BACKGROUND AND OBJECTIVE:Indoor microbial exposures influence respiratory health, yet how men and women respond differently to airborne bacterial communities remains unclear. This study aimed to assess sex-specific associations between indoor airborne bacteria, and lung function, and airway inflammation in adults. METHODS:Airborne dust was collected from the bedrooms of 1038 adults (463 men, 575 women) across five Nordic cities as part of the European Community Respiratory Health Survey (ECRHS) III. Bacterial communities were profiled using 16S rRNA amplicon sequencing. Bacterial and endotoxin loads were quantified via quantitative PCR (qPCR) and the Limulus amebocyte lysate (LAL) assay, respectively. Multivariable linear regression models stratified by sex were used to examine associations with lung function and airway inflammation. RESULTS:Greater indoor bacterial diversity was associated with higher lung function in men (FEV1 β = 0.17, 95% CI: 0.05 to 0.29, P = 0.003) and higher FeNO levels in women (β = 2.44, 95% CI: 0.73 to 4.15, P = 0.005). Endotoxin load was positively associated with FeNO in women (β = 0.37, 95% CI: 0.05 to 0.70, P = 0.02), but not in men. Genera from Actinobacteriota and Bacilli were associated with higher lung function, while Clostridia was linked to lower lung function and reduced FeNO. In contrast, several genera from Actinobacteriota and Gammaproteobacteria were positively associated with FeNO. CONCLUSIONS:Indoor bacterial exposures were associated with respiratory health in a sex-specific pattern. These findings highlight the importance of microbial composition in shaping adult lung health and underscore the need for sex-specific approaches in future epidemiological research.

Background Endotoxins are an important livestock-related respiratory health hazard. Health effects associated with ambient endotoxins are concentration-dependent, with both adverse and protective effects reported, yet little is known about their temporal variability, sources, or influencing factors. Objectives We aimed to capture short-term fluctuations in ambient endotoxin concentrations, analyze temporal trends, and identify determinants. Methods Daily PM10 samples were collected over two years using Automatic Sampling Devices at three sites in the Netherlands. The sites were located in a low-, medium-, and high-livestock density area. Endotoxin levels were analyzed using the Limulus Amebocyte Lysate assay. Associations with meteorological variables and pollen counts were examined using uni- and multivariable regression models. Results Site average endotoxin concentrations ranged from 0.18 to 0.39 EU/m3 depending on livestock density. High day-to-day variability occurred with moderate inter-site temporal correlations of daily average concentrations (r=0.6-0.7). Concentrations peaked in summer and autumn and were lowest in spring and winter. Positive associations with daily endotoxin concentrations were found for wind speed, precipitation, sunshine duration, and thunderstorms; negative associations were found for atmospheric pressure, relative humidity, and manure spreading periods. Models explained 20%-36% of the observed temporal variance (R2). Poaceae (grass) pollen was negatively associated with endotoxin levels, while Betula (birch) showed a positive association. Conclusions Ambient endotoxin concentrations peak during warmer months, likely driven by unstable weather conditions and local livestock sources. These findings highlight that endotoxin is an important component influencing air quality related to livestock farming, with potential implications for both monitoring strategies and health risk evaluations.

