A Recombinase Polymerase Amplification Lateral Flow Assay (RPA-LF) for detection of Babesia divergens

Background: Babesia divergens is a tick-borne apicomplexan parasite found predominantly in Europe, causing babesiosis in livestock and occasionally in humans, with severe disease reported in immunosuppressed or asplenic individuals. As populations of Ixodes ricinus , the principal vector, expand geographically including into urban environments, there is a growing need for rapid, sensitive, and field-deployable surveillance tools. In this study, we developed a Recombinase Polymerase Amplification Lateral Flow (RPA-LF) assay for the detection of B. divergens . Methods: RPA assays targeting B. divergens hsp70 , cox1 , and 18S rRNA genes were designed and evaluated for sensitivity and specificity using serial dilutions and control samples, including other apicomplexan parasites and tick samples spiked with B. divergens DNA. To assess field performance, 279 I. ricinus ticks were collected from urban parks in Greater London and screened by qPCR and a subset also tested using the new RPA-LF assays. Anaplasma phagocytophilum was screened by qPCR but not detected. Results: The cox1 and 18S rRNA RPA-LF assays demonstrated high specificity, with detection limits of at least 10 ^- ⁵ ng/μl and 10 ^- ³ ng/μl, respectively, under isothermal conditions (39 °C for 30 minutes). No cross-reactivity was observed with non-target DNA. Field validation using pooled samples revealed a single sample positive for Babesia divergens or the closely related Babesia capreoli by qPCR, as previously observed in UK tick studies, indicating an estimated nymphal infection prevalence of 0.4% to 1.2%. The 18S rRNA RPA-LF assay successfully detected this pool, whereas the cox1 RPA-LF did not. As with previous molecular approaches, the close genetic similarity of the selected targets prevents discrimination between B. divergens and B. capreoli . Conclusions: We report the first successful development of an RPA-LF assay for B. divergens detection in ticks. The assay demonstrates high sensitivity and specificity under isothermal conditions and provides a portable and reliable tool for field-based molecular surveillance of emerging tick-borne pathogens. Field validation revealed B. divergens / B. capreoli in questing ticks from urban green spaces in London, underscoring the importance of this tool for early detection and ongoing surveillance of tick-borne parasites in expanding urban ecosystems.