Piperaquine resistance in Plasmodium falciparum has emerged in Southeast Asia and is strongly associated with mutations in the pfcrt gene and amplification of pfplasmepsin2/3. This study assessed the frequency of pfcrt mutations and pfplasmepsin2/3 gene amplifications across Southeast Asia over 25 years, which is critical for tracking resistant parasites. A total of 898 P. falciparum isolates collected from Thailand, Myanmar, Cambodia, Laos, and Vietnam between 1995 and 2023 were analyzed for pfcrt mutations, pfplasmepsin2 copy number, and microsatellite variation around the pfcrt locus. During the study period, pfcrt mutations in Cambodia across the study period showed that H97Y had the highest prevalence at 15%, followed by T93S at 8%, while I218F was common in 24% of specimens in Srisaket and Ubon in Thailand at 24.44%, followed by G353V at 20%, T93S at 17%, F145I at 13%, and H97Y at 4%. In Tak, Thailand, a mutation was found only in 1995 with 40% at I218F and the remaining wild-type. Yala in Thailand and Kayin State in Myanmar remained wild-type. Vietnam showed frequent T93S at 21%. The prevalence of pfcrt mutations observed in this study changed throughout the study period. Over 65% of parasites with pfcrt mutations at positions 93, 97, 145, 218, 343, and 353 also had pfplasmepsin2 amplification. Temporal analysis revealed that in Cambodia, pfplasmepsin2 CNV emerged first, peaking at 88% prevalence in 2015 after the introduction of DHA-piperaquine, with the subsequent increase of pfcrt mutations H97Y and G353V. In Eastern Thailand, both pfcrt mutations and pfplasmepsin2 CNV were highly prevalent, exceeding 70% during 2015-2018. In Vietnam, rapid increases in both markers were observed after 2012, reaching peak by 2017. Microsatellite analysis revealed reduced genetic diversity around mutant pfcrt alleles, indicating selective sweeps. This study demonstrates that frequencies of pfcrt mutations and pfplasmepsin2 amplification linked to piperaquine resistance have changed over time, highlighting the importance of ongoing genetic monitoring to inform strategies for preserving artemisinin-based combination therapy efficacy.

Substandard and falsified (S F) antimalarial medicines undermine malaria control efforts across sub-Saharan Africa, with an estimated 19

Disease surveillance activities are usually resource-constrained and should be optimised to generate the most informative scientific findings, and to make the best use of time, finances, and personnel. India has a high population density, diverse geography and climatic conditions, and difficult terrain. With respect to malaria, Plasmodium falciparum and Plasmodium vivax are endemic, with substantial variability of transmission across the country. While for P. vivax, drug efficacy appears to be homogeneous within the country, for P. falciparum malaria, the drug resistance pattern varies from the northeastern region to the central region. Accounting for these complexities, we develop a decision-making framework guided by geospatial modelling outputs to identify prospective study sites for surveillance of molecular markers of antimalarial drug resistance in P. falciparum malaria in India. We first retrieve existing data on the prevalence of validated markers of resistance to artesunate and sulfadoxine-pyrimethamine from the World Wide Antimalarial Resistance Network (WWARN) Surveyor database. We then incorporate these data into a geostatistical model to estimate the prevalence of these markers across India and identify areas with high median estimated marker prevalence and high uncertainty. Finally, we create an interactive dashboard using the RShiny software package to simplify the process of selecting sites for future molecular surveillance. Our framework helps to ensure that operational decision-making is supported by data and modelling outputs. We demonstrate the utility of our framework by selecting sites for molecular surveillance of P. falciparum malaria in India.

