Bird fancier's lung serodiagnosis by automated r-IgLL1 ELISA.

CONTEXT: Bird fancier's lung (BFL) is the most prevalent form of hypersensitivity pneumonitis (HP) worldwide. The current techniques used for the serological diagnosis of BFL all use crude extracts from feathers, droppings, and blooms as test antigens, which is associated with a lack of standardization and variability of the results. An antigenic protein, immunoglobulin lambda-like polypeptide-1 (IgLL1), isolated from pigeon droppings, was recently identified to be associated with BFL. We used genetic engineering to produce IgLL1 as a recombinant antigen. AIM: We aimed to prospectively validate the use of an automated ELISA based on recombinant IgLL1 protein (r-IgLL1) as the test antigen for the serological diagnosis of BFL. METHODS: Immunoprecipitation (IP) techniques (immunodiffusion (ID), immunoelectrophoresis (IEP)) and ELISA using r-IgLL1 were performed concomitantly over 10 months on 634 sera from patients with a BFL serodiagnosis request. Questionnaires were sent to obtain details on the avian exposure, clinical data, and final diagnosis. Concordance, sensitivity (Se), and specificity (Sp) of the two techniques were compared. RESULTS: In total, 72 completed questionnaires were returned with 18 cases of BFL diagnosed and 54 of non-BFL. The concordance between the ELISA and ID+IEP precipitation techniques was 71%. The combination of immunoprecipitation techniques showed a Se of 78% and a Sp of 67%. The ELISA using r-IgLL1 showed a Se of 89% and a Sp of 91%. The automated r-IgLL1 ELISA test is sufficiently efficient to be used alone for the diagnosis of patients exposed solely to Columbidae. In cases of other avian exposure, the Se and Sp of the r-IgLL1 ELISA used for screening combined with the immunodiffusion test for confirmation were 89% and 93%, respectively. CONCLUSIONS: The automated ELISA using r-IgLL1 is a promising tool for BFL serodiagnosis. Replacing immunodiffusion by the automated ELISA using r-IgLL1 as a screening technique will be the basis of our future strategy for BFL serodiagnosis.

Pilot Study Using Recombinant Antigens r-PROE and r-IGLL1 for the Serodiagnosis of Feather Duvet Lung.

BACKGROUND: Feather duvet lung (FDL) is an underestimated form of acute and chronic hypersensitivity pneumonitis. Serological tests for FDL need to be validated. We investigated the ability of recombinant pigeon Proproteinase E (r-PROE) and Immunoglobulin-lambda-like-polypeptide-1 (r-IGLL1) proteins to support the serological diagnosis of FDL, and propose them as a serological tool for clinicians to differentiate cases from FDL and Bird fancier's lung (BFL). METHODS: Specific IgG antibodies against r-PROE and r-IGLL1, analyzed with ELISA, were measured in patients diagnosed with FDL (n=31), BFL (n=15) controls exposed (n=15) and unexposed to feathers (n=15). RESULTS: The sensitivity and specificity of the r-PROE ELISA for the serological diagnosis of FDL cases versus exposed and unexposed controls were 74.2% and 86.7% respectively, with an index threshold of 0.5 (AUC: 0.89). In addition, this serological test was effective to support the serological diagnosis of FDL and BFL cases with significantly different thresholds. The r-IGLL1 ELISA was only effective for the serological diagnosis of BFL. Also, these two serological tests were useful for the diagnosis of both chronic and acute forms. CONCLUSIONS: The new diagnostic test for FDL using r-PROE protein should help to detect overt and hidden cases of FDL. The combination of both test will help the clinician in distinguish between the etiology of birds or feathers duvet.

Pilot Study Using Recombinant Antigens r-PROE and r-IGLL1 for the Serodiagnosis of Feather Duvet Lung.

