Malaria parasite genomes have been generated predominantly using Illumina short-read sequencing that requires expensive equipment, is time-consuming with complex protocols, and does not adequately interrogate complex genomic regions that harbour important malaria virulence determinants. The portable Oxford Nanopore Technologies MinION platform generates long reads in real time and may overcome these limitations. We present compelling evidence that Nanopore sequencing delivers valuable additional information for malaria parasites with similar data fidelity for single nucleotide variant (SNV) calls compared to standard Illumina whole-genome sequencing. We demonstrate this through sequencing of pure Plasmodium falciparum DNA, mock infections and natural isolates from low-density, asymptomatic infections. Nanopore has low error rates for haploid SNV genotyping and identifies structural variants not detected with short reads. Nanopore genomes can be directly compared to publicly available genomes and produce high-quality end-to-end chromosome assemblies including complex, previously difficult-to-access regions. Nanopore sequencing could expedite whole-genome surveillance of malaria and provide new insights into parasite genome biology.

Asymptomatic Plasmodium falciparum infections sustain malaria transmission and challenge elimination efforts. School-aged children may serve as a key reservoir, yet are often overlooked in malaria control programs. A cross-sectional survey was conducted in July 2022 across Banfora, Orodara, and Gaoua districts (Burkina Faso), enrolling 1,127 children aged from 6 months to 10 years. Malaria diagnosis was performed using HRP2-based RDT, expert microscopy, and qPCR. Children with fever (> 37.5 °C) were excluded from asymptomatic analysis. Prevalence of asymptomatic Plasmodium falciparum infection was 28.0

The World Malaria Report 2025 documents operational progress while exposing systemic vulnerabilities that could reverse two decades of gains. Several biological threats are converging in Sub-Saharan Africa: artemisinin partial resistance, parasites evading rapid diagnostic tests, insecticide-resistant mosquitoes, and the spread of Anopheles stephensi into urban areas. This convergence occurs as external funding drops sharply.The resulting situation presents parallels with the period preceding chloroquine failure across Sub-Saharan Africa. A critical difference exists: molecular surveillance tools can now detect resistance before clinical failure occurs. Whether this capacity translates into effective response will depend on decisions made over the coming years.

Abstract Background Recent reports documented catastrophic sensitivity failures (18%) of Abbott-Bioline™ Malaria Ag Pf/Pv rapid diagnostic tests (RDTs) at the Thailand-Myanmar border, prompting WHO to issue an information notice on quality concerns. We evaluated the same RDT lots under controlled laboratory conditions and in a high-transmission field setting in Madagascar to assess whether performance varied across epidemiological contexts. Methods Laboratory evaluation tested four Abbott-Bioline™ Malaria Ag Pf/Pv lots (05DDI018BH, 05DDI020BA, 05DDI041AB, 05DDI040AA) using serial dilutions of cultured Plasmodium falciparum (0–60,784 parasites/µL). Field evaluation enrolled 218 consecutive febrile patients at Ranomafana health centre, southeastern Madagascar. Performance of both RDTs (Abbott-Bioline™ Malaria Ag Pf/Pv and Parascreen® Malaria Ag Pf/Pan) was assessed against microscopy and real-time PCR as reference standards. Results In laboratory testing, substantial inter-lot variability was observed, with detection failures occurring between 97 and 373 parasites/µL. Incomplete blood migration and faint test lines were noted at lower densities. In the field, malaria prevalence was 70.2% by PCR and 48.6% by microscopy. Against microscopy, Abbott-Bioline™ Malaria Ag Pf/Pv achieved sensitivity of 99.1% (95% CI 94.9–100) and specificity of 93.7% (95% CI 87.4–97.4). Parascreen® Malaria Ag Pf/Pan showed sensitivity of 100% (95% CI 96.6–100) and specificity of 92.8% (95% CI 86.3–96.8). Against PCR, sensitivity decreased to 73.2% for Abbott-Bioline™ Malaria Ag Pf/Pv and 74.5% for Parascreen® Malaria Ag Pf/Pan, while specificity remained 98.5% for both tests. No significant difference was observed between RDTs (p > 0.05). Conclusions Despite using identical lots that showed 18% sensitivity at the Thailand-Myanmar border, Abbott-Bioline™ Malaria Ag Pf/Pv RDTs achieved 99.1% sensitivity in Madagascar. This difference likely reflects higher parasite densities in the Malagasy high-transmission setting (geometric mean 10,006 parasites/µL) compared to low-transmission elimination contexts. Both RDTs met WHO performance thresholds against microscopy but missed approximately 25% of PCR-positive infections. These findings demonstrate that RDT performance is highly context-dependent and underscore the need for enhanced post-deployment surveillance.

