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.

Triatoma maculata, the Vector of Trypanosoma cruzi, in Venezuela. Phenotypic and Genotypic Variability as Potential Indicator of Vector Displacement into the Domestic Habitat.

Triatoma maculata is a wild vector of Trypanosoma cruzi, the causative agent of Chagas disease; its incursion in the domestic habitat is scant. In order to establish the possible domestic habitat of T. maculata, we evaluated wing variability and polymorphism of genotypic markers in subpopulations of T. maculata that live in different habitats in Venezuela. As markers, we used the mtCyt b gene, previously apply to evaluate population genetic structure in triatomine species, and the ß-tubulin gene region, a marker employed to study genetic variability in Leishmania subgenera. Adults of T. maculata were captured in the period 2012-2013 at domestic, peridomestic (PD), and wild areas of towns in the Venezuelan states of Anzoátegui, Bolívar, Portuguesa, Monagas, Nueva Esparta, and Sucre. The phenotypic analysis was conducted through the determination of the isometric size and conformation of the left wing of each insect (492 individuals), using the MorphoJ program. Results reveal that insects of the domestic habitat showed significant reductions in wing size and variations in anatomical characteristics associated with flying, in relation to the PD and wild habitats. The largest variability was found in Anzoátegui and Monagas. The genotypic variability was assessed by in silico sequence comparison of the molecular markers and PCR-RFLP assays, demonstrating a marked polymorphism for the markers in insects of the domestic habitat in comparison with the other habitats. The highest polymorphism was found for the ß-tubulin marker with enzymes BamHI and KpnI. Additionally, the infection rate by T. cruzi was higher in Monagas and Sucre (26.8 and 37.0%, respectively), while in domestic habitats the infestation rate was highest in Anzoátegui (22.3%). Results suggest domestic habitat colonization by T. maculata that in epidemiological terms, coupled with the presence in this habitat of nymphs of the vector, represents a high risk of transmission of Chagas disease.

Comparative analyses of the ß-tubulin gene and molecular modeling reveal molecular insight into the colchicine resistance in kinetoplastids organisms.

Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such as Leishmania spp. and Trypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site of Leishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced the ß-tubulin gene of Leishmania (Viannia) guyanensis and established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on the Leishmania ß-tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in the ß-tubulin sequence of kinetoplastid organisms such as Trypanosoma cruzi, T. brucei, and T. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of an α-helix structure and displacement to the inside of the pocket of one ß-sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access.

A PCR assay for the identification of Leishmania species of the Viannia subgenus / Un ensayo de PCR para identificar especies de Leishmania del subgénero Viannia

We have identified a novel DNA sequence of 500 bp (β500-DNA) on the Leishmania (Viannia) subgenus, located in the intergenic region of one of the loci of the β-tubulin gene family. The sequence analysis showed that this sequence has no homology to any other sequence described so far, including the β-tubulin gene. We improved a specific β500-PCR assay, which generated a PCR product of 375 bp for total genomic DNA from Leishmania strains belonging to the L. (Viannia) subgenus. In contrast, no amplification was found when using genomic DNA from species of L. (Leishmania) subgenus or other organisms. Under our PCR conditions, the lower detection limit was 1 fg when a purified DNA clone (pLgβ4), which contains one copy of the β500-DNA sequence, was used. The β500-DNA PCR assay confirmed the preliminary diagnosis of cutaneous leishmaniasis in clinical samples in which the Montenegro skin test was positive and parasite cultures were negative. The analytical specificity and the sensitivity of the PCR assay provide a tool for epidemiological studies of the disease.

En este trabajo identificamos una nueva secuencia de DNA de 500 pb (β500-DNA) en Leishmania del subgénero Leishmania (Viannia), localizada en la región intergénica de uno de los loci de la familia de los genes de la β tubulina. El análisis de secuencia mostró que β500 no tiene homología con ninguna otra secuencia previamente descrita, incluido el gen de la β tubulina. Nosotros implementamos un ensayo de PCR específico para β500, β500-PCR, que genera un producto de PCR de 375 pb a partir del DNA genómico de cepas de Leishmania pertenecientes al subgénero L. (Viannia). No hubo amplificación alguna cuando se utilizó el DNA genómico de especies del subgénero L. (Leishmania) o el de otros organismos. En las condiciones establecidas, utilizando DNA purificado del clon pLgβ4, que contiene una copia de la secuencia de DNA de β500, el límite de detección más bajo fue de 1 fentogramo. El ensayo β500-PCR confirmó el diagnóstico preliminar de leishmaniasis cutánea en muestras clínicas de pacientes positivas a la prueba de Montenegro y negativas en el cultivo de parásitos. La especificidad y sensibilidad analítica del ensayo de PCR proporciona una herramienta para estudios epidemiológicos de la enfermedad.

