Stability of Aerosolized SARS-CoV-2 on Masks and Transfer to Skin.

The potential for masks to act as fomites in the transmission of SARS-CoV-2 has been suggested but not demonstrated experimentally or observationally. In this study, we aerosolized a suspension of SARS-CoV-2 in saliva and used a vacuum pump to pull the aerosol through six different types of masks. After 1 h at 28 °C and 80% RH, SARS-CoV-2 infectivity was not detectable on an N95 and surgical mask, was reduced by 0.7 log10 on a nylon/spandex mask, and was unchanged on a polyester mask and two different cotton masks when recovered by elution in a buffer. SARS-CoV-2 RNA remained stable for 1 h on all masks. We pressed artificial skin against the contaminated masks and detected the transfer of viral RNA but no infectious virus to the skin. The potential for masks contaminated with SARS-CoV-2 in aerosols to act as fomites appears to be less than indicated by studies involving SARS-CoV-2 in very large droplets.

Shower water contributes viable nontuberculous mycobacteria to indoor air.

Nontuberculous mycobacteria (NTM) are frequently present in municipal drinking water and building plumbing, and some are believed to cause respiratory tract infections through inhalation of NTM-containing aerosols generated during showering. However, the present understanding of NTM transfer from water to air is insufficient to develop NTM risk mitigation strategies. This study aimed to characterize the contribution of shower water to the abundance of viable NTM in indoor air. Shower water and indoor air samples were collected, and 16S rRNA and rpoB genes were sequenced. The sequencing results showed that running the shower impacted the bacterial community structure and NTM species composition in indoor air by transferring certain bacteria from water to air. A mass balance model combined with NTM quantification results revealed that on average 1/132 and 1/254 of NTM cells in water were transferred to air during 1 hour of showering using a rain and massage showerhead, respectively. A large fraction of the bacteria transferred from water to air were membrane-damaged, i.e. they had compromised membranes based on analysis by live/dead staining and flow cytometry. However, the damaged NTM in air were recoverable as shown by growth in a culture medium mimicking the respiratory secretions of people with cystic fibrosis, implying a potential infection risk by NTM introduced to indoor air during shower running. Among the recovered NTM, Mycobacterium mucogenicum was the dominant species as determined by rpoB gene sequencing. Overall, this study lays the groundwork for future pathogen risk management and public health protection in the built environment.

Infectious SARS-CoV-2 Is Emitted in Aerosol Particles.

Respiratory viruses such as SARS-CoV-2 are transmitted in respiratory droplets and aerosol particles, which are released during talking, breathing, coughing, and sneezing. Noncontact transmission of SARS-CoV-2 has been demonstrated, suggesting transmission via virus carried through the air. Here, we demonstrate that golden Syrian hamsters produce infectious SARS-CoV-2 in aerosol particles prior to and concurrent with the onset of mild clinical signs of disease. The average emission rate in this study was 25 infectious virions/hour on days 1 and 2 postinoculation, with average viral RNA levels 200-fold higher than infectious virus in aerosol particles. The majority of virus was contained within particles <5 µm in size. Thus, we provide direct evidence that, in hamsters, SARS-CoV-2 is an airborne virus. IMPORTANCE SARS-CoV-2 is a respiratory virus and has been isolated from the air near COVID-19 patients. Here, using a hamster model of infection, we demonstrate that SARS-CoV-2 is emitted in aerosol particles prior to and concurrent with the onset of mild disease. Virus is contained primarily within aerosol particles <5 µm in size, which can remain airborne and be inhaled. These findings indicate that SARS-CoV-2 is an airborne virus and support the use of ventilation to reduce SARS-CoV-2 transmission.

Next generation sequencing approaches to evaluate water and wastewater quality.

