How to diagnose mould allergy? Comparison of skin prick tests with specific IgE results.

BACKGROUND: Diagnosis of mould allergy is complicated due to the heterogeneity of the test material and the decrease in the number of commercial mould skin test solutions that are currently available. OBJECTIVES: The aim of this study was to compare skin prick tests (SPT) from different manufacturers to one another and concurrently with sIgE tests for Aspergillus fumigatus (Asp f), Cladosporium herbarum (Cla h), Penicillium chrysogenum (Pen ch), Alternaria alternata (Alt a) and Aspergillus versicolor (Asp v) to ascertain a feasible diagnostic procedure for mould sensitization. METHODS: In this multi-centre study, 168 patients with mould exposure and/or mould-induced respiratory symptoms were included. Mould SPT solutions were analysed biochemically and tested in duplicate on patients' arms. Specific IgE (sIgE) concentrations to corresponding mould species and mould mix (mx1) were measured by ImmunoCAP. SPTs in accordance with one another and with sIgE were further considered. The test efficiency was calculated using receiver-operating characteristic (ROC) analysis. RESULTS: Mould sensitization was more frequently detected by the SPT (90 of 168) than by the sIgE tests (56 of 168). Concordances of double SPT positives were only sufficient (≥ 80%) for environmental allergens, two Asp f and three Alt a SPT solutions, whereas all other mould solutions revealed concordances < 80%. The antigen content of SPT solutions was positively associated with concordant SPT double values as well as with sIgE. Taking sIgE as the 'positive standard', all mould SPT solutions revealed test efficiencies > 80%, but varied up to 20% in sensitivity and positive predictive value with the exception of Alt a. CONCLUSIONS: SPT solutions are sensitive and essential diagnostic tools for the detection of mould sensitization. Our recommendation for diagnosis would be to test at least Alt a, Asp f and Pen ch using SPT and additional sIgE test to mx1.

Quantification of obeche wood allergen: Development of a sensitive sandwich-ELISA for occupational exposure assessment.

Obeche wood is a prominent cause of allergic occupational asthma. To reduce the risk of immunoglobulin E (IgE)-mediated sensitization it is important to assess airborne obeche wood allergen concentrations at exposed workplaces. Therefore, a highly sensitive obeche wood allergen immunoassay was developed and applicability was proven on airborne passive dust samples in Spanish wood workshops. Obeche wood sandwich enzyme-linked immunosorbent assay (ELISA) polyclonal antibodies (pAbs) were developed. Test specificity was verified by different wood and mold extracts. Obeche wood allergen monitoring was conducted in four Spanish wood workshops, including wood-dust-exposed and nonexposed areas inside and outside the workplaces, as well as controls. Dust was collected with electrostatic dust collectors (EDC). Measuring range of the obeche wood sandwich-ELISA was between 36 pg/ml and 1.6 µg/ml. The test system showed only marginal reactivity to other hardwoods and no reactivity to softwoods and molds. Obeche allergen was detected in all EDC from workplaces. The highest concentration was measured in the workshop with the longest obeche wood exposure (geometric mean [GM]: 7548 µg/m2); shorter obeche wood processing periods resulted in lower amounts of allergen (GM: 29 µg/m2). Obeche wood allergen transfer from exposed workplaces to nonexposed areas inside and outside the workshop was assessed. In control EDC from nonexposed facilities/homes no obeche wood allergen was found. The new obeche wood sandwich-ELISA is a valid tool to quantify obeche allergen exposure. Evidence indicates it will be possible to monitor obeche allergen exposure during different processes, as well as transfer effects in nonexposed areas.

Biochemical and immunological analysis of mould skin prick test solution: current status of standardization.

BACKGROUND: Sensitization prevalence to moulds reached from less than 10% in the general population to more than 25% in atopic and/or asthmatic subjects. To diagnose IgE-mediated mould sensitization, skin prick test (SPT) and specific IgE (sIgE) measurement are recommended. However, concordance of SPT and sIgE results is often less than 50% and standardization of the extracts is required to achieve reliable test results. OBJECTIVE: The aim of our study was to analyse mould SPT solutions (SPTs) with respect to quantity and quality of protein, antigen and human IgE-binding content as a prerequisite for further in vivo studies. METHODS: Commercial SPTs of Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum and Penicillium chrysogenum from six manufacturers as well as two in-house extracts from Aspergillus versicolor were investigated. Protein-, antigen- and IgE-binding contents were quantified by Bradford assay, sandwich ELISA and IgE-ImmunoCAP-inhibition tests. Protein composition and IgE and IgG binding were analysed by SDS-PAGE and immunoblotting, respectively. RESULTS: Median protein concentrations were similar in all mould SPT extracts (90-110 µg/mL). In contrast, antigen contents and IgE-binding capacity showed a high variability with median antigen values from 4 to 118 µg/mL and IgE inhibition results between 30 to 95%. Whereas almost all SPTs of A. alternata and A. versicolor showed complete sIgE inhibition with mean values > 80%, only three extracts for A. fumigatus, two extracts for C. herbarum and none of the tested extracts for P. chrysogenum exceeded 50% sIgE reduction. Quantitative amounts of protein, antigenic and IgE-binding structures were not comparable with the quality of the corresponding protein or immunoblot pattern, with the exception of A. alternata SPTs. CONCLUSIONS AND CLINICAL RELEVANCE: Commercially available mould SPT extracts showed high variability raising the question of comparability and reliability of SPT results. Possible consequences for diagnostic test outcome will be investigated in the next step.

