Journal of Fungi Article Aspergillus Conidia and Allergens in Outdoor Environment: A Health Hazard? Concepción De Linares 1, * , David Navarro 2,3 , Rut Puigdemunt 2,3 and Jordina Belmonte 2,3 1 Department of Botany, University of Granada, 18071 Granada, Spain 2 Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain; david.navarro@uab.cat (D.N.); rut.puigdemunt@uab.cat (R.P.); jordina.belmonte@uab.cat (J.B.) 3 Institut de Ciència i Tecnologia Ambientals (ICTA-UAB), Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain * Correspondence: delinare@ugr.es; Tel.: +34-958243394 Abstract: Aspergillus is a genus of saprophytic fungus widely distributed in the environment and associated with soil, decaying vegetation, or seeds. However, some species, such as A. fumigatus, are considered opportunistic pathogens in humans. Their conidia (asexual spores) and mycelia are associated with clinical diseases known as invasive aspergillosis (IA), mainly related to the respiratory tract, such as allergic asthma, allergic bronchopulmonary aspergillosis (ABPA), or hypersensitivity. However, they can also disseminate to other organs, particularly the central nervous system. Due to the dispersal mechanism of the conidia through the air, airborne fungal particle measurement should be used to prevent and control this mold. This study aims to measure the outdoor airborne concentration of Aspergillus conidia and the Asp f 1 allergen concentration in Bellaterra (Barcelona, Spain) during 2021 and 2022, and to compare their dynamics to improve the understanding of the biology of this genus and contribute to a better diagnosis, prevention, and therapeutic measures in the face of possible health problems. The results show that both particles were airborne nearly all year round, but their concentrations showed no correlation. Due to Asp f 1 not being present in the conidia itself but being detectable during their germination and in hyphal fragments, we report the relevance of the aero-immunological analysis as a methodology to detect the potential pathogenic hazard of this fungus. Citation: De Linares, C.; Navarro, D.; Puigdemunt, R.; Belmonte, J. Aspergillus Conidia and Allergens in Keywords: aerobiology; Asp f 1; ELISA; fungal spores; high volume sampler; hirst sampler Outdoor Environment: A Health Hazard? J. Fungi 2023, 9, 624. https://doi.org/10.3390/jof9060624 1. Introduction Academic Editors: Jorge Martínez Quesada and Idoia Postigo Airborne fungal particles are an important essential fraction of atmospheric indoor and outdoor components that can be released by active or passive processes [1]. In general, fungi Received: 28 April 2023 release large amounts of spores (sexual or asexual) and hyphal fragments into the air, which Revised: 19 May 2023 can be involved in the decomposition of food and raw materials or the deterioration of Accepted: 26 May 2023 organic matter during storage [2]. However, airborne fungi cause serious health problems, Published: 28 May 2023 such as induced inflammatory responses or respiratory tract infections [3,4]. For example, in Europe, the incidence of fungus-induced respiratory tract allergies is as high as 20–30% in the atopic population, reaching 6% in the general population [5]. Copyright: © 2023 by the authors. More than 112 fungi are recognized as allergenic sources [6]. One of the most im- Licensee MDPI, Basel, Switzerland. portant is Aspergillus, considered the major primary fungal causative agent of allergic This article is an open access article bronchopulmonary mycosis (ABPM) and respiratory allergic symptoms [7,8]. This As- distributed under the terms and comycota is a filamentous saprophytic fungus widely distributed and associated with soil, conditions of the Creative Commons decaying vegetation, or seeds. However, some species, such as A. fumigatus, are considered Attribution (CC BY) license (https:// opportunistic pathogens of immunocompromised hosts. Their conidia (asexual spores) and creativecommons.org/licenses/by/ mycelia are associated with clinical diseases related to the respiratory tract, such as allergic 4.0/). asthma, ABPM, or hypersensitivity, collectively termed ‘aspergillosis’ [9]. J. Fungi 2023, 9, 624. https://doi.org/10.3390/jof9060624 https://www.mdpi.com/journal/jof J. Fungi 2023, 9, 624 2 of 11 There are currently defined 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. Its production is related to the germination of