UNIVERSITY OF CINCINNATI

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1 UNIVERSITY OF CINCINNATI Date:_May 29, 2007 I, Nancy Clark Burton, hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Environmental Health It is entitled: Filter Sampling of Airborne Microbial Agents - Evaluation of Filter Materials for Physical Collection Efficiency, Extraction, and Comparison to Traditional Bioaerosol Sampling This work and its defense approved by: Chair: Tiina Reponen, Ph.D. Sergey Grinshpun, Ph.D. Richard Hornung, Ph.D. Daniel Oerther, Ph.D. Pasquale Scarpino, Ph.D.

2 Filter Sampling of Airborne Microbial Agents - Evaluation of Filter Materials for Physical Collection Efficiency, Extraction, and Comparison to Traditional Bioaerosol Sampling A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTOR of PHILOSOPHY In the Department of Environmental Health of the College of Medicine May 2007 By Nancy Clark Burton B.A. in Biology-Chemistry, Wells College, 1982 M.P.H. in Community Health, University of Rochester, 1987 M.S. in Industrial Hygiene, University of Rochester, 1988 ii

3 Abstract Recent events have increased interest in environmental monitoring for microbial contaminants including bioterrorism agents, fungi, viruses, and their components. The purpose of this study was to investigate the use of filter and other traditional sampling techniques for the collection of personal and area samples to evaluate microbial exposures. The first aim of this research was to determine filter materials and extraction methods appropriate for environmental sampling of Bacillus anthracis. Four filter types (mixed cellulose ester (MCE), two polytetrafluoroethylene (PTFE), and gelatin) in conjunction with Button Inhalable Aerosol samplers were tested using B. atrophaeus, a B. anthracis surrogate. Vortexing with ultrasonic agitation and vortexing with shaker agitation extraction methods were evaluated. Mean differences for culturability were not statistically significant for filter materials and extraction methods. The MCE and 1 µm PTFE filters with vortexing and shaker extraction demonstrated the best performance. The second aim determined the physical collection efficiency of commercially available filters for particles in the 10 to 900 nanometer (nm) size range. Biological and non-biological test aerosols were used: B. atrophaeus, MS2-virions, polystyrene latex (PSL) particles, and sodium chloride. The PTFE and gelatin filters showed high collection efficiencies (> 93%) for all test particles. The polycarbonate filters showed lower collection efficiency for small particles especially below 100 nm. A 4-hour loading exposure to PSL particles representing indoor dust levels did not cause significant changes in the collection efficiency. iii

4 The third aim was to determine the effectiveness of gaseous chlorine dioxide (ClO 2 ) treatment on indoor microbial contamination using different monitoring techniques. The best filter (0.3 µm pore size PTFE) from the previous experiments was used in combination with polymerase chain reaction assay. ClO 2 was effective in reducing culturable and total fungi and bacteria in indoor air. The reduction of total count on surfaces was less efficient. The treatment process appears to increase endotoxin and (1 3)-β-D-glucan concentrations. Thorough cleaning of air and surfaces is recommended to achieve acceptable re-occupancy conditions. Overall, this study shows that gelatin and PTFE filters with small vacuum pumps can be used for efficient personal and area environmental sampling for microbial agents including fungi, bacteria, and viruses. iv

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6 Acknowledgements First, I would like to express my gratitude to Dr. Tiina Reponen, for being an excellent mentor and teacher. She has great patience and I admire her dedication to science, teaching, and research. Second, I would like to thank Dr. Sergey Grinshpun for his guidance and assistance in completing the research for this dissertation. The other members of my committee, Dr. Richard Hornung, Dr. Dan Oerther, and Dr. Pat Scarpino, have been very helpful and I appreciate their constructive guidance during this process. I would also like to thank all of my friends and co-workers at the National Institute for Occupational Safety and Health (NIOSH) for their support and encouragement to finish this project. I would like to especially thank Chandran Achutan, Donnie Booher, Chad Dowell, Rick Driscoll, Kevin L. Dunn, Lynda Ewers, Ellen Galloway, Ken Martinez, Rob McCleery, John McKernan, Lauralyn McKernan, Mark Methner, Larry Reed, Robin Smith, Ron Sollberger, Dave Sundin, Aaron Sussell, Allison Tepper, Dawn Tharr, Ken Wallingford, and Martha Waters for their active interest in my progress. I would like to give special thanks to Teresa Seitz for her support in completing this dissertation and her friendship. I would like to express gratitude for my group at work for their patience in the last year Scott Brueck, Lilia Chen, Jessica Gordon, Brad King, and Dave Sylvain. vi

7 I would like to thank my fellow students and the staff at the University of Cincinnati, Center for Health-Related Aerosol Studies including Atin Adhikari, Seung-Hyun Cho, Yulia Iossifova, Shu-An Lee, Taekhee Lee, Sung Chul Seo, and Hongxia Wang for their assistance and friendship. I would like to thank the staff with Sabre Technical Services for allowing me to use their gaseous chlorine dioxide exposure chamber. Most of all, I would like to say thank you to my husband, Dave, my daughters, Rebecca, Laura, and Jessica, my mother, Betty M. Clark, and my extended family for putting up with me throughout this whole process and keeping me focused on what is truly important the ability to enjoy what each day brings. vii

8 Executive Summary The objective of this study was to investigate the use of filter sampling in the laboratory and the field to evaluate personal and area exposures to microorganisms and small particles in comparison to more traditional environmental evaluation techniques. The environmental monitoring that was conducted for the bioterrorism events in 2001 showed that there was a lack of scientific information concerning the collection and recovery efficiency of various filter media which can be used for both culturable and PCR analyses for biological agents. It is important to obtain exposure information for these agents to determine the risk of developing the disease (i.e., infectious dose) or symptoms. Another area of growing interest is the use of nanoparticles (size range: 1 to 100 nanometers [nm]) in the laboratory and industry. The following factors related to filter sampling of airborne microorganisms were evaluated: (1) extraction efficiencies between two methods (vortex-ultrasonic and vortex-shaker) (first laboratory study); (2) physical collection efficiencies of various filter materials (second laboratory study); and (3) applicability of filter sampling in field evaluation of chlorine dioxide treatment for fungi, bacteria, and microbial components in a highly contaminated indoor environment. In the field evaluation, the filter that in the laboratory studies was found to have the best extraction and collection characteristics was used to collect fungal spores in combination with polymerase chain reaction (PCR) analysis and other traditional environmental sampling techniques including culturable analysis and microscopic total counting. viii

9 The first laboratory study looked at physical collection efficiencies, sampling time, and two different extraction procedures for Bacillus atrophaeus endospores using four filter types: mixed cellulose ester (MCE), two polytetrafluoroethylene (PTFE or Teflon), and gelatin filter in conjunction with Button Inhalable samplers. B. atrophaeus, also known as Bacillus subtilis var. niger and Bacillus globigii, was chosen as the test organism since it has been used as a surrogate for B. anthracis for several decades (Burke et al., 2004). Vortexing with ultrasonic agitation and vortexing with shaker agitation extraction methods were evaluated. The results show that the MCE, 1 µm pore size PTFE, and gelatin filters provided physical collection efficiencies of 94% or greater. The 3 µm pore size PTFE filter showed high variability in physical efficiency characteristics between filters. Epifluorescence microscopic analysis of the gelatin filter extraction fluid revealed the presence of contamination by non-culturable bacteria. In summary, the MCE and 1 µm pore size PTFE filters in combination with vortexing and shaker extraction demonstrated the best performance for the filter collection and extraction of B. atrophaeus endospores. Relative culturability and the extraction of B. atrophaeus off the sampling filters were not affected by sampling times of up to 4 hours, however, the gelatin filters became brittle and difficult to handle. The purpose of the second laboratory study was to determine the physical collection efficiency of commercially available filters for collecting airborne bacteria, viruses, and other particles in the 10 to 900 nm size range. Laboratory experiments with various PTFE, polycarbonate, and gelatin filters in conjunction with Button Inhalable samplers and 3-piece cassettes were undertaken. Both biological and non-biological test aerosols were used: B. atrophaeus, MS2, polystyrene ix

10 latex (PSL), and sodium chloride (NaCl). The B. atrophaeus endospores had an aerodynamic diameter of 900 nm, whereas MS2 virion particles had a size of approximately 30 nm. Monodisperse 350 nm PSL particles were used as this size was believed to have the lowest filtration efficiency. NaCl solution (1% weight by volume) was used to create a polydisperse aerosol in the nm range. The physical collection efficiency (PCE) was determined by measuring particle concentrations size-selectively upstream and downstream of the filters. PTFE and gelatin filters showed excellent collection efficiency (> 93%) for all of the test particles. The polycarbonate filters showed lower collection efficiency for small particles especially below 100 nm. Among the tested filters, the lowest collection efficiencies, 49% and 22%, were observed for 1 and 3 µm pore size polycarbonate filters at the particle sizes of 47 and 63 nm, respectively. The results indicate that the effect of filter material is more significant for the size range of single virions than for bacteria. The effect of filter loading was examined by exposing filters to mixtures of PSL particles, which aimed at mimicking typical indoor dust levels and size distributions. A four-hour loading did not cause significant change in the PCE of the tested filters. Next, the filter that in the laboratory studies was found to have the best extraction and collection characteristics was applied to investigate the efficiency of chlorine dioxide treatment against microorganisms. The third portion of this study had two parts: a field evaluation of the use of gaseous chlorine dioxide (ClO 2 ) to treat a highly contaminated indoor environment due to an unidentified roof leak and a laboratory study that focused on two fungal organisms exposed to three gaseous ClO 2 doses (3000, 6000, and 9000 parts per million [ppm]-hours). In the field x

11 evaluation, concentrations of culturable fungi and bacteria, total fungi determined by microscopic count and polymerase chain reaction (PCR) assays, endotoxin, and (1 3)-β-Dglucan were determined before and after the house was tented and treated with ClO 2. A 0.3 µm pore size PTFE filter in conjunction with a 3-piece cassette in combination with PCR analysis was field-tested. The laboratory study was designed to evaluate the efficiency of ClO 2 treatment against known concentrations of spores of Aspergillus versicolor and Stachybotrys chartarum on surface samples. These species are commonly found in damp indoor environments and were detected in the field study. Upon analyses of the environmental data from the treated house, it was found that the culturable bacteria and fungi as well as total fungal count (as determined by microscopic count and PCR) were greatly decreased after the ClO 2 application. However, microscopic analyses of tape samples collected from surfaces after treatment showed that the fungal structures were still present on surfaces. Furthermore, there was an increase in the concentration of airborne endotoxin and (1 3)-β-D-glucan. The laboratory study supported these results also showing increase in the concentration of (1 3)-β-D-glucan after ClO 2 treatment. The 0.3 µm pore size PTFE filter performed well in the field under heavy contamination conditions. In conclusion, the gelatin and PTFE filters had the highest physical collection efficiencies. The pre-sterilized gelatin filter became brittle at 4-hour sampling times and was not useful for total microscopic counting due to non-culturable bacteria that stained and interfered with the counting procedure. However, the gelatin filter performed well for cultivation. The PTFE filters showed good loading capabilities and the resulting extraction solution can be used for a series of xi

12 different analytical techniques such as total microscopic counting, PCR analysis, or culturable count. Both filter types can be used in conjunction with sampling pumps for personal or area monitoring. In addition, gaseous ClO 2 can be used to inactivate environmental microbial contamination in the field, however, there is an increase in measured microbial components (endotoxin and (1 3)-β-D-glucan) that needs to be addressed before the space is re-occupied. xii

13 List of Peer-Reviewed Publications The results of this research have been presented in the following three peer-review journal articles: Clark Burton N, Adhikari A, Grinshpun SA, Hornung R, Reponen T [2005]. The effect of filter material on bioaerosol collection of Bacillus subtilis spores used as a Bacillus anthracis stimulant. J Environ Monit. 7(5): Clark Burton N, Grinshpun SA, Reponen T [2007]. Physical collection efficiency of filter materials for bacteria and viruses. Annals of Occupational Hygiene. 51(2): Clark Burton N, Adhikari A, Iossifova Y, Grinshpun SA, Reponen T. Effect of gaseous chlorine dioxide on indoor microbial contaminants. Journal of the Air and Waste Management Association. (to be submitted in June 2007). The full texts of the peer-reviewed publications are attached in Appendices A1 through A3. These three papers comprise the main body of this dissertation. xiii

14 Table of Contents Abstract...iii Acknowledgements... vi Executive Summary...viii List of Peer-Reviewed Publications...xiii List of Figures... xvi List of Tables...xviii Hypotheses and Specific Aims... 1 Background... 3 General Overview... 3 Overview of Environmental Monitoring Studies for B. anthracis... 5 Efficiency of Filter Sampling... 7 Chlorine Dioxide Treatment... 9 Specific Aim #1: Determine best extraction technique for various filter materials and the effect of sampling time on extraction efficiency and culturability of Bacillus atrophaeus endospores Introduction Materials and Methods Experimental Set-up Physical Collection Efficiency Experimental Protocol for Determining Culturability and Extraction Efficiency Data Analysis Results and Discussion Physical Collection Efficiency Comparison of the Total Count to the Optical Particle Count Relative Culturability Physical Extraction Efficiency Conclusions for Specific Aim # Specific Aim #2: Determine physical collection efficiencies for PC and PTFE filters for small particles using B. atrophaeus endospores, MS2 virions, polystyrene latex (PSL) particles, and sodium chloride particles Introduction Materials and Methods Test Filters Laboratory Setup Filter Sampling Pressure Drop Measurements Test Particles Physical Collection Efficiency Loading Experiment Data Analysis Results and Discussion xiv

