Rendimiento del Kit de Chagas V2.0 IICS-UNA para el tamizaje de la enfermedad

En Paraguay la enfermedad de Chagas es endémica, siendo el número de personas infectadas de aproximadamente 165.000 y la población expuesta del 30% según registros del 2012. El objetivo del trabajo fue evaluar el ELISA Chagas test IICS V2.0 para tamizaje de la enfermedad en muestras de donantes de sangre. Se realizó un estudio transversal de pruebas diagnósticas, para lo que se incluyeron 775 muestras de suero provenientes de dos bancos de sangre, a partir de cuyos resultados se calculó la sensibilidad, valores predictivos positivo, negativo y la curva ROC. También se determinó la concordancia y correlación entre el ELISA Chagas test IICS V2.0 y un ELISA comercial. De las 775 muestras de bancos de sangre analizadas se obtuvo una sensibilidad del 99%, especificidad de 96%, VPP 96%, VPN 99% y un índice kappa igual a 0,95 (0,93-0,97) Error Estándar (EE) 0.01 y p>00001 y el área ROC igual a 0,9835. Con respecto a la concordancia con el test comercial, el índice kappa fue de 0,926 IC95% (0,888-0,976), p=0,00001 y el coeficiente de correlación r=0,971 IC95% (0,962-0.978) p=0,0001. Las concordancias obtenidas fueron muy buenas con respecto a la serología de las muestras de banco de sangre como la comparada con el test comercial, pudiendo utilizarse el kit de Chagas IICS V2 para el tamizaje de la enfermedad.

In Paraguay, Chagas disease is endemic, with approximately 165,000 infected people and 30% of the exposed population according to 2012 records. The objective of this study was to evaluate the ELISA Chagas test IICS V2.0 for screening of the disease in blood donor samples. We carried out a cross-sectional study of diagnostic tests, including 775 serum samples from two blood banks, and then calculating sensitivity, positive and negative values and the ROC curve. We also determined the concordance and correlation between the ELISA Chagas test IICS V2.0 and commercial ELISA. In the 775 blood bank samples analyzed, the Chagas ELISA test IICS V2.0 obtained a sensitivity of 99%, specificity of 96%, PPV 96%, NPV 99% and a kappa index equal to 0.95 (0.93-0.97) Standard Error (SE) 0.01 and p>0.0001 and the ROC area equal to 0.9835. Regarding the concordance with the commercial test, the kappa index was 0.926 CI95% (0.888-0.976), p=0.00001 and the correlation coefficient r=0.971 CI95%(0.962-0.978) p=0.0001.The concordances obtained were very good with respect to the serology of the blood bank samples as compared to the commercial test, allowing the use of the Chagas IICS V2 kit for the disease screening.

Phytase blends for enhanced phosphorous mobilization of deoiled seeds.

Phytases are hydrolytic enzymes capable of a stepwise phosphate release from phytate which is the main phosphorous storage in seeds, cereals and legumes. Limitations such as low enzyme activity or incomplete phytate hydrolysis to inositol are a great challenge in phytase applications in food and feed. Herein we report a phytase blend of two enzymes with additive effects on phytate (InsP6) hydrolysis and its application in the enzymatic phosphorous recovery process. Blending the fast 6-phytase rPhyXT52 with the 3-phytase from Debaryomyces castellii, which is capable of fully hydrolyzing InsP6, we achieved rapid phosphate release with higher yields compared to the individual enzymes and a rapid disappearance of InsP6-3 intermediates, monitored by HPLC. NMR data suggest a nearly complete phytate hydrolysis to inositol and phosphate. The blend was applied for phosphate mobilization from phytate-rich biomass, such as deoiled seeds. For this emerging application, an up to 43% increased phosphate mobilization yield was achieved when using 1000 U of the blend per kg biomass compared to using only the E. coli phytase. Even so, the time of enzyme treatment was decreased by more than half (6 h instead of 16 h) when using 4000 U of blend, we reached a 78-90% reduction of the total phosphorous content in the explored deoiled seeds. In summary, the phytase blend of Dc phyt/rPhyXT52 was proven very efficient to obtain inositol phosphate depleted meal which has its potential application in animal feeding and is concomitant with the production of green phosphate from renewable resources.

