The EPM Society is meeting this month in California. Pathogenes is presenting 3 papers, one is of particular interest to the Society and that is Development and validation of assays offered by Pathogenes, these are tests used in our consulting services. The nuts and bolts of our tests are published, including test format, dilution factors, assay development, samples used for validation and testing, and results interpretation. A big part of testing is the sensitivity and specificity of the test. We have enough experimental and field data to allow veterinarians to use a predictive value for EPM. The predictive value depends on the amount of disease in the population. For example, disease is more prevalent in Texas, the predictive value of a positive test is more meaningful in Texas compared to Idaho, we found no EPM in Idaho. Yet, some details interest the group, such as strains of organisms and the validation protocols.
Background for non-test developers: The USDA, under animal plant health inspection service (APHIS) directs animal health and veterinary biologics. The Veterinary Biologics Program implements provisions assuring that effective diagnostics are available and appropriate standards are developed. They also issue licenses and inspect products and facilities. They issue memoranda that relate to licensing diagnostic tests. These directives outline the steps to provide sensitivity and specificity data, ruggedness, receiver operating characteristic curves (ROC) that test sensitivity versus a false positive rate. The ROC curve is useful visualizing the compromise between sensitivity and specificity for different cutoff values and for selecting a cutoff value.
Our mission is to develop patent protected technologies for licensing and we use USDA’s requirements to guide our work. For example, developing assays. A first step is understanding what you want to measure, the analyte (that can be an antigen-a protein related to a specific organism, an antibody, or a genetic sequence). The next steps are deciding on a range of concentration, sample type, and the potential for cross-reactions.
A big hurdle is the Gold Standard selected for a diagnostic test. You will read about “Gold Standards” and “EPM” testing making this topic worth understanding.
USDA’s context for a Gold Standard is to provide “an accepted means of determining the diagnostic status (positive versus negative) of a diseased animal”. These same animals should be evaluated by the test and the Gold Standard samples are used to determine the accepted “true diagnosis”. Using EPM as an example, USDA requires reference standards (serum and CSF for example) from 20 animals. They accept samples from field cases. USDA requires confirmation of disease: organisms in the brain of the animal-the accepted definition of EPM and, here is the kicker, enough of the samples (serum and CSF) to provide the reference standard over the life of the test. Our discussions with USDA confirm this is not an achievable goal.
Gold standard samples from 20 natural cases of EPM are difficult to obtain. The horses must not be treated prior to sampling because treatment changes the diagnostic analyte. S neurona has three serotypes (each analyte is unique to each serotype) that must be detected by the test. Sixty horses with natural disease are needed for SAG 1, 5, and 6 testing. Common analytes SAG 2, 3, and 4 are not specific for the disease EPM because these SAG’s are present in non-EPM causing protozoa.
A licensed EPM test is not happening.
A core issue with the disease caused by S neurona is the contribution of inflammation to the EPM syndrome. The organisms can be eliminated leaving some horses with treatable clinical signs of disease. Detecting analytes that identify S neurona in conjunction with inflammation is clinically useful. Each veterinarian must weigh the “Gold Standard” that was used for the test and it’s usefulness in their treatment decisions.
USDA will license an antibody test that detects a specific antigen. The Gold Standard for these tests can be obtained by vaccination. For example, we vaccinated 50 animals with recombinant SAG 1 to obtain enough reference material for submission to USDA. In addition, USDA requires the “master seed”, a number of vials of the analyte used in test development. The master seed is validated from serial passages of the recombinant organism, all tested and documented, so a 3rd generation seed (serial) is proven to be unchanged by passage. The requirements to license our SAG 1, 5, 6 tests are 3 master seeds (with validated serials) and 20 vaccinated animals. The test is “for the detection of antibody against serotype 1 (or 5 or 6) of S neurona in the serum or CSF of horses”. We do not diagnose the disease EPM. We report the level of antibody against the analyte SAG 1, 5, or 6 of S neurona. We also use a serum test to evaluate the inflammatory reaction to infections. This panel of tests forms the basis of our consultation.
We are pursuing a license for the ante-mortem diagnosis of equine muscular sarcocystosis due to S. fayeri in horses. The assay will detect disease (the presence of muscle cysts) so the USDA Gold Standard for disease applies. Reference standards are produced from field cases of EMS. EMS is also easy to produce experimentally, a source of material for reference standards.
Another disease that warrants a licensed diagnostic test is polyneuritis equi, a demyelinating polyneuropathy that results in ataxia (classical) or cranial nerve deficits (atypical). The USDA Gold Standard for polyneuritis equi is met with field cases. We identify cases of polyneuritis equi by clinical signs and antibody against myelin protein. The disease process, a response to an infectious organism, results in demyelination of nerves followed by remyelination of that nerve. The diagnostic we are developing identifies the demyelinating process.