BACKGROUND: Feather duvet lung (FDL) is an underestimated form of acute and chronic hypersensitivity pneumonitis. Serological tests for FDL need to be validated. We investigated the ability of recombinant pigeon Proproteinase E (r-PROE) and Immunoglobulin-lambda-like-polypeptide-1 (r-IGLL1) proteins to support the serological diagnosis of FDL, and propose them as a serological tool for clinicians to differentiate cases from FDL and Bird fancier's lung (BFL). METHODS: Specific IgG antibodies against r-PROE and r-IGLL1, analyzed with ELISA, were measured in patients diagnosed with FDL (n=31), BFL (n=15) controls exposed (n=15) and unexposed to feathers (n=15). RESULTS: The sensitivity and specificity of the r-PROE ELISA for the serological diagnosis of FDL cases versus exposed and unexposed controls were 74.2% and 86.7% respectively, with an index threshold of 0.5 (AUC: 0.89). In addition, this serological test was effective to support the serological diagnosis of FDL and BFL cases with significantly different thresholds. The r-IGLL1 ELISA was only effective for the serological diagnosis of BFL. Also, these two serological tests were useful for the diagnosis of both chronic and acute forms. CONCLUSIONS: The new diagnostic test for FDL using r-PROE protein should help to detect overt and hidden cases of FDL. The combination of both test will help the clinician in distinguish between the etiology of birds or feathers duvet.

Usefulness of bronchoalveolar lavage in a French pediatric cohort with hypersensitivity pneumonitis.

BACKGROUND: Hypersensitivity pneumonitis (HP) is a rare interstitial lung disease in children, and very little data are available on the frequency, diagnosis, and outcomes of HP. In a pediatric cohort with HP, the characteristics of the CD4/CD8 lymphocyte ratio are often described as nonspecific. METHODS: We used the National French Database (RespiRare) to collect data from the last decade on HP. The diagnosis of HP was defined by the presence of a relevant exposure, clinical symptoms, and compatible lung imaging radiology and was usually defined by positive precipitins antibodies. RESULTS: A total of 16 children with a mean age of 10 years (4-13) presented with HP. All children presented with dyspnea on exertion. Diffuse ground-glass opacity was present in all computed tomography (CT) scans. Research guided by a questionnaire and precipitins antibodies against the corresponding antigens showed that patients were positive for contact with birds with or without fungi. Bronchoalveolar lavage (BAL) was performed in 12 children. The total cell counts were elevated in BAL fluid, with a mean value of 36% lymphocytes. The CD4/CD8 lymphocyte ratio was below one for all children. CONCLUSION: BAL in our pediatric cohort with HP had the same characteristics as that of adults with HP. An HP diagnosis must be considered when dyspnea on exertion and diffuse ground-glass opacity are observed. Carrying out BAL and serological tests can help diagnose and avoid lung biopsy.

Wheezing phenotypes and risk factors in early life: The ELFE cohort.

OBJECTIVE: Different phenotypes of wheezing have been described to date but not in early life. We aim to describe wheezing phenotypes between the ages of two months and one year, and assess risk factors associated with these wheezing phenotypes in a large birth cohort. METHODS: We studied 18,041 infants from the ELFE (French Longitudinal Study of Children) birth cohort. Parents reported wheezing and respiratory symptoms at two and 12 months, and answered a complete questionnaire (exposure during pregnancy, parental allergy). RESULTS: Children with no symptoms (controls) accounted for 77.2%, 2.1% had had wheezing at two months but no wheezing at one year (intermittent), 2.4% had persistent wheezing, while 18.3% had incident wheezing at one year. Comparing persistent wheezing to controls showed that having one sibling (ORa = 2.19) or 2 siblings (ORa = 2.23) compared to none, nocturnal cough (OR = 5.2), respiratory distress (OR = 4.1) and excess bronchial secretions (OR = 3.47) at two months, reflux in the child at 2 months (OR = 1.55), maternal history of asthma (OR = 1.46) and maternal smoking during pregnancy (OR = 1.57) were significantly associated with persistent wheezing. These same factors, along with cutaneous rash in the child at 2 months (OR = 1.13) and paternal history of asthma (OR = 1.32) were significantly associated with increased odds of incident wheezing. Having one sibling (ORa = 1.9) compared to none, nocturnal cough at 2 months (OR = 1.76) and excess bronchial secretions at 2 months (OR = 1.65) were significantly associated with persistent compared to intermittent wheezing. CONCLUSION: Respiratory symptoms (cough, respiratory distress, and excessive bronchial secretion) were significantly associated with a high risk of persistent wheezing at one year. Smoking exposure during pregnancy was also a risk factor for persistent and incident wheezing.

An immunoproteomic approach revealed antigenic proteins enhancing serodiagnosis performance of bird fancier's lung.

