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Assuring a proper environment for the fulfillment of professional activities is one of the Sustainable Development Goals and is contemplated in the One Health approach assumed by the World Health Organization. This particular study is applied to an often neglected sector of our society—the conservators/restorers—despite the many health issues reported by these professionals. Three different specialties (textiles, paintings and wood sculpture) and locations were selected for evaluation by placement of electrostatic dust cloths. After treatment of the samples, bacterial and fungal contamination were assessed, as well as mycotoxin determination, the presence of azole-resistant strains and cytotoxicity of the microorganisms encountered. Bacteria were only present in one of medias used and showed relatively low numbers. The highest level of contamination by fungi was identified in one of the textiles settings. The textile area also showed the highest variability for fungi. Aspergillus sp. are one indicator of possible environmental issues, and A. sections Fumigati and Circumdati were particularly relevant in two of the settings and identified in all of them. No mycotoxins were detected and the large majority of the fungi identified were non-cytotoxic. Overall, these can be considered low-contaminated environments but attention should be given to the Aspergillus sp. contamination. Additional studies are needed not only to make these results more robust, but also to test if the environmental sampling alone is the best approach in a setting where there is very little movement and dust displacement and where professionals are in very close proximity to the artefacts being treated, which may suggest the existence of a micro-atmosphere worth evaluating and comparing to the obtained results. [ABSTRACT FROM AUTHOR]

Cemeteries are potential environmental reservoirs of pathogenic microorganisms from organic matter decomposition. This study aimed to characterize the microbial contamination in three cemeteries, and more specifically in grave diggers' facilities. One active sampling method (impingement method) and several passive sampling methods (swabs, settled dust, settled dust filters and electrostatic dust cloths—EDC) were employed. The molecular detection of Aspergillus sections and SARS-CoV-2, as well as mycotoxin analysis, screening of azole resistance, and cytotoxicity measurement were also conducted. Total bacteria contamination was 80 CFU·m−2 in settled dust samples, reached 849 CFU·m−2 in EDC and 20,000 CFU·m−2 in swabs, and ranged from 5000 to 10,000 CFU·m−2 in filters. Gram-negative bacteria (VRBA) were only observed in in settled dust samples (2.00 × 105 CFU·m−2). Regarding Aspergillus sp., the highest counts were obtained in DG18 (18.38%) and it was not observed in azole-supplemented SDA media. SARS-CoV-2 and the targeted Aspergillus sections were not detected. Mycophenolic acid was detected in one settled dust sample. Cytotoxic effects were observed for 94.4% filters and 5.6% EDC in A549 lung epithelial cells, and for 50.0% filters and 5.6% EDC in HepG2 cells. Future studies are needed in this occupational setting to implement more focused risk management measures. [ABSTRACT FROM AUTHOR]

The lack of knowledge regarding the extent of microbial contamination in Portuguese fitness centers (FC) puts attendees and athletes at risk for bioaerosol exposure. This study intends to characterize microbial contamination in Portuguese FC by passive sampling methods: electrostatic dust collectors (EDC) (N = 39), settled dust (N = 8), vacuum filters (N = 8), and used cleaning mops (N = 12). The obtained extracts were plated in selective culture media for fungi and bacteria. Filters, EDC, and mop samples' extracts were also screened for antifungal resistance and used for the molecular detection of the selected Aspergillus sections. The detection of mycotoxins was conducted using a high-performance liquid chromatograph (HPLC) system and to determine the cytotoxicity of microbial contaminants recovered by passive sampling, HepG2 (human liver carcinoma) and A549 (human alveolar epithelial) cells were employed. The results reinforce the use of passive sampling methods to identify the most critical areas and identify environmental factors that influence microbial contamination, namely having a swimming pool. The cardio fitness area presented the highest median value of total bacteria (TSA: 9.69 × 102 CFU m−2.day−1) and Gram-negative bacteria (VRBA: 1.23 CFU m−2.day−1), while for fungi it was the open space area, with 1.86 × 101 CFU m−2.day−1. Aspergillus sp. was present in EDC and in filters used to collect settled dust. Reduced azole susceptibility was observed in filters and EDC (on ICZ and VCZ), and in mops (on ICZ). Fumonisin B2 was the only mycotoxin detected and it was present in all sampling matrixes except settled dust. High and moderate cytotoxicity was obtained, suggesting that A549 cells were more sensitive to samples' contaminants. The observed widespread of critical toxigenic fungal species with clinical relevance, such as Aspergillus section Fumigati , as well as Fumonisin B2 emphasizes the importance of frequent and effective cleaning procedures while using shared mops appeared as a vehicle of cross-contamination. [Display omitted] • Passive sampling can be useful in identifying the most contaminated areas. • Having a swimming pool in the facilities increases microbial contamination. • Widespread of Aspergillus section Fumigati and species with toxigenic potential was observed. • Mops may boost cross-contamination including mycotoxins dissemination. • Frequent and effective cleaning procedures should be implemented. [ABSTRACT FROM AUTHOR]

