The Dengue Virus Nonstructural Protein 1 (NS1) Interacts with the Putative Epigenetic Regulator DIDO1 to Promote Flavivirus 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. 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.

Detection of Trypanosoma cruzi DNA in false negative samples of collected triatomines, xenodiagnosis material, and biopsies of experimentally infected animals.

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.

Ecopathogenic complexes of American trypanosomiasis in endemic areas of Venezuela: Diagnosis and variability of Trypanosoma cruzi.

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.

Genetic variability of Trypanosoma cruzi TcI isolates from rural and urban areas of Venezuela.

BACKGROUND & OBJECTIVES: Several studies have demonstrated genetic heterogeneity in populations of Trypanosoma cruzi that allowed the identification of six different discrete typing units (DTU) classified as TcI, TcII, TcIII, TcIV, TcV and TcVI. Furthermore, some characterization studies have described genetic variability within TcI isolates from endemic regions. The objective of the present study was to analyze Venezuelan T. cruzi isolates, obtained from triatomine-vectors, mammal-hosts including infected humans, detected in both rural and urban areas from diverse geographic origins. METHODS: Molecular characterization of 44 Venezuelan T. cruzi isolates, obtained from triatomine-vectors, mammalian hosts and human patients from both rural and urban areas of different geographic origins, were carried out. Samples were analyzed by PCR amplification of the intergenic region of the mini-exon gene, 24Sα rDNA and 18S rDNA, followed by sequencing of the amplification products. RESULTS: The TcI amplification pattern was found in 42 out of 44 (95.5%) isolates; a TcIII strain and one possible TcIV were also found. The sequence analysis of the TcI Venezuelan isolates showed genetic variability among them. Urban isolates formed a homogeneous group, with differences in their sequences, when compared to rural isolates. INTERPRETATION & CONCLUSION: The results showed genetic heterogeneity in Venezuelan TcI strains, probably in response to different environmental conditions.

Chagas Disease and Leishmaniasis, Sympatric Zoonoses Present in Human from Rural Communities of Venezuela.

PURPOSE: Trypanosoma cruzi and Leishmania spp. coexist in several endemic areas, and there are few studies of Chagas disease and leishmaniasis coinfection worldwide; for this reason, the objective of this work was to determine the Chagas disease and leishmaniasis coinfection in several rural communities co-endemic for these diseases. METHODS: A total of 1107 human samples from six co-endemic rural communities of Cojedes state, Venezuela, were analyzed. Serum samples were evaluated by ELISA, indirect hemagglutination, and indirect immunofluorescence for Chagas disease diagnosis, and individuals were evaluated for leishmaniasis by leishmanin skin test (LST). Approximately, 30% of the individuals were also analyzed by PCR (blood clot samples) for T. cruzi and for Leishmania spp. RESULTS: The 14.7% of the individuals were positive to Trypanosoma cruzi infection by serology, and 25.8% were positive to Leishmania spp. current or past infection by LST. Among the group with PCR results, 7.8% were positive for T. cruzi, and 9.4% for Leishmania spp. The coinfection T. cruzi/Leishmania spp. was 6.5%. The T. cruzi DTUs of the positive blood clot samples were TcI, revealed using the molecular markers: (i) intergenic region of the miniexon, (ii) D7 divergent domain of the 24Sα rDNA, (iii) size-variable domain of the 18S rDNA, and (iv) hsp60-PCR-RFLP (EcoRV). The Leishmania species identified were L. (Leishmania) mexicana and L. (Viannia) braziliensis. CONCLUSION: A high prevalence was found for T. cruzi and Leishmania spp. single and coinfections in almost all communities studied, being these results of relevance for the implementation of control programs in co-endemic areas.

[Comparison between immunological and molecular techniques for the diagnosis of Chagas disease]. / Comparación entre técnicas inmunológicas y moleculares para el diagnóstico de la enfermedad de Chagas.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Comparative Infections of Zika, Dengue, and Yellow Fever Viruses in Human Cytotrophoblast-Derived Cells Suggest a Gating Role for the Cytotrophoblast in Zika Virus Placental Invasion.

