Target Amplicon Deep Sequencing of Pfk13 gene in patients with Plasmodium falciparum malaria during the therapeutic efficacy study trials from 2020 to 2022 in Senegal

Target Amplicon Deep Sequencing of Pfk13 gene in patients with Plasmodium falciparum malaria during the therapeutic efficacy study trials from 2020 to 2022 in Senegal | 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 Research Article Target Amplicon Deep Sequencing of Pfk13 gene in patients with Plasmodium falciparum malaria during the therapeutic efficacy study trials from 2020 to 2022 in Senegal Abdoulaye Tine, Mamadou Alpha Diallo, Amy Gaye, Mamadou Samb Yade, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9272508/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background: Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment in Africa. However, the emergence of Plasmodium falciparum artemisinin partial resistance associated with mutations in the pfk13 gene in Southeast Asia and East Africa raises concern about its potential emergence or spread to West Africa. This study aimed to investigate whether therapeutic efficacy study (TES)-defined treatment failures in Senegal are associated with pfk13 mutations using targeted amplicon deep sequencing (TADS). Methods: A total of 182 P. falciparum isolates collected during TES conducted between 2020 and 2022 in Senegal were analyzed. These included paired Day 0 (D0)/Day of Failure (DF) samples (n=112) and additional D0 samples (n=70) analyzed in pools. The pfk13 gene was sequenced using TADS, and single nucleotide polymorphisms (SNPs) were identified and compared with WHO-validated artemisinin resistance markers. Results: Among the 182 samples, 23 SNPs were identified in the pfk13 gene, including both synonymous and non-synonymous variants. Six non-synonymous mutations (D547Y, V566L, A578S, V589I, E596D, and V637I) were detected at very low within-sample allele frequencies (<1%). None corresponded to validated or candidate artemisinin resistance-associated mutations. Analysis of paired D0/DF samples showed no enrichment or selection of pfk13 mutations in post-treatment samples, indicating that observed treatment failures were not associated with pfk13 -mediated artemisinin resistance. Conclusion: This study provides no evidence of pfk13 -mediated artemisinin resistance in Senegal between 2020 and 2022. The absence of validated resistance mutations and the lack of association between pfk13 variants and treatment failure support the continued efficacy of ACTs in this setting. These findings highlight the importance of integrating TES outcomes with genomic surveillance to detect early signals of resistance and inform malaria control strategies in West Africa. Malaria Plasmodium falciparum artemisinin resistance pfk13 therapeutic efficacy study targeted amplicon deep sequencing Senegal molecular surveillance Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 04 May, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 22 Apr, 2026 Editor assigned by journal 01 Apr, 2026 Submission checks completed at journal 01 Apr, 2026 First submitted to journal 30 Mar, 2026 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-9272508","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633386981,"identity":"63eace6b-305f-4a8b-b59c-0010860ce9c5","order_by":0,"name":"Abdoulaye Tine","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Abdoulaye","middleName":"","lastName":"Tine","suffix":""},{"id":633386982,"identity":"52042d3d-f421-47e5-ab19-3c0b8481774e","order_by":1,"name":"Mamadou Alpha Diallo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYLACHhBxmIHxwAMGGwY2UrQwHEhgSCNFywGwlsOEVeu2n0788KbiMAPfceYHBxIqzsvz8S9+/PEHg02+vAN2LWZncjdLzjlzmEHyMJvBgYQztw3bJJ6ZSfMwpFluPIBDy4HcDdK8bYcZDIDoQGLb7QQ2iQNmzECvGRg24NBy/u3m3xAt7B+AWs4BtRz/DHQYHi03crdBbeEB2XIggY2/x0CCB6hFHof3zW683WY550w6j+RhngKgX5KBfuEpk+YxSDMwwKXlfO7mG28qrOX4zh/f+OBDhZ28fP/xzR9/VNgYyONwGAzwIJgSCUACaIXBAfxakAA/VCkhW0bBKBgFo2DEAAAH9GIa5fFsbAAAAABJRU5ErkJggg==","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":true,"prefix":"","firstName":"Mamadou","middleName":"Alpha","lastName":"Diallo","suffix":""},{"id":633386983,"identity":"749b5b7c-78ff-4761-b41d-24f489aa4934","order_by":2,"name":"Amy Gaye","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"","lastName":"Gaye","suffix":""},{"id":633386984,"identity":"3abe5cb9-78e5-4d99-a614-960d4b41c5b5","order_by":3,"name":"Mamadou Samb Yade","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Mamadou","middleName":"Samb","lastName":"Yade","suffix":""},{"id":633386985,"identity":"b9776852-b3c2-418e-b995-9d544490a3d5","order_by":4,"name":"Yaye Dié