Definitive and Confirmatory Tests

A definitive or confirmatory test is a test that will identify with a very high degree of certainty the true identity of an agent. These tests have a very low likelihood of providing a false-positive result. Many of the definitive and confirmatory assays used today are molecular assays, which detect genetic material that is specific to a bacterium, virus, protozoa, or other organism. Nucleic-acid-based assays rely on the unique differences found in the structure of single strands of DNA and RNA. The unique pattern of bases is specific for a single organism or closely related organisms. The nucleic-acid-based assays involve the use of probes, which are strands of DNA or RNA that match distinctive DNA or RNA patterns of the organism being tested and will bind with that DNA or RNA if it is in the clinical specimen. Once the binding occurs, the binding can be detected by using electrochemical, colorimetric, and optical systems (Committee on Research and Development Needs for Improving Civilian Medical Response to Chemical and Biological Terrorism Incidents, 1999). This binding provides extreme selectivity between the known probes and the material found in clinical specimens. The use of a technique known as polymerase chain reaction (PCR) makes it is possible to amplify trace quantities of DNA or RNA in a clinical specimen to enable the detection of as few as 1,000 bacteria or viruses. The high specificity and sensitivity of molecular assays makes them especially suited for detecting minute quantities of biologic agents and toxins.

The use of genetic fingerprints has become a valuable tool for microbial forensics (Murch, 2003). By identifying distinct features of genes using sequencing techniques, it is possible to identify individual strains of organisms and to use this information as epidemiological markers. Molecular techniques allow investigators to link strains from various sources and to form associations that often unravel the mystery of disease outbreaks. Libraries of genetic patterns, or fingerprints, make it possible to trace the origin of many outbreaks. Not only does sequencing identify DNA or RNA patterns unique to a particular organism, but, in many cases, these probe technologies are simpler, faster, and less technology-dependent than are other traditional assays.

Many biotechnology companies are pursuing production of microarray systems that will test for 100 to 100,000 or more different DNA fragments simultaneously. The technology embeds the probes on a single glass or nylon substrate called a microchip. By using these arrays and various detection systems onto which the clinical specimen is applied, the individual components of the microchip will react with DNA fragments in the specimen and be detected. As this technology develops, it will become more common to use the microarray systems for screening and detection of diseases. Simultaneous PCR assays for multiple respiratory viruses now have sufficient sensitivity and specificity to be a valuable tool for diagnosis of respiratory viral infections (Hindiyeh et al., 2001). In the future, PCR assays will be able to screen a single specimen for a multitude of biologic agents.

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