Generation of phytase chimeras with low sequence identities and improved thermal stability.

Being able to recombine more than two genes with four or more crossover points in a sequence independent manner is still a challenge in protein engineering and limits our capabilities in tailoring enzymes for industrial applications. By computational analysis employing multiple sequence alignments and homology modeling, five fragments of six phytase genes (sequence identities 31-64 %) were identified and efficiently recombined through phosphorothioate-based cloning using the PTRec method. By combinatorial recombination, functional phytase chimeras containing fragments of up to four phytases were obtained. Two variants (PTRec 74 and PTRec 77) with up to 32 % improved residual activity (90 °C, 60 min) and retained specific activities of > 1100 U/mg were identified. Both variants are composed of fragments from the phytases of Citrobacter braakii, Hafnia alvei and Yersinia mollaretii. They exhibit sequence identities of ≤ 80 % to their parental enzymes, highlighting the great potential of DNA recombination strategies to generate new enzymes with low sequences identities that offer opportunities for property right claims.

Implementation of a Multiplex rRT-PCR for Zika, Chikungunya, and Dengue Viruses: Improving Arboviral Detection in an Endemic Region.

Arboviral diagnosis has been complicated throughout the tropical and subtropical Americas by the recent co-circulation of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV). The aim of this study was to implement a multiplex real-time RT-PCR (rRT-PCR) for ZIKV, CHIKV, and DENV in Paraguay to test patients who were clinically suspected of having dengue. We tested 110 sera from patients who presented to the Hospital de Clínicas in 2016 and had testing for DENV nonstructural protein 1 (NS1; 40 positive and 70 negative). Using a composite reference standard, we confirmed 51 dengue cases (46.4%): 38/40 NS1 positive and 13/70 NS1 negative. Chikungunya virus and ZIKV were detected in one sample each, both were DENV NS1 negative. The NS1 test demonstrated good agreement with rRT-PCR for DENV. However, multiplex rRT-PCR identified a subset of dengue cases and additional arboviral infections that would not be detected if NS1 assays are relied upon for diagnosis.

Engineered phytases for emerging biotechnological applications beyond animal feeding.

Phytases are important industrial enzymes able to catalyze the release of up to six phosphates from phytate in a stepwise hydrolysis reaction. Phytases are almost exclusively used as a feed supplement. However, phytases are also used in human nutrition, food processing, non-food industrial products, and emerging applications like enzymatic phosphate recovery from renewable resources. Phytate, the main phosphorus storage form in seeds, and its hydrolysis products act as a chelator and reduce protein and mineral bioavailability in intestinal absorption. Full phosphate hydrolysis from the common storage compound phytate remains a challenge. Phytate hydrolysis patterns of tailored phytases and their protein engineering campaigns are discussed. The aim of our review is to give an overview on developed and emerging application areas (animal nutrition, food processing, and environmental resource management) and thereby generate an awareness for the importance of phosphorus stewardship in a circular bioeconomy. Emphasis will be given to processes using organic-bound phosphorus and related recycling strategy of this valuable resource. In detail, the main challenge in designing phytases to completely hydrolyze phosphate from phytate to inositol and the need for engineering campaigns to broaden their industrial use are described.

KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose.