The emergence of next generation sequencing (NGS) is revolutionizing the potential to address complex microbiological challenges in the water industry. NGS technologies can provide holistic insight into microbial communities and their functional capacities in water and wastewater systems, thus eliminating the need to develop a new assay for each target organism or gene. However, several barriers have hampered wide-scale adoption of NGS by the water industry, including cost, need for specialized expertise and equipment, challenges with data analysis and interpretation, lack of standardized methods, and the rapid pace of development of new technologies. In this critical review, we provide an overview of the current state of the science of NGS technologies as they apply to water, wastewater, and recycled water. In addition, a systematic literature review was conducted in which we identified over 600 peer-reviewed journal articles on this topic and summarized their contributions to six key areas relevant to the water and wastewater fields: taxonomic classification and pathogen detection, functional and catabolic gene characterization, antimicrobial resistance (AMR) profiling, bacterial toxicity characterization, Cyanobacteria and harmful algal bloom identification, and virus characterization. For each application, we have presented key trends, noteworthy advancements, and proposed future directions. Finally, key needs to advance NGS technologies for broader application in water and wastewater fields are assessed.

Radiofrequency Ablation vs Electrocautery Blinded Randomized Trial: Impact on Clinically Meaningful Outcomes.

OBJECTIVE: To analyze patients' return to normal activity, pain scores, narcotic use, and adverse events after undergoing tonsillectomy or adenotonsillectomy with monopolar electrocautery or radiofrequency ablation. STUDY DESIGN: Randomized double-blinded clinical trial based on prospective parallel design. SETTING: Academic medical center and tertiary children's hospital between March 2018 and July 2019. METHODS: Inclusion criteria included patients aged ≥3 years with surgical indication of recurrent tonsillitis or airway obstruction/sleep-disordered breathing. Patients were randomly assigned to monopolar electrocautery or radiofrequency ablation. Patients were blinded to treatment assignment. Survey questions answered via text or email were collected daily until postoperative day 15. The primary outcome was the patient's return to normal activity. Secondary outcomes included daily pain score, total amount of postoperative narcotic use, and adverse events. RESULTS: Of the 236 patients who met inclusion criteria and were randomly assigned to radiofrequency ablation or monopolar electrocautery, 230 completed the study (radiofrequency ablation, n = 112; monopolar electrocautery, n = 118). There was no statistically significant difference between the groups in the number of days for return to normal activity (P = .89), daily pain scores over 15 postoperative days (P = .46), postoperative narcotic use (P = .61), or return to hospital for any reason (P = .60), including bleeding as an adverse event (P = .13). CONCLUSIONS: As one of the largest randomized controlled trials examining instrumentation in tonsillectomy, our data do not show a difference between monopolar electrocautery and radiofrequency ablation with regard to return to normal activity, daily pain scores, total postoperative narcotic use, or adverse events.

Building Interdisciplinary Partnerships for Community-Engaged Environmental Health Research in Appalachian Virginia.

This article describes a collaboration among a group of university faculty, undergraduate students, local governments, local residents, and U.S. Army staff to address long-standing concerns about the environmental health effects of an Army ammunition plant. The authors describe community-responsive scientific pilot studies that examined potential environmental contamination and a related undergraduate research course that documented residents' concerns, contextualized those concerns, and developed recommendations. We make a case for the value of resource-intensive university-community partnerships that promote the production of knowledge through collaborations across disciplinary paradigms (natural/physical sciences, social sciences, health sciences, and humanities) in response to questions raised by local residents. Our experience also suggests that enacting this type of research through a university class may help promote researchers' adoption of "epistemological pluralism", and thereby facilitate the movement of a study from being "multidisciplinary" to "transdisciplinary".

Viruses in the Built Environment (VIBE) meeting report.

BACKGROUND: During a period of rapid growth in our understanding of the microbiology of the built environment in recent years, the majority of research has focused on bacteria and fungi. Viruses, while probably as numerous, have received less attention. In response, the Alfred P. Sloan Foundation supported a workshop entitled "Viruses in the Built Environment (VIBE)," at which experts in environmental engineering, environmental microbiology, epidemiology, infection prevention, fluid dynamics, occupational health, metagenomics, and virology convened to synthesize recent advances and identify key research questions and knowledge gaps regarding viruses in the built environment. RESULTS: Four primary research areas and funding priorities were identified. First, a better understanding of viral communities in the built environment is needed, specifically which viruses are present and their sources, spatial and temporal dynamics, and interactions with bacteria. Second, more information is needed about viruses and health, including viral transmission in the built environment, the relationship between virus detection and exposure, and the definition of a healthy virome. The third research priority is to identify and evaluate interventions for controlling viruses and the virome in the built environment. This encompasses interactions among viruses, buildings, and occupants. Finally, to overcome the challenge of working with viruses, workshop participants emphasized that improved sampling methods, laboratory techniques, and bioinformatics approaches are needed to advance understanding of viruses in the built environment. CONCLUSIONS: We hope that identifying these key questions and knowledge gaps will engage other investigators and funding agencies to spur future research on the highly interdisciplinary topic of viruses in the built environment. There are numerous opportunities to advance knowledge, as many topics remain underexplored compared to our understanding of bacteria and fungi. Video abstract.