Ambient endotoxin, a key component of livestock-related bioaerosols, is associated with health effects, highlighting the need for improved exposure assessment in rural populations. Previous studies relied on either resource-intensive deterministic dispersion models or more practical stochastic methods. So far, land use regression (LUR) is the only stochastic approach evaluated for ambient endotoxin. We assess random forest (RF) models as an alternative for predicting residential endotoxin exposure, hypothesising that its ability to capture complex, non-linear relationships will improve performance over LUR. RF models were trained using data from the Dutch VGO research programme (Livestock Farming and Neighbouring Residents' Health Study), with repeated measurements at 61 residences (236 total) and spatial livestock-related predictors. Model performance was assessed with 10-fold cross-validation (R2 and RMSE), and variable importance was assessed with mean decrease in impurity (MDI) and Shapley Additive Explanations (SHAP). RF slightly outperformed LUR at the simplest predictor level (R2: 0.19 vs 0.10), performed comparably at the intermediate level, and was outperformed by LUR at the most detailed level (R2: 0.24 vs 0.32). Both approaches tended to underestimate concentrations at high-exposure sites, indicating limitations in capturing the upper tail of the exposure distribution. Pig, poultry, and cattle metrics were consistently key predictors; SHAP revealed non-linear links with endotoxin, highlighting RF's ability to capture complex patterns that may be overlooked by linear approaches. Overall, RF provides a viable alternative to LUR in data-constrained settings where detailed predictor data are unavailable, but does not demonstrate a general performance advantage when detailed predictor data are available.

Antibiotic resistance genes (ARGs) in airborne dust represent an emerging concern for public health, particularly in indoor environments where human exposure is prolonged. While external environmental pressures are known to shape the abundance and diversity of microorganisms in indoor dust, their role in ARG dynamics remains underexplored. This study assessed the temporal and spatial patterns of airborne ARGs in indoor dust across four Nordic cities (Aarhus, Bergen, Reykjavik, and Uppsala) using electrostatic dust collectors (EDCs) in the same households at two time points: 2012 and 2022. Shotgun metagenomic sequencing was performed to profile ARGs. National antibiotic consumption data were obtained from the European Surveillance of Antimicrobial Consumption (ESAC-Net), outdoor air pollution data (PM2.5 and PM10) from the Copernicus Atmosphere Monitoring Service (CAMS), and meteorological parameters from the NASA POWER database.Beta diversity analysis revealed city-specific differences in ARG composition (PERMANOVA, R2 = 0.18, P = 0.03), but no consistent temporal shift over the 10-year period. Macrolide, tetracycline, and aminoglycoside resistance genes were among the most abundant and persistent classes. A previously undetected macrolide resistance sequence was identified across all cities in 2022. Although national antibiotic consumption declined, cross-sectional correlations between national antibiotic consumption and ARG abundance strengthened from 2012 (Spearman's ρ = 0.25) to 2022 (ρ = 0.37), suggesting sustained ARG presence despite reduced antibiotic consumption. Several ARG classes showed associations with outdoor particulate matter, and these relationships were influenced by local meteorological conditions. For example, higher absolute humidity was associated with a weaker relationship between PM and polymyxin resistance genes, whereas stronger wind speeds were associated with stronger relationships between PM and Sulfonamide resistance genes.These findings highlight the complex environmental interplay between antibiotic consumption, air pollution, meteorological factors, and ARG dynamics in indoor air, emphasizing the need for integrated environmental and AMR surveillance, especially in the context of climate change.

In school environments, characterized by high occupancy and prolonged exposure, airborne contaminants pose risks to respiratory health and learning outcomes. Portable air cleaners (PACs) are increasingly considered as a supplement to ventilation, yet field-based evidence remains heterogeneous, with few randomized trials and limited data on microbial agents. Here, we describe the design and methodology of a large-scale cluster-randomized controlled trial evaluating PAC effectiveness in Dutch primary school classrooms. The study included 180 classrooms across 29 primary schools, with classrooms clustered within schools and randomized to HEPA-filter PACs, ionization/plasma PACs, or no PACs. The design incorporated a three-week baseline or two-week post-intervention control period, and three repeated three-week intervention periods in the main phase, totaling up to 14 weeks per school. PACs were pre-tested under standardized laboratory conditions, screened for safety, and operated at comparable clean air delivery rates (CADR). Airborne dust was collected using electrostatic dust fall collectors (EDCs) and analyzed for bacterial markers representing common human microbiome constituents, a general bacterial indicator, and viral markers for seasonal infections. In 12 classrooms, active air sampling was conducted alongside EDCs to validate and quantify passive measurements. Continuous monitoring of particulate matter (PM₁₀, PM₄, PM₂.₅, PM₁), CO₂, air temperature, relative humidity, and volatile organic compounds (VOCs) was performed. Classroom-level absenteeism and parent-reported respiratory symptoms were collected retrospectively. Weekly national infectious-disease surveillance and outdoor PM and NO₂ data will contextualize indoor measurements and health outcomes. Hierarchical mixed-effects models accounting for school, cluster, and classroom structure will analyze microbial outcomes. Bayesian hierarchical models may be applied for values outside the quantifiable range. By integrating comprehensive indoor air quality assessment with a dual-control design distinguishing pre-existing classroom differences from temporal trends, this registered trial (ClinicalTrials.gov, NCT07479420; 6 March 2026) provides a rigorous framework to evaluate PACs under real-world classroom conditions and support evidence-informed strategies to improve classroom indoor air quality.Clinical trial registration: https://clinicaltrials.gov/study/NCT07479420?term=air%20cleaner&viewType=Card&intr=air%20cleaner&rank=4, ClinicalTrials.gov, NCT07479420.

