Microencapsulation of Theobroma cacao L polyphenols: A high-value approach with in vitro anti-Trypanosoma cruzi, immunomodulatory and antioxidant activities.

Chagas disease (CHD) is the highest economic burden parasitosis worldwide and the most important cardiac infection, without therapeutic alternatives to halt or reverse its progression. In CHD-experimental models, antioxidant and anti-inflammatory compounds have demonstrated therapeutic potential in cardiac dysfunction. Theobroma cacao polyphenols are potent natural antioxidants with cardioprotective and anti-inflammatory action, which are susceptible to degradation, requiring technological approaches to guarantee their protection, stability, and controlled release. Here, 21 cocoa polyphenol-rich microencapsulates were produced by spray-drying and freeze-drying techniques using two wall materials (maltodextrin and gum arabic). Chemical (total and individual phenolic content and antioxidant activity), structural (morphology), and biological parameters (cytotoxicity, trypanocidal, antioxidant, and immunomodulatory activities) were assessed to determine the most efficient microencapsulation conditions on Trypanosoma cruzi-infected myocardioblast and macrophage cells. Significant antiproliferative properties against infected cells (superior to benznidazole) were found in two microencapsulates which also exhibited cardioprotective properties against oxidative stress, inflammation, and cell death.

Immunomodulatory, trypanocide, and antioxidant properties of essential oil fractions of Lippia alba (Verbenaceae).

BACKGROUND: Parasite persistence, exacerbated and sustained immune response, and continuous oxidative stress have been described to contribute to the development of the cardiac manifestations in Chronic Chagas Disease. Nevertheless, there are no efficient therapies to resolve the Trypanosoma cruzi infection and prevent the disease progression. Interestingly, trypanocide, antioxidant, and immunodulatory properties have been reported separately for some major terpenes, as citral (neral plus geranial), limonene, and caryophyllene oxide, presents in essential oils (EO) extracted from two chemotypes (Citral and Carvone) of Lippia alba. The aim of this study was to obtain L. alba essential oil fractions enriched with the aforementioned bioactive terpenes and to evaluate the impact of these therapies on trypanocide, oxidative stress, mitochondrial bioenergetics, genotoxicity, and inflammatory markers on T. cruzi-infected macrophages. METHODS: T. cruzi-infected J774A.1 macrophage were treated with limonene-enriched (ACT1) and citral/caryophyllene oxide-enriched (ACT2) essential oils fractions derived from Carvone and Citral-L. alba chemotypes, respectively. RESULTS: ACT1 (IC50 = 45 ± 1.7 µg/mL) and ACT2 (IC50 = 80 ± 1.9 µg/mL) exhibit similar trypanocidal effects to Benznidazole (BZN) (IC50 = 48 ± 2.5 µg/mL), against amastigotes. Synergistic antiparasitic activity was observed when ACT1 was combined with BZN (∑FIC = 0.52 ± 0.13 µg/mL) or ACT2 (∑FIC = 0.46 ± 1.7 µg/mL). ACT1 also decreased the oxidative stress, mitochondrial metabolism, and genotoxicity of the therapies. The ACT1 + ACT2 and ACT1 + BZN experimental treatments reduced the pro-inflammatory cytokines (IFN-γ, IL-2, and TNF-α) and increased the anti-inflammatory cytokines (IL-4 and IL-10). CONCLUSION: Due to its highly trypanocidal and immunomodulatory properties, ACT1 (whether alone or in combination with BZN or ACT2) represents a promising L. alba essential oil fraction for further studies in drug development towards the Chagas disease control.

Disagreement between PCR and serological diagnosis of Trypanosoma cruzi infection in blood donors from a Colombian endemic region / Desacuerdo entre la PCR y la serología en el diagnóstico de la infección por Trypanosoma cruzi en donantes de una región endémica de Colombia

