Rapid increase of A581G mutation in Southern Senegal under seasonal malaria chemoprevention pressure

Rapid increase of A581G mutation in Southern Senegal under seasonal malaria chemoprevention pressure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Rapid increase of A581G mutation in Southern Senegal under seasonal malaria chemoprevention pressure Mamadou Diallo, Djiby Sow, Abdoulaye Tine, Aita Sene, Amy Gaye, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6285854/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Seasonal malaria chemoprevention (SMC) with sulfadoxine–pyrimethamine (SP) plus amodiaquine (AQ) has reduced malaria incidence among children in many African countries, including Senegal. However, prolonged SP usage can promote the selection of drug-resistant parasites. Here, we investigated P. falciparum molecular resistance markers in two regions of southern Senegal (Kolda and Kedougou) from 2020 to 2023. The pf dhfr triple-mutant alleles (N51I, C59R, S108N) were nearly fixed throughout, confirming widespread pyrimethamine resistance. Critically, the pf dhps A581G mutation, undetected in 2020–2021, emerged at 8.1% in Kolda in 2022 and rose to 15% by 2023, while appearing at 6.5% in Kedougou in 2023. The pf dhps A437G mutation likewise remained prevalent, and K540E was sporadically detected. The rising frequency of A581G, known to confer high-level SP resistance, presents a significant threat to SMC efficacy. Enhanced molecular surveillance and adaptive policies are crucial to prevent the erosion of SMC’s protective effect in these high-burden settings. Health sciences/Medical research/Translational research Health sciences/Medical research/Outcomes research Figures Figure 1 Introduction Since 2012, Senegal has been actively implementing Seasonal Malaria Chemoprevention (SMC) to control malaria, particularly among children aged 3 to 120 months. This public health strategy, which involves the administration of Sulfadoxine-Pyrimethamine (SP) combined with Amodiaquine (AQ), has shown considerable success, achieving coverage rates of 93 to 98% in 2022 across sixteen districts in five regions 1 . However, the extensive use of SMC in the African Sahel region, including Senegal, exerts significant drug pressure on the malaria parasite, raising concerns about the potential development of drug resistance 2 . The efficacy of SMC is critically dependent on the continued susceptibility of Plasmodium falciparum to SP and AQ. While studies have indicated that resistance-associated mutations, such as PfDHPS 540E, remain relatively uncommon in regions practicing SMC, there is growing evidence that other mutations, like PfDHPS A581G, may be on the rise 3 . The presence of the A581G mutation has been shown to significantly reduce the efficacy of SP as a preventive therapy in pregnant women in Malawi. Additionally, in areas of Tanzania with a high prevalence of this mutation, SP has been found to be ineffective in intermittent preventive treatment. After several years of SMC implementation in Senegal, we aimed to leverage the Therapeutic Efficacy Study (TES) samples collected from these areas to analyze the trends in molecular markers, assessing whether any selection pressure is contributing to the emergence of drug-resistant mutations. Methods Study sites and sample collection This study was designed as an annual therapeutic efficacy study (TES) conducted in Senegal. The primary objective was to assess potential antimalarial drug resistance in Plasmodium falciparum isolates, with a particular focus on resistance markers for sulfadoxine-pyrimethamine (SP) in the dhfr and dhps genes. Samples were collected from two rural areas in Senegal: Kedougou and Kolda. The study spanned over the years 2020, 2021, 2022, 2023. Ethical considerations Written informed consent was obtained from all adult participants. For subjects younger than 18 years of age, consent was obtained from their parents or legal guardians. The study adhered to the World Health Organization (WHO) TES protocol, ensuring ethical conduct and participant safety throughout the study. Sample Collection Blood samples were collected on Day 0 from consenting outpatients who presented with a fever (temperature above 37.5°C) or reported having a fever the 24 hours before seeking care at the health facilities involved in the study. The blood samples were collected via finger prick and applied to filter paper in 5 spots. These filter papers were then air-dried, individually wrapped in plastic bags, and stored at room temperature. Patient follow-Up Following the initial sample