Defining the sizes of airborne particles that mediate influenza transmission in ferrets.

Epidemics and pandemics of influenza are characterized by rapid global spread mediated by non-mutually exclusive transmission modes. The relative significance between contact, droplet, and airborne transmission is yet to be defined, a knowledge gap for implementing evidence-based infection control measures. We devised a transmission chamber that separates virus-laden particles by size and determined the particle sizes mediating transmission of influenza among ferrets through the air. Ferret-to-ferret transmission was mediated by airborne particles larger than 1.5 µm, consistent with the quantity and size of virus-laden particles released by the donors. Onward transmission by donors was most efficient before fever onset and may continue for 5 days after inoculation. Multiple virus gene segments enhanced the transmissibility of a swine influenza virus among ferrets by increasing the release of virus-laden particles into the air. We provide direct experimental evidence of influenza transmission via droplets and fine droplet nuclei, albeit at different efficiency.

Inactivation of common hospital acquired pathogens on surfaces and in air utilizing engineered water nanostructures (EWNS) based nano-sanitizers.

Infectious diseases represent a major public health challenge worldwide. There are various modes for the transmission of these diseases, with surface and airborne transmission being two of the most important ones. The inefficiencies of current intervention methods have resulted in the emergence of nosocomial infections. Here, we report the use of a nanotechnology based antimicrobial platform using Engineered Water Nanostructures (EWNS) generated using a combined electrospray and ionization of an aqueous suspension of various active ingredients (AIs). These EWNS based nano-sanitizers were tested in terms of their ability to efficiently deliver AI and inactivate Acinetobacter baumannii and influenza H1N1/PR/8 on both surfaces and air. Results indicate a significant reduction in the concertation of the pathogens, while the delivered to pathogen AI doses required for inactivation were miniscule (nanogram level), indicating the viability of such nano-carrier platform as an intervention technology against infectious microorganisms.

Influenza virus aerosols in human exhaled breath: particle size, culturability, and effect of surgical masks.

The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza. We collected samples of exhaled particles (one with and one without a facemask) in two size fractions ("coarse">5 µm, "fine"≤5 µm) from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus. Fine particles contained 8.8 (95% CI 4.1 to 19) fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2) and in the coarse fraction by 25 fold (95% CI 3.5 to 180). Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3) reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (r = 0.17, coarse; r = 0.29, fine fraction). Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples. Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans.

Inactivation of foodborne microorganisms using engineered water nanostructures (EWNS).

Foodborne diseases caused by the consumption of food contaminated with pathogenic microorganisms or their toxins have very serious economic and public health consequences. Here, we explored the effectiveness of a recently developed intervention method for inactivation of microorganisms on fresh produce, and food production surfaces. This method utilizes Engineered Water Nanostructures (EWNS) produced by electrospraying of water vapor. EWNS possess unique properties; they are 25 nm in diameter, remain airborne in indoor conditions for hours, contain Reactive Oxygen Species (ROS) and have very strong surface charge (on average 10 e/structure). Here, their efficacy in inactivating representative foodborne bacteria such as Escherichia coli, Salmonella enterica, and Listeria innocua, on stainless steel surfaces and on organic tomatoes, was assessed. The inactivation was facilitated using two different exposure approaches in order to optimize the delivery of EWNS to bacteria: (1) EWNS were delivered on the surfaces by diffusion and (2) a "draw through" Electrostatic Precipitator Exposure System (EPES) was developed and characterized for EWNS delivery to surfaces. Using the diffusion approach and an EWNS concentration of 24,000 #/cm3, the bacterial concentrations on the surfaces were reduced, depending on the bacterium and the surface type, by values ranging between 0.7 to 1.8 logs. Using the EPES approach and for an aerosol concentration of 50,000 #/cm3 at 90 min of exposure, results show a 1.4 log reduction for E. coli on organic tomato surfaces, as compared to the control (same conditions in regards to temperature and Relative Humidity). Furthermore, for L. innocua, the dose-response relationship was demonstrated and found to be a 0.7 and 1.2 logs removal at 12,000 and 23,000 #/cm3, respectively. The results presented here indicate that this novel, chemical-free, and environmentally friendly intervention method holds potential for development and application in the food industry, as a "green" alternative to existing disinfection methods.

Mycobacteria inactivation using Engineered Water Nanostructures (EWNS).