Sulfated polysaccharides such as cellulose can mimic the functionalities of pathophysiologically important glycosaminoglycans. Enzymatic sulfation offers a green chemistry route to selective (mono)sulfation of oligosaccharides (e.g., cellobiose as a building block of cellulose) in aqueous solution, at ambient temperature, and high chemoselectivity. Here, we report the first KnowVolution campaign for the aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense to advance ASTB toward a synthetically attractive biocatalyst. The generated final recombination variant (ASTB-M5) carries two amino acid substitutions (Leu446Pro and Val579Lys) leading to an up to 7.6-fold increase in specific activity (6.15 U mg-1 ) that was obtained with one round of KnowVolution. Mass spectrometry analysis confirmed a monosulfated product of cellobiose and structure elucidation by NMR confirmed the sulfation at the positions C-3 or C-4 of GlcNAc-linker-tBoc as opposed to the preferred C-6 by chemical means. Computational analysis suggested an important role of Leu446Pro in substrate-binding and recognized Val579Lys as a distal substitution.

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High-Molecular-Weight Hyaluronic Acid.

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

Rational evolution of the unusual Y-type oxyanion hole of Rhodococcus sp. CR53 lipase LipR.

Rhodococcus sp CR-53 lipase LipR was the first characterized member of bacterial lipase family X. Interestingly, LipR displays some similarity with α/ß-hydrolases of the C. antartica lipase A (CAL-A)-like superfamily (abH38), bearing a Y-type oxyanion hole, never found before among bacterial lipases. In order to explore this unusual Y-type oxyanion hole, and to improve LipR performance, two modification strategies based on site directed or saturation mutagenesis were addressed. Initially, a small library of mutants was designed to convert LipR Y-type oxyanion hole (YDS) into one closer to those most frequently found in bacteria (GGG(X)). However, activity was completely lost in all mutants obtained, indicating that the Y-type oxyanion hole of LipR is required for activity. A second approach was addressed to modify the two main oxyanion hole residues Tyr110 and Asp111, previously described for CAL-A as the most relevant amino acids involved in stabilization of the enzyme-substrate complex. A saturation mutagenesis library was prepared for each residue (Tyr110 and Asp111), and activity of the resulting variants was assayed on different chain length substrates. No functional LipR variants could be obtained when Tyr110 was replaced by any other amino acids, indicating that this is a crucial residue for catalysis. However, among the Asp111 variants obtained, LipR D111G produced a functional enzyme. Interestingly, this LipR-YGS variant showed less activity than wild type LipR on short- or mid- chain substrates but displayed a 5.6-fold increased activity on long chain length substrates. Analysis of the 3D model and in silico docking studies of this enzyme variant suggest that substitution of Asp by Gly produces a wider entrance tunnel that would allow for a better and tight accommodation of larger substrates, thus justifying the experimental results obtained.

Seroprevalencia y factores de riesgo asociados a la toxoplasmosis en mujeres en edad reproductiva que acudieron al Hospital Distrital de Lambaré, Paraguay / Seroprevalence and associated risk factors of toxoplasmosis among women in reproductive age who attended district Hospital of Lambaré, Paraguay

La toxoplasmosis es una enfermedad zoonótica de prevalencia mundial, causada por un parásito intracelular Toxoplasma gondii que infecta a los seres vivos y tiene como hospedador definitivo a los felinos. El presente estudio tuvo como objetivo determinar la seroprevalencia de toxoplasmosis y factores de riesgo asociados en mujeres en edad reproductiva no embarazadas que asistieron al Hospital Distrital de Lambaré, Paraguay. Se obtuvieron muestras de suero de 185 mujeres y se analizaron mediante ELISA Chagas IICS-UNA para la detección de anticuerpos IgG específicos contra T. gondii. Se utilizaron encuestas diseñadas para recoger datos demográficos y factores de riesgo. Se aplicó estadística descriptiva y la prueba de chi-cuadrado y OR (odds ratio) para establecer asociación entre las variables higiénicas, alimenticias y de conocimiento con la toxoplasmosis. De las 185 participantes, 117 presentaron IgG anti-T gondii, que representa una prevalencia de 63% IC95 (56,2-69,7%). El nivel de conocimiento fue el único factor de riesgo que se asoció en forma significativa con la serología positiva para toxoplasmosis. Aunque los demás factores de riesgo no alcanzaron significancia estadística, probablemente debido a la alta seroprevalencia en esta población, sin lugar a duda los mismos contribuyen en gran medida a la propagación de la infección.

