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.

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.

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.

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.

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.

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.

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.

Inactivating influenza viruses on surfaces using hydrogen peroxide or triethylene glycol at low vapor concentrations.

BACKGROUND: Surfaces in congregate settings, such as vehicles used for mass transportation, can become contaminated with infectious microorganisms and facilitate disease transmission. We disinfected surfaces contaminated with H1N1 influenza viruses using hydrogen peroxide (HP) vapor at concentrations below 100 ppm and triethylene glycol (TEG)-saturated air containing 2 ppm of TEG at 25 degrees C. METHODS: Influenza viruses in aqueous suspensions were deposited on stainless-steel coupons, allowed to dry at ambient conditions, and then exposed for up to 15 minutes to 10 to 90 ppm of HP vapor or TEG-saturated air. Virus assays were done on the solution used to wash the viruses from these coupons and from coupons treated similarly but without exposure to HP or TEG vapor. RESULTS: After 2.5 minutes, exposure to 10-ppm HP vapor resulted in 99% inactivation. For air saturated with TEG at 25 to 29 degrees C, the disinfection rate was about 1.3 log(10) reductions per hour, about 16 times faster than the measured natural inactivation rate under ambient conditions. CONCLUSIONS: Vapor concentrations of 10 ppm HP or 2 ppm TEG can provide effective surface disinfection. At these low concentrations, the potential for damage to even the avionics of an airplane would be expected to be minimal. At a TEG vapor concentration of 2 ppm, there are essentially no health risks to people.

Inactivation of poxviruses by upper-room UVC light in a simulated hospital room environment.

In the event of a smallpox outbreak due to bioterrorism, delays in vaccination programs may lead to significant secondary transmission. In the early phases of such an outbreak, transmission of smallpox will take place especially in locations where infected persons may congregate, such as hospital emergency rooms. Air disinfection using upper-room 254 nm (UVC) light can lower the airborne concentrations of infective viruses in the lower part of the room, and thereby control the spread of airborne infections among room occupants without exposing occupants to a significant amount of UVC. Using vaccinia virus aerosols as a surrogate for smallpox we report on the effectiveness of air disinfection, via upper-room UVC light, under simulated real world conditions including the effects of convection, mechanical mixing, temperature and relative humidity. In decay experiments, upper-room UVC fixtures used with mixing by a conventional ceiling fan produced decreases in airborne virus concentrations that would require additional ventilation of more than 87 air changes per hour. Under steady state conditions the effective air changes per hour associated with upper-room UVC ranged from 18 to 1000. The surprisingly high end of the observed range resulted from the extreme susceptibility of vaccinia virus to UVC at low relative humidity and use of 4 UVC fixtures in a small room with efficient air mixing. Increasing the number of UVC fixtures or mechanical ventilation rates resulted in greater fractional reduction in virus aerosol and UVC effectiveness was higher in winter compared to summer for each scenario tested. These data demonstrate that upper-room UVC has the potential to greatly reduce exposure to susceptible viral aerosols. The greater survival at baseline and greater UVC susceptibility of vaccinia under winter conditions suggest that while risk from an aerosol attack with smallpox would be greatest in winter, protective measures using UVC may also be most efficient at this time. These data may also be relevant to influenza, which also has improved aerosol survival at low RH and somewhat similar sensitivity to UVC.

Influenza virus in human exhaled breath: an observational study.

BACKGROUND: Recent studies suggest that humans exhale fine particles during tidal breathing but little is known of their composition, particularly during infection. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a study of influenza infected patients to characterize influenza virus and particle concentrations in their exhaled breath. Patients presenting with influenza-like-illness, confirmed influenza A or B virus by rapid test, and onset within 3 days were recruited at three clinics in Hong Kong, China. We collected exhaled breath from each subject onto Teflon filters and measured exhaled particle concentrations using an optical particle counter. Filters were analyzed for influenza A and B viruses by quantitative polymerase chain reaction (qPCR). Twelve out of thirteen rapid test positive patients provided exhaled breath filter samples (7 subjects infected with influenza B virus and 5 subjects infected with influenza A virus). We detected influenza virus RNA in the exhaled breath of 4 (33%) subjects--three (60%) of the five patients infected with influenza A virus and one (14%) of the seven infected with influenza B virus. Exhaled influenza virus RNA generation rates ranged from <3.2 to 20 influenza virus RNA particles per minute. Over 87% of particles exhaled were under 1 microm in diameter. CONCLUSIONS: These findings regarding influenza virus RNA suggest that influenza virus may be contained in fine particles generated during tidal breathing, and add to the body of literature suggesting that fine particle aerosols may play a role in influenza transmission.

Characterization of UVC light sensitivity of vaccinia virus.

