Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-LepResearch in context
Summary: Background: Expansion of antimicrobial resistance monitoring and epidemiological surveillance are key components of the WHO strategy towards zero leprosy. The inability to grow Mycobacterium leprae in vitro precludes routine phenotypic drug susceptibility testing, and only limited molecula...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-07-01
|
| Series: | EBioMedicine |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352396423002141 |
| _version_ | 1797802806763061248 |
|---|---|
| author | Agathe Jouet Sofie Marijke Braet Cyril Gaudin Gaëlle Bisch Sidra Vasconcellos Rebecca Emmanuela Epaminondas Nicacio de Oliveira do Livramento Yrneh Yadamis Prado Palacios Amanda Brum Fontes Norma Lucena Patricia Rosa Milton Moraes Kevin La Nelly Badalato Esteban Lenoir Alice Ferré Marie Clément Epco Hasker Silahi Halifa Grillone Wirdane Abdou Aouladi Said Younoussa Assoumani Nissad Attoumani Yannick Laurent Emmanuelle Cambau Bouke Catherine de Jong Philip Noël Suffys Philip Supply |
| author_facet | Agathe Jouet Sofie Marijke Braet Cyril Gaudin Gaëlle Bisch Sidra Vasconcellos Rebecca Emmanuela Epaminondas Nicacio de Oliveira do Livramento Yrneh Yadamis Prado Palacios Amanda Brum Fontes Norma Lucena Patricia Rosa Milton Moraes Kevin La Nelly Badalato Esteban Lenoir Alice Ferré Marie Clément Epco Hasker Silahi Halifa Grillone Wirdane Abdou Aouladi Said Younoussa Assoumani Nissad Attoumani Yannick Laurent Emmanuelle Cambau Bouke Catherine de Jong Philip Noël Suffys Philip Supply |
| author_sort | Agathe Jouet |
| collection | DOAJ |
| description | Summary: Background: Expansion of antimicrobial resistance monitoring and epidemiological surveillance are key components of the WHO strategy towards zero leprosy. The inability to grow Mycobacterium leprae in vitro precludes routine phenotypic drug susceptibility testing, and only limited molecular tests are available. We evaluated a culture-free targeted deep sequencing assay, for mycobacterial identification, genotyping based on 18 canonical SNPs and 11 core variable-number tandem-repeat (VNTR) markers, and detection of rifampicin, dapsone and fluoroquinolone resistance-associated mutations in rpoB/ctpC/ctpI, folP1, gyrA/gyrB, respectively, and hypermutation-associated mutations in nth. Methods: The limit of detection (LOD) was determined using DNA of M. leprae reference strains and from 246 skin biopsies and 74 slit skin smears of leprosy patients, with genome copies quantified by RLEP qPCR. Sequencing results were evaluated versus whole genome sequencing (WGS) data of 14 strains, and versus VNTR-fragment length analysis (FLA) results of 89 clinical specimens. Findings: The LOD for sequencing success ranged between 80 and 3000 genome copies, depending on the sample type. The LOD for minority variants was 10%. All SNPs detected in targets by WGS were identified except in a clinical sample where WGS revealed two dapsone resistance-conferring mutations instead of one by Deeplex Myc-Lep, due to partial duplication of the sulfamide-binding domain in folP1. SNPs detected uniquely by Deeplex Myc-Lep were missed by WGS due to insufficient coverage. Concordance with VNTR-FLA results was 99.4% (926/932 alleles). Interpretation: Deeplex Myc-Lep may help improve the diagnosis and surveillance of leprosy. Gene domain duplication is an original putative drug resistance-related genetic adaptation in M. leprae. Funding: EDCTP2 programme supported by the European Union (grant number RIA2017NIM-1847 -PEOPLE). EDCTP, R2Stop: Effect:Hope, The Mission To End Leprosy, the Flemish Fonds Wetenschappelijk Onderzoek. |
| first_indexed | 2024-03-13T05:12:17Z |
| format | Article |
| id | doaj.art-afa44bec300b4e1ca706ecb98b36b085 |
| institution | Directory Open Access Journal |
| issn | 2352-3964 |
| language | English |
| last_indexed | 2024-03-13T05:12:17Z |
| publishDate | 2023-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | EBioMedicine |