To evaluated artemisinin partial resistance (ART-R) in malaria in Burundi, during December 2023-June 2024, we studied 423 children <5 years of age with uncomplicated Plasmodium falciparum malaria in 8 health facilities in the northern part of the country. After artemether/lumefantrine treatment with only the first dose directly observed, 4.5% remained parasitemic on day 3. No pfkelch13 mutations, validated or candidate markers of ART-R, were detected. However, markers of antifolate and 4-aminoquinoline resistance were widespread: the dhfr triple mutant N51I/C59R/S108N was nearly fixed (92%), dhps double and triple mutants were common (41% and 47%), and pfcrt CVIET, associated with chloroquine and amodiaquine resistance, predominated (84%). Geographic differences occurred in day-3 positivity and haplotype frequencies. Although ART-R markers were absent, delayed parasite clearance and near fixation of multidrug-resistant haplotypes serve as a warning. Strengthened efficacy monitoring and regional molecular surveillance are urgently needed to prevent drug-resistant P. falciparum from becoming established in Burundi.

BACKGROUND:Molecular surveillance is essential to detect emerging artemisinin partial resistance (ART-R) and partner drug resistance in Sub-Saharan Africa. OBJECTIVES:To describe the prevalence of Plasmodium falciparum resistance markers in two high-burden countries using artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL): the Democratic Republic of the Congo (DRC) and Tanzania. METHODS:A total of 1254 day-0 P. falciparum-positive samples were analysed: 837 from four sentinel sites of a therapeutic efficacy study (TES) in the DRC (2017) and 417 from an intermittent preventive treatment in schoolchildren (IPTsc) trial in Handeni and Kilindi districts, Tanga Region, Tanzania (2020-2021). Pfkelch13, Pfcrt and Pfmdr1 were analysed by Illumina MiSeq amplicon sequencing. RESULTS:Reliable sequences were obtained for 1193 isolates. The Pfkelch13 wild-type allele predominated (98.4%); none of the four non-synonymous mutations detected (N489Y, K568T, A578S, V589I) are classified as validated, candidate or potential ART-R markers, and the validated markers R561H, P441L and C469Y reported elsewhere in East Africa were absent. Pfcrt K76T was found in 23.3% of Tanzanian and 26.3% of DRC isolates, with substantial between-site variation in the DRC (4.3% to 93.6% at Rutshuru). Pfmdr1 haplotype profiles differed between countries: NFSND predominated in Tanzania (71.5%) while NYSND remained the most frequent in the DRC (60.9%); 86Y was twice as frequent in the DRC (11.1%) as in Tanzania (6.2%). CONCLUSIONS:No validated ART-R marker was detected, but partner-drug haplotype distributions reflected the first-line ACTs used in each country. Continued molecular surveillance is needed to track these signatures alongside the recent emergence of ART-R.

