The tissue specific tropism in Trypanosoma cruzi. Is it true?

Systematic microscopical observations on tissues from mice, inoculated with different Trypanosoma cruzi isolates, were carried out in order to assess whether the parasite expresses tissue-specific tropism, or if it can invade tissues pervasively within the mammal host. A total of ninety mice were included in the study. Sixty, subcutaneously-inoculated with 15 × 104T. cruzi-blood trypomastigotes were dissected and examined daily for detecting and counting parasites during 12 days of acute infection. Additionally, two long-term experiments using mice inoculated with 5 × 103 metacyclic-forms were performed. A group of 10 mice inoculated intraperitoneally and another group of 20 mice inoculated intradermally. Results demonstrated that T. cruzi does not exhibit a tissue-specific tropism, revealing characteristics of a paninfective species able to invade tissues of ectodermal, mesodermal, and endodermal embryonic origin, irrespective of the parasite's lineage, infective form, route of entry, or size of the inoculum causing the host's infection. Details on T. cruzi-tissue invasion, tissue-parasite load during the course time, and the hypothetical potential pseudocyst/amastigote whole-body burden in the murine model is analyzed. The importance of the findings and its interpretation related to human Chagas disease and the tissue-parasite persistence is also discussed.

Update on Chagas disease in Venezuela during the period 2003-2018. A review.

The present article reviews the status of Chagas disease in Venezuela during the period 2003-2018, based on the detection of Trypanosoma cruzi-infection in 3,343 blood samples of individuals from rural localities and 182 patients referred from health centers to confirm presumptive clinical diagnostic. The study involved samples from 81 rural localities of 17 states located at different regions and ecological life zones of the country. Analysis by parasitological (fresh microscopic observation, hemoculture and Giemsa stained blood smears), serological (DAT, IFAT-polyvalent, IgM, IgG tests) and molecular (PCR) tests, revealed 10.7% seroprevalence and 42.8% T. cruzi-infection, in individuals from rural localities and referred patients, respectively. In both groups T. cruzi-infection was detected at any age, revealing active transmission in children under 10-years-old. Clinical profile detected in referred patients, showed significantly major number of symptoms in orally infected patients than in infected by vectorial route (P<0.01). Genetic characterization of T. cruzi isolates obtained from orally and vectorial transmitted acute Chagas disease in western Venezuela, revealed the circulation of DTUI and DTUIII in the former, and DTUI, DTUII and DTUIII in patients infected by vectorial route. DTUI predominated in both cases, and haplotype Ib was the most frequently found in this genotype. Statistical analysis of clinical profile - T. cruzi DTUs - transmission route relationships did not show association among these variables and, consequently, chagasic patient's clinical condition did not depend of T. cruzi genotype or its route of transmission. In addition, differences in clinical severity may be associated with host susceptibility and/or parasite load received by the human receptor in spite of the T. cruzi genotype itself. The epidemiological implications of the present findings are discussed, and the need for developing efficient tools as well as implementation of urgent and radical changes in the public health policy to control Chagas disease transmission in the Venezuelan territory are suggested.

Aneurisma cardíaco apical en paciente chagásico agudo de avanzada edad: Reporte de un caso / Apical cardiac aneurism in acute chagasic aged patient: A case report

Se presenta el caso de un paciente de la tercera edad mostrando cuadro agudo sintomático con complicaciones cardiacas evidenciadas por detección de fibrilación auricular, miopericarditis, derrame pericárdico moderado, sin signos de taponamiento cardíaco, fracción de eyección del ventrículo izquierdo (FEVI) < 30% y aneurisma, detectados mediante estudios electro y eco cardiográficos, respectivamente. El despistaje parasitológico reveló la presencia de Trypanosoma cruzi en muestra de sangre circulante y líquido pericárdico, con hemocultivo positivo y detección de ADN específico del parásito por ensayos de PCR. Asimismo, la presencia de altos niveles de anticuerpos circulantes anti-T. cruzi detectados con dos pruebas serológicas (TAD, IFI) y la observación de altos niveles IgM, corroboran el diagnóstico de un cuadro de enfermedad de Chagas en fase aguda de la infección. Además, la procedencia del paciente de una localidad andina del occidente de Venezuela, donde la enfermedad de Chagas es endémica, unido al hallazgo de ejemplares adultos de triatominos en la vivienda del paciente confirman el diagnóstico. Se discute la significación del presente hallazgo y se detallan las observaciones durante la re-evaluación en el seguimiento post tratamiento.

