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      Detección de ADN de Trypanosoma cruzi en la placenta y fetos de ratones con infección chagásica aguda Translated title: Detection of Trypanosoma cruzi DNA in the placenta and fetuses of mice with chagasic acute infection

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          Abstract

          El objetivo del presente estudio consistió en detectar la presencia de ADN de Trypanosoma cruzi en la placenta y tejidos de fetos provenientes de ratones (Mus musculus) NMRI inoculadas con 22 × 10³ tripomastigotes metacíclicos de la cepa M/HOM/BRA/53/Y por vía intraperitoneal. Los ratones hembras fueron preñadas durante la fase aguda de la infección. El curso de la parasitemia patente por T. cruzi fue evaluada antes del apareamiento y durante el periodo de gestación. A los veinte días de gestación los animales fueron sacrificados y los fetos con sus placentas fueron removidos para evaluar la infección por T. cruzi. Las muestras de placenta, corazón y músculo esquelético fetal fueron fijadas en formalina neutra al 10%, incluidas en parafina y coloreadas con hematoxilina y eosina (HE). El estudio histopatológico de los tejidos fetales reveló la presencia de infiltrado inflamatorio de células mononucleares y polimorfonucleares y sin parasitismo tisular. La amplificación del ADN de T. cruzi por la reacción en Cadena de la Polimerasa (PCR) demostró reacción positiva en el 18% de las placentas de los ratones hembras infectadas preñadas. Las muestras de corazón y músculo esquelético de los fetos provenientes de madres infectadas con T. cruzi no mostraron la presencia de ADN del parásito. Los cortes de placenta y de músculo esquelético analizados por inmunotinción con Peroxidasa anti Peroxidasa mostraron antígeno de T. cruzi en esos tejidos. Los resultados obtenidos por PCR en los tejidos fetales pudieran relacionarse con la virulencia y tropismo asociados con las características biológicas y genética de la cepa de T. cruzi, utilizada en la infección experimental de los ratones hembras.

          Translated abstract

          The objective of the present study was to detect the presence of Trypanosoma cruzi DNA in the placenta and fetal tissues of NMRI mice (Mus musculus) inoculated with 22 × 10³ trypomastigotes metacyclic of the M/HOM/BRA/53/Y strain by intraperitoneal route. Mice were pregnant in the acute phase of the infection. The course of patent parasitemia by T. cruzi was evaluated before mating and during pregnancy. At day twenty of gestation, animals were sacrificed and the fetuses and their placentas were removed to evaluate T. cruzi infection. Samples of fetal placenta, heart and skeletal muscle were fixed in 10%, formalin, included in paraffin and stained with hematoxilin and eosin (HE). The histopathological study of sections of fetal tissues revealed inflammatory infiltrates with mononuclear and polymorphonuclear cells and without parasitism in these tissues. The amplification of T. cruzi DNA by Polymerase Chain Reaction (PCR) showed a positive reaction in 18% of placental tissue of pregnant infected mice. The samples of heart and skeletal muscle of the fetuses of mothers infected with T. cruzi did not show the presence T. cruzi DNA. The placenta and skeletal muscle of the fetuses analyzed by Peroxidase anti Peroxidase inmunostaining showed T. cruzi antigens in those tissues. Negative results by PCR in fetal tissues might be related with the virulence and tropism associated with the biological and genetic characteristic Of the T. cruzi strain used in the experimental infection of female mice.

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          Rapid quantitation of Trypanosoma cruzi in host tissue by real-time PCR.

          A real-time PCR technique that allows for accurate and sensitive quantitation of tissue parasite burden in animals infected with the protozoan parasite Trypanosoma cruzi was developed. The utility of this method was demonstrated by confirmation of higher parasite load in mice with acute infections in comparison to chronically infected mice, detection of tissue-restricted parasite persistence in different parasite:host strain combinations, and the observation of increased tissue parasite burden with higher infective doses. This method should be a useful tool for monitoring parasite burden in hosts under various treatment regimens.
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            Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease.

