Prevalence and molecular characterization of G6PD deficiency in two Plasmodium vivax endemic areas in Venezuela: predominance of the African A-(202A/376G) variant.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency causes acute haemolytic anaemia triggered by oxidative drugs such as primaquine (PQ), used for Plasmodium vivax malaria radical cure. However, in many endemic areas of vivax malaria, patients are treated with PQ without any evaluation of their G6PD status. METHODS: G6PD deficiency and its genetic heterogeneity were evaluated in northeastern and southeastern areas from Venezuela, Cajigal (Sucre state) and Sifontes (Bolívar state) municipalities, respectively. Blood samples from 664 randomly recruited unrelated individuals were screened for G6PD activity by a quantitative method. Mutation analysis for exons 4-8 of G6PD gen was performed on DNA isolated from G6PD-deficient (G6PDd) subjects through PCR-RFLP and direct DNA sequencing. RESULTS: Quantitative biochemical characterization revealed that overall 24 (3.6%) subjects were G6PDd (average G6PD enzyme activity 4.5 ± 1.2 U/g Hb, moderately deficient, class III), while DNA analysis showed one or two mutated alleles in 19 of them (79.2%). The G6PD A-(202A/376G) variant was the only detected in 17 (70.8%) individuals, 13 of them hemizygous males and four heterozygous females. Two males carried only the 376A → G mutation. No other mutation was found in the analysed exons. CONCLUSIONS: The G6PDd prevalence was as low as that one shown by nearby countries. This study contributes to the knowledge of the genetic background of Venezuelan population, especially of those living in malaria-endemic areas. Despite the high degree of genetic mixing described for Venezuelan population, a net predominance of the mild African G6PD A-(202A/376G) variant was observed among G6PDd subjects, suggesting a significant flow of G6PD genes from Africa to Americas, almost certainly introduced through African and/or Spanish immigrants during and after the colonization. The data suggest that 1:27 individuals of the studied population could be G6PDd and therefore at risk of haemolysis under precipitating factors. Information about PQ effect on G6PDd individuals carrying mild variant is limited, but since the regimen of 45 mg weekly dose for prevention of malaria relapse does not seem to be causing clinically significant haemolysis in people having the G6PD A-variant, a reasoned weighing of risk-benefit for its use in Venezuela should be done, when implementing public health strategies of control and elimination.

Oncospheral peptide-based ELISAs as potential seroepidemiological tools for Taenia solium cysticercosis/neurocysticercosis in Venezuela.

This study evaluates five synthetic peptides derived from four, potentially protective, Taenia saginata oncosphere molecules for the serodiagnosis of T. solium cysticercosis/neurocysticercosis in three distinct Venezuelan endemic regions. The peptides, all of which have been described previously, are designated HP6-3, Ts45W-1, Ts45W-5, Ts45S-10 and TEG-1. In clinically verified and seropositive hospital cases, combining the results of three of the individual peptide-based ELISAs (HP6-3, Ts45W-1 and Ts45W-5) afforded the best balance between sensitivity (85%) and specificity (83.5%), a significant improvement on the 63.6% specificity obtained with the routinely employed T. solium cyst-fluid-based ELISA. Similarly, in the seropositive Venezuelan endemic zone samples, 89.09% of Amerindians, 77.27% of symptomatic rural subjects and 67.83% of non-symptomatic rural subjects were also classed as seropositive by the combined peptide-based ELISAs. The profile of antibody recognition to individual peptides varied between the different groups of samples examined. The relevance of the above findings for the serology and prognosis of T. solium cysticercosis/neurocysticercosis in hospital- and field-based situations is discussed.

