Clinical Laboratory Testing
To obtain samples of anthrax and test them in a routine clinical microbiology laboratory, the following steps are needed to confirm the presence of anthrax:
- Gather samples -- Swabs are taken of the nasal passages and any skin lesions of persons possibly exposed to anthrax. Samples of sputum, blood and spinal fluid may also be taken from persons who have symptoms. Swabs may be taken of surfaces where the patients may have been exposed.
- Initial culture -- The samples are transported to the laboratory and plated out, placed on a medium that will support the growth of anthrax, and incubated at the appropriate temperature. This step takes 6-24 hours, depending on the purity of the sample and the concentration of bacteria in it.
- Gram stain test -- Bacteria from the culture are placed on a glass microscope slide, stained by the Gram procedure, and then examined under the microscope for their shape and color. If the bacteria are anthrax, they should be rod-shaped and "Gram-positive" (blue). This test takes 10-15 minutes.
- Repeat culture -- The bacteria from the initial culture are replated on a medium that will support the growth of anthrax, and incubated at the appropriate temperature. This step takes 24-48 hours.. The aim is to grow up larger quantities of the bacteria for biochemical testing.
- Biochemical testing -- In the laboratory, specific chemicals are added to the bacteria to identify them definitivel as anthrax. This step takes 2-3 hours.
The time elapsed from when the samples were first gathered is now between about 32 hours and 75 hours -- between one-and-a-third days and a little over three days.
To learn what strain of anthrax is in a sample and to see if two samples of anthrax contain the same or different strains, DNA fingerprinting is done. This is similar to the DNA fingerprinting used in investigations of crimes and of human remains, as after the September 11 terrorist attacks on the World Trade Center.
In DNA fingerprinting anthrax, 8 specific sites in the DNA of each sample of bacteria are analyzed and compared. These particular sites were chosen for analysis because they are very highly polymorphic (variable) and, together, they furnish great power in distinguishing strains of anthrax. This test takes approximately 6-10 hours.
How Genetic Testing of Anthrax is Done
Genetic testing utilizes an anthrax DNA fingerprinting technique first reported in 1998 in the Proceedings of the National Academy of Sciences.
To distinguish different strains of the bacteria, the technique looks at DNA fragment lengths and sequences of specific fragments. The technique uses PCR (polymerase chain reaction) and DNA sequencing. PCR replicates DNA fragments in huge volumes and makes such genetic studies possible by providing many copies of identical fragments for analysis.
In PCR, the double strands of DNA are heated to separate the strands. Then the polymerase enzyme is added along with compounds called "primers," which start or "prime" the reaction. The reaction builds a complementary strand of DNA for each original strand.
From the 2 identical copies of double-stranded DNA, 4 copies are made the same way, then 8, then 16 and so on, taking advantage of exponential duplication, rapidly resulting in thousands of copies for study. Thus, all the genetic material needed for the analysis can be gained from one or a very few bacteria (even dead bacteria) in a sample.
The technique used with anthrax is a double PCR strategy to increase the sensitivity of the procedure while specifically selecting the bacteria under investigation. DNA products of a first PCR amplification are used as templates in a second reaction primed by primers that distinguish the strains of anthrax.
Applying Genetic Testing to an Anthrax Outbreak in Russia
The technique has worked even on old samples of tissue fixed in formalin from anthrax victims. The samples came from forensic studies of an outbreak of anthrax that occurred in Sverdlovsk, USSR (now Ekaterinburg, Russia) in 1979.
At that time, Soviet officials attributed the outbreak to the consumption of contaminated meat. But genetic testing done in 1998 at the Los Alamos National Laboratory confirmed that tissues from all victims contained by an identical mixture of B anthracis strains. All natural outbreaks of anthrax so far studied have resulted from a single strain. Evidence also indicated that most of the victims worked or lived in a narrow zone extending from a military microbiology facility in the city to the southern city limit that paralleled the prevailing northerly wind. The anthrax outbreak that occurred in Russia in 1979 was clearly due to a biologic weapon accident, not to eating contaminated meat.
This report is based in part on information published by the CDC (the Centers for Disease Control and Prevention at http://www.cdc.gov) and a report from the Los Alamos National Laboratory published in the February 3, 1998 issue of the Proceedings of the National Academy of Sciences.
Medical Author: Frederick Hecht, MD
Medical Editor: Dennis Lee, MD