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.
Antibiotic resistance
Diagnostics
Gene domain duplication
Mycobacterium leprae
Surveillance
Targeted next generation sequencing
Journal
EBioMedicine
ISSN: 2352-3964
Titre abrégé: EBioMedicine
Pays: Netherlands
ID NLM: 101647039
Informations de publication
Date de publication:
Jul 2023
Jul 2023
Historique:
received:
08
02
2023
revised:
25
05
2023
accepted:
29
05
2023
medline:
17
7
2023
pubmed:
17
6
2023
entrez:
16
6
2023
Statut:
ppublish
Résumé
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. 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. 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). 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. 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.
Sections du résumé
BACKGROUND
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
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
RESULTS
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
CONCLUSIONS
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
BACKGROUND
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.
Identifiants
pubmed: 37327675
pii: S2352-3964(23)00214-1
doi: 10.1016/j.ebiom.2023.104649
pmc: PMC10279541
pii:
doi:
Substances chimiques
Dapsone
8W5C518302
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
104649Informations de copyright
Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of interests A.J., C.G., G.B., N.B., E.L., A.F., M.C. and Y.L. are employees of GenoScreen (Lille, France). P.S. is a scientific consultant for the same company. Other authors declare that they have no other competing interests.