In vitro antimalarial activity of fractions and constituents isolated from Tabebuia billbergii / Actividad antimalárica in vitro de fracciones y constituyentes aislados de Tabebuia billbergii

Introduction: in vitro antimalarial activity of naphthoquinones (1-5), isolated from Tabebuia billbergii (Bureau & K. Schum.) Standl., was investigated. Tabebuia billbergii, commonly known as guayacán, is a plant traditionally used in the Amazon in numerous conditions like bacterial and fungal infections, fever, syphilis, malaria, trypanosomiasis, as well as stomach and bladder disorders, and tumours. Objective: to study the dichloromethane extracts of both the trunk and the inner bark of Tabebuia billbergii and to demonstrate the antimalarial activity of some of its bioactive components. Methods: some bioactive components were evaluated for the antimalarial activity against Plasmodium berghei, by using the inhibition of the differentiation cycle of the parasite measure by the ³H-hypoxanthine incorporation and compared to that obtained for chloroquine. Results: conventional chromatographic techniques and bioassay-guided fractionation (Artemia salina) allowed isolating from the active fractions one naphthoquinone (lapachol) and four naphtho-furan-4,9-diones. These compounds proved to have an important antiplasmodial effect, with very encouraging IC50's, especially when compared to the results shown by Chloroquine in the same experiment. In addition, two triterpenes, β-sitosterol and stigmasterol, were obtained from the bark. Conclusions: the activity-guided fractionation (A. salina) of dichloromethane extracts of the trunk and the inner bark of Tabebuia billbergii led to the isolation and the identification of five quinonoid compounds with antiplasmodial effect. The significant inhibitory activity in vitro against Plasmodium berghei observed for compound 2-acetyl-naphtho-[2,3b]-furan-4,9-dione allow us to present them as a potential antimalarial compound.

Introducción: se evaluó la actividad antimalárica in vitro de una serie de naftoquinonas (1-5), aisladas de Tabebuia billbergii (Bureau & K. Schum.) Standl., que es conocida comúnmente como guayacán, una planta utilizada tradicionalmente en la Amazonía en numerosos problemas de salud como infecciones bacterianas y fúngicas, fiebre, sífilis, paludismo, tripanosomiasis, así como en problemas estomacales, tumores y trastornos de la vejiga. Objetivo: estudiar los extractos en diclorometano tanto del tronco como la corteza interna de Tabebuia billbergii y evaluar la actividad antimalárica de algunos de sus componentes bioactivos. Métodos: la actividad antimalárica contra Plasmodium berghei se evaluó en algunos componentes bioactivos, por la inhibición del ciclo de la diferenciación de la medida de los parásitos mediante la incorporación de 3H-hipoxantina y se comparó con la obtenida para la cloroquina. Resultados: a través de técnicas cromatográficas convencionales y el fraccionamiento guiado por bioensayo (Artemia salina) se aislaron de las fracciones activas, una naftoquinona (lapachol) y 4 nafto-furan-4,9-dionas. Estos compuestos presentaron un efecto antiplasmodial importante, con buenos valores de IC50, especialmente cuando se compara con los resultados mostrados por la cloroquina en el mismo experimento. Además, se obtuvieron de la corteza 2 triterpenos, β-sitosterol y estigmasterol. Conclusiones: el fraccionamiento guiado por Artemia salina de los extractos en diclorometano del tronco y la corteza interna de Tabebuia billbergii, condujo al aislamiento y la identificación de 5 compuestos de naturaleza quinoidal con efecto antiplasmódicol. La actividad in vitro contra Plasmodium berghei observada para el compuesto 2-acetil-nafto-[2,3 b]-furan-4,9-diona, permite proponerlo como un potencial compuesto antimalárico.

Structure of C-terminal fragment of merozoite surface protein-1 from Plasmodium vivax determined by homology modeling and molecular dynamics refinement.

One current vaccine candidate against Plasmodium vivax targeting asexual blood stage is the major merozoite surface protein-1 of P. vivax (PvMSP-1). Vaccine trials with PvMSP-1(19) and PvMSP-1(33) have succeeded in protecting monkeys and a large proportion of individuals, naturally exposed to P. vivax transmission, develop specific antibodies to PvMSP-1(19). This study presents a model for the three-dimensional structure of the C-terminal 19kDa fragment of P. vivax MSP-1 determined by means of homology modeling and molecular dynamics refinement. The structure proved to be consistent with MSP-1(19) of known crystal or solution structures. The presence of a main binding pocket, well suited for protein-protein interactions, was determined by CASTp. Corrections reported to the sequence of PvMSP-1(19) Belem strain were also inspected. Our model is currently used as a basis to understand antibody interactions with PvMSP-1(19).

