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Sample records for rt-pcr em pacientes

  1. Análise de citocinas pela RT-PCR em pacientes com rinite alérgica RT-PCR cytokine study in patients with allergic rhinitis

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    Tarcimara Moreira da Silva

    2009-02-01

    Full Text Available Rinite alérgica é uma doença que decorre de um processo inflamatório da mucosa nasal conseqüente à reação de hipersensibilidade a alérgenos inalatórios e, eventualmente, alimentares. É mediada por IgE, envolvendo diferentes células, mediadores e citocinas. OBJETIVO: Avaliar as transcrições para as seguintes citocinas: IL-4, IL-5, IL-8 e IFN-gama, particularmente importantes no processo alérgico nasal, principalmente IL-4 e IL-5. Neste estudo, optou-se por avaliar os pacientes atópicos fora das crises alérgicas, com a finalidade de se conhecer as expressões das citocinas neste período. MATERIAL E MÉTODO: Realizou-se um estudo transversal e prospectivo, selecionando-se 30 pacientes, sendo 13 pacientes portadores de rinite alérgica paucissintomáticos e 17 pacientes não-atópicos. Os grupos foram selecionados através da história, do exame clínico otorrinolaringológico e do teste alérgico cutâneo - Prick Test. O perfil das citocinas foi pesquisado nos fragmentos de mucosa nasal, através da RT-PCR semiquantitativa, escolhida por apresentar boa reprodutibilidade e especificidade, utilizando-se como referência o gene da Beta-actina. RESULTADOS: Os valores de IL-5, IL-8, IFN-gama mantiveram-se homogêneos em relação ao grupo controle. A IL-4 apresentou diferença com significância estatística. CONCLUSÃO: Os pacientes alérgicos paucissintomáticos apresentaram normalização da expressão das citocinas na mucosa nasal à exceção de IL-4.Allergic rhinitis is an inflammatory reaction of the nasal mucosa, in consequence of an IgE mediated hypersensitive reaction to inhaling allergens, involving different mediators and cytokine cells. AIM: The purpose of this study was to evaluate the transcriptions for IL-4, IL-5, IL-8 and IFN-gama, particularly important in the nasal allergy process, especially IL-4 and IL-5. For this study we decided to evaluate atopic patients who were free from allergic crises, with the purpose of

  2. Comparative Evaluation Of Conventional Rt-pcr And Real-time Rt-pcr (rrt-pcr) For Detection Of Avian Metapneumovirus Subtype A [comparação Entre As Técnicas De Rt-pcr Convencional E Rt-pcr Em Tempo Real Para A Detecção Do Metapneumovírus Aviários Subtipo A

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    Ferreira H.L.; Spilki F.R.; dos Santos M.M.A.B.; de Almeida R.S.; Arns C.W.

    2009-01-01

    Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an establis...

  3. Detection of poliovirus type 2 in oysters by using cell culture and RT-PCR Detecção de poliovírus tipo 2 em ostras através de cultura celular e RT-PCR

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    Cecília E.B. Vinatea

    2006-03-01

    Full Text Available Shellfish are readily contaminated with viruses present in water containing sewage due to the concentrating effect of filter feeding. Enteroviruses are generally used as a model for the detection of viruses from shellfish due to their public health significance. In the present work, oysters were placed in glass aquaria containing seawater plus unicellular algae. Two experiments were performed: 1 oysters bioaccumulating four different poliovirus type 2 concentrations: 5 x 10(4, 2.5 x 10(4, 5 x 10³ and 5 x 10² PFU/mL during 20h; 2 oyster tissues directly inoculated with 6.0 x 10(5 and 1.0 x 10(5 PFU/mL. After viruses seeding, tissue samples were processed by an adsorption-elution-precipitation method. Positive controls were performed by seeding 6.0 x 10(5 PFU/mL of poliovirus type 2 directly on the final oyster tissue extracts. Oyster extracts were assayed for viruses recovery by plaque assay, RT-PCR and integrated cell culture-PCR methodologies (ICC/PCR. The last one was based on the inoculation of the samples onto VERO cell monolayer followed by RT-PCR analysis of the infected cell fluid. In the first experiment (20h bioaccumulation until 5 x 10³ PFU were detected after 24 and 48h growth on VERO cells. Direct RT-PCR and ICC/PCR were able to detect 3 and 0.04 PFU of poliovirus, respectively, when bioaccumulation assay was used. When direct tissue virus seeding was performed, the plaque assays showed that polioviruses were recovered in all tested concentrations. Based on these results, it is possible to conclude that viable polioviruses can be detected in oysters after bioaccumulation and these techniques can be directly applied for monitoring virus contamination in environmental samples.Devido ao hábito alimentar filtrante, os moluscos bivalves são contaminados por vírus presentes em águas contaminadas por esgoto. Os enterovírus são geralmente usados como modelos para a detecção de vírus em moluscos bivalves devido a sua import

  4. Detecção do gene da nucleoproteína do vírus da cinomose canina por RT-PCR em urina de cães com sinais clínicos de cinomose Detection of canine distemper virus nucleoprotein gene by RT-PCR in urine of dogs with distemper clinical signs

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    C.M.S. Gebara

    2004-08-01

    Full Text Available A presença do vírus da cinomose canina (CDV foi avaliada pela reação em cadeia da polimerase, precedida de transcrição reversa (RT-PCR, em 87 amostras de urina de cães que apresentavam sinais clínicos sugestivos de cinomose. Os animais foram distribuídos em três grupos. No grupo A foram incluídos 41 cães com alterações sistêmicas; no grupo B, 37 cães com alterações neurológicas; e no grupo C, nove cães com alterações sistêmicas e neurológicas simultâneas. O grupo D (controle foi composto por 20 cães assintomáticos. Os resultados da RT-PCR foram correlacionados com a forma clínica da infecção e com as alterações hematológicas encontradas. Foi possível a amplificação parcial do gene da nucleoproteína do CDV em 41 (47,1% das 87 amostras de urina provenientes de cães com sinais clínicos sugestivos de cinomose. Todas as amostras obtidas de animais assintomáticos foram negativas na RT-PCR. Amostras positivas foram encontradas nos três grupos de animais com sinais clínicos na proporção de 51,2% (24/41, 29% (11/37 e 100% (9/9 para os grupos A, B e C, respectivamente. A leucocitose foi a alteração hematológica mais freqüente nos três grupos de cães com sinais clínicos porém, não foi possível estabelecer correlação entre o resultado da RT-PCR e as alterações hematológicas. Os resultados demonstraram que, independente da forma de apresentação clínica, a técnica da RT-PCR realizada em urina pode ser utilizada no diagnóstico ante mortem da infecção pelo CDV.The urine of 87 dogs with clinical signs suggestive of canine distemper was analyzed by RT-PCR for detection of canine distemper virus (CDV nucleoprotein gene. The samples were allotted to the following groups: group A- with 41 dogs with systemic symptoms, group B- with 37 dogs with neurological signs, and group C- with 9 dogs with simultaneous systemic and neurological clinical signs. Group D (control included 20 assymptomatic dogs. A chi2

  5. Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR for detection of avian metapneumovirus subtype A Comparação entre as técnicas de RT-PCR convencional e RT-PCR em tempo real para a detecção do metapneumovírus aviários subtipo A

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    Helena Lage Ferreira

    2009-08-01

    Full Text Available Avian metapneumovirus (AMPV belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F gene and nucleocapsid (N gene were compared with an established test for the attachment (G gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3 to 10¹ TCID50 mL-1. The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.O metapneumovírus aviário (AMPV pertence ao gênero Metapneumovirus, família Paramyxoviridae. Isolamento viral, sorologia e detecção do RNA genômico são atualmente as técnicas utilizadas para o diagnóstico desse agente. O objetivo do presente estudo foi comparar a detecção de RNA viral de seis isolados de AMPV, subtipo A (AMPV/A, utilizando diferentes métodos de RT-PCR convencional e real time RT-PCR (RRT-PCR. Duas novas técnicas de RT-PCR convencional e duas técnicas de RRT-PCR, ambas para a detecção dos genes da nucleoproteína (N e da proteína de fusão (F, foram comparadas com um RT-PCR previamente estabelecido para a detecção do AMPV (gene da glicoproteína -G. Todos esses métodos foram capazes de detectar os isolados AMPV/A. As técnicas RRT-PCR (genes F e N mostraram os menores limites de detecção (10(0.3 to 10¹ TCID50 mL-1. Os resultados sugerem que as técnicas RT-PCR convencional (gene F e as técnicas de RRT-PCR (gene F e N desenvolvidas no presente estudo podem ser utilizadas com sucesso para a detecção do

  6. Avaliação das técnicas de RT-PCR e heminested RT-PCR em cérebros de cães com sinais neurológicos compatíveis com cinomose

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    Adriana Cortez

    2015-12-01

    Full Text Available The diagnostic value of RT-PCR and hemi-nested RT-PCR (hnRT-PCR was compared in brain samples of dogs presenting neurological signs compatible with canine distemper. Samples of central nervous system (CNS were collected from 68 dogs and tested by direct immunofluorescence test (RFID and, independent of the results, they were stored at -20°C for at least three years. They were submitted to the RT-PCR and hnRT-PCR techniques aiming to determine the gene responsible for the viral nucleoprotein decoding. Fifty-nine samples were positive for RIFD, 40 for RT-PCR (Kappa = 0.358 and 54 for hnRT-PCR (Kappa = 0.740. All nine RIFD negative samples were also negative for RT-PCR and hnRT-PCR. In spite of the storage duration and proper sample conditions, the estimated accordance between hnRT-PCR and RIFD demonstrated that hnRT-PCR technique can be applied in retrospective studies.

  7. RT-PCR Detection of HIV in Republic of Macedonia

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    Golubinka Bosevska

    2008-11-01

    Full Text Available The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works.The total of 33 examined persons were divided in two groups: 1 13 persons seropositive for HIV; and 2 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5.ELFA test for combined detection of HIV p24 antigen and anti HIV-1 + 2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly.In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70°C, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20°C for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is

  8. RT-PCR Protocols - Methods in Molecular Biology

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    Manuela Monti

    2011-03-01

    Full Text Available “The first record I have of it, is when I made a computer file which I usually did whenever I had an idea, that would have been on the Monday when I got back, and I called it Chain Reaction.POL, meaning polymerase. That was the identifier for it and later I called the thing the Polymerase Chain Reaction, which a lot of people thought was a dumb name for it, but it stuck, and it became PCR”. With these words the Nobel prize winner, Kary Mullis, explains how he named the PCR: one of the most important techniques ever invented and currently used in molecular biology. This book “RT-PCR Protocols” covers a wide range of aspects important for the setting of a PCR experiment for both beginners and advanced users. In my opinion the book is very well structured in three different sections. The first one describes the different technologies now available, like competitive RT-PCR, nested RT-PCR or RT-PCR for cloning. An important part regards the usage of PCR in single cell mouse embryos, stressing how important...........

  9. Avaliação da urina e de leucócitos como amostras biológicas para a detecção ante mortem do vírus da cinomose canina por RT-PCR em cães naturalmente infectados Evaluation of the urine and leucocytes as biological samples for ante mortem detection of canine distemper virus by RT-PCR assay in naturally infected dogs

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    F.J. Negrão

    2007-02-01

    Full Text Available Urine and leucocytes were comparatively evaluated as clinical samples for ante mortem detection of the canine distemper virus (CDV by a reverse transcription-polymerase chain reaction (RT-PCR assay. One hundred and eighty eight dogs with clinical symptoms of distemper, were distributed in three groups. The group A was constituted of 93 dogs with systemic signs of distemper; the group B by 11 dogs with neurological signs, and the group C by 84 dogs that presented simultaneously systemic and neurological signs. In 66.5% (125/188 of the dogs was amplified an amplicon with 287 base pair of the CDV nucleoprotein gene. In 60.8% (76/125 of the animals the CDV was detected simultaneously in the urine and leucocytes, and in 39.2% (49/125 of the dogs just a type of clinical sample (urine: n=37; leucocytes: n=12 was positive. These results demonstrate that the different forms of clinical distemper disease can hinder the choice of only one type of clinical sample to carry out the ante mortem etiological diagnosis of CDV infection, and false-negative results can be generated.

  10. Dermatoses em pacientes com diabetes mellitus

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    Foss,N T; Polon,D P; Takada,M H; Foss-Freitas,M C; Foss,M C

    2005-01-01

    OBJETIVO: Ainda é desconhecida a relação do diabetes com fatores determinantes ou precipitantes de lesões dermatológicas em pacientes diabéticos. Assim, o objetivo do estudo foi investigar a presença de lesões cutâneas, não referidas pelo paciente diabético e sua relação com o controle metabólico da doença. MÉTODOS: Foram examinados 403 pacientes, dos quais 31% eram diabéticos do tipo 1 e 69% do tipo 2. Em ambulatório de um hospital universitário, os pacientes foram atendidos por endocrinolog...

  11. Epidemiology of Human Parechovirus Type1 in Clinical Samples from Children with Gastroenteritis Using RT-PCR

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    F Ghazi

    2012-05-01

    Full Text Available

    <em>Background and Objectives:em> Human parechovirus type-1 (HPeV-1 is a genus of picornaviridea with a single stranded positive sense RNA genome. In general it seems to be responsible for more gastrointestinal and respiratory syndromes and less responsible for central nervous system (CNS symptoms. Since there is no accurate information about diagnosis and epidemiology of HPeV-1 in Iran and it is very important to distinguish between viral and bacterial diarrhea to decrease the unnecessary use of antibiotics, this study aimed at rapid detection and epidemiology of HPeV-1 in stool samples from children with gastroenteritis using specific RT-PCR.

     

    <em>Methods:> Viral RNA was isolated from 472 stool samples from children (under 4 years old with diarrhea; CDNA was prepared and amplified using specific primers from 5untranslated region (5 UTR of HPeV-1 genome by nested RT-PCR. Amplified DNA product was electrophoresed on 1% agarose gel and a single band of 265 bp was obtained. Data were analyzed by SPSS software. We also performed a comparison between the cell culture (Vero and RT-PCR method for HPeV1 detection.

     

    <em>Results:> Out of 472 samples examined during two years, 112 samples were HpeV-1 positive (23.7%. The results showed that the prevalence of this virus was in children under one year (6-12 months old with diarrhea (p=0.036 in spring and autumn (p<0.001. Boys had more positive cases than the girls (p<0.001. Out of 20 samples which were found positive by HPeV1 RT-PCR only three of them showed CPE on Vero Cells after a week.

     

    <em>Conclusion:> The results revealed that RT-PCR is a more practical and sensitive technique for HPeV-1 detection directly from clinical samples, which is valuable for epidemiology. Also, the rapid

  12. Dislipidemia em pacientes com dermatomiosite juvenil

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    Katia Tomie Kozu

    2012-01-01

    OBJETIVO: Avaliar a presença de dislipidemia em pacientes com dermatomiosite juvenil (DMJ) e seus possíveis fatores de risco. MÉTODO: 25 pacientes com DMJ foram comparados a 25 controles de acordo com dados demográficos, composição corporal, perfil lipídico, glicêmico, autoanticorpos e enzimas musculares. Foram avaliados os instrumentos de atividade da DMJ: Disease Activity Score (DAS), Childhood Myositis Assessment Scale (CMAS), Manual Muscle Testing (MMT), Myositis Disease Activity Assessme...

  13. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Huang, S-H; Tsai, M-H; Lin, C-W; Yang, T-C; Chuang, P-H; Tsai, I-S; Lu, H-C; Wan Lei; Lin, Y-J; Lai, C-H

    2008-01-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples

  14. Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

    OpenAIRE

    Ferreira, HL; Spilki, FR; dos Santos, MMAB; de Almeida, RS; Arns, CW

    2009-01-01

    Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an establis...

  15. Comparison of RT-PCR-Dot blot hybridization based on radioisotope 32P with conventional RT-PCR and commercial ELISA Assays for blood screening of HIV-1

    International Nuclear Information System (INIS)

    Maria Lina R; Andi Yasmon

    2011-01-01

    There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32 P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked lmmunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-l RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RTPCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) were negative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has high potential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA. (author)

  16. Detection and Analysis of Circular RNAs by RT-PCR.

    Science.gov (United States)

    Panda, Amaresh C; Gorospe, Myriam

    2018-03-20

    Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.

  17. Use of RT-PCR and elisa techniques for the diagnostic of infectious bronchitis virus in broilers at slaughter

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    Davi de Oliveira Almeida

    2015-03-01

    Full Text Available ABSTRACT. Almeida D.O., Tortely R., Nascimento E.R., Khan M., Pereira V.L.A & Babapoor S. [Use of RT-PCR and elisa techniques for the diagnostic of infectious bronchitis virus in broilers at slaughter.] Uso das técnicas de RT-PCR e elisa no diagnóstico da bronquite infecciosa em frangos de corte ao abate. Revista Brasileira de Medicina Veterinária, 37(1:55-59, 2015. Departamento de Saúde Coletiva Veterinária e Saúde Pública, Universidade Federal Fluminense, Rua Vital Brazil Filho, 64, Santa Rosa, Niterói, RJ 24230-340, Brasil. E-mail: elmiro@vm.uff.br Avian infectious bronchitis (IB is a viral, acute and highly contagious disease caused by infectious bronchitis virus (IBV. The disease affects broilers improvement and performance causing major economic losses in the world poultry industry. Generally, IBV infections can be diagnosed by detection of IBV itself or the specific antibody response. This study aimed to detect IBV in broilers under Sanitary Inspection by RT-PCR and ELISA, comparing the results and relating them with the average weight of the flock. Samples were collected from 40 vaccinated broiler flocks under Sanitary Inspection at slaughter. Ten birds from every flock were randomly selected and blood samples were collected for ELISA as well three birds had their trachea and caecal tonsils collected for RT- -PCR test. From 40 flocks, 30 were IBV positive by ELISA and 26 by RT-PCR, in which 15 were detected simultaneously in trachea and caecal tonsil and 5 in each sample. There was no agreement between ELISA and RT-PCR results regarding IBV diagnosis as well positivity in both tests was not statistically significant with the average weight of the flock. There was an improvement on IBV diagnosis when caecal tonsils and tracheas were used instead of just one of them. Considering the only vaccine serotype allowed by Brazilian government is the Mass serotype and its persistence in broilers would only be detected up to 28 days

  18. Quantitative real-time RT-PCR and chromogenic in situ hybridization

    DEFF Research Database (Denmark)

    Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T

    2009-01-01

    . METHODS: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. RESULTS: The concordance...

  19. Ploidia de DNA em astrocitomas: estudo em 66 pacientes brasileiros

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    KRUTMAN-ZVEIBIL DEBORAH

    1999-01-01

    Full Text Available A determinação do conteúdo de DNA nuclear (fração de fase S e ploidia de DNA foi realizada por meio de análise de imagem em 66 astrocitomas, a partir de material fixado em formalina e seccionado em cortes de 5 micrômetros corados pela técnica de Feulgen. Nossos resultados mostraram forte relação entre a idade do paciente, grau histológico e sobrevida , com a ploidia de DNA e o percentual de células em fase de síntese. A análise da atividade proliferativa de astrocitomas intracranianos é a nosso ver muito útil no entendimento do comportamento biológico , do prognóstico e para o planejamento terapêutico dessas lesões.

  20. Simultaneous detection of three lily viruses using Triplex IC-RT-PCR.

    Science.gov (United States)

    Zhang, Yubao; Wang, Yajun; Xie, Zhongkui; Yang, Guo; Guo, Zhihong; Wang, Le

    2017-11-01

    Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily. Copyright © 2017. Published by Elsevier B.V.

  1. RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

    DEFF Research Database (Denmark)

    Dybkaer, Karen; Munch, Mette; Handberg, Kurt Jensen

    2003-01-01

    A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected wit...... of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard....

  2. Diagnóstico temprano en un brote epidémico del virus Dengue en Piura usando RT-PCR y nested-PCR

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    Oscar Nolasco

    1997-07-01

    Full Text Available Un test de diagnóstico temprano (RT-PCR y Nested-PCR fue evaluado y comparado con métodos convencionales (cultivo in vitro, IFI y MAC-ELISA. Treinta y cuatro sueros de pacientes correspondientes de un brote epidémico de la costa norte peruana (Mancora, Piura en mayo de 1997 fueron incluidos en este estudio. Todos los sueros fueron obtenidos de pacientes que presentaron en los primeros cinco días manifestaciones clínicas siendo diagnosticados luego como dengue serotipo 1. Asimismo, RT-PCR permitió diagnosticar 82% de los sueros (28/34, sin embargo Mac-ELISA y cultivo in vitro reconocieron unicamente 41% de los sueros (14/34 y 38% de los sueros (13/34 respectivamente. Por lo tanto, el uso de esta herramienta molecular (RT-PCR y Nested-PCR permitiró dar un diagnóstico temprano a estos pacientes y actuar inmediatamente ante la presencia de un brote epidémico.

  3. Oftalmoplegia internuclear em paciente com mielose funicular

    Directory of Open Access Journals (Sweden)

    J. Lamartine de Assis

    1968-06-01

    Full Text Available É relatado um caso de oftalmoplegia internuclear, tipo posterior, com manifestações oculares bilaterais e assimétricas em paciente com mielose funicular. O diagnóstico da natureza da moléstia foi comprovado pela prova de absorção da vitamina B12 radioativa marcada com 57Co (método de Schilling. Foram realizados três exames electromiográficos do músculo reto mediai mais afetado (esquerdo, com intervalos de 45 a 90 dias, não tendo havido modificação dos traçados. O electromiograma nada mostrou de específico e permitiu excluir lesão neurogênica (paresia ou paralisia do 3.° nervo e muscular propriamente dita (miopatia.

  4. Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption

    Directory of Open Access Journals (Sweden)

    Liu Chengqian

    2011-05-01

    Full Text Available Abstract Reverse transcription-polymerase chain reaction (RT-PCR has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV. One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs. The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses.

  5. Simultaneous detection of enteropathogenic viruses in buffalos faeces using multiplex reverse transcription-polymerase chain reaction (mRT-PCR

    Directory of Open Access Journals (Sweden)

    U. Pagnini

    2010-02-01

    Full Text Available A multiplex reverse transcription- polymerase chain reaction (mRT-PCR assay that detects Bovine Viral Diarrhoea Virus, Bovine Coronavirus, and Group A Rotaviruses in infected cell-culture fluids and clinical faecal samples is described. One hundred twenty faecal samples from buffalo calves with acute gastroenteritis were tested. The mRT-PCR was validated against simplex RT-PCR with published primers for Pestivirus, Coronavirus and Rotavirus. The multiplex RT-PCR was equally sensitive and specific in detecting viral infections compared with simplex RT-PCR. The mRT-PCR readily identified viruses by discriminating the size of their amplified gene products. This mRT-PCR may be a sensitive and rapid assay for surveillance of buffalo enteric viruses in field specimens. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of acute gastroenteritis.

  6. Optimisation of the RT-PCR detection of immunomagnetically enriched carcinoma cells

    International Nuclear Information System (INIS)

    Raynor, Michael; Stephenson, Sally-Anne; Walsh, David CA; Pittman, Kenneth B; Dobrovic, Alexander

    2002-01-01

    Immunomagnetic enrichment followed by RT-PCR (immunobead RT-PCR) is an efficient methodology to identify disseminated carcinoma cells in the blood and bone marrow. The RT-PCR assays must be both specific for the tumor cells and sufficiently sensitive to enable detection of single tumor cells. We have developed a method to test RT-PCR assays for any cancer. This has been investigated using a panel of RT-PCR markers suitable for the detection of breast cancer cells. In the assay, a single cell line-derived tumor cell is added to 100 peripheral blood mononuclear cells (PBMNCs) after which mRNA is isolated and reverse transcribed for RT-PCR analysis. PBMNCs without added tumor cells are used as specificity controls. The previously studied markers epidermal growth factor receptor (EGFR), mammaglobin 1 (MGB1), epithelial cell adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA) were tested. Two new epithelial-specific markers ELF3 and EphB4 were also tested. MUC1 was unsuitable as strong amplification was detected in 100 cell PBMNC controls. Expression of ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 was found to be both specific for the tumor cell, as demonstrated by the absence of a signal in most 100 cell PBMNC controls, and sensitive enough to detect a single tumor cell in 100 PBMNCs using a single round of RT-PCR. ELF3, EphB4, EpCAM, EGFR, CEA and MGB1 are appropriate RT-PCR markers for use in a marker panel to detect disseminated breast cancer cells after immunomagnetic enrichment

  7. Improvement of a real-time RT-PCR assay for the detection of enterovirus RNA

    Directory of Open Access Journals (Sweden)

    Bruynseels Peggy

    2009-07-01

    Full Text Available Abstract We describe an improvement of an earlier reported real-time RT-PCR assay for the detection of enterovirus RNA, based on the 5' exonuclease digestion of a dual-labeled fluorogenic probe by Taq DNA polymerase. A different extraction method, real-time RT-PCR instrument and primer set were evaluated. Our data show that the optimized assay yields a higher sensitivity and reproducibility and resulted in a significant reduced hands-on time per sample.

  8. Detection of Tomato black ring virus by real-time one-step RT-PCR.

    Science.gov (United States)

    Harper, Scott J; Delmiglio, Catia; Ward, Lisa I; Clover, Gerard R G

    2011-01-01

    A TaqMan-based real-time one-step RT-PCR assay was developed for the rapid detection of Tomato black ring virus (TBRV), a significant plant pathogen which infects a wide range of economically important crops. Primers and a probe were designed against existing genomic sequences to amplify a 72 bp fragment from RNA-2. The assay amplified all isolates of TBRV tested, but no amplification was observed from the RNA of other nepovirus species or healthy host plants. The detection limit of the assay was estimated to be around nine copies of the TBRV target region in total RNA. A comparison with conventional RT-PCR and ELISA, indicated that ELISA, the current standard test method, lacked specificity and reacted to all nepovirus species tested, while conventional RT-PCR was approximately ten-fold less sensitive than the real-time RT-PCR assay. Finally, the real-time RT-PCR assay was tested using five different RT-PCR reagent kits and was found to be robust and reliable, with no significant differences in sensitivity being found. The development of this rapid assay should aid in quarantine and post-border surveys for regulatory agencies. Copyright © 2010 Elsevier B.V. All rights reserved.

  9. Red blood cells in cerebrospinal fluid as possible inhibitory factor for enterovirus RT-PCR

    Directory of Open Access Journals (Sweden)

    Sérgio Monteiro de Almeida

    Full Text Available 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 (RBCs 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.

  10. Development of single step RT-PCR for detection of Kyasanur forest disease virus from clinical samples

    Directory of Open Access Journals (Sweden)

    Gouri Chaubal

    2018-02-01

    Discussion and conclusion: The previously published sensitive real time RT-PCR assay requires higher cost in terms of reagents and machine setup and technical expertise has been the primary reason for development of this assay. A single step RT-PCR is relatively easy to perform and more cost effective than real time RT-PCR in smaller setups in the absence of Biosafety Level-3 facility. This study reports the development and optimization of single step RT-PCR assay which is more sensitive and less time-consuming than nested RT-PCR and cost effective for rapid diagnosis of KFD viral RNA.

  11. Modeling qRT-PCR dynamics with application to cancer biomarker quantification.

    Science.gov (United States)

    Chervoneva, Inna; Freydin, Boris; Hyslop, Terry; Waldman, Scott A

    2017-01-01

    Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used for molecular diagnostics and evaluating prognosis in cancer. The utility of mRNA expression biomarkers relies heavily on the accuracy and precision of quantification, which is still challenging for low abundance transcripts. The critical step for quantification is accurate estimation of efficiency needed for computing a relative qRT-PCR expression. We propose a new approach to estimating qRT-PCR efficiency based on modeling dynamics of polymerase chain reaction amplification. In contrast, only models for fluorescence intensity as a function of polymerase chain reaction cycle have been used so far for quantification. The dynamics of qRT-PCR efficiency is modeled using an ordinary differential equation model, and the fitted ordinary differential equation model is used to obtain effective polymerase chain reaction efficiency estimates needed for efficiency-adjusted quantification. The proposed new qRT-PCR efficiency estimates were used to quantify GUCY2C (Guanylate Cyclase 2C) mRNA expression in the blood of colorectal cancer patients. Time to recurrence and GUCY2C expression ratios were analyzed in a joint model for survival and longitudinal outcomes. The joint model with GUCY2C quantified using the proposed polymerase chain reaction efficiency estimates provided clinically meaningful results for association between time to recurrence and longitudinal trends in GUCY2C expression.

