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1.
Med. lab ; 22(9-10): 459-478, 2016. tab, graf
Artigo em Espanhol | LILACS | ID: biblio-907820

RESUMO

Introducción: el diagnóstico de estrongiloidiasis se realiza de rutina en los laboratoriosclínicos; sin embargo, su detección se dificulta debido a la baja excreción parasitaria y la baja sensibilidad de las pruebas parasitológicas empleadas. Objetivo:diseñar y estandarizar una PCR en tiempo real (qPCR) para la detección de ADN de Strongyloides stercoralis en muestras de materia fecal. Materiales y métodos: se establecieron las condiciones de qPCR y se evaluaron: a) la especificidadanalítica mediante análisis BLASTn de secuencias obtenidas de muestras positivas para Strongyloides stercoralis, b) sensibilidad analítica mediante dilucionesseriadas de muestras que contenían larvas de Strongyloides stercoralis y c) la ocurrencia de reacciones cruzadas con otros parásitos e inhibidores de la amplificación. Resultados: se amplificó un fragmento de 101 pb del gen 18S del ARN ribosomal. El valor de Ct osciló entre 23 y 29, tomando un Ct ≤35 como el punto de corte para muestras positivas. El análisis BLASTn de las secuencias obtenidas mostró un porcentaje de identidad del 98% con secuencias 18S del ARN ribosomal de Strongyloides stercoralis reportadas en la NCBI. El límite inferiorde detección de la qPCR fue 0,9 ng/μL. No se evidenció reacción cruzada con Ascaris lumbricoides, Trichuris trichiura, Uncinarias, Hymenolepis nana, Entamoeba histolytica/Entamoeba dispar, Entamoeba hartmanni, Giardia intestinalis e Iodamoeba bütschlii. No se detectaron inhibidores en las muestras de materia fecal. Conclusiones: la sensibilidad y la especificidad analítica de la qPCR comparado con el examen directo de heces son del 100%; sin embargo, aún no es posible interpretar su utilidad clínica.


Introduction: the diagnosis of strongyloidiasis is performing routinely in clinical laboratories; however, its detection is difficult due to low parasitic excretion and low sensitivity of the parasitological tests employed. Objective: to design and standardize a real-time PCR (qPCR) for the detection of Strongyloides stercoralis DNA in stool samples. Materials and methods: qPCR conditions were established and it were assessed: a) analytical specificity by BLASTn analysis of sequences obtained from samples positive for Strongyloides stercoralis, b) analytical sensitivity by serial dilutions of samples containing Strongyloides stercoralislarvae and c) the occurrence of cross-reactions with other parasites and amplification inhibitors. Results: a 101 bp fragment of the 18S ribosomal RNA gene was amplified. The value of Ct ranged from 23 and 29, with a Ct value ≤35 as a cut-off point for positive samples. BLASTn analysis of the obtained sequences showed an identity percentage of 98% with 18S ribosomal RNA sequences of Strongyloides stercoralis reported in the NCBI. The qPCR lower limit of detection was 0.9 ng/ μL. There was no cross-reaction with Ascaris lumbricoides, Trichuristrichiura, Uncinarias, Hymenolepis nana, Entamoeba histolytica/Entamoeba dispar, Entamoeba hartmanni, Giardia intestinalis, and Iodamoeba bütschlii. No inhibitors were detected in the stool samples. Conclusion: the sensitivity and analytical specificity of qPCR compared to direct examination of feces are 100%; however, it is still not possible to interpret their clinical utility.


Assuntos
Humanos , Reação em Cadeia , Diagnóstico , Strongyloides stercoralis
2.
Acta odontol. venez ; 49(2)2011. ilus
Artigo em Espanhol | LILACS | ID: lil-678812

RESUMO

Las técnicas moleculares para recuperar DNA antiguo brindan la posibilidad de comparar la evolución molecular a través del tiempo, ya que constituye una herramienta para aclarar el diagnóstico de posibles enfermedades infecciosas del pasado. Aislar y secuenciar un fragmento de DNA de Streptococcus mutans fosilizado, considerado el principal agente infeccioso implicado en la formación de la placa bacteriana y el desarrollo de la caries dental, utilizando la reacción en cadena de polimerasa (PCR). La muestra estuvo conformada por caries y tártaro dental proveniente de dientes de diferentes colecciones de México, Barcelona e Islas Baleares. La metodología fue adaptada a las condiciones de conservación de este tipo de muestra para obtener DNA y los primers fueron específicos para la amplificación de un fragmento de 124 pb del gen de la Dextranasa del S. mutans. De las 24 muestras analizadas, 9 resultaron positivas para la amplificación y en 6 se lograron las secuencias correspondientes. Para la alineación de las secuencias obtenidas, se empleó la base de datos BLAST encontrándose una homología del 95% con el genoma del S. mutans UA159. Este estudio demuestra la primera evidencia de obtención de la secuencia de un fragmento de DNA de Streptococcus mutans recuperados a partir de caries y cálculo dental de restos humanos antiguos, presentando un 95% de homología con el DNA de S. mutans de la subespecie UA159 moderno


