Management of patients with seasonal allergic rhinitis: Diagnostic consideration of sensitization to non‐frequent pollen allergens

Abstract Background Diagnosis of pollen allergies is mainly based on test allergens for skin prick testing. In the minimum battery of test inhalant allergens recommended by the Global Allergy and Asthma European Network 10 pollen allergens are included. Complementary other pollen allergens may need to be considered; however, respective awareness may not always be granted. Furthermore, at least in Germany, the situation may be even more complicated by the fact that test allergens need regulatory approval. A decline in commercially available test allergens may result in a diagnostic gap regarding patients with non‐frequent allergies. How many patients with non‐frequent pollen allergies would be affected by this gap? The data presented here partly answer this question. Methods The study consisted of a descriptive and an analytical part. In the descriptive part, sensitization to frequent pollen allergens (alder, hazel, birch, sweet grasses; according to the German Therapy Allergen Ordinance) and to respective non‐frequent pollen allergens (cypress, Japanese cedar, ash, plane tree, olive, Bermuda grass, wall pellitory, plantain, goosefoot, mugwort, ragweed, and saltwort) was measured in adult patients with physician‐diagnosed allergic rhinitis from two German federal states, namely North‐Rhine Westphalia (n = 360) and Bavaria (n = 339), using skin prick testing and/or ISAC technology. Furthermore, respective regional pollen data were assessed. In the analytical part, sensitization data were correlated with each other and with anamnestic data on symptom periods. Results Sensitization to frequent pollen allergens ranged from 45% (sIgE to Aln g 1/Alder, NRW) to 72% (prick test reactivity to birch, NRW). Sensitization to non‐frequent pollen allergens ranged from 0% (sIgE to Amb a 1/ragweed, NRW) to 41% (prick test reactivity to olive, Bavaria). Sensitization data partly correlated with each other and in connection with symptom periods showed a partly similar seasonal pattern as pollen data. Conclusions Sensitization to non‐frequent pollen allergens have to be considered when examining patients with respective seasonal symptoms, and test (and respective therapy) allergens for non‐frequent pollen allergies need to be available. Further prerequisites for adequate patient management would be a nationwide pollen monitoring system giving continuous pollen data and a systematic sensitization monitoring at patient level.

each other and in connection with symptom periods showed a partly similar seasonal pattern as pollen data.
Conclusions: Sensitization to non-frequent pollen allergens have to be considered when examining patients with respective seasonal symptoms, and test (and respective therapy) allergens for non-frequent pollen allergies need to be available.
Further prerequisites for adequate patient management would be a nationwide pollen monitoring system giving continuous pollen data and a systematic sensitization monitoring at patient level.  1,2 In addition to respiratory symptoms, pollen allergens can induce oral symptoms due to cross-reactivities to food allergens (so-called Oral Allergy Syndrome or pollen-related food allergy syndrome) 3 and, although rather rarely, anaphylaxis. 4 At present, pollen from sweet grasses (Poaceae; including Timothy grass, excluding Bermuda grass) and the birch family (Betulaceae; including among others birch, alder and hazel) are the most common clinically relevant pollen types in Germany: Almost every fifth of the German adult population is sensitized to Timothy grass pollen and birch pollen, respectively, 5 and in adult patients with suspected allergic reaction to inhalant allergens and sensitization to sweet grasses or birch pollen, this sensitization was clinically relevant in 90% and 91%, respectively. 6 Diagnosis of pollen allergies is mainly based on test allergens for skin prick testing. In 2009, the Global Allergy and Asthma European Network (GA 2 LEN) has recommended a standardized test allergen battery for clinical use and research, which allowed the identification of the majority of sensitized subjects studied. [6][7][8] With respect to pollen, this battery included alder (Alnus), birch (Betula), cypress (Cupressus), hazel (Corylus), mugwort (Artemisia), olive (Olea europaea), plane tree (Platanus), ragweed (Ambrosia), sweet grasses (Poaceae; except corn), and wall pellitory (Parietaria judaica). 8 Complementary, occasionally other allergens beyond that pollen panel may need to be considered when examining patients with seasonal allergic symptoms, for example, goosefoot or plantain pollen, but not always respective awareness can be assumed.
In Germany, the situation may be even more difficult because of the fact that test allergens are considered medicinal products and thus need regulatory approval by the Paul-Ehrlich-Institut, the Federal Institute for Vaccines and Biomedicines, thereby ensuring its quality, efficacy and safety. This means in effect, that phase I, II and III clinical studies have to be conducted prior to their marketing authorization. 9 On the part of pharmaceutical companies, this might result in an uneconomic cost-benefit-ratio, when it comes to rare allergens, and thus, the approval of test allergens for rare allergies are possibly being pushed aside. Indeed, in the past years, a marked decrease in the number of approved test allergens has occurred in Germany. 9 Notwithstanding the above-described difficulties, patients with allergies to rare allergens have the same right for adequate diagnosis and therapy as patients with allergies to frequent allergens. 9,10 In addition, a social aspect has to be considered: Allergens rare today maybe frequent tomorrow as shown for instance for ragweed pollen and respective sensitization in Italy. 11 Thus, monitoring sensitization to rare allergens would indicate changes in the allergen spectrum as expected for instance in the course of climate change. 12 According to the European Union (EU) a disease is defined as rare if not more than 5 out of 10,000 people in the EU are affected by it. 10 This definition could also be used to define rare allergies, and indeed, the German Therapy Allergen Ordinance (TAO), in effect since 2008, seems to have used this definition. 10,13 According to the TAO, currently allergens of the following sources can be considered as frequent allergens in Germany (botanical names in brackets): pollen of alder (Alnus), pollen of hazel (Corylus), pollen of birch (Betula), pollen of sweet grasses (Poaceae; except corn), dust mite (Dermatophagoides), bee venom and wasp venom.
All other allergens including other pollen allergens and occupational allergens, for instance, would be considered as rare or-as we do here-non-frequent allergens.
The decline in the number and the spectrum of commercially available test allergens may result in a diagnostic (and respective therapeutic) gap regarding patients with non-frequent allergies. 9,10,14 With respect to pollen, this would include allergies to mugwort or ragweed, but also allergies to ash, goosefoot, or plantain, among others. How many people may be affected by this diagnostic gap? The above cited GA 2 LEN skin test study I included patient data on 10 pollen allergens, among them six non-frequent allergens, from two German cities. 7 On the basis of patient data from two German federal states, here, we expand the list of tested allergens to another six non-frequent allergens using skin prick testing and/or sIgE analysis and complete sensitization data by anamnestic data on complaint periods and regional pollen data.

