Background:Malaria transmission in southwestern Uganda is low, but persists despite ongoing control efforts. Identifying whether infections are locally sustained or imported by travelers is critical for guiding interventions. We integrated epidemiologic surveillance with parasite genomics to characterize imported malaria episodes at three health facilities in southwestern Uganda. Methods:Between January 2023 and June 2024, we enrolled microscopy-confirmed malaria cases at three health facilities, Maziba and Muko (very low transmission) and Kamwezi (low-to-moderate transmission), administered travel history questionnaires, and collected dried blood spots for genotyping. Plasmodium falciparum infections were genotyped using MAD4HatTeR, a highly sensitive multiplex amplicon sequencing panel targeting 165 diversity markers and 38 drug resistance loci. Complexity of infection and pairwise relatedness were estimated using MOIRE and Dcifer, respectively. Plasmotrack, a Bayesian transmission network framework, was used to infer network structure, transmission directionality, reproduction numbers, and importation rates. Results:Amongst malaria cases, recent overnight travel was common in Maziba (87%) and Muko (96%) but infrequent in Kamwezi (12%). Most travel in cases from Maziba and Muko was from high-transmission regions in northern and eastern Uganda. Parasites in Maziba and Muko cases exhibited higher within-host diversity and lower within-site relatedness compared to those in Kamwezi cases. Transmission network inference identified most infections in Maziba and Muko as imported, with the majority of inferred secondary transmission linked to recent travelers. In contrast, Kamwezi showed multiple highly related clusters, indicating sustained local transmission. Validated and candidate markers of artemisinin partial resistance (K13 P441L and R561H) were more prevalent in Kamwezi. Conclusion:Malaria in Maziba and Muko was driven largely by importation from other parts of Uganda, while local transmission played a larger role in Kamwezi . Tailored interventions addressing travel-associated risks and local transmission, supported by travel histories and parasite genetic data will be valuable to advance malaria elimination in this region.

Mutations in the Plasmodium falciparum genes, pfdhfr and pfdhps, drive antifolate resistance and threaten malaria control in regions where sulfadoxine-pyrimethamine (SP) is the primary chemoprevention strategy. The spatial patterns and evolutionary dynamics of these mutations in high-transmission settings remain incompletely understood. Here we genotyped 11 resistance-associated mutations in pfdhfr and pfdhps in 4,725 P. falciparum isolates collected from 16 Ugandan health facilities as part of annual surveillance between 2016 and 2022. Notably, we show that the frequency of PfDHFR I164L, which confers higher pyrimethamine resistance, increased over time from 19.4% to 32.4%. Using identity-by-descent, haplotype structure, and extended haplotype homozygosity analyses, we show that PfDHFR I164L is present on multiple haplotype backgrounds and undergoes localised expansions, without detectable signatures of recent positive selection at all but one site. Our results suggest that the evolution of antifolate resistance, driven by PfDHFR I164L, is spatially heterogeneous and complex in regions that primarily use SP chemoprevention programmes.