collection, patients were monitored for a period of 28 days or 42 days, depending on the specific antimalarial drug administered. During each follow-up visit, a clinical examination was conducted, and dried blood spots (DBS) were collected on filter paper for further analysis. Molecular analysis DNA extraction Total genomic DNA was extracted from dried blood spots (DBSs) using the QIAmp DNA Blood Mini Kit, following the manufacturer's instructions. P. falciparum confirmation To confirm P. falciparum infections and assess the quality of the DNA extracted from the DBS samples, a real-time photo-induced electron transfer-PCR (PET-PCR) method was employed as described by Lucchi et al. 4 . This technique is sensitive and specific for detecting P. falciparum DNA. Samples with a threshold cycle (Ct) value greater than 40 were considered negative for P. falciparum and were excluded from further analysis. Molecular Markers of drug resistance Analysis The analysis of malaria resistance markers followed the protocols outlined in the Malaria Resistance Surveillance (MaRS) guidelines, as recommended by Talundzic et al. 5 . This protocol provides standardized procedures for laboratory and data analysis. For detailed MaRS protocol guidelines, including laboratory procedures and data analysis techniques, refer to the official resource available at https://github.com/cdcgov/mars . Results In the Pfdhfr gene associated with pyrimethamine resistance, the N51I, C59R, and S108N mutations were observed at high frequencies in both study sites, Kolda and Kedougou, across the different study years. The frequencies remaining fixed above 90% at both sites over the years. For the Pfdhps gene associated with sulfadoxine resistance, the A437G mutation showed a high prevalence, particularly in 2021, where it reached 100% in both Kolda and Kedougou. In 2022, the frequency of this mutation slightly decreased to 76% in Kolda and 80% in Kedougou but increased to 87% and 86% respectively in 2023. In contrast, the A581G mutation was absent in 2020 and 2021 but was observed at a frequency of 8.10% in Kolda in 2022, reaching 15% in 2023, while in Kedougou, it was absent until it reached 6.50% in 2023. The K540E mutation was absent in 2020 but was observed in 2021 with a frequency of 14% in Kedougou, then showed lower frequencies in subsequent years. Regarding the quintuple and sextuple genotypes associated with increased resistance, the IRN/SGEAA genotype was observed only in 2021 in Kedougou with a frequency of 14%, while the VAGKGS genotype was detected only in 2022 in Kolda with a frequency of 6%. Neither of these genotypes was detected in other years or at other study sites. Discussion The findings from this study contribute to the growing body of evidence indicating the increasing prevalence of Plasmodium falciparum molecular markers of resistance to sulfadoxine-pyrimethamine (SP) in Senegal, specifically in the Kedougou and Kolda regions. The high prevalence of N51I, C59R, and S108N mutations in the PfDHFR gene, observed across multiple years in both regions, is consistent with previous studies that have documented widespread pyrimethamine resistance in P. falciparum populations in West Africa, including Senegal 6 , 7 . The emergence and increasing frequency of A581G and K540E mutations in the PfDHPS gene in recent years are particularly concerning. These mutations are key markers of resistance to SP, and their rise could signal a diminishing efficacy of SP-based interventions, such as Seasonal Malaria Chemoprevention (SMC), which is widely implemented in Senegal and other parts of Africa 7 , 8 . The detection of these mutations, particularly in Kolda where A581G increased from 0% in 2020 to 15% in 2023, underscores the need for ongoing molecular surveillance and potential reconsideration of SP use in these regions. The presence of A581G is particularly alarming because it is associated with high-level resistance to SP 9 . In Mali, the Pfdhps A581G allele was observed at a frequency of 11.7% following two years of implementing Seasonal Malaria Chemoprevention (SMC) 8 . Moreover, the presence of the IRN/SGEAA quintuple mutant genotype in Kedougou in 2021, albeit at a relatively low frequency, is a cause for concern. The quintuple and sextuple mutations, which include A581G and K540E, have been associated with high-level SP resistance and treatment failures in other parts of Africa 10 . The presence of these genotypes could indicate an evolving resistance profile that may eventually compromise the efficacy of SP in SMC programs. This study’s findings are