Synthesis and Structure-Activity Relationship Studies of C(13)-Desmethylene-(-)-Zampanolide Analogs.

We describe the synthesis and biochemical and cellular profiling of five partially reduced or demethylated analogs of the marine macrolide (-)-zampanolide (ZMP). These analogs were derived from 13-desmethylene-(-)-zampanolide (DM-ZMP), which is an equally potent cancer cell growth inhibitor as ZMP. Key steps in the synthesis of all compounds were the formation of the dioxabicyclo[15.3.1]heneicosane core by an intramolecular HWE reaction (67-95 % yield) and a stereoselective aza-aldol reaction with an (S)-BINOL-derived sorbamide transfer complex, to establish the C(20) stereocenter (24-71 % yield). As the sole exception, for the 5-desmethyl macrocycle, ring-closure relied on macrolactonization; however, elaboration of the macrocyclization product into the corresponding zampanolide analog was unsuccessful. All modifications led to reduced cellular activity and lowered microtubule-binding affinity compared to DM-ZMP, albeit to a different extent. For compounds incorporating the reactive enone moiety of ZMP, IC50 values for cancer cell growth inhibition varied between 5 and 133 nM, compared to 1-12 nM for DM-ZMP. Reduction of the enone double bond led to a several hundred-fold loss in growth inhibition. The cellular potency of 2,3-dihydro-13-desmethylene zampanolide, as the most potent analog identified, remained within a ninefold range of that of DM-ZMP.

First record of Cotylurus metacercariae (Trematoda:Strigeidae) in Biomphalaria straminea (Planorbidae) from Argentina: morphological and molecular identification.

In the study of the biology of trematode species, the knowledge of the larval stages in snail hosts is important to elucidate their complete life cycle. The goal of the present study was to describe a new tetracotyle-type metacercaria found in the freshwater mollusk Biomphalaria straminea sampled in a rice field from Corrientes province, Argentina. To this end, 1,768 snails were collected from the cultivated plots and irrigated channels during the flooding periods (from the time of sowing to soon after rice harvesting) between December 2016 and May 2017. We used morphological and molecular analysis to characterize the tetracotyle-type metacercariae. Its morphological traits and the internal transcribed spacers (ITS1 and ITS2 plus 5.8S; ~1200 pb) from nuclear ribosomal DNA (rDNA) were amplified and sequenced. From 1,768 specimens of B. straminea screened, 52 were found infected with metacercariae of tetracotyle type (2.9%) that were identified as Cotylurus genus. A total of 218 metacercariae were found encysted in the ovotestis or between the mantle and viscera of B. straminea. Bioinformatic analysis showed that the metacercarial rDNA sequences shared 94% identity with those of Cotylurus gallinulae from Mexico and 100% identity with those of Cotylurus sp. from Brazil. In this study, the morphological descriptions are supplemented with the first molecular identification of a metacercaria related to Cotylurus parasitizing planorbids from Argentina. Also, our study provides a new morphological description in B. straminea, thus broadening the geographical distribution. The life cycle of this Cotylurus metacercariae is unknown and there are no reports of adult stages parasitizing waterfowl in Argentina.

The life cycle of Magnivitellinum saltaensis n. sp. (Digenea: Alloglossidiidae) in Salta Province, Argentina.

We describe the alloglossiid trematode Magnivitellinum saltaensis n. sp., a parasite of the characiform fish Psalidodon endy, and its life cycle from Salta, northwest of Argentina. This is the first life cycle described for a species belonging to the genus Magnivitellinum. Cercariae emerged naturally from Biomphalaria tenagophila snails and infected experimentally exposed larvae of Diptera and Ephemeroptera as second intermediate hosts. These larvae in turn were exposed to commercially raised fish, and adults were recovered from characiform albino fish Gymnocorymbus ternetzi. Molecular analysis of natural and experimental adults showed the same genetic sequence for the partial region of 28S rDNA, thus confirming conspecificity. Comparison of these sequences with those published for M. simplex from Mexico showed 1.45% divergence, indicating that the specimens found in Salta belong to a different species, the third described of Magnivitellinum, in agreement with morphological data, geographical location, and host species composition. The new species is distinguished by its small body, vitelline follicles extending from the mid-level of the ventral sucker, Y-shaped excretory vesicle, and presence of papillae around the mouth.

Benznidazole/Poloxamer 407 Solid Dispersion as a New Strategy to Improve the Treatment of Experimental Trypanosoma cruzi Infection.