ABSTRACT Background Partial resistance to artemisinins (ART-R) has emerged in East Africa, associated with mutations in the Plasmodium falciparum kelch13 gene. It is currently unclear whether ART-R has implications for gametocyte production or for onward transmission to mosquitoes. Methods In a cohort of uncomplicated malaria patients attending Kalongo Hospital in northern Uganda, we quantified carriage of PfKelch13 mutant parasites by conventional sequencing and droplet digital PCR (ddPCR) for the C469Y and A675V mutations. Prevalence and density of gametocytes and ring-stage parasites were assessed by microscopy and quantitative reverse-transcriptase PCR (qRT-PCR). Lumefantrine concentrations, indicative of prior malaria treatment, were determined by ultra-high performance liquid chromatography-tandem mass spectrometry. Transmission potential of wild-type and PfKelch13 mutant parasites was assessed by mosquito feeding assays and complemented with molecular characterization of parasites in wild-caught mosquitoes from household resting catches. Findings We enrolled 235 patients with symptomatic P. falciparum infection; PfKelch13 C469Y or A675V mutations were detected in 35.8% (78/218) of infections by sequencing and 59.1% (136/230) by ddPCR. Gametocyte carriage was 24.0% (56/233) by microscopy and 56.6% (133/235) by qRT-PCR and not associated with the abundance of PfKelch13 mutant parasites by ddPCR (p=0.603). Among a total of 227 mosquito feeds with patient whole blood, 1.4% (120/8745) of mosquitoes became infected. Mosquito infection rates were positively associated with gametocyte density (β = 0.39, 95% CI = 0.23-0.59, p < 0.001) without an observed interaction with the abundance of PfKelch13 mutant parasites (p = 0.452). PfKelch13 C469Y or A675V mutations were detected in 40.1% (21/52) of malaria-infected bloodmeals of field-caught mosquitoes and in 28.0% (7/25) of sporozoite-positive mosquitoes. Interpretation We conclude that pfkelch13 mutations are very common in patients in northern Uganda with uncomplicated malaria, mostly in multiclonal infections. We observed no evidence that ART-R affected gametocyte production or transmission to mosquitoes. Funding Dutch Research Council (NWO) Research in context Evidence before this study Partial resistance to artemisinins (ART-R) might enhance or inhibit gametocyte formation or transmission of malaria parasites to Anopheles mosquitoes. However, few studies directly assessed P. falciparum transmission potential in relation to sensitivity to ART-R or associated mutations. We searched PubMed on January 14th 2026, with no restrictions on publication date or language, for studies assessing gametocyte carriage or transmissibility to mosquitoes in uncomplicated malaria cases in relation to artemisinin resistance using the search terms (“uncomplicated malaria” OR “patient”) AND (“gametocyte” OR “anopheles”) AND ((“artemisinin” AND “resistance”) OR “pfkelch13” OR “kelch13”). From the 101 identified articles, the majority did not report on parasite resistance or resistance markers. Nineteen articles reported original patient data, with four additional studies examining parasite isolates for gametocyte production and transmissibility in vitro . In vitro studies all reported gametocyte formation in parasite isolates with pfkelch13 mutations and, for three studies where transmission was directly determined, evidence for successful mosquito infections from ART-R parasite isolates. None of these studies demonstrated consistent differences in gametocyte production or transmissibility between ART-R and sensitive parasite isolates. One clinical study from Thailand observed higher levels of gametocyte carriage during follow-up for infections with slow asexual blood-stage parasite clearance following artemisinin-combination therapy (ACT) and an increase in gametocyte carriage at clinical presentation over a period when the prevalence of ART-R was rising. A study with patient data from 7 Asian and 3 African countries reported higher proportions of pretreatment and post-treatment gametocytemia in patients with slow parasite clearance. Another study from Cambodia reported a higher prevalence of gametocytes at enrolment in areas affected by ART-R, but no association between gametocyte carriage and either individual-level parasite clearance or treatment failure. In contrast, a meta-analysis of ACT treatment efficacy in pregnant women observed no association between gametocyte carriage at baseline and treatment failure. Only one study, directly examined gametocyte carriage at clinical presentation with pfkelch13 mutations and found no association between gametocyte carriage and molecular markers of ART-R or ex vivo drug sensitivity in Cambodia. Added value of this study We performed a direct assessment of gametocyte production, carriage of mature gametocytes and transmission to Anopheles gambiae s.s. mosquitoes in relation to validated ART-R markers in patients presenting with uncomplicated P. falciparum malaria in an area affected by ART-R in Uganda. Our work demonstrates that parasites with the PfKelch13 C469Y and A675V mutations were very common, mostly in multiclonal infections. Gametocyte carriage and gametocyte commitment were similar between patients presenting with pure or predominantly wild-type infections and those presenting with PfKelch13 mutant infections. In feeding studies, mosquito infection rates were similar for patients with wild-type or PfKelch13 mutant infections; wild-caught mosquitoes confirmed ongoing transmission of parasites with molecular signatures of ART-R. Implications of all the available evidence Taken together, there are no indications for altered gametocyte production or transmissibility to mosquitoes of infections with PfKelch13 C469Y and A675V. Future studies may examine impacts of other mutations in pfkelch13 , study transmission potential in low endemic settings where monoclonal infections dominate and aim to understand how gametocyte clearance and post-treatment transmission potential may differ between ART-R and wild-type infections upon treatment.

Artemisinin-based combination therapies are the cornerstone of malaria treatment and control. In Africa, artemether-lumefantrine is the most widely used first-line artemisinin-based combination therapy, but its efficacy in Uganda is increasingly threatened by the emergence of artemisinin partial resistance and reduced lumefantrine susceptibility. To identify loci contributing to this decreased susceptibility, here we assessed signatures of selection in 157 whole-genome sequences of Plasmodium falciparum from Uganda. Although extended haplotypes were observed around Kelch13 C469Y and A675V mutations, the strongest signal of recent selection was centered on a segment of chr. 7 encoding the phosphoinositide-binding protein (PX1, PF3D7_0720700). A haplotype, represented by three PX1 mutations (L1222P, M1701I and D1705N) and two deletions (designated PIN), was first seen in 2008 and rapidly increased, reaching a prevalence >50% in northern Uganda by 2016 and eastern Uganda by 2023. PIN-carrying parasites showed significantly decreased ex vivo susceptibilities to lumefantrine, mefloquine and dihydroartemisinin, an active metabolite of artemether. A parasite strain in which px1 was disrupted in vitro showed increased susceptibility to the three drugs. Thus, PX1 polymorphisms appear to impact on the susceptibilities of African malaria parasites to key drugs.