The Zika virus (ZIKV) is teratogenic and considered a TORCH pathogen (toxoplasmosis [Toxoplasma gondii], rubella, cytomegalovirus, herpes simplex virus [HSV], and other microorganisms capable of crossing the blood-placenta barrier). In contrast, the related flavivirus dengue virus (DENV) and the attenuated yellow fever virus vaccine strain (YFV-17D) are not. Understanding the mechanisms used by ZIKV to cross the placenta is necessary. In this work, parallel infections with ZIKV of African and Asian lineages, DENV, and YFV-17D were compared for kinetics and growth efficiency, activation of mTOR pathways, and cytokine secretion profile using cytotrophoblast-derived HTR8 cells and monocytic U937 cells differentiated to M2 macrophages. In HTR8 cells, ZIKV replication, especially the African strain, was significantly more efficient and faster than DENV or YFV-17D. In macrophages, ZIKV replication was also more efficient, although differences between strains were reduced. Greater activation of the mTORC1 and mTORC2 pathways in HTR8 cells infected with ZIKV than with DENV or YFV-17D was observed. HTR8 cells treated with mTOR inhibitors showed a 20-fold reduction in ZIKV yield, versus 5- and 3.5-fold reductions for DENV and YFV-17D, respectively. Finally, infection with ZIKV, but not DENV or YFV-17D, efficiently inhibited the interferon (IFN) and chemoattractant responses in both cell lines. These results suggest a gating role for the cytotrophoblast cells in favoring entry of ZIKV, but not DENV and YFV-17D, into the placental stroma. IMPORTANCE Zika virus acquisition during pregnancy is associated with severe fetal damage. The Zika virus is related to dengue virus and yellow fever virus, yet fetal damage has not been related to dengue or inadvertent vaccination for yellow fever during pregnancy. Mechanisms used by the Zika virus to cross the placenta need to be deciphered. By comparing parallel infections of Zika virus strains belonging to the African and Asian lineages, dengue virus, and the yellow fever vaccine virus strain YFV-17D in placenta-derived cytotrophoblast cells and differentiated macrophages, evidence was found that Zika virus infections, especially by the African strains, were more efficient in cytotrophoblast cells than dengue virus or yellow fever vaccine virus strain infections. Meanwhile, no significant differences were observed in macrophages. Robust activation of the mTOR signaling pathways and inhibition of the IFN and chemoattractant response appear to be related to the better growth capacity of the Zika viruses in the cytotrophoblast-derived cells.

Evaluation of commercial kits for the immunological and molecular diagnosis of Chagas disease in endemic areas of Venezuela.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The sensitivity and specificity of the commercial kits evaluated make them suitable for the diagnosis of Chagas disease in endemic areas of Venezuela.

Infections and Coinfections by Trypanosomatid Parasites in a Rural Community of Venezuela.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Standardization of the PCR Technique for the Detection of the Toxoplasma gondii B1 Gene in Meat and Water Samples and Cloning of the Product for Use as Control.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Septin 2 interacts with dengue virus replication complex proteins and participates in virus replication in mosquito cells.

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.

Utility of a Fluid Library with Samples of Humans, Reservoirs and Vectors Collected in Filter Paper, for Retrospective Diagnosis of American Trypanosomiasis in Endemic Areas of Venezuela.

INTRODUCTION: 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. METHODS: 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 Anzoátegui state, were analysed by PCR. RESULTS: 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. CONCLUSIONS: 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.

Flavivirus infections induce a Golgi stress response in vertebrate and mosquito cells.

The stress of the Golgi apparatus is an autoregulatory mechanism that is induced to compensate for greater demand in the Golgi functions. No 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 and increased expression of pathway targeted genes. 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 the Golgi complex imposed by virion and NS1 processing and secretion.

Molecular diagnosis of Trypanosoma cruzi/Leishmania spp. coinfection in domestic, peridomestic and wild mammals of Venezuelan co-endemic areas.