Seasonal Malaria Chemoprevention (SMC) has been adopted since 2014 in Burkina Faso to reduce malaria burden in children under 5 years. However, the intervention’s expected potential has not yet been achieved in real-life conditions, suggesting other factors may influence its effectiveness. Asymptomatic carriers, including patent and sub-patent Plasmodium falciparum infections in household members seems to be a potential factor maintaining the high malaria burden in children under SMC coverage. This study assessed the patterns of these infections in household members living around children under SMC coverage in Nanoro, Burkina Faso. A cross-sectional survey nested to a large SMC study named “SMC_RST” was conducted during the 2022 SMC campaign in Nanoro, including 745 participants. Sub-patent infections were defined as varATS qPCR-positive/RDT-negative, and patent infections as positive by both methods. Prevalence of patent and sub-patent malaria infections were presented with 95

To address the current threat of antimalarial resistance, countries need innovative solutions for timely and informed decision-making. Integrating molecular surveillance for drug-resistant malaria into routine malaria surveillance in pre-elimination contexts offers a potential early warning mechanism for further investigation and response. However, there is limited evidence on what influences the performance of such a system in resource-limited settings. From March 2018 to February 2020, a sequential mixed-methods study was conducted in primary healthcare facilities in a South African pre-elimination setting to explore factors influencing the flow, quality and linkage of malaria case notification and molecular resistance marker data. Using a process-oriented framework, we undertook monthly and quarterly data linkage and consistency analyses at different levels of the health system, as well as a survey, focus group discussions and interviews to identify potential barriers to, and enhancers of, the roll-out and uptake of this integrated information system. Over two years, 4,787 confirmed malaria cases were notified from 42 primary healthcare facilities in the Nkomazi sub-district, Mpumalanga, South Africa. Of the notified cases, 78.5% (n = 3,758) were investigated, and 55.1% (n = 2,636) were successfully linked to their Plasmodium falciparum molecular resistance marker profiles. Five tangible processes-malaria case detection and notification, sample collection, case investigation, analysis and reporting-were identified within the process-oriented logic model. Workload, training, ease of use, supervision, leadership, and resources were recognized as cross-cutting influencers affecting the program's performance. Approaching malaria elimination, linking molecular markers of antimalarial resistance to routine malaria surveillance is feasible. However, cross-cutting barriers inherent in the healthcare system can influence its success in a resource-limited setting.

Disease surveillance activities are usually resource-constrained and should be optimised to achieve spatial and real-time situational awareness. Such optimisation would help with better resource allocation, reduced logistics, and other costs. India has a high population density, diverse geography and climatic conditions, and difficult terrain. With respect to malaria, Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are endemic, with substantial variability of transmission across the country. While for Pv, drug efficacy appears to be homogenous within the country, for Pf malaria, the resistance pattern varies from the northeastern region to the central region. These factors make accurate mapping of antimalarial drug resistance difficult. To account for these complexities, we develop a targeted and adaptive methodology to identify prospective study sites for Pf antimalarial drug resistance surveillance. We retrieve existing data on the prevalence of validated markers of resistance to Artesunate (AS) and Sulfadoxine-Pyrimethamine (SP) from the WorldWide Antimalarial Resistance Network (WWARN) systematic review database. We incorporate these data into a geostatistical model to estimate the prevalence of these markers across India and identify areas with high projected median resistance marker prevalence and low uncertainty. Finally, we create an interactive dashboard using the RShiny software package to simplify the process of selecting sites for future molecular surveillance. This methodology helps to ensure that decision-making is supported by data and modelling outputs while facilitating the generation of knowledge about the current state of antimalarial drug resistance with wide geographic coverage. We demonstrate the utility of our method by selecting sites for surveillance of drug resistance in Pf malaria in India. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This research was supported by a grant from Bill & Melinda Gates Foundation (grant no. INV-004713). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The source data used for modelling is available from Artemisinin Molecular Surveyor (http://www.wwarn.org/molecular/surveyor/k13/index.html?t=201608031200#0) and SP Molecular Surveyor (http://www.wwarn.org/dhfr-dhps-surveyor/#0). R code is available on GitHub and can be access through below link (https://github.com/lu-harr/pf\_drug\_resistance). At present the dashboard has not been uploaded online due to data protection issues but a proof of concept on simulated data can be accessed through https://lucyharrison.shinyapps.io/pf\_drug\_resistance_shiny/. [https://lucyharrison.shinyapps.io/pf\_drug\_resistance_shiny/][1] [1]: https://lucyharrison.shinyapps.io/pf_drug_resistance_shiny/