15 2.4.1 Pressure Drop Measurements Physical Collection Efficiency Sodium Chloride Challenge Aerosol Effects of Filter Loading Conclusions for Specific Aim # Specific Aim #3: Comparison of microbial sampling techniques before and after chlorine dioxide treatment using known counts of fungal spores on filter surfaces in a laboratory setting and naturally occurring microbial contamination during a house remediation procedure Introduction Methods Field Evaluation Laboratory Study Data Analysis Results Field Study Laboratory Study Discussion Conclusions for Specific Aim # Overall Conclusions and Future Activities References Appendix A: Copies of Peer-Reviewed Publications Resulting From the Ph. D. Study Appendix A1. The effect of filter material on bioaerosol collection of Bacillus subtilis spores used as a Bacillus anthracis stimulant Appendix A2. Physical collection efficiency of filter materials for bacteria and viruses. Annals of Occupational Hygiene Appendix A3. Effect of gaseous chlorine dioxide on indoor microbial contaminants Appendix B: List of Other Publications (Not Included in Ph.D. Dissertation)/Conference Proceedings/Abstracts B1: Peer-Reviewed Publications B2: Conference Proceedings and Abstracts xv

16 List of Figures Page Number Figure 1-1a. Experimental Set-up 65 Figure 1-1b. Picture of Laboratory Set-up 66 Figure 1-2. Comparison of total particle concentration from microscopic count 67 to optical particle counter (OPC) count (total count was average of four replicate samples; OPC count was the average of one-minute measurements during each consecutive experimental run) for the two extraction methods Figure 1-3. Percent relative culturability comparison between vortex and 68 ultrasonic and vortex and shaker agitation extraction methods for mixed cellulose ester, polytetrafluoroethylene, and gelatin filters based on the average of at least three replicates for the two extraction methods with standard deviation bars Figure 2-1. Physical collection efficiency of different filters for three 69 challenge aerosols. Note: No result was obtained with the 0.5 µm PTFE filter challenged with virions due to pressure drop/pump failure. Gelatin filters were not tested with the 0.35 PSL particles (the PCE was assumed to be approximately 100%). The bars and error bars represent the mean values and the standard deviations, respectively (n=3) xvi

17 List of Figures (continued) Page Number Figure 2-2. Physical collection efficiency of filters challenged with NaCl 70 particles aerosolized from a 1% (w/v) suspension as a function of the particle diameter. 71 Figure 2-3. Comparison of laboratory-generated PSL mixture to fieldmeasured indoor aerosols based on number of particles Figure 2-4. Physical collection efficiency measured with 0.35 µm PSL and 72 MS2 virions before and after loading with PSL test mixture particles. Preloading measurements with MS2 virions were conducted with a different set of identical filters. The bars and error bars represent the mean values and the standard deviations, respectively (n=3) Figure Relative Efficiency of Treatment (Average and Standard 73 Deviation) for Five Most Common Fungal Species detected in the PCR Analyses xvii

18 List of Tables Page Number Table 1-1. Physical collection efficiency for different filter materials using B. 74 subtilis endospores with optical particle counter (OPC) Table 1-2. Average and standard deviation of physical extraction efficiencies 75 as a percentage for MCE and 1 µm PTFE filters Table 2-1. Characteristics of tested filters 76 Table 2-2. Measured pressure drop values for tested filters with samplers 77 Table 3-1. Sampling and analysis methods in the field study 78 Table 3-2. Geometric mean and range for indoor bioaerosol concentrations 79 before and after chlorine dioxide treatment Table 3-3. Geometric mean and range for outdoor bioaerosol concentrations 80 before and after chlorine dioxide treatment Table 3-4. Average total counts and relative efficiency with standard 81 deviations for laboratory study xviii

19 Hypotheses and Specific Aims The purpose of this study was to investigate the use of filter and other traditional sampling techniques for the collection of personal and area samples to evaluate microbial exposures. Since October 2001, when B. anthracis was used in acts of bioterrorism, there has been renewed interest in the development of validated sampling and analytical methods for biological agents to determine if they are present and at what concentration to determine the potential health hazard. This interest has been heightened by the identification of new diseases including SARS, the threat of a pandemic influenza outbreak, and the resultant microbial contamination from the flooding and storm damage in the southern United States after the 2005 hurricane season. Also, exposure issues for industrial hygienists and laboratory staff responsible for collecting and analyzing environmental samples need to be taken into consideration since occupational exposure criteria for workers exposed to microbial agents have not been developed. The results from this study will be useful in the development of standardized sampling techniques for these microbial exposures. The following hypotheses and specific aims were explored in this dissertation. Hypothesis #1: There are statistically significant differences between extraction efficiencies for vortex-ultrasonic and vortex-shaker extraction methods using mixed cellulose ester (MCE), polytetrafluoroethylene (PTFE or Teflon), and gelatin filter for Bacillus atrophaeus endospores. Specific Aim 1: Determine best extraction technique for various filter materials and the effect of sampling time on extraction efficiency and culturability of Bacillus atrophaeus endospores. 1

20 Hypothesis #2: There are statistically significant differences in the physical collection efficiency of polycarbonate (PC) and PTFE filters for particles in the size range of 10 to 900 nanometers. Specific Aim 2: Determine physical collection efficiencies of PC and PTFE filters for small particles using B. atrophaeus endospores, MS2 virions, polystyrene latex (PSL) particles, and sodium chloride particles. Hypothesis #3: Filter sampling combined with quantitative PCR analysis can be used to evaluate chlorine dioxide treatment in the laboratory and field. Specific Aim 3: Comparison of microbial sampling techniques before and after chlorine dioxide treatment using known counts of fungal spores on filter surfaces in a laboratory setting and naturally occurring microbial contamination during a house remediation procedure. 2

21 Background General Overview Bioterrorism is defined as the use or threatened use of biologic agents against individuals to obtain an advantage for a specific purpose such as intimidation, ideological principles, or disruption of everyday activities (Brachman 2002) Since October 2001 with the introduction of mail contaminated with Bacillus anthracis into several work environments, people worldwide have become increasingly aware of the potential for bioterrorism acts (Jernigan et al., 2001). Concern over airborne dissemination of viral particles, such as the coronavirus and influenza virus, as well as the growing use of engineered nanoparticles has also increased the need for additional environmental sampling techniques, especially for the nano-scale particle size range. Nano-scale particles have sizes below 100 nm (Oberdörster et al., 2005). Viruses range in size from 20 to 200 nm and can be found in droplet nuclei or attached to other airborne particles (Reponen et al., 2001). Viruses in the Orthomyxoviridae family include those associated with influenza such as the Avian flu virus and range in size from 80 to 120 nm (Mandell et al., 2005). It is estimated that globally about 5% of all adults and 20% of all children develop symptomatic influenza infections each year (Nicholson et al., 2003). It is a costly disease that results in much human suffering as well as economic impact in terms of lost time and medical expenses. Viruses in the Coronaviridae family, which includes the virus linked to severe acute respiratory syndrome (SARS), range in size from 80 to 150 nm (Mandell et al., 2004). There is also much interest in developing environmental sampling techniques for bioterrorism agents besides Bacillus anthracis (anthrax), such as bacterial agents Yersinia pestis (plague) and Francisella 3

22 tularensis (tularemia), and viral agents including variola major (smallpox) and filoviruses and arenaviruses (viral hemorrhagic fevers). Some of these agents are found in the smaller particle ranges (CDC, 2006). In addition to the concerns over potential exposures to bioterrorism and viral agents, there have also been many health problems related to exposures to indoor bioaerosols (a mixture of microbial, animal, and plant particles). The health effects are highly dependent upon individual responses to the various bioaerosols and fall into three main categories: allergic, infectious, and toxic. A wide range of symptoms have been associated with indoor biological contaminants ranging from irritation of the eyes, headache, fatigue, and respiratory tract symptoms to aggravation of asthma; however, the causal agents for these symptoms remain an area of ongoing scientific research (IOM, 2004; Mitchell et al., 2007). Suspected biological causal agents include fungi (both molds and yeasts) and bacteria, and their associated components (i.e., endotoxin and (1 3)-β-D-glucan). Endotoxin, a lipopolysaccharide complex in the cell wall of Gram-negative bacteria, has been associated with respiratory symptoms. (1 3)-β-D-glucan are the most abundant glucans from the cell walls of fungi as well as some bacteria and plants. They have been suspected to cause respiratory symptoms; however, the epidemiological data for this association are not conclusive (Douwes 2005). There is also evidence that exposure to fungi may occur through fungal fragments that can contain allergens, toxins, and (1 3)-β-D-glucan (Górny et al., 2002; Brasel et al., 2005; Reponen et al., 2006). 4

23 These events and concerns showed the need to develop validated environmental sampling and analytical methods for specific biological agents to determine whether they are present and at what concentration to determine the potential health hazard (CDC, 2002). Techniques that have been used traditionally for the collection of bioaerosols include centrifugal scrubbing, electrostatic precipitation, filtration, liquid impingement, and impaction (Otten and Burge, 1999; Sattar and Ijaz, 2002). Culturable cell counts can be affected by a variety of factors such as the type of nutrient media selected; aerosolization, collection, and assay methods; and environmental conditions (Kenny et al., 1998). Filtration utilizes impaction, interception, and diffusion as the major collection mechanisms (Henningson and Ahlberg, 1994). The primary advantages of using filtration collection for bioaerosol samples include potential to reach high collection efficiency, ease of sample collection and preparation, relatively low costs of collection equipment and supplies, and the ability to use various analysis techniques. The development of real-time quantitative polymerase chain reaction (Q-PCR) analysis techniques, which do not depend on culturability, allows for the detection of microorganisms within a short time frame. This has led to the possibility of conducting air sampling for long time periods and the ability to evaluate individual exposures using personal breathing zone filter sampling instead of using estimates based on short-term area monitoring. Overview of Environmental Monitoring Studies for B. anthracis Several environmental monitoring evaluations using various sampling techniques were conducted by other investigators to examine the level of B. anthracis contamination in the B. 5

24 anthracis affected work sites (CDC, NIOSH 2004; Small et al., 2001; Dull et al., 2002, Weis et al., 2002). Traditionally, environmental monitoring for B. anthracis has been conducted using culture-based methods (Henningson and Ahlberg, 1994). The National Institute for Occupational Safety and Health (NIOSH) provided technical assistance to the United States Postal Service at the Trenton Processing and Distribution Center in Trenton, New Jersey (CDC, NIOSH 2004). As part of the environmental assessment conducted at the facility, air samples were collected before and after a contaminated mail sorter was operated using different sampling techniques. For the gelatin filter samples, 27/36 (75%) samples were positive for B. anthracis spores after the contaminated mail sorter was operational. All the mixed cellulose ester (MCE), polytetrafluoroethylene (PTFE), and dry filter unit (DFU) air samples were positive for B. anthracis spores after the sorter was operational when the entire extraction sample was analyzed for optimum sensitivity (CDC, 2004). An environmental survey was conducted at the Brentwood Mail Processing and Distribution Center Washington, DC in October 2001 after the building had been closed and the ventilation system turned off for 3 days (Small et al., 2001). Twelve air samples were collected for a time period of about 30 hours at a flow rate of 2 liters per minute (Lpm) using open-faced 37-millimeter (mm) MCE filters and were negative for culturable B. anthracis (Small et al., 2001). Seven percent (8/114) of the sterile cotton gauze wipe samples and sixty-nine percent (27/39) of the vacuum dust samples were positive for B. anthracis. Additional monitoring was conducted on a mail sorting machine at the Brentwood facility that had handled two of the letters containing anthrax spores in October 2001 (Dull et al., 2002). Air sampling performed using slit agar samplers with TSA plates showed 1 colony forming unit (CFU) before the machine was activated and 6 CFU during simulated work tasks (Dull et al., 6

25 2002). No colonies were detected from the respirator filter samples worn by the evaluation team (Dull et al., 2002). Weis and associates investigated secondary aerosolization in an office contaminated from the October 2001 incidents (Weis et al., 2002). The investigators found that re-aerosolization did occur during both levels of activities. All 10 of the personal air samples collected on gelatin filters in 37-mm open-faced filter cassettes were positive for B. anthracis. Efficiency of Filter Sampling The use of filter sampling for microbiological agents has been the focus of many studies. Koller and Rotter looked at several issues concerning the use of gelatin filters for collecting airborne bacteria. They found that the gelatin filters had a collection efficiency of greater than 99.95% for particles between 0.5 and 3.0 µm in size (Koller and Rotter, 1974). The investigators also found that dissolving the filters in either isotonic saline or 1% peptone water yielded a higher bacterial count (1.6 and 2.3 times, respectively) when compared to bacterial counts from filters directly placed on the solid nutrient media (Koller and Rotter, 1974). They also found that shaking the dissolved filters in either isotonic saline or 1% peptone water with glass beads showed a higher bacterial count. This was not a result of the natural growth of cells during the experimentation time. The authors concluded that this was likely due to the dispersion of aggregates (Koller and Rotter, 1974). They also found that exposing gelatin filters to sterile air showed a reduction in bacteria survivability that was a function of the exposure time. Jaschhof used the gelatin filter to collect laboratory generated T1 phage and influenza A virus particles (Jaschhof, 1992). He found a retention rate of 99.76% for the T1 aerosol and was able to culture influenza A virus collected during air monitoring in the room of a patient with Influenza Type A. 7

26 Macher and First conducted a laboratory study that compared the collection efficiency of personal (designed to collect air in the worker s breathing zone) samplers including liquid impingers, spiral sampler, gelatin and membrane filters, and a personal cascade impactor (Macher and First, 1984). They used latex particles (2 µm in diameter), B. subtilis spores and Escherichia coli cells as the test particles. They reported that the gelatin filters had similar collection efficiencies when compared to membrane filters, but dehydration of the gelatin filter was a problem that affected the culturability of sensitive microorganisms. Myatt et al., (2003) used 2.0 µm pore size polytetrafluoroethylene (PTFE) filters with cassette samplers to collect airborne rhinovirus. Utilizing a reverse transcriptase polymerase chain reaction (RT-PCR), they were able to detect virus on the filter with a sampling time of 40 hours. Booth et al. (2005) examined several different monitoring techniques to determine if SARS coronavirus could be detected in environmental samples collected in Toronto hospitals. Using a slit sampler and a PCR technique, the investigators obtained two positive air samples from a room with a recovering SARS patient. Wet swab sampling yielded positive results for commonly touched surfaces, including a bed-side table, remote control, and medication refrigerator door. At the same time, traditional air sampling with PTFE filters using reverse-transcriptase (RT) PCR did not yield any results above the detection limit. Tseng and Li (2005a) investigated the collection efficiency in terms of viability for four different bacteriophages with four different samplers. They found that gelatin filter samplers, Andersen one-stage impactor samplers, and AGI-30 impingers were more suitable for the collection of 8