consistent with previous reports that has shown the spread of SP-resistant P. falciparum strains across Africa, driven in part by the widespread use of SP in malaria control programs 11 . However, the study's limitations, such as the absence of data from Kedougou in 2020 and the low sample sizes in 2021 and 2022, highlight the need for more comprehensive and continuous monitoring to fully understand the dynamics of resistance development in these regions. References WHO. SMC Alliance (2020) Seasonal malaria chemoprevention (SMC). Malar consortium 8 Nikiema S et al (2022) Seasonal Malaria Chemoprevention Implementation: Effect on Malaria Incidence and Immunity in a Context of Expansion of P. falciparum Resistant Genotypes with Potential Reduction of the Effectiveness in Sub-Saharan Africa. Infection and Drug Resistance vol. 15 Preprint at https://doi.org/10.2147/IDR.S375197 Roh ME et al (2023) Seasonal Malaria Chemoprevention Drug Levels and Drug Resistance Markers in Children with or Without Malaria in Burkina Faso: A Case-Control Study. J Infect Dis 228 Lucchi NW et al (2013) Molecular Diagnosis of Malaria by Photo-Induced Electron Transfer Fluorogenic Primers: PET-PCR. PLoS ONE 8:1–7 Talundzic E et al (2018) Next-generation sequencing and bioinformatics protocol for malaria drug resistance marker surveillance. Antimicrob Agents Chemother 62 Fall B et al (2016) Plasmodium falciparum in vitro resistance to monodesethylamodiaquine, Dakar, Senegal, 2014. Emerg Infect Dis 22 Ndiaye YD et al (2024) Two decades of molecular surveillance in Senegal reveal rapid changes in known drug resistance mutations over time. Malar J 23:205 Mahamar A et al (2022) Effect of three years’ seasonal malaria chemoprevention on molecular markers of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine and amodiaquine in Ouelessebougou, Mali. Malar J 21 Bazie VB et al (2020) Resistance of Plasmodium falciparum to Sulfadoxine-Pyrimethamine (Dhfr and Dhps) and Artemisinin and Its Derivatives (K13): A Major Challenge for Malaria Elimination in West Africa. J Biosci Med (Irvine) 08 Roux AT et al (2021) Chloroquine and Sulfadoxine–Pyrimethamine Resistance in Sub-Saharan Africa—A Review. Frontiers in Genetics vol. 12 Preprint at https://doi.org/10.3389/fgene.2021.668574 Amimo F et al (2020) Plasmodium falciparum resistance to sulfadoxine-pyrimethamine in Africa: A systematic analysis of national trends. BMJ Glob Health 5 Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6285854","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":435409441,"identity":"c2855db7-c2f1-42d3-b176-e2181f2d92c9","order_by":0,"name":"Mamadou 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1","display":"","copyAsset":false,"role":"figure","size":487304,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6285854/v1/690f075f6a401eb744367927.jpeg"},{"id":94990493,"identity":"615a6c2c-327e-42c7-a858-b58d66ddb9f5","added_by":"auto","created_at":"2025-11-03 07:17:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":914454,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6285854/v1/25973207-b4e9-4907-b85d-5c37fa9f1d46.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Rapid increase of A581G mutation in Southern Senegal under seasonal malaria chemoprevention pressure","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince 2012, Senegal has been actively implementing Seasonal Malaria Chemoprevention (SMC) to control malaria, particularly among children aged 3 to 120 months. This public health strategy, which involves the administration of Sulfadoxine-Pyrimethamine (SP) combined with Amodiaquine (AQ), has shown considerable success, achieving coverage rates of 93 to 98% in 2022 across sixteen districts in five regions \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, the extensive use of SMC in the African Sahel region, including Senegal, exerts significant drug pressure on the malaria parasite, raising concerns about the potential development of drug resistance \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe efficacy of SMC is critically dependent on the continued susceptibility of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e to SP and AQ. While studies have indicated that resistance-associated mutations, such as PfDHPS 540E, remain relatively uncommon in regions practicing SMC, there is growing evidence that other mutations, like PfDHPS A581G, may be on the rise \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe presence of the A581G mutation has been shown to significantly reduce the efficacy of SP as a preventive therapy in pregnant women in Malawi. Additionally, in areas of Tanzania with a high prevalence of this mutation, SP has been found to be ineffective in intermittent preventive treatment.