Caveolins and flotillin-2 are present in the blood stages of Plasmodium vivax.

Blood stages of Plasmodium vivax induce the development of caveolae and caveola-vesicle complexes (CVC) in the membrane of their host erythrocyte. Caveolae are found in almost all types of cells and are involved in endogenous processes as calcium and cholesterol homeostasis, cell signalling, transporting, ligand internalization and transcytosis of serum components. Major structural components of caveolae are the proteins caveolins and flotillins. The functional role of caveolae in the P. vivax-infected erythrocyte is not properly understood. As these organelles have been shown to contain malaria antigens, it has been suggested that they are involved in the transport and release of specific parasite antigens from the infected erythrocyte and in the uptake of plasma proteins. Using specific antibodies to classical caveolae proteins and an immunolocalization approach, we found caveolin-2, caveolin-3, and flotillin-2 in the vesicle profiles and some CVC of P. vivax-infected erythrocytes. Caveolin-1-3 were not found in uninfected erythrocytes. This is the first report of identification and localization of caveolins in the CVC present in erythrocytes infected with P. vivax, thereby providing evidence of the role of this particular organelle in the protein-trafficking pathway that connect parasite-encoded proteins with the erythrocyte cytoplasm and the cell surface throughout the asexual blood cycle of vivax malaria parasite.

Traffic pathways of Plasmodium vivax antigens during intraerythrocytic parasite development.

We investigated the secretory traffic of a Plasmodium vivax antigen (Pv-148) synthesised by the parasite during the blood cycle, exported into the host cell cytosol and then transported to the surface membrane of the infected erythrocyte. Studies of the ultrastructure of erythrocytes infected with P. vivax showed that intracellular schizogony is accompanied by the generation of parasite-induced membrane profiles in the erythrocyte cytoplasm. These structures are detectable soon after the parasite invades the erythrocyte and develop an elaborate organisation, leading to a tubovesicular membrane (TVM) network, in erythrocytes infected with mature trophozoites. Interestingly, the clefts formed stacked, flattened cisternae resembling a classical Golgi apparatus. The TVM network stained with the fluorescent Golgi marker Bodipy-ceramide. Specific immunolabelling showed that Pv-148 was transferred from the parasite to the erythrocyte surface membrane via the clefts and the TVM network. These findings suggest that the TVM network is part of the secretory pathways involved in parasite protein transport across the Plasmodium-infected erythrocyte and that Pv- 148 may represent a marker that links the parasite with the host cell cytoplasm and, in turn, with the extracellular milieu.

La malaria por Plasmodium Vivax (Grassi y Feletti, 1890) en los trópicos y los retos de la cura radical

Plasmodium vivax es, entre los parásitos causantes del paludismo, la especie más expandida en el mundo; predomina en Asia, América Central y del Sur y el Pacífico Occidental, y provoca unos 80 millones de casos anuales de la enfermedad. Este parásito ocasiona episodios mórbidos recurrentes o recidivas, debidos a sus estadios tisulares persistentes o hipnozoitos. El patrón de recidivas de P. vivax varía según su origen geográfico: con un ataque primario temprano seguido de varias recidivas a corto plazo, en los trópicos, o con un ataque primario temprano, seguido de una latencia prolongada y posteriormente de varias recidivas a corto plazo, en los climas templados. Actualmente el único fármaco disponible para eliminar a los hipnozoitos es la primaquina (PQ), una 8-aminoquinolina cuya pauta terapéutica de 15mg/día durante 14 días es bien tolerada en adultos. Sin embargo, durante más de 40 años se ha advertido sobre la tolerancia relativa a la PQ de los aislados tropicales de P. vivax y consecuentemente, una menor eficacia de la pauta estándar de PQ para prevenir a las recidivas de P. vivax en los trópicos. La expansión del paludismo por P. vivax en el mundo en desarrollo apremia la definición de un régimen de PQ con acción curativa radical que atienda la eficacia de la dosis total, las necesidades operacionales de la adhesión y los posibles efectos tóxicos colaterales del fármaco.

