Evaluation of two methods for detection of viable Bacillus anthracis simulant spores in maritime environmental samples.

Analytical methods exist to detect biothreat agents in environmental samples during a response to biological contamination incidents. However, the coastal zone facilities and assets of the US Coast Guard (USCG), including response boats in diverse geographical areas and maritime environmental conditions, can pose complex and unique challenges for adapting existing analytical detection methods. The traditional culture (TC) and the rapid viability polymerase chain reaction (RV-PCR) methods were evaluated for their compatibility for maritime environmental surface and grab sample analysis to detect spores of Bacillus thuringiensis subspecies kurstaki (Btk), a surrogate for Bacillus anthracis. The representative samples collected from a USCG installation included surfaces, such as aluminum on boats, nonskid tread on decks of watercraft, computer touchscreens, and concrete piers, and grab samples of boat washdown water, soil, vegetation, and gravel from surrounding areas. Replicate samples were spiked with Btk spores at two to three tenfold increasing levels and analyzed. Out of a total of 150 samples collected and analyzed, the TC method gave 10 false-positive and 19 false-negative results, while the RV-PCR method-based analysis resulted in 0 false-positive and 26 false-negative results. An abundance of microbial background and particulates in some samples interfered with true results, while both methods gave similar results for samples with low microbial background and particulates. Improved and high-throughput sample processing methods are needed for analysis of complex environmental samples.

Formaldehyde Vapor Characteristics in Varied Decontamination Environments.

INTRODUCTION: This effort investigated formaldehyde vapor characteristics under various environmental conditions by the analyses of air samples collected over a time-course. This knowledge will help responders achieve desired formaldehyde exposure parameters for decontamination of affected spaces after a biological contamination incident. METHODS: Prescribed masses of paraformaldehyde and formalin were sublimated or evaporated, respectively, to generate formaldehyde vapor. Adsorbent cartridges were used to collect air samples from the test chamber at predetermined times. A validated method was used to extract the cartridges and analyze for formaldehyde via liquid chromatography. In addition, material demand for the formaldehyde was evaluated by inclusion of arrays of Plexiglas panels in the test chamber to determine the impact of varied surface areas within the test chamber. Temperature was controlled with a circulating water bath connected to a radiator and fan inside the chamber. Relative humidity was controlled with humidity fixed-point salt solutions and water vapor generated from evaporated water. RESULTS: Low temperature trials (approximately 10°C) resulted in decreased formaldehyde air concentrations throughout the 48-hour time-course when compared with formaldehyde concentrations in the ambient temperature trials (approximately 22°C). The addition of clear Plexiglas panels to increase the surface area of the test chamber interior resulted in appreciable decreases of formaldehyde air concentration when compared to an empty test chamber. CONCLUSION: This work has shown that environmental variables and surface-to-volume ratios in the decontaminated space may affect the availability of formaldehyde in the air and, therefore, may affect decontamination effectiveness.

Decontamination of Bacillus Spores with Formaldehyde Vapor under Varied Environmental Conditions.

Introduction: This study investigated formaldehyde decontamination efficacy against dried Bacillus spores on porous and non-porous test surfaces, under various environmental conditions. This knowledge will help responders determine effective formaldehyde exposure parameters to decontaminate affected spaces following a biological agent release. Methods: Prescribed masses of paraformaldehyde or formalin were sublimated or evaporated, respectively, to generate formaldehyde vapor within a bench-scale test chamber. Adsorbent cartridges were used to measure formaldehyde vapor concentrations in the chamber at pre-determined times. A validated method was used to extract the cartridges and analyze for formaldehyde via liquid chromatography. Spores of Bacillus globigii, Bacillus thuringiensis, and Bacillus anthracis were inoculated and dried onto porous bare pine wood and non-porous painted concrete material coupons. A series of tests was conducted where temperature, relative humidity, and formaldehyde concentration were varied, to determine treatment efficacy outside of conditions where this decontaminant is well-characterized (laboratory temperature and humidity and 12 mg/L theoretical formaldehyde vapor concentration) to predict decontamination efficacy in applications that may arise following a biological incident. Results: Low temperature trials (approximately 10°C) resulted in decreased formaldehyde air concentrations throughout the 48-hour time-course when compared with formaldehyde concentrations collected in the ambient temperature trials (approximately 22°C). Generally, decontamination efficacy on wood was lower for all three spore types compared with painted concrete. Also, higher recoveries resulted from painted concrete compared to wood, consistent with historical data on these materials. The highest decontamination efficacies were observed on the spores subjected to the longest exposures (48 hours) on both materials, with efficacies that gradually decreased with shorter exposures. Adsorption or absorption of the formaldehyde vapor may have been a factor, especially during the low temperature trials, resulting in less available formaldehyde in the air when measured. Conclusion: Environmental conditions affect formaldehyde concentrations in the air and thereby affect decontamination efficacy. Efficacy is also impacted by the material with which the contaminants are in contact.

Decontamination of soil contaminated at the surface with Bacillus anthracis spores using dry thermal treatment.

