Sensitization to oil palm pollen associates with risks and severity of allergic diseases

Background Elaeis guineensis (Ela g, oil palm) pollen is one of the most predominant species of inhalant allergens in the tropical Southeast Asia region; however, its association with the manifestation of allergic diseases remains largely unexplored. This study aimed to determine the sensitization pattern of oil palm pollen and associate this with the risk and severity of allergic diseases. Methods Participants were recruited as a part of the Singapore and Malaysia cross-sectional genetic and epidemiological study (SMCSGES). Two independent cohorts were recruited: n = 564 serum samples were collected and serological assessment was performed against a panel of 16 crude inhalant allergens including house dust mite, pet, insect, pollen, and fungal allergens; n = 13 652 Singapore/Malaysia Chinese young adults were recruited and skin prick test was used to assess oil palm sensitization, which was tested for its association with the risk and severity of asthma, allergic rhinitis (AR), and atopic dermatitis (AD). Results The sensitization rate of oil palm pollen is 9.6% in the n = 564 Singapore/Malaysia cohort. In the n = 13 652 Singapore/Malaysia Chinese cohort, oil palm sensitization significantly associates with increased risks of asthma (p = 1.34x10−4), AR (p = 2.91x10−13), and AD (p = 6.95x10−7). Asthmatic patients with oil palm sensitization have increased risks of wheezing (p = 0.00995), nocturnal cough (p = 0.0122), and exacerbations (p = 0.00139) in the past 12 months. AR patients with oil palm sensitization also have an increased risk of developing moderate-to-severe symptoms (p = 0.00113). Conclusions We have identified significant associations of oil palm sensitization with increased risks, exacerbations, and the severity of symptoms of allergic diseases in the tropical Southeast Asian region (Singapore/Malaysia).


INTRODUCTION
5][6] In the tropical environment of Southeast Asia, an abundance of indoor HDM allergens was found in the home environment year-round. 7Studies also revealed a spectrum of pollen and fungal airspora that was uniquely present in the tropical environment with high allergenicity. 6,8,9Of these allergens, the pollen of Elaeis guineensis (Ela g, oil palm) was one of the most predominant types of outdoor allergens in tropical countries, 8,10 especially in Singapore during the Northeast Monsoon season (December to March). 8This is likely due to the presence of about 6.4 and 12.7 million hectares of oil palm plantations in Malaysia and Indonesia respectively. 11The oil palm pollen can be disseminated from these countries to Singapore by wind dispersal, due to their close proximities. 12l palm sensitization is an important risk factor of asthma and AR in the Southeast Asian tropical region. 6,13Multiple oil palm pollen proteins have been characterized and showed IgE reactivity, including a 31-kDa palm pollen glycoprotein (Ela g Bd 31 K), 14 a pectin esteraselike oil palm pollen protein designated as ELGU1, 15 as well as a polygalacturonase-like protein. 16While the allergenicity of these proteins has been assessed previously, the specific role of oil palm sensitization in the manifestation of allergic diseases remains to be investigated.
Among the airborne inhalant allergens commonly present in the tropical environment of Southeast Asia, HDM sensitization was frequently associated with the risk and severity of allergic airway diseases. 5,17,18Fungal sensitization was also correlated with the susceptibility and severity of asthma and AR in the Singapore/Malaysia population. 4,6However, the association between oil palm sensitization and the risk and severity of allergic diseases in this region is not well characterized.Here, we report associations of oil palm sensitization with the risk, exacerbation, and severity of allergic diseases including asthma, AR, and AD in the Singapore and Malaysia populations.Serum specific IgE (SSIgE) titers against a panel of 16 common inhalant allergens were assessed in a cohort of n ¼ 564 Singapore/ Malaysia young adults.In a separate crosssectional cohort of n ¼ 13 652 Singapore/Malaysia young adults of Chinese ethnicity, oil palm sensitization was assessed using skin prick test and associated with the risks, exacerbations, and severity of symptoms of allergic diseases.This allows understanding of the role of oil palm sensitization in the manifestation of allergic diseases in the tropical environment of Singapore and Malaysia.

