Evaluation of Real and Perceived Risk to Health Care Workers Caring for Patients With the Omicron Variant of the SARS-CoV-2 Virus in Surgery and Obstetrics.

OBJECTIVES: The Omicron variant of the SARS-CoV-2 virus is described as more contagious than previous variants. We sought to assess risk to health care workers (HCWs) caring for patients with COVID-19 in surgical/obstetrical settings, and the perception of risk among this group. METHODS: From January to April 2022, reverse transcription polymerase chain reaction was used to detect the presence of SARS-CoV-2 viral ribonucleic acid in patient, environmental (floor, equipment, passive air) samples, and HCWs' masks (inside surface) during urgent surgery or obstetrical delivery for patients with SARS-CoV-2 infection. The primary outcome was the proportion of HCWs' masks testing positive. Results were compared with our previous cross-sectional study involving obstetrical/surgical patients with earlier variants (2020-2021). HCWs completed a risk perception electronic questionnaire. RESULTS: Eleven patients were included: 3 vaginal births and 8 surgeries. In total, 5/108 samples (5%) tested positive (SARS-CoV-2 Omicron) viral ribonucleic acid: 2/5 endotracheal tubes, 1/22 floor samples, 1/4 patient masks, and 1 nasal probe. No samples from the HCWs' masks (0/35), surgical equipment (0/10), and air (0/11) tested positive. No significant differences were found between the Omicron and 2020/21 patient groups' positivity rates (Mann-Whitney U test, P = 0.838) or the level of viral load from the nasopharyngeal swabs (P = 0.405). Nurses had a higher risk perception than physicians (P = 0.038). CONCLUSION: No significant difference in contamination rates was found between SARS-CoV-2 Omicron BA.1 and previous variants in surgical/obstetrical settings. This is reassuring as no HCW mask was positive and no HCW tested positive for COVID-19 post-exposure.

Detection of SARS-CoV-2 contamination in the operating room and birthing room setting: a cross-sectional study.

BACKGROUND: The exposure risks to front-line health care workers caring for patients with SARS-CoV-2 infection undergoing surgery or obstetric delivery are unclear, and an understanding of sample types that may harbour virus is important for evaluating risk. We sought to determine whether SARS-CoV-2 viral RNA from patients with SARS-CoV-2 infection undergoing surgery or obstetric delivery was present in the peritoneal cavity of male and female patients, in the female reproductive tract, in the environment of the surgery or delivery suite (surgical instruments or equipment used, air or floors), and inside the masks of the attending health care workers. METHODS: We conducted a cross-sectional study from November 2020 to May 2021 at 2 tertiary academic Toronto hospitals, during urgent surgeries or obstetric deliveries for patients with SARS-CoV-2 infection. The presence of SARS-CoV-2 viral RNA in patient, environmental and air samples was identified by real-time reverse transcription polymerase chain reaction (RT-PCR). Air samples were collected using both active and passive sampling techniques. The primary outcome was the proportion of health care workers' masks positive for SARS-CoV-2 RNA. We included adult patients with positive RT-PCR nasal swab undergoing obstetric delivery or urgent surgery (from across all surgical specialties). RESULTS: A total of 32 patients (age 20-88 yr) were included. Nine patients had obstetric deliveries (6 cesarean deliveries), and 23 patients (14 male) required urgent surgery from the orthopedic or trauma, general surgery, burn, plastic surgery, cardiac surgery, neurosurgery, vascular surgery, gastroenterology and gynecologic oncology divisions. SARS-CoV-2 RNA was detected in 20 of 332 (6%) patient and environmental samples collected: 4 of 24 (17%) patient samples, 5 of 60 (8%) floor samples, 1 of 54 (2%) air samples, 10 of 23 (43%) surgical instrument or equipment samples, 0 of 24 cautery filter samples and 0 of 143 (95% confidence interval 0-0.026) inner surface of mask samples. INTERPRETATION: During the study period of November 2020 to May 2021, we found evidence of SARS-CoV-2 RNA in a small but important number of samples obtained in the surgical and obstetric operative environment. The finding of no detectable virus inside the masks worn by the health care teams would suggest a low risk of infection for health care workers using appropriate personal protective equipment.

Influence of seasons and sites on bioaerosols in indoor wastewater treatment plants and proposal for air quality indicators.

During wastewater treatment, bioaerosols are generated and, can either remain in suspension for several hours or settle on surfaces and workers may be exposed. The presence of pathogens in the air could contribute to an increased frequency of gastrointestinal or respiratory illness amongst workers. Due to harsh winter conditions in Eastern Canada, many of the steps in the wastewater treatment process occur indoors, leading to a greater risk of significant occupational exposure especially if there is inadequate ventilation or a lack of personal protection. This work has used stationary sampling at various indoor wastewater treatment steps both in winter and summer. Bioaerosols were evaluated using both culture and molecular methods along with ventilation characterization. Endotoxins were quantified, as well as total cultivable and gram-negative bacteria and pathogen indicators using qPCR. This study highlights the presence of potential pathogens at all steps in the treatment process, which may represent a potential occupational hazard. Comparisons between summer and winter data suggest that water temperature is an important factor for microbial activity and suggest that increasing the rate of air changes per hour in summer would be beneficial to reduce the concentration of bioaerosols during this time of the year. The screening, grit/FOGs removal and biofiltration were the most bioaerosol-loaded sites. Based on strong correlations, we suggest the reconsideration of exposure limits in WWTPs. Workers should be encouraged to use personal respiratory protection to limit the risk of health problems, especially during long-term work.Implications: The work presented herein showcases significant correlations between concentrations of endotoxins, cultivable bacteria, gram-negative bacteria, and total bacteria by qPCR from air collected in indoor wastewater treatment plants. These correlations lead us to propose new limit of exposure values, revisited to fit the endotoxin exposure limits recommendations. The results can serve as guidelines for future proposals for air quality indicators.

Surface and Air Contamination With Severe Acute Respiratory Syndrome Coronavirus 2 From Hospitalized Coronavirus Disease 2019 Patients in Toronto, Canada, March-May 2020.

BACKGROUND: We determined the burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in air and on surfaces in rooms of patients hospitalized with coronavirus disease 2019 (COVID-19) and investigated patient characteristics associated with SARS-CoV-2 environmental contamination. METHODS: Nasopharyngeal swabs, surface, and air samples were collected from the rooms of 78 inpatients with COVID-19 at 6 acute care hospitals in Toronto from March to May 2020. Samples were tested for SARS-CoV-2 ribonucleic acid (RNA), cultured to determine potential infectivity, and whole viral genomes were sequenced. Association between patient factors and detection of SARS-CoV-2 RNA in surface samples were investigated. RESULTS: Severe acute respiratory syndrome coronavirus 2 RNA was detected from surfaces (125 of 474 samples; 42 of 78 patients) and air (3 of 146 samples; 3 of 45 patients); 17% (6 of 36) of surface samples from 3 patients yielded viable virus. Viral sequences from nasopharyngeal and surface samples clustered by patient. Multivariable analysis indicated hypoxia at admission, polymerase chain reaction-positive nasopharyngeal swab (cycle threshold of ≤30) on or after surface sampling date, higher Charlson comorbidity score, and shorter time from onset of illness to sampling date were significantly associated with detection of SARS-CoV-2 RNA in surface samples. CONCLUSIONS: The infrequent recovery of infectious SARS-CoV-2 virus from the environment suggests that the risk to healthcare workers from air and near-patient surfaces in acute care hospital wards is likely limited.

Quantitative Assessment of Viral Dispersion Associated with Respiratory Support Devices in a Simulated Critical Care Environment.

