The gene expression of Leishmania infantum chagasi inside Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil

Leishmania infantum chagasi is the causative agent and Lutzomyia longipalpis is the main vector of visceral leishmaniasis in the Americas. We investigated the expression of Leishmania genes within L. longipalpis after artificial infection. mRNAs from genes involved in sugar and amino acid metabolism were upregulated at times of high parasite proliferation inside the insect. mRNAs from genes involved in metacyclogenesis had higher expression in late stages of infection. Other modulated genes of interest were involved in immunomodulation, purine salvage pathway and protein recycling. These data reveal aspects of the adaptation of the parasite to the microenvironment of the vector gut and reflect the preparation for infection in the vertebrate.

Comparación de la Muestra Salival y de Nasofaringe en la Detección de SARS-CoV-2 mediante RT-PCR / Comparison Between Saliva and Nasopharyngeal Sample in Detection of SARS-CoV-2 by RT-PCR

RESUMEN: El coronavirus tipo 2, SARS-CoV-2, que causa la enfermedad denominada por la OMS como COVID-19, se ha expandido provocando una pandemia desde 2019, sin cura hasta la fecha. El mecanismo de transmisión del SARS-CoV-2 entre humanos es mediante las secreciones generadas durante la respiración y estornudos, presentándose con un período de incubación desde 1 a 14 días. Se describen fiebre, tos y astenia como los síntomas más habituales. El diagnóstico definitivo se logra a través de la correlación entre la presentación clínica y exámenes complementarios, pero en la actualidad, el método de muestreo de preferencia para el diagnóstico de SARS-CoV-2 es mediante una muestra de nasofaringe, en donde se analiza la presencia de material genético viral por medio de RT-PCR. Debido a las complicaciones en la obtención de la muestra, tanto para el personal sanitario como para el paciente, se ha implementado la muestra de saliva con finalidad diagnóstica, como un método que proporciona una detección rápida, simple y no invasiva de la infección viral. Esta alternativa diagnóstica podría entregar información respecto a la patogenia de la enfermedad, permitiendo el manejo y control de pacientes positivos. El siguiente artículo, tiene por objetivo realizar una comparación entre las tomas de muestra de saliva y de nasofaringe para el diagnóstico de SARS-CoV-2, mediante la prueba de reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR).

SUMMARY: The type 2 coronavirus, SARS-CoV-2, named by the WHO like COVID-19, has expanded causing a pandemic since 2019, with no cure to date. The mechanism of transmission of SARS-CoV-2 between humans is through secretions generated during breathing and sneezing, presenting with an incubation period range from 1 - 14 days. Fever, cough, and fatigue are described as the most common symptoms. The definitive diagnosis is achieved through the correlation between the clinical presentation and the complementary exams, but at present, the preferred sampling method for the diagnosis of SARS-CoV-2 is through a nasopharyngeal swab specimen, where it is analyzed the presence of viral genetic material by the RT-PCR. Due to the complications in obtaining the sample, both for health personnel and for the patient, the saliva sample has been implemented, as a method that provides rapid, simple and non-invasive detection of viral infection. This diagnostic alternative could provide information on the pathogenesis of the disease, the management and control of positive patients. The following article aims to make a comparison between the saliva and nasopharyngeal samples taken for the diagnosis of SARS-CoV-2, using the reverse transcription polymerase chain reaction test (RT-PCR).

Saliva as a promising biofluid for SARS-CoV-2 detection during the early stages of infection / La saliva como biofluido promisorio para la detección del SARS-CoV-2 durante las primeras etapas de la infección

Abstract Background: Diagnostic testing for coronavirus disease (COVID)-19 is performed using nasopharyngeal swabs. This type of sampling is uncomfortable for the patient, dangerous for health workers, and its high demand has led to a global shortage of swabs. One of the alternative specimens is saliva. However, the optimal conditions for the test have not been established. Methods: Reverse transcription-polymerase chain reaction was used to detect the viral genome in saliva samples kept at room temperature, in the fridge or frozen for 2 days. In addition, the influence of brushing teeth and feeding on the detection of the virus in saliva was addressed. Finally, the efficiency of saliva in revealing the presence of the virus during the hospitalization period was determined in children. Results: The viral genome was consistently detected regardless of the storage conditions of saliva samples. Brushing teeth and feeding did not influence the sensitivity of the test. In hospitalized children, positive results were obtained only during the early days. Conclusions: These results support the idea of the use of saliva as an alternative specimen for diagnostic testing for COVID-19. The viral genome is stable and endures perturbations in the oral cavity. However, clearance of the virus from the mouth during the infection may limit the use of the test only to the early stages of the disease.