Abstract | Introduction: Chagas' disease is the leading cause of infectious myocarditis worldwide. This infection caused by Trypanosoma cruzi is usually life-long and asymptomatic; however, the third part of infected people can develop severe or even fatal cardiomyopathy. As the parasitemia in the chronic phase is both low-grade and intermittent, T. cruzi infection is principally detected by serology, although this method has sensitivity and specificity limitations. Objective: To determine the level of agreement between serologicand molecular tests in 658 voluntary blood donors from six provinces in the Colombian department of Santander. Materials and methods: We evaluated an array of diagnostic technologies by cross-section sampling performing a serological double diagnostic test for T. cruzi antibody detection (Chagas III ELISA™, BiosChile Group, and ARCHITECT Chagas CMIA™, Abbott) , and DNA detection by polymerase chain reaction (PCR). We collected the demographic, clinical, and epidemiological information of participants. The sample size was calculated using Epidat™ and the statistical analysis was done with Stata 12.1™. Results: PCR was six times more sensitive in detecting T. cruzi infection than ELISA/CMIA with prevalence values of 1.8% (12/658) and 0.3% (2/658), respectively, and kappa=0.28 (95%CI: -0.03 - 0.59). In contrast, serology showed a sensitivity of 16.7% (95%CI: 2.09 - 48.4) and a specificity of 100% (95%CI: 99.4 - 100). All seropositive samples were found to be positive by PCR. Conclusions: The implementation of PCR as a complementary method for screening donors could reduce the probability of false negative and the consequent risk of transfusional-transmission of Chagas' disease, especially in endemic regions.

Resumen | Introducción. La enfermedad de Chagas constituye la principal causa de miocarditis infecciosa en el mundo. Causada por Trypanosoma cruzi,la infección puede persistir toda la vida de manera asintomática y silenciosa, pero un tercio de los infectados desarrolla cardiomiopatía grave. Debido a que la parasitemia en la fase crónica es baja e intermitente, el diagnóstico se hace principalmente mediante la detección de anticuerpos (serología), método que tiene limitaciones de sensibilidad y especificidad. Objetivo. Determinar la concordancia entre el diagnóstico serológico y molecular de T. cruzien 658 donantes voluntarios de sangre del departamento de Santander, Colombia. Materiales y métodos. Se hizo un estudio de evaluación de tecnologías diagnósticas con muestreo transversal, utilizando un doble diagnóstico serológico para la detección de anticuerpos anti-T. cruzi (Chagas III ELISA™, BiosChile Group, y ARCHITECT ChagasCMIA™, Abbott) y la de ADN por PCR. Se recolectó la información demográfica, clínica y epidemiológica de los participantes. El tamaño de la muestra se estimó utilizando Epidat™ y el análisis estadístico se hizo mediante Stata 12.1™. Resultados. La sensibilidad de la PCR fue seis veces mayor que la de las pruebas de ELISA/CMIA, con prevalencias de 1,8 % (12/658) y 0,3 % (2/658), respectivamente, y kappa de 0,28 (IC95% -0,03 - 0,59). La sensibilidad serológica fue de 16,7 % (IC95% 2,09 - 48,4) y la especificidad de 100 % (IC95% 99,4 - 100). Todas las muestras seropositivas fueron positivas también en la PCR. Conclusiones. El uso de la PCR como método complementario para la tamización de donantes podría reducir el riesgo de falsos negativos y disminuir los casos de transmisión transfusional de la enfermedad de Chagas, especialmente en regiones endémicas.

Induction of programmed cell death in Trypanosoma cruzi by Lippia alba essential oils and their major and synergistic terpenes (citral, limonene and caryophyllene oxide).