Rationale: Patients with severe coronavirus disease (COVID-19) require supplemental oxygen and ventilatory support. It is unclear whether some respiratory support devices may increase the dispersion of infectious bioaerosols and thereby place healthcare workers at increased risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Objectives: To quantitatively compare viral dispersion from invasive and noninvasive respiratory support modalities.Methods: This study used a simulated ICU room with a breathing-patient simulator exhaling nebulized bacteriophages from the lower respiratory tract with various respiratory support modalities: invasive ventilation (through an endotracheal tube with an inflated cuff connected to a mechanical ventilator), helmet ventilation with a positive end-expiratory pressure (PEEP) valve, noninvasive bilevel positive-pressure ventilation, nonrebreather face masks, high-flow nasal oxygen (HFNO), and nasal prongs.Measurements and Main Results: Invasive ventilation and helmet ventilation with a PEEP valve were associated with the lowest bacteriophage concentrations in the air, and HFNO and nasal prongs were associated with the highest concentrations. At the intubating position, bacteriophage concentrations associated with HFNO (2.66 × 104 plaque-forming units [PFU]/L of air sampled), nasal prongs (1.60 × 104 PFU/L of air sampled), nonrebreather face masks (7.87 × 102 PFU/L of air sampled), and bilevel positive airway pressure (1.91 × 102 PFU/L of air sampled) were significantly higher than those associated with invasive ventilation (P < 0.05 for each). The difference between bacteriophage concentrations associated with helmet ventilation with a PEEP valve (4.29 × 10-1 PFU/L of air sampled) and bacteriophage concentrations associated with invasive ventilation was not statistically significant.Conclusions: These findings highlight the potential differential risk of dispersing virus among respiratory support devices and the importance of appropriate infection prevention and control practices and personal protective equipment for healthcare workers when caring for patients with transmissible respiratory viral infections such as SARS-CoV-2.

In Silico Study Suggesting the Bias of Primers Choice in the Molecular Identification of Fungal Aerosols.

This paper presents an in silico analysis to assess the current state of the fungal UNITE database in terms of the two eukaryote nuclear ribosomal regions, Internal Transcribed Spacers 1 and 2 (ITS1 and ITS2), used in describing fungal diversity. Microbial diversity is often evaluated with amplicon-based high-throughput sequencing approaches, which is a target enrichment method that relies on the amplification of a specific target using particular primers before sequencing. Thus, the results are highly dependent on the quality of the primers used for amplification. The goal of this study is to validate if the mismatches of the primers on the binding sites of the targeted taxa could explain the differences observed when using either ITS1 or ITS2 in describing airborne fungal diversity. Hence, the choice of the pairs of primers for each barcode concur with a study comparing the performance of ITS1 and ITS2 in three occupational environments. The sequence length varied between the amplicons retrieved from the UNITE database using the pair of primers targeting ITS1 and ITS2. However, the database contains an equal number of unidentified taxa from ITS1 and ITS2 regions in the six taxonomic levels employed (phylum, class, order, family, genus, species). The chosen ITS primers showed differences in their ability to amplify fungal sequences from the UNITE database. Eleven taxa consisting of Trichocomaceae, Dothioraceae, Botryosphaeriaceae, Mucorales, Saccharomycetes, Pucciniomycetes, Ophiocordyceps, Microsporidia, Archaeorhizomycetes, Mycenaceae, and Tulasnellaceae showed large variations between the two regions. Note that members of the latter taxa are not all typical fungi found in the air. As no universal method is currently available to cover all the fungal kingdom, continuous work in designing primers, and particularly combining multiple primers targeting the ITS region is the best way to compensate for the biases of each one to get a larger view of the fungal diversity.

An Overview of Bioinformatics Tools for DNA Meta-Barcoding Analysis of Microbial Communities of Bioaerosols: Digest for Microbiologists.

High-throughput DNA sequencing (HTS) has changed our understanding of the microbial composition present in a wide range of environments. Applying HTS methods to air samples from different environments allows the identification and quantification (relative abundance) of the microorganisms present and gives a better understanding of human exposure to indoor and outdoor bioaerosols. To make full use of the avalanche of information made available by these sequences, repeated measurements must be taken, community composition described, error estimates made, correlations of microbiota with covariates (variables) must be examined, and increasingly sophisticated statistical tests must be conducted, all by using bioinformatics tools. Knowing which analysis to conduct and which tools to apply remains confusing for bioaerosol scientists, as a litany of tools and data resources are now available for characterizing microbial communities. The goal of this review paper is to offer a guided tour through the bioinformatics tools that are useful in studying the microbial ecology of bioaerosols. This work explains microbial ecology features like alpha and beta diversity, multivariate analyses, differential abundances, taxonomic analyses, visualization tools and statistical tests using bioinformatics tools for bioaerosol scientists new to the field. It illustrates and promotes the use of selected bioinformatic tools in the study of bioaerosols and serves as a good source for learning the "dos and don'ts" involved in conducting a precise microbial ecology study.

