agar gel diffusion. It can be concluded that IGA is a reliable, sensitive and simple method capable of detecting both IgG and IgE antibodies against A. fumigatus in patient serum.     

Estudio comparativo de las reacciones serologicas cuantitativas con un antigeno metabolico de Aspergillus fumigatus

Summary The following quantitative serologic reactions: agar-gel immunodiffusion, complement-fixation, opposite electrophoresis and latex particle agglutination tests have been performed in 38 sera from mycologically proved pulmonary aspergillosis cases. A metabolic antigen from a strain ofAspergillus fumigatus according toAjello et al technic modified by us, has been employed. Sera from 120 subjects suffering from non-mycotic lung conditions, as well as 10 sera from histoplasmosis cases, 10 sera from S. A. blastomycosis and 2 sera from patients with lung aspergillosis produced byA. niger, gave negative results with the above mentioned seroligic reactions.One hundred per cent of positive results were obtained with the complement-fixation test (titre ranging from 1/20 to 1/1280), agar-gel immunodiffusion test (titre up to 1/64) and the opposite immunoelectrophoresis (titre ranging from 1/2 to 1/256). Twenty five per cent negative and 4 non-specific results were registered with the latex particle agglutination test.A correlation of the number of serum precipition bands obtained by the electrophoresis technic with the titre of the quantitative serologic reactions, as well as a correlation of the titre of the circulating antibodies with the severity of the clinical form of aspergillosis seems to be present.Electrophoretic motility of the specific antibody performed in 10 sera showed results like the IgM in 1 instance and an intermediate position between IgA and IgG in 9 samples.     

Synthetic biotinylated tetra β(1→5) galactofuranoside for in vitro aspergillosis diagnosis

The synthesis of a tetra ??(1→5) galactofuranoside was achieved using a thioglycoside donor with a methyl tert-butyl phenyl thio leaving group. This tetrasaccharide was conjugated to biotin and validated as antigen with the monoclonal antibody used for clinical detection of Aspergillus fumigatus galactomannan on streptavidin-coated microplates. Then we have shown its ability to detect antibodies associated with A. fumigatus induced disease by using sera from patients with Allergic broncho-pulmonary aspergillosis (ABPA) and correlated the results of antibody detection with those gained with a commercially available diagnostic test.     

Conformation of an immunoreactive undecapeptide fragment (10–20) of Asp f 1 by NMR and molecular modeling

Summary Allergic bronchopulmonary aspergillosis (ABPA), caused byAspergillus fumigatus, is a complication of allergic asthma. Asp f 1 secreted byA. fumigatus is reported to be a major allergen/antigen involved in pathogenesis of aspergillosis. A 11-mer immunodominant epitope (Leu-Asn-Pro-Lys-Thr⁵-Asn-Lys-Trp-Glu-Asp¹⁰-Lys) of Asp f 1 has shown immunoreactivity with specific IgG and IgE antibodies in the sera of patients with ABPA in ELISA inhibition assay. Various studies have suggested that the peptide has a potential use in the development of ELISA based diagnostic kit for early diagnosis of infections caused byA. fumigatus. In view of these interesting properties of the undecapeptide we have embarked on an investigation of its conformation to understand the relationship between structure and immunoreactivity. NMR and molecular modeling studies of the peptide suggest a structure with a β-turn spanning residues Asn⁶-Glu⁹ in water at pH 4.0, a β-pleated sheet in DMSO and α-helix in 40% HFA.     

Immunoreactivity of antigen extracts of Aspergillus fumigatus isolated from different sources

Allergic bronchopulmonary aspergillosis (ABPA) is the result of hypersensitivity to Aspergillus antigens in patients with long-standing atopic asthma. In the present study mycelial and culture filtrate antigens from Aspergillus fumigatus cultures isolated from diverse sources were tested against sera of 10 ABPA patients and 10 control individuals by an ELISA methodology. The results indicate higher antibody reactivity against both antigens in the sera of ABPA patients, while culture filtrate antigens also gave non-specific reactivity with control sera. Mycelial extracts, in general, were useful in the diagnosis of ABPA.     

