Diagnostics of IgE-mediated occupational allergies: Between reality, requirements, and opportunities.

Occupational skin and respiratory allergies are among the most common occupational diseases in Germany. The identification of the allergy trigger is essential for the recognition of an occupational allergy as well as for effective individual prevention. However, occupational type I allergens are among the "rare" allergens and the possibilities of guideline-compliant diagnosis using quality-tested skin test solutions is becoming increasingly difficult due to the reduction in commercially available test allergens. In order to guarantee meaningful diagnostic workup for all affected insured persons with suspected occupational type I allergies and to ensure this in the future, a durable optimization, standardization, and availability of allergy tests for occupational allergic diseases is urgently required. The need for action has been recognized by the German Social Accident Insurance (DGUV), and steps to eliminate the diagnostic gaps have been initiated by a joint research project at the Institute for Prevention and Occupational Medicine of the DGUV (IPA) and the Paul Ehrlich Institut (PEI). The evaluation of alternative methods for the production of standardized test allergen solutions can also be used for newly emerging allergens in the workplace. New allergen sources at workplaces and thus also sensitization and allergies among employees can be expected as a result of changes in work processes and the introduction of new technologies and/or working materials, which are also introduced in connection with climate change and the concept of sustainability.

Impact of climate change on allergic diseases in Germany.

Background: Allergic diseases, especially inhalation allergies, have reached epidemic levels and environmental factors play an important role in their development. Climate change influences the occurrence, frequency, and severity of allergic diseases. Methods: The contents of this article were selected by the authors and developed section by section according to their expertise and the current state of knowledge. The sections were then discussed and agreed upon amongst all authors. Results: The article highlights direct and indirect effects of climate change on allergies. It goes into detail about the connections between climate change and (new) pollen allergens as well as (new) occupational inhalation allergens, explains the effects of climate change on the clinical picture of atopic dermatitis, discusses the connections between air pollutants and allergies, and provides information about the phenomenon of thunderstorm asthma. Conclusions: There is a need for action in the field of pollen and fungal spore monitoring, allergy and sensitisation monitoring, urban planning from an allergological perspective, and changes in the working environment, among others.

Hypersensitivity pneumonitis due to metal working fluids: Detection of specific IgG antibodies to microbial antigens.

Occupational exposure to microbially contaminated metal working fluids (MWF) can cause hypersensitivity pneumonitis (HP). An important step in the diagnosis of HP is to identify the triggering antigen by detection of corresponding specific IgG antibodies (sIgG). As commercial sIgG tests are currently not available, protein antigens were prepared from MWF-workplace samples and from MWF-typical bacterial isolates. In 57 % of suspected HP-cases (n = 30) elevated sIgG concentrations were measured to at least one MWF-relevant antigen, of which Mycobacterium immunogenum was most prominent (88 %), followed by Pseudomonas oleovorans and Pseudomonas spec (82 % each), MWF-antigen mix and Pseudomonas alcaliphila (65 % each). Elevated sIgG concentrations to other microorganisms were measured to Micropolyspora faeni (82 %) and Aureobasidium pullulans (77 %). Correlation of sIgG values of all tested microbial antigens showed a significant relationship of MWF-antigen mixture to Pseudomonas antigens, but a low correlation to moulds. These newly prepared MWF-antigens are useful tools for the diagnosis of patients with suspected MWF-HP and are available for further investigations.

Impact of climate change on wood and woodworkers-Cryptostroma corticale (sooty bark disease): A risk factor for trees and exposed employees.