Airborne transmitted pathogens such as Mycobacterium tuberculosis (Mtb) cause serious, often fatal infectious disease with enormous global health implications. Due to their unique cell wall and slow growth, mycobacteria are among the most resilient microbial forms. Herein we evaluate the ability of an emerging, chemical-free, nanotechnology-based method to inactivate M. parafortuitum (Mtb surrogate). This method is based on the transformation of atmospheric water vapor into engineered water nano-structures (EWNS) via electrospray. We demonstrate that the EWNS can interact with and inactivate airborne mycobacteria, reducing their concentration levels significantly. Additionally, EWNS can inactivate M. parafortuitum on surfaces eight times faster than the control. The mechanism of mycobacteria inactivation was also investigated in this study. It was demonstrated that the EWNS effectively deliver the reactive oxygen species, encapsulated during the electrospray process, to the bacteria oxidizing their cell membrane resulting into inactivation. Overall, this is a method with the potential to become an effective intervention technology in the battle against airborne infections. FROM THE CLINICAL EDITOR: This study demonstrates the feasibility of mycobacterium inactivation in airborne form or on contact surfaces using electrospray activated water nano-structures. Given that the method is free of toxic chemicals, this might become an important tool in the prevention of mycobacterial infections, which are notoriously hard to treat.

Aerosol susceptibility of influenza virus to UV-C light.

The person-to-person transmission of influenza virus, especially in the event of a pandemic caused by a highly virulent strain of influenza, such as H5N1 avian influenza, is of great concern due to widespread mortality and morbidity. The consequences of seasonal influenza are also substantial. Because airborne transmission appears to play a role in the spread of influenza, public health interventions should focus on preventing or interrupting this process. Air disinfection via upper-room 254-nm germicidal UV (UV-C) light in public buildings may be able to reduce influenza transmission via the airborne route. We characterized the susceptibility of influenza A virus (H1N1, PR-8) aerosols to UV-C light using a benchtop chamber equipped with a UVC exposure window. We evaluated virus susceptibility to UV-C doses ranging from 4 to 12 J/m(2) at three relative humidity levels (25, 50, and 75%). Our data show that the Z values (susceptibility factors) were higher (more susceptible) to UV-C than what has been reported previously. Furthermore, dose-response plots showed that influenza virus susceptibility increases with decreasing relative humidity. This work provides an essential scientific basis for designing and utilizing effective upper-room UV-C light installations for the prevention of the airborne transmission of influenza by characterizing its susceptibility to UV-C.

Toward a Test Protocol for Surface Decontamination Using a Mobile Whole-room UVGI Device†.

Mobile whole-room UVGI devices are used in healthcare settings to control surface-borne pathogens. Unfortunately, no standard method comparing the efficacy of these devices is available. We accessed the effect of shadows on UVC 254 nm inactivation. The evaluation of a mobile whole-room UVGI device used spores of Bacillus atrophaeus as a surrogate for Clostridium difficile and Staphylococcus aureus as a surrogate for MSRA. Inactivation after 10 min of exposure varied significantly depending on whether the spores received direct UV exposure (4.3 log reduction), both direct and reflected UV exposure (3.0-4.0 log reduction) or reflected UV exposure alone (<1.0 log reduction). The susceptibility (z-value) for inactivation of B. atrophaeus spores on a glass surface was estimated to be 0.00312 m2  J-1 . Staphylococcus aureus microbial log reductions were approximately 5.5 for direct UV exposure, 3.6-5.2 for both direct and reflected UV exposure and approximately 2.75 for only reflected UV exposure. Our measurement of reflected dose ranged from 0.46% to 1.47%. Based on our findings, B. atrophaeus spores should be considered as a model organism for testing the impact of shadows on mobile whole-room UVGI device inactivation. Optimizing the reflected component of whole-room UVGI is important, especially for UVC-resistant organisms.

Role of absolute humidity in the inactivation of influenza viruses on stainless steel surfaces at elevated temperatures.

Influenza virus has been found to persist in the environment for hours to days, allowing for secondary transmission of influenza via inanimate objects known as fomites. We evaluated the efficacy of heat and moisture for the decontamination of surfaces for the purpose of preventing of the spread of influenza. Aqueous suspensions of influenza A virus were deposited onto stainless steel coupons, allowed to dry under ambient conditions, and exposed to temperatures of 55 degrees C, 60 degrees C, or 65 degrees C and relative humidity (RH) of 25%, 50%, or 75% for up to 1 h. Quantitative virus assays were performed on the solution used to wash the viruses from these coupons, and results were compared with the solution used to wash coupons treated similarly but left under ambient conditions. Inactivation of influenza virus on surfaces increased with increasing temperature, RH, and exposure time. Reductions of greater than 5 logs of influenza virus on surfaces were achieved at temperatures of 60 and 65 degrees C, exposure times of 30 and 60 min, and RH of 50 and 75%. Our data also suggest that absolute humidity is a better predictor of surface inactivation than RH and allows the prediction of survival using two parameters rather than three. Modest amounts of heat and adequate moisture can provide effective disinfection of surfaces while not harming surfaces, electrical systems, or mechanical components, leaving no harmful residues behind after treatment and requiring a relatively short amount of time.

