INTRODUCTION

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According to sentinel-based surveys and medicolegal statistics, animal-derived allergens are a major cause of occupational asthma (OA). They are among the leading causes of OA in the United Kingdom (1). Gordon and Newman-Taylor reported prevalence proportions of laboratory animal (LA) allergy in the order of 20 to 40% and of OA of approximately 10% (2). In a prospective study Cullinan and coworkers studied the occurrence of work-related symptoms in a cohort of 238 workers who had not been exposed to laboratory rats before their current employment in three institutions specializing in small-animal research (3). The symptoms were found to be related to the intensity of exposure with the relationship being stronger in atopic subjects but unrelated to smoking. Subsequently, in a case-control analysis of 342 newly employed LA workers, the majority of whom had been seen in the first study and followed at 6-mo intervals for 3 1/2 yr, Cullinan and associates showed a dose-effect gradient between exposure categories and work-related symptoms, and an increased risk of allergy to rats owing to atopy and cigarette smoking (4). However, no information was available on the immunological-atopic status of these workers at the time they started employment in these particular sites, and no assessment of nonspecific bronchial responsiveness was carried out to confirm the occurrence of asthma or OA. In another prospective study that included a limited number of subjects, Renström and coworkers reported that seven of 38 (18%) assessed over an 18-mo follow-up developed specific IgE-induced sensitization (5). Finally, Sjöstedt and coworkers reported only one of 63 (1.6%) incident cases of occupational asthma in a prospective study of 5-yr duration (6).

Atopy, and intensity of exposure, are consistent risk factors for OA (2). Hollander and coworkers have shown that the exposure-response relationship was steepest in subjects with skin reactivity or self-reported symptoms associated with exposure to cats and dogs (7). In a study that used data from three surveys conducted in different countries, Heederik and coworkers also showed that sensitization to rat was more strongly associated with sensitization to cats and dogs than other common inhalants (8). Finally, there are suggestions derived from cross-sectional data that smoking could also be related to LA allergy (4, 9).

To determine the incidence of probable OA, defined by skin reactivity to occupational allergens associated with the onset or increase in responsiveness to methacholine, and the role of preexposure host factors, including airway responsiveness, we conducted a prospective cohort study of 417 apprentices recruited when they entered a career program in animal-health technology.

	METHODS

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The subjects were 417 apprentices in animal health technology, who are part of a cohort of 769 apprentices (including 230 apprentices in pastry-making and 122 in dental hygiene technology) recruited from 14 specialized schools (five in animal health technology) upon starting their training between 1993 and 1995. Baseline characteristics of the cohort members have been reported elsewhere (10). Briefly, subjects in these schools were eligible for the cohort study provided that they had not been exposed to the relevant work-related aeroallergens in an apprenticeship or job for at least 3 mo before entering the career program, as assessed during a preliminary visit. The subjects in animal health technology were reassessed at 8, 20, 32, and 44 mo after starting the program.

At the time of entry into the apprenticeship program and at each follow-up visit, each student answered a respiratory questionnaire derived from the standardized questionnaire of the International Union against Tuberculosis and Lung Diseases (IUATLD) (11). Information was obtained on physician-diagnosed asthma, personal allergic conditions, and familial asthma. Symptoms suggestive of asthma included wheezing, chest tightness, shortness of breath, or cough under usual conditions or such conditions as exercise or exposure to cold air, strong odors, smoke, or dusts. Respiratory symptoms and rhinoconjunctivitis on contact with pets and pollens were also documented. Nasal, ocular, respiratory, and skin symptoms during exposure to the specific agents present during work in the laboratory were assessed at each visit. As the cumulated number of reported hours of exposure to rodents (rats, mice, and rabbits) was greater in one of the five schools by comparison with the others (74 h versus 52, 48, 38, and 16 h) and as attending that school was associated with an increased risk of developing specific immunological sensitization (SIS) (12), the association between school attended and risk of probable OA was investigated in the present study.

