Demonstration and evaluation of commercial and municipal equipment for urban biological decontamination.

Large-scale biological contamination incidents pose unique yet significant challenges to remediation operations. Previous incidents have demonstrated the utility of readily available commercial and municipal equipment for conducting remediation tasks. Preidentification and evaluation of such equipment could reduce lag time for response initiation and enhance overall response effectiveness and efficiency. The current study aimed to identify commercial and municipal equipment that could be beneficial in wide-area biological remediation operations. Equipment were identified by market research, their utility was assessed by a group of subject matter experts, and a subset of those equipment was observed under operational conditions in a realistic urban environment. Observations and feedback from demonstration participants are presented within the article. This information is intended to support rapid decision-making following large-scale biological incidents, broaden the universe of potentially useful equipment to support the response, enhance response operations, and reduce the impact on the public.

Monitoring spore washoff during a biological contamination incident response using automated stormwater samplers and sensors to predict contamination movement.

This study examined the washoff of Bacillus globigii (Bg) spores from concrete, asphalt, and grass surfaces by stormwater. Bg is a nonpathogenic surrogate for Bacillus anthracis, which is a biological select agent. Areas (2.74 m × 7.62 m) of concrete, grass, and asphalt were inoculated twice at the field site during the study. Spore concentrations were measured in runoff water after seven rainfall events (1.2-65.4 mm) and complimentary watershed data were collected for soil moisture, depth of water in collection troughs, and rainfall using custom-built telemetry units. An average surface loading of 107.79 Bg spores/m2 resulted in peak spore concentrations in runoff water of 102, 260, and 4.1 CFU/mL from asphalt, concrete, and grass surfaces, respectively. Spore concentrations in the stormwater runoff were greatly reduced by the third rain event after both inoculations, but still detectable in some samples. When initial rainfall events occurred longer after the initial inoculation, the spore concentrations (both peak and average) in the runoff were diminished. The study also compared rainfall data from 4 tipping bucket rain gauges and a laser disdrometer and found they performed similarly for values of total rainfall accumulation while the laser disdrometer provided additional information (total storm kinetic energy) useful in comparing the seven different rain events. The soil moisture probes are recommended for assistance in predicting when to sample sites with intermittent runoff. Sampling trough level readings were critical to understanding the dilution factor of the storm event and the age of the sample collected. Collectively the spore and watershed data are useful for emergency responders faced with remediation decisions after a biological agent incident as the results provide insight into what equipment to deploy and that spores may persist in runoff water at quantifiable levels for months. The spore measurements are also a novel dataset for stormwater model parameterization for biological contamination of urban watersheds.

Effectiveness of formaldehyde in various soil types as a wide area decontamination approach for Bacillus anthracis spores.

The purpose of this study was to evaluate and compare the decontamination efficacy of liquid formaldehyde solutions for three soil types (sand, loam, and clay) against spores of Bacillus anthracis (B.a.) and Bacillus atrophaeus. Approximately 1 x 108 colony forming units were inoculated into each sample. Through a series of six bench-scale experiments, two concentrations and two volumes of liquid formaldehyde solution were then added to the soil samples and allowed to remain in contact for either 24 or 48 hours. Decontamination efficacy was assessed at either 22° or 10°C with or without lids atop the sample jars. Complete inactivation (no spores recovered from the soil samples, typically providing > 7 log reduction) of B.a. occurred in all soil types in five of the six tests, while complete inactivation of B. atrophaeus was achieved in all soil types for three of the six tests. The results demonstrated a higher probability of complete inactivation of spores for samples that were covered, samples that received the higher volume of formaldehyde, and those contaminated with B.a. Overall, the use of liquid formaldehyde solution (2.5-5%) was highly effective in inactivating entire spore populations (typically > 107 CFU) for both B.a. and B. atrophaeus in the soil matrices studied. Covering the soil after application would allow for less formaldehyde solution to be used without impacting the overall efficacy of the process. The data from this study may aid in the selection of appropriate decontamination parameters when using liquid formaldehyde for soil remediation. The data may also aid in the decision to use B. atrophaeus as a surrogate for B.a. when performing further decontamination studies using liquid formalin solutions.

Decontamination efficacy of common liquid disinfectants against non-spore-forming biological agents in soil matrices.

