Human babesiosis is a rapidly emerging and potentially fatal tick-borne disease caused by intraerythrocytic apicomplexan parasites of the Babesia genus. Among the various species of Babesia that infect humans, B. duncani has been found to cause severe and life-threatening infections. Detection of active B. duncani infection is critical for accurate diagnosis and effective management of the disease. While molecular assays for the detection of B. duncani infection in blood are available, a reliable strategy to detect biomarkers of active infection has not yet been developed. Here, we report the development of the first B. duncani antigen capture assays that rely on the detection of two B. duncani-exported immunodominant antigens, BdV234 and BdV38. The assays were validated using blood samples from cultured parasites in human erythrocytes and B. duncani-infected laboratory mice at different parasitemia levels and following therapy. The assays display high specificity with no cross-reactivity with B. microti, B. divergens, Babesia MO1, or P. falciparum. The assay also demonstrates high sensitivity, detecting as low as 115 infected erythrocytes/µl of blood. Screening of 1,731 blood samples from diverse biorepositories, including previously identified Lyme and/or B. microtipositive human samples and new specimens from field mice, showed no evidence of B. duncani infection in these samples. The assays could be useful in diverse diagnostic scenarios, including point-of-care testing for early B. duncaniinfection detection in patients, field tests for screening reservoir hosts, and high-throughput screening such as blood collected for transfusion.

The authors have declared no competing interest.

The research described herein was supported by the Global Lyme Alliance Foundation. CBM research is also supported by NIH grants AI138139, AI123321, AI152220 and AI153100, and AI136118, the Steven and Alexandra Cohen Foundation (Lyme 62 2020), and The Blavatnik Family Foundation.

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The use of de-identified human blood samples in this study was following an approved Yale IRB protocol to Dr. Ben Mamoun.

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