Skin tests were done with the prick method (13), using 11 common inhalants: mixed trees, mixed grass, and ragweed pollens; Alternaria, Aspergillus, and Hormodendrum; feathers; Dermatophagoïdes farinae and D. pteronyssinus; and cat and dog dander (Omega, Montreal, PQ, Canada). Histamine phosphate (1/200 g/ml) was used as a positive control, and diluent (glycerine, 50%), as a negative control. The largest wheal diameter was assessed 10 to 15 min after introducing the antigen. A positive reaction was defined as a wheal  3 mm in the absence of reaction to the diluent and in the presence of a positive reaction to histamine phosphate. Atopy was defined as at least two positive reactions to the common inhalants.

In addition, skin-prick tests were performed with extracts from aeroallergens potentially present in the working areas of students and confirmed as specific to the program by the authors (12). These consisted of urinary proteins from rat, mouse, and rabbit (Pharmacia Allergon AB, Angelholm, Sweden), and rabbit danders (Omega). The criterion for a positive reaction to a specific allergen was the same as for common allergens. Subjects who demonstrated sensitization to a work-related antigen at the initial visit were also followed to detect possible new sensitization to another occupational allergen.

All extracts were obtained before the beginning of the study in sufficient amounts to perform the estimated number of skin tests throughout the study period. Each extract was prepared from the same batch. The same nurse performed the skin-prick testing during the study period.

Spirometry with the assessment of FEV₁ and FVC was carried out using a Collins apparatus (Survey/1 Plus; Collins, Braintree, MA) according to published standards (14). Methacholine inhalation tests were performed using a Wright's nebulizer (Roxon Meditech Ltd, Montreal, PQ, Canada) (output = 0.14 ml/min) at tidal volume breathing for 2 min according to guidelines slightly modified from those of the European Respiratory Society (15). The procedure for performing the methacholine test was modified as described elsewhere to take into account the absence of an onsite physician (16). The maximal concentration of methacholine administered was 32 mg/ml. The provocative concentration causing a 20% decrease in FEV₁ (PC₂₀) was interpolated from individual dose-response curves drawn on a semilogarithmic scale using noncumulative doses.

Reference values for FEV₁ and FEV₁/FVC were taken from Knudson and coworkers (17). Bronchial hyperresponsiveness (BHR) was set at 8 (18) and 16 mg/ml (19).

The methacholine challenge test was repeated at follow-up visits, and until the end of the study, for subjects who developed SIS to one of the work-related allergens as well as for a subsample of subjects with negative skin tests to the specific allergens or with no new positive test. The latter group was constituted of up to two subjects matched with cases of SIS on the basis of atopy (yes/no), baseline PC₂₀ ( 16 versus > 16 mg/ml), and school attended.

Incident cases of probable OA were defined as subjects who developed a specific immunological sensitization to at least one work- related allergen and a  3.2-fold decrease in PC₂₀ methacholine (20). If subjects had a PC₂₀ value > 32 mg/ml at entry, a change at follow-up to a value  16 mg/ml was considered equivalent to a decrease of  3.2-fold.

Three reference groups for the probable OA cases were defined (Figure 1): (1) Group 1, all cohort members not meeting the criteria for probable OA at the end of the study (n = 345); (2) Group 2, a subgroup of Group 1 including the subjects skin-prick test positive to LA and no decrease in PC₂₀ (n = 50); (3) Group 3, a subgroup of Group 1 including subjects who developed neither outcome (n = 111).

View larger version (30K): [in this window] [in a new window]   Figure 1.   Of the 391 eligible subjects at entry, 373 had skin-prick tests with LA allergens at one or another of the follow-up visits. Of these, 78 developed skin reactivity to a LA-derived allergen. These 78 were matched with 136 referents without SIS. All these subjects underwent methacholine challenges. Twenty-eight changed their responsiveness to methacholine and were defined as cases of probable OA. The rest (Group 1, n = 345) did not, including 50 with SIS (Group 2). Of the 136 referents, 25 (Group 4) changed their responsiveness to methacholine and 111 (Group 3) did not.