The collection and analysis of settled dust samples from indoor environments has become one of several environmental sampling methods used to assess bioburden indoors. The aim of the study was to characterize the bioburden in vacuumed settled dust from 10 Primary Health Care Centers by culture based and molecular methods. Results for bacterial load ranged from 1 to 12 CFU.g−1 of dust and Gram-negative bacteria ranged between 1 to 344 CFU.g−1 of dust. Fungal load ranged from 0 CFU.g−1 of dust to uncountable. Aspergillus section Fumigati was detected in 4 sampling sites where culture base-methods could not identify this section. Mucorales (Rhizopus sp.) was observed on 1 mg/L voriconazole. Three out of 10 settled dust samples were contaminated by mycotoxins. Settled dust sampling coupled with air sampling in a routine way might provide useful information about bioburden exposure. [ABSTRACT FROM AUTHOR]

The genusAspergillusis one of the most prevalent regarding fungi in several highly contaminated occupational environments. The goal of the current study was to assess the prevalence ofAspergillusspp. in different settings, focusing on those where a higher load of fungal contamination is expected according to the European Agency for Safety and Health at Work. A specific protocol to ensure a more accurate assessment of the exposure toAspergillusspp. is proposed aimed at allowing a detailed risk characterization and management. Two wastewater treatment plants, one wastewater elevation plant, four waste treatment plants, three cork industries, five slaughter houses, four feed industries, one poultry pavilion, and two swineries, all located in the outskirts of Lisbon, were assessed. In total, 125 air samples and 125 surface samples were collected and analysed by culture-based methods. Real-time polymerase chain reaction was performed to detect fungal presence in 100 samples, targeting theAspergillussectionsCircumdati, Flavi, andFumigati. The highest prevalence ofAspergillusspp. was found in wastewater treatment plants (69.3%; 31.1%), waste treatment plants (34.8%; 73.6%), and poultry feed industry (6.3%; 26.1%), in air and surfaces, respectively.Aspergillusspp. was also prevalent in cork industry (0.9%; 23.4%), slaughter houses (1.6%; 17.7%), and swineries (7.4%; 9.5%), in air and surfaces, respectively. TheAspergillussections more prevalent in the air and surfaces of all the assessed settings were theNigrisection (47.46%; 44.71%, respectively), followed byFumigati(22.28%; 27.97%, respectively) andFlavi(10.78%; 11.45%, respectively) sections.AspergillussectionFumigatiwas successfully amplified by qPCR in 18 sampling sites where the presence of this fungal species had not been identified by conventional methods. It should be highlighted that the occupational exposure burden is due not only to theAspergillusload, but also to the toxigenic potential of this genus. Based on our results, a protocol relied in the application of conventional and molecular methods in parallel is herein suggested aimed at allowing a better risk characterization and management. [ABSTRACT FROM AUTHOR]

Studies on the microbiology of coffee cherries and beans have shown that the predominant toxigenic fungal genera (AspergillusandPenicillium) are natural coffee contaminants. The aim of this study was to investigate the distribution of fungi inCoffea arabica L. (Arabica coffee) andCoffea canephora L. var. robusta (Robusta coffee) green coffee samples obtained from different sources at the pre-roasting stage. Twenty-eight green coffee samples from different countries of origin (Brazil, Timor, Honduras, Angola, Vietnam, Costa Rica, Colombia, Guatemala, Nicaragua, India, and Uganda) were evaluated. The fungal load in the contaminated samples ranged from 0 to 12330 colony forming units (CFU)/g, of which approximately 67% presented contamination levels below 1500 CFU/g, while 11% exhibited intermediate contamination levels between 1500 and 3000 CFU/g. Contamination levels higher than 3000 CFU/g were found in 22% of contaminated coffee samples. Fifteen different fungi were isolated by culture-based methods andAspergillusspecies belonging to different sections (complexes). The predominantAspergillussection detected wasNigri(39%), followed byAspergillussectionCircumdati(29%). Molecular analysis detected the presence ofAspergillussectionsFumigatiandCircumdati. The% coffee samples whereAspergillusspecies were identified by culture-based methods were 96%. Data demonstrated that green coffee beans samples were contaminated with toxigenic fungal species. Since mycotoxins may be resistant to the roasting process, this suggests possible exposure to mycotoxins through consumption of coffee. Further studies need to be conducted to provide information on critical points of coffee processing, such that fungal contamination may be reduced or eliminated and thus exposure to fungi and mycotoxins through coffee handling and consumption be prevented. [ABSTRACT FROM PUBLISHER]

