Tolerance of Plasmodium falciparum mefloquine-resistant clinical isolates to mefloquine-piperaquine: implications for triple artemisinin-based combination therapy strategies.

Tolerance of Plasmodium falciparum mefloquine-resistant clinical isolates to mefloquine-piperaquine: implications for triple artemisinin-based combination therapy strategies. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tolerance of Plasmodium falciparum mefloquine-resistant clinical isolates to mefloquine-piperaquine: implications for triple artemisinin-based combination therapy strategies. Benoit Witkowski, Roesch Camille, Anna Cosson, Melissa Mairet-Khedim, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6629488/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Nov, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Triple artemisinin-based combination therapies (TACTs) have been proposed to delay the emergence of multidrug-resistant Plasmodium falciparum by combining two partner drugs with artemisinin derivatives. Among these, mefloquine–piperaquine (MQ–PPQ) is a leading candidate, based on the assumption that resistance to both partner drugs would be difficult to develop simultaneously. Here, we assess the efficacy and resistance potential of MQ–PPQ using Cambodian clinical isolates with distinct resistance profiles. We find that MQ resistance confers significant cross-tolerance to the MQ–PPQ combination, while PPQ-resistant and sensitive strains remain susceptible. Under repeated MQ–PPQ pressures, parasites rapidly acquire MQ-PPQ tolerance, driven by pfmdr1 amplification. Mechanistic investigations reveal that MQ inhibits PPQ accumulation in a dose-dependent manner, providing a functional explanation for the compromised efficacy of the combination. These findings demonstrate that MQ resistance alone can undermine MQ–PPQ TACT efficacy, which question the strategic foundation of this combination and underscore the need for alternative combinations with lower resistance selection risk. Health sciences/Diseases/Infectious diseases/Malaria Biological sciences/Microbiology/Antimicrobials/Antiparasitic agents Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Over the past two decades, malaria control efforts have led to substantial reductions in global incidence and mortality 1 . Central to this progress has been the widespread deployment of artemisinin-based combination therapies (ACTs), which remain the cornerstone of first-line treatment for uncomplicated Plasmodium falciparum malaria. However, the emergence of artemisinin (ART) resistance (ART-R) in eastern and central Africa now poses a serious threat to these gains 2 . While partial resistance to ART alone, as previously observed in both Africa and Southeast Asia, does not necessarily result in increased ACT treatment failure rates, resistance to partner drugs remains the key driver of clinical failure 3 , 4 . Of particular concern is the potential for ART-R to act as a precursor to multidrug-resistant parasite lineages. This scenario is supported by previous observations in the Greater Mekong Subregion, where parasites harboring resistance to at least one partner drug facilitated the subsequent emergence of ACT-resistant strains 5 – 10 . The increasing detection of ART-R in Africa raises the possibility of a similar trajectory, potentially leading to a widespread loss of ACT efficacy. To mitigate this risk, an alternative approach is to develop optimized ACT formulations with a lower propensity for resistance selection, even in the presence of pre-existing drug resistance. Triple ACTs (TACTs) have been proposed as a potential solution to this challenge. By combining an ART derivative with two partner drugs, TACTs are designed to reduce the likelihood of resistance selection and maintain therapeutic efficacy in regions where conventional ACTs are failing. Two main formulations have been evaluated in clinical trials: artesunate-mefloquine-piperaquine and artesunate-amodiaquine-lumefantrine 11 – 14 . Among these, mefloquine-piperaquine (MQ-PPQ) has drawn particular interest due to a possible antagonism in the acquisition of resistance to its two partner drugs. Data from Southeast Asia suggest that resistance markers for MQ ( pfmdr1 amplification) and PPQ ( pfplasmepsin2/3 ( pfpm2/3 ) amplification) display inverse prevalence trends, depending on whether PPQ- or MQ-based ACTs are implemented 15 . Furthermore, MQ-PPQ TACT has demonstrated high efficacy in patients infected with DHA-PPQ-resistant parasites, with cure rates exceeding 95% compared to 48% for DHA-PPQ alone 14 . This finding highlights mefloquine’s ability to compensate for PPQ resistance in the context of pre-existing PPQ resistance. However, the potential of MQ-PPQ TACT in the context of pre-existing MQ resistance remains unexplored. Given its possible large-scale implementation, addressing this gap is crucial to ensure its long-term viability. In this study, we aimed to evaluate the efficacy of MQ-PPQ TACT against P. falciparum isolates exhibiting MQ resistance, providing critical insights into its operational feasibility and potential limitations. Results In vitro susceptibility of field isolates to PPQ, MQ and combined MQ-PPQ exposure The in vitro susceptibility of a panel of Cambodian P. falciparum isolates, categorized as sensitive (Sensitive, n = 5), PPQ-resistant (KEL1/PLA1, n = 9) and MQ-resistant (MQ-R, n = 17), was first evaluated for PPQ, MQ, and the combination MQ-PPQ. Based on the PSA, KEL1/PLA1 isolates exhibited higher survival rates (55·58% ± 15·67) compared to sensitive (0·00% ± 0·00, p < 0·0003, Kruskal-Wallis test followed by Dunn’s multiple comparison test) and MQ-R parasites (0·16% ± 0·37, p < 0·0001, Kruskal-Wallis test followed by Dunn’s multiple comparison test) ( Figure 1A ). As expected, the mean IC 50 values determined by [ 3 H]-hypoxanthine incorporation showed significant differences across the three groups. MQ-R isolates exhibited significantly higher IC 50 values (96·35 nM ± 22·84) than Sensitive and KEL1/PLA1 parasites (42·99 nM ± 14·60, p = 0·0148 and 30·31 nM ± 15·13, p < 0·0001 respectively, Kruskal-Wallis test followed by Dunn’s multiple comparison test) ( Figure 1B ). Finally, the susceptibility of the three groups to MQ-PPQ co-exposure was assessed using a survival assay over a range of drug concentrations, allowing for IC 50 determination. MQ-R isolates showed significantly higher IC 50 values (51·26 nM ± 16·29) compared to Sensitive and KEL1/PLA1 isolates (23·20 nM ± 2·75, p = 0·0021 and 24·29 nM ± 8·02, p = 0·0016, respectively, Kruskal-Wallis test followed by Dunn’s multiple comparison test) ( Figure 1C ). The IC 50s of MQ and the combination MQ-PPQ exposure were highly correlated (r = 0.7146, p < 0.0001, Pearson correlation test) ( Figure 1D ). Paired MQ-PPQ in vitro drug pressure To investigate the potential for stepwise adaptation to MQ-PPQ combination therapy, the P. falciparum 9097 strain was subjected to four successive rounds of drug pressure, as summarized in the supplementary figure 2. Then, the in vitro susceptibilities of the parental strain and the final selected strain ( Pressure 4 ) were compared for each drug individually and for the MQ-PPQ combination. Following drug pressure, the selected strain exhibited a significantly increased IC 50 to MQ (146·60 nM ± 33·76 vs. 63·69 nM ± 19·37, p = 0.0286, Mann-Whitney U test) ( Figure 2A ), suggesting an adaptive response to MQ exposure. However, no significant change in PPQ susceptibility was observed (29·50 nM ± 10·19 vs. 40·59 nM ± 16·29, p = 0·3152, Mann-Whitney U test) ( Figure 2B ), indicating that PPQ susceptibility remained largely unaffected by MQ-PPQ selective pressure. Interestingly, the IC 50 for MQ-PPQ co-exposure increased significantly from 21·70 nM ± 4·41 to 45·11 nM ± 15·82 ( p = 0·0286, Mann-Whitney U test) ( Figure 2C ), suggesting that adaptation to MQ-PPQ treatment primarily involved increased MQ tolerance rather than PPQ resistance. The in vitro susceptibility of both parental and selected strains to MQ and PPQ was further assessed using conventional methods, including the [ 3 H]-hypoxanthine uptake inhibition assay and PSA. The MQ IC 50 increased significantly from 48·37 nM ± 8.68 to 117·00 nM ± 18.87 ( p = 0·0040, Mann-Whitney U test), confirming the emergence of a MQ resistance phenotype. A modest but significant increase in PSA survival was observed (1·42% ± 1.34 vs 3·35% ± 0.79, p = 0·0068, Mann-Whitney U test), although the survival rate remained below the established 10% resistance threshold ( Supplementary Figure 3 ). To explore potential genetic mechanisms underlying these phenotypic changes, we assessed copy number variation in key resistance-associated genes. The Pressure 4 strain acquired an additional copy of the pfmdr1 gene compared to the parental strain (1·96 ± 0·14, n = 9 technical replicates), a known marker associated with MQ resistance. However, no change was detected in pfpm2 copy number (0·93 ± 0·08, n = 9 technical replicates), suggesting that the adaptation to MQ-PPQ was primarily mediated by pfmdr1 amplification. Comparative genomic analysis of parental and selected strains To validate our previous findings, we performed whole-genome sequencing of both the parental and Pressure 4 P. falciparum strains. Comparative genomic analysis revealed no newly acquired SNPs or indels following drug selection, suggesting the absence of specific point mutations that could directly explain the increased tolerance to the MQ-PPQ combination ( Supplementary Fig. 4 ). Notably, no mutations were detected in pfcrt , a well-characterized marker of PPQ resistance. In contrast, CNV analysis identified an amplification of a region on chromosome 5, encompassing pfmdr1 , in the Pressure 4 strain ( Figure 3 and Supplementary Table 2 ). No concomitant amplification of pfpm2 or pfpm3 was observed. Additionally, a deleted genomic region was observed on chromosome 9 following drug selection. This deletion is unlikely to be involved in drug resistance acquisition, as similar deletions have been reported in long-term in vitro cultures of P. falciparum . Altogether, the comparative genomic analysis suggests that pfmdr1 amplification alone may play a pivotal role in modulating parasite susceptibility to MQ-PPQ by potentially altering drug import or efflux mechanisms within the parasite. Evaluation of the central role of pfmdr1 in MQ-PPQ tolerance using radioactive assays To investigate the potential involvement of pfmdr1 in tolerance to the MQ-PPQ combination, we assessed the impact of MQ pre-incubation on the intracellular incorporation of radiolabeled PPQ [ 3 H(G)]. The amount of incorporated radiolabeled PPQ was significantly reduced when parasites were pre-exposed to MQ before the addition of radiolabeled PPQ (16·98 ± 2·63 vs. 4·64 ± 2·03, p = 0·0312, Wilcoxon test) ( Figure 4A ). A similar effect was observed in the parental strain, where MQ pre-incubation resulted in a significant decrease in PPQ incorporation (14·54 ± 3.99 vs. 1·82 ± 1·83, p = 0.0312, Wilcoxon test) ( Figure 4A ). These findings suggest that PPQ uptake may be influenced by pfmdr1 -mediated drug transport. Specifically, as MQ is actively exported out of the parasite via pfmdr1 , PPQ import into the parasite appears to be reduced. To further explore this hypothesis, we measured the variation in PPQ uptake among clinical isolates with different resistance phenotypes (Sensitive, MQ-R and KEL1/PLA1). In all three groups, MQ pre-incubation significantly reduced PPQ uptake ( Figure 4B ). The mean radioactive PPQ incorporation decreased from 4·72 ± 1·16 to 1·12 ± 1·80 ( p = 0.0625, Wilcoxon matched-pairs test) in Sensitive isolates, from 4·65 ± 1·52 to 0·28 ± 0·25 ( p = 0·0156, Wilcoxon matched-pairs test) in KEL1/PLA1 isolates, and from 6·84 ± 2·42 to 1·75 ± 0·90 ( p = 0.0002, Wilcoxon matched-pairs test) in MQ-R isolates. To quantify the impact of increasing MQ concentrations