Statistical Analysis

The incidence of probable OA was expressed (1) as a proportion of the cases of immunological sensitization, (2) as a proportion of the subjects who attended at least one of the follow-up visits, (3) with respect to person-years at risk in the cohort, i.e., [number of subjects who attended at least one of the follow-up visits × period for which subjects remained at risk (time to the end of the study or time to the development of probable OA or time to quitting the apprenticeship)]. A Cox proportional hazards model was used to evaluate the effect of the potential risk factors on the risk of occurrence of probable OA, with the time variable defined as the difference between time of first occurrence of a given event and time of entrance into the program. Fixed covariates in the analysis were assessed at baseline: atopy, sex, smoking, immediate skin reaction to pets and PC₂₀ ( 32 versus > 32 mg/ml), FEV₁ (% pred), exercise-induced respiratory symptoms, rhinitis on contact with pets, rhinitis in the pollen season, respiratory symptoms on contact with pets, and respiratory symptoms in the pollen season, as well as the school attended. For these fixed covariates, the proportionality assumption was tested using Harrell's procedure on Schoenfeld residuals as provided in S-PLUS. Univariate analyses were first carried out separately for comparison between the cases and each of the three reference groups. We used the 2 log likelihood test with a p value < 0.1 as a criterion to retain each variable that was included in the multivariate analysis; the risk factors for which the occurrence was rare were not considered. Rate ratios (RR) and 95% confidence intervals (CI) were estimated. Statistical analyses were performed using the SPSS software package (version 9.1 for Windows; SPSS, Chicago, IL).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Of the 417 apprentices recruited in the cohort, three had refused skin testing at entry and 24, bronchial challenge to methacholine, including one who refused both tests. This left 391 (93.8%) apprentices with baseline data on skin reactivity to specific allergens and bronchial responsiveness (Figure 1). Among these subjects, 373 attended at least one of the follow-up visits. This represents the number of apprentices at risk among whom incidence of probable OA could be detected.

Eighty-five apprentices developed SIS to at least one work-related specific allergenrat urine, mouse urine, or rabbit urine/danderduring the 3- to 4-yr prospective study (12); of those, 78 had accepted a methacholine challenge at baseline. Among the 78 subjects with SIS, 28 incident cases of probable OA were identified (Figure 1), 35.9% (28/78) among subjects with SIS, 7.5% (28/373) among subjects at risk in the whole cohort. The incidence density was 2.7% (28/1,043 person-years). Of the 136 matched referents (Figure 1) who were at risk but who did not develop SIS, 25 (18.4%) had significant changes in bronchial responsiveness (Group 4, Figure 1). Table 1 shows baseline characteristics of the 373 participants who attended at least one follow-up visit, of the cases and of the 3 groups of referents. Cases and Group 2 referents (i.e., subjects with incident SIS) had, as expected (12), a higher prevalence of atopy, skin reactivity to pets, BHR, exercise-induced respiratory symptoms, and rhinitis on contact with pets. The subjects who developed probable OA had a higher baseline FEV₁ compared with the other study subjects.

The number of subjects who developed SIS, nose/eye or chest symptoms as well as significant decrease in PC₂₀ appears in Venn diagrams (Figure 2). Among the 28 cases of probable OA, 17 (61%) reported nasal or ocular symptoms, or both, developing after the first survey, compared with 67 of 186 (36%) subjects without probable OA who had at least one bronchial challenge at follow-up. On the other hand, among cases of probable OA, eight of 28 (29%) reported new work-related respiratory symptoms (dyspnea or wheezing, or both) since the first assessment compared with 16 of 186 (8.6%) subjects without probable OA.

View larger version (20K): [in this window] [in a new window]   Figure 2.   Venn diagrams. Study outcomes among 214 subjects with a methacholine challenge test at follow-up. All subjects with chest symptoms had nose/eye symptoms; 78 subjects had none of these outcomes. As a whole, 28 subjects had probable OA and 186 were free of probable OA.

