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Toxoplasmosis is caused by the parasite Toxoplasma gondii. The infection is generally asymptomatic and the most severe cases occur in immunosuppressed patients. The main route of transmission is the ingestion of water or food contaminated with cysts of the parasite. The objective of this work was the standardization of the PCR for the detection of the T. gondii B1 gene in meat and water samples and cloning of the product for use as a control.The optimal reaction conditions of the different components of the PCR were determined and the technique was used to detect DNA from meat and water samples. Bands were purified and cloned into a pGEM-T-Easy vector and used as a control in the PCR.Optimal PCR conditions were; 100 µM dNTP, 0.4 µM primers, and 0.5 U Taq polymerase. The product obtained from the PCR was cloned with a simple cloning strategy with efficient results. With the standardized PCR and using the cloned DNA as a control, T. gondii DNA was detected in 90% of the positives samples of meat and water and there was no amplification in the negative samples.The PCR assay standardized in this study was demonstrated to be an effective technique to detect T. gondii DNA in meat and water samples. The cloning of PCR product and its application as a control in molecular diagnosis of toxoplasmosis might improve the reproducibility of this method and avoid the use of patient samples or cultures, which present several limitations.

Resumen Introduccion La sensibilidad y especificidad de las tecnicas de diagnostico para la enfermedad de Chagas dependen en gran parte de los antigenos y las dianas utilizadas y de la respuesta inmunologica y las caracteristicas de la infeccion de la poblacion donde se aplica, de alli la necesidad de la evaluacion de las tecnicas de diagnostico disponibles en un area determinada, por lo que el objetivo de este trabajo fue evaluar dos estuches comerciales para el diagnostico inmunologico y molecular de la enfermedad de Chagas en zonas endemicas de Venezuela. Metodos Se evaluaron los estuches: Chagas ELISA IgG + IgM® y Speed Oligo Chagas® (Vircell®, Granada, Espana). Se valoraron con 129 muestras (35 de pacientes en fase aguda, 33 en fase cronica, 31 de pacientes con otras enfermedades y 30 de individuos sanos). Se compararon los resultados con los obtenidos en las pruebas convencionales ELISA y PCR-ADN satelite de Trypanosoma cruzi. Resultados Con Chagas ELISA IgG + IgM® se obtuvo una sensibilidad de 94,1% y especificidad de 93,4%, con Speed Oligo Chagas® se obtuvo una sensibilidad de 92,6% y especificidad de 100%, valores similares a los obtenidos con ELISA y PCR-ADNsat convencionales. Conclusion La sensibilidad y especificidad de los estuches comerciales evaluados los hacen adecuados para el diagnostico de la enfermedad de Chagas en zonas endemicas de Venezuela.

Trypanosoma cruzi, Trypanosoma rangeli and Leishmania spp. are parasites that coexist in several endemic areas. The identification of these parasites in hosts is important for the control programs.216 samples from human blood (101), blood of other mammals (45) and triatomine intestinal content and hemolymph (70), from an endemic area of Venezuela, were analysed. The samples were evaluated by; serology (only humans) and PCR for T. cruzi in human, other mammals and triatomines, PCR for T. rangeli in mammals-including human and triatomines and PCR for Leishmania in mammals-including human.The 9.9% of the human samples were positive for T. cruzi by serology, 11.9% by PCR, 4% for T. rangeli PCR and none for Leishmania spp. PCR. 60% of the samples of other mammals showed DNA amplification for T. cruzi, 42.2% for T. rangeli and 4.4% for Leishmania spp. 61.4% of the triatomine samples showed DNA amplification for T. cruzi and 10% for T. rangeli.High T. cruzi infection was detected in mammals and triatomines compared with T. rangeli. Low leishmanial infection was detected in other mammals. It is the first time that T. cruzi/T. rangeli coinfection, in humans, Canis familiaris (dog), and Bos Taurus (cow), were reported world-wide, and that this coinfection was described in Tamandua tetradactyla (anteater) from Venezuela. The coinfection T. cruzi/T. rangeli in mammals-including humans and triatomines, and coinfection T. cruzi/Leishmania spp. in non-human mammals, show the risk for trypanosomic zoonoses in this endemic area.