\u003c/p\u003e \u003cp\u003eAfter several years of SMC implementation in Senegal, we aimed to leverage the Therapeutic Efficacy Study (TES) samples collected from these areas to analyze the trends in molecular markers, assessing whether any selection pressure is contributing to the emergence of drug-resistant mutations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy sites and sample collection\u003c/h2\u003e \u003cp\u003eThis study was designed as an annual therapeutic efficacy study (TES) conducted in Senegal. The primary objective was to assess potential antimalarial drug resistance in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e isolates, with a particular focus on resistance markers for sulfadoxine-pyrimethamine (SP) in the \u003cem\u003edhfr\u003c/em\u003e and \u003cem\u003edhps\u003c/em\u003e genes. Samples were collected from two rural areas in Senegal: Kedougou and Kolda. The study spanned over the years 2020, 2021, 2022, 2023.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthical considerations\u003c/h3\u003e\n\u003cp\u003e Written informed consent was obtained from all adult participants. For subjects younger than 18 years of age, consent was obtained from their parents or legal guardians. The study adhered to the World Health Organization (WHO) TES protocol, ensuring ethical conduct and participant safety throughout the study.\u003c/p\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eBlood samples were collected on Day 0 from consenting outpatients who presented with a fever (temperature above 37.5\u0026deg;C) or reported having a fever the 24 hours before seeking care at the health facilities involved in the study. The blood samples were collected via finger prick and applied to filter paper in 5 spots. These filter papers were then air-dried, individually wrapped in plastic bags, and stored at room temperature.\u003c/p\u003e\n\u003ch3\u003ePatient follow-Up\u003c/h3\u003e\n\u003cp\u003eFollowing the initial sample collection, patients were monitored for a period of 28 days or 42 days, depending on the specific antimalarial drug administered. During each follow-up visit, a clinical examination was conducted, and dried blood spots (DBS) were collected on filter paper for further analysis.\u003c/p\u003e\n\u003ch3\u003eMolecular analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction\u003c/h2\u003e \u003cp\u003eTotal genomic DNA was extracted from dried blood spots (DBSs) using the QIAmp DNA Blood Mini Kit, following the manufacturer's instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003econfirmation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo confirm \u003cem\u003eP. falciparum\u003c/em\u003e infections and assess the quality of the DNA extracted from the DBS samples, a real-time photo-induced electron transfer-PCR (PET-PCR) method was employed as described by Lucchi et al. \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. This technique is sensitive and specific for detecting \u003cem\u003eP. falciparum\u003c/em\u003e DNA. Samples with a threshold cycle (Ct) value greater than 40 were considered negative for \u003cem\u003eP. falciparum\u003c/em\u003e and were excluded from further analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Markers of drug resistance Analysis\u003c/h3\u003e\n\u003cp\u003e The analysis of malaria resistance markers followed the protocols outlined in the Malaria Resistance Surveillance (MaRS) guidelines, as recommended by Talundzic et al. \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This protocol provides standardized procedures for laboratory and data analysis. For detailed MaRS protocol guidelines, including laboratory procedures and data analysis techniques, refer to the official resource available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/cdcgov/mars\u003c/span\u003e\u003cspan address=\"https://github.com/cdcgov/mars\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn the \u003cem\u003ePfdhfr\u003c/em\u003e gene associated with pyrimethamine resistance, the N51I, C59R, and S108N mutations were observed at high frequencies in both study sites, Kolda and Kedougou, across the different study years. The frequencies remaining fixed above 90% at both sites over the years.