Inflammatory potential in relation to the microbial content of settled dust samples collected from moisture‐damaged and reference schools: results of HITEA study

Aiming to identify factors causing the adverse health effects associated with moisture‐damaged indoor environments, we analyzed immunotoxicological potential of settled dust from moisture‐damaged and reference schools in relation to their microbiological composition. Mouse RAW264.7 macrophages were exposed to settled dust samples (n = 25) collected from moisture‐damaged and reference schools in Spain, the Netherlands, and Finland. After exposure, we analyzed production of inflammatory markers [nitric oxide (NO), tumor necrosis factor‐α (TNF‐)α, interleukin (IL)‐6, and macrophage inflammatory protein (MIP)2] as well as mitochondrial activity, viability, apoptosis, and cell cycle arrest. Furthermore, particle counts, concentration of selected microbial groups as well as chemical markers such as ergosterol, 3‐hydroxy fatty acids, muramic acid, endotoxins, and glucans were measured as markers of exposure. Dust from moisture‐damaged schools in Spain and the Netherlands induced stronger immunotoxicological responses compared to samples from reference schools; the responses to Finnish samples were generally lower with no difference between the schools. In multivariate analysis, IL‐6 and apoptosis responses were most strongly associated with moisture status of the school. The measured responses correlated with several microbial markers and numbers of particles, but the most important predictor of the immunotoxicological potential of settled dust was muramic acid concentration, a marker of Gram‐positive bacteria.     

Stenotrophomonas,Mycobacterium, and Streptomyces in home dust and air: associations with moldiness and other home/family characteristics

Respiratory illnesses have been linked to children's exposures to water‐damaged homes. Therefore, understanding the microbiome in water‐damaged homes is critical to preventing these illnesses. Few studies have quantified bacterial contamination, especially specific species, in water‐damaged homes. We collected air and dust samples in twenty‐one low‐mold homes and twenty‐one high‐mold homes. The concentrations of three bacteria/genera, Stenotrophomonas maltophilia, Streptomyces sp., and Mycobacterium sp., were measured in air and dust samples using quantitative PCR (QPCR). The concentrations of the bacteria measured in the air samples were not associated with any specific home characteristic based on multiple regression models. However, higher concentrations of S. maltophilia in the dust samples were associated with water damage, that is, with higher floor surface moisture and higher concentrations of moisture‐related mold species. The concentrations of Streptomyces and Mycobacterium sp. had similar patterns and may be partially determined by human and animal occupants and outdoor sources of these bacteria.     

Microbial exposures in moisture-damaged schools and associations with respiratory symptoms in students: A multi-country environmental exposure study

Moisture-damaged buildings are associated with respiratory symptoms and underlying diseases among building occupants, but the causative agent(s) remain a mystery. We first identified specific fungal and bacterial taxa in classrooms with moisture damage in Finnish and Dutch primary schools. We then investigated associations of the identified moisture damage indicators with respiratory symptoms in more than 2700 students. Finally, we explored whether exposure to specific taxa within the indoor microbiota may explain the association between moisture damage and respiratory health. Schools were assessed for moisture damage through detailed inspections, and the microbial composition of settled dust in electrostatic dustfall collectors was determined using marker-gene analysis. In Finland, there were several positive associations between particular microbial indicators (diversity, richness, individual taxa) and a respiratory symptom score, while in the Netherlands, the associations tended to be mostly inverse and statistically non-significant. In Finland, abundance of the Sphingomonas bacterial genus and endotoxin levels partially explained the associations between moisture damage and symptom score. A few microbial taxa explained part of the associations with health, but overall, the observed associations between damage-associated individual taxa and respiratory health were limited.     

