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Comparison of Whole Genome Sequencing versus Standard Molecular Diagnostics for Species Identification in the Leishmania Viannia Subgenus

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  • 1 Public Health Ontario Laboratory, Toronto, Ontario, Canada;
  • | 2 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada;
  • | 3 Division of Diagnostic and Applied Microbiology, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada;
  • | 4 Alberta Precision Laboratories–Public Health, Edmonton, Alberta, Canada;
  • | 5 Faculty of Nursing, University of Toronto, Toronto, Ontario, Canada;
  • | 6 Kirby Institute, University of New South Wales, Sydney, Australia;
  • | 7 Instituto de Medicina Tropical Alejandro von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru;
  • | 8 Facultad de Salud Pública y Administración, Universidad Peruana Cayetano Heredia, Lima, Peru;
  • | 9 Tropical Disease Unit, Toronto General Hospital, Toronto, Canada;
  • | 10 Department of Medicine, University of Toronto, Toronto, Ontario, Canada

ABSTRACT.

The prognosis and treatment of New World tegumentary leishmaniasis is dependent on the infecting species, yet such species identification in the Leishmania Viannia subgenus poses a diagnostic challenge. Currently, speciation relies on standard molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing (SS). Whole-genome sequencing (WGS) is a robust and increasingly cost-efficient tool that may improve Leishmania species identification. We evaluated WGS versus standard RFLP-SS for species identification in three reference and five clinical strains of Leishmania Viannia spp. Internal transcribed spacer1 (its1), cysteine proteinase b (cpb), and heat shock protein 70 (hsp70) polymerase chain reaction-restriction fragment length polymorphism (RFLP) was performed, followed by SS of the its2, cpb, hsp70, and mannose phosphate isomerase (mpi) loci. After de novo assembly, sequences were mapped, and homology compared with both reference strains and reference genomes on National Center for Biotechnology Information. All American Type Culture Collection strains were confirmed to be single-species of L. V. braziliensis, L. V. guyanensis, or L. V. panamensis by WGS. Conversely, RFLP-SS was able to definitively identify one of three isolates to the species level. Clinical samples were identified as either single-species (N = 3), mixed (N = 1), or hybrid (N = 1) infections by WGS, while standard molecular diagnosis required multi-target composite analysis for identification due to loci-dependent results by RFLP-SS. We have corroborated the utility of WGS as a diagnostic tool to speciate members of the L. Viannia subgenus and to discriminate between mixed and hybrid infections. WGS is a potentially useful complement to multistaged RFLP-SS for species identification in Leishmania infections.

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Author Notes

Address correspondence to Andrea K. Boggild, Tropical Disease Unit, Toronto General Hospital, 200 Elizabeth Street, 13EN-218, Toronto, ON, Canada, M5G2C4. E-mail: andrea.boggild@utoronto.ca

Disclosure: This study was approved by the Ethics Research Board of Public Health Ontario.

Financial support: This work was funded by Public Health Ontario via a Project Initiation Fund.

Authors’ addresses: Rachel Lau, Public Health Ontario Laboratory, Toronto, Ontario, Canada, E-mail: rachel.lau@oahpp.ca. Avinash N. Mukkala, Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada, E-mail: avinash.mukkala@mail.utoronto.ca. Ruwandi Kariyawasam, University of Alberta, Edmonton, Alberta, Canada, E-mail: ruwandi@ualberta.ca. Shareese Clarke, Faculty of Nursing, University of Toronto, Toronto, Ontario, Canada, E-mail: shareese.clarke@mail.utoronto.ca. Braulio M. Valencia, Kirby Institute, New South Wales, Australia, E-mail: braulio.valencia@upch.pe. Alejandro Llanos-Cuentas, Universidad Peruana Cayetano Heredia, Lima, Peru, E-mail: alejandro.llanos.c@upch.pe. Andrea K. Boggild, Institute of Medical Science and the Department of Medicine, University of Toronto, Toronto, Ontario, Canada, E-mail: andrea.boggild@utoronto.ca,

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