Aspergillus terreus Antibody Serosurveillance in Tyrol: A Population-Based, Cross-Sectional Study of a Healthy Population

The rare, but emerging mold Aspergillus terreus is an important pathogen in some geographical areas, like Tyrol (Austria) and Houston (Texas). The reason for this high prevalence is unknown. The present serosurveillance study aimed to evaluate the trends in levels of A. terreus-specific IgG antibodies in various regions of Tyrol and to compare the results to the environmental spread of A. terreus in Tyrol. Therefore, 1058 serum samples from healthy blood donors were evaluated. Data revealed a significant difference between the Tyrolean Upland and Lowland. Moreover, female participants had higher A. terreus IgG antibody levels than male participants. The differences found in our study are consistent with the distributional differences in environmental and clinical samples described in previous studies, supporting that A. terreus IgG antibody levels reflect the environmental epidemiology of A. terreus in Tyrol.

The Aspergillus fumigatus species complex is the most common etiological agent within the genus of Aspergillus, followed by less common species complexes such as A. flavus, A. niger and A. terreus [3,6].Depending on the geographical region, A. terreus can be either the third or the fourth most common etiological agent of IA [7].Areas with a higher A. terreus abundance are Tyrol (Austria), Houston (Texas), Spain and Israel [8,9].
The significance of infections caused by less common species such as A. terreus (contributing to 4-5.2% of IA cases worldwide) has increased as the epidemiology of invasive fungal infections (IFIs) continues to change, with a wider range of patients appearing to be at risk [8,10,11].In addition to a changing epidemiology and an expanding at-risk population, A. terreus presents clinical challenges due to its unique characteristics.Infections from A. terreus are represented with a high tendency for dissemination and a mortality up to 70% in IA [3,12].Moreover, the natural lower susceptibility of A. terreus to amphotericin J. Fungi 2023, 9, 1008 2 of 8 B and increasing azole resistance, even in some less common species of section Terrei, are of particular concern, which may result in complications in patient treatment [10,[13][14][15].Furthermore, in Tyrol (Austria), A. terreus plays an important role due to the significantly higher number of clinical cases when compared globally [3,9,16].Since 1994, infections caused by A. terreus have been detected at the University Hospital Innsbruck, which were mainly related to construction activities [9].
A. terreus has a significant, uneven environmental distribution between the Tyrolean Upland (West) and Lowland (East), with the capital city of Innsbruck located in the middle of Tyrol [17].Dietl et al. found that 61.9% of environmental samples came from the Tyrolean Lowland, whereas only 11.6% of the Tyrolean Upland samples resulted in the isolation of A. terreus [17].Furthermore, the distribution of patients with proven infection or colonization with A. terreus at the Institute of Hygiene and Medical Microbiology of the Medical University of Innsbruck correlated well with these findings (Upland: 9.43%; Lowland: 56.6%) [17].Dietl et al. have evaluated several environmental, economic and microclimatic variables, like temperature, vegetation, population density and wind, concluding that none of these factors resulted in significant differences within the area of Tyrol [17].
As Aspergillus spores are constantly found in the air and human respiratory tract, antibodies against an invading or colonizing pathogen are formed both in healthy as well as in immunocompromised humans [4,18].Mold-specific immunoglobulin G (IgG) antibodies are widely used as biomarkers in research of environmental molds [19].Further, it has been shown that levels of mold-specific IgG antibodies reflect exposure to the specific pathogen [20].Serosurveillance is known as an effective tool for measuring infection prevalence and incidence [21] and could give valuable insights into the differences among the environmental spread of A. terreus in Tyrol.
The aim of this study was to assess the trends in A. terreus-specific IgG antibody levels in a serosurveillance analysis of a healthy population in Tyrol and to determine whether the data reflect the different distribution of A. terreus between the Tyrolean Upland and Lowland.

Ethics Statement
Serum samples were collected from Tyrolean blood donors for this population-based, cross-sectional study.The Human Ethics Committee of the Medical University of Innsbruck evaluated and approved the protocol for this study in 2022 (EK No. 1284/2022).

Study Population
Between January 2023 and April 2023, a total of 1058 blood samples were collected-362 samples from blood donors in the Tyrolean Upland and 696 samples from blood donors in the Tyrolean Lowland-in cooperation with the Central Institute for Blood Transfusion and Immunology of the University Hospital Innsbruck and the Tyrolean Red Cross blood donation service.Samples were collected as part of routine infection serology testing.This study included only participants registered as residents of Tyrol.

