Fundamentos y aplicaciones biomédicas de las principales tecnologías de secuenciación: una revisión de literatura / Biomedical Foundations and Applications of Major Sequencing Technologies: A Literature Review / Fundamentos e aplicações biomédicas das principais tecnologias de sequenciamento: uma re-visão da literatura

Introducción: el objetivo de la secuenciación es determinar la composición de los nucleótidos presentes en el ADN o el ARN. Desde la finalización del proyecto genoma humano, surgieron diversas tecnologías de secuenciación rápida como Roche 454, SOLiD, Illumina, Ion Torrent, PacBio y Oxford Nanopore, más precisas y costoeficientes, que permiten desarrollar proyectos a gran escala y estudiar genes y genomas, la composición de microbiomas, enfermedades metabólicas y enfermedades genéticas que afectan a la población. Objetivo: describir los fundamentos de los métodos de secuenciación de ADN y sus aplicaciones en las ciencias biomédicas. Métodos: revisión descriptiva de las principales estrategias de secuenciación de ADN de primera, segunda y tercera generación y su aplicación en el entorno biomédico, a partir de la búsqueda de artículos en bases de datos electró-nicas especializadas en investigación científica. Se encontraron 118 documentos, de los cuales se excluyeron 6, por no cumplir con los criterios de inclusión, y se seleccionaron 112, por cumplir con todos los requisitos. Conclusiones:el surgimiento de los métodos de secuenciación de siguiente generación arroja una gran canti-dad de datos, incluidos genomas secuenciados completamente de varias especies, con un rendimiento extenso, tiempos reducidos y costoeficiencia, que lleva a la completa transformación de las ciencias de la vida y logra un progreso sin precedentes en el análisis de genomas, la evaluación de la ecología microbiana y el diagnóstico de enfermedades.

Introduction: The purpose of sequencing is to determine the composition of the nucleotides present in DNA or RNA. Since the completion of the human genome project, several sequencing technologies such as Roche 454, SOLiD, Illumina, Ion Torrent, PacBio and Oxford Nanopore have emerged as tools for rapid sequencing, with greater precision and cost-efficiency, allowing the development of lar-ge-scale projects and the study of genes and genomes, along with the composition of microbiomes and the study of metabolic and genetic diseases that affect the population. Objective: To describe the foundations of the methods of DNA sequencing and their applications in the biomedical sciences. Methods: Descriptive review of the main strategies of first, second and third generation DNA sequencing and their application in the biomedical environment. This review was carried out by searching articles in electronic databases specialized in scientific research. A total of 118 papers were found, of which 6 were excluded as they did not meet the inclusion criteria and 112 were selected as meeting all the requirements. Conclusions: The emergence of next-generation sequencing methods yielding a wealth of data, including fully sequenced genomes of various species, with extensive throughput, reduced time and cost-effec-tiveness that has led to the complete transformation of the life sciences, achieving unprecedented progress in genome analysis, assessment of microbial ecology and disease diagnosis

Introdução: o objetivo do sequenciamento é determinar a composição dos nucleotídeos presentes no DNA ou RNA. Desde a conclusão do projeto do genoma humano, surgiram diversas tecnologias de sequenciamento rápida como Roche 454, SOLiD, Illumina, Ion Torrent, PacBio e Oxford Nanopore, mais precisas e econômicas, que permitem o desenvolvimento de projetos de grande escala e estudo de genes e genomas, composição de microbiomas, doenças metabólicas e genéticas que afetam a popula-ção. Objetivo: descrever os fundamentos dos métodos de sequenciamento de DNA e suas aplicações nas ciências biomédicas. Métodos: revisão descritiva das principais estratégias de sequenciamento de DNA de primeira, segunda e terceira geração e sua aplicação no ambiente biomédico, a partir da busca de artigos em bases de dados eletrônicas especializadas em pesquisa científica. Foram encontrados 118 documen-tos, dos quais 6 foram excluídos por não atenderem aos critérios de inclusão e 112 fo-ram seleciona-dos por atenderem a todos os requerimentos. Conclusões: o surgimento de métodos de sequenciamento de próxima geração rende uma riqueza de dados, incluindo genomas totalmente se-quenciados de várias espécies, com produção extensa, tempos reduzidos e eficiência de custo, levando à transformação completa das ciências da vida e alcançando um progresso sem precedentes no genoma análise, avaliação de ecologia microbiana e diagnóstico de doenças.