BACKGROUNDZika virus (ZIKV) is highly teratogenic, in contrast with dengue virus (DENV) or the yellow fever virus vaccine (YFV-17D). The mechanisms employed by ZIKV to cross the placenta need to be elucidated.METHODSParallel infections with ZIKV, DENV and YFV-17D were compared in terms of efficiency, activation of mTOR pathways and cytokine secretion profile in human cytotrophoblastic HTR8 cells (CTB), and monocytic U937 cells, differentiated to M2 macrophages (M2-MØ).RESULTSIn CTB, ZIKV replication was significantly more efficient than DENV or YFV-17D. In M2-MØ, ZIKV replication continued to be more efficient, although differences between strains were reduced. Significantly greater activation of Phospho-S6r and Phospho-AKT/Ser473 fractions in CTB infected with ZIKV than with DENV or YFV-17D, was observed. CTB treated with the mTOR inhibitors rapamycin or AZD8055, showed a 20-fold-reduction in ZIKV yield, versus 5 and 3.5-fold for DENV and YFV-17D, respectively. Finally, we detected that ZIKV infection, but not DENV or YFV-17D, efficiently inhibited the interferon response of CTB cells.CONCLUSIONSThese results suggest that CTB cells are permissive and act favoring ZIKV entry into the placental stroma, over DENV and YFV-17D and that the mTOR complex is a switch that enhances the replication of ZIKV in CTB cells.

Las infecciones con el virus del dengue, transmitidas al humano por mosquitos del género Aedes sp., constituyen un problema de salud pública para las regiones tropicales y subtropicales del planeta. El ciclo de replicación del virus del dengue se lleva a cabo principalmente en el citoplasma de las células, en estrecha asociación con organelos membranosos como el retículo endoplasmático rugoso y el complejo de Golgi. En esta revisión se analiza la respuesta al estrés de estos organelos, ante la infección viral, tanto en células de vertebrados como de mosquitos, y se discuten las implicaciones de esta respuesta en la biología de estos agentes.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. A total of 817 mosquito targets were identified as protein–protein interacting with DENV NS1. Approximately 14% of them coincide with interactomes previously obtained in vertebrate cells, including the oligosaccharide transferase complex, the chaperonin containing TCP-1, vesicle localization, and ribosomal proteins. Notably, other protein pathways not previously reported in vertebrate cells, such as epigenetic regulation and RNA silencing, were also found in the NS1 interactome in mosquito cells. Due to the novel and strong interactions observed for NS1 and the epigenetic regulator DIDO1 (Death-Inducer Obliterator 1), the role of DIDO1 in viral replication was further explored. Interactions between NS1 and DIDO1 were corroborated in infected mosquito cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected cells silenced for DIDO1 revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD, and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response necessary for flavivirus replication in mosquito cells. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication, and add to the understanding of NS1 as a multifunctional protein. IMPORTANCE Dengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as transcription and epigenetic regulation, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1 revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