Bacterial and fungal ecology on air conditioning cooling coils is influenced by climate and building factors.

The presence of biofilms on the cooling coils of commercial air conditioning (AC) units can significantly reduce the heat transfer efficiency of the coils and may lead to the aerosolization of microbes into occupied spaces of a building. We investigated how climate and AC operation influence the ecology of microbial communities on AC coils. Forty large-scale commercial ACs were considered with representation from warm-humid and hot-dry climates. Both bacterial and fungal ecologies, including richness and taxa, on the cooling coil surfaces were significantly impacted by outdoor climate, through differences in dew point that result in increased moisture (condensate) on coils, and by the minimum efficiency reporting value (MERV 8 vs MERV 14) of building air filters. Based on targeted qPCR and sequence analysis, low efficiency upstream filters (MERV 8) were associated with a greater abundance of pathogenic bacteria and medically relevant fungi. As the implementation of air conditioning continues to grow worldwide, better understanding of the factors impacting microbial growth and ecology on cooling coils should enable more rational approaches for biofilm control and ultimately result in reduced energy consumption and healthier buildings.

Seasonal dynamics of DNA and RNA viral bioaerosol communities in a daycare center.

BACKGROUND: Viruses play an important role in ecosystems, including the built environment (BE). While numerous studies have characterized bacterial and fungal microbiomes in the BE, few have focused on the viral microbiome (virome). Longitudinal microbiome studies provide insight into the stability and dynamics of microbial communities; however, few such studies exist for the microbiome of the BE, and most have focused on bacteria. Here, we present a longitudinal, metagenomic-based analysis of the airborne DNA and RNA virome of a children's daycare center. Specifically, we investigate how the airborne virome varies as a function of season and human occupancy, and we identify possible sources of the viruses and their hosts, mainly humans, animals, plants, and insects. RESULTS: Season strongly influenced the airborne viral community composition, and a single sample collected when the daycare center was unoccupied suggested that occupancy also influenced the community. The pattern of influence differed between DNA and RNA viromes. Human-associated viruses were much more diverse and dominant in the winter, while the summertime virome contained a high relative proportion and diversity of plant-associated viruses. CONCLUSIONS: This airborne microbiome in this building exhibited seasonality in its viral community but not its bacterial community. Human occupancy influenced both types of communities. By adding new data about the viral microbiome to complement burgeoning information about the bacterial and fungal microbiomes, this study contributes to a more complete understanding of the airborne microbiome.

Coordinated Sampling of Microorganisms Over Freshwater and Saltwater Environments Using an Unmanned Surface Vehicle (USV) and a Small Unmanned Aircraft System (sUAS).