Benznidazole and nifurtimox are the only drugs specifically approved for the treatment of Chagas disease. Both compounds are given orally in tablets, but occasionally are ineffective and cause adverse effects. Benznidazole, the first-line treatment in many countries, is a compound with low solubility in water that is administered at high doses for long periods of time. To improve its solubility, we developed a new liquid formulation on the basis of solid dispersions (SD) using the amphiphilic polymer poloxamer 407. Herein we present data on its trypanocidal performance in mouse models of acute and chronic Trypanosoma cruzi infection. SD at doses of 60 or 15 mg/kg per day given with different administration schedules were compared with the commercial formulation (CF; 50 mg/kg per day) and vehicle. The SD performance was assessed by direct parasitemia, total anti-T. cruzi antibodies, and parasitic burden in tissues after 4 or 6 mo posttreatment. The efficacy of the SD was equivalent to the CF but without manifest side effects and hepatotoxicity. Considering our previous data on solubility, together with these on efficacy, this new liquid formulation represents a promising alternative for the treatment of Chagas disease, particularly in cases when dosing poses a challenge, as in infants.

Antigen-Based Nano-Immunotherapy Controls Parasite Persistence, Inflammatory and Oxidative Stress, and Cardiac Fibrosis, the Hallmarks of Chronic Chagas Cardiomyopathy, in A Mouse Model of Trypanosoma cruzi Infection.

Chagas cardiomyopathy is caused by Trypanosoma cruzi (Tc). We identified two candidate antigens (TcG2 and TcG4) that elicit antibodies and T cell responses in naturally infected diverse hosts. In this study, we cloned TcG2 and TcG4 in a nanovector and evaluated whether nano-immunotherapy (referred as nano2/4) offers resistance to chronic Chagas disease. For this, C57BL/6 mice were infected with Tc and given nano2/4 at 21 and 42 days post-infection (pi). Non-infected, infected, and infected mice treated with pcDNA3.1 expression plasmid encoding TcG2/TcG4 (referred as p2/4) were used as controls. All mice responded to Tc infection with expansion and functional activation of splenic lymphocytes. Flow cytometry showed that frequency of splenic, poly-functional CD4+ and CD8+ T cells expressing interferon-γ, perforin, and granzyme B were increased by immunotherapy (Tc.nano2/4 > Tc.p2/4) and associated with 88%-99.7% decline in cardiac and skeletal (SK) tissue levels of parasite burden (Tc.nano2/4 > Tc.p2/4) in Chagas mice. Subsequently, Tc.nano2/4 mice exhibited a significant decline in peripheral and tissues levels of oxidative stress (e.g., 4-hydroxynonenal, protein carbonyls) and inflammatory infiltrate that otherwise were pronounced in Chagas mice. Further, nano2/4 therapy was effective in controlling the tissue infiltration of pro-fibrotic macrophages and established a balanced environment controlling the expression of collagens, metalloproteinases, and other markers of cardiomyopathy and improving the expression of Myh7 (encodes ß myosin heavy chain) and Gsk3b (encodes glycogen synthase kinase 3) required for maintaining cardiac contractility in Chagas heart. We conclude that nano2/4 enhances the systemic T cell immunity that improves the host's ability to control chronic parasite persistence and Chagas cardiomyopathy.

Preparation and Characterization of Poloxamer 407 Solid Dispersions as an Alternative Strategy to Improve Benznidazole Bioperformance.

Benznidazole (BZL), the first line drug for Chagas disease treatment, presents a low solubility, limiting the possibilities for its formulation. In this work, solid dispersions' (SDs) technology was exploited to increase BZL kinetic solubility and dissolution rate, seeking for an improvement in its bioperformance. A physical mixture (PM) and an SD using Poloxamer 407 as carrier were prepared and characterized. Dissolution tests were performed, and data were analyzed with the lumped model, which allowed to calculate different parameters of pharmaceutical relevance. A bioactivity assay was also carried out to probe the SD anti-trypanocidal activity. Among the most relevant results, the initial dissolution rate of the BZL SD was near 3, 4 and about 400-fold faster than the PM, a commercial formulation (CF) and an extracted BZL, respectivley. The times needed for an 80% of drug dissolution were 3.6 (SD), 46.4 (PM), and 238.7 min (CF); while the dissolution efficiency values at 30 min were 85.2 (SD), 71.2 (PM), and 65.0% (CF). Survival curves suggested that using Poloxamer 407 as carrier did not alter the anti-trypanocidal activity of BZL. These results allow to conclude that SDs can be an effective platform for immediate release of BZL in an oral administration.