The molecular techniques for ancient DNA recovering, offers the possibility to compare the molecular evolution through time as these are tools which make clear possible infectious diseases from the ancient times Objective: To isolate and sequence fossilized Streptococcus mutans DNA fragments, considered the infectious agent involved with dental caries and plaque formation and development, by using the polymerase chain reaction (PCR). Dental caries and tartar samples of teeth collections from Mexico, Barcelona and Balearic Islands. The methodology was adapted to the conservation conditions of this type of DNA samples, and primers were specific to amplify a fragment of 124 bp of S. mutans dextranase gene. Results: 24 samples were analyzed, 9 were positive for amplification and 6 were obtained with its corresponding sequences. To alignment the sequences obtained, we used the BLAST database, giving us the 95% homology with the S. mutans UA159 genome. This study shows us the first evidence of Streptococcus mutans DNA sequence fragment recovered from dental caries and tartar from ancient human remains, presenting a 95% homology with S. mutans UA159 modern subspecies DNA


Assuntos
Humanos , Masculino , Feminino , Rastreamento de Células , Reação em Cadeia , DNA , DNA Polimerase I , Streptococcus mutans/isolamento & purificação , Odontologia
3.
Braz. j. microbiol ; 41(3): 749-756, Oct. 2010. graf, tab
Artigo em Inglês | LILACS | ID: lil-549417

RESUMO

Cervical cancer is an important health problem in women living in developing countries. Infection with some genotypes of human papillomavirus (HPV) is the most important risk factor associated with cervical cancer. Little information exists about HPV genotype distribution in rural and suburban regions of Mexico. Thus, we determined the prevalence of HPV genotypes in women from Tlaxcala, one of the poorest states in central Mexico, and we evaluated age infection prevalence and risk factors associated with cervical neoplasm. A cross-sectional study was conducted in 236 women seeking gynecological care at the Mexican Institute for Social Security in Tlaxcala, Mexico. Cervical scrapings were diagnosed as normal, low-grade, and high-grade squamous intraepithelial lesions (LGSIL, HGSIL). Parallel samples were used to detect HPV genotypes by PCR assays using type-specific primers for HPV 6, 11, 16, 18, and 31. An epidemiological questionnaire was applied. Prevalence of HPV infection was 31.3 percent. From the infected samples, prevalence of HPV 16 was 45.9 percent; HPV 18, 31.1 percent; HPV 31, 16.2 percent; HPV 6, 10.8 percent; HPV 11, 6.7 percent. With regard to age, the highest HPV prevalence (43.5 percent) was found in the 18- to 24-year-old group and the lowest (19 percent) in the 45- to 54-year-old group. None of the risk factors showed association with cervical neoplasia grade. HPV 16 was the most common in cervical lesions. HPV was present in 22 percent of normal samples and, of these, 82.6 percent represented high-risk HPVs. Tlaxcala showed HPV prevalence comparable to that of the largest cities in Mexico, with higher prevalence for HPV 31.


Assuntos
Humanos , Feminino , Adulto , Reação em Cadeia , Técnicas In Vitro , Infecções por Papillomavirus , Papillomaviridae/isolamento & purificação , Papillomaviridae/patogenicidade , Métodos Epidemiológicos , Genótipo , Prevalência , Fatores de Risco , Técnicas
4.
São Paulo; s.n; 2009. [118] p. ilus, tab, graf, mapas.
Tese em Português | LILACS | ID: lil-587330

