Species typing in dermal leishmaniasis

Abstract

Leishmania is an infectious protozoan parasite related to African and American trypanosomes. All Leishmania species that are pathogenic to humans can cause dermal disease. When one is confronted with cutaneous leishmaniasis, identification of the causative species is relevant in both clinical and epidemiological studies, case management, and control. This review gives an overview of the currently existing and most used assays for species discrimination, with a critical appraisal of the limitations of each technique. The consensus taxonomy for the genus is outlined, including debatable species designations. Finally, a numerical literature analysis is presented that describes which methods are most used in various countries and regions in the world, and for which purposes.

FIG 1

MLEE-based taxonomy of the Leishmania genus as listed by Schönian et al. (21), but with the addition of L. siamensis (22), L. martiniquensis (23), L. adleri, and L. hoogstraali (24). The various levels are indicated by their respective colors. Several species (black) are grouped into “species complexes,” or “complexes” (blue), whereby the complex is named after one of its species. The underlined species are those documented in the studies analyzed in Currently Applied Methods, which are those relevant for human and domestic animal diseases. Species names between brackets are not recognized as separate entities by most authors and in fact are part of the species listed above them. L. chagasi is a synonym of L. infantum of the New World. °, some L. pifanoi strains are more related to L. amazonensis than to L. mexicana (61, 62); *, several authors have reported L. major-like parasites from the New World (62, 79–83). The figure does not represent a dendrogram with evolutionary relationships but a practical classification system.

FIG 2

Multilocus sequence analysis of Van der Auwera et al. (60), based on sequences of 7 housekeeping genes. Each species and species complex are indicated, as in Fig. 1. L. braziliensis outliers are discussed in “Clinically Relevant Taxa: Certainties and Doubts.” L. shawi was not included in this analysis, and L. panamensis was represented by a single strain. The dissimilarity scale is depicted in the top left corner, in substitutions per nucleotide.

FIG 3

Schematic overview of common techniques to discriminate between two species (A and B). See “PCR-Based Methods” for details.

FIG 4

rRNA gene array of L. major strain MHOM/IL/81/Friedlin. The sequence and annotations were taken from www.tritrypdb.org (LmjF chromosome 27, nucleotides 989640 to 998595; accessed on 10 August 2014). Arrows in blue indicate the 5′-to-3′ direction of the genes. The lower panel represents a more detailed view of the most relevant fragment for species typing. Regions used by different authors are indicated above (top) or below (bottom) each panel (24, 77, 102, 111, 112, 114, 116–118, 120–124, 126). The scale of the upper panel is given on the left. SSU, small ribosomal subunit rRNA gene (18S rRNA); LSU, large ribosomal subunit rRNA gene; ITS, internal transcribed spacer.

FIG 5

In silico RFLP analysis of the ITS1 sequences of Van der Auwera et al. (60), covering the LITSR-L5,8S fragment used by Schönian et al. (Fig. 4) (112). For each of the 3 enzymes, the expected fragments are plotted as a function of size in base pairs, whereby each data point on the abscissa represents a different strain. Species abbreviations: aet, L. aethiopica; tro, L. tropica; maj, L. major; don, L. donovani; inf, L. infantum; ama, L. amazonensis; me, L. mexicana; bra, L. braziliensis; per, L. peruviana; o, L. braziliensis outliers (Fig. 2); nai, L. naiffi; guy, L. guyanensis; p, L. panamensis; lai, L. lainsoni.

FIG 6

Primers used for species discrimination by size differences of the variable region of kDNA minicircles (134–138). The primers are mapped to a composite sequence obtained from GenBank accession numbers AF308685 and JF831926 (L. major). The arrows indicate the 5′-to-3′ orientation. The variable region is not drawn to scale.

FIG 7

(A) Cytochrome b coding sequence of L. major (GenBank accession number AB095961) (163). The editing region is shown in red, and the arrow indicates the sense direction. The two overlapping fragments sequenced for typing of clinical samples, covering 887 bp in total, are depicted in green (164). Other fragments amplified for typing of both clinical samples and sand flies are shown in brown, the smallest of which (817 bp) is sequenced (165). The larger brown fragment is the outer PCR amplicon in a nested PCR approach needed for human sample analysis (166). (B) Dendrogram constructed on the basis of cytochrome b gene sequences of reference isolates reported by Luyo-Acero et al. (163), Asato et al. (82), Foulet et al. (164), and Leelayova et al. (22), complemented with the sequences under GenBank accession numbers AB433279, AB433280, AB433282, AB566382, AB566381, and AB566380. The analyzed fragment is indicated in panel A (pink fragment). The dendrogram was constructed by the neighbor-joining method and is based on uncorrected p-distances (the scale is shown below, in substitutions per nucleotide). Bootstrap values from a 2,000-replicate analysis are depicted in percentages at the internodes, when higher than 70%. The dendrogram was constructed with the software package MEGA5 (253).

FIG 8

Mapping of hsp70 fragments used in different studies to the complete coding region of the HSP70 gene (GenBank accession number XM001684512.1) (the arrow indicates the sense direction). These regions were used in either RFLP or sequence analysis (66, 194, 196, 197, 200).

FIG 9

Miniexon repeat of L. major (GenBank accession number X69449). The nontranscribed spacer varies in length and sequence between species, from 51 to 341 bp (209). The PCR product amplified in the assay of Marfurt et al. (209) is indicated, whereby primers are shown with arrows in the 5′-to-3′ direction. All regions are drawn to scale.

FIG 10

Relative use of different species typing methods and targets in the 161 studies where parasite typing was done. The numbers give the percentages of studies using the different methods. The total percentage is more than 100, as many studies used more than one technique.