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Background: Equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona remains an antemortem diagnostic challenge in some horses. Recent work suggested the use of real-time PCR (rtPCR) on cerebrospinal fluid (CSF) as a promising diagnostic tool., Objective: To evaluate the sensitivity and specificity of S. neurona rtPCR on CSF for EPM diagnosis using horses with EPM and S. neurona-seropositive horses with other neurologic conditions., Animals: Ninety-nine horses with neurologic disease that underwent complete neurologic examination, CSF collection, and, if euthanized, necropsy including the central nervous system (CNS)., Methods: Retrospective case-control study using banked CSF samples. Samples from horses with neurologic abnormalities and necropsy-confirmed EPM diagnosis, presumptive EPM diagnosis using strict criteria (SnSAG2/4/3 ELISA serum:CSF titer ratios <50) and horses diagnosed with other neurologic diseases were used., Results: Fifty-two horses had EPM; 23 were confirmed on necropsy, and 29 were presumptive clinical diagnoses. The other 47 horses all had necropsy-confirmed diagnoses. Four of the 47 horses had normal neurologic findings on necropsy and the remaining 43 horses had neurologic diseases including equine degenerative myeloencephalopathy (EDM), cervical vertebral stenotic myelopathy, trauma, and other miscellaneous conditions. One CSF sample was weakly positive for S. neurona by rtPCR, this sample was obtained from a horse with confirmed EDM. Samples from the other 98 horses were negative for S. neurona by rtPCR., Conclusions and Clinical Importance: Our study contradicts previous conclusions that S. neurona rtPCR is potentially useful for EPM diagnosis, because our results indicate that the assay has a low sensitivity (0%) for EPM., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)