Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 10/19/2006
Publication Date: 10/19/2006
Citation: Suarez, D.L. 2006. Rapid molecular diagnostic tools for avian influenza [abstract]. 2006 European Directorate for the Quality of Medicines Symposium, Requirements for Production and Control of Avian Influenza Vaccines, October 19-20, 2006, Strasbourg, France. p. 14.
Technical Abstract: An accurate and early diagnosis of a foreign animal disease is crucial for rapid control and eradication of an outbreak in a country previously free of the disease. Historically many animal diseases have been controlled based solely on clinical signs of disease. However with avian influenza virus (AIV), the disease expression is extremely variable in different species of birds making clinical signs an ineffective diagnostic tool. Serologic tools are also widely available and are useful to show freedom of disease in a population, particularly for trade purposes. However, serology is a retrospective tool and not a useful control tool. Diagnostic methods that directly detect the AIV virus, antigen, or RNA are critical to identify infected birds and institute control measures. A test that is both sensitive and rapid is needed for optimal control. Virus isolation is sensitive, but requires a delay of several days until a diagnosis can be made. Two other tools that are rapid are commonly used for the diagnosis of AIV, including molecular diagnostics and antigen capture ELISA (immunoassay) tests. Several molecular diagnostic tools are available for AIV that all amplify the viral RNA (or cDNA) to detectable levels, but the most commonly used molecular test is the real-time reverse transcription polymerase reaction test (RRT-PCR). The test used in the U.S. screens with Type A influenza matrix primers and probe, and positive samples are confirmed with a subtype specific test (for H5 and H7 viruses). This test is standardized and is used throughout the U.S. in the National Animal Health Laboratory Network (NAHLN) that is overseen by the USDA’s National Veterinary Services Laboratories (NVSL). The use of local or regional laboratories has been successfully used to rapidly diagnosis outbreaks of AIV in the U.S. and aid in their rapid control. The antigen capture ELISA tests can also be valuable in an AIV outbreak because they are rapid, easy to use and require little equipment. These tests are not as sensitive as the molecular tests, but in birds sick or dead from AIV can provide a reliable diagnosis. These tests can be good screening tools, but positives are typically confirmed by other methods. It is important for all molecular tests that they be validated for “fitness for use”, which includes purpose of test, sample type, and species tested. Validation requires comparison data with a performance standard. The RNA extraction step has proven to be a key control point for successful RRT-PCR. Some sample types, including cloacal swabs or feces, have proven to be difficult samples to extract both because of RNA extraction efficiency issues and the presence of PCR inhibitors. Typically several protocols will be needed to handle tracheal swabs, cloacal swabs, and tissues, and each method will need to be validated for purpose.