on PPQ incorporation, we performed a dose-response experiment using pre-incubation with MQ at concentrations ranging from 25 nM to 750 nm before the addition of [ 3 H]-PPQ ( Figure 5A ). The concentration of MQ required to inhibit 50% of PPQ incorporation was lowest for KEL1/PLA1 (15·57 nM) and Sensitive isolates (33·52 nM), whereas MQ-R parasites required a significantly higher MQ concentration (115·33 nM) to achieve the same level of inhibition ( p = 0·0312 and p = 0·0008, respectively, Mann-Whitney U test). Finally, we examined the relationship between pfmdr1 copy number and PPQ uptake. Isolates with multiple pfmdr1 copies exhibited significantly higher incorporation of [ 3 H]-PPQ compared to single-copy isolates, even when pre-incubated with 200 nM MQ ( p = 0·0160, Mann-Whitney U test) ( Figure 5B ). These findings reinforce the hypothesis that pfmdr1 plays a central role in modulating the intracellular balance of MQ and PPQ. MQ pre-incubation reduces PPQ susceptibility in MQ-R isolates Given the observed interaction between MQ and PPQ uptake, we next assessed whether MQ pre-incubation could modulate PPQ susceptibility in MQ-R parasites. Cambodian clinical isolates classified as MQ-R (n = 12), as well as the Parental and Pressure 4 laboratory strains, were subjected to the PSA under standard conditions and with prior exposure to 200 nM MQ pre-incubation. PSA survival rates varied according to genotype ( Figure 6 ). The addition of MQ had no significant impact on PSA levels in the Parental strain. However, in the Pressure 4 strain, MQ pre-incubation resulted in a significant increase in survival rate, from 3·35 ± 0·79 to 6·38 ± 2·06 ( p = 0·0312, Wilcoxon test). A similar effect was observed for MQ-R parasites, where pre-incubation with MQ significantly increased PSA survival from 0·29 ± 0·37 to 4·36 ± 4·79 ( p = 0·0142, Paired t -test). These findings provide further evidence that MQ exposure influences PPQ susceptibility, particularly in MQ-resistant isolates. Discussion The primary objective of this study was to assess the activity of paired MQ-PPQ against the different resistance profiles frequently observed in Southeast Asia. In vitro susceptibility testing confirmed that sensitive and KEL1/PLA1 parasites were highly susceptible to MQ, whereas WT and MQ-R parasites remained susceptible to PPQ. Conversely, KEL1/PLA1 and MQ-R strains displayed increased tolerance to PPQ and MQ. Evaluation of the combined MQ-PPQ activity against sensitive and KEL1-PLA1 parasites demonstrated high susceptibility, consistent with clinical studies reporting excellent therapeutic efficacy of this combination in infections with similar parasite genotypes 14 , 16 . In sharp contrast, MQ-PPQ activity against MQ-R parasites revealed significantly higher tolerance compared to sensitive and KEL1/PLA1 strains. This result contrasts with the proposed rationale for TACTs, which assumes that the presence of two partner drugs should mitigate resistance to one of them 17 . A possible explanation for this unexpected finding could be the presence of parasites with co-existing PPQ and MQ resistance, as previously observed in Cambodia 18 . However, our data ruled out this hypothesis, as all MQ-R strains retained full susceptibility to PPQ when tested individually. A key observation from our study was that MQ-PPQ tolerance in MQ-R parasites was directly correlated with their intrinsic MQ resistance levels. This was confirmed by a strong and significant correlation between MQ and MQ-PPQ IC 50 values. Based on these findings, we hypothesized that pfmdr1 gene amplification – previously implicated in MQ resistance – plays a central role in the acquisition of a tolerance phenotype to paired MQ-PPQ. To test this hypothesis, we evaluated the selection potential of MQ-PPQ in a parasite lineage that had previously exhibited intra-host acquisition of MQ resistance. Upon repeated MQ-PPQ exposure, parasites acquired pfmdr1 amplification, which was associated with increased tolerance to both MQ and MQ-PPQ. In contrast, no molecular markers or phenotypic evidence of PPQ resistance were observed. Whole-genome analysis of the parental and derived strains confirmed amplification of a region on chromosome 5, encompassing pfmdr1 , as the only genomic change. These results strongly support the role of pfmdr1 amplification in MQ-PPQ tolerance and suggest that PPQ does not effectively reach its intracellular target in these parasites. The acquisition pfmdr1 amplification upon MQ-PPQ pressure was relatively rapid, which is consistent with previous observations of in vitro resistance selection through MQ alone pressure. Yet, the fitness cost and the persistence of the amplification detected in this study were not evaluated, but it can be suspected to be similar with what Preechapornkul et al. observed 19 . This hypothesis was further validated through radioactive PPQ incorporation assays. Our results showed that PPQ uptake into infected erythrocytes was directly dependent on pfmdr1 copy number, with pfmdr1 -amplified parasites exhibiting significantly higher intracellular PPQ concentrations. This phenomenon explains the observed “over-sensibilization” of MQ-R parasites to PPQ, highlighting PfMDR1 as a key determinant of PPQ intracellular accumulation and activity. Furthermore, pre-incubation with increasing concentrations of MQ resulted in a dose-dependent reduction in PPQ uptake, suggesting that MQ, a known PfMDR1 substrate, competitively inhibits PPQ uptake. This competition likely reduces intracellular PPQ concentrations to subtherapeutic levels, thereby impairing its efficacy in MQ-R parasites. Finally, the phenotypic discrepancies observed between MQ-R parasites and the other groups can be explained by the ability of pfmdr1 -amplified parasites to efficiently expel MQ, thus maintaining resistance, as summarized in Fig. 7 . This mechanism would explain the paradoxical loss of MQ-PPQ activity against MQ-R parasites, whereas in sensitive and KEL1/PLA1 strains, MQ remains at therapeutic concentrations, ensuring the continued efficacy of the combination. In conclusion, this study demonstrates that the inherent strategy of MQ-PPQ TACT, which relies on the antagonistic resistance mechanisms of the two partner drugs, inadvertently leads to impaired drug action due to altered intracellular pharmacodynamic. This phenomenon facilitates the emergence of paradoxical resistance, driven by pfmdr1 amplification. Although these findings are based on in vitro observations, they underscore the need for active surveillance of pfmdr1 -amplified parasites following MQ-PPQ TACT implementation. Methods Clinical isolate selection and culture conditions Clinical isolates were obtained from Therapeutic Efficacy Studies (TES) conducted in Cambodia between 2016 and 2019. Samples were selected based on their pfk13 , pfmdr1 , pfpm2 and pfcrt genotypes according to the following classification: sensitive, characterized by pfk13 wild-type, pfmdr1 monocopy, pfpm2 monocopy and without mutation on pfcrt codons known to modulate PPQ susceptibility (amino acid positions 88, 93, 97, 145, 218, 343 and 353); KEL1/PLA1, a genetically characterized group of P. falciparum parasites that has emerged in the Greater Mekong Subregion, characterized by pfk13 C580Y and pfpm2 amplification 20 with or without polymorphisms at the above pfcrt amino acid positions; and MQ-R, characterized by pfk13 mutation, pfmdr1 amplification and without polymorphisms at the above pfcrt amino acid positions. Characteristics and in vitro results of the selected samples are summarized in the supplementary table 1. Genetic characterization was performed using the following methodology: polymorphism of the pfk13 propeller domain (PF3D7_1343700 from codon 445 to 680) was determined by dideoxy-sequencing according to Ariey et al. 3 ; pfmdr1 and pfpm2 copy number variations were determined by quantitative PCR according to Witkowski et al. 4 with modification of the hybridization temperature to 63°C. The presence of pfcrt (PF3D7_0709000) polymorphisms (codons 93, 97, 145, 343 and 353) was determined by dideoxy sequencing using primers described in Mairet et al. 18 or WGS data. These isolates were adapted to in vitro continuous culture at 2% haematocrit (O+ human blood, Centre de Transfusion Sanguine, Phnom Penh, Cambodia) in RPMI 1640 supplemented with 0·5% AlbuMAX II, 2·5% human plasma (mixed serogroups), 5% CO2 and 5% O2, at 37°C. 18 Phenotypic assays Piperaquine tetraphosphate (provided by the Worldwide Antimalarial Resistance Network (WWARN) and prepared in 0·5% lactic acid) was used for Piperaquine Survival Assay (PSA) as previously described 4 . MQ (provided by the WWARN and dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich, Singapore)) was used for in vitro MQ susceptibility of P. falciparum isolates using the [ 3 H]-hypoxanthine uptake inhibition assay, as previously described 18 . Susceptibility to MQ was also measured using a survival test similar to the PSA, in which PPQ doses were replaced by MQ doses at defined concentrations. Half-maximal inhibitory concentrations (IC 50s ) and half-maximal survival rates (SR 50s ; defined as the drug concentration inhibiting 50% of parasite survival) were determined using the ICEstimator software (http://www.antimalarial-icestimator.net/) or the non-linear regression analysis tool in GraphPad Prism 7.0. In vitro co-susceptibility to PPQ and MQ was assessed in 0-3 hours ring-stage parasites, which were exposed to a combination of seven increasing concentrations of PPQ and MQ (ranging from 12·5 to 800 nM for each drug) for 48 hours. Following drug removal, parasite cultures were maintained for an additional 24 hours in drug-free medium. After a total of 72 hours, Giemsa-stained blood smears were prepared, and P. falciparum survival was quantified as the ratio of viable parasites in the drug-exposed condition relative to the drug-free control. Paired MQ-PPQ in vitro selection pressure A P. falciparum strain (9097), collected in western Cambodia (Pursat) in 2019, was selected for continuous in vitro exposure to MQ and PPQ. This strain was isolated from a patient who experienced late treatment failure on day 34 after receiving the recommended ACT (artesunate-mefloquine), as showed in the supplementary figure 1. Genomic characterization of the recrudescent parasite showed a duplication of the pfmdr1 gene, associated with increased mefloquine IC 50 . Baseline genotypic analysis of strain 9097 identified the pfk13 Y493H mutation associated with ART-R, a single-copy of genes pfmdr1 and pfpm2 , and the absence of mutations in pfcrt . Phenotypic characterization was consistent with these genotypes, showing a ring-stage survival assay (RSA) slightly above 1% and no baseline in vitro resistance to MQ, PPQ, or amodiaquine. The culture conditions and drug concentrations used for selection are summarized in the supplementary figure 2. Briefly, strain 9097 was first cultured in the presence of 40 nM MQ + 40 nM PPQ until no viable parasites were microscopically detectable ( Pressure 1 ). At this stage, the cultures were returned to drug-free medium until parasitemia reached 2% ( Pressure 1 recovery ). A second drug exposure of 40 nM MQ + 40 nM PPQ had no observable effect, so the drug concentration was increased to 60 nM for each compound ( Pressure 2 ). Once no viable parasites were microscopically detected, the cultures were returned to drug-free medium until full recovery ( Pressure 2 recovery ). This was followed by a third round of drug pressure at 60 nM MQ + 60 nM PPQ ( Pressure 3 ), followed by a recovery period ( Pressure 3 recovery ). Finally, a fourth and final pressure of 80 nM MQ + 80 nM PPQ ( Pressure 4 ) was performed. Between each round of drug pressure, parasites were cryopreserved, genotyped ( pfmdr1 and pfpm2 copy number variation) and phenotyped (mefloquine IC 50 , PSA, and survival rates under MQ and PPQ co-exposure). Whole-genome sequencing Genomic DNA extracted from both the parental and Pressure 4 P. falciparum strains were used for library preparation. The libraries were prepared following the DNA Prep protocol from Illumina, starting from 250 ng of high-quality genomic DNA followed by sequencing of libraries using paired end mode (2X149) on a Nextseq 500 (Illumina). Raw reads were aligned to the P. falciparum 3D7 reference genome (PlasmoDB release 39) using the BWA-MEM algorithm (Burrows-Wheeler Aligner; default parameters). Alignment files were processed with SAMtools (version 1.4) and genome-wide coverage statistics were assessed using Qualimap (version 2.2.1). Duplicate reads were removed with Picard MarDuplicates (version 2.26.10). Single nucleotide polymorphisms (SNPs) and indels were identified using a custom analysis pipeline. A pileup file, containing information on matches, mismatches, insertions, deletions and mapping quality, was generated using the SAMtools mpileup function (version 1.13). This file was used as input to custom Python scripts for genomic variant detection. Comparative analysis between the parental and Pressure 4 strains was performed using custom R scripts (version 4.1), allowing identification of mutations acquired under drug pressure. To assess copy number variations (CNVs), we used PlasmoCNVScan, a read-depth-based strategy specifically optimized for Plasmodium genomes. Piperaquine incorporation assay Tritium-labeled PPQ (PPQ-[ 3 H(G)]) was purchased from American Radiolabeled Chemicals, Inc. (USA) and prepared in pure ethanol according to the manufacturer’s instructions. Synchronized trophozoites (21-24 hours post-invasion) were obtained by centrifugation on a 75% Percoll gradient, followed by sorbitol treatment (5% D-sorbitol) to remove remaining ring-stage parasites. Cultures were adjusted to a haematocrit of approximately 4%, with a minimum parasitemia of 2·5%. Trophozoites were pre-incubated for 10 min with MQ (25 to 750 nM, depending on the experimental conditions). Then, PPQ-[ 3 H(G)] was added to a final concentration of 200 nM, and the parasites were incubated for 5 hours at 37°C under controlled atmospheric conditions (5% CO 2 , 5% O 2 ). PPQ-[ 3 H(G)] incorporation was quantified by calculating the ratio of intracellular to extracellular radioactivity. After incubation, infected erythrocytes were lysed using a solution containing 0·015% saponin and 0·1% Triton X-100. The intracellular fraction (PPQ* in ) and extracellular fraction (PPQ* out ) were measured using a β-scintillation counter (MicroBeta TriLux, Perkin-Elmer, Waltham, USA). The degree of PPQ incorporation was expressed as the PPQ* in /PPQ* out ratio. Statistical analyses Statistical analyses were performed with the GraphPad Prism 7.0 software. A p -value < 0·05 was considered statistically significant. The normality of each dataset was tested using the Shapiro-Wilk test. The Mann-Whitney or the one-way ANOVA statistical test with Tukey’s or Dunn’s multiple comparison tests were used to compare medians. The IC 50 values of the MQ-PPQ combination were determined for each P. falciparum strain using the non-linear regression analysis in GraphPad Prism 7.0 and the ICEstimator software (http://www.antimalarial-icestimator.net). Declarations Ethical clearance All P. falciparum isolates were collected during therapeutic efficacy studies conducted in compliance with the ethical guidelines of the Cambodian National Ethical Committee for Human Research (identifiers: NECHR #086, NECHR #087, NECHR #092 and NECHR #106) and WPRO Ethical Review Committee. Written informed consent was obtained from all study participants or their legal guardians. Data availability The data that support the findings of this study are available from the corresponding authors upon reasonable request. The source data underlying Figs. 1a-d, 2a-c, 3, 4a-b, 5a-b, 6, Supplementary Figs. 2, 3 and 4 are provided as a Source Data file. Next-generation sequence files are available from the European Nucleotide Archive under the accession numbers ERR14369052 and ERR14369053 (project: PRJEB85790). All the scripts developed for this study were deposited in the following GitHub repository: https://github.com/Rcoppee/MQ-PPQ_pressure. Acknowledgements We are grateful to the patients who accepted to participate in the therapeutic efficacy studies and to the staff of the Cambodian Health Centers for their contribution. This study was supported by the Global Fund RAI3E initiative grant through WHO. Author information Authors and Affiliations Malaria Unit, Institut Pasteur du Cambodge, Institut Pasteur, Phnom Penh, Cambodia Camille Roesch, Melissa Mairet Khedim, Nimol Khim, Nimol Kloeung, Sopheakvatey Ke, Sreynet Srun, Rotha Eam, Chanra Kean, Chanvong Kul, Jean Popovici Benoit Witkowski Laboratoire de parasitologie-mycologie, UR 7510 ESCAPE, Université de Rouen Normandie, Rouen, France Anna Cosson & Romain Coppée National Center for Parasitology, Entomology, and Malaria Control, Ministry of Health, Phnom Penh, 120801, Cambodia Rithea Leang Mekong Malaria Elimination Programme, WHO, Phnom Penh, Cambodia Pascal Ringwald INSERM U1344, MERIT IRD, Université Paris Cité, Paris, France Frédéric Ariey Service de Parasitologie-Mycologie, Hôpital Cochin, Paris, France Frédéric Ariey Centre National de Référence du Paludisme, Laboratoire de Parasitologie-Mycologie, Hôpital Bichat-Claude Bernard, Paris, France Romain Coppée GENOM’IC, INSERM U1016 Institut Cochin, Paris France Lucie Adoux Present affiliation : Genetic and Biology of Plasmodium Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar & PV-ESMEE Pasteur International Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar ; Pasteur Institute, Paris, France ; Pasteur Institute of Cambodia, Phnom Penh, Cambodia. Camille Roesch & Benoit Witkowski Contributions Conceptualization: C.R. and B.W. Methodology: C.R., A.C., J.P., R.C. and B.W. Validation: C.R., A.C., M.M.K., R.C. and B.W. Formal analysis: C.R., A.C., F.A., R.C. and B.W. Investigation: C.R., A.C., M.M.K., N.Kh., N.Kl., S.K., S.S., R.E., C.Ke. and C.Ku. Resources: L.A., R.L., P.R. and B.W. Original draft preparation: C.R. and B.W. Review and editing of the manuscript: C.R., A.C., M.M.K., J.P., P.R., F.A., R.C., and B.W. Visualization: C.R., A.C., R.C. and B.W. Project administration and supervision: B.W. Funding acquisition: B.W. Corresponding authors Correspondence to Benoit Witkowski. Ethics declarations Competing interests The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. None of the authors declared financial conflict of interest. P.R. is a staff member of the World Health Organization. The authors are solely responsible for the views expressed in this publication, which do not necessarily represent the decisions, policies, or views of the World Health Organization References World Health Organization. World Malaria Report 2022 . (2022). Ishengoma, D. S. et al. Urgent action is needed to confront artemisinin partial resistance in African malaria parasites. Nat Med 30 , 1807–1808 (2024). Ariey, F. et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505 , 50–55 (2014). Witkowski, B. et al. A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype-genotype association study. The Lancet Infectious Diseases 17 , 174–183 (2017). Amaratunga, C. et al. Dihydroartemisinin–piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study. The Lancet Infectious Diseases 16 , 357–365 (2016). Imwong, M. et al. The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong Subregion: a molecular epidemiology observational study. Lancet Infect Dis (2017) doi:10.1016/S1473-3099(17)30048-8. Leang, R. et al. Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment. Antimicrobial Agents and Chemotherapy 59 , 4719–4726 (2015). Na-Bangchang, K., Ruengweerayut, R., Mahamad, P., Ruengweerayut, K. & Chaijaroenkul, W. Declining in efficacy of a three-day combination regimen of mefloquine-artesunate in a multi-drug resistance area along the Thai-Myanmar border. Malar J 9 , 273 (2010). Phuc, B. Q. et al. Treatment Failure of Dihydroartemisinin/Piperaquine for Plasmodium falciparum Malaria, Vietnam. Emerg. Infect. Dis. 23 , 715–717 (2017). Saito, M. et al. A randomized controlled trial of dihydroartemisinin-piperaquine, artesunate-mefloquine and extended artemether-lumefantrine treatments for malaria in pregnancy on the Thailand-Myanmar border. BMC Med 19 , 132 (2021). Hamaluba, M. et al. Arterolane–piperaquine–mefloquine versus arterolane–piperaquine and artemether–lumefantrine in the treatment of uncomplicated Plasmodium falciparum malaria in Kenyan children: a single-centre, open-label, randomised, non-inferiority trial. The Lancet Infectious Diseases 21 , 1395–1406 (2021). Mahamar, A. et al. Artemether-lumefantrine-amodiaquine or artesunate-amodiaquine combined with single low-dose primaquine to reduce Plasmodium falciparum malaria transmission in Ouélessébougou, Mali: a five-arm, phase 2, single-blind, randomised controlled trial. Lancet Microbe 6 , 100966 (2025). Peto, T. J. et al. Triple therapy with artemether–lumefantrine plus amodiaquine versus artemether–lumefantrine alone for artemisinin-resistant, uncomplicated falciparum malaria: an open-label, randomised, multicentre trial. The Lancet Infectious Diseases 22 , 867–878 (2022). Van Der Pluijm, R. W. et al. Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated Plasmodium falciparum malaria: a multicentre, open-label, randomised clinical trial. The Lancet 395 , 1345–1360 (2020). Imwong, M. et al. Evolution of Multidrug Resistance in Plasmodium falciparum : a Longitudinal Study of Genetic Resistance Markers in the Greater Mekong Subregion. Antimicrob Agents Chemother 65 , e01121-21 (2021). Hanboonkunupakarn, B. et al. Sequential Open-Label Study of the Safety, Tolerability, and Pharmacokinetic Interactions between Dihydroartemisinin-Piperaquine and Mefloquine in Healthy Thai Adults. Antimicrob Agents Chemother 63 , e00060-19 (2019). Rossi, G., De Smet, M., Khim, N., Kindermans, J.-M. & Menard, D. Emergence of Plasmodium falciparum triple mutant in Cambodia. The Lancet Infectious Diseases 17 , 1233 (2017). Mairet-Khedim, M. et al. Prevalence and characterization of piperaquine, mefloquine and artemisinin derivatives triple-resistant Plasmodium falciparum in Cambodia. Journal of Antimicrobial Chemotherapy 78 , 411–417 (2023). Preechapornkul, P. et al. Plasmodium falciparum pfmdr1 Amplification, Mefloquine Resistance, and Parasite Fitness. Antimicrob Agents Chemother 53 , 1509–1515 (2009). Amato, R. et al. Origins of the current outbreak of multidrug-resistant malaria in southeast Asia: a retrospective genetic study. The Lancet Infectious Diseases 18 , 337–345 (2018). Additional Declarations There is NO Competing Interest. Supplementary Files SUPPLEMENTARYAPPENDIX.docx supplementary figure and tables Cite Share Download PDF Status: Published Journal Publication published 27 Nov, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6629488","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":458957538,"identity":"217f48fa-2b64-4013-819e-a2c161f682bb","order_by":0,"name":"Benoit 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Control","correspondingAuthor":false,"prefix":"","firstName":"Rithea","middleName":"","lastName":"Leang","suffix":""},{"id":458957552,"identity":"fd2791a0-b46e-4db3-9b22-03b303282f62","order_by":14,"name":"Pascal Ringwald","email":"","orcid":"","institution":"World Health Organization","correspondingAuthor":false,"prefix":"","firstName":"Pascal","middleName":"","lastName":"Ringwald","suffix":""},{"id":458957553,"identity":"e00d3f62-41aa-4fa7-a3a4-7b0f2aede356","order_by":15,"name":"Frederic Ariey","email":"","orcid":"","institution":"Institut Cochin, INSERM U:1016, Parasitology-Mycology Unit, Cochin Hospital Paris Descartes University","correspondingAuthor":false,"prefix":"","firstName":"Frederic","middleName":"","lastName":"Ariey","suffix":""},{"id":458957554,"identity":"a9843a1d-fb0c-476e-8be0-c5ee25cb2de5","order_by":16,"name":"Romain Coppée","email":"","orcid":"https://orcid.org/0000-0002-3024-5928","institution":"Université de Rouen Normandie","correspondingAuthor":false,"prefix":"","firstName":"Romain","middleName":"","lastName":"Coppée","suffix":""}],"badges":[],"createdAt":"2025-05-09 14:20:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6629488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6629488/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-65629-8","type":"published","date":"2025-11-27T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83350661,"identity":"fc474407-9572-4b51-99a1-52feb8f95b38","added_by":"auto","created_at":"2025-05-23 13:59:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56053,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esusceptibility of sensitive (Sensitive, n = 5), piperaquine-resistant (KEL1/PLA1, n = 9) and mefloquine-resistant (MQ-R, n = 17) parasites to piperaquine (A), mefloquine (B) and paired MQ-PPQ (C).