BACKGROUND:In India, Plasmodium falciparum and Plasmodium vivax remain in circulation. Accurate detection of the parasite species remains crucial for prompt initiation of treatment and reducing onward transmission. METHODS:A cross-sectional study across 12 sites of varying malaria endemicities was conducted from September 2023 to April 2024. Febrile participants were tested for malaria using rapid diagnostic tests (RDTs) and microscopy. Malaria positivity proportions along with 95% confidence intervals (95% CI) were presented separately by parasite species. The diagnostic performance of the RDT was compared against microscopy. RESULTS:A total of 10,290 febrile participants were tested by both RDT and microscopy: 1,516 (14.7%, 95% CI 7.7-21.8%) malaria cases were identified by RDT and 1,436 (14.0%, 95% CI 6.9-21.1%) by microscopy. Of the 1,516 RDT positives, 1,105 (72.9%) had P. falciparum mono-infection, 290 (19.1%) had P. vivax mono-infection, and 121 (8.0%) had P. falciparum and P. vivax mixed infections. The sensitivity and specificity of RDT were 95.0% [95% CI 94-96%] and 99% [95% CI 98-99%], respectively, for detecting P. falciparum mono-infection, 83% [95% CI 78-87%] and 100% [95% CI 99-100%] for detecting P. vivax mono-infection, and 88% [95% CI 80-93%] and 100% for detecting a mixed infection of P. falciparum and P. vivax. Overall, 43 (0.4%) participants who were RDT negative were found to have malaria on subsequent microscopic examination. CONCLUSION:Approximately 15% of the febrile participants tested were identified as malaria positive by RDT, of which nearly one-fifth were P. vivax mono-infections and 8% harboured P. falciparum and P. vivax mixed infections. Low sensitivity of the RDTs for identifying P. vivax underscores an urgent need for developing reliable diagnostics.

AIMS:Resistance to the artemisinins and the artemisinin-based combination therapy (ACT) partner drugs has developed in Southeast Asia, and artemisinin resistance has also emerged in eastern Africa. Triple ACTs (triple artemisinin-based combination therapies, TACT), consisting of two partner drugs with different mechanisms of action and similar pharmacokinetic profiles, combined with an artemisinin derivative can help to delay or prevent artemisinin resistance and prolong the useful lifetime of the partner drugs. This study aims to characterize the pharmacokinetic properties of a recommended TACT, artemether-lumefantrine plus amodiaquine, using data from two large clinical trials. METHODS:We analysed data from two randomized, controlled intervention trials conducted between 2015 and 2020 in one African country and two Southeast Asian countries, in which artemether-lumefantrine was administered alone (n = 443) or together with amodiaquine (n = 442) to patients with uncomplicated P. falciparum malaria. Both studies included a sub-cohort with dense pharmacokinetic sampling, combined with sparse data in the other patients. Concentration-time data of artemether, dihydroartemisinin, lumefantrine, desbutyllumefantrine, amodiaquine and desethylamodiaquine were analysed using nonlinear mixed-effects modelling. RESULTS:Pharmacokinetic models were developed for all drugs and demonstrated good predictive performance and goodness-of-fit diagnostics. Coadministered amodiaquine was not a significant covariate on pharmacokinetic properties of artemether-lumefantrine. Model-predicted Cmax and AUC (median [95% confidence interval, CI]) for artemether were 256 (159-407) ng/mL and 2850 (1820-4920) h·ng/mL for artemether-lumefantrine alone, and 230 (123-391) ng/mL and 2800 (1570-4570) h·ng/mL for artemether-lumefantrine-amodiaquine. For dihydroartemisinin, values were 135 (54.5-214) ng/mL and 1870 (813-3015) h·ng/mL for artemether-lumefantrine alone, and 116 (40.8-186) ng/mL and 1580 (547-2680) h·ng/mL for artemether-lumefantrine-amodiaquine. For lumefantrine, values were 15.2 (2.90-31.3) μg/mL and 600 (275-1230) h·μg/mL for artemether-lumefantrine alone, and 14.1 (2.72-31.4) μg/mL and 586 (269-1070) h·μg/mL for artemether-lumefantrine-amodiaquine. Day 7 concentrations of lumefantrine were 452 (215-1240) and 438 (204-1030) μg/mL for artemether-lumefantrine alone and artemether-lumefantrine-amodiaquine, respectively. All geometric mean ratios (GMRs) for the drug-drug interaction (DDI) effect on key pharmacokinetic parameters of artemether, dihydroartemisinin and lumefantrine fell within the 0.80-1.25 range, with the majority of the corresponding 90% CI also contained within this range. This indicates no clinically relevant DDIs between artemether-lumefantrine and amodiaquine. CONCLUSIONS:The DDI effect of amodiaquine on the pharmacokinetics of artemether-lumefantrine is expected to be minimal, the based on the current analysis. However, further large-scale clinical trials are needed to confirm this finding.