Climate changes have promoted an increased fungal infection of maple trees with Cryptostroma corticale, the causative agent of sooty bark disease. The hosts of C. corticale are maples, and since the early 2000s the fungus has been appearing more frequently in European forests, due to the droughts and hot summers of recent years. Infestation by C. corticale discolors the wood and makes it unusable for further processing, which leads to considerable economic damage in the timber industry. Therefore, the occurrence and spread of sooty bark disease raise serious problems. In addition to forestry and economic problems, the conidiospores of C. corticale can also cause health problems in exposed wood workers and they can trigger hypersensitivity pneumonitis (HP). Since the spores, which are deposited over the entire area under the bark of infected trees, can spread during processing, exposed workers must take special precautions to protect themselves against exposure. If an occupational disease is nevertheless suspected following exposure to C. corticale, valid diagnostics are required to confirm possible HP and derive appropriate therapies and exposure reduction or avoidance. Diagnosis of HP is based on several criteria, one of them is the detection of specific IgG in patient's serum against the potentially triggering antigens, in this case C. corticale antigens. To produce a diagnostic tool to measure C. corticale specific IgG, which is not commercially available so far, spores and mycelial material from ITS-sequenced strains of C. corticale was prepared and analyzed. These biochemically characterized extracts of spore and mycelial antigens were biotinylated and coupled to Streptavidin-ImmunoCAPs. To validate these diagnostic test tools the first step is to measure the concentration of C. corticale specific IgG in sera of healthy non-exposed and healthy exposed subjects to establish cut-off values. Suitable participants were recruited and the individual exposure to C. corticale and symptoms experienced during or after working with infected maple trees were recorded using questionnaires. Finally, diagnostic tools for serological testing in suspected cases of HP by C. corticale were created and evaluated. The following article provides recommendations for the treatment and disposal of infected damaged wood and for occupational health protection procedures. Secondly, the diagnosis of HP induced by exposure to C. corticale as an occupational disease is described including the verification of newly developed serological test tools for antigens of C. corticale.

Is in vitro cytokine release a suitable marker to improve the diagnosis of suspected mold-related respiratory symptoms? A proof-of-concept study.

Indoor mold infestation can lead to a variety of adverse health effects, including allergic and non-allergic respiratory complaints. Especially if no evidence of an allergic reaction can be found for the complaints, diagnostic tools that might explain mold-associated health problems are missing. As a proof-of-concept, in the present study whole blood assay (WBA) was used to determine cellular response by measuring cytokine release (IL-1ß and IL-8) after in vitro stimulation. Blood was available from a total of 48 subjects. By questionnaire, complaints and possible mold exposure were documented. Specific in vitro blood stimulation was tested with Escherichia coli endotoxin and extracts of different molds (Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Cladosporium herbarum). To characterize the relevance of WBA in describing the mold-induced immune response, we compared the following groups: asthmatics vs. non-asthmatics, mx1-sensitized vs. non-mx1-sensitized, mold-exposed vs. non-mold-exposed. In response to endotoxin stimulation, a significantly higher IL-1ß release was found in mx1-sensitized than in non-mx1-sensitized subjects. Furthermore, the blood of asthmatics showed significantly higher IL-8 and IL-1ß release after stimulation with Penicillium chrysogenum and endotoxin, respectively, compared to non-asthmatics. However, no significant difference in the level of cytokine release was observed between the mold-exposed and non-exposed group, neither after endotoxin nor mold stimulation. In conclusion, the WBA used in this study is not a suitable tool for clinical routine diagnostic workup. Our data suggests that WBA reflects cellular differences that are disease-related but not directly attributable to mold exposure. However, in combination with further data, WBA will be a helpful und interesting tool in research, e.g., in description of the complex immune response to molds.

What should be tested in patients with suspected mold exposure? Usefulness of serological markers for the diagnosis.

The associations of mold exposure, IgE-mediated sensitization, inflammatory markers, and respiratory symptoms were analyzed in 46 exposed and 23 non-exposed individuals. Both exposure and clinical symptoms were assessed by questionnaire. Specific (s)IgE to mold mixture (mx1) was significantly higher and found more frequently in exposed (41%) than non-exposed individuals (17%), which was not observed for sIgG to mold mix (Gmx6). Notably, exposed asthmatics were more frequently sensitized to molds (55%) compared to exposed non-asthmatics (18%). In addition, the serum concentrations of club cell protein (CC16) were significantly lower in exposed subjects, especially in asthmatics. Positive associations were observed among mold sensitization, asthma, and mold exposure, but not in subjects with predominantly environmental sensitizations without mold sensitization. Thus, sIgE to mx1 but not sIgG to Gmx6 is a useful diagnostic marker to verify mold-associated respiratory symptoms.

Quantitative measurement of IgG to SARS-CoV-2 antigens using monoclonal antibody-based enzyme-linked immunosorbent assays.

