Multidimensional in vitro assay for antimalarial combination testing and pharmacodynamic modeling – the MULT-i 2 assay

Abstract Malaria remains a major global health challenge, with emerging partial resistance to first-line therapies in Africa threatening current control efforts. Drug combinations are essential to improve treatment efficacy and restrain resistance development. However, in vitro assays that quantify parasite viability after drug exposure and characterize pharmacodynamic drug interactions are labor- and resource-intensive, with standard approaches such as the parasite reduction ratio assay limiting systematic, high-resolution evaluation of drug combinations. We present the MUltidimensional Luminescence Test for integration of interactions (MULT-i2), an in vitro assay that enables scalable, high-resolution assessment of parasite viability across multidimensional drug concentration spaces. For dual drug combinations, the MULT-i2 assay characterizes interaction surfaces while requiring ∼50-fold fewer resources and more than two-fold less time than conventional methods, enabling exploration of broader combination scenarios. The assay combines a highly sensitive chemiluminescence readout with inducible reporter expression in Plasmodium falciparum, supporting potential extension to multidimensional combination testing. Using the general pharmacodynamic interaction (GPDI) model, the MULT-i2 assay quantified interaction potency and directionality, confirming and refining the known synergy between atovaquone and proguanil, and revealing detailed interaction patterns for additional drug combinations. Overall, this approach provides an efficient framework for testing and characterizing pharmacodynamic drug interactions and supports the rational development of antimalarial combination therapies. Competing Interest Statement The authors have declared no competing interest. Abbreviations - ACTs - artemisinin-based combination therapies - PRR - parasite reduction ratio - PCT - parasite clearance time - EC50 - half-maximal effective concentration - Emax - maximal drug effect - HRP-2 ELISA - histidine-rich protein 2 sandwich enzyme-linked immunosorbent assay - MULT-i2 - multidimensional luminescence test for integration of interactions - GPDI - general pharmacodynamic interaction model - gfp or GFP - green fluorescent protein - hsp70 - heat shock protein 70 - cam - calmodulin - loxPint - loxP-intron - DMSO - dimethyl sulfoxide - uRBC - uninfected red blood cells - iRBC - infected red blood cells - rapa - rapamycin - GFP - green fluorescent protein - LOQ - limit of quantification - PRR v2 - parasite reduction ratio version 2 assay published by Walz et al. - cPRR - combination PRR assay - AIC - Akaike Information Criterion - CM - culture medium - E - effect - C - concentration - Ecomb - combination effect - H - hill coefficient - INT - interaction parameter - EC50INT - half-maximal effective concentration of interaction - HINT - hill coefficient - IC50 - half-maximal inhibitory concentration - LLOQ - lower limit of quantification - ULOQ - upper limit of quantification.