  12. Molecular analysis of dolphin morbillivirus: A new sensitive detection method based on nested RT-PCR.

    Science.gov (United States)

    Centelleghe, Cinzia; Beffagna, Giorgia; Zanetti, Rossella; Zappulli, Valentina; Di Guardo, Giovanni; Mazzariol, Sandro

    2016-09-01

    Cetacean Morbillivirus (CeMV) has been identified as the most pathogenic virus for cetaceans. Over the past three decades, this RNA virus has caused several outbreaks of lethal disease in odontocetes and mysticetes worldwide. Isolation and identification of CeMV RNA is very challenging in whales because of the poor preservation status frequently shown by tissues from stranded animals. Nested reverse transcription polymerase chain reaction (nested RT-PCR) is used instead of conventional RT-PCR when it is necessary to increase the sensitivity and the specificity of the reaction. This study describes a new nested RT-PCR technique useful to amplify small amounts of the cDNA copy of Cetacean morbillivirus (CeMV) when it is present in scant quantity in whales' biological specimens. This technique was used to analyze different tissues (lung, brain, spleen and other lymphoid tissues) from one under human care seal and seven cetaceans stranded along the Italian coastline between October 2011 and September 2015. A well-characterized, 200 base pair (bp) fragment of the dolphin Morbillivirus (DMV) haemagglutinin (H) gene, obtained by nested RT-PCR, was sequenced and used to confirm DMV positivity in all the eight marine mammals under study. In conclusion, this nested RT-PCR protocol can represent a sensitive detection method to identify CeMV-positive, poorly preserved tissue samples. Furthermore, this is also a rather inexpensive molecular technique, relatively easy to apply. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Direct recovery of infectious Pestivirus from a full-length RT-PCR amplicon

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Hoffmann, Bernd

    2008-01-01

    This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro, and the result......This study describes the use of a novel and rapid long reverse transcription (RT)-PCR for the generation of infectious full-length cDNA of pestiviruses. To produce rescued viruses, full-length RT-PCR amplicons of 12.3 kb, including a T7-promotor, were transcribed directly in vitro......, and the resulting RNA transcripts were electroporated into ovine cells. Infectious virus was obtained after one cell culture passage. The rescued viruses had a phenotype similar to the parental Border Disease virus strain. Therefore, direct generation of infectious pestiviruses from full-length RT-PCR cDNA products...

  14. Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix® RT-PCR.

    Science.gov (United States)

    Wagner, Karoline; Springer, Burkard; Imkamp, Frank; Opota, Onya; Greub, Gilbert; Keller, Peter M

    2018-04-01

    Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix ® multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix ® RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix ® multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was

  15. Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus.

    Science.gov (United States)

    Puglia, Ana L P; Rezende, Alexandre G; Jorge, Soraia A C; Wagner, Renaud; Pereira, Carlos A; Astray, Renato M

    2013-11-01

    Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SFV RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Manifestações bucais em pacientes submetidos à quimioterapia

    OpenAIRE

    Hespanhol,Fernando Luiz; Tinoco,Eduardo Muniz Barretto; Teixeira,Henrique Guilherme de Castro; Falabella,Márcio Eduardo Vieira; Assis,Neuza Maria de Souza Picorelli

    2010-01-01

    Em decorrência da quimioterapia, alterações na cavidade oral podem ser observadas e levar a complicações sistêmicas importantes, podendo aumentar o tempo de internação hospitalar, os custos do tratamento e afetar diretamente a qualidade de vida dos pacientes. Este trabalho teve como objetivo realizar uma pesquisa em um hospital de oncologia da cidade de Juiz de Fora (MG), sendo realizado através de coleta de dados nos prontuários de pacientes que estiveram em tratamento oncológico, onde foram...

  17. Desempenho cognitivo em pacientes operados de aneurisma cerebral

    Directory of Open Access Journals (Sweden)

    FOBE JEAN-LUC

    1999-01-01

    Full Text Available Vinte e cinco pacientes operados de aneurisma cerebral foram submetidos a avaliação cognitiva pós operatória tardia para linguagem, praxia, orientação, lógica, compreensão, memória, depressão, demência e gnosia visual. Os resultados foram correlacionados com idade, escala de Hunt-Hess à admissão, lado, local e tamanho do aneurisma, sangue à TC (Fisher, vaso-espasmo angiográfico (George. Déficit cognitivo tardio estava ausente em 8 pacientes (32%, era leve em 5 (20%, moderado em 6 (24% e grave em 6 (24%. A função cognitiva isoladamente mais alterada foi a da lógica com acometimento moderado e grave em 7 pacientes (28%, seguida de praxia em 6 (24%, orientação em 5 (20%, linguagem e memória em 4 (16%. A alteração gnósica visual, demência e depressão foram pouco frequentes. A compreensão estava levemente alterada em 3 pacientes. Na faixa etária 25-50 anos ocorreu melhor resultado cognitivo com sequela inexistente ou leve em 9 pacientes (75%. Os aneurismas da artéria comunicante posterior direita apresentaram déficit cognitivo tardio ausente ou leve em 5 pacientes (71,42%, os da artéria cerebral média direita apresentaram resultado equivalente em 2 (66,66%. Os aneurismas de artéria cerebral média esquerda apresentaram o pior resultado com sequela cognitiva moderada e grave em 5 pacientes (71,42%. As sequelas neuropsicológicas tardias apresentaram correlação direta com a idade, Hunt-Hess, sangue à TC, vaso-espasmo angiográfico e localização anatômica do aneurisma.

  18. Miastenia gravis: resultados de timectomia em 52 pacientes

    Directory of Open Access Journals (Sweden)

    José Teotonio de Oliveira

    1995-06-01

    Full Text Available Durante o período de 1971 a 1993, 52 miastênicos sem timoma (7 homens e 45 mulheres foram timectomizados por via esternal. Os resultados foram classificados em remissão e não-remissão . A taxa de remissão foi 48%. No grupo de remissões havia 5 homens e 20 mulheres. O tempo de seguimento foi de 5,5 anos em ambos os grupos. Os pacientes foram classificados clinicamente segundo a classificação de Osserman. De 16 pacientes na categoria II A, 11 entraram em remissão; de 36 pacientes nas categorias II B e III, 14 entraram em remissão. O tempo de duração de doença foi de 1,8 e 4,3 anos nos grupos de remissões e não-remissões, respectivamente. Todos os pacientes que entraram em remissão tinham menos de quatro anos de doença. Dos 43 pacientes com menos de quatro anos de doença, 42% não apresentaram remissão significando que um tempo de doença de menos de quatro anos não é indicador de remissão. Nossos dados indicam um melhor prognóstico quando a timectomia é feita nos primeiros quatro anos de doença.

  19. Table 1. Primer sequences used for real-time qRT-PCR analysis of ...

    Indian Academy of Sciences (India)

    User

    TGTCCCAGTAAACCGCTC. GAATCCAGCACGATACCAGT. Figure 1. Expression analysis of candidate CsActin and CsFbox genes by qRT-PCR in response to 4°C treatment. The y-axis indicates Cq values, and error bars represent standard deviations of the mean values of four replicates. Rt, roots; St, stems; Le, leaves; Fl ...

  20. Detecting the Presence of Nora Virus in "Drosophila" Utilizing Single Fly RT-PCR

    Science.gov (United States)

    Munn, Bethany; Ericson, Brad; Carlson, Darby J.; Carlson, Kimberly A.

    2015-01-01

    A single fly RT-PCR protocol has recently been developed to detect the presence of the persistent, horizontally transmitted Nora virus in "Drosophila." Wild-caught flies from Ohio were tested for the presence of the virus, with nearly one-fifth testing positive. The investigation presented can serve as an ideal project for biology…

  1. RT-PCR for detection of all seven genotypes of Lyssavirus genus.

    Science.gov (United States)

    Vázquez-Morón, S; Avellón, A; Echevarría, J E

    2006-08-01

    The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of the RT-PCR standardised in this study with a published RT-PCR optimised for EBLV1 detection and capable of amplifying RNA from all seven lyssaviruses. All seven genotypes were detected by both RT-PCRs, however, the sensitivity was higher with the new version of the test. Twenty samples submitted for rabies diagnosis were tested by the new RT-PCR. Eight out of 20 samples from six dogs, one horse and one bat were found positive, in agreement with immunofluorescence results. Seven samples from terrestrial mammals were genotype 1 and one from a bat was genotype 5. In conclusion, this method can be used to complement immunofluorescence for the diagnosis of rabies, enabling the detection of unexpected lyssaviruses during rabies surveillance.

  2. RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza

    DEFF Research Database (Denmark)

    Dybkaer, Karen; Munch, Mette; Handberg, Kurt Jensen

    2003-01-01

    A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected...

  3. Identification and characterization of a novel tospovirus species using a new RT-PCR approach

    NARCIS (Netherlands)

    Cortez, I.; Saaijer, J.; Wonjkaew, K.S.; Pereira, A.M.; Goldbach, R.W.; Peters, D.; Kormelink, R.

    2001-01-01

    Summary. A novel tospovirus serologically distinct from all established tospo- virus species was found in Thailand in Physalis minima L. The S RNA of this virus was cloned by a new RT-PCR approach revealing a nucleotide sequence of 3257 nucleotides. The ambisense RNA segment encoded a nonstructural

  4. Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.

    Science.gov (United States)

    Klungthong, Chonticha; Manasatienkij, Wudtichai; Phonpakobsin, Thipwipha; Chinnawirotpisan, Piyawan; Rodpradit, Prinyada; Hussem, Kittinun; Thaisomboonsuk, Butsaya; Ong-ajchaowlerd, Prapapun; Nisalak, Ananda; Kalayanarooj, Siripen; Buddhari, Darunee; Gibbons, Robert V; Jarman, Richard G; Yoon, In-Kyu; Fernandez, Stefan

    2015-02-01

    AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods. Published by Elsevier B.V.

  5. Analysis of gene expression in small numbers of purified hemopoietic progenitor cells by RT-PCR.

    Science.gov (United States)

    Ziegler, B L; Lamping, C P; Thoma, S J; Fliedner, T M

    1995-05-01

    Primitive hemopoietic stem cells represent the most probable targets for genetic alterations due to exposure to ionizing irradiation or chemical carcinogens. We have applied a two-step protocol for the purification of CD34+HLA-DR-/low hemopoietic progenitor cells from cord blood (CB). CD34+ cells were isolated by monoclonal antibody (mAb) against CD34 (My10) and immunomagnetic beads. Beads were cleaved off the CD34+ cells by enzymatic treatment with chymopapain. Due to chymopapain-resistance of epitopes recognized by the used mAbs purity control of CD34+ cells and separation into CD34+HLA-DR-/low and CD34+HLA-DR+ subsets could be performed by using flow cytometry. Two miniaturized procedures were applied to isolate poly(A)+ mRNA for the reverse transcription polymerase chain reaction (RT-PCR) from small numbers of CD34+HLA-DR-/low cells. In five experiments, the mean purity of immunomagnetically isolated CD34+ cells was 93.8% +/- 3.9. Flow cytometry sorting of CD34+ cells resulted in pure CD34+HLA-DR-/low populations (purity > 98.8%; range 98.8% to 99.9%; viability > 96%) with an average yield of 2600 +/- 800 cells/5 x 10(7) low density CB cells. By RT-PCR using both poly(A)+ mRNA isolation procedures, sequences corresponding to CD34 and beta 2-microglobulin were amplified from as few as 20 cells. Furthermore, a sequence-independent RT-PCR (SIP-RT-PCR) was applied to amplify the cDNA derived from five erythroblasts isolated from a burst-forming unit-erythroid (BFU-E). Upon hybridization, full-length c-fos message was detected in the SIP-RT-PCR amplified material. Our data demonstrate that gene expression can be detected at the transcriptional level in small numbers of hemopoietic progenitor cells. In addition, the SIP-RT-PCR may allow the amplification of unique mRNA species when subtractive hybridization procedures are performed. The presented data should be useful to analyze gene expression in rare subsets of radiation-exposed immature hemopoietic stem

  6. Investigations for a multi-marker RT-PCR to improve sensitivity of disseminated tumor cell detection.

    NARCIS (Netherlands)

    Vlems, F.A.; Diepstra, J.H.S.; Cornelissen, I.M.; Ligtenberg, M.J.L.; Wobbes, Th.; Punt, C.J.A.; Krieken, J.H.J.M. van; Ruers, T.J.M.; Muijen, G.N.P. van

    2003-01-01

    BACKGROUND: In order to develop a multi-marker RT-PCR, which as such may be more sensitive than a single marker assay for the detection of disseminated tumor cells, we evaluated six RT-PCR markers: cytokeratin 20 (CK20), carcinoembryonic antigen (CEA), guanylyl cyclase C (GCC), epidermal growth

  7. Investigations for a multi-marker RT-PCR to improve sensitivity of disseminated tumor cell detection

    NARCIS (Netherlands)

    Vlems, F. A.; Diepstra, J. H. S.; Cornelissen, I. M. H. A.; Ligtenberg, M. J. L.; Wobbes, Th; Punt, C. J. A.; van Krieken, J. H. J. M.; Ruers, T. J. M.; van Muijen, G. N. P.

    2003-01-01

    In order to develop a multi-marker RT-PCR, which as such may be more sensitive than a single marker assay for the detection of disseminated tumor cells, we evaluated six RT-PCR markers: cytokeratin 20 (CK20), carcinoembryonic antigen (CEA), guanylyl cyclase C (GCC), epidermal growth factor receptor

  8. Outcome on EPIZONE Extension on VER/ VNN: Diagnostics, proficiency test and qRT-PCR validation

    DEFF Research Database (Denmark)

    Bigarré, Laurent; Panzarin, Valentina; Baud, Marine

    2012-01-01

    1 is to bring valuable genetic information on the second genome component of a given isolate. ANSES has developed new DNA probes targeting RNA1 with the goal of detecting all genotypes of nodaviruses. The aim of the project is thus: - To organize, conduct and report an inter-laboratory proficiency...... test for detection of aquatic nodaviruses by real time RT-PCR targeting RNA1 and RNA2, respectively. - To test a newly developed real-time RT-PCR targeting RNA1: sensitivity, specificity, range of detection and genetic information provided by sequencing the PCR product. Materials & methods Primers...... to be extracted by each partner. In the meantime, ANSES produced and distributed RNA extracted from healthy or infected fish (2 genogroups), or cell culture; one sample from cell culture had to be serially diluted to test the sensitivity of each method in partners’ hands. Samples were tested in duplicates...

  9. Real time RT-PCR assay for detection of different serotypes of FMDV in Egypt

    Directory of Open Access Journals (Sweden)

    Laila El-Shehawy

    Full Text Available Aim: The present study indicated that rRT-PCR could be provided for the detection of FMDV in infected, contact and carrier cattle and also provide a rapid sensitive tool aiming to aid in rapid disease detection and control. Foot and Mouth disease virus serotypes O and A still existing in Egypt. In January 2012, sever outbreaks struck the animal population in most Egyptian 1 governorates. The causative virus was identified as FMDV SAT2. Material and Methods: Five samples of tongue epithelium (ET and five oesophageal-pharyngeal (OP fluid samples were collected from FMD suspected cattle in infected farm at El-Fayoum and 20 OP samples from in-contact cattle at the same farm in addition to 30 OP samples from apparently healthy cattle at three different localities in El-Fayoum governorate (12 from Fayoum; 9 from Sinoras and 9 from Edsa in order to detect carrier cattle. All of these samples were collected during November and December 2011 and January 2012. Results: All the ET and OP samples were inoculated on BHK cell culture and baby mice. The obtained results were identified using complement fixation test in addition to real-time reverse transcriptase polymerase chain reaction (rRT-PCR. In the infected farm at El-Fayoum FMDV type SAT2 was detected in cattle which are considered as the first introduction of this type while FMDV type O and SAT2 were detected in the in-contact cattle in the same farm. The sensitivity of rRT-PCR was cleared in the in-contact cattle as 13 out of 20 OP samples were positive to FMDV by rRT-PCR while 11 out of 20 OP samples were positive to FMDV by CFT. The OP samples collected from apparently healthy cattle from Fayoum, Sinoras and Edsa localities in Fayoum governorate demonstrate the circulation of the FMDV type A, O and the recent SAT2 in carrier cattle which threaten cattle population in Fayoum governorate. Also the sensitivity of real time RT-PCR over the CFT in detection of FMDV carrier cattle was clearly noticed in

  10. Development of duplex RT-PCR-ELISA for the simultaneous detection of hepatitis A virus and hepatitis E virus.

    Science.gov (United States)

    Tahk, Hongmin; Lee, Min Hwa; Lee, Kang Bum; Cheon, Doo-Sung; Choi, Changsun

    2011-07-01

    This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1 ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Detection of enteroviruses and hepatitis a virus in water by consensus primer multiplex RT-PCR

    Science.gov (United States)

    Li, Jun-Wen; Wang, Xin-Wei; Yuan, Chang-Qing; Zheng, Jin-Lai; Jin, Min; Song, Nong; Shi, Xiu-Quan; Chao, Fu-Huan

    2002-01-01

    AIM: To develop a rapid detection method of enteroviruses and Hepatitis A virus (HAV). METHODS: A one-step, single-tube consensus primers multiplex RT-PCR was developed to simultaneously detect Poliovirus, Coxsackie virus, Echovirus and HAV. A general upstream primer and a HAV primer and four different sets of primers (5 primers) specific for Poliovirus, Coxsacki evirus, Echovirus and HAV cDNA were mixed in the PCR mixture to reverse transcript and amplify the target DNA. Four distinct amplified DNA segments representing Poliovirus, Coxsackie virus, Echovirus and HAV were identified by gel electrophoresis as 589-, 671-, 1084-, and 1128 bp sequences, respectively. Semi-nested PCR was used to confirm the amplified products for each enterovirus and HAV. RESULTS: All four kinds of viral genome RNA were detected, and producing four bands which could be differentiated by the band size on the gel. To confirm the specificity of the multiplex PCR products, semi-nested PCR was performed. For all the four strains tested gave positive results. The detection sensitivity of multiplex PCR was similar to that of monoplex RT-PCR which was 24 PFU for Poliovrus, 21 PFU for Coxsackie virus, 60 PFU for Echovirus and 105 TCID50 for HAV. The minimum amount of enteric viral RNA detected by semi-nested PCR was equivalent to 2.4 PFU for Poliovrus, 2.1 PFU for Coxsackie virus, 6.0 PFU for Echovirus and 10.5 TCID50 for HAV. CONCLUSION: The consensus primers multiplex RT-PCR has more advantages over monoplex RT-PCR for enteric viruses detection, namely, the rapid turnaround time and cost effectiveness. PMID:12174381

  12. Real-Time RT-PCR for the Detection of Lyssavirus Species

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    A. Deubelbeiss

    2014-01-01

    Full Text Available The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV. Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

  13. Ring trial 2016 for Bluetongue virus detection by real-time RT-PCR in France.

    Science.gov (United States)

    Sailleau, Corinne; Viarouge, Cyril; Breard, Emmanuel; Vitour, Damien; Zientara, Stephan

    2017-05-01

    Since the unexpected emergence of BTV-8 in Northern Europe and the incursion of BTV-8 and 1 in France in 2006-2007, molecular diagnosis has considerably evolved. Several real-time RT-PCR (rtRT-PCR) methods have been developed and published, and are currently being used in many countries across Europe for BTV detection and typing. In France, the national reference laboratory (NRL) for orbiviruses develops and validates 'ready-to-use' kits with private companies for viral RNA detection. The regional laboratories network that was set up to deal with a heavy demand for analyses has used these available kits. From 2007, ring tests were organized to monitor the performance of the French laboratories. This study presents the results of 63 regional laboratories in the ring trial organized in 2016. Blood samples were sent to the laboratories. Participants were asked to use the rtRT-PCR methods in place in their laboratory, for detection of all BTV serotypes and specifically BTV-8. The French regional laboratories are able to detect and genotype BTV in affected animals. Despite the use of several methods (i.e. RNA extraction and different commercial rtRT-PCRs), the network is homogeneous. The ring trial demonstrated that the French regional veterinary laboratories have reliable and robust BTV diagnostic tools for BTV genome detection.

  14. Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.

    Science.gov (United States)

    Mehta, Rohini; Birerdinc, Aybike; Hossain, Noreen; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair; Baranova, Ancha

    2010-05-21

    Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

  15. Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

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    Afendy Arian

    2010-05-01

    Full Text Available Abstract Background Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Results Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. Conclusions We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

  16. Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

    Science.gov (United States)

    McMillan, Mary; Pereg, Lily

    2014-01-01

    Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data.

  17. Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.

    Directory of Open Access Journals (Sweden)

    Mary McMillan

    Full Text Available Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA is sufficient for effective normalisation of qRT-PCR data.

  18. Detection of avian metapneumovirus subtypes in turkeys using RT-PCR.

    Science.gov (United States)

    Ongor, H; Karahan, M; Kalin, R; Bulut, H; Cetinkaya, B

    2010-03-20

    This study investigated the prevalence of avian metapneumovirus (aMPV) and the detection of molecular subtypes of field strains of the virus using RT-PCR in clinically healthy turkeys and those showing signs of respiratory disease. In the RT-PCR examination of 624 tracheal tissue samples collected from a local turkey abattoir, 2.9 per cent (18/624) of samples tested positive. In the examination of tracheal swab samples collected from flocks with respiratory problems, 18 of 20 samples tested positive. When the results were assessed at flock level, aMPV infection was detected in only one of the 23 clinically healthy turkey flocks, whereas all four flocks with respiratory problems were infected. Molecular typing using primers specific to the attachment glycoprotein (G) gene showed that all 36 positive samples belonged to subtype B. Partial sequence analysis of DNA samples showed 95 per cent homology between the field types and the reference strain aMPV subtype B. Whereas clinically healthy turkeys had been vaccinated with a subtype A virus vaccine, the flocks with respiratory problems had been vaccinated with a subtype B virus vaccine. Despite four blind passages of RT-PCR-positive samples on Vero and chicken embryo fibroblast cells, no cytopathic effect was detected by microscopic examination.

  19. Development and implementation of the quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1 surveillance network in mainland China

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    Wang Wei

    2011-03-01

    Full Text Available Abstract Background Reverse transcription PCR (RT-PCR and real time RT-PCR (rRT-PCR have been indispensable methods for influenza surveillance, especially for determination of avian influenza. The movement of testing beyond reference lab introduced the need of quality control, including the implementation of an evaluation system for validating personal training and sample proficiency testing. Methods We developed a panel with lysates of seasonal influenza virus (H1N1, H3N2 and B, serials of diluted H5N1 virus lysates, and in-vitro transcribed H5 hemaglutinin (HA and an artificial gene RNAs for RT-PCR and rRT-PCR quality control assessment. The validations of stability and reproducibility were performed on the panel. Additionally, the panel was implemented to assess the detection capability of Chinese human avian influenza networks. Results The panel has relatively high stability and good reproducibility demonstrated by kappa's tests. In the implementation of panel on Chinese human avian influenza networks, the results suggested that there were a relatively low number of discrepancies for both concise and reproducibility in Chinese avian influenza virus net works. Conclusions A quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1 surveillance network was developed. An availably statistical data, which are used to assess the detection capability of networks on avian influenza virus (H5N1, can be obtained relatively easily through implementation of the panel on networks.

  20. Biomarker discovery for colon cancer using a 761 gene RT-PCR assay

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    Hackett James R

    2007-08-01

    Full Text Available Abstract Background Reverse transcription PCR (RT-PCR is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan® RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX™ assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis and the likelihood of tumor response to standard chemotherapy regimens (prediction. We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. Results RNA was extracted from formalin fixed paraffin embedded (FPE tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan® reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. Conclusion We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of

  1. Mutação BRAF em pacientes idosos submetidos à tireoidectomia BRAF mutation in the elderly submitted to thyroidectomy

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    Antonio Augusto T. Bertelli

    2013-04-01

    Full Text Available OBJETIVO: Avaliar a frequência da mutação V600E do gene BRAF em pacientes com mais de 65 anos de idade submetidos à tireoidectomia, correlacionando sua presença ou ausência com as diferentes lesões histológicas, com as variantes e com fatores prognósticos do carcinoma papilífero. MÉTODOS: Foram avaliados 85 pacientes com mais de 65 anos de idade submetidos à tireoidectomia, analisando a mutação BRAF V600E através de reação de PCR-RT realizada após a extração do DNA dos blocos de parafina. RESULTADOS: Detectou-se ausência ou presença da mutação BRAF V600E em 47 pacientes (55,3%. Entre os 17 carcinomas papilíferos estudados, sete apresentavam a mutação (41,2%. Demonstrou-se associação estatística entre a presença desta mutação e a variante clássica do carcinoma papilífero, além de tendência de associação com o extravasamento tireoideano. CONCLUSÃO: A mutação BRAF nos pacientes idosos também é exclusiva do carcinoma papilífero e tem frequência expressiva. Além disso, está relacionada à variante clássica e, possivelmente, ao extravasamento tireoideano.OBJECTIVE: To evaluate the frequency of the BRAF V600E mutation in patients over 65 years of age undergoing thyroidectomy, correlating its presence or absence with the different histologic lesions, their variants and with prognostic factors of papillary carcinoma. METHODS: We evaluated 85 patients over 65 years of age who underwent thyroidectomy, analyzing the BRAF V600E mutation by RT-PCR performed after DNA extraction from the paraffin blocks. RESULTS: The study detected the presence or absence of BRAF V600E mutation in 47 patients (55.3%. Among the 17 papillary carcinomas studied, seven had the mutation (41.2%. There was a statistical association between the presence of this mutation and the classic variant of papillary carcinoma, and a trend of association with thyroid extravasation. CONCLUSION: BRAF mutation in the elderly is also exclusive of

  2. Psicoterapia em grupo de pacientes com transtorno afetivo bipolar

    OpenAIRE

    Gomes,Bernardo Carramão; Lafer,Beny

    2007-01-01

    CONTEXTO: Vem crescendo nos últimos anos o número de estudos com abordagens psicoterápicas no tratamento de pacientes com transtorno afetivo bipolar. Contudo, pouco ainda se sabe sobre o efeito que tem estas abordagens nestes pacientes. OBJETIVO: Avaliar a efetividade da terapia de grupo no tratamento do transtorno afetivo bipolar. MÉTODO: Levantamento bibliográfico no Medline, Lilacs, PubMed e ISI de artigos publicados em língua inglesa no período de 1975 a 2005 e busca manual com base na bi...

  3. Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.

    Science.gov (United States)

    De La Cruz Hernández, Sergio Isaac; Anaya Molina, Yazmin; Gómez Santiago, Fabián; Terán Vega, Heidi Lizbeth; Monroy Leyva, Elda; Méndez Pérez, Héctor; García Lozano, Herlinda

    2018-04-01

    Rotavirus produces diarrhea in children under 5 years old. Most of those conventional methods such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR) have been used for rotavirus detection. However, these techniques need a multi-step process to get the results. In comparison with conventional methods, the real-time RT-PCR is a highly sensitive method, which allows getting the results in only one day. In this study a real-time RT-PCR assay was tested using a panel of 440 samples from patients with acute gastroenteritis, and characterized by PAGE and RT-PCR. The results show that the real-time RT-PCR detected rotavirus from 73% of rotavirus-negative samples analyzed by PAGE and RT-PCR; thus, the percentage of rotavirus-positive samples increased to 81%. The results indicate that this real-time RT-PCR should be part of a routine analysis, and as a support of the diagnosis of rotavirus in Mexico. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach.