Modeling the airborne survival of influenza virus in a residential setting: the impacts of home humidification.

BACKGROUND: Laboratory research studies indicate that aerosolized influenza viruses survive for longer periods at low relative humidity (RH) conditions. Further analysis has shown that absolute humidity (AH) may be an improved predictor of virus survival in the environment. Maintaining airborne moisture levels that reduce survival of the virus in the air and on surfaces could be another tool for managing public health risks of influenza. METHODS: A multi-zone indoor air quality model was used to evaluate the ability of portable humidifiers to control moisture content of the air and the potential related benefit of decreasing survival of influenza viruses in single-family residences. We modeled indoor AH and influenza virus concentrations during winter months (Northeast US) using the CONTAM multi-zone indoor air quality model. A two-story residential template was used under two different ventilation conditions - forced hot air and radiant heating. Humidity was evaluated on a room-specific and whole house basis. Estimates of emission rates for influenza virus were particle-size specific and derived from published studies and included emissions during both tidal breathing and coughing events. The survival of the influenza virus was determined based on the established relationship between AH and virus survival. RESULTS: The presence of a portable humidifier with an output of 0.16 kg water per hour in the bedroom resulted in an increase in median sleeping hours AH/RH levels of 11 to 19% compared to periods without a humidifier present. The associated percent decrease in influenza virus survival was 17.5 - 31.6%. Distribution of water vapor through a residence was estimated to yield 3 to 12% increases in AH/RH and 7.8-13.9% reductions in influenza virus survival. CONCLUSION: This modeling analysis demonstrates the potential benefit of portable residential humidifiers in reducing the survival of aerosolized influenza virus by controlling humidity indoors.

Author Correction: Respiratory virus shedding in exhaled breath and efficacy of face masks.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Respiratory virus shedding in exhaled breath and efficacy of face masks.

We identified seasonal human coronaviruses, influenza viruses and rhinoviruses in exhaled breath and coughs of children and adults with acute respiratory illness. Surgical face masks significantly reduced detection of influenza virus RNA in respiratory droplets and coronavirus RNA in aerosols, with a trend toward reduced detection of coronavirus RNA in respiratory droplets. Our results indicate that surgical face masks could prevent transmission of human coronaviruses and influenza viruses from symptomatic individuals.

An optimized method to detect influenza virus and human rhinovirus from exhaled breath and the airborne environment.

Respiratory viruses are difficult to characterize in the airborne environment due to their low concentration and the presence of a wide range of inhibitors. As a first step in studying airborne viruses, we optimized molecular biology methods to quantify influenza viruses and human rhinovirus. Quantitative PCR was used as an endpoint to evaluate RNA extraction techniques and reverse transcription protocols. We found that a Trizol-chloroform extraction and MultiScribe RT increased virus detection 10-fold compared to methods used in published field studies of airborne respiratory viruses. Virus was recovered without inhibition from samples contaminated with up to 50 microg/sample of particulate matter. The methods developed can be used in studies of airborne respiratory viruses.

Occupant UV exposure measurements for upper-room ultraviolet germicidal irradiation.

The threshold limit value (TLV) guideline for ultraviolet (UV) radiation specifies that irradiance measurements to ensure occupant safety be taken over an angle of 80° at the sensor. The purpose of this study was to evaluate the effect of an 80° field of view (FOV) tube on lower room UV-C irradiation measurements. Measurements were made in an experimental chamber at a height of 1.73m with and without an FOV tube. The FOV tube reduced the lower room irradiance readings by 18-34%, a statistically significant reduction compared to the bare sensor. An 80° FOV tube should be used for lower room irradiance measurements to comply with the TLV guideline. The resulting lower readings would allow more UV-C radiation in the upper room without compromising occupant safety. More UV-C radiation in the upper room could increase efficacy of UVGI systems for reducing transmission of airborne infectious diseases. In addition, recommendations are made to standardize lower room irradiance measurement techniques.

Optimization of a nanotechnology based antimicrobial platform for food safety applications using Engineered Water Nanostructures (EWNS).