| Patients
The present analysis was based on patient data collected in the context of a study on climate change and allergy funded by the For the present analysis, only patients with physician-diagnosed allergic rhinitis and valid skin prick testing (criteria see below) were included (n = 699; NRW: n = 360, minimum/median/maximum age 20/ 39/65 years, female 65%; Bavaria: n = 339, minimum/median/maximum age 20/45/65 years, female 63%). Patients were attributed with "physician-diagnosed allergic rhinitis," if they had answered both of the consecutive questions affirmatively: (i) "Did you suffer or do you suffer from hay fever or allergic rhinitis?," (ii) "Has the hay fever or allergic rhinitis ever been diagnosed or confirmed by a doctor?" Questions, given in German, were adopted from a standardized physician-administered computer-assisted personal interview (CAPI) used in the German health interview and examination survey for adults (DEGS1). 16

| Sensitization data
Sensitization data were analyzed by skin prick testing and/or measurement of sIgE in serum using ISAC technology.
Skin prick testing was performed according to the GA 2 LEN guidelines on harmonization of skin prick testing in Europe. 17 Histamine dihydrochloride (10 mg/ml; ALK-Abelló) was used as positive control, diluent (ALK-Abelló) as negative control. Results were assessed after 15 min. Valid negative and positive controls provided (i.e. largest diameter of the negative control <2 mm and largest diameter of the positive control ≥3 mm, respectively), skin prick testing to an allergen extract was evaluated positive if the largest diameter of the wheal was ≥3 mm. Pollen allergen extracts to which prick test reactions were assessed are given in Table 1. In addition, dust mite allergen was included as perennial control allergen.
ISAC technology (Fisher Scientific GmbH, Schwerte, Germany; 112 allergen components) was run in the study center in Aachen. 18,19 IgE data were displayed in ISU, and ISU-values ≥0.3 were evaluated positive. The pollen allergen components to which sIgE levels were assessed are given in Table 2

| Data on symptom periods and their relation to sensitization data
Information on symptomatic periods was gained from the medical interviews. Patients were asked to highlight all the months over the course of the year with symptoms they would relate to their hay fever/allergic rhinitis disease, hereinafter called "months with symptoms." The question was applied from the Mini Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ). 20 To relate specific sensitization to months with symptoms, for each month of the year the number of respective prick test negative patients with symptoms was set 100% and the number of respective prick test positive patients with symptoms was related to it. For a given test allergen and a given month, this would result in values below, around, or above 100% indicating that the proportion of sensitized patients with symptoms was either lower, equivalent or higher than in unsensitized patients. Taken together with respective exposure periods, the course of the year of this relation would give an idea about the "overall" clinical relevance of the respective sensitization.

| Pollen data
Pollen data were provided by the German Pollen Information Service Foundation (PID). Data of 12 pollen types, displayed in Table 3, were acquired from the PID reference monitoring stations Mönchengladbach (NRW) and Munich (Bavaria).
Pollen was collected using 7 days volumetric traps (Hirst type) and samples were analyzed according to the national standard HÖFLICH ET AL.
Note: Botanical names are given in brackets.
DIN EN 16868. 21  If pollen data were missing at one pollen station, the respective days of the other station were coded "missing" as well to allow better comparison of both stations. The resulting magnitudes of days with no pollen data, specified for low, pre/post, and main season of each pollen type, are given in Table S1.