The precipitin test in human systemic aspergillosis

Summary The sera of healthy women and of adult men either with chest disease or some other condition associated with a mycosis have been tested for precipitins against Aspergillus extracts. Although these precipitins have been demonstrated in the serum of patients suffering from systemic aspergillosis, they have also been detected in antenatal sera and in the sera of patients with no clinical evidence of aspergillosis. The significance of the presence of precipitins againstA. fumigatus is discussed.     

Conformation of an immunoreactive undecapeptide fragment (10–20) of Asp f 1 by NMR and molecular modeling

Allergic bronchopulmonary aspergillosis (ABPA), caused by Aspergillus fumigatus, is a complication of allergic asthma. Asp f 1 secreted by A. fumigatus is reported to be a major allergen/antigen involved in pathogenesis of aspergillosis. A 11-mer immunodominant epitope (Leu-Asn-Pro-Lys-Thr⁵-Asn-Lys-Trp-Glu-Asp¹⁰-Lys) of Asp f 1 has shown immunoreactivity with specific IgG and IgE antibodies in the sera of patients with ABPA in ELISA inhibitionassay. Various studies have suggested that the peptide has a potential use in the development of ELISA based diagnostic kit for early diagnosis of infections caused by A. fumigatus.In view of these interesting properties of the undecapeptide wehave embarked on an investigation of its conformation to understand the relationship between structure and immunoreactivity. NMR and molecular modeling studies of the peptide suggest a structure with a -turn spanning residuesAsn⁶ – Glu⁹ in water at pH 4.0, a -pleated sheet in DMSO and a -helix in 40% HFA.     

A comparison of euglobulin fractions and whole serum in the hemagglutination and latex fixation tests

One hundred and thirty-two sera were investigated with the Waaler-Rose and latex fixation reactions. The reactions were performed with serum, with acid-precipitated euglobulin, and with cold-precipitated euglobulin. The material consisted of 35 sera from healthy persons, 23 from patients with various diseases, 28 from patients with joint symptoms not due to rheumatoid arthritis, and 46 from patients with classical rheumatoid arthritis.In rheumatoid arthritis sera, an increase in positive reactions was obtained in the Waaler-Rose test from 70 per cent in serum to 83 per cent in acid-precipitated euglobulin. This increase was due to a greater specificity of reactions with low titers. The cold-precipitated euglobulin gave less positive Waaler-Rose reactions than the acid-precipitated euglobulin. With the latex fixation test an increase from 65 per cent positive reactions in serum to about 71 per cent with both cold- and acid-precipitated euglobulin fractions was obtained. Here, the increase consisted of reactions negative in serum but positive in the euglobulin fractions, but again with low titers. Because the increase in positive reactions consists merely of low titer values, fractionation of sera only slightly enhances the reliability of the serological tests.Negatively reacting rheumatoid arthritis sera often had low values of the _2A globulin.     

Azole Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> in Patients with Cystic Fibrosis: A Matter of Concern?

Aspergillus fumigatus is the most frequent filamentous fungus isolated from respiratory specimens from patients with cystic fibrosis (CF). Triazoles are the most widely used antifungals in the treatment of allergic bronchopulmonary aspergillosis (ABPA) and invasive aspergillosis (IA) in CF patients. Treatment success could be severely compromised by the occurrence of azole-resistant A. fumigatus (ARAf), which is increasingly reported worldwide from both clinical samples and the environment. In previous studies, ARAf has been detected in up to 8% of CF patients. Isolates from CF patients requiring antifungal treatment should therefore be routinely subjected to antifungal susceptibility testing. The optimal treatment of ABPA or IA in CF patients with azole-resistant isolates has not been established; treatment options include liposomal amphotericin B i.v. and/or echinocandins i.v.     

Specific antibodies to recombinant allergens of Aspergillus fumigatus in cystic fibrosis patients with ABPA

Background

Aspergillus fumigatus, a widely distributed fungus, has been implicated in causing life threatening infections as well as severe asthma and allergic diseases in man. Allergic affliction like allergic bronchopulmonary aspergillosis (ABPA) is a disabling lung disease frequently seen in patients with asthma and cystic fibrosis. Immunodiagnosis of the former is comparatively easier due to the availability of purified antigens and sensitive methods. However, this is not true with cystic fibrosis patients where the prevalence of ABPA is fairly high and the morbidity and mortality are significant.