AIMS: The purpose of this study was to evaluate decontamination efficacy, within three soil types, against Yersinia pestis, Burkholderia pseudomallei, and the Venezuelan Equine Encephalitis virus (VEEV). METHODS AND RESULTS: One of three liquid disinfectants (dilute bleach, Virkon-S or Klozur One) was added to three soil types (sand, loam, or clay) and allowed contact for four pre-spike durations: 0, 15, 30 and 60 min. Y. pestis, B. pseudomallei, or VEEV was then spiked into the soil (10 microliters or approx. 1 × 107  CFU or PFU into 1 g soil) and decontamination efficacy assessed at post-spike contact times of 10 or 60 min at ambient environmental conditions. Across all soil types, sandy soil resulted in the least quenching to all three disinfectants tested as shown by sustained decontamination efficacy across all pre-spike and post-spike timepoints. Clay and loam soil types exhibited quenching effects on the hypochlorite and peroxygen based disinfectants (dilute bleach and Virkon S) and in general resulted in decreased efficacy with increased pre-spike contact time. The sodium persulfate (Klozur One) performance was the most consistent across all soil types and pre-spike contact times, resulting in greater efficacy with increased post-spike time. CONCLUSIONS: Liquid disinfectants can provide high levels of decontamination in soil for both viral and non-spore-forming bacterial select agents. Hypochlorite and peroxygen based disinfectants used in soils containing higher organic content (loam or clay) may require extended contact times or re-application of liquid disinfectant, in as little as 15 min of application, to achieve a 6-log reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information for the performance of three disinfectants in soil against non-spore-forming select agents. These data may aid response decision makers following a biological contamination incident by informing the selection of disinfectant as well as the re-application time to achieve effective site remediation.

Residual antimicrobial coating efficacy against SARS-CoV-2.

AIMS: This study evaluated the residual efficacy of commercially available antimicrobial coatings or films against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on non-porous surfaces. METHODS AND RESULTS: Products were applied to stainless steel or ABS plastic coupons and dried overnight. Coupons were inoculated with SARS-CoV-2 in the presence of 5% soil load. Recovered infectious SARS-CoV-2 was quantified by TCID50 assay. Tested product efficacies ranged from <1.0 to >3.0 log10 reduction at a 2-h contact time. The log10 reduction in recovered infectious SARS-CoV-2 ranged from 0.44 to 3 log10 reduction on stainless steel and 0.25 to >1.67 log10 on ABS plastic. The most effective products tested contained varying concentrations (0.5%-1.3%) of the same active ingredient: 3-(trihydroxysilyl) propyldimethyloctadecyl ammonium chloride. Products formulated with other quaternary ammonium compounds were less effective against SARS-CoV-2 in this test. CONCLUSIONS: The residual antimicrobial products tested showed varied effectiveness against SARS-CoV-2 as a function of product tested. Several products were identified as efficacious against SARS-CoV-2 on both stainless steel and ABS plastic surfaces under the conditions evaluated. Differences in observed efficacy may be due to variation in active ingredient formulation; efficacy is, therefore, difficult to predict based upon listed active ingredient and its concentration. SIGNIFICANCE AND IMPACT: This study highlights the formulation-specific efficacy of several products against SARS-CoV-2 and may inform future development of residual antiviral products for use on non-porous surfaces. The identification of antimicrobial coatings or films showing promise to inactivate SARS-CoV-2 suggests that these products may be worth future testing and consideration.

Efficacy of chemical disinfectants against SARS-CoV-2 on high-touch surface materials.

AIMS: This study aimed to provide operationally relevant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface disinfection efficacy information. METHODS AND RESULTS: Three EPA-registered disinfectants (Vital Oxide, Peroxide, and Clorox Total 360) and one antimicrobial formulation (CDC bleach) were evaluated against SARS-CoV-2 on material coupons and were tested using Spray (no touch with contact time) and Spray & Wipe (wipe immediately post-application) methods immediately and 2 h post-contamination. Efficacy was evaluated for infectious virus, with a subset tested for viral RNA (vRNA) recovery. Efficacy varied by method, disinfectant, and material. CDC bleach solution showed low efficacy against SARS-CoV-2 (log reduction < 1.7), unless applied via Spray & Wipe. Additionally, mechanical wiping increased the efficacy of treatments against SARS-CoV-2. The recovery of vRNA post-disinfection suggested that vRNA may overestimate infectious virus remaining. CONCLUSIONS: Efficacy depends on surface material, chemical, and disinfection procedure, and suggests that mechanical wiping alone has some efficacy at removing SARS-CoV-2 from surfaces. We observed that disinfectant treatment biased the recovery of vRNA over infectious virus. SIGNIFICANCE AND IMPACT OF STUDY: These data are useful for developing effective, real-world disinfection procedures, and inform public health experts on the utility of PCR-based surveillance approaches.

Influence of wash aids on Bacillus spore removal from an asphalt parking lot using two spray-based washing methods.

AIMS: The goal of this study was to measure the removal efficacy of Bacillus atrophaeus spores from a parking lot using spray-based washing methods (a pressure washer and a garden hose) and wash aids. B. atrophaeus is a commonly used nonpathogenic surrogate for B. anthracis, the causative agent of anthrax and a deadly bioterrorism agent that would cause major disruptions and damage to public health should it be disseminated over an urban area. METHODS AND RESULTS: Five wash aids (1 mM sodium chloride, an Instant Ocean® seawater solution, 0.01% Tween 20, 0.01% sodium dodecyl sulfate, and unamended tap water) were used along with two different spray sequences in this study. Across all treatment conditions, 3.7-6.4 log10  colony forming unit were recovered in the runoff water, and 0.15%-23% of spores were removed from the surface of the parking lot. CONCLUSIONS: Pressure washing removed more spores than the garden hose, and for both types of washing methods, the first pass removed more spores than the subsequent passes. The Instant Ocean and Tween 20 wash aids were found to significantly increase the percentage of spore removal when using the pressure washer, but the overall increase was only 1%-2% compared to the tap water alone. SIGNIFICANCE AND IMPACT OF STUDY: This study provides public officials and emergency responders with baseline spore physical removal information for situations where a corrosive disinfectant might have a negative impact on the environment and washing is being considered as an alternative remediation approach.

