Evaluation of four single-locus markers for Leishmania species discrimination by sequencing

Abstract

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.

FIG 1

MLST analysis and comparisons with individual markers. The neighbor-joining dendrogram was built from a concatenated alignment of 7 household genes (20). The numbers shown at the internodes indicate the bootstrap support in percentages, whereby values <70% are not shown. The recognized species are indicated by their bootstrap values and separated by dotted horizontal lines. The dissimilarity scale is depicted in the top left corner in substitutions per nucleotide. The vertical black lines on the right-hand side depict the clusters as seen in dendrograms from the 4 typing markers analyzed in this paper, which are indicated on top (see Fig. S1 to S4 in the supplemental material). For hsp70 and 7SL RNA, the numbers accompanying these lines indicate the bootstrap support of the respective clades. For rDNA ITS1 and ME, the size range of the PCR products is given for each species, as determined from the complete nucleotide sequences. For L. lainsoni, no complete sequences were obtained from the miniexon; the size range is an estimate from agarose gel analysis of the PCR amplicons. Strains are identified using their WHO code, as in Table S1 in the supplemental material, followed by the species as determined by MLEE, if available (maj, L. major; ger, L. gerbilli; tur, L. turanica; aet, L. aethiopica; tro, L. tropica; don, L. donovani; arc, L. archibaldi; inf, L. infantum; mex, L. mexicana; ama, L. amazonensis; gar, L. garnhami; nai, L. naiffi; bra, L. braziliensis; guy, L. guyanensis; pan, L. panamensis). #, strain was not included in the 7SL RNA analysis because no sequences were obtained, ∼, a lacking ITS1 sequence, *, no complete ME sequence could be determined, °, some 5′ nucleotides were lacking from the ME sequence. OWL, Old World L. (Leishmania) subgenus; NWL, New World L. (Leishmania) subgenus; NWV, New World L. (Viannia) subgenus; as indicated on the branch leading to these taxa.

FIG 2

Chromosomal location of the genetic typing markers. The locations of the 7 MLST markers and the 4 single-gene species typing markers analyzed in this paper are shown relative to those of the L. major chromosomes. The 7 MLST genes are depicted in gray below the indicated mapped positions; the 4 targets used as individual typing markers are in black. The sizes of the PCR products without the primers are indicated after the gene names. nt, nucleotides.