A Clinical case from an aged patient with evidence of suffering heart failure showing arrhythmia, atrial fibrillation and pericarditis, pericardial effusion and left ventricle aneurism detected by electrocardiogram and echocardiogram respectively, is reported. Parasitological examination revealed presence of Trypanosoma cruzi in blood and pericardial fluid samples. In addition, hemoculture was positive and PCR assay revealed specific T. cruzi DNA. Serological techniques (DAT, IFAT) showed anti-T.cruzi circulating antibodies, including high level IgM. The sero-parasitological and molecular (PCR) findings confirmed the presumptive clinical diagnosis for Chagas disease acute infection. The significance of the present findings is discussed and clinical details observed during re-evaluation post-treatment, are included.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease.

Brote de enfermedad de Chagas agudo de posible transmisión oral en Mérida, Venezuela / Acute Chagas disease outbreak of possible oral transmission in Merida, Venezuela

Se registra un brote de enfermedad de chagas agudo, en una localidad rural de Mérida-Venezuela, asociado con transmisión oral por ingesta contaminada con trypanosoma cruzi dada la masiva y simultanea infección detectada en 5 miembros de una familia. La masiva transmisión de T. cruzi generó cuadros clínicos severos causando miocarditis aguda en 2 pacientes (40%) incluyendo un caso fatal. La severidad clínica en todos los pacientes involucrados mostró correspondencia con los hallazgos parasitológicos, serológicos y moleculares (PCR) con los cuales se evidenció presencia de tripomastigotes sanguícolas de T. cruzi, anticuerpos anti-T. cruzi con niveles variables de IgM e IgG específicas y ADN de T. cruzi, respectivamente. Asimismo, el perfil clínico reveló 17 signos o síntomas con un promedio por paciente de 12±3 (rango=9-16) con simultaneidad en 8 de los síntomas más frecuentemente detectados. Resalta en la sintomatología la presencia de edema facial interno con manifiesta tumefacción y parestesia lingual en ausencia del cuadro típico de edema bipalpebral (signo de Romaña) y/o chagoma de inoculación comúnmente registrados en la infección chagásica por vía vectorial. Se argumenta la posibilidad de la transmisión oral y se discute la potencial importancia epidemiológica del presente hallazgo.

An acute Chagas disease outbreak associated with oral transmission, detected in a rural village of Merida- Venezuela, is reported. The massive transmission due to ingestion of food contaminated with Trypanosoma cruzi generated, in 5 members of the same family, severe clinical features causing acute myocarditis in two of them (40%) including a fatal case. Parasitological, serological and molecular (PCR) examination of samples from the 5 patients involved in the outbreak, revealed T.cruzi blood circulating tripomastigotes, anti-T. cruzi specific antibodies (IgM; IgG) and the presence of T. cruzi respectively. The recorded clinical pattern detected a total of 17 signs or symptoms with an average ± SD 12±3 per patients (range = 9-16) showing simultaneity in 8 symptoms and a 20% mortality. A remarkable characteristic detected during the study was the presence of internal facial swollen with paresthesia in tongue in absence of the typical Romaña's sign or chagoma commonly found in Chagas disease-infection caused by vector transmission. The possible oral transmission and the potential of the findings from the epidemiological viewpoint are discussed.

