Background

The discovery of broad-spectrum antiparasitic agents requires in vitro systems that allow direct comparison of compound efficacy across multiple species under uniform conditions. However, existing intraerythrocytic parasite culture platforms rely on species-specific media with differing nutrient compositions, serum sources, and undefined protein content. These inconsistencies affect parasite growth dynamics and drug potency measurements (e.g., IC₅₀ values), making it difficult to determine whether observed efficacy differences are due to true biological variation or culture-dependent artifacts. As a result, cross-species prioritization of chemical scaffolds with pan-apicomplexan potential remains challenging.

The clinical and epidemiological burden of intraerythrocytic apicomplexan parasites underscores the importance of developing such comparative approaches. According to the World Malaria Report 2024, there were an estimated 263 million malaria cases and approximately 597,000 deaths in 2023, with 94% of cases and 95% of deaths occurring in the WHO African Region [1]. Children under five accounted for approximately 76% of all malaria deaths, highlighting their disproportionate vulnerability [1]. Human babesiosis, caused by tick-transmitted Babesia species such as B. microti, B. duncani, B. divergens, and B. MO1, is an emerging zoonosis with increasing incidence in North America, Europe, and Asia [2–7]. According to the Centers for Disease Control and Prevention (CDC), tick-borne apicomplexan parasitic diseases have increased globally, with over 2,000 annual cases in the United States alone [8]. Resistance to first-line therapies has been documented in both genera: in Plasmodium, through mutations in PfCRT, DHFR, DHPS, and ATP4 [9–13], and in Babesia, notably in CytB and ribosomal proteins [14–17]. These shared trends of rising incidence and resistance highlight the need for new broad-spectrum antiparasitic drugs and standardized platforms that enable cross-species drug efficacy evaluation under harmonized conditions.

Plasmodium and Babesia species share key biological and pharmacological features that make cross-species drug development viable. They are obligate intraerythrocytic parasites that invade host erythrocytes using specialized cellular machineries, undergo clonal asexual replication (schizogony in Plasmodium and binary fission in Babesia), and depend on conserved metabolic pathways localized in essential organelles such as the apicoplast and mitochondrion [18–23] (Figure 1). The single mitochondrion is indispensable in both genera and serves as a validated pan-apicomplexan drug target. Mitochondrial bc₁ complex inhibitors such as atovaquone and endochin-like quinolones (ELQs) exhibit potent activity against both Plasmodium and Babesia species [14,22–27]. Resistance to these agents has been linked to conserved mutations in the cytochrome b (CytB) gene in both genera, confirming shared druggable vulnerabilities and resistance trajectories [14, 17,26,28–31]. Likewise, conserved enzymes such as DHFR-TS and core mitochondrial metabolic enzymes represent shared vulnerabilities that have been pharmacologically targeted in both parasites using antifolate and glycolytic inhibitor chemotypes [18,26,32–35]. These and additional shared, yet underexplored, vulnerabilities highlight the need for a unified in vitro system for drug screening and resistance testing (Figure 1).

Despite these shared vulnerabilities, current culture methodologies are highly fragmented. P. falciparum and B. divergens are typically cultured in RPMI-1640 supplemented with human serum or lipid-based substitutes such as Albumax [36,37]. In contrast, the first in vitro culture assays for B. duncani relied exclusively on HL-1 and Claycomb media [22,26,38], both of which are proprietary and inconsistently available. A major advance occurred in 2021, when B. duncani was shown to grow continuously in human erythrocytes using DMEM/F-12 supplemented with 20% fetal bovine serum (DFS20) [39,40], providing a defined and widely accessible alternative to these media. Although B. MO1 displays growth characteristics similar to B. divergens [34], it has not yet been incorporated into a unified culture system. Of particular importance, B. duncani remains the only Babesia species for which both continuous in vitro culture in human erythrocytes and a lethal in vivo murine infection model are available [28,39–41], making it uniquely suited for translational validation of drug candidates.

To address these limitations, we evaluated DMEM/F-12 supplemented with 20% fetal bovine serum (DFS20) as a common culture medium (CCM) capable of supporting the growth of Plasmodium and Babesia. We demonstrate that DFS20 supports robust asexual replication of P. falciparum (3D7, Dd2, HB3, V1/S), B. duncani, B. divergens (Rouen 87), and B. MO1 with growth kinetics and morphology comparable to those observed in their respective traditional media [33–35]. In addition, we emphasize that DFS20 functions as a unified and practical solution for comparative drug evaluation by eliminating media-dependent variability across species. This enables consistent readouts, improves assay reproducibility, and positions DFS20 as a scalable platform for pan-apicomplexan drug screening. This unified culture system enables standardized, cross-species drug susceptibility testing and facilitates screening of compounds targeting conserved metabolic vulnerabilities. Furthermore, when integrated with the in-culture and in-mouse (ICIM) model, currently applicable only to B. duncani, it serves as a proof-of-concept for how this standardized system could support parallel assessment of pharmacodynamics, pharmacokinetics, toxicity, and resistance emergence. By replacing fragmented media systems with a single, scalable, and reproducible platform, this protocol establishes a foundation for cross-species efficacy assessment, resistance profiling, and the identification and prioritization of next-generation pan-apicomplexan therapeutics.

Figure 1. Schematic representation of a high-throughput screening workflow used to evaluate pan-antiparasitic drugs targeting the in vitro asexual life cycle stages of Plasmodium and Babesia parasites. The diagram illustrates the major intraerythrocytic developmental stages [ring, trophozoite, and schizont for Plasmodium falciparum (3D7, Dd2, HB1, and V1/S); ring, filament, and tetrad stages for Babesia (B. duncani, B. divergens Rouen87, and B. MO1)] and depicts merozoite invasion into fresh human red blood cells (RBCs) and continuous asexual replication. Additionally, the figure highlights the potential development of gametocytes in both parasites. A screening funnel illustrates the evaluation of diverse candidate compounds across the erythrocytic stages of malaria and babesiosis parasites.