Antibiotic resistance genes (ARGs) are prevalent in livestock environments due to antimicrobial use, yet their airborne dispersal into human-occupied indoor spaces remains poorly characterized. We investigated whether airborne ARGs disperse from livestock stables into farmers' homes and surrounding outdoor environments. Electrostatic dust collectors were deployed in paired pig and cow stables and their associated homes in Jutland, Denmark, to collect settled airborne dust. Pooled samples were analyzed using shotgun metagenomic sequencing. ARG dispersal patterns were assessed using FEAST source tracking and ecological similarity metrics, including shared ARG ratios and Jaccard indices. Pig production systems exhibited higher antibiotic use and stronger resistome continuity with farmers' homes than cow systems, reflected by greater FEAST contributions (P = 0.029) and Jaccard similarity (P = 0.029). Beta-diversity analysis supported higher compositional similarity between pig stables and homes (PERMANOVA R2 = 0.23, p = 0.052), whereas cow environments showed greater divergence (R2 = 0.41, P = 0.035). Across environments, tetracycline, macrolide-lincosamide-streptogramin B, and aminoglycoside resistance genes dominated, consistent with livestock-specific antibiotic use patterns. Supplementary indoor-outdoor comparisons across cow, pig, and chicken stables (from an independent 2024 sampling campaign not directly comparable to the 2008 EDC-based survey) revealed contrasting dispersal dynamics, with higher bacterial species spillover from cow stables but stronger ARG overlap from pig stables. Collectively, these findings are consistent with airborne ARG connectivity across occupational and environmental interfaces and support consideration of air as a potential pathway in One Health AMR surveillance.

INTRODUCTION:Intensive livestock farming exposes workers to elevated levels of particulate matter (PM) and endotoxins, increasing respiratory health risks. Although personal exposure has occasionally been investigated in the 3 major livestock sectors-cattle, pigs, and poultry-emerging sectors such as intensive goat farming remain understudied. This study aimed to quantify personal exposure to inhalable dust, PM10, and endotoxins among workers on Dutch dairy goat farms and to explore associations between exposure levels, specific work tasks, and farm characteristics. METHODS:Repeated personal air sampling was conducted among 41 participants working at 15 goat farms. Inhalable dust and PM10 samples were collected using filter-based methods attached to portable air pumps. For all inhalable dust and PM10 samples, PM mass concentrations were determined by gravimetrical analyses and endotoxin concentrations with the Limulus-Amebocyte-Lysate assay. Determinants of exposure levels were analyzed by linear mixed modeling. RESULTS:Inhalable dust concentrations showed a median of 0.966 mg/m3 (range: 0.228 to 3.093), with a median endotoxin concentration of 612 EU/m3 (range: 48 to 7,818). For the PM10 concentrations, a median of 0.376 mg/m3 (range: 0.070 to 1.233) was observed, with a median endotoxin concentration of 700 EU/m3 (range: 8 to 2,886). In total, 90% of the samples exceeded recommended occupational exposure limits for endotoxin (>90 EU/m3). PM10 and inhalable dust concentrations were strongly correlated (Pearson r = 0.71), as were endotoxin concentrations in both fractions (Pearson r = 0.71). Exposure to PM and endotoxin varied significantly between farms, within farms and within workers. Overall, highest exposures were recorded for workers with milking as a primary job task. DISCUSSION AND CONCLUSIONS:Goat farm workers are exposed to substantial levels of PM and endotoxins during routine work activities. On average, concentrations exceeded those reported for dairy cattle farm workers, yet remained lower than levels typically observed in pig and poultry farming. The observed considerable variation in exposure both between farms and among individual workers necessitates future research and more detailed microbiological characterization of air samples on determinants of exposure, to guide appropriate measures in husbandry and practices at goat farms. This research highlights that in emerging intensive livestock farming sectors, increased exposure to PM and endotoxins-and related health effects-can be expected among workers unless reduction of these exposures is explicitly addressed in the development of agricultural policies and practices.