Estudio epidemiológico sobre leishmaniasis visceral en la región semiárida del occidente de Venezuela con especial referencia a la detección de infecciones inaparentes / Epidemiological study on visceral leishmaniasis in the semiarid region of western Venezuela with special reference to inapparent infections

Se registra un estudio epidemiológico sobre leishmaniasis visceral (LV) llevado a cabo en localidades de la región semiárida noroccidental de Venezuela, donde previamente se había detectado casos activos de esta dolencia. Un total de 1036 individuos sin manifestaciones clínicas aparentes (SMCA) domiciliados en 25 localidades, y 67 perros, de 8 localidades, fueron examinados por métodos serológicos (TAD, IFI, ELISA) y ensayos de PCR. Los resultados revelan una seropositividad a la presencia de anticuerpos circulantes anti-Leishmania infantum (=L. chagasi) del 16,9% y 76,1% en humanos asintomáticos y perros, respectivamente. Los hallazgos serológicos fueron corroborados por la reactividad a la prueba de PCR, arrojando valores de 17,7% en humanos y 29,9% en caninos. Se demuestra la alta prevalencia de infecciones inaparentes por L. infantum en individuos asintomáticos, resultando más frecuentes y ampliamente distribuidas que las infecciones activas. Se discute su rol en la dinámica de transmisión de la LV. Se propone considerar el perro como factor importante en el mantenimiento de L. infantum como fuente de infección humana, corroborando su rol de reservorio de LV en el semiárido venezolano. Se identifican 12 especies flebotominas entre 6000 especímenes colectados en 12 localidades, detectándose un franco predominio de Lutzomyia evansi (66,6%) sobre Lu. longipalpis (14,9%). Se detecta en ambas especies infección natural por L. infantum, utilizando ensayos de PCR. Se concluye que la asociación de personas con LV, activas o SMCA; perros infectados con L. infantum y Lu. evansi, presente en todas las localidades; aunada a las pobres condiciones sanitarias y/o nutricionales, son factores de riesgo suficientes para explicar la alta endemicidad de la LV en la región semiárida del occidente de Venezuela.

An epidemiological study on visceral leishmaniasis (VL) in the semiarid region of western Venezuela, where some acute cases have been previously reported, is recorded. A total of 1036 symptomless people's sera samples from 25 villages, and 67 dogs from 8 different localities, were tested by using 3 different serologic methods (DAT, IFAT, ELISA) and the polymerase chain reaction assay (PCR). Serological tests revealed 16.9% and 76.1% seropositive to Leishmania infantum in asymptomatic humans and dogs, respectively. In addition, a PCR assay detected the presence of L. infantumspecific DNA in 17.7% of the symptomless sampled individuals and in 29.9% of the tested dogs. The combined analyses of serologic and molecular findings demonstrate the presence of subclinical or inapparent VL infections in local people from this part of western Venezuela. The observed prevalence of VL in asymptomatic people is by itself a matter of concern from the epidemiological point of view. The present results also serve to call the attention to the presence of infected dogs as a risk factor in the maintenance of L. infantum as a source for infection to humans in the studied semiarid region. A total of 12 sand fly species were identified from 6000 specimens collected in 12 sampled villages, with a predominance of Lu. evansi (66.6%) over Lu. longipalpis (14.9%). In both sand fly species natural infection by L. infantum was detected using a PCR assay. It is concluded that the association among VL infected people, L. infantum-infected dogs and the presence of Lu. evansi, together with the observed poor sanitary and/or nutritional conditions, are risk factors to explain the high endemicity by VL at the semiarid region of western Venezuela.