Current drug therapy and pharmaceutical challenges for Chagas disease.

One of the most significant health problems in the American continent in terms of human health, and socioeconomic impact is Chagas disease, caused by the protozoan parasite Trypanosoma cruzi. Infection was originally transmitted by reduviid insects, congenitally from mother to fetus, and by oral ingestion in sylvatic/rural environments, but blood transfusions, organ transplants, laboratory accidents, and sharing of contaminated syringes also contribute to modern day transmission. Likewise, Chagas disease used to be endemic from Northern Mexico to Argentina, but migrations have earned it global. The parasite has a complex life cycle, infecting different species, and invading a variety of cells - including muscle and nerve cells of the heart and gastrointestinal tract - in the mammalian host. Human infection outcome is a potentially fatal cardiomyopathy, and gastrointestinal tract lesions. In absence of a vaccine, vector control and treatment of patients are the only tools to control the disease. Unfortunately, the only drugs now available for Chagas' disease, Nifurtimox and Benznidazole, are relatively toxic for adult patients, and require prolonged administration. Benznidazole is the first choice for Chagas disease treatment due to its lower side effects than Nifurtimox. However, different strategies are being sought to overcome Benznidazole's toxicity including shorter or intermittent administration schedules-either alone or in combination with other drugs. In addition, a long list of compounds has shown trypanocidal activity, ranging from natural products to specially designed molecules, re-purposing drugs commercialized to treat other maladies, and homeopathy. In the present review, we will briefly summarize the upturns of current treatment of Chagas disease, discuss the increment on research and scientific publications about this topic, and give an overview of the state-of-the-art research aiming to produce an alternative medication to treat T. cruzi infection.

Morphological and ITS2 Molecular Characterization of Ribeiroia Cercariae (Digenea: Psilostomidae) from Biomphalaria spp. (Gastropoda: Planorbidae) in Northern Argentina.

Species of Ribeiroia use planorbid snails as intermediate host. Since there is little information about these digenean parasites in South America, we aimed to assess whether Ribeiroia cercariae from 3 north Argentina locations belonged to the same species and differed from Ribeiroia cercariae described elsewhere. Specimens were obtained from Biomphalaria tenagophila and Biomphalaria orbignyi (Salta Province), and Biomphalaria occidentalis (Corrientes Province). Morphological traits of cercariae were analyzed, as well as their sequence of the ribosomal internal transcribed spacer 2 (ITS2). The ITS2 region consisted of 426 nucleotides identical in all samples, suggesting that all specimens belong to the same species in spite of their morphological differences and first intermediate host species. Comparison of the ITS2 region with GenBank database records showed that specimens from Argentina were different from Ribeiroia ondatrae (0.9% divergence), Ribeiroia marini (0.7% divergence), and Cercaria lileta (0.2% divergence). In summary, morphological, ecological, and ITS2 molecular data suggest that specimens from Argentina belong to a different species.

Detección de Trypanosoma cruzi en tejido y sangre murina por PCR convencional y en tiempo real / Detection of Trypanosoma cruzi in murine blood and tissue by conventional and real time PCR / Detecção de Trypanosoma cruzi em tecido e sangue murinos por PCR convencional e em tempo real

El objetivo del presente trabajo fue comparar la detección de ADN de Trypanosoma cruzi mediante PCR en tiempo real (qPCR) y PCR convencional en sangre periférica (n=25) y músculo esquelético (n=20) de ratones tratados con drogas tripanomicidas luego de 6 meses post-tratamiento. En las muestras de sangre se detectaron un total de 7 positivas por qPCR, mientras que por PCR convencional sólo se detectaron 2. En músculo esquelético, 15 muestras fueron positivas por qPCR y 3 por PCR convencional. Los resultados obtenidos demuestran que la fuerza de concordancia es débil entre las técnicas de PCR utilizadas para la detección de ADN de T. cruzi (k=0,37; 49% positivas por qPCR vs. 11% por PCR convencional, p=0,0001). En las muestras de sangre, los valores diagnósticos de qPCR con respecto a la PCR convencional fueron: 100% sensibilidad; 78% especificidad; 30% VPP; 100% VPN; 4,6 RVP; 0 RVN. Para las muestras de músculo esquelético se obtuvieron los siguientes valores diagnósticos de qPCR: 100% sensibilidad; 29% especificidad; 20% VPP; 100% VPN; 1,4 RVP; 0 RVN. Ambas técnicas fueron igualmente sensibles en el rango de mediana-alta concentración, pero qPCR fue más efectiva para detectar bajas cargas parasitarias, en particular en las muestras de tejido.

