The Hidden Enemy

• By Jim DiPeso
• Mar 01, 2002

Whether it's pulmonary or cutaneous anthrax -- diseases caused by the Bacillus anthracis organism -- the bacterium is a living threat we're not so sure about. Our human nature has exhibited a reliable "fear of the unknown" against a real, live natural born killer.

The anthrax bacterium is closely related to a variety of harmless bacteria that are commonly found on skin and in the soil, which makes specific detection in the environment very difficult.

An aerobic organism, the bug requires molecular oxygen to flourish, and in the presence of high oxygen concentrations - as in the air - the organism changes to endospores. The formation of endospores makes Bacillus anthracis particularly appealing as a biological weapon, because the spores are resistant to sunlight, heat and most disinfectants, and can remain active in soil and water for years.

Five people have died since October, 2001, and more than a dozen people were infected by anthrax in New York, New Jersey, Florida and Washington. The Hart Senate Office Building reopened in January, 2002, after a three-month cleanup that cost at least $14 million due to an anthrax-laced letter that was sent to U.S. Senate Majority Leader Tom Daschle (D-S.D).

Ever since anthrax spread through U.S. territory, pressure has been placed on public and private entities to develop efficient detection technologies, rapid sampling and testing techniques, and to find foolproof methods of decontamination. Dozens of companies are working to vanquish the uncertainty, and over the last several months there has been a surge in ammunition production against biological warfare -- developments in everything from super shelters to small detection devices.

The air is filled with biological material from pollen to pathogens. Engineering a machine that can distinguish between the normal "background" particles and lethal weapons is difficult. Where exactly to place these machines is a mystery as well.

Ideally, a device like a smoke detector is needed to continuously sniff the air and warn about an attack. If authorities knew immediately, they could begin evacuations, quarantines and treatments. For now, sick people seem to be the difinitive means of detecting an attack.

Hand-held assays (analyses to determine the quantity of a components present in a sample) are sold commercially for the detection of Bacillus anthracis, but are intended only for the screening of environmental samples. First responder and law enforcement communities are using these as instant screening devices, but confirm positive samples with additional testing.

At this time, the utility and validity of the assays are unknown, and the U.S. Centers for Disease Control (CDC) do not have enough scientific data to recommend their use. Analytical sensitivity is limited by the technology, and data provided by manufacturers indicate that a minimum of 10,000 spores is required to generate a positive signal -- a negative result does not necessarily rule out all levels of contamination. CDC is currently evaluating the sensitivity and specificity of these assays.

CDC conducts the definitive testing for major threat agents - such as anthrax, smallpox and botulism -- and is engaging its partners in the Laboratory Response Network (LRN) all across the United States. A multilevel system linking 100 state and local public health laboratories with advanced capacity laboratories, LRN rapidly identifies threat agents.

LRN has been using a validated real-time polymerase chain reaction (PCR) assay for some time. PCR is a laboratory method used to detect and amplify genetic material. When any cell divides, enzymes called polymerases make a copy of all the DNA in each chromosome. It can be used to diagnose disease by identifying DNA strands commonly found in all Bacillus anthracis strains. The PCR process can often be done in less than an hour, and tests can be done in the field, but are usually performed in a diagnostic laboratory. Currently, PCR assays are not considered confirmatory tests for anthrax.

This field detection method cracks open a spore or biological organism and looks at the DNA to identify it. The tests work in minutes, and if there's only a small amount of a sample, copies of DNA pieces help make the analysis more accurate. However, a little dirt or water can throw off the results, and methods to purify samples aren't refined enough.

Dr. Jeanne Small, an Eastern Washington University biophysicist, is working on a new device that shines a laser into an air sample and records the sound waves given off by a specific bacteria from one to 10 microns in size. The system would work constantly to monitor the air.

"The idea is to see when you hit a particle with a pulse of laser light, whether that particle will give off an ultra-sound signature that will identify what the particle is," said Dr. Small.

The detection process still has a long way to go before Americans can have absolute confidence in a reliable warning of a biological attack. No one has yet to figure out how to make equipment that provides continuous, automatic and accurate detection. False positives often result with the quick equipment -- such as the assay -- which are mistaken identifications of killer germs from common ones. Additionally, detectors can clog up because they can't filter out dust and other small particles from the germs being looked for. Plus, samples of air or water can easily be contaminated by dust, pollen, dirt or other particles in the air.