We are surrounded, in both indoor and outdoor environments, by air containing particles of biological origin (bioaerosols). We constantly inhale them, and, depending upon their size, they deposit in different parts of our airways. Despite their ubiquitous nature and our constant exposure, bioaerosol diversity and composition of the environment are not well characterized, and we understand little about which bioaerosols we are exposed to and how this impacts our health, either positively or negatively. Indoor/Outdoor Bioaerosols Interface and Relationships Network (BioAirNet), a Clean Air Programme-funded network, has recognized the need for the bioaerosol community to reflect on the current challenges facing bioaerosol exposure assessment and the determination of the associated cellular/molecular responses driving specific health outcomes. A series of online workshops for the bioaerosol community were hosted by BioAirNet in September 2022, which aimed to bring together global expertise to discuss the current challenges impeding improved assessment of bioaerosol exposure and understanding of the downstream cellular and molecular mechanisms driving health outcomes by discussing these challenges; considering where we need to be, where we are now and how we get there. Professional facilitation was key to their success, enabling the multidisciplinary bioaerosol community to explore and address these challenges within a focused and productive environment to prioritize themes and agree on action plans for continued momentum following the workshops. These themes were as follows: (1) conceptual model; (2) stakeholder mapping; (3) knowledge transfer; (4) writing project and (5) conference-type event, collectively covering research, knowledge mobilization and networking activities. A subsequent in-person follow-up workshop was held in November 2023. It provided an opportunity to share progress on the five themes, critique what had already been done and act as a launch-pad to progress the actions further. Delegates also had the opportunity to share ongoing or upcoming work, particularly projects requiring input from others, to encourage collaborative working and sharing expertise. The use of facilitated workshops is a valuable tool for all scientific communities to collectively explore and successfully address key issues within their field.

OBJECTIVE:To assess personal exposure to respirable and inhalable dust and its components endotoxin, black carbon and crystalline silica among sugarcane workers in Nicaragua. METHODS:Individual exposures to respirable (measurements=98) and inhalable (measurements=36) dust were collected in January and March 2020, with the month of March generally being hotter and less humid. Respirable dust and its components black carbon and crystalline silica, as well as inhalable dust and its component endotoxin, were personally measured. Linear mixed models were used to identify the determinants of occupational dust exposure considering different job tasks and meteorological conditions. RESULTS:Respirable dust and black carbon concentrations were higher in March among burned cane cutters compared with the other job groups (respirable dust geometric mean (GM)=1.9 mg m-3; black carbon GM=13.7 µg m-3), with considerably lower levels in January (respirable dust GM=0.2 mg m-3; black carbon GM=3.4 µg m-3). Almost all respirable crystalline silica measurements were below the limit of detection, except for four measurements, which ranged from 8 µg m-³ to 15 µg m-³. Seed cutters (GM=3.1 mg m-3) and weeders (GM=2.5 mg m-3) had the highest exposure to inhalable dust, while endotoxin concentrations were higher among seed cutters (GM=100 EU m-3) and burned cane cutters (GM=63 EU m-3) than the other work groups. CONCLUSIONS:Overall, exposure levels to the assessed agents varied across work groups, with higher levels observed among burned cane and seed cutters.