OBJECTIVE: Standardised quantitative analysis of the humoral immune response to SARS-CoV-2 antigens may be useful for estimating the extent and duration of immunity. The aim was to develop enzyme-linked immunosorbent assays (ELISAs) for the quantification of human IgG antibodies against SARS-CoV-2 antigens. METHODS: Enzyme-linked immunosorbent assays were developed based on monoclonal antibodies against human IgG and recombinant SARS-CoV-2 antigens (Spike-S1 and Nucleocapsid). The WHO 67/086 immunoglobulin and WHO 20/136 SARS-CoV-2 references were used for standardisation. Sera of a study group of COVID-19-positive subjects (n = 144), pre-pandemic controls (n = 135) and individuals vaccinated with BioNTech-Pfizer BNT162b2 vaccine (n = 48) were analysed. The study group sera were also tested using EuroImmun SARS-CoV-2-ELISAs and a quantitative S1-specific fluorescence enzyme immunoassay (FEIA) from Thermo Fisher. RESULTS: The ELISA results were repeatable and traceable to international units because of their parallelism to both WHO references. In the study group, median anti-S1-IgG concentrations were 102 BAU mL-1, compared to 100 and 1457 BAU mL-1 in the vaccination group after first and second vaccination, respectively. The ELISAs achieved an area under the curve (AUC) of 0.965 (S1) and 0.955 (Nucleocapsid) in receiver operating characteristic (ROC) analysis, and a specificity of 1 (S1) and 0.963 (Nucleocapsid) and sensitivity of 0.903 (S1) and 0.833 (Nucleocapsid) at the maximum Youden index. In comparison, the commercial assays (S1-FEIA, S1 and Nucleocapsid ELISA EuroImmun) achieved sensitivities of 0.764, 0.875 and 0.882 in the study group, respectively. CONCLUSIONS: The quantitative ELISAs to measure IgG binding to SARS-CoV-2 antigens have good analytical and clinical performance characteristics and units traceable to international standards.

Microbial contamination in water-based metalworking fluid as trigger for occupational hypersensitivity pneumonitis - development of specific IgG tools for a suspected clinical case.

Microbially contaminated metal-working fluid (MWF) can cause respiratory symptoms in exposed workers in the form of exogenous allergic alveolitis/hypersensitivity pneumonitis (HP). The diagnosis of HP is based, among others, on the identification of the culprit and the detection of corresponding specific IgG antibodies (sIgG) in the patient's serum. Commercial antigen tools for the detection of these HP triggers are rarely available; therefore, antigens from contaminated MWF workplace samples were isolated exemplarily for diagnosis of a suspected HP case. Various MWF-specific bacteria were identified in the workplace samples, including Pseudomonas oleovorans, Pseudomonas alcaliphila, Pseudomonas spec., Paenibacillus glucanolyticus, and Corynebacterium amycolatum. The sIgG antigen binding, detected by ImmunoCAP system against MWF antigens from workplace samples and against the identified bacterial antigens, was much stronger in the patient serum compared to selected reference sera. The highest sIgG concentrations in the patient's serum could be determined against Pseudomonas antigens. Inhibition tests showed cross-reactions of MWF and Pseudomonas antigens, whereby the Pseudomonas antigens cross-reacted less with each other. For in-vitro diagnosis in case of suspected HP caused by contaminated MWF, workplace-related antigens are now available.

Mould allergens: Where do we stand with molecular allergy diagnostics?: Part 13 of the series Molecular Allergology.

The official WHO/IUIS database (www.allergen.org) currently lists 77 mould allergens from a variety of protein families. To date, only eight recombinant single allergens from three mould species are available for molecular allergy diagnosis of mould sensitization. These include rAlt a 1, the major allergen in Alternaria alternata-sensitized individuals, and enolase rAlt a 6 with it potential cross-reactivity to mould, food and natural latex allergens. rAsp f 1, 2, 3, 4 and 6 from Aspergillus fumigatus are available for diagnostic purposes; specific IgE to rAsp f 2, 4 and 6 is often positive in allergic bronchopulmonary aspergillosis (ABPA). The dehydrogenase rCla h 8 is considered a major allergen of Cladosporium herbarum with possible cross-reactivity to other dehydrogenase allergens. The narrow range of commercially available individual mould allergens should be expanded to include marker allergens typical for mould (e.g., serine proteases). In addition, standardization of total extracts needs to be improved in the future to guarantee valid mould products with defined allergen content for diagnostic and therapeutic purposes.