One of the occupational environments that need intervention to overcome the lack of information concerning exposure to biological agents is the Firefighter Headquarters' (FFH). This study intends to assess the microbial contamination in Portuguese FFH with a multi-approach protocol for sampling and analyses using active (air samples by impaction and impingement) and passive (surface swabs collected as swabs, settled dust, cleaning cloths and mops, electrostatic dust cloths and identification badges) sampling methods. The fungal contamination was also characterized through molecular detection of toxigenic species, antifungal resistance and mycotoxins profile. Cytotoxicity assessment was included in specific cells line using the MTT assay in order to recognize the possible health effects. It was raised concern regarding the following findings: a) microbial contamination didn't comply with Portuguese IAQ legal requirements in most of the FFH (bacteria load 63.63%, fungal load 45.45%); b) fungal exposure through inhalation underlining a possible risk factor for respiratory diseases; c) prevalence of Aspergillus sp. in cleaning materials that might represent a potential source of cross-contamination; d) widespread of Aspergillus section Fumigati in all the FFH analysed; e) trend of multidrug resistance found among fungi with focus on Fumigati isolates; f) detection of several mycotoxins (fumonisin B2, nivalenol, mycophenolic acid and sterigmatocystin) and; g) potential role of Aspergillus genus in the cytotoxicity found, particularly in lung cells. The multi-approach on sampling methods (active and passive) and laboratory assays (culture based-methods, molecular tools, mycotoxins detection, cytotoxicity evaluation) improved data findings, enabling a more detailed and accurate risk characterization. ⁃ Microbial contamination didn't comply with Portuguese IAQ legal requirements ⁃ Fungal exposure through inhalation underlining a possible risk factor for respiratory diseases ⁃ Widespread of Aspergillus section Fumigati in all the FFH analysed ⁃ Multidrug resistance found among fungi with focus on Aspergillus section Fumigati isolates ⁃ Detection of several mycotoxins in the FFH analysed [ABSTRACT FROM AUTHOR]

The higher education sector represents a unique environment and it acts as a work environment, a learning environment for students, and frequently, also a home environment. The aim of this study was to determine the microbial contamination (SARS-CoV-2, fungi, and bacteria) in Higher Education Facilities (HEI) by using active and passive sampling methods and combining culture-based methods with molecular tools targeting Aspergillus section Fumigati. In addition, the resistance to azole profile was also assessed. Surface samples showed a range of total bacterial contamination between 1 × 103 to 3.1 × 106 CFU·m−2, while Gram-negative bacteria ranged from 0 to 1.9 × 104 CFU·m−2. Fungal contamination ranged from 2 × 103 to 1.8 × 105 CFU·m−2 on MEA, and from 5 × 103 to 1.7 × 105 CFU·m−2 on DG18. The most prevalent species found on both media was Cladosporium sp. (47.36% MEA; 32.33% DG18). Aspergillus genera was observed on MEA (3.21%) and DG18 (14.66%), but not in the supplemented media used for the azole screening. Aspergillus section Fumigati was detected in 2 air samples (2.22%, 2 out of 90 samples) by qPCR. When testing for SARS-CoV-2 all results were negative. The present study showed that although cleaning and disinfection procedures are done regularly due to the COVID-19 pandemic, being effective in eliminating SARS-CoV-2, surfaces were often contaminated with microorganisms other than SARS-CoV-2. This can be a result of increasing resistance to biocides, and to the wide range of environmental factors that can contribute to the dissemination of microbial contamination indoors. [ABSTRACT FROM AUTHOR]

