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Keywords:

  • allergens;
  • baker's asthma;
  • bronchial hyperresponsiveness;
  • methacholine;
  • occupational asthma;
  • skin sensitivity;
  • specific inhalation challenge

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background:  Quantitative relationships between immunological reactivity, non-specific bronchial responsiveness and bronchial responsiveness to allergens have scarcely been investigated in occupational asthma.

Methods:  We assessed the above relationships in 24 subjects with baker's asthma. The skin endpoint titration to bakery allergens as a measure of immunological reactivity, together with the methacholine PC20 and allergen PC20 during early asthmatic reaction were determined.

Results:  All patients had positive skin tests to some bakery allergens (wheat and rye flour, soybean flour, fungal enzymes and egg white proteins) and bronchial hyperresponsiveness to methacholine. Specific inhalation challenge (SIC) tests were performed with aqueous allergen extracts of cereal flour (n = 14), soybean (n = 8), baking enzymes (n = 12), and egg white proteins (n = 8) in sensitized workers. A positive asthmatic reaction was observed in 84% of the inhalation challenges. SIC elicited isolated early asthmatic reactions in 62%, dual reactions in 32% and isolated late reactions in 5%. Multiple linear regression analysis showed allergen PC20 as a function of skin sensitivity to allergen and methacholine PC20, yielding the following highly significant regression formula: log-allergen PC20 = 0.18 + 0.99 log(skin sensitivity) + 0.343 log(methacholine PC20) (r = 0.89, P < 0.001). This formula predicted allergen PC20 to within one double concentration in 67%, to within two double concentrations in 85% and within three double concentrations in 97%.

Conclusion:  The main determinant of bronchial responsiveness to allergen in patients with baker's asthma is the degree of sensitization to occupational allergens as determined by skin reactivity, modulated to a lesser extent by non-specific bronchial hyperresponsiveness.

The relationship between the degree of allergen sensitization, airway hyperresponsiveness to bronchoconstrictive mediators (such as methacholine or histamine) and bronchial responsiveness to allergen was initially proposed by Tiffenau (1). This association was subsequently confirmed by other groups with common aeroallergens (2–4). Cockcroft et al. reported that the severity of the early asthmatic reaction to common aeroallergens can be predicted from skin sensitivity to the allergen and airway responsiveness to histamine (4). A recent study confirmed the dependence of the allergen-induced early asthmatic reaction upon the level of allergen skin test sensitivity and the degree of airway hyperresponsiveness as assessed by methacholine (5).

This relationship, however, has rarely been investigated with occupational allergens. Shirai et al. (6) demonstrated the links between specific and non-specific airway hyperresponsiveness and skin test reactivity to the low-molecular-weight agent epigallocatechin galate, the causative agent of green tea-induced asthma. However, no data are available regarding the relationship between these parameters in occupational asthma (OA) caused by high-molecular-weight agents.

The gold standard in the diagnosis of OA is specific inhalation challenge (SIC) (7). Laboratory-specific inhalation testing should only be performed in specialized centers with trained personnel in order to ensure safety for patients and reliable results. The prediction of the magnitude of airway response to an occupational allergen relying on the assessment of specific skin reactivity and airway responsiveness to methacholine is highly relevant for safety purposes.

The aim of this study was to evaluate the relationship between skin test sensitivity, airway hyperresponsiveness to methacholine and bronchial responsiveness to occupational allergens among bakery workers with asthma.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Subjects

This study included 24 consecutive employees (92% males) in the baking industry (bakers and pastry makers) with suspected OA on the basis of a suggestive clinical history. These patients were working at different bakeries or confectioners and were referred for evaluation to the Allergy Department, Fundación Jiménez Díaz in Madrid, between 1998 and 2005. Their ages ranged between 22 and 60 years (mean 38 years). These subjects had developed work-related symptoms of cough, chest tightness, shortness of breath and wheezing. All of them improved during long holidays and most of them during weekends. Sneezing, itching, and runny nose preceded the development of asthma symptoms in 22 of these workers. All subjects but two (one had retired 2 years ago and another had a new job 8 months before evaluation) were still working at the time of the study.

These workers routinely handled cereal flour (wheat, rye), soybean flour and flour additives containing fungal enzymes (alpha-amylase, hemicellulase and glucoamylase). Pastry makers also used liquid egg at work. All the subjects could eat bread and baking products without any symptoms. They were taking inhaled corticosteroids on a regular basis and short-acting and/or long-acting beta-agonists at the time of the study. All subjects gave written inform consent to participate in the study and to undergo at least one SIC.