    Science.gov (United States)

    Yu, Y; Zhao, Z; Jiang, D; Wu, Z; Li, S

    2013-10-01

    A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282 bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously. The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach. © 2013 The Society for Applied Microbiology.

  5. Anestesia para craniotomia em paciente acordado: relato de caso

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    Nelson Davi Bolzani

    2013-12-01

    Full Text Available JUSTIFICATIVA E OBJETIVOS: Alguns procedimentos intracranianos são possíveis com pacientes acordados e os desafios vão da cooperação do paciente até a homeostasia. O objetivo é apresentar um caso de cirurgia intracraniana para exérese de tumor em lobo parietal esquerdo com o paciente em estado vígil. RELATO DE CASO: Após seleção do paciente e preparo psicológico, foi esclarecida e aceita a proposta de exérese de lesão parietal esquerda em estado vígil. Administraram-se propofol e remifentanil em perfusão contínua para manter o escore de Ramsay entre 2-3. Foi feito um bloqueio bilateral do escalpo com ropivacaína. Foi instalado o fixador de Mayfield e os campos cirúrgicos foram ajustados para manter vias aéreas e olhos acessíveis para o mapeamento com eletroestimulação e exérese da lesão. Para incisão da dura-máter foi aplicada uma compressa com lidocaína 2% por três minutos. A cirurgia transcorreu sem intercorrências. O paciente recebeu alta hospitalar no sétimo dia de internação sem apresentar complicação. CONCLUSÃO: Apesar de ser um desafio manter analgesia e estabilidade hemodinâmica com o paciente acordado, a infusão alvo-controlada do propofol estabeleceu o nível de consciência desejado; a do remifentanil titulou a analgesia e a sedação sem o acúmulo da droga e o bloqueio com a ropivacaína, uma analgesia satisfatória. Concluímos que a técnica anestésica foi satisfatória para nosso paciente.

  6. Sangramento intracraniano em paciente hemofílico

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    Maria Carolina Pereira da Rocha

    2016-10-01

    Full Text Available Introdução: A hemofilia é uma coagulopatia congênita ou adquirida na qual há deficiência de fatores VIII (Hemofilia A e IX (Hemofilia B. A doença não tem cura e é classificada em leve, moderada ou grave conforme o grau de atividade coagulante do fator deficiente. O quadro clínico clássico é a presença e hematomas e hemartroses, mas também pode haver hematúria, hemorragia de rinofaringe ou língua, epistaxe, melena, hematêmese, sangramentos abdominais, torácicos e no sistema nervoso central. Objetivos: Relatar um caso de hemorragia intracraniana em um paciente portador de hemofilia A e comparar com os dados da literatura. Metodologia: As informações foram obtidas por meio de revisão do prontuário e acompanhamento da evolução do paciente, entrevista com o acompanhante do paciente e revisão da literatura. Relato do Caso: Paciente de 20 meses, portador de hemofilia A moderada, é levado ao Pronto Atendimento com queixa de irritabilidade, sonolência e náuseas há 3 dias. É encaminhado ao Conjunto Hospitalar de Sorocaba por suspeita de sangramento intracraniano. Conclusão: O diagnóstico de hemorragia intracraniana em pacientes hemofílicos deve ser questionado sempre que há alterações do estado de consciência ou do estado neurológico normal, mesmo que discretas. As medidas apropriadas aumentam as chances de bom prognóstico e ausência de sequelas neurológicas.

  7. A novel and highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus.

    Science.gov (United States)

    Shen, Xin-Xin; Qiu, Fang-Zhou; Zhao, Huai-Long; Yang, Meng-Jie; Hong, Liu; Xu, Song-Tao; Zhou, Shuai-Feng; Li, Gui-Xia; Feng, Zhi-Shan; Ma, Xue-Jun

    2018-03-01

    The sensitivity of qRT-PCR assay is not adequate for the detection of the samples with lower viral load, particularly in the cerebrospinal fluid (CSF) of patients. Here, we present the development of a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of human enterovirus (HEV). The clinical performance of both RTN RT-PCR and qRT-PCR was also tested and compared using 140 CSF and fecal specimens. The sensitivities of RTN RT-PCR assay for EV71, Coxsackievirus A (CVA)16, CVA6 and CVA10 achieved 10 -8 dilution with a corresponding Ct value of 38.20, 36.45, 36.75, and 36.45, respectively, which is equal to traditional two-step nested RT-PCR assay and approximately 2-10-fold lower than that of qRT-PCR assay. The specificity of RTN RT-PCR assay was extensively analyzed insilico and subsequently verified using the reference isolates and clinical samples. Sixteen qRT-PCR-negative samples were detected by RTN RT-PCR and a variety of enterovirus serotypes was identified by sequencing of inner PCR products. We conclude RTN RT-PCR is more sensitive than qRT-PCR for the detection of HEV in clinical samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. RT-PCR for confirmation of echovirus 30 isolated in Belém, Brazil

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    Maria de Lourdes C. Gomes

    Full Text Available Echovirus (Echo 30 or human enterovirus B is the most frequent enterovirus associated with meningitis cases. Epidemics and outbreaks of this disease caused by Echo 30 have occurred in several countries. In Brazil, Echo 30 has been isolated from sporadic cases and outbreaks that occurred mainly in the south and southeast regions. We used RT-PCR to examine Echo 30 isolates from meningitis cases detected from March 2002 to December 2003 in Belém, state of Pará, in northern Brazil. The patients were attended in a Basic Health Unit (State Health Secretary of Pará, where cerebrospinal fluid (CSF was collected and stored in liquid nitrogen. Weekly visits were made by technicians from Evandro Chagas Institute to the health unit and samples were stored at -70ºC in the laboratory until use. HEp-2 and RD cell lines were used for viral isolation and neutralization with specific antisera for viral identification. RNA extraction was made using Trizol reagent. The RT-PCR was made in one step, and the total mixture (50 µL was composed of: RNA, reaction buffer, dNTP, primers, Rnase inhibitor, reverse transcriptase, Taq polymerase and water. The products were visualized in agarose gel stained with ethidium bromide, visualized under UV light. Among the 279 CSF samples examined, 30 (10.7% were EV positive, 29 being Echo 30 and one was Cox B. Nineteen Echo 30 were examined with RT-PCR; 18 tested positive (762 and 494 base pairs. The use of this technique permitted viral identification in less time than usual, which benefits the patient and is of importance for public-health interventions.

  9. DETECTION OF CLASSICAL SWINE FEVER VIRUS BY RT-PCR IN WEST BENGAL, INDIA

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    Sumit Chowdhury

    2016-12-01

    Full Text Available Classical swine fever is a deadly disease of swine, caused by a RNA virus. The present study has identified presence of the classical swine fever virus (CSFV in pigs of West Bengal by one step reverse transcriptase PCR (RT-PCR performed using 5’ NTR specific primers. Internal organs from clinically affected pigs were examined from three districts of West Bengal. RT-PCT has identified presence of CSFV in all the tissues examined confirming presence of CSFV in different parts of the state.

  10. Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue

    DEFF Research Database (Denmark)

    Green, Tina Marie; de Stricker, Karin; Møller, Michael Boe

    2009-01-01

    Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue...... was 0.93 (Pnormalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes....

  11. Performance of nested RT-PCR on CSF for tuberculous meningitis diagnosis in HIV-infected patients.

    Science.gov (United States)

    Gualberto, F A S; Gonçalves, M G; Fukasawa, L O; Santos, A M Ramos Dos; Sacchi, C T; Harrison, L H; Boulware, D R; Vidal, J E

    2017-10-01

    Timely diagnosis of tuberculous meningitis (TBM) in patients with human immunodeficiency virus (HIV) infection remains a challenge. Despite the current scale-up of the Xpert® MTB/RIF assay, other molecular diagnostic tools are necessary, particularly in referral centres in low- and middle-income countries without Xpert testing. To determine the diagnostic performance of nested real-time polymerase chain reaction (nRT-PCR) in HIV-infected TBM patients categorised according to standardised clinical case definitions. Based on clinical, laboratory and imaging data, HIV-infected patients with suspected TBM were prospectively categorised as 'definite TBM', 'probable TBM', 'possible TBM' or 'not TBM'. We evaluated nRT-PCR sensitivity and specificity in diagnosing TBM among definite TBM cases, and among definite + probable TBM cases. Ninety-two participants were enrolled in the study. nRT-PCR sensitivity for definite TBM (n = 8) was 100% (95%CI 67-100) and 86% (95%CI 60-96) for both definite and probable TBM (n = 6). Assuming that 'not TBM' patients (n = 74) were true-negatives, nRT-PCR specificity was 100% (95%CI 95-100). The possible TBM group (n = 4) had no nRT-PCR positives. The nRT-PCR is a useful rule-in test for HIV-infected patients with TBM according to international consensus case definitions. As nRT-PCR cannot exclude TBM, studies comparing and combining nRT-PCR with other assays are necessary for a rule-out test.

  12. A simple RT-PCR-based strategy for screening connexin identity

    Directory of Open Access Journals (Sweden)

    M. Urban

    1999-08-01

    Full Text Available Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

  13. Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

    Science.gov (United States)

    Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C

    2009-06-01

    Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P cancer group (P prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.

  14. Strategy for the maximization of clinically relevant information from hepatitis C virus, RT-PCR quantification.

    LENUS (Irish Health Repository)

    Levis, J

    2012-02-03

    BACKGROUND: The increasing clinical application of viral load assays for monitoring viral infections has been an incentive for the development of standardized tests for the hepatitis C virus. OBJECTIVE: To develop a simple model for the prediction of baseline viral load in individuals infected with the hepatitis C virus. METHODOLOGY: Viral load quantification of each patient\\'s first sample was assessed by RT-PCR-ELISA using the Roche MONITOR assay in triplicate. Genotype of the infecting virus was identified by reverse line probe hybridization, using amplicons resulting from the qualitative HCV Roche AMPLICOR assay. RESULTS: Retrospective evaluation of first quantitative values suggested that 82.4% (n=168\\/204) of individuals had a viral load between 4.3 and 6.7 log(10) viral copies per ml. A few patients (3.4%; n=7\\/204) have a serum viremia less than the lower limit of the linear range of the RT-PCR assay. Subsequent, prospective evaluation of hepatitis C viral load of all new patients using a model based on the dynamic range of viral load in the retrospective group correctly predicted the dynamic range in 75.9% (n=33\\/54). CONCLUSION: The dynamic range of hepatitis C viremia extends beyond the linear range of the Roche MONITOR assay. Accurate determination of serum viremia is substantially improved by dilution of specimens prior to quantification.

  15. Evaluation of gamma-sterilization (60Co) by RT-PCR by DHFR expression detection

    International Nuclear Information System (INIS)

    Converso, Ana Paula G.; Andrade Junior, Heitor F. de; Vieira, Daniel P.

    2007-01-01

    The improvement of techniques to detect pathogen agents in blood had reduced significantly the contamination mechanisms by hemocomponents in blood transfusion procedures. Ionizing radiation is a method that has presented several applications on medicine and in currently days has been showing special attention on blood banks which has been applied to avoid TA-GVHD development. DHFR is an enzyme constitutive in Plasmodium protozoa and has an important role in folate metabolism on these parasites. Detecting the expression of RNAm coder for this enzyme is possible to evaluate the viability of this parasite in blood samples. Plasmodium chabaudi AJ is a parasite that induces lethal malaria in rodents similar to human malaria In this work, the objective was to detect the presence of plasmodium protozoa in irradiated blood samples, infected experimentally, through the application of a RT-PCR using primers for the coder sequence of DHFR's mRNA. We studied doses of ionizing radiation between 0 and 75 Gy. The irradiation procedures were accomplished in Center of Radiation Technology of IPEN-CNEN in a 60 Co panoramic source. Our results had demonstrated that RT-PCR is a sensible method to evaluate the viability of plasmodium in blood samples because the technique could detect low parasite burden in all tested samples. (author)

  16. Evaluation of gamma-sterilization ({sup 60}Co) by RT-PCR by DHFR expression detection

    Energy Technology Data Exchange (ETDEWEB)

    Converso, Ana Paula G.; Andrade Junior, Heitor F. de [Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, SP (Brazil)]. E-mail: anapaulagconverso@gmail.com; hfandrad@usp.br; Vieira, Daniel P. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Instituto de Medicina Tropical de Sao Paulo, Sao Paulo, SP (Brazil); E-mail: dperezv@usp.br

    2007-07-01

    The improvement of techniques to detect pathogen agents in blood had reduced significantly the contamination mechanisms by hemocomponents in blood transfusion procedures. Ionizing radiation is a method that has presented several applications on medicine and in currently days has been showing special attention on blood banks which has been applied to avoid TA-GVHD development. DHFR is an enzyme constitutive in Plasmodium protozoa and has an important role in folate metabolism on these parasites. Detecting the expression of RNAm coder for this enzyme is possible to evaluate the viability of this parasite in blood samples. Plasmodium chabaudi AJ is a parasite that induces lethal malaria in rodents similar to human malaria In this work, the objective was to detect the presence of plasmodium protozoa in irradiated blood samples, infected experimentally, through the application of a RT-PCR using primers for the coder sequence of DHFR's mRNA. We studied doses of ionizing radiation between 0 and 75 Gy. The irradiation procedures were accomplished in Center of Radiation Technology of IPEN-CNEN in a {sup 60}Co panoramic source. Our results had demonstrated that RT-PCR is a sensible method to evaluate the viability of plasmodium in blood samples because the technique could detect low parasite burden in all tested samples. (author)

  17. Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR

    Directory of Open Access Journals (Sweden)

    Hoseong Choi

    2013-03-01

    Full Text Available To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea.

  18. Detection of canine distemper virus nucleoprotein gene by RT-PCR in urine of dogs with distemper clinical signs

    International Nuclear Information System (INIS)

    Gebara, C.M.S.; Wosiachi, S.R.; Negrao, F.J.; Oliveira, D.B. de; Beloni, S.N.E.; Alfieri, A.A.; Alfieri, A.F.

    2004-01-01

    The urine of 87 dogs with clinical signs suggestive of canine distemper was analyzed by RT-PCR for detection of canine distemper virus (CDV) nucleoprotein gene. The samples were allotted to the following groups: group A- with 41 dogs with systemic symptoms, group B- with 37 dogs with neurological signs, and group C- with 9 dogs with simultaneous systemic and neurological clinical signs. Group D (control) included 20 assymptomatic dogs. A c2 was used to test RT-PCR results according to clinical form and hematological characteristics. The RT-PCR was positive for CDV in 47% (41/87) of the urine samples from dogs with clinical signs. All samples from assymptomatic dogs were RT-PCR negatives. Positive samples were found in all groups of dogs with distemper symptoms according to the following propositions: 51.2% (21/41), 29% (11/37) and 100% (9/9) for groups A, B and C, respectively. In all clinical forms (groups A, B and C) leucocytosis was the most frequent observed hematological alteration. No relationship between RT-PCR results and hematological changes was observed. The results showed that independently of the clinical stage of the illness the RT-PCR based on urine sample can be applied for ante mortem diagnosis of CDV

  19. Unequal distribution of RT-PCR artifacts along the E1-E2 region of Hepatitis C virus.

    Science.gov (United States)

    Domingo-Calap, Pilar; Sentandreu, Vicente; Bracho, Maria Alma; González-Candelas, Fernando; Moya, Andrés; Sanjuán, Rafael

    2009-10-01

    Although viral variability studies have focused traditionally on consensus sequences, the relevance of molecular clone sequences for studying viral evolution at the intra-host level is being increasingly recognized. However, for this approach to be reliable, RT-PCR artifacts do not have to contribute excessively to the observed variability. Molecular clone sequences were obtained from an in vitro transcript to estimate the maximum error rate associated to RT-PCR for the Hepatitis C virus (HCV) E1-E2 region. On average, the frequency of RT-PCR errors was one order of magnitude lower than the level of intra-host genetic variability observed in samples from an HCV outbreak. However, RT-PCR errors were not distributed evenly along the E1-E2 region and were concentrated heavily in the hypervariable region 2 (HVR 2). Although it is concluded that RT-PCR molecular clone sequences are reliable, these results warn against extrapolation of RT-PCR error rates to different genome regions. The data suggest that the RNA sequence context or secondary structure can determine the fidelity of in vitro transcription or reverse transcription. Potentially, these factors might also modify the fidelity of the viral polymerase.

  20. Massa óssea em pacientes com anorexia nervosa

    Directory of Open Access Journals (Sweden)

    Santos Evaldo dos

    2004-01-01

    Full Text Available OBJETIVO: avaliar a ingestão diária de cálcio e estabelecer sua correlação com a densidade óssea de pacientes com anorexia nervos. PACIENTES E MÉTODOS: quatorze mulheres com anorexia nervosa registraram, em diário alimentar padronizado, sua ingestão em 24 horas e foram submetidas ao exame de densitometria óssea. A análise estatística foi feita pelos testes do c² e correlação de Pearson, adotando-se como significância estatística p<0,05. RESULTADOS: a média da densidade óssea na coluna lombar foi de 0,95 ± 0,15 e no colo do fêmur foi de 0,88 ± 0,26. Houve correlação significativa entre o tempo de amenorréia e a perda de massa óssea, tanto na coluna lombar (r=-0,65; p=0,01 como no fêmur (r=-0,71; (p=0,0068. Com exceção de uma paciente, todas apresentavam ingestão de cálcio inferior ao recomendado pelo RDA, com média de 554,5 mg/dia (variando de 120 a 840 mg/dia. Observou-se que 64% das pacientes apresentavam algum grau de perda de massa óssea (osteopenia ou osteoporose na coluna e 57% no fêmur. Das seis pacientes com déficit na ingestão de cálcio inferior a 60%, nenhuma apresentou osteoporose, ao passo que das oito pacientes com déficit superior a 60% na ingestão de cálcio, três tinham osteoporose. CONCLUSÃO: Pacientes com anorexia nervosa têm baixa ingestão de cálcio e perda significativa de massa óssea, que estão diretamente relacionadas entre si. A baixa densidade óssea está, também, correlacionada com o tempo de amenorréia.

  1. Fatores de risco da sepse em pacientes queimados

    Directory of Open Access Journals (Sweden)

    Jefferson Lessa S. de Macedo

    Full Text Available OBJETIVO: Os avanços no tratamento de queimados têm reduzido as taxas de mortalidade e melhorado a qualidade de vida das vítimas de queimaduras. Entretanto, a sepse continua sendo um desafio e umas das principais causas de óbito no queimado. O objetivo deste trabalho é investigar, através de um estudo caso-controle, os fatores de risco da sepse em pacientes queimados. MÉTODO: O estudo caso-controle foi conduzido durante 12 meses, compreendendo os pacientes que foram tratados em regime de internação hospitalar na Unidade de Queimados do Hospital Regional da Asa Norte (HRAN, Brasília-DF. RESULTADOS: Quarenta e nove (19,4% pacientes tiveram sepse, de um total de 252 queimados internados na Unidade de Queimados durante o periodo do estudo. Eles tiveram um ou no maximo tres episodios de sepse durante a internacao, totalizando 62 episodios. Vinte e seis (53,1% eram homens e a media de idade foi de 21,9 ± 18,9 anos (variacao de um a 89 anos. A superficie corporal queimada dos pacientes que tiveram sepse variou de sete a 84%, com uma media de 37,7 ± 18,4%, sendo significativamente superior aos controles. As principais bacterias causadoras de sepse foram Staphylococcus aureus (46,5%, Staphylococcus coagulase negativo (20,7%, Acinetobacter baumannii (12,1% e Enterobacter cloacae (12,1%. Trinta (61,2% pacientes tiveram seu primeiro episodio de sepse na primeira semana de internacao. Quanto aos fatores de risco para a ocorrencia de sepse, destacam-se os seguintes, conforme seu poder de associacao "odds ratio": o uso de tres ou mais cateteres, a presenca de duas ou mais complicacoes, a superficie corporal queimada > 30%, o agente chama aberta e o sexo feminino. No geral, a taxa de letalidade por sepse foi de 24,5%. CONCLUSÃO: Um melhor conhecimento dos fatores de risco da sepse no paciente queimado permite o tratamento precoce dessa complicação, com antibioticoterapia sistêmica adequada, contribuindo para reduzir a morbidade e a mortalidade

  2. Psoríase em paciente HIV positivo

    Directory of Open Access Journals (Sweden)

    Mario León Silva-Vergara

    1996-12-01

    Full Text Available Dois pacientes com síndrome de imunodeficiência adquirida (SIDA desenvolveram psoríase, um com quadro mais grave e outro com apresentação mais benigna, sendo tratados com zidovudine na dose oral de 200mg a cada oito horas. No primeiro caso, a resposta terapêutica foi completa e, após nove meses em esquema de manutenção, o paciente se apresenta sem nenhuma lesão. No segundo, a despeito de resposta evidente, após seis meses de tratamento, o paciente ainda apresenta descamação furfurãcea a nível dos membros inferiores, mesmo em uso de medicação.Two patients with the acquired immunodeficiency syndrome (AIDS developed psoriasis, one of them presenting the more severe form and the other one the milder form of the disease, were treated with zidovudine per oral via, 200mg 3 times a day. In the first case the therapeutical response was complete. No lesion was verified in the patient after 9 months under maintenance schedule. In the second case, despite the response being clear, after 6 months of treatment, the patient still presented furfuraceous scalings at limbs even under the medication schedule.

  3. Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

    Science.gov (United States)

    Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

    2017-07-25

    Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an

  4. Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated associated virus 3 variant groups I, II, III and VI

    Directory of Open Access Journals (Sweden)

    Bester Rachelle

    2012-09-01

    Full Text Available Abstract Background Grapevine leafroll-associated virus 3 (GLRaV-3 is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. Methods In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. Results A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM

  5. Free cancer cell detection in peritoneal cavity in gastric cancer patients by RT-PCR for CEA

    International Nuclear Information System (INIS)

    Lee, Jong Inn; Moon, Nan Mo; Paik, Nam Sun; Choi, Dong Wook; Bang, Ho Yun; Hong, Seok Il

    1997-12-01

    Authors applied RT-PCR assay to detecting CEA expressing free cancer cells in peritoneal cavity of 114 gastric cancer patients to find an indication for prophylactic treatment to prevent peritoneal recurrence. Sixty-three of 114 cases were positive for RT-PCR, of which 16 cases were positive for cytologic examination and 47 cases were negative. Forty-nine of 51 cases who were negative for RT-PCR were negative for cytologic examination. Positivity for RT-PCR according to the depth of invasion were as follows : two (28.6 %) of seven cases whose cancer invaded mucosal or submucosal layer were positive. Ten (45.5 %) of 22 cases whose cancer invaded muscular or subserosal layer were positive. Forty-one (57.7 %) of 71 serosa involved cases were positive. Eleven (78.6 %) of cases who had grossly perioneal seedings were positive (p=0.026). However, all of 7 EGC cases, 19 of 22 cases whose cancer invaded to muscle layer or to subserosa were negative for cytologic examination, and eight of 13 cases who had had peritoneal seedings were positive. Positivity for RT-PCR according to cell differentiation were as follows: forty-two (61.8 %) of 68 cases who cancer were poorly differentiated type were positive. (p=0.163) Serum level of CEA of RT-PCR positive group and that of negative group were not statistically different. It was revealed that RT-PCR was more sensitive than cytologic examination in detecting free tumor cells, especially in pm, ss and serosa positive cancers, so if further study with more cases and longer follow-up is performed, its role as prognostic factor and an indication of prophylactic therapy will be clarified. (author). 22 refs., 5 tabs

  6. Free cancer cell detection in peritoneal cavity in gastric cancer patients by RT-PCR for CEA

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jong Inn; Moon, Nan Mo; Paik, Nam Sun; Choi, Dong Wook; Bang, Ho Yun; Hong, Seok Il [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1997-12-01

    Authors applied RT-PCR assay to detecting CEA expressing free cancer cells in peritoneal cavity of 114 gastric cancer patients to find an indication for prophylactic treatment to prevent peritoneal recurrence. Sixty-three of 114 cases were positive for RT-PCR, of which 16 cases were positive for cytologic examination and 47 cases were negative. Forty-nine of 51 cases who were negative for RT-PCR were negative for cytologic examination. Positivity for RT-PCR according to the depth of invasion were as follows : two (28.6 %) of seven cases whose cancer invaded mucosal or submucosal layer were positive. Ten (45.5 %) of 22 cases whose cancer invaded muscular or subserosal layer were positive. Forty-one (57.7 %) of 71 serosa involved cases were positive. Eleven (78.6 %) of cases who had grossly perioneal seedings were positive (p=0.026). However, all of 7 EGC cases, 19 of 22 cases whose cancer invaded to muscle layer or to subserosa were negative for cytologic examination, and eight of 13 cases who had had peritoneal seedings were positive. Positivity for RT-PCR according to cell differentiation were as follows: forty-two (61.8 %) of 68 cases who cancer were poorly differentiated type were positive. (p=0.163) Serum level of CEA of RT-PCR positive group and that of negative group were not statistically different. It was revealed that RT-PCR was more sensitive than cytologic examination in detecting free tumor cells, especially in pm, ss and serosa positive cancers, so if further study with more cases and longer follow-up is performed, its role as prognostic factor and an indication of prophylactic therapy will be clarified. (author). 22 refs., 5 tabs.

  7. Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing

    DEFF Research Database (Denmark)

    Wu, Jia Qian; Shteynberg, David; Arumugam, Manimozhiyan

    2004-01-01

    an alternative approach: reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially...... in the single-intron experiment. Spliced sequences were amplified in 46 cases (34%). We conclude that this procedure for elucidating gene structures with native cDNA sequences is cost-effective and will become even more so as it is further optimized.......The publication of a draft sequence of a third mammalian genome--that of the rat--suggests a need to rethink genome annotation. New mammalian sequences will not receive the kind of labor-intensive annotation efforts that are currently being devoted to human. In this paper, we demonstrate...

  8. Detection panel for identification of twelve hemorrhagic viruses using real-time RT-PCR.

    Science.gov (United States)

    Fajfr, M; Neubauerová, V; Pajer, P; Kubíčková, P; Růžek, D

    2014-09-01

    Viral hemorrhagic fevers are caused by viruses from four viral families and develop diseases with high fatality rates. However, no commercial diagnostic assay for these pathogens is available. We developed real-time RT-PCR assays for viruses Ebola, Marburg, Lassa, Guanarito, Machupo, Junin, Sabiá, Seoul, Puumala, Hantaan, Crimean-Congo hemorrhagic fever virus and Rift Valley fever virus. The assays were optimized for identical reaction conditions and can be performed using several types of real-time PCR instruments, both capillary and plate, including a portable Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.) (Idaho Technology, Inc.). In combination with primers and probes from previously published studies, we present a simple system for rapid identification of hemorrhagic filoviruses, arenaviruses and bunyaviruses with sufficient sensitivity for first contact laboratory and diagnosis under field conditions.

