Molecular allergen sensitization of Aspergillus fumigatus between allergic bronchopulmonary aspergillosis and A fumigatus‐sensitized asthma in Guangzhou, Southern China

Abstract Background Few studies have assessed the sensitization of mycotic allergens and Aspergillus fumigatus molecular allergens. This study aimed to investigate the relationships of A fumigatus components and mycotic allergens in allergic bronchopulmonary aspergillosis (ABPA) patients and A fumigatus (Af)‐sensitized asthma patients. Methods Serum sIgE levels of Penicillium chrysogenum, Cladosporium herbarum, Mucor racemosus, Candida albicans, Alternaria alternata, Helminthosporium halodes, and A fumigatus allergen components (Asp f 1, Asp f 2, Asp f 3, Asp f 4, and Asp f 6) were measured via the ImmunoCAP assay in 18 ABPA and 54 Af‐sensitized asthma patients in Guangzhou city, China. Results 94.44% of ABPA patients and 87.04% of Af‐sensitized asthma patients were co‐sensitized to at least one other fungal allergen. The positive rates of Asp f 1 (88.89% vs 59.26%, P < .05), Asp f 2 (66.67% vs 33.33%, P < .05), Asp f 4 (61.11% vs 33.33%, P < .05), and Asp f 6 (66.67% vs 14.81%, P < .001) in ABPA patients were higher than those in Af‐sensitized asthma patients. IgE levels of Asp f 1 (P < .05), Asp f 4 (P < .05), and Asp f 6 (P < .001) were higher in ABPA patients than in Af‐sensitized asthma patients. Optimal scale analysis showed that ABPA was more relevant to Af components (Cronbach's alpha = 90.7%). Conclusion The A fumigatus components and their relationships with various mycotic allergens were different in ABPA and Af‐sensitized asthma patients. This finding may help local doctors in the diagnosis and immunotherapy of fungal allergies.


| INTRODUC TI ON
Fungi are the most common microorganisms in the human living environment, and they not only readily cause respiratory tract infections and induced inflammatory responses but also cause severe allergic reactions. Studies have shown that Aspergillus, Alternaria, Candida, Cladosporium, and Epicoccum are considered major sources of allergens worldwide. 1,2 During reproduction, fungi release large amounts of spores and hyphal fragments into the air, which may cause immunoglobulin E (IgE)-mediated respiratory allergic diseases, 3

especially
Aspergillus fumigatus (Af)-sensitized asthma and allergic bronchopulmonary aspergillosis (ABPA). 4 Investigations in Europe have shown that the incidence of fungus-induced respiratory tract allergies is as high as 20%-30% in atopic populations, reaching 6% in the general population. 5,6 Allergic bronchopulmonary aspergillosis is a pulmonary disease caused by A fumigatus, and pathogenesis is caused by the allergic response against A fumigatus colonizing the airways rather than saprophytic or invasive of the fungi. 7,8 When patients with ABPA are exposed to fungi in the environment, they display repeated wheezing and dyspnea; this condition can even be life-threatening in severe cases. 9 Due to the lack of effective clinical treatment, patients have heavy economic burden and poor quality of life. A European study showed that Af-sensitized asthma can easily develop into ABPA, 10,11 which may be induced by molecular allergens of A fumigatus. There are five major molecular allergens of A fumigatus (Asp f 1, Asp f 2, Asp f 3, Asp f 4, and Asp f 6). Among them, Asp f 1 is the most important protein of A fumigatus. It secretes a lot after spore germination and early fungal invasion, which is related to fungal colonization and the saprophytic nature of the fungi. Asp f 2 is a fibronectin, Asp f 3 is an epitope of the peroxidase membrane protein, Asp f 4 is a glycosylated hydrolase, and Asp f 6 is a manganese superoxide dismutase. 12,13 Our previous research showed that more than 18% of asthmatic patients are sensitized to Asp f 3. 14 Although several studies have focused on A fumigatus sensitization in Southern China, 15,16 the studies investigating the connection between various fungal allergens and A fumigatus components are still limited, especially in Guangzhou, a beautiful and unique cultural city in China. In addition, various fungal allergens and A fumigatus components may be co-pathogenic and play an important role in ABPA or Af-sensitized asthma. Accordingly, in this study, we compared various fungal allergens and A fumigatus major components between ABPA and Af-sensitized asthma patients, and our findings are expected to provide meaningful evidence for more accurate diagnosis and guiding disease treatment.

| Patients
This study included 18 ABPA patients and 54 Af-sensitized asthma patients, all of whom had undergone A fumigatus allergen sIgE There were no significant differences in age and sex of patients be-

| Statistical analyses
Data analyses were performed using the statistical software package SPSS 22.0 (Chicago, IL, USA). Nonparametric quantitative data were described as medians (interquartile ranges) and between-group comparisons of numerical data were performed using Mann-Whitney U tests or Kruskal-Wallis tests. Parametric quantitative data were depicted as means ± standard deviations.
To show the proportion of positive results, categorical data were reported as percentages. Chi-square (χ 2 ) tests or F tests were used to demonstrate differences in proportions between groups.
Correlation analyses among the groups were performed by calculating the Spearman correlation coefficient (r s ). The correlation between components was calculated with optimal scale analysis.
Differences were regarded as statistically significant if the P value was lower than .05.