spores and growth of the mycelia’s early fungal invasion [10], which is related to fungal colonization and the saprophytic nature of the fungi. Nowadays, there is no universal, recommended method for continuous air sampling that can accurately show the entire aeromycobiota of a location [11]. The volumetric methods based on Hirst-type samplers offer a continuous sampling from the air, with the reliable determination of fungal spores. However, it is less effective in the case of particles with a diameter below 5 µm, as occurs with Aspergillus, Penicillium, and some basidiospores, which may lead to the underestimation of the number of spores in the air [11]. Another problem is the identification of fungal spores. In the case of Aspergillus and Penicillium, the conidia of both fungi genera present similar morphology and are indistinguishable under a light microscope. Therefore, in aerobiological studies, both fungi are grouped together as Aspergillus/Penicillium spore type [12]. Moreover, this methodology cannot identify smaller fungal fragments and hyphae [13]. The viable methods (cultivation on the medium) can resolve the identification of these genera; they even can identify the order of the species [14]. However, continuous samples are not possible. In contrast to these traditional methods, the immunological test may detect the presence of fungi based on the detection of their major allergen. Although this methodology depends on the commercial availability of specific allergens [13], it could be an alternative method for continuous airborne control of these fungi. This study compares the airborne dynamics of Aspergillus/Penicillium spore type and the concentration of Asp f 1 allergen in order to contribute to better diagnoses, prevention, and therapeutic measures for aspergillosis patients. 2. Materials and Methods 2.1. Sampling Site The aerobiological station is located at the Universitat Autònoma de Barcelona (Bel- laterra, Barcelona, NE, Spain), on the rooftop of the C building, at 23 m.a.g.l. (41◦ 300 2000 N, 02◦ 060 2800 E and 245 m.a.s.l.). The climate of this locality can be described as fresh (mean annual temperature 12.0–15.5 ◦ C) and humid (total annual precipitation 400–700 mm), and the corresponding phytoclimate is Fresh-Continental Oriental-semihumid [15]. 2.2. Aerobiological Methodology A Hirst-type volumetric spore trap (VPPS 2000 spore trap, Lanzoni s.r.l.) was used for the aerobiological fungal spore study. This collector aspires at a flow rate of 10 L/min, comparable with the respiration of an average human adult, and was designed specifically for the intake of spores, pollen, and other particles suspended in the air [16]. It is a continuous volumetric sampler with wind orientation of the intake orifice, and, thanks to an aspiration system, the collected particles are deposited on a plastic band coated with silicone oil tightly deposited around a drum that is displaced at a rate of 2 mm/hour using a clockwise mechanism. Once a week, the plastic band in the drum is replaced, and the segments corresponding to each day (0–24 h UTC) are mounted on a slide that is analyzed at the light microscope. The spore monitoring was performed continuously between 1 January 2021 and 31 December 2022 under the Spanish Aerobiological Network (Red Española de Aerobiología, REA) [17] and following the minimum requirements of the European Aerobiology Society, EAS [18]. The terminology used in this paper follows the International Association for Aerobiology (IAA) and EAS recommendations [19]. The data used in this study were expressed as the daily average of spores per cubic meter of air (spores/m3 ) and Annual Spores Integral (ASIn, spore * day/m3 ), or the addition of the average daily spores’ concentration over the year. The fungal taxa considered were the Aspergillus/Penicillium spore type. In both cases, the asexual spores (conidia) extend out in long chains from the vegetative mycelium and readily detach to become airborne [20]. J. Fungi 2023, 9, 624 (spores/m3) and Annual Spores Integral (ASIn, spore * day/m3), or the addition of t J. Fungi 2023, 9, 624 3 of 11 erage daily spores’ concentration over the year. The fungal taxa considered were t pergillus/Penicillium spore type. In both cases, the asexual spores (conidia) extend long chains from the vegetative mycelium and readily detach to become airborne [20 The size of thissize spore of type is around this