A new immunoassay to quantify fungal antigens from the indoor mould Aspergillus versicolor.

Aspergillus versicolor is among the most commonly found moulds in moisture-damaged buildings and can be associated with adverse health effects in humans. This paper reports the development, validation and application of an enzyme immunoassay to quantify A. versicolor antigens. A sandwich ELISA was developed using polyclonal antibodies that recognize a broad range of A. versicolor proteins present in fungal spores and in mycelia fragments. To validate the new method, A. versicolor antigens were quantified in samples collected from homes with visible mould growth, including dust from vacuumed walls and bulk samples of building materials. Antigen concentrations were compared to the results of a commercial ELISA based on monoclonal antibodies (AveX ELISA, Indoor Biotechnologies, Charlottesville, USA) and correlated with colony forming units (CFU) of A. versicolor. The A. versicolor ELISA was very sensitive with a lower detection limit of 120 pg ml(-1). The assay also showed some reactivity to other moulds with strongest reactions with other Aspergillus species (1-3% reactivity). The new assay detected A. versicolor antigens in a much higher percentage of dust samples (88% vs. 27%) and bulk samples (89% vs. 24%) than the AveX assay. A significant correlation (r = 0.67, and p < 0.0001) was found between antigen concentrations and CFU of A. versicolor. Based on its low detection limit and good correlation with the culture-based method, this new immunoassay seems to be a useful tool for the measurement of A. versicolor exposure levels and a reliable complement to the traditional monitoring techniques, such as mould cultivation or microscopy.

Development and application of mold antigen-specific enzyme-linked immunosorbent assays (ELISA) to quantify airborne antigen exposure.

The aim of our study was to develop specific enzyme-linked immunosorbent assays (ELISAs) and apply these to assess mold antigen exposure in composting plants. Sandwich ELISAs based on polyclonal antibodies to Aspergillus fumigatus (Af), Penicillium chrysogenum (Pc), and Cladosporium herbarum (Ch) antigens were developed and validated. Reactivity to 18 different mold species was tested. To optimize extraction procedure, inhalable dust samples taken by a parallel sampler were extracted with or without homogenization. In 31 composting plants stationary pumps were installed at 4 sites to collect 124 inhalable dust samples. The newly developed ELISAs were used in addition to an anti ß-1,3-glucan ELISA to quantify mold antigens. The Cladosporium ELISA showed less than 0.04% reactivity to extracts from other fungal genera, while the Af ELISA demonstrated a reactivity of up to 3.6% and the Pc ELISA reacted up to 11% to other mold species. Extraction of parallel sampled filters gave higher antigen amounts with homogenization. The increase was highest for Pc-antigens, followed by Af-antigens, and lowest for Ch-antigens. Mean lower detection limits of homogenized inhalable dust samples were 5 ng/m(3) (Af), 0.6 ng/m(3) (Pc), 0.2 ng/m(3) (Ch), and 0.6 ng/m(3) (ß-1,3-glucan). The ELISAs were able to detect antigens in 43% (Af), 37% (Pc), 94% (Ch), or 100% (ß-1,3-glucan) of the 124 airborne dust samples. Inhalable dust, ß-1,3-glucan, and Af-, Pc-, and Ch-antigen concentrations were significantly correlated. The newly developed mold antigen ELISAs are thus able to measure airborne exposure levels in composting plants and differentiate between distinct fungi genera.

Occupational IgE-mediated softwood allergy: characterization of the causative allergen.

Allergic reactions to wood dust allergens are rare, and only few in vitro diagnostic tools and information about relevant allergens are available. To differentiate between protein-based allergy and probably clinically silent glycogenic sensitization, it is helpful to characterize the relevant protein allergens and specify IgE binding. The current case report deals with the occupational softwood allergy of a carpenter exposed to different wood dusts. Skin tests and IgE tests against wood were performed with specifically tailored ImmunoCAPs and cross-reactive carbohydrate determinants. Potential allergens were identified by IgE blots and tandem mass spectrometry. The clinical relevance was verified by challenge tests. Specific IgE to softwood (spruce, pine and larch wood), beech wood, natural rubber latex (NRL) and horseradish peroxidase (HRP) were detected. Allergens in spruce wood, the dominant allergen source, were identified as peroxidases. Softwood were the strongest inhibitors. HRP reduced IgE binding to softwood to <50%, indicating predominantly proteinogenic epitopes, whereas IgE binding to NRL and beech wood was reduced to >50% by HRP, indicating predominantly glycogenic IgE epitopes. Skin and challenge tests underlined that softwoods were the source of sensitization. For the polysensitized patient, a clinically relevant softwood allergy was diagnosed, not only by challenge tests but also with specifically tailored in vitro tools.