RESUMO

A leishmaniose tegumentar americana (LTA) é doença endêmica no Brasil onde apresenta aumento contínuo do número de casos bem como expansão da área geográfica de detecção de casos. Na Amazônia, a situação epidemiológica da LTA é ainda mais complexa, pois há várias espécies de leishmânia como agente causal da doença, cada espécie com seus reservatórios e vetores próprios podendo determinar diferentes quadros clínicos com diversas respostas ao tratamento medicamentoso. O município de Santarém situa-se no oeste do estado do Pará, onde há alta endemicidade da LTA. A participação de cada espécie de leishmânia nos casos de leishmaniose cutânea (LC) na região não é ainda conhecida através de estudos definitivos. Este trabalho tem como objetivo principal identificar as espécies de leishmânia nos casos de pacientes portadores de LC atendidos no Centro de Controle de Zoonoses de Santarém (CCZS) no ano de 2003, e estudar a possibilidade de diferenças significativas na apresentação clínica das lesões causadas pelas diferentes espécies do parasito. Através de duas técnicas moleculares, uma através de identificação de sequência de nucleotídeos que codifica a subunidade menor do RNA ribossomal e outra que identifica sequência de gene que codifica a enzima glicose-6-fosfato desidrogenase, aplicadas em material de biópsia de 93 pacientes atendidos no CCZS, durante o ano de 2003, identificamos espécies de leishmânias. Com os métodos realizados consegue-se diferenciar Leishmania (V.) braziliensis, Leishmania (L.) amazonensis e o grupo de outras espécies do gênero Leishmania (V.) que não Leishmania (V.) braziliensis a que chamamos Leishmania (L.) outras espécies. As espécies encontradas e suas proporções foram: Leishmania (V.) braziliensis 57%, Leishmania (V.) outras espécies 37% e Leishmania (L.) amazonensis 5%. Encontrou-se maior proporção de lesões em membros inferiores nos casos ocasionados por Leishmania (V.) braziliensis. Não foram encontradas diferenças significativas...


Tegumentary leishmaniasis (TL) is an endemic disease in Brazil, where it shows a continual rise in the number of cases, as well as geographic expansion in the area of detected cases. In the Amazon, the epidemiological situation of TL is even more complex because there are several species of leishmania as causal agent of the disease, each species with its own reservoir hosts and sandfly vectors being able to determine different clinical expressions with diverse responses to medical treatment. The municipality of Santarem is located in the western part of the state of Pará, where there are high endemic levels of TL. The participation of each species of leishmania in the cases of cutaneous leishmaniasis (CL) in the region has not yet been determined through definitive studies. The principal object of this study is to identify the species of leishmania in the cases of carriers who were patients at the Santarem Zoonosis Control Center in the year 2003, and to study the possibility of significant differences in the clinical presentation of lesions caused by different species of the parasite. We identified species of Leishmania using two molecular techniques, the first through identifying the sequence of nucleotides that encodes the smaller subunit of ribossomal RNA and another that identifies the gene sequence that encodes the enzyme glicose-6-phosphate desidrogenasis applied to biopsy material from 93 patients of the Santarem Zoonosis Control Center, during the year 2003. Using these methods we were able to differentiate Leishmania (V.) braziliensis, Leishmania (L.) amazonensis and the other species of the genus Leishmania (V.) that were not Leishmania (V.) braziliensis which we call Leishmania (L.) of other species. The species found and their proportions were: Leishmania (V.) braziliensis 57%, Leishmania (V.) other species 37%, and Leishmania (L.) amazonensis 5%. A greater proportion of lesions was found in inferior members in the cases of...


Assuntos
Humanos , Masculino , Feminino , Ecossistema Amazônico , Reação em Cadeia , Leishmaniose Cutânea , Leishmaniose/classificação
5.
Med. U.P.B ; 24(1): 23-27, abr. 2005.
Artigo em Espanhol | LILACS | ID: lil-594284

RESUMO

Se hace una revisión de la confirmación del diagnóstico de la fiebre tifoidea por la seroaglutinación de Widal (1896) y por el método moderno y específico de la Reacción en Cadena de la Polimerasa, método inventado por Kary Banks Mullis (1983). Esta revisión se lleva a cabo con ocasión de la validación de este último método en Medellín por Sánchez y Cardona. También, se describen datos biográficos de Widal y Mullis y se comenta el cuadro clínico de la enfermedad.


A review ofthe Widal test (1896) and the polymerase chain reaction (PCR) method invented by Kary BanksMullis (1983) for the diagnosis of typhoid fever is made. This is done due to the validation of this method in Medellín by Sánchez and Cardona.Biographies ofWidal and Mullis, as well as the c1inical manifestations of typhoid fever are also described.


Assuntos
Humanos , Febre Tifoide , Reação em Cadeia
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