spore type 2.5 µm in diameter, is around 2.5 µm in with a spherical diameter, with a morphology spherical morpholog that may have faint may cinereous have faint pigmentation or be hyaline. cinereous pigmentation or beOn their surface hyaline. On theirappear surfaceslight appear slight ornamentation mentation similar to minute dots similar to at thedots minute perimeter. Under a light at the perimeter. Undermicroscope, it is a light microscope, it frequently observed that the spores are in short chains or small groups adhered together quently observed that the spores are in short chains or small groups adhered to (Figure 1). Due to the difficulties (Figure 1). Due tointhethe differentiation difficulties in the of this morphology differentiation and of this because theand becau morphology conidia may be hidden along with other airborne particles, it is very plausible conidia may be hidden along with other airborne particles, it is very that theyplausible are tha underestimated.are underestimated. Figure 1. Aspergillus/Penicillium spore type. (A) Spores observed on aerobiological samples; (B Figure 1. Aspergillus/Penicillium spore type. (A) Spores observed on aerobiological samples; parative image between the size of Aspergillus/Penicillium spores and Cupressaceae pollen; (B) comparative image between the size of Aspergillus/Penicillium spores and Cupressaceae pollen; nidiophore with conidiospores of A. fumigatus. (C) conidiophore with conidiospores of A. fumigatus. Added to the information generated during this study, the daily concentrati Added to the information generated during this study, the daily concentrations of airborne Aspergillus/Penicillium spore type in the Bellaterra sampling site for the ten airborne Aspergillus/Penicillium spore type in the Bellaterra sampling site for the ten years (2011–2020) were used to analyze the representativity of the results obtained for (2011–2020) were used to analyze the representativity of the results obtained for years 2021 and 2022. These data were extracted from the database of the Catalan Aerobiol 2021 and 2022. These data were extracted from the database of the Catalan Aerobiological Network (Xarxa Aerobiològica de Catalunya, XAC) and are summarized in Table 1 Network (Xarxa Aerobiològica de Catalunya, XAC) and are summarized in Table 1. Table 1. Main aerobiological parameters of Aspergillus/Penicillium spore type for the period Table 1. Main aerobiological parameters of Aspergillus/Penicillium spore type for the period 2011– 2020 (data from the Xarxa Aerobiològica de Catalunya-XAC) database and summary of th 2020 (data from the Xarxa Aerobiològica meteorological de ASIn: parameters. Catalunya-XAC) Annual Sporedatabase Indice;and summary ATSIn: AnnualofTotal the main Spore Indice; meteorological parameters. ASIn: Annual Spore Indice; ATSIn: perature; P: annual rainfall; RH: related humidity. Annual Total Spore Indice; T: temperature; P: annual rainfall; RH: related humidity. Mean 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 Mean 20 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 ASIn (spore * day/m3) 16,106 9730 8462 11,421 9817 8660 6964 9724 2011–2020 6759 8812 96 3 ASIn (spore * day/m )Peak (spores/m 16,106 3) 9730 624 8462 591 11,421 918 9817 395 8660 361 6964 252 9724 207 6759 392 8812 274 9645 708 4 Peak (spores/m3 ) Peak624day 591 21-nov 918 28-sep 395 31-oct 361 26-nov 252 09-apr207 13-mar 392 04-oct 274 17-jun708 29-may472 05-apr Peak day 21-nov 28-sep 31-oct 26-nov 09-apr 13-mar 04-oct 17-jun 29-may 05-apr – Nr of days with spores 222 190 178 246 259 249 237 250 227 209 227 J. Fungi 2023, 9, 624 4 of 11 Table 1. Cont. Mean 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2011–2020 Nr of analyzed days 364 364 357 362 359 360 361 364 361 340 359 ATSIN (spore * day/m3 ) 1,873,455 2,065,498 1,986,474 2,567,732 1,427,132 1,898,641 1,401,089 2,867,906 1,259,152 1,644,947 1,899,202 % vs. Total Spores 0.86 0.47 0.43 0.44 0.69 0.46 0.50 0.34 0.54 0.54 0.52 T Max (◦ C) 21.1 21.0 20.6 21.2 21.8 21.5 21.7 21.2 21.7 21.4 21.3 T Min (◦ C) 9.6 8.9 8.8 9.7 9.7 9.7 9.4 9.9 9.3 9.6 9.5 T Mean (◦ C) 15.3 15.0 147 15.5 15.7 15.6 15.6 15.6 15.5 15.5 15.4 P 0–24 h (mm) 809.3 445.6 563.7 639.0 326.6 441.6 468.8 919.4 553.0 847.2 601.4 RH (%) 74.2 69.6 71.8 74.4 68.9 69.7 68.8 73.3 68.6 73.8 71.3 *: daily spores per cubic meter. 