Biological aerosols (bioaerosols) are ubiquitous in terrestrial and aquatic environments and may influence cloud formation and precipitation processes. Little is known about the aerosolization and transport of bioaerosols from aquatic environments. We designed and deployed a bioaerosol-sampling system onboard an unmanned surface vehicle (USV; a remotely operated boat) to collect microbes and monitor particle sizes in the atmosphere above a salt pond in Falmouth, MA, United States and a freshwater lake in Dublin, VA, United States. The bioaerosol-sampling system included a series of 3D-printed impingers, two different optical particle counters, and a weather station. A small unmanned aircraft system (sUAS; a remotely operated airplane) was used in a coordinated effort with the USV to collect microorganisms on agar media 50 m above the surface of the water. Samples from the USV and sUAS were cultured on selective media to estimate concentrations of culturable microorganisms (bacteria and fungi). Concentrations of microbes from the sUAS ranged from 6 to 9 CFU/m3 over saltwater, and 12 to 16 CFU/m3 over freshwater (over 10-min sampling intervals) at 50 m above ground level (AGL). Concentrations from the USV ranged from 0 (LOD) to 42,411 CFU/m3 over saltwater, and 0 (LOD) to 56,809 CFU/m3 over freshwater (over 30-min sampling intervals) in air near the water surface. Particle concentrations recorded onboard the USV ranged from 0 (LOD) to 288 µg/m3 for PM1, 1 to 290 µg/m3 for PM2.5, and 1 to 290 µg/m3 for PM10. A general trend of increasing concentration with an increase in particle size was recorded by each sensor. Through laboratory testing, the collection efficiency of the 3D-printed impingers was determined to be 75% for 1 µm beads and 99% for 3 µm beads. Additional laboratory tests were conducted to determine the accuracy of the miniaturized optical particle counters used onboard the USV. Future work aims to understand the distribution of bioaerosols above aquatic environments and their potential association with cloud formation and precipitation processes.

Influenza Virus Infectivity Is Retained in Aerosols and Droplets Independent of Relative Humidity.

Pandemic and seasonal influenza viruses can be transmitted through aerosols and droplets, in which viruses must remain stable and infectious across a wide range of environmental conditions. Using humidity-controlled chambers, we studied the impact of relative humidity on the stability of 2009 pandemic influenza A(H1N1) virus in suspended aerosols and stationary droplets. Contrary to the prevailing paradigm that humidity modulates the stability of respiratory viruses in aerosols, we found that viruses supplemented with material from the apical surface of differentiated primary human airway epithelial cells remained equally infectious for 1 hour at all relative humidities tested. This sustained infectivity was observed in both fine aerosols and stationary droplets. Our data suggest, for the first time, that influenza viruses remain highly stable and infectious in aerosols across a wide range of relative humidities. These results have significant implications for understanding the mechanisms of transmission of influenza and its seasonality.

Survival of the Enveloped Virus Phi6 in Droplets as a Function of Relative Humidity, Absolute Humidity, and Temperature.

Infectious diseases caused by enveloped viruses, such as influenza, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS), cause thousands of deaths and billions of dollars of economic losses per year. Studies have found a relationship among temperature, humidity, and influenza virus incidence, transmission, or survival; however, there are contradictory claims about whether absolute humidity (AH) or relative humidity (RH) is most important in mediating virus infectivity. Using the enveloped bacteriophage Phi6, which has been suggested as a surrogate for influenza viruses and coronaviruses, we designed a study to discern whether AH, RH, or temperature is a better predictor of virus survival in droplets. Our results show that Phi6 survived best at high (>85%) and low (<60%) RHs, with a significant decrease in infectivity at mid-range RHs (∼60 to 85%). At an AH of less than 22 g · m-3, the loss in infectivity was less than 2 orders of magnitude; however, when the AH was greater than 22 g · m-3, the loss in infectivity was typically greater than 6 orders of magnitude. At a fixed RH of 75%, infectivity was very sensitive to temperature, decreasing two orders of magnitude between 19°C and 25°C. We used random forest modeling to identify the best environmental predictors for modulating virus infectivity. The model explained 83% of variation in Phi6 infectivity and suggested that RH is the most important factor in controlling virus infectivity in droplets. This research provides novel information about the complex interplay between temperature, humidity, and the survival of viruses in droplets.IMPORTANCE Enveloped viruses are responsible for a number of infectious diseases resulting in thousands of deaths and billions of dollars of economic losses per year in the United States. There has been a lively debate in the literature over whether absolute humidity (AH) or relative humidity (RH) modulates virus infectivity. We designed a controlled study and used advanced statistical modeling techniques specifically to address this question. By providing an improved understanding of the relationship between environmental conditions and virus infectivity, our work will ultimately lead to improved strategies for predicting and controlling disease transmission.

Microbial community structure of sea spray aerosols at three California beaches.