Methods

In the present study, we have evaluated purified recombinant allergens from A. fumigatus, namely Asp f 1, f 2, f 3, f 4, and f 6 using ELISA and a semi-automated method (ImmunoCAP). We studied 17 patients each from cystic fibrosis with ABPA, and cystic fibrosis with asthma, 22 cystic fibrosis with no ABPA or asthma, and 11 age matched controls.

Results

The results indicate that no antigen, antibody or method is capable of differentiating cystic fibrosis (CF) with ABPA from other CF patients, although some allergens showed strong reaction or showed more prevalence among the patients studied.

Conclusion

When results of several allergens such as Asp f 1, f 2, f 3, f 4, and f 6 in their binding to IgA, IgG, and IgE antibodies were analyzed, a more strong discrimination of CF patients with ABPA was possible from the other groups studied.     

Serologic diagnosis of syphilis; the use of Treponema pallidum immobilization and immune adherence tests

TPI tests were carried out on 4,060 sera. Among 3,934 patients with reactive STS and no history of syphilis, 2,148 or 54.6 per cent had negative reaction to TPI tests, 1,695 or 43.1 per cent had positive reaction and 91 or 2.3 per cent had doubtful reaction. Two hundred and ninety-two or 73.0 per cent of 400 pregnant women with reaction to STS in the absence of a history of syphilis showed negative results by TPI test, 103 or 25.8 per cent had positive results and five or 1.2 per cent had doubtful reaction.Ninety-five or 75.4 per cent of 126 patients with a history of treated syphilis had positive reaction to TPI tests, 20 or 20.2 per cent had negative reaction and nine or 9.1 per cent had doubtful reaction.TPI and TPIA tests were done on 143 sera carefully selected for the study. Among 102 sera subjected to the TPI test, 46 or 100 per cent of these positive were also positive by TPIA tests, while 52 or 94.5 per cent of 55 TPI-negative sera were also nonreactive by TPIA test. One serum gave doubtful TPI test reaction and positive TPIA test reaction.     

SEROLOGIC DIAGNOSIS OF SYPHILIS—The Use of Treponema Pallidum Immobilization and Immune Adherence Tests

TPI tests were carried out on 4,060 sera. Among 3,934 patients with reactive STS and no history of syphilis, 2,148 or 54.6 per cent had negative reaction to TPI tests, 1,695 or 43.1 per cent had positive reaction and 91 or 2.3 per cent had doubtful reaction.Two hundred and ninety-two or 73.0 per cent of 400 pregnant women with reaction to STS in the absence of a history of syphilis showed negative results by TPI test, 103 or 25.8 per cent had positive results and five or 1.2 per cent had doubtful reaction.Ninety-five or 75.4 per cent of 126 patients with a history of treated syphilis had positive reaction to TPI tests, 20 or 20.2 per cent had negative reaction and nine or 9.1 per cent had doubtful reaction.TPI and TPIA tests were done on 143 sera carefully selected for the study. Among 102 sera subjected to the TPI test, 46 or 100 per cent of these positive were also positive by TPIA tests, while 52 or 94.5 per cent of 55 TPI-negative sera were also nonreactive by TPIA test. One serum gave doubtful TPI test reaction and positive TPIA test reaction.     

Allergic Bronchopulmonary Aspergillosis with Aspergilloma: An Immunologically Severe Disease with Poor Outcome

Background and Aims

The association between allergic bronchopulmonary aspergillosis (ABPA) and aspergilloma has been proposed as a severe form of ABPA. However, this conclusion is based on single-patient case reports. In this study, we describe the clinical details and immunological findings of this association and compare patients of ABPA with aspergilloma and those without.

Methods

This is a retrospective analysis of data of patients with ABPA managed in the Chest Clinic. We compared the clinical, radiological and immunological profile of patients with ABPA and central bronchiectasis, with and without the presence of aspergilloma on HRCT scan.