Detección de Leishmania braziliensis en lesión mucosa con 16 años de evolución: Registro de un caso / Detection of Leishmania braziliensis in a mucosal lesion with 16 years of evolution: A case report

Se registra, en un paciente masculino de 48 años, la detección de Leishmania (Viannia) braziliensis en muestra de lesión mucosa crónica con 16 años de evolución clínica. El caso presentado cursa con perforación del tabique nasal no evidenciándose lesión cutánea primaria sugestiva de leishmaniasis. Por su ocupación el paciente ha frecuentado, por largos períodos, bosques en áreas endémicas para leishmaniasis en el occidente de Venezuela. Continuas evaluaciones clínicas, inmunológicas e histopatológicas realizadas durante varios años fallaron en establecer un diagnóstico certero incrementando la severidad del caso. Exámenes recientes realizados en nuestro laboratorio revelan la presencia de amastigotes en muestras de la lesión nasal activa en extendidos coloreados con tinción de Giemsa. La identificación del parásito como L. braziliensis y la verificación de la infección por este parásito en el paciente, fue lograda por ensayos de PCR y Western Blot, respectivamente. Se concluye que el caso presentado sufre una leishmaniasis mucocutánea de vieja data con una baja respuesta inmunológica, a juzgar por la frecuente negatividad en pruebas de IDR y/o la detección de anticuerpos circulantes anti-Leishmania. Se discute sobre la imprecisión diagnóstica en el caso presentado y se advierte sobre el riesgo epidemiológico potencial de casos similares.

The detection of Leishmania (Viannia) braziliensis from a chronic mucosal lesion with 16 years of clinical evolution in a 48 years old male patient is reported. The patient showed destruction of the nasal septal cartilage with no evidence of primary leishmanial lesion. Due to his professional work he frequently visited areas of western Venezuela where leishmaniasis is endemic. Frequent clinical, immunologic and histopathologic evaluations carried out during several years failed to establish a right diagnosis, increasing the severity of the mucosal lesion. The finding of a sparse number of amastigotes in a sample from the mucosal lesion stained by Giemsa stain, suggested a mucosal infection by Leishmania. The identification of the parasite as L. braziliensis and the verification of the infection by this parasite in the reported case were carried out using a PCR assay and a Western Blot test, respectively. It is concluded that the patient was suffering a long-lasting, reluctant to heal and severe lesion with a low immunological response due to infection by L. braziliensis causing mucocutaneous leishmaniasis (MCL). The fail in the diagnosis of this particular case of MCL and the potential epidemiological risk in similar cases are discussed.

Detección de infecciones inaparentes de la enfermedad de Chagas en individuos asintomáticos de la etnia Yukpa del occidente de Venezuela / Chagas disease inapparent infection in asymptomatic individuals from a Yukpa ethnic community in western Venezuela

Inapparent infections by Trypanosoma cruzi were detected, for the first time, in symptomless seropositive healthy individuals from a Yukpa ethnic community in western Venezuela where Chagas disease had not been previously reported. Seropositivity was detected in 24 out of 173 (13.9%) sera samples taken from asymptomatic people by using three serological methods (DAT, IFAT, ELISA). Complementary analyses by IFAT revealed a low level of anti-T. cruzi specific IgM and IgG in all seropositive people when compared with detected levels in acute and chronic chagasic patients included as positive controls. In addition, 100% of the sampled dogs (8) were seropositive showing a level of anti-T. cruzi IgG similar to that detected in humans. Genotyping of a T. cruzi isolate obtained from an infected wild specimen of Rhodnius pictipes, revealed the circulation of lineage1 (DTU1) in the study area. The importance of the detection of asymptomatic T.cruzi-infections in this indigenous community and its potential epidemiological implications are discussed.

Infecciones inaparentes por Trypanosoma cruzi fueron detectadas, por vez primera, en individuos seropositivos asintomáticos muestreados en una comunidad Yukpa del occidente de Venezuela donde no se ha registrado hasta el presente enfermedad de Chagas. De un total de 173 muestras de suero examinadas 24 (13,9%) fueron registradas como seropositivas utilizando tres pruebas serológicas. Análisis complementarios por IFI revelaron bajos niveles de IgM e IgG cuando las muestras de individuos seropositivos asintomáticos fueron comparadas con casos agudos y crónicos incluidos como controles positivos. La totalidad de perros examinados (8) reveló seropositividad, registrándose niveles de IgG similares a los detectados en el grupo humano. La caracterización molecular de un aislado de T. cruzi obtenido de un espécimen silvestre de Rhodnius pictipes reveló la circulación del linaje 1 (DTU1) en el área de estudio. Se discute la importancia de infecciones asintomáticas por T. cruzi y sus implicaciones epidemiológicas en la comunidad indígena estudiada.