The continuing spread of partially artemisinin-resistant Plasmodium falciparum in Africa is a health challenge that requires urgent attention. The World Health Organization has recommended multiple first-line therapies (MFT) as a response strategy. Implementing a response is critical for Uganda where four artemisinin resistance mutations are at local allele frequencies >0.20 and partner-drug efficacy may be at risk. Using a Uganda-calibrated individual-based mathematical model of P. falciparum transmission and evolution, we evaluate 53 public-sector deployment strategies for artemisinin-based combination therapies and report projected reductions in drug-resistance associated treatment failure from 2025 to 2031. Changing first-line therapy from artemether-lumefantrine (AL) to artesunate-amodiaquine (ASAQ) is projected to reduce treatment failures by 34.7% to 38.3% (90% range) while a first-line policy change to dihydroartemisinin-piperaquine (DHA-PPQ) is projected to reduce treatment failures by 10.0% to 12.9%. Optimal MFT deployments and cycling approaches balance their treatment distribution to higher ASAQ use and lower DHA-PPQ use, with projected treatment failure reduction at ~36% when compared to status quo AL use. Deployment of the triple therapy artemether-lumefantrine-amodiaquine is projected to reduce treatment failures by ~42% if enacted immediately. Increased adoption of and coverage with ASAQ is projected to play a large near-term role in reducing malaria treatment failure counts in Uganda.

ABSTRACT The emergence and spread of drug resistance threaten malaria control in Uganda. This study reports new data on key polymorphisms associated with antimalarial drug sensitivity at 32 malaria reference centers in Uganda in 2023 and 2024. Ten thousand thirty samples were collected from patients aged >6 months presenting with uncomplicated falciparum malaria and sequenced using the MAD 4 HatTeR panel and Illumina platforms; of these, 8,518 passed quality control and were analyzed. Multiple validated or candidate K13 mutations associated with artemisinin partial resistance were detected at ≥20% prevalence at one or more sites across multiple timepoints. The K13 mutations A675V and C469Y predominated in northern Uganda, P441L was most common in western Uganda, and R561H and C469F were confined to southwestern Uganda. After rapid increases in earlier years, prevalences of K13 mutations plateaued at most sites, with A675V, C469Y, and P441L reaching maximum site prevalences of 40%, 58%, and 46%, respectively. Prevalence of the chloroquine resistance marker CRT K76T was generally low but increased substantially at three sites in northwestern Uganda, rising to 38% at the last timepoint, with CRT H97L, newly reported in Africa, following a similar trend. The antifolate resistance quintuple mutant haplotype remained highly prevalent nationwide. In addition, DHPS A581G and DHFR I164L, markers of higher-level antifolate resistance, were most common in southwestern Uganda with prevalences up to 64% and 76%, respectively; DHFR I164L also expanded into central and eastern regions. These results highlight continued geographic heterogeneity and underscore the need for continued, nationwide molecular surveillance to guide treatment and chemoprevention policies.

The emergence of Plasmodium falciparum parasites partially resistant to artemisinins (ART-R) poses a significant threat to recent gains in malaria control. ART-R has been associated with PfKelch13 (K13) mutations, which differ in fitness costs. This study investigates the gametocyte production and transmission fitness of African and Asian P. falciparum isolates with different K13 genotypes across multiple mosquito species. We tested three ART-sensitive (ART-S) isolates (NF54, NF135, and NF180) and three ART-R isolates (ARN1G, 3815, and PAT-023) for sexual conversion and transmission to Anopheles stephensi, An. gambiae, and An. coluzzii. ART-R levels were quantified in vitro using the Ring-stage Survival Assay (RSA), and the transmission-reducing effects of dihydroartemisinin (DHA) on mature gametocytes were assessed. Results showed that ART-S parasite lines consistently produced gametocytes and transmitted effectively in all three mosquito species. ART-R isolates showed variability: ARN1G maintained high transmission levels, whereas 3815 showed limited transmission potential despite higher sporozoite loads in An. coluzzii. The African ART-R isolate PAT-023 demonstrated low gametocyte commitment but was transmitted efficiently in both An. gambiae and An. coluzzii. DHA exposure reduced mosquito infectivity for all isolates, regardless of K13 genotype. These findings, based on a limited number of field isolates, suggest that ART-R parasites remain transmissible across different Anopheles species. However, ART-R does not appear to confer a direct transmission advantage. This study highlights the complexity of ART-R dynamics and underscores the need for further research to inform malaria control strategies in regions where ART-R parasites are circulating.