| spelling | doaj.art-afa44bec300b4e1ca706ecb98b36b0852023-06-16T05:10:04ZengElsevierEBioMedicine2352-39642023-07-0193104649Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-LepResearch in contextAgathe Jouet0Sofie Marijke Braet1Cyril Gaudin2Gaëlle Bisch3Sidra Vasconcellos4Rebecca Emmanuela Epaminondas Nicacio de Oliveira do Livramento5Yrneh Yadamis Prado Palacios6Amanda Brum Fontes7Norma Lucena8Patricia Rosa9Milton Moraes10Kevin La11Nelly Badalato12Esteban Lenoir13Alice Ferré14Marie Clément15Epco Hasker16Silahi Halifa Grillone17Wirdane Abdou18Aouladi Said19Younoussa Assoumani20Nissad Attoumani21Yannick Laurent22Emmanuelle Cambau23Bouke Catherine de Jong24Philip Noël Suffys25Philip Supply26GenoScreen, Lille, FranceInstitute of Tropical Medicine, Antwerp, Belgium; Department of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Antwerp, Belgium; Research Foundation Flanders, Brussels, BelgiumGenoScreen, Lille, FranceGenoScreen, Lille, FranceLaboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, BrazilLaboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, BrazilLaboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, BrazilCPqAM/Fiocruz Recife, Pernambuco, BrazilCPqAM/Fiocruz Recife, Pernambuco, BrazilInstituto Lauro de Souza Lima, Bauru, São Paulo, BrazilLaboratório de Hanseníase, IOC, Fiocruz RJ, BrazilAPHP-GHU Paris Nord Hôpital Bichat, Service de mycobactériologie spécialisée et de référence, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux - Laboratoire Associé, Paris, France; Université Paris Cité, INSERM, IAME UMR1137, Paris, FranceGenoScreen, Lille, FranceGenoScreen, Lille, FranceGenoScreen, Lille, FranceGenoScreen, Lille, FranceInstitute of Tropical Medicine, Antwerp, BelgiumDamien Foundation, ComorosDamien Foundation, ComorosDamien Foundation, ComorosDamien Foundation, Comoros; National Tuberculosis and Leprosy Control Program, Moroni, ComorosDamien Foundation, ComorosGenoScreen, Lille, FranceAPHP-GHU Paris Nord Hôpital Bichat, Service de mycobactériologie spécialisée et de référence, Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux - Laboratoire Associé, Paris, France; Université Paris Cité, INSERM, IAME UMR1137, Paris, FranceInstitute of Tropical Medicine, Antwerp, BelgiumLaboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, BrazilUniv. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, F-59000 Lille, France; Corresponding author.Summary: Background: Expansion of antimicrobial resistance monitoring and epidemiological surveillance are key components of the WHO strategy towards zero leprosy. The inability to grow Mycobacterium leprae in vitro precludes routine phenotypic drug susceptibility testing, and only limited molecular tests are available. We evaluated a culture-free targeted deep sequencing assay, for mycobacterial identification, genotyping based on 18 canonical SNPs and 11 core variable-number tandem-repeat (VNTR) markers, and detection of rifampicin, dapsone and fluoroquinolone resistance-associated mutations in rpoB/ctpC/ctpI, folP1, gyrA/gyrB, respectively, and hypermutation-associated mutations in nth. Methods: The limit of detection (LOD) was determined using DNA of M. leprae reference strains and from 246 skin biopsies and 74 slit skin smears of leprosy patients, with genome copies quantified by RLEP qPCR. Sequencing results were evaluated versus whole genome sequencing (WGS) data of 14 strains, and versus VNTR-fragment length analysis (FLA) results of 89 clinical specimens. Findings: The LOD for sequencing success ranged between 80 and 3000 genome copies, depending on the sample type. The LOD for minority variants was 10%. All SNPs detected in targets by WGS were identified except in a clinical sample where WGS revealed two dapsone resistance-conferring mutations instead of one by Deeplex Myc-Lep, due to partial duplication of the sulfamide-binding domain in folP1. SNPs detected uniquely by Deeplex Myc-Lep were missed by WGS due to insufficient coverage. Concordance with VNTR-FLA results was 99.4% (926/932 alleles). Interpretation: Deeplex Myc-Lep may help improve the diagnosis and surveillance of leprosy. Gene domain duplication is an original putative drug resistance-related genetic adaptation in M. leprae. Funding: EDCTP2 programme supported by the European Union (grant number RIA2017NIM-1847 -PEOPLE). EDCTP, R2Stop: Effect:Hope, The Mission To End Leprosy, the Flemish Fonds Wetenschappelijk Onderzoek.http://www.sciencedirect.com/science/article/pii/S2352396423002141Mycobacterium lepraeTargeted next generation sequencingAntibiotic resistanceGene domain duplicationDiagnosticsSurveillance |
