Table 1.
Sequences and concentrations of primers and probes used for the Multiplex Taqman qPCR assay.
Table 2.
Inclusivity assay for T. cruzi DTUs.
Table 3.
Exclusivity assay with other Trypanosomatids.
Figure 1.
Anticipated reportable range and linearity of qPCR method.
Multiplex TaqMan qPCR strategy was carried out with spiked GEB samples containing parasite stocks belonging to TcI and TcVI in ten concentrations spanning 106 to 0.625 par. eq./10 mL, tested in triplicate. Assigned values were plotted on the x axis versus measured values (converted to log10) on the y axis using SigmaPlot 10.0 for Windows (SPSS, Chicago, IL). Linear regression analysis rendered the equation y = 1.013x+0.058 (R2 = 0.992) for TcI, and y = 1.001x+0.005 (R2 = 0.998) for TcVI.
Table 4.
Estimation of Precision of the qPCR assay.
Figure 2.
Estimation of the Limit of quantification of qPCR method.
The LOQ was derived from a 20% threshold value for the coefficient of variation (CV) of measurements obtained in the precision experiments reported in Table 4. Linear least squares curve fit for relationship between CV and parasite concentration (log10 par. eq./10 mL) using SigmaPlot 10.0 for Windows (SPSS, Chicago, IL). The derivation of LOQ20%CV is illustrated by dotted lines.
Figure 3.
Distribution of parasitic loads in different patients' groups.
Detectable qPCR findings obtained from peripheral blood samples of Chagas disease patients: G1, orally-acquired infected patients from Chacao, Venezuela (n = 14); G2, chronic Chagas disease patients from Cochabamba, Bolivia (n = 38); G3, chronic Chagas disease patients from endemic regions of Argentina (n = 26); G4, congenitally infected newborns to seropositive women (n = 3). LOQ: Limit of quantification. •: Quantifiable samples above LOQ, ○: Detectable samples below LOQ (1.185 log10 par. eq./10 mL).
Table 5.
Parasitic loads in Chagas disease clinical groups.
Table 6.
Estimation of IAC amplification in blood specimens from different clinical groups.
Figure 4.
Follow-up of T. cruzi infected patients using qPCR.
A. Follow-up of orally infected cases from Chacao, Caracas, Venezuela. Years pos-treatment (ys pos-T) are represented in the x-axis. Parasite equivalents (par. eq.) were estimated using a Silvio X-10 (TcI) calibration curve. Case 1- Pre-T: 5.23 log10 par. eq./10 mL; 2 ys pos-T: 1.88 log10 par. eq./10 mL. Case 2- Pre-T: 3.78 log10 par. eq./10 mL; 2 ys pos-T: 1.83 log10 par. eq./10 mL. Case 3- Pre-T: 2.94 log10 par. eq./10 mL; 2 ys pos-T: 1.88 log10 par. eq./10 mL. B. A 42 year-old seronegative man received kidney transplantation from a seropositive cadaveric donor. Progression of parasitic load after transplantation is shown as well as post-treatment follow-up. The quantification was estimated using a Cl-Brener (TcVI) calibration curve. Days pos-Transplantation (Tx) are represented in the x-axis. The number of par. eq./10 mL of blood is represented in the y-axis, in a log-scale. Arrow marks initiation of Benznidazole treatment. ND: not detectable. The line indicates LOQ (1.185 log10 par. eq./10 mL) derived from analysis of CL-Brener (TcVI) spiked samples. Discontinued line: parasitic loads in Chacao patients were estimated with Silvio X-10 (TcI) calibration curves.