Quantifying Intracellular Parasite Loads

Intracellular parasite loads were analyzed as previously described [19 (link)]. Briefly, cells were infected and treated with nucleotides and were fixed onto slides, stained using a panoptic stain (Laborclin®, PR, Brazil), and counted using a Primo Star light microscope (Zeiss, Germany), with a 40x objective (100x for representative pictures). Images were acquired using a Bx51 camera (Olympus, Tokyo Japan) operated using the Cell^F software. We calculated the “infection index,” representing the overall infection load, based on the count of about 100 cells in a total of five fields to obtain the number of infected macrophages and the average number of parasites per macrophage. Individual amastigotes were clearly visible in the cytoplasm of infected macrophages. The results were expressed as the infection index II = (%infected macrophages) × (amastigotes/infected macrophage)/100.