Beyond quantitative indicators: Comprehensive characterization of indoor fungal contamination in Portuguese...

Children are particularly vulnerable to environmental pollutants, making indoor air quality in schools a key health determinant. This study addresses regulatory gaps and the lack of standardized methods for assessing fungal contamination in Portuguese elementary schools, particularly regarding pathogenicity, toxigenicity, and antifungal resistance. Fungal contamination was assessed in 11 Portuguese elementary schools using surveys on building context and operation, active and passive air sampling, molecular detection of Aspergillus sections by qPCR, mycotoxin and azole resistance screening, and PM measurements. Results:Schools showed heterogeneous ventilation, with some relying on natural ventilation and others using mechanical systems. Airborne fungal contamination was dominated by respirable fractions, reaching 3.3 × 10² CFU m^-3. Culture-based analyses revealed distinct fungal profiles, with Cladosporium sp. and Penicillium sp. frequently prevailing, while Aspergillus detection increased at higher incubation temperatures. Seasonal variability was observed, with contamination patterns differing between seasons. Rural and urban schools showed contrasting fungal distributions, with higher concentrations in rural schools, particularly in EDCs on DG18 at 27 °C (1.67 × 10³ CFU/m²). Azole resistance screening detected resistant Aspergillus sections across environments. In gymnasiums, the Fumigati section represented 100% of sections detected on ITZ in filters and EDCs. In classrooms, Circumdati and Fumigati accounted for 100% of sections detected on ITZ in EDCs, while the Candidi section represented 100% in filters. The Fumigati section also grew on VOZ and POZ in EDCs. Mycotoxins, including ochratoxin A, mycophenolic acid and sterigmatocystin, were detected in 1.93% of dust samples. Particle concentrations occasionally exceeded reference thresholds in warm (47.1% PM2.5; 58.8% PM10) and cold seasons (47.4% PM2.5; 23.1% PM10). Regulatory thresholds did not prevent the detection of relevant fungal hazards, including Aspergillus species and azole-tolerant growth. These findings show that quantitative indicators alone may not capture exposure complexity, highlighting the need for complementary data, and the limitations in the current legal framework.