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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-LEP
Jouet, Agathe et al. | Date Issued: 2023
Author
Jouet, Agathe
Braet, Sofie Marijke
Gaudin, Cyril
Bisch, Gaëlle
Vasconcellos, Sidra Ezidio Gonçalves
Livramento, Rebecca Emmanuela Epaminondas Nicacio de Oliveira do
Palacios, Yrneh Yadamis Prado
Fontes, Amanda Nogueira Brum
Silva, Norma Lucena Cavalcanti Licinio da
Rosa, Patrícia Sammarco
Moraes, Milton Ozório
La, Kevin
Badalato, Nelly
Lenoir, Esteban
Ferré, Alice
Clément, Marie
Hasker, Epco
Grillone, Silahi Halifa
Abdou, Wirdane
Said, Aouladi
Assoumani, Younoussa
Attoumani, Nissad
Laurent, Yannick
Cambau, Emmanuelle
Jong, Bouke Catherine de
Suffys, Philip Noël
Supply, Philip
Affilliation
GenoScreen. Lille, France.
Institute of Tropical Medicine. Antwerp, Belgium / University of Antwerp. Department of Pharmaceutical, Biomedical and Veterinary Sciences. Antwerp, Belgium / Research Foundation Flanders. Brussels, Belgium.
GenoScreen. Lille, France.
GenoScreen. Lille, France.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.
Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.
Instituto Lauro de Souza Lima. Bauru, SP, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Hanseníase. Rio de Janeiro, RJ, Brasil.
APHP-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. Paris, France.
GenoScreen. Lille, France.
GenoScreen. Lille, France.
GenoScreen. Lille, France.
GenoScreen. Lille, France.
Institute of Tropical Medicine. Antwerp, Belgium.
Damien Foundation. Union of the Comoros.
Damien Foundation. Union of the Comoros.
Damien Foundation. Union of the Comoros.
Damien Foundation. Union of the Comoros / National Tuberculosis and Leprosy Control Program. Moroni, Union of the Comoros.
Damien Foundation. Union of the Comoros.
GenoScreen. Lille, France.
Université Paris Cité. Paris, France.
Institute of Tropical Medicine. Antwerp, Belgium.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular Aplicada a Micobactérias. Rio de Janeiro, RJ, Brasil.
Institut Pasteur de Lille. Center for Infection and Immunity of Lille. Lille, France.
Abstract
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.
Keywords
Mycobacterium leprae
Targeted next generation sequencing
Antibiotic resistance
Gene domain duplication
Diagnostics
Surveillance
 
Publisher
Elsevier
Citation
JOUET, Agathe et al. 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-Lep. EBioMedicine, v. 93, n. 104649, p. 1-14, 14 June 2023.
DOI
10.1016/j.ebiom.2023.104649
ISSN
2352-3964
Notes
Produção científica do Laboratório de Biologia Molecular Aplicada a Micobactérias.
Produção científica do Laboratório de Hanseníase.