Can Surface Environmental DNA Reveal Emerging Leishmaniasis Risk? A Testable Hypothesis for Spatial Risk Surveillance

Climate-driven expansion of Leishmania infantum and its phlebotomine vectors is reshaping the geography of leishmaniasis across Europe, while surveillance systems remain largely dependent on labour-intensive trapping and pathogen screening. We propose a hypothesis-driven framework in which surface environmental DNA (eDNA) collected from sand fly resting microhabitats is analysed using duplex droplet digital PCR (ddPCR) targeting both vector and parasite DNA. The approach is not intended to replace conventional entomological surveillance but to function as a first-tier triage tool for identifying locations where targeted trapping and pathogen investigations should be prioritised.The hypothesis is based on three observations: (i) adult sand flies aggregate at predictable sheltered resting sites; (ii) infected sand flies may harbour substantial parasite burdens, with each Leishmania parasite containing thousands of kinetoplast minicircle copies; and (iii) biological residues deposited at resting sites may contribute detectable quantities of vector and parasite DNA to environmental surfaces. We therefore hypothesise that repeated occupation of resting microhabitats could generate environmental DNA signals reflecting local vector–parasite co-occurrence.We outline a tiered interpretation framework and a falsifiable validation pathway combining controlled insectarium experiments and field investigations. Particular emphasis is placed on distinguishing environmental detection of parasite DNA from evidence of active transmission and on defining the analytical and ecological conditions required for reliable signal interpretation.If validated, surface eDNA surveillance could provide a scalable spatial risk surveillance component within integrated One Health surveillance systems, helping to prioritise entomological investigations in newly exposed or under-surveyed regions. Beyond operational surveillance, this framework may contribute to addressing a recurrent challenge in neglected tropical diseases: distinguishing true emergence from delayed detection in areas where transmission has historically been overlooked.