Resumen Introducción: El diagnóstico de COVID-19 (enfermedad por coronavirus 2019) se realiza con un hisopado nasofaríngeo. El procedimiento de toma de muestra es molesto para el paciente y peligroso para el personal de salud, y la alta demanda de análisis ha conducido a la escasez de hisopos. Una alternativa es el uso de saliva, pero las condiciones óptimas para realizar el estudio no han sido establecidas. Métodos: Se usó la reacción en cadena de la polimerasa con transcriptasa reversa para detectar el genoma viral en muestras de saliva mantenidas a temperatura ambiente, en refrigeración o congeladas. Además, se evaluó la influencia del aseo bucal y de la ingesta de alimento en la detección del virus. Finalmente, se determinó el desempeño de la saliva para reportar la presencia del virus durante el periodo de hospitalización en niños. Resultados: El genoma viral fue estable durante 2 días a las diferentes temperaturas ensayadas. El aseo bucal y la ingesta de alimento no influyeron en la detección del virus. En los niños hospitalizados solo se obtuvieron resultados positivos durante los primeros días. Conclusiones: Los resultados coinciden con la idea del uso de la saliva como biofluido alternativo para el diagnóstico de COVID-19. El genoma viral es estable y no se ve afectado por perturbaciones en la cavidad oral; sin embargo, la dinámica de la infección puede provocar que el ensayo solo sea útil durante las primeras etapas de la enfermedad.

Evaluación diagnóstica preoperatoria para COVID-19 / Preoperative diagnostic evaluation for COVID-19

CONTEXTO CLÍNICO: La enfermedad por el Coronavirus 2019 (COVID­19, por su sigla en inglés Coronavirus Disease 2019) es una enfermedad respiratoria de humanos producida por un nuevo coronavirus identificado con la sigla SARS-CoV-2. El 11 de marzo de 2020 la Organización Mundial de la Salud (OMS) declaro la COVID-19 como uma pandemia. Desde ese momento hasta el 30 agosto 2020 su circulación se ha reportado en más de 200 países reportándose más de 25.000.000 casos activos. La tasa de letalidad del COVID-19 a la fecha, sobre los casos cerrados es del 9% (846.000 muertes). El período de incubación de la infección por 2019­nCoV es de 2 a 14 días. La mayor parte de los contagios se producen persona a persona, siendo altamente transmisible. La clínica varía desde casos asintomáticos a cuadros febriles con tos y dificultad respiratoria, neumonía y distrés respiratorio. Debido a la falta de tratamiento o vacunas para la epidemia por COVID-19 se han establecido distintas medidas para evitar la propagación del virus y el auto cuidado de la gente como el distanciamento social, el lavado de manos o el uso de tapa bocas a nivel social. TECNOLOGÍA: La prueba Polimerasa Transcriptasa Inversa (RT- PCR) utiliza una enzima llamada RT que es capaz de convertir el ARN en ADN por retro transcripción dado que el virus SARS-CoV-2 no tiene ADN en su interior sino ARN. Se realiza en laboratorios con equipos especializados en tres tipos de muestras: el esputo, que es una secreción procedente de la nariz, la garganta o los bronquios; de un lavado bronco alveolar o aspirado traqueal (cuando sea posible) y/o hisopado de garganta o nariz. El sistema de PCR a tiempo real permite cuantificar la muestra, es decir, saber cuántas copias del virus hay por mililitro. OBJETIVO: El objetivo del presente informe es evaluar la evidencia disponible acerca de la utilidad de la evaluación diagnóstica preoperatoria para COVID-19. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas, en buscadores genéricos de internet, y financiadores de salud. Se priorizó la inclusión de revisiones sistemáticas (RS), ensayos clínicos controlados aleatorizados (ECAs), evaluaciones de tecnologías sanitarias (ETS), evaluaciones económicas y guías de práctica clínica (GPC) y recomendaciones de diferentes sistemas de salud. RESULTADOS: Se incluyeron una ETS y 14 GPC o recomendaciones, acerca del uso del cribado preoperatorio del SARS­CoV­ 2. CONCLUSIONES: No se encontró evidencia de la eficacia ni la seguridad de la implementación de la evaluación diagnostica preoperatoria para la detección del SARS-CoV-2 / COVID-19. Las recomendaciones sobre la evaluación prequirúrgica con PCR en tiempo real para pacientes asintomáticos son heterogéneas y dependen de los niveles de casos a nivel nacional, políticas en cada institución para la creación de protocolos específicos para la evaluación de pacientes quirúrgicos consensuados com las normativas sobre COVID-19. La Sociedad de Argentina de Cirugía sugiere que ante la necesidad de una cirugía urgente testear a todos los pacientes que cumplan los criterios de caso sospechosos o contacto estrecho de COVID-19. En el caso de que un paciente con diagnóstico de COVID-19 requiera de una cirugía de urgencia, usar los protocolos de cada institución sobre sobre el manejo de casos COVID-19 en área quirúrgica médica y si es posible, se recomienda disponer de un quirófano específico sólo para pacientes COVID-19 positivos. También recomiendan que ante una curva en aumento de casos positivos suspender toda consulta y cirugía programada. Las asociaciones de cirugía de España, Australia, las asociaciones de cirujanos, anestesistas y financiadores privados de salud estadounidenses recomiendan el testeo prequirúrgico para COVID-19 en todo paciente que requiera cirugía electiva. En el caso de cirugías de urgencia evaluar la posibilidad de reprogramar la cirugía hasta tener el resultado del hisopado para COVID-19.

