Publications (14)33.94 Total impact
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Article: Trypanosoma cruzi persistence at oral inflammatory foci in chronic chagasic patients.
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ABSTRACT: The persistence of Trypanosoma cruzi in seropositive individuals, previously diagnosed as chronic chagasic patients (CCP), was detected for the first time in biopsies taken from gingival inflammatory foci processed by polymerase chain reaction (PCR). Seven out of 31 (22.5%) gum samples from selected unquestionably CCP showing different degrees of gingival inflammation revealed T. cruzi-DNA using 3 specific PCR assays. All the included CCP had been diagnosed in previous studies carried out over the last 19 years. Samples of inflamed gums were recently taken from the indicated patients at: an outpatient hospital cardiac unit; a village where Chagas disease is endemic; and a specialized diagnostic research center, showing molecular evidence of parasite persistence in 17.6%, 42.8% and 14.3% of them, respectively. The relatively frequent parasite persistence, demonstrated here in oral inflammatory processes of treated and/or untreated patients bearing long term T. cruzi-infection, suggests the establishment of secondary small foci for the maintenance of hidden or inapparent chagasic infection. The easy and low-risk, non-invasive method to get the sample may add the use of gingival biopsy as a potential alternative diagnostic tool to confirm T. cruzi-infection in CCP. The significance of T. cruzi persistence as a primary cause of chronic Chagas disease and the proposal of this mechanism to explain the pathogenesis in CCP are considered.Acta tropica 03/2011; 117(3):207-11. · 2.22 Impact Factor -
Article: International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.
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ABSTRACT: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05-0.5 parasites/mL whereas specific kDNA tests detected 5.10(-3) par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3-94.4%, specificity of 85-95%, accuracy of 86.8-89.5% and kappa index of 0.7-0.8 compared to consensus PCR reports of the 16 good performing tests and 63-69%, 100%, 71.4-76.2% and 0.4-0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.PLoS Neglected Tropical Diseases 01/2011; 5(1):e931. · 4.69 Impact Factor -
Article: Trypanosoma cruzi congenital transmission in wild bats.
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ABSTRACT: Trypanosoma cruzi congenital transmission in wild bats (Molossus molossus), associated with infected Rhodnius prolixus in a natural habitat from a rural locality in western Venezuela, is reported. T. cruzi blood circulating trypomastigotes in a pregnant bat were detected by parasitological methods. Polymerase chain reaction (PCR) assays carried out in samples from the heart and the fetus of the same infected female, revealed the presence of T. cruzi-specific DNA in both of the tissues, demonstrating transmission of the infection from the mother to the offspring. Eighty percent of the captured bats and 100% of the examined fetuses from pregnant specimens were shown to be infected by T. cruzi, indicating that M. molossus is a very susceptible species for this parasite, and that T. cruzi congenital transmission is a common phenomenon in nature. To our knowledge, this seems to be the first report on congenital T. cruzi transmission in wild bats in Venezuela. The circulation of T. cruzi lineage I in the study area was demonstrated by typing the isolates from bats and triatomine bugs captured in the same habitat. The potential epidemiological implication of these findings in areas where Chagas disease is endemic is discussed.Acta tropica 10/2008; 109(1):78-80. · 2.22 Impact Factor -
Article: Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis.
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ABSTRACT: GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.Acta Tropica 03/2008; 105(2):139-44. · 2.72 Impact Factor -
Article: Expression of GP82 and GP90 surface glycoprotein genes of Trypanosoma cruzi during in vivo metacyclogenesis in the insect vector Rhodnius prolixus.
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ABSTRACT: Trypanosoma cruzi, the parasite causing Chagas' disease, relies on triatomines for its transmission. T. cruzi metacyclic trypomastigotes express GP82 and GP90, which are developmentally regulated surface proteins that have been implicated in host cell invasion. We used quantitative RT-PCR to quantify GP90 and GP82 mRNA levels expressed by T. cruzi in the digestive tract of experimentally infected Rhodnius prolixus at different times post infection. Translation of these transcripts was assessed by immunofluorescence using specific monoclonal antibodies against GP90 and GP82. We found that although GP82 and GP90 proteins were not detected in epimastigote cells by immunofluorescence, transcripts were present at lower levels. Increased levels of GP90 and GP82 transcripts and the appearance of these proteins on the parasite surface were accompanied by morphological differentiation from epimastigotes into metacyclic forms. Our data suggest that during in vivo metacyclogenesis there is a coordinated mechanism that links stabilization of GP90 and GP82 mRNAs with their translation.Acta Tropica 02/2008; 105(1):87-91. · 2.72 Impact Factor -
Article: Infected dogs as a risk factor in the transmission of human Trypanosoma cruzi infection in western Venezuela.