| Fungal sensitization between ABPA patients and Af-sensitized asthma patients
Overall, 31.9% of patients were sensitive to A fumigatus in Class 3.
( Table 1). There was no significant difference in A fumigatus sIgE levels between ABPA and Af-sensitized asthma patients (P > .05).
Moreover, 94.44% of ABPA patients and 87.04% of Af-sensitized asthma patients were sIgE positive to at least one fungal allergen among P chrysogenum, C herbarum, M racemosus, C albicans, A alternata, and H halodes. High positive rates to P chrysogenum were found in 94.44% of ABPA patients and 77.78% of Af-sensitized asthma patients. The positivity rates of C herbarum (88.89% vs 62.96%, P < .05) and A alternata (72.22% vs 44.44%, P < .05) were higher in ABPA patients than in Af-sensitized asthma patients (Figure 1). Although sIgE levels of P chrysogenum, C herbarum, M racemosus, C albicans, A alternata, and H halodes were higher in ABPA patients than in Afsensitized asthma patients, there were no significant differences between the two groups ( Figure 2A).
As shown in Figure 2B Table 2) and 10 (18.52%) Af-sensitized asthma patients co-sensitized to Asp f 1, Asp f 2, Asp f 3, Asp f 4, and Asp f 6 at the same time. Interestingly, all of the 7 ABPA patients were co-sensitized to P chrysogenum and C albicans, but Af-sensitized asthma patients were not.

| Correlation analysis between A fumigatus components and various mycotic allergens in ABPA patients and Af-sensitized asthma patients
Spearman correlation analysis showed that tIgE (r s = 0.586, P < .05),

| D ISCUSS I ON
Although A fumigatus-specific precipitins (Ouchterlony immunodiffusion test) have been widely used in the diagnosis of ABPA, their positive rates in patients with ABPA range widely from 27% to 87%. 20 Moreover, their diagnostic value is limited. Currently, component-resolved diagnosis (CRD) has been applied to the diagnosis of A fumigatus allergy, which is conducive to the accurate diagnosis of ABPA.
In our study, we found that 31.9% of patients were sensitized to A fumigatus in Class 3. Guangzhou city is influenced by the East Asian monsoon season and has a humid and warm subtropical climate, with a relative air humidity of 68% and annual precipitation of more than 1700 mm. 21 This contributes greatly to the proliferation

Characteristic (n, %)
Af-sensitized asthma ABPA  and growth of fungi, which prefer humid and warm environments.
Therefore, a high concentration of mycotic spores in indoor and outdoor air is one of the most important causes of allergic respiratory tract diseases in Guangzhou. 22  and 0.87, respectively) and strong correlations with C herbarum, C albicans, and A alternata (0.60 ≤ r s < 0.80). 26 However, in the present study, A fumigatus was strongly correlated with C herbarum (r s = 0.688) in ABPA and A alternata (r s = 0.692) in Af-sensitized asthma patients but not correlated with C albicans (P > .05).
The difference between fungal and non-fungal allergens is that fungal allergens are more complex. They contain proteases, glycosidases, and protein products, which can easily lead to cross-reactions. Therefore, exposure to a single mycotic spore is equivalent to exposure to all fungal allergens. 27 For example, P chrysogenum and

| CON CLUS ION
In summary, this study is the first to demonstrate the complex relationship between A fumigatus components and various mycotic allergens in ABPA and Af-sensitized asthma patients from Guangzhou, Southern China. Asp f 1, Asp f 2, Asp f 4, and Asp f 6 in ABPA patients were significantly higher than those in Af-sensitized asthma patients and were connected with various mycotic allergens. This finding is expected to help local doctors in the diagnosis of fungal allergies, particularly in differential diagnosis between ABPA and Af-sensitized asthma.

ACK N OWLED G M ENTS
We thank Shiquan Wu and the doctors and nurses at the department of Allergy and Clinical Immunology of the First Affiliated Hospital of Guangzhou Medical University, China for their great support.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R ' S CO NTR I B UTI O N S
BQS conceived and designed the experiments. NLW, HMH, PYZ, and YL performed the experiments. WTL and ZHW analyzed the data. HSH and WTL wrote the article. All authors read and approved the final manuscript.

E TH I C A L S TATEM ENT
Approval was obtained from the ethics committee of The First Affiliated Hospital of Guangzhou Medical University (Reference number: GYFYY-2016-73).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support these findings are available on reasonable request from the corresponding author BQS. Data are not publicly available due to concerns regarding research participant privacy.