BACKGROUND:Malaria in pregnancy is a major cause of maternal and neonatal morbidity in sub-Saharan Africa. Intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP), reduces malaria-associated adverse outcomes but routine diagnostics miss low-density infections, and coverage remains suboptimal in many endemic settings. METHODS:We conducted an observational study (June 2023-June 2024) at a health centre in Koubri, central Burkina Faso, combining a cross-sectional assessment at the first antenatal care (ANC) visit with retrospective abstraction of routine ANC and delivery records. At enrolment, pregnant women were tested by real-time PCR; microscopy and rapid diagnostic test (RDT) were performed when available. IPTp-SP doses, follow-up testing, and birth outcomes were extracted from facility registers. Modified Poisson regression with robust variance was used to identify predictors of PCR-confirmed infection, factors associated with low birthweight (LBW, <2500 g) and the association between cumulative IPTp-SP doses and PCR positivity. RESULTS:At first ANC, PCR detected Plasmodium infection in 30.1% (155/515) of women. Among 183 women tested by all three methods, PCR prevalence was 23.5% versus 8.2% by microscopy and 8.7% by RDT, with approximately two-thirds of infections missed by routine diagnostics. Primigravidae had twice the infection risk of multigravidae (aPR 2.14, 95% CI 1.21-3.79),). Each additional IPTp-SP dose was associated with lower infection prevalence (aPR 0.89, 95% CI 0.80-0.99). Most women (78.6%) received ≥ 3 doses, but only 18.9% reached ≥ 5 doses, Among 225 deliveries, LBW prevalence was 14.7% ( PCR-confirmed infection at first ANC was associated with increased LBW risk (aPR 1.52, 95% CI 1.01-2.28). CONCLUSION:Routine ANC diagnostics substantially underestimated malaria prevalence among pregnant women Higher IPTp-SP dosing was associated with reduced infection during pregnancy. but few women achieved five or more doses. Primigravidae and women living far from facilities had both lower coverage and higher infection risk. Earlier and more frequent ANC attendance, is essential to maximize the protective potential of IPTp-SP.

Therapeutic efficacy studies (TESs) are the standard to evaluate antimalarial drug efficacy and guide malaria treatment policy. TESs are particularly relevant now, with resistance to first-line regimens emerging in sub-Saharan Africa. For TESs, a range of parasite genotyping and data analyses are available for genotype correction, a process to distinguish whether recurrent parasitemia after therapy is due to recrudescence of initially infecting parasites (treatment failure) or a new infection. The choice of methods for laboratory genotyping and data analyses can have a large effect on how outcomes are classified, and thereby on trial results. The currently recommended and most widely used laboratory and analytical methods for TES genotyping do not incorporate recent methodological advances and can produce biased results. As such current TES results can be difficult to interpret, especially in areas with high malaria transmission, such as much of sub-Saharan Africa. Thus, improving the accuracy and reliability of TES genotyping and data analysis are a major priority. To that end, we present target product profiles that outline key specifications for genetic data generation, processing, and data analysis, with the goal of creating rigorous and consistent community standards. Primary recommended specifications for laboratory methods include high sensitivity, specificity, and reproducibility, and guidance on the number and genetic diversity of targets; criteria which are best and likely only met by amplicon sequencing. Primary recommendations for data analysis methods include high classification accuracy, accounting for errors in genotyping, and accounting for alleles matching by chance. All laboratory and data analysis methods used should be systematically validated and publicly documented so that TES results, which have major policy implications, can be relied upon for sound programmatic decision making.

Genomic data are increasingly leveraged to enable malaria genomic surveillance, with many teams now generating Plasmodium falciparum sequence data for routine analysis. However, downstream analysis remains dependent on ad-hoc scripts and local infrastructure. This reliance limits reproducibility, and scalability, while obstructing the deployment of standardized pipelines across diverse high-performance computing (HPC) and cloud environments. This review describes the main workflow management systems and bioinformatics tools that are currently used for malaria genomic surveillance, with potential applicability to P. falciparum. We focus on the four most prominent workflow management systems (Nextflow, Snakemake, WDL/Cromwell, and Galaxy) and the core components of established pipelines in the field. Therefore, we summarize which tools are most widely used and how they are assembled into end-to-end workflows for surveillance. We organize these tools and pipelines around eight standardized PlasmoGenEPi use cases and propose a four-layer framework for pipeline design (data, pipeline structure, software environment, execution platform). Building on this, we introduce a pragmatic decision matrix that links sample volumes, infrastructure profiles and surveillance objectives to concrete choices of sequencing strategy and workflow system. We then discuss practical challenges that directly affect implementation in endemic settings, including heterogeneous computing infrastructure, long-term pipelines maintenance, staff turnover, and the systemic vulnerability of shared infrastructure, notably exposed by the recent VEuPathDB funding crisis. Finally, we highlight emerging directions, including community efforts such as PlasmoGenEPi, the increasing use of targeted Oxford Nanopore amplicon sequencing, and the move towards more standardized, portable and well-documented workflows that can be adapted to other pathogen surveillance systems.