Skin prick tests

Skin prick tests (SPT) were performed with in-house extracts of wheat flour, rye flour and soybean flour that were prepared in saline solution at 10% w/v as previously described (8). The Aspergillus-derived enzymes alpha-amylase, hemicellulase and glucoamylase (Bial-Aristegui, Bilbao, Spain) were prepared at 1% w/v. Egg white proteins (ovalbumin, ovomucoid, conalbumin and lysozyme) were purchased from Sigma Chemical Co. (St Louis, MO, USA) and tested at 1% w/v in all pastry makers. The extracts were filtered through a 0.22-μm membrane (Millipore Corp., Bedford, MA, USA).

A panel of common inhalant allergens, including pollen (grass, trees and weeds), mites (Dermatophagoides pteronyssinus and D. farinae), animal dander (cat and dog), and moulds (Alternaria, Aspergillus and Penicillium) (ALK-Abelló, Madrid, Spain) was also tested. Atopy was defined as the presence of a positive skin reaction to at least one of these common aeroallergens.

Histamine phosphate at 10 mg/ml and normal saline were used as positive and negative controls, respectively. The response was read 15 min after puncture, and the results were expressed as the mean of two largest orthogonal diameters (mm). A wheal diameter 3 mm or greater with erythema, compared with the saline control, was defined as a positive reaction. Allergen SPT endpoint titration was performed using aqueous extracts of the same allergen preparations to be used for inhalation. On the control day, duplicate SPT were carried out with doubling dilutions of the stock allergen extract from 1 : 2 to 1 : 32 768. The skin endpoint was defined as the threshold concentration producing a 2-mm wheal diameter (3).

Serum IgE determinations

Total serum IgE was measured by Pharmacia CAP system IgE fluoroenzyme immunoassay (FEIA). The determination of specific IgE antibodies to wheat, rye and soybean flour, alpha-amylase, egg white, ovalbumin, ovomucoid and lysozyme was performed using Pharmacia CAP system FEIA (Pharmacia Diagnostics, Uppsala, Sweden). IgE levels higher than 0.35 kU/l were regarded as positive, as recommended by the manufacturer.

Inhalation challenge tests

A spirometer (MasterScope; Jaeger, Höchberg, Germany) was used for pulmonary function measurements. Bronchial hyperresponsiveness to methacholine was assessed by using the procedure described by Cockcroft et al. (9) with some modifications. The aerosolized particles were generated by a continuous pressurized nebulizer model DeVilbiss 646 (DeVilbiss Co., Somerset, PA, USA) with an output of 0.13 ml/min. Methacholine PC20 values <16 mg/ml were considered to reflect significant bronchial hyperresponsiveness. The result of this test was expressed as the provocative concentration of methacholine causing a 20% fall (PC20) in forced expiratory volume in 1 s (FEV1) and it was determined by interpolation of the last two concentrations (10). Methacholine inhalation test was performed the day before the allergen challenge.

Specific inhalation challenge was carried out following the method previously described (11, 12). The patient inhaled the aerosolized allergen using the nebulizer method mentioned above in progressive concentrations at tidal breathing for 2 min. A control challenge with isotonic saline solution was carried out before antigen provocation. Increasing concentrations of allergen were given by inhalation starting with a concentration that induced a 2-mm wheal on SPT (3, 11, 12). When several consecutive dilutions induced a 2-mm wheal, the least concentrated was selected (4). The dose was increased in twofold increments at intervals of 10 min and FEV1 was measured at 5 and 10 min after inhalation of each concentration. Inhalation challenge test was discontinued when there was a fall in FEV1 of ≥20% from the lowest post-saline value, or when the highest concentration had been given. At the end of the inhalation test, spirometry was performed at 20, 30, 40, 60 and 120 min after challenge. From that moment, FEV1 measurements were performed hourly with a portable computerized Asthma Monitor (AM1, Jaeger) for 24 h after challenge, respecting sleeping time. A fall in FEV1 of ≥20% from the lowest post-saline within 60 min of challenge was considered a positive early asthmatic reaction (EAR), and a similar fall between 2 and 24 h after challenge was considered a late asthmatic reaction (LAR) if no change was observed during the control day. A dual asthmatic reaction (DAR) was an EAR followed by a LAR. Patients were monitored in the laboratory for at least 6 h after the challenge. Written instructions were given to the patients to treat an LAR with inhaled beta-2 agonists and oral prednisone (0.5 mg/kg) in the event this type of reaction developed after they had left the hospital.

