**3. Demonstration (microscopy) and isolation of parasite (culture) (Figure 6)**

Direct visualization of amastigotes in clinical specimens is the diagnostic gold standard in regions where tissue aspiration is feasible and microscopy and technical skill are available. [24] Microscopy of bone marrow aspirates is the safest diagnostic approach for paediatric patients, with amastigotes seen in more than 90% of cases by an experienced observer. The higher diagnostic efficacy of the bone marrow examination in children is probably related to the heavier parasitisation encountered in children. Microscopic examination of splenic aspirates offers the highest sensitivity (up to 98%), but is assoiated with the risk of lfe-threatening haemorrhage in cases with profound thrombocytopenia. [1] The results of culture in Novy-McNeal-Nicole (NNN) and RPMI 1640 media have been disappointing in one study. [24]

**Figure 6.** Amastigotes forms of *Leishmania spp* by Giemsa stain