Dengue diagnostics: serious inaccuracies are likely to occur if pre-analytical conditions are not strictly followed

BACKGROUND The heat-labile nature of Dengue virus (DENV) in serum samples must be considered when applying routine diagnostic tests to avoid issues that could impact the accuracy of test results with direct implications for case management and disease reporting. OBJECTIVES To check if pre-analytical variables, such as storage time and temperature, have an impact on the accuracy of the main routine diagnostic tests for dengue. METHODS Virus isolation, reverse transcription real-time polymerase chain reaction (RT-PCR) and NS1 enzyme-linked immunosorbent assay (ELISA) were evaluated using 84 samples submitted to different pre-analytical conditions. FINDINGS Sensitivity and negative predictive value were directly affected by sample storage conditions. RT-PCR and virus isolation showed greater dependence on well-conserved samples for an accurate diagnosis. Interestingly, even storage at -30ºC for a relatively short time (15 days) was not adequate for accurate results using virus isolation and RT-PCR tests. On the other hand, NS1 ELISA showed no significant reduction in positivity for aliquots tested under the same conditions as in the previous tests. MAIN CONCLUSIONS Our results support the stability of the NS1 marker in ELISA diagnosis and indicate that the accuracy of routine tests such as virus isolation and RT-PCR is significantly affected by inadequate transport and storage conditions of serum samples.

RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens / 生物医学与环境科学（英文）

Objective@#To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.@*Methods@#A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID @*Results@#The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID @*Conclusion@#This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Comparison of rRNA-based reverse transcription PCR and rDNA-based PCR for the detection of streptococci in root canal infections