Primoinfección por Trypanosoma cruzi previene re-infecciones severas en ratones / Trypanosoma cruzi primary infection prevents severe re-infection in mice

The effect of Trypanosoma cruzi re-inoculations on experimentally infected mice was evaluated. Mice received a primary infection by intradermal inoculation of 5x103 T. cruzi metacyclic infective forms from laboratory infected Rhodnius prolixus. From 200 mice initially infected, 52 survived the course of the infection during 23 weeks. From these, 45 mice were re-inoculated and seven used as infected control. Two re-inoculations were performed using the same conditions as in the prime-infection. Observations on re-inoculated mice revealed a parasitemia level lower than that detected during the primary-infection. Serologic evaluation showed no variation in the immunoglobulin profile, maintaining similar IgM and IgG levels after re-inoculations. Similar mortality rates were observed in primary-infected, re-inoculated and infected control mice. No remarkable histopathological changes attributable to re-inoculation were detected. These results lead us to conclude that T. cruzi re-inoculation in mice previously infected with the same parasite does not produce a reactivation of infection similar to the typical acute clinical or immunological profiles. This also suggests that T. cruzi primary infection may prevent severe re-infection, establishing a protective stage making infected mice resistant to a second infection. Epidemiological implications of the present findings are discussed.

Se evalúa el efecto de reinoculaciones por Trypanosoma cruzi en ratones experimentalmente infectados. De un total de 200 ratones que fueron infectados por vía intradérmica con un inóculo de 5x103 tripomastigotes metacíclicos provenientes de especimenes de Rhodnius prolixus, 52 sobrevivieron a la primo-infección luego de 23 semanas. De estos 45 fueron re-inoculados en la misma forma como en la infección primaria y siete fueron utilizados como controles infectados no re-inoculados. Observaciones sistemáticas llevadas a cabo en muestras de los ratones re-inoculados revelaron parasitemias significativamente menores que las detectadas durante la primo-infección. La evaluación serológica no mostró diferencias en los niveles de IgM e IgG entre ratones primo-infectados y los re-inoculados. Observaciones histopatológicas no mostraron cambios atribuibles al efecto de las re-inoculaciones en muestras de corazón y músculo esquelético. Una tasa similar de mortalidad fue observada en ratones primo-infectados, re-inoculados y controles infectados. Se concluye que re-inoculaciones con T. cruzi en ratones previamente infectados con el mismo parásito no producen reactivación de la infección similar al típico cuadro agudo. Asimismo, se sugiere que la primo-infección por T. cruzi previene una reinfección severa estableciendo un estado de protección y resistencia a subsecuentes infecciones. Se discuten las implicaciones epidemiológicas de los presentes hallazgos.

International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.

BACKGROUND: 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. METHODOLOGY/FINDINGS: 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. CONCLUSION/SIGNIFICANCE: This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples.

Trypanosoma cruzi persistence at oral inflammatory foci in chronic chagasic patients.

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.

Valoración comparativa de pruebas serodiagnósticas utilizadas para detectar enfermedad de Chagas en Venezuela / Comparative evaluation of serologic diagnostic tests used to detect Chagas disease in Venezuela