Investigation of A. terreus IgG Antibody Levels in Serum Samples of a Healthy Tyrolean Population
Samples from participants with signed consent for sample use for scientific purposes were collected and further processed by an ETI-Max 3000 (DiaSorin S.p.A., Centralino, Italy) to obtain 950 µL of serum.Samples were stored at −80 • C prior to use.
All serum samples were analyzed on a PhadiaTM 250 laboratory system (Thermo Fisher Scientific Inc., Waltham, MA, USA) using an ImmunoCap allergen m36 for A. terreus (Thermo Fisher Scientific Inc., Waltham, MA, USA).Specimens were analyzed according to the manufacturer's protocol.As our aim was to investigate serum antibody levels in a healthy population without any bias on diseases, we did not use a positive cut-off value.All antibody levels above the detection limit (0.02 mg/L) were enrolled.

Statistical Analysis
Data were checked for normality and homogeneity prior to analysis.The statistical analysis was performed using GraphPad Prism9 (GraphPad Software, Boston, MA, USA).A Mann-Whitney U test was used to compare the data collected between the Tyrolean Upland and Lowland.
Data for the comparison among the different blood donation sites were analyzed using a Kruskal-Wallis test with Dunn's correction for multiple comparisons.Sex-specific data for the Tyrolean Upland and Lowland were analyzed via a Mann-Whitney U test and two-way analysis of variance (ANOVA) with Bonferroni's correction for multiple comparisons.IgG values are presented as mean ± SD.A p value of less than 0.05 (p < 0.05) was considered statistically significant.

Patient Population Data
In total, 1058 serum samples from blood donors all over Tyrol were collected and analyzed for their levels of IgG antibodies against A. terreus.A total of 1046 samples were used from this 1058 sample set, with 355 samples coming from the Tyrolean Upland and 691 from the Tyrolean Lowland.Twelve samples were excluded from the study because they were not registered as residents of Tyrol.

Serosurveillance of IgG Antibody Levels in the Tyrolean Upland and Lowland
The results of the comparison of A. terreus IgG antibody levels between the Tyrolean Upland and Lowland showed a statistical difference (p = 0.0468) (Figure 1).Inhabitants of the Tyrolean Upland had lower A. terreus IgG antibody levels (12.90 ± 8.53 mg/L) than inhabitants of the Tyrolean Lowland (14.88 ± 11.76 mg/L) (Figure 1).Only 15 study participants from the Tyrolean Upland and 10 participants from the Tyrolean Lowland showed A. terreus IgG antibody levels lower than the detection limit.These values were therefore set to the detection limit of 0.02 mg/L for the statistics.
When samples were grouped into the different blood donation sites, several statistically significant results could be found (Figure 2).Regarding the Tyrolean Upland, no significant differences in the levels of A. terreus IgG antibodies between the separate donation sites could be observed.However, there was a tendency for participants from Umhausen to have the lowest and participants from Mieming to have the highest A. terreus IgG antibody levels within the Tyrolean Upland.Concerning the Tyrolean Lowland, A. terreus IgG antibody levels were significantly higher among donors in Buch in Tirol (Buch i. T., 18.59 ± 11.51 mg/L) when compared to different locations in the Tyrolean Lowland, specifically Kirchbichl, Kitzbühel and Thiersee.Concerning the whole of Tyrol, blood donors in the Tyrolean Upland from Imst and Umhausen showed significantly lower antibody levels than donors from Buch i. T. in the Tyrolean Lowland.
With the influence of sex taken into consideration, female study participants showed significantly higher A. terreus IgG antibody levels than male participants (p < 0.0001) (Figure 3A).Female participants had A. terreus IgG antibody levels of 15.77 ± 10.75 mg/L, whereas male participants showed levels of 13.05 ± 10.72 mg/L.Statistical significance was sustained for both female and male groups when separated into Tyrolean Upland and Lowland (Upland: p = 0.0049; Lowland: p = 0.0177) (Figure 3B).significantly higher A. terreus IgG antibody levels than male participants (p < 0.0001) (Figure 3A).Female participants had A. terreus IgG antibody levels of 15.77 ± 10.75 mg/L, whereas male participants showed levels of 13.05 ± 10.72 mg/L.Statistical significance was sustained for both female and male groups when separated into Tyrolean Upland and Lowland (Upland: p = 0.0049; Lowland: p = 0.0177) (Figure 3B).