A Comparison of Whole Genome Sequencing of SARS-CoV-2 Using Amplicon-Based Sequencing, Random Hexamers, and Bait Capture.

Genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is increasingly important to monitor the transmission and adaptive evolution of the virus. The accessibility of high-throughput methods and polymerase chain reaction (PCR) has facilitated a growing ecosystem of protocols. Two differing protocols are tiling multiplex PCR and bait capture enrichment. Each method has advantages and disadvantages but a direct comparison with different viral RNA concentrations has not been performed to assess the performance of these approaches. Here we compare Liverpool amplification, ARTIC amplification, and bait capture using clinical diagnostics samples. All libraries were sequenced using an Illumina MiniSeq with data analyzed using a standardized bioinformatics workflow (SARS-CoV-2 Illumina GeNome Assembly Line; SIGNAL). One sample showed poor SARS-CoV-2 genome coverage and consensus, reflective of low viral RNA concentration. In contrast, the second sample had a higher viral RNA concentration, which yielded good genome coverage and consensus. ARTIC amplification showed the highest depth of coverage results for both samples, suggesting this protocol is effective for low concentrations. Liverpool amplification provided a more even read coverage of the SARS-CoV-2 genome, but at a lower depth of coverage. Bait capture enrichment of SARS-CoV-2 cDNA provided results on par with amplification. While only two clinical samples were examined in this comparative analysis, both the Liverpool and ARTIC amplification methods showed differing efficacy for high and low concentration samples. In addition, amplification-free bait capture enriched sequencing of cDNA is a viable method for generating a SARS-CoV-2 genome sequence and for identification of amplification artifacts.

Comparison of the performance of ITS1 and ITS2 as barcodes in amplicon-based sequencing of bioaerosols.

This paper presents the performance of two eukaryotic genomic ribosomal regions, ITS1 and ITS2, in describing fungal diversity in aerosol samples using amplicon-based High-Throughput Sequencing (HTS). Composting sites, biomethanization facilities, and dairy farms, all affected by the presence of fungi, were visited to collect air samples. The amplicon-based HTS approach is a target enrichment method that relies on the amplification of a specific target using particular primers before sequencing. Thus, the results are highly dependent on the quality of amplification. For this reason, the authors of this paper used a shotgun metagenomic approach to compare its outcome with the amplicon-based method. Indeed, shotgun metagenomic does not rely on any amplification prior to sequencing, because all genes are sequenced without a specific target. In addition, culture methods were added to the analyses in biomethanization and dairy farms samples to validate their contribution to fungal diversity of aerosols. The results obtained are unequivocal towards ITS1 outperformance to ITS2 in terms of richness, and taxonomic coverage. The differential abundance analysis did demonstrate that some taxa were exclusively detected only by ITS2, and vice-versa for ITS1. However, the shotgun metagenomic approach showed a taxonomic profile more resembling to ITS1 than ITS2. Based on these results, neither of the barcodes evaluated is perfect in terms of distinguishing all species. Using both barcodes offers a broader view of the fungal aerosol population. However, with the actual knowledge, the authors strongly recommend using ITS1 as a universal fungal barcode for quick general analyses of diversity and when limited financial resources are available, primarily due its ability to capture taxonomic profiles similar to those obtained using the shotgun metagenomic. The culture comparison with amplicon-based sequencing showed the complementarity of both approaches in describing the most abundant taxa.

Field sampling of indoor bioaerosols.