Se presenta la valoración comparativa de 8 técnicas de uso común en el despistaje serológico de la enfermedad de Chagas utilizando muestras de pacientes de diversas procedencias del occidente de Venezuela. En el estudio fueron utilizados métodos serológicos convencionales (TAD, IFI, ELISA) y pruebas de diagnóstico comercialmente expendidas en el país (Pharmatest®, Chagatest ELISA®, Chagatest ELISA Recombinante V.3.0®, Test ELISA para Chagas III® y Chagas Stat PakTM Assay®). La comparación de los resultados no reveló diferencias estadísticamente significativas entre los niveles de sensibilidad y especificidad de las técnicas evaluadas (P>0.05). Se sugiere, para la obtención de un diagnóstico preciso, la implementación de pruebas de rutina y pruebas confirmatorias. El análisis de la relación costo-beneficio reflejó diferencias entre las técnicas evaluadas, sugiriendo su uso de acuerdo a las condiciones de servicio presentes en cada centro diagnóstico, para lo cual se proponen pruebas candidatas para la rutina y la confirmación diagnóstica. Se demuestra la disminución progresiva de reactividad, con el consecuente incremento de resultados erróneos, en una prueba comercial sometida a sucesivos cambios de temperatura en el tiempo

The comparative evaluation of conventional serologic methods (DAT, IFAT, ELISA) and commercial tests (Pharmatest®, Chagatest ELISA®, Chagatest ELISA Recombinant V.3.0®, Test ELISA for Chagas III®, Chagas stat pakTM assay®) commonly used to detect Chagas disease is presented. Sera samples were taken from chagasic patients and controls from different rural localities of western Venezuela. Statistical comparison of the sensitivity and specificity level, revealed no-significant differences among methods (P > 0.05). To warrant a secure sero-diagnosis the implementation of a routine and a confirmatory test is suggested. The cost-benefit analysis showed significant differences among methods, suggesting its use according to the condition of each diagnostic center. Considering the results obtained in the present study a combination of candidate tests is proposed to be used either as a routine or confirmatory method according to the circumstances. The effect of thermo-shock on the loss of reactivity of a commercial test is demonstrated

Detección de transmisión transplacentaria de Anaplasma Marginale en bovinos asintomáticos / Detection of Anaplasma marginale Transplacental Transmission in Asymptomatic Cattle / The presence of Anaplasma marginale in calves descendants from cows with asymptomatic infections, was detected. Polymerase chain reaction (PCR) assays carried out in blood samples from 31 healthy cows and their respective calves, revealed the presence of A. marginale-specific DNA in 70 and 40% of them, respectively. The detection of a portion of A. marginale genome in newborn calves, suggests a transplacental mother-offspring transmission. The epizootiological implications of A. marginale-infection in asymptomatic animals and the potential of transplacental transmission in the maintenance of A. marginale were considered. Results lead to conclude that A. marginale transplacental transmission might be of frequent occurrence in the study area, so more specific techniques, such as PCR, are suggested for a more thorough diagnosis.

Se detecta la presencia de Anaplasma marginale en becerros hijos de vacas con infección asintomática. Ensayos de reacción en cadena de la polimerasa (PCR) llevados a cabo en muestras de sangre de 31 vacas lactantes, en aparente buen estado físico y sus respectivas crías, revelaron la presencia de ADN específico de A. marginale en el 70 y 40%, respectivamente. La detección de parte del genoma de A. marginale en becerros recién nacidos, hijos de vacas asintomáticas PCR positivas, sugiere la transmisión de la infección madre-hijo por vía transplacentaria. Se discute las implicaciones epizootiológicas de la infección por A. marginale en animales asintomáticos y se advierte sobre la potencialidad de esta forma de transmisión en el mantenimiento del ciclo de este organismo. Se concluye que la transmisión por vía transplacentaria podría ser un evento de frecuente ocurrencia en el área de estudio y se sugiere la utilización de técnicas producto de la nueva biotecnología como la PCR para incrementar la especificidad en el diagnóstico.

