Sarcocystis neurona SAG2/3/4 ELISA Educational Bulletin Revised December 2013

Diagnosis of equine  protozoal myeloencephalitis (EPM), a neurologic disease in horses caused by the parasites  Sarcocystis neurona  and less commonly Neospora hughesi, has historically been a challenge.  Tests currently available detect IgG antibodies against S. neurona in serum or CSF. However, due to the high exposure rate of horses to the parasite many asymptomatic animals carry moderate to high levels of serum antibodies, resulting in false positive blood tests.  CSF testing is much more predictive of active disease, but results can be affected by blood contamination and normal passive transfer of antibodies across the blood-brain barrier.

Test description and validation

Quantitative enzyme-linked immunosorbent assays (ELISAs), were developed at the Maxwell H. Gluck Equine Research Center at the University of Kentucky and  are now exclusively available at Equine Diagnostic Solutions, LLC (EDS).   These assays incorporate three  unique  surface  antigens (SAGs) of S. neurona, SAG2, SAG3 and SAG4, for the measurement of an IgG response to them (Hoane et al., 2005 and Yeargan and Howe, 2011); results are expressed as endpoint titers.  While similar antigens are found in other Sarcocystis species, only S. neurona  is known to infect horses and raise an immune response.  Blood contamination of CSF was determined to be confounding only with RBC counts greater than 10,000/ul.

Validation was based on analysis of paired serum and CSF samples (tested ante-mortem) from 128 necropsy-characterized clinical cases (Reed et al. 2013; JVIM 27:1193-1200) from a complete set of 402 field cases.  Neurologic status was monitored at presentation and on follow-up examination for most patients.   After all lab and clinical data were tabulated, cases were grouped by diagnosis and the data analyzed.   While endpoint serum titers alone were not specifically predictive of an EPM diagnosis, endpoint CSF titers of ≥1:20 had a higher correlation with an EPM diagnosis.  Using these assays, the serum/CSF titer ratio was the most informative and predictive criterion for the differential diagnosis.  This calculation is based on the premise of proportionality of antibodies across the blood-brain barrier.  Movement of antibodies across the BBB occurs by normal passive transfer.  However, with CNS infection, proportionally higher levels of antibodies are found in the CSF, reflecting intrathecal antibody production. Utilizing these three S. neurona specific antigens, this ratio becomes very predictive, with ratios of ≤100 strongly correlating to an EPM diagnosis. Using the ≤100 ratio value, test sensitivity = 93% and specificity = 83%.

Additionally, a S. neurona specific antibody  index or C-value can also be determined using the serum and CSF endpoint titers in conjunction with either albumin levels  (antibody index) or  total IgG  levels (C-value). These calculations are useful in determining intrathecal antibody production versus blood contamination or increased blood brain barrier permeability.

Test comparison studies

Study 1 consisted of 19 of the validation study cases,  including 11 of the necropsy cases  (6 EPM and 5 CVM) and 8  field cases with well defined diagnoses (6 EPM and 2 CVM). The sample submitted for testing was based on the recommendations of the testing laboratory  for making an EPM diagnosis: Serum for IFA and SAG1 ELISA, CSF for western blot, and serum plus CSF for SAG 2, 4/3 ELISA.

 

  • IFA serum results were interpreted as positive EPM for those samples with titers of >1:80,  interpreted as >55% probability.  3  (2 were necropsy) of the 7 CVM cases tested as false EPM positive and 5 (4 were necropsy) of the 12 EPM cases tested as false EPM negative.
  • SAG1 ELISA serum results were interpreted as positive EPM  for those samples with titers of ≥1:32.   2  (neither were necropsy) of the 7 CVM cases tested as false EPM positive and 11 (6 were necropsy) of 12 EPM cases tested as false EPM negative.
  • Standard western blot CSF results of positive or low positive were interpreted as positive EPM.  1  (not a necropsy case) of the 7 CVM cases tested false EPM positive and 1 (a necropsy case) of the 12 EPM cases tested false EPM negative.
  • SAG 2, 4/3 ELISA serum/CSF titer ratios were interpreted as positive EPM for those samples with titer ratios of ≤100.  None of the 7 CVM cases tested false EPM positive while 1 (a necropsy case) of the 12 EPM cases tested as false EPM negative.
  • One case tested as false negative by all four assays.

 

Study 2 consisted of  a  university hospital  set of  57 cases (37 with postmortem  and 20 clinically well characterized) including 15  (9 with postmortem) EPM cases (Johnson et al. 2013; JVIM 27:596-599).  Serum and CSF samples were tested by IFA and SAG 2, 4/3 ELISA.  The individual serum and CSF titers and the serum/CSF titer ratios were analyzed for both test formats and ROC analysis was used to select cut-offs (not necessarily those of the testing labs) that maximized sensitivity and specificity. The use of serum results alone for either test format significantly decreased diagnostic accuracy.  The IFA CSF titer and titer ratio had accuracy = 88%, with sensitivity = 65% and specificity = 98%.  The SAG 2, 4/3 ELISA titer ratio had the highest accuracy = 97%, highest sensitivity = 88% and highest specificity = 100%.

Evaluation of the data for clinical usefulness of the SAG2/3/4 ELISAs and diagnostic capability concluded:

  • Serum titers do not independently correlate with an EPM diagnosis, while CSF titers and serum/CSF titer ratios are highly predictive.
  • Serum/CSF titer ratios were the most accurate diagnostically when compared to the three previously most common S. neurona IgG tests.
  • The importance and diagnostic value of performing CSF taps is reaffirmed.

