Modern diagnostics in equine medicine–don't throw out the old (Proceedings)
Issues facing practitioners
Understanding the older and the newer formats for detection of infection
Table 1 is a listing of the basic definitions of PCR methods. Several previous presentations at the ACVIM and at other veterinary meetings have discussed PCR, and a recent paper indicates that a basic understanding of the different PCR test formats is needed for continuing education of practitioners and diplomates. While it is not necessary for the practitioner to be a molecular biologist, this table provides a short review of the definitions of molecular testing.Proper procedures: collection, transport, storage
The main advantage of genomic strategies for testing is that a live organism is not necessarily needed for diagnosis. While the fact that diagnosis based on molecular techniques does not require live organism, DNA and RNA are subject to the same microbiological, biochemical and physical factors as live organism for degradation. Although in some situations, genomic DNA may be robust in its survival for forensic purposes, overgrowth and chemical contamination of a small microbiological sample will result in false negative reactions for any of the aforementioned molecular techniques. A sample collected using the correct media that is solely dedicated for PCR testing should be taken. For blood, EDTA or purple top tubes are best. Use of EDTA as an anticoagulant for testing of virus in plasma or buffy coat is applicable for most genomic techniques and viral culture techniques. However, if one wishes to culture plasma (such as that of a foal) for bacteria, EDTA is bacteriostatic and a blood culture bottle must also be collected. Anticoagulants such as heparin can inhibit PCR reactions especially when a kit extraction method is used which will likely the typical formats in most diagnostic laboratories.
For detection of nasal/respiratory pathogens, wooden swabs contain formalin which can inhibit PCR (fixed tissue methods are performed on paraffin and formalin has been removed in the paraffin process). Thus, plastic, polypropylene sticks with Dacron or rayon swabs, not those with calcium alginate, are essential for nasal swabbing. Furthermore, cotton allows bacteria and virus to become embedded in fibers, and frequently the extraction method calls for direct extraction off the swab. This step will inactivate most viruses and bacteria because of the detergent used in the first lysis step. Finally, use of a transport media is best for viruses because this will inhibit overgrowth of bacteria that may inactivate and break down nucleic acids needed for the successful detection of viruses. The proteins in the viral media will also assist in keep the virus in a biologically active state, so this is preferable for viral culture also.
Most testing has actually been validated on specimens collected under the best conditions without inhibitors and contamination and these samples have been stored appropriately. Specimens requiring storage before shipping to a laboratory should not be stored and the MOST optimal time for testing by PCR is less than 3 days in a sample stored consistently at 4°C. If one can not ship a sample within 3 days we recommend that the sample be allowed to sit in a refrigerator for 20 minutes and then the plasma drawn off and placed in a new tube without any anti-coagulants. For horses, do not centrifuge the sample! The white blood cells of horses settle in this time and do not form a buffy coat that is adhered to red blood cells. This allows collection of as few RBCs as possible.8 This is essential because as RBCs lyse in older samples, heavy protein contamination occurs and these particular proteins (iron) are toxic to many PCR reactions. Many laboratories historically have indicated shipment at either 4°C or not to exceed 75°C. The latter is not appropriate for blood or nasal specimens due to the potential for hemolysis and bacterial overgrowth, respectively. Ship all samples on ice packs (NO WET ICE) and then overnight for 24 hour arrival. Do not ship expecting successful Saturday delivery and it is our experience that samples become lost more frequently with weekend deliveries.
Interpretation of pcr results
So given, the problems with inappropriate sampling and handling and lack of standards for these new techniques, what can be said regarding interpretation of test results? There is no question that molecular medicine has dramatically revolutionized infectious disease diagnosis, treatment, and, especially biosecurity. In the long run molecular based assays are more efficient and allow for minimal exposure of laboratory personnel and veterinarians to many infectious agents. Nothing will ultimately take the place of isolation of an infectious agent as confirmation of active infection from a properly collected and handled sample, but the efficiency and accuracy with good sampling and laboratory standardization make PCR diagnostics how most infectious diseases will be tested in the future.
Given that sampling, handling and all quality assurance for a particular laboratory is reliable, one must understand what a positive or a negative genomic-based test means. For instance, a positive PCR test results means that nucleic acids that belong to the genome of that particular pathogen was detected in the sample. This agent may or may not be live, infectious or capable of replication in that sample. At least three different scenarios may be occurring in regards to the sample tested: 1) the pathogen is present and directly is causing the clinical signs observed, 2) the pathogen is present but is not responsible for the clinical signs observed or 3) the pathogen is not present but the reaction mixture is binding to some other target in the assay. By the same token, a negative sample has failed to detect the nucleic acids of the infectious agent. A negative result can reflect at least three different scenarios' in regards to the sample tested: 1) the pathogen was absent at the time of testing, 2) the pathogen is present but not detectable within the limits of sensitivity, and 3) there was some type of inhibition of the positive reaction. The mere results cannot be interpreted without understanding the context in which testing was performed in the first place unless this is used for regulatory purposes. In the absence of case criteria, the results by themselves are not confirmatory for disease causation. This is especially true for negative tests; hence repeated sampling is recommended should the case criteria create a high degree of suspicion for that disease. In the end, a comprehensive investigation using multiple samples utilizing different detection formats may be the only way to confirm disease causation in an outbreak or new emergence of disease in a group of animals. There is no magic bullet when it comes to testing.