  9. Emulating a crowded intracellular environment in vitro dramatically improves RT-PCR performance

    International Nuclear Information System (INIS)

    Lareu, Ricky R.; Harve, Karthik S.; Raghunath, Michael

    2007-01-01

    The polymerase chain reaction's (PCR) phenomenal success in advancing fields as diverse as Medicine, Agriculture, Conservation, or Paleontology is based on the ability of using isolated prokaryotic thermostable DNA polymerases in vitro to copy DNA irrespective of origin. This process occurs intracellularly and has evolved to function efficiently under crowded conditions, namely in an environment packed with macromolecules. However, current in vitro practice ignores this important biophysical parameter of life. In order to more closely emulate conditions of intracellular biochemistry in vitro we added inert macromolecules into reverse transcription (RT) and PCR. We show dramatic improvements in all parameters of RT-PCR including 8- to 10-fold greater sensitivity, enhanced polymerase processivity, higher specific amplicon yield, greater primer annealing and specificity, and enhanced DNA polymerase thermal stability. The faster and more efficient reaction kinetics was a consequence of the cumulative molecular and thermodynamic effects of the excluded volume effect created by macromolecular crowding

  10. Detection of bacterial species involved in perimplantitis concerned with cultural and RT-PCR

    Directory of Open Access Journals (Sweden)

    Marcello Gatti

    2010-06-01

    Full Text Available Dental implants offer new treatment options for edentulous either partially or completely, now represent a viable alternative to conventional fixed protheses. Dental implants are colonized by a flora dominated by Gram-positive facultative aerobic, while in patients with bone loss and formation of pockets peri-implant diseases was found a significant difference in the composition of microflora, bacteria, Gram-negative anaerobes in particular Fusobacterium spp., Treponema denticola (Spirochetes, Tannerella forsythensis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia as interim black-pigmented bacteria, Porphyromonas gingivalis, often in high concentrations. Aims. The purpose of this study was to identify those at risk of perimplantitis using 2 techniques: RT-PCR examination of trade and culture. The results were compared taking into consideration the advantages and disadvantages of both methods. Materials and methods.We studied 24 patients (14 women and 10 men, aged, women between 43 and 76 years, with an average of 63.8 + / - 10.9 years, men between 45 and 88 years with a average of 64.3 years + / - 12.5 years. Was performed a double levy of sub-gingival plaque at multiple sites that had an implant CAL (clinical attachment level> 4mm in order to assess the microbiological identification with the two techniques: Examining culture and Real-Time PCR of Commerce ( Gum-Sunstar that identifies 4 bacterial species: A. actinomycetemcomitans (A.a., P.gingivalis (P.g., T.forsythensis (T.f., and T.denticola (T.d.. Results. All patients studied were positive to both tests with charger high: the consideration of tenure, with CFU / ml > 105, was positive in 66.6% of samples by:T.f., and P.g., in 12.5% for A.a., while T.d. not been sought by examining culture, the RT-PCR was positive, with high loads, in 95.8% of samples for T.f., in 79.1% for P.g., in 12.5% for A.a. and 20.8% for T.d.The test crop showed the presence of even P.intermedia in 91

  11. Design and analysis of Q-RT-PCR assays for haematological malignancies using mixed effects models

    DEFF Research Database (Denmark)

    Bøgsted, Martin; Mandrup, Charlotte; Petersen, Anders

    2009-01-01

    research use and needs qualit control for accuracy and precision. Especially the identification of experimental variations and statistical analysis has recently created discussions. The standard analytical technique is to use the Delta-Delta-Ct method. Although this method accounts for sample specific...... developed based on a linear mixed effects model for factorial designs. The model consists of an analysis of variance where the variation of each fixed effect of interest and identified experimental and biological nuisance variations are split. Hereby it accounts for varying efficiency, inhomogeneous......The recent WHO classification of haematological malignancies includes detection of genetic abnormalities with rognostic significance. Consequently, an increasing number of specific real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) based assays are in clinical...

  12. Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

    DEFF Research Database (Denmark)

    Lyng, Maria B; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

    2008-01-01

    BACKGROUND: Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various...... means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent...... of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER...

  13. No control genes required: Bayesian analysis of qRT-PCR data.

    Directory of Open Access Journals (Sweden)

    Mikhail V Matz

    Full Text Available Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process.In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts. Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests.Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been implemented as the MCMC.qpcr package in R.

  14. No control genes required: Bayesian analysis of qRT-PCR data.

    Science.gov (United States)

    Matz, Mikhail V; Wright, Rachel M; Scott, James G

    2013-01-01

    Model-based analysis of data from quantitative reverse-transcription PCR (qRT-PCR) is potentially more powerful and versatile than traditional methods. Yet existing model-based approaches cannot properly deal with the higher sampling variances associated with low-abundant targets, nor do they provide a natural way to incorporate assumptions about the stability of control genes directly into the model-fitting process. In our method, raw qPCR data are represented as molecule counts, and described using generalized linear mixed models under Poisson-lognormal error. A Markov Chain Monte Carlo (MCMC) algorithm is used to sample from the joint posterior distribution over all model parameters, thereby estimating the effects of all experimental factors on the expression of every gene. The Poisson-based model allows for the correct specification of the mean-variance relationship of the PCR amplification process, and can also glean information from instances of no amplification (zero counts). Our method is very flexible with respect to control genes: any prior knowledge about the expected degree of their stability can be directly incorporated into the model. Yet the method provides sensible answers without such assumptions, or even in the complete absence of control genes. We also present a natural Bayesian analogue of the "classic" analysis, which uses standard data pre-processing steps (logarithmic transformation and multi-gene normalization) but estimates all gene expression changes jointly within a single model. The new methods are considerably more flexible and powerful than the standard delta-delta Ct analysis based on pairwise t-tests. Our methodology expands the applicability of the relative-quantification analysis protocol all the way to the lowest-abundance targets, and provides a novel opportunity to analyze qRT-PCR data without making any assumptions concerning target stability. These procedures have been implemented as the MCMC.qpcr package in R.

  15. Qualidade do sono em pacientes fibromiálgicos

    Directory of Open Access Journals (Sweden)

    Florinda Freire Moro

    2014-03-01

    Full Text Available Objetivo: Analisar a qualidade do sono em paciente fibromiálgicos, identificando seus principais hábitos noturnos e verificando os possíveis fatores que influenciam na qualidade do sono. Métodos: Pesquisa observacional, transversal, de caráter quantitativo, realizada em ambulatório de fisioterapia no período março a abril de 2012. Participaram 24 pacientes fibromiálgicos, independente do sexo e idade. Aplicou-se um questionário baseado no Pittsburgh Sleep Quality Index (PSQI para avaliar a qualidade e hábitos noturnos. As informações foram analisadas através de estatística descritiva. Resultados: Em relação à latência do sono, apenas 1 (4,2% leva para dormir um tempo menor ou igual a 15 min e 19 (79,2% apontaram dificuldade de “não adormecer em até 30 min” por mais de 3 vezes por semana. O componente “sentir dor” e o “acordar no meio da noite” tiveram maiores influências no distúrbio do sono destes pacientes. Quanto à duração do sono, 7 (29,2% dormem menos que 5 horas. Sobre a eficiência do sono, 12 (50,0% possuíam uma eficiência do sono superior a 85%, 10 (41,7% possuíam eficiência do sono entre 75-84% e apenas 2 (8,33% eficiência de 65-74%. Quanto ao uso de medicação para dormir, 12 (50% não usaram durante o mês e 12 (50% usavam entre 3 ou mais vezes durante a semana. Conclusão: Os pacientes com fibromialgia no presente estudo apresentaram um declínio na qualidade e eficiência do sono, sendo influenciados pela latência e duração do sono, presença de dor e despertar noturno. doi: 10.5020/18061230.2014.p72

  16. Qualidade do sono em pacientes fibromiálgicos

    Directory of Open Access Journals (Sweden)

    Florinda Freire Moro

    2014-03-01

    Full Text Available Objetivo: Analisar a qualidade do sono em paciente fibromiálgicos, identificando seus principais hábitos noturnos e verificando os possíveis fatores que influenciam na qualidade do sono. Métodos: Pesquisa observacional, transversal, de caráter quantitativo, realizada em ambulatório de fisioterapia no período março a abril de 2012. Participaram 24 pacientes fibromiálgicos, independente do sexo e idade. Aplicou-se um questionário baseado no Pittsburgh Sleep Quality Index (PSQI para avaliar a qualidade e hábitos noturnos. As informações foram analisadas através de estatística descritiva. Resultados: Em relação à latência do sono, apenas 1 (4,2% leva para dormir um tempo menor ou igual a 15 min e 19 (79,2% apontaram dificuldade de “não adormecer em até 30 min” por mais de 3 vezes por semana. O componente “sentir dor” e o “acordar no meio da noite” tiveram maiores influências no distúrbio do sono destes pacientes. Quanto à duração do sono, 7 (29,2% dormem menos que 5 horas. Sobre a eficiência do sono, 12 (50,0% possuíam uma eficiência do sono superior a 85%, 10 (41,7% possuíam eficiência do sono entre 75-84% e apenas 2 (8,33% eficiência de 65-74%. Quanto ao uso de medicação para dormir, 12 (50% não usaram durante o mês e 12 (50% usavam entre 3 ou mais vezes durante a semana. Conclusão: Os pacientes com fibromialgia no presente estudo apresentaram um declínio na qualidade e eficiência do sono, sendo influenciados pela latência e duração do sono, presença de dor e despertar noturno.

  17. The development and application of the two real-time RT-PCR assays to detect the pathogen of HFMD.

    Directory of Open Access Journals (Sweden)

    Aili Cui

    Full Text Available Large-scale Hand, Foot, and Mouth Disease (HFMD outbreaks have frequently occurred in China since 2008, affecting more than one million children and causing several hundred children deaths every year. The pathogens of HFMD are mainly human enteroviruses (HEVs. Among them, human enterovirus 71 (HEV71 and coxsackievirus A16 (CVA16 are the most common pathogens of HFMD. However, other HEVs could also cause HFMD. To rapidly detect HEV71 and CVA16, and ensure detection of all HEVs causing HFMD, two real-time hybridization probe-based RT-PCR assays were developed in this study. One is a multiplex real-time RT-PCR assay, which was developed to detect and differentiate HEV71 specifically from CVA16 directly from clinical specimens within 1-2 h, and the other is a broad-spectrum real-time RT-PCR assay, which targeted almost all HEVs. The experiments confirmed that the two assays have high sensitivity and specificity, and the sensitivity was up to 0.1 TCID50/ml for detection of HEVs, HEV71, and CVA16, respectively. A total of 213 clinical specimens were simultaneously detected by three kinds of assays, including the two real-time RT-PCR assays, direct conventional RT-PCR assay, and virus isolation assay on human rhabdomyosarcoma cells (RD cells. The total positive rate of both HEV71 and CVA16 was 69.48% with real-time RT-PCR assay, 47.42% with RT-PCR assay, and 34.58% with virus isolation assay. One HFMD clinical specimen was positive for HEV, but negative for HEV71 or CVA16, which was identified as Echovirus 11 (Echo11 by virus isolation, RT-PCR, and sequencing for the VP1 gene. The two real-time RT-PCR assays had been applied in 31 provincial HFMD labs to detect the pathogens of HFMD, which has contributed to the rapid identification of the pathogens in the early stages of HFMD outbreaks, and helped to clarify the etiologic agents of HFMD in China.

  18. Selection of reference genes for quantitative real time RT-PCR during dimorphism in the zygomycete Mucor circinelloides.

    Science.gov (United States)

    Valle-Maldonado, Marco I; Jácome-Galarza, Irvin E; Gutiérrez-Corona, Félix; Ramírez-Díaz, Martha I; Campos-García, Jesús; Meza-Carmen, Víctor

    2015-03-01

    Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus.

  19. Combination of RT-PCR and proteomics for the identification of Crimean-Congo hemorrhagic fever virus in ticks

    Directory of Open Access Journals (Sweden)

    Isabel G. Fernández de Mera

    2017-07-01

    Full Text Available Crimean-Congo hemorrhagic fever (CCHF is an emerging tick-borne zoonotic disease caused by the CCHF virus (CCHFV. In this study, an experimental approach combining RT-PCR and proteomics was used for the identification and characterization of CCHFV in 106 ticks from 7 species that were collected from small ruminants in Greece. The methodological approach included an initial screening for CCHFV by RT-PCR followed by proteomics analysis of positive and control negative tick samples. This novel approach allowed the identification of CCHFV-positive ticks and provided additional information to corroborate the RT-PCR findings using a different approach. Two ticks, Dermacentor marginatus and Haemaphysalis parva collected from a goat and a sheep, respectively were positive for CCHFV. The sequences for CCHFV RNA segments S and L were characterized by RT-PCR and proteomics analysis of tick samples, respectively. These results showed the possibility of combining analyses at the RNA and protein levels using RT-PCR and proteomics for the characterization of CCHFV in ticks. The results supported that the CCHFV identified in ticks are genetic variants of the AP92 strain. Although the AP92-like strains probably do not represent a high risk of CCHF to the population, the circulation of genetically diverse CCHFV strains could potentially result in the appearance of novel viral genotypes with increased pathogenicity and fitness.

  20. Application and evaluation of RT-PCR-ELISA for the nucleoprotein and RT-PCR for detection of low-pathogenic H5 and H7 subtypes of avian influenza virus

    DEFF Research Database (Denmark)

    Dybkær, Karen; Munch, Mette; Handberg, Kurt J.

    2004-01-01

    Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples...... originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT...

  1. Perfil cardiovascular em pacientes com apneia obstrutiva do sono

    Directory of Open Access Journals (Sweden)

    Fátima Dumas Cintra

    2011-04-01

    Full Text Available FUNDAMENTO: Apneia Obstrutiva do Sono (AOS é um fator de risco para várias condições cardiovasculares incluindo aumento na mortalidade cardiovascular. Sendo assim, é essencial o conhecimento das principais repercussões cardiovasculares dos distúrbios respiratórios do sono durante uma avaliação clínica. OBJETIVO: Analisar as características cardiovasculares de pacientes com AOS. MÉTODOS: Pacientes submetidos a polissonografia basal foram consecutivamente selecionados do banco de dados do Instituto do Sono entre março de 2007 e março de 2009. Todos os pacientes foram orientados a comparecer ao ambulatório para coleta de sangue, exame físico, eletrocardiograma de 12 derivações, espirometria, teste cardiopulmonar em esteira ergométrica e ecocardiograma transtorácico. O estudo foi aprovado pelo comitê de ética e pesquisa e registrado no site http://clinicaltrials.gov/ sob o número: NCT00768625. RESULTADOS: Foram analisados 261 pacientes e 108 controles. As principais características dos pacientes com AOS foram: obesidade, hipertensão, baixos níveis plasmáticos de lipoproteínas de alta densidade (HDL e aumento no diâmetro do átrio esquerdo quando comparados com controles (3,75 ± 0,42; 3,61 ± 0,41, p = 0,001, respectivamente. Essas características associadas correspondem a um acréscimo de 16,6 vezes na probabilidade de ocorrência de AOS independentemente do relato de algum sintoma dessa desordem, como sonolência ou ronco. CONCLUSÃO: Na amostra avaliada, o perfil cardiovascular dos pacientes com AOS mais encontrado foi: obesidade, hipertensão arterial, baixos níveis plasmáticos de HDL e átrio esquerdo com diâmetro aumentado.

  2. Doenca renal policistica autossomica dominante em pacientes em hemodialise no sul do Brasil

    Directory of Open Access Journals (Sweden)

    Everton Fernando Alves

    2014-03-01

    Full Text Available Introdução: A doença renal policística autossômica dominante é a enfermidade renal hereditária mais comum em seres humanos. Objetivo: Analisar a prevalência, características clínicas e laboratoriais de pacientes com rins policísticos e relacionar as manifestações da doença por gênero. Métodos: Trata-se de um estudo observacional e retrospectivo. Foram revisados todos os prontuários médicos de pacientes com rins policísticos admitidos para hemodiálise entre 1995 e 2012, em quatro centros que atendem a área de abrangência da 15ª regional de saúde do Paraná, Brasil. Resultados: Fizeram parte do estudo 48 pacientes com rins policísticos, causa primária da doença renal crônica (DRC estágio 5. A prevalência da doença foi de um em 10.912 habitantes. A média de idade de ingresso na hemodiálise (50,7 anos e o tempo de seguimento em hemodiálise até o transplante (36,5 meses foi menor nos homens. A hipertensão arterial foi o diagnóstico mais frequente em 73% dos pacientes, com predominância em mulheres (51,4%. O cisto hepático foi a manifestação extrarrenal mais frequente nos homens (60,0%. Foram a óbito 10,4% dos pacientes que faziam uso de hemodiálise, sendo 60% de homens. A classe de droga anti-hipertensiva mais utilizada foi a que atua no sistema renina-angiotensina, com maior frequência de uso nas mulheres (53,3%. A ureia pós-diálise foi significativamente maior em homens. Conclusão: A prevalência da doença é baixa entre pacientes em hemodiálise no sul do Brasil. As diferenças observadas entre os gêneros, com exceção da ureia pós, não foram significantes. Os dados encontrados são diferentes dos reportados na América do Norte e Europa.

  3. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.

    Directory of Open Access Journals (Sweden)

    Gilberto A Santiago

    Full Text Available Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV. There are four genetically distinct DENVs (DENV-1-4 that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

  4. Enhancing the sensitivity of Dengue virus serotype detection by RT-PCR among infected children in India.

    Science.gov (United States)

    Ahamed, Syed Fazil; Vivek, Rosario; Kotabagi, Shalini; Nayak, Kaustuv; Chandele, Anmol; Kaja, Murali-Krishna; Shet, Anita

    2017-06-01

    Dengue surveillance relies on reverse transcription-polymerase chain reaction (RT-PCR), for confirmation of dengue virus (DENV) serotypes. We compared efficacies of published and modified primer sets targeting envelope (Env) and capsid-premembrane (C-prM) genes for detection of circulating DENV serotypes in southern India. Acute samples from children with clinically-diagnosed dengue were used for RT-PCR testing. All samples were also subjected to dengue serology (NS1 antigen and anti-dengue-IgM/IgG rapid immunochromatographic assay). Nested RT-PCR was performed on viral RNA using three methods targeting 654bp C-prM, 511bp C-prM and 641bp Env regions, respectively. RT-PCR-positive samples were validated by population sequencing. Among 171 children with suspected dengue, 121 were dengue serology-positive and 50 were dengue serology-negative. Among 121 serology-positives, RT-PCR detected 91 (75.2%) by CprM654, 72 (59.5%) by CprM511, and 74 (61.1%) by Env641. Among 50 serology-negatives, 10 (20.0%) were detected by CprM654, 12 (24.0%) by CprM511, and 11 (22.0%) by Env641. Overall detection rate using three methods sequentially was 82.6% (100/121) among serology-positive and 40.0% (20/50) among serology-negative samples; 6.6% (8/120) had co-infection with multiple DENV serotypes. We conclude that detection of acute dengue was enhanced by a modified RT-PCR method targeting the 654bp C-prM region, and further improved by using all three methods sequentially. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus.

    Science.gov (United States)

    Santiago, Gilberto A; Vergne, Edgardo; Quiles, Yashira; Cosme, Joan; Vazquez, Jesus; Medina, Juan F; Medina, Freddy; Colón, Candimar; Margolis, Harold; Muñoz-Jordán, Jorge L

    2013-01-01

    Dengue is an acute illness caused by the positive-strand RNA dengue virus (DENV). There are four genetically distinct DENVs (DENV-1-4) that cause disease in tropical and subtropical countries. Most patients are viremic when they present with symptoms; therefore, RT-PCR has been increasingly used in dengue diagnosis. The CDC DENV-1-4 RT-PCR Assay has been developed as an in-vitro diagnostic platform and was recently approved by the US Food and Drug Administration (FDA) for detection of dengue in patients with signs or symptoms of mild or severe dengue. The primers and probes of this test have been designed to detect currently circulating strains of DENV-1-4 from around the world at comparable sensitivity. In a retrospective study with 102 dengue cases confirmed by IgM anti-DENV seroconversion in the convalescent sample, the RT-PCR Assay detected DENV RNA in 98.04% of the paired acute samples. Using sequencing as a positive indicator, the RT-PCR Assay had a 97.92% positive agreement in 86 suspected dengue patients with a single acute serum sample. After extensive validations, the RT-PCR Assay performance was highly reproducible when evaluated across three independent testing sites, did not produce false positive results for etiologic agents of other febrile illnesses, and was not affected by pathological levels of potentially interfering biomolecules. These results indicate that the CDC DENV-1-4 RT-PCR Assay provides a reliable diagnostic platform capable for confirming dengue in suspected cases.

  6. Virus isolation and molecular characterization of canine distemper virus by RT-PCR from a mature dog with multifocal encephalomyelit Isolamento e caracterização molecular do vírus da cinomose canina por RT-PCR a partir de um cão adulto com encefalomielite multifocal

    Directory of Open Access Journals (Sweden)

    Alexandre Mendes Amude

    2007-06-01

    Full Text Available A case of multifocal distemper encephalomyelitis in a mature dog is described. In the presented case the ante mortem clinical diagnosis of canine distemper virus (CDV infection could not be ideally performed due to the absence of typical signs of distemper, such as myoclonus and systemic signs accompanying the nervous signs. The definitive diagnosis of distemper encephalomyelitis was only carried out at post mortem through virus isolation in cell culture from fresh central nervous system (CNS fragments and CDV nucleoprotein gene detection in the CNS by RT-PCR.Descreve-se um caso de encefalomielite multifocal pela cinomose em um cão adulto. No caso apresentado o diagnóstico clínico da infecção pelo vírus da cinomose canina (CDV não pode ser adequadamente realizado devido à ausência de sinais típicos da enfermidade, tais como mioclonia e sinais sistêmicos. O diagnóstico definitivo somente foi possível post mortem pelo isolamento do CDV em cultivo celular a partir dos fragmentos frescos do sistema nervoso central (SNC e pela detecção do gene da nucleoproteína do CDV em fragmentos do SNC por meio da RT-PCR.

  7. Detection of ROS1 Gene Rearrangement in Lung Adenocarcinoma: Comparison of IHC, FISH and Real-Time RT-PCR

    OpenAIRE

    Shan, Ling; Lian, Fang; Guo, Lei; Qiu, Tian; Ling, Yun; Ying, Jianming; Lin, Dongmei

    2015-01-01

    Aims To compare fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and quantitative real-time reverse transcription-PCR (qRT-PCR) assays for detection of ROS1 fusion in a large number of ROS1-positive lung adenocatcinoma (ADC) patients. Methods Using IHC analysis, sixty lung ADCs including 16 cases with ROS1 protein expression and 44 cases without ROS1 expression were selected for this study. The ROS1 fusion status was examined by FISH and qRT-PCR assay. Results Among 60 ca...

  8. Simultaneous detection of Plasmodium vivax and Plasmodium falciparum gametocytes in clinical isolates by multiplex-nested RT-PCR.

    Science.gov (United States)

    Kuamsab, Napaporn; Putaporntip, Chaturong; Pattanawong, Urassaya; Jongwutiwes, Somchai

    2012-06-10

    Gametocyte carriage is essential for malaria transmission and endemicity of disease; thereby it is a target for malaria control strategies. Malaria-infected individuals may harbour gametocytes below the microscopic detection threshold that can be detected by reverse transcription polymerase chain reaction (RT-PCR) targeting gametocyte-specific mRNA. To date, RT-PCR has mainly been applied to the diagnosis of Plasmodium falciparum gametocytes but very limited for that of Plasmodium vivax. A multiplex-nested RT-PCR targeting Pfs25 and Pvs25 mRNA specific to mature gametocytes of P. falciparum and P. vivax, respectively, was developed. The assay was evaluated using blood samples collected in rainy and dry seasons from febrile patients,in a malaria-endemic area in Thailand. Malaria diagnosis was performed by Giemsa-stained blood smears and 18S rRNA PCR. The multiplex-nested RT-PCR detected Pfs25 mRNA in 75 of 86 (87.2%) P. falciparum-infected individuals and Pvs25 mRNA in 82 of 90 (91.1%) P. vivax malaria patients diagnosed by 18S rRNA PCR. Gametocytes were detected in 38 (eight P. falciparum and 30 P. vivax) of 157 microscopy positive samples, implying that a large number of patients harbour sub-microscopic gametocytaemia. No seasonal differences in gametocyte carriage were observed for both malaria species diagnosed by multiplex-nested RT-PCR. With single-nested RT-PCR targeting Pfs25 or Pvs25 mRNA as standard, the multiplex-nested RT-PCR offered sensitivities of 97.4% and 98.9% and specificities of 100% and 98.8% for diagnosing mature gametocytes of P. falciparum and P. vivax, respectively. The minimum detection limit of the multiplex-nested PCR was 10 copies of templates. The multiplex-nested RT-PCR developed herein is useful for simultaneous assessment of both P. falciparum and P. vivax gametocyte carriage that is prevalent and generally sympatric in several malaria-endemic areas outside Africa.

  9. Sobrecarga de trabalho da Enfermagem e incidentes e eventos adversos em pacientes internados em UTI

    Directory of Open Access Journals (Sweden)

    Marcia Cristina Zago Novaretti

    2014-10-01

    Full Text Available Estudo prospectivo, tipo coorte que visou identificar a influência da sobrecarga de trabalho da Enfermagem na ocorrência de incidentes sem lesão e eventos adversos em 399 pacientes internados em Unidades de Terapia Intensiva (UTI. Para coleta de dados, foi aplicado um questionário estruturado e feita análise de prontuários. Nessas admissões, aproximadamente 78% dos incidentes sem lesão e eventos adversos em pacientes foram relacionados à esfera da Enfermagem. Essas ocorrências foram atribuídas à sobrecarga de trabalho, aumentaram o número de dias de internação e o risco de óbito dos pacientes estudados. É fundamental que os gerentes de enfermagem atuem no processo de gestão de pessoas no âmbito hospitalar, evitando a sobrecarga de trabalho e proporcionando, consequentemente, aumento da segurança do paciente.

  10. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

    Directory of Open Access Journals (Sweden)

    Porceddu Enrico

    2009-02-01

    Full Text Available Abstract Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability

  11. Iatrogenia em pacientes idosos hospitalizados Iatrogeny in hospitalized elderly patients

    Directory of Open Access Journals (Sweden)

    Eurico T. Carvalho-Filho

    1998-02-01

    Full Text Available Analisar as complicações iatrogênicas apresentadas por idosos hospitalizados. Estudo retrospectivo dos prontuários de 96 pacientes, 48 do sexo masculino e 48 do feminino, com idades variando de 60 a 93 anos (média: 75,7 anos, hospitalizados durante o ano de 1995 em enfermaria geriátrica. A análise da evolução dos pacientes durante o período de hospitalização permitiu evidenciar: 1 em 42 (43,7% pacientes ocorreram uma ou mais complicações iatrogênicas, num total de 56 episódios; 2 manifestações relacionadas aos procedimentos diagnósticos corresponderam a 17,9% das iatrogenias; 3 alterações relacionadas às medidas terapêuticas corresponderam a 58,9%, sendo 32,1% referentes à terapêutica farmacológica e 26,8% a outros procedimentos terapêuticos; 4 manifestações iatrogênicas não relacionadas diretamente às afecções (úlceras de decúbito, quedas e fraturas corresponderam a 23,2%; 5 a presença de manifestações iatrogênicas correlacionou-se com período mais prolongado de internação; 6 cinco pacientes faleceram em conseqüência direta de complicações iatrogênicas. A iatrogenia é freqüente em pacientes idosos hospitalizados, podendo determinar manifestações graves e mesmo fatais. Como uma significativa proporção dessas complicações pode ser evitada através de medidas adequadas, deve-se procurar identificar suas causas e desenvolver métodos para previni-la ou reduzir seus efeitos.PURPOSE: To evaluate the iatrogenic complications in hospitalized elderly patients. MATERIAL AND METHOD: Review of the medical records of 96 patients, 48 men and 48 women, aged 60 to 93 years (75.7 years on average, hospitalized in a geriatric ward during 1995. RESULTS: The study of the medical records of the patients showed: 1 forty-two (43.7% of the elderly had one or more iatrogenic illnesses, with a total of 56 occurrences; 2 complications due to diagnostic tests corresponded to 17.9% of the iatrogenic disorders; 3

  12. Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

    Directory of Open Access Journals (Sweden)

    Stephanie L Angione

    Full Text Available As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR. Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic, H1N1s (seasonal, and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 10(4 copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.