27 viral particles than open-faced cassette samplers equipped with nucleopore (membrane) filters using a plaque assay. Alternative sampling methodologies have also been developed, e.g., a new personal bioaerosol sampler that allows collecting bioaerosol particles through porous media immersed in a collection fluid (Agranovski et al., 2004 a,b). Two different collection fluids, sterile water and Hank's solution (for virus maintenance), were used. The different collection fluids were used in various analyses including viral plaque assays and titration on chicken embryos (Agranovski et al., 2004 a,b). Extraction methodology is very important when working with microorganisms collected on a filter medium. Wang et al. (1999) evaluated the effectiveness of low frequency shaking, vortexing, and ultrasonic vibrating in eluting bacteria from respirator filter materials. Vortexing was found to extract the highest total and culturable bacteria counts from the filters. Another series of experiments by Wang et al. (2001) explored the effect of sampling time, humidity, and extraction technique using five different microorganisms (B. subtilis endospores, Penicillium melinii, Aspergillus versicolor, Pseudomonas fluorescens, and Serratia marcescens). They achieved the highest extraction efficiency (96%-98%) with 2 minutes of vortexing followed by 15 minutes of ultrasonic agitation with polycarbonate filters (Wang et al., 2001). Increased sampling time was associated with a decrease in the culturability of the bacterial cells and spores. Chlorine Dioxide Treatment There has been increased interest in the use of newer technology for the remediation of mold and bacteria since the prolonged flooding after Hurricanes Katrina and Rita in the New Orleans area resulted in highly contaminated buildings (Rao et al., 2007; Chew et al., 2006; Brandt et al.; 9

28 2006). One technique that is being explored is the use of gaseous chlorine dioxide (ClO 2 ) in the remediation of structures that have been impacted by microbial growth. Using a gas is beneficial, since it can potentially penetrate into building cavities. ClO 2 has been approved by the U.S. Environmental Protection Agency (EPA) as a disinfectant, sanitizer, and sterilant (U.S. EPA, 2003) Gaseous ClO 2 is used as a disinfectant and sterilant in the paper, fruit, vegetable, dairy, poultry, and beef processing industries, as well as in industrial wastewater processing (Sy et al., 2005a, 2005b; S.-Y. Lee et al., 2006). Aqueous ClO 2 has been frequently used to treat drinking water and for wood pulp bleaching in the paper industry. It has also been used to control mold in libraries (Southwell 2002; Weaver-Meyers et al., 1998). Under a crisis exemption from the U.S. EPA, ClO 2 gas was used to treat Bacillus anthracis spores in 2001 and 2002 in contaminated buildings and the exterior of mail packages (U.S. EPA 2006; Canter et al., 2005). Additional studies have been completed on the efficiency of ClO 2 for inactivation of various Bacillus endospores, as surrogates for B. anthracis spores (Buttner et al., 2004, Cortezzo et al., 2004, Han et al, 2003, Young and Setlow, 2003). Wilson and associates conducted a laboratory study investigating the effect of ClO 2 gas on the colonies of four fungal species (Chaetomium globosum, Cladosporium cladosporioides, Penicillium chrysogenum, and Stachybotrys chartarum), ascospores of Chaetomium globosum, and mycotoxins produced by S. chartarum (Wilson et al., 2005a). The investigators exposed fungal colonies grown on filter paper and purified ascospores to ClO 2 concentrations of either 500 ppm or 1000 ppm in a sealed chamber for 24 hours. Both ClO 2 exposure concentrations were effective in rendering C. cladosporioides, P. chrysogenum, and S. chartarum colonies non- 10

29 culturable. C. globosum colonies showed a reduction of 91% at the 500 ppm concentration and 87% at the 1000 ppm ClO 2 concentration. The C. globosum spore count decreased indicating that some ascospores were destroyed by the treatment. The ClO 2 did not detoxify the S. chartarum mycotoxins as determined by a yeast toxicity assay. This study did not look at the effect of ClO 2 on total fungal count (except for C. globosum ascospores), (1 3)-β-D-glucan concentrations, or bacteria commonly found in indoor environments. The ClO 2 generation method and concentrations were also different than those used in the anthrax remediation projects. ClO 2 is a strong oxidizing agent which has been shown in laboratory studies to interact with amino acids, proteins, and viral ribonucleic acid (RNA) (U.S. EPA, 1999). The cellular mechanisms that are affected by exposure to ClO 2 are not totally understood. Young and Setlow found that liquid ClO 2 did not damage the DNA of Bacillus subtilis endospores (Young and Setlow, 2003). It has been proposed that the oxidation process damages the inner membrane of bacterial endospores. Furthermore, ClO 2 has been shown to preferentially inactivate the outer protein layers rather than the nucleic acids for viruses (McDonneell and Russell, 1999). Implications for This Study As described in the investigations referred to above, there is a need to develop environmental sampling methods that can be used in the field for both area and personal monitoring and to validate new remediation techniques. Each of the traditional environmental monitoring techniques provides a different piece of information that can be used to characterize the degree of exposure or contamination. It is important to determine what question needs to be addressed 11

30 before picking the appropriate sampling technique. This study serves to provide more information on what sampling techniques can work for various size particles under different sampling conditions both in the laboratory and field settings. 12

31 Specific Aim #1: Determine best extraction technique for various filter materials and the effect of sampling time on extraction efficiency and culturability of Bacillus atrophaeus endospores 1.1 Introduction In Specific Aim 1, protocols for processing of filter samples of B. atrophaeus endospores using different filter materials that have not previously been compared in a laboratory setting were investigated. The extraction efficiency of two standard methods was studied and compared: vortexing with ultrasonic agitation and vortexing with mechanical shaking. The effect of sampling time on the extraction efficiency and culturability of the endospores was evaluated. The results from this study can be used to select an appropriate filter and extraction method for bacterial endospores. 1.2 Materials and Methods Experimental Set-up Four commercially available 25-mm filters were used for this study: MCE filters with a pore size of 3 µm (Millipore Corporation, Billerica, Massachusetts); Zefon Corporation PTFE (Zefluor ) filters with a 1 µm pore size (obtained from SKC Inc., Eighty-Four, Pennsylvania); Pall Corporation PTFE (Teflo ) filters with a PMP support ring with a 3 µm pore size (obtained 13

32 from SKC Inc.); and Sartorius gelatin filters with 3 µm pore size (obtained from SKC Inc.). The porosity of all filters ranged from 60% to 80%. Selected experiments were also conducted using polycarbonate filters with a pore size of 3 µm (GE Osmonics, Inc., Minnetonka, Minnesota). Each filter was used with the SKC Button Inhalable Aerosol Sampler operated at a flow rate of 4 Lpm by the SKC Universal sampling pump (Model 224), which was connected to the sampler by Tygon tubing. The Button Sampler was chosen for this study because it follows the ACGIH/ISO inhalability curve at 4 Lpm and can be used to collect both stationary (area) and personal (breathing zone) air samples (Aizenberg et al., 2000). The Button sampler was used as designed with one o-ring above and one o-ring below the backing plate. The volumetric flow rate for each sampler was pre- and post-calibrated after each laboratory run using a Buck calibrator (A.P. Buck, Inc., Orlando, Florida). The samples were collected for 15-minute, 1-hour, and 4- hour intervals. The laboratory chamber system was housed in a Biosafety Level II cabinet (SterilchemGARD, Baker Co., Sanford, Maine). The set-up is similar to the one used by Wang et al. (2001). A diagram of the experimental set-up is shown in Figure 1.1a and a picture of the laboratory setting is presented in Figure 1.1b. A 6-jet Collison-type air-jet nebulizer (BGI Inc., Waltham, Massachusetts) generated viable aerosols for B. atrophaeus endospores at 10 Lpm. The B. atrophaeus endospores (also known as Bacillus globigii [BG]), frequently used as a simulant for B. anthracis, were provided by the U.S. Army Edgewood Laboratories, Aberdeen Proving Ground, Maryland, in 2000 in a dried, powder form. The endospores have an aerodynamic diameter of approximately 0.9 µm (Wang et al., 2001). The B. atrophaeus endospores were 14

33 activated by suspending the dry spores in sterile deionized water, heating for 25 minutes at 55 C and then washing by centrifugation twice in sterile deionized water at 7000 rpm. An initial concentration of 10 6 to 10 7 endospores per milliliter of the Collison nebulizer solution was established for the laboratory experiments and verified using a hemacytometer (Hausser Scientific, Horsham, Pennsylvania). If clumping was identified with the hemacytometer, the initial solution was agitated using a vortex for three minutes and re-checked with the haemocytometer before use. The generated bioaerosol was mixed with high efficiency particulate air (HEPA) filtered laboratory air at 30 Lpm. The mixture passed through an electrostatic charge neutralizer (TSI Aerosol Neutralizer Model 3012, TSI Incorporated, Shoreview, Minnesota) before entering the bioaerosol chamber. Temperature and relative humidity were monitored by a direct reading thermohygrometer (Fisher Scientific International Inc., Hampton, New Hampshire) during the experiments. The tests were performed at ambient conditions: the average temperature in the setup was 24.2 ± 1.8 C and the average relative humidity (RH) was 33 ± 4.5% Physical Collection Efficiency The physical collection efficiency (E pce ) of the filters was determined by measuring the B. atrophaeus endospore concentration upstream (C up ) and downstream (C down ) of the filter sampler using an optical particle counter (OPC) (Grimm Model 1.108, Grimm Technologies Inc., Douglasville, Georgia). In each test, C up was determined first, and then a directional switch was made to measure C down. The initial two measurements collected when the direction was switched 15

34 were not included in the analyses to allow the instrument to reach a consistent flow. A typical aerosol monitoring time was 2 to 3 minutes with each filter undergoing three consecutive replications to determine the average E pce. Three different filters were used for each series of experiments with the exception of the 3 µm PTFE for which eight filters were tested due to high variability between filters. To ascertain whether desiccation affected the E pce for the gelatin filter the E pce was additionally determined for gelatin filters after 4-hour sampling of HEPA filtered air. The E pce was calculated as follows: E pce = [1 - (C down /C up )] 100%. (1-1) Experimental Protocol for Determining Culturability and Extraction Efficiency When determining the effect of sampling time on the measured bioaerosol concentrations and culturability, the test organisms were generated during a fixed period of 10 minutes in all the experiments to obtain similar loading for all filters. Clean HEPA-filtered air then was aspirated through the filters. Each filter was removed from the Button Sampler immediately after sampling and soaked for 10 minutes in 20 milliliters of an extraction fluid of 0.1% (w/v) sterile peptone water with 0.01% Tween 80 (Wang et al., 2001) The samples in extraction fluid were vortexed for 2 minutes 16

35 (Vortex-Fisher Scientific Inc.). The samples in solution then underwent an agitation step in either an ultrasonic bath (Fisher Ultrasonic Cleaners, Model FS20, 3 qt., 120V 50/60Hz, 1A, 80W, without heater, Fisher Scientific Inc.), or in a shaker (Burrell Wrist Action Shaker, Burrell Scientific, Pittsburgh, Pennsylvania) for 15 minutes. The extraction fluid was then decanted into a new centrifuge tube to remove the filter and serial dilutions (10-1 ) were made from each extraction fluid. The samples were analyzed for culturable and total microbial count as explained below. For each set, a series of control and blank samples were collected. To check for contamination in the chamber set-up, a 15-minute sample of HEPA filtered air was collected on the appropriate filter without aerosolizing endospores. The resultant filter was then processed using the same laboratory techniques as for real samples. A filter media blank was also included and analyzed for each experimental set. Aliquots of the extraction fluid were also analyzed to assess potential laboratory contamination Culturable Count For the culture-based analysis, aliquots (0.1 ml) of the original extraction fluid solutions, 10-1 dilution extraction fluid solutions, 10-3 and 10-4 dilutions of nebulizer solution, and extraction solutions from the blank samples were placed on TSA agar (Becton, Dickinson and Company, Sparks, Maryland) plates. Three replicates were made for each solution. The plates were incubated at 28 C for 18 hours and the resultant colonies were counted on each culture plate that 17

36 had the dilution of colonies. The culturable counts in the extraction fluid (N cfu-extraction ) and in the Collison nebulizer fluid (N cfu-collison ) were calculated as follows: N cfu = (cfu/10 -n ) (V 1 /V 2 ), (1-2) where cfu is the average number of colony-forming units on the three replicate agar plates, n is the dilution factor, V 1 is the extraction fluid volume, and V 2 is the volume of dilution applied to the plate Total Count Two methods were used to determine the total count of bacterial endospores on the filter: OPC and microscopic counting. An OPC was used to determine the aerosol concentration of endospores in the laboratory chamber based on the reading for the spore particle size range obtained during the experiment as a 1-minute average value using all particles greater than 0.65 µm. N Total-OPC is the total particle count for the generation period: N Total-OPC = C Total-OPC Q t (1-3) where C Total-OPC is an integrated particle concentration for the sampling period based on the average of C up over the sampling time, t is the sampling time in minutes, and Q is the sampling collection flow rate (4 Lpm). 18

