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 that can be used to unravel cellular heterogeneity when applied to unicellular eukaryotes, including Plasmodium parasites. scRNA-seq is particularly useful to study complex infections like malaria, with mixed life stages or clones, yet until now it has been primarily limited to in vitro and animal malaria models due to challenges associated with working with malaria natural infections in endemic settings. We validated a novel single-cell RNA sequencing technology for use with Plasmodium parasites by combining optimized sample preparation methods with RNA preservation integrated in a sample capture device, which allows for the separation of the sample collection and library preparation and sequencing. We recovered 22,345 P. knowlesi single-cell transcriptomes from 6 samples, the most extensive P. knowlesi dataset to date. Regardless of preparation methods used, all samples resulted in reproducible circular UMAP projections with consistent cluster localization and high gene expression correlation, which were confirmed by biomarker expression and annotating life stages using the Malaria Cell Atlas P. knowlesi reference dataset. Some variation in life stage recovery was observed, especially for the ring stage. This could be attributed to the inherent differences between sample preparation methods, or as a result of gene and transcript filtering thresholds which should be further investigated and optimized based on what is known about parasite biology. In conclusion, by combining parasite enrichment with a novel scRNA-seq and preservation technology, scRNA-seq can be expanded to field settings, even when limited resources are available. Overall design: To validate the application of HIVE technology for the analysis of Plasmodium natural infections, we tested different sample processing and loading methods, and assessed the single-cell RNA sequencing results. To create a mock natural infection sample with mixed parasite life stages, we diluted in vitro-cultured P. knowlesi A1-H.1 parasites to 0.8% parasitemia in whole blood. Tested methods for sample processing include two combinations of leukodepletion and parasite enrichment techniques tailored to optimally isolate all life stages present in blood circulation and eliminate uninfected human cells from natural infections, which would otherwise dominate sequencing reads. A schematic representation of the protocols of the 6 samples is provided in Figure 1. For the PNyco method, which only requires a tube centrifuge, we used a Plasmodipur filter to remove human leukocytes, followed by a Nycodenz density gradient to enrich for parasite life stages. Here we found an enrichment to 22% parasitemia after the Nycodenz gradient by microscopy. In the second method, MACSPS, we used a MACS column to enrich for trophozoite and schizont life stages in the magnetic column based on the paramagnetic properties of hemozoin, similar to the MCA method (2). The first elution contains leukocytes, uninfected RBCs, and ring stages which have passed through without binding to the magnetic column. Subsequently, the first elution underwent further processing using a Plasmodipur filter to remove leukocytes, followed by a saponin lysis to remove uninfected RBCs, resulting in the isolation of the ring stage. In the second elution, the trophozoite and schizont stages bound to the magnetic column were released and recovered. The ring stages were then combined with trophozoites and schizonts recovered to obtain an enriched sample containing all life stages. For the HIVE loading, we evaluated two different methods for loading enriched parasite into the HIVEs: one utilizing a plate centrifuge to facilitate the sinking of parasites into the pico-wells, and the other allowing parasites to sink by gravity, eliminating the need for a plate centrifuge if unavailable (C and G, respectively, Figure 1). In addition, we also tested cultured P. knowlesi samples without mixing with whole blood. Parasite enrichment was conducted using two different density gradient centrifugation methods. Half of the cultured parasites were purified using a Percoll gradient (PkCultureP), typically employed to enrich samples for schizonts stages (40), while the other half underwent purification using a Nycodenz gradient (PkCultureN), commonly used for P. knowlesi (33), which recovered a broader range of parasite life stages compared to Percoll. Both methods led to a similar level of enrichment, around 50%, and samples were loaded using centrifugation.