The aim of this work was to compare detection of Trypanosoma cruzi DNA by real time (qPCR) and conventional PCR in peripheral blood (n=25), and skeletal muscle (n=20) of mice treated with trypanocidal compounds after 6 months post-treatment. A total of 7 blood samples were positive by qPCR; whereas, by conventional PCR only 2 were detected. In skeletal muscle, 15 samples were regarded positive by qPCR and 3 by conventional PCR. These results showed a weak concordance strength among PCR techniques employed to detect T. cruzi DNA in the studied samples (k=0.37; 49% positives by qPCR vs. 11% by conventional PCR, p=0.0001). In blood samples, qPCR diagnostic values in comparison with conventional PCR were: 100% sensibility; 78% specificity; 30% PPV; 100% NPV; 4.6 PVR; 0 NVR. For skeletal muscle samples, qPCR diagnostic values were: 100% sensibility; 29% specificity; 20% PPV; 100% NPV; 1.4 PVR; 0 NVR. Both techniques were equally sensitive in the medium-high concentration range, but qPCR was more effective to detect low parasitic burden, particularly in skeletal muscle samples.

O objetivo deste estudo foi comparar a detecção de DNA de Trypanosoma cruzi por PCR em tempo real (qPCR) e PCR convencional no sangue periférico (N=25) e músculo esquelético (N= 20) de camundongos tratados com medicamentos tripanomicidas depois de 6 meses de pós-tratamento. Nas amostras de sangue foi detectado um total de sete positivas por qPCR; enquanto que apenas foram encontradas 2 por PCR convencional. No músculo esquelético, 15 amostras foram positivas por qPCR e 3 por PCR convencional. Os resultados mostram que a força de concordância é fraca entre as técnicas de PCR utilizadas para a detecção de DNA de T. cruzi (k=0,37, 49% positivas por qPCR vs. 11% para a PCR convencional, p=0,0001). Nas amostras de sangue, os valores diagnósticos de qPCR em relação a PCR convencional foram de 100% sensibilidade; 78% de especificidade; 30% de VPP; 100% VPN; 4,6 RVP; 0 RVN. Para as amostras de músculo esquelético, os seguintes valores diagnósticos de qPCR foram obtidos: 100% sensibilidade; 29% de especificidade; 20% de VPP; 100% VPN; 1,4 RVP; 0 RVN. Ambas as técnicas são igualmente sensíveis na faixa de concentração média-alta, mas qPCR foi mais eficaz na detecção de baixas cargas parasitárias, especialmente em amostras de tecido.

Detección de Trypanosoma cruzi en tejido y sangre murina por PCR convencional y en tiempo real / Detection of Trypanosoma cruzi in murine blood and tissue by conventional and real time PCR / DetecþÒo de Trypanosoma cruzi em tecido e sangue murinos por PCR convencional e em tempo real

El objetivo del presente trabajo fue comparar la detección de ADN de Trypanosoma cruzi mediante PCR en tiempo real (qPCR) y PCR convencional en sangre periférica (n=25) y músculo esquelético (n=20) de ratones tratados con drogas tripanomicidas luego de 6 meses post-tratamiento. En las muestras de sangre se detectaron un total de 7 positivas por qPCR, mientras que por PCR convencional sólo se detectaron 2. En músculo esquelético, 15 muestras fueron positivas por qPCR y 3 por PCR convencional. Los resultados obtenidos demuestran que la fuerza de concordancia es débil entre las técnicas de PCR utilizadas para la detección de ADN de T. cruzi (k=0,37; 49% positivas por qPCR vs. 11% por PCR convencional, p=0,0001). En las muestras de sangre, los valores diagnósticos de qPCR con respecto a la PCR convencional fueron: 100% sensibilidad; 78% especificidad; 30% VPP; 100% VPN; 4,6 RVP; 0 RVN. Para las muestras de músculo esquelético se obtuvieron los siguientes valores diagnósticos de qPCR: 100% sensibilidad; 29% especificidad; 20% VPP; 100% VPN; 1,4 RVP; 0 RVN. Ambas técnicas fueron igualmente sensibles en el rango de mediana-alta concentración, pero qPCR fue más efectiva para detectar bajas cargas parasitarias, en particular en las muestras de tejido.(AU)