37 The following samples were analyzed for total count using an epifluorescence microscope (Model Laborlux S, W. Nuhsbaum Inc., McHenry, Illinois) at a magnification of 1000 : the original extraction fluids from the filter samples, 10-3 and 10-4 dilutions of the Collison nebulizer solution, and the blank samples. The slides were prepared by first filtering sterile phosphate buffer through a black 25 mm polycarbonate filter with a pore size of 0.2 µm in a filter holder using a vacuum. A 1 ml extraction fluid subsample was then filtered and stained using 3 ml of acridine orange solution for 10 minutes. The excess stain was removed by adding sterile phosphate buffer through the filter. The filter was mounted on a glass slide using immersion oil and a cover slip, and the edges were sealed with clear nail polish. The counting procedure was based on the acridine orange method described by Palmgren et al. (1986). Forty randomly chosen fields were counted on each slide. The total microbial count in the extraction fluid (N Total- Extraction) and in the Collison nebulizer fluid (N Total-Collison ) was calculated using the same expression from the average microscopic field count (N): N Total = N (πr 2 /A) (V 1 /V 3 ), (1-4) where R is the effective filter radius (8.5 mm), A is the microscopic field area ( mm 2 for Laborlux S, Leitz Inc. microscope), V 1 is the extraction fluid volume (20 ml), and V 3 is the volume of dilution used for analysis (1 ml). The total microbial concentration (C Total-Microscope ) in the air sample (endospores m -3 ) was calculated as: C Total-Microscope = N Total-Extraction /(Q t), (1-5) 19

38 and related to the C Total-OPC Relative Culturability The relative culturability (RC) was defined as: RC = [CF Extract /CF orig ] 100%, (1-6) where CF Extract is the culturable fraction in the extraction fluid (N cfu-extraction /N Total-OPC ) and CF orig is the initial culturable fraction in the Collison nebulizer (N cfu-collison /N Total-Collison ). The initial culturability for this experiment ranged from to with an average of 0.25 and a standard deviation of Physical Extraction Efficiency The physical extraction efficiency (E E ) was defined as: E E = [N Total-Extraction /N Total-OPC ] 100%, (1-7) E E was determined to compare the mechanical extraction of the B. subtilis endospores from the filter materials. 20

39 1.2.4 Data Analysis Data analysis was performed using the SAS statistical package version 8 (SAS Institute, Inc., Cary, North Carolina). A general linear model (GLM) procedure was used to look at the differences in relative culturability between filters, sampling times, and extraction efficiencies because there were unequal observations for some experimental conditions. Paired t-tests were performed to compare the average total count obtained by the OPC to the one obtained by microscopic counting. Standard t-tests were used to compare the physical collection efficiencies of gelatin filters obtained during two sampling periods. A one-way ANOVA was used to compare E pce -values obtained with different filter types. General linear models were utilized to assess the effects of sampling time, filter type, and extraction method for the E E -values. A significance level of 0.05 was used for all statistical tests Sample Size Calculation for Extraction Study The extraction efficiency for vortexing (2 minutes) plus ultrasonic agitation (15 minutes) for polycarbonate membrane filters for 10 minutes sampling time for B. atrophaeus endospores was 98 ± 1%. The extraction efficiency for vortexing (2 minutes) was 85 ± 3%. Each experiment was repeated three times (Wang et al., 2001). A difference of 5% or less would be acceptable for the extraction efficiency for this experiment. Using the formula, given below for a continuous variable with two independent samples with an alpha (Type 1 Error of 0.05) and Type 2 Error (power) of 80% and the data from the polycarbonate filter study, the estimated sample size for each set of experimental conditions will be: 21

40 N = (σ σ 2 2 ) x (Z α/2 +Z β ) 2 = ( )( ) 2 = 3.16 round up to 4. (1-8) (µ 1 - µ 2 ) 2 (98-93) 2 Therefore, the sample size for this factorial study will be 96 filter samples (4 filter types, 2 extraction methods, 3 sampling periods, and 4 replicates). 1.3 Results and Discussion Physical Collection Efficiency Table 1.1 presents the physical collection efficiencies (E pce ) for the five filters used in this study. The MCE, 1 µm PTFE, and gelatin filters had similar average physical collection efficiencies of 94% or greater. Due to the low collection efficiency of the 3 µm PTFE filter along with the wide range of variability between the filters, this filter type was not used for the rest of the experiments. Further laboratory investigation showed that there was leakage around the filter when used with the Button Sampler metal back-up pad and two o-rings. Almost 100% collection efficiency was obtained for one filter out of six using two o-rings that had a snug fit. A one-way ANOVA analysis of the MCE, 1 µm PTFE, and gelatin filters found no significant differences between the three filter types for E PCE (p = 0.41). No significant differences were found when the E PCE of the gelatin filters, measured directly after insertion into the Button Sampler, was compared to the E PCE measured after the filtered laboratory hood air passed through the filter for 4 hours (t-test: p = 0.47). However, the gelatin filters were found to be brittle after the 4-hour air sampling period and could be easily broken. The 3 µm polycarbonate filter showed an average E PCE of 61%. 22

41 1.3.2 Comparison of the Total Count to the Optical Particle Count Figure 1.2 shows the concentrations obtained by the OPC and by the microscopic counting for MCE and 1 µm PTFE filters. Paired t-tests comparing the two enumeration techniques showed no significant differences (p = 0.13 and p = 0.37 for vortex and ultrasonic agitation and vortex and shaker agitation, respectively). Linear regression models also showed strong positive correlations between the two enumeration methods (r 2 = 0.96 for vortex and ultrasonic agitation and r 2 = 0.89 for vortex and shaker agitation). This demonstrates that the average OPC particle count can be used instead of microscopic total count as the denominator in the relative culturability calculations. The microscopic analysis of the extraction fluid obtained from the gelatin filters revealed the presence of bacteria, other than B. atrophaeus, which were also found in the media blanks. These species, however, were not present in the samples collected using other filters. No growth other than B. atrophaeus was found on the culture plates, indicating that the bacteria were rendered non-viable during the gamma sterilization process. This microbial contamination made the gelatin filter samples unsuitable for performing accurate total counts under the microscope. Thus, the data were not used. The microscopic analysis of some MCE filter extraction samples showed stray fibers that obscured some of the bacteria cells but did not interfere with counting procedures. 23

42 1.3.3 Relative Culturability Figure 1.3A shows that the relative culturability using the vortex and ultrasonic extraction method for the MCE, 1 µm PTFE, and gelatin filters ranged from 72% to 130%; 93% to 100%; and 87% to 126%, respectively. The corresponding values when using the vortex and shaker extraction method ranged from 24% to 88%; 59% to 130%; and 72% to 100% (Figure 1.3B). The data were examined for outliers and one data point was removed. The general linear model analyses indicated that mean differences were not statistically significant for time, extraction technique, or filter material. The vortex and shaker method showed more variability than the vortex and ultrasonic extraction method for the three filter types, but the differences between the two extraction methods were not statistically significant (p = 0.071). A relative culturability count of up to 126% was found for gelatin filters when dissolved in extraction fluid. Koller and Rotter also found a higher bacterial count than expected when comparing dissolved gelatin filter extract counts to traditional culture techniques (Koller and Rotter, 1974). The investigators used two extraction fluids (isotonic saline and 1% peptone water) in their experiments. The culturability did not decrease over a 4-hour period for MCE, 1 µm PTFE, and gelatin filters as was expected from the polycarbonate (PC) results reported by Wang et al. (2001). Overall, culturability obtained in this study was higher than that observed by Wang et al. (2001) who collected B. subtilis endospores on 0.2 µm PC filters and found that the culturability decreased from 17% to 5% with an increase in sampling time from 5 minutes to 4 hours at RH = 30%. The referenced study utilized a different filter and different batch of B. subtilis endospores than those 24

43 tested in this experiment, which could partially explain the difference in results. In addition, the Wang study used endospores with a wider initial culturability rate (30% to 70%). In this study, PC filters of 3 µm pore size using the vortex and ultrasonic extraction method in conjunction with a 15-minute sampling time were evaluated. The relative culturability was 18 ± 17%. Thus, the relative culturability results with 3 µm PC filters are in general agreement with those reported by Wang et al. (2001). The data obtained in the two studies suggest that collection onto PC filters results in a lower microbial culturability compared to other filters Physical Extraction Efficiency The E E -values for the MCE and 1 µm PTFE filters are presented in Table 1.2 for three sampling periods: 15-minute, 1-hour, and 4-hour. Each number represents the average percentage of endospores that were extracted from the filters relative to the total number of endospores collected on the filters. The data were examined for outliers and three data points were removed. The GLM analysis showed that the extraction method had a significant effect on the total number of spores extracted from the filter (p < 0.001). The vortex with shaker method showed higher extraction efficiencies for both filters over the three sampling times. This may be due to the higher mechanical forces that the filter undergoes when the shaker device is used. As described above, the relative culturability did not differ between the extraction methods. Thus, the two methods had similar effects on the culturability of B. atrophaeus, but the overall efficiency of vortexing with shaker agitation extraction was higher than that of vortexing with ultrasonic 25

44 extraction. The E E -values for the gelatin filters was assumed to be 100% because the gelatin filters dissolved into the extraction fluid. The extraction efficiency for the 3 µm PC filter was 88 ± 12% when using the vortexing with ultrasonic agitation extraction method was used. Wang et al. (2001) found that the extraction efficiency was 98 ± 1% for 0.2 µm PC filters when vortexing for 2 minutes and agitating ultrasonically for 15 minutes. 1.4 Conclusions for Specific Aim #1 The two extraction methods showed a similar effect on the bacterial culturability, but the vortex with shaker agitation extraction method showed a significantly higher physical extraction efficiency for MCE and 1 µm PTFE filters than the vortex with ultrasonic agitation extraction method. Relative culturability and the extraction of B. atrophaeus off the sampling filters were not affected by sampling times of up to 4 hours. The 3 µm PTFE filters, which were thinner than the other filters, showed a wide range of E PCE -values that would limit the ability to collect consistent environmental samples. The gelatin filter extraction fluid contained contamination by non-culturable bacterial cells that made total microscopic counting unfeasible. The results show that among the tested filters and extraction methods, the MCE and 1 µm PTFE filters and the vortex with shaker agitation extraction method had the best performance when sampling and analyzing B. atrophaeus endospores. 26

45 Specific Aim #2: Determine physical collection efficiencies for PC and PTFE filters for small particles using B. atrophaeus endospores, MS2 virions, polystyrene latex (PSL) particles, and sodium chloride particles 2.1 Introduction As described in the background section and in Specific Aim #1, filter sampling appears to be a promising method for sampling of viruses and bacteria. There is lack of information, however, on the collection characteristics of commonly used filters for bioaerosol sampling for the smaller bacteria and viral particles. The objective of Specific Aim 2 was to determine the PCE of PTFE, gelatin, and PC filters for biological and non-biological particles in the nanometer size range; to determine if the mass and particle size distribution found in household dust could be recreated in the laboratory using PSL particles; and to examine the effect that loading has on PCE for nanometer-sized particles. 2.2 Materials and Methods Test Filters The following commercially available filters were tested for this study: Sartorius gelatin filters with 3 µm pore size, GE Osmonics, Inc. PC filters with 0.4, 1, and 3 µm pore sizes, BHA Technologies PTFE with 0.3 µm pore size preloaded in 3-part 37-mm plastic cassettes, Pall PTFE filters with 0.5 µm pore size, Zefon Corporation PTFE filters with 1 µm pore size, and Fluoropore PTFE filters with 3 µm pore size. These filters have been used successfully for 27

46 bioaerosol sampling in previous studies (Willeke and Macher, 1999; Wang et al., 2001; CDC, 2004; Burton et al., 2005; Hung et al., 2005). Filter characteristics are presented in Table Laboratory Setup The same laboratory setup as described in Specific Aim #1, Section was used Filter Sampling Seven of the filters had a diameter of 25-mm and were used in conjunction with the SKC Button Inhalable Aerosol Sampler (SKC Inc.) operated at a flow rate of 4 Lpm provided by an SKC Universal sampling pump (Model 224). The Button Sampler was chosen for this study since it can be used in a stationary as well as personal mode and its sampling efficiency closely follows the ACGIH/ISO inhalability curve at 4 Lpm (Aizenberg et al., 2000). The 0.3 µm pore size PTFE filters preloaded in 37-mm cassettes were also used with the SKC Universal sampling pumps but at a lower flow rate of 2 Lpm because that is the manufacturer s recommended flow rate. A radioactive neutralizer was used when filters were loaded into the Button Sampler to neutralize electrostatic charges. The volumetric flow rate for each sampler was pre- and postcalibrated after each laboratory run using a mini-buck calibrator. The filters were placed inside the chamber as shown in Figure Pressure Drop Measurements Pressure drop measurements were performed with a Magnehelic gauge (range: 0-80 water) to determine the air resistance through the filter and sampler (Model 2080, Dwyer Instruments, Michigan City, Indiana). Measurements for each type of filter/sampler combination were 28

47 repeated using three different filters. A GAST Model 1532 rotary vane pump (Gast Manufacturing, Inc., Benton Harbor, Michigan) was used with the 0.4 µm PC filters to achieve a consistent flow rate of 4 Lpm in order to obtain an accurate pressure drop measurement. The pressure drop measurements were conducted independently from the chamber experiments Test Particles Bacillus atrophaeus endospores were selected to represent bacteria. A suspension containing about 10 6 cells/ml was prepared as described above in Chapter The tests were also conducted with MS2, a small RNA virus with an aerodynamic diameter of about 28 nm (Hogan et al., 2004). This is a bacteriophage that infects only male Escherichia coli bacteria (Fiers 1967; Valegard et al., 1990; Golmohammadi et al., 1993). The small size and simple structure of MS2 virions, their single-stranded RNA genome, as well as its harmlessness to humans, animals, plants, and other higher organisms, have made MS2 particularly useful in simulating RNA viruses such as Ebola, Marburg, and the equine encephalitis alphaviruses (O Connell et al., 2006). In addition, MS2 bacteriophages have previously been used a surrogate for poliovirus and many other enteric viruses due to similarity of their characteristics. Several investigators have utilized MS2 as a simulant of pathogenic viral strains (Belgrader et al., 1998; Alvarez et al., 2000; Shin and Sobsey, 2003; Thomas et al., 2004; and Tseng and Li, 2005b). The MS2 bacteriophage stock solution was prepared from a freeze-dried phage stock vial (ATCC B1) by adding 9 ml of Luria-Bertani broth, which had been made using ultra-filtered deionized water. The resulting suspension was serially diluted and the final suspension resulted in 10 8 to 10 9 plaque-forming units of MS2 per milliliter of solution. 29