Virucidal efficacy of antimicrobial surface coatings against the enveloped bacteriophage Φ6.

AIMS: Antimicrobial coatings, for use in combination with routine cleaning and disinfection, were evaluated for their effectiveness in reducing virus concentration on stainless steel surfaces. METHODS: Twenty antimicrobial coating products, predominantly composed of organosilane quaternary ammonium compounds, were applied to stainless steel coupons, dried overnight and evaluated for efficacy against Φ6, an enveloped bacteriophage. Additionally, two peel and stick polymer-based films, a copper-based film and three copper alloys were evaluated. Efficacy was determined by comparison of recoveries from uncoated (positive control) and coated (test) surfaces. RESULTS: The results indicated that some of the coating products initially demonstrated >3-log reduction of Φ6; no direct correlation of efficacy was observed with an active ingredient or its concentration. The peel and stick films and copper alloys each demonstrated efficacy in initial testing. However, none of the spray-based products retained efficacy after subjecting the coating to abrasion with either a hypochlorite or quaternary ammonium-based solution applied in accordance with EPA Interim Guidance for Evaluating the Efficacy of Antimicrobial Surface Coatings. Of the products tested for this durability, only one peel and stick polymeric film retained efficacy; the copper alloys were not tested for their durability in this study. CONCLUSIONS: These results suggest that while some organosilane quaternary ammonium compound-based products demonstrate antiviral efficacy, more research and development is needed to understand effective formulations with sufficient durability to perform as supplements to routine cleaning and disinfection.

Low-concentration hydrogen peroxide decontamination for Bacillus spore contamination in buildings.

Remediation and recovery efforts after a release of Bacillus anthracis (anthrax) spores may be difficult and costly. In addition, response and recovery technologies may be focused on critical resources, leaving the small business or homeowner without remediation options. This study evaluates the efficacy of relatively low levels of hydrogen peroxide vapor (HPV) delivered from off-the-shelf equipment for the inactivation of Bacillus spores within an indoor environment. Decontamination evaluations were conducted in a house using both Bacillus atrophaeus var. globigii (Bg; as surrogates for B. anthracis ) inoculated on the carpet and galvanized metal as coupons and Geobacillus stearothermophilus (Gs) as biological indicators on steel. The total decontamination time ranged from 4 to 7 days. Using the longer exposure times, low concentrations of HPV (average levels below 20 parts per million) effectively inactivated Bg and Gs spores on the materials tested. The HPV was generated with commercial humidifiers and household-strength hydrogen peroxide solutions. The presence of home furnishings did not have a significant impact on HPV efficacy. This simple, inexpensive, and effective decontamination method could have significant utility for remediation following a B. anthracis spore release, such as following a terrorist attack.

Composite sampling of a Bacillus anthracis surrogate with cellulose sponge surface samplers from a nonporous surface.

A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm2 (four separate 929 cm2 areas), larger than the 645 cm2 prescribed by the standard Centers for Disease Control (CDC) and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261). Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720) for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001). The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.

Evaluation of peracetic acid fog for the inactivation of Bacillus anthracis spore surrogates in a large decontamination chamber.

The purpose of this study was to evaluate the sporicidal (inactivation of bacterial spores) effectiveness and operation of a fogging device utilizing peracetic acid/hydrogen peroxide (PAA). Experiments were conducted in a pilot-scale 24 m(3) stainless steel chamber using either biological indicators (BIs) or bacterial spores deposited onto surfaces via aerosolization. Wipe sampling was used to recover aerosol-deposited spores from chamber surfaces and coupon materials before and after fogging to assess decontamination efficacy. Temperature, relative humidity, and hydrogen peroxide vapor levels were measured during testing to characterize the fog environment. The fog completely inactivated all BIs in a test using a 60 mL solution of PAA (22% hydrogen peroxide/4.5% peracetic acid). In tests using aerosol-deposited bacterial spores, the majority of the post-fogging spore levels per sample were less than 1 log colony forming units, with a number of samples having no detectable spores. In terms of decontamination efficacy, a 4.78 log reduction of viable spores was achieved on wood and stainless steel. Fogging of PAA solutions shows potential as a relatively easy to use decontamination technology in the event of contamination with Bacillus anthracis or other spore-forming infectious disease agents, although additional research is needed to enhance sporicidal efficacy.