Aims
To add a spore germination step in order to reduce decontamination temperature and time requirements compared to the current hot, humid air decontamination parameters, which are 75–80°C, ≥72 h, ...70–90% RH, down to ≤60°C and ≤24 h total decontamination time.
Methods and Results
Bacillus anthracis spore germination with l‐alanine+inosine+calcium dipicolinate (CaDPA) was quantified at 0–40°C, several time points and spore concentrations of 5–9 log10 per ml. Germination efficiency at 0–40°C was >99% at <8 log10 spores per ml. The temperature optimum was 20°C. Germination efficiency was significantly higher but slower at 0°C compared to ≥30°C at ≥8 log10 spores per ml. A single germinant application followed by 60°C, 1‐h treatment consistently inactivated >2 log10 (>99%) of spores. However, a repeat application of germinant was needed to achieve the objective of ≥6 log10 spore inactivation out of a 7 log10 challenge (≥99·9999%) for ≤24 h total decontamination time for nylon and aircraft performance coating.
Conclusions
l‐alanine+inosine+CaDPA stimulated germination across wide temperature and spore concentration ranges.
Significance and Impact of the Study
Germination expands the scope of spore decontamination to include materials from any industry sector that can be sprayed with an aqueous germinant solution.
Aim
To develop test methods and evaluate survival of Bacillus thuringiensis kurstaki cry− HD‐1 and B. thuringiensis Al Hakam spores after exposure to hot, humid air inside of a C‐130 aircraft.
...Methods and Results
Bacillus thuringiensis spores were either pre‐inoculated on 1 × 2 or 2 × 2 cm substrates or aerosolized inside the cargo hold of a C‐130 and allowed to dry. Dirty, complex surfaces (10 × 10 cm) swabbed after spore dispersal showed a deposition of 8–10 log10 m−2 through the entire cargo hold. After hot, humid air decontamination at 75–80°C, 70–90% relative humidity for 7 days, 87 of 98 test swabs covering 0·98 m2, showed complete spore inactivation. There was a total of 1·67 log10 live CFU detected in 11 of the test swabs. Spore inactivation in the 98 test swabs was measured at 7·06 log10 m−2.
Conclusions
Laboratory test methods for hot, humid air decontamination were scaled for a large‐scale aircraft field test. The C‐130 field test demonstrated that hot, humid air can be successfully used to decontaminate an aircraft.
Significance and Impact of the Study
Transition of a new technology from research and development to acquisition at a Technology Readiness Level 7 is unprecedented.
AIMS: To develop test methods and evaluate survival of Bacillus anthracis ∆Sterne or Bacillus thuringiensis Al Hakam on materials contaminated with dirty spore preparations after exposure to hot, ...humid air using response surface modelling. METHODS AND RESULTS: Spores (>7 log₁₀) were mixed with humic acid + spent sporulation medium (organic debris) or kaolin (dirt debris). Spore samples were then dried on five different test materials (wiring insulation, aircraft performance coating, anti‐skid, polypropylene, and nylon). Inoculated materials were tested with 19 test combinations of temperature (55, 65, 75°C), relative humidity (70, 80, 90%) and time (1, 2, 3 days). The slowest spore inactivation kinetics was on nylon webbing and/or after addition of organic debris. CONCLUSIONS: Hot, humid air effectively decontaminates materials contaminated with dirty Bacillus spore preparations; debris and material interactions create complex decontamination kinetic patterns; and B. thuringiensis Al Hakam is a realistic surrogate for B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Response surface models of hot, humid air decontamination were developed which may be used to select decontamination parameters for contamination scenarios including aircraft.
Aims To develop test methods and evaluate survival of Bacillus anthracis Delta Sterne or Bacillus thuringiensis Al Hakam on materials contaminated with dirty spore preparations after exposure to hot, ...humid air using response surface modelling. Methods and Results Spores (>7 log sub(10)) were mixed with humic acid + spent sporulation medium (organic debris) or kaolin (dirt debris). Spore samples were then dried on five different test materials (wiring insulation, aircraft performance coating, anti-skid, polypropylene, and nylon). Inoculated materials were tested with 19 test combinations of temperature (55, 65, 75 degree C), relative humidity (70, 80, 90%) and time (1, 2, 3 days). The slowest spore inactivation kinetics was on nylon webbing and/or after addition of organic debris. Conclusions Hot, humid air effectively decontaminates materials contaminated with dirty Bacillus spore preparations; debris and material interactions create complex decontamination kinetic patterns; and B. thuringiensis Al Hakam is a realistic surrogate for B. anthracis. Significance and Impact of the Study Response surface models of hot, humid air decontamination were developed which may be used to select decontamination parameters for contamination scenarios including aircraft.
In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of
that contained live spores. In response, a systematic ...evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of
spores containing 3 × 10
CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D
) values and irradiation doses required to ensure sterility (D
) to the point at which the probability of detecting a viable spore is 10
Furthermore, spores of a genetically tagged strain of
strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of
for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques.
The inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live
(anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated
spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.
To develop infectious (live/dead) enveloped virus test indicators and response surface methodology (RSM) models that evaluate survival of an enveloped ribonucleic acid (RNA) virus on contaminated ...aircraft materials after exposure to hot, humid air (HHA).
Enveloped RNA bacteriophage Phi6 (Φ6) was dried on wiring insulation, aircraft performance coating (APC), polypropylene, and nylon at ≥ 8 log
plaque-forming units (PFU) test coupon
. Only 2.4 log
inactivation was measured on APC at 70°Celsius (°C), 5% relative humidity (RH) after 24 h. In contrast, HHA RSM models showed a 90% probability of a 7 log
inactivation at ≥63°C, 90% RH after 1 h, and decontamination kinetics were similar across different materials. HHA decontamination of C-130 and C-17 aircraft showed >7 log
and ≥5.9 log
inactivation of enveloped virus on 100 and 110 test indicators, respectively, with a 1-h treatment, excluding ramp-up and ramp-down times.
Enveloped RNA virus test indicators were successfully developed, lab tested for HHA decontamination, analyzed for RSM, and field-tested in aircraft demonstrations.
The utility of HHA decontamination was demonstrated after inactivating enveloped RNA virus on aircraft with a 1-h HHA treatment within aircraft temperature and RH limits.
Subscale testing of prompt agent defeat formulations
SHOCK COMPRESSION OF CONDENSED MATTER - 2015: Proceedings of the Conference of the American Physical Society Topical Group on Shock Compression of Condensed Matter,
01/2017
Conference Proceeding
PDF
