Anthrax Testing, Part 2

by CDC

(Page 5 of 10)

What is the turnaround time for an anthrax test of an environmental sample?

Before testing can begin, samples must be collected and arrive in a form suitable for testing. The length of time necessary to get results of tests depends on transportation to the laboratory and the specific tests to be done. Testing is a two-step process. Initial screening tests (such as Gram stain) may be positive within two hours if the sample is large and the concentration of bacteria is high. These tests are used to narrow the definition of the sample. The confirmation tests take much longer, depending in part on how fast the bacteria grow, but are usually available 24-48 hours after the sample is received by the laboratory. (See Basic Diagnostic Testing Protocols for Level A Laboratories for the Presumptive Identification of Bacillus anthracis.)

Is the Mayo Clinic/Roche Rapid Anthrax Test a new test?

This is not a "new test." The Laboratory Response Network (LRN) has been using a validated real-time polymerase chain reaction (PCR) assay on the LightCycler for some time. CDC has also developed and validated real-time PCR assays for Bacillus anthracis for the SmartCycler™, ABI/PE 7700 and 5700. In addition, Idaho Technology has a real-time PCR assay for B. anthracis that can be used with the R.A.P.I.D.™, which is similar to the LightCycler™. SmartCycler is a trade name of Cepheid; R.A.P.I.D. is a trade name of Idaho Technology; LightCycler is a trade name of Roche; Idaho Technology is the name of a company.

Is the Mayo Clinic assay the same as the assay available to Laboratory Response Network (LRN) laboratories?

No. The Mayo Clinic assay targets the Lethal Factor (lef) gene on the virulence plasmid p0X1 and the Protective Antigen gene (pag) on p0X2. This assay has been tested with DNA from 32 strains of Bacillus anthracis, 26 Bacillus species, and 21 different bacterial genera commonly encountered in human specimens. The Mayo Clinic assay has not been validated in multiple laboratories.

The CDC assay uses targets on p0X1, p0X2, and the chromosome. A total of 100 Bacillus anthracis isolates were used to evaluate the sensitivity of the assay. Of the 100, 77 were selected to provide the best possible representation with respect to geographic origin and date isolated. The strains were obtained from infected animals, humans, and from industrial sites associated with anthrax outbreaks; they span 58 years (1939-1997) and are from various countries. In addition, five p0X1-cured strains (including the Sterne strain) and one p0X2-cured strain (Pasteur strain) were included. For evaluation of the specificity of the assay, 54 Bacillus species were used (B. subtilis, 9; B. cereus, 23; B. thuringiensis, 12; and B. megaterium, 10) as well as 250 other DNAs of various viruses and bacteria from human, animal, and insect sources. The assay was validated in a multicenter study by using state public health laboratories that had the specific platform.

What are the limitations of the Mayo Clinic test?

Because the Mayo Clinic assay uses only two plasmid targets, it cannot identify Bacillus anthracis strains such as Sterne or Pasteur that may be present in environmental specimens. This would not be a problem if the assay were used to confirm the identity of a gram-positive, non-motile, non-hemolytic rod.

Is CDC going to validate this assay?

CDC is testing samples from the current anthrax outbreak. When we have sufficient time, we will study this and other anthrax assays.

Are we aware of any sensitivity or specificity issues with the Mayo Clinic test? Should we expect a large number of false positive/negative results?

On the basis of the data provided by the Mayo Clinic, the assay appears to be sensitive and specific. However, the results are only as good as the method used to prepare the sample for analysis. There have been no data provided to indicate the types of samples that can be assayed or how they are to be processed. The Food and Drug Administration (FDA) has not seen the package insert for this test. The CDC assay has been validated for different types of samples and sample processing methods.

Are we furnishing CDC-tested equipment and reagents to laboratories?

Through the bioterrorism cooperative agreement, CDC has funded the purchase of platforms for real-time polymerase chain reaction (PCR) assays for the Laboratory Response Network (LRN). To date, 61 instruments have been purchased or ordered. Among these are 17 LightCyclers,™ 23 SmartCyclers™, 13 7700s, and 5 5700s. Reagents for real-time anthrax assays are made at CDC and placed in inventory. They are available at no charge to LRN laboratories. Currently, reagents for the LightCycler™ anthrax assay are available; reagents for the other platforms will be available soon. All of the assays have undergone the same rigorous validation procedure.

If a laboratory asks our opinion on whether to use Mayo Clinic/Roche Rapid test, what is our answer?

The Food and Drug Administration (FDA) considers this an investigational assay. As such it should be used only in conjunction with other tests, such as culture tests. Currently, polymerase chain reaction (PCR) assays are not considered confirmatory assays.

Will CDC accept results from laboratories that use this assay?

Currently, polymerase chain reaction (PCR) assays are not considered confirmatory tests for anthrax. PCR-positive specimens (or cultures) should be forwarded to the nearest LRN laboratory for confirmation.

About the Author

www.cdc.gov
The Centers for Disease Control and Prevention (CDC) is one of the 13 major operating components of the Department of Health and Human Services (HHS), which is the principal agency in the United States government for protecting the health and safety of all Americans and for providing essential human services, especially for those people who are least able to help themselves.