2.3. Immunochemical Methodology Besides the Hirst sampler, the Asp f 1 sampling was performed during 2021 and 2022, using a high-volume sampler (MCV CAV-A/mb, ©MCV) designed in principle for the sampling of atmospheric particulate matter. It is a volumetric sampler, working at a flow intake rate of 400 L/min. and impelling the air against a fiberglass filter (UNE- EN12341:2015; UNE-EN 14907:2006a, b) that will retain the particles. Based on MCV S.L. Company, this collector complies with the specifications for high-volume collectors in the UNE-EN 12,341 and UNE-EN 14,907 standards. The total diameter of the filter is 15 cm, but the diameter of the impacted area is 12 cm. The daily samples were carried out from 0 to 24 h, and the sampling was carried out on alternate days to permit manual replacement. The samples were stored at 2 ◦ C until their analysis. Before the analysis, a representative part of the daily filters, consisting of 8 circles of 0.5 cm in diameter obtained in selected areas, was equally distributed in two Eppendorf vials with 500 µL PBS solution and hydrated for 6 h [21]. Once hydrated, the samples obtained were analyzed according to commercial ELISA kits to detect Asp f 1 (Inbio, Indoor Biotechnologies, Inc, Cardiff, United Kingdom) according to the protocol provided on the supplier’s website [22]. The allergen standards used were provided with the kits. The microplates used were NUNC™ mi- croplates. Absorbance measurements were made at a 492 nm filter in a plate reader model Halo MpR-96 from Dynamica® . The results were expressed as daily allergen concentrations in nanograms of allergen per cubic meter of air (ng/m3 ) and as Annual Allergen Inte- gral (AAIn) obtained by summing the average daily allergen concentration over the year (ng * day/m3 ). 2.4. Meteorological Data The meteorological variables considered in this study were maximum and minimum daily temperatures (expressed in degrees Celsius), daily rainfall (expressed in millimeters), and relative humidity (expressed in percentages). Data were obtained from the Servei Mete- orològic de Catalunya and correspond to the Sabadell meteorological station (41◦ 330 5600 N, 02◦ 040 1000 E, 258 m.a.s.l.). 2.5. Statistical Analysis Spearman’s correlation coefficients were calculated between the daily concentrations of Aspergillus/Penicillium spore type and Asp f 1 allergen and daily meteorological variables. The analyses were carried out by using the IBM SPSS Statistic version 25. J. Fungi 2023, 9, 624 5 of 11 3. Results 3.1. Aerobiological and Meteorological Results Based on the data of the 10-year period previous to the years of this study, As- pergillus/Penicillium spore type accounted for 0.52% of all monitored fungal spores in the year (varying from 0.86% to 0.34%, Table 1). During the years 2021 and 2022, As- pergillus/Penicillium spore type showed a higher proportion with regard to the ASIn of all the spore types registered, accounting for 0.79 and 0.75%, respectively (Table 2). Table 2. Main aerobiological parameters of Aspergillus/Penicillium spore type and Asp f 1 allergen and main meteorological conditions in Bellaterra, Barcelona, Spain, during the period studied. T: temperature; P: annual rainfall; and RH: relative humidity. 2021 2022 Aspergillus/Penicillium spores. ASIn (spore * day/m3 ) 10,912 9957 Peak (spore/m3 ) Hirst sampler 319 386 Peak day 23-may 28-may Nr of days with Aspergillus/Penicillium spore type 237 235 Nr of analyzed days 364 356 Total spores. ASIn (spore * day/m3 ) 1,380,260 1,329,525 % vs. Total spores 0.79 0.75 Asp f 1. AAIn (ng * day/m3 ) 506,7 505,0 Meteorological High-volume Peak (ng/m3 ) sampler 9.0 14.4 Peak day 18-feb 11-feb Nr of days with Asp f 1 179 163 Nr of analyzed days 179 163 T Max (◦ C) 21.1 22.7 T Min (◦ C) 9.4 10.3 data T Mean (◦ C) 15.3 16.5 P 0–24h (mm) 417.9 393.7 RH (%) 72.6 70.6 *: daily spores per cubic meter. The dynamics along the year of the mean daily airborne Aspergillus/Penicillium spore type for the period 2011–2020 and the daily concentrations during 2021 and 2022 were studied (Figure 2). As can be observed, considering the mean values of the 10-year period results in this fungal spore type being present in the atmosphere for all 365 days of the year, while this is not the case in any of the years analyzed (Tables 1 and 2, Figure 2). Moreover, the mean sporulation tended to be more important in autumn. An important aspect to consider is that when daily averages for the ten-year period are plotted, the concentrations