BACKGROUND: Bird fancier's lung (BFL) caused by repeated inhalation of avian proteins is the most common form of hypersensitivity pneumonitis. However, the exact identification of proteins involved is unknown, and serological test use for diagnosis need to be standardized. The objectives of this study were (i) to identify antigenic proteins from pigeon droppings (ii) to provide information about their location in avian matrices and (iii) to produce them in recombinant proteins to evaluate their diagnostic performances. METHOD: Antigenic proteins of pigeon dropping extracts were investigated using 2-dimensional immunoblotting with sera from patients with BFL, asymptomatic exposed controls and healthy volunteers. We investigated the origin of these antigenic proteins by analyzing droppings, blooms and sera using a shotgun proteomic analysis. BFL-associated proteins were produced as recombinant antigens in E. coli and were assessed in ELISA with sera from patients (n=25) and subject exposed controls (n=30). These diagnostic performances were compared with those obtained by precipitin techniques (agar gel double diffusion, immunoelectrophoresis). RESULTS: We identified 14 antigenic proteins mainly located in droppings and blooms. These proteins were involved in either the digestive or immune systems of pigeons. Using the recombinant BFL-associated proteins: Immunoglobulin lambda-like polypeptide-1 (IGLL1: sensitivity: 76%; specificity: 100%; AUC: 0.93) and Proproteinase E (ProE: sensitivity: 84%; specificity: 80%; AUC: 0.85), the ELISA test showed better performance than precipitin assays with pigeon dropping extracts (sensitivity: 60%; specificity: 93.3%; AUC: 0.76). CONCLUSION: IGLL1 and ProE were identified as the biomarkers of the disease. The use of these highly standardized antigens discriminates BFL cases from exposed subjects in serological assays. The results of this study offer new possibilities for the serological diagnosis of the disease. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: Identifier NCT03056404.

Hypersensitivity pneumonitis in a beautician.

A 52-year-old non-smoking beautician using a skincare device spraying steam and ozone (a "vapozone" facial steamer) was referred for progressive dyspnea and dry cough during working periods. Although spirometry was normal, she had decreased diffusing capacity of the lung for carbon monoxide, bronchiolitis with air trapping on high-resolution CT scan and 60% lymphocytosis by bronchoalveolar lavage. Twenty-six antigens were tested and serum-specific precipitins were found mainly against Pseudomonas sp. and Mycobacterium mucogenicum. Cultures from her skincare device isolated Pseudomonas sp. Outcome was favorable with cessation of occupational exposure to the device, without any medication. This is the first report of a case of HP in a beautician due to steam contaminated by Pseudomonas sp. from a vapozone. HP, and not only asthma and contact dermatitis, should be suspected in beauticians with respiratory symptoms. Am. J. Ind. Med. 59:1041-1045, 2016. © 2016 Wiley Periodicals, Inc.

New Commercially Available IgG Kits and Time-Resolved Fluorometric IgE Assay for Diagnosis of Allergic Bronchopulmonary Aspergillosis in Patients with Cystic Fibrosis.

Allergic bronchopulmonary aspergillosis (ABPA) is difficult to diagnose; diagnosis relies on clinical, radiological, pathological, and serological criteria. Our aim was to assess the performance of two new commercially available kits and a new in-house assay: an Aspergillus fumigatus enzyme-linked immunosorbent assay (ELISA) IgG kit (Bordier Affinity Products), an Aspergillus Western blotting IgG kit (LDBio Diagnostics), and a new in-house time-resolved fluorometric IgE assay (dissociation-enhanced lanthanide fluorescent immunoassay, or DELFIA) using recombinant proteins from an Aspergillus sp. recently developed by our laboratory for ABPA diagnosis in a retrospective study that included 26 cystic fibrosis patients. Aspergillus fumigatus-specific IgG levels measured by a commercial ELISA kit were in accordance with the level of precipitins currently used in our lab. The ELISA kit could accelerate and help standardize ABPA diagnosis. Aspergillus fumigatus-specific IgE levels measured by ImmunoCAP (Phadia) with A. fumigatus M3 antigen and by DELFIA with a purified protein extract of A. fumigatus were significantly correlated (P < 10(-6)). The results with recombinant antigens glucose-6-phosphate isomerase and mannitol-1-phosphate dehydrogenase were encouraging but must be confirmed with sera from more patients. The DELFIA is an effective tool that can detect specific IgE against more fungal allergens than can be detected with other commercially available tests.