The aim of this work was to compare detection of Trypanosoma cruzi DNA by real time (qPCR) and conventional PCR in peripheral blood (n=25), and skeletal muscle (n=20) of mice treated with trypanocidal compounds after 6 months post-treatment. A total of 7 blood samples were positive by qPCR; whereas, by conventional PCR only 2 were detected. In skeletal muscle, 15 samples were regarded positive by qPCR and 3 by conventional PCR. These results showed a weak concordance strength among PCR techniques employed to detect T. cruzi DNA in the studied samples (k=0.37; 49% positives by qPCR vs. 11% by conventional PCR, p=0.0001). In blood samples, qPCR diagnostic values in comparison with conventional PCR were: 100% sensibility; 78% specificity; 30% PPV; 100% NPV; 4.6 PVR; 0 NVR. For skeletal muscle samples, qPCR diagnostic values were: 100% sensibility; 29% specificity; 20% PPV; 100% NPV; 1.4 PVR; 0 NVR. Both techniques were equally sensitive in the medium-high concentration range, but qPCR was more effective to detect low parasitic burden, particularly in skeletal muscle samples.(AU)

O objetivo deste estudo foi comparar a detecþÒo de DNA de Trypanosoma cruzi por PCR em tempo real (qPCR) e PCR convencional no sangue periférico (N=25) e músculo esquelético (N= 20) de camundongos tratados com medicamentos tripanomicidas depois de 6 meses de pós-tratamento. Nas amostras de sangue foi detectado um total de sete positivas por qPCR; enquanto que apenas foram encontradas 2 por PCR convencional. No músculo esquelético, 15 amostras foram positivas por qPCR e 3 por PCR convencional. Os resultados mostram que a forþa de concordÔncia é fraca entre as técnicas de PCR utilizadas para a detecþÒo de DNA de T. cruzi (k=0,37, 49% positivas por qPCR vs. 11% para a PCR convencional, p=0,0001). Nas amostras de sangue, os valores diagnósticos de qPCR em relaþÒo a PCR convencional foram de 100% sensibilidade; 78% de especificidade; 30% de VPP; 100% VPN; 4,6 RVP; 0 RVN. Para as amostras de músculo esquelético, os seguintes valores diagnósticos de qPCR foram obtidos: 100% sensibilidade; 29% de especificidade; 20% de VPP; 100% VPN; 1,4 RVP; 0 RVN. Ambas as técnicas sÒo igualmente sensíveis na faixa de concentraþÒo média-alta, mas qPCR foi mais eficaz na detecþÒo de baixas cargas parasitárias, especialmente em amostras de tecido.(AU)

Hepatotoxicity in mice of a novel anti-parasite drug candidate hydroxymethylnitrofurazone: a comparison with Benznidazole.

BACKGROUND: Treatment of Chagas disease, caused by Trypanosoma cruzi, relies on nifurtimox and benznidazole (BZL), which present side effects in adult patients, and natural resistance in some parasite strains. Hydroxymethylnitrofurazone (NFOH) is a new drug candidate with demonstrated trypanocidal activity; however, its safety is not known. METHODS: HepG2 cells dose response to NFOH and BZL (5-100 µM) was assessed by measurement of ROS, DNA damage and survival. Swiss mice were treated with NFOH or BZL for short-term (ST, 21 d) or long-term (LT, 60 d) periods. Sera levels of cellular injury markers, liver inflammatory and oxidative stress, and fibrotic remodeling were monitored. RESULTS: HepG2 cells exhibited mild stress, evidenced by increased ROS and DNA damage, in response to NFOH, while BZL at 100 µM concentration induced >33% cell death in 24 h. In mice, NFOH ST treatment resulted in mild-to-no increase in the liver injury biomarkers (GOT, GPT), and liver levels of inflammatory (myeloperoxidase, TNF-α), oxidative (lipid peroxides) and nitrosative (3-nitrotyrosine) stress. These stress responses in NFOH LT treated mice were normalized to control levels. BZL-treated mice exhibited a >5-fold increase in GOT, GPT and TNF-α (LT) and a 20-40% increase in liver levels of MPO activity (ST and LT) in comparison with NFOH-treated mice. The liver inflammatory infiltrate was noted in the order of BZL>vehicle≥NFOH and BZL>NFOH≥vehicle, respectively, after ST and LT treatments. Liver fibrotic remodeling, identified after ST treatment, was in the order of BZL>vehicle>NFOH; lipid deposits, indicative of mitochondrial dysfunction and in the order of NFOH>vehicle>BZL were evidenced after LT treatment. CONCLUSIONS: NFOH induces mild ST hepatotoxicity that is normalized during LT treatment in mice. Our results suggest that additional studies to determine the efficacy and toxicity of NFOH are warranted.