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Susceptibility of clinical isolates to piperaquine as measured by PSA. KEL1/PLA1 isolates have a significantly higher survival rate than Sensitive and MQ-R parasites. \u003cstrong\u003eB.\u003c/strong\u003e Susceptibility of clinical isolates to mefloquine measured by 3H-hypoxanthine incorporation. MQ-R isolates have significantly higher IC\u003csub\u003e50s\u003c/sub\u003e than Sensitive and KEL1/PLA1 parasites. \u003cstrong\u003eC.\u003c/strong\u003e Susceptibility of clinical isolates to piperaquine and mefloquine co-exposure. IC\u003csub\u003e50s\u003c/sub\u003e were calculated after measuring the survival rate of parasites at increasing concentrations of mefloquine + piperaquine. MQ-R isolates have significantly higher IC\u003csub\u003e50s\u003c/sub\u003e then Sensitive and KEL1/PLA1 parasites. Mean and standard deviation are shown on the graphs. One-way ANOVA was used as statistical test followed by Dunn’s multiple comparison test. \u003cstrong\u003eD.\u003c/strong\u003e Correlation between MQ and MQ-PPQ IC\u003csub\u003e50\u003c/sub\u003e. The IC\u003csub\u003e50s\u003c/sub\u003e of MQ and the combination MQ-PPQ exposure are highly correlated (r = 0.7146, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 Pearson correlation test).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/34b9b9f012da26550af9c9aa.png"},{"id":83350663,"identity":"78b69089-2454-463c-9ea7-81cea1a36168","added_by":"auto","created_at":"2025-05-23 13:59:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolution of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e susceptibility of the parent strain and the selected strain after continuous exposure to MQ+PPQ.\u003c/strong\u003e Each dot represents the mean of four independent experiments. Error bars represent the standard deviation. All data were generated using a derived version of PSA, where 0-3hrs rings were exposed to increasing concentrations of drug (0-800nM). Drug was washed after 48hrs and experiment was stopped after 72hrs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Mefloquine susceptibility evolution. The susceptibility of the selected strain to MQ has decreased (* \u003cem\u003ep\u003c/em\u003e = 0.0286, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test). \u003cstrong\u003eB.\u003c/strong\u003e Piperaquine susceptibility evolution. The susceptibility to PPQ of the selected strain has not changed (\u003cem\u003ep\u003c/em\u003e = 0.3152, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test). \u003cstrong\u003eC.\u003c/strong\u003e Mefloquine-piperaquine co-exposure susceptibility evolution. The susceptibility to MQ-PPQ co-exposure of the selected strain has decreased (* \u003cem\u003ep\u003c/em\u003e = 0.0286, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/722f4d36d664d6d2f7a21ab3.png"},{"id":83350664,"identity":"dadeca61-a77d-41f5-a569-53618a4c1ac3","added_by":"auto","created_at":"2025-05-23 13:59:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":88444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene copy number variations between parental and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePressure 4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains.\u003c/strong\u003e Each dot represents a gene. Green and red dots represent amplified (ratio ³ 1.5) and deleted (ratio £ 0.5) genes in \u003cem\u003ePressure 4\u003c/em\u003e strain, respectively. Grey dots show genes with relatively stable copy numbers. \u003cem\u003epfmdr1\u003c/em\u003e, \u003cem\u003epfpm2\u003c/em\u003eand \u003cem\u003epfpm3\u003c/em\u003e, previously associated with drug resistance, are labeled. The whole list of amplified and deleted genes in \u003cem\u003ePressure 4\u003c/em\u003e strain are listed in the Supplementary Table 2.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/d36c2d7120dc47326e508362.png"},{"id":83350665,"identity":"ebe7fd47-aea5-479e-a504-263a3ad2a858","added_by":"auto","created_at":"2025-05-23 13:59:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRadioactive PPQ incorporation assay.\u003c/strong\u003e Mean and standard deviation are represented on the graphs and each dot represents one independent replicate. The incorporation of piperaquine [3H(G)] is measured on synchronized trophozoites (21-24hrs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Variation of radioactive PPQ incorporation with and without 200 nM mefloquine pre-incubation before addition of 3H-PPQ. Comparison between the parental and the selected strain (* \u003cem\u003ep\u003c/em\u003e = 0.0312, Wilcoxon test). \u003cstrong\u003eB.\u003c/strong\u003e Variation of piperaquine incorporation among clinical isolates with different phenotypes. In both cases, pre-incubation with 200nM MQ before addition of 3H-PPQ significantly decreased the incorporation of PPQ by parasites (* \u003cem\u003ep\u003c/em\u003e = 0.0156 and *** \u003cem\u003ep\u003c/em\u003e = 0.0002, Wilcoxon matched-pairs test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/23fe4f436d7334443cdc2eca.png"},{"id":83350920,"identity":"65d80b64-9217-4e08-9664-f284806fc44e","added_by":"auto","created_at":"2025-05-23 14:07:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":58314,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncorporation of radioactive PPQ among clinical isolates.\u003c/strong\u003e The incorporation of piperaquine [3H(G)] is measured on synchronized trophozoites (21-24hrs).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Variation of radioactive PPQ incorporation with increasing mefloquine concentrations pre-incubation (25 nM to 750 nM) before addition of 3H-PPQ. Mean and standard deviation are represented on the graphs and each dot represents one independent replicate. The solid line represents the non-linear regression of the mefloquine concentration vs piperaquine uptake, 95% confidence interval is also shown (WT, n = 2; KEL1/PLA1, n = 2 and MQ-R, n = 5). \u003cstrong\u003eB.\u003c/strong\u003e Difference in radioactive PPQ incorporation between \u003cem\u003epfmdr1\u003c/em\u003emono- and multicopy isolates. Isolates with multiple copies of the \u003cem\u003epfmdr1\u003c/em\u003egene incorporate significantly more 3H-PPQ than isolates with a single-copy even when 3H-PPQ is added after 200 nM of mefloquine preincubation (* \u003cem\u003ep\u003c/em\u003e = 0.0160, Mann-Whitney \u003cem\u003eU\u003c/em\u003etest).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/76fd15f40cf01af40f9778ce.png"},{"id":83351991,"identity":"03616414-b2e3-49f5-8ea4-051be388ccb5","added_by":"auto","created_at":"2025-05-23 14:15:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":47424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the survival rate of the parental line, the selected strain and clinical isolates with mefloquine resistance to piperaquine with or without mefloquine pre-incubation. \u003c/strong\u003eMean and standard deviation are shown on the graphs and each dot represents one independent replicate.\u003c/p\u003e\n\u003cp\u003eThe median survival of parasites to 200 nM PPQ is unchanged in presence of MQ pre-incubation before exposure to PPQ in the parental strain (\u003cem\u003ep\u003c/em\u003e = 0.4688, Wilcoxon test). The pre-incubation with MQ significantly increased the survival of the selected strain (* \u003cem\u003ep\u003c/em\u003e = 0.0312, Wilcoxon test) and of clinical MQ-R parasites (* \u003cem\u003ep\u003c/em\u003e = 0.0142, Paired \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/3b74879c9b750ba7170faa56.png"},{"id":83350673,"identity":"d70b3ea4-fe68-42c5-b2cb-bd25143b4444","added_by":"auto","created_at":"2025-05-23 13:59:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":265184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypothesis supporting the mechanism of resistance of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. falciparum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e to paired MQ-PPQ by PfMDR1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePPQ treatment. \u003c/strong\u003ePPQ enters the cell through the PfMDR1 channel, which may explain why parasites with \u003cem\u003epfmdr1 \u003c/em\u003eamplification exhibit increased sensitivity to PPQ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePaired MQ-PPQ treatment.\u003c/strong\u003e Parasites use PfMDR1 to export MQ out of the cell. In the presence of both MQ and PPQ, PfMDR1 becomes saturated with MQ, thereby preventing PPQ from entering the cell. As a result, PPQ activity is significantly reduced in the combination. This mechanism mirrors the action of MQ alone, revealing that MQ is the primary active molecule in this combination when parasites carry \u003cem\u003epfmdr1\u003c/em\u003eamplification.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/e9b7923dfa58a2fc8fe1240e.png"},{"id":96974296,"identity":"810edfc0-776c-4008-96a4-6740906ab526","added_by":"auto","created_at":"2025-11-28 08:12:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1835301,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/141ecc4f-cd8c-452b-a833-4b6a8f70ca2c.pdf"},{"id":83350666,"identity":"a15caba0-970d-4b53-bc04-9e74fdc90b60","added_by":"auto","created_at":"2025-05-23 13:59:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":701217,"visible":true,"origin":"","legend":"supplementary figure and tables","description":"","filename":"SUPPLEMENTARYAPPENDIX.docx","url":"https://assets-eu.researchsquare.com/files/rs-6629488/v1/ff7db5817da84f63723ae9e4.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Tolerance of Plasmodium falciparum mefloquine-resistant clinical isolates to mefloquine-piperaquine: implications for triple artemisinin-based combination therapy strategies.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOver the past two decades, malaria control efforts have led to substantial reductions in global incidence and mortality\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Central to this progress has been the widespread deployment of artemisinin-based combination therapies (ACTs), which remain the cornerstone of first-line treatment for uncomplicated \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria. However, the emergence of artemisinin (ART) resistance (ART-R) in eastern and central Africa now poses a serious threat to these gains\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. While partial resistance to ART alone, as previously observed in both Africa and Southeast Asia, does not necessarily result in increased ACT treatment failure rates, resistance to partner drugs remains the key driver of clinical failure\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOf particular concern is the potential for ART-R to act as a precursor to multidrug-resistant parasite lineages. This scenario is supported by previous observations in the Greater Mekong Subregion, where parasites harboring resistance to at least one partner drug facilitated the subsequent emergence of ACT-resistant strains\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The increasing detection of ART-R in Africa raises the possibility of a similar trajectory, potentially leading to a widespread loss of ACT efficacy. To mitigate this risk, an alternative approach is to develop optimized ACT formulations with a lower propensity for resistance selection, even in the presence of pre-existing drug resistance.