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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.

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.

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.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.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.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.

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 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.

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.

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