7ee9bce984b17295e94fa38056ab5e15 pmc_9697816.pdf fb148bfd15dc47aba1abd08f862b3e0c064e936a pmc_9697816.pdf bafc16744d9f60fe6140cf40d809aebd67cdeb045c572005db56e6426dac19a8 pmc_9697816.pdf Title: Identification of Leishmania infantum Epidemiology, Drug Resistance and Pathogenicity Biomarkers with Nanopore Sequencing Subject: The emergence of drug-resistant strains of the parasite Leishmania infantum infecting dogs and humans represents an increasing threat. L. infantum genomes are complex and unstable with extensive structural variations, ranging from aneuploidies to multiple copy number variations (CNVs). These CNVs have recently been validated as biomarkers of Leishmania concerning virulence, tissue tropism, and drug resistance. As a proof-of-concept to develop a novel diagnosis platform (LeishGenApp), four L. infantum samples from humans and dogs were nanopore sequenced. Samples were epidemiologically typed within the Mediterranean L. infantum group, identifying members of the JCP5 and non-JCP5 subgroups, using the conserved region (CR) of the maxicircle kinetoplast. Aneuploidies were frequent and heterogenous between samples, yet only chromosome 31 tetrasomy was common between all the samples. A high frequency of aneuploidies was observed for samples with long passage history (MHOM/TN/80/IPT-1), whereas fewer were detected for samples maintained in vivo (MCRI/ES/2006/CATB033). Twenty-two genes were studied to generate a genetic pharmacoresistance profile against miltefosine, allopurinol, trivalent antimonials, amphotericin, and paromomycin. MHOM/TN/80/IPT-1 and MCRI/ES/2006/CATB033 displayed a genetic profile with potential resistance against miltefosine and allopurinol. Meanwhile, MHOM/ES/2016/CATB101 and LCAN/ES/2020/CATB102 were identified as potentially resistant against paromomycin. All four samples displayed a genetic profile for resistance against trivalent antimonials. Overall, this proof-of-concept revealed the potential of nanopore sequencing and LeishGenApp for the determination of epidemiological, drug resistance, and pathogenicity biomarkers in L. infantum. Keywords: Leishmania infantum; leishmaniosis; drug resistance; treatment; nanopore sequencing; copy number variation; aneuploidy; maxicircle; LeishGenApp Author: Joan Martí-Carreras, Marina Carrasco, Marcel Gómez-Ponce, Marc Noguera-Julián, Roser Fisa, Cristina Riera, Maria Magdalena Alcover, Xavier Roura, Lluís Ferrer and Olga Francino Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Nov 21 02:43:15 2022 CET ModDate: Mon Nov 21 02:50:11 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1513480 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- NRLUCH+URWPalladioL-Bold Type 1 Custom yes yes no 10 0 XTDCWV+URWPalladioL-Roma Type 1 Custom yes yes no 15 0 XNMOUS+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 JSSGKG+URWPalladioL-BoldItal Type 1 Custom yes yes no 23 0 IGDFIY+VnURWPalladioL Type 1 Custom yes yes no 27 0 PRJKGQ+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 SIXRFS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 0 OVNEXW+EURM10 Type 1 Builtin yes yes no 75 0 FLPZNY+CMSY10 Type 1 Builtin yes yes no 82 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 97 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 102 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 107 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 112 0 BCDKEE+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 118 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 134 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 139 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 144 0 BCDJEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 149 0 BCDLEE+ArialMT CID TrueType Identity-H yes yes yes 167 0 BCDMEE+Calibri-Bold CID TrueType Identity-H yes yes yes 176 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-12-08 02:27:53 CET RepresentationInformation: pmc_9697816.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-12-01 02:19:10 CET Size: 1513480 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 434 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4923ab24 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Children: Item: Title: Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: DNA Extraction and Sequencing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Bioinformatic Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Children: Item: Title: Identification, Typing, and Phylogeny Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Aneuploidy and Gene Copy Number Variation Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Data Availability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Children: Item: Title: Identification, Typing, and Phylogeny Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Aneuploidy Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: CNV for Drug Resistance and Pathogenicity Biomarkers in L. infantum Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: Identification, Typing, and Phylogeny Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Detection of Aneuploidy and Gene Copy Number Variation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Info: Title: Identification of Leishmania infantum Epidemiology, Drug Resistance and Pathogenicity Biomarkers with Nanopore Sequencing Author: Joan Martí-Carreras, Marina Carrasco, Marcel Gómez-Ponce, Marc Noguera-Julián, Roser Fisa, Cristina Riera, Maria Magdalena Alcover, Xavier Roura, Lluís Ferrer and Olga Francino Subject: The emergence of drug-resistant strains of the parasite Leishmania infantum infecting dogs and humans represents an increasing threat. L. infantum genomes are complex and unstable with extensive structural variations, ranging from aneuploidies to multiple copy number variations (CNVs). These CNVs have recently been validated as biomarkers of Leishmania concerning virulence, tissue tropism, and drug resistance. As a proof-of-concept to develop a novel diagnosis platform (LeishGenApp), four L. infantum samples from humans and dogs were nanopore sequenced. Samples were epidemiologically typed within the Mediterranean L. infantum group, identifying members of the JCP5 and non-JCP5 subgroups, using the conserved region (CR) of the maxicircle kinetoplast. Aneuploidies were frequent and heterogenous between samples, yet only chromosome 31 tetrasomy was common between all the samples. A high frequency of aneuploidies was observed for samples with long passage history (MHOM/TN/80/IPT-1), whereas fewer were detected for samples maintained in vivo (MCRI/ES/2006/CATB033). Twenty-two genes were studied to generate a genetic pharmacoresistance profile against miltefosine, allopurinol, trivalent antimonials, amphotericin, and paromomycin. MHOM/TN/80/IPT-1 and MCRI/ES/2006/CATB033 displayed a genetic profile with potential resistance against miltefosine and allopurinol. Meanwhile, MHOM/ES/2016/CATB101 and LCAN/ES/2020/CATB102 were identified as potentially resistant against paromomycin. All four samples displayed a genetic profile for resistance against trivalent antimonials. Overall, this proof-of-concept revealed the potential of nanopore sequencing and LeishGenApp for the determination of epidemiological, drug resistance, and pathogenicity biomarkers in L. infantum. 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