Currently, no environmental or occupational exposure standards exist for Bacillus anthracis spores. Investigators who review and interpret the results of environmental sampling for such spores consider these uncertainties and use professional judgment in interpreting any findings. The "gold standard" remains taking samples and waiting for them to grow in a lab, yet the positive or negative results are only as good as the method used to prepare the sample for analysis in the first place.

According to the CDC, sample testing is a two-step process. The first test, a screening test, may be positive within two hours if the sample is large and contains a lot of Bacillus anthracis. However, a positive reading on this first test must be confirmed with a second, more accurate test. This confirmation test, conducted by a more sophisticated laboratory, takes much longer. The length of time needed depends in part on how fast the bacteria grow, but results are usually available one to three days after the sample is received in the laboratory.

There are many kinds of anthrax tests, and the reliability of each has not been determined. In general, findings from actual culturing of environmental samples are specific; that is, a positive result reflects the true presence of Bacillus anthracis, and a negative result likely means that no Bacillus anthracis is present. However, it is best to confirm the results of any test with another test that uses a different detection scheme.

The U.S. Environmental Protection Agency (EPA) has lead responsibility for issues related to environmental cleanup of hazardous materials and weapons of mass destruction with the assistance of 16 different federal agencies and departments working with state and local agencies. The decision for a most efficient approach to cleanup will be defined based upon factors such as sampling results, cleanup options and the environmental media.

Decontamination of anthrax is a rapidly evolving field, with new technologies continually being advanced and tested. In developing a strategy for decontamination, EPA consults with a variety of scientific experts. Several different antimicrobial pesticides and devices are being currently used by qualified experts under carefully controlled conditions in anthrax cleanups. Currently, three methods dominate the decontamination techniques available.

Chlorine dioxide (ClO₂) is an antimicrobial pesticide recognized for its disinfectant properties and is used to control harmful microorganisms on inanimate objects and surfaces. It kills microorganisms by disrupting transport of nutrients across the cell wall and can be generated in a gas or liquid form.

Decontamination foam is a formulation consisting of two active ingredients that, when combined, act exclusively for the decontamination of biological warfare agents. These active ingredients include Alkyl Dimethyl Benzyl Ammonium Chloride, and Hydrogen Peroxide, which is an oxidizing agent. When combined, the active ingredients disrupt and destroy anthrax spores.

Ethylene oxide is an industrial chemical used in sterilizing medical items, fumigating spices, and manufacturing other chemicals. Ethylene oxide kills microorganisms by denaturing their proteins and subsequently modifying their molecular structure.

A new system being tested delivers successive pulses of ultraviolet light to bacteria for only one second, which appears to damage spores so extensively that the spores are not able to reproduce.

A major problem experts are tackling is making sure contaminated buildings are technically clean. Officials do not know what a safe level of exposure may be.

Containing an agent is the most troublesome part of using biological weapons, and one of the most important reasons they have not been widely used. The bacteria and viruses don't discriminate between an ally and a foe, and the "boomerang effect" -- the biological agent affecting those who released it -- is a common occurrence.

Delivering a biological agent is difficult, as well. Spreading a disease through the air would most likely involve delivering it in an aerosol cloud. Any change in the weather would make the behavior of that cloud completely unpredictable. Despite the attention given to crop duster planes, in actuality, they are best suited to discharge relatively large particles 100 microns in diameter, while the anthrax spores found in Daschle's office were only 1.5 to 3 microns in diameter.

On January 12, the head of CDC told postal workers that medical experts are providing their best advice on anthrax, but the disease isn't well enough understood to tell people exactly what to do. Experts had originally thought it takes thousands of spores to cause the inhaled form of the disease, but now think they may be wrong - they don't know how much exposure is needed for the disease to develop.

Until a rapid detection and response are possible, the United States must concentrate on simply deterring attackers armed with biological weapons.

John Hopkins University, Center for Civilian Biodefense Strategies -- www.hopkins-biodefense.org/

U.S. Centers for Disease Control, Public Health Emergency Preparedness and Response -- www.bt.cdc.gov/

U.S. Environmental Protection Agency -- www.epa.gov/epahome/hi-anthrax.htm

This article originally appeared in the 03/01/2002 issue of Environmental Protection.