Interest in airborne smallpox transmission has been renewed because of concerns regarding the potential use of smallpox virus as a biothreat agent. Air disinfection via upper-room 254-nm germicidal UV (UVC) light in public buildings may reduce the impact of primary agent releases, prevent secondary airborne transmission, and be effective prior to the time when public health authorities are aware of a smallpox outbreak. We characterized the susceptibility of vaccinia virus aerosols, as a surrogate for smallpox, to UVC light by using a benchtop, one-pass aerosol chamber. We evaluated virus susceptibility to UVC doses ranging from 0.1 to 3.2 J/m(2), three relative humidity (RH) levels (20%, 60%, and 80%), and suspensions of virus in either water or synthetic respiratory fluid. Dose-response plots show that vaccinia virus susceptibility increased with decreasing RH. These plots also show a significant nonlinear component and a poor fit when using a first-order decay model but show a reasonable fit when we assume that virus susceptibility follows a log-normal distribution. The overall effects of RH (P < 0.0001) and the suspending medium (P = 0.014) were statistically significant. When controlling for the suspending medium, the RH remained a significant factor (P < 0.0001) and the effect of the suspending medium was significant overall (P < 0.0001) after controlling for RH. Virus susceptibility did not appear to be a function of virus particle size. This work provides an essential scientific basis for the design of effective upper-room UVC installations for the prevention of airborne infection transmission of smallpox virus by characterizing the susceptibility of an important orthopoxvirus to UVC exposure.

Use of green fluorescent protein-expressing Aspergillus fumigatus conidia to validate quantitative PCR analysis of air samples collected on filters.

This study used green fluorescent protein (GFP)-expressing Aspergillus fumigatus conidia to compare quantitative PCR (qPCR) enumeration with direct epifluorescent microscopic filter counts of conidia collected on filters in a test chamber. In separate experiments this study initially compared white versus fluorescent light microscopy for counting A. fumigatus conidia, then compared fluorescent microscopy counting of corresponding filter halves, and finally compared qPCR enumeration to counting by fluorescent light microscopy. The use of GFP-expressing conidia with epifluorescent microscopy yielded significantly higher conidia counts (p = 0.026, n = 41, mean of 4.1 conidia per counting field) and 40% faster counting times when compared to conventional counting using white light microscopy. GFP-expressing conidia were aerosolized in a test chamber and collected onto filters. Filters were divided in half and GFP-expressing conidia enumerated. There was no significant difference in the average conidia count per field between corresponding filter halves (p = 0.3, n = 9 filters, mean of 7.8 conidia per counting field). Thus, one filter half could be counted optically and would provide a reliable estimate of filter loading of the corresponding half, which could then be analyzed by qPCR. Filters (n = 38) loaded with GFP conidia in the aerosol chamber were divided in half and analyzed by either fluorescent microscopy or qPCR. The estimated filter loadings ranged from 15-30,000 conidia per filter. There was a linear relationship with a nearly 1:1 ratio between qPCR and direct microscopic estimates of filter loading (y = 1.06x + 404; R(2) = 0.91) showing that the outlined qPCR analysis method is in agreement with an external reference method and is reliable for enumerating A. fumigatus conidia collected on filters. The comparative data derived using GFP-expressing conidia confirmed that qPCR provides sensitive and accurate quantification of DNA from airborne conidia collected on filters.

Development of a method to detect and quantify Aspergillus fumigatus conidia by quantitative PCR for environmental air samples.

Exposure to Aspergillus fumigatus is linked with respiratory diseases such as asthma, invasive aspergillosis, hypersensitivity pneumonitis, and allergic bronchopulmonary aspergillosis. Molecular methods using quantitative PCR (qPCR) offer advantages over culture and optical methods for estimating human exposures to microbiological agents such as fungi. We describe an assay that uses lyticase to digest A. fumigatus conidia followed by TaqMan qPCR to quantify released DNA. This method will allow analysis of airborne A. fumigatus samples collected over extended time periods and provide a more representative assessment of chronic exposure. The method was optimized for environmental samples and incorporates: single tube sample preparation to reduce sample loss, maintain simplicity, and avoid contamination; hot start amplification to reduce non-specific primer/probe annealing; and uracil-N-glycosylase to prevent carryover contamination. An A. fumigatus internal standard was developed and used to detect PCR inhibitors potentially found in air samples. The assay detected fewer than 10 A. fumigatus conidia per qPCR reaction and quantified conidia over a 4-log10 range with high linearity (R2 >0.99) and low variability among replicate standards (CV=2.0%) in less than 4 h. The sensitivity and linearity of qPCR for conidia deposited on filters was equivalent to conidia calibration standards. A. fumigatus DNA from 8 isolates was consistently quantified using this method, while non-specific DNA from 14 common environmental fungi, including 6 other Aspergillus species, was not detected. This method provides a means of analyzing long term air samples collected on filters which may enable investigators to correlate airborne environmental A. fumigatus conidia concentrations with adverse health effects.

Development of a PCR-enzyme immunoassay oligoprobe detection method for Toxoplasma gondii oocysts, incorporating PCR controls.

Infections caused by Toxoplasma gondii are widely prevalent in animals and humans throughout the world. In the United States, an estimated 23% of adolescents and adults have laboratory evidence of T. gondii infection. T. gondii has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease. Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to T. gondii oocysts. Because of the lack of information regarding the prevalence of T. gondii in surface waters, there is a clear need for a rapid, sensitive method to detect T. gondii from water. Currently available animal models and cell culture methods are time-consuming, expensive, and labor-intensive, requiring days or weeks for results to be obtained. Detection of T. gondii nucleic acid by PCR has become the preferred method. We have developed a PCR amplification and detection method for T. gondii oocyst nucleic acid that incorporates the use of hot-start amplification to reduce nonspecific primer annealing, uracil-N-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a nonradioactive DNA hybridization immunoassay. This method can provide positive, confirmed results in less than 1 day. Fewer than 50 oocysts can be detected following recovery of oocyst DNA. Development of a T. gondii oocyst PCR detection method will provide a useful technique to estimate the levels of T. gondii oocysts present in surface waters.

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.