Toxoplasmosis is a zoonotic disease of worldwide prevalence, caused by an intracellular parasite Toxoplasma gondii that infects living beings and has as its final host the felines. The present study aimed to determine the seroprevalence of toxoplasmosis and associated risk factors in non-pregnant reproductive age women attending the Hospital District of Lambaré, Paraguay. Serum samples were obtained from 185 women and analyzed by ELISA for the detection of IgG antibodies specific for T. gondii. Surveys designed to collect demographic data and risk factors were used. Descriptive statistics were applied and chi2 and OR (odds ratio) were calculated for the analysis of hygienic, nutritional and knowledge variables. Of the 185 participants, 117 presented positive serology for anti-T. gondii IgG, yielding a prevalence of 63% IC95 (56,2-69,7). The level of knowledge was The only risk factor significantly related to to positive serology for toxoplasmosis. Although the other risk factors were not statistically significant, probably due to the high seroprevalence in this population, there is no doubt that they contributgreatly to the spread of the disease.

Caracterización del perfil antigénico de extractos proteicos solubles de cepas de Trypanosoma cruzi / Antigenic profile characterization of soluble protein extracts from two Trypanosoma cruzi strains

El parásito Trypanosoma cruzi es el agente causal de la enfermedad de Chagas. El mismo presenta una amplia diversidad biológica y genética, por lo cual se lo agrupa en 6 unidades taxonómicas discretas. Las cepas Y, CL Brener y una aislada en Paraguay aún no caracterizada en su totalidad fueron empleadas en este estudio con el fin de determinar sus perfiles proteico y antigénico para aportar en la investigación sobre esta enfermedad en nuestro país, aplicando técnicas como SDS-PAGE, ELISA y Western blot. Se observaron semejanzas en los perfiles proteicos de los extractos solubles de Y Lote 73 e Y Lote 74, siendo ambos obtenidos a partir de la misma cepa, así como perfil diferente de éstos con los de CL Brener y Py. Se detectaron bandas de proteínas de pesos moleculares comunes entre extractos, principalmente las consideradas de bajo peso molecular. Se constató la capacidad antigénica al ensayar los 4 extractos frente a sueros chagásicos, no chagásicos, controles positivos y negativos, se evidenció la importancia de confirmar el diagnóstico con pruebas de principios diferentes. Además, se detectaron proteínas antigénicas comunes a los 3 extractos empleados en el Western blot, las que podrían ser estudiadas con mayor profundidad debido a su potencial antigénico, característica de interés para fines diagnósticos.

The parasite Trypanosoma cruzi is the causal agent of Chagas disease.It presents a wide biological and genetic diversity, therefore it is grouped into 6 taxonomic units..The strains Y, CL Brener and one strain isolated in Paraguaywere used in this study in order to determine their protein and antigenic profiles, applying techniques such as SDS-PAGE, ELISA and Western blot. Similarities were observed in the protein prifles of the soluble extracts of Lot 73 Y and Lot 74 Y both obtained from the same strain, and the different profile of these with those of CL Brener and Py strains. Protein bands of common molecular weights, manly those consideredlow, were detected between extracts. By testing the four extracts against chagasic and non-chagasic sera, positive and negative controls, the antigenic capacity was verified and the importance of utilizing different test to confirm diagnosis was shown. In addition antigenic proteins common to the three extracts used in the Western blot were detected and can be further investigated due to their antigenic potential, an interesting characteristic for diagnostic purpose.

Alternative Oils Tested as Feedstocks for Enzymatic FAMEs Synthesis: Toward a More Sustainable Process.

