James McDevitt

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 µm 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.

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.

Airborne influenza virus detection with four aerosol samplers using molecular and infectivity assays: considerations for a new infectious virus aerosol sampler.

As a first step in conducting studies of airborne influenza transmission, we compared the collection performance of an SKC Biosampler, a compact cascade impactor (CCI), Teflon filters, and gelatin filters by collecting aerosolized influenza virus in a one-pass aerosol chamber. Influenza virus infectivity was determined using a fluorescent focus assay and influenza virus nucleic acid (originating from viable and non-viable viruses) was measured using quantitative PCR. The results showed that the SKC Biosampler recovered and preserved influenza virus infectivity much better than the other samplers - the CCI, Teflon, and gelatin filters recovered only 7-22% of infectious viruses compared with the Biosampler. Total virus collection was not significantly different among the SKC Biosampler, the gelatin, and Teflon filters, but was significantly lower in the CCI. Results from this study show that a new sampler is needed for virus aerosol sampling, as commercially available samplers do not efficiently collect and conserve virus infectivity. Applications for a new sampler include studies of airborne disease transmission and bioterrorism monitoring. Design parameters for a new sampler include high collection efficiency for fine particles and liquid sampling media to preserve infectivity. Practical Implications New air samplers are needed to study infectious airborne viruses and learn about airborne disease transmission. As a first step in designing a new air sampler to collect influenza virus we evaluated four commercial samplers and determined necessary design parameters for a new collector.

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

ABSTRACT 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°C, 60°C, or 65°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°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.

Development of a PCR-Enzyme Immunoassay Oligoprobe Detection Method for Toxoplasma gondii Oocysts, Incorporating PCR Controls

ABSTRACT 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.

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.

Characterization of UVC Light Sensitivity of Vaccinia Virus

ABSTRACT 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/m2, 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.

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.

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.

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°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°C, the disinfection rate was about 1.3 log10 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.