The EAR was calculated as the maximum per cent reduction in FEV1 (% fall from baseline) occurring in the first hour after challenge. PC20 allergen was calculated as described above (10). The tests were undertaken when the patients were absent from work (for at least 48 h), starting at 9:00 hours, always by the same physician. When patients underwent more than one SIC, the allergen challenges were separated for at least 1 month. Asthma drugs were withheld for the generally recommended interval (short-acting and long-acting inhaled beta-2 agonists were stopped at least 8 and 48 h before the challenge, respectively) (13). The dose of inhaled corticosteroids was taken the night before SIC (13).

When the patient was sensitized to more than one bakery-derived allergen on SPT, the order in which SIC were carried out was based on the largest wheal size observed on SPT with the stock allergen solutions. Some patients refused to undergo more than one or two SIC.

Statistical analysis

Logarithmic transformations of skin sensitivity (skin test endpoint), specific IgE levels, allergen PC20 and methacholine PC20 data were used for analyses. Correlations among skin sensitivity and allergen PC20, specific IgE levels and allergen PC20, methacholine PC20 and allergen PC20, and skin sensitivity and specific IgE levels with methacholine PC20 were studied using Pearson's correlation coefficient with confidence interval (CI) at the 95% level.

A multiple linear regression analysis was performed to identify if skin sensitivity to allergen, specific IgE levels or methacholine PC20 could explain bronchial responsiveness to allergen. Allergen PC20 was expressed as a function of both, skin sensitivity and methacholine PC20, by means of an equation. Correlation coefficient (r) was calculated for all regressions. A P-value of <0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Clinical characteristics

Demographic and clinical characteristics of these patients are shown in Table 1. These employees had been working in the baking industry between 2.5 and 45 years. The mean duration of asthma symptoms in these subjects was 6.7 years (range 0.4–29). All workers but two also had rhinitis symptoms.

Table 1.   Demographic, occupational and clinical data of study subjects.
  1. PC20: provocative concentration of methacholine causing a 20% fall in forced expiratory volume in 1 second (FEV1).

  2. BDP, beclomethasone dipropionate equivalent dose daily.

n24
Gender
 Male22 (91.7%)
 Female2 (8.3%)
Mean age ± SD (years)38.1 ± 12.40
Atopy66.7%
Smoking
 Never54.2%
 Smoker29.2%
 Ex-smoker16.7%
Job
 Bakers58.3%
 Pastry makers29.2%
 Bakers and pastry makers12.5%
Duration of exposure (years)16.1 ± 11.74
Duration of symptoms
 Rhinitis7.4 ± 7.34
 Asthma6.7 ± 7.92
Work-related symptoms
 Asthma8.3%
 Rhinoconjunctivitis and asthma79.2%
 Rinoconjuntivitis, asthma and contact urticaria12.5%
Pulmonary function tests
 FVC (% predicted)97.9 ± 16.06
 FEV1 (% predicted)88.9 ± 19.8
 FEV1/FVC (%)74.8 ± 10.8
PC20 methacholine (mg/ml)
 <0.1253 (13.1%)
 0.125–115 (65.2%)
 >1–165 (21.7%)
Asthma medication
 Inhaled corticosteroids (median BDP equivalent)100% (730 μg)
 Long-acting beta-agonists79.2%
 Leukotriene receptor antagonists25%

Skin prick tests and IgE determinations

The results of SPT to common aeroallergens revealed that 16 subjects (67%) were atopics. The percentage of workers with a positive SPT to the occupational allergens was: wheat flour 75%, rye flour 71%, soybean flour 42%, soybean trypsin inhibitor 10%, alpha-amylase 67%, hemicellulase 43%, glucoamylase 19%. In addition, the percentage of positive SPT with egg proteins among pastry makers (n = 15) was: egg white 33%, ovalbumin 27%, ovomucoid 13%, lysozyme 33%, and conalbumin 0%. All workers showed positive SPT to at least one of the bakery-derived allergens. Positive skin tests to more than two occupational allergens was observed in 83%. Two workers had positive skin tests only to cereal allergens (wheat and rye) and other two workers only to alpha-amylase.

The percentage of positive IgE determinations to occupational allergens was as follows: wheat flour 75% (geometric mean of positive determinations 9.22, range 0.8–100 kU/l), rye flour 67% (6.96, 0.37–100 kU/l), soybean flour 34% (2.49, 0.71–10.7 kU/l), alpha-amylase 55% (2.87, 0.63–32.2 kU/l). The frequency of positive IgE to egg proteins among pastry makers was: egg white 27% (3.05, 0.60–51.3 kU/l), ovalbumin 20% (4.11, 0.45–29.2 kU/l), ovomucoid 13% (0.60, 0.45–29.2), lysozyme 20% (1.32, 0.76–1.82 kU/l) and conalbumin 0%. Total serum IgE ranged from 17.7 to 2509 kU/l (geometric mean 268.33 kU/l).

