Quantifying Parasite Load in Leishmaniasis

The parasite load was determined by limiting dilution assay [21 (link),52 (link)]. In VL, parasitism was quantified in the spleen and liver, while in cutaneous leishmaniasis, it was analyzed in the skin at the point of parasite inoculation. Briefly, the skin, spleen, and liver were individually weighed, homogenized in S10, and then diluted 1:2000 (skin) or 1:500 (spleen and liver). An initial homogenized suspension was placed into the first well (200 μL) and serial dilutions (1:4) were distributed in a 96-multiwell plate (Nunc, Germany) and subjected to 12 serial dilutions with four replicate wells. After 10 days at 25 °C, each well was examined by optical microscopy and the final titer was set as the highest dilution for which the well contained at least one parasite. The viable parasitic load per gram of homogenized organ was calculated as follows: (reciprocal titer of the last positive well per total volume of homogenized tissue x dilution factor) divided by the weight (gram) of the homogenized tissue. The parasite load was expressed as the number of parasites per gram of homogenized organ.