Assessment of occupational exposure to viruses is crucial to identify virus reservoirs and sources of dissemination at an early stage and to help prevent spread between employees and to the general population. Measuring workers' exposure can facilitate assessment of the effectiveness of protective and mitigation measures in place. The aim of this scoping review is to give an overview of available methods and those already implemented for airborne virus' exposure assessment in different occupational and indoor environments. The results retrieved from the different studies may contribute to the setting of future standards and guidelines to ensure a reliable risk characterization in the occupational environments crucial for the implementation of effective control measures. The search aimed at selecting studies between January 1st 2010 and June 30th 2023 in the selected databases. Fifty papers on virus exposure assessment fitted the eligibility criteria and were selected for data extraction. Overall, this study identified gaps in knowledge regarding virus assessment and pinpointed the needs for further research. Several discrepancies were found (transport temperatures, elution steps, …), as well as a lack of publication of important data related to the exposure conditions (contextual information). With the available information, it is impossible to compare results between studies employing different methods, and even if the same methods are used, different conclusions/recommendations based on the expert judgment have been reported due to the lack of consensus in the contextual information retrieved and/or data interpretation. Future research on the field targeting sampling methods and in the laboratory regarding the assays to employ should be developed bearing in mind the different goals of the assessment.

Endotoxins released from poultry feces have been associated with impaired human health. Because endotoxins are released from gram-negative intestinal bacteria, it was hypothesized that dietary strategies may influence endotoxin excretion via modulation of gut microbiota. We therefore tested dietary strategies that could potentially reduce cloacal endotoxin levels in broiler chickens. One-day-old male Ross 308 (N = 1,344) broilers were housed in 48 pens (N = 8 pens/treatment, 28 chickens per pen) and fed 1 of 6 diets for 35 days (d) in a 3-phase feeding program: a basic diet (CON) that served as the reference diet, or basic diet supplemented with butyrate (BUT), inulin (INU), medium-chain fatty acids (MCFA) or Original XPC (TM) LS (XPC), or a high-fiber-low-protein (HF-LP) diet. A significant (P < 0.05) increase in cloacal endotoxin concentration at d 35 was observed in BUT as compared to CON. Analysis of cloacal microbiota showed a trend (P < 0.07) for a higher gram-negative/gram-positive ratio and for a higher relative abundance of gram-negative bacteria at d 35 (P <= 0.08) in BUT and HF-LP as compared to CON. A significant (P < 0.05) increase in average daily gain (ADG) and improved feed conversion ratio (P < 0.05) were observed in MCFA during the grower phase (d 14-28), and a significant (P < 0.05) increase in average daily feed intake (ADFI) was observed in MCFA during d 0 to 28. Broilers fed HF-LP had a significantly (P < 0.05) higher FCR and lower ADG throughout the rearing period. No treatment effects were found on footpad dermatitis, but BUT had worst hock burn scores at d 35 (P < 0.01) and MCFA had worst cleanliness scores at d 21 but not at d 35 (treatment*age P < 0.05), while INU had better cleanliness as compared to CON at d 35 (P < 0.05). In conclusion, especially BUT and HF-LP were able to modulate resident microbiota and BUT also increased cloacal endotoxin levels, which was opposite to our hypothesis. The present study indicates that cloacal endotoxin release can be affected by the diet but further study is needed to find dietary treatments that can reduce cloacal endotoxin release.