In the event of a large, aerosol release of Bacillus anthracis spores in a major metropolitan area, soils and other outdoor materials may become contaminated with the biological agent. A study was conducted to assess the in-situ remediation of soil using a dry thermal treatment approach to inactivate a B. anthracis spore surrogate inoculated into soil samples. The study was conducted in two phases, using loam, clay and sand-based soils, as well as biological indicators and spore-inoculated stainless-steel coupons. Initial experiments were performed in an environmental test chamber with temperatures controlled between 80 and 110 °C, with and without added humidity, and with contact times ranging from 4 h to 7 weeks. Tests were then scaled up to assess the thermal inactivation of spores in small soil columns, in which a heating plate set to 141 °C was applied to the soil surface. These column tests were conducted to assess time requirements to inactivate spores as a function of soil depth and soil type. Results from the initial phase of testing showed that increasing the temperature and relative humidity reduced the time requirements to achieve samples in which no surrogate spores were detected. For the test at 80 °C with no added humidity, 49 days were required to achieve soil samples with no spores detected in clay and loam. At 110 °C, 24 h were required to achieve samples in which no spores were detected. In the column tests, no spores were detected at the 2.5 cm depth at four days and at the 5.1 cm depth at 21 days, for two of the three soils. The experiments described in the study demonstrate the feasibility of using dry thermal techniques to decontaminate soils that have been surficially contaminated with B. anthracis spores.

Comparison of surface sampling methods for an extended duration outdoor biological contamination study.

Bacillus anthracis, the causative agent for anthrax, is a dangerous pathogen to humans and has a history as a bioterrorism agent. While sampling methods have been developed and evaluated for characterizing and clearing contaminated indoor sites, the performance of these sampling methods is unknown for use in outdoor environments. This paper presents surface sampling data for Bacillus atrophaeus spores, a surrogate for B. anthracis, from a 210-day outdoor study that evaluated the detection and recovery of spores using five different sampling methods as follows: sponge sticks, 37-mm vacuum filter cassettes, residential wet vacuums, robotic floor cleaners, and grab samples of soil, leaves, and grass. The spores were applied by spraying a liquid suspension onto the surfaces. Both asphalt and concrete surfaces were sampled by all the surface sampling methods, excluding grab sampling. Stainless steel coupons placed outdoors were additionally sampled using sponge sticks. Sampling methods differed in their ability to collect detectable spores over the duration of the study. The 37-mm vacuums and sponge sticks consistently detected spores on asphalt through day 37 and robots through day 99. The wet vacuums detected spores on asphalt for days 1 and 4, but not again until day 210. On concrete, all samplers detected spores until day 210 except for sponge stick samplers that detected spores only up until the day 99 time point. For all sampling methods, spore recoveries were higher from concrete than from asphalt surfaces. There was no statistically significant difference in recoveries of sponge sticks and 37-mm vacuums from either asphalt or concrete surfaces. Processing of grab samples was challenging due to non-target background microorganisms resulting in high detection limits for the samples.

Evaluating the Environmental Persistence and Inactivation of MS2 Bacteriophage and the Presumed Ebola Virus Surrogate Phi6 Using Low Concentration Hydrogen Peroxide Vapor.

Ebola virus (EBOV) disease outbreaks, as well as the ability of EBOV to persist in the environment under certain conditions, highlight the need to develop effective decontamination techniques against the virus. We evaluated the efficacy of hydrogen peroxide vapor (HPV) to inactivate MS2 and Phi6 bacteriophages, the latter a recommended surrogate for EBOV. The phages were inoculated onto six material types with and without the presence of whole human blood. The inoculated materials were then exposed to either a high or low concentration of HPV for various elapsed times. The phages were also recovered from positive controls at these same elapsed times, to assess environmental persistence and decontamination efficacy. Low concentration hydrogen peroxide vapor (LCHP; 25 ppm) was effective against both phages on all materials without the presence of blood at 2 h. LCHP was ineffective against the phages in the presence of blood, on all materials, even with a 3-day contact time. Higher concentrations of HPV (>400 ppm) with contact times of 24-32 h achieved approximately 2-6 log reduction of the phages in the presence of blood.

Evaluation of standardized sample collection, packaging, and decontamination procedures to assess cross-contamination potential during Bacillus anthracis incident response operations.

Sample collection procedures and primary receptacle (sample container and bag) decontamination methods should prevent contaminant transfer between contaminated and non-contaminated surfaces and areas during bio-incident operations. Cross-contamination of personnel, equipment, or sample containers may result in the exfiltration of biological agent from the exclusion (hot) zone and have unintended negative consequences on response resources, activities and outcomes. The current study was designed to: (1) evaluate currently recommended sample collection and packaging procedures to identify procedural steps that may increase the likelihood of spore exfiltration or contaminant transfer; (2) evaluate the efficacy of currently recommended primary receptacle decontamination procedures; and (3) evaluate the efficacy of outer packaging decontamination methods. Wet- and dry-deposited fluorescent tracer powder was used in contaminant transfer tests to qualitatively evaluate the currently-recommended sample collection procedures. Bacillus atrophaeus spores, a surrogate for Bacillus anthracis, were used to evaluate the efficacy of spray- and wipe-based decontamination procedures. Both decontamination procedures were quantitatively evaluated on three types of sample packaging materials (corrugated fiberboard, polystyrene foam, and polyethylene plastic), and two contamination mechanisms (wet or dry inoculums). Contaminant transfer results suggested that size-appropriate gloves should be worn by personnel, templates should not be taped to or removed from surfaces, and primary receptacles should be selected carefully. The decontamination tests indicated that wipe-based decontamination procedures may be more effective than spray-based procedures; efficacy was not influenced by material type but was affected by the inoculation method. Incomplete surface decontamination was observed in all tests with dry inoculums. This study provides a foundation for optimizing current B. anthracis response procedures to minimize contaminant exfiltration.

