Figure 5.

Repeat blood sample collection and deep-sampling PCR of fragmented DNA can quantify infection load over a minimum of 8 orders of magnitude. A) Frequent blood sampling demonstrates the sampling error involved in the detection of T. cruzi DNA in infected macaques with low parasite burden. Duplicate blood samples were collected twice weekly for 4 weeks from six macaques with the lowest overall PCR positive rate in the monthly sampling study (Figure 3 and S1 Fig). Bleed 1 was used in the experiments in Figure 4; the results of bleeds 2–8 are shown here. DNA was extracted from the duplicate samples at each bleed point and subjected to fragmentation before aliquots were used in 184 replicate PCR reactions/sample. The bottom of each subfigure shows the Cq value of each replicate reaction and the top plots the % positive reactions. B) Replicate PCR analysis of fragmented macaque blood DNA spiked with known parasite equivalents (PE) of T. cruzi DNA. Insets show the linear relationship between Cq values and PE/reaction over the high range of PE and percent positive reactions and PE/reaction on the lower range of inputs. 10 to 388 replicate reactions were conducted for each dilution. NTC = no T. cruzi DNA