Calmodulin Polymerase Chain Reaction–Restriction Fragment Length Polymorphism for Leishmania Identification and Typing

Abstract

Abstract. A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.Leishmaniasis is a major neglected tropical disease affecting around 2 million people worldwide every year. 1 The infections tend to cluster among the poorest and most isolated increasing the socioeconomic burden upon these vulnerable populations. There are more than 20 species of Leishmania capable of infecting humans and each having distinct epidemiological/demographic patterns, and nearly all have a zoonotic transmission with one main sylvatic reservoir.2 In the absence of vaccines and other effective preventive measures, the control of leishmaniasis is currently based on accurate diagnosis and prompt treatment.A precise identification of the species has epidemiological, and particularly clinical importance, since the Leishmania species involved in human infections can influence the treatment decision and outcome.3 Furthermore, species identification is particularly relevant now that the disease appears to be currently underestimated and on the rise in several countries.

Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama

“…Local studies focus mainly on discriminating Leishmania species on vectors, hosts, and humans rather than inferring their genetic diversity. (10,20,21) Therefore, it is important to conduct further studies in our country using molecular markers that can differentiate Leishmania at the subspecies level. Since haplotypes might arise as an intrinsic attribute of population genetic variation, it is feasible to use these targets as molecular markers in studies aiming to assess the genetic structure of a particular etiological agent.…”

Insights into the Genetic Diversity of Leishmania (Viannia) panamensis in Panama, Inferred via Multilocus Sequence Typing (MLST)

“…Additionally, a few cases of TL caused by L. mexicana have been reported in the country [ 6 , 9 ]. The initial reports of Leishmania species in the country were made using the gold standard approach, multilocus enzyme electrophoresis (MLEE), and after the nineties, reports were made via molecular protocols based on PCR-RFLP or PCR sequencing of specific genes such as calmodulin [ 10 , 11 ], HSP70 [ 8 ], cytochrome b [ 12 ], and kinetoplast minicircles [ 7 ]. Among the Leishmania species reported using these tools, L. panamensis was the most prevalent species found in human cases of TL in Panama.…”