Plasmodium vivax is the most widespread species causing human malaria; it prevails in Asia, Central and South America and the Western Pacific, and its global burden is of approximately 80 million cases annually. Although rarely fatal, the parasite causes recurrent morbid epishowed a well tolerated regimen in adults. However, clinical data from several tropical countries indicate that for an unfailing radical cure of tropical infections a higher dose of PQ should be given. It seems therefore, that clinical and epidemiological management of P. vivax malaria in the tropics is complicated by the wide spread of PQ tolerant parasites producing short-term relapses in an ecological environment where transmission is favored. Studies on the more appropriate therapeutic regimen of PQ for radical cure of P. vivax in the tropics are required without delay.

Plasmodium vivax é, entre os parasitos que causam o paludismo, a espécie mais expandida no mundo; predomina na Ásia, América Central e do Sul e o Pacífico Ocidental, e provoca uns 80 milhões de casos anuais da enfermidade. Este parasito ocasiona episódios mórbidos recorrentes ou recidivas, devidos a seus estágios tisulares persistentes ou hipnozoitos. O padrão de recidivas de P. vivax varia segundo sua origem geográfica: com um ataque primário prematuro seguido de várias recidivas a curto prazo, nos trópicos, ou com um ataque primário prematuro, seguido de uma latência prolongada e posteriormente de várias recidivas a curto prazo, nos climas temperados. Atualmente o único fármaco disponível para eliminar os hipnozoitos é a primaquina (PQ), uma 8-aminoquinolina cuja pauta terapêutica de 15mg/dia durante 14 dias é bem tolerada em adultos. No entanto, durante mais de 40 anos tem-se advertido sobre a tolerância relativa à PQ dos isolados tropicais de P. vivax e conseqüentemente, uma menor eficácia da pauta estándar de PQ para prevenir às recidivas de P. vivax nos trópicos. A expansão do paludismo por P. vivax no mundo em desenvolvimento requer com urgência a definição de um regime de PQ com ação curativa radical que atenda a eficácia da dose total, as necessidades operacionais da adesão e os possíveis efeitos tóxicos colaterais do fármaco.

Lymph node cell responsiveness in BALB/C mice infected with Leishmania mexicana

No presente trabalho medimos a resposta blastogênica de células de nódulos linfáticos de camundongos BALB/c inoculados com Leishmania mexicana, no decurso da infecçäo. Os resultados mostraram que os animais infectados exibem respostas blastogênicas normais nos nódulos linfáticos até vinte semanas depois da infecçäo. Daí por diante houve uma supressäo gradual. A comparaçäo da capacidade de resposta imunitária no baço e nos nódulos linfáticos mostrou respostas normais nesses nódulos várias semanas depois da ocorrência da supressäo no baço. A supressäo das respostas blastogênicas nos nódulos linfáticos estava relacionada a uma célula aderente de tipo macrófago, que suprimia ativamente as respostas proliferativas normais aos agentes mitogênicos

El inmunoensayo enzimático (Elisa) en el diagnóstico serológico de Plasmodium vivax / The immunoenzymatic assay (Elisa) in the serodiagnosis of plasmodium vivax

Comunicamos nuestra experiencia con la aplicación del inmunoensayo enzimático al diagnóstico serológico de Plasmodium vivax con antígeno homólogo. Este se obtuvo a partir de una muestra de sangre de un paciente y luego de lisar los glóbulos rojos con detergente (NP-40). en un "pool" conformado con 11 sueros de pacientes con malaria por P. vivax se encontraron anticuerpos de las clases IgM, IgG e IgA. Se introdujo además la proteína A como sonda secundaria para determinar la respuesta anti-P. vivax en una muestra de 30 sueros de pacientes con un primer episodio palúdico. Se encontró un 93% de correlación con el diagnóstico parasitológico y la prueba específica y reproducible

