As the third wave of the COVID-19 pandemic hit Portugal, it forced the country to reintroduce lockdown measures due to hospitals reaching their full capacities. Under these circumstances, environmental contamination by SARS-CoV-2 in different areas of one of Portugal’s major Hospitals was assessed between 21 January and 11 February 2021. Air samples (n = 44) were collected from eleven different areas of the Hospital (four COVID-19 and seven non-COVID-19 areas) using Coriolis® μ and Coriolis® Compact cyclone air sampling devices. Surface sampling was also performed (n = 17) on four areas (one COVID-19 and three non-COVID-19 areas). RNA extraction followed by a one-step RT-qPCR adapted for quantitative purposes were performed. Of the 44 air samples, two were positive for SARS-CoV-2 RNA (6575 copies/m3 and 6662.5 copies/m3, respectively). Of the 17 surface samples, three were positive for SARS-CoV-2 RNA (200.6 copies/cm2, 179.2 copies/cm2, and 201.7 copies/cm2, respectively). SARS-CoV-2 environmental contamination was found both in air and on surfaces in both COVID-19 and non-COVID-19 areas. Moreover, our results suggest that longer collection sessions are needed to detect point contaminations. This reinforces the need to remain cautious at all times, not only when in close contact with infected individuals. Hand hygiene and other standard transmission-prevention guidelines should be continuously followed to avoid nosocomial COVID-19.

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