Background:The treatment and control of malaria in Africa is challenged by drug resistance, including Plasmodium falciparum transporter, folate pathway, and PfK13 mutations that mediate resistance to aminoquinolines, antifolates, and artemisinins, respectively. Characterization of drug susceptibility informs optimal control strategies. Methods:We characterized ex vivo susceptibilities to nine drugs of isolates collected from individuals presenting with uncomplicated falciparum malaria in eastern (2019-2024) and northern (2021-2024) Uganda using a growth inhibition assay and the dihydroartemisinin (DHA) ring survival assay (RSA). Genetic polymorphisms were characterized by molecular inversion probe and dideoxy sequencing. We assessed drug susceptibilities over time and evaluated associations between susceptibilities and potential resistance markers for samples studied since 2016. Results:Of 1,297 collected, 724/828 eastern and 390/469 northern Uganda isolates were successfully evaluated for ex vivo drug susceptibilities. Median half-maximal inhibitory concentrations (IC 50 s) were low-nanomolar for chloroquine, monodesethylamodiaquine, piperaquine, pyronaridine, lumefantrine, mefloquine, and DHA, but higher for quinine and pyrimethamine. Over time, susceptibilities improved for chloroquine, decreased for lumefantrine, mefloquine, and DHA, and were unchanged for other drugs. Changes in prevalences of known markers of altered drug susceptibility followed the same patterns. Genotypes associated with drug susceptibility were those previously identified for aminoquinolines and pyrimethamine. For lumefantrine, susceptibility was decreased with wild-type PfCRT K76T or PfMDR1 N86Y, mutant PfK13 C469Y or A675V, the newly identified PfCARL D611N mutation, which increased in prevalence over time, and a number of other polymorphisms. For DHA, RSA results were not associated with PfK13 mutations, but susceptibilities based on IC 50 s were decreased in parasites with the PfK13 C469Y or A675V mutations and the newly identified PfMDR1 Y500N mutation. Interpretation:Susceptibilities to antimalarial drugs were mostly excellent, but decreased activities of lumefantrine and DHA over time suggest potential loss of efficacies of leading regimens. Funding:National Institutes of Health, Medicines for Malaria Venture, Gates Foundation. Research in Context:Evidence before this study: We searched PubMed for combinations of the terms "antimalarial resistance", "malaria", " Plasmodium ", "Africa", "ex vivo", "pfmdr1", "pfcrt", "kelch", or "K13" and identified papers published between Jan 1, 2020, and Dec 30, 2024 on antimalarial drug sensitivity and resistance in Africa. A prior identical search was conducted for papers published from Jan 1, 2000 to Dec 31, 2020 in preparation for an earlier publication. We reviewed and included any relevant articles cited in those references. Our search identified many studies on antimalarial drugs and molecular markers of resistance, but few combining ex vivo drug susceptibility with genotyping results. Added value of this study: This study provides a comprehensive assessment of ex vivo susceptibility of Ugandan Plasmodium falciparum parasites to nine antimalarial drugs from July, 2019 to June, 2024. It also characterized genotype-phenotype associations based on these ex vivo data and sequencing of 80 genes identified as potential resistance mediators. Our findings add value to the existing literature by providing comprehensive data on antimalarial drug susceptibility in Uganda, including ex vivo drug susceptibilities for >1100 isolates from two regions of the country, description of changes in drug susceptibilities over time, and characterisation of genotype-phenotype associations, considering genetic polymorphisms previously associated with resistance to various antimalarials and potential novel resistance mediators. Implication of all the available evidence: Malaria parasites circulating in eastern and northern Uganda over the past five years were mostly sensitive to commonly used antimalarial drugs. However, parasite genotypes and phenotypes have changed over time. Most importantly, susceptibilities to dihydroartemisinin and lumefantrine, the components of the first-line antimalarial therapy in Uganda, have decreased over time, although the magnitudes of these decreases are modest, and the clinical implications of the results are uncertain. Continued performance of parasitological and genomic surveillance for evidence of antimalarial drug resistance and institution of policy changes to limit resistance selection and treatment failure should be high priorities.