Supervivencia e infectividad de formas metacíclicas de Tripanosoma cruzi en alimentos experimentalmente contaminados / Survival and infectivity of Trypanosoma cruzi metacyclic forms in experimentally contaminated food

Se registra el tiempo de supervivencia de formas metacíclicas de Trypanosoma cruzi, obtenidas del tracto digestivo de Rhodnius prolixus, sobre alimentos contaminados experimentalmente.Observaciones entre 1h y 18h post-contaminación revelaron la presencia de abundantes y activosmetacíclicos en los alimentos contaminados durante las primeras 6 horas, declinando la poblaciónde los mismos sobre la décima hora. La actividad de las formas metacíclicas no mostró diferencias en alimentos contaminados en fase sólida o líquida. Asimismo, se demuestra la alta infectividad de metacíclicos de T. cruzi que sobreviven en alimento contaminado luego de ser ingeridos por un hospedador vertebrado

The survival of Trypanosoma cruzi metacyclic forms in contaminated food is reported. Observationscarried out 1-18h after contaminating food withmetacyclics from infected Rhodnius prolixus, revealedabundant and active flagellates during the first 6h postcontamination.The same activity was observed inmetacyclics maintained on liquid or solid contaminatedfood. In addition, the infectivity of surviving T. cruzimetacyclic forms in contaminated food after being ingested by a vertebrate host is demonstrated

Trypanosoma cruzi congenital transmission in wild bats.

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.

Supervivencia de formas de cultivo de Trypanosoma cruzi en alimentos experimentalmente contaminados / Survival of culture forms of Trypanosoma cruzi in experimentally contaminated food

Se registra la supervivencia de formas de cultivo de Trypanosoma cruzi en alimentos experimentalmente contaminados. En 8 de 11 (73%) muestras de frutas y hortalizas utilizadas en los experimentos los parásitos permanecieron vivos por períodos entre 6 y 72 horas. Se estima que el mayor número de parásitos vivos está entre las 6 y las 18 horas post-contaminación. Se discute las implicaciones epidemiológicas potenciales de estos hallazgos.

Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis.

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.

Expression of GP82 and GP90 surface glycoprotein genes of Trypanosoma cruzi during in vivo metacyclogenesis in the insect vector Rhodnius prolixus.

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.

Estudio de un nuevo foco de lishmaniasis visceral en el estado Falcón Venezuela: registro de un caso / Study on a new focus of visceral leishmaniasis in Falcón state Venezuela: record of a case

Se registró un caso infantil de leishmaniasis visceral procedente del caserío “Los Pozones”, municipio Buchivacoa ubicado al occidente del Estado Falcón, Venezuela, a finales del año 2002. Se presentan resultados de un estudio epidemiológico en la localidad, realizado durante el año siguiente la detección del caso. El estudio incluyó un muestreo serológico a 110 humanos de diversos grupos etarios y a 10 perros, utilizando tres pruebas inmunodiagnósticas (TAD, IFI y ELISA). Además se realizaron capturas de flebotominos en refugios naturales y con trampa lumínica de Shannon cada dos meses durante un año, detectándose la presencia dominante de Lutzomyia evansi (62.7%) sobre Lutzomyia longipalpis (37.3%). La zona de vida donde se enclava la localidad rural estudiada corresponde a un bosque seco espinoso. El estudio serológico arrojó valores de seroprevalencia humana y canina para Leishmania del 17,3% y 50%, respectivamente. Estos hallazgos sugieren la existencia de un nuevo foco de LV en la parte occidental del estado Falcón, Venezuela, el cual requiere más investigación.

Uso de proteínas como alternativa diagnóstica para discriminar infecciones entre Trypanosoma vivax y Trypanosoma evansi / Use of proteins as diagnostic tool to discriminate between Trypanosoma vivax and Trypanosoma evansi infections

Tripomastigotes sanguícolas de Trypanosoma vivax y Trypanosoma evansi obtenidos de infecciones experimentales en ovejas y ratones respectivamente, fueron utilizados para purificar y caracterizar proteínas citosólicas mediante el método de partición con Tritón X-114. Los resultados revelan diferencias en los patrones proteicos entre las dos especies. Asimismo, las reacciones antigénicas mediante Western blot utilizando suero de animales naturalmente infectados, permitió discriminar las infecciones entre ambos parásitos. Se sugiere la utilización de esta metodología como una prueba diagnóstica confiable.