Microbial growth in building material samples and occupants’ health in severely moisture‐damaged homes

There is no commonly approved approach to detect and quantify the health‐relevant microbial exposure in moisture‐damaged buildings. In 39 single‐family homes with severe moisture damage, we studied whether concentrations of viable microbes in building material samples are associated with health among 71 adults and 68 children, and assessed with symptoms questionnaires, exhaled NO, and peak expiratory flow (PEF) variability. Symptoms were grouped into three scores: upper respiratory symptoms, lower respiratory symptoms, and general symptoms. The homes were divided into three groups based on viable counts of fungi, actinomycetes, and total bacteria cultivated from building material samples. Highest group of actinomycete counts was associated with more general symptoms, worse perceived health, and higher daily PEF variability (aOR 12.51; 1.10‐141.90 as compared to the lowest group) among adults, and with an increase in lower respiratory symptoms in children, but the confidence intervals were wide. We observed significant associations of fungal counts and total microbial score with worse perceived health in adults. No associations with exhaled NO were observed.     

Observational scores of dampness and mold associated with measurements of microbial agents and moisture in three public schools

We examined associations between observational dampness scores and measurements of microbial agents and moisture in three public schools. A dampness score was created for each room from 4‐point‐scale scores (0–3) of water damage, water stains, visible mold, moldy odor, and wetness for each of 8 room components (ceiling, walls, windows, floor, ventilation, furniture, floor trench, and pipes), when present. We created mixed microbial exposure indices (MMEIs) for each of 121 rooms by summing decile ranks of 8 analytes (total culturable fungi; total, Gram‐negative, and Gram‐positive culturable bacteria; ergosterol; (1→3)‐β‐D‐glucan; muramic acid; and endotoxin) in floor dust. We found significant (P ≤ 0.01) linear associations between the dampness score and culturable bacteria (total, Gram‐positive, and Gram‐negative) and the MMEIs. Rooms with dampness scores greater than 0.25 (median) had significantly (P < 0.05) higher levels of most microbial agents, MMEIs, and relative moisture content than those with lower scores (≤0.25). Rooms with reported recent water leaks had significantly (P < 0.05) higher dampness scores than those with historical or no reported water leaks. This study suggests that observational assessment of dampness and mold using a standardized form may be valuable for identifying and documenting water damage and associated microbial contamination.     

Microbial secondary metabolites in homes in association with moisture damage and asthma

We aimed to characterize the presence of microbial secondary metabolites in homes and their association with moisture damage, mold, and asthma development. Living room floor dust was analyzed by LC‐MS/MS for 333 secondary metabolites from 93 homes of 1‐year‐old children. Moisture damage was present in 15 living rooms. At 6 years, 8 children had active and 15 lifetime doctor‐diagnosed asthma. The median number of different metabolites per house was 17 (range 8–29) and median sum load 65 (4–865) ng/m². Overall 42 different metabolites were detected. The number of metabolites present tended to be higher in homes with mold odor or moisture damage. The higher sum loads and number of metabolites with loads over 10 ng/m² were associated with lower prevalence of active asthma at 6 years (aOR 0.06 (95% CI <0.001–0.96) and 0.05 (<0.001–0.56), respectively). None of the individual metabolites, which presence tended (P < 0.2) to be increased by moisture damage or mold, were associated with increased risk of asthma. Microbial secondary metabolites are ubiquitously present in home floor dust. Moisture damage and mold tend to increase their numbers and amount. There was no evidence indicating that the secondary metabolites determined would explain the association between moisture damage, mold, and the development of asthma.     

Measured moisture in buildings and adverse health effects: A review

It has not yet been possible to quantify dose‐related health risks attributable to indoor dampness or mold (D/M), to support setting specific health‐related limits for D/M. An overlooked target for assessing D/M is moisture in building materials, the critical factor allowing microbial growth. A search for studies of quantified building moisture and occupant health effects identified 3 eligible studies. Two studies assessed associations between measured wall moisture content and respiratory health in the UK. Both reported dose‐related increases in asthma exacerbation with higher measured moisture, with 1 study reporting an adjusted odds ratio of 7.0 for night‐time asthma symptoms with higher bedroom moisture. The third study assessed relationships between infrared camera‐determined wall moisture and atopic dermatitis in South Korea, reporting an adjusted odds ratio of 14.5 for water‐damaged homes and moderate or severe atopic dermatitis. Measuring building moisture has, despite extremely limited available findings, potential promise for detecting unhealthy D/M in homes and merits more research attention. Further research to validate these findings should include measured “water activity,” which directly assesses moisture availability for microbial growth. Ultimately, evidence‐based, health‐related thresholds for building moisture, across specific materials and measurement devices, could better guide assessment and remediation of D/M in buildings.     