Objectives To provide insights into exposure of Dutch dairy farmers to formaldehyde derived from formalin footbaths used for cows. Dutch safety norms are set at a limit of 0.122 ppm during an 8-hour time-weighted average (TWA) and 0.407 ppm for a 15-min TWA.Methods At 20 farms formaldehyde air concentrations were determined using stationary active air sampling with impingers next to the footbath and in the milking parlour during footbath usage. Formalin footbath concentrations were tested and meteorological conditions were collected using a climate monitor to assess associations with formaldehyde concentrations. A structured interview inquired on potential exposure routes and exposure duration.Results Formaldehyde concentrations next to the footbath ranged from <0.003 to 0.316 ppm, with seven measurements exceeding the 8-hour TWA threshold. None of the measurements exceeded the 15-min TWA threshold at either location. Formaldehyde air concentrations in the milking parlour were generally lower, yet at two farms exceeded the 8-hour TWA limit during sampling. Self-reported exposure time of the dairy farmers to the formalin footbath never exceeded 15 min. Although due to the small sample size, no significant associations between most predictor variables and formaldehyde levels in the air were found, the direction of effects were as expected.Conclusions The exposure of Dutch dairy farmers presumably falls within the established safety norms. Nonetheless, substantial levels of formaldehyde could be detected. This study further emphasises the importance of substitution of formalin in dairy practice and the relevance of informing dairy farmers on proper handling of formalin to reduce exposure.

Background Sex is a significant epidemiological factor in the incidence of respiratory disease. However, the role of sex in indoor bacterial exposure remains largely unexplored. Objective Our objective was to investigate the association between indoor bacterial exposure and lung function and airway inflammation, focusing on sex-specific differences. Methods Airborne dust samples from the bedrooms of 463 men and 575 women in the European Community Respiratory Health Survey (ECRHS) III from five Nordic cities were subjected to 16S rRNA amplicon sequencing to characterize bacterial communities. Limulus amebocyte lysate (LAL) assay and qPCR were used to measure endotoxin and bacterial load, respectively. Sex stratified adjusted linear regression analysis were used to examine the association between bacterial profile and lung function and FeNO measurements. Results Higher indoor bacterial diversity were significantly associated with higher lung function (FVC and FEV1 Z-scores) in men only, and with higher FeNO levels in women only. Indoor endotoxin levels were significantly associated with higher FeNO levels in women only. No clear associations were found between endotoxin levels and lung function. Most bacterial genera associated with higher lung function were affiliated with the Actinobacteriota phylum. Higher relative abundance of Bacteroidia, and Patescibacteria were associated with lower lung function, and the same was seen for several bacterial genera considered to be part of the core oral microbiome, including Streptococcus and Veillonella. More bacterial genera were associated with high FeNO levels in women than in men, among them Campylobacter, Fusebactrium, and several Bacteroidia genera. Conclusions The results of our study indicate that there might be distinct sex-specific differences in how individuals respond to indoor bacterial community exposure and its impact on lung health. This underlines the importance of talking sex into consideration in future evaluations of health implications associated with various indoor microbial communities.

Adverse health effects have been linked with exposure to livestock farms, likely due to airborne microbial agents. Accurate exposure assessment is crucial in epidemiological studies, however limited studies have modelled bioaerosols. This study used measured concentrations in air of livestock commensals (Escherichia coli (E. coli) and Staphylococcus species (spp.)), and antimicrobial resistance genes (tetW and mecA) at 61 residential sites in a livestock-dense region in the Netherlands. For each microbial agent, land use regression (LUR) and random forest (RF) models were developed using Geographic Information System (GIS)-derived livestock-related characteristics as predictors. The mean and standard deviation of annual average concentrations (gene copies/m3) of E. coli, Staphylococcus spp., tetW and mecA were as follows: 38.9 (±1.98), 2574 (±3.29), 20991 (±2.11), and 15.9 (±2.58). Validated through 10-fold cross-validation (CV), the models moderately explained spatial variation of all microbial agents. The best performing model per agent explained respectively 38.4%, 20.9%, 33.3% and 27.4% of the spatial variation of E. coli, Staphylococcus spp., tetW and mecA. RF models had somewhat better performance than LUR models. Livestock predictors related to poultry and pig farms dominated all models. To conclude, the models developed enable enhanced estimates of airborne livestock-related microbial exposure in future epidemiological studies. Consequently, this will provide valuable insights into the public health implications of exposure to specific microbial agents.

