A highly sensitive Leishmania infantum chagasi isolation method from bone marrow and peripheral blood of adults and children.

Dear Editor, Visceral leishmaniasis (VL) is the most severe form of a neglected tropical diseases group transmitted by phlebotomine sandflies harboring promastigotes forms of donovani complex Leishmania species with distinct eco-epidemiological patterns. The anthroponotic L. donovani in East Africa and India; and the zoonotic L. infantum in Europe and North Africa and in America after its introduction during European colonization, being named L. infantum chagasi [1]. Promastigotes regurgitated by sandfly vector into the mammalian host dermis are phagocytosed by monocyte cells lineage, replicating as amastigotes [2]. Infected macrophages migrate through lymphatic and vascular systems infiltrating lymph nodes (LN) bone marrow (BM), spleen (S) and liver, causing a strict fatal systemic disease, if untreated. According to World Health Organization last review on global VL incidence, 90% of 0.2 to 0.4 million cases per year, worldwide, occur in six countries: India, Bangladesh, Sudan, South Sudan, Ethiopia and Brazil (90% of American human cases); with a mortality rate of 10-20% [3]. In endemic and new affected areas, early and accurate VL diagnosis are shown to decrease mortality rates [4]. VL classical diagnosis correspond to microscopic examination of amastigotes in BM, LN and S cells smears, Giemsa or Leishman stained, obtained through aspiration; a painful, invasive and risky procedure, principally in children. Accuracy of this method depends on the tissue examined, the quality of reagents used and the technician identification ability [5]. Serologic and nucleic acid based VL diagnostic methods present several drawbacks like cross reaction with others American endemic trypanosomiasis including tegumentar leishmaniasis species [6]. Presence of macrophages in peripheral blood (PB) makes culture isolation suitable, as showed for children and adult patients infected with L. infantum from Mediterranean basin [7-9]. Most prominent L. infantum culture isolation techniques from PB presenting high sensitivity depends on sample washing with culture medium and concentration, or isolation of peripheral blood mononuclear cells (PBMC) [7,9], which are hard to be performed in laboratory routine, principally due to contamination risk during sample manipulation. Low efficiency of L. infantum isolation methods are probably related to immune complexes mediated parasite lysis [10,9]. Therefore, by using a BM and PB cells extensive saline washing approach, we obtained a 100% sensitive L. i. chagasi isolation method directly from adult and children patients samples.