Previously isolated and characterized Pseudomonas lipases were immobilized in a low-cost MP-1000 support by a re-loading procedure that allowed a high activity per weight of support. Immobilized LipA, LipC, and LipCmut lipases, and commercial Novozym® 435 were tested for fatty acid methyl ester (FAMEs) synthesis using conventional and alternative feedstocks. Triolein and degummed soybean oils were used as model substrates, whereas waste cooking oil and M. circinelloides oil were assayed as alternative, low cost feedstocks, whose free fatty acid (FFA), and acylglyceride profile was characterized. The reaction conditions for FAMEs synthesis were initially established using degummed soybean oil, setting up the best water and methanol concentrations for optimum conversion. These conditions were further applied to the alternative feedstocks and the four lipases. The results revealed that Pseudomonas lipases were unable to use the FFAs, displaying a moderate FAMEs synthesis, whereas a 44% FAMEs production was obtained when M. circinelloides oil was used as a substrate in the reaction catalysed by Novozym® 435, used under the conditions established for degummed soybean oil. However, when Novozym® 435 was tested under previously described optimal conditions for this lipase, promising values of 85 and 76% FAMEs synthesis were obtained for waste cooking oil and M. circinelloides oil, respectively, which might result in promising, nonfood, alternative feedstocks for enzymatic biodiesel production. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1209-1217, 2017.

Desempeño del kit ELISA chagas IICS V.1 para el diagnóstico de la enfermedad de chagas / ELISA chagas test IICS V.1 evaluation for the diagnosis of chagas disease

En Paraguay, el tamizaje serológico para la enfermedad de Chagas en bancos de sangre es necesario, por lo cual es importante un método diagnóstico con alta sensibilidad. El ELISA Chagas test IICS V.1 es un ELISA indirecto sensibilizados con antígeno soluble de epimastigote de T.cruzide la cepa Ypsilon. El objetivo del presente trabajo fue evaluar el desempeño del ELISA Chagas test IICS V.1 en comparación con kits comerciales, ademásanalizar los resultados de la evaluación externa e interna de calidad del kit. En este estudio observacional de prueba diagnóstica se analizaron 56 muestras de suerospositivos y negativos para antígenos de Trypanosoma cruzi, testados por el ELISA BiosChile, obteniéndose una concordancia excelente entre el ELISA Chagas test IICS V.1y los kits comerciales: Chagatest ELISA-Wiener, con Índice kappa: 0,89 IC de 95% (0,76-1) y Test ELISA para Chagas III-Grupo BiosChile con Índice kappa: 0,92 IC 95% (0,82-1).En la evaluación externa de calidad realizada por la Fundação Pró-Sangue/Hemocentro de São Paulo, Brasil en el periodo 2001 al 2012 se analizaron 450 muestras: 372 negativas y 78 positivas para T. cruzi, obteniéndose en dicha evaluación la calificación "A" que indica ausencia de falsos positivos y negativos. Además, en el mismo periodo los valores del control interno se encontraron dentro del rango permitido de ±2DS. Los resultados obtenidos en el estudio demuestran la alta calidad de este test de producción nacional, que sumado al bajo costo del mismo, pueden ser utilizados en trabajos de campo, donde no necesita de instrumentación y las lecturas pueden realizarse a simple vista, constituyendo una herramienta válida y útil para el apoyo al diagnóstico de la enfermedad de Chagas.