Domestic exposure to endotoxin and respiratory morbidity in former smokers with COPD

Indoor air pollution has been linked to adverse chronic obstructive pulmonary disease (COPD) health, but specific causative agents have not yet been identified. We evaluated the role of indoor endotoxin exposure upon respiratory health in former smokers with COPD. Eighty‐four adults with moderate to severe COPD were followed longitudinally and indoor air and dust samples collected at baseline, 3 and 6 months. Respiratory outcomes were repeatedly assessed at each time point. The associations between endotoxin exposure in air and settled dust and health outcomes were explored using generalizing estimating equations in multivariate models accounting for confounders. Dust endotoxin concentrations in the main living area were highest in spring and lowest in fall, while airborne endotoxins remained steady across seasons. Airborne and dust endotoxin concentrations were weakly correlated with one another (r_s = +0.24, P = 0.005). Endotoxin concentrations were not significantly associated with respiratory symptoms, rescue medication use, quality of life, or severe exacerbations. In vitro whole‐blood assays of the pro‐inflammatory capacity of PM₁₀ filters with and without endotoxin depletion demonstrated that the endotoxin component of indoor air pollution was not the primary trigger for interleukin‐1β release. Our findings support that endotoxin is not the major driver in the adverse effects of indoor PM upon COPD morbidity.     

Indoor air microbes and respiratory symptoms of children in moisture damaged and reference schools

Microbial indoor air quality and respiratory symptoms of children were studied in 24 schools with visible moisture and mold problems, and in eight non-damaged schools. School buildings of concrete/brick and wooden construction were included. The indoor environment investigations included technical building inspections for visible moisture signs and microbial sampling using six-stage impactor for viable airborne microbes. Children's health information was collected by questionnaires. The effect of moisture damage on concentrations of fungi was clearly seen in buildings of concrete/brick construction, but not in wooden school buildings. Occurrence of Cladosporium, Aspergillus versicolor, Stachybotrys, and actinobacteria showed some indicator value for moisture damage. Presence of moisture damage in school buildings was a significant risk factor for respiratory symptoms in schoolchildren. Association between moisture damage and respiratory symptoms of children was significant for buildings of concrete/brick construction but not for wooden school buildings. The highest symptom prevalence was found during spring seasons, after a long exposure period in damaged schools. The results emphasize the importance of the building frame as a determinant of exposure and symptoms.     

Allergy and respiratory health effects of dampness and dampness‐related agents in schools and homes: a cross‐sectional study in Danish pupils

Little is known about the health effects of school‐related indoor dampness and microbial exposures. In this study, we investigated dampness and dampness‐related agents in both homes and schools and their association with allergy and respiratory health effects in 330 Danish pupils. Classroom dampness was identified based on technical inspection and bedroom dampness on parents' self‐report. Classroom and bedroom dust was analyzed for seven microbial components. Skin prick testing determined atopic sensitization. Lung function was expressed as z‐scores for forced expiratory volume in one‐second (zFEV₁), forced vital capacity (zFVC) and the ratio zFEV₁/zFVC using GLI‐2012 prediction equations. The parents reported children's allergies, airway symptoms, and doctor‐diagnosed asthma. High classroom dampness, but not bedroom dampness, was negatively associated with zFEV₁ (β‐coef. ?0.71; 95% CI ?1.17 to ?0.23) and zFVC (β‐coef. ?0.52; 95% CI ?0.98 to ?0.06) and positively with wheezing (OR 8.09; 95% CI 1.49 to 43.97). No consistent findings were found between any individual microbial components or combination of microbial components and health outcomes. Among other indoor risk factors, environmental tobacco smoke (ETS) decreased zFEV₁ (β‐coef. ?0.22; 95% CI ?0.42 to ?0.02) and zFEV₁/zFVCratio (β‐coef. ?0.26; 95% CI ?0.44 to ?0.07) and increased upper airway symptoms (OR 1.66; 95% CI 1.03–2.66). In conclusion, dampness in classrooms may have adverse respiratory health effects in pupils, but microbial agents responsible for this effect remain unknown.     