We characterized the microbial communities in sea spray aerosols (SSA), water and sand of three beaches in central California (Cowell Beach, Baker Beach and Lovers Point) by sequencing the V4 region of the 16S rRNA gene. Average concentrations of 16S rRNA genes in SSA ranged from 2.4 × 104 to 1.4 × 105 gene copies per m3 of air. A total of 9781 distinct OTUs were identified in SSA and of these, 1042 OTUs were found in SSA of all beaches. SSA microbial communities included marine taxa, as well as some associated with the terrestrial environment. SSA taxa included organisms that play important roles in biogeochemical cycling of elements such as Planctomyces and Synechococcus, as well as those representing potential pathogens and fecal indicator bacteria including Staphylococcus epidermidis and Enterococcus spp. There were a large number of shared OTUs among SSA and water, and there was relatively high similarity between SSA and water communities. Results are consistent with a conceptual model where SSA is generated by breaking waves and bubble bursting in marine waters and that enables the transport of microorganisms from the sea to sand or other environments.

Ten Questions Concerning the Aerosolization and Transmission of Legionella in the Built Environment.

Legionella is a genus of pathogenic Gram-negative bacteria responsible for a serious disease known as legionellosis, which is transmitted via inhalation of this pathogen in aerosol form. There are two forms of legionellosis: Legionnaires' disease, which causes pneumonia-like symptoms, and Pontiac fever, which causes influenza-like symptoms. Legionella can be aerosolized from various water sources in the built environment including showers, faucets, hot tubs/swimming pools, cooling towers, and fountains. Incidence of the disease is higher in the summertime, possibly because of increased use of cooling towers for air conditioning systems and differences in water chemistry when outdoor temperatures are higher. Although there have been decades of research related to Legionella transmission, many knowledge gaps remain. While conventional wisdom suggests that showering is an important source of exposure in buildings, existing measurements do not provide strong support for this idea. There has been limited research on the potential for Legionella transmission through heating, ventilation, and air conditioning (HVAC) systems. Epidemiological data suggest a large proportion of legionellosis cases go unreported, as most people who are infected do not seek medical attention. Additionally, controlled laboratory studies examining water-to-air transfer and source tracking are still needed. Herein, we discuss ten questions that spotlight current knowledge about Legionella transmission in the built environment, engineering controls that might prevent future disease outbreaks, and future research that is needed to advance understanding of transmission and control of legionellosis.

Factors Shaping the Human Exposome in the Built Environment: Opportunities for Engineering Control.

The "exposome" is a term describing the summation of one's lifetime exposure to microbes and chemicals. Such exposures are now recognized as major drivers of human health and disease. Because humans spend ∼90% of their time indoors, the built environment exposome merits particular attention. Herein we utilize an engineering perspective to advance understanding of the factors that shape the built environment exposome and its influence on human wellness and disease, while simultaneously informing development of a framework for intentionally controlling the exposome to protect public health. Historically, engineers have been focused on controlling chemical and physical contaminants and on eradicating microbes; however, there is a growing awareness of the role of "beneficial" microbes. Here we consider the potential to selectively control the materials and chemistry of the built environment to positively influence the microbial and chemical components of the indoor exposome. Finally, we discuss research gaps that must be addressed to enable intentional engineering design, including the need to define a "healthy" built environment exposome and how to control it.

Seasonal Dynamics of the Airborne Bacterial Community and Selected Viruses in a Children's Daycare Center.

Children's daycare centers appear to be hubs of respiratory infectious disease transmission, yet there is only limited information about the airborne microbial communities that are present in daycare centers. We have investigated the microbial community of the air in a daycare center, including seasonal dynamics in the bacterial community and the presence of specific viral pathogens. We collected filters from the heating, ventilation, and air conditioning (HVAC) system of a daycare center every two weeks over the course of a year. Amplifying and sequencing the 16S rRNA gene revealed that the air was dominated by Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes that are commonly associated with the human skin flora. Clear seasonal differences in the microbial community were not evident; however, the community structure differed when the daycare center was closed and unoccupied for a 13-day period. These results suggest that human occupancy, rather than the environment, is the major driver in shaping the microbial community structure in the air of the daycare center. Using PCR for targeted viruses, we detected a seasonal pattern in the presence of respiratory syncytial virus that included the period of typical occurrence of the disease related to the virus; however, we did not detect the presence of adenovirus or rotavirus at any time.