BACKGROUND: Chagas Disease caused by Trypanosoma cruzi infection, is one of the most important neglected tropical diseases (NTD), without an effective therapy for the successful parasite eradication or for the blocking of the disease's progression, in its advanced stages. Due to their low toxicity, wide pharmacologic spectrum, and potential synergies, medicinal plants as Lippia alba, offer a promising reserve of bioactive molecules. The principal goal of this work is to characterize the inhibitory properties and cellular effects of the Citral and Carvone L. alba chemotype essential oils (EOs) and their main bioactive terpenes (and the synergies among them) on T. cruzi forms. METHODS: Twelve L. alba EOs, produced under diverse environmental conditions, were extracted by microwave assisted hydrodistillation, and chemically characterized using gas chromatography coupled mass spectrometry. Trypanocidal activity and cytotoxicity were determined for each oil, and their major compounds, on epimastigotes (Epi), trypomastigotes (Tryp), amastigotes (Amas), and Vero cells. Pharmacologic interactions were defined by a matrix of combinations among the most trypanocidal terpenes (limonene, carvone; citral and caryophyllene oxide). The treated cell phenotype was assessed by fluorescent and optic microscopy, flow cytometry, and DNA electrophoresis assays. RESULTS: The L. alba EOs displayed significant differences in their chemical composition and trypanocidal performance (p = 0.0001). Citral chemotype oils were more trypanocidal than Carvone EOs, with Inhibitory Concentration 50 (IC50) of 14 ± 1.5 µg/mL, 22 ± 1.4 µg/mL and 74 ± 4.4 µg/mL, on Epi, Tryp and Amas, respectively. Limonene exhibited synergistic interaction with citral, caryophyllene oxide and Benznidazole (decreasing by 17 times its IC50) and was the most effective and selective treatment. The cellular analysis suggested that these oils or their bioactive terpenes (citral, caryophyllene oxide and limonene) could be inducing T. cruzi cell death by an apoptotic-like mechanism. CONCLUSIONS: EOs extracted from L. alba Citral chemotype demonstrated significant trypanocidal activity on the three forms of T. cruzi studied, and their composition and trypanocidal performance were influenced by production parameters. Citral, caryophyllene oxide, and limonene showed a possible induction of an apoptotic-like phenotype. The best selective anti-T. cruzi activity was achieved by limonene, the effects of which were also synergic with citral, caryophyllene oxide and benznidazole.

Nueva reacción en cadena de la polimerasa múltiple para el diagnóstico específico de especies implicadas en la candidiasis humana / A new multiplex PCR for species-specific diagnosis of human candidiasis

RESUMEN Introducción. Las candidiasis son un grupo de infecciones oportunistas causadas por levaduras del género Candida. Candida albicans es la especie de mayor prevalencia en las infecciones superficiales y profundas. Sin embargo, en la última década, la frecuencia de especies diferentes a C. albicans ha aumentado y, por ende, su relevancia clínica, lo cual exige la utilización de técnicas diagnósticas que permitan su detección y el tratamiento adecuado de los pacientes afectados. Objetivo. Diseñar y optimizar una técnica de reacción en cadena de la polimerasa múltiple (PCR múltiple) considerando parámetros termodinámicos para la detección simultánea de cinco especies de Candida relevantes en la etiología de la candidiasis humana. Materiales y métodos. Para el diseño de los cebadores se consideraron restricciones físicas y termodinámicas que afectan la PCR múltiple, usando el programa Gene Runner y la herramienta Mult-PSOS. Como plantillas se utilizaron la región transcrita interna 2 (ITS2) (AJ249486.1) para C. albicans y la topoisomerasa II (TOPII) para C. parasilopsis (AB049144.1), C. krusei (AB049139.1), C. tropicalis (AB049141.1) y C. guillermondii (AB049145.1), y como moldes, extractos de ADN total obtenidos de cepas ATCC y de aislamientos clínicos de las especies de Candida. Resultados. Se diseñaron diez cebadores para la amplificación simultánea de las especies de Candida. Se obtuvo el siguiente patrón de bandas: C. albicans (206 pb), C. guillermondii (244 pb), C. tropicalis (474 pb), C. parasilopsis (558 pb) y C. krusei (419 pb). Conclusión. El ensayo diseñado de PCR múltiple permitió la amplificación simultánea y eficiente de todos los amplicones correspondientes a las especies estudiadas de Candida, así como su adecuada resolución en gel de agarosa al 1,3 %.

ABSTRACT Introduction: Candidiases is a group of opportunistic infections caused by yeasts belonging to the genus Candida. Candida albicans is the most prevalent species in both superficial and deep infections, however, the clinical importance of non-albicans Candida has increased during the last decade, driving an urgent need for diagnostic tests that allow for species-level resolution and selection of the optimum therapeutic approach. Objective: To design and to optimize a new multiplex PCR assay for the simultaneous identification of the five most relevant species of Candida involved in human candidiasis etiology. Materials and methods: For primers design, the physical and thermodynamic restrictions that affect multiplex PCR performance were analyzed using Gene Runner and Mult-PSOS. As templates, the internal transcribed region 2 (ITR2) was selected for C. albicans (AJ249486.1), and topoisomerase II (TOPII) for C. parasilopsis (AB049144.1), C. krusei (AB049139.1), C. tropicalis (AB049141.1), and C. guillermondii (AB049145.1). We used ATCC strains of all these five species and clinical isolates as templates. Results: We designed ten oligonucleotides for the simultaneous amplification of the Candida species. The electrophoresis band profile was: C. albicans (206 bp), C. guillermondii (244 bp), C. tropicalis (474 bp), C. parasilopsis (558 bp), and C. krusei (419 bp). Conclusion: The new multiplex PCR assay designed in this study allowed a simultaneous and efficient amplification of the amplicons corresponding to the five species of Candida under study, with an adequate resolution in standard agarose gel.