48 Monodisperse 0.35 µm PSL particles (Bangs Laboratory Inc., Fishers, IN) were used in the physical collection efficiency measurements to represent the 0.3 µm particle size, which is believed to be the most penetrating through filters (CDC/NIOSH, 1996). One-tenth of a milliliter of re-constituted PSL particles were mixed with 50 ml of sterilized, deionized water to create stock solutions In order to determine a typical concentration and particle size distribution in indoor air to use for the loading experiment, aerosol measurements were carried out for 23 hours with the OPC in a home that was part of a larger indoor air quality study (T. Lee et al., 2006a). Then, three separate PSL test mixtures were created using aliquots of 0.35, 0.54, 0.69, 0.61, 1.08, 2.43, and 5.05 µm stock solutions. The comparison of the particle size distributions of the fieldmeasured indoor aerosol and the laboratory-generated PSL mixtures (with fractions from 0.35 to 5.05 µm) is presented in Figure 2.1 as a numeric percentage. This figure shows that the fieldmeasured levels can be reproduced in the laboratory using a mixture of monodisperse PSL particles of different sizes. The second PSL test solution showed a higher percentage of 0.5 µm particles which may be a result of incomplete mixing or clumping in the Collison nebulizer. Sodium chloride (NaCl), which was also used as a challenge aerosol, was aerosolized from a 1% weight by volume (w/v) solution. It formed a log-normal particle size distribution in a range of nm with the number concentration peak of nm. The size range of NaCl aerosol included MS2 virions as well as 0.35 μm PSL particles. 30

49 2.2.6 Physical Collection Efficiency The physical collection efficiency (E pce ) for the filters was determined by measuring the particle concentration upstream (C up ) and downstream (C down ) of the filter sampler. When testing with B. atrophaeus and 0.35-μm PSL particles, the real-time particle size resolve measurements were performed with an optical particle counter (OPC) (Grimm Model 1.108), which sorts particles in 15 channels in the size range from 0.3 to greater than 20 µm. For the B. atrophaeus endospores, all particles greater than 0.65 µm and less than 2.0 µm were used to calculate initial concentrations to include any agglomerates that might have occurred (Burton et al., 2005). For the 0.35-μm PSL particles, the particles that were detected in the channel greater than 0.3 µm and less than 0.4 µm were used for the calculations. When testing with smaller particles, including MS2 virus and NaCl, we used a Wide-range Particle Spectrometer (WPS ) (Model 1000XP, configuration A, MSP Corporation, Shoreview, Minnesota). With three devices built in (the condensation particle counter [CPC], differential mobility analyzer [DMA], and light particle spectrometer [LPS]), this instrument size-selectively measures particles starting from 10 nm. The CPC and DMA measure from 10 to 500 nm (up to 96 channels) and the LPS covers particles from 350 to 10,000 nm (24 channels). The CPC and DMA were set to collect measurements in 48 channels for this study. The particle diameter range of 10 to 80 nm was used for the MS2 virions to take agglomeration into consideration (Bałazy et al., 2006). In each test, once the C up was determined, a directional switch was used on the sampling line to measure C down (Jankowska et al., 2000). A typical sampling time was 3 minutes with each filter undergoing three consecutive C up and C down measurements. Three different filters were used for 31

50 each series of experiments and the results were averaged to determine the E pce. The instrument reading during the first minute was always omitted to eliminate confounding of any material that might be left in the sampling tube. The E pce was calculated using equation Loading Experiment Loading experiments were performed to investigate if the collection efficiency of filters increases after collecting a specific amount of particles on these filters. The four-hour loading experiments were conducted using 0.3 µm pore size PTFE, 1 µm pore size PC, and 3 µm pore size PTFE filters. One test PSL mixture was used for each set of filters to mimic the observed concentration and size distribution of indoor air particles. These filters were selected for the loading tests because they showed the highest physical collection efficiency in conjunction with a pressure drop suitable for personal (breathing zone) samples. Three identical filters were loaded for each filter type. The filters were equilibrated for seven days under control temperature and relative humidity conditions before weighing on a Mettler balance (Mettler-Toledo AT20, Mettler-Toledo, Inc., Columbus, OH) before and after loading. The E pce was determined for each filter before loading using 0.35 µm PSL particles and after loading (E pce-loaded ) using 0.35 µm PSL and MS2 particles. The E pce before loading measurements for the MS2 bacteriophage aerosol was determined using a separate set of identical filters to avoid additional loading of the set of filters used for loading. 32

51 2.3 Data Analysis Data analysis was performed using the SAS statistical package version 9.1 (SAS Institute, Inc.). Paired t-tests were performed to compare the average E pce (before) and E pce-loaded (after filter loading) for 0.35 µm PSL particles and MS2 bacteriophages. A one-way ANOVA was used to compare E pce -values obtained with different filter types for each particle type. Multiple comparisons of the means were conducted using the Scheffé procedure as the most conservative analysis. A significance level of 0.05 was used for all statistical tests. 2.4 Results and Discussion Pressure Drop Measurements The pressure drop values through the tested filter loaded in the Button Sampler or 3-piece cassette are presented in Table 2.2. They range from 0.3 kpa for the 0.3 µm pore size PTFE filters to 15.2 kpa for 0.4 µm pore size PC filters. Due to the high pressure drop observed for 0.4 µm pore size PC at the 4 Lpm flow rate, this filter was not used in further testing. The 0.5 µm pore size PTFE filters also showed a high pressure drop at 4 Lpm, which occasionally caused pump failure during sampling. When the collection filters are used with the Button Sampler, the overall pressure drop can be reduced by replacing the manufacturer-provided metal filter support with an autoclavable metal mesh support as described by Lee and associates (Lee et al., 2006b) Physical Collection Efficiency Figure 2.1 presents the collection efficiencies obtained for the three test aerosols. For the airborne B. atrophaeus, the test filters showed an average E pce of 94% or higher. The 1 µm PTFE 33

52 filter showed a statistically significantly lower E pce (average 94%) when compared to the other filters. This information, however, could not be verified by additional experiments since this filter is no longer manufactured and additional filters could not be obtained. The collection of 0.35 µm PSL particles on the 3 µm pore size PC filters (average 69%) was significantly less efficient than that obtained with the other filters. For the MS2 virions, the 1 µm and 3 µm pore size PC filters had E pces = 68% and 27%, respectively. These were statistically significantly lower than the collection efficiencies obtained for the PTFE and gelatin filters, which were > 96%. Furthermore, the collection efficiency of the 3 µm PC filter was the lowest among the tested filters. The high collection efficiency for the gelatin filters for the MS2 virions agrees with the results of prior investigations (Koller and Rotter, 1974 and Jaschhof, 1992) Sodium Chloride Challenge Aerosol Figure 2.2 presents the particle size selective data on the collection efficiency of the 1 µm and 3 µm pore size PC filters and 0.3 µm and 3 µm pore size PTFE filters challenged with NaCl particles. The minimum E pces for the 1 µm and 3 µm pore size PC were observed at the particle average diameter of 47 µm and 63 µm, respectively. The respective E pces values were 49% and 22%. In contrast, the 0.3 µm and 3 µm pore size PTFE filters showed minimum E pces of 99.7% and 98.4%, respectively. The data obtained with the NaCl particles is in agreement with those obtained with PSL particles of 0.35 µm and MS2 virions measured within a size range of nm (see Figure 2.2). The low E pce for the PC filters also agrees with the reported performance of PC filters since the 1980s. Hinds and Liu and associates reported that minimum filter efficiencies for membrane filters were at approximately 50 nm, which agrees with the data shown in Figure 2.3 (Hinds 1999; Liu, 1983). This was predicted in work by Spurný and associated with the 34

53 initial work with Nucleopore filters (Spurný et al., 1969). In our tests, we observed that when loading and unloading the PC filters in the Button Samplers it was important to ensure that the filters were not wrinkled. Smith et al. (1993) noted difficulty with polycarbonate filters in terms of static charges and problems with folding and wrinkling during filter loading. It should be noted that previous studies have found that gelatin filters dried out over time and were of limited use for long-term sampling (Burton et al., 2005; Tseng and Li, 2005a) Effects of Filter Loading The comparison of the particle size distributions of the field-measured indoor aerosol and the laboratory-generated PSL mixture (with fractions from 0.35 to 5.05 µm) is presented in Figure 2.3. This figure shows that the field-measured levels can be reproduced in the laboratory using a mixture of monodisperse PSL particles of different sizes. In order to see if this was reproducible, three separate mixtures using the same concentration of PSL particles of the different sizes were created and used in the loading experiments. When 1 µm pore size PC filters, 0.3 µm pore size PTFE filters, and 3 µm pore size PTFE filters were loaded for 4 hours with PSL particles, the average tared filter weights were 56 ± 7, 45 ± 10, and 47±8 µg, respectively, corresponding to average airborne concentrations of 58 ± 7.3 µg/m 3, 93 ± 22 µg/m 3 ; and 49 ± 8.3µg/m 3. These concentration levels are comparable to the average indoor dust concentrations (geometric mean: ± µg/m 3 ) determined previously during a four-season study in North Carolina (Wallace et al., 2006). 35

54 Figure 2.4 presents the physical collection efficiency of three filters before (E pce ) and after (E pceloaded) particle loading as measured with 0.35 µm PSL particles and MS2 virions. The collection efficiencies obtained for the pre- and post-loading for both particle types are very similar to those presented in Figure 2.2. Similar to E pce, the E pce-loaded was higher for the 0.35 µm PSL particles (>90%) than for the MS2 particles (>67%) when tested with 1 µm pore size PC filters. The 0.3 µm pore size PTFE filters exhibited high E pces-loaded (>99%) with both challenge aerosols. The 3 µm pore size PTFE filters showed E pce-loaded of >99% for the 0.35µm PSL particles and >96% for the MS2. The PTFE filters had much higher E pce-loaded for the MS2 particles than the 1 µm pore size PC which is consistent with the data collected for unloaded filters. The small increase in E pce-loaded that was observed for the majority of the filters was anticipated. Paired t-tests, however, showed that the collection efficiencies for loaded and unloaded filters were not statistically significantly different. 2.5 Conclusions for Specific Aim #2 The PTFE filters were found to be efficient for collecting submicrometer and nano-scale aerosol particles, including bacteria and viruses. The 0.3 µm PTFE filter used with the 37-mm threepiece cassette exhibited the lowest pressure drop and highest physical collection efficiency for B. atrophaeus and MS2 particles. The other PTFE filters also showed very good physical collection efficiencies across the size range of 10 to 900 nm with relatively low pressure drop. PTFE filters were found to have good recovery of aerosolized bacteria when used in Button samplers (Specific Aim #1; Burton et al., 2005). Additional work, however, needs to be conducted to investigate the recovery efficiency for smaller particles from the PTFE filters. The tested gelatin filter also had good physical collection efficiency, but may not be suitable for long-term 36

55 sampling due to potential drying out (Burton et al., 2005; Tseng and Li, 2005a). The PC filters made of a thin layer of material appear to have little internal capture capability when compared to the fibrous membrane filters in the nano-scale particle size range. At the same time, the PC filters exhibited acceptable E pce with the B. atrophaeus bacteria (the largest aerosol particles tested in this study). The findings suggest that the PTFE filters are the best option among the tested ones for long-term personal sampling of nano-scale particles and virions in terms of collection efficiency. Several of the tested filters were found to be equally appropriate for the collection of bacteria including the 1 µm PC, 0.3 µm PTFE, and 3 µm PTFE filters. 37

56 Specific Aim #3: Comparison of microbial sampling techniques before and after chlorine dioxide treatment using known counts of fungal spores on filter surfaces in a laboratory setting and naturally occurring microbial contamination during a house remediation procedure 3.1 Introduction The purpose of Specific Aim 3 was to apply the filter that was found to have the best performance in Specific Aims 1 and 2 to a bioaerosol investigation. This included studying the effect of gaseous ClO 2 exposure on bioaerosol contaminants in a contained indoor environment in a field setting, using traditional and modern bioaerosol enumeration techniques: culture-based assay, microscopic counting, quantitative polymerase chain reaction (PCR), and Limulus Amebocyte lysate assay (LAL). Additional laboratory data were obtained to determine the effect of ClO 2 on known concentrations of fungal spores commonly associated with damp indoor environments (Aspergillus versicolor and Stachybotrys chartarum). 3.2 Methods Field Evaluation The field study was performed in a 1890s Victorian house located in a small city in Upstate New York. The house had three stories and a dirt/stone basement and had been purchased by a nonprofit organization because of its location and structure. The house was to be renovated to use as a shelter for women and children, and different agencies volunteered their services to help with 38