Immunoglobulin E-mediated sensitization to pine and beech dust in relation to wood dust exposure levels and respiratory symptoms in the furniture industry.

OBJECTIVES: Wood dust exposure may cause Immunoglobulin E (IgE)-mediated allergic diseases. Our objectives were to estimate pine and beech dust sensitization rates among woodworkers and a reference group, explore the association between exposure and sensitization and between sensitization and respiratory symptoms, and finally investigate the impact of proteinogenic specific IgE (sIgE) epitopes on respiratory symptoms. METHODS: In a Danish study among 52 furniture factories and 2 reference factories, we evaluated the workers' asthma and rhinitis status using questionnaires and blood samples collected from 1506 woodworkers and 195 references. Workers with asthma symptoms (N=298), a random study sample (N=399) and a random rhinitis sample (N=100) were evaluated for IgE-mediated sensitization to pine and beech dust. RESULTS: The prevalence of pine and beech sensitization among current woodworkers was 1.7 and 3.1%, respectively. No differences in sensitization rates were found between woodworkers and references, but the prevalence of wood dust sensitization was dose-dependently associated with the current level of wood dust exposure. No relation was observed between wood dust sensitization per se and respiratory symptoms. Only symptomatic subjects had proteinogenic IgE epitopes to pine. Increased odds ratios for sIgE based on proteinogenic epitopes to beech and respiratory symptoms were found, although they were not statistically significant. CONCLUSIONS: Sensitization rates to pine and beech were the same for woodworkers and references but dependent on the current wood dust exposure level. The importance of beech and pine wood sensitization is limited, but may be of clinical significance for a few workers if the IgE epitopes are proteinogenic.

Development of an obeche wood allergen quantification assay for the assessment of allergen exposure in workplaces.

OBJECTIVES: The purpose of this study was to develop a quantification assay to measure airborne concentrations of obeche wood allergen at workplaces. METHODS: Specific polyclonal antibodies to obeche wood were produced in rabbit and used to develop an inhibition enzyme immunoassay (EIA). Inhalable dust samples from three wood-processing companies were taken with a stationary sampling device (Gravicon VC25). The loaded dust filters were extracted under standardized conditions and measured with the assay. In addition, the antigen and allergen contents of obeche wood from different sources (Cameroon, N=5; Ghana, N=4) were analyzed from immunoblots, detected with rabbit immunoglobulin (Ig) G and human Ig E. RESULTS: Polyclonal antibodies specific for obeche wood allergens, without cross-reactivity to other woods, were used to establish the inhibition enzyme immunoassay. The assay is able to quantify allergen concentrations from 30 to 300 ng/ml. With inhibition enzyme immunoassay, exposure to airborne obeche wood allergen can be monitored in wood-processing companies. Inhalable dust samples from workplaces contained an average allergen concentration of 15 microg/g dust. Significantly lower protein and allergen contents were measured for obeche wood from Cameroon (ayous) in one company. IgE immunoblots indicated that the lower antigen and allergen contents of the ayous wood may be the result of its lacking the major obeche wood allergen, Trip s 1. CONCLUSIONS: The data showed that the total dust concentration in workplaces containing obeche wood dust did not correspond to the assessed allergen concentration. To estimate exposure limits regarding sensitization risk, it is necessary to measure the allergen content directly.

Sensitization due to gum arabic (Acacia senegal): the cause of occupational allergic asthma or crossreaction to carbohydrates?

