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Therefore, it is necessary to carry out periodical environmental microbiological assessments that evaluate the fungal bioburden in air and surfaces from different hospital zones. Current microbiological monitoring protocols at healthcare settings are mostly based on cultivation, while environmental DNA (eDNA) assessments are still scarce and should be further evaluated. To fill this gap, this study combines a large sampling scheme, comprising\u2009&gt;\u2009200 samples (air, surface, dust and soil) collected from four zones at three Spanish hospitals in two campaigns (winter and autumn), with two eDNA approaches (DNA metabarcoding and quantitative PCR) to characterize the hospital mycobiomes (diversity, community composition and airborne load), compared to a parallel culture-dependent study. DNA metabarcoding revealed a much more comprehensive inventory of hospital fungi compared to culturing; however, both approaches found similar dominant taxa including a variety of potentially opportunistic human pathogens. Hospital mycobiomes were affiliated to 4 phyla (mostly\n                    <jats:italic>Ascomycota<\/jats:italic>\n                    and\n                    <jats:italic>Basidiomycota<\/jats:italic>\n                    ), 35 classes, 114 orders, 305 families, 643 genera and 535 species. The dominant genera, in both air and surfaces from the three hospitals, were\n                    <jats:italic>Cladosporium<\/jats:italic>\n                    ,\n                    <jats:italic>Alternaria<\/jats:italic>\n                    ,\n                    <jats:italic>Aureobasidium<\/jats:italic>\n                    ,\n                    <jats:italic>Penicillium<\/jats:italic>\n                    ,\n                    <jats:italic>Neodidymelliopsis<\/jats:italic>\n                    ,\n                    <jats:italic>Aspergillus<\/jats:italic>\n                    ,\n                    <jats:italic>Pseudopithomyces<\/jats:italic>\n                    and\n                    <jats:italic>Stemphylium<\/jats:italic>\n                    . The yeasts\n                    <jats:italic>Candida<\/jats:italic>\n                    and\n                    <jats:italic>Clavispora<\/jats:italic>\n                    were particularly abundant on high-touch surfaces indoors. The most important explanatory factors for the variance in community composition were the hospital and zone where samples were collected, the type of sample and the sampling campaign. DNA metabarcoding can assist hospital managers by providing an in-depth characterization of the baseline hospital mycobiome during normal operating conditions, as well as identifying and controlling community imbalances and associated health risks under demanding situations such as construction works or reported clinical outbreaks.\n                  <\/jats:p>","DOI":"10.1007\/s00248-025-02626-w","type":"journal-article","created":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T19:24:50Z","timestamp":1764012290000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Contribution of DNA Metabarcoding to the Environmental Fungal Assessments in Hospitals"],"prefix":"10.1007","volume":"89","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-0621-1490","authenticated-orcid":false,"given":"Laura","family":"Garc\u00eda-Guti\u00e9rrez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9801-0260","authenticated-orcid":false,"given":"Emilia","family":"Mellado","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2674-8650","authenticated-orcid":false,"given":"Pedro M.","family":"Martin-Sanchez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,11,24]]},"reference":[{"key":"2626_CR1","doi-asserted-by":"publisher","first-page":"e428","DOI":"10.1016\/S1473-3099(23)00692-8","volume":"24","author":"DW Denning","year":"2024","unstructured":"Denning DW (2024) Global incidence and mortality of severe fungal disease. 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