Research Article

Standardization of a molecular method for epidemiologic identification of Leishmania strains

Abstract

Molecular studies of the evolutionary relationships among Leishmania species suggest the presence of high genetic variation within this genus, which has a direct effect on public health in many countries. The coexistence of species in a particular region can result in different leishmaniasis clinical forms and treatment responses. We aimed to standardize the kinetoplast DNA (kDNA) enterobacterial repetitive intergenic consensus (ERIC) sequence polymerase chain reaction (PCR) method for molecular epidemiological identification of Leishmania strains, and estimate existing inter-strain genomic differences and kDNA signatures using this technique. ERIC-PCR of genomic DNA revealed genetic polymorphisms between species, although some strains shared many DNA fragments. Leishmania guyanensis, L. amazonensis, and L. braziliensis clustered together in a dendrogram with similarities ranging from 42.0 to 61.0%, whereas L. chagasi grouped with these three species with a similarity of 28.0%. After amplification of kDNA, 780-bp bands were extracted from an agarose gel and purified for analysis of its genetic signature. kDNA ERIC-PCR electrophoretic patterns consisted of 100- to 600- bp fragments. Using these profiles, L. braziliensis and L. guyanensis grouped with a similarity of 26.0%, and L. amazonensis and L. chagasi clustered based on a similarity of 100%. The electrophoretic profiles and dendrograms showed that, for epidemiological identification by ERIC-PCR, genomic DNA had greater discriminatory power than kDNA did. More strains need to be analyzed to validate the kDNA ERIC-PCR method. The genomes of these strains should be sequenced for better epidemiological identification of Leishmania species.

Molecular studies of the evolutionary relationships among Leishmania species suggest the presence of high genetic variation within this genus, which has a direct effect on public health in many countries. The coexistence of species in a particular region can result in different leishmaniasis clinical forms and treatment responses. We aimed to standardize the kinetoplast DNA (kDNA) enterobacterial repetitive intergenic consensus (ERIC) sequence polymerase chain reaction (PCR) method for molecular epidemiological identification of Leishmania strains, and estimate existing inter-strain genomic differences and kDNA signatures using this technique. ERIC-PCR of genomic DNA revealed genetic polymorphisms between species, although some strains shared many DNA fragments. Leishmania guyanensis, L. amazonensis, and L. braziliensis clustered together in a dendrogram with similarities ranging from 42.0 to 61.0%, whereas L. chagasi grouped with these three species with a similarity of 28.0%. After amplification of kDNA, 780-bp bands were extracted from an agarose gel and purified for analysis of its genetic signature. kDNA ERIC-PCR electrophoretic patterns consisted of 100- to 600- bp fragments. Using these profiles, L. braziliensis and L. guyanensis grouped with a similarity of 26.0%, and L. amazonensis and L. chagasi clustered based on a similarity of 100%. The electrophoretic profiles and dendrograms showed that, for epidemiological identification by ERIC-PCR, genomic DNA had greater discriminatory power than kDNA did. More strains need to be analyzed to validate the kDNA ERIC-PCR method. The genomes of these strains should be sequenced for better epidemiological identification of Leishmania species.