Targeted amplicon sequencing is a powerful and efficient tool for interrogating the Plasmodium falciparum genome, generating actionable data from infections to complement traditional malaria epidemiology. For maximum impact, genomic tools should be multi-purpose, robust, sensitive, and reproducible. We developed, characterized, and implemented MAD4HatTeR, an amplicon sequencing panel based on Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing, along with a bioinformatic pipeline for data analysis. Additionally, we introduce an analytical approach to detect gene duplications and deletions from amplicon sequencing data. Laboratory control and field samples were used to demonstrate the panel’s high sensitivity and robustness. MAD4HatTeR targets 165 highly diverse loci, focusing on multiallelic microhaplotypes, key markers for drug and diagnostic resistance (including duplications and deletions), and CSP and potential vaccine targets. The panel can also detect non-falciparum Plasmodium species. MAD4HatTeR successfully generated data from low-parasite-density dried blood spot and mosquito midgut samples and detected minor alleles at within-sample allele frequencies as low as 1% with high specificity in high-parasite-density dried blood spot samples. Gene deletions and duplications were reliably detected in mono- and polyclonal controls. Data generated by MAD4HatTeR were highly reproducible across multiple laboratories. The successful implementation of MAD4HatTeR in five laboratories, including three in malaria-endemic African countries, showcases its feasibility and reproducibility in diverse settings. MAD4HatTeR is thus a powerful tool for research and a robust resource for malaria public health surveillance and control.

The recent discovery of genetic mutations in Plasmodium falciparum —the most lethal malaria parasite—that enable it to overcome the protective effects of sickle cell trait, raises fundamental questions about the underlying biological and evolutionary interactions. Here we develop a geostatistical model to compare sickle haemoglobin genotype frequencies to the Plasmodium falciparum sickle-associated alleles across global populations, and find a robust association at multiple geographical scales, implying that sickle drives positive selection for these parasite mutations. A model of parasite evolution and an analysis of local haplotype patterns suggest that key features of these mutations – that they are polymorphic in all African populations and are mutually correlated despite lying in different genome regions - are caused by geographical variation in selection pressure, and that the alleles may have been maintained by balancing selection over timescales comparable to the age of the sickle mutation itself. The predicted impact of this host-parasite interaction on disease outcomes varies widely across populations, and functional data are needed to discover the biological mechanisms involved. ### Competing Interest Statement The authors have declared no competing interest. Wellcome Trust, https://ror.org/029chgv08, 304926/Z/23/Z National Natural Science Foundation of China, T2350610281, 82273731 Zhejiang University Education Foundation Global Partnership Fund, 188170-11103

The treatment and control of malaria in Africa is challenged by drug resistance. We characterized ex vivo susceptibilities to nine drugs of isolates collected from individuals presenting with uncomplicated falciparum malaria in eastern (2019-2024) and northern (2021-2024) Uganda and performed deep sequencing, with analysis of 80 Plasmodium falciparum genes, to evaluate associations between susceptibilities and potential resistance markers for samples studied since 2016. For 1114 evaluated isolates, median half-maximal inhibitory concentrations (IC50s) were low-nanomolar for chloroquine, monodesethylamodiaquine, piperaquine, pyronaridine, lumefantrine, mefloquine, and DHA, but higher for quinine and pyrimethamine. Over time, susceptibilities improved for chloroquine, decreased for lumefantrine, mefloquine, and DHA, and were unchanged for other drugs. Changes in prevalences of known markers of altered drug susceptibility followed the same patterns. Genotypes associated with drug susceptibility were those previously identified for aminoquinolines and pyrimethamine. For lumefantrine, susceptibility was decreased with wild-type PfCRT K76T or PfMDR1 N86Y, mutant PfK13 C469Y or A675V, mutant PfCARL D611N, and other polymorphisms. For DHA, susceptibility was decreased with the PfK13 C469Y or A675V and PfMDR1 Y500N mutations. Decreasing activities of lumefantrine and DHA suggest potential loss of efficacies of leading regimens, although the clinical consequences of these changes are, to date, uncertain.

The coronavirus disease 2019 pandemic showcased the power of genomic surveillance in tracking infectious diseases, driving rapid public health responses, and global collaboration. This same infrastructure is being leveraged for malaria molecular surveillance (MMS) in Africa to address challenges such as artemisinin partial resistance and deletions in the Plasmodium falciparum histidine-rich protein 2 and 3 genes. However, variability in reporting sequencing methods and data reporting currently limits the validation, comparability, and reuse of data. To maximize the impact of MMS, minimal and optimal data that are key for validation and maximizing transparency and findable, accessible, interoperable, and reusable principles are proposed for reporting. Rather than focusing on specific data formats, in the current study, the authors propose what should be reported and why. Progressing to reporting individual infection-level polymorphism or microhaplotype data is central to maximizing the impact of MMS. Reporting must adhere to local regulatory practices and ensure proper data oversight and management, preventing data colonialism and preserving opportunities for data generators. With malaria's challenges transcending borders, reporting and adopting standardized practices are essential to advancing research and strengthening global public health efforts.

