New Primers for Detection and Differentiation between  Leishmania viannia and L. leishmania Subgenera by Polymerase Chain Reaction

Manuel Calvopiña; David Fonseca-Carrera; Irina  Villacrés-Granda; Alberto  Toapanta; Carlos  Chiluisa-Guacho; Carlos  Bastidas-Caldes

doi:10.18502/ijpa.v18i3.13758

Manuel Calvopiña One Health Research Group, Faculty of Medicine, Universidad de las Américas (UDLA), Quito, Ecuador
David Fonseca-Carrera Biotechnology Engineering, Faculty of Engineering and Applied Sciences (FICA), Universidad de las Américas (UDLA), Quito, Ecuador
Irina Villacrés-Granda Biotechnology Engineering, Faculty of Engineering and Applied Sciences (FICA), Universidad de las Américas (UDLA), Quito, Ecuador
Alberto Toapanta Biotechnology Engineering, Faculty of Engineering and Applied Sciences (FICA), Universidad de las Américas (UDLA), Quito, Ecuador
Carlos Chiluisa-Guacho National Institute for Investigation in Public Health “Leopoldo Izquieta Pérez (INSPI)”, Tena, Ecuador
Carlos Bastidas-Caldes One Health Research Group, Faculty of Medicine, Universidad de las Américas (UDLA), Quito, Ecuador

DOI: https://doi.org/10.18502/ijpa.v18i3.13758

Keywords: Cpb gene; Diagnosis; Leishmania; Naga gene; New world; Primers; Ecuador

Abstract

Background: Leishmania is the parasitic protozoan responsible for leishmaniases, a disease that can cause a range of cutaneous, mucosal, and visceral infections. Two subgenera L. Viannia and L. Leishmania are known to infect humans in the tropics and subtropics of the Americas. The aim of the present study was to develop a new pair of primers for the two subgenera and test in clinical samples.

Methods: We designed two new pairs of primers for a PCR method from two conserved genes, cysteine proteinase B (cpb) and N-acetylglucosamine-6-phosfate deacetylase-like protein (nagA), as specific markers for those two respective subgenera. Primers were tested with 16 microscopical positive clinical samples from the Amazon region of Ecuador obtained in 2010-2020 period.

Results: The cpb presented a band of 172 bp and the nagA a band of 300 bp, thus clearly differentiating L. viannia from L. leishmania. Additionally, primers identified and differentiated the clinical samples in the two subgenera.

Conclusion: The new primers targeting different two genes and standardized in a PCR assay could identified and differentiated Leishmania parasites at subgenus level. This protocol could be used for Leishmania genus identification and diagnosis at the subgenus level and for determining the parasite's geographical distribution where different Leishmania subgenera are found in the same area.