Comparación de métodos de extracción de ADN de Giardia spp. medidos por PCR convencional / Comparison of methods of DNA extraction from Giardia spp. measured by conventional PCR

RESUMEN Objetivos . Comparar diferentes métodos de extracción de ADN a partir de quistes y trofozoítos de Giardia spp. mediante la técnica de reacción en cadena de la polimerasa (PCR) convencional. Materiales y métodos. Se aislaron quistes de Giardia spp. a partir de 65 muestras coprológicas procedentes de hospitales de referencia nacional, obteniéndose una carga promedio de 5x104 parásitos. Asimismo, se cultivaron trofozoítos de Giardia intestinalis (ATCC® 30957™) obteniéndose una carga parasitaria de 5x106. Se compararon once métodos de extracción para quistes y seis para trofozoítos. La concentración y pureza del ADN extraído se determinó por espectrofotometría y el rendimiento de la extracción se evaluó mediante la amplificación de los genes beta giardina (bg) y glutamato deshidrogenasa (gdh) por PCR semi-anidada. Resultados. Se observó que el método I mostró la mayor concentración de ADN a partir de quistes (12,24 ng/µL), pureza (1,4) y mejor rendimiento (100% amplificación bg, 60% gdh) en comparación con los otros métodos evaluados. En el caso de los trofozoítos el método que no tuvo pretratamientos presentó la mayor concentración de ADN, pureza y rendimiento (26,56 ng/µL; 1,85; 100% amplificación bg y gdh). Conclusiones. Los pretratamientos mecánicos, de choque térmico y enzimáticos son necesarios para la ruptura de la pared quística de Giardia spp., siendo el marcador molecular bg de mayor rendimiento para detección de ADN de quistes. Los trofozoítos no requieren pretratamientos para lograr resultados satisfactorios. Se cuenta con una metodología reproducible para la extracción de ADN de Giardia spp. a partir de cualquier estadio evolutivo.

ABSTRACT Objectives. To compare different methods of DNA extraction from cysts and trophozoites of Giardia spp. using the conventional polymerase chain reaction (PCR) technique. Materials and Methods. Cysts of Giardia spp. were isolated from 65 coprological samples from national reference hospitals, obtaining an average load of 5x104 parasites. In addition, Giardia intestinalis trophozoites (ATCC® 30957™) were cultured obtaining a 5x106 parasitic load. Eleven extraction methods for cysts and six for trophozoites were compared. The concentration and purity of the extracted DNA were determined by spectrophotometry and the extraction yield was assessed by amplification of the ß-giardin (bg) and glutamate dehydrogenase (gdh) genes with a semi nested PCR assay. Results. It was observed that method 1 showed the highest concentration of DNA from cysts (12.24 ng/µL), purity (1.4) and best performance (bg: 100% amplification; gdh: 60% amplification) compared to the other methods evaluated. In the case of trophozoites, the method without pre treatment showed the highest level of DNA concentration, purity, and yield (26.56 ng/µL; 1.85; 100% amplification of bg and gdh, respectively). Conclusions . Mechanical, thermal shock, and enzymatic pre-treatments are necessary for the rupture of the cystic wall of Giardia spp. making it the highest-yielding bg molecular marker for detecting cyst DNA. Trophozoites do not require pre-treatment to achieve satisfactory results. A reproducible methodology for the extraction of DNA from Giardia spp. from any evolutionary stage is available.

El intersticio pulmonar y sus enfermedades en la infancia. Confusión diagnóstica en el recién nacido. Enfermedades genéticas del surfactante pulmonar. A propósito de un caso clínico: clínica, radiología, estudios y diagnóstico diferencial / The pulmonary interstitium and its diseases in childhood. Diagnostic confusion in the new born. Genetic diseases of pulmonary surfactant. About a clinical case: clinical, radiology, studies and differential diagnosis

Se presenta el caso clínico de un lactante fallecido a los siete meses de edad con cuadro intersticial persistente. Objetivos: describir detalladamente el camino diagnóstico; alertar sobre posibles confusiones en recién nacidos con diagnósticos más frecuentes; detallar los hallazgos clínicos, radiológicos y de anatomía patológica (consultas en el exterior). Metodología: sumatoria de estudios complejos para descartar causas más frecuentes de patología intersticial en el lactante; consultas radiológicas, de anatomía patológica y genética en el exterior del país. Resultado: con diagnóstico de PAP (proteinosis alveolar pulmonar) se encontró una duplicación de material genético a nivel de cromosoma X, correspondiente al gen CSF2RA (colony stimulating factor 2-subunidad a). Este gen codifica al receptor CSF2 cuya citoquina controla la producción, diferenciación y función de granulocitos/macrófagos. (AU)