The emergence of Plasmodium falciparum parasites resistant to artemisinins compromises the efficacy of Artemisinin Combination Therapies (ACTs), the global first-line malaria treatment. Artemisinin resistance is a complex genetic trait in which nonsynonymous SNPs in PfK13 cooperate with other genetic variations. Here, we present population genomic/transcriptomic analyses of P. falciparum collected from patients with uncomplicated malaria in Cambodia and Vietnam between 2018 and 2020. Besides the PfK13 SNPs, several polymorphisms, including nonsynonymous SNPs (N1131I and N821K) in PfRad5 and an intronic SNP in PfWD11 (WD40 repeat-containing protein on chromosome 11), appear to be associated with artemisinin resistance, possibly as new markers. There is also a defined set of genes whose steady-state levels of mRNA and/or splice variants or antisense transcripts correlate with artemisinin resistance at the base level. In vivo transcriptional responses to artemisinins indicate the resistant parasite's capacity to decelerate its intraerythrocytic developmental cycle (IDC), which can contribute to the resistant phenotype. During this response, PfRAD5 and PfWD11 upregulate their respective alternatively/aberrantly spliced isoforms, suggesting their contribution to the protective response to artemisinins. PfRAD5 and PfWD11 appear under selective pressure in the Greater Mekong Sub-region over the last decade, suggesting their role in the genetic background of the artemisinin resistance.

Surveillance for genetic markers of resistance can provide valuable information on the likely efficacy of antimalarials but needs to be targeted to ensure optimal use of resources. We conducted a systematic search and review of publications in seven databases to compile resistance marker data from studies in India. The sample collection from the studies identified from this search was conducted between 1994 and 2020, and these studies were published between 1994 and 2022. In all, Plasmodium falciparum Kelch13 (PfK13), P. falciparum dihydropteroate synthase, and P. falciparum dihydrofolate reductase (PfDHPS) genotype data from 2,953, 4,148, and 4,222 blood samples from patients with laboratory-confirmed malaria, respectively, were extracted from these publications and uploaded onto the WorldWide Antimalarial Resistance Network molecular surveyors. These data were fed into hierarchical geostatistical models to produce maps with a predicted prevalence of the PfK13 and PfDHPS markers, and of the associated uncertainty. Zones with a predicted PfDHPS 540E prevalence of > 15% were identified in central, eastern, and northeastern India. The predicted prevalence of PfK13 mutants was nonzero at only a few locations, but were within or adjacent to the zones with > 15% prevalence of PfDHPS 540E. There may be a greater probability of artesunate-sulfadoxine-pyrimethamine failures in these regions, but these predictions need confirmation. This work can be applied in India and elsewhere to help identify the treatments most likely to be effective for malaria elimination.

