Representative of optimization for the multiplex qPCR assay using primers-probe concentration for each target at (i) 250 nM primers/100 nM probe, (ii) 125 nM primers/50 nM probe, and (iii) 62.5 nM primers/25 nM probe and 102 fg/reaction for (A) L. martiniquensis, (B) L. orientalis, and (C) Crithidia sp. (CLA-KP1) as templates. The qPCR reactions were conducted according to the recommended thermal cycling protocol: 1 cycle of polymerase activation at 95 °C for 5 min, followed by 45 cycles of PCR at 95 °C for 15 s, and 60 °C for 1 min.