Evaluation of a novel Tc-24 recombinant antigen ELISA for serologic testing for Trypanosoma cruzi in dogs

Chagas disease is a parasitic infection caused by Trypanosoma cruzi. Diagnosis of chronic Chagas disease in dogs relies on limited serological test options. This study used a new Tc-24 recombinant antigen ELISA on an archival set of 70 dog serum samples from multi-dog kennel environments in Texas subjected to three existing Chagas serological tests. Tc-24 ELISA produced a quantitative result and could detect anti-T. cruzi antibodies in dogs with high sensitivity and specificity. Comparing individual tests to Tc-24 ELISA resulted in strong associations and correlations, which suggest that Tc-24 ELISA is a reliable and accurate diagnostic tool for dogs with a single test.

These differences exist in both human and canine testing.Dogs are a known reservoir for T. cruzi across various endemic areas, particularly the southern U.S., including Texas, where infection prevalence between 20.3 -31.6% and as high as 57.6% have been documented in domestic dogs and kennel environments, respectively (6,7).Currently, there are limited immunodiagnostic tests specific for veterinary use.Therefore, many epidemiological studies in domestic and wild animal reservoirs rely on an IFA test, often used with commercial immunodiagnostic tests approved and intended for human use (8).
A concern surrounding Chagas diagnostics is that different diagnostic tests may perform differently in humans or animals infected with the seven known discrete typing units (9) and can potentially cross-react with related protozoans that may be co-endemic/or co-occur (e.g., Leishmania) (10).Our study aims to compare the performance of three commercial diagnostic tests to our Tc-24 recombinant antigen ELISA in samples of dogs from an endemic region.Tc-24 is a 24 kDa calcium-binding protein ubiquitously expressed in all stages of T. cruzi (11).It is an immunodominant protein during infection and conservative across all geographical strains of T. cruzi (97% homologous) but not present in other protozoa or helminths (12), suggesting that it can detect antibodies induced by all strains of T. cruzi.Our study examined 70 archival serum samples collected from dogs in ten large kennels in south Texas.The dogs were sampled up to three times each between May 2018 and September 2019 to initially collect data for a study to quantify the incidence of canine T. cruzi infections (6).Dogs enrolled in the study were between the ages of 13 months and 12.2 years (mean = 6.1 years; median = 6.1 years) and included hound dogs, Belgian Malinois, German Shorthair Pointers, Labrador Retrievers, Brittany Spaniels, and English Pointers.Samples were kept frozen at -80°C until processing.
A Tc-24 recombinant antigen ELISA protocol previously used for human samples (unpublished) was adapted for canine samples.Briefly, a capture ELISA was developed based on a Strongyloides antigen but using Tc-24 antigen at the same concentration instead of the Strongyloides antigen (13).A different secondary antibody was used to detect canine IgG, Rabbit anti-canine, Fc fragment, alkaline phosphatase at 1:2500 dilution (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA).For the Tc-24 ELISA, the threshold of positivity was estimated using positives and negatives for the respected commercial tests and calculating the receiver operating characteristic curve (ROC).All Tc-24 ELISA plates were run on the same day to minimize inter-plate variability.
As previously reported( 6), serum samples were tested for T. cruzi antibodies using three  (8,14,15).For the current study, the strength of the response on the rapid tests was scored on a scale from 0-4, in which 0 was a negative result (no color development or when a very faint incomplete band developed) and scores of 1-4 were positive results with increasing intensity of the reaction line.The IFA was run at the Texas A&M Veterinary Medical Diagnostic Laboratory with endpoint titers determined.Both the IFA and Tc-24 ELISA produced a quantitative result.
Results were compared using Mann-Whitney tests for nonparametric variables.Spearman correlation tests were used to determine relationships between continuous variables.Analysis of covariance was utilized for comparison of linear correlations.P < .05 was considered significant.
These tests were performed using GraphPad Prism 8.2.1 (GraphPad Software, San Diego, California, USA).
The results showed that SP and IB strips had a significant association and high correlation to Tc-24 ELISA.IFA also showed a significant association and moderate correlation to Tc-24 ELISA.Moreover, comparing any two positive commercial tests to the Tc-24 ELISA revealed a higher sensitivity and specificity than a single commercial test.Comparing high IFA titers and high immunochromatographic values to positive Tc-24 showed high sensitivity and specificity, respectively.These results indicate that Tc-24 ELISA may have the potential to be a new serological assay to diagnose T. cruzi infection in dogs with a single test.
The use of Tc-24 in diagnosing Chagas disease in dogs has not been widely studied.This study is significant as it suggests that Tc-24 recombinant antigen ELISA can potentially improve Chagas disease diagnosis in dogs.While this study provides promising results, several limitations need to be addressed.Firstly, the study had a small sample size, and the results must be replicated in larger studies.Secondly, the study only evaluated dogs from multi-dog kennel environments in Texas, and it is unknown whether the results can be generalized to other geographical locations or different populations of dogs.The study did not compare the Tc-24 recombinant antigen ELISA with other diagnostic assays for Chagas disease, such as Western blot or PCR-based tests.
Lastly, the subjective grading of immunochromatographic tests may have introduced potential bias in the results.

Summary
In conclusion, Chagas disease diagnosis in dogs is challenging due to the limitations of existing serological tests.This study suggests that Tc-24 recombinant antigen ELISA has potential as a new serological assay to aid in diagnosing T. cruzi infection in dogs with a singular test.These findings have implications as early diagnosis and treatment are crucial to preventing the development of chronic disease, reducing transmission, and broadening epidemiological surveillance in endemic areas.
Acknowledgments: American Kennel Club Canine Health Foundation Grant No 02448 provided funding for dog sample collection.The funders had no role in study design, data collection and analysis, publication decisions, or manuscript preparation.
serological tests: Chagas Stat-Pak ('SP'; ChemBio, Medford, NY, USA), Chagas Detect Plus Rapid Test ('IB'; InBios International, Inc., Seattle, WA, USA), and an indirect fluorescent antibody (IFA) test.The Chagas Stat-Pak and Chagas Detect Plus are rapid tests that, while not labeled for use in dogs, are commonly used for research purposes to detect T. cruzi antibodies