Ndiaye","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Yaye","middleName":"Dié","lastName":"Ndiaye","suffix":""},{"id":633386986,"identity":"102366b4-f5f4-494b-afed-8a8ea7aff5e9","order_by":5,"name":"Jules François Gomis","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Jules","middleName":"François","lastName":"Gomis","suffix":""},{"id":633386987,"identity":"f9f76151-57b6-4eab-856f-f6cf3b49cac7","order_by":6,"name":"Mouhamad Sy","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Mouhamad","middleName":"","lastName":"Sy","suffix":""},{"id":633386988,"identity":"9a3b7203-96a3-449a-830e-d6c9d57bf70d","order_by":7,"name":"Bassirou Ngom","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Bassirou","middleName":"","lastName":"Ngom","suffix":""},{"id":633386990,"identity":"83a4fd0c-173b-4582-bf17-936ccdf53938","order_by":8,"name":"Djiby Sow","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Djiby","middleName":"","lastName":"Sow","suffix":""},{"id":633386991,"identity":"1b88a4bc-2a00-4665-a066-8c9622f6c1b6","order_by":9,"name":"Chérif Younouss Diouf","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Chérif","middleName":"Younouss","lastName":"Diouf","suffix":""},{"id":633386992,"identity":"789e77d3-75a6-4615-899d-27e4b8e09b90","order_by":10,"name":"Mamane N. Garba","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Mamane","middleName":"N.","lastName":"Garba","suffix":""},{"id":633386995,"identity":"a7d0b2ac-2471-4f3d-af62-24350dd29fe8","order_by":11,"name":"Aïta Sène","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Aïta","middleName":"","lastName":"Sène","suffix":""},{"id":633386996,"identity":"cffa7b34-f485-4ad0-afb0-214108aa0c38","order_by":12,"name":"Mariama Touré","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Mariama","middleName":"","lastName":"Touré","suffix":""},{"id":633386997,"identity":"ce677ab8-11ff-495f-9a46-a47a357ede71","order_by":13,"name":"Awa Fall","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Awa","middleName":"","lastName":"Fall","suffix":""},{"id":633386998,"identity":"38f41f7d-86d6-4a82-8fc1-875eec678aa1","order_by":14,"name":"Tolla Ndiaye","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Tolla","middleName":"","lastName":"Ndiaye","suffix":""},{"id":633387000,"identity":"cd3a4264-dbcc-4cf4-a1b4-c4d6f1c2f46b","order_by":15,"name":"Ibrahima M. Ndiaye","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Ibrahima","middleName":"M.","lastName":"Ndiaye","suffix":""},{"id":633387001,"identity":"d19fb87f-37ec-48cc-a68f-2c6a44ee141e","order_by":16,"name":"Baba Dièye","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Baba","middleName":"","lastName":"Dièye","suffix":""},{"id":633387002,"identity":"fda778a7-3466-4516-b6a7-7ebb2f41cad5","order_by":17,"name":"Awa B. Dème","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Awa","middleName":"B.","lastName":"Dème","suffix":""},{"id":633387003,"identity":"492f96ff-7346-402b-b253-1679b5a16bab","order_by":18,"name":"Khadim Diongue","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Khadim","middleName":"","lastName":"Diongue","suffix":""},{"id":633387004,"identity":"0b10865e-1214-4b89-89be-c6357b753039","order_by":19,"name":"Mame C. Seck","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Mame","middleName":"C.","lastName":"Seck","suffix":""},{"id":633387005,"identity":"eccdc8c5-4b3d-4d3a-a807-bc73b27f7a46","order_by":20,"name":"Mouhamadou Ndiaye","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Mouhamadou","middleName":"","lastName":"Ndiaye","suffix":""},{"id":633387006,"identity":"198c968b-d64f-4b58-ac41-f2d6f20e81b7","order_by":21,"name":"Aliou Thiongane","email":"","orcid":"","institution":"Programme National de Lutte contre le Paludisme","correspondingAuthor":false,"prefix":"","firstName":"Aliou","middleName":"","lastName":"Thiongane","suffix":""},{"id":633387007,"identity":"7ecbb0b8-35f3-49f5-ad76-304838ca30a3","order_by":22,"name":"Ibrahima Diallo","email":"","orcid":"","institution":"Programme National de Lutte contre le Paludisme","correspondingAuthor":false,"prefix":"","firstName":"Ibrahima","middleName":"","lastName":"Diallo","suffix":""},{"id":633387008,"identity":"68d81989-b1a3-4c11-af59-041bf4852ddd","order_by":23,"name":"Aïda S. Badiane","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Aïda","middleName":"S.","lastName":"Badiane","suffix":""},{"id":633387009,"identity":"ce5e3aa3-075f-4da2-b899-f4698928a9ba","order_by":24,"name":"Daouda Ndiaye","email":"","orcid":"","institution":"Cheikh Anta Diop University","correspondingAuthor":false,"prefix":"","firstName":"Daouda","middleName":"","lastName":"Ndiaye","suffix":""}],"badges":[],"createdAt":"2026-03-31 00:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9272508/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9272508/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108494213,"identity":"21fb34ea-7edc-472a-9d61-b99ee500f5a2","added_by":"auto","created_at":"2026-05-05 10:03:08","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":630568,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscriptTESlongit2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9272508/v1_covered_f3e10108-74d5-4df0-a46d-42fb13f1a73e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Target Amplicon Deep Sequencing of Pfk13 gene in patients with Plasmodium falciparum malaria during the therapeutic efficacy study trials from 2020 to 2022 in Senegal","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Malaria, Plasmodium falciparum, artemisinin resistance, pfk13, therapeutic efficacy study, targeted amplicon deep sequencing, Senegal, molecular surveillance","lastPublishedDoi":"10.21203/rs.3.rs-9272508/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9272508/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment in Africa. However, the emergence of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e artemisinin partial resistance associated with mutations in the \u003cem\u003epfk13\u003c/em\u003egene in Southeast Asia and East Africa raises concern about its potential emergence or spread to West Africa. This study aimed to investigate whether therapeutic efficacy study (TES)-defined treatment failures in Senegal are associated with \u003cem\u003epfk13\u003c/em\u003e mutations using targeted amplicon deep sequencing (TADS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A total of 182 \u003cem\u003eP. falciparum\u003c/em\u003e isolates collected during TES conducted between 2020 and 2022 in Senegal were analyzed. These included paired Day 0 (D0)/Day of Failure (DF) samples (n=112) and additional D0 samples (n=70) analyzed in pools. The \u003cem\u003epfk13\u003c/em\u003e gene was sequenced using TADS, and single nucleotide polymorphisms (SNPs) were identified and compared with WHO-validated artemisinin resistance markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Among the 182 samples, 23 SNPs were identified in the \u003cem\u003epfk13\u003c/em\u003e gene, including both synonymous and non-synonymous variants. Six non-synonymous mutations (D547Y, V566L, A578S, V589I, E596D, and V637I) were detected at very low within-sample allele frequencies (\u0026lt;1%). None corresponded to validated or candidate artemisinin resistance-associated mutations. Analysis of paired D0/DF samples showed no enrichment or selection of \u003cem\u003epfk13\u003c/em\u003e mutations in post-treatment samples, indicating that observed treatment failures were not associated with \u003cem\u003epfk13\u003c/em\u003e-mediated artemisinin resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e This study provides no evidence of \u003cem\u003epfk13\u003c/em\u003e-mediated artemisinin resistance in Senegal between 2020 and 2022. The absence of validated resistance mutations and the lack of association between \u003cem\u003epfk13\u003c/em\u003e variants and treatment failure support the continued efficacy of ACTs in this setting. These findings highlight the importance of integrating TES outcomes with genomic surveillance to detect early signals of resistance and inform malaria control strategies in West Africa.\u003c/p\u003e","manuscriptTitle":"Target Amplicon Deep Sequencing of Pfk13 gene in patients with Plasmodium falciparum malaria during the therapeutic efficacy study trials from 2020 to 2022 in Senegal","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 23:07:54","doi":"10.21203/rs.3.rs-9272508/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-04T08:54:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"327827309524625720435783531123364783265","date":"2026-04-23T09:31:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265308413314704943019122770152715277322","date":"2026-04-23T09:06:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T14:48:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T19:34:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-01T19:33:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2026-03-31T00:05:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6b118898-ae01-4fc7-ab10-338bac2b86cb","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-04T08:54:22+00:00","index":20,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T23:07:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 23:07:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9272508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9272508","identity":"rs-9272508","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}