become smoother, and important peak values, as the one for 31st October 2013 (918 s/m3 ), are decreased (in this case to approximately 150 spores/m3 ). In relation to the years analyzed in this study (2021–2022), this spore type followed similar dynamics than in the previous 10-year period, although in the study period, the peak of the years was detected in May (23rd and 28th, respectively), and they varied from March to November (occurring each month with the only exception of July and August) between 2011 and 2020 (Figure 2). Therefore, we assumed that the results for the period studied can be considered comparable and be extrapolated to other years. (918 s/m3), are decreased (in this case to approximately 150 spores/m3). In relation to the years analyzed in this study (2021–2022), this spore type followed similar dynamics than in the previous 10-year period, although in the study period, the peak of the years was detected in May (23rd and 28th, respectively), and they varied from March to November J. Fungi 2023, 9, 624 (occurring each month with the only exception of July and August) between 2011 and6 2020 of 11 (Figure 2). Therefore, we assumed that the results for the period studied can be considered comparable and be extrapolated to other years. Figure 2. Airborne Aspergillus/Penicillium spore type daily dynamics along the year. Mean data of Figure 2. Airborne Aspergillus/Penicillium spore type daily dynamics along the year. Mean data of the the period 2011–2020 (blue area and left y-axis) vs. 2021 (black line and right y-axis) and 2022 (yellow period 2011–2020 (blue area and left y-axis) vs. 2021 (black line and right y-axis) and 2022 (yellow line and right y-axis). line and right y-axis). The aerobiological parameters studied for Aspergillus/Penicillium spore type, Asp f The aerobiological parameters studied for Aspergillus/Penicillium spore type, Asp f 1, and for for 1, and meteorological meteorological datadata showed showed thatthat the aerobiological the aerobiological behavior behavior of spore of this this spore type type was was similar similar in thein the years years 2021 and 20212022and(Table 2022 (Table 2). Incases, 2). In both both ASIn cases,did ASInnotdid not12,000 reach reachspores 12,000 spores *3 day/m 3, the peak was similar (319 and 386 spore/m3), the peak dates were ob- 3 * day/m , the peak was similar (319 and 386 spore/m ), the peak dates were observed in servedwith spring in spring withoffive five days days of difference, difference, and the numberand the of number of days days of the of theofpresence presence this sporeof this spore type in the air was 237 days (over 364) in 2021 type in the air was 237 days (over 364) in 2021 and 235 (over 356) in 2022. and 235 (over 356) in 2022. Asfor As forAsp Aspf f1,1,ititwas wasalsoalsodetected detected atat similar similar levels levels in in both both years, years, with with AAIn AAIn values values of of 507 and 505 ng * day/m 3 3, respectively. The peak day in both cases 507 and 505 ng * day/m , respectively. The peak day in both cases occurred in February occurred in February (23rdand (23rd and28th), 28th),andandthe thepresence presenceofofAspAspf f11waswascontinuous continuousfor forall allthe thedays daysstudied. studied. In relation to the meteorological parameters, data from the In relation to the meteorological parameters, data from the study period showed study period showed slightly higher temperatures in the year 2022, and much lower precipitation slightly higher temperatures in the year 2022, and much lower precipitation in both years in both years thanin than inthe theperiod period2011–2020. 2011–2020.However, However,between between2021 2021and and2022, 2022,the thevalues valuesreached reachedwerewere similarwithout similar withoutsignificant significantdifferences. differences. 