Diagnóstico de infección por Trypanosoma cruzi en Centros de Atención Primaria de Salta, Argentina / Diagnosis of Trypanosoma cruzi infection in Primary Public Health Centers of Salta, Argentina / Diagnóstico de infecção por Trypanosoma cruzi nos Postos de Saúde de Salta, Argentina

La Enfermedad de Chagas causada por el parásito hemoflagelado Trypanosoma cruzi, constituye un grave problema de Salud Pública. En Argentina, se controla sistemáticamente la sangre a transfundir, la donación de órganos y se ha disminuido notablemente la transmisión vectorial. El objetivo de este proyecto fue implementar el uso del equipo para recolección de sangre capilar y conservación en glicerina (Serokit) en los servicios de enfermería de los Centros de Atención Primaria de la Salud en la ciudad de Salta a fin de conocer la seroprevalencia de infección por Trypanosoma cruzi en pacientes que concurren a los mismos. Para ello se realizó el par serológico HAI y ELISA en las muestras conservadas en Serokit y luego en muestras de sangre tomadas por punción venosa para confirmación. Durante dos años de trabajo se analizaron 1647 pacientes que concurrieron a 28 Centros de Salud, resultando 1,7% (29/1647) seropositivos. El Valor Predictivo Positivo fue 93,50% y el Valor Predictivo Negativo fue 99,8%. Todos los niños seropositivos fueron tratados con Benznidazol. Se concluye que el uso de Serokit para la toma y conservación de muestras para posterior diagnóstico de infección por Trypanosoma cruzi es recomendable en Centros de Atención Primaria de la Salud que no cuentan con laboratorio.

Chagas disease is caused by the hemoflagelate parasite Trypanosoma cruzi, and represents a main concern in public health. In Argentina, blood transfusion and organ donation are systematically controlled for T. cruzi infection, and vectorial transmission has dropped significantly. The aim of this project was to implement the use of the equipment for collecting capillary blood and conservation in glycerine (Serokit) in the nursery service of Primary Health Care Centers (PHCC) in the city of Salta, and to know the seroprevalence of T. cruzi infection in PHCC patients. To that aim, the serological pair HAI and ELISA was carried out in samples preserved in Serokit, followed by analysis of blood samples taken by venous punction to confirm the results. During a two-year period, 1647 patients that were assisted at 28 PCHC were analyzed, resulting in 1.7% (29/1647) seropositive samples. Positive Predictive Value was 93.5% and Negative Predictive Value was 99.8%. Every seropositive child was given Benznidazole treatment. It can be concluded that use of Serokit for taking and preserving samples to diagnose serologically T. cruzi infection is recommended in PHCC that lack their own biochemistry laboratory.

A doença de Chagas produzida pelo protozoário flagelado Trypanosoma cruzi constitui um grave problema para a Saúde Pública. Na Argentina, o controle sistemático do sangue para transfusão e o do órgão para transplantar, fez com que diminuísse notavelmente a transmissão vetorial. O objetivo deste projeto foi implementar o uso do equipamento para coleta de sangue capilar e conservação em glicerina (Serokit) nos serviços de enfermagem dos Postos de Atenção Primaria da Saúde da cidade de Salta a fim de conhecer a soroprevalência de infecção com Trypanosoma cruzi em pacientes que são atendidos nos mesmos. Foi realizado o par sorológico HAI e ELISA nas amostras conservadas em Serokit e depois nas amostras de sangue retiradas através de uma punção venosa para a confirmação. Durante dois anos de trabalho, foram analisados 1647 pacientes que foram atendidos nos 28 Postos de Saúde, resultando 1.7% (29/1647) soropositivos. O Valor Preditivo Positivo foi Tripae 93.50% e o Valor Preditivo Negativo foi de 99.8%. Todas as crianças soropositivas foram medicadas com Benznidazol. A conclusão é que o uso de Serokit para a tomada e conservação de amostras para posterior diagnóstico de infecção por Trypanosoma cruzi é recomendável nos Postos de Atenção Primária da Saúde onde não existe a disponibilidade de laboratórios.

