First isolation of Leishmania from Northern Thailand:case report, identification as Leishmania martiniquensis and phylogenetic position within the Leishmania enriettii complex

Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as "L. siamensis" also appear to be L. martiniquensis

Giemsa-stained promastigote forms from culture.

<p>A–H, examples showing morphological variation of forms observed, all at the same magnification, bar in F represents 5 µm. Procyclic-like promastigotes can be observed in A (arrows);, leptomonad-like promastigotes in B and C (arrows); nectomonad-like promastigotes in D, E and F; and rosettes and aggregates in G and H.</p

Light micrograph of <i>Leishmania</i> amastigotes in bone marrow aspirate.

<p>Two infected macrophages are stained (large arrows), each with a nucleus (N) and numerous amastigotes (examples with small arrows) within the cytoplasm. The specimen was stained using Wright's stain. The bar represents 20 µm.</p

Phylogenetic analysis by ML method of <i>Leishmania</i> strain CM1 using RNA Pol II sequences.

<p>Tree with 25 species of <i>Leishmania</i> and <i>Endotrypanum monterogeii</i> using <i>Trypanosoma brucei</i> as an outgroup, based on alignment of 1191-1206 homologous nucleotide sequences. AM-2004 is <i>Leishmania</i> from Australia, PCM2 is the “<i>L. siamensis</i>” PCM2 Trang isolate. Bootstrap values from 1000 replicates are given at the nodes.</p

Diagnosis of leishmaniasis by PCR.

<p>Agarose gel electrophoresis showing PCR products using the LeF/LeR primers for <i>Leishmania</i> rRNA ITS-1 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003339#pntd.0003339-Spanakos1" target="_blank">[17]</a> from patient bone marrow aspirate samples compared to five <i>Leishmania</i> species, 1% gel, stained with ethidium bromide. Lane 1, negative control – no DNA; lane 2, <i>L. donovani</i>; lane 3, <i>L. infantum</i>; lane 4, <i>L. tropica</i>; lane 5, <i>L. major</i>; lane 6, “<i>L. siamensis</i>”PCM2 Trang; lane 7, BM1 is bone marrow collected 2 days after commencement of treatment; lane 8, BM2 is bone marrow collected 14 days after commencement of treatment. Size markers (MW), 100 bp ladder, are as shown.</p