Histidine Rich Protein 2 (HRP2)/pan-Lactate Dehydrogenase (pLDH) combination rapid diagnostic tests (RDTs) may address the shortcomings of RDTs that detect HRP2 alone. However, the relative contribution of the possible causes of discordant results (RDT-negative and microscopy-positive) and performance in field settings across Uganda are poorly quantified. This study utilized samples from two cross-sectional surveys conducted in 32 districts at 64 sites across Uganda between November 2021 and March 2023 that enrolled 6354 febrile participants ≥ two years of age. Discordant samples (negative by HRP2/pLDH RDT and positive by microscopy) underwent quantitative PCR (qPCR) to detect and quantify parasitaemia. Those confirmed to be positive for Plasmodium falciparum at > 1 parasites/microlitre (p/µL) were tested for pfhrp2 and pfhrp3 deletions using digital PCR. Those that were negative or had P. falciparum detected at ≤ 1 p/µL underwent Plasmodium species testing using nested PCR. The performance of the Bioline Malaria Ag P.f/Pan combination RDT was evaluated by comparison with microscopy and qPCR. There were 166 (8.4

Molecular surveillance of drug-resistant Plasmodium falciparum is crucial for malaria control in endemic regions. Two targeted-resequencing tools, the Molecular Inversion Probe (MIP) drug resistance panel DR23K and the Multiplexed Amplicons for Drugs, Diagnostics, Diversity, and Differentiation using High-Throughput Targeted Resequencing (MAD4HatTeR) panel, are widely used to detect resistance genotypes. However, comparisons of their performance for genotyping drug resistance polymorphisms in malaria parasites and their comparative utility for other use cases is lacking. To compare the performance of DR23K and MAD4HatTeR in terms of sequencing depth, sensitivity to minor alleles, and precision, each platform was used to evaluate SNP alleles and microhaplotypes in double- and triple-strain mixtures of well-characterized laboratory parasites at densities of 10, 100, 1000, and 10,000 parasites/μL. In addition, 67 Ugandan field samples collected in 2022 were genotyped using each platform to assess performance and concordance. Across the four parasite densities of 10, 100, 1000, and 10,000 parasites/μL, MAD4HatTeR exhibited superior sequencing depth (mean reads per locus: 144, 992, 1153, and 1300) compared to DR23K (mean unique molecular identifiers [UMIs] per locus: 1, 4, 49, and 364). For SNP detection, MAD4HatTeR achieved 100

BACKGROUND:Anti-malarial artemisinin-based combination therapies (ACTs) might be losing efficacy in east Africa, with the spread of artemisinin partial resistance and reduced partner drug activity. Our trial aimed to measure the efficacies of artemether-lumefantrine, artesunate-amodiaquine, dihydroartemisinin-piperaquine, and artesunate-pyronaridine in three sites in Uganda. METHODS:This randomised, open-label, phase 4 clinical trial was carried out at three sites in the Agago, Arua, and Busia districts of Uganda. Children aged 6 months to 10 years with uncomplicated Plasmodium falciparum malaria were randomly assigned to receive either artemether-lumefantrine (20 mg artemether; 120 mg lumefantrine; twice a day for 3 days) in all sites or dihydroartemisinin-piperaquine (40 mg dihydroartemisinin and 320 mg piperaquine, once a day for 3 days) in Agago, artesunate-amodiaquine (25 mg artesunate and 67·5 mg amodiaquine for children <9 kg or 50 mg artesunate and 135 mg amodiaquine for children ≥9 kg, once a day for 3 days) in Busia; and artesunate-pyronaridine (60 mg artesunate and 180 mg pyronaridine for children >15 kg or 20 mg artesunate and 60 mg pyronaridine for children <15 kg, once a day for 3 days) in Arua, with follow-up to 42 days. Participants were not blinded to group assignments; however, investigators and those assessing outcome were masked. The primary outcome was parasitaemia, assessed by microscopy, either uncorrected or PCR-corrected to distinguish recrudescence from new infection. All participants who received the treatment per protocol and were not lost to follow-up were included in the primary outcome. All participants who were randomly allocated to treatment groups were included in the safety analyses. This study is registered with the Pan African Clinical Trials Registry, number PACTR202301796134887, and is complete. FINDINGS:Between Nov 7, 2022, and March 24, 2023, 808 participants (437 [54%] female) were enrolled and assigned to treatment groups; 15 (2%) were lost to follow-up and 793 (98%) completed follow-up. The uncorrected adequate clinical and parasitological response for artemether-lumefantrine was 87 (51·8%; 95% CI 44·0-59·5) of 168 participants in Arua, 88 (51·8%; 44·0-59·4) of 170 and Busia, and 131 (79·4%; 72·3-85·1) of 165 in Agago. This response for artemether-lumefantrine was lower than that of the other ACTs at all sites: 97 (98·0%; 92·2-99·6) of 99 for dihydroartemisinin-piperaquine in Agago, 95 (99·0%; 93·5-99·9) of 96 for artesunate-amodiaquine in Busia, and 73 (73·7%; 63·8-81·8) of 99 for artesunate-pyronaridine in Arua. PCR-corrected 28-day efficacies were 88 (81·5%; 72·6-88·1) of 108 for artemether-lumefantrine and 95 (100%; 95·2-100·0) of 95 for artesunate-amodiaquine in Busia; 131 (97·0%; 92·1-99·0) of 135 for artemether-lumefantrine and 97 (100%; 95·3-100·0) of 97 for dihydroartemisinin-piperaquine in Agago; and 87 (82·1%; 73·2-88·6) of 106 for artemether-lumefantrine and 73 (92·4%; 83·6-96·9) of 79 for artesunate-pyronaridine in Arua. All regimens were well tolerated. The most common adverse events were upper respiratory tract infection, diarrhoea, and anaemia. None of the reported adverse events were attributed to the study drugs. There were two serious adverse events, both cases of severe malaria in Arua, one in each of the treatment groups. Parasite clearance half-lives were prolonged with parasites carrying the PfK13 Cys469Tyr (median 4·2 h; IQR 3·4-4·9) and Ala675Val (4·9 h; 3·4-5·7) mutations compared with wild-type parasites (2·8 h; 2·3-3·6; p<0·0001). INTERPRETATION:Artemether-lumefantrine was associated with a higher risk of recurrent malaria than other antimalarial combinations tested, and K13 mutations were associated with delayed parasite clearance. Changes in first-line therapy for uncomplicated malaria must be considered in response to suboptimal efficacy of artemether-lumefantrine. FUNDING:US President's Malaria Initiative, US Agency for International Development, through the Uganda Malaria Reduction Activity and the National Institutes of Health (AI075045 and AI117001). TRANSLATION:For the Swahili translation of the abstract see Supplementary Materials section.

