A Set of Rapid Diagnostic Tool for Babesia microti Infection

Background Human babesiosis caused by Babesia microti is an emerging tick‐borne zoonosis, with a global pooled prevalence of 2.23% and regional peaks in Europe (4.17%) and North America (1.54%). Traditional diagnostics like microscopy and polymerase chain reaction (PCR) suffer from low sensitivity in low‐parasitemia cases or high costs ($230/test), necessitating accessible, rapid assays for resource‐limited regions. Methods A cross‐priming amplification combined with vertical flow visualization (CPA‐VF) assay, a straightforward molecular method targeting the 18S rRNA gene of B. microti , requires minimal equipment and facilitates rapid detection. Results Sensitivity/Specificity: The CPA‐VF assay detected 2.56 fg/reaction (equivalent to 0.000004% parasitic red blood cells), with a sensitivity of 95.5% matching that of RT‐PCR but at a 60‐fold lower cost ($3.8/test). It showed no cross‐reactivity with B. duncani , B . divergens , or Plasmodium . Clinical Validation: Testing 49 positive samples (19 experimentally infected mice +30 artificially spiked) and 492 field samples, CPA‐VF demonstrated 95.5% sensitivity (95% CI: 88.2–98.7) and 95.5% specificity compared to nested PCR (nPCR). Intra‐assay coefficients of variation (CV) was 2.1%–7.2% and inter‐assay kappa coefficient was 0.94, confirming reliability. Conclusion CPA‐VF is a rapid, low‐cost ($3.8/test), and instrument‐free diagnostic tool for B. microti , particularly suitable for endemic regions where timely diagnosis reduces mortality risks from misdiagnosis as malaria. Its portability and visual readout address critical gaps in resource‐constrained settings.