American trypanosomiasis and leishmaniases are diseases caused by protozoans of the Trypanosomatidae family. In Venezuela, although several endemic foci of both diseases coincide, there are no reports of coinfection in mammals. The molecular diagnosis of the coinfection T. cruzi-Leishmania spp. was done in 527 blood samples collected on filter paper of several species of mammals (Canis familiaris, Equus asinus, Didelphis marsupialis, Equus mulus, Rattus rattus, Equus caballus, Artibeus fraterculus, Felis catus, Sus scrofa, Bos taurus, Capra hircus and Sciurus granatensis) from the states Cojedes, Aragua, Anzoátegui, Guárico, Miranda and Capital District. The T. cruzi infection was determined through PCR amplification of DNA of kinetoplast minicircles (kDNA) and satellite DNA (sDNA). The Leishmania spp. infection was detected by Leishmania nested PCR (Ln-PCR), and ribosomal DNA internal transcribed spacer 1 PCR (ITS1-PCR). The percentage of infection by T. cruzi was 23.5%, by Leishmania spp. 12.9% and coinfection was 5.7%. D. marsupialis was the species with the highest percentage of infection for each parasitosis (T. cruzi 34.3%, Leishmania spp. 20.0%) and coinfection (14.3%). Anzoátegui was the state with the highest percentage of infection for each parasitosis (T. cruzi 64.9%, Leishmania spp. 64.9%) and coinfection (43.2%). Infections were determined in species not reported as natural reservoirs of T. cruzi (E. asinus and E. mulus) and of Leishmania spp. (E. mulus and S. scrofa). Coinfection was a frequent phenomenon in mammals in several co-endemic zones evaluated.

[Standardization of a direct agglutination test for the immunodiagnosis of Chagas disease]. / Estandarización de la técnica de aglutinación directa para el inmunodiagnóstico de la enfermedad de Chagas.

INTRODUCTION: Chagas' disease is caused by the parasite Trypanosoma cruzi and its immunological diagnosis is mainly based on the detection of antibodies against T. cruzi using tests such as the ELISA, the indirect fluorescence antibody test (IFAT) and the indirect hemagglutination test (IHAT). The main disadvantage of the IHAT is the need to prepare sheep erythrocytes, whose availability is limited and they have a short duration once prepared. However, there are alternative tests, such as the direct agglutination test (DAT). OBJECTIVE: To standardize the direct agglutination test for the diagnosis of Chagas disease. MATERIALS AND METHODS: Trypanosoma cruzi epimastigotes were prepared using two protocols, with and without trypsin treatment. The parasites were stained and optimal conditions for parasitic concentration and serum dilutions were determined. We evaluated the technique using sera from patients with Chagas disease, from healthy individuals and from individuals with other parasitic diseases. RESULTS: The optimal parasitic concentration was 500 x 10(6) parasites/ml using stained parasites without trypsin treatment. The optimal serum dilutions were 1/25, 1/50 y 1/100 and the cut-off point was the 1/50 dilution. The diagnostic indices for the standardized technique were as follows: Sensitivity, 94.3% (95% CI: 79.5-99.0) and specificity, 96.3% (95% CI: 88.8-99.0), with positive and negative predictive values of 91.7% (95% CI: 76.4-97.8) and 97.5% (95% CI: 90.4-99.6), respectively. Cross-reaction was observed only in three sera from individuals with visceral leishmaniasis. The results were compared with those obtained by IHA, ELISA, and IFA, and the concordance rate was 96% and the kappa index, 0.90 (95% CI: 0.81-0.99). CONCLUSION: The standardized direct agglutination test could be useful for immunodiagnosis of Chagas disease.

[Comparing two protocols of DNA extraction of Trypanosoma cruzi cultured in axenic medium]. / Comparación de dos protocolos de extracción de ADN de Trypanosoma cruzi cultivados en medio axénico.

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 (Chelex100), 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 Chelex100 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 Chelex100 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.

Estandarización de la técnica de PCR para la detección de ADN de Leishmanía sp. en muestras de sangre de caninos / Standardization of the PCR technique for detection of Leishmania sp. DNA in canine blood samples

