Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

Abstract

BackgroundThe 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 FindingsWe 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/SignificanceThe 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.

Evaluation of Nifurtimox Treatment of Chronic Chagas Disease by Means of Several Parasitological Methods

“…Some PCR protocols have been described that have led to unequal results, probably due to differences in the volume of blood processed and in the DNA extraction procedure (38,41,42,43). The true potential of real-time PCR has been well recognized in situations such as treatment of congenital infections (41,44), monitoring parasitemia during and after treatment (13,38,42,45), early detection of relapses after heart transplantation (46), and other immunosuppressive circumstances (47). In this study, the PCR assay directed to minicircles of kDNA from triatomine XD fed from each patient allowed identification of live T. cruzi genotypes amplified in the midgut of the triatomines.…”

Antitrypanosomal Activity of Sterol 14α-Demethylase (CYP51) Inhibitors VNI and VFV in the Swiss Mouse Models of Chagas Disease Induced by the Trypanosoma cruzi Y Strain

“…Guanidine-EDTA blood (GEB) samples were processed with the QIAamp DNA minikit (Qiagen) as previously described (39). Multiplex real-time qPCR assays targeting the T. cruzi satellite nuclear DNA and the exogenous internal amplification control (plasmid pZErO-2 containing an insert from the Arabidopsis thaliana aquaporin gene, 40 amplification cycles) were performed as previously described (40). The standard curves for absolute quantification were constructed with 1/10 serial dilutions of total DNA obtained from a negative GEB sample spiked with 10 5 parasite (Y strain) equivalents/ml of blood.…”

“…The data represent means Ϯ standard deviations from two experiments run in duplicate, and statistical analysis was performed by analysis of variance with the level of significance set at P Յ 0.05 (40).…”

Different Therapeutic Outcomes of Benznidazole and VNI Treatments in Different Genders in Mouse Experimental Models of Trypanosoma cruzi Infection

“…For qPCR, 500 l blood was diluted 1:2 in a guanidine solution (6 M guanidine-HCl-0.2 M EDTA) and heated for 90 s in boiling water in order to promote the denaturation of the parasite kinetoplast DNA network-associated minicircles (12). Guanidine-EDTA blood samples (GEB) were processed using the QIAamp DNA minikit (Qiagen), as described by in reference 13. qPCR multiplex assays were performed targeting the T. cruzi satellite nuclear DNA and the internal amplification control (IAC) (plasmid pZErO-2 containing an insert from the Arabidopsis thaliana aquaporin gene), as described by Duffy et al (14). The standard curves for the absolute quantification were constructed with serial 1/10 dilutions of total DNA obtained from a negative GEB sample spiked with 10 5 parasite equivalents per milliliter of blood.…”