Study design
This present study belongs to a part of an ongoing Singapore and Malaysia cross-sectional genetic and epidemiological study (SMCSGES).Participants were recruited from this ongoing epidemiological collection in Singapore and Malaysia universities: National University of Singapore, Singapore (NUS, from Aug 2005 to Sep 2019), University Tunku Abdul Rahman, Malaysia (UTAR, from Feb 2016 to Oct 2018), and Sunway University, Malaysia (SU, Nov 2019).[21][22] Two independent cross-sectional cohorts were reported in this study: 1) serum samples of n ¼ 564 participants were collected from UTAR and SU for serological assessment using the immune-dot blot approach; 2) detailed information on demographics, medical history, and severity of allergic diseases of n ¼ 13 652 Chinese individuals were collected from NUS, UTAR, and SU using an investigator-administered questionnaire.Participants from both cohorts underwent a skin prick test (SPT), using a panel of 4 types of allergens, including 2 HDM species (Dermatophagoides pteronyssinus and Blomia tropicalis), oil palm pollen (Elaeis guineensis), and a fungus species (Curvularia lunata).A positive SPT was defined as having a wheal of at least 3 mm in diameter 15 min after the skin prick.The SPT was performed during the recruitment process.These allergens used in the SPT were the most common inhalant allergen present in the Singapore environment with high allergenicity, as demonstrated previously. 6,23Sensitizations to HDM and fungal allergens have already been discussed in our previous publications. 4,5In this manuscript, we focus on sensitization against oil palm pollen due to its high abundance in the atmosphere of the tropical Southeast Asia region. 23The demographics of both cohorts are summarized in Table S1.

Cloning, expression, and purification of Ela g profilin
The Ela g profilin gene was cloned into a modified pET28b (þ) vector and overexpressed as a 6 Â His-tag fusion protein.The construct was transformed into Escherichia coli strain BL21 (DE3) to produce the Ela g profilin protein.Transformed bacteria cells were grown overnight in Luria Bertani (LB, Bio Basic Inc., Canada) containing kanamycin (100 mg/mL) at 37 C. Bacteria cultures were then inoculated and grown in 1L LB until OD600 reached 0.6.Recombinant protein expression was induced by the addition of 0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG, 1st Base, Singapore) to the bacterial cultures, which were grown at 37 C for 4 h before harvesting by centrifugation at 5465g.The pellet was resuspended in binding buffer (20 mM Tris-HCl pH 7.9, 0.5 M NaCl, 20 mM imidazole, and 6 M urea) and lysed by sonication at an amplitude of 38% for 15 min (30 s pulse on and 30 s pulse off) followed by centrifugation at 14 000 g.The supernatant containing the profilin protein was filter-sterilized using a 0.22 mm PVDF membrane filter and then applied onto an Ni-NTA-coupled HisTrap HP 5 ml column (GE Healthcare, UK) which had been preequilibrated with 20 mL binding buffer.The Ela g profilin protein was eluted using a linear gradient of washing buffer (20 mM Tris-HCl pH 7.9, 0.5 M NaCl, 0.5 M imidazole, and 6 M urea).Fraction containing soluble Ela g profilin protein was used for subsequent immuno-dot blot assay.

Immuno-dot blot assay
SSIgE titers were measured using the immunodot blot approach.To collect serum samples, 10 mL of whole blood were obtained from the participants and then centrifuged at 2600 g and 4 C for 15 min.5][26][27] SSIgE titers against these allergens were also previously measured by our group using the ImmunoCAP system approved by the United States Food and Drug Administration (FDA), 5 and these prior findings were in concordance with the present immunodot blot results.We also performed the immunedot blot assay to assess SSIgE titers against the Ela g profilin recombinant protein.All immuno-dot blot measurements were conducted at the same time to avoid batch variation.A positive sensitization to the tested allergen was defined as SSIgE titers >0.35IU/ml.