La leishmaniasis es una enfermedad con diversidad clínica y epidemiológica, producida por varias especies de protozoarios parásitos del género Leishmania. Estos parásitos infectan una amplia variedad de hospedadores mamíferos y son transmitidos por insectos del género Lutzomyia, en nuestro país. Los caninos han sido implicados como posibles reservorios del parásito. Para el diagnóstico de leishmaniasis se utilizan técnicas parasitológicas que generalmente tienen baja sensibilidad e inmunológicas, con pobre especificidad. Debido a las limitaciones en el diagnóstico, y lo difícil de la obtención, transporte y almacenamiento de las muestras, en este trabajo se planteó estandarizar de una técnica de PCR anidada (Leishmania nested PCR, Ln-PCR) para la detección de ADN de Leishmania sp. en muestras de sangre de caninos colectadas en papel de filtro. Para ello se titularon las concentraciones de reactivos de la PCR para la amplificación del ADN del parásito y se determinó la sensibilidad analítica y la especificidad de la técnica. Se evaluaron 36 muestras de sangre de caninos (6 infectados y 30 no infectados). Las condiciones óptimas de reacción fueron 0,2 mM de dNTPs, 0,4 µM de cada cebador y 1 U de Taq polimerasa. La sensibilidad analítica de Ln-PCR fue de 10 fg y la especificidad fue de 100% en la detección de ADN de Leishmania sp., ya que no se observó amplificación con ADN de otros parásitos, ni con ADN humano, ni de perro. De las muestras de caninos evaluadas los seis controles infectados todos amplificaron por la PCR, mientras que los 30 no infectados, en ninguno se observó amplificación. La extracción de ADN de muestras de sangre colectadas en papel de filtro fue eficiente para la amplificación por la PCR, técnica que puede ser muy útil para el diagnóstico de leishmaniasis en animales y su implicación como posibles reservorios.

Leishmaniasis is a disease with clinical and epidemiological diversity, caused by several species of protozoan parasites of the genus Leishmania. These parasites infect a wide variety of mammalian hosts, and are transmitted by insects of the genus Lutzomyia in our country. Canines have been implicated as potential reservoirs of the parasite. For the diagnosis of leishmaniasis, parasitological techniques generally with low sensitivity and immunological techniques, with poor specificity, are used. Because of limitations in the diagnosis, and the difficulty to obtain, transport, and store samples, the aim of this research was to standardize a Leishmania nested PCR test (Ln-PCR), for the detection of Leishmania sp. DNA in blood samples from dogs collected in filter paper. For that purpose, concentrations of the PCR reagent for DNA parasite amplification were titrated and the analytical sensitivity and specificity of the technique were determined. A total of 36 canine blood samples (6 infected and 30 uninfected) were evaluated.  The optimal reaction conditions were: 0.2 mM dNTPs; 0.4 µM of each primer; and 1 U of Taq polymerase. The analytical sensitivity of the Ln-PCR was 10 fg and the specificity was 100% in detecting Leishmania sp. DNA, since no amplification was observed with DNA from other parasites, human DNA or canine DNA. The six samples from infected canines evaluated amplified Leishmania sp. DNA by PCR, whereas in none of the 30 samples from uninfected canines the amplification was observed. The DNA extraction from blood samples collected in filter paper was efficient for the PCR amplification, a technique that can be very useful for the diagnosis of leishmaniasis in animals and their involvement as potential reservoirs.

Leishmaniasis.

dogs.

diagnosis.

PCR.

Estandarización de la técnica de aglutinación directa para el inmunodiagnóstico de la enfermedad de Chagas / Standardization of a direct agglutination test for the immunodiagnosis of Chagas disease

Introducción. La enfermedad de Chagas es causada por el parásito Trypanosoma cruzi y su diagnóstico inmunológico se basa principalmente en la detección de anticuerpos contra T. cruzi mediante pruebas tales como ELISA, inmunofluorescencia indirecta (IFI) y hemaglutinación indirecta (HAI). Esta última tiene el inconveniente de requerir la preparación de eritrocitos de carnero, difíciles de obtener y de poca duración. Sin embargo, existen pruebas alternativas, como la técnica de aglutinación directa. Objetivo. Estandarizar la técnica de aglutinación directa para el diagnóstico de la enfermedad de Chagas. Materiales y métodos. Se prepararon parásitos epimastigotes de T. cruzi mediante dos protocolos, con tratamiento con tripsina y sin él. Los parásitos se colorearon, y se determinaron las condiciones óptimas de concentración parasitaria y diluciones de suero. Se utilizaron sueros de pacientes con enfermedad de Chagas, de individuos sanos y con otras parasitosis. Resultados. La concentración parasitaria óptima fue de 500 x10 6 parásitos/ml, utilizando parásitos coloreados y sin tratamiento con tripsina. Las diluciones de suero óptimas fueron de 1/25, 1/50 y1/100, y el punto de corte, la dilución de 1/50. La técnica estandarizada mostró índices diagnósticos de sensibilidad de 94,3 % (IC 95% 79,5-99,0) y de especificidad de 96,3 % (IC 95% 88,8-99,0); se encontró reacción cruzada en tres sueros de individuos con leishmaniasis visceral, con valores pronósticos positivo y negativo de 91,7 % (IC 95% 76,4-97,8) y de 97,5 % (IC 95% 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 índice kappa de 0,90 (IC 95% 0,81-0,99). Conclusión. La técnica de aglutinación directa estandarizada podría ser útil para el inmunodiagnóstico de la enfermedad de Chagas.