Results

There were 98 men and 81 women with a mean (SD) age of 33.6 (12.2) years. Eight patients were diagnosed to have aspergilloma. Sputum cultures grew Aspergillus fumigatus in all these eight patients. The aspergilloma was solitary in six patients, and two each in two patients. Patients with aspergilloma had higher IgE levels (both total and A. fumigatus specific) than those without aspergilloma. Bronchiectasis was also more extensive in patients with aspergilloma. Overall, 70?% of the ABPA patients experienced relapse during the median (interquartile range) follow-up of 27 (19?C39) months. The number of relapses was significantly higher in patients with aspergilloma (p?=?0.0001). On a multivariate linear regression analysis, high-attenuation mucus and aspergilloma were independent predictors of relapse frequency.

Conclusions

The concurrent presentation of ABPA and aspergilloma is associated with an immunologically severe disease and risk of recurrent relapses.     

Animal models of allergic bronchopulmonary aspergillosis

Among the allergic fungi, Aspergillus fumigatus, a saprophytic mold, distributed widely in the environment is a frequently recognized etiologic agent in a number of allergic conditions. Among the different allergic diseases caused by this fungus, allergic bronchopulmonary aspergillosis (ABPA) is by far the most significant one. The immunopathogenesis of this disease is not fully understood. Although several immunomodulatory treatments are available for allergic disease, none of them are applicable or relevant or useful in fungal induced allergy. It is essential to understand the pathogenesis of the disease including the antigen induced immunoregulation and the resulting factors, such as cytokine, chemokines, pathways activating factors, inflammatory and airway remodeling factors need to be understood for intervening with appropriate treatment. Animal models are essential in understanding these features of the disease. Several models of allergic aspergillosis have been developed in recent years in various animals. However, murine models have been studied more carefully and extensively. The exposure to antigen in mice leads to allergy very similar to ABPA with high IgE, elevated peripheral blood and lung eosinophils, pulmonary inflammation, and airway hyperreactivity. The role of various cytokines and chemokines and their receptors were also studied. In addition, immunotherapy and vaccination have been attempted in recent years using the murine model of ABPA. This review covers the murine model of Aspergillus induced allergy and asthma and presented critically our current understanding of the subject and the potential application of such a model in future for developing treatment modalities. This revised version was published online in June 2006 with corrections to the Cover Date.     

Aerobiological,biochemical and immunological studies on some of the dominant <Emphasis Type="Italic">Aspergillus</Emphasis> species of South Assam (India)

Two years atmospheric survey of air-borne Aspergillus was carried out in the environmental conditions of South Assam. The survey revealed a total of 16 different species of Aspergillus with marked seasonal and annual variations. Aspergillus fumigatus was found to be the dominant atmospheric fungal species followed by Aspergillus flavus, Aspergillus niger, etc. Among the sample extracts tested, highest quantity of soluble protein was recorded in Aspergillus fumigatus (95.0 mg/g) whereas highest quantity of soluble carbohydrate (40.8 mg/g) and free amino acid (135.0 mg/g) was recorded in the sample extract of Aspergillus niger per gram of dry weight, respectively. The highest numbers of protein polypeptide bands were detected in the sample extract of Aspergillus fumigatus followed by Aspergillus flavus and lowest in Aspergillus niger. The maximum numbers of immunoglobulin E binding protein fractions were found in Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus clavatus, etc.     

Evaluation of an indirect hemagglutination inhibition technique for identifying antibody in animals experimentally and naturally exposed to fungi

Rabbits were exposed to aerosols containing spores of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, or of a Penicillium sp. Sera from these rabbits were tested by indirect hemagglutination (IHA) and by IHA inhibition. The serologic reactions with the rabbit sera were compared to reactions with sera from cattle naturally exposed to airborne microorganisms. By three months of age, most cattle had positive IHA reactions to A. fumigatus and Penicillium antigens. The IHA inhibition tests indicated that antibody production in 12 of the 20 cattle probably resulted from exposure to A. flavus. One calf reacted as if sensitized by A. niger. Two were totally nonreactive. Five of the cattle had reactions that were not identifiable relative to the reactions in rabbits.Purchased by U.S. Department of Agriculture for Official Use.     