Does dampness and mold in schools affect health? Results of a meta‐analysis

This paper provides meta‐analyses of the published findings relating the respiratory health of occupants of schools with visible dampness, water damage, visible mold, and/or mold odor. Random effects models were used to develop central estimates and confidence limits for the associations of respiratory health effects with school dampness and mold. Eleven studies, all with cross‐sectional designs, were included in the meta‐analyses; however, analyses for some health outcomes were based on as few as four studies. Analyses were performed using data from adults and children combined, using only data from children, and using data from adults and children after excluding two studies. The central estimates of odds ratios from the meta‐analyses were consistently above unity. The evidence of adverse health effects was strongest for cough and wheeze, which had confidence limits excluding unity in some or all analyses. The odds ratios of 1.32 for cough and 1.68 for wheeze suggest moderate increases in health risk. Studies not included in the meta‐analyses provide additional evidence that dampness and mold in schools are associated with adverse health outcomes. These meta‐analyses and the published literature not included in the meta‐analyses suggest that dampness and mold in schools are associated with adverse respiratory health effects.     

Teachers' work-related non-literature-known building-related symptoms are also connected to indoor toxicity: A cross-sectional study

A previous study showed that classical building-related symptoms (BRS) were related to indoor dust and microbial toxicity via boar sperm motility assay, a sensitive method for measuring mitochondrial toxicity. In this cross-sectional study, we analyzed whether teachers’ most common work-related non-literature-known BRS (nBRS) were also associated with dust or microbial toxicity. Teachers from 15 schools in Finland completed a questionnaire evaluating 20 nBRS including general, eye, respiratory, hearing, sleep, and mental symptoms. Boar sperm motility assay was used to measure the toxicity of extracts from wiped dust and microbial fallout samples collected from teachers’ classrooms. 231 teachers answered a questionnaire and their classroom toxicity data were recorded. A negative binomial mixed model showed that teachers’ work-related nBRS were 2.9-fold (95% CI: 1.2-7.3) higher in classrooms with highly toxic dust samples compared to classrooms with non-toxic dust samples (p = 0.024). The RR of work-related nBRS was 1.8 (95% CI: 1.1-2.9) for toxic microbial samples (p = 0.022). Teachers’ BRS appeared to be broader than reported in the literature, and the work-related nBRS were associated with toxic dusts and microbes in classrooms.     

Chloroanisoles may explain mold odor and represent a major indoor environment problem in Sweden

Indoor mold odor is associated with adverse health effects, but the microbial volatiles underlying mold odor are poorly described. Here, chloroanisoles were studied as potential key players, being formed by microbial metabolism of chlorophenols in wood preservatives. Using a three‐stage approach, we (i) investigated the occurrence of chloroanisoles in buildings with indoor air quality problems, (ii) estimated their frequency in Sweden, and (iii) evaluated the toxicological risk of observed chloroanisole concentrations. Analyses of 499 building materials revealed several chloroanisole congeners in various types of buildings from the 1950s to 1970s. Evaluation of Swedish records from this time period revealed three coinciding factors, namely an unprecedented nationwide building boom, national regulations promoting wood preservatives instead of moisture prevention, and use of chlorophenols in these preservatives. Chlorophenols were banned in 1978, yet analysis of 457 indoor air samples revealed several chloroanisole congeners, but at median air levels generally below 15 ng/m³. Our toxicological evaluation suggests that these concentrations are not detrimental to human health per se, but sufficiently high to cause malodor. Thereby, one may speculate that chloroanisoles in buildings contribute to adverse health effects by evoking odor which, enhanced by belief of the exposure being hazardous, induces stress‐related and inflammatory symptoms.     