Sampling campaigns indoors have shown that occupants exposed to contaminated air generally exhibit diverse health outcomes. This study intends to assess the deposition rates of total settleable dust and bioburden in the indoor air of dwellings onto quartz fiber filters and electrostatic dust collectors (EDCs), respectively. EDC extracts were inoculated onto malt extract agar (MEA) and dichloran glycerol (DG18) agar-based media used for fungal contamination characterization, while tryptic soy agar (TSA) was applied for total bacteria assessment, and violet red bile agar (VRBA) for Gram-negative bacteria. Azole-resistance screening and molecular detection by qPCR was also performed. Dust loading rates ranged from 0.111 to 3.52, averaging 0.675 μg cm−2 day−1. Bacterial counts ranged from undetectable to 16.3 colony-forming units (CFU) m−2 day−1 and to 2.95 CFU m−2 day−1 in TSA and VRBA, respectively. Fungal contamination ranged from 1.97 to 35.4 CFU m−2 day−1 in MEA, and from undetectable to 48.8 CFU m−2 day−1 in DG18. Penicillium sp. presented the highest prevalence in MEA media (36.2%) and Cladosporium sp. in DG18 (39.2%). It was possible to observe: (a) settleable dust loadings and fungal contamination higher in dwellings with pets; (b) fungal species considered indicators of harmful fungal contamination; (c) Aspergillus section Candidi identified in supplemented media with voriconazole and posaconazole; (d) specific housing typologies and (e) specific housing characteristics influencing the microbial contamination. [ABSTRACT FROM AUTHOR]

The assessment and control of microbial contamination in health care facilities is presently a mandatory and vital part of strategies to prevent and control hospital-acquired infections. This study aims to assess the bioburden with two passive sampling methods (30 ventilations grids swabs and 16 electrostatic dust collectors (EDCs)) at Clinical Pathology Services. The fungal burden was characterized through molecular tools, antifungal resistance, and the mycotoxins and cytotoxicity profile. Total bacteria presented the highest prevalence in both matrixes, whereas Gram-bacteria presented the lowest. Swabs presented a higher prevalence (27.6%) for fungal burden. Chrysonilia sitophila presented the highest prevalence in swabs, whereas for EDCs, C. sitophila and Mucor sp. were the most prevalent. Concerning Aspergillus genera on swabs, section Flavi was the one with the highest prevalence (58.02%), whereas, for EDCs, section Versicolores was the only section observed (100%). Aspergillus section Fumigati was detected in 10 swabs and 7 EDC samples and Aspergillus section Versicolores was detected in one EDC sample. Fungal growth on azole-supplemented media was observed in eight EDC samples. No mycotoxins were detected in any of the samples. A low cytotoxic effect was observed in two sites upon incubation of collected samples with A549 and SK cells and in two other sites upon incubation of collected samples with SK cells only. A medium cytotoxic effect was observed with one EDC sample upon incubation with A549 cells. This study reinforces the need of determination of the azole resistance profile for fungal species and allowed a preliminary risk characterization regarding the cytotoxicity. An intervention including the use of a ultraviolet with wavelength between 200 nm and 280 nm (UVC)—emitting device and an increased maintenance and cleaning of the central heating, ventilation, and air conditioning (HVAC) systems should be ensured to promote the reduction of microbial contamination. [ABSTRACT FROM AUTHOR]

Previous studies anticipated that microorganisms and their metabolites in waste will increase as a consequence of a decreased collection frequency and due to differences in what kind of waste is bagged before collection leading to an increased exposure of workers handling the waste. This study aim was to investigate the microbial contamination present in the waste collection trucks (WCT) and in the support facilities (waste collection station - WCS). It was applied a multi-approach protocol using active (air sampling by impingement and impaction) and passive (surface swabs, electrostatic dust cloths and settled dust) sampling methods. The screening of azole-resistance, the investigation of mycotoxins and the assessment of the elicited biological responses in vitro were also carried out aiming recognizing the possible health effects of waste collection drivers. SARS-CoV-2 detection was also performed. In WCS only air samples had contamination in all the four sampling sites (canteen, operational removal core, operational removal center, and administrative service). Among all the analyzed matrices from the WCT a higher percentage of total bacterial counts and Gram-was detected in swabs (66.93%; 99.36%). In WCS the most common species were Penicillium sp. (43.98%) and Cladosporium sp. (24.68%), while on WCT Aspergillus sp. (4.18%) was also one of the most found. In the azole resistance screening Aspergillus genera was not observed in the azole-supplemented media. SARS-CoV-2 was not detected in any of the environmental samples collected, but Aspergillus section Fumigati was detected in 5 samples. Mycotoxins were not detected in EDC from WCS, while in WCT they were detected in filters (N = 1) and in settled dust samples (N = 16). In conclusion, our study reveals that a comprehensive sampling approach using active and passive sampling (e.g. settled dust sampling for a representative mycotoxin evaluation) and combined analytic methods (i.e., culture-based and molecular) is an important asset in microbial exposure assessments. Concerning the waste collection exposure scenario, the results of this study unveiled a complex exposure, particularly to fungi and their metabolites. Aspergillus section Fumigati highlight the significance of targeting this section in the waste management industry as an indicator of occupational health risk. • Broad sampling and analyses approach is needed for microbial exposure assessments. • Aspergillus section Fumigati was suggested as an indicator of occupational health risk. • Waste collection might be a hotspot for the development of azole resistance. • Mycotoxins were detected in the waste collection trucks. [ABSTRACT FROM AUTHOR]