The results from the Cox regression analyses are presented in Tables 2 and 3. Table 2 shows the rate ratios and 95% CI for the effect of suspected risk factors on the incidence of probable OA obtained by univariate analysis. In the first comparison (cases versus all subjects remaining at risk at the end of the study, n = 345), most factors, with the exception of sex, smoking, rhinitis, and respiratory symptoms in the pollen season, were significantly associated with an increased risk of developing probable OA. In the comparison of probable OA cases with subjects with incident SIS and no decrease in bronchial responsiveness (Group 2, n = 50 in Figure 1), only immediate skin reaction to pets was significantly associated with the risk of probable OA, and a lower FEV₁ had an apparent protective effect for the incidence of probable OA. In the third comparison (cases of probable OA versus Group 3, n = 111 in Figure 1), atopy, immediate skin reaction to pets, and rhinitis on contact with pets significantly increased the risk of developing probable OA, whereas having a lower FEV₁ was associated with a decreased risk.

Table 3 shows the results of the multivariate Cox regression analysis. In the first comparison (i.e., cases versus Group 1 subjects), baseline immediate skin reaction to pets, PC₂₀  32 mg/ml, and attending School 1 remained significantly related to the incidence of probable OA, and a lower FEV₁ remained significantly related to a decreased risk of probable OA. These factors were independently associated with the development of probable OA. Immediate skin reactivity to pets was significantly associated with the incidence of probable OA, and a lower baseline FEV₁ had an independent protective effect when cases were compared with Group 2 and Group 3 referents.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This prospective study, conducted in a cohort of 417 apprentices starting exposure to animal-derived antigens, shows that (1) 28 of 373 (7.5%) developed skin sensitization and BHR to methacholine over a 32- to 44-month-long program, which fits the authors' definition for the development of probable OA; (2) baseline immediate skin reactivity to pets, methacholine responsiveness, and being a student in one school were the most significant determinants for the development of probable OA. These factors were all independently associated with the incidence of probable OA as revealed from the multivariate analysis. The association between attending School 1 and the incidence of OA was not confounded by atopy as the proportion of atopic students in School 1 was not greater than in the other schools.

Most of the previous studies in subjects exposed to high-molecular-weight allergens and LA have been of the cross-sectional type, as reviewed by Gordon and Newman-Taylor (2). In these studies, exposure and atopy are consistently identified as risk factors. Few longitudinal studies have been carried out. In the study by Cullinan and coworkers (3), no assessment was carried out on entering the workplace and collection of some of the data was retrospective. These investigators found that 21 of 238 subjects (8.8%) had evidence of immunological reactivity to rat urine and 16 of 238 (6.7%) developed symptoms during the 4-yr interval since starting exposure. Ten subjects of 219 (4%) had sensitization to rat urine and chest symptoms. Atopy was a determinant for development of chest symptoms, but the investigators did not comment on the role of sensitization to pets.

In the subsequent case-control analysis of their prospective study, the same investigators found that 46 subjects (13.5%) developed skin reactivity to rat urine and 36 (10.5%), work-related chest symptoms (4). Cullinan and associates mention that 37 subjects who reported chest, oculonasal, or skin symptoms also had a positive skin-prick test to rat urine, but they do not give information on the number of subjects who had both skin reactivity and chest symptoms. Renström and coworkers found that seven of 38 subjects (18%) prospectively assessed in an 18-mo follow-up survey developed specific IgE sensitization to LA; four of these seven subjects also had significant changes in bronchial responsiveness (the incidence of occupational asthma according to our definition would be 10.5%) (5). Fuortes and coworkers prospectively examined 98 university employees exposed to laboratory animals by comparison with 90 workers in wet laboratory (21). However, the participation rate declined to only 10% at the 36-mo assessment. They found only three cases of skin sensitization to LA antigens during this period in the exposed group (and none in the control group), whereas 4.5% of subjects in the exposed group had symptoms suggesting work-related asthma 24 mo after starting exposure. This incidence was not, however, superior to that found in the nonexposed control group. The present study offers the advantages of having a prospective design, of including apprentices who had not been exposed previously, of assessing both immunological sensitization and bronchial responsiveness, and of having a sustained high participation rate.