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ABSTRACT: A total of 565 mongrel dogs from rural localities of Venezuela were examined by serological (DAT, IFAT and ELISA) and parasitological tests to address the status of Trypanosoma cruzi infection and to evaluate their role in the transmission of the infection to human population. The overall percentage of sero-positive infected dogs shown to be 67.6% (382/565):253 (61.7%) from 47 villages belonging to 8 states located at 4 different geographical regions of western Venezuela and 129 (33.5%) dogs from 48 households located in areas where Chagas disease is endemic. From 101 sampled dogs living in close proximity to 30 acute chagasic patients, 84% expressed specific anti-T. cruzi antibodies (Ab) with 12 of them (14%) showing blood circulating parasites (BCP). In these houses a high proportion of sero-positive people (20%) and frequent indoor infestation by triatomine-bugs (70%) was also recorded. The analysis revealed that from the 47 rural villages sampled during the study, 91.5% had the presence of T. cruzi sero-positive dogs, ranging from 62% positive localities at the states of Falcon and Cojedes to 100% in the other six studied Venezuelan states. This demonstrates that T. cruzi-infected dogs are found throughout all the geographical regions of western Venezuela irrespective of their ecological differences. Molecular typing of T. cruzi isolates from infected dogs using ribosomal and mini-exon gene markers, revealed the presence of both T. cruzi I and T. cruzi II lineages. The coincidence in the circulation of T. cruzi II in dog and human populations at the same locality and at the same time is reported and its significance is discussed. The combined serological, parasitological, epidemiological and molecular data is gathered here to call the attention on the presence of infected dogs as a risk factor in the maintenance of T. cruzi as a source for infection to humans.Acta Tropica 08/2006; 98(3):247-54. · 2.72 Impact Factor -
Article: Isolation, purification and characterization of GPI-anchored membrane proteins from Trypanosoma rangeli and Trypanosoma cruzi.
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ABSTRACT: GPI-anchored proteins from plasma membrane of Trypanosoma rangeli and Trypanosoma cruzi epimastigotes were isolated and characterized using the partition Triton X-114 method. The detection by Western blot of specific proteins of 90, 85 and 56 kDa molecular mass in T. rangeli compared to those of 30, 70 and 100 kDa detected in T. cruzi demonstrates specific discrimination between these two species of Trypanosoma. The potential diagnostic value of the here reported proteins to differentiate mixed infections by T. cruzi and T. rangeli is evaluated and its potential for epidemiological studies of Chagas disease in endemic areas is also discussed.Acta Tropica 03/2006; 97(2):140-5. · 2.72 Impact Factor -
Article: Trypanosoma rangeli expresses a gene of the group II trans-sialidase superfamily.
Molecular and Biochemical Parasitology 08/2005; 142(1):133-6. · 2.55 Impact Factor -
Article: Expression of fluorescent genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): its application to parasite-vector biology.
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ABSTRACT: Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites.Journal of Medical Entomology 02/2005; 42(1):48-56. · 1.76 Impact Factor -
Article: Predominance of lineage I among Trypanosoma cruzi isolates from Venezuelan patients with different clinical profiles of acute Chagas' disease.