A clinical case of a deceased seven month old infant presenting persistent interstitial lung compromise is presented. Objectives. Detailed description of the diagnostic pathway used; to alert about possible confusion with other ­ more frequent ­ pathologies in the new borninfant age; to present clinical, radiological, genetic and pathology findings (consultations abroad). Methodology. A complete description of complex studies to rule out other more frequent pathologies is presented together with radiological, pathological and genetic results from consultations abroad. Results. A diagnosis of PAP (pulmonary alveolar proteinosis) was confirmed with duplication of genetic material at CSF2RA gene (colony stimulating factor 2-subunit a). This gene codifies the CSF2 receptor whose cytokine controls production, differentiation and function of granulocytes/macrophages. (AU)

Patologia morfológica e molecular aplicadas à inovação diagnóstica e vigilância da leishmaniose visceral / Morphological and molecular pathology applied to diagnostic innovation and surveillance of visceral leishmaniasis

O conceito de Saúde Única surgiu para ressaltar a união indissociável entre a saúde animal, humana e ambiental. Nesse contexto, a leishmaniose visceral americana (LVA) é considerada uma importante doença de saúde pública no Brasil, devido a sua crescente expansão geográfica e aumento na incidência de casos humanos. A LVA é uma doença parasitária, zoonótica, causada pela Leishmania (Leishmania) infantum (syn. chagasi) e transmitida por flebotomíneos do gênero Lutzomyia. Os cães são considerados os principais reservatórios do parasito nas áreas urbanas. O diagnóstico da LVA é baseado em aspectos epidemiológicos, clínicos e laboratoriais. A demonstração da presença do parasito através de exames diretos em tecidos biológicos do hospedeiro é o diagnóstico de escolha, principalmente, em municípios em que a transmissão de LVA ainda não tenha sido confirmada. Diversas metodologias podem ser aplicadas com essa finalidade. O objetivo desse trabalho é apresentar as técnicas citológicas, anatomo-patológicas e moleculares em amostras fixadas em formalina e incluídas em parafina para o diagnóstico da leishmaniose visceral em humanos e cães. Esses dados são complementares à apresentação realizada no I Simpósio Internacional de Leishmaniose Visceral, realizado nos dia 23 e 24 de Abril de 2018, e organizado pelo Instituto Adolfo Lutz em São Paulo-SP, Brasil. (AU)

The One Health concept emerged to highlight the inseparable link between animal, human and environmental health. In this context, American Visceral Leishmaniasis (AVL) is considered an important public health disease in Brazil, due to its increasing geographic expansion and in the incidence of human cases. AVL is a parasitic and zoonotic disease caused by Leishmania (Leishmania) infantum (syn. chagasi) and transmitted by sandflies of the genus Lutzomyia. Dogs are considered the main reservoirs of the parasite in urban areas. The diagnosis of AVL is based on epidemiological, clinical and laboratory aspects. The demonstration of the presence of the parasite through direct examinations in biological tissues of the host is the diagnosis of choice, mainly in municipalities where the transmission of AVL has not yet been confirmed. Several methodologies can be applied for this purpose. The objective of this work is to present the cytological, anatomopathological and molecular techniques in formalin fixed and paraffin embedded samples for the diagnosis of visceral leishmaniasis in humans and dogs. These data are complementary to the present study at the First International Symposium on Visceral Leishmaniasis, held on April 23 and 24, 2018, and organized by Adolfo Lutz Institute in São Paulo, Brazil. (AU)

Theranostic applications of phage display to control leishmaniasis: selection of biomarkers for serodiagnostics, vaccination, and immunotherapy

Phage display is a high-throughput subtractive proteomic technology used for the generation and screening of large peptide and antibody libraries. It is based on the selection of phage-fused surface-exposed peptides that recognize specific ligands and demonstrate desired functionality for diagnostic and therapeutic purposes. Phage display has provided unmatched tools for controlling viral, bacterial, fungal, and parasitic infections, and allowed identification of new therapeutic targets to treat cancer, metabolic diseases, and other chronic conditions. This review presents recent advancements in serodiagnostics and prevention of leishmaniasis -an important tropical parasitic disease- achieved using phage display for the identification of novel antigens with improved sensitivity and specificity. Our focus is on theranostics of visceral leishmaniasis with the aim to develop biomarker candidates exhibiting both diagnostic and therapeutic potential to fight this important, yet neglected, tropical disease.

