03590nas a2200529   4500000000100000008004100001260001200042653002600054653001600080653002800096653001500124653002500139653004000164100001200204700001200216700001300228700001200241700001900253700001700272700001500289700001300304700001300317700001100330700001300341700000900354700001500363700001300378700001300391700001500404700001300419700001500432700001200447700001100459700001600470700001600486700001400502700001300516700001400529700001300543700001300556245020100569856007100770300000900841490000700850520218900857022001403046       2023                            d  c06/202310aAntibiotic resistance10aDiagnostics10aGene domain duplication10atratamento10aContact surveillance10aTargeted next generation sequencing1 aJouet A1 aBraet S1 aGaudin C1 aBisch G1 aVasconcellos S1 aLivramento R1 aPalacios Y1 aFontes A1 aLucena N1 aRosa P1 aMoraes M1 aLa K1 aBadalato N1 aLenoir E1 aFerré A1 aClément M1 aHasker E1 aGrillone S1 aAbdou W1 aSaid A1 aAssoumani Y1 aAttoumani N1 aLaurent Y1 aCambau E1 ade Jong B1 aSuffys P1 aSupply P00aHi-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.  uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC10279541/pdf/main.pdf  a1-140 v933 a<p><strong>Background: </strong>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&nbsp;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 <em>nth</em>.</p>

<p><strong>Methods: </strong>The limit of detection (LOD) was determined using DNA of M.&nbsp;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.</p>

<p><strong>Findings: </strong>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).</p>

<p><strong>Interpretation: </strong>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.&nbsp;leprae.</p>

<p><strong>Funding:&nbsp;</strong>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.</p>
  a2352-3964