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]

Sex-linked peptidase-1 patterns in Pleurodeles waltlii Michah. (Urodele Amphibian) : genetic evidence for a new codominant allele on the W sex chromosome and identification of ZZ, ZW and WW sexual genotypes

International audienc

Candidate targets for Multilocus Sequence Typing of Trypanosoma cruzi: validation using parasite stocks from the Chaco Region and a set of reference strains.

A Multilocus Sequence Typing (MLST) scheme was designed and applied to a set of 20 Trypanosoma cruzi stocks belonging to three main discrete typing units (T. cruzi I, V and VI) from a geographically restricted Chagas disease endemic area in Argentina, 12 reference strains comprising two from each of the six main discrete typing units of the parasite (T. cruzi I-VI), and one T. cruzi marinkellei strain. DNA fragments (≅400-bp) from 10 housekeeping genes were sequenced. A total of 4178 bp were analyzed for each stock. In all, 154 polymorphic sites were identified. Ninety-five sites were heterozygous in at least one analyzed stock. Seventeen diploid sequence types were identified from 32 studied T. cruzi stocks (including the reference strains). All stocks were correctly assigned to their corresponding discrete typing units. We propose this MLST scheme as provisional, with scope for improvement by studying new gene targets on a more diverse sample of stocks, in order to define an optimized MLST scheme for T. cruzi. This approach is an excellent candidate to become the gold standard for T. cruzi genetic typing. We suggest that MLST will have a strong impact on molecular epidemiological studies of Chagas disease and the phylogenetics of its causative agent

Genetic mapping of three human homologues of murine t-complex genes localizes TCP10 to 6q27, 15 cM distal to TCP1 and PLG.

International audienceHuman homologues of mouse t-complex genes have been cloned and localized physically to chromosome 6p or 6q. TCP1, TCP10, and PLG are human homologues of genes located in the proximal portion of the t-complex on mouse chromosome 17. We present here results of genetic mapping of these human t-complex homologues previously localized to 6q25-q27, 6q21-q27, and 6q26-q27, respectively, by physical techniques. TCP1 and PLG do not recombine with each other and are separated from TCP10 by about 15 cM, while the corresponding mouse genes are no more than 4 cM apart. Genetic mapping with markers well localized cytogenetically places TCP1 and PLG proximal to TCP10 and localizes the latter to the cytogenetic band 6q27. It is likely that the organization of human t-complex homologues on 6q is similar to that of t haplotypes rather than that of wildtype murine chromosome 17

Preponderant clonal evolution of Trypanosoma cruzi I from Argentinean Chaco revealed by Multilocus Sequence Typing (MLST)

Trypanosoma cruzi has been historically classified as a species with preponderant clonal evolution (PCE). However, with the advent of highly polymorphic markers and studies at geographically reduced scales,the PCE in T. cruzi was challenged. In fact, some studies have suggested that recombination in T. cruzi lineage I (TcI) is much more frequent than previously believed. Further analyses of TcI populations from different geographical regions of Latin America are needed to examine this hypothesis. In the present study,we contribute to this topic by analyzing the population structure of TcI from a restricted geographical area in the Chaco region, Argentina. We analyzed TcI isolates from different hosts and vectors using a Multilocus Sequence Typing (MLST) approach. These isolates were previously characterized by sequencing the spliced leader intergenic region (SL-IR). Low levels of incongruence and well-supported clusters for MLST dataset were obtained from the analyses. Moreover, high linkage disequilibrium was found and five repeated and overrepresented genotypes were detected. In addition, a good correspondence between SL-IR and MLST was observed which is expected under PCE. However, recombination is not ruled out because five out of 28 pairs of loci were incompatible with strict clonality and one possible genetic exchange event was detected. Overall, our results represent evidence of PCE in TcI from the study area.Finally, considering our findings we discuss the scenario for the genetic structure of TcI.Fil: Tomasini, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Lauthier, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Monje Rumi, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Ragone, Paula Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Alberti D'amato, Anahi M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Perez Brandan, Cecilia Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Basombrio, Miguel Angel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; ArgentinaFil: Diosque, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Patología Experimental; Argentina. Universidad Nacional de Salta; Argentin

Candidate targets for multilocus sequence typing of Trypanosoma cruzi: validation using parasite stocks from the Chaco Region and a set of reference strains

A Multilocus Sequence Typing (MLST) scheme was designed and applied to a set of 20 Trypanosoma cruzi stocks belonging to three main discrete typing units (T. cruzi I, V and VI) from a geographically restricted Chagas disease endemic area in Argentina, 12 reference strains comprising two from each of the six main discrete typing units of the parasite (T. cruzi I-VI), and one T. cruzi marinkellei strain. DNA fragments (congruent to 400-bp) from 10 housekeeping genes were sequenced. A total of 4178 bp were analyzed for each stock. In all, 154 polymorphic sites were identified. Ninety-five sites were heterozygous in at least one analyzed stock. Seventeen diploid sequence types were identified from 32 studied T. cruzi stocks (including the reference strains). All stocks were correctly assigned to their corresponding discrete typing units. We propose this MLST scheme as provisional, with scope for improvement by studying new gene targets on a more diverse sample of stocks, in order to define an optimized MLST scheme for T. cruzi. This approach is an excellent candidate to become the gold standard for T. cruzi genetic typing. We suggest that MLST will have a strong impact on molecular epidemiological studies of Chagas disease and the phylogenetics of its causative agent