Because bioaerosols are related to adverse health effects in exposed humans and indoor environments represent a unique framework of exposure, concerns about indoor bioaerosols have risen over recent years. One of the major issues in indoor bioaerosol research is the lack of standardization in the methodology, from air sampling strategies and sample treatment to the analytical methods applied. The main characteristics to consider in the choice of indoor sampling methods for bioaerosols are the sampler performance, the representativeness of the sampling, and the concordance with the analytical methods to be used. The selection of bioaerosol collection methods is directly dependent on the analytical methods, which are chosen to answer specific questions raised while designing a study for exposure assessment. In this review, the authors present current practices in the analytical methods and the sampling strategies, with specificity for each type of microbe (fungi, bacteria, archaea and viruses). In addition, common problems and errors to be avoided are discussed. Based on this work, recommendations are made for future efforts towards the development of viable bioaerosol samplers, standards for bioaerosol exposure limits, and making association studies to optimize the use of the big data provided by high-throughput sequencing methods.

Bioaerosols Play a Major Role in the Nasopharyngeal Microbiota Content in Agricultural Environment.

Background: Bioaerosols are a major concern for public health and sampling for exposure assessment purposes is challenging. The nasopharyngeal region could be a potent carrier of long-term bioaerosol exposure agents. This study aimed to evaluate the correlation between nasopharyngeal bacterial flora of swine workers and the swine barns bioaerosol biodiversity. Methods: Air samples from eight swine barns as well as nasopharyngeal swabs from pig workers (n = 25) and from a non-exposed control group (n = 29) were sequenced using 16S rRNA gene high-throughput sequencing. Wastewater treatment plants were used as the industrial, low-dust, non-agricultural environment control to validate the microbial link between the bioaerosol content (air) and the nasopharynxes of workers. Results: A multivariate analysis showed air samples and nasopharyngeal flora of pig workers cluster together, compared to the non-exposed control group. The significance was confirmed with the PERMANOVA statistical test (p-value of 0.0001). Unlike the farm environment, nasopharynx samples from wastewater workers did not cluster with air samples from wastewater treatment plants. The difference in the microbial community of nasopharynx of swine workers and a control group suggest that swine workers are carriers of germs found in bioaerosols. Conclusion: Nasopharynx sampling and microbiota could be used as a proxy of air sampling for exposure assessment studies or for the determination of exposure markers in highly contaminated agricultural environments.

On the interpretation of bioaerosol exposure measurements and impacts on health.

Bioaerosols are recognized as one of the main transmission routes for infectious diseases and are responsible for other various types of health effects through inhalation and potential ingestion. Associating exposure with bioaerosol and health problems is challenging, and adequate exposure monitoring is a top priority for aerosol scientists. The multiple factors affecting bioaerosol content, the variability in the focus of each bioaerosol exposure study, and the variations in experimental design and the standardization of methods make bioaerosol exposure studies very difficult. Therefore, the health impacts of bioaerosol exposure are still poorly understood. This paper presents a brief description of a state-of-the-art development in bioaerosol exposure studies supported by studies on several related subjects. The main objective of this paper is to propose new considerations for bioaerosol exposure guidelines and the development of tools and study designs to better interpret bioaerosol data. The principal observations and findings are the discrepancy of the applicable methods in bioaerosol studies that makes result comparison impossible. Furthermore, the silo mentality helps in creating a bigger gap in the knowledge accumulated about bioaerosol exposure. Innovative and original ideas are presented for aerosol scientists and health scientists to consider and discuss. Although many examples cited herein are from occupational exposure, the discussion has relevance to any human environment. This work gives concrete suggestions for how to design a full bioaerosol study that includes all of the key elements necessary to help understand the real impacts of bioaerosol exposure in the short term. The creation of the proposed bioaerosol public database could give crucial information to control the public health. Implications: How can we move toward a bioaerosol exposure guidelines? The creation of the bioaerosol public database will help accumulate information for long-term association studies and help determine specific exposure biomarkers to bioaerosols. The implementation of such work will lead to a deeper understanding and more efficient utilization of bioaerosol studies to prevent public health hazards.

Recovery of Fungal Cells from Air Samples: a Tale of Loss and Gain.