BACKGROUND: A pharmaceutical industry worker was exposed to dust of gum arabic in the tablet coating plant and complained of work-related shortness of breath, chest tightness, runny nose, itching and redness of the eyes. This case was investigated for allergy to gum arabic and compared with a control group. The aim of the study was to identify the IgE-binding components responsible for the work-related symptoms. METHODS: Skin prick tests (SPTs)and specific IgE (sIgE) measurements with environmental and occupational allergens, spirometry and a specific bronchial challenge with gum arabic were performed. One hundred and nineteen control subjects underwent SPT with gum arabic and 43 controls were tested for sIgE. Crossreactivity between gum arabic and horse radish peroxidase was investigated by IgE CAP inhibition. A combined procedure of immunoblotting and periodate treatment was applied to identify the epitope nature of gum arabic. RESULTS: Allergy to gum arabic was shown by SPT, presence of sIgE and a positive bronchial challenge with gum arabic. Sensitization to gum arabic was demonstrated by SPT or sIgE in 7 and 5 controls, respectively. The results of inhibition with horse radish peroxidase, immunoblotting and periodate treatment suggest that gum arabic sIgE of the patient and 1 SPT-positive control subject were directed to the polypeptide chains of gum arabic. In contrast, gum arabic sIgE of the other controls reacted to carbohydrate components. CONCLUSIONS: Sensitization to gum arabic carbohydrate structures occurs casually in atopic patients with pollen sensitization without obvious exposure to gum arabic. This study suggests that allergy to gum arabic is mediated preferentially by IgE antibodies directed to polypeptide chains of gum arabic.

Bites of the European pigeon tick (Argas reflexus): Risk of IgE-mediated sensitizations and anaphylactic reactions.

BACKGROUND: Local and systemic reactions can occur after bites of Argas reflexus (Argas), a soft tick parasitizing pigeons. OBJECTIVE: Risk assessment of IgE-mediated sensitizations and systemic reactions after Argas bites. METHODS: Case histories, skin prick tests (SPTs) with a whole-body extract of Argas containing major allergen Arg r 1, and common inhalants and specific IgE measurements were obtained from 148 subjects who had had Argas bites and 20 volunteers as a control group. RESULTS: Systemic reactions (urticaria, angioedema, dyspnea, cardiovascular dysregulation, unconsciousness) were reported in 12 of 148 (8%); 146 of 148 (99%) had local reactions. Atopy was found in 37 of 146 (25%) with local reactions and 3 of 12 (25%) with systemic reactions. SPT to Argas was positive in 24 of 148 (16%) with a high proportion of atopics 10 of 24 (42%); specific IgE to Argas was detectable in 12 of 135 (8% of 148) with moderate concordance to systemic reactions. No positive SPT or specific IgE results to Argas were obtained in the control group. Immunoblotting of 23 sera revealed an IgE-binding protein in 19 of 23 sera (82%) at 22 kd, indicating a major allergen of Argas. CONCLUSION: Severe anaphylactic reactions were infrequently (approximately 8%) found after bites of the soft tick Argas reflexus. Atopy is a risk factor for skin sensitizations to Argas, but not for systemic reactions after bites by Argas. Using a whole-body extract of Argas, diagnosis through SPT and specific IgE is hampered by false-negative and irrelevant positive results, particularly in atopy.

A new method to bind allergens for the measurement of specific IgE antibodies.

BACKGROUND: Detection of allergen-specific IgE antibodies in patients' sera plays a key role for the diagnosis of IgE-mediated allergy. If no validated test system is available, diagnostic tools must be developed, usually by coupling or binding the allergens to a solid phase. Streptavidin ImmunoCAP is a new solid phase for binding of allergens which can be used in the Pharmacia CAP system. OBJECTIVE: It was the aim of this study to assess the diagnostic validity of Streptavidin ImmunoCAP. METHODS: Biotinylation and allergen concentration for binding to Streptavidin ImmunoCAP were optimized and IgE obtained with natural rubber latex, obeche wood, wheat and rye flour Streptavidin ImmunoCAP were compared with the results of ImmunoCAP and Enzyme Allergo-Sorbent Test (EAST) using sera from patients complaining of workplace-related respiratory symptoms. RESULTS: While the relation of biotin-label and protein was critical (best results were obtained with a 5- fold molar excess), labelled protein for coupling to streptavidin ImmunoCAP was applicable in a wide concentration range. On average, IgE values with streptavidin ImmunoCAP were as high as with ImmunoCAP but considerably higher than values obtained by EAST. CONCLUSION: Streptavidin ImmunoCAP is a valuable tool for sensitive and specific measurement of IgE binding to new allergens superior to cellulose disk-based methods.