Evaluation of Four Single-Locus Markers for Leishmania Species Discrimination by Sequencing

Abstract

f Several genetic markers have been described for discriminating Leishmania species. In most reported cases, one or a few polymorphisms are the basis of species identification, and the methods were validated on a limited number of strains from a particular geographical region. Therefore, most techniques may underestimate the global intraspecies variability and are applicable only in certain areas. In addition, interlaboratory standardization is mostly absent, complicating comparisons among different studies. Here, we compared species typing results from all sequence polymorphisms found in four popular markers that can be applied directly on clinical samples: the miniexon or spliced leader, the internal transcribed spacer of the ribosomal DNA array, the 7SL RNA gene, and the heat shock protein 70 gene. Clustering was evaluated among 74 Leishmania strains, selected to represent a wide geographic distribution and genetic variability of the medically relevant species of the genus. Results were compared with a multilocus sequence typing (MLST) approach using 7 single-copy household genes and with multilocus enzyme electrophoresis (MLEE), still considered the gold standard by some. We show that strain groupings are highly congruent across the four different single-locus markers, MLST, and MLEE. Overall, the heat shock protein 70 gene and the miniexon presented the best resolutions for separating medically relevant species. As gene sequence analysis is validated here on a global scale, it is advocated as the method of choice for use in genetic, clinical, and epidemiological studies and for managing patients with unknown origins of infection, especially in Western infectious disease clinics dealing with imported leishmaniasis.T he parasitic protozoa of the genus Leishmania cause a spectrum of diseases in humans, collectively called the leishmaniases. In its most benign form, referred to as cutaneous leishmaniasis, the disease manifests itself as a localized skin ulcer at the site of infection by the bite of a female infectious sandfly. Sometimes the parasites spread to other parts of the body, causing secondary lesions. In more severe cases, when the mucosa is infected, a condition known as mucosal leishmaniasis, the disease leads to disfiguring lesions of the nose and mouth. Finally, when the parasite colonizes internal organs such as the spleen, liver, and bone marrow, a condition referred to as visceral leishmaniasis, the disease becomes lethal. As the manifestation of disease depends, to a large extent, on the infecting species, so do the treatment options (1, 2).According to a recent estimate (3), leishmaniasis is endemic in 98 countries and 3 territories. Besides the indigenous population being at risk of infection, many active transmission areas are frequently visited by tourists, military personnel, expatriates, and people visiting friends and relatives. They can potentially import leishmaniasis into their home countries, and management of such cases calls for a globally applicable reliable species typing approa...

Species Typing in Dermal Leishmaniasis

“…Some minor sequence variation between the different copies has been observed, which occasionally complicates interpretation of sequence reads (60,111). It is not clear, however, whether these differences are biological or caused by enzymes in PCR or sequencing reaction mixtures.…”

“…Nevertheless, in silico analysis of the global data set of Van der Auwera et al (60) disproved the possibility of identifying the individual species or species complexes (Fig. 5).…”

“…2), comprising parasites from different countries (33,60). This group has been given various names, such as "outlier L. braziliensis" (66), "atypical L. braziliensis" (67), and "L. braziliensis type 2" (60).…”

“…To assess this, we undertook an in silico RFLP analysis of the data set of Van der Auwera et al (60), and the results are depicted in Fig. 5.…”

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

“…[18][19][20] However, the establishment of MLST or any combination of molecular markers as a global and acceptable typing system requires a detailed evaluation of the number and type of Leishmania genes needed to achieve consistent typing results, as well as the validation of the methodology analyzing a wider range of Leishmania species from different geographical regions. In light of our results, the calmodulin intergenic spacer could be an interesting molecular marker to be considered in the standardization of a global system to type Leishmania.…”