Evaluation of mold exposure in cystic fibrosis patients' dwellings and allergic bronchopulmonary risk.

Very few studies have been conducted on cystic fibrosis (CF) patients' exposure to the indoor environment and, to our knowledge, there are no studies dealing with the link between specific fungal environmental exposure at home and fungal colonization resulting in allergic bronchopulmonary aspergillosis (ABPA). Fungal exposure of CF adult patients with ABPA (n=4) with fungal sensitization (n=7) and with no ABPA (n=5) was assessed in 16 homes by dust sampling with electrostatic dust fall collectors (EDCs). Aspergillus fumigatus was specifically quantified by real-time quantitative polymerase chain reactions (qPCRs), and A. fumigatus DNA concentrations were significantly higher in homes of ABPA patients (p<0.001). Results indicate that indoor fungal contamination could be a factor favoring ABPA and suggest that environmental surveys could help in preventing fungal risk in CF patients.

Occupational hypersensitivity pneumonitis in a baker: a new cause.

Bakers are exposed daily to flour and may be susceptible to immunologic occupational diseases. A 30-year-old, nonsmoking, female baker was referred for progressive dyspnea on exertion, basal crackles on auscultation, restrictive lung function, decreased diffusing capacity of the lung for carbon monoxide, ground glass hyperdensities with a mosaic pattern on high-resolution CT scan, 25% lymphocytosis by BAL, and cellular chronic bronchiolitis with peribronchiolar interstitial inflammation by lung biopsy specimen. Cultures from flours isolated nine species, including Aspergillus fumigatus. Twenty-six antigens were tested. Serum-specific precipitins were found against A fumigatus, the flour mite Acarus siro, and total extracts from maize and oat. Outcome was favorable with cessation of occupational exposure to flours and transient therapy with prednisone and immunosuppressive agents. To our knowledge, this report is the first of a well-documented case of hypersensitivity pneumonitis due to sensitization to fungi- and mite-contaminated flours. Hypersensitivity pneumonitis--and not only asthma and allergic rhinitis--should be suspected in bakers with respiratory symptoms.

Azole-resistant Aspergillus fumigatus isolate with the TR34/L98H mutation in both a fungicide-sprayed field and the lung of a hematopoietic stem cell transplant recipient with invasive aspergillosis.

A French farmer developed invasive aspergillosis with azole-resistant Aspergillus fumigatus with the TR34/L98H mutation following a hematopoietic stem cell transplantation. He had worked in fungicide-sprayed fields where a non-genetically related A. fumigatus TR34/L98H isolate was collected. If azole resistance detection increases, voriconazole as first-line therapy might be questioned in agricultural areas.

Health effects of occupational exposure in a dairy food industry, with a specific assessment of exposure to airborne lactic acid bacteria.

OBJECTIVE: Lactic acid bacteria (LAB) are used in food industries as probiotic agents. The aim of this study is to assess the potential health effects of airborne exposure to a mix of preblend (LAB and carbohydrate) and milk powder in workers. METHODS: A medical questionnaire, lung function tests, and immunologic tests were carried out on 50 workers. Occupational exposure to inhalable dust and airborne LAB was measured. RESULTS: Workers not using respiratory masks reported more symptoms of irritation than workers using protection. Workers from areas with higher levels of airborne LAB reported the most health symptoms and the immune responses of workers to LAB was higher than the immune responses of a control population. CONCLUSIONS: Measures to reduce exposure to airborne LAB and milk powder in food industries are recommended.

Factors influencing the microbial composition of metalworking fluids and potential implications for machine operator's lung.

