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.
Highlights
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), remains a major public health concern in 21 endemic countries of America, with an estimated prevalence of 8 million infected people [1].The human disease occurs in two stages: an acute stage, which occurs shortly after an initial infection, and a chronic stage that develops over many years
It is transmitted to humans by blood-sucking triatomine bugs, congenital transmission, blood transfusion, organ transplantation and by consuming food and juice contaminated with the parasite
This study focused on the evaluation of a novel quantitative PCR assay for the diagnosis and follow-up of patients with Chagas disease, on the basis of international guidelines for analytical validation of molecular diagnostic methods
Summary
Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), remains a major public health concern in 21 endemic countries of America, with an estimated prevalence of 8 million infected people [1]. The human disease occurs in two stages: an acute stage, which occurs shortly after an initial infection, and a chronic stage that develops over many years. Individuals from different endemic regions are infected with distinct parasite populations, recently classified into six Discrete Typing Units (DTUs), designated as T. cruzi I (TcI) to T. cruzi VI (TcVI) [3], initially defined as ‘‘sets of stocks that are genetically more related to each other than to any other stock and that are identifiable by common genetic, molecular or immunological. 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
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