Discussion
An increasing number of vulnerable individuals is making invasive fungal infections (IFIs) more prevalent [22].IFIs result in approximately 1.5 million deaths globally each year, with 70% resulting from an infection with fungi of the genera Aspergillus, Candida, Cryptococcus and Histoplasma [23].Aspergillosis, usually caused by A. fumigatus, can lead to severe pulmonary infections [3,4,6].
Despite the global dominance of A. fumigatus, A. terreus shows a higher infection rate in some global areas, such as Tyrol, Austria [8,9,16].A prospective international study with respect to A. terreus showed a global prevalence of 5.2% of mold infections [8]. A. terreus is restricted geographically to some areas with higher infection rates, like Tyrol (Austria), Spain, Israel and Houston (Texas) [9,16].There is no clear explanation for this distributional difference, which requires further investigation [13].
A. terreus known to be a unique representative of the Aspergillus genus because of its low susceptibility to amphotericin B, which makes the treatment of infections from this pathogen challenging [9,[24][25][26][27].Furthermore, A. terreus is commonly used as a producer of natural statins, such as compactin, mevastatin and lovastatin or itaconic acid [28].
In a study by Dietl et al., 5.4% of environmental samples (soil, air, plant material, etc.) were culture-positive for A. terreus.These results indicate an increased environmental

Discussion
An increasing number of vulnerable individuals is making invasive fungal infections (IFIs) more prevalent [22].IFIs result in approximately 1.5 million deaths globally each year, with 70% resulting from an infection with fungi of the genera Aspergillus, Candida, Cryptococcus and Histoplasma [23].Aspergillosis, usually caused by A. fumigatus, can lead to severe pulmonary infections [3,4,6].
Despite the global dominance of A. fumigatus, A. terreus shows a higher infection rate in some global areas, such as Tyrol, Austria [8,9,16].A prospective international study with respect to A. terreus showed a global prevalence of 5.2% of mold infections [8]. A. terreus is restricted geographically to some areas with higher infection rates, like Tyrol (Austria), Spain, Israel and Houston (Texas) [9,16].There is no clear explanation for this distributional difference, which requires further investigation [13].
A. terreus is known to be a unique representative of the Aspergillus genus because of its low susceptibility to amphotericin B, which makes the treatment of infections from this pathogen challenging [9,[24][25][26][27].Furthermore, A. terreus is commonly used as a producer of natural statins, such as compactin, mevastatin and lovastatin or itaconic acid [28].
In a study by Dietl et al., 5.4% of environmental samples (soil, air, plant material, etc.) were culture-positive for A. terreus.These results indicate an increased environmental burden with respect to A. terreus when compared to other regions like Cologne and Madrid, with only 0.2% and 0.5% of samples, respectively [17,18,29].The study by Dietl et al. further revealed that, even within Tyrol, an uneven distribution can be observed, with the Tyrolean Upland (11.6%) resulting in a lower prevalence than the Tyrolean Lowland (61.9%) [17].Currently, there are no known factors of significant difference between the two regions of Tyrol that could result in the different environmental spread of A. terreus.In their study, Dietl et al. evaluated several environmental, economic and climatic variables, such as temperature, vegetation, altitude, population density, traffic and wind, and concluded that all of these factors are comparable within the area of Tyrol [17].Other possible factors, like microclimate, topography, geographical diversity, humidity, agricultural use of antifungals and socio-economic factors, need to be analyzed further in subsequent studies.
In our project, we used serum samples from healthy blood donors in Tyrol.The study population consisted of 446 female and 600 male participants and showed a mean age of 47 ± 14, ranging from 18 to 70 years.Some studies suggest that immunosenescence has no impact on the quantity of antibody production, but the elderly show lower production of specific antibodies against invading pathogens [30,31].Our results are somewhat contradictory as the highest levels of A. terreus IgG antibodies derived from study participants who were 50 years of age or older.A study including 100 healthy Ugandan blood donors (mean age of 19 years) displayed a median Aspergillus-specific IgG level of 5 mg/L, with no participants having higher than 40 mg/L [32].The present study showed a mean A. terreus IgG antibody level of 14.29 ± 11.14 mg/L (Upland: 12.90 ± 8.53 mg/L; Lowland: 14.88 ± 11.76 mg/L); comparable data and literature on A. terreus are largely absent.Lee et al. showed that in healthy blood donors from Taiwan, mean IgG antibody levels for A. fumigatus and A. niger were 28.6 mg/L and 20.3 mg/L [33].In their study, age, female sex, tuberculosis and rural residency were associated with A. fumigatus-specific IgG >50 mg/L; as well, a lower risk of cancer was associated with an IgG >50 mg/L.The prevalence of specific antibodies seems to be widely influenced.
Our data revealed that blood donors from the Tyrolean Upland have significantly lower A. terreus IgG antibody levels than study participants from the Tyrolean Lowland (Figure 1).As antibodies against an invading or colonizing pathogen are formed both in healthy as well as in immunocompromised humans, IgG antibodies can be used as biomarkers in research [4,19].Our results imply that humans living in an area of high A. terreus prevalence, like the Tyrolean Lowland, also develop high levels of IgG antibodies against this mold.The serosurveillance of A. terreus IgG antibodies further shows that, even within the Tyrolean Lowland, differences can be observed.Blood donors from Buch i. T. had significantly higher A. terreus IgG antibody levels than blood donors from several other donation sites.These findings are in agreement with a previous study [17].Both Buch i. T. as well as Mayrhofen are located in the district of Schwaz, which is known to have a higher prevalence of A. terreus in the environment, followed by the district of Kufstein [17].
Besides the significant difference between the Tyrolean Upland and Lowland, a difference in A. terreus IgG antibody levels between female and male participants could be observed (Figure 3A).Female blood donors showed higher levels of IgG antibodies against A. terreus.So far, this sex-specific difference is novel for A. terreus and could be related to the fact that adult females tend to have higher antibody responses than males, as has been mentioned for different species [34][35][36].As shown by Klein and Flanagan, females have a higher B-cell count, higher immunoglobulin levels and a better antibody response than males, which could be associated with female hormones or the X chromosome [36].Our results are also in concordance with findings from Lee et al., showing an odds ratio of 1.49 (95% CI: 1.14-1.93)for antibody formation in females [33].Significant differences between female and male participants persisted even when they were split into the Tyrolean Upland and Lowland (Figure 3B).

