Deep-sampling (replicate PCR) allows detection of T. cruzi in a decreasing frequency of replicate reactions to at least 4 orders of magnitude below the normal limit of quantitation (10−3) used for single PCR reactions. % positive is the percentage of replicate reactions that gave a detectable amplification in 40 cycles (Cq value <40). NTC = no T. cruzi DNA

A. Protocol for the collection and PCR analysis of NHP blood samples. B. The combined PCR results of samples A and B at the first of monthly sampling points, plotting Cq values for all replicates (bottom) and percent positive replicates (top).

Monthly tally of representative macaques with A) rarely, B) variably or C) frequently positive PCR reactions over the one year of sampling. Animals in groups A) and B) were sampled all 12 months while those in C) were sampled only for the first 6 months then in the 12th month. Percentages indicate the overall percentage of positive PCR reactions over all sampling points. D) Macaque P4 switched from 100% negative to 50% positive PCR reactions coincident with a change in health status. E) and F) Pearson correlation analysis indicated a strong positive correlation between the overall frequencies of positive PCRs and hemocultures and a negative correlation between these frequencies and Cq values, but no significant correlation between age or length of infection with any of the three parasite parameters.

DNA Fragmentation increases the sensitivity of PCR detection of T. cruzi DNA. A) The quantity of parasite DNA required to yield 50% positive PCR reactions in replicate assays is reduced by >10-fold by cuphorn fragmentation of DNA from blood. A total of 72 to 96 replicate PCR reactions were conducted for each sample. B) The increased sensitivity of consistent detection of T. cruzi DNA achieved by prior fragmentation is evident in samples from infected macaques. Blood samples were collected as described in Figure 2A for samples A and B but in this case, sample C was also used for DNA isolation and that DNA fragmented by cuphorn sonication. N=the number of replicate PCR

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

T. cruzi DNA can be detected in whole blood, the cell pellet of heparinzed blood, or plasma, but is at the highest and most consistently detected in the blood cell pellet. Replicate blood samples taken at the same time were either processed as whole blood (sample 1) or separated into the cell pellet and plasma (sample 2) by centrifugation, for DNA purification and fragmentation. Twelve replicate aliquots of 125 ng of DNA each were amplified by PCR for each fraction. Mean and standard deviation are shown.

Deep sampling PCR of A) fragmented DNA from whole blood from 9 not-treated, chronically infected humans and B) fragmented blood cell pellet DNA from 20 seropositive dogs. An additional 10 seropositive dogs from this study group were negative for up to 384 replicate PCR reactions (not shown).

Monitoring by deep sampling PCR of T. cruzi DNA from whole blood in dogs during treatment with benznidazole. Dogs were treated with 18-20 mg/kg benznidazole twice (initially) or thrice (if remaining PCR positive) per week.