Airborne bacteria and endotoxin may affect asthma and allergies. However, there is limited understanding of the environmental determinants that influence them. This study investigated the airborne microbiomes in the homes of 1038 participants from five cities in Northern Europe: Aarhus, Bergen, Reykjavik, Tartu, and Uppsala. Airborne dust particles were sampled with electrostatic dust fall collectors (EDCs) from the participants' bedrooms. The dust washed from the EDCs' clothes was used to extract DNA and endotoxin. The DNA extracts were used for quantitative polymerase chain (qPCR) measurement and 16S rRNA gene sequencing, while endotoxin was measured using the kinetic chromogenic limulus amoebocyte lysate (LAL) assay. The results showed that households in Tartu and Aarhus had a higher bacterial load and diversity than those in Bergen and Reykjavik, possibly due to elevated concentrations of outdoor bacterial taxa associated with low precipitation and high wind speeds. Bergen-Tartu had the highest difference (ANOSIM R = 0.203) in β diversity. Multivariate regression models showed that α diversity indices and bacterial and endotoxin loads were positively associated with the occupants' age, number of occupants, cleaning frequency, presence of dogs, and age of the house. Further studies are needed to understand how meteorological factors influence the indoor bacterial community in light of climate change.

Introduction Our aim was to gain insight into the effect of COVID-19 measures on SARS-CoV-2 incidence in secondary schools and the association with classroom CO 2 concentration and airborne contamination. Methods Between October 2020—June 2021, 18 schools weekly reported SARS-CoV-2 incidence and completed surveys on school-initiated COVID-19 measures (e.g. improving hygiene or minimizing contacts). CO 2 was measured in occupied classrooms twice, and SARS-CoV-2 air contamination longitudinally using electrostatic dust collectors (EDC) and analyzed using RT-qPCR. National COVID-19 policy measures varied during pre-lockdown, lockdown and post-lockdown periods. During the entire study, schools were recommended to improve ventilation. SARS-CoV-2 incidence rate ratios (IRR) were estimated by Generalized Estimating Equation (GEE) models. Results During 18 weeks follow-up (range: 10–22) SARS-CoV-2 school-incidence decreased during national lockdown (adjusted IRR: 0.41, 95%CI: 0.21–0.80) and post-lockdown (IRR: 0.60, 0.39–0.93) compared to pre-lockdown. School-initiated COVID-19 measures had no additional effect. Pre-lockdown, IRRs per 10% increase in time CO 2 exceeded 400, 550 and 800 ppm above outdoor level respectively, were 1.08 (1.00–1.16), 1.10 (1.02–1.19), and 1.08 (0.95–1.22). Post-lockdown, CO 2- concentrations were considerably lower and not associated with SARS-CoV-2 incidence. No SARS-CoV-2 RNA was detected in any of the EDC samples. Conclusion During a period with low SARS-CoV-2 population immunity and increased attention to ventilation, with CO 2 levels most of the time below acceptable thresholds, only the national policy during and post-lockdown of reduced class-occupancy, stringent quarantine, and contact testing reduced SARS-CoV-2 incidence in Dutch secondary schools. Widespread SARS-CoV-2 air contamination could not be demonstrated in schools under the prevailing conditions during the study.