| spellingShingle | Agathe Jouet Sofie Marijke Braet Cyril Gaudin Gaëlle Bisch Sidra Vasconcellos Rebecca Emmanuela Epaminondas Nicacio de Oliveira do Livramento Yrneh Yadamis Prado Palacios Amanda Brum Fontes Norma Lucena Patricia Rosa Milton Moraes Kevin La Nelly Badalato Esteban Lenoir Alice Ferré Marie Clément Epco Hasker Silahi Halifa Grillone Wirdane Abdou Aouladi Said Younoussa Assoumani Nissad Attoumani Yannick Laurent Emmanuelle Cambau Bouke Catherine de Jong Philip Noël Suffys Philip Supply Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-LepResearch in context EBioMedicine Mycobacterium leprae Targeted next generation sequencing Antibiotic resistance Gene domain duplication Diagnostics Surveillance |
| title | Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-LepResearch in context |
| title_full | Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-LepResearch in context |
| title_fullStr | Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-LepResearch in context |
| title_full_unstemmed | Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-LepResearch in context |
| title_short | Hi-plex deep amplicon sequencing for identification, high-resolution genotyping and multidrug resistance prediction of Mycobacterium leprae directly from patient biopsies by using Deeplex Myc-LepResearch in context |
| title_sort | hi plex deep amplicon sequencing for identification high resolution genotyping and multidrug resistance prediction of mycobacterium leprae directly from patient biopsies by using deeplex myc lepresearch in context |
| topic | Mycobacterium leprae Targeted next generation sequencing Antibiotic resistance Gene domain duplication Diagnostics Surveillance |
| url | http://www.sciencedirect.com/science/article/pii/S2352396423002141 |
| work_keys_str_mv | AT agathejouet hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT sofiemarijkebraet hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT cyrilgaudin hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT gaellebisch hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT sidravasconcellos hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT rebeccaemmanuelaepaminondasnicaciodeoliveiradolivramento hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT yrnehyadamispradopalacios hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT amandabrumfontes hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT normalucena hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT patriciarosa hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT miltonmoraes hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT kevinla hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT nellybadalato hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT estebanlenoir hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT aliceferre hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT marieclement hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT epcohasker hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT silahihalifagrillone hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT wirdaneabdou hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT aouladisaid hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT younoussaassoumani hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT nissadattoumani hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT yannicklaurent hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT emmanuellecambau hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT boukecatherinedejong hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT philipnoelsuffys hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext AT philipsupply hiplexdeepampliconsequencingforidentificationhighresolutiongenotypingandmultidrugresistancepredictionofmycobacteriumlepraedirectlyfrompatientbiopsiesbyusingdeeplexmyclepresearchincontext |