Direct test over the gut material from triatomine vectors and xenodiagnosis over mammalian hosts are classical techniques for Trypanosoma cruzi parasitological diagnosis. Nevertheless, negative results can be a source of uncertainty. Experimental models have allowed evaluating the tissue invasion of different strains of T. cruzi, but conventional techniques for tissue biopsies involve time-consuming and elaborated procedures and have low sensitivity. Gut material of collected triatomines (microscopically negative) (n = 114), material of mammal xenodiagnoses (microscopically negative) (n = 138), and biopsy material (microscopically negative) from experimentally infected animals (n = 34) with isolates from endemic areas of Chagas' disease from Venezuela were used for DNA extraction and PCR for the amplification of kinetoplast DNA (kDNA) and satellite DNA (sDNA) of T. cruzi. Positive PCR was observed in 53.6% of collected triatomine material, 15.8% of parasitological negative xenodiagnosis material, and 70.6% in biopsies, revealing underestimation by the parasitological tests and the valour of this analysis with preserved material. Anzoátegui was the state with the highest percentage of infection, and the triatomine species Rhodnius prolixus and Panstrongylus geniculatus had the highest percentages of infection. Didelphis marsupialis and Canis familiaris were the most infected by T. cruzi revealed by PCR of xenodiagnosis material. In addition, the PCR technique allowed demonstrating the invasion of T. cruzi in all tissues analyzed, constituting a molecular marker of tissue invasion.

Septins are a family of GTP-binding proteins identified in insects and mammals. Septins are components of the cytoskeleton and participate in cytokinesis, chromosomal segregation, intracellular vesicular traffic, and response to pathogens. Human septin 6 was identified as necessary for hepatitis C virus replication. Information about host factors necessary for flavivirus replication in mosquitoes is scarce. Thus, the role of septins in the replicative cycle of dengue virus in Aedes spp. derived cells was investigated. Through bioinformatic analysis, sequences of septin-like proteins were identified. Infected mosquito cells showed increased expression of Sep2. Colocalization analysis, proximity ligation and immunoprecipitation assays indicated that Sep2 interacts with proteins E, NS3 and NS5, but not NS1. Immunoelectron microscopy evidenced the presence of AalSep2 in replicative complexes. Finally, silencing of Sep2 expression resulted in a significant decrease in virus progeny, indicating that Sep2 is a host factor participating in dengue virus replication in mosquito cells.

Dengue virus (DENV) NS1 is a multifunctional protein essential for viral replication. To gain insights into NS1 functions in mosquito cells, the protein interactome of DENV NS1 in C6/36 cells was investigated using a proximity biotinylation system and mass spectrometry. Approximately 14% of the 817 identified proteins coincide with interactomes obtained in vertebrate cells, including ontology groups of the oligosaccharide transferase complex, the chaperonin containing TCP-1, and nuclear import and export, vesicle localization and ribosomal proteins. Notably, other protein pathways such as epigenetic regulation and RNA silencing, not previously reported in vertebrate cells, were also found in the NS1 interactome in mosquito cells. Due to the novel interaction observed for NS1 and DIDO1 (Death Inducer-Obliterator 1), we further explored the role of DIDO1 in viral replication. Interactions between NS1 and DIDO1were corroborated in infected C6/36 and Aag2 cells, by colocalization and proximity ligation assays. Silencing DIDO1 expression in C6/36 and Aag2 cells results in a significant reduction in DENV and ZIKV replication and progeny production. Comparison of transcription analysis of mock or DENV infected C6/36 silenced for DIDO1, revealed variations in multiple gene expression pathways, including pathways associated with DENV infection such as RNA surveillance, IMD and Toll. These results suggest that DIDO1 is a host factor involved in the negative modulation of the antiviral response and necessary for flavivirus replication. Our findings uncover novel mechanisms of NS1 to promote DENV and ZIKV replication and add to the understanding of NS1 as a multifunctional protein.ABSTRACT IMPORTANCEDengue is the most important mosquito-borne viral disease to humans. Dengue virus NS1 is a multifunctional protein essential for replication and modulation of innate immunity. To gain insights into NS1 functions, the protein interactome of dengue virus NS1 in Aedes albopictus cells was investigated using a proximity biotinylation system and mass spectrometry. Several protein pathways, not previously observed in vertebrate cells, such as epigenetic regulation and RNA silencing, were found as part of the NS1 interactome in mosquito cells. Among those, DIDO1 was found to be a necessary host factor for dengue and Zika virus replication in vertebrate and mosquito cells. Transcription analysis of infected mosquito cells silenced for DIDO1, revealed alterations of the IMD and Toll pathways, part of the antiviral response in mosquitoes. The results suggest that DIDO1 is a host factor involved in modulation of the antiviral response and necessary for flavivirus replication.

