A Highly Sensitive Multiplex Antibody Assay Reduces Window Period for Detection of Babesia microti Infection

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Background The health burden of Babesia microti, the primary causative agent of human babesiosis in the United States, is significant and increasing. Diagnosis of clinical babesiosis still remains challenging, resulting in misdiagnosis and underreporting. The gold standard for detection of B. microti–specific antibody, immunofluorescence assay (IFA), is cumbersome and resource-intensive. A high-throughput assay to detect serological biomarkers of B. microti exposure would facilitate epidemiological studies and clinical diagnosis. Methods We developed a multiantigen, high-throughput, and highly sensitive Luminex bead-based assay (LBA) for detection of Babesia microti­–specific antibodies in babesiosis patients and endemic populations. Serum samples from 191 individuals who had confirmed B. microti exposure (IFA or polymerase chain reaction [PCR] positive) were screened for antibody reactivity to 4 immunodominant antigens—MCFRP1, BAHCS1, SERA1, and PiβS1—by LBA. Results Among the 4 antigens evaluated, MCFRP1 and BAHCS1 were the most sensitive biomarkers for B. microti exposure, detecting 96.6% and 100% of IFA+/PCR+ and 75.3% and 87.6% of IFA+/PCR- samples, respectively. The “window period” before IFA-detectable seroconversion is of particular concern for clinical diagnosis using serological detection methods. Importantly, combining all 4 antigens allowed detection of 6/13 (46.2%) PCR-positive cases that were missed by IFA. No single antigen yielded reactivity to more than 3/13 (23.1%) IFA-/PCR+ cases in our LBA, indicating diversity in the polarization of early immune responses following B. microti exposure. Conclusions Combination of these antigens in our LBA would reduce the window period before IFA-detectable seroconversion of detection in Babesia microti–exposed individuals.