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
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
Affiliations
Agathe Jouet
GenoScreen, Lille, France
Sofie Marijke Braet
Institute of Tropical Medicine, Antwerp, Belgium; Department of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Antwerp, Belgium; Research Foundation Flanders, Brussels, Belgium
Cyril Gaudin
GenoScreen, Lille, France
Gaëlle Bisch
GenoScreen, Lille, France
Sidra Vasconcellos
Laboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
Rebecca Emmanuela Epaminondas Nicacio de Oliveira do Livramento
Laboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
Yrneh Yadamis Prado Palacios
Laboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
Amanda Brum Fontes
CPqAM/Fiocruz Recife, Pernambuco, Brazil
Norma Lucena
CPqAM/Fiocruz Recife, Pernambuco, Brazil
Patricia Rosa
Instituto Lauro de Souza Lima, Bauru, São Paulo, Brazil
Milton Moraes
Laboratório de Hanseníase, IOC, Fiocruz RJ, Brazil
Kevin La
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, INSERM, IAME UMR1137, Paris, France
Nelly Badalato
GenoScreen, Lille, France
Esteban Lenoir
GenoScreen, Lille, France
Alice Ferré
GenoScreen, Lille, France
Marie Clément
GenoScreen, Lille, France
Epco Hasker
Institute of Tropical Medicine, Antwerp, Belgium
Silahi Halifa Grillone
Damien Foundation, Comoros
Wirdane Abdou
Damien Foundation, Comoros
Aouladi Said
Damien Foundation, Comoros
Younoussa Assoumani
Damien Foundation, Comoros; National Tuberculosis and Leprosy Control Program, Moroni, Comoros
Nissad Attoumani
Damien Foundation, Comoros
Yannick Laurent
GenoScreen, Lille, France
Emmanuelle Cambau
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, INSERM, IAME UMR1137, Paris, France
Bouke Catherine de Jong
Institute of Tropical Medicine, Antwerp, Belgium
Philip Noël Suffys
Laboratório de Biologia Molecular Aplicada a Micobactérias, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil
Philip Supply
Univ. 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.
