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Background: This study aimed to investigate the influence of patient selection criteria, i.e., mite-allergic individuals exposed and not exposed to Dermatophagoides siboney and Blomia tropicalis, on the biologic activity of mite extracts. Determination of the potency of mite extracts in vivo requires selection of patients with a clinical history of mite allergy. In Scandinavia, there are some anamnestic criteria for mite allergy, whereas in the tropics, where patients are continuously exposed to high levels of mites, selection of patients with mite allergy by clinical history is difficult.
Methods: A total of 210 Cuban asthmatics with continuous symptoms, and 43 Swedes with a clinical history of mite allergy were investigated. Skin prick tests were performed with D. siboney, D. pteronyssinus, D. farinae, B. tropicalis, Acarus siro, Lepidoglyphus destructor, and Tyrophagus putrescentiae extracts. For analysis of the biologic activity of mite extracts, Cuban patients were divided into four groups:
1)all patients skin-test-positive to mites
2)patients positive to mites, but not to other inhalant allergens
3)patients reacting most to the mite species analyzed
4)patients reactive only to mites and reacting most to the mite species analyzed.
The biologic potency was calculated according to the Nordic Guidelines.
Results: Due to cross-reactivity between mites, Swedish mite-sensitive patients, with a clear clinical history of mite allergy, but not exposed to D. siboney and B. tropicalis, were more skin reactive to these mites than were Cubans. The estimated potency increased gradually to >200% in group 4. In group 1 Cubans, the reactivity to all mites but B. tropicalis was lower than that in mite-sensitive Swedes.
Conclusions: According to the influence of patient selection criteria on the estimation of the potency of mite extracts, the determination of the biologic activity of allergenic extracts in subjects without a clear-cut clinical history should be replaced by new methods when available.
Biologic standardization (BS) by skin prick test (SPT) is recommended in the Nordic Guidelines for registration of allergen preparations ( 1). The concept implies that the biologic potency of allergenic extracts from different species, labeled with the same biologic units (BU)/ml, should be similar in equally sensitive patients ( 2). Patient inclusion criteria in BS should be clinical symptoms at exposure and a positive SPT or in vitro IgE test to the offending allergen. Reproducible results have been reported from Europe ( 2). If there is no obvious clinical history, the selection will be mainly based on diagnostic tests, and the sensitivity of these tests will determine the inclusion of patients ( 3).
In the tropics, where there is a continuously high level of exposure to mite allergens, symptoms of asthma and rhinitis are present throughout the year in atopic patients. Such patients are sensitized to many inhalant allergens. Therefore, selection of mite-sensitive patients, based on the clinical history, is very difficult.
Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) are found in temperate as well as in warm and humid climates ( 4). Other mites such as D. siboney (Ds) and Blomia tropicalis (Bt) are restricted to tropical countries (5–7).
This study aimed to investigate the influence of patient selection criteria on the biologic activity of Ds and Bt extracts.
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- Material and methods
The prevalence of sensitization, as measured by positive skin tests to mite allergens, in Cubans and Swedes, is shown in Fig. 1. For all Dermatophagoides species, the prevalence degree of positive reactions in Swedes was higher than that in Cubans. In contrast, the skin reactivity to Bt was higher in the Cubans than in the Swedes.
Figure 1. Median size of SPT to different mite extracts in asthmatic Cuban (░) and Swedish (▒) mite-allergic adults. Vertical lines show 95% confidence intervals (CI) of medians.
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The labeled potency of the Dp and Df extracts, 10 HEP, was confirmed in the Swedish patients selected according to the Nordic Guidelines. The potency in BU of Ds, Df, Dp, and Bt extracts, as calculated with results from Swedes and Cubans, is shown in Fig. 2. The Ds extract showed the highest potency in the Swedish group. The potency of the Bt extract was low, as calculated in the Swedish patients, despite the high prevalence of sensitization.
Figure 2. Calculated potency (BU/ml) of mite extracts in Swedes (SW) and in four groups of Cubans (C1–C4). Vertical lines represent 95% CI of medians. Significant differences are indicated by ** (P<0.01). Solid horizontal lines in Df and Dp graphs represent labeled potency and tolerance limits of these extracts.