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin as a first-line treatment against malaria. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multidrug-resistant KEL1/PLA1 lineage isolate (KH004) and a drug-susceptible Malawian parasite (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and multiple copies of pm2/3. We recovered 104 unique recombinant parasites and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3. We performed a detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes with these progenies, including PPQ survival assay, area under the dose response curve, and a limited point IC50. We find that inheritance of the KH004 pfcrt allele is required for reduced PPQ sensitivity, whereas copy number variation in pm2/3 further decreases susceptibility but does not confer resistance in the absence of additional mutations in pfcrt. A deep investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background to a range of PPQ-related traits. Additionally, we find that the resistance phenotype associated with parasites inheriting the G367C substitution in pfcrt is consistent with previously validated PPQ resistance mutations in this transporter.IMPORTANCEResistance to piperaquine, used in combination with dihydroartemisinin, has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and has the distinct advantage of quantifying the effects of naturally evolved genetic variation. Our study was strengthened since the full range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of the pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multigene model suggests an important role for both loci in the evolution of this multidrug-resistant parasite lineage.

BACKGROUND:Hydroxychloroquine (HCQ) has proved ineffective in treating patients hospitalised with Coronavirus Disease 2019 (COVID-19), but uncertainty remains over its safety and efficacy in chemoprevention. Previous chemoprevention randomised controlled trials (RCTs) did not individually show benefit of HCQ against COVID-19 and, although meta-analysis did suggest clinical benefit, guidelines recommend against its use. METHODS AND FINDINGS:Healthy adult participants from the healthcare setting, and later from the community, were enrolled in 26 centres in 11 countries to a double-blind, placebo-controlled, randomised trial of COVID-19 chemoprevention. HCQ was evaluated in Europe and Africa, and chloroquine (CQ) was evaluated in Asia, (both base equivalent of 155 mg once daily). The primary endpoint was symptomatic COVID-19, confirmed by PCR or seroconversion during the 3-month follow-up period. The secondary and tertiary endpoints were: asymptomatic laboratory-confirmed Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection; severity of COVID-19 symptoms; all-cause PCR-confirmed symptomatic acute respiratory illness (including SARS-CoV-2 infection); participant reported number of workdays lost; genetic and baseline biochemical markers associated with symptomatic COVID-19, respiratory illness and disease severity (not reported here); and health economic analyses of HCQ and CQ prophylaxis on costs and quality of life measures (not reported here). The primary and safety analyses were conducted in the intention-to-treat (ITT) population. Recruitment of 40,000 (20,000 HCQ arm, 20,000 CQ arm) participants was planned but was not possible because of protracted delays resulting from controversies over efficacy and adverse events with HCQ use, vaccine rollout in some countries, and other factors. Between 29 April 2020 and 10 March 2022, 4,652 participants (46% females) were enrolled (HCQ/CQ n = 2,320; placebo n = 2,332). The median (IQR) age was 29 (23 to 39) years. SARS-CoV-2 infections (symptomatic and asymptomatic) occurred in 1,071 (23%) participants. For the primary endpoint the incidence of symptomatic COVID-19 was 240/2,320 in the HCQ/CQ versus 284/2,332 in the placebo arms (risk ratio (RR) 0.85 [95% confidence interval, 0.72 to 1.00; p = 0.05]). For the secondary and tertiary outcomes asymptomatic SARS-CoV-2 infections occurred in 11.5% of HCQ/CQ recipients and 12.0% of placebo recipients: RR: 0.96 (95% CI, 0.82 to 1.12; p = 0.6). There were no differences in the severity of symptoms between the groups and no severe illnesses. HCQ/CQ chemoprevention was associated with fewer PCR-confirmed all-cause respiratory infections (predominantly SARS-CoV-2): RR 0.61 (95% CI, 0.42 to 0.88; p = 0.009) and fewer days lost to work because of illness: 104 days per 1,000 participants over 90 days (95% CI, 12 to 199 days; p < 0.001). The prespecified meta-analysis of all published pre-exposure RCTs indicates that HCQ/CQ prophylaxis provided a moderate protective benefit against symptomatic COVID-19: RR 0.80 (95% CI, 0.71 to 0.91). Both drugs were well tolerated with no drug-related serious adverse events (SAEs). Study limitations include the smaller than planned study size, the relatively low number of PCR-confirmed infections, and the lower comparative accuracy of serology endpoints (in particular, the adapted dried blood spot method) compared to the PCR endpoint. The COPCOV trial was registered with ClinicalTrials.gov; number NCT04303507. INTERPRETATION:In this large placebo-controlled, double-blind randomised trial, HCQ and CQ were safe and well tolerated in COVID-19 chemoprevention, and there was evidence of moderate protective benefit in a meta-analysis including this trial and similar RCTs. TRIAL REGISTRATION:ClinicalTrials.gov NCT04303507; ISRCTN Registry ISRCTN10207947.