\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003ePfdhps\u003c/em\u003e gene associated with sulfadoxine resistance, the A437G mutation showed a high prevalence, particularly in 2021, where it reached 100% in both Kolda and Kedougou. In 2022, the frequency of this mutation slightly decreased to 76% in Kolda and 80% in Kedougou but increased to 87% and 86% respectively in 2023. In contrast, the A581G mutation was absent in 2020 and 2021 but was observed at a frequency of 8.10% in Kolda in 2022, reaching 15% in 2023, while in Kedougou, it was absent until it reached 6.50% in 2023. The K540E mutation was absent in 2020 but was observed in 2021 with a frequency of 14% in Kedougou, then showed lower frequencies in subsequent years.\u003c/p\u003e \u003cp\u003eRegarding the quintuple and sextuple genotypes associated with increased resistance, the IRN/SGEAA genotype was observed only in 2021 in Kedougou with a frequency of 14%, while the VAGKGS genotype was detected only in 2022 in Kolda with a frequency of 6%. Neither of these genotypes was detected in other years or at other study sites.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe findings from this study contribute to the growing body of evidence indicating the increasing prevalence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e molecular markers of resistance to sulfadoxine-pyrimethamine (SP) in Senegal, specifically in the Kedougou and Kolda regions. The high prevalence of N51I, C59R, and S108N mutations in the PfDHFR gene, observed across multiple years in both regions, is consistent with previous studies that have documented widespread pyrimethamine resistance in \u003cem\u003eP. falciparum\u003c/em\u003e populations in West Africa, including Senegal \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe emergence and increasing frequency of A581G and K540E mutations in the PfDHPS gene in recent years are particularly concerning. These mutations are key markers of resistance to SP, and their rise could signal a diminishing efficacy of SP-based interventions, such as Seasonal Malaria Chemoprevention (SMC), which is widely implemented in Senegal and other parts of Africa \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The detection of these mutations, particularly in Kolda where A581G increased from 0% in 2020 to 15% in 2023, underscores the need for ongoing molecular surveillance and potential reconsideration of SP use in these regions. The presence of \u003cb\u003eA581G\u003c/b\u003e is particularly alarming because it is associated with high-level resistance to SP \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In Mali, the \u003cb\u003ePfdhps\u003c/b\u003e \u003cb\u003eA581G\u003c/b\u003e allele was observed at a frequency of 11.7% following two years of implementing Seasonal Malaria Chemoprevention (SMC) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoreover, the presence of the IRN/SGEAA quintuple mutant genotype in Kedougou in 2021, albeit at a relatively low frequency, is a cause for concern. The quintuple and sextuple mutations, which include A581G and K540E, have been associated with high-level SP resistance and treatment failures in other parts of Africa \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The presence of these genotypes could indicate an evolving resistance profile that may eventually compromise the efficacy of SP in SMC programs.\u003c/p\u003e \u003cp\u003eThis study\u0026rsquo;s findings are consistent with previous reports that has shown the spread of SP-resistant \u003cem\u003eP. falciparum\u003c/em\u003e strains across Africa, driven in part by the widespread use of SP in malaria control programs \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, the study's limitations, such as the absence of data from Kedougou in 2020 and the low sample sizes in 2021 and 2022, highlight the need for more comprehensive and continuous monitoring to fully understand the dynamics of resistance development in these regions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWHO. SMC Alliance (2020) Seasonal malaria chemoprevention (SMC). Malar consortium 8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikiema S et al (2022) Seasonal Malaria Chemoprevention Implementation: Effect on Malaria Incidence and Immunity in a Context of Expansion of P. falciparum Resistant Genotypes with Potential Reduction of the Effectiveness in Sub-Saharan Africa. \u003cem\u003eInfection and Drug Resistance\u003c/em\u003e vol. 15 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IDR.S375197\u003c/span\u003e\u003cspan address=\"10.2147/IDR.S375197\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoh ME et al (2023) Seasonal Malaria Chemoprevention Drug Levels and Drug Resistance Markers in Children with or Without