A chemical free, nanotechnology-based, antimicrobial platform using Engineered Water Nanostructures (EWNS) was recently developed. EWNS have high surface charge, are loaded with reactive oxygen species (ROS), and can interact-with, and inactivate an array of microorganisms, including foodborne pathogens. Here, it was demonstrated that their properties during synthesis can be fine tuned and optimized to further enhance their antimicrobial potential. A lab based EWNS platform was developed to enable fine-tuning of EWNS properties by modifying synthesis parameters. Characterization of EWNS properties (charge, size and ROS content) was performed using state-of-the art analytical methods. Further their microbial inactivation potential was evaluated with food related microorganisms such as Escherichia coli, Salmonella enterica, Listeria innocua, Mycobacterium parafortuitum, and Saccharomyces cerevisiae inoculated onto the surface of organic grape tomatoes. The results presented here indicate that EWNS properties can be fine-tuned during synthesis resulting in a multifold increase of the inactivation efficacy. More specifically, the surface charge quadrupled and the ROS content increased. Microbial removal rates were microorganism dependent and ranged between 1.0 to 3.8 logs after 45 mins of exposure to an EWNS aerosol dose of 40,000 #/cm(3).

Comparison of extremely low frequency (ELF) magnetic field personal exposure monitors.

The MultiWave System III (MW III), a recently developed personal monitor for extremely low frequency (ELF) magnetic fields, was compared with the standard EMDEX Lite (Electric and Magnetic Field Digital Exposure System), the type of monitor widely used in epidemiology and other exposure assessments. The MW III captures three-axis magnetic field waveforms for the calculation of many exposure metrics, while the EMDEX monitors measure only the root-mean-squared (RMS) vector magnitude (or resultant). Thirty-eight partial period personal samples were monitored in six different job classifications. The sampling time for each personal sample ranged from 90 to 133 min, with a mean sample time of 110 min. The EMDEX Lite and MW III were evaluated by comparing the maximum and partial period time-weighted average (TWA) of the ELF magnitude. TWA exposures measured for the 38 partial period samples by the EMDEX Lite ranged from 1.2 to 65.3 mG, with a mean of 18.1 mG, while corresponding values for the MW III ranged from 1.1 to 65.8 mG, with a mean of 17.7 mG. The maximum magnetic field exposures measured for the 38 partial period personal samples by the EMDEX Lite ranged from 27.0 to 420.2 mG, with a mean of 216.3 mG, while corresponding values for the MW III ranged from 40.2 to 1311.8 mG, with a mean of 368.4 mG. The maximum and TWA ELF magnetic field exposures measured by the EMDEX Lite and MW III were compared using a two-tailed, paired t-test. Analyses indicate that there was no significant difference in the TWA magnetic field magnitude measured by the EMDEX Lite and MW III. On the other hand, the EMDEX Lite reported significantly lower (P=0.002) maximum magnetic field measurements compared to the MW III. From a detailed analysis of the time traces, the EMDEX Lite appears to measure the ELF magnitude inaccurately when the field changes rapidly over a 4-s sampling interval. The results of this comparison suggest that the standard EMDEX Lite and MW III provide similar measure of the TWA magnetic field in a variety of occupational settings and ELF magnetic field magnitudes. However, the EMDEX Lite underestimates maximum exposures when compared to the MW III.

Influence of Bioaerosol Source Location and Ceiling Fan Direction on Eggcrate Upper-room Ultraviolet Germicidal Irradiation.

BACKGROUND: Eggcrate upper-room ultraviolet germicidal irradiation (UVGI), an engineering control method for reducing the airborne transmission of infectious diseases, was recently developed as an alternative to conventional upper-room UVGI using conventional louvered fixtures. A UV screen, which is composed of open-cell eggcrate panels supported in a frame designed for a conventional suspended ceiling, was used to minimize UV radiation in the lower room. A ceiling fan, which was blowing upward directly above the microbiological source, provided vertical air exchange between the upper and lower room. This system has been shown to be significantly more effective than conventional upper-room UVGI. STUDY DESIGN: In the present study, the microbiological source location and the airflow direction due to the ceiling fan were varied in order to evaluate their impact on germicidal efficacy. RESULTS: The test results clearly showed that placing an aerosol source directly underneath an upward blowing ceiling fan produces the maximum efficacy. CONCLUSIONS: The likely explanation for this outcome is that the fan sucks the microorganisms emitted by the source into the UV beam before being mixed with the air in the room. This is somewhat analogous to local exhaust ventilation in which the contaminant is removed prior to being mixed with the air in the room. Thus, when possible, the ceiling fan should be blowing upward and directly above the source. However, for experimental testing, the source location should be varied in order to access the range of germicidal efficacies that can be expected.