Asymptomatic carriers of Plasmodium falciparum represent important parasite reservoirs maintaining malaria transmission in the community. This study aimed on the one hand to screen the other household members living with children under seasonal malaria chemoprevention (SMC) coverage in order to determine the level of malaria infection in this population and on the other hand to determine the appropriate type of rapid diagnostic test (RDT) for this screening to detect these asymptomatic carriers in the community. During the 2022 SMC campaign (July to October), a cross-sectional survey was carried out in 745 participants who were screened by ultrasensitive rapid diagnostic test (usRDT), standard rapid diagnostic test (rRDT) and microscopy. Out of them, 395 had microscopy results available and were included in the data analysis. The prevalence of asymptomatic carriers of asexual forms of Plasmodium falciparum was 26.58% (105/395) while sexual forms were found in 5.32% (21/395) of the study population. Children from 5 to 15 years had the highest prevalence of P. falciparum asexual forms 35.76% (59/165) compared with older participants. Malaria positivity rate for rRDT and usRDT was 29.40% (219/745) and 40.49% (305/745) respectively. The usRDT had a higher sensitivity than the rRDT (72.38% (95% CI 62.8-80.66) vs. 60.95% (95% CI 50.94-70.33)). In terms of specificity, rRDT had a higher specificity 82.41% (95% CI 77.53-86.62) versus 69.66% (95% CI 64.01-74.89) for usRDT. This study reports a high prevalence of parasite carriers in household members of children under SMC coverage in Nanoro, Burkina Faso. In conclusion, usRDT seems more appropriate for strategies based on detection and treatment of parasite carriers within the community.

Investment in community health workers is essential.

AbstractPiperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in thePlasmodium falciparum chloroquine resistance transporter(pfcrt) and increased copies ofplasmepsin II/III(pm2/3). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrtallele and a single copy ofpm2/3,while KH004 has a chloroquine-resistant (Dd2-like) pfcrtallele with an additional G367C substitution and four copies ofpm2/3. We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants atpfcrt and pm2/3for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC50(LP-IC50). We find that inheritance of the KH004pfcrtallele is required for PPQ resistance, whereas copy number variation inpm2/3further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations inpfcrt. Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributionsof pfcrt, pm2/3,and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance.ImportanceResistance to PPQ used in combination with DHA has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention, and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and have the distinct advantage of assaying the effects of naturally evolved genetic variation. Our study was significantly strengthened because the full a range of copies of KH004pm2/3was inherited among the progeny clones, allowing us to directly test the roleof pm2/3copy number on resistance-related phenotypes in the context of a uniquepfcrtallele. Our multi-gene model suggests an important role for both loci in the evolution of this ACT resistant parasite lineage.

Increasing levels of artemisinin and partner drug resistance threaten malaria control and elimination globally. Triple artemisinin-based combination therapies (TACTs) which combine artemisinin derivatives with two partner drugs are efficacious and well tolerated in clinical trials, including in areas of multidrug-resistant malaria. Whether early TACT adoption could delay the emergence and spread of antimalarial drug resistance is a question of vital importance. Using two independent individual-based models of Plasmodium falciparum epidemiology and evolution, we evaluated whether introduction of either artesunate-mefloquine-piperaquine or artemether-lumefantrine-amodiaquine resulted in lower long-term artemisinin-resistance levels and treatment failure rates compared with continued ACT use. We show that introduction of TACTs could significantly delay the emergence and spread of artemisinin resistance and treatment failure, extending the useful therapeutic life of current antimalarial drugs, and improving the chances of malaria elimination. We conclude that immediate introduction of TACTs should be considered by policy makers in areas of emerging artemisinin resistance.