Education to a Healthy Lifestyle Improves Symptoms and Cardiovascular Risk Factors – AsuRiesgo Study / Educação para um Estilo de Vida Saudável Melhora Sintomas e Fatores de Risco Cardiovasculares – Estudo AsuRiesgo

Background: Cardiovascular diseases are the current leading causes of death and disability globally. Objective: To assess the effects of a basic educational program for cardiovascular prevention in an unselected outpatient population. Methods: All participants received an educational program to change to a healthy lifestyle. Assessments were conducted at study enrollment and during follow-up. Symptoms, habits, ATP III parameters for metabolic syndrome, and American Heart Association’s 2020 parameters of cardiovascular health were assessed. Results: A total of 15,073 participants aged ≥ 18 years entered the study. Data analysis was conducted in 3,009 patients who completed a second assessment. An improvement in weight (from 76.6 ± 15.3 to 76.4 ± 15.3 kg, p = 0.002), dyspnea on exertion NYHA grade II (from 23.4% to 21.0%) and grade III (from 15.8% to 14.0%) and a decrease in the proportion of current active smokers (from 3.6% to 2.9%, p = 0.002) could be documented. The proportion of patients with levels of triglycerides > 150 mg/dL (from 46.3% to 42.4%, p < 0.001) and LDL cholesterol > 100 mg/dL (from 69.3% to 65.5%, p < 0.001) improved. A ≥ 20% improvement of AHA 2020 metrics at the level graded as poor was found for smoking (-21.1%), diet (-29.8%), and cholesterol level (-23.6%). A large dropout as a surrogate indicator for low patient adherence was documented throughout the first 5 visits, 80% between the first and second assessments, 55.6% between the second and third assessments, 43.6% between the third and fourth assessments, and 38% between the fourth and fifth assessments. Conclusion: A simple, basic educational program may improve symptoms and modifiable cardiovascular risk factors, but shows low patient adherence. .

Fundamentos: As doenças cardiovasculares são, atualmente, as maiores causas de óbito e incapacitação em todo o mundo. Objetivos: Avaliar os efeitos de um programa educativo básico para prevenção cardiovascular em uma população de pacientes ambulatoriais não selecionados. Métodos: Todos os participantes frequentaram um programa educativo de mudança para um estilo de vida saudável. Foram realizadas avaliações à admissão no estudo e durante o acompanhamento. Foram avaliados sintomas, hábitos, parâmetros do ATP III para síndrome metabólica e parâmetros da American Heart Association 2020 para saúde cardiovascular. Resultados: Foram incluídos no estudo 15.073 participantes com idade ≥ 18 anos. Foi feita a análise de dados dos 3.009 pacientes que completaram a segunda avaliação. Foram documentados perda de peso (de 76,6 ± 15,3 para 76,4 ± 15,3 kg, p = 0,002), melhora da dispneia aos esforços graus II-NYHA (de 23,4% para 21,0%) e III (de 15,8% para 14,0%), e redução na proporção de fumantes ativos atuais (de 3,6% para 2,9%, p = 0,002). Houve melhora na proporção de pacientes com níveis de triglicérides > 150 mg/dL (de 46,3% para 42,4%, p < 0,001) e de colesterol LDL > 100 mg/dL (de 69,3% para 65,5%, p < 0,001). Houve melhora ≥ 20% na métrica AHA 2020 no nível classificado como ruim para tabagismo (-21,1%), alimentação (-29,8%), e nível de colesterol (23,6%). Foi documentada grande evasão como indicador substituto para baixa adesão de paciente nas primeiras 5 consultas, sendo 80% entre a primeira e a segunda avaliação, 55,6% entre a segunda e a terceira, 43,6% entre a terceira e a quarta, e 38% entre a quarta e a quinta. Conclusão: Um programa educativo básico e simples pode melhorar os sintomas e fatores de risco cardiovasculares modificáveis, mas conta com pouca adesão por parte dos pacientes. .

Identificação de mutações nos genes APC e MUTYH em famílias com polipose adenomatosa intestinal / Mutational screening of APC and MUTYH genes in families with adenomatous polyposis

Atualmente, pacientes com múltiplos adenomas colorretais são avaliados para mutações germinativas em dois genes, APC e MUTYH. Pacientes com mutações em APC apresentam Polipose Adenomatosa Familiar Clássica ou Atenuada (FAP/AFAP), enquanto que pacientes portadores de mutações bialélicas em MUTYH apresentam Polipose Associada ao MUTYH (MAP). O espectro das mutações em APC e MUTYH, assim como as correlações genótipo-fenótipo nestas síndromes, apresentam importante impacto clínico e podem ser distintas em cada população, tornando necessária a obtenção de dados genéticos e clínicos de diferentes populações. Além disso, cerca de 10-15% dos pacientes com polipose não apresentam mutações nesses genes, o que sugere a existência de outros genes de predisposição ainda desconhecidos. Assim, os objetivos deste estudo foram caracterizar mutações germinativas nos genes APC e MUTYH em pacientes Brasileiros com polipose, além de identificar novos genes associados com a síndrome através de sequenciamento de exoma dos pacientes negativos. No total, 23 pacientes não relacionados foram avaliados para mutações pontuais na região codificante dos genes APC e MUTYH através de sequenciamento capilar, e para rearranjos genômicos nos mesmos genes por meio de MLPA (Multiplex Ligation-Dependent Probe Amplification), arrays de hibridação genômica comparativa (CGH-array), e PCR duplex quantitativo. Este último método de avaliação do número de cópias genômicas foi desenvolvido e validado no presente estudo. Foram identificados 21 pacientes mutados nesta coorte (91%) - 6 pacientes apresentaram mutações patogênicas em MUTYH, 14 apresentaram mutações patogênicas em APC e um paciente foi portador de uma nova variante missense de significado clínico desconhecido em APC (p.Val1789Leu); seis mutações foram descritas pela primeira vez neste trabalho. Em um destes pacientes identificamos a primeira grande deleção genômica descrita no gene MUTYH. Correlações genótipo-fenótipo dos dados...