Introduction: Chagas´ disease is caused by the parasite Trypanosoma cruzi and its immunological diagnosis is mainly based on the detection of antibodies against T. cruzi using tests such as the ELISA, the indirect fluorescence antibody test (IFAT) and the indirect hemagglutination test (IHAT). The main disadvantage of the IHAT is the need to prepare sheep erythrocytes, whose availability is limited and they have a short duration once prepared. However, there are alternative tests, such as the direct agglutination test (DAT). Objective: To standardize the direct agglutination test for the diagnosis of Chagas disease. Materials and methods: Trypanosoma cruzi epimastigotes were prepared using two protocols, with and without trypsin treatment. The parasites were stained and optimal conditions for parasitic concentration and serum dilutions were determined. We evaluated the technique using sera from patients with Chagas disease, from healthy individuals and from individuals with other parasitic diseases. Results: The optimal parasitic concentration was 500 x 10 6 parasites/ml using stained parasites without trypsin treatment. The optimal serum dilutions were 1/25, 1/50 y 1/100 and the cut-off point was the 1/50 dilution. The diagnostic indices for the standardized technique were as follows: Sensitivity, 94.3% (95% CI: 79.5-99.0) and specificity, 96.3% (95% CI: 88.8-99.0), with positive and negative predictive values ?? of 91.7% (95% CI: 76.4-97.8) and 97.5% (95% CI: 90.4-99.6), respectively. Cross-reaction was observed only in three sera from individuals with visceral leishmaniasis. The results were compared with those obtained by IHA, ELISA, and IFA, and the concordance rate was 96% and the kappa index, 0.90 (95% CI: 0.81-0.99). Conclusion: The standardized direct agglutination test could be useful for immunodiagnosis of Chagas disease.

Comparación de dos protocolos de extracción de ADN de Trypanosoma cruzi cultivados en medio axénico / Comparing two protocols of DNA extraction of Trypanosoma cruzi cultured in axenic medium

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.

Primer registro de triatóminos naturalmente infectados por Trypanosoma cruzi en el estado Bolívar, Venezuela / First record of naturally infected triatomines with trypanosoma cruzi, Bolivar state, Venezuela

La enfermedad de chagas o tripanosomiasis americana está adquiriendo cada vez mayor importancia en la región amazónica de Venezuela, en la cual hasta ahora se carecía de información de la presencia de trypanosoma cruzi en triatóminos, reservorios y humanos, particularmente para el estado Bolívar, el estado de mayor superficie del país, situado al sur del río Orinoco. Cuatro ejemplares de triatoma maculata fueron recolectados en el peridomicilio de la comunidad de Maniapure, en el municipio Cedeño. En dos de los ejemplares se determinó la infección natural por trypanosoma cruzi al examen al fresco de heces con presencia de tripomastigotos metacíclicos infectantes. Los parásitos fueron inoculados en modelos murinos y aislados en cultivos para su caracterización molecular. Se confirmó el diagnóstico de este parásito por pruebas parasitológicas y moleculares, caracterizándose los aislados como TcI. La importancia de estos hallazgos pioneros podría motivar el estudio de la transmisión vectorial de la enfermedad de chagas en este estado catalogado como no endémico.

American trypanosomiasis or chagas disease is becoming increasingly important in the Amazon region of Venezuela, in which up to now information was lacking of the presence of trypanosoma cruzi in triatomins, reservoirs and humans, particularly for Bolivar state, the state with the largest territory of the country, located in the south of the Orinoco river. Four specimens of triatoma maculata were collected in the peri-domiciliar area of the community of Maniapure, in the Cedeño municipality. Two of these individuals were found naturally infected with trypanosoma cruzi upon examination of fresh stool with presence of metacyclic trypomastigotes. Parasites were inoculated in murine models and isolated for molecular characterization. Parasite isolates were molecularly characterized as TcI. The importance of these pioneering findings should motivate the study of the vector or oral transmission of Chagas disease in this non endemic State of Venezuela.