In this Phase II project, development and clinical validation of an ELISA test for in vitro diagnosis of Babesia microti infection will be completed. Babesiosis is an emerging tick-borne parasitic disease which may cause severe to fatal illness in immunocompromised or otherwise weakened patients, and may be carried in the blood subclinically in up to 1% of the population in endemic areas. Recent cases of fatal transfusion- transmitted babesiosis have also led to the identification of this pathogen as a significant threat to the blood supply. However, currently no commercial, validated and FDA approved tests are available for B. microti. Babesiosis is currently diagnosed by immunofluorescence staining, microscopy of blood smears, and/or PCR. None of these procedures are easily adaptable to routine clinical laboratory use. In Phase I, we developed a microplate-based ELISA using a mixture of 5 novel synthetic B. microti peptide antigens comprising 6 immunodominant epitopes that were demonstrated to be diagnostically significant markers of infection. The five peptide sequences were identified through screening of libraries of overlapping peptides derived from members of the BMN1 family of B. microti proteins, a group previously identified as highly immunoreactive, against babesiosis patient sera. Several of these peptides and epitopes have not previously been recognized as immunodominant, and represent new discoveries. Sensitivity of 100% among 60 confirmed babesiosis cases was achieved, along with specificity above 99.5% in low-risk blood donors and other controls. The 5 peptides have been combined in a single well ELISA format using an innovative immobilization chemistry. The prototype ELISA will be optimized with respect to peptide stoichiometry and all other assay parameters and scaled up for manufacturing in Phase II. To validate assay performance, a clinical study will be carried out in Phase II, in which the B. microti ELISA will be compared with PCR and blood smear, which are considered gold standard assays for Babesia infection. A retrospective clinical study will evaluate assay performance on well-characterized samples from confirmed babesiosis cases, while a prospective study will examine undiagnosed patients seen at tick-borne disease clinics at three medical centers in regions of the Northeast and Midwest that are highly endemic for B. microti. The clinical study will determine the performance of the B. microti ELISA vs. PCR, blood smear and immunofluorescence for diagnosis of B. microti infection. Results will be submitted in a 510(k) application to FDA for clearance of the B. microti ELISA for in vitro diagnostic use.
Babesiosis is an emerging, tick-borne parasitic infection which can produce severe to fatal illness, especially in immunocompromised or weakened patients. However, no FDA approved tests are currently available to aid in clinical diagnosis of this disease. This project will lead to the development, validation, FDA approval and commercial availability of the first simple, accurate and user-friendly diagnostic test for babesiosis.