BACKGROUND:Artemisinin partial resistance (ART-R) has been confirmed in four sub-Saharan African countries since 2020, but evidence from Ethiopia is limited to molecular surveys without phenotypic confirmation. We aimed to determine whether ART-R met WHO criteria in Ethiopian Plasmodium falciparum populations during 2024-25, integrating day-3 parasite positivity after artemether-lumefantrine treatment, Pfkelch13 genotyping, and the ring-stage survival assay (RSA)0-3 h on culture-adapted field isolates. METHODS:We conducted a prospective, multisite, surveillance study at five sentinel health facilities in Ethiopia (Rama, Werkamba, Mehoni, Bako, and Metehara). Patients aged 6 months or older with uncomplicated P falciparum malaria confirmed by microscopy received artemether-lumefantrine according to bodyweight (six doses during 3 days). Pfkelch13 was genotyped by Nanopore sequencing and Pfhrp2/Pfhrp3 deletions were assessed by quantitative PCR. The RSA0-3 h was performed on culture-adapted field isolates. Follow-up occurred on days 0 and 3. The main outcome was day-3 parasite positivity rate (defined as microscopically detectable asexual P falciparum parasitaemia on day 3 after initiation of artemether-lumefantrine). This study is registered with ClinicalTrials.gov, NCT07527182 (completed). FINDINGS:Patients were enrolled from June 6 to Dec 8, 2024, during the 2024 P falciparum transmission season at all five sentinel sites and from July 1 to Nov 8, 2025, during the 2025 transmission season at Werkamba. 3207 febrile patients were assessed for malaria, of whom 2771 were excluded and 436 (14%) were P falciparum-positive on microscopy. 277 (64%) patients were enrolled, of whom 153 (55%) returned for the day-3 parasitological assessment and 124 (45%) were lost to follow-up. 172 (62%) of 277 patients were male and 105 (38%) were female. The median age was 20·0 years (IQR 10·0-30·0). 243 (88%) had P falciparum monoinfection by PCR and 34 (12%) had P falciparum and Plasmodium vivax co-infections undetected by microscopy at enrolment. The day-3 parasite positivity rate was 19·6% ([95% CI 14·1-26·6] in 30 of 153 patients with available data). 26 (9%) of 277 enrolled patients carried a validated Pfkelch13 mutation and were positive on day 3, exceeding the WHO 5% threshold for confirmation of ART-R. A strong age-dependent gradient was observed in a post-hoc analysis, with six (38%) of 16 patients younger than 5 years, 14 (35%) of 40 aged 5-15 years, and ten (10%) of 97 older than 15 years (p=0·00035). R622I was detected in 143 (53%) samples of 271 genotyped isolates. Carrying an R622I mutation was associated with day-3 positivity (26 [33%] of 79) in a univariate analysis (crude odds ratio [OR] 7·85 [95% CI 2·58-23·91]; p=0·0003). After adjustment, the association remained strong and independent (adjusted OR 9·96 [95% CI 3·39-36·35]; p<0·0001). Of 70 P falciparum field isolates on day 0 collected for culture adaptation, only six were successfully maintained. Five R622I isolates carried the Pfkelch13 R622I mutation and exceeded the 1% in vitro threshold for ART-R (mean survival rates of 1·41% [SD 0·27] for EW04, 2·69% [0·90] for EW14, 1·07% [0·52] for EW23, 3·76% [0·35] for EW38, and 1·29% [0·04] for EW56). Pooled with the wild-type strains, all five R622I isolates exceeded the 1% threshold compared with three wild-type isolates that did not exceed this threshold (p=0·018). Pfhrp3 was the most frequent deletion (39·8% [95% CI 32·6-47·4]; in 66 of 166 patients), followed by double Pfhrp2/Pfhrp3 deletion (23·5% [17·7-30·5]; in 39), wild-type (23·5% [17·7-30·5]; in 39), and Pfhrp2 deletion (13·3% [8·9-19·3]; in 22). R622I prevalence was similar across all four deletion categories (range 42-56%; p=0·47). INTERPRETATION:Ethiopia is the fifth sub-Saharan African country now meeting WHO confirmation criteria for ART-R. High R622I prevalence and double deletion rates, consistent with distinct selective pressures, represent a dual threat to treatment and HRP2-based diagnosis of P falciparum malaria in this region. FUNDING:Fondation pour la Recherche Médicale, Institut Universitaire de France, Agence Nationale de la Recherche, Université de Strasbourg, and the National Natural Science Foundation of China. TRANSLATION:For the Amharic translation of the abstract see Supplementary Material section.