All patients with a positive specific IgE determination also showed a positive SPT to the same allergen. However, in some cases, patients with a positive skin test to some bakery-derived allergens showed a negative IgE determination to the same allergens, indicating that the sensitivity of SPT is higher than serum IgE measurements.

Inhalation challenges

Twenty-three subjects had bronchial hyperresponsiveness to methacholine (data from one patient was missing). The geometric mean of methacholine PC20 was 0.39 mg/ml (range 0.08–15.9).

Specific inhalation challenge tests with aqueous extracts were performed with wheat flour (n = 12), rye flour (n = 2), soybean flour (n = 6), soybean trypsin inhibitor (n = 2), alpha-amylase (n = 4), hemicellulase (n = 5), glucoamylase (n = 3), ovalbumin (n = 2), ovomucoid (n = 1) and lysozyme (n = 5) in sensitized workers. Thus, a total of 42 SIC were carried out with occupational allergens in aqueous form (13 subject underwent one SIC, 10 two SIC, and 3 three SIC). Of these, 37 elicited a positive asthmatic response (27 EAR, eight DAR, two LAR) and five a negative reaction (two with hemicellulase, one wheat flour, one soybean flour and one lysozyme). The mean fall in FEV1 during the EAR in the positive responses was 25.5% (range 20–41%).

Correlations

A total of 33 EAR (isolated or the early component of the DAR) observed in 24 subjects were suitable for correlation analysis. Two EAR observed in one subject after SIC were not included in the correlation analysis because methacholine inhalation test results from this patient was missing.

The skin sensitivity to bakery-derived allergens had a positive correlation with allergen PC20 (r = 0.88, 95% CI 0.77–0.94, P < 0.001; Fig. 1) and methacholine PC20 had a weak correlation with allergen PC20 that approached statistically significance (r = 0.30, 95% CI = −0.06 to 0.59, P = 0.07). There was no correlation between skin sensitivity and methacholine PC20 (r = 0.22, 95% CI = −0.14 to 0.53, P = 0.22). No correlation was found between the levels of specific IgE to occupational allergens and neither methacholine PC20 nor allergen PC20.

image

Figure 1.  The correlation between skin sensitivity to bakery allergens (skin endpoint titration) and allergen PC20 (r = 0.88, P < 0.001).

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Multiple linear regression which expressed allergen PC20 as a function of skin sensitivity (threshold allergen concentration producing a 2-mm wheal diameter) and methacholine PC20, yielded the following highly significant regression line:

  • image

.

This formula predicted allergen PC20 to within one double concentration in 67%, to within two double concentrations in 85% and within three double concentrations in 97%. Only one of 33 SIC (3%) was outside the three double concentrations. A highly significant correlation (r = 0.92, P < 0.001) was found between the measured allergen PC20 and the predicted allergen PC20 calculated with this formula (Fig. 2). A similar formula in which immunological reactivity was assessed by the level of allergen-specific IgE instead of skin sensitivity could not be established.

image

Figure 2.  The correlation between measured allergen PC20 and predicted allergen PC20 calculated with the multiple regression formula (r = 0.92, P < 0.001).

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The results of this study provide evidence that the magnitude of the EAR to bakery-derived allergens can be satisfactory predicted by skin sensitivity and bronchial hyperresponsiveness to methacholine. Thus, the information obtained from skin endpoint titration and methacholine inhalation test can be used to safely select a starting concentration for allergen inhalation (4–6), but it should not replace the performance of SIC.

The aforementioned equations (4–6) are derived from data of asthmatic patients who have a positive EAR upon allergen inhalation challenge, but do not take into account the results of negative inhalation challenges in sensitized subjects with airway hyperresponsiveness (14). Nonetheless, a recent meta-analysis has concluded that SPT in workers with suspected OA caused by high-molecular-weight agents and bronchial hyperresponsiveness to methacholine correlates with SIC (high specificity, moderate sensitivity) (15).

It has been shown with different high-molecular-weight occupational allergens that the combination of immediate skin reactivity to an allergen and increased bronchial hyperresponsiveness does not necessarily prove that the subject will develop an asthmatic reaction when exposed to this agent (13). For instance, about 20% of subjects with skin reactivity to psyllium (16) or guar gum (17) and bronchial hyperresponsiveness did not experience an asthmatic reaction after inhalation challenge with the specific agent. Carletti et al. (18) reported that the specific bronchial response to flour dust among subjects with suspected OA caused by wheat flour could not be adequately predicted by any clinical, allergological or functional data, and therefore, an accurate aetiological diagnosis needs to be established by SIC.