[new multiplex PCR for species-specific diagnosis of human candidiasis].

INTRODUCTION: Candidiases is a group of opportunistic infections caused by yeasts belonging to the genus Candida. Candida albicans is the most prevalent species in both superficial and deep infections, however, the clinical importance of non-albicans Candida has increased during the last decade, driving an urgent need for diagnostic tests that allow for species-level resolution and selection of the optimum therapeutic approach. OBJECTIVE: To design and to optimize a new multiplex PCR assay for the simultaneous identification of the five most relevant species of Candida involved in human candidiasis etiology. MATERIALS AND METHODS: For primers design, the physical and thermodynamic restrictions that affect multiplex PCR performance were analyzed using Gene Runner and Mult-PSOS. As templates, the internal transcribed region 2 (ITR2) was selected for C. albicans (AJ249486.1), and topoisomerase II (TOPII) for C. parasilopsis (AB049144.1), C. krusei (AB049139.1), C. tropicalis (AB049141.1), and C. guillermondii (AB049145.1). We used ATCC strains of all these five species and clinical isolates as templates. RESULTS: We designed ten oligonucleotides for the simultaneous amplification of the Candida species. The electrophoresis band profile was: C. albicans (206 bp), C. guillermondii (244 bp), C. tropicalis (474 bp), C. parasilopsis (558 bp), and C. krusei (419 bp). CONCLUSION: The new multiplex PCR assay designed in this study allowed a simultaneous and efficient amplification of the amplicons corresponding to the five species of Candida under study, with an adequate resolution in standard agarose gel.

A New Multiplex-PCR for Urinary Tract Pathogen Detection Using Primer Design Based on an Evolutionary Computation Method.

This work describes a new strategy for optimal design of Multiplex-PCR primer sequences. The process is based on the Particle Swarm Optimization-Simplex algorithm (Mult-PSOS). Diverging from previous solutions centered on heuristic tools, the Mult-PSOS is selfconfigured because it does not require the definition of the algorithm's initial search parameters. The successful performance of this method was validated in vitro using Multiplex- PCR assays. For this validation, seven gene sequences of the most prevalent bacteria implicated in urinary tract infections were taken as DNA targets. The in vitro tests confirmed the good performance of the Mult-PSOS, with respect to infectious disease diagnosis, in the rapid and efficient selection of the optimal oligonucleotide sequences for Multiplex-PCRs. The predicted sequences allowed the adequate amplification of all amplicons in a single step (with the correct amount of DNA template and primers), reducing significantly the need for trial and error experiments. In addition, owing to its independence from the initial selection of the heuristic constants, the Mult-PSOS can be employed by non-expert users in computational techniques or in primer design problems.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference.

The kinetoplast genetic code deviates from the universal code in that 90% of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.

Effects of Trypanosoma brucei tryptophanyl-tRNA synthetases silencing by RNA interference

The kinetoplast genetic code deviates from the universal code in that 90 percent of mitochondrial tryptophans are specified by UGA instead of UGG codons. A single nucleus-encoded tRNA Trp(CCA) is used by both nuclear and mitochondria genes, since all kinetoplast tRNAs are imported into the mitochondria from the cytoplasm. To allow decoding of the mitochondrial UGA codons as tryptophan, the tRNA Trp(CCA) anticodon is changed to UCA by an editing event. Two tryptophanyl tRNA synthetases (TrpRSs) have been identified in Trypanosoma brucei: TbTrpRS1 and TbTrpRS2 which localize to the cytoplasm and mitochondria respectively. We used inducible RNA interference (RNAi) to assess the role of TbTrpRSs. Our data validates previous observations of TrpRS as potential drug design targets and investigates the RNAi effect on the mitochondria of the parasite.