Abstract
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.