Introduction Plasmodium falciparum histidine-rich protein 2/3 (pfhrp2/3) gene deletions threaten the effectiveness of HRP2-based malaria rapid diagnostic tests (RDTs), which are the primary malaria diagnostic tool in Uganda. Methods We collected 200 dried blood spot (DBS) samples from patients with uncomplicated malaria from 30 sentinel sites across Uganda in Jan-Apr 2023, Jul-Sep 2023, Feb-Apr 2024, and Aug-Oct 2024. From each site and round, 100 samples with parasitemia >= 1000 parasites/microliter by varATS qPCR were selected for targeted sequencing with the MAD4HatTeR assay. Data was analyzed with the MAD4HatTeR pipeline to generate allele calls data across diversity, drug and diagnostic resistance loci. Copy number variation (CNV) of pfhrp2 and pfhrp3 genes was estimated using a generalized additive model to correct for amplification bias and coverage, calculating fold-change in read depth relative to Pf3D7 controls lacking hrp2/hrp3 deletions. CNV fold change of <= 0.5 was used to screen samples for deletions in Pfhrp2&Pfhrp3 genes. Results 7,524 samples (3,999 samples in year 2023 and 3,525 samples in year 2024) passed quality filtering. Pfhrp3suspected deletions were detected in 61 samples representing 0.81% (95% CI: 0.63–1.04%) aggregated across all rounds of collections. The prevalence of Pfhrp3 single gene deletions remained very similar between 2023 and 2024 of 0.83% (95% CI: 0.59–1.16%) and 0.79% (95% CI: 0.55–1.15%) respectively. Site-level analysis revealed heterogeneous distribution, with low suspected Pfhrp3 gene deletion prevalence ranging from 0 to 4.7% across different sites and regions of the country. No suspected Pfhrp2 or Pfhrp2/3 gene deletions were observed in the samples at the set threshold. Conclusion Pfhrp2 and Pfhrp3 gene deletions remain rare in Uganda which makes the HRP2-based RDTs remain useful for diagnosis of malaria in Uganda currently. Digital PCR genotyping of samples with suspected deletions is ongoing to confirm and to estimate the true prevalences of the Pfhrp2&3 deletions.

