Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon
BackgroundResistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular ma...
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2025-02-01
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| author | Jacob M. Sadler Alfred Simkin Valery P. K. Tchuenkam Isabela Gerdes Gyuricza Isabela Gerdes Gyuricza Abebe A. Fola Kevin Wamae Ashenafi Assefa Ashenafi Assefa Karamoko Niaré Kyaw Thwai Samuel J. White William J. Moss Rhoel R. Dinglasan Sandrine Eveline Nsango Christopher B. Tume Jonathan B. Parr Jonathan B. Parr Jonathan B. Parr Innocent Mbulli Ali Jeffrey A. Bailey Jeffrey A. Bailey Jonathan J. Juliano Jonathan J. Juliano Jonathan J. Juliano Jonathan J. Juliano |
| author_facet | Jacob M. Sadler Alfred Simkin Valery P. K. Tchuenkam Isabela Gerdes Gyuricza Isabela Gerdes Gyuricza Abebe A. Fola Kevin Wamae Ashenafi Assefa Ashenafi Assefa Karamoko Niaré Kyaw Thwai Samuel J. White William J. Moss Rhoel R. Dinglasan Sandrine Eveline Nsango Christopher B. Tume Jonathan B. Parr Jonathan B. Parr Jonathan B. Parr Innocent Mbulli Ali Jeffrey A. Bailey Jeffrey A. Bailey Jonathan J. Juliano Jonathan J. Juliano Jonathan J. Juliano Jonathan J. Juliano |
| author_sort | Jacob M. Sadler |
| collection | DOAJ |
| description | BackgroundResistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing.MethodsPlasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing (Pf-SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach.ResultsWe evaluated Pf-SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf-SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf-SMARRT’s hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed.ConclusionOverall, Pf-SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible. |
| format | Article |
| id | doaj-art-22c30792347f461ea4d76e9314a5bd3c |
| institution | Kabale University |
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| publishDate | 2025-02-01 |
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| spelling | doaj-art-22c30792347f461ea4d76e9314a5bd3c2025-02-05T07:32:48ZengFrontiers Media S.A.Frontiers in Parasitology2813-24242025-02-01310.3389/fpara.2024.15092611509261Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, CameroonJacob M. Sadler0Alfred Simkin1Valery P. K. Tchuenkam2Isabela Gerdes Gyuricza3Isabela Gerdes Gyuricza4Abebe A. Fola5Kevin Wamae6Ashenafi Assefa7Ashenafi Assefa8Karamoko Niaré9Kyaw Thwai10Samuel J. White11William J. Moss12Rhoel R. Dinglasan13Sandrine Eveline Nsango14Christopher B. Tume15Jonathan B. Parr16Jonathan B. Parr17Jonathan B. Parr18Innocent Mbulli Ali19Jeffrey A. Bailey20Jeffrey A. Bailey21Jonathan J. Juliano22Jonathan J. Juliano23Jonathan J. Juliano24Jonathan J. Juliano25Institute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesDepartment of Pathology and Laboratory Medicine, Warren Alpert Medical School, Brown University, Providence, RI, United StatesDepartment of Biochemistry, Faculty of Science, University of Dschang, Dschang, CameroonInstitute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesCurriculum in Genetics and Molecular Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesDepartment of Pathology and Laboratory Medicine, Warren Alpert Medical School, Brown University, Providence, RI, United StatesKenyan Medical Research Institute (KEMRI)-Wellcome Trust Research Programme, Kilifi, KenyaInstitute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesMalaria and Neglected Tropical Disease Directorate, Ethiopian Public Health Institute, Addis Ababa, EthiopiaDepartment of Pathology and Laboratory Medicine, Warren Alpert Medical School, Brown University, Providence, RI, United StatesInstitute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesInstitute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesDepartment of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United StatesDepartment of Infectious Diseases & Immunology, College of Veterinary Medicine & Emerging Pathogens Institute, University of Florida, Gainesville, FL, United StatesMalaria Research Unit, Centre Pasteur du Cameroun, Yaoundé, CameroonDepartment of Biochemistry, Faculty of Science, University of Dschang, Dschang, CameroonInstitute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesCurriculum in Genetics and Molecular Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States0Division of Infectious Diseases, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesDepartment of Biochemistry, Faculty of Science, University of Dschang, Dschang, CameroonDepartment of Pathology and Laboratory Medicine, Warren Alpert Medical School, Brown University, Providence, RI, United States1Data Science Institute, Center for Computational and Molecular Biology, Brown University, Providence, RI, United StatesInstitute for Global Health and Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesCurriculum in Genetics and Molecular Biology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States0Division of Infectious Diseases, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States2Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, Chapel Hill, NC, United StatesBackgroundResistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing.MethodsPlasmodium falciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing (Pf-SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach.ResultsWe evaluated Pf-SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf-SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf-SMARRT’s hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed.ConclusionOverall, Pf-SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible.https://www.frontiersin.org/articles/10.3389/fpara.2024.1509261/fullmalariaPlasmodium falciparumantimalarial resistancegenetic relatednessamplicon sequencing |
| spellingShingle | Jacob M. Sadler Alfred Simkin Valery P. K. Tchuenkam Isabela Gerdes Gyuricza Isabela Gerdes Gyuricza Abebe A. Fola Kevin Wamae Ashenafi Assefa Ashenafi Assefa Karamoko Niaré Kyaw Thwai Samuel J. White William J. Moss Rhoel R. Dinglasan Sandrine Eveline Nsango Christopher B. Tume Jonathan B. Parr Jonathan B. Parr Jonathan B. Parr Innocent Mbulli Ali Jeffrey A. Bailey Jeffrey A. Bailey Jonathan J. Juliano Jonathan J. Juliano Jonathan J. Juliano Jonathan J. Juliano Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon Frontiers in Parasitology malaria Plasmodium falciparum antimalarial resistance genetic relatedness amplicon sequencing |
| title | Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon |
| title_full | Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon |
| title_fullStr | Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon |
| title_full_unstemmed | Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon |
| title_short | Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon |
| title_sort | application of a new highly multiplexed amplicon sequencing tool to evaluate plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from dschang cameroon |
| topic | malaria Plasmodium falciparum antimalarial resistance genetic relatedness amplicon sequencing |
| url | https://www.frontiersin.org/articles/10.3389/fpara.2024.1509261/full |
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