Patients with multiple colorectal adenomas are currently screened for germline mutations in two genes, APC and MUTYH. APC-mutated patients present classic or attenuated familial adenomatous polyposis (FAP/ AFAP), while patients carrying biallelic MUTYH mutations exhibit MUTYH-associated polyposis (MAP). The spectrum of APC and MUTYH mutations as well as the genotype-phenotype correlations in polyposis syndromes present clinical impact and can be population specific, making important to obtain genetic and clinical data from different populations. Furthermore, up to 10-15% of polyposis patients do not harbor mutations in these genes, suggesting that other yet unknown polyposis-predisposing genes could exist. Thus, the aim of this study was to characterize germline mutations in APC and MUTYH genes in Brazilian polyposis and to investigate novel susceptibility genes by exome sequencing of negative patients. At total, 23 unrelated polyposis patients were screened for APC/MUTYH point mutations through DNA capillary sequencing, and for APC and MUTYH genomic rearrangements through MLPA (multiplex ligation-dependent probe amplification), array-comparative genomic hybridization, and duplex quantitative PCR. This last gene dosage method was developed and validated in this study. We identified 21 mutated patients in this cohort (91%) – 6 patients carried MUTYH pathogenic mutations, 14 carried APC pathogenic mutations and one carried a novel APC missense variant of unknown clinical significance (p.Val1789Leu); six mutations were described for the first time in this series. One of these patients harbored the first large genomic deletion identified in MUTYH gene...

Diagnóstico molecular da infecção por hemoplasmas em gatos domésticos naturalmente infectados da cidade de Belém, Pará / Molecular diagnosis of haemoplasmas infection in naturally infected domestic cats from Belém, Pará

Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' e 'Candidatus Mycoplasma turicensis' são os agentes causadores da micoplasmose felina, que podem causar anemia aguda ou crônica. O objetivo deste trabalho foi determinar a ocorrência de hemoplasmas em gatos domésticos de Belém, Pará. Para isso, 201 gatos foram divididos em três grupos: Grupo A foi composto por 101 gatos de rua capturados pelo Centro de Controle de Zoonoses, o grupo B foi composto por 62 gatos domiciliados e saudáveis e o grupo C foi composto por 38 gatos domiciliados que apresentavam alguma afecção clínica. Foram coletadas amostras de sangue para a realização de Reação em Cadeia pela Polimerase (PCR) para detectar o DNA destes agentes, os quais foram sequenciados e alinhados. A análise estatística foi realizada para detectar a associação entre a infecção, o sexo dos animais e os grupos experimentais. O DNA de pelo menos uma das espécies de hemoplasmas pesquisados foi detectado em 19,9% (40/201) das amostras, sendo o DNA de 'Candidatus M. haemominutum' encontrado em 7,96% (16/201) das amostras, M. haemofelis em 1,49% (3/201) das amostras, enquanto que o DNA de 'Candidatus M. turicensis' foi detectado em 12,93% (26/201) das amostras. O DNA destes três agentes foi detectado em gatos dos grupos A e C, enquanto que no grupo B foi detectado apenas 'Candidatus M. turicensis' e 'Candidatus M. haemominutum' Foi detectada a influência do sexo sobre a infecção hemoplasmas apenas entre 'Candidatus M. haemominutum' e machos. Estes resultados mostraram que os hemoplasmas circulam entre os gatos domésticos em Belém e 'Candidatus M. turicensis' e 'Candidatus M. haemominutum' foram mais comuns do que M. haemofelis, especialmente em gatos vadios.

Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' and 'Candidatus Mycoplasma turicensis' are the causative agent of the feline mycoplasmosis, which could cause acute or chronic anemia. The aim of this work was to determine the occurrence of hemoplamas in domestic cats from Belém, Pará. To this, 201 cats were divided into three groups: Group A were composed by 101 stray cats captured by Zoonosis Control Center, group B were composed by 62 owners healthy cats and group C were composed by 38 owners cats that were suffering by some medical condition. Blood samples were collected to perform Polymerase Chain Reaction (PCR) to detect the DNA of these agents, which were sequenced and aligned. Statistical analysis was performed to detect association between the infection, the sex of the animals and experimental groups. The DNA of at least one of the hemoplasmas studied were detected in 19,9% (40/201) of the samples, being the DNA of 'Candidatus M. haemominutum' was found in 7.96% (16/201) of samples, M. haemofelis in 1.49% (3/201) of samples, while 'Candidatus M. turicensis' in 12.93% (26/201) of the samples. The DNA of these three agents was detected in cats from groups A and C, while in Group B was detected only 'Candidatus M. turicensis' and 'Candidatus M. haemominutum'. The influence of sex on hemoplasma infection was detected only between 'Candidatus M. haemominutum' and males. These findings showed that hemoplasma circulate among domestic cats in Belém, and 'Candidatus M. turicensis' and 'Candidatus M. haemominutum' were more common than M. haemofelis, especially in stray cats.

Micrometástasis: estrategias para su detección / Micrometastases: strategies for detection

La micrometástasis o enfermedad mínima residual ha adquirido una importancia trascendental en oncología al representar un verdadero problema clínico que debe ser solucionado, ya que aún se desconoce la respuesta de estos focos tumorales a los diferentes tratamientos que se usan para el control del cáncer. Aun cuando este es un problema específico fundamental a ser solucionado, ya existen métodos de ensayo inmunohistoquímicos y de biología molecular, que han permitido la ubicación de microfocos de células tumorales en diferentes órganos y tejidos, existiendo diferentes técnicas para determinar y cuantificar estas lesiones. Dentro de estas técnicas destacan la citometría de flujo y diferentes técnicas moleculares que van desde las ya tradicionales hasta las más nuevas y sofisticadas. El objetivo de la presente revisión está dirigido evaluar los nuevos métodos de diagnóstico que permitan la identificación de esta enfermedad residual, lo cual serviría para establecer tratamientos individualizados que pudieran prevenir la recurrencia de la enfermedad en los pacientes de cáncer bajo tratamiento.

Micrometastasis or minimal residual disease has become critically important in oncology since it represents a true clinical problem that must be solved, as the response of these tumor foci to the different treatments that are used for the control of cancer, is still unknown. Even though this is a fundamental specific problem to be solved, there are already immunohistochemical and molecular biology diagnostic methods that have allowed microfoci location of tumor cells in various organs and tissues, and different techniques are available to determine and quantify these lesions. Within these techniques, flow cytometry and different molecular methods are included, and they range from the traditional to the newest and most sophisticated. The goal of this review was aimed to evaluate new diagnostic methods that permit the identification of this residual disease, which would serve to establish individualized treatments and prevent the recurrence of the disease in cancer patients under treatment.

Utilização de modelos de norma de reação com variância residual heterogênea para estudo de valores genéticos de peso de codornas de corte em função de níveis de proteína bruta na dieta / Using reaction norm models with heterogeneous residual variance to study genetic values of meat type quail weight in function of crude protein levels of diet

Avaliou-se a sensibilidade dos valores genéticos do peso de codornas de corte, mensurados ao 21º e 35º dias de idade, a dietas contendo diferentes níveis de proteína bruta. Observações obtidas de animais provenientes de duas linhagens (EV1 e EV2) foram utilizadas no ajuste de modelos de regressão aleatória, considerando-se heterogeneidade de variância residual. Os coeficientes de regressão aleatória do intercepto (b0) e linear (b1) apresentaram correlação positiva entre si em todas as análises, porém a linhagem EV2 apresentou maior magnitude dos valores deste parâmetro para ambas as idades. Houve heterogeneidade de variância genética aditiva e alteração na herdabilidade com a mudança no nível proteico da dieta para ambos os grupos genéticos e em todas as idades avaliadas. As normas de reação do grupo EV1 indicam presença de interação entre genótipo e ambiente (G x E) em ambas as idades, com alteração na ordem dos efeitos genéticos de peso em função do nível proteico da dieta. Modificação da dispersão dos valores genéticos em função do nível de proteína indica presença de G x E na linhagem EV2. Portanto, a avaliação genética de codornas de corte sob dietas contendo determinado nível de proteína bruta não permite a predição de valores genéticos para outros níveis proteicos da dieta.

