Submitted to: American Association of Veterinary Laboratory Diagnosticians
Publication Type: Abstract Only
Publication Acceptance Date: 8/17/2006
Publication Date: 10/14/2006
Citation: Suarez, D.L. 2006. Rapid diagnostics for avian influenza -- Advances in testing [abstract]. American Association of Veterinary Laboratory Diagnosticians. p. 25.
Technical Abstract: A variety of tools are available for the diagnosis of avian influenza virus. They can be generally divided into the serologic diagnostic tests and direct virus detection tests. The serologic tests are important primarily for active surveillance to assure our poultry flocks are free of avian influenza for trade purposes. However in an outbreak, serologic detection plays only a limited role for identifying infected flocks because of the delay in antibody production. In an outbreak the rapid diagnosis of direct diagnostic tests are more crucial to identify and quarantine (or euthanize) infected flocks to limit the spread of the virus. In the U.S. three methods of direct diagnosis are commonly used, virus isolation, real-time RT-PCR (RRT-PCR), and antigen capture tests. Virus isolation is still a critical tool for the initial diagnosis and official reporting of an avian influenza outbreak, but because of the time required for a diagnosis, the use of virus isolation plays more of a supportive role for outbreak control. The primary frontline tests for avian influenza in an outbreak are the RRT-PCR and antigen capture tests. The RRT-PCR was first used to control an avian influenza outbreak in Virginia in 2002. Since its successful use in Virginia, the test has become the National Animal Health Laboratory Network standard for testing and is currently available in approved labs in 49 of the 50 states. This test has two steps, first screening with the matrix primers to identify any Type A influenza virus, and identification of subtype. Currently only primers to the H5 and H7 subtype are in common use, although primer sets for the other subtypes are in development. The basic test has not changed in the last 5 years, but several enhancements to the procedure are available. The RNA extraction step, critical for success of the test, has been a major area of research emphasis. This has included the validation of magnetic bead RNA extraction procedures that are amenable to robotics. Robotics to increase processing throughput has become a priority goal in the NAHLN. Several robotic systems are commercially available, but considerable work will be needed to validate a system that provides high quality RNA from a variety of samples at a reasonable cost. The equipment costs in particular need to be low enough to allow most or all NAHLN labs to work with the same standard. A second area of research is how to extract RNA from complex and dirty samples, including wild bird cloacal samples and tissue samples that commonly contain PCR inhibitors that can cause false negative results. Another area of improvement is the availability of an internal positive control for both quality control purposes and to help identify false negatives caused by PCR inhibition. The development of lyophilized or bead reagents have also been developed to improve quality control.