In Paraguay, the serological screening for Chagas disease is mandatory in pregnant women and blood banks, therefore a high sensitivity diagnostic method is required. The aimof this study was to evaluate the ELISA Chagas test IICS V.1 by comparison with commercial kits and to analyze the external and internal quality evaluation results. In this descriptive observational study, 56 seropositive and seronegative to Trypanosoma cruzisamples were analyzed, obtaining an excellent concordance between the ELISA Chagas test IICS V.1 and these commercial kits: Chagatest ELISA-Wiener, Argentina (kappa index:0.89) and Test ELISA Chagas III-Grupo Bios, Chile (kappa index: 0.92).In the externalquality assessment carried out by the Fundação Pró-Sangue /Blood Center of São Paulo,Brazil in the period 2001 to 2012, 450 samples were analyzed: 372 seronegative and 78seropositive for T. cruzi. In this evaluation, an “A” score was obtained indicating theabsence of false positives and negatives. Additionally, in the same period of time theinternal control values were within the accepted range of ± 2SD, with a confidence intervalof 95%. The results obtained in the present study demonstrate the high quality of this locally produced test which added to its low cost, making it a valid and useful tool tosupport the diagnosis of Chagas disease.

Perfil clínico, hematológico y serológico en pacientes con sospecha de dengue del IICS-UNA, 2009-2013 / Clinical, hematological and serological profile in suspected dengue patients at IICS-UNA, 2009-2013

El dengue constituye una de las enfermedades transmitidas por mosquitos más importante a nivel mundial. La enfermedad puede cursar con un cuadro asintomático, presentarse con un amplio rango de manifestaciones clínicas inespecíficas o cierto porcentaje puede derivar en casos graves. Este estudio observacional descriptivo de corte transverso tuvo como objetivo determinar las características clínicas, parámetros hematológicos y presencia de IgM en 92 pacientes que acudieron al IICS-UNA con sospecha clínica de dengue en el periodo 2009 al 2013. Se utilizó el MAC-ELISA desarrollado en el IICS-UNA, se registraron los datos clínicos-epidemiológicos a través de una encuesta y se determinaron los parámetros hematológicos. Se obtuvieron resultados positivos para IgM en 51/92 (55%) pacientes y resultados negativos en 41/92 (45%). Las características clínicas más frecuentes fueron: fiebre, cefalea, mialgias y artralgias. Entre los pacientes con IgM positiva, 14/51 (27%) manifestaron dolores abdominales, 19/51 (37%) reportaron letargo o postración y 20/51 (39%) declararon tener náuseas y/o vómitos, 4/51 (8%) presentaron leucopenia, 10/51 (20%) valores de hematocrito disminuido y 6/51 (12%) plaquetopenia. Sólo 13/92 (14%) pacientes declararon haber cursado con la enfermedad anteriormente. Del total de pacientes, 4/92 (4%) manifestaron haber presentado algún tipo de hemorragia. Los resultados obtenidos en el estudio refuerzan la importancia de integrar todos los parámetros posibles: detección de IgM, perfil hematológico y la clínica del paciente con sospecha de dengue para brindar un mejor diagnóstico. Así también, es necesario resaltar en cuanto a los signos de alarma para una intervención rápida a fin de evitar complicaciones.

Dengue is one of the most important mosquito-borne diseases worldwide. The disease may present as asymptomatic, with a wide range of non-specific clinical manifestations or acertain percentage can result in severe cases. This observational, descriptive, crosssectional study aimed to determine the clinical features, hematological parameters and the presence of IgM in 92 patients who attended to IICS-UNA with clinical suspicion of denguefrom 2009 to 2013. The MAC-ELISA developed at IICS-UNA was used, clinical and epidemiological data were recorded through a survey and hematological parameters were determined. Positive results for IgM in 51/92 (55%) patients were obtained and negative results in 41/92 (45%). The most frequent clinical features were fever, headache, muscleand joint pains. Among patients with positive IgM, 14/51 (27%) reported abdominal pain,19/51 (37%) reported lethargy or prostration and 20/51 (39%) reported having nausea and/or vomiting, 4/51(8%) had leucopenia, 10/51 (20%) decreased hematocrit values and6/51 (12%) presented thrombocytopenia. Only 13/92 (14%) patients reported a previous DENV infection. Of the total, 4/92 (4%) presented some type of bleeding. The results of thestudy reinforce the importance of integrating all possible parameters: IgM detection, hematological and clinical profile of patients with suspected dengue to provide better diagnosis. It is also necessary to emphasize warning signs for a quick intervention to avoid complications.

