Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

Abstract

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Combined parasitological and molecular-based diagnostic tools improve the detection of Trypanosoma cruzi in single peripheral blood samples from patients with Chagas disease

“…Despite this, the absence of a reliable method to detect and quantify parasitemia is still an obstacle for improving our understanding of the impact of persistent parasitemia in the natural history of ChD and to characterize the parasite load in order to evaluate prognosis and therapy 28 . Recent findings have demonstrated a reliable protocol based on real-time PCR for validation and quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment in patients with ChD 56 .…”

Molecular characterization of trypanosomatid infections in wild howler monkeys ( Alouatta caraya ) in northeastern Argentina

“…in all of the study sites, whereas only 6% (n = 7) of these samples were also positive according to SatDNA-PCR, confirming T. cruzi infection. This result was expected because SatDNA-PCR is less sensitive than kDNA-PCR, especially for infections with T. cruzi I or T. cruzi IV (Duffy et al., 2009, Schijman et al., 2011, Ramírez et al., 2015). Therefore, the low prevalence of T. cruzi infection obtained with SatDNA-PCR might have been due to a low parasite burden.…”

New Scheme of Intermittent Benznidazole Administration in Patients Chronically Infected with Trypanosoma cruzi: a Pilot Short-Term Follow-Up Study with Adult Patients

“…These techniques were carried out according to the international workshop sponsored by the Special Program PAHO/WHO Research and Training in Tropical Diseases for validation and standardization of laboratory quantitative PCR (15). The limits of quantification were 0.90 parasitic equivalent in 1 ml of blood (par.eq/ml) and 1.53 par.eq/ml for the kDNA qPCR and SatDNA qPCR, respectively.…”