The 6-phosphogluconate dehydrogenase of Leishmania (Leishmania) mexicana: gene characterization and protein structure prediction.

6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we describe the cloning, sequencing and characterization of a 6PGDH gene from Leishmania (Leishmania) mexicana. The gene encodes a polypeptide chain of 479 amino acid residues with a predicted molecular mass of 52 kDa and a pI of 5.77. The recombinant protein possesses a dimeric quaternary structure and displays kinetic parameter values intermediate between those reported for Trypanosoma brucei and T. cruzi with apparent K(m) values of 6.93 and 5.2 µM for 6PG and NADP(+), respectively. The three-dimensional structure of the enzymes of Leishmania and T. cruzi were modelled from their amino acid sequence using the crystal structure of the enzyme of T. brucei as template. The amino acid residues located in the 6PGDH C-terminal region, which are known to participate in the salt bridges maintaining the protein dimeric structure, differed significantly among the enzymes of Leishmania, T. cruzi, and T. brucei. Our results strongly suggest that 6PGDH can be selected as a potential target for the development of new therapeutic drugs in order to improve existing chemotherapeutic treatments against these parasites.

Amiodarone and miltefosine act synergistically against Leishmania mexicana and can induce parasitological cure in a murine model of cutaneous leishmaniasis.

Leishmaniasis is parasitic disease that is an important problem of public health worldwide. Intramuscularly administered glucantime and pentostam are the most common drugs used for treatment of this disease, but they have significant limitations due to toxicity and increasing resistance. A recent breakthrough has been the introduction of orally administered miltefosine for the treatment of visceral, cutaneous, and mucocutaneous leishmaniasis, but the relative high cost and concerns about teratogenicity have limited the use of this drug. Searching for alternative drugs, we previously demonstrated that the antiarrhythmic drug amiodarone is active against Leishmania mexicana promastigotes and intracellular amastigotes, acting via disruption of intracellular Ca(2+) homeostasis (specifically at the mitochondrion and the acidocalcisomes of these parasites) and through inhibition of the parasite's de novo sterol biosynthesis (X. Serrano-Martín, Y. García-Marchan, A. Fernandez, N. Rodriguez, H. Rojas, G. Visbal, and G. Benaim, Antimicrob. Agents Chemother. 53:1403-1410, 2009). In the present work, we found that miltefosine also disrupts the parasite's intracellular Ca(2+) homeostasis, in this case by inducing a large increase in intracellular Ca(2+) levels, probably through the activation of a plasma membrane Ca(2+) channel. We also investigated the in vitro and in vivo activities of amiodarone and miltefosine, used alone or in combination, on L. mexicana. It was found that the drug combination had synergistic effects on the proliferation of intracellular amastigotes growing inside macrophages and led 90% of parasitological cures in a murine model of leishmaniasis, as revealed by a PCR assay using a novel DNA sequence specific for L. mexicana.

Identificación de una secuencia de ADN genómico de Leishmania especifica del subgénero Leishmania / Identification of a genomic DNA sequence of Leishmania, specific of Leishmania subgenus

Leishmania es el agente causante de la compleja enfermedad conocida como leishmaniasis. Las distintas especies de este parásito protozoario se encuentran agrupadas en dos subgéneros, Viannia y Leishmania, de acuerdo a su desarrollo en el mosquito vector. Un ensayo de PCR, β500-PCR, específico del subgénero Viannia, ha sido desarrollado utilizando la secuencia de ADN genómico denominada β500. En este trabajo se presenta el aislamiento e identificación de una secuencia genómica de 280 pb, L280, a partir del ADN genómico de Leishmania (Leishmania) mexicana luego de aplicar el ensayo β500-PCR en condiciones de baja rigurosidad. La secuenciación parcial de L280 permitió diseñar un ensayo de PCR (L280-PCR) que generó un producto de amplificación de 260 pb, en distintas condiciones de rigurosidad, cuando se utilizó el ADN genómico de distintas especies pertenecientes al subgénero Leishmania. El ensayo L280-PCR resultó negativo para el ADN genómico de distintas especies del subgénero Viannia al igual que para el ADN de otros organismos kinetoplastidos o humano. Los resultados sugieren que el ensayo L280-PCR es específico del subgénero Leishmania.