| Data management and statistical analysis
In the context of the abovementioned study on climate change and al-
Data on sensitization to pollen allergens detected by ISAC technology are given in Figure 1B.  were sIgE positive to at least one of them. Exclusion of these patients from analysis did not significantly change the pollen sensitization pattern ( Figure S1).

| Correlation of sensitization data
Partially, sensitization data highly correlated which each other, also across both test principles. Data pairs with a correlation coefficient >0.7 are given in Table 4. Correlation coefficients of all data pairs are given in Table S2.

| Relation between sensitization and months with symptoms
Data on the relation between sensitization and months with symptoms are given in Table 5A,B (prick test data) and Table 5C,D (sIgE data). However, it did not apply to all of the tested allergens and especially not to those with sensitization numbers below n = 10.
For prick test diagnosed tree pollen sensitization (Table 5A, Respective raw data are given in Table S3 (prick test data) and Table S4 (sIgE data), and all p values are given in Table S5.  (Table 6).

| Pollen data
Yearly data are given in Table S6.
As expected, tree pollen types were mainly detectable in the first months of the year, whereas sweet grasses and weed pollen were mainly detectable in the mid of the year and in late summer.

| DISCUSSION
Here, we show data on sensitization to 16 pollen allergens in patients with physician-diagnosed allergic rhinitis from two German federal states. We correlate sensitization data with each other and relate them to months with symptoms. Furthermore, we show respective data on regional pollen load.

| Sensitization to frequent versus non-frequent allergens-pharmaceutical and clinical implications
From the 16 allergens analyzed, four are explicitely within the scope of the German TAO and from this point of view can be considered as frequent allergens: alder, hazel, birch and sweet grasses. Populationbased data from DEGS1 showed that these allergens were indeed the most frequent ones with respect to sensitization in German adults. 5 However, the same study also showed sensitization to other pollen allergens albeit to a lesser extent, and concluding from patient-based data the GAL 2 EN network has recommended a prick test panel of 10 pollen allergens to be tested in European patients. Apart from pollen of the birch family and sweet grasses this panel included cypress, mugwort, olive or ash, plane tree, ragweed and wall pellitory. 5,17 The prick test data provided by our study confirm the clinical relevance of the GAL 2 EN prick test panel: Each of the test allergens recommended by GAL 2 EN induced positive prick test reactions in at least some of our study patients even if the number greatly varied (range from 3 Cypress-reactive to 258 Birch-reactive patients).
Additional to the GAL 2 EN prick test panel both olive and ash were included in our study. More than 20% of our patients showed positive reactions to olive and ash, respectively, but not all of these positive reactions can be explained by "cross-reactivity" to ash and olive, respectively, as already described elsewhere. 15 Currently, in Germany field-grown olive does not exist and significant olive pollen load has not been found so far. So, olive-but-not-ash reactive patients may have become sensitized to olive on the basis of travelling to respective exposure regions or may cross-react for instance to other pollen of the family Oleaceae like the insect-pollinated forsythia or Jasmine. 15 In contrast to olive, ash is endemic in Germany and pollen load is By using ISAC technology we were able to extent our sensitization study to Japanese cedar, Bermuda grass, plantain, goosefoot, and saltwort. Japanese cedar, Bermuda grass and saltwort are not widespread in Germany but plantain and goosefoot are. 22 For each of these allergens we could see at least one positive reaction in our patient cohort. The number of positive patients ranged from 1 Pla l 1 (plantain)-positive to 177 Cyn d 1 (Bermuda grass)positive patients. The high sensitization rates to Bermuda grass are very likely due to cross reactivity to Timothy grass, which is endemic in Germany. 22 The correlation data support this hypothesis.
Our sensitization data support the need for approved test and therapy allergens for both the diagnosis and treatment of frequent but also of non-frequent pollen allergies. To counteract the current decline in the number of approved test allergens in Germany, pharmaceutical companies can now propose a substantial reduction of the fees needed to approve a given test allergen provided it belongs to the group of non-frequent allergens. 9,23 Support for the diagnostic and therapeutic needs of patients with non-frequent pollen allergies may also come from the initiative "National action group for people with rare allergies"  Note: For each month of the year, the number of respective prick test negative patients with symptoms was set 100% and the number of respective prick test positive patients with symptoms was related to it. (A) Prick test data from NRW, (B) prick test data from Bavaria, (C) sIgE data from NRW, and (D) sIgE data from Bavaria. The data field colors indicate the position of a given value within the data range: The redder a field, the higher the value in relation to 100%, the bluer, the lower. Proportions based on test positive rates below n = 10 and proportions related to dust mite sensitization are highlighted in grey. Proportions based on significant group differences appear in bold. n.d., no data. HÖFLICH ET AL. Institute. 5 At the patient level systematic monitoring would also be of use but so far is not established in Germany.

| CONCLUSIONS
Sensitization to non-frequent pollen allergens has to be considered when examining patients with respective seasonal symptoms, and test and therapy allergens for non-frequent pollen allergies need to be available. Further prerequisites for adequate patient management would be a nationwide pollen monitoring system giving continuous pollen data and a systematic sensitization monitoring at patient level.