Hypersensitivity pneumonitis, also known as "machine operator's lung" (MOL), has been related to microorganisms growing in metalworking fluids (MWFs), especially Mycobacterium immunogenum. We aimed to (i) describe the microbiological contamination of MWFs and (ii) look for chemical, physical, and environmental parameters associated with variations in microbiological profiles. We microbiologically analyzed 180 MWF samples from nonautomotive plants (e.g., screw-machining or metal-cutting plants) in the Franche-Comté region in eastern France and 165 samples from three French automotive plants in which cases of MOL had been proven. Our results revealed two types of microbial biomes: the first was from the nonautomotive industry, showed predominantly Gram-negative rods (GNR), and was associated with a low risk of MOL, and the second came from the automotive industry that was affected by cases of MOL and showed predominantly Gram-positive rods (GPR). Traces of M. immunogenum were sporadically detected in the first type, while it was highly prevalent in the automotive sector, with up to 38% of samples testing positive. The use of chromium, nickel, or iron was associated with growth of Gram-negative rods; conversely, growth of Gram-positive rods was associated with the absence of these metals. Synthetic MWFs were more frequently sterile than emulsions. Vegetable oil-based emulsions were associated with GNR, while mineral ones were associated with GPR. Our results suggest that metal types and the nature of MWF play a part in MWF contamination, and this work shall be followed by further in vitro simulation experiments on the kinetics of microbial populations, focusing on the phenomena of inhibition and synergy.

Variable beta-glucans production by different states of Eurotium amstelodami explains differences in inflammatory responses in airway cells.

Eurotium amstelodami, a mold frequently identified in housing and farm air samples, is a suspected cause of respiratory diseases such as allergic alveolitis, atopic asthma, and organic dust toxic syndrome. This fungus is present in the air in three different states (ascospores, conidia, and hyphae). The aim of this study was to test in vitro the differential inflammatory response of airway cells exposed to 1,3 betaglucanase-treated protein extract (BGPE), from E. amstelodami ascospores, conidia, and hyphae. Confluent cells from the A549 cell line were inoculated with calibrated BGPE issued from the three fungal forms. The levels of eight cytokines and chemokines involved in inflammatory responses were measured after 8 h of exposure. Beta-d-glucan (BDG) was quantified in total fungal extract as well as in the BGPE from the three fungal states. Hyphal BGPE were the only ones to induce a marked inflammatory response and they contain higher quantities of BDG. The present study adds to the growing body of evidence that beta-glucan from fungal hyphae play a crucial role in respiratory diseases.

Hypersensitivity pneumonitis due to molds in a saxophone player.

This 48-year-old patient was evaluated for an interstitial pneumonia. An open-lung biopsy showed a pattern of nonspecific interstitial pneumonia. The CT scan appearance, showing mosaic ground-glass opacities in the ventilated parts of the lung, the centrolobular predominance of inflammation on the lung sections, and the presence of a lymphocytic alveolitis at BAL suggested a hypersensitivity pneumonitis. The patient was a white-collar worker and had no contact with pets, birds, drugs, or molds at home. He used to play the saxophone as a hobby. Two molds, Ulocladium botrytis and Phoma sp, were detected in the saxophone. Precipitating antibodies to these molds were present in his serum. An additional study confirmed the frequent colonization of saxophones with potentially pathogenic molds, such as Fusarium sp, Penicillium sp, and Cladosporium sp. Respiratory physicians should be aware of the risk of hypersensitivity pneumonitis in saxophone or perhaps other wind instrument players.

New evidence of the involvement of Lichtheimia corymbifera in farmer's lung disease.

Farmer's lung disease (FLD) is a form of hypersensitivity pneumonitis resulting from recurrent exposure to moldy plant materials. We investigated and compared the initial response of respiratory epithelium after exposure to extracts of Sacharopolyspora rectivirgula, Lichtheimia corymbifera (formerly Absidia corymbifera), Eurotium amstelodami and Wallemia sebi. The two criteria for selection of these species were their high prevalence in the hay handled by FLD patients and the presence of high levels of specific precipitins to these molds in FLD patients’ sera. Hydrosoluble extracts were prepared from spores and hyphae grown in culture under optimal conditions for each of the four species. Confluent A549 cells were inoculated with one of the four calibrated soluble extracts. Two mediators, one inflammatory (Interleukin (IL)-8) and one allergic (IL-13), were quantified using real-time PCR and ELISA assay, after four exposure periods (30 min, 2 h, 4 h and 8 h). S. rectivirgula and L. corymbifera extracts were the only ones which induced a marked upregulation of IL-8, as shown by both real-time PCR and ELISA assay 8 h after the initial contact. This study adds to the growing body of evidence that L. corymbifera should be recognized as an etiologic agent of FLD along with S. rectivirgula.