Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene
Abstract Global control of hookworm infections relies on periodic Mass Drug Administration of benzimidazole drugs to high-risk groups, regardless of infection status. Mutations in the isotype-1 β-tubulin gene have been identified in veterinary nematodes, resulting in structural changes and reduced d...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-15718-1 |
| _version_ | 1811224118287138816 |
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| author | Santosh George Peter Suwondo Jewelna Akorli Joseph Otchere Lisa M. Harrison Kaya Bilguvar James R. Knight Debbie Humphries Michael D. Wilson Adalgisa Caccone Michael Cappello |
| author_facet | Santosh George Peter Suwondo Jewelna Akorli Joseph Otchere Lisa M. Harrison Kaya Bilguvar James R. Knight Debbie Humphries Michael D. Wilson Adalgisa Caccone Michael Cappello |
| author_sort | Santosh George |
| collection | DOAJ |
| description | Abstract Global control of hookworm infections relies on periodic Mass Drug Administration of benzimidazole drugs to high-risk groups, regardless of infection status. Mutations in the isotype-1 β-tubulin gene have been identified in veterinary nematodes, resulting in structural changes and reduced drug-binding. In Ghana, previous studies have demonstrated significant variability in albendazole effectiveness among people infected with the hookworm Necator americanus, although the mechanisms underlying deworming response have not been defined. Using hookworm egg samples from a cross-sectional study in Ghana, we developed a multiplex amplicon deep sequencing (MAD-seq) method to screen genomic regions encapsulating putative drug-resistance markers in N. americanus isotype-1 β-tubulin gene. Three single nucleotide polymorphisms (SNPs) corresponding to resistance-associated mutations (F167Y, E198A, F200Y) within the coding region of the isotype-1 β-tubulin gene were characterized using MAD-seq in 30 matched pre- and post-treatment samples from individuals with persistent infection following therapy. Post-sequence analysis showed that the highest mean alternative nucleotide allele at each PCR amplicon was 0.034% (167amplicon) and 0.025% (198/200amplicon), suggesting minimal allelic variation. No samples contained the F167Y SNP, while one contained low-frequency reads associated with E198A (3.15%) and F200Y (3.13%). This MAD-seq method provides a highly sensitive tool to monitor the three putative benzimidazole resistance markers at individual and community levels. Further work is required to understand the association of these polymorphisms to treatment response. |
| first_indexed | 2024-04-12T08:44:50Z |
| format | Article |
| id | doaj.art-3286679ca0d94f1dab115c96dc5d8987 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T08:44:50Z |
| publishDate | 2022-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-3286679ca0d94f1dab115c96dc5d89872022-12-22T03:39:45ZengNature PortfolioScientific Reports2045-23222022-07-0112111010.1038/s41598-022-15718-1Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin geneSantosh George0Peter Suwondo1Jewelna Akorli2Joseph Otchere3Lisa M. Harrison4Kaya Bilguvar5James R. Knight6Debbie Humphries7Michael D. Wilson8Adalgisa Caccone9Michael Cappello10Department of Pediatrics, Yale School of MedicineDepartment of Pediatrics, Yale School of MedicineDepartment of Parasitology, Noguchi Memorial Institute for Medical Research, University of GhanaDepartment of Parasitology, Noguchi Memorial Institute for Medical Research, University of GhanaDepartment of Pediatrics, Yale School of MedicineYale Center for Genome Analysis, Yale School of MedicineYale Center for Genome Analysis, Yale School of MedicineDepartment of Chronic Disease Epidemiology, Yale School of Public HealthDepartment of Parasitology, Noguchi Memorial Institute for Medical Research, University of GhanaDepartment of Ecology and Evolutionary Biology, Yale UniversityDepartment of Pediatrics, Yale School of MedicineAbstract Global control of hookworm infections relies on periodic Mass Drug Administration of benzimidazole drugs to high-risk groups, regardless of infection status. Mutations in the isotype-1 β-tubulin gene have been identified in veterinary nematodes, resulting in structural changes and reduced drug-binding. In Ghana, previous studies have demonstrated significant variability in albendazole effectiveness among people infected with the hookworm Necator americanus, although the mechanisms underlying deworming response have not been defined. Using hookworm egg samples from a cross-sectional study in Ghana, we developed a multiplex amplicon deep sequencing (MAD-seq) method to screen genomic regions encapsulating putative drug-resistance markers in N. americanus isotype-1 β-tubulin gene. Three single nucleotide polymorphisms (SNPs) corresponding to resistance-associated mutations (F167Y, E198A, F200Y) within the coding region of the isotype-1 β-tubulin gene were characterized using MAD-seq in 30 matched pre- and post-treatment samples from individuals with persistent infection following therapy. Post-sequence analysis showed that the highest mean alternative nucleotide allele at each PCR amplicon was 0.034% (167amplicon) and 0.025% (198/200amplicon), suggesting minimal allelic variation. No samples contained the F167Y SNP, while one contained low-frequency reads associated with E198A (3.15%) and F200Y (3.13%). This MAD-seq method provides a highly sensitive tool to monitor the three putative benzimidazole resistance markers at individual and community levels. Further work is required to understand the association of these polymorphisms to treatment response.https://doi.org/10.1038/s41598-022-15718-1 |
| spellingShingle | Santosh George Peter Suwondo Jewelna Akorli Joseph Otchere Lisa M. Harrison Kaya Bilguvar James R. Knight Debbie Humphries Michael D. Wilson Adalgisa Caccone Michael Cappello Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene Scientific Reports |
| title | Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene |
| title_full | Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene |
| title_fullStr | Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene |
| title_full_unstemmed | Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene |
| title_short | Application of multiplex amplicon deep-sequencing (MAD-seq) to screen for putative drug resistance markers in the Necator americanus isotype-1 β-tubulin gene |
| title_sort | application of multiplex amplicon deep sequencing mad seq to screen for putative drug resistance markers in the necator americanus isotype 1 β tubulin gene |
| url | https://doi.org/10.1038/s41598-022-15718-1 |
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