• Exposure to microbial contaminants should be considered. • In vitro toxicology provides relevant data on the potential health effects. • Interior layers exhibited higher cytotoxicity effect than exhalation valves. • Higher cytotoxicity among exhalation valves correlated with bacteria. • Training and education on the proper use of FRPD is needed. Filtering respiratory protection devices (FRPD) are mandatory for workers to wear in the Portuguese waste-sorting industry. Previous results regarding microbial contamination found on FRPD interior layer raised the question of whether microbial contamination from the exhalation valve would also have cytotoxicity effects. Since the FRPD exhalation valves are very close to workers' nose and mouth, they represent a source of exposure to bioburden by inhalation. This study aimed to evaluate the cytotoxicity of the microbial contamination present in the FRPD exhalation valves. For this purpose, the cytotoxicity effects were determined through the MTT assay in two different cell lines (human A549 epithelial lung cells, and swine kidney cells) and compared with previous results obtained with FRPD interior layers. The contamination present in the FRPD exhalation valves presented some cytotoxicity on epithelial lung cells, suggesting the inhalation route as a potential route of exposure through the use of FRPD in the waste-sorting industry. Half-maximal (50%) inhibitory concentration (IC50) values were lower for FRPD interior layer than exhalation valves in lung cells, with overall cytotoxicity lower in exhalation valves when compared to interior layer (z = −4.455, p = 0.000). Higher bacterial counts in TSA were correlated with lower IC50 values, thus, higher cytotoxicity effect in lung cells. No statistically significant differences were detected among different workplaces. [ABSTRACT FROM AUTHOR]

The use of Filtering Respiratory Protective Devices (FRPD) is mandatory in Portugal to protect workers from the waste industry of harmful exposures. Deleterious health effects of exposure to bioburden via inhalation and/or ingestion include respiratory symptoms and nephrotoxicity. Between January and February 2019, 118 FRPD samples were collected in one waste sorting industry and characterized regarding microbial contamination and cytotoxicity, defined as cell metabolic activity, through the MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide). Cytotoxic effect was classified according to percentage of extinction values with respect to the control group, as follows: absent (≥90); low (80%–90%, +); medium (60%–79%, ++); and high (below 60%, +++). For 113 samples the MTT assay revealed a cytotoxic effect in A549 cells, of which 81 presented high cytotoxicity. In SK cells, a cytotoxic effect was observed in 56 samples, of which five displayed a high cytotoxic effect. Several moderate (p < 0.05) to strong (p < 0.01) correlations were found between higher bacterial and fungal counts both in interior layers (fungi and bacteria) and in exhalation valves (fungi) of FRPD samples and reduced cell metabolic activity of SK cells. On the basis of the obtained results for the cytotoxic effect of FRPD samples on two different cells lines, it was determined that A549 cells exhibited a cytotoxic effect for a higher number of FRPD, whereas the SK cells model correlated better with the other assessed parameters, namely, bacterial and fungal counts and conditions of FRPD use. Although the results are not conclusive on the most appropriate cell line to assess FRPD cytotoxicity, they reinforce the importance of in vitro toxicology in exposure assessments to determine the cytotoxicity of mixtures of contaminants, for better risk characterization and selection of appropriate risk management measures. • Swine kidney (SK) cells reveal higher sensibility to contaminants present at FRPD. • A moderate cytotoxic effect was observed in A549 and in SK cells. • SK cells revealed moderate to strong correlations between higher microbial counts. • In vitro tools allowed to recognize cytotoxicity linked with the mixture of contaminants. [ABSTRACT FROM AUTHOR]