Malaria in Burkina Faso: A Case-Control Study. J Infect Dis 228\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLucchi NW et al (2013) Molecular Diagnosis of Malaria by Photo-Induced Electron Transfer Fluorogenic Primers: PET-PCR. PLoS ONE 8:1\u0026ndash;7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalundzic E et al (2018) Next-generation sequencing and bioinformatics protocol for malaria drug resistance marker surveillance. Antimicrob Agents Chemother 62\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFall B et al (2016) Plasmodium falciparum in vitro resistance to monodesethylamodiaquine, Dakar, Senegal, 2014. Emerg Infect Dis 22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNdiaye YD et al (2024) Two decades of molecular surveillance in Senegal reveal rapid changes in known drug resistance mutations over time. Malar J 23:205\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahamar A et al (2022) Effect of three years\u0026rsquo; seasonal malaria chemoprevention on molecular markers of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine and amodiaquine in Ouelessebougou, Mali. Malar J 21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBazie VB et al (2020) Resistance of Plasmodium falciparum to Sulfadoxine-Pyrimethamine (Dhfr and Dhps) and Artemisinin and Its Derivatives (K13): A Major Challenge for Malaria Elimination in West Africa. J Biosci Med (Irvine) 08\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoux AT et al (2021) Chloroquine and Sulfadoxine\u0026ndash;Pyrimethamine Resistance in Sub-Saharan Africa\u0026mdash;A Review. \u003cem\u003eFrontiers in Genetics\u003c/em\u003e vol. 12 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fgene.2021.668574\u003c/span\u003e\u003cspan address=\"10.3389/fgene.2021.668574\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmimo F et al (2020) Plasmodium falciparum resistance to sulfadoxine-pyrimethamine in Africa: A systematic analysis of national trends. BMJ Glob Health 5\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6285854/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6285854/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeasonal malaria chemoprevention (SMC) with sulfadoxine\u0026ndash;pyrimethamine (SP) plus amodiaquine (AQ) has reduced malaria incidence among children in many African countries, including Senegal. However, prolonged SP usage can promote the selection of drug-resistant parasites. Here, we investigated \u003cb\u003eP. falciparum\u003c/b\u003e molecular resistance markers in two regions of southern Senegal (Kolda and Kedougou) from 2020 to 2023. The \u003cb\u003epf\u003c/b\u003edhfr triple-mutant alleles (N51I, C59R, S108N) were nearly fixed throughout, confirming widespread pyrimethamine resistance. Critically, the \u003cb\u003epf\u003c/b\u003edhps A581G mutation, undetected in 2020\u0026ndash;2021, emerged at 8.1% in Kolda in 2022 and rose to 15% by 2023, while appearing at 6.5% in Kedougou in 2023. The \u003cb\u003epf\u003c/b\u003edhps A437G mutation likewise remained prevalent, and K540E was sporadically detected. The rising frequency of A581G, known to confer high-level SP resistance, presents a significant threat to SMC efficacy. Enhanced molecular surveillance and adaptive policies are crucial to prevent the erosion of SMC\u0026rsquo;s protective effect in these high-burden settings.\u003c/p\u003e","manuscriptTitle":"Rapid increase of A581G mutation in Southern Senegal under seasonal malaria chemoprevention pressure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-01 11:44:54","doi":"10.21203/rs.3.rs-6285854/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-medicine","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmed","sideBox":"Learn more about [Communications Medicine](http://www.nature.com/commsmed)","snPcode":"43856","submissionUrl":"https://mts-commsmed.nature.com/cgi-bin/main.plex","title":"Communications Medicine","twitterHandle":"@commsmedicine","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e31e6196-6a1f-43fe-a0bd-48264bb8e002","owner":[],"postedDate":"November 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":46365380,"name":"Health sciences/Medical research/Translational research"},{"id":46365381,"name":"Health sciences/Medical research/Outcomes research"}],"tags":[],"updatedAt":"2025-11-02T20:55:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-01 11:44:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6285854","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6285854","identity":"rs-6285854","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}