  13. RT-PCR analysis of dystrophin mRNA in DND/BMD patients

    Energy Technology Data Exchange (ETDEWEB)

    Ciafaloni, E.; Silva, H.A.R. de; Roses, A.D. [Duke Univ. Medical Center, Durham, NC (United States)

    1994-09-01

    Duchenne and Becker muscular dystrophies (DMD, BMD) are X-linked recessive disorders caused by mutations in the dystrophin (dys) gene. The majority of these mutations are intragenic deletions of duplications routinely detected by Southern biots and multiplex PCR. The remainder are very likely, smaller mutations, mostly point-mutations. Detection of these mutations is very difficult due to the size and complexity of the dys gene. We applied RT-PCR to analyse the entire dys mRNA of three DMD patients with no detectable genomic defect. In two unrelated patients, a duplication of the 62 bp exon 2 was identified. This causes a frameshift sufficient to explain the DMD phenotype. In the third patient, who had congenital DMD and severe mental retardation, a complex pattern of aberrant splicing at the 3-prime exons 67-79 was observed. Sural nerve biopsy in this patient showed the complete absence of Dp116. PCR-SSCP studies are presently in progress to identify the mutations responsible for the aberrant splicing patterns.

  14. Multiplex RT-PCR and Automated Microarray for Detection of Eight Bovine Viruses.

    Science.gov (United States)

    Lung, O; Furukawa-Stoffer, T; Burton Hughes, K; Pasick, J; King, D P; Hodko, D

    2017-12-01

    Microarrays can be a useful tool for pathogen detection as it allow for simultaneous interrogation of the presence of a large number of genetic sequences in a sample. However, conventional microarrays require extensive manual handling and multiple pieces of equipment for printing probes, hybridization, washing and signal detection. In this study, a reverse transcription (RT)-PCR with an accompanying novel automated microarray for simultaneous detection of eight viruses that affect cattle [vesicular stomatitis virus (VSV), bovine viral diarrhoea virus type 1 and type 2, bovine herpesvirus 1, bluetongue virus, malignant catarrhal fever virus, rinderpest virus (RPV) and parapox viruses] is described. The assay accurately identified a panel of 37 strains of the target viruses and identified a mixed infection. No non-specific reactions were observed with a panel of 23 non-target viruses associated with livestock. Vesicular stomatitis virus was detected as early as 2 days post-inoculation in oral swabs from experimentally infected animals. The limit of detection of the microarray assay was as low as 1 TCID 50 /ml for RPV. The novel microarray platform automates the entire post-PCR steps of the assay and integrates electrophoretic-driven capture probe printing in a single user-friendly instrument that allows array layout and assay configuration to be user-customized on-site. © 2016 Her Majesty the Queen in Right of Canada.

  15. Single reaction, real time RT-PCR detection of all known avian and human metapneumoviruses.

    Science.gov (United States)

    Lemaitre, E; Allée, C; Vabret, A; Eterradossi, N; Brown, P A

    2018-01-01

    Current molecular methods for the detection of avian and human metapneumovirus (AMPV, HMPV) are specifically targeted towards each virus species or individual subgroups of these. Here a broad range SYBR Green I real time RT-PCR was developed which amplified a highly conserved fragment of sequence in the N open reading frame. This method was sufficiently efficient and specific in detecting all MPVs. Its validation according to the NF U47-600 norm for the four AMPV subgroups estimated low limits of detection between 1000 and 10copies/μL, similar with detection levels described previously for real time RT-PCRs targeting specific subgroups. RNA viruses present a challenge for the design of durable molecular diagnostic test due to the rate of change in their genome sequences which can vary substantially in different areas and over time. The fact that the regions of sequence for primer hybridization in the described method have remained sufficiently conserved since the AMPV and HMPV diverged, should give the best chance of continued detection of current subgroups and of potential unknown or future emerging MPV strains. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Diagnosis of intestinal parasites in a rural community of Venezuela : Advantages and disadvantages of using microscopy or RT-PCR

    NARCIS (Netherlands)

    Incani, Renzo Nino; Ferrer, Elizabeth; Hoek, Denise; Ramak, Robbert; Roelfsema, Jeroen; Mughini-Gras, Lapo; Kortbeek, Titia M.; Pinelli, Elena

    2017-01-01

    A cross-sectional study was carried out to determine the prevalence and diagnostic performance of microscopy and real time PCR (RT-PCR) for 14 intestinal parasites in a Venezuelan rural community with a long history of persistent intestinal parasitic infections despite the implementation of regular

  17. Detection of canine distemper virus nucleocapsid protein gene in canine peripheral blood mononuclear cells by RT-PCR.

    Science.gov (United States)

    Shin, Y; Mori, T; Okita, M; Gemma, T; Kai, C; Mikami, T

    1995-06-01

    For a rapid diagnosis of canine distemper virus (CDV) infection, the reverse transcription-PCR (RT-PCR) was carried out to detect CDV nucleoprotein (NP) gene from canine peripheral blood mononuclear cells (PBMCs). Two sets of primers were targeted to two regions of NP gene of CDV Onderstepoort strain. The NP gene fragments were well amplified by the RT-PCR in each of the RNA extracts from Vero cells infected with 6 laboratory strains of CDV including Onderstepoort strain, and from PBMCs of a dog experimentally infected with CDV. The amplified NP gene was detected in 17 of 32 samples from dogs which were clinically suspected for CDV infection at veterinary hospitals. No RT-PCR product was found in 52 samples from healthy dogs including 40 specific pathogen free beagles vaccinated with an attenuated live virus-vaccine for CDV and 12 stray dogs. The RT-PCR provides a fast, sensitive, and supplementary method for the diagnosis of CDV infection in dogs.

  18. Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.

    Science.gov (United States)

    Singh, R P; Nie, X; Singh, M; Coffin, R; Duplessis, P

    2002-01-01

    Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.

  19. Detection of EML4-ALK in lung adenocarcinoma using pleural effusion with FISH, IHC, and RT-PCR methods.

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    Leilei Liu

    Full Text Available Anaplastic lymphoma kinase (ALK and echinoderm microtubule-associated protein-like 4 (EML4 gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC, leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH, reverse transcription-polymerase chain reaction (RT-PCR, and immunohistochemistry (IHC. EML4-ALK was detected in three of 66 patient samples (4.5% with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients.

  20. Molecular detection of infectious bronchitis and Newcastle disease viruses in broiler chickens with respiratory signs using Duplex RT-PCR.

    Science.gov (United States)

    Saba Shirvan, Aylar; Mardani, Karim

    2014-01-01

    Infectious bronchitis (IB) and Newcastle disease (ND) are highly contagious and the most economically important diseases of the poultry affecting respiratory tract and causing economic losses in poultry industry throughout the world. In the present study, the simultaneous detection and differentiation of causative agents of these diseases were investigated using duplex-RT-PCR. RNA was extracted from vaccinal and reference strains of infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) and then cDNA was synthesized. Using two universal primer sets for detection of IBV and NDV, the duplex-RT-PCR was developed. In order to assess the efficiency of the developed duplex RT-PCR, a number of 12 broiler farms with the symptoms of respiratory tract infection was sampled (trachea, lung and kidney were sampled from affected birds suspicious for IBV and NDV infections). After RNA extraction from tissues and cDNA synthesis, the presence of IBV and NDV genome were investigated using duplex-PCR. The results showed that three of twelve examined broiler farms were positive for IBV and two farms were positive for NDV and IBV. The results revealed that the duplex-RT-PCR is a quick and sensitive procedure for simultaneously detecting IBV and NDV in birds with respiratory infections.

  1. Effect of specific or random c-DNA priming on sensitivity of tyrosinase nested RT-PCR : Potential clinical relevance

    NARCIS (Netherlands)

    Calogero, A; Hospers, GAP; Timmer-Bosscha, H; Mulder, NH; Schraffordt Koops, H.

    2000-01-01

    The reverse transcriptase polymerase chain reaction (RT-PCR) can be of clinical relevance in identifying malignant melanoma cells in blood or tissues of patients at risk for disseminated melanoma. The diagnostic value of this marker however, is still controversial. The objective of this study was to

  2. Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

    Directory of Open Access Journals (Sweden)

    Parida Manmohan

    2008-01-01

    Full Text Available Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Japanese encephalitis, West Nile, Yellow fever and alphavirus (Chikungunya. The feasibility of M-RT-PCR assay for clinical diagnosis was validated with 620 acute phase dengue patient sera samples of recent epidemics in India. The comparative evaluation vis a vis conventional virus isolation revealed higher sensitivity. None of the forty healthy serum samples screened in the present study revealed any amplification, thereby establishing specificity of the reported assay for dengue virus only. Conclusion These findings clearly suggested that M-RT-PCR assay reported in the present study is the rapid and cost-effective method for simultaneous detection as well as typing of the dengue virus in acute phase patient serum samples. Thus, the M-RT-PCR assay developed in this study will serve as a very useful tool for rapid diagnosis and typing of dengue infections in endemic areas.

  3. Simple, specific molecular typing of dengue virus isolates using one-step RT-PCR and restriction fragment length polymorphism.

    Science.gov (United States)

    Ortiz, Alma; Capitan, Zeuz; Mendoza, Yaxelis; Cisneros, Julio; Moreno, Brechla; Zaldivar, Yamitzel; Garcia, Mariana; Smith, Rebecca E; Motta, Jorge; Pascale, Juan Miguel

    2012-10-01

    A one-step RT-PCR and one-enzyme RFLP was used to detect and distinguish among flaviviruses, including the four serotypes of dengue and the St. Louis Encephalitis, West Nile and Yellow Fever viruses in cultured virus samples or acute-phase human serum. Using a previously described RT-PCR, but novel RFLP procedure, results are obtained in 24 h with basic PCR and electrophoresis equipment. There is 95% agreement between RT-PCR/RFLP results and those achieved by indirect immunofluorescence assays, and 100% agreement between RT-PCR/RFLP results and gene sequencing. This method is more rapid than tests of cytopathic effect based on virus isolation in tissue culture, and simpler than real-time PCR. It does not require specialized equipment, radioisotopes or computer analysis and is a method that can be applied widely in the developing world. It allows for prompt determination of whether a flavivirus is the cause of illness in a febrile patient, rapid identification of dengue serotypes in circulation, and improved patient management in cases where prior dengue exposure make dengue hemorrhagic fever or dengue shock syndrome a risk. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

    OpenAIRE

    Arun, Alok; Bauml?, V?ronique; Amelot, Ga?l; Nieberding, Caroline M.

    2015-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at ident...

  5. Proteomic analysis and qRT-PCR verification of temperature response to Arthrospira (Spirulina platensis.

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    Wang Huili

    Full Text Available Arthrospira (Spirulina platensis (ASP is a representative filamentous, non-N2-fixing cyanobacterium that has great potential to enhance the food supply and possesses several valuable physiological features. ASP tolerates high and low temperatures along with highly alkaline and salty environments, and can strongly resist oxidation and irradiation. Based on genomic sequencing of ASP, we compared the protein expression profiles of this organism under different temperature conditions (15°C, 35°Cand 45°C using 2-DE and peptide mass fingerprinting techniques. A total of 122 proteins having a significant differential expression response to temperature were retrieved. Of the positively expressed proteins, the homologies of 116 ASP proteins were found in Arthrospira (81 proteins in Arthrospira platensis str. Paraca and 35 in Arthrospira maxima CS-328. The other 6 proteins have high homology with other microorganisms. We classified the 122 differentially expressed positive proteins into 14 functions using the COG database, and characterized their respective KEGG metabolism pathways. The results demonstrated that these differentially expressed proteins are mainly involved in post-translational modification (protein turnover, chaperones, energy metabolism (photosynthesis, respiratory electron transport, translation (ribosomal structure and biogenesis and carbohydrate transport and metabolism. Others proteins were related to amino acid transport and metabolism, cell envelope biogenesis, coenzyme metabolism and signal transduction mechanisms. Results implied that these proteins can perform predictable roles in rendering ASP resistance against low and high temperatures. Subsequently, we determined the transcription level of 38 genes in vivo in response to temperature and identified them by qRT-PCR. We found that the 26 differentially expressed proteins, representing 68.4% of the total target genes, maintained consistency between transcription and

  6. Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth disease virus and look-alike disease viruses

    Energy Technology Data Exchange (ETDEWEB)

    Hindson, B J; Reid, S M; Baker, B R; Ebert, K; Ferris, N P; Bentley Tammero, L F; Lenhoff, R J; Naraghi-Arani, P; Vitalis, E A; Slezak, T R; Hullinger, P J; King, D P

    2007-07-26

    A high-throughput multiplexed assay was developed for the differential laboratory diagnosis of foot-and-mouth disease virus (FMDV) from viruses which cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses using multiplexed reverse transcription PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the seventeen primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR was evaluated using 287 field samples, including 248 (true positive n= 213, true negative n=34) from suspect cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true negative samples collected from healthy animals. The mRT-PCR assay results were compared with two singleplex rRT-PCR assays, using virus isolation with antigen-ELISA as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% [95% C.I. 89.8-96.4%], compared to 98.1% [95% C.I. 95.3-99.3%] for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n=2) and bovine viral diarrhea virus (n=2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized using focused single-target rRT-PCR assays.

  7. Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba skin biopsies

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    Casini Silvia

    2006-09-01

    Full Text Available Abstract Background Odontocete cetaceans occupy the top position of the marine food-web and are particularly sensitive to the bioaccumulation of lipophilic contaminants. The effects of environmental pollution on these species are highly debated and various ecotoxicological studies have addressed the impact of xenobiotic compounds on marine mammals, raising conservational concerns. Despite its sensitivity, quantitative real-time PCR (qRT-PCR has never been used to quantify gene induction caused by exposure of cetaceans to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. A systematic evaluation of potential reference genes in cetacean skin biopsies is presented, in order to validate future qRT-PCR studies aiming at using the expression of selected genes as non-lethal biomarkers. Results Ten commonly used housekeeping genes (HKGs were partially sequenced in the striped dolphin (Stenella coeruleoalba and, for each gene, PCR primer pairs were specifically designed and tested in qRT-PCR assays. The expression of these potential control genes was examined in 30 striped dolphin skin biopsy samples, obtained from specimens sampled in the north-western Mediterranean Sea. The stability of selected control genes was determined using three different specific VBA applets (geNorm, NormFinder and BestKeeper which produce highly comparable results. Glyceraldehyde-3P-dehydrogenase (GAPDH and tyrosine 3-monooxygenase (YWHAZ always rank as the two most stably expressed HKGs according to the analysis with geNorm and Normfinder, and are defined as optimal control genes by BestKepeer. Ribosomal protein L4 (RPL4 and S18 (RPS18 also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC, phosphoglycerate kinase 1 (PGK1, hypoxanthine ribosyltransferase (HPRT1 and β-2-microglobin (B2M show variable expression

  8. Epidemiology of Human Parechovirus Type1 in Clinical Samples from Children with Gastroenteritis Using RT-PCR

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    Dabirmanesh B

    2011-03-01

    Full Text Available Background and Objectives: Human parechovirus type-1 (HPeV-1 is a genus of picornaviridea with a single stranded positive sense RNA genome. In general it seems to be responsible for more gastrointestinal and respiratory syndromes and less responsible for central nervous system (CNS symptoms. Since there is no accurate information about diagnosis and epidemiology of HPeV-1 in Iran and it is very important to distinguish between viral and bacterial diarrhea to decrease the unnecessary use of antibiotics, this study aimed at rapid detection and epidemiology of HPeV-1 in stool samples from children with gastroenteritis using specific RT-PCR. Methods: Viral RNA was isolated from 472 stool samples from children (under 4 years old with diarrhea; CDNA was prepared and amplified using specific primers from 5′untranslated region (5′ UTR of HPeV-1 genome by nested RT-PCR. Amplified DNA product was electrophoresed on 1% agarose gel and a single band of 265 bp was obtained. Data were analyzed by SPSS software. We also performed a comparison between the cell culture (Vero and RT-PCR method for HPeV1 detection.Results: Out of 472 samples examined during two years, 112 samples were HpeV-1 positive (23.7%. The results showed that the prevalence of this virus was in children under one year (6-12 months old with diarrhea (p=0.036 in spring and autumn (p<0.001. Boys had more positive cases than the girls (p<0.001. Out of 20 samples which were found positive by HPeV1 RT-PCR only three of them showed CPE on Vero Cells after a week.Conclusion: The results revealed that RT-PCR is a more practical and sensitive technique for HPeV-1 detection directly from clinical samples, which is valuable for epidemiology. Also, the rapid detection of HPeV1 by RT-PCR can decrease both the unnecessary use of antibiotics and the costs in clinical practice

  9. Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process

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    Kohn Michael

    2007-10-01

    Full Text Available Abstract Background Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants. Results We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/ Conclusion We present proof of the principle that a mechanistically based model can be fit to observations

  10. Selection of Housekeeping Genes for Transgene Expression Analysis in Eucommia ulmoides Oliver Using Real-Time RT-PCR

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    Ren Chen

    2010-01-01

    Full Text Available In order to select appropriate housekeeping genes for accurate calibration of experimental variations in real-time (RT- PCR results in transgene expression analysis, particularly with respect to the influence of transgene on stability of endogenous housekeeping gene expression in transgenic plants, we outline a reliable strategy to identify the optimal housekeeping genes from a set of candidates by combining statistical analyses of their (RT- PCR amplification efficiency, gene expression stability, and transgene influences. We used the strategy to select two genes, ACTα and EF1α, from 10 candidate housekeeping genes, as the optimal housekeeping genes to evaluate transgenic Eucommia ulmoides Oliver root lines overexpressing IPPI or FPPS1 genes, which are involved in isoprenoid biosynthesis.

  11. Rapid detection of bovine coronavirus by a semi-nested RT-PCR Detecção rápida do Coronavírus Bovino (BCoV por meio de uma semi-nested RT-PCR

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    Karen M. Asano

    2009-11-01

    Full Text Available Bovine coronavirus (BCoV is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256 in DEPC treated ultra-pure water, in fetal bovine serum (FBS and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694. The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.O Coronavírus bovino (BCoV pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doen

  12. An Evidence-Based Approach to Detection by DASI-ELISA and RT-PCR in Dormant Period

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    Antonio Olmos

    2008-01-01

    Full Text Available An evidence-based approach, such as those developed in clinical and veterinary medicine, was applied to the detection of Plum pox virus (PPV during the dormant period. A standardized methodology was used for the calculation of parameters of the operational capacity of DASI-ELISA and RT-PCR in wintertime. These methods are routinely handled to test the sanitary status of plants in national or international trading and in those cases concerning export-import of plant materials. Diagnosis often has to be performed during the dormant period, when plant material is commercialized. Some guidelines to interpret diagnostic results of wintertime are provided in an attempt to minimize risks associated with the methods and over-reliance on the binary outcome of a single assay. In order to evaluate if a complementary test increased the confidence of PPV diagnosis when discordant results between DASI-ELISA and RT-PCR are obtained, NASBA-FH also was included. Likelihood ratios of each method were estimated based on the sensitivity and specificity obtained in wintertime. Subsequently, a Bayesian approach was performed to calculate post-test probability of PPV infection in spring. Results of evidence-based approach show that different PPV prevalences require different screening tests. Thus, at very low PPV prevalence levels DASI-ELISA should be used as the election method, whilst at the highest PPV prevalence levels RT-PCR should be performed. NASBA-FH could be used at medium prevalences to clarify discordances between DASI-ELISA and RT-PCR.

  13. A community resource for high-throughput quantitative RT-PCR analysis of transcription factor gene expression in Medicago truncatula

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    Redman Julia C

    2008-07-01

    Full Text Available Abstract Background Medicago truncatula is a model legume species that is currently the focus of an international genome sequencing effort. Although several different oligonucleotide and cDNA arrays have been produced for genome-wide transcript analysis of this species, intrinsic limitations in the sensitivity of hybridization-based technologies mean that transcripts of genes expressed at low-levels cannot be measured accurately with these tools. Amongst such genes are many encoding transcription factors (TFs, which are arguably the most important class of regulatory proteins. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is the most sensitive method currently available for transcript quantification, and one that can be scaled up to analyze transcripts of thousands of genes in parallel. Thus, qRT-PCR is an ideal method to tackle the problem of TF transcript quantification in Medicago and other plants. Results We established a bioinformatics pipeline to identify putative TF genes in Medicago truncatula and to design gene-specific oligonucleotide primers for qRT-PCR analysis of TF transcripts. We validated the efficacy and gene-specificity of over 1000 TF primer pairs and utilized these to identify sets of organ-enhanced TF genes that may play important roles in organ development or differentiation in this species. This community resource will be developed further as more genome sequence becomes available, with the ultimate goal of producing validated, gene-specific primers for all Medicago TF genes. Conclusion High-throughput qRT-PCR using a 384-well plate format enables rapid, flexible, and sensitive quantification of all predicted Medicago transcription factor mRNAs. This resource has been utilized recently by several groups in Europe, Australia, and the USA, and we expect that it will become the 'gold-standard' for TF transcript profiling in Medicago truncatula.

  14. Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

    OpenAIRE

    Parida Manmohan; Shrivastava Ambuj; Santhosh SR; Dash Paban; Saxena Parag; Rao PV

    2008-01-01

    Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Jap...

  15. qRT-PCR quantification of the biological control agent Trichoderma harzianum in peat and compost-based growing media.

    Science.gov (United States)

    Beaulieu, Robert; López-Mondéjar, Rubén; Tittarelli, Fabio; Ros, Margarita; Pascual, José Antonio

    2011-02-01

    To ensure proper use of Trichoderma harzianum in agriculture, accurate data must be obtained in population monitoring. The effectiveness of qRT-PCR to quantify T. harzianum in different growing media was compared to the commonly used techniques of colony counting and qPCR. Results showed that plate counting and qPCR offered similar T. harzianum quantification patterns of an initial rapid increase in fungal population that decreased over time. However, data from qRT-PCR showed a population curve of active T. harzianum with a delayed onset of initial growth which then increased throughout the experiment. Results demonstrated that T. harzianum can successfully grow in these media and that qRT-PCR can offer a more distinct representation of active T. harzianum populations. Additionally, compost amended with T. harzianum exhibited a lower Fusarium oxysporum infection rate (67%) and lower percentage of fresh weight loss (11%) in comparison to amended peat (90% infection rate, 23% fresh weight loss). Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

    Science.gov (United States)

    Del Prete, D; Forino, M; Gambaro, G; D'Angelo, A; Baggio, B; Anglani, F

    1998-01-01

    Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

  17. Profilaxia para úlcera de estresse em pacientes internados em UTI pediátrica

    Directory of Open Access Journals (Sweden)

    Taisa E. Araujo

    2010-12-01

    Full Text Available OBJETIVO: Avaliar a utilização de profilaxia para úlcera de estresse (UE, em pacientes internados, de cinco unidades de terapia intensiva pediátrica (UTIP de Porto Alegre (RS. MÉTODOS: Estudo multicêntrico, prospectivo, transversal, observacional. Foram avaliados os prontuários dos pacientes internados em dia definido para visitação, entre abril de 2006 e fevereiro de 2007, excluindo os avaliados em visitas anteriores e aqueles com hemorragia digestiva alta na admissão. Foram avaliados a idade, o gênero, o diagnóstico na admissão, a gravidade da doença, o uso de profilaxia para UE, a sua justificativa e o medicamento profilático utilizado como primeira escolha. As variáveis foram descritas como frequências absoluta e relativa, ou média e desvio padrão/mediana, e intervalo interquartil (IQ. Os testes qui-quadrado de Pearson, de tendência linear, ou exato de Fisher foram utilizados para avaliar as associações. O nível de significância adotado foi de 5%, sendo estatisticamente significativo p < 0,05. RESULTADOS: Foram avaliados 398 pacientes, sendo 57% do gênero masculino. A mediana de idade foi de 16 meses (IQ4-65 e mediana de permanência em UTIP foi de 4 dias (IQ1-9. O principal motivo de internação foi doença respiratória (32,7%. Usaram profilaxia 77,5% dos pacientes, variando de 66 a 91%; a ventilação mecânica (22,3% foi a justificativa mais prevalente, seguida de rotina informal do serviço (21,4%. Apenas uma das UTIP tinha protocolo assistencial para profilaxia de UE. A ranitidina foi o medicamento mais empregado (84,5%. CONCLUSÕES: O uso de profilaxia para UE foi prática frequente nas UTIP avaliadas, sendo a ranitidina a droga de escolha. Entre as justificativas, a ventilação mecânica e o uso baseado em rotinas institucionais foram as mais prevalentes.

  18. EFEITOS DAS PISTAS VISUAIS NA MARCHA EM PACIENTES COM PARKINSON

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    Ana Sousa

    2015-05-01

    Full Text Available A  doença de Parkinson (DP é uma doença neurodegenerativa crónica, resultante de disfunções nos gânglios basais, localizados no cérebro, relacionados com o controle motor. Um dos sintomas mais incapacitantes da DP é a festinação, caracterizada pela pobreza dos movimentos, passos curtos, pés rentes ao chão e uma diminuição da velocidade. Investigações recentes recorrem ao treino da marcha com pistas sensoriais, tais como as visuais, para a reabilitação do congelamento ou freezing da marcha parkinsoniana, com o objetivo de desviar a função dos núcleos da base com o fim de regular a função motora deficitáriaObjetivo: Esta revisão sistémica da Literatura foi desenvolvida com o objetivo de analisar os efeitos das pistas visuais na marcha em pacientes com DP.

  19. A novel RT-PCR for the detection of Helicobacter pylori and identification of clarithromycin resistance mediated by mutations in the 23S rRNA gene.

    Science.gov (United States)

    Redondo, Javier Jareño; Keller, Peter M; Zbinden, Reinhard; Wagner, Karoline

    2018-01-01

    In this study we evaluated the commercially available LightMix® RT-PCR assay for Helicobacter pylori detection and identification of clarithromycin (CLR) resistance in culture and clinical specimens (gastric biopsies and stool). The H. pylori LightMix® RT-PCR detects a 97bp long fragment of the 23S rRNA gene and allows the identification of 3 distinct point mutations conferring CLR resistance via melting curve analysis. The performance of the H. pylori LightMix® RT-PCR was evaluated using a set of 60 H. pylori strains showing phenotypical CLR susceptibility or CLR resistance (Minimum inhibitory concentrations from 0.016 to 256mg/L). We found high concordance (95%) between phenotypical CLR resistance screening by E-Test® and the Lightmix® RT-PCR. Discrepant results were verified by sequencing of the 23S rRNA gene that always confirmed the results obtained by Lightmix® RT-PCR. Furthermore, H. pylori was detected in clinical biopsy and stool specimens by Lightmix® RT-PCR that identified the correct H. pylori genotype. The LightMix® RT-PCR is an accurate, sensitive and easy to use test for H. pylori and CLR resistance detection and can therefore be readily implemented in any diagnostic laboratory. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique.

    Science.gov (United States)

    Nicoś, M; Krawczyk, P; Wojas-Krawczyk, K; Bożyk, A; Jarosz, B; Sawicki, M; Trojanowski, T; Milanowski, J

    2017-12-01

    RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

  1. Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler's murine encephalomyelitis virus and rat theilovirus.

    Science.gov (United States)

    Yuan, Wen; Wang, Jing; Xu, Fengjiao; Huang, Bihong; Lian, Yuexiao; Rao, Dan; Yin, Xueqin; Wu, Miaoli; Zhu, Yujun; Zhang, Yu; Huang, Ren; Guo, Pengju

    2016-10-01

    Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation duplex real-time RT-PCR and conventional RT-PCR. For 439 clinical samples,95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Interações Medicamentosas Potenciais em Pacientes de Unidades de Terapia Intensiva

    OpenAIRE

    Liliana Batista Vieira; Adriano Max Moreira Reis; Rhanna Emanuela Fontenele Lima de Carvalho; Leila Márcia Pereira de Faria; Silvia Helena de Bortoli Cassiani

    2012-01-01

    O objetivo do estudo é determinar a frequência de interações fármaco-fármaco em prescrições de pacientes internados em unidades de terapia intensiva e analisar os fatores associados relativos à farmacoterapia e ao paciente. O estudo descritivo transversal foi realizado em dois hospitais de ensino da região centro oeste do Brasil. As interações medicamentosas potenciais foram identificadas nas prescrições do primeiro dia (24 horas) e do quinto dia (120 horas) de interna...

  3. Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib.