Activación de celulas supresoras de la respuesta de hipersensibilidad tardía en ratones Balb/c infectados o vacunados con Leishmania mexicana pifanoi / Activation of supressor cells of delayed hypersensitivity response in BALB/C mice infected or immunized with Leishmania mexicana pifanoi

Los ratones BALB/c inmunizados SC con promastigotes no viables de Leishmania mexicana pifanoi, desarrollaron respuestas de hipersensibilidad tardia (RHT) contra el parásito. En contraposición, los ratones BALB/c infectados crónicamente con L.m. pifanoi y aquellos inmunizados IV con una dosis supraóptima de parásitos muertos, mostraron inhibición de la RHT. La supresión de la RHT en los animales infectados, estuvo precedida por un estado transitorio de inmunidad celular, manifestado durante la fase inicial del desarrollo de las lesiones (4-12 semanas). La inhibición de la RHT observada en los animales infectados o inmunizados con dosis supraóptimas, fue causada por un mecanismo activo, transferible a receptores singénicos a través de las células esplénicas de los donantes suprimidos. La población de células supresoras fue no adherente a "nylon", sensible al tratamiento con un suero anti-timocitos de ratón y C', y de acción específica sobre la RHT contra antígenos leishmánicos. Se propone que la ocurrencia de células T supresoras de la RHT en los animales infectados crónicamente o inmunizados con dosis supraóptimas, es causada por el exceso de carga antigénica. Esta última, en el caso de los animales infectados, secundaria a una falla primaria en el control inmunológico de la población parasitaria

Depressed mixed lymphocyte responses in chronic experimental cutaneous leishmaniasis

Los ratones BALB/c infectados con Leishmania mexicana presentan lesiones crónicas y progresivas. El estudio de las células esplénicas durante la fase avanzada de la enfermedad reveló una depresión acentuada de la linfoblastogénesis estimulada por los mitógenos concanavalina A y fitohemaglutinina, y de aquella provocada por células alogénicas provenientes de ratones incompatibles a nivel del locus H-2. La remoción de las células esplénicas adherentes a plástico condujo a una reversión de los índices de estimulación provocados por concanavalina A y fitohemaglutinina, hacia valores normales. Sin embargo, el mismo procedimiento tuvo poco o ningún efecto sobre las reducidas respuestas de las células esplénicas de animales infectados en cultivos mixtos. Dichas células resultaron además, defectuosas en cuanto a su capacidad para estimular respuestas alogénicas en cultivos mixtos

La respuesta inmunológica a Trypanosoma venezuelense (T evansi) en ratas curadas / The immune response to Trypanosoma venezuelense (T evansi),in cured rats

Se estudió la respuesta de ratas albinas a la infección con Trypanosoma venezuelense (T. evansi). Los animales inoculados con 1x10 7 tripomastigotos, mostraron una o más ondas de una parasitemia que fue siempre fatal, pero aquellos que recibieron una dosis curativa de babenil eliminaron la parasitemia y en sus sueros encontramos anticuerpos protectores muy eficientes contra los parásitos homólogos, y más aún contra otros heterólogos

Suppressed induction and expression of delayed hypersensitivity responses in BALB/c mice infected with trypanosoma venezuelense

Following inoculation with 1x10E4 trypomastigotes of Trypanosome venezuelense BALB/c mice showed a progressive, fatal parasitemia and an impaired capability to develop delayed hypersensitivity responses (DHR) as induced by IV immunization with sheep red blood cells (SRBC). Depressed DHR were seen in mice immunized at the time of inoculation and challenged 4 days later. A local cell transfer of immune cells at the site of challenge in the footpad showed that T cells obtained from infected mice immunized with an optimal dose of SRBC on the day of inoculation failed to transfer DHR to naive recipients. However, significant DHR were transfered by T cells from infected mice immunized with a sub-optimal dose of SRBC. By the other hand, 4 days-infected- mice receiving a local transfer of immune T cells taken from non-infected donors failed to express DHR ascompared to controls. It is suggested that in BALB/c mice infected with T. venezuelense both the induction and expression of DHR are impared