Abstract Objective The rDNA-based method is unable to distinguish between alive and dead cells. Alternatively, bacterial viability can be assessed by molecular methods based on ribosomal RNA (rRNA). Therefore, this study aimed to detect viable streptococci in root canal samples using rRNA-based reverse transcription polymerase chain reaction (RT-PCR), compared to an rDNA-based PCR assay. Methodology Microbiological root canal samples were obtained from 32 teeth with primary endodontic infections before (S1) and after chemomechanical preparation (S2), and after removal of intracanal medication (S3). RNA and DNA were extracted, and complementary DNA (cDNA) was synthesized from RNA using RT reaction. cDNA and genomic DNA were subjected to PCR with primers complementary to the 16S rRNA sequences of Streptococcus spp. McNemar's test was used to compare the detection rate of both assays (P<0.05). Results Streptococci were detected in 28.12% (9/32) and 37.5% (12/32) of S1 samples using rRNA- and rDNA-based PCR assays, respectively. In contrast, they were detected in only 6.25% (2/32) of S2 samples using rRNA-based RT-PCR, compared to 15.62% (5/32) using rDNA-based PCR. Finally, in S3 samples, streptococci were not detected by rRNA, whereas rDNA-based PCR still detected the bacteria in 12.5% (4/32) of the samples. The total number of PCR-positive reactions in the rDNA-based PCR was higher than in the rRNA-based assay (P<0.05). Conclusions The rRNA-based RT-PCR showed a lower detection rate of streptococci when compared to the rDNA-based PCR, suggesting that the latter may have detected dead cells of streptococci in root canal samples.

Disponibilidad de técnicas moleculares para niños con tumores de difícil caracterización. Un problema en la Oncopatología pediátrica. Aplicación y difusión de técnicas de diagnóstico complementarias en el ámbito público / Availability of molecular techniques for children with difficult-to-characterize tumors. A problem in pediatric oncopathology. Application and diffusion of complementary diagnostic techniques in the public setting

La revolución de la biología molecular y el desarrollo de la investigación biomédica básica para el diagnóstico y posterior manejo del cáncer infantil han llevado a la necesidad de organización de grupos interdisciplinarios de profesionales, los cuales se encargan de afrontar los nuevos desafíos diagnósticos y terapéuticos. Los sarcomas indiferenciados pediátricos constituyen un grupo heterogéneo de neoplasias malignas de aspecto primitivo y polifenotípico. La categorización de gran parte de este tipo de tumores es posible gracias a la aplicación de técnicas moleculares complementarias al estudio histopatológico. El objetivo del presente estudio fue recategorizar sarcomas indiferenciados mediante la implementación de una nueva metodología diagnóstica. Se efectuaron técnicas de inmunohistoquimica (IHQ), FISH de interfase y RT-PCR a partir de tejido fijado en formol e incluido en parafina en 144 casos de sarcomas indiferenciados. Se logró la recategorización del 95.1% de los casos, arribando a 24 diagnósticos diferentes. Sólo un 4.9% permanece aún como sarcoma indiferenciado o inclasificable. Los resultados alcanzados por este estudio demuestran la importancia de contar con nuevas herramientas diagnósticas a nivel molecular y recursos humanos especializados que posibiliten su correcta implementación para el diagnóstico de neoplasias de difícil caracterización (AU)

The revolution of molecular biology and the development of basic medical research for the diagnosis and subsequent management of childhood cancer have led to a need to organize interdisciplinary groups of professionals in charge of facing new diagnostic and treatment challenges. Childhood undifferentiated sarcomas are a heterogeneous group of malignant neoplasms that are primitive in appearance and have polyphenotypic features. Categorization of a large part of this type of tumor has become possible with molecular techniques as a complement to histopathological studies. The aim of this study was to categorize undifferentiated sarcomas using new diagnostic tools. Immunohistochemistry (IHC), interfase FISH, and RT-PCR techniques were used on formalin-fixed and paraffin-embedded tissues of 144 cases of undifferentiated sarcomas. Overall, 95.1% of the cases could be recategorized resulting in 24 different diagnoses. In only 4.9% the diagnosis of undifferentiated or unclassifiable sarcoma was maintained. These results emphasize the importance of the availability of new diagnostic tools at the molecular level and specialized human resources enabling adequate implementation for the diagnosis of difficult-to-characterize neoplasms (AU)

Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum

ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

Detection of Circulating Tumor Cells in Breast Cancer Patients Using Cytokeratin-19 Real-Time RT-PCR

