Novel single-cell preservation and RNA sequencing technology unlocks field studies for Plasmodium natural infections.

Single-cell RNA sequencing (scRNA-seq) is a powerful technology used to investigate cellular heterogeneity. When applied to unicellular eukaryotes such as Plasmodium, scRNA-seq provides a single-cell resolution valuable to study complex infections comprised of mixed lifecycle stages and clones. Until now, the application of scRNA-seq has been mainly limited to in vitro and animal malaria models due to the challenges of working with Plasmodium natural infections in endemic settings. We validated sample preparation methods and a single-cell RNA preservation and sequencing technology in P. knowlesi for the future implementation in low-resource settings. We recovered 22,345 P. knowlesi single-cell transcriptomes containing all asexual blood stages from 6 samples to generate the most extensive P. knowlesi single-cell dataset to date. All 6 samples produced reproducible circular UMAP projections with consistent cluster localization and high gene expression correlation, regardless of the sample preparation method. Biomarker expression and life stage annotation using the Malaria Cell Atlas P. knowlesi reference dataset further confirmed these results. In conclusion, adaptable sample preparation methods combined with novel scRNA-seq preservation technology has the potential to unlock scRNA-seq for field studies which will lead to additional insights into Plasmodium biology and fundamentally transform how we study malaria natural infections.