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. Few examples of Golgi stress responses due to physiological stimuli are known. Furthermore, the impact on this organelle of viral infections that occupy the vesicular transport during replication is unknown. In this work, we evaluated if a Golgi stress response is triggered during dengue and Zika viruses replication, two flaviviruses whose replicative cycle is heavily involved with the Golgi complex, in vertebrate and mosquito cells. Using GM-130 as a Golgi marker, and treatment with monensin as a positive control for the induction of the Golgi stress response, a significant expansion of the Golgi cisternae was observed in BHK-21, Vero E6 and mosquito cells infected with either virus. Activation of the TFE3 pathway was observed in the infected cells as indicated by the translocation from the cytoplasm to the nucleus of TFE3. Of note, no sign of activation of the stress response was observed in CRFK cells infected with Feline Calicivirus (FCV), a virus released by cell lysis, not requiring vesicular transport. Finally, dilatation of the Golgi complex and translocation of TFE3 was observed in vertebrate cells expressing dengue and Zika viruses NS1, but not NS3. These results indicated that infections by dengue and Zika viruses induce a Golgi stress response in vertebrate and mosquito cells due to the increased demand on Golgi complex imposed by virion and NS1 processing and secretion.

Background & objectives: Trypanosoma cruzi and Leishmania spp. are protozoans that cause American trypanosomiasis and leishmaniasis, respectively. In endemic foci where both diseases coincide, coinfection can occur. The objective of this work was the characterization of the parasites involved in coinfection in several endemic areas of Venezuela. Methods: Molecular characterization was done in 30 samples of several species of mammals (Didelphis marsupialis, Equus mulus, Rattus rattus, Canis familiaris, Felis catus, and Sciurus granatensis) from the states of Anzoategui, Cojedes and Capital District diagnosed with T. cruzi and Leishmania spp. coinfections. For the typing of T. cruzi DTUs, the markers of miniexon, 24Sa rDNA, 18Sa rDNA, and hsp60-PCR-RFLP (EcoRV) were used. Infection by Leishmania spp. was characterized by miniexon multiplex PCR for complexes of Leishmania and ITS1-PCR-RFLP (HaeIII, HhaI, and RsaI) for the identification of the species. Results: The T. cruzi TcI was present in 100% of the coinfected mammals, which included 76.7% of triple infection by T. cruzi TcI-complex–L. (L) mexicana–L. infantum/chagasi, 13.3% of double infection by T. cruzi TcI-L. mexicana and 10% of double infection by T. cruzi Tcl—L. infantum/chagasi. Interpretation & conclusion: These results suggest that the double or triple infection is a phenomenon existing in almost all the coendemics areas and mammals studied, which might influence the mechanisms of adaptation and pathogenicity of these parasites.

The sensitivity and specificity of diagnostic techniques for Chagas disease depend largely on the antigens and targets used and on the immune response and characteristics of the infection of the population where it is applied, hence the need for evaluation of the diagnostic techniques available in a given area. So, the objective of this work was to evaluate two commercial kits for the immunological and molecular diagnosis of Chagas disease in endemic areas of Venezuela.The evaluated kits were: Chagas ELISA IgG+IgM® and Speed Oligo Chagas® (Vircell®, Granada, Spain). They were evaluated with 129 samples (35 from patients in the acute phase, 33 in the chronic phase, 31 from patients with other diseases, and 30 from healthy individuals). The results were compared with those obtained in the conventional ELISA and PCR-satellite DNA tests for Trypanosoma cruzi.With Chagas ELISA IgG+IgM® a sensitivity of 94.1% and specificity of 93.4% were obtained, with Speed Oligo Chagas® a sensitivity of 92.6% and specificity of 100% were achieved, values similar to those showed by conventional ELISA and satDNA-PCR.The sensitivity and specificity of the commercial kits evaluated make them suitable for the diagnosis of Chagas disease in endemic areas of Venezuela.