Aspectos clínico-epidemiológicos de la toxoplasmosis en pacientes que consultan por problemas de visión / Clinical and epidemiological aspects of toxoplasmosis in patients with vision problems

Introduction: Toxoplasmosis is a worldwide disease; it can cause decreased vision or even blindness. The route of transmission in humans may vary according to the habits of the region; probably the ingestion of raw or undercooked meat is the main source of infection. Objective: To determine the seroprevalence of toxoplasmosis in an eye clinic, the frequency of ocular toxoplasmosis (OT) and risk habits for acquiring the infection. Materials and Methods: Adult patients consulting in the Retina Department of the Teaching Hospital of the National University of Asuncion, Paraguay between August and September, 2014 were included. Prior informed consent, socio-demographic and epidemiological data related to T. gondii infection were obtained. In addition a blood sample for the determination of anti T. gondii IgG antibodies by the ELISA method was taken and ophthalmologic evaluation for the diagnosis of OT was made. Results: A total of 80 patients with mean ± SD age of 53 ± 20 years were studied, with slight predominance of women (55%). The seroprevalence of toxoplasmosis was 84% (67/80) and OT was detected in 8.9% of the 67 seropositive persons. The habit of not washing vegetables with sodium hypochlorite and eat meat from wild animals was related to higher risk of infection in this population. Conclusion: It is important to conduct research at the population level to establish the epidemiology of toxoplasmosis in our country. Information on prophylactic measures to prevent infection by T. gondii should be given to the population.

Introducción: La toxoplasmosis es una enfermedad de distribución mundial, que puede ocasionar disminución de la visión hasta ceguera. La vía de transmisión en el hombre puede variar de acuerdo a los hábitos de cada región, siendo probablemente la ingestión de carne cruda o mal cocida la principal vía de contagio. Objetivo: Determinar la seroprevalencia de toxoplasmosis en una clínica oftalmológica, la frecuencia de toxoplasmosis ocular (TO) y los hábitos de riesgo para adquirir la enfermedad. Pacientes y Métodos: Fueron incluidos 80 pacientes adultos que consultaron en el Departamento de Retina de la Cátedra de Oftalmología del Hospital de Clínicas entre agosto y septiembre de 2014. Previo consentimiento informado, se obtuvieron los datos socio-demográficos y epidemiológicos relacionados a la infección por Toxoplasma gondii. Además se tomó una muestra de sangre para la determinación de anticuerpos del tipo IgG anti T. gondii por el método de ELISA y se realizó la evaluación oftalmológica para el diagnóstico de TO. Resultados: La edad promedio ± DE fue de 53 ± 20 años, con leve predominio de mujeres (55%). La seroprevalencia de toxoplasmosis fue de 84% (67/80) y la TO se detectó en 8,9% de los 67 seropositivos. Se observó que el hábito de no lavar las verduras con hipoclorito de sodio y comer carne silvestre presentó mayor riesgo de contraer la infección en esta población. Conclusión: Es importante realizar trabajos de investigación a nivel poblacional para establecer la epidemiología de la toxoplasmosis en nuestro país. Se debe dar a conocer a la población las medidas de profilaxis para evitar la infección por T. gondii.

[Clinical and epidemiological aspects of toxoplasmosis in patients with vision problems]. / Aspectos clínico-epidemiológicos de la toxoplasmosis en pacientes que consultan por problemas de visión.

