Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background: Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).Methods/Principal Findings: The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.Conclusions/Significance: Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.This work received financial support from the Ministry of Science and Technology of Argentina [PICT 2011-0207 to AGS] and the National Scientific and Technical Research Council in Argentina (CONICET) [PIP 112 2011-010-0974 to AGS]. Work related to evaluation of biological samples was partially sponsored by the Pan-American Health Organization (PAHO) [Small Grants Program PAHO-TDR]; the Drugs and Neglected Diseases Initiative (DNDi, Geneva, Switzerland), Wellcome Trust (London, United Kingdom), SANOFI-AVENTIS (Buenos Aires, Argentina) and the National Council for Science and Technology in Mexico (CONACYT) [FONSEC 161405 to JMR]

Neuroscience and End-of-Life Decisions: What Kind of Coexistence?

One of the most far-reaching challenges posed to the law by the development of neuroscience is the possibility to unveil some of the interior elements of human beings in the absence of external manifestations. The perception of pain, the presence of consciousness and the wishes of incompetent patients, once unfathomable because they could not be externalized, are nowadays accessible also from persons unable to express themselves (such as infants, people in comas, severely disabled people, etc.). These new frontiers raise an anthropological question and place heavy demands for lawyers embroiled in end-of-life matters, especially for constitutional lawyers. The present paper aims to highlight how the search for a balanced relationship between neuroscientific acquisitions and end-of-life decisions is weakened by at least two risks: the utmost deference towards science and scientific authority and the maximization of self-determination. In view of all this, the paper will provide, at the beginning, a framework of case law and end-of-life regulatory attempts with a specific focus on the recent law on informed consent and living will approved by the Italian Parliament; it will follow the analysis of the main challenges posed to law by advances in neuroscience. In the final part of this paper, we will offer food for thought on the role of neuroscience and\u2014in a broader perspective\u2014of science in law