Evaluation of the Efficacy of Methyl Bromide in the Decontamination of Building and Interior Materials Contaminated with Bacillus anthracis Spores.

The primary goal of this study was to determine the conditions required for the effective inactivation of Bacillus anthracis spores on materials by using methyl bromide (MeBr) gas. Another objective was to obtain comparative decontamination efficacy data with three avirulent microorganisms to assess their potential for use as surrogates for B. anthracis Ames. Decontamination tests were conducted with spores of B. anthracis Ames and Geobacillus stearothermophilus, B. anthracis NNR1Δ1, and B. anthracis Sterne inoculated onto six different materials. Experimental variables included temperature, relative humidity (RH), MeBr concentration, and contact time. MeBr was found to be an effective decontaminant under a number of conditions. This study highlights the important role that RH has when fumigation is performed with MeBr. There were no tests in which a ≥6-log10 reduction (LR) of B. anthracis Ames was achieved on all materials when fumigation was done at 45% RH. At 75% RH, an increase in the temperature, the MeBr concentration, or contact time generally improved the efficacy of fumigation with MeBr. This study provides new information for the effective use of MeBr at temperatures and RH levels lower than those that have been recommended previously. The study also provides data to assist with the selection of an avirulent surrogate for B. anthracis Ames spores when additional tests with MeBr are conducted.

Capture of methyl bromide emissions with activated carbon following the fumigation of a small building contaminated with a Bacillus anthracis spore simulant.

A wide-area Bacillus anthracis spore contamination incident will present immense challenges related to decontamination capacity. For this reason, fumigation with methyl bromide (MeBr) has been proposed as a potential remediation option. Although a few bench-scale laboratory studies have been conducted to evaluate activated carbon for the capture of MeBr, these studies were conducted at conditions replicating commodity fumigation using relatively low MeBr concentrations, temperatures, and/or relative humidity (RH) levels. The more rigorous MeBr fumigation requirements to fully inactivate B. anthracis spores are much more of a challenge for an activated carbon system (ACS) to capture MeBr, and warrant their own investigation. Further, while the aforementioned studies have shown activated carbon to be a possible option for the capture of MeBr in gas streams, these tests were conducted at laboratory bench scale, and thus lack operational perspective and data. Thus, we present for the first time the results of a full-scale study to evaluate an ACS employed for the capture of MeBr at conditions that would be used for decontaminating a building structure contaminated with B. anthracis spores. Airflow rate, temperature, RH, and MeBr levels were measured within the ACS during its operation. Despite the relatively high humidity, temperature, and MeBr levels, the MeBr capture efficiency of the ACS was demonstrated to be more than 99%. The concentration of MeBr exhausted from the structure was reduced from 41,000 to 136 ppmv in 3.5 hr, corresponding to an overall atmospheric emission rate of less than 2 kg. The practical adsorption rate of the ACS was determined to be 4.83 kg MeBr/100 kg carbon. The information and data presented here will facilitate future use of this technology when fumigating with MeBr.

Evaluation of surface sampling for Bacillus spores using commercially available cleaning robots.

Five commercially available domestic cleaning robots were evaluated on their effectiveness for sampling aerosol-deposited Bacillus atrophaeus spores on different indoor material surfaces. The five robots tested include three vacuum types (R1, R2, and R3), one wet wipe (R4), and one wet vacuum (R5). Tests were conducted on two different surface types (carpet and laminate) with 10(6) colony forming units of B. atrophaeus spores deposited per coupon (35.5 cm × 35.5 cm). Spores were deposited on the center surface (30.5 × 30.5 cm) of the coupon's total surface area (71.5 × 71.5 cm), and the surfaces were sampled with an individual robot in an isolation chamber. Chamber air was sampled using a biofilter sampler to determine the potential for resuspension of spores during sampling. Robot test results were compared to currently used surface sampling methods (vacuum sock for carpet and sponge wipe for laminate). The test results showed that the average sampling efficacies for R1, R2, and R3 on carpet were 26, 162, and 92% of vacuum sock sampling efficacy, respectively. On laminate, R1, R2, R3, R4, and R5 average sampling efficacies were 8, 11, 2, 62, and 32% of sponge wipe sampling efficacy, respectively. We conclude that some robotic cleaners were as efficacious as the currently used surface sampling methods for B. atrophaeus spores on these surfaces.