\u003c/p\u003e \u003cp\u003eTriple ACTs (TACTs) have been proposed as a potential solution to this challenge. By combining an ART derivative with two partner drugs, TACTs are designed to reduce the likelihood of resistance selection and maintain therapeutic efficacy in regions where conventional ACTs are failing. Two main formulations have been evaluated in clinical trials: artesunate-mefloquine-piperaquine and artesunate-amodiaquine-lumefantrine\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Among these, mefloquine-piperaquine (MQ-PPQ) has drawn particular interest due to a possible antagonism in the acquisition of resistance to its two partner drugs. Data from Southeast Asia suggest that resistance markers for MQ (\u003cem\u003epfmdr1\u003c/em\u003e amplification) and PPQ (\u003cem\u003epfplasmepsin2/3\u003c/em\u003e (\u003cem\u003epfpm2/3\u003c/em\u003e) amplification) display inverse prevalence trends, depending on whether PPQ- or MQ-based ACTs are implemented\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, MQ-PPQ TACT has demonstrated high efficacy in patients infected with DHA-PPQ-resistant parasites, with cure rates exceeding 95% compared to 48% for DHA-PPQ alone\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This finding highlights mefloquine\u0026rsquo;s ability to compensate for PPQ resistance in the context of pre-existing PPQ resistance.\u003c/p\u003e \u003cp\u003eHowever, the potential of MQ-PPQ TACT in the context of pre-existing MQ resistance remains unexplored. Given its possible large-scale implementation, addressing this gap is crucial to ensure its long-term viability. In this study, we aimed to evaluate the efficacy of MQ-PPQ TACT against \u003cem\u003eP. falciparum\u003c/em\u003e isolates exhibiting MQ resistance, providing critical insights into its operational feasibility and potential limitations.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;susceptibility of field isolates to PPQ, MQ and combined MQ-PPQ exposure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e susceptibility of a panel of Cambodian \u003cem\u003eP. falciparum\u003c/em\u003e isolates, categorized as sensitive (Sensitive, n = 5), PPQ-resistant (KEL1/PLA1, n = 9) and MQ-resistant (MQ-R, n = 17), was first evaluated for PPQ, MQ, and the combination MQ-PPQ. Based on the PSA, KEL1/PLA1 isolates exhibited higher survival rates (55\u0026middot;58% \u0026plusmn; 15\u0026middot;67) compared to sensitive (0\u0026middot;00% \u0026plusmn; 0\u0026middot;00, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0\u0026middot;0003, Kruskal-Wallis test followed by Dunn\u0026rsquo;s multiple comparison test) and MQ-R parasites (0\u0026middot;16% \u0026plusmn; 0\u0026middot;37, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0\u0026middot;0001, Kruskal-Wallis test followed by Dunn\u0026rsquo;s multiple comparison test) (\u003cstrong\u003eFigure 1A\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs expected, the mean IC\u003csub\u003e50\u003c/sub\u003e values determined by [\u003csup\u003e3\u003c/sup\u003eH]-hypoxanthine incorporation showed significant differences across the three groups. MQ-R isolates exhibited significantly higher IC\u003csub\u003e50\u003c/sub\u003e values (96\u0026middot;35 nM \u0026plusmn; 22\u0026middot;84) than Sensitive and KEL1/PLA1 parasites (42\u0026middot;99 nM \u0026plusmn; 14\u0026middot;60, \u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0148 and 30\u0026middot;31 nM \u0026plusmn; 15\u0026middot;13, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0\u0026middot;0001 respectively, Kruskal-Wallis test followed by Dunn\u0026rsquo;s multiple comparison test) (\u003cstrong\u003eFigure 1B\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFinally, the susceptibility of the three groups to MQ-PPQ co-exposure was assessed using a survival assay over a range of drug concentrations, allowing for IC\u003csub\u003e50\u003c/sub\u003e determination. MQ-R isolates showed significantly higher IC\u003csub\u003e50\u003c/sub\u003e values (51\u0026middot;26 nM \u0026plusmn; 16\u0026middot;29) compared to Sensitive and KEL1/PLA1 isolates (23\u0026middot;20 nM \u0026plusmn; 2\u0026middot;75, \u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0021 and 24\u0026middot;29 nM \u0026plusmn; 8\u0026middot;02, \u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0016, respectively, Kruskal-Wallis test followed by Dunn\u0026rsquo;s multiple comparison test) (\u003cstrong\u003eFigure 1C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe IC\u003csub\u003e50s\u003c/sub\u003e of MQ and the combination MQ-PPQ exposure were highly correlated (r = 0.7146, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, Pearson correlation test) (\u003cstrong\u003eFigure 1D\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePaired MQ-PPQ\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;drug pressure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential for stepwise adaptation to MQ-PPQ combination therapy, the\u003cem\u003e\u0026nbsp;P. falciparum\u0026nbsp;\u003c/em\u003e9097 strain was subjected to four successive rounds of drug pressure, as summarized in the supplementary figure 2. Then, the \u003cem\u003ein vitro\u003c/em\u003e susceptibilities of the parental strain and the final selected strain (\u003cem\u003ePressure 4\u003c/em\u003e) were compared for each drug individually and for the MQ-PPQ combination.\u003c/p\u003e\n\u003cp\u003eFollowing drug pressure, the selected strain exhibited a\u0026nbsp;significantly increased IC\u003csub\u003e50\u003c/sub\u003e to MQ (146\u0026middot;60 nM \u0026plusmn; 33\u0026middot;76\u0026nbsp;vs. 63\u0026middot;69 nM\u0026nbsp;\u0026plusmn; 19\u0026middot;37,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e = 0.0286, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test) (\u003cstrong\u003eFigure 2A\u003c/strong\u003e), suggesting an adaptive response to MQ exposure. However, no significant change in PPQ susceptibility was observed (29\u0026middot;50 nM\u0026nbsp;\u0026plusmn; 10\u0026middot;19\u0026nbsp;vs. 40\u0026middot;59 nM\u0026nbsp;\u0026plusmn; 16\u0026middot;29,\u0026nbsp;\u003cem\u003ep\u003c/em\u003e = 0\u0026middot;3152,\u0026nbsp;Mann-Whitney \u003cem\u003eU\u003c/em\u003e test) (\u003cstrong\u003eFigure 2B\u003c/strong\u003e), indicating that PPQ susceptibility remained largely unaffected by MQ-PPQ selective pressure.\u003c/p\u003e\n\u003cp\u003eInterestingly, the IC\u003csub\u003e50\u003c/sub\u003e for MQ-PPQ co-exposure increased significantly from 21\u0026middot;70 nM \u0026plusmn; 4\u0026middot;41 to 45\u0026middot;11 nM\u0026nbsp;\u0026plusmn;\u0026nbsp;15\u0026middot;82 (\u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0286,\u0026nbsp;Mann-Whitney \u003cem\u003eU\u003c/em\u003e test) (\u003cstrong\u003eFigure 2C\u003c/strong\u003e), suggesting that adaptation to MQ-PPQ treatment primarily involved increased MQ tolerance rather than PPQ resistance.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e susceptibility of both parental and selected strains to MQ and PPQ was further assessed using conventional methods, including the [\u003csup\u003e3\u003c/sup\u003eH]-hypoxanthine uptake inhibition assay and PSA. The MQ IC\u003csub\u003e50\u003c/sub\u003e increased significantly from 48\u0026middot;37 nM \u0026plusmn; 8.68 to 117\u0026middot;00 nM \u0026plusmn; 18.87 (\u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0040, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test), confirming the emergence of a MQ resistance phenotype. A modest but significant increase in PSA survival was observed (1\u0026middot;42% \u0026plusmn; 1.34 vs 3\u0026middot;35% \u0026plusmn; 0.79, \u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0068, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test), although the survival rate remained below the established 10% resistance threshold (\u003cstrong\u003eSupplementary Figure 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eTo explore potential genetic mechanisms underlying these phenotypic changes, we assessed copy number variation in key resistance-associated genes. The \u003cem\u003ePressure 4\u003c/em\u003e strain acquired an additional copy of the \u003cem\u003epfmdr1\u003c/em\u003e gene compared to the parental strain (1\u0026middot;96 \u0026plusmn; 0\u0026middot;14, n = 9 technical replicates), a known marker associated with MQ resistance. However, no change was detected in \u003cem\u003epfpm2\u003c/em\u003e copy number (0\u0026middot;93 \u0026plusmn; 0\u0026middot;08, n = 9 technical replicates), suggesting that the adaptation to MQ-PPQ was primarily mediated by \u003cem\u003epfmdr1\u003c/em\u003e amplification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparative genomic analysis of parental and selected strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate our previous findings, we performed whole-genome sequencing of both the parental and \u003cem\u003ePressure 4\u003c/em\u003e \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003estrains. Comparative genomic analysis revealed no newly acquired SNPs or indels following drug selection, suggesting the absence of specific point mutations that could directly explain the increased tolerance to the MQ-PPQ combination (\u003cstrong\u003eSupplementary Fig. 4\u003c/strong\u003e). Notably, no mutations were detected in \u003cem\u003epfcrt\u003c/em\u003e, a well-characterized marker of PPQ resistance.\u003c/p\u003e\n\u003cp\u003eIn contrast, CNV analysis identified an amplification of a region on chromosome 5, encompassing \u003cem\u003epfmdr1\u003c/em\u003e, in the \u003cem\u003ePressure 4\u0026nbsp;\u003c/em\u003estrain (\u003cstrong\u003eFigure 3\u003c/strong\u003e and \u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e). No concomitant amplification of \u003cem\u003epfpm2\u003c/em\u003e or \u003cem\u003epfpm3\u003c/em\u003e was observed. Additionally, a deleted genomic region was observed on chromosome 9 following drug selection. This deletion is unlikely to be involved in drug resistance acquisition, as similar deletions have been reported in long-term \u003cem\u003ein vitro\u003c/em\u003e cultures of \u003cem\u003eP. falciparum\u003c/em\u003e. Altogether, the comparative genomic analysis suggests that \u003cem\u003epfmdr1\u003c/em\u003e amplification alone may play a pivotal role in modulating parasite susceptibility to MQ-PPQ by potentially altering drug import or efflux mechanisms within the parasite.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of the central role of \u003cem\u003epfmdr1\u003c/em\u003e in MQ-PPQ tolerance using radioactive assays\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential involvement of\u0026nbsp;\u003cem\u003epfmdr1\u003c/em\u003e in tolerance to the MQ-PPQ combination, we assessed the impact of MQ pre-incubation on the intracellular incorporation of radiolabeled PPQ\u0026nbsp;[\u003csup\u003e3\u003c/sup\u003eH(G)]. The amount of incorporated radiolabeled\u0026nbsp;PPQ was significantly reduced when parasites were pre-exposed to MQ before the addition of radiolabeled PPQ\u0026nbsp;(16\u0026middot;98 \u0026plusmn; 2\u0026middot;63 vs. 4\u0026middot;64 \u0026plusmn; 2\u0026middot;03, \u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0312, Wilcoxon test) (\u003cstrong\u003eFigure 4A\u003c/strong\u003e).