Last Updated on Friday, 17 January 2014 02:46
 
Sarcocystis neurona SAG2/3/4 ELISA Educational Bulletin Revised December 2013

Diagnosis of equine  protozoal myeloencephalitis (EPM), a neurologic disease in horses caused by the parasites  Sarcocystis neurona  and less commonly Neospora hughesi, has historically been a challenge.  Tests currently available detect IgG antibodies against S. neurona in serum or CSF. However, due to the high exposure rate of horses to the parasite many asymptomatic animals carry moderate to high levels of serum antibodies, resulting in false positive blood tests.  CSF testing is much more predictive of active disease, but results can be affected by blood contamination and normal passive transfer of antibodies across the blood-brain barrier.

Test description and validation

Quantitative enzyme-linked immunosorbent assays (ELISAs), were developed at the Maxwell H. Gluck Equine Research Center at the University of Kentucky and  are now exclusively available at Equine Diagnostic Solutions, LLC (EDS).   These assays incorporate three  unique  surface  antigens (SAGs) of S. neurona, SAG2, SAG3 and SAG4, for the measurement of an IgG response to them (Hoane et al., 2005 and Yeargan and Howe, 2011); results are expressed as endpoint titers.  While similar antigens are found in other Sarcocystis species, only S. neurona  is known to infect horses and raise an immune response.  Blood contamination of CSF was determined to be confounding only with RBC counts greater than 10,000/ul.

Validation was based on analysis of paired serum and CSF samples (tested ante-mortem) from 128 necropsy-characterized clinical cases (Reed et al. 2013; JVIM 27:1193-1200) from a complete set of 402 field cases.  Neurologic status was monitored at presentation and on follow-up examination for most patients.   After all lab and clinical data were tabulated, cases were grouped by diagnosis and the data analyzed.   While endpoint serum titers alone were not specifically predictive of an EPM diagnosis, endpoint CSF titers of ≥1:20 had a higher correlation with an EPM diagnosis.  Using these assays, the serum/CSF titer ratio was the most informative and predictive criterion for the differential diagnosis.  This calculation is based on the premise of proportionality of antibodies across the blood-brain barrier.  Movement of antibodies across the BBB occurs by normal passive transfer.  However, with CNS infection, proportionally higher levels of antibodies are found in the CSF, reflecting intrathecal antibody production. Utilizing these three S. neurona specific antigens, this ratio becomes very predictive, with ratios of ≤100 strongly correlating to an EPM diagnosis. Using the ≤100 ratio value, test sensitivity = 93% and specificity = 83%.

Additionally, a S. neurona specific antibody  index or C-value can also be determined using the serum and CSF endpoint titers in conjunction with either albumin levels  (antibody index) or  total IgG  levels (C-value). These calculations are useful in determining intrathecal antibody production versus blood contamination or increased blood brain barrier permeability.

Test comparison studies

Study 1 consisted of 19 of the validation study cases,  including 11 of the necropsy cases  (6 EPM and 5 CVM) and 8  field cases with well defined diagnoses (6 EPM and 2 CVM). The sample submitted for testing was based on the recommendations of the testing laboratory  for making an EPM diagnosis: Serum for IFA and SAG1 ELISA, CSF for western blot, and serum plus CSF for SAG 2, 4/3 ELISA.

 

  • IFA serum results were interpreted as positive EPM for those samples with titers of >1:80,  interpreted as >55% probability.  3  (2 were necropsy) of the 7 CVM cases tested as false EPM positive and 5 (4 were necropsy) of the 12 EPM cases tested as false EPM negative.
  • SAG1 ELISA serum results were interpreted as positive EPM  for those samples with titers of ≥1:32.   2  (neither were necropsy) of the 7 CVM cases tested as false EPM positive and 11 (6 were necropsy) of 12 EPM cases tested as false EPM negative.
  • Standard western blot CSF results of positive or low positive were interpreted as positive EPM.  1  (not a necropsy case) of the 7 CVM cases tested false EPM positive and 1 (a necropsy case) of the 12 EPM cases tested false EPM negative.
  • SAG 2, 4/3 ELISA serum/CSF titer ratios were interpreted as positive EPM for those samples with titer ratios of ≤100.  None of the 7 CVM cases tested false EPM positive while 1 (a necropsy case) of the 12 EPM cases tested as false EPM negative.
  • One case tested as false negative by all four assays.

 

Study 2 consisted of  a  university hospital  set of  57 cases (37 with postmortem  and 20 clinically well characterized) including 15  (9 with postmortem) EPM cases (Johnson et al. 2013; JVIM 27:596-599).  Serum and CSF samples were tested by IFA and SAG 2, 4/3 ELISA.  The individual serum and CSF titers and the serum/CSF titer ratios were analyzed for both test formats and ROC analysis was used to select cut-offs (not necessarily those of the testing labs) that maximized sensitivity and specificity. The use of serum results alone for either test format significantly decreased diagnostic accuracy.  The IFA CSF titer and titer ratio had accuracy = 88%, with sensitivity = 65% and specificity = 98%.  The SAG 2, 4/3 ELISA titer ratio had the highest accuracy = 97%, highest sensitivity = 88% and highest specificity = 100%.

Evaluation of the data for clinical usefulness of the SAG2/3/4 ELISAs and diagnostic capability concluded:

  • Serum titers do not independently correlate with an EPM diagnosis, while CSF titers and serum/CSF titer ratios are highly predictive.
  • Serum/CSF titer ratios were the most accurate diagnostically when compared to the three previously most common S. neurona IgG tests.
  • The importance and diagnostic value of performing CSF taps is reaffirmed.

Last Updated on Friday, 17 January 2014 02:46