There are limitations in establishing a direct link between fungal exposure and health effects due to the methodology used, among other reasons. Culture methods ignore the nonviable/uncultivable fraction of airborne fungi. Molecular methods allow for a better understanding of the environmental health impacts of microbial communities. However, there are challenges when applying these techniques to bioaerosols, particularly to fungal cells. This study reveals that there is a loss of fungal cells when samples are recovered from air using wet samplers and aimed to create and test an improved protocol for concentrating mold spores via filtration prior to DNA extraction. Results obtained using the new technique showed that up to 3 orders of magnitude more fungal DNA was retrieved from the samples using quantitative PCR. A sequencing approach with MiSeq revealed a different diversity profile depending on the methodology used. Specifically, 8 fungal families out of 19 families tested were highlighted to be differentially abundant in centrifuged and filtered samples. An experiment using laboratory settings showed the same spore loss during centrifugation for Aspergillus niger and Penicillium roquefortii strains. We believe that this work helped identify and address fungal cell loss during processing of air samples, including centrifugation steps, and propose an alternative method for a more accurate evaluation of fungal exposure and diversity.IMPORTANCE This work shed light on a significant issue regarding the loss of fungal spores when recovered from air samples using liquid medium and centrifugation to concentrate air particles before DNA extraction. We provide proof that the loss affects the overall fungal diversity of aerosols and that some taxa are differentially more affected than others. Furthermore, a laboratory experiment confirmed the environmental results obtained during field sampling. The filtration protocol described in this work offers a better description of the fungal diversity of aerosols and should be used in fungal aerosol studies.

Fungal aerosols at dairy farms using molecular and culture techniques.

Occupational exposure to harmful bioaerosols in industrial environments is a real threat to the workers. In particular, dairy-farm workers are exposed to high levels of fungal bioaerosols on a daily basis. Associating bioaerosol exposure and health problems is challenging and adequate exposure monitoring is a top priority for aerosol scientists. Using only culture-based tools does not express the overall microbial diversity and underestimate the large spectrum of microbes in bioaerosols and therefore the extended fungal profile that farmers are exposed to. The aim of this study was to provide an in-depth characterization of fungal exposure at Eastern Canadian dairy farms using qPCR and high-throughput sequencing methods. Specific primers were used for the quantification of Penicillium/Aspergillus and Aspergillus fumigatus in dairy farms air samples. Illumina Miseq sequencing of the ITS1 region provided sequences for the diversity analyses. The minimum and maximum concentration of Penicillium/Aspergillus ranged from 4.6 × 106 to 9.4 × 106 gene copies/m3 and from 1 × 104 gene copies/m3 to 4.8 × 105 gene copies/m3 for Aspergillus fumigatus, respectively. Differences in the diversity profiles of the five dairy farms support the idea that the novel approach identifies a large number of fungal taxa. The most striking differences include Microascus, Piptoporus, Parastagonospora, Dissoconium, Microdochium, Tubilicrinis, Ganoderma, Ustilago, Phlebia and Wickerhamomyces. The presence of a diverse portrait of fungi in air may represent a health risk for workers who are exposed on a daily basis. The broad spectrum of fungi detected in this study includes many known pathogens like Aspergillus, Acremonium, Alternaria and Fusarium. Adequate monitoring of bioaerosol exposure is necessary to evaluate and minimize risks.

Bioaerosol Sampler Choice Should Consider Efficiency and Ability of Samplers To Cover Microbial Diversity.

