Serial ‘deep-sampling’ PCR of fragmented DNA reveals the wide range of Trypanosoma cruzi burden among chronically infected human, macaque, and canine hosts, and allows accurate monitoring of parasite load following treatment
- Center for Tropical and Emerging Global Disease, United States
- Michale E. Keeling Center for Comparative Medicine and Research, The University of Texas MD Anderson Cancer Center, United States
- Department of Veterinary Integrative Biosciences, School of Veterinary Medicine and Biomedical Sciences, Texas A&M University, United States
- Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, United States
- Research Department, Instituto Nacional de Parasitología "Dr. Mario Fatala Chaben", Buenos Aires, Argentina. Chagas Disease Unit, Hospital Interzonal General de Agudos Eva Perón, Argentina
- Department of Statistics, University of Georgia, United States
- Department of Cellular Biology, University of Georgia, United States
Figures
Figure 1
Deep-sampling (replicate PCR) allows detection of T. cruzi in a decreasing frequency of replicate reactions to at least four 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.
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Figure 1—source data 1
Source data for Figure 1.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig1-data1-v1.xlsx
Figure 2
Monthly screening protocol for macaques.
(A) Protocol for the collection and PCR analysis of non-human primate (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).
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
Monthly tally of representative macaques with (A) rarely, (B) variably, or (C) frequently positive PCR reactions over the 1 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 and then in the 12th month. Percentages indicate the overall percentage of positive PCR reactions across all sampling points. (D) Macaque P4 switched from 100% negative to 50% positive PCR reactions coincident with a change in health status. (E, 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.
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
The monthly pattern of detection of T. cruzi in replicate PCR reactions in DNA from macaque blood collected over 1 year of sampling.
The positive reactions (Cq values <40) plotted for each month are the total from two blood samples collected each month (see Figure 2A). The total number of PCR reactions, the average Cq values and the % positive PCR reactions are provided in Supplementary file 1.
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 2 supplements
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 72–96 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.
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Figure 4—source data 1
Source data for Figure 4A.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig4-data1-v1.xlsx
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Figure 4—source data 2
Source data for Figure 4B.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Fragmentation of DNA increases the frequency of positive replicate PCR reactions in a DNA sample.
(A) Agarose gel profile of DNA isolated from macaque blood spiked with a known number of T. cruzi epimastigotes, either without fragmentation (Unfrag) or using Covaris settings to achieve an average fragment size of 300, 500, or 1000 bases. (B) Four DNA samples from seropositive dogs pre- and post-fragmentation using a sonicator and cuphorn attachment as described in the Materials and methods. Arrows indicate 500 bp band in DNA standards. (C) Fragmentation at all three Covaris settings increased the frequency of PCR positive replicate aliquots with an average of 10–5 parasite equivalents per aliquot. A total of 78–81 replicates were amplified for each condition. (D) The Covaris- and the cuphorn-fragmented DNA provided comparable increases in the frequency of product amplification at different parasite equivalents per aliquot as compared to unfragmented (Unfrag) DNA. A total of 72–90 aliquots were amplified for each condition.
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1C, D.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig4-figsupp1-data1-v1.xlsx
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Figure 4—figure supplement 1—source data 2
Image files of original gels for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig4-figsupp1-data2-v1.zip
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Figure 4—figure supplement 1—source data 3
Marked image files of original gels for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig4-figsupp1-data3-v1.pdf
Figure 4—figure supplement 2
DNA fragmentation results in more consistent PCR amplification and detection, even in samples where T. cruzi DNA may be detectable using a single PCR assay.
Replicate aliquots (10 each) of non-fragmented and sonication-fragmented blood DNA from three macaques at three sampling times were amplified for detection of T. cruzi DNA.
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Figure 4—figure supplement 2—source data 1
Source data for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig4-figsupp2-data1-v1.xlsx
Figure 5
Repeat blood sample collection and deep-sampling PCR of fragmented DNA can quantify infection load over a minimum of eight 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, Figure 3—figure supplement 1, and Supplementary file 1). 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. 10–388 replicate reactions were conducted for each dilution. NTC = no T. cruzi DNA.
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Figure 5—source data 1
Source data for Figure 5A.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig5-data1-v1.xlsx
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Figure 5—source data 2
Source data for Figure 5B.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig5-data2-v1.xlsx
Figure 6
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.
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Figure 6—source data 1
Source data for Figure 6.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig6-data1-v1.xlsx
Figure 7 with 1 supplement
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).
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Figure 7—source data 1
Source data for Figure 7A.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig7-data1-v1.xlsx
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Figure 7—source data 2
Source data for Figure 7B.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig7-data2-v1.xlsx
Figure 7—figure supplement 1
Detection of T. cruzi infection by deep-sampling PCR of blood- or plasma-derived DNA is corroborated by the PCR detection of T. cruzi DNA in individual tissue samples from skeletal muscle, heart, or other organs including liver, spleen, and gut.
The indicated percentages reflect the fraction of PCR positive aliquots of a single DNA sample prepared from blood or plasma, or DNA purified from multiple individual sites in the case of muscle and tissues.
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Figure 7—figure supplement 1—source data 1
Source data for Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig7-figsupp1-data1-v1.xlsx
Figure 8
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.
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Figure 8—source data 1
Source data for Figure 8.
- https://cdn.elifesciences.org/articles/104547/elife-104547-fig8-data1-v1.xlsx
Additional files
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Supplementary file 1
Historical and year one PCR and hemoculture summary for macaques.
N1 and N2 are seronegative controls and macaques T1–T5 were previously infected but cured of T. cruzi infection using benzoxaborole AN15368 (Padilla et al., 2022).
- https://cdn.elifesciences.org/articles/104547/elife-104547-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/104547/elife-104547-mdarchecklist1-v1.pdf
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Serial ‘deep-sampling’ PCR of fragmented DNA reveals the wide range of Trypanosoma cruzi burden among chronically infected human, macaque, and canine hosts, and allows accurate monitoring of parasite load following treatment
eLife 14:RP104547.
https://doi.org/10.7554/eLife.104547.2