Residential microbial composition likely contributes to the development of lower respiratory tract infections (LRTI) among children, but the association is poorly understood. We aimed to study the relationship between the indoor airborne dust bacterial and fungal microbiota and childhood LRTI in Ibadan, Nigeria. Ninety-eight children under the age of five years hospitalized with LRTI were recruited and matched by age (±3 months), sex, and geographical location to 99 community-based controls without LRTI. Participants' homes were visited and sampled over a 14-day period for airborne house dust using electrostatic dustfall collectors (EDC). In airborne dust samples, the composition of bacterial and fungal communities was characterized by a meta-barcoding approach using amplicons targeting simultaneously the bacterial 16S rRNA gene and the internal-transcribed-spacer (ITS) region-1 of fungi in association with the SILVA and UNITE database respectively. A 100-unit change in house dust bacterial, but not fungal, richness (OR 1.06; 95%CI 1.03-1.10) and a 1-unit change in Shannon diversity (OR 1.92; 95%CI 1.28-3.01) were both independently associated with childhood LRTI after adjusting for other indoor environmental risk factors. Beta-diversity analysis showed that bacterial (PERMANOVA p < 0.001, R2 = 0.036) and fungal (PERMANOVA p < 0.001, R2 = 0.028) community composition differed significantly between homes of cases and controls. Pair-wise differential abundance analysis using both DESEq2 and MaAsLin2 consistently identified the bacterial phyla Deinococcota (Benjamini-Hochberg (BH) adjusted p-value <0.001) and Bacteriodota (BH-adjusted p-value = 0.004) to be negatively associated with LRTI. Within the fungal microbiota, phylum Ascomycota abundance (BH adjusted p-value <0.001) was observed to be directly associated with LRTI, while Basidiomycota abundance (BH adjusted p-value <0.001) was negatively associated with LRTI. Our study suggests that early-life exposure to certain airborne bacterial and fungal communities is associated with LRTI among children under the age of five years.

There is an interest in impact of national COVID-19 measures and ventilation regimes on potential SARS-CoV-2 exposure in school environments. Since quantitative SARS-CoV-2 exposure information is lacking, we explored associations between total bacterial load (16S), as a marker of microbial exposure, and national COVID-19 measures, ventilation regimes and occupancy. Longitudinal air monitoring for presence of 16S was conducted through repeated collection of airborne settling dust samples in classrooms of 18 secondary schools between 10-2020 until 06-2021. Total bacterial load was assessed through 16S rRNA qPCR. Information on occupancy and ventilation was collected through questionnaires. A Bayesian linear regression model was used to analyse the data. In the 335 samples collected, differences in 16S levels between ventilation regimes were small and not statistically significant, possibly due to additional natural ventilation by opening windows in classrooms. A trend for lower 16S levels with mechanical supply and exhaust compared to natural ventilation was observed. Repeated measures in pre- and post-lockdown, with looser versus stricter national COVID-19 measures at schools, showed significantly higher 16S levels pre- compared to post-lockdown. 16S levels were positively associated with higher occupancy, independently from COVID-19 measures. Occupancy was also higher during pre-lockdown when less national school COVID-19 measures were applied. Total bacterial load was associated with higher classroom occupancy and less COVID-19 measures, suggesting that both variables may improve air quality. Whether associations observed for 16S also exist for SARS-CoV-2 in school air needs to be confirmed in separate studies.

Collecting and obtaining sufficient amount of airborne particles for multiple microbial component assessments can be challenging. A passive dust sampling device, the electrostatic dust fall collector (EDC) has been established for assessing airborne exposures including endotoxin and glucans. Recently, with advances in next-generation sequencing techniques, EDCs were used to collect microbial cells for DNA sequencing analysis to promote the study of airborne bacterial and fungal communities. However, low DNA yields have been problematic when employing passive sampling with EDC. To address this challenge, we attempted to increase the efficiency of extraction. We compared DNA extraction efficiency of bacterial components from EDCs captured on filters through filtration using five extraction techniques. By measuring the abundance, diversity and structure of bacterial communities using qPCR and amplicon sequencing targeting 16S rRNA genes, we found that two techniques outperformed the rest. Furthermore, we developed protocols to simultaneously extract both DNA and endotoxin from a single EDC cloth. Our technique promotes a high quality to price ratio and may be employed in large epidemiological studies addressing airborne bacterial exposure where a large number of samples is needed.