Impact of cross-reactive carbohydrate determinants on wood dust sensitization.

BACKGROUND: Occupational wood dust exposure can induce allergy and may be one cause of respiratory health problems among woodworkers. OBJECTIVE: The objective was to determine the prevalence and quantitative level of specific immunoglobulin E (sIgE) to beech and pine wood in exposed workers. Wood sensitization was specified with regard to cross-reactivity and was correlated to the reported symptoms. METHODS: Danish workers (n=701) were investigated for sIgE to beech and pine. Wood samples from workplaces were analysed and coupled to ImmunoCAPs. Workers sensitized to wood were tested for cross-reactive carbohydrate determinants (CCDs) and environmental allergens. IgE binding was specified for glycogenic vs. proteinogenic epitopes by inhibition tests. RESULTS: The prevalence of wood sensitization among all workers was 3.7%. There was no association between sensitization prevalence or sIgE concentrations and self-reported allergic symptoms. Beech- and pine-sensitized workers showed a high prevalence of CCD sensitization (73%). However, workers with a single sensitization to wood had no sIgE to CCDs. Specifying IgE epitopes demonstrated that sera of workers reporting allergic symptoms recognized proteinogenic IgE-epitopes on wood allergens, whereas workers without allergic symptoms had primarily sIgE-epitopes to glycogenic structures. Although 96% of the wood-sensitized workers were atopic, no significant correlation was found between wood sensitization and sIgE to beech and birch pollen, but an association was found between sIgE against CCDs and pine pollen. CONCLUSION: Sensitization prevalence to beech and pine wood measured by tailored ImmunoCAPs was not correlated to allergic symptoms. We recommend the application of CCD tools to assess the relevance of individual wood sensitization.

Identification and characterization of cross-reactive natural rubber latex and Ficus benjamina allergens.

BACKGROUND: An association between allergy to Ficus benjamina and natural rubber latex (NRL) has been suspected based on clinical and immunological observations. The responsible cross-reactive allergens have not been identified yet. This study was undertaken to investigate the cross-reactivity between hevein (Hev b 6.02, 4.7 kD), a major allergen of NRL, and F. benjamina and identify its counterpart in F. benjamina. METHODS: 89 serum samples from subjects allergic to NRL were used in the study. Skin prick tests were performed with highly purified hevein and sap extract of F. benjamina. Specific IgE antibodies to NRL, F. benjamina and Hev b 6.02 were determined by the Pharmacia CAP method. Cross-reactivity among these allergens was investigated by means of CAP and immunoblot inhibition experiments. Two-dimensional gel electrophoresis separation and protein microsequencing were performed to identify the cross-reactive allergens in F. benjamina. RESULTS: 67 out of 89 (75%) sera showed elevated IgE to hevein. Specific IgE to Ficus were found in 22 (24.7%) sera, and with 1 exception, all these sera also had IgE to Hev b 6.02. Results of CAP inhibition assays using 11 sera showing IgE to both Hev b 6.02 and Ficus demonstrated that the IgE to Ficus could be completely inhibited by Hev b 6.02 in 6 of 11 sera. Immunoblots and immunoblot inhibition assays revealed that a protein of about 45 kD in F. benjamina is strongly recognized by serum IgE. In addition, the IgE-binding reactivity to this 45-kD protein could be completely inhibited by preincubation of the sera with Hev b 6.02. N-terminal protein sequencing of 14 amino acids indicated that this 45-kD protein has a hevein-like domain at the N-terminal region and may belong to the endochitinase family. CONCLUSION: Latex-allergic patients are at higher risk of becoming sensitized to Ficus. Hev b 6.02 in latex is a major cross-reactive allergen and its counterpart in F. benjamina is an acidic protein with a molecular weight of about 45 kD and a hevein-like N-terminal domain.