3.2. Airborne Fungal Aeroallergens vs. Spores and vs. Meteorology Throughout the whole period studied, the annual patterns of the daily concentrations of the airborne Aspergillus/Penicillium spore type and Asp f 1 in 2021 and 2022 were present in the air an important number of days per year (over 65% of analyzed days), showing its maximum sporulation in spring and autumn (Figure 3). In relation to the dynamics of the aeroallergen Asp f 1, it was characterized as being present on all the days analyzed (alternate days) in 2021 and 2022, although with abundant oscillations in the concentration values. In both years, when airborne spores registered the highest concentrations, the values of the aeroallergen were lower. Moreover, in both years, the maximum airborne allergen concentrations were registered in winter (mainly in January and February, with the annual absolute peaks occurring in February). The results of the Spearman correlation test applied to the daily values of Aspergillus/Penicillium spore type, Asp f 1 allergen, and the meteorological parame- ters showed no influence between them (Table 3). No significant correlations were found in the period studied. Only in 2021 did the spore type show a positive and significant correla- tion (p < 0.01) with precipitation and relative humidity. In the case of Asp f 1 allergen, both years showed a negative and significant correlation (p < 0.05; p < 0.01) with temperature. J. Fungi 2023, 9, 624 7 of 11 J. Fungi 2023, 9, 624 7 of 11 Figure 3. Daily dynamics of airborne Aspergillus/Penicillium spore type and Asp f 1 allergen during Figure Daily dynamics of airborne Aspergillus/Penicillium spore type and Asp f 1 allergen during 3. 2022. 2021 and 2021 and 2022. Table 3. Correlation coefficients for Aspergillus/Penicillium spore type, Asp f 1, and meteorological Table 3. Correlation coefficients for Aspergillus/Penicillium spore type, Asp f 1, and meteorological variables. variables. 2021 2022 Asp./Pen. Spore 2021 Type Asp f 1 Asp./Pen. Spore Type 2022 Asp f 1 Spearman T. Asp./Pen. Spore N Spearman T. N Spearman T. Asp./Pen. Spore N Spearman T. N Asp./Pen. spore type 1 367 Asp f1 −0.098 181 1 359 Asp f 1 −0.008 163 Type Type Asp f 1 −0.098 Spearman T. N 181 Spearman T.1 N 182 −0.008 Spearman T. N 163 1 Spearman T. 166 N TMax (°C) −0.043 367 −0.186 * 182 0.057 359 −0.348 ** 166 Asp./Pen. TMin (°C) 1−0.021 367367 −0.098−0.207 ** 181182 1 0.043 359359 −0.008 −0.408 ** 163 166 spore type TMean (°C) −0.033 367 −0.189 * 182 0.050 359 −0.409 ** 166 PAsp 0–24f◦(mm) 1 −0.098 0.234 ** 181367 10.040 182182 −0.008 0.039 163359 1 0.061 166 166 TMax ( C) −0.043 367 −0.186 * 182 0.057 359 −0.348 ** 166 RH ◦(%) 0.182 ** 367 0.104 182 0.101 359 −0.067 166 TMin ( C) −0.021 367 −0.207 ** 182 0.043 359 −0.408 ** 166 TMean ( C) ◦ −0.033 367 −0.189 * 182 Spearman 0.050 T. 359 N −0.409 ** 166 PAsp./Pen. 0–24 (mm)spore type (2021) 0.234 ** vs. Asp./Pen. 367 spore 0.040 type (2022) 182 0.145 ** 359358 0.039 0.061 166 RH (%) Asp f0.182 1 (2021) ** vs. Asp 367 f 1 (2022) 0.104 182 −0.022 0.101 359 31 −0.067 166 ** p < 0.01; * p < 0.05. The blue and red shadows and bold format Spearman T. indicate N more relevant results. the Asp./Pen. =spore (Asp./Pen. type (2021) vs. Asp./Pen. spore type (2022) Aspergillus/Penicillium). 0.145 ** 358 Asp f 1 (2021) vs. Asp f 1 (2022) −0.022 31 ** p < 0.01; * p < 0.05. The blue and red shadows and bold format indicate the more relevant results. (Asp./Pen. = As- pergillus/Penicillium). 4. Discussion The study of the daily dynamics of the Aspergillus/Penicillium spore type along the ten years previous to the analysis of Asp f 1 (Table 1, Figure 2) evidenced that it does not follow a clear seasonal pattern, that the spores are observed in the aerobiological plates a critical number of days per year (between 50% and 72% of the days analyzed per year) but not continuously, and that they are distributed over several months. However, the J. Fungi 2023, 9, 624 8 of 11 maximum airborne spore concentration period in autumn (October and November) can be intuitively established. This yearly phenomenon has been reported by other studies, such as Oliveira et al. [23] in Portugal and Rosas et al. [24] in Mexico. However, the dynamics of this spore type around the world are variable. Several authors have shown that the main period occurs during spring, as in Central Spain, Salamanca [25]. In contrast, others reported a two-season occurrence (spring and autumn), as in Poland [11] or Ireland [26]. In the USA [27] and Finland [28], the main period was observed during summer. Finally, some studies demonstrated that the Aspergillus/Penicillium spore type is independent of seasonal changes, such as in Denmark [29]. The correlation study between Aspergillus/Penicillium spore type and meteorological variables showed positive and significant correlations with precipitation and relative humidity. So, their sporulation could be favored by the rainfall and increasing humidity, as has occurred in other studies [11,23]. Although the period of high