Precipitating antibodies againstMicropolyspora faeni in equines in north-western India

Prevalence of serum precipitins againstMicropolyspora faeni, Thermoactinomyces vulgaris andAspergillus fumigatus, employing the counterimmunoelectrophoresis (COTE) and Ouchterlony's double diffusion (DD) techniques, is reported in 162 of the equines stationed at two military installations in northwestern India.M. faeni specific precipitins were demonstrable in 58 of 112 mules from site I in the mountainous region whereas the results were negative for all of the 50 horses examined from site IT located in the plains. Of the 58M. faeni positive mules, 45 (78%) had signs and symptoms suggestive of chronic obstructive pulmonary disease (COPD) while the remaining 13 (22%) were apparently free from any respiratory disorder. The more frequent occurrence ofM. faeni precipitins in the symptomatic than in the asymptomatic group of mules was found to be statistically significant (P < 0.01). Precipitins againstA. fumigatus were concomitantly demonstrated in 5 of the mules afflicted with COPD and found to be positive forM. faeni.     

A comparison of aspergillus fumigatus antigens by counterimmunoelectrophoresis

Counterimmunoelectrophoresis (CIE) has been evaluated for routine diagnostic work, using antigens prepared from 2 different isolates of Aspergillus fumigatus, at 2 different concentrations. Additional antigens prepared in a variety of ways and 2 commercially available antigens (Bencard, London; Institut Pasteur, Paris, France) have been compared with the routine antigens. It has been shown that the use of the routine antigens will detect the majority of positive reactions. At optimal reacting concentrations antibodies were detected in 75% of asthmatic patients. In sera from patients with a presumptive diagnosis of allergic aspergillosis, CIE will detect twice as many positive reactions as a conventional agar gel diffusion test.     

Specificity of antigens from pathogenic Aspergillus species. II. Studies with line immunoelectrophoresis

Somatic (mycelial) and metabolic (culture filtrate) antigens of Aspergillus flavus, A. fumigatus, A. nidulans, A. niger and A. terreus were compared by line immunoelectrophoresis with sera from patients with allergic bronchopulmonary aspergillosis (ABPA) or aspergilloma, or from immunized animals. Number of lines observed when tested with human sera were similar for somatic and metabolic preparations of A. fumigatus, but up to 33 lines were present when both types of antigens were tested simultaneously. Cross-reactions between heterologous antigens and sera from patients with aspergilloma or ABPA were uncommon. In contrast, cross-reactions were common when standard antisera prepared in animals against heterologous species of Aspergillus were tested against A. fumigatus antigens. Lines of identity between homologous antigens and those from A. fumigatus were observed in 5 of 9 lines obtained with A. flavus, 4 of 16 lines of A. nidulans, 4 of 9 lines of A. niger and 4 of 8 lines of A. terreus.     

Detection of a Serum Siderophore by LC-MS/MS as a Potential Biomarker of Invasive Aspergillosis

Invasive aspergillosis (IA) is a life-threatening systemic mycosis caused primarily by Aspergillus fumigatus. Early diagnosis of IA is based, in part, on an immunoassay for circulating fungal cell wall carbohydrate, galactomannan (GM). However, a wide range of sensitivity and specificity rates have been reported for the GM test across various patient populations. To obtain iron in vivo, A. fumigatus secretes the siderophore, N,N'',N"-triacetylfusarinine C (TAFC) and we hypothesize that TAFC may represent a possible biomarker for early detection of IA. We developed an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for TAFC analysis from serum, and measured TAFC in serum samples collected from patients at risk for IA. The method showed lower and upper limits of quantitation (LOQ) of 5 ng/ml and 750 ng/ml, respectively, and complete TAFC recovery from spiked serum. As proof of concept, we evaluated 76 serum samples from 58 patients with suspected IA that were investigated for the presence of GM. Fourteen serum samples obtained from 11 patients diagnosed with probable or proven IA were also analyzed for the presence of TAFC. Control sera (n = 16) were analyzed to establish a TAFC cut-off value (≥6 ng/ml). Of the 36 GM-positive samples (≥0.5 GM index) from suspected IA patients, TAFC was considered positive in 25 (69%). TAFC was also found in 28 additional GM-negative samples. TAFC was detected in 4 of the 14 samples (28%) from patients with proven/probable aspergillosis. Log-transformed TAFC and GM values from patients with proven/probable IA, healthy individuals and SLE patients showed a significant correlation with a Pearson r value of 0.77. In summary, we have developed a method for the detection of TAFC in serum that revealed this fungal product in the sera of patients at risk for invasive aspergillosis. A prospective study is warranted to determine whether this method provides improved early detection of IA.