Association of indoor microbial aerosols with respiratory symptoms among under-five children: a systematic review and meta-analysis

Background Despite the recognition of the importance of indoor microbial exposures on children’s health, the role of different microbial agents in development and aggravation of respiratory symptoms and diseases is only poorly understood. This study aimed to assess whether exposure to microbial aerosols within the indoor environment are associated with respiratory symptoms among children under-5 years of age. Methods A systematic literature search was conducted on PubMed, Web of Science, GreenFILE, ScienceDirect, EMBASE and Cochrane library through February 2020. Studies that investigated the exposure–response relationship between components of the indoor microbial communities and respiratory symptoms among under-five children were eligible for inclusion. A random-effect meta-analysis was applied to estimate pooled relative risk (RR) and 95% confidence interval (CI) for study specific high versus low microbial exposures. The potential effect of individual studies on the overall estimate was evaluated using leave-one-out analysis, while heterogeneity was evaluated by I2 statistics using RevMan 5.3. Results Fifteen studies were eligible for inclusion in a meta-analysis. The pooled risk estimate suggested that increased microbial exposure was associated with an increased risk of respiratory symptoms [pooled relative risk (RR): 1.24 (1.09, 1.41), P = 0.001]. The association was strongest with exposure to a combination of Aspergillus, Penicillium, Cladosporium and Alternaria species [pooled RR: 1.73 (1.30, 2.31), P = 0.0002]. Stratified analysis revealed an increased risk of wheeze [pooled RR: 1.20 (1.05, 1.37), P = 0.007 and allergic rhinitis [RR: 1.18 (0.94, 1.98), P = 0.16] from any microbial exposure. Conclusions Microbial exposures are, in general, associated with risk of respiratory symptoms. Future studies are needed to study the indoor microbiome more comprehensively, and to investigate the mechanism of these associations. Supplementary Information The online version contains supplementary material available at 10.1186/s12940-021-00759-2.

or prior water damage [13]. The interest in the indoor microbiome has increased over the last few decades [1]. This is largely because of the wider recognition that exposures to microbes in the residential indoor environment are associated with a vast number of adverse health outcomes with major public health importance, including infectious diseases, acute toxic effects, allergies and cancer [1].
Most epidemiological studies have been heterogeneous in assessing different respiratory health effects associated with exposure to specific microbial components, especially endotoxins, and culturable molds [14][15][16][17] while very few have investigated indoor microbial communities [18,19]. These studies have shown inconsistent findings, suggesting protective, detrimental, and no health effects in relation to asthma and allergy [14]. Greater diversity of fungal and bacterial agents has been shown to reduce the risk of asthma and wheeze in children [17,20,21] while others found a positive association between elevated levels of total viable mold and risk of rhinitis with persistent cough [22][23][24].
Respiratory health effects have been the subject of recent research among preschool children [25,26] and children under the age of 5 years [27]. Children under the age of 5 years are more at risk of respiratory outcomes from exposure to indoor microbial agents due to the fact that they spend a considerable proportion of time in the home environment during a period of intense growth and development of the immunologic and respiratory systems [28,29]. Despite the recognition of the importance of exposure to the indoor microbiome on children's health, the precise role of different microbial agents in the development and aggravation of symptoms and diseases is only poorly understood. It is therefore not clear which specific component(s) primarily contribute to the presumed respiratory health effects. To the best of our knowledge, there has been no systematic review or meta-analysis exploring the role of the indoor microbial exposure on respiratory health outcomes among children under 5. Therefore, this systematic review and meta-analysis aims to summarize evidence of associations between different indoor microbial agents and their combined role in the incidence of respiratory allergies and asthma, thereby providing opportunities to improve future respiratory health interventions among under-five children.

Literature search
The protocol for this meta-analysis was reported using MOOSE guideline [30] and registered in the International Prospective Register of Systematic Reviews (Reg ID: CRD42020178514). Two reviewers independently explored PUBMED, WEB OF SCIENCE, GREEN FILE (EBSCO), ScienceDirect, EMBASE and Cochrane databases independent of date through February 2020 to identify appropriate previously published studies using the following search terms; "home" OR "house" OR "dwelling" OR "residence" OR "residential" OR "indoor" OR "domicile" OR "living unit" OR "property" OR "build" OR "built environment" OR "domestic environment" OR "bedroom" OR "living room" OR "wall" OR "floor" OR "ceiling" OR "construction material" AND "damp" OR "fungi" OR "mold" OR "mould" OR "fungal" OR "fungus" OR "bacteria" OR "virus" OR "microbial" OR "microbiome" OR "microbial diversity" OR "microbial load" OR "microbial burden" OR "microbiota" OR "biodiversity" AND "respiratory symptoms" OR "allergy" OR "hay fever" OR "cough" OR "fever" OR "difficulty breathing" OR "wheeze" OR "allergic rhinitis" OR "sinusitis" OR "asthma". Title and abstract of each article was evaluated independently and differences on which publication(s) to include were clarified by recourse to a third reviewer. A snowball search was also carried out by screening reference lists of publications and reviews.

