Project Summary/Abstract Toxocariasis, caused by the dog and cat roundworm (Toxocara canis or T. cati), is the most common zoonotic helminth infection in children living in the United States (US). Toxocariasis disproportionately affects children from underserved communities in the US, with the highest prevalence of disease in non-Hispanic Black children (up to 21%). Toxocariasis manifests as distinct clinical syndromes leading to life-long morbidity from epilepsy, asthma, and blindness. Thus, toxocariasis is an important contributor to child health disparities in the US. Despite the significant life-long morbidity, toxocariasis remains underdiagnosed in the US in large part due to limitations of the standard-of-care Toxocara diagnostic assay, an enzyme immunoassay (EIA) that detects host antibody against T. canis excretory-secretory (TES) crude antigen. An improved Toxocara diagnostic assay would facilitate accurate diagnosis and evaluate treatment response in children to prevent life-long morbidity. Given the scalability and reproducibility, recombinant proteins are an exciting technology for improving Toxocara diagnostics. Our state-of-the art laboratory, which routinely creates recombinant proteins, generated a T. canis cDNA library to evaluate Toxocara antigens. Immunoscreening of the T. canis cDNA library with Toxocara infected human sera identified T. canis C-type lectins (Tc-CTL-1 and Tc-CTL-2) as the most immunodominant antigens. We subsequently identified the Tc-CTL-1 homolog in T.cati (Tca-CTL-1) with 93% sequence identity. Using recombinant protein technology, we developed a multiplex recombinant protein Luminex immunoassay and enzyme linked immunosorbent assay (ELISA) using Tc-CTL-1, Tc-CTL-2, Tca-CTL-1. The overall objective of this project is to evaluate the diagnostic performance and potential for post-treatment monitoring of two new multiplex immunoassays based on three, scalable recombinant proteins. We hypothesize that both multi-plex immunoassays, utilizing the three recombinant proteins (rTc-CTL-1, rTc-CTL-2 and rTca-CTL-1), will have improved test performance for diagnosis of toxocariasis compared to TES EIA and will permit the evaluation of anthelminthic treatment monitoring in children. To evaluate this hypothesis, we will first determine the performance characteristics of the two multiplex immuno-assays compared to TES EIA (standard of care) and TES-immunoblot (TES-Blot; gold standard). Second, using both immunoassays we will quantitate total IgG and IgG subtypes in peri-treatment sera collected from a longitudinal cohort of children with toxocariasis living in Houston, TX. Achievement of our aims will result in at least one accurate, low-cost, scalable multiplex immunoassay for the diagnosis and post-treatment monitoring of toxocariasis in children.