    Science.gov (United States)

    Xu, Chun-Wei; Wang, Wen-Xian; Chen, Yan-Ping; Chen, Yu; Liu, Wei; Zhong, Li-Hua; Chen, Fang-Fang; Zhuang, Wu; Song, Zheng-Bo; Chen, Xiao-Hui; Huang, Yun-Jian; Guan, Yan-Fang; Yi, Xin; Lv, Tang-Feng; Zhu, Wei-Feng; Lu, Jian-Ping; Wang, Xiao-Jiang; Shi, Yi; Lin, Xian-Dong; Chen, Gang; Song, Yong

    2018-04-11

    ALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements. A total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent. Among 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion. VENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for

  4. Clinical utility of RT-PCR in assessing HER 2 gene expression versus traditional IHC and FISH in breast cancer patients.

    Science.gov (United States)

    Suryavanshi, Moushumi; Mehta, Anurag; Jaipuria, Jiten; Kumar, Dushyant; Vishwakarma, Gayatri; Panigrahi, Manoj Kumar; Verma, Haristuti; Saifi, Mumtaz; Sharma, Sanjeev; Tandon, Simran; Doval, D C; Das, Bhudev C

    2018-02-09

    IHC and FISH are used for categorizing HER 2 status in breast cancer at the protein and DNA level, respectively. HER 2 expression at the RNA level is quantitative, cheaper, easier to standardize and free from interobserver variation. 115 consecutive patients were tested by IHC, FISH and RT-PCR (test cohort). Assuming FISH result to be the response variable, ROC curves for RT-PCR ratio were analyzed to label HER 2 negative, equivocal and positive cases as RT-PCR score 1, 2 and 3, respectively. Inter-relationships between RT-PCR, IHC and FISH were defined. 'Clinical benefit' of a test was defined as proportion of patients labeled unequivocally as HER 2 positive or negative. Population for 1 year was simulated constraint to previous reports of HER 2 positivity and IHC category distribution by a meta-analysis of previous studies that evaluated concordance between IHC and FISH to determine HER 2 status (simulation cohort). Four diagnostic pathways in the simulation cohort were defined-(1) initial IHC, followed by FISH (conventional pathway); (2) initial RT-PCR, followed by FISH; (3) initial IHC, followed by RT-PCR and then by FISH; (4) initial RT-PCR, followed by IHC and then by FISH. The clinical benefit of IHC and RT-PCR in the four pathways was analyzed and sensitivity analysis for incremental cost-effectiveness ratio and cost-benefit comapring RT-PCR against IHC, both as first-line tests and among those with IHC score 2 as a reflex second-line test was performed by the Monte Carlo technique. 115 patients comprised the study population. While none with IHC score of 0 or 1 was FISH positive for HER 2, all cases with IHC score of 3 were FISH positive. 43 cases were assigned IHC score of 2. Thus, 72 patients benefited from the initial IHC testing [clinical benefit 62.6%], with the overall concordance between IHC and FISH being 100% for those with IHC score of 0, 1 and 3 (conclusive IHC categories). For RT-PCR with 100% concordance, 15.7% (115-97 = 18) patients

  5. Establishment of realtime RT-PCR assay to detect polio virus in the Acute Flaccid Paralysis laboratory surveillance

    Directory of Open Access Journals (Sweden)

    Nike Susanti

    2016-07-01

    Full Text Available AbstrakLatar belakang: Virus polio indigenous terakhir ditemukan di Indonesia tahun 1995 tetapi ancaman viruspolio impor dan mutasi virus dari Oral Polio Vaccine (OPV menjadi Vaccine Derived Poliovirus (VDPVmasih berlanjut. Tahun 1991 WHO mengembangkan Surveilans Acute Flaccid Paralysis (AFP dan tahun2014, identifikasi virus polio dengan real-time reverse transcriptase Polymerase Chain Reaction (rRTPCRmulai digunakan di Laboratorium Nasional Polio Pusat Biomedis dan Teknologi Dasar Kesehatan.Tujuan dari penggunaan rRT-PCR untuk mendapatkan metode yang cepat dan lebih baik dalam memantausirkulasi dan mutasi virus polio.Metode: Isolat polio positif diidentifikasi menggunakanan rRT PCR dengan kombinasi primer dan probeyang ditetapkan WHO. RNA virus di konversi ke cDNA menggunakan reverse transcriptase lalu diamplifikasimenggunakan taq polymerase. Produk PCR di deteksi dan diidentifikasi dengan hibridisasi menggunakanprobe spesifik. Sintesis cDNA dan reaksi PCR menggunakan primer yang dilekatkan di probe. Kombinasiprimer dan probe menghasilkan identifikasi serotipe dan intratypic differentiation (ITD dari isolat virus.Hasil: Selama tahun 2014, NPL Jakarta menerima 604 kasus AFP dari surveilans dan lima kasusterdeteksi positif mengandung virus polio. Semua spesimen positif mengandung virus polio yang berasaldari vaksin. Dua kasus positif virus polio tipe P2 (40%, satu kasus jenis virus polio P1 (20%, 1 kasusjenis virus polio P3 (20% dan satu kasus virus polio campuran jenis P1 + P2 (20%.Kesimpulan: Real-time PCR dapat digunakan di Laboratorium Polio Jakarta untuk membantu identifikasivirus Polio secara cepat. Tes ini dapat digunakan untuk memantau sirkulasi virus polio pada populasiyang rutin diimunisasi dengan OPV. (Health Science Journal of Indonesia 2016;7:27-31Kata kunci: ITD, Poliovirus, Identification, rRT-PCR AbstractBackground: The last indigenous polio was detected in 1995 but the threat of wild type polio viruses and themutation of Oral

  6. Crises parciais motoras induzidas por movimentos em pacientes diabéticos

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    Glória M. A. S. Tedrus

    1991-12-01

    Full Text Available Levando em conta o número restrito de casos em âmbito internacional e a ausência de publicações na literatura nacional, os autores apresentam três casos de crises motoras focais induzidas por movimentos em pacientes diabéticos com mais de 50 anos de idade. As crises eram numerosas, sendo também desencadeadas ao serem assumidas determinadas posturas. Em dois casos havia hiperglicemia não cetótica. Todos os pacientes apresentavam hemiparesia e, em dois, tal déficit desapareceu após o cotnrole das crises. No outro paciente a hemiparesia era decorrente de acidente vascular cerebral isquêmico instalado 6 meses antes. Síndrome parietal transitória foi observada nos três pacientes. O EEG crítico mostrou, em dois pacientes, descargas na região parieto-occipital e, em um, na região temporal média contralateral. As crises foram resistentes aos anticonvulsivantes, só havendo controle com o tratamento dos distúrbios metabólicos. São discutidos aspectos clínico-eletren-cefalográficos e etiopatogênicos. A pesquisa do caráter reflexo das crises e a dosagem da glicemia devem ser feitas em todos os pacientes idosos com crises motoras focais, face às implicações de ordem terapêutica e prognóstica.

  7. Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses.

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    Dongli Wan

    Full Text Available Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2 were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.

  8. Penerapan Metode Diagnosis Cepat Virus Avian Influenza H5N1 dengan Metode Single Step Multiplex RT-PCR

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    Aris Haryanto

    2010-12-01

    Full Text Available Avian influenza (AI virus is a segmented single stranded (ss RNA virus with negative polarity andbelong to the Orthomyxoviridae family. Diagnose of AI virus can be performed using conventional methodsbut it has low sensitivity and specificity. The objective of the research was to apply rapid, precise, andaccurate diagnostic method for AI virus and also to determine its type and subtype based on the SingleStep Multiplex Reverse Transcriptase-Polymerase Chain Reaction targeting M, H5, and N1 genes. In thismethod M, H5 and NI genes were simultaneously amplified in one PCR tube. The steps of this researchconsist of collecting viral RNAs from 10 different AI samples originated from Maros Disease InvestigationCenter during 2007. DNA Amplification was conducted by Simplex RT-PCR using M primer set. Then, bysingle step multiplex RT-PCR were conducted simultaneously using M, H5 and N1 primers set. The RTPCRproducts were then separated on 1.5% agarose gel, stained by ethidum bromide and visualized underUV transilluminator. Results showed that 8 of 10 RNA virus samples could be amplified by Simplex RTPCRfor M gene which generating a DNA fragment of 276 bp. Amplification using multiplex RT-PCRmethod showed two of 10 samples were AI positive using multiplex RT-PCR, three DNA fragments weregenerated consisting of 276 bp for M gene, 189 bp for H5 gene, and 131 bp for N1. In this study, rapid andeffective diagnosis method for AI virus can be conducted by using simultaneous Single Step Multiplex RTPCR.By this technique type and subtype of AI virus, can also be determined, especially H5N1.

  9. Development of an in situ magnetic beads based RT-PCR method for electrochemiluminescent detection of rotavirus

    Science.gov (United States)

    Zhan, Fangfang; Zhou, Xiaoming

    2012-12-01

    Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL. RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs. Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2'-bipyridyl) ruthenium (TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs. Then the MBs-TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or post-incubation which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study, rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be 104copies/μL. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in food safety field and clinical diagnosis.

  10. Achados oculares em pacientes com mielomeningocele: 72 casos Ocular findings in 72 patients with meningomyelocele

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    Mônica Fialho Cronemberger

    2000-10-01

    Full Text Available Objetivo: Estudar as alterações oculares em pacientes com mielomeningocele. Material e Método: Realizado estudo retrospectivo em 72 pacientes com mielomeningocele, descrevendo as alterações oculares presentes nessa população, além de correlacionar com a presença ou não de hidrocefalia. Resultados: Dos 72 pacientes com mielomeningocele, com idade variando de 5 meses a 18 anos, 64 (88,9% pacientes tinham hidrocefalia. Destes 64 pacientes, 36 (56,3% tinham estrabismo, sendo que 21 (58,3% eram endotrópicos, 13 (36,1% exotrópicos, 1 (2,8% exofórico e 1 (2,8% com estrabismo discinético. Do total dos 72 pacientes estudados 38 (52,8% eram estrábicos. A anisotropia foi encontrada em 16 (22,2% pacientes, sendo que em 14 (87,5% em A e em 2 (12,5% em V. Todos os 16 pacientes com anisotropia apresentavam hidrocefalia. O erro refrativo mais freqüente foi a hipermetropia encontrada em 64 (44,4% olhos. Atrofia óptica foi encontrada em 9 (12,5% pacientes. Conclusão: A mielomeningocele, associada a hidrocefalia, apresenta uma porcentagem de estrabismo maior do que o encontrado na população normal.Purpose: To study ocular findings in patients with meningomyelocele. Methods: A retrospective study of 72 patients with myelomeningocele was performed to evaluate ocular motility disorders, refractive errors and indirect ophthalmoscopy findings. Results: In a group of 72 patients with myelomeningocele, aged 5 month -- 18 years, 64 (88.9% had hydrocephalus. Of this group of 64 patients, 36 (56.3% had strabismus, 21 (58.3% of whom were esotropic, and 13 (36.1% exotropic, 1 (2.8% exophoric and 1 (2.8% presented sometimes esotropia and sometimes exotropia. Of the total of 72 studied patients, 38 (52.8% had strabismus. Anisotropia was found in 16 (22.2% patients, 14 (87.5% with an A pattern and 2 (12.5% with a V pattern. The most frequent refractive error was hyperopia, found in 64 (44.4% eyes. All the 16 patients with anisotropia had hydrocephalus

  11. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

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    Borges-Pérez Andrés

    2008-12-01

    Full Text Available Abstract Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC, SGN-U321250 (TIP41, SGN-U346908 ("Expressed" and SGN-U316474 (SAND genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real

  12. One-step triplex PCR/RT-PCR to detect canine distemper virus, canine parvovirus, and canine kobuvirus.

    Science.gov (United States)

    Liu, Dafei; Liu, Fei; Guo, Dongchun; Hu, Xiaoliang; Li, Zhijie; Li, Zhigang; Ma, Jianzhang; Liu, Chunguo

    2018-01-23

    To rapidly distinguish Canine distemper virus (CDV), canine parvovirus (CPV), and canine kobuvirus (CaKoV) in practice, a one-step multiplex PCR/RT-PCR assay was developed, with detection limits of 10 2.1 TCID 50 for CDV, 10 1.9 TCID 50 for CPV and 10 3 copies for CaKoV. This method did not amplify nonspecific DNA or RNA from other canine viruses. Therefore, the assay provides a sensitive tool for the rapid clinical detection and epidemiological surveillance of CDV, CPV and CaKoV in dogs.

  13. Evaluation of FTA cards as a laboratory and field sampling device for the detection of foot-and-mouth disease virus and serotyping by RT-PCR and real-time RT-PCR.

    Science.gov (United States)

    Muthukrishnan, Madhanmohan; Singanallur, Nagendrakumar B; Ralla, Kumar; Villuppanoor, Srinivasan A

    2008-08-01

    Foot-and-mouth disease virus (FMDV) samples transported to the laboratory from far and inaccessible areas for serodiagnosis pose a major problem in a tropical country like India, where there is maximum temperature fluctuation. Inadequate storage methods lead to spoilage of FMDV samples collected from clinically positive animals in the field. Such samples are declared as non-typeable by the typing laboratories with the consequent loss of valuable epidemiological data. The present study evaluated the usefulness of FTA Classic Cards for the collection, shipment, storage and identification of the FMDV genome by RT-PCR and real-time RT-PCR. The stability of the viral RNA, the absence of infectivity and ease of processing the sample for molecular methods make the FTA cards a useful option for transport of FMDV genome for identification and serotyping. The method can be used routinely for FMDV research as it is economical and the cards can be transported easily in envelopes by regular document transport methods. Live virus cannot be isolated from samples collected in FTA cards, which is a limitation. This property can be viewed as an advantage as it limits the risk of transmission of live virus.

  14. Variações no posicionamento dos eixos visuais em pacientes submetidos a cirurgias de estrabismo

    Directory of Open Access Journals (Sweden)

    Graciela Scalco Brum

    2011-02-01

    Full Text Available OBJETIVOS: Determinar as variações no ângulo de posicionamento ocular pós-operatório em pacientes submetidos a cirurgias para correção de estrabismo e identificar possíveis fatores de risco associados a tal ocorrência. MÉTODOS: Foi realizado estudo retrospectivo de 819 pacientes portadores de estrabismo submetidos à cirurgia para correção do desvio ocular entre janeiro de 1995 e dezembro de 2005 no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo. Os pacientes foram divididos em quatro grupos quanto ao tipo de desvio pré-operatório (esotropia alternante, esotropia monocular, exotropia alternante e exotropia monocular e as variações no posicionamento ocular foram quantificadas em cada grupo. RESULTADOS: A prevalência de variações no posicionamento ocular (pós-operatório maiores que 10∆ entre o total de pacientes estudado foi de 33,5% (274 pacientes. Entre estes, foi verificada maior frequência de desvios no sentido exotrópico (178 pacientes ou 65,0% do que no sentido esotrópico (96 pacientes ou 35,0%, diferença esta que foi estatisticamente significativa (teste do qui-quadrado; p<0,001. CONCLUSÕES: Instabilidade no posicionamento ocular pode ocorrer ao longo do tempo em pacientes submetidos a cirurgias de estrabismo. Tal ocorrência reforça a necessidade do desenvolvimento de alternativas terapêuticas a fim de proporcionar maior estabilidade ao sistema oculomotor no pós-operatório de cirurgias de estrabismo.

  15. Fatores terapêuticos em um grupo de apoio para pacientes psiquiátricos ambulatoriais

    Directory of Open Access Journals (Sweden)

    Guanaes Carla

    2001-01-01

    Full Text Available INTRODUÇÃO/OBJETIVOS: O emprego da psicoterapia de grupo no atendimento em saúde mental, sobretudo em contextos institucionais, tem se expandido aceleradamente, não havendo uma expansão correlata de pesquisas na área, conforme aponta a literatura especializada. Objetivou-se compreender algumas possibilidades e alguns limites de um grupo de apoio de curta duração (16 sessões para pacientes psiquiátricos ambulatoriais, investigando os fatores terapêuticos que operam nesse grupo, segundo a percepção de seus participantes. MÉTODOS: Questionários do Incidente Crítico (QIC, observação e registro do grupo, consultas aos prontuários dos pacientes e notas de campo constituíram as fontes de dados. A análise das respostas dos pacientes ao QIC foi realizada por procedimentos de avaliação categorial de conteúdo, tendo como base um sistema descritivo dos fatores terapêuticos proposto na literatura. As demais fontes foram tomadas como dados de contexto dos sentidos produzidos pelos pacientes em suas respostas ao QIC. RESULTADOS: Os resultados remetem a dez categorias derivadas da percepção dos pacientes, indicativas de vivências terapêuticas e não-terapêuticas no grupo. DISCUSSÃO/CONCLUSÃO: Este estudo possibilita uma compreensão sobre o modo como os pacientes vivenciaram o processo desse grupo, tendo em vista os sentidos que produziram a partir de sua participação neste, e, em conjunção com a compreensão de seus quadros clínicos, situa algumas possibilidades e alguns limites desse tipo de tratamento em saúde mental, considerando o contexto em que ocorre e a clientela que usualmente o integra.

  16. Comparison of ELISA and RT-PCR for the detection of Prunus necrotic ring spot virus and prune dwarf virus in almond (Prunus dulcis).

    Science.gov (United States)

    Mekuria, Genet; Ramesh, Sunita A; Alberts, Evita; Bertozzi, Terry; Wirthensohn, Michelle; Collins, Graham; Sedgley, Margaret

    2003-12-01

    A technique based on the reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect the presence of Prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) simultaneously in almond. This paper presents the results of a 3-year study comparing both enzyme-linked immunosorbent assay (ELISA) and RT-PCR for the detection of PNRSV and PDV using 175 almond leaf samples. Multiplex RT-PCR was found to be more sensitive than ELISA, especially when followed by nested PCR for the detection of PDV. The RT-PCR technique has the added advantage that plant material can be tested at any time throughout the growing season.

  17. Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

    DEFF Research Database (Denmark)

    Knüsel, R.; Bergmann, S. M.; Einer-Jensen, Katja

    2007-01-01

    in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus......This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools...... (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and...

  18. Simultaneous Detection of Mixed Infection of Onion yellow dwarf virus and an Allexivirus in RT-PCR for Ensuring Virus Free Onion Bulbs.

    Science.gov (United States)

    Kumar, Sandeep; Baranwal, V K; Joshi, Subodh; Arya, Meenakshi; Majumder, S

    2010-06-01

    Reduced seed production in onion is associated with Onion yellow dwarf virus (OYDV), a filamentous Potyvirus. Onion is also infected with other filamentous virus particles suspected to be Allexivirus. RT-PCR was used to detect mixed infection of both the viruses in leaves and bulbs. A duplex RT-PCR was developed, which simultaneously detected the presence of these two viruses in winter (Rabi) onion bulb. In summer (Kharif) onion bulbs only Allexivirus was detected. The absence of OYDV in summer crop is discussed. The sequencing of RT-PCR amplified products confirmed the identity of OYDV and Allexivirus, the latter showing closer identity to Garlic virus C (GVC)/Garlic mite-borne mosaic virus. This makes the first detection of an Allexivirus in onion crop in India. The duplex RT-PCR to detect these viruses (OYDV and Allexivirus) would be an improvement for indexing of viruses in onion bulbs for seed production.

  19. Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization.

    Science.gov (United States)

    Ceol, M; Forino, M; Gambaro, G; Sauer, U; Schleicher, E D; D'Angelo, A; Anglani, F

    2001-01-01

    Gene expression can be examined with different techniques including ribonuclease protection assay (RPA), in situ hybridisation (ISH), and quantitative reverse transcription-polymerase chain reaction (RT/PCR). These methods differ considerably in their sensitivity and precision in detecting and quantifying low abundance mRNA. Although there is evidence that RT/PCR can be performed in a quantitative manner, the quantitative capacity of this method is generally underestimated. To demonstrate that the comparative kinetic RT/PCR strategy-which uses a housekeeping gene as internal standard-is a quantitative method to detect significant differences in mRNA levels between different samples, the inhibitory effect of heparin on phorbol 12-myristate 13-acetate (PMA)-induced-TGF-beta1 mRNA expression was evaluated by RT/PCR and RPA, the standard method of mRNA quantification, and the results were compared. The reproducibility of RT/PCR amplification was calculated by comparing the quantity of G3PDH and TGF-beta1 PCR products, generated during the exponential phases, estimated from two different RT/PCR (G3PDH, r = 0.968, P = 0.0000; TGF-beta1, r = 0.966, P = 0.0000). The quantitative capacity of comparative kinetic RT/PCR was demonstrated by comparing the results obtained from RPA and RT/PCR using linear regression analysis. Starting from the same RNA extraction, but using only 1% of the RNA for the RT/PCR compared to RPA, significant correlation was observed (r = 0.984, P = 0.0004). Moreover the morphometric analysis of ISH signal was applied for the semi-quantitative evaluation of the expression and localisation of TGF-beta1 mRNA in the entire cell population. Our results demonstrate the close similarity of the RT/PCR and RPA methods in giving quantitative information on mRNA expression and indicate the possibility to adopt the comparative kinetic RT/PCR as reliable quantitative method of mRNA analysis. Copyright 2001 Wiley-Liss, Inc.

  20. Anestesia em paciente portadora de síndrome de Mckusick-Kaufman: relato de caso

    OpenAIRE

    Hobaika,Adriano Bechara de Souza; Borges,Ziltomar Donizetti; Teixeira,Vera Coelho

    2004-01-01

    JUSTIFICATIVA E OBJETIVOS: A síndrome de Mckusick-Kaufman é uma doença rara, caracterizada tipicamente por hidrometrocolpos, polidactilia e defeitos cardíacos congênitos. Pacientes portadores desta doença podem ser submetidos a diversos procedimentos cirúrgicos durante a sua vida e o anestesiologista deve estar preparado para possíveis alterações. O objetivo deste artigo é relatar a conduta anestésica adotada em uma paciente portadora desta síndrome. RELATO DO CASO: Paciente do sexo feminino ...

  1. Evaluation of RIDA®GENE norovirus GI/GII real time RT-PCR using stool specimens collected from children and adults with acute gastroenteritis.

    Science.gov (United States)

    Kanwar, N; Hassan, F; Barclay, L; Langley, C; Vinjé, J; Bryant, P W; George, K St; Mosher, L; Matthews-Greer, J M; Rocha, M A; Beenhouwer, D O; Harrison, C J; Moffatt, M; Shastri, N; Selvarangan, R

    2018-04-10

    Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. To evaluate RIDA ® GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014-2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5'end of the norovirus ORF2 gene. A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5-93.5) and 99.1% (98.0-99.6) and for GII was 94.8% (90.8-97.2) and 98.6% (96.9-99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE. Copyright © 2018. Published by Elsevier B.V.

  2. Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

    Directory of Open Access Journals (Sweden)

    Soares Fernando A

    2009-03-01

    Full Text Available Abstract Background HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC and fluorescence in situ hybridization (FISH. These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas. Methods To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. Results The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4% and HER-2 transcript overexpression (20%. Moreover, 2+ immunostaining cases presented nonamplified status (50% by CISH and HER-2 downexpression (38.5% by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350. Conclusion Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene.

  3. Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

    International Nuclear Information System (INIS)

    Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia GT; Bérgamo, Nádia A; Neto, Francisco A Moraes; Domingues, Maria AC; Soares, Fernando A; Caldeira, José RF; Rogatto, Silvia R

    2009-01-01

    HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas. To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and HER-2 downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350). Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene

  4. Hipotermia em pacientes no período perioperatório

    Directory of Open Access Journals (Sweden)

    Ana Lúcia De Mattia

    2012-02-01

    Full Text Available O objetivo deste estudo foi identificar os fatores que desencadeiam a hipotermia em pacientes em sala de operação e as manifestações em sala de recuperação anestésica. O estudo foi realizado em um hospital de grande porte da rede mista da cidade de Santos. A coleta de dados foi realizada em 30 pacientes adultos, com temperatura corpórea entre de 36ºC e 37,2ºC na entrada da sala de operações com procedimentos cirúrgicos eletivos e anestesia geral. Os resultados demonstraram que os métodos preventivos de hipotermia mais utilizados foram a infusão venosa aquecida e a manta térmica. Na saída da sala de operação e até 30 minutos na sala de recuperação anestésica, os pacientes permaneceram hipotérmicos. As manifestações de hipotermia foram demonstradas pela hipoxemia e por tremores. Neste estudo, evidenciou-se que a hipotermia no paciente é desencadeada na sala de operação pela falta de medidas preventivas adequadas, acarretando complicações em período de recuperação anestésica.

  5. A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries.

    Science.gov (United States)

    Dobhal, Shefali; Olson, Jennifer D; Arif, Mohammad; Garcia Suarez, Johnny A; Ochoa-Corona, Francisco M

    2016-06-01

    Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

    Science.gov (United States)

    Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

    2014-11-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the β-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was β-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was α-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

  7. Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus.

    Science.gov (United States)

    Faye, Martin; Dacheux, Laurent; Weidmann, Manfred; Diop, Sylvie Audrey; Loucoubar, Cheikh; Bourhy, Hervé; Sall, Amadou Alpha; Faye, Ousmane

    2017-05-01

    Rabies virus (RABV) remains one of the most important global zoonotic pathogens. RABV causes rabies, an acute encephalomyelitis associated with a high rate of mortality in humans and animals and affecting different parts of the world, particularly in Asia and Africa. Confirmation of rabies diagnosis relies on laboratory diagnosis, in which molecular techniques such as detection of viral RNA by reverse transcription polymerase chain reaction (RT-PCR) are increasingly being used. In this study, two real-time quantitative RT-PCR assays were developed for large-spectrum detection of RABV, with a focus on African isolates. The primer and probe sets were targeted highly conserved regions of the nucleoprotein (N) and polymerase (L) genes. The results indicated the absence of non-specific amplification and cross-reaction with a range of other viruses belonging to the same taxonomic family, i.e. Rhabdoviridae, as well as negative brain tissues from various host species. Analytical sensitivity ranged between 100 to 10 standard RNA copies detected per reaction for N-gene and L-gene assays, respectively. Effective detection and high sensitivity of these assays on African isolates showed that they can be successfully applied in general research and used in diagnostic process and epizootic surveillance in Africa using a double-check strategy. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Generic detection of poleroviruses using an RT-PCR assay targeting the RdRp coding sequence.

    Science.gov (United States)

    Lotos, Leonidas; Efthimiou, Konstantinos; Maliogka, Varvara I; Katis, Nikolaos I

    2014-03-01

    In this study a two-step RT-PCR assay was developed for the generic detection of poleroviruses. The RdRp coding region was selected as the primers' target, since it differs significantly from that of other members in the family Luteoviridae and its sequence can be more informative than other regions in the viral genome. Species specific RT-PCR assays targeting the same region were also developed for the detection of the six most widespread poleroviral species (Beet mild yellowing virus, Beet western yellows virus, Cucurbit aphid-borne virus, Carrot red leaf virus, Potato leafroll virus and Turnip yellows virus) in Greece and the collection of isolates. These isolates along with other characterized ones were used for the evaluation of the generic PCR's detection range. The developed assay efficiently amplified a 593bp RdRp fragment from 46 isolates of 10 different Polerovirus species. Phylogenetic analysis using the generic PCR's amplicon sequence showed that although it cannot accurately infer evolutionary relationships within the genus it can differentiate poleroviruses at the species level. Overall, the described generic assay could be applied for the reliable detection of Polerovirus infections and, in combination with the specific PCRs, for the identification of new and uncharacterized species in the genus. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections.

    Science.gov (United States)

    Díaz de Arce, Heidy; Pérez, Lester J; Frías, Maria T; Rosell, Rosa; Tarradas, Joan; Núñez, José I; Ganges, Llilianne

    2009-11-18

    Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5' non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89TCID(50). The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.

  10. Detecting Rice stripe virus (RSV) in the small brown planthopper (Laodelphax striatellus) with high specificity by RT-PCR.