The sensitivity of genetic values for body weight of meat type quails predicted at 21 and 35 days of age under diets with different crude protein levels was evaluated. Data from subjects belonging to two strains (EV1 and EV2) were used to fit a random regression model under heterogeneity of residual variance assumption. The random regression coefficients for intercept (bo) and slope (b1) were positively correlated in all analyses results, but the correlation was higher in the EV2 data analyses for both ages. Results indicated that additive genetic variance and heritability change as a function of the environment gradient for both genetic strains and ages. The reaction norms for EV1 strain suggest there is genotype by environment interaction (G x E) for both ages as there were remarkable changes in the ranking of body weight breeding values for different crude protein levels. Furthermore, changes in the magnitude of the genetic effects dispersion as a function of protein level of diet indicates there is G x E in EV1 and EV2 strains. Therefore, the prediction of breeding values for body weight of quails under a specific level of crude protein in the diet does not hold for different environments regarding the level of this nutrient.

Diagnosis of the pulmonary tuberculosis by polymerase chain reaction: a comparative study between HIV - positive and - negative individuals

This study was performed to assess the efficiency of polymerase chain reaction (PCR) directly from sputum for the diagnosis of pulmonary tuberculosis by comparison between HIV-positive and HIV-negative individuals. Sputum samples were collected from hospitalized patients admitted with a clinical diagnosis of pulmonary tuberculosis, and subjected to smear microscopy, culture on LJ medium and detection of M. tuberculosis by PCR. Sensitivity, specificity, and predictive values (positive and negative) were calculated using smear and/or culture at day 42 as the gold standard, by comparing the yield in HIV-positive and HIV-negative individuals. Regardless of serostatus, the technique's yield had 62% sensitivity, 70% specificity, 79% positive predictive value, 50% negative predictive value, and 65% accuracy. HIV-negative had 64% sensitivity, 74% specificity, 75% positive predictive value, 63% negative predictive value, and 68% accuracy. HIV-positive had 59% sensitivity, 33% specificity, 87% positive predictive value, 10% negative predictive value, and 56% accuracy. The PCR showed a higher yield in HIV-negative individuals compared to HIV-positive individuals.

Phenotypic and genotypic diversity of Pseudomonas aeruginosa strains isolated from hospitals in siedlce (Poland)

A total of 62 Pseudomonas aeruginosa strains isolated from two hospitals in Siedlce (Poland) were studied by repetitive element based PCR (rep-PCR) using BOX primer. BOX-PCR results revealed the presence of 7 numerous genotypes and 31 unique patterns among isolates. Generally, the strains of P. aeruginosa were characterized by resistance to many antibiotics tested and by differences in serogroups and types of growth on cetrimide agar medium. However, the P. aeruginosa strains isolated from faeces showed much lower phenotypic and genotypic variations in comparison with strains obtained from other clinical specimens. It was observed that genetic techniques supported by phenotypic tests have enabled to conduct a detailed characterization of P. aeruginosa strains isolated from a particular environment at a particular time.

Detection of integrons among multi-drug resistant (MDR) Escherichia coli strains isolated from clinical specimens in northern west of Iran

Transference of resistance determinants by integrons is one of the important factors that can contribute to the increase in multi-resistant bacteria. We determined the prevalence and class of integrons among multi-drug resistant (MDR) Escherichia coli strains isolated from clinical specimens in Tabriz teaching hospitals. Firstly, susceptibility of 140 isolates to 13 antibiotics was determined using the disc diffusion method. Then, prevalence and class of integrons was detected in MDR strains by PCR-RFLP. One hundred five (75 percent) of total 140 isolates were uropathogenic Escherichia coli (UPEC). Other pathotypes included were: diarrheagenic Escherichia coli (13; 9.3 percent), sepsis-associated E. coli (5; 3.6 percent) and newborn meningitis-associated E. coli (2; 1.4 percent). Antibiotic resistance patterns were as follows: amoxicillin 99.3 percent, gentamicin 33.6 percent, tetracycline 72.8 percent, ceftazidime 46.4 percent, co-trimoxazole 75 percent, imipenem 1.4 percent, ciprofloxacin 47.6 percent, norfloxacin 50.7 percent, cephalothin 77.8 percent, amikacin 12.1 percent, nitrofurantoin 12.9 percent, chloramphenicol 20.7 percent and nalidixic acid 60.7 percent. One hundred eighteen (84.2 percent) of tested isolates were multi-drug resistant. Prevalence of integrons was confirmed in 27.1 percent of MDR isolates. intI1 and intI2 were detected respectively in 22.05 percent and 5.08 percent of MDR strains. No intI3 was detected. Resistance to gentamicin, amikacin and chloramphenicol was significantly associated with the presence of integrons. These results showed high resistance of E. coli to routine antibiotics, however, in consideration of low prevalence of integrons among these strains, we can conclude that antibiotic resistance genes in these strains presumably carried on elements other than integrons.