\u0026nbsp;A similar effect was observed in the parental strain, where MQ pre-incubation resulted in a significant decrease in PPQ incorporation (14\u0026middot;54 \u0026plusmn; 3.99 vs. 1\u0026middot;82 \u0026plusmn; 1\u0026middot;83, \u003cem\u003ep\u003c/em\u003e = 0.0312, Wilcoxon test) (\u003cstrong\u003eFigure 4A\u003c/strong\u003e). These findings suggest that PPQ uptake may be influenced by \u003cem\u003epfmdr1\u003c/em\u003e-mediated drug transport. Specifically, as MQ is actively exported out of the parasite via \u003cem\u003epfmdr1\u003c/em\u003e, PPQ import into the parasite appears to be reduced.\u003c/p\u003e\n\u003cp\u003eTo further explore this hypothesis, we measured the variation in PPQ uptake among clinical isolates with different resistance phenotypes (Sensitive, MQ-R and KEL1/PLA1). In all three groups, MQ pre-incubation significantly reduced PPQ uptake (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). The mean radioactive PPQ incorporation decreased from 4\u0026middot;72 \u0026plusmn; 1\u0026middot;16 to 1\u0026middot;12 \u0026plusmn; 1\u0026middot;80 (\u003cem\u003ep\u003c/em\u003e = 0.0625, Wilcoxon matched-pairs\u0026nbsp;test)\u0026nbsp;in Sensitive isolates, from 4\u0026middot;65\u0026nbsp;\u0026plusmn; 1\u0026middot;52 to 0\u0026middot;28 \u0026plusmn; 0\u0026middot;25 (\u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0156,\u0026nbsp;Wilcoxon matched-pairs\u0026nbsp;test) in KEL1/PLA1 isolates, and from 6\u0026middot;84\u0026nbsp;\u0026plusmn; 2\u0026middot;42 to 1\u0026middot;75 \u0026plusmn; 0\u0026middot;90 (\u003cem\u003ep\u003c/em\u003e = 0.0002,\u0026nbsp;Wilcoxon matched-pairs\u0026nbsp;test) in MQ-R isolates.\u003c/p\u003e\n\u003cp\u003eTo quantify the impact of increasing MQ concentrations on PPQ incorporation, we performed a dose-response experiment using pre-incubation with MQ at concentrations ranging from 25 nM to 750 nm before the addition of [\u003csup\u003e3\u003c/sup\u003eH]-PPQ (\u003cstrong\u003eFigure 5A\u003c/strong\u003e). The concentration of MQ required to inhibit 50% of PPQ incorporation was lowest for KEL1/PLA1 (15\u0026middot;57 nM)\u0026nbsp;and Sensitive isolates (33\u0026middot;52 nM), whereas MQ-R parasites required a significantly higher MQ concentration (115\u0026middot;33 nM) to achieve the same level of inhibition (\u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0312 and \u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0008, respectively, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test).\u003c/p\u003e\n\u003cp\u003eFinally, we examined the relationship between \u003cem\u003epfmdr1\u003c/em\u003e copy number and PPQ uptake. Isolates with multiple \u003cem\u003epfmdr1\u003c/em\u003e copies exhibited significantly higher incorporation of [\u003csup\u003e3\u003c/sup\u003eH]-PPQ compared to single-copy isolates, even when pre-incubated with 200 nM MQ (\u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0160, Mann-Whitney \u003cem\u003eU\u003c/em\u003e test) (\u003cstrong\u003eFigure 5B\u003c/strong\u003e). These findings reinforce the hypothesis that \u003cem\u003epfmdr1\u003c/em\u003e plays a central role in modulating the intracellular balance of MQ and PPQ.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMQ pre-incubation reduces PPQ susceptibility in MQ-R isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the observed interaction between MQ and PPQ uptake, we next assessed whether MQ pre-incubation could modulate PPQ susceptibility in MQ-R parasites. Cambodian clinical isolates classified as MQ-R (n = 12), as well as the \u003cem\u003eParental\u003c/em\u003e and \u003cem\u003ePressure 4\u003c/em\u003e laboratory strains, were subjected to the PSA under standard conditions and with prior exposure to 200 nM MQ pre-incubation. PSA survival rates varied according to genotype (\u003cstrong\u003eFigure 6\u003c/strong\u003e). The addition of MQ had no significant impact on PSA levels in the \u003cem\u003eParental\u003c/em\u003e strain. However, in the \u003cem\u003ePressure 4\u003c/em\u003e strain, MQ pre-incubation resulted in a significant increase in survival rate,\u0026nbsp;from 3\u0026middot;35\u0026nbsp;\u0026plusmn; 0\u0026middot;79 to 6\u0026middot;38 \u0026plusmn; 2\u0026middot;06 (\u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0312, Wilcoxon test). A similar effect was observed for MQ-R parasites, where pre-incubation with MQ significantly increased PSA survival\u0026nbsp;from 0\u0026middot;29\u0026nbsp;\u0026plusmn; 0\u0026middot;37 to 4\u0026middot;36 \u0026plusmn; 4\u0026middot;79 (\u003cem\u003ep\u003c/em\u003e = 0\u0026middot;0142, Paired \u003cem\u003et\u003c/em\u003e-test). These findings provide further evidence that MQ exposure influences PPQ susceptibility, particularly in MQ-resistant isolates.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe primary objective of this study was to assess the activity of paired MQ-PPQ against the different resistance profiles frequently observed in Southeast Asia. \u003cem\u003eIn vitro\u003c/em\u003e susceptibility testing confirmed that sensitive and KEL1/PLA1 parasites were highly susceptible to MQ, whereas WT and MQ-R parasites remained susceptible to PPQ. Conversely, KEL1/PLA1 and MQ-R strains displayed increased tolerance to PPQ and MQ.\u003c/p\u003e \u003cp\u003eEvaluation of the combined MQ-PPQ activity against sensitive and KEL1-PLA1 parasites demonstrated high susceptibility, consistent with clinical studies reporting excellent therapeutic efficacy of this combination in infections with similar parasite genotypes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In sharp contrast, MQ-PPQ activity against MQ-R parasites revealed significantly higher tolerance compared to sensitive and KEL1/PLA1 strains. This result contrasts with the proposed rationale for TACTs, which assumes that the presence of two partner drugs should mitigate resistance to one of them\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. A possible explanation for this unexpected finding could be the presence of parasites with co-existing PPQ and MQ resistance, as previously observed in Cambodia\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, our data ruled out this hypothesis, as all MQ-R strains retained full susceptibility to PPQ when tested individually.\u003c/p\u003e \u003cp\u003eA key observation from our study was that MQ-PPQ tolerance in MQ-R parasites was directly correlated with their intrinsic MQ resistance levels. This was confirmed by a strong and significant correlation between MQ and MQ-PPQ IC\u003csub\u003e50\u003c/sub\u003e values. Based on these findings, we hypothesized that \u003cem\u003epfmdr1\u003c/em\u003e gene amplification \u0026ndash; previously implicated in MQ resistance \u0026ndash; plays a central role in the acquisition of a tolerance phenotype to paired MQ-PPQ. To test this hypothesis, we evaluated the selection potential of MQ-PPQ in a parasite lineage that had previously exhibited intra-host acquisition of MQ resistance. Upon repeated MQ-PPQ exposure, parasites acquired \u003cem\u003epfmdr1\u003c/em\u003e amplification, which was associated with increased tolerance to both MQ and MQ-PPQ. In contrast, no molecular markers or phenotypic evidence of PPQ resistance were observed. Whole-genome analysis of the parental and derived strains confirmed amplification of a region on chromosome 5, encompassing \u003cem\u003epfmdr1\u003c/em\u003e, as the only genomic change. These results strongly support the role of \u003cem\u003epfmdr1\u003c/em\u003e amplification in MQ-PPQ tolerance and suggest that PPQ does not effectively reach its intracellular target in these parasites. The acquisition \u003cem\u003epfmdr1\u003c/em\u003e amplification upon MQ-PPQ pressure was relatively rapid, which is consistent with previous observations of \u003cem\u003ein vitro\u003c/em\u003e resistance selection through MQ alone pressure. Yet, the fitness cost and the persistence of the amplification detected in this study were not evaluated, but it can be suspected to be similar with what Preechapornkul \u003cem\u003eet al.\u003c/em\u003e observed \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis hypothesis was further validated through radioactive PPQ incorporation assays. Our results showed that PPQ uptake into infected erythrocytes was directly dependent on \u003cem\u003epfmdr1\u003c/em\u003e copy number, with \u003cem\u003epfmdr1\u003c/em\u003e-amplified parasites exhibiting significantly higher intracellular PPQ concentrations. This phenomenon explains the observed \u0026ldquo;over-sensibilization\u0026rdquo; of MQ-R parasites to PPQ, highlighting PfMDR1 as a key determinant of PPQ intracellular accumulation and activity. Furthermore, pre-incubation with increasing concentrations of MQ resulted in a dose-dependent reduction in PPQ uptake, suggesting that MQ, a known PfMDR1 substrate, competitively inhibits PPQ uptake. This competition likely reduces intracellular PPQ concentrations to subtherapeutic levels, thereby impairing its efficacy in MQ-R parasites.\u003c/p\u003e \u003cp\u003eFinally, the phenotypic discrepancies observed between MQ-R parasites and the other groups can be explained by the ability of \u003cem\u003epfmdr1\u003c/em\u003e-amplified parasites to efficiently expel MQ, thus maintaining resistance, as summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. This mechanism would explain the paradoxical loss of MQ-PPQ activity against MQ-R parasites, whereas in sensitive and KEL1/PLA1 strains, MQ remains at therapeutic concentrations, ensuring the continued efficacy of the combination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that the inherent strategy of MQ-PPQ TACT, which relies on the antagonistic resistance mechanisms of the two partner drugs, inadvertently leads to impaired drug action due to altered intracellular pharmacodynamic. This phenomenon facilitates the emergence of paradoxical resistance, driven by \u003cem\u003epfmdr1\u003c/em\u003e amplification. Although these findings are based on \u003cem\u003ein vitro\u003c/em\u003e observations, they underscore the need for active surveillance of \u003cem\u003epfmdr1\u003c/em\u003e-amplified parasites following MQ-PPQ TACT implementation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eClinical isolate selection and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical isolates were obtained from Therapeutic Efficacy Studies (TES) conducted in Cambodia between 2016 and 2019. Samples were selected based on their \u003cem\u003epfk13\u003c/em\u003e, \u003cem\u003epfmdr1\u003c/em\u003e, \u003cem\u003epfpm2\u003c/em\u003e and \u003cem\u003epfcrt\u003c/em\u003e genotypes according to the following classification: sensitive, characterized by \u003cem\u003epfk13\u003c/em\u003e wild-type, \u003cem\u003epfmdr1\u003c/em\u003e monocopy, \u003cem\u003epfpm2\u003c/em\u003e monocopy and without mutation on \u003cem\u003epfcrt\u003c/em\u003e codons known to modulate PPQ susceptibility (amino acid positions 88, 93, 97, 145, 218, 343 and 353); KEL1/PLA1, a genetically characterized group of \u003cem\u003eP. falciparum\u003c/em\u003e parasites that has emerged in the Greater Mekong Subregion, characterized by \u003cem\u003epfk13\u003c/em\u003e C580Y and \u003cem\u003epfpm2\u0026nbsp;\u003c/em\u003eamplification\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003csup\u003e20\u003c/sup\u003e with or without polymorphisms at the above \u003cem\u003epfcrt\u003c/em\u003e amino acid positions; and MQ-R, characterized by \u003cem\u003epfk13\u003c/em\u003e mutation, \u003cem\u003epfmdr1\u003c/em\u003e amplification and without polymorphisms at the above \u003cem\u003epfcrt\u003c/em\u003e amino acid positions. Characteristics and \u003cem\u003ein vitro\u003c/em\u003e results of the selected samples are summarized in the supplementary table 1. Genetic characterization was performed using the following methodology: polymorphism of the \u003cem\u003epfk13\u003c/em\u003e propeller domain (PF3D7_1343700 from codon 445 to 680) was determined by dideoxy-sequencing according to Ariey \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e3\u003c/sup\u003e; \u003cem\u003epfmdr1\u003c/em\u003e and \u003cem\u003epfpm2\u003c/em\u003e copy number variations were determined by quantitative PCR according to Witkowski \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e4\u003c/sup\u003e with modification of the hybridization temperature to 63\u0026deg;C. The presence of \u003cem\u003epfcrt\u003c/em\u003e (PF3D7_0709000) polymorphisms (codons 93, 97, 145, 343 and 353) was determined by dideoxy sequencing using primers described in Mairet \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e18\u003c/sup\u003e or WGS data. These isolates were adapted to \u003cem\u003ein vitro\u003c/em\u003e continuous culture at 2% haematocrit (O+ human blood, Centre de Transfusion Sanguine, Phnom Penh, Cambodia) in RPMI 1640 supplemented with 0\u0026middot;5% AlbuMAX II, 2\u0026middot;5% human plasma (mixed serogroups), 5% CO2 and 5% O2, at 37\u0026deg;C.