Cincinnati, OH: U.S. Environmental Protection Agency, EPA/600/R-10/127, 2010 Dec; :1-28
The U.S. Environmental Protection Agency (EPA) has identified the detection of pathogenic airborne microorganisms following a terrorist attack as a critical component of an effective response. Detection of such pathogens would require validated sampling techniques that could be used by multiple laboratories following a homeland security event. To meet this requirement, EPA's National Homeland Security Research Center (NHSRC), along with other EPA divisions and sister agencies, published Standardized Analytical Methods for Use During Homeland Security Events, Revision 1.0 (September 2004, EPA/600/R-04/126) and Revision 2.0 (September 2005, EPA/600/R-04/126B). It was retitled in 2007 and published as Standardized Analytical Methods for Environmental Restoration Following Homeland Security Events (SAM) in revision 3.0 (February 2007,EPA/600/R-07/015), 3.1 (November 2007, EPA/600/R-07/136), 4.0 (September 2008, EPA/600/R-07/126D), and 5.0 (September 2009, EPA/600/R-07/126E) which contain suggested assays for use by laboratories tasked with performing confirmatory analysis of environmental samples following a homeland security event. Concern regarding human exposure to bioaerosols laden with toxic agents has led to the development of a variety of air sampling devices. However, data addressing the efficacy of current samplers to detect viable and infectious airborne viruses is sparse and points to the need of a more efficient sampler. A two-stage cyclone bioaerosol sampler has recently been developed by the National Institute for Occupational Safety and Health (NIOSH). The NIOSH sampler is unique in that it size-fractionates bioaerosols and collects them in disposable centrifuge tubes, facilitating direct processing of samples. As air is drawn into an inlet at 3.5 L/min, the first stage of the NIOSH sampler, particles that are >4 um are collected into a 15 ml centrifuge tube. In the second stage, 1 to 4 um particles are collected into a 1.5 ml microcentrifuge tube, and particles that are <1 um are collected onto a 37 mm polytetrafluoroethylene (PTFE) filter. In previous studies employing a calm air chamber to evaluate the performance of the NIOSH bioaerosol cyclone personal sampler, a nebulized suspension of the FluMistŪ vaccine (Medimmune, Gaithersburg, MD), which contains live, attenuated influenza virus, (3) was tested. Quantitative polymerase chain reaction (qPCR) analysis results demonstrated that the sampler effectively captured and separated viral-laden particles based on their aerodynamic size. Similarly, while conducting a field study during the February 2008 influenza season, aerosol samples were successfully collected and size-fractionated by both personal and stationary samplers situated in the West Virginia University Hospital Emergency Department. With qPCR analysis, 53% of the detectable viral RNA was found in the respirable fraction of the aerosol. Respirable particles are defined as those small enough to be drawn down into the alveolar region of the lungs. Collectively, these studies suggest the potential for airborne transmission of influenza. However, the viability and potential infectivity of the captured viral aerosols were not addressed during either study. Development of the methodologies to assess viability and infectivity would directly address any dangers posed by virus-containing particles and improve the utility of the NIOSH bioaerosol sampler for the collection of airborne viruses. Numerous reports have shown that the viability of airborne viruses is dependent on the virus type, environmental conditions, and on the methods of collection and handling of bioaerosol samples. The survival of airborne influenza, for example, has been shown to greatly depend on the relative humidity, as well as on ambient air temperature and ultraviolet radiation levels. The number of viable influenza, measles, and mumps virus recovered from a bubbling sampler increases when a virus maintenance fluid is used in the sampler rather than distilled water. Airborne bacteriophages have been shown to retain viability longer after collection when they refrigerated rather than stored at room temperature. The objectives of this project were to examine the ability of the NIOSH bioaerosol sampler to collect viable airborne viruses and to devise techniques to preserve the viability of airborne viruses during and following collection. During experimentation, influenza A virus was used as the surrogate virus due to the potential of newly emerging strains to create a pandemic. In this study, we showed that viable infectious virus were present in all three fractions of collected particles. The highest percentages of viable influenza virus were found in the 1-4 um fraction (48-55%) and the <1 um fraction (26-41%) while the smallest percentages were found in the >4 um fraction (11-19%). Further, significantly more total and more viable viral particles were collected in the <1 um range by increasing the air sampling time beyond 15 min. Attempts to increase the viability of aerosolized viruses by collecting them onto test tube walls coated with mucin or agar, or into test tubes filled with various amounts of Hank's balanced salt solution (HBSS), did not further improve viability.
Don Beezhold, Allergy and Clinical Immunology BranchHealth Effects Laboratory Division, Centers for Disease Control, National Institute for Occupational Safety and Health, Morgantown, WV 26505