With all COVID-19 restrictions being lifted in Norway, and no obligation for mask use, not even in healthcare facilities, the present study aimed to assess the presence of SARS-CoV-2 in air samples from various indoor and outdoor spaces, including both healthcare and non-healthcare related facilities. We found that, out of 31 air samples, only four showed the presence of SARS-CoV-2 RNA by RT-qPCR, with no amplification after RNAse pre-treatment, strongly suggesting that no viable virus was present in these four samples.

The four positive samples came from a kindergarten (n = 2), a nursing home (n = 1) and from the pediatrics waiting room in a health clinic (n = 1). It should be noted that on the day of the sampling in the kindergarten, most of the children were showing respiratory disease symptoms but none was using facemasks. Moreover, at the nursing home one of the nurses’ staff had tested positive for COVID-19 the previous day.

It is important to mention that the number of people present in each sampled location varied throughout sampling duration in every location, as sampled locations were either outdoor public spaces with a constant and heavy influx of people such as public parks or the train station, or indoor spaces with constant but not so heavy movement of people such as the health clinic, where patients would come and go constantly. Nonetheless, in the locations where SARS-CoV-2 RNA could be recovered from the air samples, the density of people present was low (that is, less than 15 people present at the locations during sampling).

When looking at these results, although they indicate that non-infectious viruses were present in air samples, we cannot exclude the possibility that the collection process might have contributed to the inactivation of SARS-CoV-2 [24]. Multiple factors can potentially contribute to the decline in virus viability, including the collection process and prolonged airborne state (which increases the likelihood of surface damage due to impaction, exposure to harmful airborne contaminants, desiccation, or degradation) [25]. In the case of the Coriolis Compact, which is a cyclone sampler, a film of liquid is injected close to the cyclone’s inlet to wet the cyclone walls, which are subsequently collected at the cyclone’s base for analysis. This is thought to increase viability of collected viral or other microorganism particles that are being sampled, however shear forces may still decrease particles ‘viability [26,27,28].

As the probability of detecting infectious SARS-CoV-2 is directly correlated with the amount of viral RNA detected by RT-qPCR, and infectious viruses are more likely to be detected when viral RNA is present in concentrations greater than 1 × 106 gene copies/mL [29], viable viruses were not expected to be present in our study based on the low numbers of gene copies/m3 found. This is in keeping with previous studies on SARS-CoV-2 that identified viable virus in air samples [30,31,32], with 1000 gene copies/m3 [31]. Of note, in another study it was estimated that an RNA concentration of 2.5 × 105 RNA copies/mL had less than a 5% success rate for isolating infectious virus [33]. Within this < 5% success rate of isolating infectious virus, another study reported a limit of 4.3 × 106 RNA copies/mL [34]. All these values are in accordance with other studies which have shown that samples with Ct values > 24 are unlikely to be virus positive after cultured [35]. However, it is important to emphasize that Ct measurements depend on the RT-qPCR assay and platform used [36].

Information about viability of SARS-CoV-2 viruses in air samples is very important when discussing risk assessment, as risk assessment studies are used by policymakers and health agencies to develop mitigation and preventative strategies to control viral transmission in the community, as well as its applications in occupational health [37]. In this context, the nuclease pre-treatment method offers an alternative to estimate the presence of infectious viruses in air samples when viral culture is not possible, allowing for a better interpretation of studies on SARS-CoV-2 presence in ar. Moreover, this method has the potential to enhance analyses aimed at supporting risk-based investigations, whether for preventing new outbreaks or managing recurrent ones, and can be applied across a wide range of scenarios [38].

SARS-CoV-2 typically causes mild illness and few deaths in children and adolescents when compared to adults [39]. However, these groups still remain susceptible to SARS-CoV-2 infection and may transmit the virus to other people, such as the elderly parcel of the population, which increases the burden of the disease on public health systems (World Health Organization (WHO) [40]). Interestingly, our results point to a link between SARS-CoV-2 presence in air in environments with more children under the age of three present, which, until this day, still makes up the age-group worldwide with more unvaccinated people against COVID-19 [41, 42].

Nonetheless, the sequenced sampled presented the nucleocapsid mutations R203K (28881G >A, 28882G>A) and G204R (28883G>C) that have been reported to increase the infectivity, fitness, and virulence of SARS-CoV-2 [43], being associated with increased infectivity of SARS-CoV-2 strains in the United States (USA), as well as being predominant in both the USA and Europe [44]. The mutation G204R is non-conservative, and the R203K mutation has been pointed to function as a non-conservative substitution due to the different size of the arginine (R) versus lysine (K) residues and the considerably different chemical features of the side-chain guanidinium group (arginine) versus the primary amine (lysine). It has been hypothesized that these mutations may influence disease severity by altering linker region flexibility and dynamics, which would in turn alter nucleocapsid function [45].

