Identification of six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay.
BACKGROUND
Leishmaniases are tropical zoonotic diseases, caused by parasites from the genus Leishmania. New World (NW) species are related to sylvatic cycles although urbanization processes have been reported in some South American Countries such as Colombia. This eco-epidemiological complexity imposes a challenge to the detection of circulating parasite species, not only related to human cases but also infecting vectors and reservoirs. Currently, no harmonized methods have been deployed to discriminate the NW Leishmania species.


RESULTS
Herein, we conducted a systematic and mechanistic High-Resolution Melting (HRM) assay targeted to HSP70 and ITS1. Specific primers were designed that coupled with a HRM analyses permitted to discriminate six NW Leishmania species. In order to validate the herein described algorithm, we included 35 natural isolates obtained from human cases, insect vectors and mammals. Our genotyping assay allowed the correct assignment of the six NW Leishmania species (L. mexicana, L. infantum (chagasi), L. amazonensis, L. panamensis, L. guyanensis and L. braziliensis) based on reference strains. When the algorithm was applied to a set of well-characterized strains by means of PCR-RFLP, MLEE and monoclonal antibodies (MA) we observed a tailored concordance between the HRM and PCR-RFLP/MLEE/MA (KI = 1.0). Additionally, we tested the limit of detection for the HRM method showing that this is able to detect at least 10 equivalent-parasites per mL.


CONCLUSIONS
This is a rapid and reliable method to conduct molecular epidemiology and host-parasite association studies in endemic areas.
Grupo de Parasitología, Instituto Nacional de Salud, Bogotá, Colombia. dcahernandezc@gmail.com.|Grupo de Investigaciones Microbiológicas - UR (GIMUR), Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia. dcahernandezc@gmail.com.
Carolina
C
Hernández
Centro de Investigaciones en Microbiología y Parasitología Tropical (CIMPAT), Departamento de Ciencias Biológicas, Facultad de Ciencias, Universidad de los Andes, Bogotá, Colombia. mc.alvarez68@uniandes.edu.co.
Catalina
C
Alvarez
Centro de Investigaciones en Microbiología y Parasitología Tropical (CIMPAT), Departamento de Ciencias Biológicas, Facultad de Ciencias, Universidad de los Andes, Bogotá, Colombia. c.gonzalez2592@uniandes.edu.co.
Camila
C
González
Grupo de Parasitología, Instituto Nacional de Salud, Bogotá, Colombia. mayala@ins.gov.co.
Martha Stella
MS
Ayala
Grupo de Investigaciones Microbiológicas - UR (GIMUR), Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia. cmlr8527@gmail.com.
Cielo Maritza
CM
León
Grupo de Investigaciones Microbiológicas - UR (GIMUR), Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia. juand.ramirez@urosario.edu.co.
Juan David
JD
Ramírez
D000818:Animals; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D007303:Insect Vectors / Q000469:parasitology*; D007891:Leishmania / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000469:parasitology*; D008322:Mammals / Q000469:parasitology*; D044366:Transition Temperature
D016054:DNA, Protozoan