Leishmania is the causal agent of the leishmaniasis disease. The different species of this protozoa parasite are grouped in two subgenera, Viannia and Leishmania, according to their development in the sandfly vector. A specific PCR assay, β500-PCR, has been developed for the Viannia subgenus using the genomic β500 DNA sequence. In the present work we present the isolation and identification of a genomic sequence of 280 bp, L280, obtained from genomic DNA of Leishmania (Leishmania) mexicana after application of the β500-PCR assay at low stringency. After partial sequencing of L280 a PCR assay was generated, L280-PCR, this yielded a product of 260 bp at different conditions of stringency, when genomic DNA of different species of Leishmania subgenus was used. The L280-PCR assay was negative to genomic DNA of species belonging to the Viannia subgenus and also to other kinetoplastid organisms and human. The results suggest specificity of the L280-PCR assay for the Leishmania subgenus.

Glibenclamide, a blocker of K+(ATP) channels, shows antileishmanial activity in experimental murine cutaneous leishmaniasis.

Glibenclamide reduced the rate of lesion growth in BALB/c mice infected with Leishmania (Leishmania) mexicana, the effect was dose dependent, and the highest dose proved more effective than glucantime. Cross-resistance to glucantime was found in animals infected with a glibenclamide-resistant line, but combined therapy reduced lesion progression even in the glibenclamide-resistant strain.

Detección parsitológica y molecular de infecciones naturales por trypanosoma evansy y trypanosoma vivax en búfalos de agua (bubalus bubalis) y chigüires (hydrochoerus hydrochaeris) en los estados Apure Cojedes y Guárico Venezuela / Parasitological and molecular detection of natural infection by trypanosoma evansi and trypanosoma vivax in water buffaloes (bubalus bubalis) and capybaras (hydrochoerus hydrochaeris) in Apure Cojedes and Guarico states Venezuela

Este estudio evaluó la presencia de Trypanosoma evansi y Trypanosoma vivax en búfalos de agua (Bubalus bubalis) y chigüires (Hydrochoerus hydrochaeris) de tres estados de Venezuela por técnicas parasitológicas y moleculares (frotis teñido: FT; microcentrifugación capilar: TMC; reacción en cadena de la Polimerasa: PCR), estableciéndose el porcentaje de infecciones activas. En 316 muestras sanguíneas de búfalos las tasas de detección para FT, TMC y PCR fueron de 20 (6,33 por ciento), 36 (11,39 por ciento) y 60 (18,98 por ciento),respectivamente. Por PCR se caracterizó a T. vivax como la especie responsable de todas las infecciones, no detectándose presencia de T. evansi. En 186 muestras de chigüires FT y TMC identificaron positividad en 36 (19,35 por ciento) y 71 (38,17 por ciento) de estas, respectivamente, con altas parasitemias; en 27 de estas se observaron tripanosomas del Subgénero Megatrypanum. Por PCR se caracterizaron como T. evansi 51 muestras de chigüires, seis con resultados TMC-negativo. No se detectó T. vivax en chigüires. Un análisis de t-student estableció diferencias (p<0.05) entre los valores de hematocrito (Ht) de búfalos positivos y negativos a tripanosomas; mientras que por análisis de varianza se detectó efecto (p< 0.05) del grado de parasitemia sobre el Ht. No hubo ningún efecto (p>0.05) al realizar los mismos análisis para las muestras de chigüires.

Detección diferencial de trypanosoma evansi y trypanosoma vivax mediante un ensayo de reacción en cadena de la polimerasa / Differential detection of trypanosoma evansi and trypanosoma vivax through a polymerase chain reaction assay