fungal spore concentrations does not show a unique pattern, it is indisputable that the existence of this spore type in the air has implications for patients with problems in the respiratory system [20]. Even so, there is no general agreement on analyzing the presence of airborne fungal particles in the atmosphere. Some authors analyzed the air using viable sampling methods that resulted in the calculation of colony-forming units (CFU), which means brief periods of sampling sporadically through the year [30–32]. Other authors use aerobiological methods based on palynology (with the identification of the particles through an optical microscope) [23,25]. In this case, the air sampling is continuous, but there is no consensus on the surface of the sample analyzed. These aerobiological studies have a substantial limitation in counting and identifying the numerous and diverse fungal spore types present in the atmosphere, especially in the case of Aspergillus/Penicillium spore type, due to their small size and hyaline color. Furthermore, this methodology cannot identify smaller fungal fragments or hyphae, considered a signifi- cant source of allergens [33], so the actual allergenic risk due to Aspergillus would still not be observed. In recent decades, aerobiological studies have evolved, incorporating molecular an- alytical methods, such as detecting and quantifying the allergen load in the atmosphere through immunological techniques. This new methodology allows the comparison of their results with those from traditional sampling, achieving a better knowledge of the natural aerobiological dynamics of pollen, spores, and allergens. Therefore, the information obtained with both methodologies can help assess pollen/spore exposure and improve research on the epidemiology and clinical trials. The present study offers an analysis of the airborne Aspergillus/Penicillium spore type and the allergenic load of Asp f 1 in Bellaterra (Barcelona) to determine if the traditional aerobiological method reflects the real risk period of Asp f 1 in the air. This study recorded airborne dynamics of Aspergillus/Penicillium spore type in 2021 and 2022. As shown in our results and other research [34,35], the levels of this spore type can be variable throughout the year, with high values in some seasons and low values in others. During the period studied, high daily concentrations of spores were registered on certain days of the year exceeding 200 spores/m3 . While in 2021, this occurred on some days of April, May, and June, in 2022, this was registered in May, June, and October. This fact, and the lack of correlation with meteorological variables, could be explained by the sporadic behavior of these spores [11]. However, the positive and significant correlation between the two years studied (0.145; p < 0.01) shows how this spore type presents similar annual airborne dynamics, although some days randomly register high concentrations. In this study, the authors used a high-volume sampler with a suction capacity of 400 L/min. The election of this sampler was based on the hypothesis that this high-volume collector could collect biological particles, such as allergens, at the same time as inorganic particles. As shown in De Linares et al. [21], sample collection analysis and quantification of fungal allergens are possible with this sampler and using the ELISA technique. Despite several studies on airborne allergens, they are mainly focused on allergens from pollen, and there need to be more studies on fungal spores. The research of aeroal- J. Fungi 2023, 9, 624 9 of 11 lergens from fungal spores was mainly based on the detection and quantification of Alt a 1 [21,36,37], because it is the most critical fungal aeroallergen that provokes respiratory tract symptoms. Studies related to outdoor Aspergillus spore and their major allergen (Asp f 1) are scarce. In this case, we found one article using a similar methodology: a volumetric sampler with a flow rate of 40 L/min [38] to quantify the airborne Asp f 1. As in this study, we found that the seasonal distribution of airborne spores did not correlate with the aeroallergen load. In our case, during the two-year study (2021–2022), the higher air- borne spores’ concentrations were registered in spring and autumn, with the peak days on 23 May and 28 May, respectively. The fact that the maximum spore concentrations were registered in the more humid season