BACKGROUND:Serological surveillance is a vital component in the control and elimination of Plasmodium vivax, particularly during low-transmission or elimination phases. Immunoassays are core tools for malaria serological surveillance. Among them, protein arrays, dot blot, enzyme-linked immunosorbent assay (ELISA), and Western blot are widely used, each with distinct diagnostic and operational characteristics. METHODS:We evaluated a recombinant His-tagged PvMSP1-42 protein using sera from immunized mice, P. vivax-infected patients, and healthy individuals across the platforms under standardized conditions. RESULTS:All immunoassays demonstrated high reproducibility but varied in sensitivity and sample requirements. Protein arrays enabled a broad detection range (3.13-200 ng/μl) with minimal protein and antibody use, with performance further enhanced using a protein stabilizing diluent. Dot blot and Western blot exhibited similar detection thresholds (6.25-200 ng/μl), although dot blot showed improved sensitivity at low serum dilutions when using Protein Microarray Spot Diluent. ELISA achieved the highest sensitivity, detecting specific antibodies at dilutions up to 1:128,000, but plateaued beyond 50 ng/μl of antigen. Apart from the dot blot, the other methods demonstrated both sensitivity and specificity exceeding 90%, whereas dot blot sensitivity remained lower at 80%. CONCLUSIONS:Protein arrays offered a favorable balance between sensitivity, throughput capability and sample conservation. ELISA was the most effective for detecting low antibody titers. Dot blot and Western blot remained useful, depending on resource availability and experimental goals. These findings provide a practical reference for selecting appropriate immunoassay platforms for malaria serology and for developing scalable serosurveillance strategies in elimination-phase settings.

Malaria remains a health problem, with Plasmodium falciparum accounting for 96% of cases in Africa and 15% in Brazil. The growing threat of drug resistance to artemisinin-based combination therapies (ACTs) jeopardizes progress toward elimination. This study examined P. falciparum samples collected from 141 patients in Brazil (2013-2023) by PCR and DNA sequencing to identify single-nucleotide polymorphisms in the pfcrt, pfmdr1, and pfk13 genes. Half of the samples carried the SVMNTMCGI haplotype in pfcrt, and none of the samples showed C350R mutations. In pfmdr1, the NYCDY haplotype was dominant (70%), with low occurrences of N86Y (4%) and no Y184F polymorphisms. No mutations linked to artemisinin partial resistance were detected in pfk13. Only one Amazonas sample exhibited wild-type haplotypes across all genes. Genetic diversity was more pronounced in pfcrt than pfmdr1, reflecting selective drug pressure. Significant linkage disequilibrium (LD) was observed within pfcrt (C72S and K76T) and pfmdr1 (S1034C and N1042D), but not between the two genes. The absence of pfk13-resistant mutations and the low prevalence of key pfmdr1 markers support the efficacy of ACTs. The persistence of diverse haplotypes and intragenic LD reflects ongoing drug pressure, underscoring the need for continuous genetic surveillance to anticipate emerging resistance.

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.

We conducted a hospital-based cross-sectional study of Plasmodium falciparum in 2017 in southeastern Sudan. Among 257 P. falciparum samples, we found 22% harbored the pfkelch13 R622I mutation and 10.7% showed hrp2/3 gene deletions. Our findings highlight the urgent need for enhanced surveillance of drug- and diagnostic-resistant parasites in the Horn of Africa.