57 the remediation. The house had been occupied until approximately 6 months prior to the ClO 2 treatment. A major roof leak was identified and repaired after an asbestos abatement project was completed. Visible mold was present in most areas of the first, second, and third floors. Some rooms had bird and cat droppings present on the floor. After a walk-through survey of the house, eight sampling stations were chosen for the study (two on each floor and the basement). Floor fans were turned off or re-directed to prevent interference with the collection of air samples. An outdoor sampling location was set-up in the backyard of the house. A sampling/monitoring equipment facility was established in a tent in the backyard. The entire structure was tented (using the standard procedure for whole house pesticide treatment) and the interior was heated, ventilated, and humidified prior to the application of ClO 2 gas to maintain the tent under positive pressure and provide optimal conditions for the ClO 2 treatment (NIOSH, 2007). The ClO 2 solution was created on-site using household bleach (5-6% sodium hypochlorite), 6 N hydrochloric acid, 25% sodium chlorite, and distilled water. The Sabre ClO 2 gas generator used a sparging column into which the ClO 2 solution was pumped. Air from the house chamber was pumped counter-current from the ClO 2 solution in the sparging column which picked up the ClO 2 from the solution. The ClO 2 laden air was then returned to the house. When the ClO 2 concentration reached the desired level (650 ppm), the pumping of the liquid solution into the gas generator was stopped. The ClO 2 concentration was monitored during the treatment and additional ClO 2 was added to the house using this method to keep the ClO 2 at the desired level. The air inside the house was neutralized using a negative air scrubbing system after the target 39

58 exposure level had been obtained. The spent liquid and remaining ClO 2 solution was treated with 10% sodium hydroxide (USEPA, 2006). During the treatment process, ClO 2 concentrations were monitored inside the house on each floor and outside the house every 15 minutes. Samples were collected into a midget impinger containing 5% potassium iodide phosphate buffer solution in conjunction with a Gillen sampling pump at 1 Lpm. Pre- and post-calibration of the impinger/pump were performed using a mini- Buck calibrator. The samples were analyzed using a sodium thiolsulfate titration method (US EPA 1999; OSHA 2007). An average total exposure level of 10,351 parts per million hours (ppm-hrs), calculated as CT = ppm hrs, was achieved for treatment. The average concentrations over the 12.5-hour treatment period for the basement, first floor, second floor, and third floor were 739 ppm, 902 ppm, 845 ppm, and 821 ppm. Relative humidity and temperature inside the house were measured in real-time by HOBO units (Onset Computer Corporation, Bourne, Massachusetts). The house was maintained at about 75 F temperature and 70% relative humidity. Dräger colorimetric tubes were used to determine the remaining concentration of ClO 2 48 hours after treatment. The 48 hour time period was similar to that used after the anthrax building treatments to allow any remaining ClO 2 gas inside the house to off-gas and react with any remaining organic materials (US EPA, 2005). The tent was kept under positive pressure in order to prevent fungi and bacteria from entering the house from the outside environment. 40

59 Environmental microbial sampling was conducted by using the same protocol before and after ClO 2 treatment. As presented in Table 3.1, microbial contamination was assessed using a series of standardized microbial monitoring techniques. Airborne culturable count was determined by collecting samples with an Andersen N-6 single stage impactor (Andersen Instruments, Inc., Smyrna, Georgia) on malt extract agar (MEA) and tryptic soy agar (TSA) in triplicate. Air samples for total microbial counting were collected using an Air-O-Cell spore trap sampling (Zefon International, Inc., Ocala, Florida). Three parallel filter samples were collected from the air: one for PCR, one for endotoxin, and one for (1 3)-β-D-glucan assay. PCR samples were collected on a 0.3 μm pore-size 37-mm PTFE filter. The PCR analysis was conducted to determine spore equivalent count of 23 selected fungal species using standard protocols and prepared primer sequences for biological agents as patented by the US EPA (Haugland et al., 2002). Endotoxin and (1 3)-β-D-glucan samples were collected on 5.0 μm pore size 37-mm polycarbonate and 0.3 μm pore size 37-mm PTFE filters, respectively. The samples were analyzed by the LAL-assay. Microscopic analysis of tape samples collected from surfaces was performed to determine the level and form of fungal growth. The laboratory analyses for the field samples were conducted by Aerotech/P&K Laboratories, Inc. (Cherry Hill, New Jersey) with the exception of the endotoxin analysis which was conducted by DataChem Laboratories, Inc. (Salt Lake City, Utah). The relative efficiency of the treatment for each of the measured microbial sample type was calculated as: Relative Efficiency = Concentration before - Concentration after 100% (3-1) Concentration before 41

60 3.2.2 Laboratory Study Two fungal species were used for the laboratory study: (1) Aspergillus versicolor (RTI 367, Research Triangle Institute International, Research Triangle Park, NC), and (2) Stachybotrys chartarum (No , National Institute for Occupational Safety and Health, Morgantown, WV). The two test organisms were chosen since they were found in culture-based and PCR samples collected before and after ClO 2 treatment in the field study. Pure cultures of A. versicolor were grown on MEA for 7 days at room temperature. The resultant A. versicolor spores were harvested from the plates using glass beads with sterilized, deionized water, as described by Schmechel et al. (2003). Pure cultures of S. chartarum were grown on MEA for 4 weeks at room temperature. The S. chartarum spores were collected using sterilized, deionized water. Glass beads were not used because of the sticky nature of the mature spores. The spore count of the original fungal spore suspension was determined using a hemacytometer and was adjusted to 10 6 spores/ml. To mimic spores on surfaces, one milliliter of the fungal spore suspension was vacuum filtered through a 0.2 µm 25-mm-cassette polycarbonate (PC) filter. The loaded PC filters were placed in 37-mm cassettes for transport to the field. The laboratory exposure was conducted in an exposure chamber that was filled with gaseous ClO 2. The loaded filter samples were taken out of the cassettes and loaded into a tray which was slid into the exposure chamber. The gaseous ClO 2 was manufactured on-site using a Saber patented generator using the same ClO 2 generation and monitoring procedures that were used for the field project. Concentrations were monitored in the exposure chamber every fifteen minutes 42

61 for the twelve hours of the exposure experiment using the sodium thiolsulfate titration method similarly as in the field study (US EPA 1999; OSHA 2007). The spores were exposed to ClO 2 gas using three time periods (4, 8, and 12 hours) at 750 ppm to achieve total exposure levels of ClO 2 of 3,000, 6,000, and 9,000 ppm-hours. The chamber was purged and opened at 4, 8, and 12 hours to remove the sequentially exposed samples. Three replicate filters were used for each exposure level and for the controls. The control samples were handled similarly as the other samples, except that they were not exposed. The average temperature and relative humidity in the chamber were 78.5 F and 84.4%. The exposed and control filters were placed in sterile, 50-mL centrifuge tubes containing 10 ml extraction fluid of 0.1% (weight/volume) sterile peptone water with 0.01% Tween 80. The filters were allowed to soak for 10 minutes. An extraction method of vortexing for two minutes followed by 15 minutes shaker agitation under ambient temperatures was used as this protocol was found to result in highest extraction efficiency in Specific Aim 1 (Burton et al., 2005). The extraction suspensions were analyzed for total spore count, spore-equivalent count (PCR) and (1 3)-β-D-glucan. The total spore count was determined using an epifluorescence microscope after 1 ml of the extraction fluid was stained by acridine orange and filtered through a 25-mm black polycarbonate filter (Palmgren et al., 1986). Quantitative PCR assays for the two organisms was performed from 5 ml of the extraction fluid using the same protocol as for field samples by Aerotech/P&K Laboratories, Inc. One ml of extraction fluid was used for (1 3)-β-D-glucan analysis using a LAL kinetic Glucatell Test kit (Associates of Cape Cod Incorporated, East 43

62 Falmouth, MA) by the University of Cincinnati, Department of Environmental Health. The (1 3)-β-D-glucan samples were corrected for (1 3)-β-D-glucan contamination in the sterile reagent water used for extraction fluid. 3.3 Data Analysis Data analysis was performed using the SAS statistical package version 9.1. Paired t-tests were performed to compare the average sample concentrations before and after the ClO 2 treatment in the field study. A one way ANOVA was used to compare the microbial concentrations and relative efficiency values obtained for the three ClO 2 exposure levels for the two fungal species in the laboratory study. Scheffé s test was used to locate the difference that ANOVA indicated. For samples with non-detectable concentrations, one-half of the limit of detection was used in the analyses. A significance level of 0.05 was used for all statistical tests. 3.4 Results Field Study Table 3.2 shows the results for the microbial sampling that was conducted at the house before and after ClO 2 treatment. Initial concentrations culturable fungi in the house were extremely high. All plates were over-grown with a laboratory estimate of over 400 colonies per plate, which after adjusting for multiple particle impaction, yields with an estimate of about 1,000,000 colony forming units per cubic meter (CFU/m 3 ). After treatment, the geometric mean for culturable fungi was 252 CFU/m 3. Paired t-tests comparing the culturable fungal concentrations before and after treatment showed statistically significant difference (p = ). The average 44

63 relative efficiency against culturable fungi was 97.4%. The predominant fungal type in the house before ClO 2 treatment were Aspergillus niger, Aspergillus versicolor, Cladosporium sp., Mucor sp., and Penicillium sp.; whereas after ClO 2 treatment, Aspergillus versicolor, Penicillium sp., and Sporobolomyces sp. dominated. The data for the outside samples are presented in Table 3.3. The geometric means for the outside samples were 548 CFU/m 3 before treatment and 144 CFU/m 3 after treatment. The predominant genera/class for the outside samples prior to treatment were Basidiomycetes, Cladosporium sp., Penicillium sp., and Epicoccum nigrum and, after treatment, were Aspergillus fumigatus, Aureobasidium pullulans, Basidiomycetes, Cladosporium sp., Epicoccum nigrum, Penicillium sp., and Pithomyces chartarum. The geometric mean for indoor total spore counts determined from Air-O-Cell samples was 73,454 spores per cubic meter (S/m 3 ) before the ClO 2 treatment, and 1,552 S/m 3 after treatment, and this difference was statistically significant (p = ). The average relative efficiency against total fungi was 97.55%. Aspergillus/Penicillium, Stachybotrys, Basidiospores, Cladosporium, and Chaetomium were the most commonly detected fungal spores before treatment in the spore trap samples. After treatment, Ascospores, Aspergillus/Penicillium, Basidiospores, and Cladosporium were found in the air samples. Outside concentrations of total fungi were 3,556 S/m 3 before treatment and 444 S/m 3 after treatment. The predominant genera/classes for the outside samples both before and after treatment were Ascospores, Basidiomycetes, Cladosporium, and Aspergillus/Penicillium. Curvularia and Mxomycetes were also detected before treatment and Torula after treatment. 45

64 The geometric means for the PCR samples before and after ClO 2 treatment were 73,454 and 1,552 spore equivalents per cubic meter (SE/m 3 ), respectively. These concentration were significantly different (p = ), and the average relative efficiency was 90.45%. The five most commonly detected fungal species in the house using PCR analyses were A. versicolor, Eurotium (Aspergillus) amstelodami, Cladosporium cladosporioides, Penicillium brevicompactum, and S. chartarum. Figure 3.1 shows the relative efficiency of the treatment processes for these five species. A. versicolor and S. chartarum showed the highest relative efficiency (approximately 100%), followed by E. (Aspergillus) amstelodami (95%), P. brevicompactum (90%), and C. cladosporioides (85%). Outside fungal spore concentrations were 375 SE/m 3 (before) and 76 SE/m 3 (after). The geometric means for (1 3)-β-D-glucan samples before and after ClO 2 treatment were below the limit of detection (LOD) and 736 picograms per cubic meter (pg/m 3 ), respectively. Paired t-tests comparing the (1 3)-β-D-glucan concentrations before and after treatment showed no significant difference (p = ). The average relative efficiency against (1 3)-β-D-glucan was %. The bacterial species detected were highly variable between the sampling locations in the house and included both Gram-negative and Gram-positive organisms. Most commonly found species both before and after treatment were Aeromonas caviae, Bacillus mycoides, Bacillus sphaericus, Brevibacillus brevis, Brevibacterium casei, Brevundimonas vesicularis, Chryseobacterium indologenes, Comamonas testosteroni, Enterococcus durans, Flavimonas orzyhabicans, Micrococcus luteus, Pseudomonas fluorescens, Psychrobacter phenylpyruvicus, Rhizobium 46

65 radiobacter, Sphingomonas paucimobilis, Staphyococcus xylosus, and Streptomyces. The majority of these bacteria are environmental species. The geometric means for indoor culturable bacteria samples before and after ClO 2 treatment were 1,077 and 158 CFU/m 3, respectively. These concentrations were significantly different (p = ) resulting in an average relative efficiency of 84.93%. Endotoxin concentrations before and after ClO 2 treatment were and endotoxin units per cubic meter (EU/m 3 ), respectively. Paired t-tests comparing endotoxin concentrations before and after treatment showed no significant difference (p = 0.23). The average relative efficiency against endotoxin was -96%. Outside endotoxin levels were 0.74 EU/m 3 before and EU/m 3 after the gas application. Tape sampling results showed the presence of spores, hyphae, and conidiophores of Aspergillus, Cladosporium, Penicillium, Scopulariopsis, and S. chartarum on the surfaces before treatment. After treatment, it was still possible to identify spores, hyphae, and conidiophores of Aspergillus, Cladosporium, and Penicillium using microscopic techniques at the same levels of contamination as found before ClO 2 treatment Laboratory Study Table 3.4 presents the total count, PCR, and (1 3)-β-D-glucan results for the laboratory study. Spores were easily visible using the acridine orange stain for total counting. A one-way ANOVA analyses of the total count concentrations for A. versicolor found no significant differences between the control samples and samples treated with the three ClO 2 exposure levels (p = 47