Survey questionnaires and disease definition
In the n ¼ 13 652 Singapore/Malaysia Chinese cohort, participants were requested to complete an investigator-administered questionnaire collecting information on demographics and medical history, which was based on the Allergic Rhinitis Impact on Asthma (ARIA) 28 guidelines and International Study of Asthma and Allergies in Childhood (ISAAC) 29 questionnaire.Ethnicity was self-reported through survey questionnaires and confirmed in a previously performed principal component analysis. 30thma was defined as ever having asthma positively diagnosed by a physician.AR was defined as having at least 2 major AR-related symptoms that include nasal congestion, rhinorrhea, nasal itching, and sneezing (based on 2008 guidelines set by the ARIA consortium). 28Moderate-severe AR was defined as having at least 1 of the following ARrelated symptoms: disturbed sleep, impaired daily activities including sport, leisure, impaired work and school, and troublesome symptoms (based on 2008 guidelines set by the ARIA consortium). 28AD was defined as having a persistent itchy rash that affected flexural areas.

Statistical analysis
Association analyses between oil palm sensitization and all allergic disease-related phenotypes were performed using logistic regression analysis in the R program version 3.6.1 (R Foundation for Statistical Computing, Vienna, Austria), and p < 0.05 was considered as statistically significant.Spearman correlation analyses of SSIgE and total IgE levels were also performed using the R program.
In this cohort, the sensitization rates of 5 pollen allergens were measured, including oil palm (Elaeis guineensis, 9.6%), chloridoids (Cynodon dactylon, 13.5%), panicoids (Sorghum halepense, 11.9%), pooids (Phleum pratense, Festuca pratensis, and Lolium perenne, 16.7%), and weeds (Brassica spp., Ambrosia artemisifolia, and Helianthus annus, 9.4%, Fig. 1).SSIgE titers against all of these pollen allergens were also correlated with each other (Spearman's Rho ranged from 0.505 to 0.800, all p < 0.0001; Fig. S2).By contrast, SSIgE titers against 4 common types of fungal allergens were generally lower than all the other types of allergen tested (Aspergillus sp.: 5.3%, Cladosporium sp.: 6.0%, Curvularia sp.: 6.6%, and Penicilium sp.: 4.6%, Fig. 1).Pairwise correlations were observed across serum IgE titers specific to all fungal allergens tested (Spearman's Rho ranged from 0.391 to 0.765, all p < 0.0001; Fig. S3).This is in agreement with previous findings. 4rrelation between total IgE and serum sIgE levels specific to HDM and oil palm pollen HDM sensitization was previously correlated to the atopy condition, as well as the serum total IgE titer in the tropical Southeast Asian population. 5To confirm these findings, SSIgE titers against all 16 common allergens in this present cohort were combined and used as an estimation of the total IgE titer.As shown in Fig. 2, SSIgE titers against 3 HDM species were all strongly correlated with the total IgE titer (Spearman's Rho ranged from 0.717 to 0.778, all p < 0.0001).A significant correlation was also observed between the SSIgE titer against oil palm and the total IgE titer (Spearman's Rho ¼ 0.466, p < 0.0001).Thus, both HDM and oil palm sensitizations might be indicative of a higher total IgE titer, which also suggest a correlation with atopy in the Singapore/Malaysia population.Besides, individuals sensitized to HDM also showed a higher sensitization rate to oil palm pollen (14.7%), as compared to the HDM-sIgE-negative individuals (4.4% sensitized to oil palm pollen).This suggests polysensitization against both HDM and oil palm allergens was more prevalent in the Singapore/ Malaysia population, as compared to monosensitization against either type of these allergens.