Diagnóstico de infección por Trypanosoma cruzi en Centros de Atención Primaria de Salta, Argentina / Diagnosis of Trypanosoma cruzi infection in Primary Public Health Centers of Salta, Argentina / Diagnóstico de infecþÒo por Trypanosoma cruzi nos Postos de Saúde de Salta, Argentina

La Enfermedad de Chagas causada por el parásito hemoflagelado Trypanosoma cruzi, constituye un grave problema de Salud Pública. En Argentina, se controla sistemáticamente la sangre a transfundir, la donación de órganos y se ha disminuido notablemente la transmisión vectorial. El objetivo de este proyecto fue implementar el uso del equipo para recolección de sangre capilar y conservación en glicerina (Serokit) en los servicios de enfermería de los Centros de Atención Primaria de la Salud en la ciudad de Salta a fin de conocer la seroprevalencia de infección por Trypanosoma cruzi en pacientes que concurren a los mismos. Para ello se realizó el par serológico HAI y ELISA en las muestras conservadas en Serokit y luego en muestras de sangre tomadas por punción venosa para confirmación. Durante dos años de trabajo se analizaron 1647 pacientes que concurrieron a 28 Centros de Salud, resultando 1,7% (29/1647) seropositivos. El Valor Predictivo Positivo fue 93,50% y el Valor Predictivo Negativo fue 99,8%. Todos los niños seropositivos fueron tratados con Benznidazol. Se concluye que el uso de Serokit para la toma y conservación de muestras para posterior diagnóstico de infección por Trypanosoma cruzi es recomendable en Centros de Atención Primaria de la Salud que no cuentan con laboratorio.(AU)

Chagas disease is caused by the hemoflagelate parasite Trypanosoma cruzi, and represents a main concern in public health. In Argentina, blood transfusion and organ donation are systematically controlled for T. cruzi infection, and vectorial transmission has dropped significantly. The aim of this project was to implement the use of the equipment for collecting capillary blood and conservation in glycerine (Serokit) in the nursery service of Primary Health Care Centers (PHCC) in the city of Salta, and to know the seroprevalence of T. cruzi infection in PHCC patients. To that aim, the serological pair HAI and ELISA was carried out in samples preserved in Serokit, followed by analysis of blood samples taken by venous punction to confirm the results. During a two-year period, 1647 patients that were assisted at 28 PCHC were analyzed, resulting in 1.7% (29/1647) seropositive samples. Positive Predictive Value was 93.5% and Negative Predictive Value was 99.8%. Every seropositive child was given Benznidazole treatment. It can be concluded that use of Serokit for taking and preserving samples to diagnose serologically T. cruzi infection is recommended in PHCC that lack their own biochemistry laboratory.(AU)

A doenþa de Chagas produzida pelo protozoário flagelado Trypanosoma cruzi constitui um grave problema para a Saúde Pública. Na Argentina, o controle sistemático do sangue para transfusÒo e o do órgÒo para transplantar, fez com que diminuísse notavelmente a transmissÒo vetorial. O objetivo deste projeto foi implementar o uso do equipamento para coleta de sangue capilar e conservaþÒo em glicerina (Serokit) nos serviþos de enfermagem dos Postos de AtenþÒo Primaria da Saúde da cidade de Salta a fim de conhecer a soroprevalÛncia de infecþÒo com Trypanosoma cruzi em pacientes que sÒo atendidos nos mesmos. Foi realizado o par sorológico HAI e ELISA nas amostras conservadas em Serokit e depois nas amostras de sangue retiradas através de uma punþÒo venosa para a confirmaþÒo. Durante dois anos de trabalho, foram analisados 1647 pacientes que foram atendidos nos 28 Postos de Saúde, resultando 1.7% (29/1647) soropositivos. O Valor Preditivo Positivo foi Tripae 93.50% e o Valor Preditivo Negativo foi de 99.8%. Todas as crianþas soropositivas foram medicadas com Benznidazol. A conclusÒo é que o uso de Serokit para a tomada e conservaþÒo de amostras para posterior diagnóstico de infecþÒo por Trypanosoma cruzi é recomendável nos Postos de AtenþÒo Primária da Saúde onde nÒo existe a disponibilidade de laboratórios.(AU)

[Comparison of nucleic acid extraction efficiency using different commercial kits and qPCR. Effect of inhibitors]. / Comparación de la eficiencia de extracción de ácidos nucleicos utilizando distintos kits comerciales y empleando la qPCR. Efecto de las sustancias inhibidoras.

The detection of specific nucleic acid (NA) sequences by PCR has revolutionized the biological and medical sciences. Real-time PCR (qPCR) opened up the possibility of obtaining quantitative results. NA extraction is a decisive step prior to qPCR since it may produce either the removal or co-extraction of inhibitory substances of the enzymatic reaction, which in turn affects the amplification efficiency. In the present work we compared the commercial NA extraction kits from Qiagen, Invitrogen and Macherey-Nagel, which were used to extract DNA from mice blood artificially infected with Trypanosoma cruzi and PP7 RNA, Pseudomonas aeruginosa bacteriophage, in spiked aqueous matrices. NA recovery efficiency in samples without inhibitors was similar for the three extraction kits. However, the Invitrogen kit was the only one that remained unaffected in the presence of inhibitors in the samples.

