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. 2013;7(1):e2000.
doi: 10.1371/journal.pntd.0002000. Epub 2013 Jan 17.

Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples

Affiliations

Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples

Tomas Duffy et al. PLoS Negl Trop Dis. 2013.

Abstract

Background: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy.

Methods/principal findings: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject.

Conclusions/significance: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
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.
Figure 2
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
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, ○しろまる: Detectable samples below LOQ (1.185 log10 par. eq./10 mL).
Figure 4
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.

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