One of the significant unanswered questions about the COVID-19 epidemiology is related to the role of children in the transmission of SARS-CoV-2 [46], which is a group that comprises a significant share of the population in many countries (Charumilind et al. [47]). With milder symptoms, children are tested less often and cases may go unreported (World Health Organization (WHO) [40]), allowing the virus to reach more susceptible groups such as the elderly and immune-compromised. Recent reports have also suggested that the Omicron variant and its sub-lineages may lead to more frequent hospitalization in children, as children make up a larger part of patients hospitalized with COVID-19 than in previous infection waves caused by other variants of concern [41]. Moreover, when considering that SARS-CoV-2 RNA could be detected in a nursing home facility, it puts into question the prevention guidelines in place at the time, when it comes to the safety of the elderly population, as previous epidemiological data from Norway show that the majority of COVID-19 related deaths are among this risk group [48].

However, evidence to date still suggests that children are not among the main drivers of the pandemic [49]. That added to the fact that asymptomatic children have significantly lower viral loads compared to children with symptomatic infections [50] have resulted in less effort trying to understand the role of children in the airborne transmission of SARS-CoV-2 [51].

In another study, it was highlighted that although estimates of children’s susceptibility and infectivity are lower than those of adults within a household, it is important to remember that their role in the spread of SARS-CoV-2 is also affected by different contact patterns and hygiene habits outside the household (Dattner et al. [46]). More intense contact and mixing among children compared to adults in schools, e.g., could offset the effect of reduced susceptibility. In a review it is brought to attention the fact that roughly half of the United States population goes to school, works in a school, or is a first-degree contact of individuals that frequent these environments, suggesting that in-school transmission can impact the disease burden in surrounding communities [52]. This raises the alarm for the higher probability of virus spreading in school settings, as they have one of the main elements for a superspreading event, which is prolonged indoor exposure to other people. Whether these schools have appropriate ventilation or not is another factor that should be taken into consideration when evaluating exposure risk in school settings. Another important factor concerning children and increased risk of SARS-CoV-2 infection is that households with children in low income, urban communities have an extremely high household secondary attack rate, with children playing important roles as index cases [51].

This study sheds light on the presence of SARS-CoV-2 in indoor and outdoor environments, particularly focusing on healthcare and non-healthcare settings. With lifted COVID-19 restrictions and a significant percentage of the population vaccinated, our findings provide valuable insights into the dynamics of SARS-CoV-2 transmission at a time where all restrictions have been lifted and vaccination coverage is high.

Our air sampling in Norway took place in two cities, namely Oslo and Ås, in the end of April and beginning of May 2022. During this period, there had been a steady decline in the number of new patients admitted to Norwegian hospitals with COVID-19, with the number of new patients admitted per 100.000 people being highest in the age groups 75–84 and ≥ 85 years [19]. Of note, during this period, 58% of people who deceased due to COVID-19 complications died in a health institution other than a hospital, primarily in nursing homes. The Omicron variant BA.2 was the dominant sub-lineage in the country, accounting for 95–100% of all whole-genome sequenced samples during that period [19].

Considering that our sampling campaign took place at a moment of low-transmission in Norway, and that sampling covered various indoor and outdoor environments (public parks, public transport stations, university restaurants, a nursing home, a kindergarten, and a health clinic), the fact that SARS-CoV-2 RNA could be detected only in environments related to children and the elderly raises some issues when it comes to mitigation guidelines and prevention strategies.

First of all, as vaccination coverage is increasing around the world and we have started to normalize life towards the pre-COVID-19 time, COVID-19 cases are escalating and SARS-CoV-2 surveillance is reduced, painting out a coming challenging winter in Europe [53]. That being said, it is urgent that countries in the EU region relaunch mitigation efforts and are ready to respond to an increased burden on their health-care systems.

The application of what the WHO calls “Five Pandemic Stabilizers” (increased vaccination rates, a second booster dose to immunocompromised people and their close contacts, mask wearing indoors and in public transports, improving ventilation in crowded and public spaces, and applying rigorous therapeutic protocols for those at risk of severe disease) will be of the utmost importance in order to control virus transmission during autumn and winter [53].

When considering that children still represent the majority of the unvaccinated people not only in the EU region but in a global scale, and the fact that this group is often asymptomatic and less frequently tested, special attention should be given to the importance of re-implementing mask wearing indoors and in public transports, as this is still one of the most efficient interventions against SARS-CoV-2 airborne transmission [54, 55]. This would reduce transmission of the virus to children, which in turn would help prevent the virus spread from this group to at-risk groups such as the elderly and immunocompromised. Furthermore, considering the surge of Omicron and its sub-lineages that are more easily transmissible, reinstating mask mandates might be the best strategy to control community transmission of COVID-19 [55].