Association between oil palm sensitization and risks of allergic diseases in the Singapore/ Malaysia population
To evaluate the association between oil palm sensitization and the risks of allergic diseases in the Singapore/Malaysia population, we recruited  1).IgE sensitization to oil palm pollen was also associated with having at least one allergic disease (asthma, AR, and/or AD, p ¼ 7.86 x 10 À11 , OR ¼ 2.08, 95% CI: 1.68-2.61,Table 1).Oil palm sensitization was associated with a higher risk of disease multimorbidity (affected by more than one allergic disease, p ¼ 8.35 x 10 À16 , OR ¼ 2.80, 95% CI: 2.19-3.61,Table 1).

Association between Elaeis guineensis sensitization and severity of allergic diseases in the Singapore/Malaysia population
In the n ¼ 13 652 Singapore/Malaysia Chinese cohort (Table S1), we next evaluated if oil palm   3).Collectively, we also observed significant association between oil palm sensitization and any asthma exacerbation event in the past 12 months (p ¼ 0.00139, OR ¼ 2.07, 95% CI: 1.31-3.21,Table 3).Among the ARaffected individuals (n ¼ 5453), oil palm sensitization was associated with the presence of itchy nose symptoms in past 12 months (p ¼ 0.0181, OR ¼ 1.41, 95% CI: 1.07-1.90,Table 3).Further, AR patients sensitized against oil palm pollen were more likely to be affected by moderate-to-severe AR symptoms as compared to non-sensitized patients (p ¼ 0.00113, OR ¼ 1.53, 95% CI: 1.19-1.98,Table 3).
Association between oil palm sensitization and the severity of asthma and AR was also significant in the stratified cohort of HDM-sensitized individuals (n ¼ 8089, Table 4).In the sub-cohort of HDM sensitized asthma patients (n ¼ 1842), oil palm sensitization was associated with the presence of recent (past 12 months) wheezing (p ¼ 0.0116, OR ¼ 1.67, 95% CI:  4).In the sub-cohort of HDM-sensitized AR patients (n ¼ 3849), oil palm sensitization was associated with the presence of recent (past 12 months) itchy nose symptoms (p ¼ 0.0415, OR ¼ 1.38, 95% CI: 1.02-1.89)and moderate-to-severe AR symptoms (p ¼ 0.00266, OR ¼ 1.53, 95% CI: 1.16-2.02,Table 4).Overall, these results suggest an independent influence of oil palm pollen sensitization on the symptoms and severity of asthma and AR.
Lastly, we also examined the associations between oil palm sensitization and severity of AD symptoms among AD-affected individuals (n ¼ 2447); however, the results were not significant (Table S2).
Sensitization to the Ela g profilin allergen is not associated with oil palm pollen sensitization in the Singapore/Malaysia population Accumulating evidences have demonstrated profilin as one of the most important tree pollen panallergens (reviewed in Asam et al 31 ).Therefore, we sought to investigate the prevalence of Ela g profilin sensitization in the present study population.Serological assessment of SSIgE titers against Ela g profilin was performed on n ¼ 228 recruited subjects from the serological assessment cohort (Table S1).As shown in Table S3, the frequency of SSIgE sensitization against Ela g profilin (>0.35IU/ml) was not significantly different between oil palm pollensensitized subjects (17.4%) and oil palm pollen non-sensitized subjects (7.3%, p ¼ 0.076, Table S3 and Fig. S4).This suggests the Ela g profilin is not a major allergen of the oil palm pollen.Further, we also examined the associations between Ela g profilin sensitization and the risk of allergic diseases in this cohort, however, the results were not significant (data not shown).

