Comparison of Three Antigenic Extracts of Eurotium amstelodami in Serological Diagnosis of Farmer's Lung Disease

Abstract

In France and Finland, farmer's lung disease (FLD), a hypersensitivity pneumonitis common in agricultural areas, is mainly caused by Eurotium species. The presence of antibodies in patients' serum is an important criterion for diagnosis. Our study aimed to improve the serological diagnosis of FLD by using common fungal particles that pollute the farm environment as antigens. Fungal particles of the Eurotium species were observed in handled hay. A strain of Eurotium amstelodami was grown in vitro using selected culture media; and antigen extracts from sexual (ascospores), asexual (conidia), and vegetative (hyphae) forms were made. Antigens were tested by enzyme-linked immunosorbent assay (ELISA), which was used to test for immunoglobulin G antibodies from the sera of 17 FLD patients, 40 healthy exposed farmers, and 20 nonexposed controls. The antigens were compared by receiver operating characteristic analysis, and a threshold was then established. The ascospores contained in asci enclosed within cleistothecia were present in 38% of the hay blades observed; conidial heads of aspergillus were less prevalent. The same protocol was followed to make the three antigen extracts. A comparison of the results for FLD patients and exposed controls showed the area under the curve to be 0.850 for the ascospore antigen, 0.731 for the conidia, and 0.690 for the hyphae. The cutoffs that we determined, with the standard deviation for measures being taken into account, showed 67% for sensitivity and 92% for specificity with the ascospore antigen. In conclusion, the serological diagnosis of FLD by ELISA was improved by the adjunction of ascospore antigen.Eurotium amstelodami is known to cause hypersensitivity pneumonitis (HP), of which farmer's lung disease (FLD) is a common form (24,29). Eurotium species, widely isolated from hay harvested in several European countries (22, 30), constitute a major etiological agent of the disease in France and Finland (13,26). Thus far, the diagnosis of HP has most often relied on an array of unspecific clinical symptoms and signs developed in an appropriate setting and the demonstration of interstitial markings on chest radiographs, serum antibodies against offending antigens, a lymphocytic alveolitis on bronchoalveolar lavage (BAL), and the granulomatous reaction on lung biopsy specimens (17, 21). Enzyme-linked immunosorbent assay (ELISA) is one of the techniques that can be used to look for specific serum antibodies (6). Although ELISA indicates significantly higher immunoglobulin G antibody levels in patients with FLD than in control groups, no clear threshold has been established due to an extreme variability in the results that have been obtained (6,25). The antigens used in most studies derive from the sonication of crude fungi.E. amstelodami can be found in any one of three forms (sexual, asexual, or vegetative), and the one that is predominant in the agricultural environment is not known to date. Sexual reproduction is characterized by the production of cleistothecia containing asci wit...

Airborne ascospores in Mérida (SW Spain) and the effect of rain and other meteorological parameters on their concentration

“…Allergy or asthma caused by ascospores has been reported for Daldinia (Herxheimer et al 1966), Leptosphaeria (Ganderton 1968;Burge et al 1985;Hasnain 1993a;Newson et al 2000;GrinnGofroń and Mika 2008), Didymella (Frankland and Gregory 1973;Jenkins et al 1981; Ackroyd and Fry 1983;Harries et al 1985;Allitt 1986;Richardson 1996;Newson et al 2000;Grinn-Gofroń and Mika 2008), Leptosphaerulina, Gibberella, Gnomonia and Chaetomium (Burge 1986) and Pleospora (Newson et al 2000). Moreover, the presence of ascospores and other fungal spores has been associated with increases in the number of emergency visits and hospital admissions for asthma (Atkinson et al 2006), and a recent study (Roussel et al 2010) shows that antigens are more abundant in ascospores than in conidia or hyphae in one species of Eurotium.…”

Aspergillus species recombinant antigens for serodiagnosis of farmer's lung disease

“…1,3-b-Glucanase-treated protein extract (BGPE) from E amstelodami ascospores was recently demonstrated to be the most efficient antigen for differentiating between patients with FLD and healthy control subjects by using an ELISA serologic test. 6 Developing a battery of standardized antigens known to cause FLD and making them available to clinicians for use in diagnosis is recommended in the report from the National Heart, Lung, and Blood Institute/Office of Rare Diseases Workshop. 7 The aim of the present study was to characterize immunogenic proteins of ascospores and to synthesize standardized recombinant antigens to improve the serologic diagnosis of FLD.…”

“…The ability to differentiate between patients with FLD and exposed control subjects by using ELISAs with ascospore crude extract was evaluated in a previous study 6 and was demonstrated as follows: sensitivity, 71%; specificity, 87%, positive likelihood ratio, 5.6; negative likelihood ratio, 0.3; and area under the curve, 0.85. Sensitivity was at 89% when a score combining the results of ELISAs with the 2 recombinant antigens was used.…”

Hypersensitivity pneumonitis and metalworking fluids contaminated by mycobacteria

“…The structures of M. immunogenum were recovered by scraping and filtration. Two M. immunogenum Ag extracts were produced: a total aqueous lyophilised extract (somatic Ag) used for ES on cellulose acetate [10,11] and a protein extract used for ELISA tests [12].…”

“…As described previously [10,12], 96-well plates (PolySorp Immunomodule; Nalge Nunc1, Rochester, NY, USA) were coated with 200 mL 1 mg?mL -1 protein Ag solution in 50 mM K 2 HPO 4 buffer (Sigma-Aldrich1), pH 8.5, at 4uC for 48 h. Excess binding sites were blocked at 37uC for 1 h with 250 mL of 50 mM NaH 2 PO 4 buffer (Sigma-Aldrich1) containing 0.5% bovine serum albumin (Sigma-Aldrich1) and 60 g?L -1 sorbitol (Sigma-Aldrich1). 100-mL serum samples diluted 1:200 were added in triplicate to all wells.…”