Anthrax Detection
the faster the better
 
 

SANDRA A. PARTAMIAN
MICROBIOLOGY 12: BIOTERRORISM AND EMERGING INFECTIONS
HONORS WEBPAGE
PROFESSOR ROBINSON
SPRING 2001

What is Anthrax?
Conventional Methods of Anthrax Detection
Modern Modes of Anthrax Detection
DNA chip analysis
Bibliography
 
 
 
 

"A suspicious package discovered three years ago in the mail room of B'nai B'rith's Washington headquarters sparked fears of biological terrorism.  Authorities had no way to quickly tell if the oozing red substance found in a Manila envelope at the Jewish community organization was really the deadly bacterium anthrax, as its label indicated. Thus, workers were barricaded in their offices for more than eight hours, two city blocks were shut down and two employees were stripped to their underwear and hosed with chemicals on the sidewalk.
In the end, the incident turned out be a hoax--scientists analyzed the material and discovered it to be a common household bacterium."

__excerpt from "Countering Germ Warfare"  by Terence Chea (Washington Post Staff Writer)
 
 

Over recent years, there have been numerous incidences, such as the one cited above, of false biological threats all around the world, and many individuals, in the scientific community as well as the political arena seem to agree upon the imminent possibility of a major biological attack or bioterrorist act becoming a reality sometime in the near future.  As Nightline's Ted Koppel might say, and as others have said, it is not a question of "if" such a disastrous event might take place, it is only a question of "when" and "where."  For this reason, it is becoming increasingly critical/imperative to develop  reliable, but quick methods by which anthrax can be detected so that proper measures and precautions can be carried out by the medical, political, and legal authorities in due time if anyone is infected by the disease or even to avoid this likelihood entirely.  In a case of an anthrax scare, whether it be a hoax or not, timing can make all  the difference;  quick methods of detection, which is the focus of this web page, particularly detection by DNA chip methods, can spare unnecessary losses and aid us in the fight against, possibly even help us win, the war against biological weapons.
 
 

WHAT IS ANTHRAX?

• Anthrax is a disease caused by the the gram positive, rod-shaped bacterium, Bacillus anthracis.
• It is commonly found in plant eating animals, including goats, sheep, and cattle.  It can be passed into the soil and to other plant eating organisms via the fecal-oral route of transmission.
• It is a disease which has been around for a very long time.
• While the bacilli themselves are very vulnerable, anthrax spores have the ability to remain viable for several decades, in the extremist of conditions,  suffering no reduction in their ability to develop into bacilli or to cause fatal anthrax; thus an absence of cases does not mean there is an absence of risk.
• There are 3 ways by which humans can contract anthrax, including cutaneous (contact with skin), gastrointestinal (consumption of infected meat), and inhalational (breathing anthrax spores)--which is  the most dangerous and deadly of the 3.  Victims of inhalational anthrax must be treated with antibiotics immediately following exposure/prior to the onset of symptoms; otherwise they have little, usually no chance of survival.  In addition, treatment must include vaccination before discontinuing the use of antibiotics.
• A person must contract somewhere between 8,000-10,000 spores in order to be infected with anthrax (Illustration).
• Symptoms of inhalational anthrax usually begin after a 1 to 6 day incubation period following exposure.  After the incubation period, the infected individual experiences a flulike illness, low-grade fever, dry hacking cough, headache, and mild chest discomfort. The person may briefly improve after 2 to 4 days; however, within 24 hours of this temporary improvement, respiratory distress occurs resulting in shock and death shortly thereafter.
• Although an anthrax vaccine exists, supplies are limited and none are available for civilian use.
• Anthrax is the preferred biological warfare agent because:

            (a) easy and cheap to produce
            (b) can be stored almost indefinitely as a dry powder
            (c) silent, invisible killer

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Robert Koch's original micrographs of the anthrax bacillus
 

CONVENTIONAL METHODS OF DETECTION

Initially confirming the identity of B. anthracis was solely dependent on inoculating the suspect culture or specimen into an animal which was then observed for development of anthrax. Gradually, this approach became largely replaced by standard bench tests.  In some countries, where the basic culture materials and reagents are not available, this is still the approach taken.
One well known, conventional method of disease agent detection is known as the enzyme-linked immunosorbent assay (ELISA) method.  ELISA is used in many laboratories to determine whether a particular antibody (which of course is produced by a person's immune system in response to the presence of a foreign organism in the body) is present in a patient's blood sample.  However, this general test has the following important limitations:
First, a positive result correctly confirming the presence of antibody does not necessarily mean the patient is sick. The body can continue to produce antibodies even though the person may have had the disease earlier and recovered.
Second, some people may be poor producers of antibody or may have some interfering substance in their blood. The amount of antibody, consequently, may be too low to measure accurately or may go undetected. This result is termed a false negative.
Third, a positive result may occur if an unrelated antibody reacts with the antigen nonspecifically. Unlike a true positive result where the specific antibody is detected, however, this positive reaction is false.  To avoid simple experimental mistakes leading to incorrect results, scientists conduct tests using duplicate (or, sometimes, more than two) samples.

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MODERN MODES OF DETECTION

Concerns about the use of anthrax spores as a weapon of mass destruction have motivated the development of portable instruments capable of detecting and monitoring a suspected release of the agent. Typically, local authorities must wait 24 to 48 hours for laboratory test results to be returned before determining whether an event was a hoax or an actual biological threat.  With recent on-site screening equipment, however, local officials can confidently determine whether a threat exists or not within a matter of minutes.