Elaeis guineensis
Positive SPT  1. Association between Elaeis guineensis (oil palm) pollen sensitization and the risk of allergic diseases in the Singapore/Malaysia Chinese population.CI: confidence interval; OR: odds ratio; SPT: skin prick test.All data were evaluated based on skin prick test results of the Elaeis guineensis (oil palm) pollen allergen in a cross-sectional cohort of Singapore/Malaysia Chinese individuals (n ¼ 13 652).P-value, odds ratio, and 95% CI were calculated using a logistic regression analysis with adjustment for age and gender.*Logisticp < 0.05 is considered as significant.a Allergic asthma case was defined as ever having asthma positively diagnosed by a physician (Based on ISAAC questionnaire).Non-asthma control was defined as no symptoms and history of asthma.b Allergic rhinitis (AR) case is defined by 2 or more self-reported rhinitis symptoms.Non-AR control was defined as no symptoms and history of AR. c Atopic Dermatitis (AD) case is defined as having a persistent itchy rash that affected flexural areas.Non-AD control was classified based on no symptoms and history of AD HDM-sensitized individuals (n ¼ 8089)

Elaeis guineensis
Positive SPT Table 2. Association between Elaeis guineensis (oil palm) pollen sensitization and the susceptibility of allergic diseases among house dust mite-sensitized individuals in the Singapore/ Malaysia Chinese population.CI: confidence interval; HDM: house dust mite; OR: odds ratio; SPT: skin prick test.All data were evaluated based on skin prick test results of the Elaeis guineensis (oil palm) pollen allergen in a cross-sectional cohort of Singapore/Malaysia Chinese individuals with positive sensitization to house dust mite allergens (n ¼ 8089).P-value, odds ratio, and 95% CI were calculated using a logistic regression analysis with adjustment for age and gender.*Logisticp < 0.05 is considered as significant.a Allergic asthma case was defined as ever having asthma positively diagnosed by a physician (Based on ISAAC questionnaire).Non-asthma control was defined as no symptoms and history of asthma.b Allergic rhinitis (AR) case is defined by 2 or more self-reported rhinitis symptoms.Non-AR control was defined as no symptoms and history of AR. c Atopic Dermatitis (AD) case is defined as having a persistent itchy rash that affected flexural areas.Non-AD control was classified based on no symptoms and history of AD Table 3. Associations of Elaeis guineensis (oil palm) pollen sensitization with the severity and frequency of symptoms of allergic diseases in the Singapore/Malaysia Chinese population.A&E: accident and emergency department of a hospital; AR: allergic rhinitis; CI: confidence interval; GP: general practitioner; OR: odds ratio; SPT: skin prick test.All data were evaluated based on skin prick test results of the Elaeis guineensis (oil palm) pollen allergen in a cross-sectional cohort of Singapore/Malaysia Chinese individuals (n ¼ 13 652).P-value, odds ratio, and 95% CI were calculated using a logistic regression analysis with adjustment for age and gender.*p< 0.05 is considered as significant.a Asthma-related symptoms and exacerbation: cases are defined as asthma-affected individuals with the presence of asthma-related symptoms or exacerbations in the past 12 months.Controls are defined as asthma-affected individual with the absence of respective symptoms or exacerbations in the past 12 months.b Any exacerbation event includes school absence, GP/specialist visits, A&E admission, or hospitalization due to asthma exacerbation.c AR-related symptoms: cases are defined as AR-affected individuals with the presence of AR-related symptoms or exacerbations in the past 12 months.Controls are defined as AR-affected individual with the absence of respective symptoms in the past 12 months.d Severity of AR symptoms: Moderate-to-severe AR was defined as AR-affected individuals having at least one of the following disturbances due to AR-related symptoms in the past 12 months: disturbed sleep, impaired daily activities including sport, leisure, impaired work and school, and troublesome symptoms.Mild AR was defined as AR-affected individuals with the absence of these disturbances