66 0.6676). The average relative efficiencies for the exposure levels of 3000, 6000, and 9000 ppmhours were 51.7, 17.7 and 23.6%, respectively, and these values were not significantly different (p=0.6454). The S. chartarum spores were not evenly distributed on the filters and, therefore, the direct count totals from the filters were considered unreliable and are not reported. The PCR samples for both species showed lower counts as the ClO 2 exposure increased. The PCR total count was especially low for the S. chartarum samples. One-way ANOVA analyses of the PCR concentrations for A. versicolor showed only a borderline significant difference between the controls and the three ClO 2 exposure levels (p = ), whereas for S. chartarum, this difference was more pronounced (p = 0.041). Posthoc analysis of S. chartarum results showed that the means for the three ClO 2 exposure levels were similar to each other but significantly lower from the control. The average relative efficiencies for the exposure levels of 3000, 6000, and 9000 ppm-hours were 99%, 99.8%, and 100% for A. versicolor, which were not significantly different (p = ). For S. chartarum, the relative efficiency value was already 99.99% for the exposure level of 3000, and 100% for the two higher levels. The (1 3)-β-D-glucan concentrations increased with increasing exposure to ClO 2. (1 3)-β-Dglucan levels for A. versicolor ranged from 8.48 to ng/ml and for S. chartarum from to ng/ml. A one-way ANOVA analysis of the (1 3)-β-D-glucan concentrations for A. versicolor found significant differences between the controls and the three ClO 2 exposure levels (p = ), but did not show significant differences for S. chartarum (p = ). The posthoc analysis of A. versicolor results showed that the control values were significantly lower than the 6000 and 9000 ppm-hours exposure levels. The average relative efficiencies against (1 3)- 48

67 β-d-glucan in A. versicolor spores were -30.1, , and %, for the exposure levels of 3000, 6000, and 9000 ppm-hours, respectively. These differences were not significant (p = ). The corresponding values for the S. chartarum were 33.3, , and %, which were not significantly different (p = ). 3.5 Discussion In the field study, the relative efficiency for culturable fungi and bacteria, total fungal spore counts from spore traps, and total fungal spore counts from PCR analyses ranged from 84.9% to 97.6%. In contrast, the relative efficiency for the (1 3)-β-D-glucan and endotoxin showed negative values (-515% and -96%). These results indicate that the ClO 2 treatment efficiently decreased both the culturable and total counts of airborne microorganisms but increased the concentrations of their components, i.e., (1 3)-β-D-glucan and endotoxin. The outside fungal concentrations after the treatment process may have been lower than anticipated due to a snow event prior and during the sampling period. However, the initial indoor culturable fungal concentrations indoors were much higher than outside concentrations and the species profile in indoor air differed from that in outdoor air. Therefore, the decrease in the indoor air concentrations cannot be explained by the decrease in the outdoor air concentration. The field samples were collected from the air to gain information on inhalation exposures. The laboratory study simulated fungal spores on surfaces which is the worst-case scenario for ClO 2 treatment because it is harder to deactivate spores on surfaces than in the air. Compared to the field study, the laboratory study showed similar, although less significant trends for relative 49

68 efficiencies in terms of decrease in PCR total counts of fungi and increase in (1 3)-β-D-glucan concentrations after exposure to ClO 2 gas. No clear trend was seen in the total spore counts obtained in the laboratory study, which may indicate variability in the extraction process or in the initial spore suspension. The three exposure levels were used in the chamber experiment to investigate the effect of increasing dose on the fungal spores in terms of total count, PCR count, and (1 3)-β-D-glucan. The 6000 ppm-hours exposure level was reflective of the original exposure level approved by waiver by US EPA for the treatment of B. anthracis. This was later revised to 9000 ppm-hours. The current laboratory study showed a trend of higher relative efficiency with increasing exposure level for total fungi and opposite trend for (1 3)-β-Dglucan. Our results are well in line with those obtained in a previous laboratory-based study by Wilson and associates who used initial ClO 2 concentrations of 1,000 ppm in an enclosed chamber against fungal colonies and spores (Wilson et al., 2005a). They found a reduction of > 87% in the culturable spore count. They determined total spore count using a hemacytometer and did not find any differences in total count between control and exposed samples when treating fungal colonies, but a significant decrease when treating purified spores. Furthermore, the gaseous ClO 2 exposure did not decrease the activity of two trichothecene mycotoxins (roridin A and verrucarin A) or trichothecene mycotoxins from S. chartarum spores (Wilson et al., 2005a; Wilson et al., 2005b). These results are consistent with the results of the present field study, which did not show a decrease in the surface contamination evaluated by semi-quantitative microscopic method, but demostrated a clear decrease was seen in the culturable and total count of airborne spores. Furthermore, an increasing trend was observed for endotoxin and (1 3)-β-D-glucan 50

69 both in the laboratory and field study supporting the results of Wilson et al. (2005a,b) on the inefficiency of ClO 2 treatment in reducing the concentrations of fungal components. Rendering microorganism non-culturable will prevent microbial growth and further production of harmful microbial components, such as endotoxin, (1 3)-β-D-glucan, mycotoxin and allergens. However, the destruction of bacterial cells, fungal spores, and hyphae during the treatment appears to release microbial components and thereby increase their concentrations. Exposure to endotoxin has been associated with respiratory complaints in indoor environments and a wide-range of symptoms such as fever, cough, and shortness of breath. There are some studies that have shown (1 3)-β-D-glucan to have pro-inflammatory capacities and are associated with adverse non-allergic respiratory health effects (Douwes, 2005; Chew et al., 2001; Rylander 1999). Therefore, attention has to be paid on cleaning the air and surfaces after treatment to achieve acceptable conditions before re-occupancy. Assessment of microbial components, such as endotoxin or (1 3)-β-D-glucan, should be part of clearance sampling after ClO 2 treatment. Before the treatment, the levels of total fungi, culturable fungi, and endotoxin found in the house were similar or lower when compared to those found in two recent studies performed in flooded homes in New Orleans. Rao and associates reported a geometric mean of 2.8 x 10 5.spores/m 3 for total fungi, CFU/m 3 for culturable fungi, and 22.3 EU/m 3 for endotoxin in moderately (n=5) to heavily flooded homes (n=15) in New Orleans (Rao et al., 2007). Chew et al. (2006) found the following ranges for total fungi, culturable fungi, and endotoxin in three homes before renovation: spores/m 3, CFU/m 3, and EU/m 3, respectively. 51

70 In this study, the respective concentrations were about spores/m 3, CFU/m 3 and 18.6 EU/m 3. Park and fellow researchers (2000) reported lower endotoxin levels, geometric mean 0.64 EU/m 3 (range: EU/m 3 ) from bedrooms of 15 homes located in the greater Boston, Massachusetts, area. Outdoor levels of endotoxin before treatment were comparable to the ones in outdoor air in California (0.44 EU/m 3, Mueller-Anneling et al. 2004) and Denmark (0.33 EU/m 3, Madsen A, 2006). It should be noted; however, that short-term exposure to endotoxin levels above 45 EU/m 3 have been associated with decreases in lung function and respiratory inflammation (Milton et al., 1996; Rylander R. 2002) which are higher than the levels found in this study. The (1 3)-β-D-glucan concentrations in the field and laboratory studies were similar to the ones reported in other studies that used LAL for (1 3)-β-D-glucan analysis (Douwes 2005). The reported concentrations ranged from non-detected to 19 ng/m 3 in indoor environments. In this study, the average (1 3)-β-D-glucan concentration was ng/m 3 in the house after treatment. Bacteria concentrations in the house were much higher (1,077 CFU/m 3 ) than those found in non-problem buildings in the United States (average 102 CFU/m 3 ) (Tsai and Macher 2005). Levels of bacteria in microbially contaminated homes are not readily available for the United States. Traditionally, monitoring for bioaerosols has consisted of culturing and microscopic counting of fungi and bacteria using short-term samples (Martinez et al., 2004). There are many advantages of using newer PCR technologies for indoor air environments including quick turn around of sample results, accurate identification and reproducibility, and the detection of non-viable fungi 52

71 and fungal spores. The technology also allows for a long sampling time to get a better understanding of environmental exposures (Meklin et al., 2004). The PCR-method was used in this study along with traditional cultivation and microscopic counting techniques to assess the efficiency of ClO 2 treatment against fungi. The predominant species for fungal contamination in the house were similar for these three methods. Aspergillus, Penicillium, and Cladosporium species were among the five most common detected by the three methods before the treatment. Stachybotrys was detected both by microscopic counting and PCR, but not with cultivation. For the relative efficiency of ClO 2 treatment, these methods showed similar trends, but the highest efficiency was found with the culture-based technique. In the field study, both the total microscopic count and the PCR count obtained for air samples decreased significantly. This could be caused by direct reduction of spores in the air or reduction of spores on surfaces that would serve as the source for the airborne spores. However, no decrease was observed in the semi-quantitative analysis of amount of spores and hyphae in the sticky tape samples collected from surfaces. Furthermore, laboratory study, which evaluated the efficiency of ClO 2 treatment on spores on surfaces, did not show any decrease in the total microscopic count of spores. In contrast, a decrease was observed for PCR count on surfaces. This discrepancy between the microscopic count and PCR-count could be caused by deactivation of DNA or inhibition of the PCR assay by the ClO 2 gas. Previous studies have shown that environmental contaminants in the indoor environment can inhibit PCR analyses, which may also give a false negative result (Buttner et al., 2001; Keswani et al., 2005, Peccia and Hernandez, 2006). Buttner and fellow investigators also identified the issue of inhibition of PCR for environmental samples in their surface disinfection study using gaseous ClO 2 and foam 53

72 decontaminant (Buttner et al., 2004). They also found that DNA and other compounds capable of producing immune responses were still present after treatment. 3.5 Conclusions for Specific Aim #3 This study showed that gaseous ClO 2 treatment can be used to prevent the growth of fungi and bacteria in a field setting. The treatment also reduced the total fungi in the air of the treated house. The field study showed that the 0.3 μm PTFE filter could be used effectively in the field for PCR analysis using a long sampling period (> 6 hours). The laboratory study supported the results obtained in the field study in terms of reduction of PCR total counts but it remains unclear if this was due to inhibition of the PCR assay caused by ClO 2 gas. The fungal spores were visible using microscopic techniques both in the field and laboratory settings. It appears that the treatment makes some of the cells, spores and hyphae break into fragments based upon the measured increase of endotoxin and (1 3)-β-D-glucan. To document the effectiveness of the ClO 2 treatment of microbially contaminated houses, environmental sampling techniques should include the collection of samples for culturable microorganisms as well as endotoxin and (1 3)- β-d-glucan before and after the treatment process. The results call for additional clean-up techniques such as use of air cleaners and cleaning surfaces with vacuums using high efficiency particle air (HEPA) filters to reduce exposures to remaining spores and microbial components after gaseous ClO 2 treatment in microbially contaminated indoor environments. 54

73 Overall Conclusions and Future Activities This study showed that the MCE and 1 µm pore-size PTFE filters in combination with vortexing and shaker extraction demonstrated the best performance in terms of extraction and culturability. Gelatin filters had high culturability rates. Gelatin and PTFE filters had very high physical collection efficiencies for particles between 10 and 900 nm. There was no effect on physical collection efficiency for PTFE filters after loading of particles that represented typical indoor dust levels. These filters can be used for long-term exposure monitoring with small personal sampling pumps to obtain a better estimate of individual exposures instead of relying on area samples to estimate exposure. This would be most effective using either microscopic counting or PCR technology since filter sampling is known to effect culturability rates. The best filter (0.3 µm pore size PTFE) in terms of extraction efficiency and PCE from the first two studies was used for the sample collection in combination with sample analysis by polymerase chain reaction to determine the effectiveness of gaseous chlorine dioxide (ClO 2 ) on indoor microbial contamination. ClO 2 was effective in reducing culturable and total fungi and bacteria in indoor air. The reduction of total count on surfaces was less efficient. The treatment process appears to increase endotoxin and (1 3)-β-D-glucan concentrations, therefore, thorough cleaning of air and surfaces would be recommended to achieve acceptable conditions before reoccupancy. 55

74 Overall, this study shows that gelatin filters and PTFE filters in conjunction with small vacuum pumps can be used effectively to determine personal and area exposures to microbial agents including fungi, bacteria, and viral particles. Gelatin filters are recommended for short-term (< 4 hours) sampling in combination with the culture-based assay, whereas PTFE filters are recommended in combination with non-culture assays. The latter combination can be used for long-term sampling. Future Activities The collection efficiency of the 0.3 µm pore-size PTFE and gelatin filters will be verified by the U.S. Army Dugway Proving Ground Test Center using B. anthracis endospores to ensure that the filter methodology results obtained in this study can be replicated with the agent of interest and to determine a limit of detection. This is being undertaken as a contract monitored by the Industry Wide Studies Branch, Division of Surveillance, Hazard Evaluations, and Field Studies/NIOSH. The US EPA has entered into a cooperative research and development agreement (CRADA) with Sabre Technologies, Inc. to further investigate the use of gaseous ClO 2 in mold remediation. 56

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83 HEPAfiltered Air Neutralizer Test Chamber Filter Chamber Measurement Aerosolized particles Switch Pump Particle Counter Figure 1-1a. Experimental Set-up 65

84 Figure 1-1b. Picture of Laboratory Set-up 66

85 35 30 A. Vortex/Ultrasonic Extraction 25 r 2 = Total count based on OPC ( 10 3 particles L -1 ) B. Vortex/Shaker Extraction 1:1 Ratio Simple Regression 15 r 2 = Total count based on microscopy ( 10 3 cells L -1 ) Figure 1-2. Comparison of total particle concentration from microscopic count to optical particle counter (OPC) count (total count was average of four replicate samples; OPC count was the average of one-minute measurements during each consecutive experimental run) for the two extraction methods 67

86 250 A. Vortex and Ultrasonic Agitation Extraction Relative Culturability (%) µm MCE µm PTFE Gelatin B. Vortex and Shaker Agitation Extraction Sampling Time (hr) Figure 1-3. Percent relative culturability comparison between vortex and ultrasonic and vortex and shaker agitation extraction methods for mixed cellulose ester, polytetrafluoroethylene, and gelatin filters based on the average of at least three replicates for the two extraction methods with standard deviation bars 68

87 100 Physical Collection Efficiency (%) µm Bacteria 0.35 µm PSL < 80 nm Virions 1 µm PC 3 µm PC 0.3 µm PTFE 0.5 µm PTFE 1 µm PTFE 3 µm PTFE Gelatin Particle Type Figure 2-1. Physical collection efficiency of different filters for three challenge aerosols. Note: No result was obtained with the 0.5 µm PTFE filter challenged with virions due to pressure drop/pump failure. Gelatin filters were not tested with the 0.35 PSL particles (the PCE was assumed to be approximately 100%). The bars and error bars represent the mean values and the standard deviations, respectively (n=3). 69