PURPOSE: The roles of circulating tumor cells (CTCs) as predictive and prognostic factors, as well as key mediators in the metastatic cascade, have been investigated. This study aimed to validate a method to quantify CTCs in peripheral blood using a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for cytokeratin (CK)-19 and to evaluate the utility of this assay in detecting CTCs in breast cancer patients. MATERIALS AND METHODS: Real-time monitoring PCR of fluorescently labeled specific hybridization probes for CK-19 mRNA was established. Peripheral blood samples from 30 healthy donors, 69 patients with early breast cancer, 47 patients with locally advanced breast cancer, and 126 patients with metastatic breast cancer were prospectively obtained and analyzed for CTC detection. RESULTS: CK-19 mRNA was not detectable in healthy subjects using the real-time RT-PCR method. The detection rates of CK-19 mRNA in breast cancer patients were 47.8% for early breast cancer (33/69), 46.8% for locally advanced breast cancer (22/47), and 61.1% for metastatic breast cancer (77/129). The detection rate of CK-19-positive CTCs in metastatic disease was slightly higher than early or locally advanced breast cancer; however, the detection rate according to disease burden was not statistically different (p=0.097). The detection rate was higher in patients with pleural metastasis (p=0.045). CTC detection was associated with poor survival (p=0.014). CONCLUSION: A highly specific and sensitive CK-19 mRNA-based method to detect CTCs in peripheral blood in breast cancer patients can be used in further prospective studies to evaluate the predictive and prognostic importance of CTCs.

Descrição dos primeiros casos de febre pelo vírus Zika investigados em municípios da região Nordeste do Brasil, 2015 / Descripción de los primeros casos de fiebre por el virus Zika investigados en los municipios de la región noreste de Brasil, 2015 / Description of the first cases of Zika virus fever investigated in municipalities of the Brazilian Northeastern Region, 2015

OBJETIVO: Confirmar a circulação do vírus Zika e descartar outros agentes etiológicos em surto ocorrido no Rio Grande do Norte (RN), Maranhão (MA) e Paraíba (PB), em maio/2015. MÉTODOS: estudo descritivo de série de casos com residentes em Natal-RN, Barra do Corda-MA, São Luís-MA e João Pessoa-PB, 20 em cada estado, com exantema e ausência de febre ou febre baixa e um dos seguintes sinais/sintomas, hiperemia conjuntival, artralgia ou edema de membros; realizou-se RT-PCR/isolamento para Zika, enterovírus e vírus respiratórios, e sorologias (dengue, rubéola e parvovírus B19). RESULTADOS: os principais sintomas foram exantema (n=60), prurido (n=54) e artralgia (n=47); 51 indivíduos não apresentaram febre; identificou-se vírus Zika em 18 casos (12 na PB, quatro no MA e dois no RN) e anticorpos para dengue em 14. CONCLUSÃO: os sintomas foram compatíveis com febre pelo vírus Zika; houve confirmação laboratorial de Zika e dengue.

OBJETIVOS: confirmar la circulación del virus Zika y descartar otros agentes etiológicos en el brote ocurrido en Rio Grande do Norte (RN), Maranhão (MA) y Paraíba (PB), en mayo/2015. MÉTODOS: estudio descriptivo de serie de casos con residentes de Natal-RN, Barra do Corda-MA, São Luís-MA y João Pessoa-PB, 20 en cada estado, con exantema y ausencia de fiebre o fiebre baja y uno de los siguientes signos/síntomas, hiperemia conjuntival, artralgia o edema de miembros; se realizaron RT-PCR/aislamiento para Zika, enterovirus y virus respiratorios y serologías (dengue, rubéola y parvovirus B19). RESULTADOS: los principales síntomas fueron exantema (n=60), prurito (n=54) y artralgia (n=47); 51 individuos no presentaron fiebre, se identificó virus Zika en 18 casos (12 en PB, cuatro en MA y dos en RN) y anticuerpos para dengue en 14. CONCLUSIÓN: Los síntomas fueron compatibles con fiebre por el virus Zika; hubo confirmación por laboratorio de Zika y dengue.