DISCUSSION
7,18 However, the association of pollen sensitization with the risk and severity of allergic diseases in this region was not well characterized.To address this, the present study first detected a substantial sensitization rate (9.4-16.7%) of 5 types of tree and grass pollen allergens (Elaeis guineensis, chloridoids, panicoids, pooids, and weeds) in the Singapore/ Malaysia population, as well as a high crossreactivity among these pollen allergens.In a separate cross-sectional cohort of n ¼ 13 652 Singapore/Malaysia young adults of Chinese ethnicity, oil palm sensitization was significantly associated with the risks, exacerbations, and severity of symptoms of allergic diseases.
The tropical climatic condition of Singapore and Malaysia favors the growth of vegetation all year round, resulting in a rich and diverse spectrum of fungi, fern spore, and pollen presented in this environment. 8,23In Singapore, a high abundance of oil palm pollen was detected in the atmosphere, especially during the Northeast Monsoon season (December to March). 8Due to the abundance of this pollen in the atmosphere, a high frequency of oil palm sensitization (11.8%-48%) was reported in the Singapore population. 6This has also been evaluated in the present study, using a large-scale cross-sectional cohort of Singapore/Malaysia young adults that is independent of the previous cohort.We report an oil palm sensitization rate of 9.6% in Singapore and Malaysia, similar to the rate reported in Thailand (8.3% among pediatric asthmatics), 17 but lower than the rate reported in Indonesia (22.43%). 13udies have suggested oil palm sensitization may associate with the development of allergyrelated phenotypes in the Asian population.For instance, a higher frequency of oil palm sensitization was found in both Singaporean and Indonesian patients affected by asthma and/or AR. 6,13 Also, the incidence of asthma and AR was significantly higher among palm tree garden workers, compared to office workers in Iran. 32ur current results have thus confirmed these findings, where increased risks, exacerbation, and severity of symptoms of asthma, AR, and AD were observed in oil palm sensitized individuals.Significantly increased nasal symptoms were also observed in persistent AR patients 5 min after nasal challenge with Elaeis guineensis (unpublished data), using a previously reported approach performed by our group. 33 our work, skin prick test was performed using a panel of 4 types of allergens: 2 house dust mite (HDM) species (Dermatophagoides pteronyssinus and Blomia tropicalis), oil palm pollen (Elaeis guineensis), and a fungus species (Curvularia lunata).These allergens were previously shown to be the most common indoor and outdoor inhalant allergen presented in the Singapore environment with high allergenicity. 5,6We have previously shown that allergic response is largely dominated by HDM allergens: HDM sensitization was strongly associated with the risk and severity of asthma, AR, and AD. 5 At least 70% of Singaporean/Malaysian individuals were skin prick test (SPT) positive against HDM, 5 whereas about 4% of individuals were SPT positive against oil palm pollen (Table S1).Despite this, the present stratified analysis has also shown oil palm pollen sensitization can influence the manifestation of allergic diseases, independent of HDM sensitization.This suggested both indoor (HDM) and outdoor (oil palm pollen) allergens contribute independently in the development of allergic disorders in this region.Further, in this Singapore/Malaysia population, polysensitization against HDM and oil palm pollen is more prevalent and is associated with an increased risk and severity of allergic diseases compared to monosensitization (unpublished data).This will be discussed in a separate publication.
Accumulating evidences have demonstrated profilin as one of the most important tree pollen panallergens (reviewed in Asam et al 31 ).A study in Spain has showed 56% of date palm pollenallergic patients had a specific IgE-reactivity to natural profilin Pho d 2. 34 Positive SPT to the date palm pollen profilin was also observed among 25% of pollen-allergic children in Italy. 35In the present study, the frequency of SSIgE sensitization against Ela g profilin (>0.35IU/ml) in the oil palm-sensitized individuals was not significantly higher than the non-sensitized controls (p ¼ 0.076).This suggests the Ela g profilin is not a major allergen of the oil palm pollen.Individuals sensitized to oil palm pollen may show sensitizations towards other sources of novel tree pollen allergens.
A seasonal effect on the atmospheric concentration of oil palm pollen was previously observed. 8Its concentration peaks during the Northeast Monsoon season (December to March). 8The present study has not investigated whether this can influence the association between oil palm sensitization and allergy-related phenotype, due to the limitation of a crosssectional study.Further study using a longitudinal cohort with multiple follow-up time points across different seasons within a year might help to address this question.Besides, we cannot determine whether age can influence the allergenicity of oil palm in our study population because the recruited cross-sectional cohort comprised mostly young adults (age: 21.96 AE 5.004 in the large-scale epidemiological assessment cohort, Table S1).Given the pollen sensitization rate was previously shown to increase from childhood to adulthood, 6 future studies involving participants across age groups are therefore required to validate and investigate this.As a limitation of this study, we were unable to directly measure the total IgE levels in serum due to the restrictions in the volume of serum available.Total IgE levels were derived from the summation of SSIgE levels of individual allergens.This might confound the correlation analysis between total IgE and SSIgE levels.However, in this population, we have previously demonstrated that measured total IgE levels were correlated with the ssIgE levels of house dust mite (Blomia tropicalis and Dermatophagoides pteronyssinus). 5These correlations have now been replicated in this present study (Fig. 2A-C).A direct measurement of total IgE should also be performed in the future study to validate our present findings.
Overall, the present findings emphasized.animportant role of oil palm pollen allergen in the manifestation of allergic disorders in this region.In allergy patients with oil palm pollen sensitization, implementation of allergen-specific immunotherapy might help to reduce morbidity.This approach has been proven to be effective in the treatment of moderate-severe seasonal AR. 36llergen avoidance has also been suggested to be effective in managing asthma-related phenotypes. 12,37By reducing the exposure of oil palm pollen allergen during the Northeast Monsoon season (December to March), the prevalence of oil palm allergy can be reduced in this population.