Introduction:The use of next-generation sequencing technologies (NGS) to study parasite populations and their response and evolution to interventions is important to support malaria control and elimination efforts. While whole-genome sequencing (WGS) is optimal in terms of assessing the entire genome, it is costly for numerous samples. Targeted approaches selectively enriching for the sequence of interest are more affordable and have higher throughput but sometimes lack adequate information content for key analyses. Methods:We have developed a highly multiplexed molecular inversion probe (MIP) panel (IBC2FULL) targeting 4,264 single-nucleotide polymorphisms (SNPs) with ≥5% minor allele frequency (MAF) in Sub-Saharan African regions from publicly available Plasmodium falciparum WGS (n = 3,693). We optimized the panel alone and in combination with antimalarial drug resistance MIPs in laboratory P. falciparum strains at different parasitemias and validated it by sequencing field isolates from the Democratic Republic of Congo, Ethiopia, Ghana, Mali, Rwanda, Tanzania, and Uganda and evaluating the population structure, identity-by-descent (IBD), signals of selection, and complexity of infection (COI). Results:The new panel IBC2FULL consisted of 2,128 MIPs (containing 4,264 common SNPs) spaced by 5.1-18.4 kb across the entire genome. While these microhaplotypes were developed based on variations from Sub-Saharan African WGS data, 59.3% (2,529) of SNPs were also common in Southeast Asia. The MIPs were balanced to produce more a uniform and higher depth of coverage at low parasitemia (100 parasites/μL) along with MIPs targeting antimalarial drug resistance genes. Comparing targeted regions extracted from public WGS, we observed that IBC2FULL provided a higher resolution of the local population structure in Sub-Saharan Africa than current PCR-based targeted sequencing panels. For sequencing field samples (n = 140), IBC2FULL approximated WGS measures of relatedness, population structure, and COI. Interestingly, genome-wide analysis of extended haplotype homozygosity detected the same major peaks of selection as WGS. We also chose a subset of 305 high-performing MIPs to create a core panel (IBC2CORE) that produced high-quality data for basic population genomic analysis and accurate estimation of COI. Discussion:IBC2FULL and IBC2CORE panels have been designed to provide an improved platform for malaria genomic epidemiology and biology that can approximate WGS for many applications and is deployable for malaria molecular surveillance in resource-limited settings.

BACKGROUND:Artemisinin partial resistance, mediated by mutations in the Plasmodium falciparum kelch13 gene (k13), rapidly spread in southeast Asia, undermining the antimalarial effectiveness of artemisinin-based combination therapies. k13 mutations have also arisen in Africa, but their rates of increase are not well characterised. We aimed to quantify the selection of k13 mutations in Africa and compare the selection with that in southeast Asia. METHODS:In this modelling study, we investigated k13 mutation allele frequency at 16 sites in Uganda (2016-22) and five sites in southeast Asia (in Cambodia, Thailand, and Viet Nam; 2003-14). The Ugandan data were obtained from annual clinical surveillance studies and the southeast Asian data were obtained from the MalariaGEN Pf7 dataset. We investigated five validated and candidate k13 mutations: Pro441Leu, Cys469Phe, Cys469Tyr, Arg561His, and Ala675Val. We calculated annual selection coefficients using Bayesian mixed-effect linear models. We then tested whether the k13 mutation allele frequency in southeast Asia could have been forecast accurately using up to the first 5 years of available data and forecast future k13 mutation allele frequency in Uganda. FINDINGS:We used data from 7564 samples from Uganda and 6568 samples from southeast Asia. The annual selection coefficient of evaluable k13 mutations (Pro441Leu, Cys469Phe/Tyr, Arg561His, and Ala675Val) across all sites was estimated at 0·381 (95% credible interval 0·298 to 0·472) per year, a 38% increase in relative allele frequency. Selection coefficients across Uganda were 0·494 (-0·462 to 1·410) for Pro441Leu, 0·324 (-0·629 to 1·150) for Cys469Phe, 0·383 (0·207 to 0·591) for Cys469Tyr, and 0·237 (0·087 to 0·403) for Ala675Val. In southeast Asia, the selection coefficients were 0·627 (-0·088 to 1·312) for Cys580Tyr, 0·224 (-0·903 to 1·397) for Arg539Thr, and 0·330 (-0·075 to 0·683) for all validated k13 mutations. Compared with out-of-sample data, the forecasts for southeast Asia underestimated mutation allele frequency and were of variable accuracy. Overall, forecast allele frequencies for Uganda, assuming constant selection, neared fixation (>0·95 allele frequency) within a decade (between 2031 and 2033) for combined k13 mutations. INTERPRETATION:k13 mutation selection in Uganda was similar to that observed in southeast Asia, suggesting that frequencies of k13 mutations will continue to increase quickly in Uganda. These commensurate levels of selection indicate a high potential for rapid transmission across other parts of Africa, underscoring the urgent need for treatments and policies to mitigate the spread and impact of k13 mutations. FUNDING:US National Institutes of Health.