A chemical free, nanotechnology-based method for airborne bacterial inactivation using engineered water nanostructures.

Airborne pathogens are associated with the spread of infectious diseases and increased morbidity and mortality. Herein we present an emerging chemical free, nanotechnology-based method for airborne pathogen inactivation. This technique is based on transforming atmospheric water vapor into Engineered Water Nano-Structures (EWNS) via electrospray. The generated EWNS possess a unique set of physical, chemical, morphological and biological properties. Their average size is 25 nm and they contain reactive oxygen species (ROS) such as hydroxyl and superoxide radicals. In addition, EWNS are highly electrically charged (10 electrons per particle on average). A link between their electric charge and the reduction of their evaporation rate was illustrated resulting in an extended lifetime (over an hour) at room conditions. Furthermore, it was clearly demonstrated that the EWNS have the ability to interact with and inactivate airborne bacteria. Finally, inhaled EWNS were found to have minimal toxicological effects, as illustrated in an acute in-vivo inhalation study using a mouse model. In conclusion, this novel, chemical free, nanotechnology-based method has the potential to be used in the battle against airborne infectious diseases.

Development and Performance Evaluation of an Exhaled-Breath Bioaerosol Collector for Influenza Virus.

The importance of the aerosol mode for transmission of influenza is unknown. Understanding the role of aerosols is essential to developing public health interventions such as the use of surgical masks as a source control to prevent the release of infectious aerosols. Little information is available on the number and size of particles generated by infected persons, which is partly due to the limitations of conventional air samplers, which do not efficiently capture fine particles or maintain microorganism viability. We designed and built a new sampler, called the G-II, that collects exhaled breath particles that can be used in infectivity analyses. The G-II allows test subjects to perform various respiratory maneuvers (i.e. tidal breathing, coughing, and talking) and allows subjects to wear a mask or respirator during testing. A conventional slit impactor collects particles > 5.0 µm. Condensation of water vapor is used to grow remaining particles, including fine particles, to a size large enough to be efficiently collected by a 1.0 µm slit impactor and be deposited into a buffer-containing collector. We evaluated the G-II for fine particle collection efficiency with inert particle aerosols and evaluated infective virus collection using influenza A virus aerosols. Testing results showed greater than 85% collection efficiency for particles greater than 50nm and influenza virus collection comparable with a reference SKC BioSampler®. The new design will enable determination of exhaled infectious virus generation rate and evaluate control strategies such as wearing a surgical type mask to prevent the release of viruses from infected persons.

Collection of aerosolized human cytokines using Teflon® filters.

BACKGROUND: Collection of exhaled breath samples for the analysis of inflammatory biomarkers is an important area of research aimed at improving our ability to diagnose, treat and understand the mechanisms of chronic pulmonary disease. Current collection methods based on condensation of water vapor from exhaled breath yield biomarker levels at or near the detection limits of immunoassays contributing to problems with reproducibility and validity of biomarker measurements. In this study, we compare the collection efficiency of two aerosol-to-liquid sampling devices to a filter-based collection method for recovery of dilute laboratory generated aerosols of human cytokines so as to identify potential alternatives to exhaled breath condensate collection. METHODOLOGY/PRINCIPAL FINDINGS: Two aerosol-to-liquid sampling devices, the SKC® Biosampler and Omni 3000™, as well as Teflon® filters were used to collect aerosols of human cytokines generated using a HEART nebulizer and single-pass aerosol chamber setup in order to compare the collection efficiencies of these sampling methods. Additionally, methods for the use of Teflon® filters to collect and measure cytokines recovered from aerosols were developed and evaluated through use of a high-sensitivity multiplex immunoassay. Our results show successful collection of cytokines from pg/m(3) aerosol concentrations using Teflon® filters and measurement of cytokine levels in the sub-picogram/mL concentration range using a multiplex immunoassay with sampling times less than 30 minutes. Significant degradation of cytokines was observed due to storage of cytokines in concentrated filter extract solutions as compared to storage of dry filters. CONCLUSIONS: Use of filter collection methods resulted in significantly higher efficiency of collection than the two aerosol-to-liquid samplers evaluated in our study. The results of this study provide the foundation for a potential new technique to evaluate biomarkers of inflammation in exhaled breath samples.