Influence of housing characteristics on bacterial and fungal communities in homes of asthmatic children

Variations in home characteristics, such as moisture and occupancy, affect indoor microbial ecology as well as human exposure to microorganisms. Our objective was to determine how indoor bacterial and fungal community structure and diversity are associated with the broader home environment and its occupants. Next‐generation DNA sequencing was used to describe fungal and bacterial communities in house dust sampled from 198 homes of asthmatic children in southern New England. Housing characteristics included number of people/children, level of urbanization, single/multifamily home, reported mold, reported water leaks, air conditioning (AC) use, and presence of pets. Both fungal and bacterial community structures were non‐random and demonstrated species segregation (C‐score, P < 0.00001). Increased microbial richness was associated with the presence of pets, water leaks, longer AC use, suburban (vs. urban) homes, and dust composition measures (P < 0.05). The most significant differences in community composition were observed for AC use and occupancy (people, children, and pets) characteristics. Occupant density measures were associated with beneficial bacterial taxa, including Lactobacillus johnsonii as measured by qPCR. A more complete knowledge of indoor microbial communities is useful for linking housing characteristics to human health outcomes. Microbial assemblies in house dust result, in part, from the building's physical and occupant characteristics.     

Co-occurrence of toxic bacterial and fungal secondary metabolites in moisture-damaged indoor environments

Toxic microbial secondary metabolites have been proposed to be related to adverse health effects observed in moisture-damaged buildings. Initial steps in assessing the actual risk include the characterization of the exposure. In our study, we applied a multi-analyte tandem mass spectrometry-based methodology on sample materials of severely moisture-damaged homes, aiming to qualitatively and quantitatively describe the variety of microbial metabolites occurring in building materials and different dust sample types. From 69 indoor samples, all were positive for at least one of the 186 analytes targeted and as many as 33 different microbial metabolites were found. For the first time, the presence of toxic bacterial metabolites and their co-occurrence with mycotoxins were shown for indoor samples. The bacterial compounds monactin, nonactin, staurosporin and valinomycin were exclusively detected in building materials from moist structures, while chloramphenicol was particularly prevalent in house dusts, including settled airborne dust. These bacterial metabolites are highly bioactive compounds produced by Streptomyces spp., a group of microbes that is considered a moisture damage indicator in indoor environments. We show that toxic bacterial metabolites need to be considered as being part of very complex and diverse microbial exposures in 'moldy' buildings. PRACTICAL IMPLICATIONS: Bacterial toxins co-occur with mycotoxins in moisture-damaged indoor environments. These compounds are measurable also in settled airborne dust, indicating that inhalation exposure takes place. In attempts to characterize exposures to microbial metabolites not only mycotoxins but also bacterial metabolites have to be targeted by the analytical methods applied. We recommend including analysis of samples of outdoor air in the course of future indoor assessments, in an effort to better understand the outdoor contribution to the indoor presence of microbial toxins. There is a need for a sound risk assessment concerning the exposure to indoor microbial toxins at concentrations detectable in moisture-damaged indoor environments.     

Modeling microbial growth in carpet dust exposed to diurnal variations in relative humidity using the “Time-of-Wetness” framework

Resuspension of microbes in floor dust and subsequent inhalation by human occupants is an important source of human microbial exposure. Microbes in carpet dust grow at elevated levels of relative humidity, but rates of this growth are not well established, especially under changing conditions. The goal of this study was to model fungal growth in carpet dust based on indoor diurnal variations in relative humidity utilizing the time-of-wetness framework. A chamber study was conducted on carpet and dust collected from 19 homes in Ohio, USA and exposed to varying moisture conditions of 50%, 85%, and 100% relative humidity. Fungal growth followed the two activation regime model, while bacterial growth could not be evaluated using the framework. Collection site was a stronger driver of species composition (P = 0.001, R² = 0.461) than moisture conditions (P = 0.001, R² = 0.021). Maximum moisture condition was associated with species composition within some individual sites (P = 0.001-0.02, R² = 0.1-0.33). Aspergillus, Penicillium, and Wallemia were common fungal genera found among samples at elevated moisture conditions. These findings can inform future studies of associations between dampness/mold in homes and health outcomes and allow for prediction of microbial growth in the indoor environment.     