    Science.gov (United States)

    Lijun, Cai; Xizhi, Ma; Lin, Kang; Kejing, Deng; Shouyuan, Zhao; Changben, Li

    2003-09-01

    Rice stripe disease, caused by Rice stripe virus (RSV), may lead to severe or even crippling losses in many rice-cultured countries and regions. As the most important vector of RSV, the small brown planthopper (SBPH) (Laodelphax striatellus) is largely responsible for the epidemic phase of the disease. Therefore, a rapid identification of RSV in the SBPH is of a great need for disease forecasting. A reverse transcription polymerase chain reaction (RT-PCR) assay is described to amplify a RSV gene in individual L. striatellus. By using primers matched to the viral RNA dependent RNA polymerase gene in RNA1, a 445 bp product was detected in viruliferous SBPHs. Meanwhile, the PCR products produced by the SBPH actin primers constructed across the boundary of an intron and an exon were used as RNA specific positive control for each stage of the experiment to ensure the validity of the negative results. Duplex RT-PCR conditions were established for the simultaneous detection of RSV and actin. This approach can be used for the early detection of RSV in L. striatellus and the subsequent rice stripe disease forecasting.

  11. Comparison of ELISA and dual stage real time RT-PCR to differentiate Sabin like and non-Sabin like poliovirus isolates.

    Science.gov (United States)

    Kaundal, Nirmal; Sarkate, Purva; Prakash, Charu; Rishi, Narayan

    2017-06-01

    Environmental surveillance of polioviruses has been used as an important tool in monitoring circulation of wild polioviruses and/or Vaccine derived polioviruses in sewage samples. It is important to distinguish Sabin like isolates from non-Sabin like; ELISA & dual stage real time RT-PCR have been used for the same. Current study was carried out on sewage isolates to compare ELISA & RT-PCR with sequencing to distinguish Sabin like from non-Sabin like. Out of 468 sewage specimens, 91 (19.44%) were non-polio enteroviruses positive and 377 (80.56%) were polio positive by virus isolation method. A total of 488 polio virus isolates were detected by L20B and RD route which were further subjected to ELISA and RT-PCR. The results were compared with sequencing. On comparison, the specificity of ELISA was only 66.67% in spite of very low sensitivity (3.43%). The sensitivity of RT-PCR was 97.71% which makes it a good primary screening test for detection of non-Sabin like viruses. However, the specificity was only 33.33%. RT-PCR appears to be a sensitive tool for detecting non-Sabin like viruses however; the isolates which are non-Sabin like by RT-PCR may not necessarily be mutated viruses. ELISA cannot be used for differentiation of Sabin likes from non-Sabin likes due to low sensitivity.

  12. Detection of rabies in camel, goat and cattle in Sudan using Fluorescent antibody test (FAT and hemi nested Polymerase Chain Reaction (hnRT-PCR

    Directory of Open Access Journals (Sweden)

    Baraa Abdalaziz Ahmed

    2016-09-01

    Full Text Available Objective: The objective of this study was to identify rabies virus in camels and other animals in Sudan. Materials and methods: Four camel samples were collected from Garraht Elzawia, Kab-kabia and North Darfur areas in Sudan. The samples were collected based on clinical signs. In addition, two camel samples were obtained from Khartoum and Tambool, one goat sample was collected from El-Fashir, and one cattle sample was obtained from Atbara. The samples were transported to the Veterinary Research Institute (VRI at Khartoum, Sudan for further studies. The samples were subjected for nested and hemi nested RT-PCR (hnRT-PCR along with the gold standard Fluorescent antibody test (FAT to diagnose rabies. Results: Out of eight samples, seven were found to be positive by both FAT and RT-PCR methods. The remaining one sample was positive by FAT but negative by hnRT-PCR indicating the suitablity of hnRT-PCR along with FAT for accurate diagnosis of rabies in animals. Conclusion: The study concluded that FAT and RT-PCR are useful tools for research and diagnosis of rabies. [J Adv Vet Anim Res 2016; 3(3.000: 274-277

  13. Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Hout, David R; Schweitzer, Brock L; Lawrence, Kasey; Morris, Stephan W; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly; Saltman, David L

    2017-08-01

    Patients with lung cancers harboring an activating anaplastic lymphoma kinase ( ALK ) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off Δ C t of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

  14. Diagnosis of intestinal parasites in a rural community of Venezuela: Advantages and disadvantages of using microscopy or RT-PCR.

    Science.gov (United States)

    Incani, Renzo Nino; Ferrer, Elizabeth; Hoek, Denise; Ramak, Robbert; Roelfsema, Jeroen; Mughini-Gras, Lapo; Kortbeek, Titia; Pinelli, Elena

    2017-03-01

    A cross-sectional study was carried out to determine the prevalence and diagnostic performance of microscopy and real time PCR (RT-PCR) for 14 intestinal parasites in a Venezuelan rural community with a long history of persistent intestinal parasitic infections despite the implementation of regular anthelminthic treatments. A total of 228 participants were included in this study. A multiplex RT-PCR was used for the detection of Dientamoeba fragilis, Giardia intestinalis, Cryptosporidium sp. and a monoplex RT-PCR for Entamoeba histolytica. Furthermore, a multiplex PCR was performed for detection of Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus and Ancylostoma duodenale. Combined microscopy-PCR revealed prevalences of 49.3% for A. lumbricoides, 10.1% for N. americanus (no A. duodenale was detected), 2.0% for S. stercoralis, 40.4% for D. fragilis, 35.1% for G. intestinalis, and 7.9% for E. histolytica/dispar. Significant increases in prevalence at PCR vs. microscopy were found for A. lumbricoides, G. intestinalis and D. fragilis. Other parasites detected by microscopy alone were Trichuris trichiura (25.7%), Enterobius vermicularis (3.4%), Blastocystis sp. (65.8%), and the non-pathogenic Entamoeba coli (28.9%), Entamoeba hartmanni (12.3%), Endolimax nana (19.7%) and Iodamoeba bütschlii (7.5%). Age- but no gender-related differences in prevalences were found for A. lumbricoides, T. trichiura, G. intestinalis, and E. histolytica/dispar. The persistently high prevalences of intestinal helminths are probably related to the high faecal pollution as also evidenced by the high prevalences of non-pathogenic intestinal protozoans. These results highlight the importance of using sensitive diagnostic techniques in combination with microscopy to better estimate the prevalence of intestinal parasites, especially in the case of D. fragilis trophozoites, which deteriorate very rapidly and would be missed by microscopy. In addition, the differentiation between

  15. Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula.

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    Wun S Chao

    Full Text Available Quantitative real-time polymerase chain reaction (qRT-PCR is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4 were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100 identified from the analyses of leafy spurge microarray data; (2 3 orthologs of Arabidopsis "general purpose" traditional reference genes (GAPDH_1, GAPDH_2, and UBC; and (3 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the C(T values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ΔC(T method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes

  16. Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.

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    Qiusheng Kong

    Full Text Available Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT-PCR is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT-PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT-PCR analyses involving watermelon.

  17. Estudo epidemiológico de pacientes com queimaduras por eletricidade atendidos em unidade de queimados em Recife – PE

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    Ana Claudia Siqueira Torquato

    2015-09-01

    Full Text Available Objetivo: verificar a frequência das queimaduras por eletricidade e perfil clínico-epidemiológico de pacientes atendidos na Unidade de Queimados em Recife-PE. Métodos: estudo retrospectivo, transversal e descritivo de pacientes com queimaduras por eletricidade atendidos na Unidade de Queimados do Hospital da Restauração (HR em Recife-PE, no período de janeiro 2007 a dezembro de 2008. Resultados: foram atendidos 115 pacientes com queimaduras por eletricidade no período de realização do estudo (média de 57,5 pacientes/ano, correspondendo a quase 8% de todas as queimaduras atendidas no serviço. Quanto ao sexo, 83,5% eram do sexo masculino e 16,5% eram do sexo feminino. A idade média foi de 27 anos. Ocorreu predominância do Médio queimado (70%. O acidente doméstico foi mais encontrado. Quanto ao tempo de hospitalização, aproximadamente 90% dos pacientes receberam alta com menos de dois meses de internação. Durante a evolução, 42,6% dos pacientes necessitaram de enxerto e 13% sofreram amputação. Nenhum óbito foi notificado no período de realização do estudo. Conclusões: a frequência de queimaduras por eletricidade em nosso estudo foi maior que as relatadas em outras séries. O perfil clínico- epidemiológico encontrado no presente estudo foi similar ao descrito na literatura.

  18. Vasculite mesentérica em paciente com lúpus eritematoso sistêmico juvenil

    OpenAIRE

    Albuquerque-Netto, Adão F.; Cavalcante, Erica G.; Sallum, Adriana M. E.; Aikawa, Nádia E.; Tannuri, Uenis; Silva, Clovis Artur Almeida da

    2013-01-01

    A vasculite mesentérica lúpica (VML) é uma rara causa de dor abdominal aguda. Há poucos relatos de caso demonstrando VML em adultos e, particularmente, em crianças e adolescentes. No entanto, para o nosso conhecimento, a prevalência dessa grave vasculite em uma população pediátrica com lúpus ainda não foi estudada. Portanto, dados de 28 anos consecutivos foram revisados e incluídos 5.508 pacientes em seguimento no Hospital da Faculdade de Medicina da Univesidade de São Paulo (FMUSP). Identifi...

  19. Cuidar de pacientes em fase terminal: a experiência de alunos de medicina

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    Maria Lúcia Araújo Sadala

    2008-03-01

    Full Text Available O objetivo deste estudo foi compreender os significados que alunos de medicina atribuem à experiência de cuidar de pacientes em fase terminal. Os dados foram coletados mediante entrevistas individuais com vinte e quatro estudantes de medicina de uma universidade paulista. As convergências dos depoimentos dos participantes foram agrupadas em três categorias: a deparando-se com o mundo da doença terminal/morte; b relação com o paciente e sua família; c reflexão sobre o cuidar do paciente terminal. Nos depoimentos, os estudantes expressam ansiedade ao cuidar deste tipo de paciente, assim como as dificuldades em lidar com os próprios sentimentos. Consideram-se pouco preparados para relacionar-se com esses pacientes, mas aprenderam com a experiência vivida. Os resultados do estudo apontam para a necessidade de se introduzirem, na formação médica, conteúdos visando desenvolver competências interpessoais e capacidade de reflexão sobre questões de ética e deontologia médica, envolvendo a terminalidade da vida.

  20. Terapia em paciente com disfunção temporomandibular muscular

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    Aline Avrella

    2015-11-01

    Full Text Available Objetivo. Relatar um caso clínico de uma paciente jovem com disfunção temporomandibular muscular (DTM. Materiais e métodos. O estudo foi realizado em uma clínica odontológica de ensino superior, utilizando a aplicação de calor úmido, exercícios ativos e agulhamento seco. Resultados. Paciente feminina, 31 anos, relatando dor na região parotídea-massetérica bilateral, sendo diagnosticada com DTM muscular bilateral em masseter com presença de pontos gatilho. Para o tratamento foi realizado a termoterapia pela utilização do calor úmido, fisioterapia com exercícios ativos de relaxamento, agulhamento seco utilizando agulhas filiformes sólidas e, Terapia Cognitiva Comportamental, com melhora importante referida pela paciente. Discussão. Dentre as formas de tratamento para DTM muscular encontram-se as técnicas utilizadas com a paciente em estudo, as quais proporcionando uma melhora biopsicossocial. Conclusão. As técnicas de tratamento realizadas foram efetivas para o tratamento da paciente, proporcionando alívio da dor e melhora nos sintomas.

  1. Varicela hemorrágica em paciente com Doença de Still

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    Maria Beatriz Bray Beraldo

    2010-03-01

    Full Text Available A artrite idiopática juvenil (AIJ é um processo inflamatório crônico em uma ou mais articulações que pode apresentar manifestações extra-articulares; o subtipo com comprometimento sistêmico é denominado Doença de Still. A infecção pelo Herpesvirus varicelae, apesar de apresentar caráter benigno na maioria das vezes, pode complicar-se em pacientes imunossuprimidos. Este relato trata-se de uma paciente com AIJ de início sistêmico em uso de medicamentos que levam à imunossupressão e em consequência disto, após ser acometida pelo vírus da varicela, desenvolveu complicações hemorrágicas, porém, apesar da alta taxa de mortalidade nestas ocasiões, teve uma boa evolução.

  2. Development of duplex real-time RT-PCR based on Taqman technology for detecting simultaneously the genome of pan-enterovirus and enterovirus 71.

    Science.gov (United States)

    Hwang, Seoyeon; Kang, Byunghak; Hong, Jiyoung; Kim, Ahyoun; Kim, Hyejin; Kim, Kisang; Cheon, Doo-Sung

    2013-07-01

    Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks. Copyright © 2013 Wiley Periodicals, Inc.

  3. EQUOTERAPIA E COGNIÇÃO EM PACIENTES AUTISTAS: UM ESTUDO DE CASO

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    Rose–Lídice Holanda

    2017-06-01

    Full Text Available O autismo é uma síndrome comportamental com etiologias diferentes, onde a sintomatologia foi organizada por uma tétrade clínica. A equoterapia é considerada como um conjunto de técnicas reeducativas que agem para superar danos sensoriais, motores e comportamentais, que tem como meio o cavalo. O estudo teve como objetivo avaliar á intervenção da equoterapia em pacientes com autismo. Foi realizado um estudo com um paciente autista praticante de equoterapia na cavalaria da Polícia Militar do Ceará, onde foi aplicado o teste MoCA em dois momentos, para avaliar a cognição. A pesquisa foi realizada em 2008.. O projeto foi submetido ao Comitê de Ética em Pesquisa, do Hospital Geral de Fortaleza com parecer aprovado sob nº de protocolo 070801/08. Como resultado verificou-se no primeiro momento uma pontuação de 16 pontos e num segundo momento, pontuou 14 pontos, totalizando 30 pontos. Houve um aumento de pontos em alguns aspectos, como também uma diminuição, durante aplicação do teste MoCA. Concluímos que o paciente não apresentou uma melhora significativa devido a variável tempo e psicológico. Podendo este fato está relacionado com o quadro de alteração psicológica, notando-se um estado depressivo com uma diminuição de interesses do paciente. É relevante a realização da equoterapia em pacientes autistas, melhorando assim sua autoestima, proporcionando uma melhora e uma qualidade de vida.

  4. Pancreatite crônica: resultados do tratamento cirúrgico em 74 pacientes

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    Olívio Louro Costa

    Full Text Available OBJETIVO: Analisar as indicações, técnicas e resultados do tratamento cirúrgico de 74 pacientes operados por complicações da pancreatite crônica. MÉTODO: Foram pacientes consecutivos, estudados prospectivamente pelo preenchimento de um protocolo individual, no período de 1971 a 2000. Foram realizadas cirurgias de derivação e ressecção. O acompanhamento foi feito pelo agendamento de consultas no ambulatório, por convocação por cartas e telefonemas. RESULTADOS: Dos 270 pacientes com pancreatite crônica, acompanhados pelo Serviço, 74 (27,4% foram operados. Destes 74 pacientes, 97,7% eram do sexo masculino e a idade variou de 15 a 63 anos, com média de 39,4 anos para alcoolistas e 33,1 para aqueles com outras etiologias. O alcoolismo foi a causa da doença em 68 pacientes (90,5% e os outros casos estiveram relacionados a hiperparatiroidismo(1, pancreatite hereditária (1, fibrose retroperitoneal (1 e em três casos a etiologia não foi definida. As seguintes causas únicas ou associadas definiram a indicação cirúrgica:1. dor em 44,6% dos pacientes; 2. compressão de vias biliares em 28,4%; 3. pseudocistos em 12,2%; 4. fístulas internas em 10,8%. Cinqüenta pacientes (67,5% foram submetidos a operações de derivação e 24 (32,5% a cirurgias de ressecção. Oito pacientes derivados (16,0% complicaram, ocorrendo três óbitos; dez ressecados (41,6% complicaram, com cinco óbitos. Os óbitos estiveram relacionados a abscessos, às deiscências e às hemorragias cirúrgicas. CONCLUSÕES: As cirurgias de derivação mostraram-se mais seguras e as complicações que evoluíram com infecção foram as mais graves e ocorreram com mais freqüência nas ressecções.

  5. Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR.

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    Ruby Chandna

    Full Text Available The real time quantitative reverse transcription PCR (qRT-PCR is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes, ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes, in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

  6. Identification of Suitable Endogenous Normalizers for qRT- PCR Analysis of Plasma microRNA Expression in Essential Hypertension

    Science.gov (United States)

    Solayman, Mohamed Hassan M.; Langaee, Taimour; Patel, Archanakumari; El-Wakeel, Lamia; El-Hamamsy, Manal; Badary, Osama; Johnson, Julie A.

    2016-01-01

    Circulating microRNAs (miRNAs) are promising biomarkers for many diseases. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a gold standard for miRNA expression profiling that requires proper data normalization. Since there is no universal normalizer, it is recommended to evaluate normalizers under every experimental condition. This study describes the identification of suitable endogenous normalizer(s) (ENs) for plasma miRNA expression in essential hypertension. Expression levels of 5 candidate ENs and 2 plasma quality markers were determined by qRT-PCR in plasma samples from 18 hypertensive patients and 10 healthy controls. NormFinder, GeNorm, and DataAssist software programs were used to select the best EN(s). Expression levels of the 5 candidate ENs were also analyzed in urine samples from hypertensive patients and compared to the plasma samples of the hypertensive patients. Among the analyzed candidates, hsa-miR-92a-3p was identified as the best EN, and hsa-miR-21-5p and hsa-miR-16-5p as next best. Moreover, hsa-miR-92a-3p showed the most consistent expression between plasma and urine In conclusion, this study showed that hsa-miR-92a-3p, hsa-miR-21-5p, and hsa-miR-16-5p may be used as normalizers for plasma miRNA expression data in essential hypertension studies. PMID:26798072

  7. Airborne rhinovirus detection and effect of ultraviolet irradiation on detection by a semi-nested RT-PCR assay

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    Rudnick Stephen

    2003-01-01

    Full Text Available Abstract Background Rhinovirus, the most common cause of upper respiratory tract infections, has been implicated in asthma exacerbations and possibly asthma deaths. Although the method of transmission of rhinoviruses is disputed, several studies have demonstrated that aerosol transmission is a likely method of transmission among adults. As a first step in studies of possible airborne rhinovirus transmission, we developed methods to detect aerosolized rhinovirus by extending existing technology for detecting infectious agents in nasal specimens. Methods We aerosolized rhinovirus in a small aerosol chamber. Experiments were conducted with decreasing concentrations of rhinovirus. To determine the effect of UV irradiation on detection of rhinoviral aerosols, we also conducted experiments in which we exposed aerosols to a UV dose of 684 mJ/m2. Aerosols were collected on Teflon filters and rhinovirus recovered in Qiagen AVL buffer using the Qiagen QIAamp Viral RNA Kit (Qiagen Corp., Valencia, California followed by semi-nested RT-PCR and detection by gel electrophoresis. Results We obtained positive results from filter samples that had collected at least 1.3 TCID50 of aerosolized rhinovirus. Ultraviolet irradiation of airborne virus at doses much greater than those used in upper-room UV germicidal irradiation applications did not inhibit subsequent detection with the RT-PCR assay. Conclusion The air sampling and extraction methodology developed in this study should be applicable to the detection of rhinovirus and other airborne viruses in the indoor air of offices and schools. This method, however, cannot distinguish UV inactivated virus from infectious viral particles.

  8. Simultaneous Expression of GUS and Actin Genes by Using the Multiplex RT-PCR and Multiplex Gold Nanoparticle Probes.

    Science.gov (United States)

    Ghazi, Yaser; Vaseghi, Akbar; Ahmadi, Sepideh; Haddadi, Fatemeh

    2018-04-23

    Gene expression analysis is considered to be extremely important in many different biological researches. DNA-based diagnostic test, which contributes to DNA identification, has higher specificity, cost, and speed than some biochemical and molecular methods. In this study, we try to use the novel nano technology approach with Multiplex RT-PCR and Gold nano particular probes (GNPs-probes) in order to get gene expression in Curcumas melons. We used Agrobacterium tumefactions for gene transfer and GUS reporter gene as a reporter. After cDNA synthesis, Multiplex PCR and Multiplex RT-PCR techniques were used. Finally, probes were designed for RNA of GUS and Actin genes, and then the analysis of the gene expression using the probes attached to GNPs was carried out and the color changes in the GNPs were applied. In the following, probes hybridization was checked with DNA between 400 to 700 nm wavelengths and the highest rate was observed in the 550 to 650 nm. The results show that the simultaneous use of GNP-attached detectors and Multiplex RT-PCRcan reduce time and costmore considerably than somelaboratory methods for gene expiration investigation. Additionally, it can be seen thatthere is an increase in sensitivity and specificity of our investigation. Based on our findings, this can bea novel study doneusingMultiplex RT-PCRand unmodified AuNPs for gene transfer and expression detection to plants. We can claim that this assay has a remarkable advantage including rapid, cost-effectiveness, specificity and accuracy to detect transfer and expression genes in plants. Also,we can use this technique from other gene expressionsin many different biology samples.

  9. Application of F⁺RNA Coliphages as Source Tracking Enteric Viruses on Parsley and Leek Using RT-PCR.

    Science.gov (United States)

    Shahrampour, Dina; Yavarmanesh, Masoud; Najafi, Mohammad Bagher Habibi; Mohebbi, Mohebbat

    2015-12-01

    The objective of this study was to identify sources of fecal contamination in leek and parsley, by using four different F(+)RNA coliphage genogroups (IV, I indicate animal fecal contamination and II, III indicate human fecal contamination). Three different concentrations (10(2), 10(4), 10(6) pfu/ml) of MS2 coliphage were inoculated on the surface of parsley and leek samples for detection of phage recovery efficiency among two methods of elution concentration (PEG-precipitation and Ultracentrifugation) by performing double agar layer (DAL) assay in three replications. Highest recovery of MS2 was observed in PEG method and in 10(6) inoculation concentration. Accordingly, the PEG method was used for washing and isolation of potentially contaminated phages of 30 collected samples (15 samples from the market and 15 samples from the farm). The final solutions of PEG method were tested for the enumeration of plaques by DAL assay. Total RNA was then extracted from recovered phages, and RT-PCR was performed by using four primer sets I, II, III, and IV. Incidence of F(+)RNA coliphages was observed in 12/15 (80 %) and 10/15 (66/6 %) of samples were obtained from farm and market, respectively, using both DAL and RT-PCR test methods. Different genotypes (I, II, and IV) of F(+)RNA coliphages were found in farm samples, while only genotype I was detected in market samples by using the primer sets. Due to the higher frequency of genotype I and IV, the absence of genotype III, and also the low frequency of genotype II, it is concluded that the contamination of vegetable (parsley and leek) in Neyshabour, Iran is most likely originated from animal sources.

  10. The incidence and distribution characteristics of MLL rearrangements in Chinese acute myeloid leukemia patients by multiplex nested RT-PCR.

    Science.gov (United States)

    Yang, Hua; Cao, Tingting; Gao, Li; Wang, Lili; Zhu, Chengying; Xu, Yuanyuan; Jing, Yu; Zhu, Haiyan; Lv, Na; Yu, Li

    2017-07-20

    Occurrence of MLL (Mixed Lineage Leukemia) gene rearrangements indicates poor prognosis in acute myeloid leukemia (AML) patients. This is the first study to report the positive rate and distribution characteristics of MLL rearrangements in AML patients in north China. We used multiplex nested real time PCR (RT-PCR) to screen for incidence of 11 MLL rearrangements in 433 AML patients. Eleven MLL rearrangements included (MLL-PTD, MLL-AF9, MLL-ELL, MLL-AF10, MLL-AF17, MLL-AF6, MLL-ENL, MLL-AF1Q, MLL-CBP, MLL-AF1P, MLL-AFX1). There were 68 AML patients with MLL rearrangements, and the positive rate was 15.7%. MLL-PTD (4.84%) was detected in 21 patients, MLL-AF9 in 15, (3.46%), MLL-ELL in 10 (2.31%), MLL-AF10 in 8 (1.85%), MLL-AF1Q in 2 (0.46%), 3 cases each of MLL-AF17, MLL-AF6, MLL-ENL (0.69% each), a and single case each of MLL-CBP, MLL-AF1P, and MLL-AFX1 (0.23% each). The highest rate of MLL rearrangements was found in 24 patients with M5 subtype AML, occurring in 24 cases (35.3%). MLL rearrangements occurred in 21 patients with M2 subtype AML (30.9%), and in 10 patients with M4 subtype AML (14.7%). Screening fusion genes by multiplex nested RT-PCR is a convenient, fast, economical, and accurate method for diagnosis and predicting prognosis of AML.

  11. Avaliação e tratamento da hiperglicemia em pacientes graves

    Directory of Open Access Journals (Sweden)

    Marina Verçoza Viana

    2014-03-01

    Full Text Available A hiperglicemia é um problema frequentemente encontrado em pacientes graves em ambiente de terapia intensiva. Sua presença se associa ao aumento da morbidade e da mortalidade, independentemente da causa da admissão (infarto agudo do miocárdio, condição após cirurgia cardiovascular, acidente vascular cerebral e sepse. Entretanto, permanecem muitas dúvidas com relação à fisiopatologia e, particularmente, em relação ao tratamento da hiperglicemia no paciente graves. Na prática clínica, devem ser levados em consideração diversos aspectos para o controle desses pacientes, inclusive os alvos de glicemia, o histórico de diabetes mellitus, a via de nutrição (enteral ou parenteral e o equipamento de monitoramento disponível, o que aumenta substancialmente a carga de trabalho dos profissionais envolvidos nesse tratamento. Esta revisão descreveu a epidemiologia, a fisiopatologia, o tratamento e o monitoramento da hiperglicemia no paciente adulto grave.

  12. Investigação da queixa de disfagia em pacientes afásicos

    Directory of Open Access Journals (Sweden)

    Karin Zazo Ortiz

    2013-12-01

    Full Text Available OBJETIVOS: verificar a presença e evolução da queixa de deglutição em pacientes afásicos pós- AVE e, a partir do relato dos familiares em relação à melhora das manifestações, analisar se a ocorrência da afasia interferiu no prognóstico da disfagia. MÉTODO: 30 pacientes afásicos pós-AVE na fase crônica foram entrevistados e, juntamente com seus familiares, responderam a um questionário sobre a presença e a permanência de queixas de deglutição pós-AVE e aspectos relacionados, bem como melhoras ocorridas em relação aos quadros de disfagia e afasia. Para verificar se a disfagia parece ter interferido no prognóstico da afasia, comparou-se as curvas de Kaplan-Meier dos pacientes que referiram queixas de disfagia com os que não referiram. RESULTADOS: 48% dos pacientes tiveram queixas de dificuldades de deglutição pós-AVE. Destes, 93% apresentaram mudanças positivas (melhora parcial ou total do quadro. O tempo médio para que o paciente apresentasse qualquer tipo de mudança (espontânea ou não foi de 76 dias. 60% referiu melhora total da disfagia, sendo que 47% foram submetidos à terapia fonoaudiológica. Em relação à afasia, 87% dos pacientes referiram melhora, apesar de nenhum paciente ter referido melhora total. 57% haviam sido expostos à terapia fonoaudiológica para a afasia. O tempo médio referido para que o paciente apresentasse qualquer tipo de mudança positiva nas manifestações foi de 183 dias. Não houve diferença significante na melhora da afasia entre o grupo com e sem queixa de deglutição. CONCLUSÕES: dos pacientes afásicos avaliados neste estudo, 48% apresentou queixa de disfagia. Verificou-se, a partir do relato dos familiares, que a ocorrência da afasia parece não ter interferido no prognóstico da disfagia.