OBJECTIVE: to confirm Zika virus circulation and discard other etiological agents in an outbreak occurred in the states of Rio Grande do Norte, Maranhão and Paraíba, in May, 2015. METHODS: this is a case series descriptive study with residents in Natal-RN, Barra do Corda-MA, São Luis-MA and João Pessoa-PB, with 20 cases in each state, presenting rash, absent or mild fever and one of the following signs/symptoms: conjunctival hyperemia, arthralgia or limb edema; RT-PCR/isolation tests for Zika, enterovirus and respiratory viruses, and serology tests (dengue, rubella and parvovirus B19) were performed. RESULTS: the main symptoms were rash (n=60), pruritus (n=54), and arthralgia (n=47); 51 individuals did not present fever; Zika virus was identified in 18 cases (12 in Paraíba, four in Maranhão and two in Rio Grande do Norte), and antibodies to dengue, in 14 cases. CONCLUSION: the symptoms were consistent with Zika virus fever; there was laboratory confirmation for Zika and dengue.

Red blood cells in cerebrospinal fluid as possible inhibitory factor for enterovirus RT-PCR / Presença de hemácias no líquido cefalorraquidiano como possível fator inibitório da RT-PCR para detecção de enterovirus

ABSTRACT The presence of hemoglobin in samples are considered an important inhibitory factor for polymerase chain reaction (PCR). The aim of this study was to examine the influence of red blood cells (RBC)s in cerebrospinal fluid (CSF) as an inhibitory factor to reverse transcription polymerase chain reaction (RT-PCR) for enteroviruses (EV). Forty-four CSF samples from patients showing characteristics of viral meningitis were assessed for EV by RT-PCR. Viral RNA extracted with guanidine isothyocianate buffer and virus detection was performed by in-house nested PCR. Positivity for EV RT-PCR was higher in CSF samples without RBCs than in samples with RBCs: 13(26%) and 36(9.2%), p = 0.001. In the group with positive EV RT-PCR, the mean + SD CSF RBC was 37 ± 183 cell/mm3; the group with negative results had 580 + 2,890 cell/mm3 (p = 0.007). The acceptable upper limit for CSF RBCs that could not influence RT-PCR was 108 cells/mm3. CSF samples with negative results for EV RT-PCR have more erythrocytes.

RESUMO A presença de hemoglobina em amostras de fluidos corporais é considerada um fator inibitório importante da reação em cadeia da polimerase (PCR). O objetivo deste estudo era examinar a influencia de hemácias no líquido cefalorraquidiano (LCR) como um fator inibitório da RT-PCR para enterovirus (EV). Quatrocentos e quarenta amostras de LCR de pacientes com características de meningite viral foram avaliados para enterovirus por RT-PCR. RNA viral foi extraído com tampão de isotiocianato de guanidina e a detecção viral foi feita com nested PCR in-house. A positividade do EV RT-PCR no LCR foi maior nas amostras de LCR sem hemácias do que as amostras com hemácias: 13 (26%) e 36 (9,2%), respectivamente (p = 0,001). No grupo com resultados EV RT-PCR positivo, a media ± DP do número de hemácias no LCR foi 37 ± 183 cell/mm3 e no grupo com resultados negativos foi 580 ± 2.890 cell/mm3 (p = 0,007). O limite superior aceitável de hemácias no LCR para não inibir o resultado do PCR foi 108 cells/mm3. As amostras de LCR com resultados negativos para RT-PCR EV tem mais eritrócitos em comparação com amostras com resultados positivos.

Monitoramento de vírus respiratórios na região metropolitana de Belo Horizonte, 2011 a 2013 / Monitoreo de vírus respiratórios en la región metropolitana de Belo Horizonte, 2011-2013 / Monitoring respiratory virus infection in the metropolitan area of Belo Horizonte, Brazil, 2011-2013

OBJETIVO: analisar a circulação dos vírus respiratórios em residentes na região metropolitana de Belo Horizonte, Brasil, hospitalizados em Belo Horizonte, de 2011 a 2013. MÉTODOS: estudo descritivo de 5.158 indivíduos com síndrome respiratória aguda grave; foram comparadas as características dos casos confirmados com casos descartados ou sem coleta de swab. RESULTADOS: metade dos vírus isolados foi da influenza A, especialmente os subtipos A(H1N1)pdm09 em pessoas de 20-59 anos e A(H3N2) naquelas com 60 anos ou mais; crianças menores de cinco anos tiveram identificado, com maior frequência, o vírus sincicial respiratório (65,6%), seguido pelo vírus da influenza A (21,2%); o vírus da influenza circulou em todas as estações do ano, e seus períodos de maior incidência intercalaram-se com os de maior atividade do vírus sincicial respiratório. CONCLUSÃO: o monitoramento dos vírus respiratórios contribui para o conhecimento dos períodos de circulação viral e a adoção de medidas de controle específicas.