Inclusion criteria and study selection
Studies eligible for inclusion in the meta-analysis were epidemiological reports in humans with exposureresponse relationship between indoor microbial aerosols and respiratory symptoms. Inclusion criteria were: (I) qualitative or quantitative assessment of indoor microbial aerosols. Studies that reported exposure to specific microbial communities (bacteria, fungi, viruses, and/or microbial by-products) or presence/absence of visible molds were included; (II) studies conducted among children aged ≤ 5 years; (III) respiratory symptoms (including wheeze/allergic rhinitis) and/or asthma adequately defined and described; (IV) full text articles in English originally published in peer reviewed journals. Literature reviews, abstracts, letters to the editor, case reports, and non-human studies were excluded.
Assessment of articles was performed in EndNote databases. All duplicates were removed and studies were selected based on title or abstract for full text-screening. For studies that were excluded, the reasons for exclusion were listed.

Data extraction
From the studies that met the inclusion criteria, the following information was extracted: (1) name and initials of the first author, (2) year of publication, (3) country, (4) type of sample, (5) sampling equipment, (6) microbial agent(s), (7) analytical method, (8) level and measure of exposure, (9) respiratory outcome definition, (10) number of cases, (11) sample size and (12) effect estimate. All incongruities from the data extracted were resolved by a third author.

Quality assessment of included studies
Two team members (AGF and NJ) assessed the methodological quality and risk of bias of the included studies using the Cochrane Collaboration guidelines [31] and the Newcastle-Ottawa scales [32]. The quality of the studies was graded by rating nine items representing the study selection procedure, comparability, and outcome/ exposure definition. Each item was scored as 'yes' (if present) and 'no' (if absent) in the included studies and the overall scores were presented as percentages. Studies with median a score ≥ 80% (median in our study) were arbitrarily considered to have a low risk of bias while those with a score < 80% were considered to have a high risk of bias.

Statistical analyses
All statistical analysis was carried out using Review Manager 5.3. We applied the inverse of variance method for weighting and computed the summary effect estimates by first log-transforming all relative risk (RR) and 95% confidence interval (CI) for high vs low category (as reference) of microbial exposure. The standard error (SE) was estimated based on the formula: The extent of variability across studies and heterogeneity of the summary effect estimates were evaluated using I 2 test statistics. Where I 2 statistics ≥ 50%, a random effect model was employed, otherwise, a fixed effect model [30,33]. The area of the black square in forest plots implies the weighted contribution by each study. Sensitivity analyses of the results and publication bias was evaluated using leave-one-out and funnel plot techniques respectively. P < 0.05 (two-tailed) was considered statistically significant [34].
The funnel plots showed no significant evidence of publication bias among the studies included in the meta-analyses (Supplementary Figures S1, S2 and S3). We tested the effect of excluding individual studies on the stability of the pooled effect estimates and found no single study exerted significant effect on the overall effect estimate of the meta-analysis. Details of the sensitivity analysis are provided in Supplementary Table S2.