An in-house multiplex pcr method to detect of putative virulence factors in Aeromonas species

A pentaplex PCR was developed and optimised to detect the genes that encode the five most important putative virulence factors in Aeromonas isolates. It seems to be more efficient than previously reported techniques and promises to be a powerful tool for more accurate risk assessments and for monitoring pathogenic strains.

Opportunistic microorganisms in patients undergoing antibiotic therapy for pulmonary tuberculosis

Antimicrobial therapy may cause changes in the resident oral microbiota, with the increase of opportunistic pathogens. The aim of this study was to compare the prevalence of Candida, Staphylococcus, Pseudomonas and Enterobacteriaceae in the oral cavity of fifty patients undergoing antibiotic therapy for pulmonary tuberculosis and systemically healthy controls. Oral rinsing and subgingival samples were obtained, plated in Sabouraud dextrose agar with chloramphenicol, mannitol agar and MacConkey agar, and incubated for 48 h at 37ºC. Candida spp. and coagulase-positive staphylococci were identified by phenotypic tests, C. dubliniensis, by multiplex PCR, and coagulase-negative staphylococci, Enterobacteriaceae and Pseudomonas spp., by the API systems. The number of Candida spp. was significantly higher in tuberculosis patients, and C. albicans was the most prevalent specie. No significant differences in the prevalence of other microorganisms were observed. In conclusion, the antimicrobial therapy for pulmonary tuberculosis induced significant increase only in the amounts of Candida spp.

BEAUVERIA BASSIANA: quercetinase production and genetic diversity

Beauveria bassiana genetic diversity and ability to synthesize quercetin 2,3-dioxygenase (quercetinase) were analyzed. B. bassiana isolates, obtained from Brazilian soil samples, produced quercetinase after induction using 0.5 g/L quercetin. B. bassiana ATCC 7159 (29.6 nmol/mL/min) and isolate IP 11 (27.5 nmol/ml/min) showed the best performances and IP 3a (9.5 nmol/mL/min) presented the lowest level of quercetinase activity in the culture supernatant. A high level of polymorphism was detected by random amplified polymorphic DNA (RAPD) analysis. The use of internal-transcribed-spacer ribosomal region restriction fragment length polymorphism (ITS-RFLP) did not reveal characteristic markers to differentiate isolates. However, the ITS1-5.8S-ITS2 region sequence analysis provided more information on polymorphism among the isolates, allowing them to be clustered by relative similarity into three large groups. Correlation was tested according to the Person's correlation. Data of our studies showed, that lower associations among groups, level of quercetinase production, or geographical origin could be observed. This study presents the production of a novel biocatalyst by B. bassiana and suggests the possible industrial application of this fungal species in large-scale biotechnological manufacture of quercetinase.

Structural interaction between GFP-labeled diazotrophic endophytic bacterium Herbaspirillum seropedicae RAM10 and pineapple plantlets 'Vitória'

The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets 'Vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae.

Characterization of Lactobacillus from Algerian goat's milk based on phenotypic, 16S rDNA sequencing and their technological properties

Nineteen strains of Lactobacillus isolated from goat's milk from farms in north-west of Algeria were characterized. Isolates were identified by phenotypic, physiological and genotypic methods and some of their important technological properties were studied. Phenotypic characterization was carried out by studying physiological, morphological characteristics and carbohydrate fermentation patterns using API 50 CHL system. Isolates were also characterized by partial 16S rDNA sequencing. Results obtained with phenotypic methods were correlated with the genotypic characterization and 13 isolates were identified as L. plantarum, two isolates as L. rhamnosus and one isolate as L. fermentum. Three isolates identified as L. plantarum by phenotypic characterization were found to be L. pentosus by the genotypic method. A large diversity in technological properties (acid production in skim milk, exopolysaccharide production, aminopeptidase activity, antibacterial activity and antibiotic susceptibility) was observed. Based on these results, two strains of L. plantarum (LbMS16 and LbMS21) and one strain of L. rhamnosus (LbMF25) have been tentatively selected for use as starter cultures in the manufacture of artisanal fermented dairy products in Algeria.

Campylobacter insulaenigrae: first isolation report from South American sea lion (Otaria flavescens, (Shaw, 1800)

Campylobacter insulaenigrae have been isolated from different pinnipeds but not from South American sea lion (Otaria flavescens). The aim of this work is to report the first isolation of C. insulaenigrae from South American sea lion (Otaria flavescens). The isolate, identified by its phenotypic and molecular characteristics, allow recognizing O. flavescens as a new host for C. insulaenigrae.