\u003csup\u003e18\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotypic assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePiperaquine\u0026nbsp;tetraphosphate (provided by the Worldwide Antimalarial Resistance Network (WWARN) and prepared in 0\u0026middot;5% lactic acid) was used for\u0026nbsp;Piperaquine\u0026nbsp;Survival Assay (PSA) as previously described\u003csup\u003e4\u003c/sup\u003e. MQ (provided by the WWARN and dissolved in dimethyl sulfoxide (DMSO; Sigma Aldrich, Singapore)) was used for \u003cem\u003ein vitro\u003c/em\u003e MQ susceptibility of \u003cem\u003eP. falciparum\u003c/em\u003e isolates using the [\u003csup\u003e3\u003c/sup\u003eH]-hypoxanthine uptake inhibition assay, as previously described\u003csup\u003e18\u003c/sup\u003e.\u0026nbsp;Susceptibility to MQ was also measured using a survival test similar to the PSA, in which PPQ doses were replaced by MQ doses at defined concentrations.\u003c/p\u003e\n\u003cp\u003eHalf-maximal inhibitory concentrations (IC\u003csub\u003e50s\u003c/sub\u003e) and half-maximal survival rates (SR\u003csub\u003e50s\u003c/sub\u003e; defined as the drug concentration inhibiting 50% of parasite survival) were determined using the ICEstimator software (http://www.antimalarial-icestimator.net/) or the non-linear regression analysis tool in GraphPad Prism 7.0. \u003cem\u003eIn vitro\u003c/em\u003e co-susceptibility to PPQ and MQ was assessed in 0-3 hours ring-stage parasites, which were exposed to a combination of seven increasing concentrations of PPQ and MQ (ranging from 12\u0026middot;5 to 800 nM for each drug) for 48 hours. Following drug removal, parasite cultures were maintained for an additional 24 hours in drug-free medium. After a total of 72 hours, Giemsa-stained blood smears were prepared, and \u003cem\u003eP. falciparum\u003c/em\u003e survival was quantified as the ratio of viable parasites in the drug-exposed condition relative to the drug-free control.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePaired MQ-PPQ \u003cem\u003ein vitro\u003c/em\u003e selection pressure\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA \u003cem\u003eP. falciparum\u003c/em\u003e strain (9097), collected in western Cambodia (Pursat) in 2019, was selected for continuous \u003cem\u003ein vitro\u003c/em\u003e exposure to MQ and PPQ. This strain was isolated from a patient who experienced late treatment failure on day 34 after receiving the recommended ACT (artesunate-mefloquine), as showed in the supplementary figure 1. Genomic characterization of the recrudescent parasite showed a duplication of the \u003cem\u003epfmdr1\u003c/em\u003e gene, associated with increased mefloquine IC\u003csub\u003e50\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eBaseline genotypic analysis of strain 9097 identified the \u003cem\u003epfk13\u003c/em\u003e Y493H mutation associated with ART-R, a single-copy of genes \u003cem\u003epfmdr1\u003c/em\u003e and\u0026nbsp;\u003cem\u003epfpm2\u003c/em\u003e, and the absence of mutations in \u003cem\u003epfcrt\u003c/em\u003e. Phenotypic characterization was consistent with these genotypes, showing a ring-stage survival assay (RSA) slightly above 1% and no baseline \u003cem\u003ein vitro\u003c/em\u003e resistance to MQ, PPQ, or amodiaquine.\u003c/p\u003e\n\u003cp\u003eThe culture conditions and drug concentrations used for selection are summarized in the supplementary figure 2. Briefly, strain 9097 was first cultured in the presence of 40 nM MQ + 40 nM PPQ until no viable parasites were microscopically detectable (\u003cem\u003ePressure 1\u003c/em\u003e). At this stage, the cultures were returned to drug-free medium until parasitemia reached 2% (\u003cem\u003ePressure 1 recovery\u003c/em\u003e). A second drug exposure of 40 nM MQ + 40 nM PPQ had no observable effect, so the drug concentration was increased to 60 nM for each compound (\u003cem\u003ePressure 2\u003c/em\u003e). Once no viable parasites were microscopically detected, the cultures were returned to drug-free medium until full recovery (\u003cem\u003ePressure 2 recovery\u003c/em\u003e). This was followed by a third round of drug pressure at 60 nM MQ + 60 nM PPQ (\u003cem\u003ePressure 3\u003c/em\u003e), followed by a recovery period (\u003cem\u003ePressure 3 recovery\u003c/em\u003e). Finally, a fourth and final pressure of 80 nM MQ + 80 nM PPQ (\u003cem\u003ePressure 4\u003c/em\u003e) was performed.\u003c/p\u003e\n\u003cp\u003eBetween each round of drug pressure, parasites were cryopreserved, genotyped (\u003cem\u003epfmdr1\u003c/em\u003e and \u003cem\u003epfpm2\u003c/em\u003e copy number variation) and phenotyped (mefloquine IC\u003csub\u003e50\u003c/sub\u003e, PSA, and survival rates under MQ and PPQ co-exposure).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole-genome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA extracted from both the parental and \u003cem\u003ePressure 4\u003c/em\u003e \u003cem\u003eP. falciparum\u003c/em\u003e strains were used for library preparation. The libraries were prepared following the DNA Prep protocol from Illumina, starting from 250 ng of high-quality genomic DNA followed by sequencing of libraries using paired end mode (2X149) on a Nextseq 500 (Illumina). Raw reads were aligned to the \u003cem\u003eP. falciparum\u003c/em\u003e 3D7 reference genome (PlasmoDB release 39) using the BWA-MEM algorithm (Burrows-Wheeler Aligner; default parameters). Alignment files were processed with SAMtools (version 1.4) and genome-wide coverage statistics were assessed using Qualimap (version 2.2.1). Duplicate reads were removed with Picard MarDuplicates (version 2.26.10).\u003c/p\u003e\n\u003cp\u003eSingle nucleotide polymorphisms (SNPs) and indels were identified using a custom analysis pipeline. A pileup file, containing information on matches, mismatches, insertions, deletions and mapping quality, was generated using the SAMtools \u003cem\u003empileup\u003c/em\u003e function (version 1.13). This file was used as input to custom Python scripts for genomic variant detection. Comparative analysis between the parental and \u003cem\u003ePressure 4\u0026nbsp;\u003c/em\u003estrains was performed using custom R scripts (version 4.1), allowing identification of mutations acquired under drug pressure.\u003c/p\u003e\n\u003cp\u003eTo assess copy number variations (CNVs), we used PlasmoCNVScan, a read-depth-based strategy specifically optimized for \u003cem\u003ePlasmodium\u003c/em\u003e genomes.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePiperaquine incorporation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTritium-labeled PPQ (PPQ-[\u003csup\u003e3\u003c/sup\u003eH(G)]) was purchased from American Radiolabeled Chemicals, Inc. (USA) and prepared in pure ethanol according to the manufacturer\u0026rsquo;s instructions. Synchronized trophozoites (21-24 hours post-invasion) were obtained by centrifugation on a 75% Percoll gradient, followed by sorbitol treatment (5% D-sorbitol) to remove remaining ring-stage parasites. Cultures were adjusted to a haematocrit of approximately 4%, with a minimum parasitemia of 2\u0026middot;5%.\u003c/p\u003e\n\u003cp\u003eTrophozoites were pre-incubated for 10 min with MQ (25 to 750 nM, depending on the experimental conditions). Then, PPQ-[\u003csup\u003e3\u003c/sup\u003eH(G)] was added to a final concentration of 200 nM, and the parasites were incubated for 5 hours at 37\u0026deg;C under controlled atmospheric conditions (5% CO\u003csub\u003e2\u003c/sub\u003e, 5% O\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003ePPQ-[\u003csup\u003e3\u003c/sup\u003eH(G)] incorporation was quantified by calculating the ratio of intracellular to extracellular radioactivity. After incubation, infected erythrocytes were lysed using a solution containing 0\u0026middot;015% saponin and 0\u0026middot;1% Triton X-100. The intracellular fraction (PPQ*\u003csub\u003ein\u003c/sub\u003e) and extracellular fraction (PPQ*\u003csub\u003eout\u003c/sub\u003e) were measured using a \u0026beta;-scintillation counter (MicroBeta TriLux, Perkin-Elmer, Waltham, USA). The degree of PPQ incorporation was expressed as the PPQ*\u003csub\u003ein\u003c/sub\u003e/PPQ*\u003csub\u003eout\u003c/sub\u003e ratio.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed with the GraphPad Prism 7.0 software. A \u003cem\u003ep\u003c/em\u003e-value \u0026lt; 0\u0026middot;05 was considered statistically significant. The normality of each dataset was tested using the Shapiro-Wilk test. The Mann-Whitney or the one-way ANOVA statistical test with Tukey\u0026rsquo;s or Dunn\u0026rsquo;s multiple comparison tests were used to compare medians.\u003c/p\u003e\n\u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e values of the\u0026nbsp;MQ-PPQ\u0026nbsp;combination were determined for each \u003cem\u003eP. falciparum\u003c/em\u003e strain using the non-linear regression analysis in GraphPad Prism 7.0 and the ICEstimator software (http://www.antimalarial-icestimator.net).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical clearance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll \u003cem\u003eP. falciparum\u003c/em\u003e isolates were collected during therapeutic efficacy studies conducted in compliance with the ethical guidelines of the Cambodian National Ethical Committee for Human Research (identifiers: NECHR #086, NECHR #087, NECHR #092 and NECHR #106) and WPRO Ethical Review Committee.\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all study participants or their legal guardians. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding authors upon reasonable request. The source data underlying Figs. 1a-d, 2a-c, 3, 4a-b, 5a-b, 6, Supplementary Figs. 2, 3 and 4 are provided as a Source Data file.