Although allergen inhalation challenge tests are rarely indicated with common aeroallergens and it has a limited role clinically (4, 14), SIC tests are considered the gold standard in the diagnosis of OA (7, 19). Laboratory inhalation challenge tests with occupational agents are used for both diagnostic and research purposes in specialized centers across Europe and Canada, whereas in the USA they are limited to research settings (20). The reasons why this test is not widely used in the diagnosis of OA have been reviewed (21). To carry out SIC, extensive precautions are needed for safety requirements (13). Improvements in the safety of this test is of crucial importance; thereby, the prediction of the severity of the EAR to occupational agents has clinical relevance. It has been suggested that high-molecular-weight occupational allergens ought to behave in the same way as common aeroallergens, but this had not been established yet.

The choice of baker's asthma as a model to assess these relationships has strengths and limitations. On the one hand, it still is a common occupational disease and the agents implicated in this type of OA are high-molecular-weight allergens that act through an IgE-mediated mechanism (22), and therefore induce EAR on SIC. On the other hand, a caveat to this model is that bakers are usually polysensitized to occupational allergens (8, 22–24) and it is unknown how sensitization and simultaneous exposure to multiple allergens affect the relationships between the specific and non-specific bronchial responsiveness. In our study, we challenged the subjects with different occupational allergens and, in some cases, more than one SIC was carried out in order to identify the causative allergens of their asthma. Although this may have had some influence on the results, it also resembles a real world situation. In fact, one of the indications of SIC is to identify the agent responsible for asthma when there are multiple possible agents in the workplace (13, 19), and in that scenario it may be necessary to perform several challenges to precisely identify the offending agent. Other pitfalls are that flour allergens are not standardized (25), and they may experience some deactivation during the challenge (26), affecting the results of the provocations tests (27).

It has been shown that inhaled corticosteroids reduce bronchial hyperresponsiveness to pharmacological agents in a dose-dependent manner (28). In addition, prolonged administration of inhaled corticosteroids causes some or null inhibition of the EAR to high-molecular-weight allergens, and reduces the magnitude of the LAR (29). The way how inhaled corticosteroids may affect the relationship between bronchial responsiveness to allergen, airway responsiveness to methacholine and skin sensitivity to allergen is unknown. However, in the original study reporting the regression formula, several subjects were receiving inhaled beclomethasone (3).

In spite of these limitations, we were able to demonstrate that bronchial responsiveness to bakery-derived allergens can be satisfactorily predicted by assessing skin sensitivity to allergen and bronchial hyperresponsiveness to methacholine.

Airway hyperresponsiveness is thought to be important in modifying the response to inhaled allergens (30). However, no correlation has been found between specific and non-specific bronchial responsiveness in OA because of platinum salts (31) and with other low-molecular-weight agents the association is inconsistent (32). Lemière et al. (33) examined specific bronchial responsiveness to occupational agents in 16 workers who still showed skin reactivity but had normal airway responsiveness to methacholine after removal from work. They found that the main determinant of specific bronchial responsiveness (including seven SIC with cereal flour) was the level of specific immunization to the allergen (33). In the green tea-induced asthma model, it was also found that the key element for developing an asthmatic reaction was the degree of skin sensitivity (6). Walusiak et al. (34) reported that skin reactivity to common and occupational allergens is the main risk factor of baker's asthma in a cohort of apprentice bakers.

Choudat et al. (35) reported a significant correlation between bronchial hyperresponsiveness to methacholine and the provocative dose of flour dust causing a decrease in FEV1 of 15%, but not with the dose of flour provoking a FEV1 fall of 20%. In the studies by Cockcroft et al. (3–5) the correlation between allergen PC20 and skin sensitivity was closer than that between allergen PC20 and methacholine PC20. Our results also show that the level of specific bronchial responsiveness to bakery-derived aeroallergens is conditioned by specific immunological reactivity to this allergen, as determined by skin sensitivity, modulated by non-specific bronchial hyperresponsiveness, the latter being less essential.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors wish to thank Cristina García Fernández, from Dirección General de Salud Pública, Comunidad de Madrid, for her assistance in statistical analyses. This study was funded by Red Temática RESPIRA, FIS C03/011, Instituto de Salud Carlos III, Spain.

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  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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