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Illuminating Anthrax

The Defense Threat Reduction Agency’s Chemical and Biological Technologies Department (DTRA CB) has a new test that reduces the time needed to determine clearance decontamination for anthrax (anthrax clearance). Anthrax clearance means that anthrax spores are no longer detectable in a laboratory test of an environmental sample. Clearance testing ensures that decontamination efforts have reduced the hazard to a level that does not threaten the warfighter. Current clearance tests can take up to two days.

DTRA CB’s faster test, called the Bacteriophage Rapid Anthrax Viability Evaluation System (BRAVES), produces results in 6–8 hours. BRAVES is an adaptation of a commercial, off-the-shelf technology. DTRA CB supported the adaptation effort, which was performed by government researchers at the U.S. Army Corps of Engineers Engineer Research and Development Center Construction Engineering Research Laboratory and the Naval Surface Warfare Center Dahlgren Division.

BRAVES rapidly detects living anthrax spores in multiple environmental samples at one time. Additional research is necessary to evaluate the new test’s efficacy in the environmental conditions where warfighters operate. Researchers will also compare the new test’s ability to identify viable anthrax spores across various samples (plywood, concrete, fabric, water, soil), collected before and after a decontamination. Outstanding assessments aside, BRAVES is showing promise as a new anthrax clearance test.

BRAVES overcomes disadvantages associated with current anthrax clearance methods that include the standard polymerase chain reaction (PCR) test, the plate culture assay, and quantitative PCR. Standard PCR takes 12–18 hours to complete and does not differentiate dead spores from living or viable spores that can become infectious. The plate culture assay test requires 1–2 days to complete, is demanding in labor, and needs its results verified by sophisticated laboratory equipment that, in turn, needs skilled technicians to operate and an environmentally controlled setting to store. Quantitative PCR also needs equipment that is impractical for the field, and its results take over 24 hours to yield.

Unlike other tests, BRAVES produces light to signal the number of viable spores present in a sample. Here’s how the new test functions: soil or another environmental sample is added to a broth containing nutrients for germinating viable spores and growing the parent bacterium Bacillus anthracis. The broth also includes antibiotics and a genetically engineered bacteriophage (a small virus) called the Wβ. Bacteriophages infect certain bacteria, and the Wβ infects B. anthracis.

If a sample has viable anthrax spores, then the spores germinate into vegetative cells that are capable of causing the anthrax infection. Wβ infects the vegetative cells and replicates itself and, in the process, makes the luciferase protein, the same type of protein that makes a firefly glow. The mixture of sample and broth incubate for 6–8 hours, and the luciferase protein produces bioluminescence, thereby causing each living anthrax cell to illuminate. The antibiotic in the broth kills all nonanthrax bacteria in the sample, so the light generated is only due to viable anthrax spores.

Typically, researchers expect an anthrax clearance test to detect spores at a number below the amount required to infect humans. BRAVES exceeds this expectation. Laboratory tests reveal that the new test identifies as few as 1,000 B. anthracis viable spores (about 10 times less than a human infectious dose) in a gram of normal soil.
The new Wβ-mediated bioluminescent technology achieves anthrax clearance at 10% of the cost, labor, and laboratory space needed by current methods — a logistical improvement.

BRAVES is faster in determining anthrax clearance than competing state-of-the-art methods, thereby enabling warfighters to more quickly establish the success of their decontamination efforts. For warfighters, a quicker achievement of clearance means a quicker return to the mission and a quicker return home.


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