\u003c/p\u003e\n\u003cp\u003eNext-generation sequence files are available from the European Nucleotide Archive under the accession numbers ERR14369052 and ERR14369053 (project: PRJEB85790). All the scripts developed for this study were deposited in the following GitHub repository: https://github.com/Rcoppee/MQ-PPQ_pressure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the patients who accepted to participate in the therapeutic efficacy studies and to the staff of the Cambodian Health Centers for their contribution. This study was supported by the Global Fund RAI3E initiative grant through WHO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMalaria Unit, Institut Pasteur du Cambodge, Institut Pasteur, Phnom Penh, Cambodia\u003c/p\u003e\n\u003cp\u003eCamille Roesch, Melissa Mairet Khedim, Nimol Khim, Nimol Kloeung, Sopheakvatey Ke, Sreynet Srun, Rotha Eam, Chanra Kean, Chanvong Kul, Jean Popovici Benoit Witkowski\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLaboratoire de parasitologie-mycologie, UR 7510 ESCAPE, Universit\u0026eacute; de Rouen Normandie, Rouen, France\u003c/p\u003e\n\u003cp\u003eAnna Cosson \u0026amp; Romain Copp\u0026eacute;e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNational Center for Parasitology, Entomology, and Malaria Control, Ministry of Health, Phnom Penh, 120801, Cambodia\u003c/p\u003e\n\u003cp\u003eRithea Leang\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMekong Malaria Elimination Programme, WHO, Phnom Penh, Cambodia\u003c/p\u003e\n\u003cp\u003ePascal Ringwald\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eINSERM U1344, MERIT IRD, Universit\u0026eacute; Paris Cit\u0026eacute;, Paris, France\u003c/p\u003e\n\u003cp\u003eFr\u0026eacute;d\u0026eacute;ric Ariey\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eService de Parasitologie-Mycologie, H\u0026ocirc;pital Cochin, Paris, France\u003c/p\u003e\n\u003cp\u003eFr\u0026eacute;d\u0026eacute;ric Ariey\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCentre National de R\u0026eacute;f\u0026eacute;rence du Paludisme, Laboratoire de Parasitologie-Mycologie, H\u0026ocirc;pital Bichat-Claude Bernard, Paris, France\u003c/p\u003e\n\u003cp\u003eRomain Copp\u0026eacute;e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGENOM\u0026rsquo;IC, INSERM U1016 Institut Cochin, Paris France\u003c/p\u003e\n\u003cp\u003eLucie Adoux\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePresent affiliation\u003csup\u003e\u0026nbsp;\u003c/sup\u003e: Genetic and Biology of Plasmodium Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar \u0026amp;\u003csup\u003e\u0026nbsp;\u003c/sup\u003ePV-ESMEE Pasteur International Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar\u0026nbsp;; Pasteur Institute, Paris, France\u0026nbsp;; Pasteur Institute of Cambodia, Phnom Penh, Cambodia.\u003c/p\u003e\n\u003cp\u003eCamille Roesch \u0026amp; Benoit Witkowski\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: C.R. and B.W. Methodology: C.R., A.C., J.P., R.C. and B.W. Validation: C.R., A.C., M.M.K., R.C. and B.W. Formal analysis: C.R., A.C., F.A., R.C. and B.W. Investigation: C.R., A.C., M.M.K., N.Kh., N.Kl., S.K., S.S., R.E., C.Ke. and C.Ku. Resources: L.A., R.L., P.R. and B.W. Original draft preparation: C.R. and B.W. Review and editing of the manuscript: C.R., A.C., M.M.K., J.P., P.R., F.A., R.C., and B.W. Visualization: C.R., A.C., R.C. \u0026nbsp;and B.W. Project administration and supervision: B.W. Funding acquisition: B.W.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Benoit Witkowski.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. None of the authors declared financial conflict of interest. P.R. is a staff member of the World Health Organization. The authors are solely responsible for the views expressed in this publication, which do not necessarily represent the decisions, policies, or views of the World Health Organization\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWorld Health Organization. \u003cem\u003eWorld Malaria Report 2022\u003c/em\u003e. (2022).\u003c/li\u003e\n\u003cli\u003eIshengoma, D. S. \u003cem\u003eet al.\u003c/em\u003e Urgent action is needed to confront artemisinin partial resistance in African malaria parasites. \u003cem\u003eNat Med\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 1807\u0026ndash;1808 (2024).\u003c/li\u003e\n\u003cli\u003eAriey, F. \u003cem\u003eet al.\u003c/em\u003e A molecular marker of artemisinin-resistant \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e505\u003c/strong\u003e, 50\u0026ndash;55 (2014).\u003c/li\u003e\n\u003cli\u003eWitkowski, B. \u003cem\u003eet al.\u003c/em\u003e A surrogate marker of piperaquine-resistant \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria: a phenotype-genotype association study. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 174\u0026ndash;183 (2017).\u003c/li\u003e\n\u003cli\u003eAmaratunga, C. \u003cem\u003eet al.\u003c/em\u003e Dihydroartemisinin\u0026ndash;piperaquine resistance in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria in Cambodia: a multisite prospective cohort study. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 357\u0026ndash;365 (2016).\u003c/li\u003e\n\u003cli\u003eImwong, M. \u003cem\u003eet al.\u003c/em\u003e The spread of artemisinin-resistant \u003cem\u003ePlasmodium falciparum\u003c/em\u003e in the Greater Mekong Subregion: a molecular epidemiology observational study. \u003cem\u003eLancet Infect Dis\u003c/em\u003e (2017) doi:10.1016/S1473-3099(17)30048-8.\u003c/li\u003e\n\u003cli\u003eLeang, R. \u003cem\u003eet al.\u003c/em\u003e Evidence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 4719\u0026ndash;4726 (2015).\u003c/li\u003e\n\u003cli\u003eNa-Bangchang, K., Ruengweerayut, R., Mahamad, P., Ruengweerayut, K. \u0026amp; Chaijaroenkul, W. Declining in efficacy of a three-day combination regimen of mefloquine-artesunate in a multi-drug resistance area along the Thai-Myanmar border. \u003cem\u003eMalar J\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 273 (2010).\u003c/li\u003e\n\u003cli\u003ePhuc, B. Q. \u003cem\u003eet al.\u003c/em\u003e Treatment Failure of Dihydroartemisinin/Piperaquine for \u003cem\u003ePlasmodium falciparum\u003c/em\u003e Malaria, Vietnam. \u003cem\u003eEmerg. Infect. Dis.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 715\u0026ndash;717 (2017).\u003c/li\u003e\n\u003cli\u003eSaito, M. \u003cem\u003eet al.\u003c/em\u003e A randomized controlled trial of dihydroartemisinin-piperaquine, artesunate-mefloquine and extended artemether-lumefantrine treatments for malaria in pregnancy on the Thailand-Myanmar border. \u003cem\u003eBMC Med\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 132 (2021).\u003c/li\u003e\n\u003cli\u003eHamaluba, M. \u003cem\u003eet al.\u003c/em\u003e Arterolane\u0026ndash;piperaquine\u0026ndash;mefloquine versus arterolane\u0026ndash;piperaquine and artemether\u0026ndash;lumefantrine in the treatment of uncomplicated \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria in Kenyan children: a single-centre, open-label, randomised, non-inferiority trial. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 1395\u0026ndash;1406 (2021).\u003c/li\u003e\n\u003cli\u003eMahamar, A. \u003cem\u003eet al.\u003c/em\u003e Artemether-lumefantrine-amodiaquine or artesunate-amodiaquine combined with single low-dose primaquine to reduce \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria transmission in Ou\u0026eacute;less\u0026eacute;bougou, Mali: a five-arm, phase 2, single-blind, randomised controlled trial. \u003cem\u003eLancet Microbe\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 100966 (2025).\u003c/li\u003e\n\u003cli\u003ePeto, T. J. \u003cem\u003eet al.\u003c/em\u003e Triple therapy with artemether\u0026ndash;lumefantrine plus amodiaquine versus artemether\u0026ndash;lumefantrine alone for artemisinin-resistant, uncomplicated falciparum malaria: an open-label, randomised, multicentre trial. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 867\u0026ndash;878 (2022).\u003c/li\u003e\n\u003cli\u003eVan Der Pluijm, R. W. \u003cem\u003eet al.\u003c/em\u003e Triple artemisinin-based combination therapies versus artemisinin-based combination therapies for uncomplicated \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria: a multicentre, open-label, randomised clinical trial. \u003cem\u003eThe Lancet\u003c/em\u003e \u003cstrong\u003e395\u003c/strong\u003e, 1345\u0026ndash;1360 (2020).\u003c/li\u003e\n\u003cli\u003eImwong, M. \u003cem\u003eet al.\u003c/em\u003e Evolution of Multidrug Resistance in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e: a Longitudinal Study of Genetic Resistance Markers in the Greater Mekong Subregion. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, e01121-21 (2021).\u003c/li\u003e\n\u003cli\u003eHanboonkunupakarn, B. \u003cem\u003eet al.\u003c/em\u003e Sequential Open-Label Study of the Safety, Tolerability, and Pharmacokinetic Interactions between Dihydroartemisinin-Piperaquine and Mefloquine in Healthy Thai Adults. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e \u003cstrong\u003e63\u003c/strong\u003e, e00060-19 (2019).\u003c/li\u003e\n\u003cli\u003eRossi, G., De Smet, M., Khim, N., Kindermans, J.-M. \u0026amp; Menard, D. Emergence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e triple mutant in Cambodia. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 1233 (2017).\u003c/li\u003e\n\u003cli\u003eMairet-Khedim, M. \u003cem\u003eet al.\u003c/em\u003e Prevalence and characterization of piperaquine, mefloquine and artemisinin derivatives triple-resistant \u003cem\u003ePlasmodium falciparum\u003c/em\u003e in Cambodia. \u003cem\u003eJournal of Antimicrobial Chemotherapy\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 411\u0026ndash;417 (2023).\u003c/li\u003e\n\u003cli\u003ePreechapornkul, P. \u003cem\u003eet al.\u003c/em\u003e \u003cem\u003ePlasmodium falciparum pfmdr1\u003c/em\u003e Amplification, Mefloquine Resistance, and Parasite Fitness. \u003cem\u003eAntimicrob Agents Chemother\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 1509\u0026ndash;1515 (2009).\u003c/li\u003e\n\u003cli\u003eAmato, R. \u003cem\u003eet al.\u003c/em\u003e Origins of the current outbreak of multidrug-resistant malaria in southeast Asia: a retrospective genetic study. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 337\u0026ndash;345 (2018).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6629488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6629488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTriple artemisinin-based combination therapies (TACTs) have been proposed to delay the emergence of multidrug-resistant \u003cem\u003ePlasmodium falciparum\u003c/em\u003e by combining two partner drugs with artemisinin derivatives. Among these, mefloquine\u0026ndash;piperaquine (MQ\u0026ndash;PPQ) is a leading candidate, based on the assumption that resistance to both partner drugs would be difficult to develop simultaneously. Here, we assess the efficacy and resistance potential of MQ\u0026ndash;PPQ using Cambodian clinical isolates with distinct resistance profiles. We find that MQ resistance confers significant cross-tolerance to the MQ\u0026ndash;PPQ combination, while PPQ-resistant and sensitive strains remain susceptible. Under repeated MQ\u0026ndash;PPQ pressures, parasites rapidly acquire MQ-PPQ tolerance, driven by \u003cem\u003epfmdr1\u003c/em\u003e amplification. Mechanistic investigations reveal that MQ inhibits PPQ accumulation in a dose-dependent manner, providing a functional explanation for the compromised efficacy of the combination. These findings demonstrate that MQ resistance alone can undermine MQ\u0026ndash;PPQ TACT efficacy, which question the strategic foundation of this combination and underscore the need for alternative combinations with lower resistance selection risk.\u003c/p\u003e","manuscriptTitle":"Tolerance of Plasmodium falciparum mefloquine-resistant clinical isolates to mefloquine-piperaquine: implications for triple artemisinin-based combination therapy strategies.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-23 13:59:38","doi":"10.21203/rs.3.rs-6629488/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"80727724-b857-487d-9ec7-1524b39e67cd","owner":[],"postedDate":"May 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48757065,"name":"Health sciences/Diseases/Infectious diseases/Malaria"},{"id":48757066,"name":"Biological sciences/Microbiology/Antimicrobials/Antiparasitic agents"}],"tags":[],"updatedAt":"2025-11-28T08:12:29+00:00","versionOfRecord":{"articleIdentity":"rs-6629488","link":"https://doi.org/10.1038/s41467-025-65629-8","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-11-27 05:00:00","publishedOnDateReadable":"November 27th, 2025"},"versionCreatedAt":"2025-05-23 13:59:38","video":"","vorDoi":"10.1038/s41467-025-65629-8","vorDoiUrl":"https://doi.org/10.1038/s41467-025-65629-8","workflowStages":[]},"version":"v1","identity":"rs-6629488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6629488","identity":"rs-6629488","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}