A set of diagnostic tests for detection of active Babesia duncani infection

•Developed enzyme-linked immunosorbent assays—Babesia duncani-specific antigen capture assays (BdACA)38 and BdACA234 to detect Babesia duncani, causing human babesiosis.•Both BdV234 and BdV38 are specific to B. duncani and detect 115 infected red blood cells/µl with high sensitivity.•Assays were validated in vitro and in vivo and screened 1731 human and field mice blood samples.•Assays hold promise for various diagnostics, such as early patient detection and reservoir animal screening. Human babesiosis is an emerging and potentially fatal tick-borne disease caused by intraerythrocytic parasites of the Babesia genus. Among these, Babesia duncani is particularly notable for causing severe and life-threatening illness in humans. Accurate diagnosis and effective disease management hinge on the detection of active B. duncani infections. While molecular assays are available to detect the parasite in blood, a reliable method for identifying biomarkers of active infection remains elusive. We developed the first B. duncani antigen capture assays, targeting two immunodominant antigens, BdV234 and BdV38. These assays were validated using established in vitro and in vivo B. duncani infection models, and following drug treatment. The assays demonstrated no cross-reactivity with other species such as B. microti, B. divergens, Babesia MO1, or Plasmodium falciparum, and can detect as few as 115 infected erythrocytes/µl of blood. Screening of 1731 blood samples from various biorepositories, including samples previously identified as Lyme and/or B. microti-positive, as well as new specimens from wild mice, revealed no evidence of B. duncani infection or cross-reactivity. These assays hold significant promise for various applications, including point-of-care testing for the early detection of B. duncani in patients, field tests for screening reservoir hosts, and high-throughput screening of blood samples intended for transfusion.