We define a fluid library as a library of samples of different biological fluids (from humans, animals or vectors) collected and properly stored on filter paper, which allows retrospective studies, especially of diagnosis or detection of infectious agents in these samples, using different techniques. The objective of this work was the retrospective diagnosis of American trypanosomiasis by PCR in a Venezuelan endemic area using a fluid library. A fluid library with samples that had been collected on filter paper, 5 years ago, was used for the detection of Trypanosoma cruzi DNA. 165 blood samples of humans, 30 samples of 25 animals (Didelphis marsupialis, Canis familiaris, Equus asinus and Felis catus) and 8 samples of vectors from endemic areas of Anzoategui state, were analysed by PCR. The results revealed that 16.4% of the humans samples were positive, 11.1% of those detected positive were children younger than 10 years old, and 26.72% young people aged 11–20 years, suggesting that T. cruzi infection has been active for the past two decades. 56% of the animal samples showed amplification; Didelphis marsupialis 66%, Canis familiaris 54.5%, Equus asinus 50%, and Felis catus 33.3%. On the other hand, positivity (50%) was detected in the studied vectors, of which the 3 most important species in Venezuela (Rhodnius prolixus, Triatoma maculata and Panstrongylus geniculatus) were involved. The PCR using a fluid library allowed the detection of T. cruzi DNA in old samples from the three host of the epidemiological chain, suggesting that retrospective diagnosis can be made through this strategy and demonstrate that there has been active transmission, which helps to clarify the epidemiological situation in areas where there are no previous reports.

Background & objectives: Trypanosoma cruzi, the causative agent of American trypanosomiasis, has been reported in 180 mammalian species and 154 triatomines species of Neotropic. This is a clonal parasite with variable biological behaviour, associated with the genetics of the parasite and its hosts. To know the eco-pathogenic complex of this zoonosis, it was proposed to characterize T. cruzi isolates obtained from triatomines and domestic, peridomestic and wild mammals of the eastern and central-western regions of Venezuela. Methods: The positivity to T. cruzi was established and the isolates were genetically characterized by PCR amplification of the mini-exon gene, the DNA coding for 24Sa and 18S rRNA, and polymorphic sequences-RFLPs. The sampling sites were georeferenced using the MapSource Software and ArcGis 9.3 programs to generate distribution maps of the isolates. Results: Of the 460 hosts (205 triatomines and 255 mammals), 49% were positive for the parasite. On the other hand, 38 isolates obtained from the triatomines and 23 isolates obtained from mammals were evaluated. The TcI genotype predominated in most of the isolates; however, in those obtained from triatomines the presence of the TcIII genotype in single infections and TcI + TcIII or TcI + TcIV in mixed infections was also evidenced. Interpretation & conclusion: There is a possibility that the triatomines act as biological syringes for these genotypes associated exclusively to them. The heterogeneity in T. cruzi isolates demonstrated the complexity of parasitosis in these regions, presenting its control and prevention as a challenge.

Chlamydia psittaci (Cp) es una bacteria intracelular obligada transmitida a través de aerosoles derivados de secreciones nasales y ópticas, tejidos, heces y plumas. Comúnmente es identificada en aves, sin embargo, han emergido genotipos capaces de infectar nuevos reservorios mamíferos. Por ello, se buscó ADN de Cp en muestras de cinco individuos de Mustella putorios furo y un hisopado cloacal de un individuo de Colinus cristatus en cautiverio en Venezuela a través de la PCR-anidada, amplificando un segmento del gen 16S ADNr. Se demostró la presencia de Cp en un Colinus cristatus con signos de clamidiosis y en cuatro Mustella putorios furo sin signos clínicos de clamidiosis. Se indica un posible nuevo reservorio para Cp, donde el contacto con productos de excreción de Colinus cristatus con manifestaciones clínicas de clamidiosis, hacinamiento, inadecuada ventilación, contacto con productos de excreción de Psittaciformes y condiciones sanitarias deficientes favoreció la infección por Cp. Se desconoce el total de reservorios de Cp, por ello la notificación de los aislados permite el entendimiento, distribución y diversidad de agentes clamidiales en fauna silvestre y en cautiverio.