• BioThreat Alert (BTA) test strips

This technology, which can be used to identify anthrax in the environment within a time period of 15 minutes, was invented by the Maryland-based biotech company, Tetracore.  It is a small plastic device which works like a home pregnancy test in which a substance of questionable identity is placed.  The device contains anthrax antibodies, proteins which fight the anthrax bacterium.  If the substance turns out to be anthrax, then it will bind to the antibodies, causing a white paper strip to change colors.  Two solid bands (one on the Control Window and one on the Sample Window) indicates a positive result (Look at figure below), while one solid band (Control Window) indicates a negative result.  Any other combination of lines means that there has been a mistake and the test must be repeated.   Important to note, however, is that a negative result on an Anthrax BTA strip does not necessarily mean that there are no anthrax bacteria at all; A negative result on the BTA strip indicates only that anthrax bacteria was not detectable within the range that can be measured by the test.  The detectable level or "threshold concentration" of bacteria for the strip is referred to as the "sensitivity" level of the test.
The company is presently undergoing efforts to make test strips which would detect the presence of other disease agents, besides anthrax, in the environment, such as botulinum toxin, ricin, and Yersinia pestis (which causes plague).  Tetracore also makes DNA-based tests that detect agents by recognizing their genetic sequences.  These tests are much more sensitive than the BTA strips and in turn can detect bacteria long after they have died.  For more detail on the procedures of the BTA strip test method Click here
 

Drawing of a BTA strip

• Guardian Biothreat Alert Test Strip Reader

Yet another biotech company, Alexeter Technologies, has joined forces with Tetracore, and created the Guardian Bio-Threat Alert System, which utilizes  Tetracore's technology of BTA strips alongside its own BTA strip readers.  The Test Strip Reader is designed to accept and analyze BTA Test Strips. The Reader offers greater accuracy, as its optical technology can recognize positive results that might be missed by the human eye.  Finally, it provides a printout of the test results and date.
 
 

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The Latest and Most Promising Method of Quickly Identifying Anthrax
 

• DNA chip analysis:

Several companies are working on developing DNA chips/biochips which can be used to detect Anthrax in a matter of minutes. These companies include such names as Alexeter, Argonne, Cepheid and Tetracore.  The research team at Argonne is headed by one of the world's leading biologists, Andrei Mirzabekov.  Advocating the growing need for fast detection of biological agents, Mirzabekov states: "One reason these biological agents pose such a great threat today is because they are difficult to detect and identify quickly and reliably.  The biological microchips we are producing....could be a solution to the problem."  The DNA chip is a wafer made of silicon or glass on which DNA strands are immobilized.  These DNA strands will bind to, or hybridize, with any complementary DNA strands in the sample that is being tested.  Then a specially designed microscope detects where the DNA hybridized.  A computer attached to the microscope analyzes the data, and in a few seconds will reveal the identity of the sample in question.  A technique known as polymerase chain reaction (PCR) can be used to amplify or multiply the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.  In the biological agent detection program, the substances being immobilized onto the chip would be key DNA strands from a particular biological agent such as anthrax, whose sequences are known, and also anthrax antibodies.  Argonne is presently working on a chip which not only contains immobilized strands of anthrax on its surface but also immobilized strands of other biological agents such as Yersinia pestis (the bacterium which causes plague) on it as well.  Therefore, if combat units or anti-terrorism forces were to be exposed to any one of these agents, the Argonne biochip would identify its presence in the environment and change color as a result.  According to an October 1999 article from USA Today, Argonne is also trying to develop a second type of chip which will function to detect genetically engineered biowarfare organisms.  Harvey Drucker, associate director at Argonne, said that there are about 10 toxins that bioterrorists would favor for engineering into harmless organisms.  Biochips for these toxins are under development.

 
 

My illustration of how a DNA chip with the help of PCR would work:

 
 
 
 
 
 

Bacillus Anthracis

Below are a few examples of the overwhelming number of books that dedicate their pages to the discussion of the increasingly popular topic of bioterrorism and/or biological warfare.

 
 
 

Bibliography:

Alexeter Technologies, LLC.  "Guardian BTA Summary."  http://www.alexeter.com/guardian_bta_summary.htm (April 2001):  2 pp.  Online.  Internet.  26 May, 2001.

Argonne News.  "Biochips may lessen germ war threat." www.anl.gov/OPA/local/news97/an970519.html (May 1997): 2pp.  Online.  Internet.  20 May, 2001

Chea, Terence.  "Countering Germ Warfare. "http://www.tetracore.com/countering_germ_warfare.htm (November 2000): 2 pp.  Online.  Internet.  11 May, 2001

Cieslak, Theodore J., Eitzen, Edward M.  "Clinical and Epidemiologic Principles of Anthrax."  Emerging Infectious Diseases.  July-August 1999: pp. 552-555.

Gibbs, Wayt W. "Shrinking to Enormity."  Scientific American.  February 2001: pp. 33-34.

Johns Hopkins Center for Civilian Biodefense Studies.  "Facts about Anthrax and Its Potential as a Bioweapon." http://www.hopkins-biodefense.org/pages/news/quarter1_2.html#Anchor-facts
(September 1999): 10 pp.  Online.  Internet.  28 May, 2001.

Koch, Robert.  Essays of Robert Koch.  Westport: Greenwood Press, 1987.

University of Arizona.  "Introduction to ELISA Activity." http://www.biology.arizona.edu/immunology/activities/elisa/main.html (May 2000): pp. 1.  Online.  Internet.  29 May, 2001.

Turnbull, P.C.B.  "Definitive identification of Bacillus anthracis—a review."  Journal of Applied Microbiology.  August 1999: pp.237-240.
 
 

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