Development and Field Evaluation of a Rapid Detection Method for Babesia microti using fluorescent recombinase polymerase amplification(RPA) technology

Background The frequent occurrences of Babesia microti ( B. microti ) infection have emerged as a significant global public health safety concern. The diagnosis of patients constitutes a crucial component in the prevention and control of babesiosis, necessitating the urgent establishment of efficient and accurate molecular biology diagnostic methods for B. microti detection. Purpose This study aimed to develop a sensitive, specific, and rapid fluorescent recombinase polymerase amplification (RPA) technique for the detection of B. microti ( B. microti ) and assess its suitability for field use. Methods This study developed and optimized a fluorescent RPA assay targeting the B. microti 18S rRNA gene fragment. The method's sensitivity (detection limit down to 10 fg/µL genomic DNA), specificity (distinguishing Plasmodium samples), and detection limit for spiked mouse blood samples (across varying parasitemia levels) were evaluated. The detection capability was validated using samples from an infected mouse model (tail-tip blood collected 3–21 days post-infection) and two confirmed human cases. Furthermore, 119 blood samples from patients presenting with fever accompanied by thrombocytopenia were tested using both nested PCR and the newly developed fluorescent RPA assay. The agreement between the two methods was analyzed using the Kappa value, and the significance of their difference was assessed using McNemar's test. Results A fluorescent RPA assay was developed targeting two B. microti 18S rRNA fragments (243 bp/191 bp). The 191 bp fragment demonstrated superior sensitivity (detection limit: 1 fg/µL genomic DNA) and was selected for assay establishment. Optimized at 39°C for 20 min, its detection threshold was defined by the lowest detectable concentration (1 fg/µL). Specificity testing with 8 P. falciparum and 8 P. ovale samples showed fluorescence below this threshold. The assay achieved a detection limit of 0.046 parasites/µL blood, demonstrating a 600-fold increase in sensitivity compared to nested PCR, which detects 28.49 parasites/µL. In infected mice, both RPA and nested PCR detected parasites by day 9 post-infection (microscopy: day 15), with RPA showing higher positivity. The method confirmed infection in 2 patients. Among 119 febrile patients with thrombocytopenia from Xinyang City, Henan Province, nested PCR detected 20 B. microti- positive cases while fluorescent RPA detected 21. The Kappa value for agreement between the methods was 97.05% (91.31% − 100%), and McNemar's test indicated no statistically significant difference in their detection rates (S = 1, P > 0.1). Based on the combined results of both tests, the molecular positivity rate for B. microti in this sample set was 17.65%, with co-infections involving Bunya virus present, accounting for 10.08% of the tested samples. Conclusion The fluorescence RPA method provides a rapid, sensitive, and specific tool for detecting B. microti and is effective for field screening.