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For all mite allergens, the potency was higher when calculated with data from the subsamples of Cuban patients that best met the selection criteria according to the Nordic Guidelines. In group 1, the “all individuals” group, 82% of the patients also reacted to the other inhalant allergens tested. Group 2 had a biologic activity 66% higher than that of group 1. All four extracts showed an increased potency of more than 200% in Cubans from groups 1 to 4. The potency of all mite extracts was higher when calculated with Swedes than group 1 Cubans.
The Df extract had a lower potency in the nonselected Cubans than the labeled potency of the extract as calculated by the manufacturer. The Bt extract had a low potency in nonselected Cubans, and in the Swedish sample of mite-sensitive patients. The Ds and Dp extracts showed the highest potency in the Swedes, whereas the highest potency of the Df and Bt extracts was found in the group 4 Cubans.
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The pattern of sensitization of Cuban asthmatics to mites largely resembled that reported previously ( 7). All Swedish patients reacted to Dp and the majority to Df. The nonselected Cubans also reacted to these mite extracts, but to a lesser extent. Furthermore, Swedish patients reacted not only to these extracts but also to Ds, and most of them also to Bt; i.e., mites not found in Scandinavia.
The Swedish patients met the inclusion criteria of the Nordic Guidelines. In Cuba, we could not define the clinical history of mite sensitivity better than “sensitive to house dust”, meaning indoor allergens, or “perennial allergic airway symptoms”. Moreover, in Cuba, patients are continuously exposed to mites and many other perennial allergens. Thus, symptoms do not vary with the season. Diagnostic tests were used to identify mite-sensitive patients. The sensitivity of the SPT, i.e., the criteria for selection of patients, varies with the potency and composition of the allergenic extract used and with the SPT technique ( 10). Thus, the potency of the extract varies with the sensitivity of the patients selected for testing.
In Scandinavia, on the other hand, there is some seasonal variation in mite exposure and mite-related problems ( 11). Thus, mite-allergic patients can be identified by clinical history and selected for biologic standardization by this history in addition to positive SPT or in vitro IgE test. With these inclusion criteria, the results of biologic standardization of mite extracts have been shown to be repeatable between regions of Europe ( 2).
When we used the Swedish patients for biologic standardization, the 95% CI of the median potency of the Dp and Df extracts exceeded the tolerated variation in potency in relation to the labeled potency; i.e., 50–200% of the labeled potency, as defined by the Nordic Guidelines ( 1) and the Notes for Guidance on Allergen Products of the European Union ( 12). However, when we calculated the results of all SPT-positive patients (group 1) in the Cuban sample, the activity of these extracts was significantly lower than with the Swedes and lower than the labeled potency. We think the discrepancy occurred because the Cuban patients were not as clinically sensitive to mites as the Swedes. Because we included only the Cuban patients most sensitive to each of the mite species, the median activity approached that obtained in the Swedish sample; i.e., a sample with proper selection criteria.
The extracts of Ds and Bt, two mites that are not present in Sweden, behaved differently. Bt induced weak reactions in the Swedish sample. However, the percentage of positive reactions in Swedes was high. IgE binding to several Bt allergens in sera from Swedish allergic subjects has been recently reported, and cross-reactivity with Ld has been demonstrated ( 13). Because of the low reactivity to the Ld extract used in our study, the reactivity of the Swedes to Bt was possibly explained by cross-reactivity with Dermatophagoides species (14, 15). That patients reacted at all to Ds must be due to the reported cross-reactivity between Ds and the mite species found in Scandinavia ( 15). The Dp andDf extracts had a potency adjusted (diluted) to 10 000 BU/ml, whereas mite extracts prepared without adjustment of the potency but prepared according to modern principles, such as the Bt and Ds extracts, are mostly much more potent ( 16).
In conclusion, biologic standardization cannot be achieved if the patient inclusion criteria cannot be strictly followed. If this is not possible, other methods of potency adjustment must be sought. Determination of major allergen content, which is commonly done, has been shown to reflect the biologic activity of allergenic extracts ( 17) and has been recommended for biologic standardization ( 18). However, this method does not always give correct information about the biologic activity in man, since the monoclonal antibodies used in such assays probably do not often recognize all isoallergens and thus do not determine the total amount of allergen present. Furthermore, they often do not recognize IgE epitopes; therefore, they do not determine the biologic activity of extracts.