OBJETIVOS: analizar la circulación de virus respiratorios en residentes de la región metropolitana de Belo Horizonte, Brasil, hospitalizados entre 2011 y 2013. MÉTODOS: estudio descriptivo de 5.158 individuos con infección respiratoria aguda grave; fueron comparadas las características de los casos confirmados con los descartados o sin colecta de swab. RESULTADOS: la mitad de los virus aislados fueron influenza A, especialmente subtipos A(H1N1)pdm09 en personas entre 20-59 años, y A(H3N2) en personas de 60 años o más; los niños menores de cinco años presentaron, con mayor frecuencia, el virus sincicial respiratorio (65,6%), seguido por influenza tipo A (21,2%); el virus de la Influenza circuló en todas las estaciones y los periodos de mayor incidencia se intercalaron con los de mayor actividad del virus sincicial. CONCLUSIÓN: el monitoriamente del virus respiratorio contribuyo para el conocimiento de los periodos de circulación viral y la adopción de medidas de control específicas.

OBJECTIVE: to analyze the circulation of respiratory viruses in people living in the metropolitan area of Belo Horizonte, Brazil, and hospitalized in Belo Horizonte from 2011 to 2013. METHODS: this is a descriptive study of 5,158 patients with Severe Acute Respiratory Syndrome; a comparison was made between the characteristics of confirmed cases and those of discarded cases or cases without swab samples. RESULTS: Influenza A virus accounted for half the isolated viruses, especially subtype A(H1N1)pdm09 among patients aged 20-59 years old, and subtype A(H3N2) in those aged 60 or over; the most frequently identified respiratory virus among children under five years old was respiratory syncytial virus (65.6%), followed by influenza A virus (21.2%); influenza virus circulated in all seasons of the year and its periods of greatest incidence were interspersed with those of higher Respiratory Syncytial Virus activity. CONCLUSION: monitoring respiratory viruses contributes to knowledge about periods of virus circulation and the adoption of specific control measures.

Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

Isolation and gene expression analysis of Phospholipase C in response to abiotic stresses from Vigna radiata (L.) Wilczek.

Phosphatidylinositol (PtdIns) is a major phospholipid in eukaryotic cells. Many studies have revealed that the phosphoinositide (PI) signaling pathway plays an important role in plant growth and development. Phospholipase C (PLC) is reported to have a crucial role in the PI pathway. This work focuses on the isolation and investigation of PLC in response to abiotic stress factors in green gram. The PLC cDNA, designated VrPLC, encoding a protein of 591 amino acids was cloned and expressed in E. coli.The predicted isoelectric point (pI) and molecular weight were 5.96 and 67.3 kDa, respectively. The tertiary structure of the PLC was also predicted and found to be mainly composed of random coils. In addition, VrPLC expression analysis was performed under environmental stress and the results showed that the expression of VrPLC was rapidly induced in an abscisic acid independent manner in response to drought and salt stress. PLC expression was found to be up-regulated by SA and down-regulated by wound in leaf tissues; however, there was no significant difference in the expression of PLC in plants subjected to high temperature and H2O2. Our results suggest that a close link/relationship between PLC expression and stress responses in green gram.

ß-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria / ß-defensina 2 no leite materno mostra uma ampla atividade antimicrobiana contra bactérias patogênicas

OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA). Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n = 61) than in mature milk samples (n = 39), as follows: median and range, 8.52 (2.6-16.3) µg/ml versus 0.97 (0.22-3.78), p < 0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs) of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL) was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients. .