Resumen Introduccion La enfermedad de Chagas es causada por el parasito Trypanosoma cruzi. Esta enfermedad comprende una fase aguda y una cronica. El diagnostico presenta limitaciones en las tecnicas parasitologicas y en las inmunologicas. Las tecnicas moleculares son una alternativa, pero estas deben ser evaluadas. El objetivo de este trabajo fue comparar la eficacia de las tecnicas inmunologicas con la de las moleculares en el diagnostico de la enfermedad de Chagas en las diferentes fases. Metodos Se utilizaron las pruebas inmunologicas ELISA, HAI e IFI, y como ensayos moleculares las PCR para la amplificacion de ADN de minicirculo de cinetoplasto y ADN satelite de T. cruzi. Se evaluaron 39 muestras de sangre de pacientes en fase aguda y 42 muestras de pacientes en fase cronica de la enfermedad. Ademas se analizaron 20 muestras de individuos sanos y 10 de pacientes con otras enfermedades. Resultados Con las tecnicas inmunologicas resultaron positivas el 69,2% de las muestras de pacientes en fase aguda, mientras que en la fase cronica resultaron positivas el 95,2%. Con las pruebas moleculares resultaron positivas el 79,5% de las muestras de pacientes en fase aguda, mientras que con las muestras de pacientes en fase cronica resultaron positivas el 23,8%. Ninguna de las muestras de los individuos sanos resulto positiva por ninguna tecnica, mientras que en las pruebas inmunologicas 2 muestras de pacientes con otras enfermedades resultaron positivas. Conclusion La eficacia diagnostica de las tecnicas moleculares es elevada en la fase aguda, mientras que en la fase cronica son mas eficaces los ensayos inmunologicos.

Introduccion. La enfermedad de Chagas es causada por el parasito Trypanosoma cruzi y su diagnostico inmunologico se basa principalmente en la deteccion de anticuerpos contra T. cruzi mediante pruebas tales como ELISA, inmunofluorescencia indirecta (IFI) y hemaglutinacion indirecta (HAI). Esta ultima tiene el inconveniente de requerir la preparacion de eritrocitos de carnero, dificiles de obtener y de poca duracion. Sin embargo, existen pruebas alternativas, como la tecnica de aglutinacion directa. Objetivo. Estandarizar la tecnica de aglutinacion directa para el diagnostico de la enfermedad de Chagas. Materiales y metodos. Se prepararon parasitos epimastigotes de T. cruzi mediante dos protocolos, con tratamiento con tripsina y sin el. Los parasitos se colorearon, y se determinaron las condiciones optimas de concentracion parasitaria y diluciones de suero. Se utilizaron sueros de pacientes con enfermedad de Chagas, de individuos sanos y con otras parasitosis. Resultados. La concentracion parasitaria optima fue de 500 x106 parasitos/ml, utilizando parasitos coloreados y sin tratamiento con tripsina. Las diluciones de suero optimas fueron de 1/25, 1/50 y1/100, y el punto de corte, la dilucion de 1/50. La tecnica estandarizada mostro indices diagnosticos de sensibilidad de 94,3 % (IC95% 79,5-99,0) y de especificidad de 96,3 % (IC95% 88,8-99,0); se encontro reaccion cruzada en tres sueros de individuos con leishmaniasis visceral, con valores pronosticos positivo y negativo de 91,7 % (IC95% 76,4-97,8) y de 97,5 % (IC95% 90,4-99,6), respectivamente. Se compararon los resultados con los obtenidos por HAI, ELISA e IFI y la concordancia fue de 96 % con un indice kappa de 0,90 (IC95% 0,81-0,99). Conclusion. La tecnica de aglutinacion directa estandarizada podria ser util para el inmunodiagnostico de la enfermedad de Chagas.