Figure 1 .
Figure 1.Differences in A. terreus antibody levels (mg/L) between the Tyrolean Upland and Lowland.The measurements are depicted as a box-and-whiskers Tukey plot.The detection limit was 0.02 mg/L.A Mann-Whitney U test resulted in a p value of 0.0468 (* p < 0.05).

Figure 1 . 9 Figure 1 .
Figure 1.Differences in A. terreus antibody levels (mg/L) between the Tyrolean Upland and Lowland.The measurements are depicted as a box-and-whiskers Tukey plot.The detection limit was 0.02 mg/L.A Mann-Whitney U test resulted in a p value 0.0468 (* p < 0.05).

Figure 3 .
Figure 3. Differences in A. terreus IgG antibody levels between (A) female and male study participants; (B) female and male study participants grouped into the Tyrolean Upland and Lowland.Measurements are depicted as a box-and-whiskers Tukey plot.The detection limit was 0.02 mg/L.Statistical differences were determined via (a) a Mann-Whitney U test; (b) two-way ANOVA with Bonferroni's correction for multiple comparisons.Statistical analysis resulted in a p value of (a) p < 0.0001; (b) Upland: p = 0.0049, Lowland: p = 0.0177 (**** p < 0.0001; ** p < 0.01; * p < 0.05).

Figure 3 .
Figure 3. Differences in A. terreus IgG antibody levels between (A) female and male study participants; (B) female and male study participants grouped into the Tyrolean Upland and Lowland.Measurements are depicted as a box-and-whiskers Tukey plot.The detection limit was 0.02 mg/L.Statistical differences were determined via (a) a Mann-Whitney U test; (b) two-way ANOVA with Bonferroni's correction for multiple comparisons.Statistical analysis resulted in a p value of (a) p < 0.0001; (b) Upland: p = 0.0049, Lowland: p = 0.0177 (**** p < 0.0001; ** p < 0.01; * p < 0.05).