Bioaerosol studies aim to describe the microbial content and increase understanding of the aerosolization processes linked to diseases. Air samplers are used to collect, identify, and quantify bioaerosols. Studies comparing the performances of air samplers have typically used a culture approach or have targeted a specific microorganism in laboratory settings. The objective of this study was to use environmental field samples to compare the efficiencies of 3 high-airflow-rate samplers for describing bioaerosol diversity using a next-generation sequencing approach. Two liquid cyclonic impactors and one electrostatic filter dry sampler were used in four wastewater treatment plants to target bacterial diversity and in five dairy farms to target fungal diversity. The dry electrostatic sampler was consistently more powerful in collecting more fungal and bacterial operational taxonomic units (OTUs). Substantial differences in OTU abundances between liquid and dry sampling were revealed. The majority of the diversity revealed by dry electrostatic sampling was not identified using the cyclonic liquid impactors. The findings from this work suggest that the choice of a bioaerosol sampler should include information about the efficiency and ability of samplers to cover microbial diversity. Although these results suggest that electrostatic filters result in better coverage of the microbial diversity among the tested air samplers, further studies are needed to confirm this hypothesis. While it is difficult to determine a single universally optimal air sampler, this work provides an in-depth look at some of the considerations that are essential when choosing an air sampler for studying the microbial ecology of bioaerosols.IMPORTANCE Associating bioaerosol exposure and health problems is challenging, and adequate exposure monitoring is a priority for scientists in the field. Conclusions that can be drawn from bioaerosol exposure studies are highly dependent on the design of the study and the methodologies used. The air sampling strategy is the first methodological step leading to an accurate interpretation of what is present in the air. Applying new molecular approaches to evaluate the efficiencies of the different types of samplers used in the field is necessary in order to circumvent traditional approaches and the biases they introduce to such studies. The results and conclusions provided in this paper should be taken in consideration when conducting a bioaerosol study.

Fungal bioaerosols in biomethanization facilities.

Biomethanization is a new technology used for green-waste valorization where organic waste is biodegraded by microbial communities under anaerobic conditions. The main product of this type of anaerobic digestion is a biogas used as an energy source. Moving and handling organic waste may lead to the emission of high concentrations of bioaerosols. High exposure levels are associated with adverse health effects amongst green environment workers. Fungal spores are suspected to play a role in many respiratory illnesses. There is a paucity of information related to the detailed fungal diversity in biomethanization facilities. The aim of this study was to provide an in-depth description of fungal bioaerosols in biomethanization work environments using a next-generation sequencing approach combined with real-time polymerase chain reaction (PCR). Two biomethanization facilities treating different wastes were visited during the sampling campaign (n = 16). Quantification of Penicillium/Aspergillus and Aspergillus fumigatus revealed a greater exposure risk during summer for both facilities visited. Concentrations of Penicillium and Aspergillus were similar in all work areas in both biomethanization facilities. Taxonomy analyses showed that the type of waste treated affects the fungal diversity of aerosols emitted. Although eight classes were evenly distributed in all samples, Eurotiomycetes were more dominant in the first facility and Agaricomycetes were dominant in the second one. A large diversity profile was observed in bioaerosols from both facilities showing the presence of pathogenic fungi. The following fungi detected are known allergens and/or are opportunistic pathogens: Aspergillus, Malassezia, Emericella, Fusarium, Acremonium, and Candida. Daily exposure to these fungi may put workers at risk. The information from this study can be used as a reference for minimizing occupational exposure in future biomethanization facilities. Implications: Biomethanization is a new technology used for green-waste valorization where organic waste is biodegraded by microbial communities. Effective waste management is increasingly recognized as a strategic approach for achieving newly created regulations concerning the disposal of organic residues; therefore, an expansion of facilities is expected. Workers' exposure to diverse fungal communities is certain, as fungi are ubiquitous and necessary in organic matter decomposition. Monitoring this occupational exposure is important in order to prevent workers' health problems.

Bioaerosol sampling and detection methods based on molecular approaches: No pain no gain.

Bioaerosols are among the less studied particles in the environment. The lack of standardization in sampling procedures, difficulties related to the effect of sampling processes on the integrity of microorganisms, and challenges associated with the application of environmental microbiology analyses and molecular and culture methods frighten many young scientists. Every microorganism has its own particularities and acts differently when aerosolized in various conditions. Because the air is an extremely biologically diluted environment, it is necessary to concentrate its content before any analysis is performed. Challenges faced when applying molecular methods to air samples reveal the need for a better standardization of approaches for cell and nucleic acid recovery, the choice of genetic markers, and interpretation of data. This paper presents a few of the limits and difficulties tackled when molecular methods are applied to bioaerosols, suggests some improvements by specifying the critical stages that should be considered when studying the microbial ecology of bioaerosols, and provides thoughtful insights on how to overcome the challenges encountered.