INTRODUCTION: Toxoplasmosis is a worldwide disease; it can cause decreased vision or even blindness. The route of transmission in humans may vary according to the habits of the region; probably the ingestion of raw or undercooked meat is the main source of infection. OBJECTIVE: To determine the seroprevalence of toxoplasmosis in an eye clinic, the frequency of ocular toxoplasmosis (OT) and risk habits for acquiring the infection. MATERIALS AND METHODS: Adult patients consulting in the Retina Department of the Teaching Hospital of the National University of Asuncion, Paraguay between August and September, 2014 were included. Prior informed consent, socio-demographic and epidemiological data related to T. gondii infection were obtained. In addition a blood sample for the determination of anti T. gondii IgG antibodies by the ELISA method was taken and ophthalmologic evaluation for the diagnosis of OT was made. RESULTS: A total of 80 patients with mean ± SD age of 53 ± 20 years were studied, with slight predominance of women (55%). The seroprevalence of toxoplasmosis was 84% (67/80) and OT was detected in 8.9% of the 67 seropositive persons. The habit of not washing vegetables with sodium hypochlorite and eat meat from wild animals was related to higher risk of infection in this population. CONCLUSION: It is important to conduct research at the population level to establish the epidemiology of toxoplasmosis in our country. Information on prophylactic measures to prevent infection by T. gondii should be given to the population.

Unusual carboxylesterase bearing a GGG(A)X-type oxyanion hole discovered in Paenibacillus barcinonensis BP-23.

Strain Paenibacillus barcinonensis BP-23, previously isolated from Ebro's river delta (Spain), bears a complex hydrolytic system showing the presence of at least two enzymes with activity on lipidic substrates. EstA, a cell-bound B-type carboxylesterase from the strain was previously isolated and characterized. The gene coding for a second putative lipase, located upstream cellulase Cel5A, was obtained using a genome walking strategy and cloned in Escherichia coli for further characterization. The recombinant clone obtained displayed high activity on medium/short-chain fatty acid-derivative substrates. The enzyme, named Est23, was purified and characterized, showing maximum activity on pNP-caprylate (C8:0) or MUF-heptanoate (C7:0) under conditions of moderate temperature and pH. Although Est23 displays a GGG(A)X-type oxyanion hole, described as an important motif for tertiary alcohol ester resolution, neither conversion nor enantiomeric resolution of tertiary alcohols could be detected. Amino acid sequence alignment of Est23 with those of known bacterial lipase families and with closely related proteins suggests that the cloned enzyme does not belong to any of the described bacterial lipase families. A phylogenetic tree including Est23 and similar amino acid sequences showed that the enzyme belongs to a differentiated sequence cluster which probably constitutes a new family of bacterial lipolytic enzymes.

Fast and economic immobilization methods described for non-commercial Pseudomonas lipases.

BACKGROUND: There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. RESULTS: To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite®545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI.3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I.3, requiring a refolding step, was poorly immobilized on all supports tested (best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. CONCLUSIONS: The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes.

Aeromonas hydrophila motY is essential for polar flagellum function, and requires coordinate expression of motX and Pom proteins.

By the analysis of the Aeromonas hydrophila ATCC7966(T) genome we identified A. hydrophila AH-3 MotY. A. hydrophila MotY, like MotX, is essential for the polar flagellum function energized by an electrochemical potential of Na(+) as coupling ion, but is not involved in lateral flagella function energized by the proton motive force. Thus, the A. hydrophila polar flagellum stator is a complex integrated by two essential proteins, MotX and MotY, which interact with one of two redundant pairs of proteins, PomAB and PomA(2)B(2). In an A. hydrophila motX mutant, polar flagellum motility is restored by motX complementation, but the ability of the A. hydrophila motY mutant to swim is not restored by introduction of the wild-type motY alone. However, its polar flagellum motility is restored when motX and -Y are expressed together from the same plasmid promoter. Finally, even though both the redundant A. hydrophila polar flagellum stators, PomAB and PomA(2)B(2), are energized by the Na(+) ion, they cannot be exchanged. Furthermore, Vibrio parahaemolyticus PomAB and Pseudomonas aeruginosa MotAB or MotCD are unable to restore swimming motility in A. hydrophila polar flagellum stator mutants.