agrees with other studies [11,34,35,38,39]. However, the maximum concentration of the Asp f 1 allergen was found during winter, peaking on 18 February and 11 February, as occurred in Vermani et al. [33], which detected the highest outdoor Asp f 1 concentration of A. fumigatus and A. niger in February too. Statistical results showed no significant correlation between meteorology and As- pergillus/Penicillium spore type. Only in 2021 were positive correlations registered with precipitation and relative humidity. The same lack of correlation with temperature was obtained by Grinn-Gofron [11], Sadys et al. [40], and Akgül et al. [41]. In the case of Asp f 1 allergen, non-correlation was detected in any year, so it could indicate that the weather conditions do not influence these small particles and depend on other mechanisms that must be studied. Consequently, based on the discrepancies in the moments of these two particles being present in the air and the similitudes in the date of maximum concentrations in both years and with other studies, we postulate that (a) The coincidence of the values of ASIn, AAIn, and the dates of the peaks of spores and allergens, as well as the dynamic found during the two years studied, confirm that the detection of the Aspergillus/Penicillium spore type and Asp f 1 allergen is possible with both aerobiological techniques, and follow a defined behavior along the time; (b) If we accept the postulate of Sporik et al. [30] that Asp f 1 is not present in the spore but in hyphae fragments, and as previous studies have shown, we accept too that the level of the fungal hyphae can be relatively high in the air [42]; (c) Aspergillus/Penicillium spore type is conditioned to precipitation and relative humidity, which promote the production and release of the spores of these fungi. On the other hand, the aeroallergens registered in winter could be derived from hyphal fragments, which, under the light microscope, are extremely difficult to distinguish but can be detected through the allergens with immunological techniques. 5. Conclusions Asp f 1 daily concentration measurements have been obtained, for the first time in Catalonia, at the Bellaterra (Barcelona) aerobiological station, for the years 2021 and 2022, showing that the MCV CAV-A/mb high-volume sampler has proven to be valuable and sensitive enough to the detection and quantification of the allergen Asp f 1 using ELISA Kits. Despite being unable to explain specific temporal differences observed between con- centrations of spores and allergens, we recommend using these two methodologies (quan- tification of fungal spores under a light microscope and immunochemical quantification to detect allergens) to detect, control, prevent, and diagnose this fungal taxon. Further studies must focus on establishing the origin of the aeroallergen registered in periods without airborne fungal spores and determine which moment the exposure is more hazardous when there are high concentrations of spores or the allergen. Author Contributions: C.D.L. and J.B. conceived the project and participated equally in its develop- ment and in the interpretation of the data and writing of the paper. C.D.L. and D.N. performed the allergen analyses. D.N. and R.P. performed the spore counting. All authors have read and agreed to the published version of the manuscript. J. Fungi 2023, 9, 624 10 of 11 Funding: This research was carried out within the framework of the research projects funded by the Ministry of Science and Technology I + D + I of Spain CGL2012-39523-C02-01 and CTM2017-89565- C2-1-P. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy. Acknowledgments: The authors wish to thank the entities and projects that made it possible to obtain the database used in this study: Laboratorios LETI PHARMA; Servei Meteorològic de Catalunya; SCAIC; SEAIC; J. Uriach y Cia; Spanish Ministry of Science and Technology I+D+I for “CGL2012- 39523-C02-01,” “CTM2017-89565-C2-1-P”; the Catalan Government AGAUR for “2017SGR1692” and “2021SGR00519”; and the Andalusian Government for RNM 0110 PAIDI. This research contributes to the “María de Maeztu” Programme for Units of Excellence of the Spanish Ministry of Science and Innovation (CEX2019-000940-M). Conflicts of Interest: The authors declare that they have no conflicts of interest. The funders had no role in the design of the study. 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