88 100 Physical Collection Efficiency (%) Particle Diameter (nanometers) 1 µm PC 3 µm PC 0.3 µm PTFE 3 µm PTFE Figure 2-2. Physical collection efficiency of filters challenged with NaCl particles aerosolized from a 1% (w/v) suspension as a function of the particle diameter. 70

89 1000 Percent Aerosol (%) Indoor Dust Levels PSL Test Mixture 1 PSL Test Mixture 2 PSL Test Mixture Particle Size (μm) Figure 2-3. Comparison of laboratory-generated PSL mixture to filed-measured indoor aerosols based on number of particles 71

90 100 Physical Collection Efficiency (%) µm PC 0.3 µm PTFE 3 µm PTFE Filter Type 0.35 µm PSL Pre-loading 0.35 µm PSL Post-loading MS2 Pre-loading MS2 Post-loading Figure 2-4. Physical collection efficiency measured with 0.35 µm PSL and MS2 virions before and after loading with PSL test mixture particles. Pre-loading measurements with MS2 virions were conducted with a different set of identical filters. The bars and error bars represent the mean values and the standard deviations, respectively (n=3). 72

91 100 Relative Efficiency (%) Aspergillus versicolor Eurotium amstelodami Cladosporium cladosporioides Pencillium brevicompactum Stachybotrys chartarum Figure Relative Efficiency of Treatment (Average and Standard Deviation) for Five Most Common Fungal Species detected in the PCR Analyses. 73

92 Table 1-1. Physical collection efficiency for different filter materials using B. atrophaeus endospores with optical particle counter (OPC) Filter Material (pore size) Physical Collection Efficiency (%) Mixed Cellulose Ester (3 µm) a 97.6 ± 4.1 Polytetrafluoroethylene (1 µm) a 94.2 ± 2.3 Polytetrafluoroethylene (3 µm) b 63.6 ± 32.3 Gelatin (3 µm) a 97.9 ± 3.7 Gelatin (3 µm) c 94.3 ± 6.5 Polycarbonate (3 µm) a 61.4 ± 24.6 a - average of 3 repeats for 3 different filters b - average of 3 repeats for 8 different filters showed leakage around filter when used with metal back-up pad and two o-rings c - four-hour sampling period prior to measurement average of 3 repeats for 3 different filters with standard deviation 74

93 Table 1-2. Average and standard deviation of physical extraction efficiencies as a percentage for MCE and 1 µm PTFE filters Filter Physical Extraction Efficiency Vortex with Ultrasonic Agitation (%) a Vortex with Shaker Agitation (%) a 1-hour 4-hour 15-minute 1-hour sampling sampling sampling sampling 15-minute sampling time 4-hour sampling time time time time time MCE 66 ± 8 72 ± ± ± ± ± 25 1µm PTFE 77 ± ± ± ± ± ± 54 a - Average of four sample replicates with standard deviation 75

94 Table 2-1. Characteristics of tested filters Filter Manufacturer Sartorius (obtained from SKC Inc., Eighty-Four, Pennsylvania) GE Osmonics, Inc., Minnetonka, Minnesota GE Osmonics, Inc., Minnetonka, Minnesota GE Osmonics, Inc., Minnetonka, Minnesota BHA Technologies Kansas City, Missouri (obtained from SKC Inc.) Pall (obtained from SKC Inc.) Zefon Corporation (obtained from SKC Inc.) Millipore Corporation, Bedford, Massachusetts Material Filter diameter (mm) Pore Size (µm) Gelatin Membrane PC^ PC^ PC^ PTFE* with back-up pad PTFE* with laminated PTFE support Zefluor PTFE* Fluoropore (PTFE*) filters with back-up pad ^PC = Poretics Polycarbonate membrane *PTFE = Polytetrafluoroethylene Thickness (µm) 76

95 Table 2-2. Measured pressure drop values for tested filters with samplers Filter Type Pressure (kpa) Pressure (inches of H 2 O) 0.4 µm PC b µm PC c µm PC µm PTFE c µm PTFE µm PTFE µm PTFE c µm Gelatin a Measurements were conducted using three different filters in conjunction with the Button Inhalable Aerosol Sampler at a flow rate of 4 Lpm with the exception of the 0.3 µm PTFE filters which used a 3-piece 37 mm cassettes at 2 Lpm. b - used Gast Pump to hold flow at 4 Lpm c - used in loading experiments 77

96 Table 3-1. Sampling and analysis methods in the field study Analyte Sampler Media Analysis Method Air Samples Culturable fungi Andersen N-6 MEA Cultivation (ID based on colony morphology) Total fungi Air-O-Cell Slide Microscopic Counting (ID based on spore morphology) Average Sampling Time (minutes) Flow Rate (Lpm)* PCR fungi 3 piece Cassette Filter (0.3 µm PTFE^^) Real-time PCR^ (DNA) (1 3)-β-Dglucan 3 piece Cassette Filter (0.3 µm PTFE) LAL** Culturable bacteria Andersen N-6 TSA Culturability (ID by MIDI- Gas Chromatography) Endotoxin Cassette Filter (5 µm PVC) LAL Surface samples Total Fungi Sticky Tape Slide Microscopic Counting N/A N/A Note: *- Lpm liters per minute; ^- PCR polymerase chain reaction; **- LAL Limulus amebocyte lysate; ^^- PTFE polytetrafluoroethylene; - PVC polyvinyl chloride N/A not applicable 78

97 Table 3-2. Geometric mean and range for indoor bioaerosol concentrations before and after chlorine dioxide treatment Analysis Culturable fungi (CFU/m 3 *) Number of Samples before and after treatment Geometric Mean (Range) Before ClO 2 Treatment After ClO 2 Treatment > 1,000, ( ) Relative Efficiency (%) ± Standard Deviation ± 0.45 Total fungi (S/m 3 **) ,454 (16, ,289) 1,552 (978-2,267) ± 2.45 PCR fungi (SE/m 3^) ,535 (943-23,598) 332 ( ) ± 11.2 (1 3)-β-Dglucan (pg/m 3^^) < 125 (Limit of Detection) 736 (580-1,100) ± 230 Culturable bacteria (CFU/m 3 ) (718 1,319) 158 (82-353) ± 7.74 Endotoxin (EU/m 3 ) ( ) ( ) -96 ± 218 Note: *CFU/m 3 Colony forming units per cubic meter; **S/m 3 Spores per cubic meter; ^SE/m 3 Spore equivalents per cubic meter; ^^pg/m 3 Picograms per cubic meter; # EU/m 3 Endotoxin units per cubic meter 79

98 Table 3-3. Geometric Mean and Range for Outdoor Bioaerosol Concentrations Before and After Chlorine Dioxide Treatment Analysis Culturable fungi (CFU/m 3 )* Total fungi (S/m 3 )** PCR fungi (SE/m 3 )^ Number of Samples Geometric Mean (Range) before and after treatment Before ClO 2 After ClO 2 Treatment Treatment ( ) (94-188) Culturable bacteria (CFU/m 3 ) ( ) 67 (35-94) Endotoxin# (EU/m 3 ) Note: *CFU/m 3 Colony forming units per cubic meter; **S/m 3 Spores per cubic meter; ^SE/m 3 Spore equivalents per cubic meter; # EU/m 3 Endotoxin units per cubic meter 80

99 Table 3-4. Average Total Counts and Relative Efficiency with Standard Deviations for Laboratory Study A.versicolor* Total fungi 3.39x10 6 ± 2.12x x10 6 ± 7.65x x10 6 ± 1.05 x x10 6 ± 2.35x ± ± ± Concentration (average ± standard deviation) Relative Efficiency (average ± standard deviation) Control 3000 ppm-hours 6000 ppm-hours 9000 ppm-hours 3000 ppm-hours 6000 ppm-hours 9000 ppmhours PCR fungi (#/ml)^ (1 3)-β-Dglucan (ng/ml)^^ 3.20 x10 5 ± 2.79 x x10 3 ± 5.76 x x10 2 ± 1.20 x ± ± ± ± ± ± ± ± ± ± ± 29.4 S. chartarum** PCR fungi 2.89 x10 4 ± 2.38 x ± ± 0 1 ± ± ± ± 0 (1 3)-β-D-glucan ± ± ± ± ± ± ± Note: * - Aspergillus versicolor; ** - Stachybotrys chartarum; ^ - number of spores per milliliter of solution; ^^ - nanograms per milliliter of solution; Three filters were used for each set of samples. 81

100 Appendix A: Copies of Peer-Reviewed Publications Resulting From the Ph. D. Study 82

101 Appendix A1. The effect of filter material on bioaerosol collection of Bacillus subtilis spores used as a Bacillus anthracis stimulant 83

102 PAPER The effect of filter material on bioaerosol collection of Bacillus subtilis spores used as a Bacillus anthracis simulant Nancy Clark Burton,* ab Atin Adhikari, b Sergey A. Grinshpun, b Richard Hornung c and Tiina Reponen b a Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Division of Surveillance, Hazard Evaluations, and Field Studies, 4676 Columbia Parkway, Cincinnati, OH 45226, USA b Center for Health-Related Aerosol Studies, Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, Cincinnati, OH , USA c Institute for Health Policy & Health Services Research, University of Cincinnati, 275 East French, Cincinnati, OH Received 5th January 2005, Accepted 18th March 2005 First published as an Advance Article on the web 14th April 2005 The objective of this study was to determine filter materials and extraction methods that are appropriate to use for environmental sampling of B. anthracis. Four types of filters were tested: mixed cellulose ester (MCE) with a pore size of 3 mm, polytetrafluoroethylene (PTFE) with pore sizes of 1 and 3 mm, and gelatin with a pore size of 3 mm. Bacillus subtilis var. niger endospores (also known as Bacillus globigii [BG]) were used as a surrogate for B. anthracis. Endospores were collected into Button Inhalable Aerosol Samplers with sampling times of 15 minutes, 1 hour, and 4 hours. Physical collection efficiency was determined by measuring upstream and downstream B. subtilis concentrations with an optical particle counter. Vortexing with ultrasonic agitation and vortexing with shaker agitation extraction methods were evaluated. The MCE, 1 mm PTFE, and gelatin filters provided physical collection efficiencies of 94% or greater. The 3 mm PTFE filter showed inconsistent physical efficiency characteristics between filters. Epifluorescence microscopic analysis of the gelatin filter extraction fluid revealed the presence of contamination by non-culturable bacteria. Mean differences for microbial culturability were not statistically significant for filter materials and extraction methods. However, the vortexing with shaker agitation extraction method resulted in higher total microbial counts in the extraction fluids for MCE and 1 mm PTFE filters when compared to vortexing with ultrasonic agitation. In summary, the MCE and 1 mm PTFE filters in combination with vortexing and shaker extraction demonstrated the best performance for the filter collection and extraction of BG spores. DOI: /b500056d 1. Introduction Bioterrorism is defined as the use or threatened use of biologic agents against individuals to obtain an advantage for a specific purpose such as intimidation, ideological principles, or disruption of everyday activities. 1 Since October 2001 with the introduction of mail contaminated with Bacillus anthracis into several work environments, people worldwide have become increasingly aware of the potential for bioterrorism acts. 2 These events revealed the need to develop validated environmental sampling and analytical methods for specific biological agents to determine whether they are present and at what concentration to determine the potential health hazard. 3 Traditionally, environmental monitoring for B. anthracis has been conducted using culture-based methods. 4 Culturable cell counts can be affected by a variety of factors such as the type of nutrient media selected; aerosolization, collection, and assay methods; and environmental conditions. 5 Filtration utilizes impaction, interception, and diffusion as the major collection mechanisms. 4 The primary advantages of using filtration collection for bioaerosol samples include potential to reach high collection efficiency, ease of sample collection and preparation, relatively low costs of collection equipment and supplies, and the ability to use various analysis techniques. Several environmental monitoring evaluations using various sampling techniques were conducted to investigate the level of B. anthracis contamination in the B. anthracis affected work sites. 6 9 The National Institute for Occupational Safety and Health (NIOSH) provided technical assistance to the United States Postal Service at the Trenton Processing and Distribution Center in Trenton, New Jersey. 6 As part of the environmental assessment conducted at the facility, air samples were collected before and after a contaminated mail sorter was operated using different sampling techniques. For the gelatin filter samples, 27/36 (75%) samples were positive for B. anthracis spores after the contaminated mail sorter was operational. All the mixed cellulose ester (MCE), polytetrafluoroethylene (PTFE), and dry filter unit (DFU) air samples were positive for B. anthracis spores after the sorter was operational when the entire extraction sample was analyzed for optimum sensitivity. 6 An environmental survey was conducted at the Brentwood Mail Processing and Distribution Center Washington, DC in October 2001 after the building had been closed and the ventilation system turned off for 3 days. 7 Twelve air samples were collected for a time period of about 30 hours at a flow rate of 2 litres per minute (L min 1 ) using openfaced 37-mm MCE filters and were negative for culturable B. anthracis. 7 Seven percent (8/114) of the sterile cotton gauze wipe samples and sixty-nine percent (27/39) of the vacuum dust samples were positive for B. anthracis. Additional monitoring was conducted on a mail sorting machine at the Brentwood facility that had handled two of the letters containing anthrax spores in October Air sampling performed using slit agar samplers with TSA plates showed 1 colony forming unit (CFU) before the machine was activated and 6 CFU during simulated work tasks. 8 No colonies were detected from the respirator filter samples worn by the evaluation team. 8 Weis and associates investigated secondary aerosolization in an office contaminated from the October 2001 incidents. 9 The investigators found that This journal is & The Royal Society of Chemistry 2005 J. Environ. Monit., 2005, 7,