A novel mitochondrial quantitative PCR duplex for the detection of Babesia microti and Babesia odocoilei

Tick samples and host blood are commonly screened for Babesia microti, the causative agent of human babesiosis in the northeastern United States, using qPCR assays that target the 18S rRNA gene. While screening ticks for B. microti with multiple 18S-based assays, we observed inconsistent results that suggested potential cross-reactivity with non-target organisms, limited sensitivity, or both. These observations highlighted the need for a more sensitive and species-specific qPCR assay for B. microti and for improved tools to detect and differentiate other Babesia species circulating in Maine ticks and vertebrate hosts. We identified Babesia odocoilei as the other Babesia spp. most commonly found in Ixodes scapularis ticks in Maine and sequenced ~5000 bp regions of B. microti and B. odocoilei mitochondrial DNA. We then developed and evaluated a duplex mitochondrial qPCR assay for simultaneous detection and differentiation of these two species. Using the new assay, we tested 4,464 Ixodes spp. ticks collected from both active and passive surveillance in Maine. We detected B. microti in 449 ticks (10.1%), B. odocoilei in 803 ticks (18.0%), and mixed infections in 92 ticks (2.1%). Prevalence varied by collection method, tick life stage, and location. In 46 B. microti -positive ticks, the mitochondrial assay produced Cq values 5.9–7.1 cycles lower than two published 18S rDNA assays (Dunnett-adjusted p < 0.0001). The mitochondrial duplex qPCR assay provided improved sensitivity and detection of B. microti and B. odocoilei compared to published 18S rDNA assays. These results support the use of mitochondrial targets to improve differentiation of co-occurring Babesia species and to strengthen the interpretation of tick-borne pathogen surveillance data.