Since 2012, the WHO has recommended a single low dose of primaquine (SLDPQ, 0.25 mg/kg) alongside artemisinin-based combination therapies (ACTs) to block Plasmodium falciparum transmission and combat artemisinin resistance. Despite its proven benefits, SLDPQ adoption in African malaria policies remains limited. We conducted a systematic review of studies published between 2012 and 2023 on the safety, efficacy and implementation of SLDPQ in Africa. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, we searched 7 databases and screened 819 records. Eligible studies focused on SLDPQ co-administered with ACTs for treating uncomplicated P. falciparum malaria in African contexts. Data were extracted and analysed from 41 studies, including 15 randomised controlled trials (RCTs) and 26 non-trial studies. SLDPQ was found to be safe and well-tolerated, including in glucose-6-phosphate dehydrogenase deficiency individuals and children under 5. Eight RCTs confirmed significant reductions in gametocyte carriage, validating SLDPQ’s individual-level efficacy. However, evidence on community-level impact remains limited. Key implementation barriers include persistent misconceptions about primaquine toxicity, absence of paediatric formulations and operational challenges in health systems. Most studies used the WHO-recommended dose (0.25 mg/kg), but higher doses and age-based regimens were also investigated. This review supports SLDPQ as a safe and effective tool for malaria transmission reduction in Africa. Addressing barriers to implementation, through health worker training, community sensitisation and operational research, is essential to accelerate its adoption. The ongoing Implementing Primaquine Single Low Dose in Africa project aims to generate real-world evidence across three countries, with a focus on paediatric use and health system integration. SLDPQ scale-up should be prioritised within malaria elimination strategies across sub-Saharan Africa.

Artemisinin (ART) production faces bottlenecks due to low and variable yields from its natural source, Artemisia annua. This limitation, coupled with expanding therapeutic potential beyond malaria, highlights the need for innovative production solutions. This systematic review aims to synthesize the evidence on alternative production platforms for ART. We searched PubMed, Scopus, Web of Science, and Google Scholar for studies published primarily between 2020 and 2025. Some search terms included "Artemisinin", "Artemisia annua", "biosynthesis", "in vitro culture", and "artificial intelligence". We included primary research articles reporting on strategies for ART production. We narratively synthesized data by production theme. Our review of 30 studies identified four frontiers for ART production: (1) Enhancement in A. annua ART content; (2) In vitro platforms focusing on callus and cell suspension cultures, which offer precise control but face scale-up bottlenecks; (3) Heterologous expression in non-Artemisia plants; and (4) Scalable semi-synthetic routes using microbially fermented precursors and chemical conversion. Furthermore, the review highlights the emerging role of AI-driven predictive modeling in source discovery and process optimization. By integrating these innovations, a robust roadmap exists for sustainable ART production.

Plasmodium vivax malaria is a major public health problem outside sub-Saharan Africa. However, an increasing number of P. vivax infections in Duffy-negative individuals has been reported across Africa in recent years, raising concerns that the parasites may have evolved alternative pathways to invade reticulocyte and overcome Duffy-negativity. Here, we investigated the global genetic structure and diversity of sub-Saharan African P. vivax populations, exploring possible molecular signatures of adaptation to Duffy-negative hosts. We analyzed 204 previously published P. vivax genome sequences from Africa, Southeast Asia, the Pacific Coral Triangle, and South America and generated whole-genome sequences of 133 P. vivax field isolates collected from 10 sub-Saharan African countries. Our analysis revealed four distinct geographic clusters, with clear contrasts between East/West Africa and the Indian Ocean populations. Despite the limited number of interpretable sequences from Duffy-negative individuals - attributable to low parasitemia - and the lack of clear evidence of selective pressure acting on invasion-related genes of the P. vivax parasite populations circulating in sub-Saharan Africa, our study offers valuable insights into the genetic diversity of P. vivax and lays the groundwork for future research exploring parasite adaptation to Duffy-negative hosts.

BACKGROUND:The World Health Organization (WHO) recommends parasite-based diagnosis of malaria before treatment. The use of nucleic-acid amplification (NAAT) for detection of Plasmodium spp. has expanded rapidly in recent years, for epidemiological research globally and clinical care in high-resource settings. Data from NAATs are frequently used to inform policy decisions, so quality control is essential to ensure results are reliable and comparable. Therefore, robust quality control, including an external quality assessment (EQA) scheme targeting malaria NAATs, is essential. The WHO Global Malaria Programme and the UK National External Quality Assessment Service (UK NEQAS) have collaborated since 2017 to implement a global malaria NAAT EQA scheme. METHODS:Panels of specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were validated by expert referee laboratories prior to distribution. Between 37 and 51 laboratories participated in each distribution and submitted results online. Participants were scored based on their laboratory's stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers. Change in performance over time was calculated using a generalized mixed model with a logit link function. RESULTS:Participating laboratories were located in 42 countries. Sample format (DBS or LB) and parasite density were found to significantly affect performance, while referee labs performed better at identifying P. falciparum samples than non-referee labs. Performance of laboratories improved significantly over time, especially for lower density and P. falciparum samples. CONCLUSIONS:Results from the first eleven distributions indicate that the EQA scheme has facilitated improved performance of laboratories over time, highlighting the value of implementing such programmes. EQA schemes are critical to safeguarding the reliability of data and diagnoses, especially in situations where NAAT methodologies and protocols are used. In future, funders should make participation in an EQA scheme a requirement for laboratories, and countries can take initiatives to embed such schemes into their own national assessment programmes.