El propósito de este trabajo fue optimizar un ensayo de reacción en cadena de la polimerasa (PCR) para efectuar el diagnóstico diferencial de Trypanosoma evansi y Trypanosoma vivax, usando cebadores previamente descritos: 21-mer/22-mer e ILO1264/ILO1265. La especificidad se evaluó efectuando pruebas preliminares sobre muestras de ADN purificado de diversas especies de parásitos y sobre mezclas de éstas. La sensibilidad se valoró empleando diluciones de ADN de aislados de referencia de T. evansi y T. vivax y concentraciones variables de cebadores. El ensayo optimizado mostró una sensibilidad de 10 pg de ADN, con especificidad para el Subgénero Trypanozoon (cebadores 21-mer/22-mer) y especificidad absoluta para T. vivax (cebadores ILO1264/ILO1265). Ambos grupos de cebadores mostraron potencialidad para detectar infecciones mixtas T. evansi/T. vivax. Un análisis de hibridación sobre el patrón de cariotipo de diversos Kinetoplastida, usando la sonda Te-ADN, mostró su carácter repetitivo y la distribución de su secuencia en diferentes cromosomas de T. evansi TE0. La PCR fue evaluada como herramienta diagnóstico de la presencia de tripanosomas en muestras sanguíneas de animales con infecciones experimentales, demostrando alta sensibilidad al detectar positividad hasta 72 horas antes que lo hicieran los métodos parasitológicos. Este ensayo también fue evaluado sobre muestras sanguíneas de búfalos de agua con infecciones naturales, mostrando alta sensibilidad y especificidad al detectar 9/86 muestras como positivas a T. vivax, revelando una tasa de infección activa de 10,47 por ciento; valor tres veces superior a los resultados de la evaluación microscópica de frotis teñidos (3,49 por ciento) y casi dos veces superior (6,98 por ciento) a la técnica parasitológica de microcentrifugación capilar. En ninguna de las muestras de búfalos evaluadas se detectó T. evansi.

Molecular markers for species identification in the Leishmania subgenus Viannia.

We have previously identified a novel genomic sequence of 500 bp, the beta 500-DNA sequence, in the subgenus Leishmania (Viannia). This sequence was localized upstream of the beta-tubulin gene. Restriction fragment length polymorphism and hybridization analysis has shown that the beta 500-DNA sequence is specific to this subgenus. A polymerase chain reaction (PCR) assay confirmed this specificity. The beta 500-DNA sequence was apparently absent from the genomic deoxyribonucleic acid of L. colombiensis and L. equatoriensis. These results indicate that a PCR assay based on the beta 500-DNA sequence is likely to be of use to detect and identify Leishmania parasites of this subgenus in clinical samples with high sensitivity, specificity and reliability. The beta 500-DNA sequence can be considered a molecular marker for the subgenus Viannia.

Biología Molecular: diagnóstico parasitológico / Molecular biology: diagnosis parasitology

La identificación de un organismo patógeno a distintos niveles de agrupación, es de gan importancia en términos de prevención, diagnosis tratamiento y control de la enfermedad infecciosa a la cual puede estar asociado. La detección e identificación molecular de un agente patógeno puede llevarse a cabo a través de su fenotipo o de su genotipo. En el primer caso, los métodos más informativos han sido el desarrollo de anticuerpos monocloneales e isoenzimas. Al nivel genotípico, la detección e identificación del agente patógeno está basada en la estabilidad y variabilidad de su material genético (ADN o ARN). El estudio y caracterización de secuencias de ADN, ha permitido la implementación y desarrollo de técnicas moleculares de gran especificidad y sensibilidad para la detección y el diagnóstico de organismos patógenos. Un ejemplo de ello lo constituye la evaluación de sondas de ADN a través de la técnica de hibridación molecular y la técnica de PCR. La aplicabilidad de estas técnicas moleculares en el estudio de parasitosis, ha tenido un gran impacto en la identificación del organismo patógeno con una gran sensibilidad y especificidad, de igual manera en la confirmación del diagnóstico de la enfermedad, así como también estudios epidemiológicos destinados a la identificación precisa e reservorios y vectores

Sensitivity of Leishmania spp. to glibenclamide and 4-Aminopiridine: a tool for the study of drug resistance development

We have demonstrated that Leishmania spp. grown as promastigotes, are sensitive to the K+ channel inhibitors 4-aminopyridine and glibenclamide. Their host cells, the macrophages, are not affected by similar concentrations of the drugs. We have also initiated the molecular characterization of the mechanisms involved in the development of drug resistance to glibenclamide by the parasite. Therefore, we have selected experimentally and begun to characterize the Venezuelan Leishmania (Leishmania) strain, NR resistant to glibenclamide [NR(Gr)]. The analysis of genomic DNA evidenced the existence of a fragment which apparently is amplified in NR(Gr). The fragment recognized by the pgpA probe, related to the Leishmania P-glycoprotein family and which was originally isolated from L. tarentolae, showed a size polymorfism between the sensitive and the resistant strain. These results suggest that the development of resistance to glibenclamide in the strain NR(Gr) might be associated with the amplification of the ItpgpA or related gene(s).