2.10. Estimation of parasitic loads

The kit was designed for qualitative amplification of T. cruzi DNA. However, parasitic loads were quantified to further characterize those qPCR positive samples. Thus, a standard quantitative curve was constructed as follows: a pellet of epimastigotes (CL-Brener stock) quantitated using a Neubauer chamber was suspended in EDTA-Blood and mixed with DNAgard to a final concentration of 10⁵ parasites/mL. DNA extraction was performed as described above and diluted serially (1/10) in DNA matrix negative for T. cruzi, which was extracted from a seronegative PVB sample mixed with DNAgard. Quantification curves ranged from 10,000 to 0·5 par.eq/mL and were amplified in each plate together with samples and controls. The qPCR conditions were the same as those for clinical samples and the parasitic loads were automatically calculated by the thermocycler software and expressed in par.eq/mL.