OBJETIVO: Descrever a atividade antimicrobiana da defensina-beta 2 na glândula mamária e secretada no leite materno humano. MÉTODOS: A produção de peptídeos foi realizada por clonagem de DNA. Os níveis de defensina-beta 2 foram quantificados em 61 amostras de colostro e 39 de leite maduro de doadoras saudáveis pelo teste ELISA indireto. Por um ensaio de halo de inibição, avaliamos a atividade contra sete isolados clínicos diarreicos de crianças entre 0 e 2 anos. A atividade da defensina 2 contra três patógenos oportunistas que podem causar infecções nosocomiais foi determinada pelo teste de microdiluição. RESULTADOS: Os níveis de peptídeos estavam significativamente maiores nas amostras de colostro (n = 61) que de leite maduro (n = 39), como segue: 8,52 (2,6-16,3 µg/mL) mediana e faixa em comparação a 0,97 (0,22-3,78), p < 0,0001; teste de Mann-Whitney. O peptídeo recombinante foi obtido da alta atividade antimicrobiana demonstrada contra uma ampla gama de bactérias patogênicas. Sua atividade antibacteriana foi demonstrada em um disco contendo entre 1-4 µg, que produziu zonas de inibição entre 18 e 30 mm contra três isolados de Salmonella spp. e quatro de E. coli. A defensina-beta 2 demonstrou concentrações inibitórias mínimas (CIMs) de 0,25 µg/mL e 0,5 µg/mL para S. marcescen and P. aeruginosa, ao passo que uma CIM maior (4 µg/mL) foi obtida contra um isolado de cepa multirresistente de A. baumannii. CONCLUSÕES: Até onde sabemos, este estudo é o primeiro a relatar níveis de defensina em mulheres da América Latina. A produção e a atividade da defensina 2 no leite materno comprovam sua importância como uma molécula de defesa para a saúde intestinal em pacientes pediátricos. .

Serum Dickkopf-1 as a Biomarker for the Diagnosis of Hepatocellular Carcinoma

PURPOSE: Dickkopf-1 (DKK-1) is a Wnt/beta-catenin signaling pathway inhibitor. We investigated whether DKK-1 is related to progression in hepatocellular carcinoma (HCC) cells and HCC patients. MATERIALS AND METHODS: In vitro reverse-transcription polymerase chain reaction (RT-PCR), wound healing assays, invasion assays, and ELISAs of patient serum samples were employed. The diagnostic accuracy of the serum DKK-1 ELISA was assessed using receiver operating characteristic (ROC) curves and area under ROC (AUC) analyses. RESULTS: RT-PCR showed high DKK-1 expression in Hep3B and low in 293 cells. Similarly, the secreted DKK-1 concentration in the culture media was high in Hep3B and low in 293 cells. Wound healing and invasion assays using 293, Huh7, and Hep3B cells showed that DKK-1 overexpression promoted cell migration and invasion, whereas DKK-1 knock-down inhibited them. When serum DKK-1 levels were assessed in 370 participants (217 with HCC and 153 without), it was significantly higher in HCC patients than in control groups (median 1.48 ng/mL vs. 0.90 ng/mL, p0.05). When three biomarkers were combined (DKK-1 plus AFP plus DCP), they showed significantly higher AUC (AUC=0.952) than single marker, DKK-1 plus AFP, or DKK-1 plus DCP (all p<0.001). CONCLUSION: DKK-1 might be a key regulator in HCC progression and a potential therapeutic target in HCC. Serum DKK-1 could complement the diagnostic accuracy of AFP and DCP.

A Melting Method for RNA Extraction from the Mucosal Membrane of the Mouse Middle Ear

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.

A Melting Method for RNA Extraction from the Mucosal Membrane of the Mouse Middle Ear

PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.

Analysis on the arcelin expression in bruchid pest resistant wild pulses using real time RT-qPCR.

Arcelin, the antimetabolic protein from wild pulses is a known natural insecticidal molecule. Wild pulses with high arcelin content could serve as potential source to increase the levels of insect resistance in cultivated pulse crops. In this study, arcelin (Arl) gene expression was screened in seven stored product insect pest resistant wild pulse varieties using real time RT-qPCR. Arcelin gene specific real time PCR primers were synthesized from arcelin mRNA sequence of the wild pulse variety, Lablab purpureus. The results revealed different levels of arcelin gene expression in the tested varieties. Canavalia virosa registered significantly high content indicating its suitability for utilization of arcelin gene in developing stored product insect pest resistance with other cultivated pulses.