Discussion
This systematic review and meta-analysis summarizes the current knowledge on the association between qualitative and quantitative estimates of microbial agents within the indoor environment and respiratory symptoms among children under the age of 5 years. To the best of our knowledge, our meta-analysis is the most comprehensive overview to investigate whether exposure to indoor microbial aerosols is associated with respiratory health of children under the age of 5 years. First, we observed that exposure to a combination of Aspergillus, Penicillium and Cladosporium species increased the risk of wheeze by 67%. Secondly, the association of indoor microbial exposure with allergic rhinitis was not significant. Thirdly, exposure to microbial agents such as endotoxins was protective against asthma, although the estimate was from two studies.
The combined analysis revealed an increased risk of wheeze when children under 5 were exposed to a combination of Aspergillus and Penicillium species, which was reduced by the addition of Cladosporium species to the model. This suggests that microbial interaction within the indoor environment may play a role in the respiratory health of children under the age of 5 years. These specific fungi within the indoor environment have also been shown to be associated with an increased risk of wheeze [17,36] and asthma [24,35,50] in longitudinal studies. A similar meta-analysis on fungal exposure and respiratory health in children aged 6-12 years [51] compares well to the pooled estimate reported in our meta-analyses. Relevant studies both in vitro and in vivo have demonstrated that repeated activation of immune responses and inflammation from fungal exposures may contribute to inflammation-related diseases,    and the resulting inflamed mucosal tissue may provide a diminished barrier to respiratory infections [17]. Also, prolonged exposure to aerosolized fungal components mainly target the respiratory and nervous system causing specific pathological changes in the host characterized by inflammation and continuous activation of immune responses as a result of fungal exposures may contribute to inflammation-related diseases [52]. Our review considered exposure to visible molds only or in association with dampness, but excluded studies considering dampness alone. Indeed, mold and dampness exposures are often connected, leading to increased fungal growth and correlated microbial exposures, such as fungal spores, hyphae, fragments [53], microbial volatile organic compounds [54,55], mycotoxins [56], house dust mite allergens [57] and endotoxins [58,59]. The definitions of respiratory outcomes among children < 6 years of age are often poorly described and confusing, thereby making the diagnosis of the disease in preschool children difficult [60]. As a result, the European Respiratory Society task force proposed the use of terms such as "episodic (viral) wheeze" (among children with recurrent wheeze and who are well between episodes) and "multiple-trigger wheeze" (among children who wheeze both during and after discrete episodes) [61]. In fact, some other definitions have also been used to describe the different phenotypes of preschool wheezing disorders such as the presence of transient early wheezing in children < 3 years, non-atopic wheezing in children aged 3-6 years, and IgE-mediated wheeze in older children [62]. More recent studies have suggested that these definitions may reflect disease severity and that they are likely to vary with time [63]. Specific factors responsible for the development of respiratory diseases in children < 6 years has not been identified; however, interactions between the environment and genetic factors of each individual play a vital role [64]. These factors include infections, atopy, prematurity, exposure to tobacco smoke, exposure to elevated levels of air pollution or family history of asthma [62,65,66]. A recent meta-analysis reported an increased risk of lower respiratory tract infection among under-five children as a result of increased exposure to indoor microbes with emphasis on detailed microbial characterization using modern molecular techniques [67].
It was interesting to discover that none of the studies included in the present meta-analysis employed molecular-based techniques in the analysis of microbial agents. Although, studies have investigated the burden of indoor microbial exposures using sequencing-based assessment [9,[68][69][70] but very few demonstrated a link with disease epidemiology such as respiratory outcomes as revealed in the present study. This is probably due to the complexity     Recent studies have emphasized the protective effect of exposure to endotoxins [25,26,[71][72][73] against respiratory allergies and allergic asthma. Our findings corroborate these reports but contradicts some findings among older children [74][75][76] and adults [77][78][79]. A previous study reported that exposure to endotoxin has been associated with reduced risk of childhood atopy but an increased risk of wheeze [80]. A comprehensive meta-analysis confirmed this contradiction with respect to endotoxin exposure [73]. Among pre-school children, in whom asthma is more strongly associated with atopy, endotoxin exposure resulted in reduced risk, whereas among infants and toddlers with virus-triggered wheeze, endotoxin increased the risk [73]. Similar findings were reported from the survey carried out by the National Health and Nutrition Examination Survey study enrolling more than 6,000 subjects across the United States. They found that endotoxin was a risk for wheeze but not asthma [59]. Less is known about other bacterial and fungal exposures in indoor urban homes. In the Boston cohort increased levels of muramic acid as a marker for gram-positive bacteria were inversely associated with current asthma but not with allergic sensitization [80]. However, the mechanisms are still not fully understood. Possible explanation indicate that endotoxin is a potent inducer of interleukin-12 and interferon gamma, which downregulate the production of T-lymphocyte helper 2 (Th2) cells involved in the development of allergic diseases [81]. Besides, the potential of T-lymphocyte helper 1 (Th1) inducers like endotoxin and other microbial exposures to mitigate allergy and asthma is consistent with clinical studies. Overall, evidence of the mechanism of association between exposure to indoor microbes and respiratory outcomes is limited. Nevertheless, further longitudinal studies of the effect of early-life exposure to endotoxins on subsequent child health will be needed to understand this mechanism more fully.
In addition, studies included in the meta-analyses have used proxy measures for estimating respiratory health outcome, such as outcome obtained from parental or other caregiver interviews, questionnaires, and medical records. None of the studies quantified indoor microbial contamination to the species level using molecular techniques, which restricted analyses to the fungal genera and potentially underestimate exposures. Also, the differences in the RRs across the included studies could have also contributed to the high heterogeneity. Regardless of these limitations, this study has provided sufficient evidence required in designing future longitudinal studies to further investigate and explain the mechanism involved in the exposure-response relationship between indoor microbial exposure and respiratory symptoms among children under 5.

Conclusions
Indoor microbial aerosol exposures increase the risk of respiratory symptoms such as wheeze and allergic rhinitis but protect against asthma in children under 5. More research regarding these relationships is required using modern analytical approaches such as molecularbased sequencing techniques to better inform/advise parents, form guidelines to reduce exposure to microbial agents within the indoor environment and provide useful intervention strategies for managing the impact of exposure to microbial agents in association with respiratory symptoms among children under the age of 5 years.