School attendance and daily respiratory symptoms in children: influence of moisture damage

We investigated the effect of weekends and school holidays on the daily frequency and severity of respiratory and other symptoms among children attending schools with (index) or without (reference) moisture damage in Spain, the Netherlands, and Finland. Throughout 1 year, parents of 419 children with a respiratory condition attending index (n=15) or reference (n=10) primary schools completed three symptom diaries. We assessed associations between lower respiratory tract, upper respiratory tract or allergy, and other symptom scores and school day, weekend, or summer holiday using mixed regression models stratified by country and moisture damage. We evaluated interactions between moisture damage and type of day. We combined country‐specific estimates (incidence rate ratios [IRRs] and 95% confidence interval [CI]) in meta‐analyses. Symptom scores were lower during weekends and holiday. Lower respiratory tract symptoms were statistically significantly less common during holiday with strongest effect in index schools (IRR=0.7; CI=0.6–0.8). Reporting of other symptoms was more reduced during holiday in index (IRR=0.6; CI=0.4–0.9) than in reference (IRR=0.95; CI=0.8–1.2) schools (interaction P<.01). In conclusion, symptoms were less frequent and/or severe during summer holiday and weekends. This pattern was stronger among children attending moisture‐damaged schools, suggesting potential improvement in moisture damage‐related symptoms during school breaks.     

Indoor visible mold and mold odor are associated with new‐onset childhood wheeze in a dose‐dependent manner

Evidence is accumulating that indoor dampness and mold are associated with the development of asthma. The underlying mechanisms remain unknown. New Zealand has high rates of both asthma and indoor mold and is ideally placed to investigate this. We conducted an incident case‐control study involving 150 children with new‐onset wheeze, aged between 1 and 7 years, each matched to two control children with no history of wheezing. Each participant's home was assessed for moisture damage, condensation, and mold growth by researchers, an independent building assessor and parents. Repeated measures of temperature and humidity were made, and electrostatic dust cloths were used to collect airborne microbes. Cloths were analyzed using qPCR. Children were skin prick tested for aeroallergens to establish atopy. Strong positive associations were found between observations of visible mold and new‐onset wheezing in children (adjusted odds ratios ranged between 1.30 and 3.56; P ≤ .05). Visible mold and mold odor were consistently associated with new‐onset wheezing in a dose‐dependent manner. Measurements of qPCR microbial levels, temperature, and humidity were not associated with new‐onset wheezing. The association between mold and new‐onset wheeze was not modified by atopic status, suggesting a non‐allergic association.     

Detection of streptomycetes in house dust--comparison of culture and PCR methods

Streptomycetes are gram-positive, spore producing, filamentous bacteria common in soil, but also present in indoor environments. They are potent producers of secondary metabolites and inducers of inflammatory responses in vitro. Polymerase chain reaction (PCR)- and culture-based detection methods for streptomycetes in house dust samples were compared. A total of 47 dust samples were investigated, and the presence of streptomycetes was determined by cultivation on tryptone-yeast-extract-glucose agar and PCR. The 16S rRNA gene of actinomycete isolates from house dust was partially sequenced to investigate if they belong to the genus Streptomyces. Both PCR and culture showed more frequent occurrence of streptomycetes in moisture-damaged homes, although the results did not correlate well. The occurrence of streptomycetes in house dust was associated with moisture damage of the home. The amount of Streptomyces-specific PCR amplification product was significantly higher in dust from moisture-damaged homes than in homes with no moisture damage (P < 0.05, Mann-Whitney U test). A correlation between streptomycetes and moisture damage, although not statistically significant, was also observed when using binary data, e.g. presence or absence of streptomycetes or moisture damage (P = 0.054 for PCR, and P = 0.127 for culture, Fisher's exact test). Altogether, the presence of streptomycetes in house dust seems to indicate the presence of moisture damage in the building.