Chagas disease is caused by the parasite Trypanosoma cruzi. The disease involves an acute and chronic phases. The diagnosis has limitations, both in parasitological and immunological techniques. Molecular assays are an alternative, but these must be evaluated to determine its diagnostic usefulness. The aim of this study was to compare the effectiveness of immunological techniques with molecular assays in the diagnosis of Chagas disease in its different phases.The immunological techniques used were ELISA, HAI and IFI and the molecular techniques used were PCR for amplification of kinetoplast minicircles, and satellite DNA of T. cruzi. Thirty-nine blood samples from patients in the acute phase of Chagas disease, and 42 samples from patients in the chronic phase were evaluated. In addition, 20 samples from healthy individuals and 10 patients with other diseases were also studied.With immunological techniques were positive, 69.2% of samples from patients in the acute phase, while in the chronic phase were positive 95.2%. Using molecular techniques 79.5% of samples from patients in the acute phase were positive, while 23.8% of the samples from patients in the chronic phase were positive. None of the samples from healthy individuals was positive for any technique, while two samples from patients with other diseases were positive by the immunological assays.The diagnostic efficacy of molecular techniques is high in the acute phase of Chagas disease, while in the chronic phase the immunological techniques are more effective.

Objetivos. Comparar dos protocolos de extracción de ADN de Trypanosoma cruzi para su uso en la amplificación de ADN de minicírculos de kinetoplasto (ADNk) mediante la técnica de Reacción en Cadena de Polimerasa (PCR). Materiales y métodos. Se cultivaron epimastigotas de T. cruzi en condiciones exénicas obteniéndose masas entre 1,5 hasta 100 x 10(6) parásitos. A partir de estas se procedió a la extracción de ADN mediante dos protocolos: extracción con solventes orgánicos (fenol/cloroformo), y empleo de resina (Chelex®100), a partir de los diferentes sedimentos parasitarios. La concentración y pureza del ADN se determinó por espectrofotometría y la integridad se evaluó mediante electroforesis en geles de agarosa. Se realizó el análisis de varianza y comparaciones de medias mediante la prueba de Tukey, utilizando el software Statistix 8.0. Resultados. Se realizaron diez extracciones de ADN de cada una de las diferentes cantidades de parásitos sedimentados. En la extracción de ADN con la resina Chelex®100 se obtuvo mayor rendimiento, pero menor pureza e integridad respecto a la extracción con solventes orgánicos. Sin embargo, permitió la amplificación del producto de 330 pb de ADNk de T. cruzi. Conclusiones. Aun cuando la técnica de Chelex®100 proporcionó menor pureza e integridad del ADN, permitió la amplificación con éxito de ADNk por PCR, evitando el uso de técnicas laboriosas y solventes orgánicos tóxicos Objectives. To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR). Materials and methods. Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex®100), from different parasitic sediments. Concentration and purity of DNA was determined by spectrophotometry, and integrity was assessed by agarose gel electrophoresis. Analysis of variance and comparisons of means were performed through Tukey’s test, using the Statistix 8.0 software. Results. Ten DNA extractions were done of each one of the different amounts of parasitic sediments. In the DNA extraction with Chelex®100 resin, a higher performance was obtained but a lower purity and integrity compared to the extraction with organic solvents. However, it allowed a product amplification of 330 bp of T. cruzi kDNA. Conclusions. Although the technique of Chelex®100 provided less purity and integrity of DNA, it allowed a successful amplification of kDNA by PCR, avoiding the use of laborious techniques and toxic organic solvents