Sources of airborne microorganisms in the built environment.

Each day people are exposed to millions of bioaerosols, including whole microorganisms, which can have both beneficial and detrimental effects. The next chapter in understanding the airborne microbiome of the built environment is characterizing the various sources of airborne microorganisms and the relative contribution of each. We have identified the following eight major categories of sources of airborne bacteria, viruses, and fungi in the built environment: humans; pets; plants; plumbing systems; heating, ventilation, and air-conditioning systems; mold; dust resuspension; and the outdoor environment. Certain species are associated with certain sources, but the full potential of source characterization and source apportionment has not yet been realized. Ideally, future studies will quantify detailed emission rates of microorganisms from each source and will identify the relative contribution of each source to the indoor air microbiome. This information could then be used to probe fundamental relationships between specific sources and human health, to design interventions to improve building health and human health, or even to provide evidence for forensic investigations.

Total Virus and Bacteria Concentrations in Indoor and Outdoor Air.

Viruses play important roles in microbial ecology and some infectious diseases, but relatively little is known about concentrations, sources, transformation, and fate of viruses in the atmosphere. We have measured total airborne concentrations of virus-like and bacteria-like particles (VLPs between 0.02 µm and 0.5 µm in size and BLPs between 0.5 µm and 5 µm) in nine locations: a classroom, a daycare center, a dining facility, a health center, three houses, an office, and outdoors. Indoor concentrations of both VLPs and BLPs were ~105 particles m-3, and the virus-to-bacteria ratio was 0.9 ± 0.1 (mean ± standard deviation across different locations). There were no significant differences in concentration between different indoor environments. VLP and BLP concentrations in outdoor air were 2.6 and 1.6 times higher, respectively, than in indoor air. At the single outdoor site, the virus-to-bacteria ratio was 1.4.

Challenges of studying viral aerosol metagenomics and communities in comparison with bacterial and fungal aerosols.

Despite the obvious importance of viral transmission and ecology to medicine, epidemiology, ecology, agriculture, and microbiology, the study of viral bioaerosols and community structure has remained a vastly underexplored area, due to both unresolved technical challenges and unrecognized importance. High-throughput, culture-independent techniques such as viral metagenomics are beginning to revolutionize the study of viral ecology. With recent developments in viral metagenomics, characterization of viral bioaerosol communities provides an opportunity for high-impact future research. However, there remain significant challenges for the study of viral bioaerosols compared with viruses in other matrices, such as water, the human gut, and soil. Collecting enough biomass is essential for successful metagenomic analysis, but this is a challenge with viral bioaerosols. Herein, we provide a perspective on the importance of studying viral bioaerosols, the challenges of studying viral community structure, and the potential opportunities for improvements in methods to study viruses in indoor and outdoor air.

Fitness of Erysiphe necator with G143A-Based Resistance to Quinone Outside Inhibitors.

Management of grape powdery mildew (Erysiphe necator) using quinone outside inhibitors (QoIs) has eroded in an increasing number of regions due to resistance development. To determine persistence of resistance when QoIs are withdrawn, competition assays were conducted on unsprayed grape plants (Vitis vinifera 'Chardonnay') by cycling mixtures of resistant and sensitive isolates characterized as genetically diverse based on microsatellite analyses. Under laboratory conditions, %G143A, quantified by quantitative polymerase chain reaction (qPCR), increased significantly, indicating competitiveness of the resistant fraction. To confirm competitiveness in the field, trials using potted plants were conducted. Percent G143A tended to decrease in one growing season, probably due to spore migration and mixing of populations with natural background inoculum. In a second season, QoI resistance persisted at high frequency for 4 weeks. Resistant populations were also found to persist in one vineyard without QoI application for four consecutive years. The frequency was still about 25% in the fourth year, with higher frequency (36%) in a hotspot section. QoI-resistant populations with >5% G143A also harbored Y136F in the cyp51 gene that confers some resistance to sterol demethylation inhibitors, another fungicide class for powdery mildew control. Double resistance could have been partly responsible for persistence of QoI resistance at this location.