In Portugal, mechanical protection gloves (MPG) are of mandatory use and during their use sweat is released and, consequently, the humidity of the material increases leading to conditions favorable to the growth of microorganisms. However, no studies have been conducted in MPG to assess the bioburden. This study intended to determine the bioburden present in MPG and their biological effects, and to discuss the possibility to use MPG as a passive method to assess occupational exposure to microbial contamination. Fungal burden was characterized through molecular tools for fungal toxigenic species, and antifungal resistance and mycotoxins profiles were determined. Cell viability was determined in swine kidney (SK) monolayer and hepatocellular carcinoma (Hep G2) cell lines. All MPG samples presented Gram-negative bacteria. The fungal contamination ranged from 0 CFU.m−2 in both MEA and DG18, to 5.09 × 106 and 2.75 × 106 and the most commonly fungi found was Aspergillus spp. (50.46%). Azole resistant Aspergillus sections were found in azole supplemented media. Aspergillus sections (Circumdati , Flavi , Fumigati and Versicolores) were detected by molecular tools in 66 out of 67 samples. The most reported mycotoxin was mycophenolic acid (89.6%). HepG2 cells appear to be more sensitive to MPG contamination, with high cytotoxicity (IC50 < 0.05 mm2/ml) observed for 18 out of 57 gloves. MPG can be used in passive sampling to assess occupational exposure to bioburden in waste sorting industries and contribute for risk characterization. Some contaminants of MPG had cytotoxic potential and affected the biology of hepatic cells more than renal cells. • Gram-negative bacteria were found in all the Mechanic Protection Gloves (MPG). • Aspergillus sections with toxigenic and azole resistance potential were observed. • Mycotoxins exposure through ingestion route by hand-mouth contact is possible. • Cytotoxic potential affecting hepatic cells more than renal cells was observed. • MPG can be an adequate passive sampling method to estimate workers exposure. [ABSTRACT FROM AUTHOR]

The bioburden in a Hospital building originates not only from patients, visitors and staff, but is also disseminated by several indoor hospital characteristics and outdoor environmental sources. This study intends to assess the exposure to bioburden in one central Hospital with a multi-approach protocol using active and passive sampling methods. The microbial contamination was also characterized through molecular tools for toxigenic species, antifungal resistance and mycotoxins and endotoxins profile. Two cytotoxicity assays (MTT and resazurin) were conducted with two cell lines (Calu-3 and THP-1), and in vitro pro-inflammatory potential was assessed in THP-1 cell line. Out of the 15 sampling locations 33.3% did not comply with Portuguese legislation regarding bacterial contamination, whereas concerning fungal contamination 60% presented I/O > 1. Toxigenic fungal species were observed in 27% of the sampled rooms (4 out of 15) and qPCR analysis successfully amplified DNA from the Aspergillus sections Flavi and Fumigati, although mycotoxins were not detected. Growth of distinct fungal species was observed on Sabouraud dextrose agar with triazole drugs, such as Aspergillus section Versicolores on 1 mg/L VORI. The highest concentrations of endotoxins were found in settled dust samples and ranged from 5.72 to 23.0 EU.mg−1. While a considerable cytotoxic effect (cell viability < 30%) was observed in one HVAC filter sample with Calu-3 cell line, it was not observed with THP-1 cell line. In air samples a medium cytotoxic effect (61–68% cell viability) was observed in 3 out of 15 samples. The cytokine responses produced a more potent average cell response (46.8 ± 12.3 ρg/mL IL-1β; 90.8 ± 58.5 ρg/mL TNF-α) on passive samples than air samples (25.5 ± 5.2 ρg/mL IL-1β and of 19.4 ± 5.2 ρg/mL TNF-α). A multi-approach regarding parameters to assess, sampling and analysis methods should be followed to characterize the biorburden in the Hospital indoor environment. This study supports the importance of considering exposure to complex mixtures in indoor environments. • The multi-parameter approach on sampling methods and assays allowed to assess the biorburden. • Toxigenic fungal species were identified by culture based-methods and detected by qPCR. • Aspergillus section Versicolores was identified in the resistance to azoles screening. • Low levels of endotoxins were observed in the environmental matrices. • Low to moderate cytotoxic and pro-inflammatory effects were found. [ABSTRACT FROM AUTHOR]