            Congenital transmission of Trypanosoma cruzi may occur in some or all the gestations from a T. cruzi-infected mother. Variable rates of congenital transmission have been reported in different geographical areas where different parasitic strains predominate, suggesting that parasitic genotypes might play a role in the risk of congenital transmission. Moreover, in cases of transmission it is unknown if the whole maternal T. cruzi population or certain clones are preferentially transmitted by the transplacental route. In this study, bloodstream T. cruzi lineages were identified in blood samples from congenitally infected children, transmitting and non-transmitting mothers and unrelated Chagas disease patients, using improved PCR strategies targeted to nuclear genomic markers. T. cruzi IId was the prevalent genotype among 36/38 PCR-positive congenitally infected infants, 5/5 mothers who transmitted congenital Chagas disease, 12/13 mothers who delivered non-infected children and 28/34 unrelated Chagas disease patients, all coming from endemic localities of Argentina and Bolivia. These figures indicate no association between a particular genotype and vertical transmission. Furthermore, minicircle signatures from the maternal and infants' bloodstream trypanosomes were profiled by restriction fragment length polymorphism of the 330-bp PCR-amplified variable regions in seven cases of mothers and congenitally infected infants. Minicircle signatures were nearly identical between each mother and her infant/s and unique to each mother-infant/s case, a feature that was also observed in twin deliveries. Moreover, allelic size polymorphism analysis of microsatellite loci from populations transmitted to twins showed that all clones from the maternal polyclonal population were equally infective to both siblings.
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              Aetiological treatment of congenital Chagas' disease diagnosed and monitored by the polymerase chain reaction.

              This prospective study focused on the evaluation of anti-parasitic therapy in congenital Chagas' disease, diagnosed and monitored by PCR and conventional diagnosis. We studied 152 children born to seroreactive mothers, living in a non-endemic area. Fifty infants aged 0-6 months (GA) were diagnosed by microhaematocrit and PCR and 102 children aged 7 months to 17 years (GB) were diagnosed by serology and PCR. Forty treated patients were monitored for 2 or 3 years by PCR and conventional methods. A competitive-quantitative PCR was used to determine pre-therapy parasitic loads and follow their post-treatment evolution. In GA, the sensitivities of the PCR and microhaematocrit were 100% and 82.4% and their specificities 97% and 100%, respectively. In GB, the sensitivity of the PCR was 73.8% with a specificity of 100%. Pre-therapy parasitic loads ranged from 12.5 to 125,000 and 12.5 to 125 parasite genomic equivalents/mL of blood in GA and GB, respectively. PCR turned negative in all treated pre-therapy PCR positive patients before or at the end of treatment, which was followed by their seronegativation in 10/10 GA, in 3/5 children initiating therapy at 7 months to 2 years of age but in 0/16 initiating therapy at an older age. Two out of the latter patients were occasionally PCR positive during post-treatment, suggesting no parasitological response. Out of nine pre-therapy PCR negative patients, four turned seronegative after treatment, suggesting that in undetermined patients, undetectable parasitic burdens may lead to better post-treatment prognosis. PCR was useful for sensitive diagnosis and therapy monitoring, allowing early detection of refractory cases.
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                Author and article information

                Contributors
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
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                Role: ND
                Journal
                ic
                Investigación Clínica
                Invest. clín
                Universidad del Zulia (Maracaibo )
                0535-5133
                September 2009
                : 50
                : 3
                : 335-345
                Affiliations
                [1 ] Universidad de los Andes Venezuela
                [2 ] Universidad de los Andes Venezuela
                [3 ] Universidad de los Andes Venezuela
                Article
                S0535-51332009000300008
                13e1ec7e-7f2a-49ae-8706-b46a5dbcfe83

                http://creativecommons.org/licenses/by/4.0/

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                Product

                SciELO Venezuela

                Self URI (journal page): http://www.scielo.org.ve/scielo.php?script=sci_serial&pid=0535-5133&lng=en
                Categories
                MEDICINE, RESEARCH & EXPERIMENTAL

                Medicine
                Trypanosoma cruzi,NMRI mice,congenital infection,fetuses,PCR,ratón NMRI,infección congénita,fetos
                Medicine
                Trypanosoma cruzi, NMRI mice, congenital infection, fetuses, PCR, ratón NMRI, infección congénita, fetos

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