Leukocyte Depletion in Dried Blood Spot Cards Enables Enrichment of Parasite DNA for Improved Sequencing

Expanding access to simple blood collection tools is essential to monitor, control, and eliminate malaria in low-resource settings where the disease is endemic. The most common method to preserve blood is depositing fingerstick samples onto filter paper─the dried blood spot (DBS) card. While DBS cards offer more optimal storage solutions than venous blood in vacutainers, they do not provide sample cleanup or enrichment of Plasmodium DNA. These samples retain high host-to-parasite DNA ratios, which negatively affect the quality of downstream sequencing. We developed a Leukocyte Depletion Card (LDC) that substantially depletes host white blood cells from whole blood to enrich Plasmodium-infected red blood cells in a hematocrit-independent volume (9.0 ± 0.5 μL). Using quantitative PCR, we evaluate the performance of the LDC using blood collected from 16 Plasmodium falciparum (P. falciparum)-infected patients at a clinic in Cape Coast, Ghana. The LDC achieved an average 32.5-fold parasite enrichment over venous blood. Promisingly, the LDC also provides a 36.6-fold parasite enrichment over a DBS card. Initial testing of targeted sequencing demonstrates significant (p < 0.01) improvement in P. falciparum read counts and coverage for the LDC. The LDC represents a unique microsampling device with potential applications in epidemiological studies of malaria. Drug resistance hinders malaria control efforts and makes population surveillance crucial. Dried blood spot (DBS) cards support these efforts, but host DNA makes collected samples inadequate for molecular analysis. A Leukocyte Depletion Card (LDC), which separates parasitized red blood cells from white blood cells, provides superior sequencing results over the traditional DBS card.