The protein synthesis inhibitors mycalamides A and E have limited susceptibility toward the drug efflux network.

The mycalamides belong to a family of protein synthesis inhibitors noted for antifungal, antitumour, antiviral, immunosuppressive, and nematocidal activities. Here we report a systematic analysis of the role of drug efflux pumps in mycalamide resistance and the first isolation of mycalamide E. In human cell lines, neither P-glycoprotein overexpression nor the use of efflux pump inhibitors significantly modulated mycalamide A toxicity in the systems tested. In Saccharomyces cerevisiae, it appears that mycalamide A is subject to efflux by the principle mediator of xenobiotic efflux, Pdr5p along with the major facilitator superfamily pump Tpo1p. Mycalamide E showed a similar efflux profile. These results suggest that future drugs based on the mycalamides are likely to be valuable in situations where efflux pump-based resistance leads to failure of other chemotherapeutic approaches, although efflux may be a mediator of resistance in antifungal applications.

Hydroxymethylnitrofurazone is active in a murine model of Chagas' disease.

The addition of a hydroxymethyl group to the antimicrobial drug nitrofurazone generated hydroxymethylnitrofurazone (NFOH), which had reduced toxicity when its activity against Trypanosoma cruzi was tested in a murine model of Chagas' disease. Four groups of 12 Swiss female mice each received 150 mg of body weight/kg/day of NFOH, 150 mg/kg/day of nitrofurazone (parental compound), 60 mg/kg/day of benznidazole (BZL), or the solvent as a placebo. Treatments were administered orally once a day 6 days a week until the completion of 60 doses. NFOH was as effective as BZL in keeping direct parasitemia at undetectable levels, and PCR results were negative. No histopathological lesions were seen 180 days after completion of the treatments, a time when the levels of anti-T. cruzi antibodies were very low in mice treated with either NFOH or BZL. Nitrofurazone was highly toxic, which led to an overall rate of mortality of 75% and necessitated interruption of the treatment. In contrast, the group treated with its hydroxymethyl derivative, NFOH, displayed the lowest mortality (16%), followed by the BZL (33%) and placebo (66%) groups. The findings of histopathological studies were consistent with these results, with the placebo group showing the most severe parasite infiltrates in skeletal muscle and heart tissue and the NFOH group showing the lowest. The present evidence suggests that NFOH is a promising anti-T. cruzi agent.

In vivo studies of 5-arylethenylbenzofuroxans in acute murine models of Chagas' disease.

5-arylethenylbenzofuroxan derivatives with high in vitro anti-Trypanosoma cruzi activity were studied in vivo using acute murine models of Chagas' disease. The selected compounds, as pure isomeric forms, 1, 2, 3 and 4, or as equimolecular mixture of geometric isomers, 1:2, 3:4, 5:6 were studied against different T. cruzi strains. Consequently, Tulahuen 2 strain, Colombiana strain (resistant to Nifurtimox and Benznidazole), and two different wild strains, one isolated from the wild reservoir Didelphis marsupialis and another one from Uruguayan patients, were selected. No relevant signs of in vivo toxicity were observed with the benzofuroxans orally administered. Compound 1 and the mixture of isomers 1:2 were the best for treating infection against the four studied strains.

In vivo anti-Chagas vinylthio-, vinylsulfinyl-, and vinylsulfonylbenzofuroxan derivatives.

New benzofuroxans were developed and studied as antiproliferative Trypanosoma cruzi agents. Compounds displayed remarkable in vitro activities against different strains, Tulahuen 2, CL Brener and Y. Its unspecific cytotoxicity was evaluated using human macrophages being not toxic at a concentration at least 8 times, and until 250 times, that of its T. cruzi IC50. Some biochemical pathways were studied, namely parasite respiration, cysteinyl active site enzymes and reaction with glutathione, as target for the mechanism of action. Not only T. cruzi respiration but also Cruzipain or trypanothione reductase were not affected, however the most active derivatives, the vinylsulfinyl- and vinylsulfonyl-containing benzofuroxans, react with glutathione in a redox pathway. Furthermore, the compounds showed good in vivo activities when they were studied in an acute murine model of Chagas' disease. The compounds were able to reduce the parasite loads of animals with fully established T. cruzi infections.