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ABSTRACT: Trypanosoma cruzi isolates from 23 acute chagasic patients from localities of Western Venezuela (state of Barinas) where Chagas' disease is endemic were typed using ribosomal and mini-exon gene markers. Results showed that isolates of the two major phylogenetic lineages, T. cruzi I and T. cruzi II, were isolated from these patients. Six isolates (26%) were typed as T. cruzi II and 17 (74%) as belonging to T. cruzi lineage I. Analysis of random amplified polymorphic DNA (RAPD) patterns confirmed these two groups of isolates, but did not disclose significant genetic intra-lineage polymorphism. Patients infected by both T. cruzi I or T. cruzi II showed different clinical profiles presenting highly variable signs and symptoms of acute phase of Chagas' disease ranging from totally asymptomatic to severe heart failure. The predominance of T. cruzi I human isolates in Venezuela allied to the higher prevalence of severe symptoms of Chagas' disease (heart failure) in patients infected by this lineage do not corroborate an innocuousness of T. cruzi I infection to humans. To our knowledge, this is the first study describing predominance of T. cruzi lineage I in a large number of acute chagasic patients with distinct and well-characterized clinical profiles.Tropical Medicine & International Health 01/2005; 9(12):1319-26. · 2.80 Impact Factor -
Article: Update on Chagas disease in Venezuela--a review.
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ABSTRACT: The present article reviews the status of Chagas disease in Venezuela based on the detection of Trypanosoma cruzi infections both in referred patients with clinical presumptive diagnosis (1988-2002) and in individuals sampled from rural localities representative of the different geographical regions of the country (1995-2002). In the former group from 306 individuals examined, 174 (56.8%) were seropositive to T. cruzi; 73 (42%) in the acute phase with 52 (71%) showing blood circulating parasites, and from these 38% were children under 10 years old. The other 101 (58%) showed chronic infection at different degrees of cardiac complication. In addition, serologic examination of 3835 individuals from rural areas revealed 11.7% seroprevalence. From these, 8.5% (38/448) were children aged from 0 to 10 years old. These figures suggest that Chagas disease may be re-emerging in Venezuela judging for the active transmission detected during the last decade. The success of the Venezuelan anti-chagasic campaign during the last 40 years is evaluated in the frame of the present results. The epidemiological situation is discussed and recommendation to consider Chagas disease as a national priority is given.Memórias do Instituto Oswaldo Cruz 01/2005; 99(8):781-7. · 2.15 Impact Factor -
Article: Detection of Trypanosoma cruzi and Trypanosoma rangeli infection by duplex PCR assay based on telomeric sequences.
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ABSTRACT: We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.Clinical and Diagnostic Laboratory Immunology 10/2003; 10(5):775-9. · 2.51 Impact Factor -
Article: Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major.
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ABSTRACT: A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV) and the thoracic mid-gut (TMG) of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG). Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads), paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.Memórias do Instituto Oswaldo Cruz 07/2003; 98(4):495-8. · 2.15 Impact Factor -
Article: Infected dogs as a risk factor in the transmission of human Trypanosoma cruzi infection in western Venezuela
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ABSTRACT: A total of 565 mongrel dogs from rural localities of Venezuela were examined by serological (DAT, IFAT and ELISA) and parasitological tests to address the status of Trypanosoma cruzi infection and to evaluate their role in the transmission of the infection to human population. The overall percentage of sero-positive infected dogs shown to be 67.6% (382/565):253 (61.7%) from 47 villages belonging to 8 states located at 4 different geographical regions of western Venezuela and 129 (33.5%) dogs from 48 households located in areas where Chagas disease is endemic. From 101 sampled dogs living in close proximity to 30 acute chagasic patients, 84% expressed specific anti-T. cruzi antibodies (Ab) with 12 of them (14%) showing blood circulating parasites (BCP). In these houses a high proportion of sero-positive people (20%) and frequent indoor infestation by triatomine-bugs (70%) was also recorded. The analysis revealed that from the 47 rural villages sampled during the study, 91.5% had the presence of T. cruzi sero-positive dogs, ranging from 62% positive localities at the states of Falcon and Cojedes to 100% in the other six studied Venezuelan states. This demonstrates that T. cruzi-infected dogs are found throughout all the geographical regions of western Venezuela irrespective of their ecological differences. Molecular typing of T. cruzi isolates from infected dogs using ribosomal and mini-exon gene markers, revealed the presence of both T. cruzi I and T. cruzi II lineages. The coincidence in the circulation of T. cruzi II in dog and human populations at the same locality and at the same time is reported and its significance is discussed. The combined serological, parasitological, epidemiological and molecular data is gathered here to call the attention on the presence of infected dogs as a risk factor in the maintenance of T. cruzi as a source for infection to humans.Acta Tropica.
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2006
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University of the Andes (Venezuela)
- Departamento de Biología
Mérida, Estado Merida, Venezuela
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