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: The skin stage of malaria is a vital and vulnerable migratory life stage of the parasite. It has been characterised in rodent models, but remains wholly uninvestigated for human malaria parasites. To enable in depth analysis of not genetically modified (non-GMO) Plasmodium falciparum (Pf) sporozoite behaviour in human skin, we devised a labelling technology (Cy5M 2 , targeting the sporozoite mitochondrion) that supports tracking of individual non-GMO sporozoites in human skin. Methods : Sporozoite labelling with Cy5M 2 was performed in vitro as well as via the feed of infected Anopheles mosquitos. Labelling was validated using confocal microscopy and flow cytometry and the fitness of labelled sporozoites was determined by analysis of infectivity to human hepatocytes in vitro , and in vivo in a rodent infection model . Using confocal video microscopy and custom software, single-sporozoite tracking studies in human skin-explants were performed. Results : Both in vitro and in mosquito labelling strategies yielded brightly fluorescent sporozoites of three different Plasmodium species. Cy5M 2 uptake colocalized with MitoTracker ® green and could be blocked using the known Translocator protein (TSPO)-inhibitor PK11195. This method supported the visualization and subsequent quantitative analysis of the migration patterns of individual non-GMO Pf sporozoites in human skin and did not affect the fitness of sporozoites. Conclusions : The ability to label and image non-GMO Plasmodium sporozoites provides the basis for detailed studies on the human skin stage of malaria with potential for in vivo translation. As such, it is an important tool for development of vaccines based on attenuated sporozoites and their route of administration.

A tracer-based method enables tracking of Plasmodium falciparum malaria parasites during human skin infection