CONCLUSION
In conclusion, the present study has provided an up-to-date sensitization profile of common inhalant allergen and the associations of oil palm sensitization with the risk and severity of allergic diseases in the tropical environment of Singapore and Malaysia.The associations of oil palm sensitization with allergic diseases are independent of HDM or profilin sensitization.Our findings emphasize an important role of pollen sensitization in the manifestation of allergic disease in this region, which also suggests a need for primary prevention of sensitization against oil palm in this tropical environment of Southeast Asia.

Table 4 .
Associations of Elaeis guineensis (oil palm) pollen sensitization with the severity and frequency of symptoms of allergic diseases among house dust mite-sensitized individuals in the Singapore/Malaysia Chinese population.A&E: accident and emergency department of a hospital; AR: allergic rhinitis; CI: confidence interval; GP: general practitioner; HDM: house dust mite; OR: odds ratio; SPT: skin prick test.All data were evaluated based on skin prick test results of the Elaeis guineensis (oil palm) pollen allergen in a cross-sectional cohort of Singapore/Malaysia Chinese individuals with positive sensitization to house dust mite allergens (n ¼ 8089).P-value, odds ratio, and 95% CI were calculated using a logistic regression analysis with adjustment for age and gender.*p< 0.05 is considered as significant.a Asthma-related symptoms and exacerbation: cases are defined as asthmaaffected individuals with the presence of asthma-related symptoms or exacerbations in the past 12 months.Controls are defined as asthma-affected individual with the absence of respective symptoms or exacerbations in the past 12 months.b Any exacerbation event includes school absence, GP/specialist visits, A&E admission, or hospitalization due to asthma exacerbation.c AR-related symptoms: cases are defined as AR-affected individuals with the presence of AR-related symptoms or exacerbations in the past 12 months.Controls are defined as AR-affected individual with the absence of respective symptoms in the past 12 months.d Severity of AR symptoms: Moderate-to-severe AR was defined as AR-affected individuals having at least one of the following disturbances due to ARrelated symptoms in the past 12 months: disturbed sleep, impaired daily activities including sport, leisure, impaired work and school, and troublesome symptoms.Mild AR was defined as AR-affected individuals with the absence of these disturbances