BACKGROUND:Since 2006, artemisinin-based combination therapies (ACTs) have been introduced in Senegal in response to chloroquine resistance (CQ-R) and have shown high efficacy against Plasmodium falciparum. However, the detection of the PfKelch13R515K mutation in Kaolack, which confers artemisinin resistance in vitro, highlights the urgency of strengthening antimalarial drug surveillance to achieve malaria elimination by 2030. OBJECTIVE:To assess the proportion of P. falciparum parasites carrying molecular signatures associated with antimalarial resistance (PfKelch13, Pfmdr1, Pfcrt, dhfr and dhps) in isolates collected at Kédougou using multiplex amplicon deep sequencing. METHODS:Venous blood samples were collected from patients diagnosed with P. falciparum infection over a 3-year period (2021, 2022 and 2023). Parasite DNA was extracted, and multiplex amplicon sequencing was used to investigate gene polymorphisms. RESULTS:Analysis of PfKelch13 did not reveal any non-synonymous mutations. Pfcrt mutations were present in 45% of the samples, mainly K76T (44%) and I356T (36%). The dominant Pfmdr-1 allele was Y184F (62%). The sextuple mutant 51I/59R/108N + 436A/437G/613S dhfr/dhps was observed in 10% of the samples. CONCLUSION:The absence of PfKelch13 mutants suggests that ACT efficacy remains uncompromised, although clinical outcome studies are required to confirm this. Analysis of Pfcrt and Pfmdr-1 shows that CQ-R alleles, probably from previous CQ use, are slowly decreasing. Likewise, the detection of the dhfr/dhps sextuple mutant highlights the need to monitor sulfadoxine-pyrimethamine resistance and the emergence of 581G. There is therefore a need for continued antimalarial resistance surveillance in Senegal.

BACKGROUND:The recent emergence of Plasmodium falciparum parasites harbouring kelch 13 mutations associated with artemisinin partial resistance (ART-R) in sub-Saharan Africa is a major concern. Regular molecular epidemiological surveys are recommended to monitor the level of mutant parasites associated with drug resistance. OBJECTIVE:To analyse single nucleotide polymorphisms (SNPs) in P. falciparum genes associated with resistance to antimalarial drugs in blood samples collected from malaria patients prior to treatment in Busia County, Western Kenya, using a highly multiplexed deep amplicon sequencing protocol. METHODS:Parasite DNA was extracted from dried blood spots (DBS) collected from patients attending health facilities along the Kenya-Uganda border, in Busia County. A highly multiplexed deep amplicon sequencing protocol was applied to analyze SNPs in genes associated with antimalarial drugs resistance. RESULTS:Out of 118 analysed samples, Pfkelch13 mutations were identified in 6 (5.1%) samples. All the six samples harboured A578S, one of the most frequently detected Pfkelch13 mutations in Africa, which does not confer ART-R. The data also indicated that 11% (13) of the samples had mutant Pfcrt alleles, with 62% harbouring the triple mutant haplotype, CVIET. In contrast, 98% of the samples had dhfr mutations at positions N51I, C59R, or S108N alleles. All the samples carried mutations in the dhps gene with a high prevalence of the single A437G mutation (84%) and a lower frequency of double or triple mutations. Further analysis revealed absence of deletions in the Pfhrp2, with no mutations at Pfmdr1 codons 86 or 1246, both of which are associated with chloroquine resistance. CONCLUSION:These findings show absence of validated Pfkelch13 markers associated with ART-R in Busia County, Western Kenya. Although chloroquine-sensitive (wild-type) parasites are widely circulating, sulfadoxine-pyrimethamine-sensitive parasites remain rare. Continued large scale genomic surveillance studies are essential to detect emerging resistance and guide malaria treatment strategies in the region.