Skin sensitization to pets, and atopy per se were found to increase significantly the likelihood of developing probable OA. However, in the multivariate analysis including both factors, only skin reactivity to pets remained a significant determinant. There are cross-sectional data that previously suggested that sensitization to pets was more strongly associated to sensitization to rats than to other common inhalants (7).

The definition of probable OA adopted in our study was based on both immunological evidence and objective testing for bronchial responsiveness. It has been shown that having immediate skin reactivity to an inhalant and a positive PC₂₀ results in an approximately 80% likelihood of developing an asthmatic reaction during a specific inhalation test using the relevant inhalant (22, 23). Indeed, the squared regression coefficient value of the equation which relates the diameter of immediate skin test to common inhaled allergens and BHR to methacholine expressed by the PC₂₀ was 0.8 in the study by Cockcroft and coworkers (22). We also showed that eight of 10 (80%) nurses with PC₂₀ values  16 mg/ml and skin reactivity to psyllium, a high-molecular-weight allergen, demonstrated an asthmatic reaction on laboratory challenges to psyllium (23). It is therefore probable that students who developed immunological sensitization and BHR had OA although only eight of 28 (29%) of these apprentices also reported respiratory symptoms and 17 of 28 (61%), rhinoconjunctivitis, the latter condition being occasionally associated with BHR (18). It is known that a questionnaire is not a satisfactory tool for the diagnosis of OA (24). Moreover, it can be assumed that many apprentices who entered an apprenticeship that they selected denied having symptoms that could preclude continuation of studies and entering a career. Another possibility, which we are currently examining, is that these subjects with probable OA will develop work-related respiratory symptoms on exposure to animals at a later stage.

Interestingly, of 78 apprentices identified prospectively as developing immunological sensitization, 28 (36.9%) developed probable OA. Therefore, the development of IgE-dependent immunological sensitization results in a high risk of having probable OA in the case of animal-derived allergens. By comparison, five incident cases of probable OA were identified among seven apprentices who developed SIS in the dental hygiene program (12, 25), the proportion being 4.8% among the 104 subjects at risk. No one in the pastry-making program developed probable OA.

It is generally accepted that a previous history of asthma is not a risk factor for the development of OA (26). This assertion is based on results from cross-sectional studies of OA cases with retrospective questionnaire data. This study adds some evidence that asthma is not a risk factor for the incidence of probable OA, and also suggests that having a high FEV₁ does not preclude the development of probable OA. On the other hand, this study shows that having a measurable PC₂₀ ( 32 mg/ml) (but neither a PC₂₀  16 [RR = 1.8, 95% CI = 0.87 to 3.77], nor a PC₂₀  8 mg/ml [RR = 1.22, 95% CI = 0.50 to 2.98], in the comparison cases versus all) was a determinant for the development of probable OA. It might therefore be interesting to consider including the assessment of both immunological reactivity and bronchial responsiveness in OA screening programs among animal handlers. This would allow detecting onset or increase of nonspecific bronchial responsiveness.

It is also interesting to note that the only determinants distinguishing those with immunological sensitization to LA-derived antigens who also developed changes in PC₂₀ from those who developed immunological sensitization but did not develop changes in PC₂₀ are the presence of skin sensitization to pets and a higher baseline FEV₁. These characteristics, in particular baseline skin reactivity to pets, could therefore be useful in screening programs to distinguish among those who acquire specific sensitization to LA, those who are more likely to develop OA.

Other prospective longitudinal studies should be undertaken in apprentices starting exposure to agents that represent risk for the development of immunological sensitization and OA. This includes other proteinaceous agents and low-molecular-weight agents.