  13. A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment

    Directory of Open Access Journals (Sweden)

    Tian Ding

    2017-05-01

    Full Text Available This study firstly developed a multiplex real-time PCR (RT-PCR technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus, Listeria monocytogenes (L. monocytogenes and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water in one reaction. Brain heart infusion (BHI broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

  14. A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

    Directory of Open Access Journals (Sweden)

    Qin E-de

    2010-06-01

    Full Text Available Abstract A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009 influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same as that of each single-target RT-PCR for pandemic H1N1 and even more sensitive for H5N1 with the same primers and probes. No cross reactivity of detecting other subtype influenza viruses or respiratory tract viruses was observed. Two hundred and thirty-six clinical specimens were tested by comparing with single real-time RT-PCR and result from the duplex assay was 100% consistent with the results of single real-time RT-PCR and sequence analysis.

  15. Development of an In-House Multiplex Nested RT-PCR Method for Detecting Acute HIV-1 Infection in High Risk Populations.

    Science.gov (United States)

    Liu, Zhiying; Li, Wei; Xu, Meng; Sheng, Bo; Yang, Zixuan; Jiao, Yanmei; Zhang, Tong; Mou, Danlei; Chen, Dexi; Wu, Hao

    2015-01-01

    The detection of acute HIV infection (AHI) among high risk populations can help reduce secondary transmission of HIV. The nucleic acid testing (NAT) can shorten the test window period by up to 7-12 days. In this study, we describe an in-house NAT based on the multiplex nested RT-PCR method to detect the HIV RNA. We also evaluated it in a high risk cohort in Beijing. Four primer pairs were designed and evaluated for the detection of different HIV-1 subtypes in group M. Multiplex RT-PCR and nested PCR were performed. The sensitivity, specialty, primers compatibility among HIV subtypes were evaluated simultaneously. In an MSM cohort in Beijing during a 3-year period, a total of 11,808 blood samples that were negative by ELISA or indeterminate by Western blot were analyzed by this multiplex nested RT-PCR with pooling strategy. The multiplex nested RT-PCR was successfully applied for the detection of at least six HIV-1 subtypes. The sensitivity was 40 copies/ml and the specificity was 100%. A total of 29 people were tested HIV-1 positive with acute infection in a MSM cohort of Beijing during a 3 years period. This multiplex nested RT-PCR provides a useful tool for the rapid detection of acute HIV-1 infection. When used in combination with the 3(rd) generation ELISA, it can improve the detection rate of HIV infection, especially in the source limited regions.

  16. Marcadores práticos de função renal em pacientes com cistinose

    Directory of Open Access Journals (Sweden)

    Luciana Pache de Faria Guimaraes

    2012-09-01

    Full Text Available INTRODUÇÃO: Cistinose é uma doença sistêmica, autossômica recessiva, que leva à insuficiência renal crônica na infância, a não ser que o tratamento com cisteamina seja iniciado precocemente. Mesmo nestas condições, os pacientes evoluem para doença renal crônica terminal por volta da segunda década da vida. Portanto, a avaliação da função renal é essencial neste grupo de pacientes. OBJETIVO: Avaliar e correlacionar a cistatina C, creatinina sérica e o clearance de creatinina pela Fórmula de Schwartz em pacientes com cistinose, com diferentes graus de função renal. MÉTODOS: Foram incluídos pacientes com menos de 18 anos de idade, com diferentes níveis de função renal, de acordo com o KDOQI em estágios 1 a 4. Nenhum dos pacientes estava em terapia de substituição renal. Foram medidos os seguintes parâmetros: cistatina C, creatinina sérica e o clearance de creatinina pela fórmula de Schwartz. RESULTADOS: Foram analisadas 103 amostras de sangue de 26 pacientes. Foi detectada correlação significativa entre creatinina sérica e cistatina C (r = 0,81, p < 0,0001, cistatina C e o clearance de creatinina pela fórmula de Schwartz (r = -0,84, p < 0,0001 e creatinina sérica e clearance de creatinina (r = -0,97, p < 0,0001. CONCLUSÕES: A medida da cistatina não mostrou nenhuma vantagem sobre a creatinina sérica e o clearance de creatinina pela fórmula de Schwartz em pacientes com cistinose para avaliar o ritmo de filtração glomerular. Este é o primeiro relato sobre o valor da creatinina sérica, do clearance de creatinina pela fórmula de Schwartz e da cistatina C em pacientes com cistinose.

  17. Prevalência de Fatores de Risco Cardiovascular em Pacientes em Hemodiálise - O Estudo CORDIAL

    Directory of Open Access Journals (Sweden)

    Jayme Eduardo Burmeister

    2014-06-01

    Full Text Available Fundamentos: Há uma carência de dados epidemiológicos sobre o perfil de risco cardiovascular nos pacientes renais crônicos em hemodiálise no Brasil. Objetivo: O estudo CORDIAL foi planejado para avaliar fatores de risco cardiovascular e acompanhar a evolução de uma população em programa de hemodiálise numa cidade metropolitana do Brasil. Métodos: Todos os pacientes em hemodiálise por doença renal crônica nos quinze centros de nefrologia de Porto Alegre foram considerados para inclusão na fase inicial do estudo CORDIAL. Dados clínicos, laboratoriais e demográficos foram obtidos nos registros médicos, e em entrevistas individuais estruturadas realizadas com todos os pacientes por pesquisadores treinados. Resultados: Foram incluídos 1215 pacientes (97,3% de todos os que estavam em hemodiálise na cidade de Porto Alegre. A média de idade era 58,3 anos, 59,5% eram homens e 62,8% eram brancos. A prevalência de fatores de risco cardiovascular encontrada foi 87,5% para hipertensão, 84,7% para dislipidemia, 73,1% para sedentarismo, 53,7% para tabagismo e 35,8% para diabetes. Em uma análise multivariada ajustada, sedentarismo (p = 0,032; RP 1,08 - IC95%: 1,01-1,15, dislipidemia (p = 0,019; RP 1,08 - IC95%: 1,01-1,14, e obesidade (p < 0,001; RP 1,96 - IC95%: 1,45-2,63 foram mais frequentes em mulheres; e hipertensão (p = 0,018; PR 1,06 - IC95%: 1,01-1,11 e tabagismo (p = 0,006; RP 2,7 - IC95%: 1,79-4,17 foram mais frequentes naqueles com menos de 65 anos. Sedentarismo apresentou uma associação independente com tempo em diálise inferior a 12 meses (p < 0,001; RP 1,23 - IC95%: 1,14-1,33. Conclusão: Pacientes em hemodiálise nesta metrópole do sul do Brasil apresentaram uma prevalência elevada de fatores de risco cardiovascular similar a diversos países do hemisfério norte.

  18. Vulnerabilidades presentes no percurso vivenciado pelos pacientes com HIV/AIDS em falha terapêutica

    Directory of Open Access Journals (Sweden)

    Petra Kelly Rabelo de Sousa

    2013-04-01

    Full Text Available Um grande avanço no tratamento da AIDS deu-se com o surgimento da terapia antiretroviral. Entretanto, diversas vulnerabilidades podem estar presentes no percurso vivenciado pelos pacientes durante o tratamento. Objetivou-se analisar as vulnerabilidades dos pacientes com HIV/AIDS em falha terapêutica. Estudo qualitativo realizado com sete usuários com AIDS considerados em falha terapêutica, em uma Unidade de Serviço Ambulatorial Especializado em HIV/AIDS, em Fortaleza-CE. Para interpretação dos dados, utilizou-se uma técnica de análise de conteúdo. Nos discursos, percebeu-se que os depoentes enfrentavam conflitos na família e, no ambiente de trabalho, dificuldades de relacionamento com as pessoas e estigma. Observou-se que os pacientes haviam vivenciado diversas situações que os tornaram mais suscetíveis à infecção por HIV e ao adoecimento.

  19. Manifestações musculoesqueléticas em pacientes submetidos à hemodiálise

    OpenAIRE

    Vieira,Walber Pinto; Gomes,Kirla Wagner Poti; Frota,Niedja Bezerra; Andrade,José Eyorand Castelo Branco; Vieira,Rejane Maria Rodrigues de Abreu; Moura,Francisca Edwiges Araújo; Vieira,Francisco José Fernandes

    2005-01-01

    As anormalidades musculoesqueléticas em pacientes submetidos à hemodiálise são numerosas e freqüentes, sendo mais prevalentes naqueles em tratamento dialítico de longa duração. A artralgia é o sintoma mais comum, ocorrendo em mais de 70% dos pacientes. O hiperparatireoidismo secundário é a principal causa da doença renal óssea, e as manifestações clínicas são: dor óssea, artralgia e prurido. A amiloidose, decorrente da deposição de proteína beta2-microglobulina nos tecidos, manifesta-se pela ...

  20. Candidíase em pacientes aidéticos

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    C.E.O.P. Campos

    1992-09-01

    Full Text Available Trinta e cinco aidéticos entre 19 e 55 anos admitidos e tratados de candidíase no Hospital Emílio Ribas, SP, com ELISA positivo para HIV e confirmado pelo Western Blot. Tuberculose em 9 sendo 2 com pericardite; neurotoxoplasmose em 6; neurocriptococose em 5; herpes labial em 4; pneumocistose em 3 e sarcoma de Kaposi em 2, achavam-se associadas. A concentração inibitória mínima 50% (MIC 50% para os azoles foi: ketoconazol= 2,2 µg/ml; itraconazol- 21,0 µg/ml; fluconazol = 19,0 µg/ml. O MIC 50% para ospolienos: nistatina- 50,0 µg/ml; anfotericina B= 0,12 µg/ml e para 5 fluorcitosina= 1,6 µg/ml nas 35 amostras de Candida isoladas. Testes não paramétricos de Siegel revelaram significante identificação (80% das Candida albicans na candidíase, e que a dose de AMB não modificou o número de óbitos, precoce e tardio, ocorridos nesses aidéticos. O uso prévio dos azoles e da nistatina explicaria, talvez, o elevado MIC 50% observado nas amostras de Candida isoladas.

  1. Prevalência e fatores associados à obesidade abdominal em pacientes em hemodiálise em Goiânia - GO

    Directory of Open Access Journals (Sweden)

    Ana Tereza Vaz de Souza Freitas

    2013-12-01

    Full Text Available INTRODUÇÃO: A presença de excesso de peso, especialmente obesidade visceral, contribui para o maior risco de complicações metabólicas e cardiovasculares em pacientes com doença renal crônica. OBJETIVO: Determinar a prevalência e os fatores associados à obesidade abdominal em pacientes em hemodiálise (HD. MÉTODOS: Estudo transversal com 344 pacientes maiores de 18 anos. A obesidade abdominal foi definida pela circunferência da cintura > 94 cm nos homens e > 80 cm nas mulheres. As variáveis independentes envolveram aspectos socioeconômicos, demográficos, hábitos de vida, tempo em HD, consumo alimentar e índice de massa corporal (IMC. A análise dos fatores associados foi realizada por regressão de Poisson múltipla, permanecendo no modelo final as variáveis com p 25 kg/m². Nos homens, a classe econômica D/E também permaneceu associada à obesidade abdominal, p < 0,05. CONCLUSÃO: Observou-se alta prevalência de obesidade abdominal em pacientes em hemodiálise. Idade superior a 40 anos, classes econômicas mais baixas, ingestão proteica inferior ao recomendado e excesso de peso foram associados à obesidade abdominal.

  2. Cultura e clima organizacional para segurança do paciente em Unidades de Terapia Intensiva

    OpenAIRE

    Santiago, Thaiana Helena Roma; Turrini, Ruth Natalia Teresa

    2015-01-01

    RESUMO Objetivo Avaliar a percepção dos profissionais de saúde sobre o clima e a cultura de segurança do paciente em Unidades de Terapia Intensiva (UTI) e a relação entre os instrumentos Hospital Survey on Patient Safety Culture (HSOPSC) e o Safety Attitudes Questionnaire (SAQ). Método Estudo transversal realizado em hospital de ensino no interior do estado de São Paulo, Brasil, em março/abril de 2014. Aplicaram-se o HSOPSC, o SAQ e um instrumento para levantamento das informações sociode...

  3. Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa

    DEFF Research Database (Denmark)

    Bachanek-Bankowska, Katarzyna; Mero, Herieth R.; Wadsworth, Jemma

    2016-01-01

    Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping...... methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within...... sequencing. Samples (n = 71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious...

  4. Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

    DEFF Research Database (Denmark)

    Hakhverdyan, Mikhayil; Hägglund, Sara; Larsen, Lars Erik

    2005-01-01

    understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format......, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. According to comparative analyses, the detection limit of the fRT-PCR was on the same level as that of a nested PCR and the sensitivity relatively higher than that of a conventional PCR......, antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can...

  5. Distúrbios de memória em pacientes epilépticos

    Directory of Open Access Journals (Sweden)

    STELLA FLORINDO

    1999-01-01

    Full Text Available O autor estudou distúrbios de memória em pacientes epilépticos com crises parciais complexas (CPC, com os objetivos de: 1 identificar estes distúrbios; e 2 comparar os resultados dos pacientes com os dos controles. Foram estudados 50 pacientes adultos e 20 sujeitos sem enfermidades neuropsiquiátricas. Os métodos consistiram em: 1 investigação da atividade mnemônica através do Teste de Memória de Wechsler (subtestes: Armazenamento e Recuperação, Memória Recente e Memória Imediata; e 2 comparação entre os resultados de ambos os grupos. 3 associação entre SPECT Cerebral e atividade mnemônica. Nos três subtestes, os pacientes apresentaram desempenho cognitivo significativamente inferior ao dos controles (p <0,05. Constatou-se a associação entre hipofluxo em região temporal esquerda e distúrbio da memória nos três subtestes. Concluiu-se que há associação entre CPC e distúrbios da atividade mnemônica.

  6. Ambliopia por estrabismo: estudo retrospectivo de pacientes em hospital universitário

    OpenAIRE

    Costa,Deborah Salerno; Klein,Rita de Cássia Andrade; Leite,Cristiane de Almeida; Ginguerra,Maria Antonieta da Anunciação; Polati,Mariza

    2006-01-01

    OBJETIVO: Avaliar a influência da adesão ao tratamento, da gravidade da ambliopia e da idade de início do tratamento em pacientes com ambliopia por estrabismo submetidos à terapia oclusiva. MÉTODOS: Analisaram-se 569 prontuários de pacientes com ambliopia por estrabismo atendidos no Hospital das Clínicas da Faculdade de Medicina da USP no período de 1983 a 2000. Os critérios de exclusão foram: perda de seguimento, idade maior que 12 anos, presença de nistagmo e outras doenças oculares. Todos ...

  7. Acuidade visual e eletrorretinografia de campo total em pacientes com síndrome de Usher

    OpenAIRE

    Mendieta,Luana; Berezovsky,Adriana; Salomão,Solange Rios; Sacai,Paula Yuri; Pereira,Josenilson Martins; Fantini,Sérgio Costa

    2005-01-01

    A síndrome de Usher (SU) é doença autossômica recessiva caracterizada por perda auditiva neuro-sensorial acompanhada de retinose pigmentária (RP). OBJETIVO: Analisar a eletrorretinografia de campo total (ERG) e a acuidade visual (AV) em pacientes com síndrome de Usher tipos I e II. MÉTODOS: Foram estudadas as respostas da eletrorretinografia de campo total e a acuidade visual de 22 pacientes (idade média = 26,8±16,8 anos). Destes, 17 tinham síndrome de Usher tipo I e 5 tinham síndrome ...

  8. Anestesia em paciente com síndrome de Rubinstein-Taybi: relato de caso

    OpenAIRE

    Oliveira, Carlos Rogério Degrandi; Elias, Luciana

    2005-01-01

    JUSTIFICATIVA E OBJETIVOS: A síndrome de Rubinstein-Taybi (SRT) é uma doença genética causada por uma mutação ou apagamento do cromossomo 16, caracterizada por retardo físico e mental, anormalidades craniofaciais e hálux e polegares largos. Há pouca informação sobre esta síndrome na literatura anestésica. O objetivo deste relato foi apresentar a conduta anestésica em paciente submetido à cirurgia odontológica e discutir as características de interesse para a anestesia nesses pacientes. RELATO...

  9. Rosuvastatina e ciprofibrato no tratamento da dislipidemia em pacientes com HIV

    Directory of Open Access Journals (Sweden)

    Hamilton Domingos

    2012-11-01

    Full Text Available FUNDAMENTO: A dislipidemia secundária à terapia antirretroviral potente nos pacientes com HIV está associada à significativa elevação da morbimortalidade cardiovascular por doença aterosclerótica, sendo, portanto, necessário tratamento imediato e eficaz. OBJETIVO: Demonstrar a efetividade e a segurança da rosuvastatina e do ciprofibrato no tratamento da dislipidemia associada à terapia antirretroviral potente em pacientes com HIV. MÉTODOS: Trezentos e quarenta e seis pacientes com dislipidemia foram submetidos a tratamento farmacológico: 200 pacientes com hipertrigliceridemia receberam ciprofibrato (Grupo I; 79 pacientes com hipercolesterolemia receberam rosuvastatina (Grupo II; e 67 pacientes com dislipidemia mista receberam ciprofibrato associado a rosuvastatina (Grupo III. O perfil lipídico foi avaliado antes e após o tratamento hipolipemiante, sendo feita comparação estatística pelo teste de Wilcoxon. Transaminases hepáticas e creatinofosfoquinase foram dosadas para controle de toxicidade hepática e muscular. RESULTADOS: As concentrações séricas de triglicérides e de colesterol total foram significativamente menores do que as obtidas antes do tratamento, para os três grupos experimentais (p < 0,002. Observou-se aumento significativo do HDL colesterol nos grupos experimentais I e III (p < 0,002. Nos grupos I e II, o LDL-colesterol foi significativamente menor (p < 0,001. Nenhum dos pacientes apresentou elevações de transaminases ou de creatinofosfoquinase a níveis de toxicidade significativa. CONCLUSÃO: Os resultados deste estudo demonstram que ciprofibrato, rosuvastatina ou a combinação de ambos pode ser considerada tratamento hipolipemiante efetivo, seguro e com boa tolerância nos pacientes com Aids submetidos à terapia antirretroviral potente.

  10. PACIENTE EM UNIDADES MÉDICO-CIRÚRGICAS

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    Gisele Hespanhol Dorigan

    2015-01-01

    Full Text Available Este estudio tuvo como objetivo identificar los factores predictivos de la experiencia y la satisfacción del paciente con la atención de enfermería y comparar las propiedades psicometricas de la versión brasileña y la versión original del Newcastle Satisfaction with Nursing Scales. Se trata de un estudio transversal con 351 pacientes en unidades médico-quirúrgicas de un hospital universitario. Para el análisis de los dados se utilizó el método de regresión lineal múltiple. La satisfacción con la atención de enfermería en general fue el predictor de las experiencias (p<0.0001 y la satisfacción con el internamiento (p<0,0001. Otros predictores de la satisfacción con la atención fueron la edad (p=0,027, el nivel de educación (p=0,024 y la satisfacción con la hospitalización (p=0,021. Llegamos a la conclusión de que la versión brasileña del instrumento demostró confiabilidad y validez satisfactorios, así como ser fácil de usar y menos costoso.

  11. Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay.

    Science.gov (United States)

    Chen, Jingfang; Zhang, Rusheng; Ou, Xinhua; Yao, Dong; Huang, Zheng; Li, Linzhi; Sun, Biancheng

    2017-06-01

    A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

    OpenAIRE

    Kang, Xiao-ping; Jiang, Tao; Li, Yong-qiang; Lin, Fang; Liu, Hong; Chang, Guo-hui; Zhu, Qing-yu; Qin, E-de; Qin, Cheng-feng; Yang, Yin-hui

    2010-01-01

    Abstract A duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was improved for simultaneous detection of highly pathogenic H5N1 avian influenza virus and pandemic H1N1 (2009) influenza virus, which is suitable for early diagnosis of influenza-like patients and for epidemiological surveillance. The sensitivity of this duplex real-time RT-PCR assay was 0.02 TCID50 (50% tissue culture infective dose) for H5N1 and 0.2 TCID50 for the pandemic H1N1, which was the same a...

  13. Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.

    Directory of Open Access Journals (Sweden)

    Thrush Anthony

    2010-01-01

    Full Text Available Abstract Background Perennial ryegrass (Lolium perenne L. is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2 were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L. samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h, a moderately, but stably expressed eEF1A (s, and combined expression of multigene eEF1A (m. NormFinder identified eEF1A (s and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples

  14. Dermatite associada à incontinência: estudo de coorte em pacientes críticos

    Directory of Open Access Journals (Sweden)

    Tânia Couto Machado Chianca

    2017-03-01

    Full Text Available RESUMO Objetivos Estimar incidência, determinar fatores de risco e propor modelo de predição de risco para desenvolvimento de dermatite associada a incontinência em pacientes adultos críticos. Método Trata-se de um estudo de coorte concorrente realizado com 157 pacientes críticos. A coleta de dados foi conduzida diariamente entre fevereiro e julho de 2015 em hospital público e de ensino de Belo Horizonte, MG. Os dados foram lançados em banco de dados, submetidos a análise descritiva de sobrevida e multivariada. Resultados Obteve-se uma incidência global de 20,4%. Foram encontrados 19 fatores de risco que apresentaram associação significativa com o problema. As variáveis encontradas no modelo de predição de risco foram: sexo masculino, trauma, uso de hipnótico/sedativos, lactulona, suporte nutricional, fezes pastosas e queixa de ardência (local. Conclusão Os resultados mostraram que a dermatite é um achado clínico comum em pacientes adultos críticos e merece atenção especial para maior qualidade da assistência de enfermagem.

  15. Qualidade de vida em pacientes submetidos à prostatectomia radical

    OpenAIRE

    Goulart, Débora Moura Miranda

    2012-01-01

    Introdução: O câncer de próstata (CaP) é entre os homens o segundo tumor maligno mais prevalente e a segunda neoplasia em causa de morte. Duas características são peculiares: a incidência aumenta com a idade e o caráter indolente na maioria dos casos. Estudos sobre o CaP são relevantes frente à elevada incidência e às altas taxas de cura quando há o diagnóstico em fases iniciais. A prostatectomia radical (PR) é o padrão ouro em tumores localizados. Como complicações cirúrgicas destacam-se,...

  16. Manifestações bucais em pacientes com hipogamaglobulinemia

    OpenAIRE

    Karin Sá Fernandes

    2010-01-01

    A hipogamaglobulinemia é uma alteração da imunidade humoral caracterizada por baixos níveis séricos de anticorpos podendo ter causas primárias e secundárias. Estes pacientes apresentam uma susceptibilidade a infecções bacterianas de repetição ou crônicas, principalmente do trato respiratório. Além disso, há alta prevalência de doenças gastrointestinais infecciosas e inflamatórias, hepatite C, doenças auto-imunes, doenças linfoproliferativas e granulomatosas. Ainda que a doença seja conhecida ...

  17. Candidíase em pacientes aidéticos

    Directory of Open Access Journals (Sweden)

    C.E.O.P. Campos

    1992-09-01

    Full Text Available Trinta e cinco aidéticos entre 19 e 55 anos admitidos e tratados de candidíase no Hospital Emílio Ribas, SP, com ELISA positivo para HIV e confirmado pelo Western Blot. Tuberculose em 9 sendo 2 com pericardite; neurotoxoplasmose em 6; neurocriptococose em 5; herpes labial em 4; pneumocistose em 3 e sarcoma de Kaposi em 2, achavam-se associadas. A concentração inibitória mínima 50% (MIC 50% para os azoles foi: ketoconazol= 2,2 µg/ml; itraconazol- 21,0 µg/ml; fluconazol = 19,0 µg/ml. O MIC 50% para ospolienos: nistatina- 50,0 µg/ml; anfotericina B= 0,12 µg/ml e para 5 fluorcitosina= 1,6 µg/ml nas 35 amostras de Candida isoladas. Testes não paramétricos de Siegel revelaram significante identificação (80% das Candida albicans na candidíase, e que a dose de AMB não modificou o número de óbitos, precoce e tardio, ocorridos nesses aidéticos. O uso prévio dos azoles e da nistatina explicaria, talvez, o elevado MIC 50% observado nas amostras de Candida isoladas.A total of 35 in patients admitted at Emilio Ribas Hospital - São Paulo, Brazil, with digestive candidiasis and AIDS clinical diagnostic were evaluated 10 month later, being 29 male and 6 female; white outnumbering black with age ranged from 30 to 50 years old. Agar Sabouraud culture and tube germinative tests identified 28 (80% Candida albicans out 35 strains. Minimum inhibitory concentration (MIC 50% was against azoles (ketoconazole= 2.2 µg/ml; itraconazole = 21.0 µg/ml and fluconazole- 19.0 µg/ml; polyenes (ny statine - 50.0 µg/ml and amphotericin B= 0.12 µg/ml and 5 fluorcytosine= 1.6µg/ml. Siegeltests showed significant Candida albicans proportions in strains isolated from 35 AIDS patients. There was no significant relation between AMB dosis and early or late death. Conclusions: candidiasis in AIDS patients showed high MIC 50% to azoles and nystatine and significant Candida albicans proportion in all strains isolated from AIDS patients. Previous amphotericin B

  18. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    Science.gov (United States)

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    JUAN P.P. LLERENA

    Full Text Available 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.

  20. A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2).

    Science.gov (United States)

    Duarte, Margarida Dias; Carvalho, Carina L; Barros, Silvia C; Henriques, Ana M; Ramos, Fernanda; Fagulha, Teresa; Luís, Tiago; Duarte, Elsa L; Fevereiro, Miguel

    2015-07-01

    A specific real time RT-PCR for the detection of RHDV2 was developed and validated using RHDV and RHDV2 RNA preparations from positive field samples. The system was designed to amplify a 127 nucleotide-long RNA region located within the vp60 gene, based on the alignment of six sequences originated in Portugal, obtained in our laboratory, and 11 sequences from France and Italy. The primers and probe target sequences are highly conserved in the vast majority of the RHDV2 sequences presently known. In the sequences showing variability, only one mismatch is found per strain, usually outlying the 3' end of the primer or probe hybridization sequences. The specificity of the method was demonstrated in vitro with a panel of common rabbit pathogens. Standardization was performed with RNA transcripts obtained from a recombinant plasmid harboring the target sequence. The method was able to detected nine RNA molecules with an efficiency of 99.4% and a R(2) value of 1. Repeatability and reproducibility of the method were very high, with coefficients of variation lower than 2.40%. The assay was proven a valuable tool to diagnose most of RDVH2 circulating strains, and may be also useful to monitor viral loads, and consequently, disease progression and vaccination efficacy. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Identification of Dobrava, Hantaan, Seoul, and Puumala viruses by one-step real-time RT-PCR.

    Science.gov (United States)

    Aitichou, Mohamed; Saleh, Sharron S; McElroy, Anita K; Schmaljohn, C; Ibrahim, M Sofi

    2005-03-01

    We developed four assays for specifically identifying Dobrava (DOB), Hantaan (HTN), Puumala (PUU), and Seoul (SEO) viruses. The assays are based on the real-time one-step reverse transcriptase polymerase chain reaction (RT-PCR) with the small segment used as the target sequence. The detection limits of DOB, HTN, PUU, and SEO assays were 25, 25, 25, and 12.5 plaque-forming units, respectively. The assays were evaluated in blinded experiments, each with 100 samples that contained Andes, Black Creek Canal, Crimean-Congo hemorrhagic fever, Rift Valley fever and Sin Nombre viruses in addition to DOB, HTN, PUU and SEO viruses. The sensitivity levels of the DOB, HTN, PUU, and SEO assays were 98%, 96%, 92% and 94%, respectively. The specificity of DOB, HTN and SEO assays was 100% and the specificity of the PUU assay was 98%. Because of the high levels of sensitivity, specificity, and reproducibility, we believe that these assays can be useful for diagnosing and differentiating these four Old-World hantaviruses.

  2. Combined Metabonomic and Quantitative RT-PCR Analyses Revealed Metabolic Reprogramming Associated with Fusarium graminearum Resistance in Transgenic Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Fangfang Chen

    2018-01-01

    Full Text Available Fusarium head blight disease resulting from Fusarium graminearum (FG infection causes huge losses in global production of cereals and development of FG-resistant plants is urgently needed. To understand biochemistry mechanisms for FG resistance, here, we have systematically investigated the plant metabolomic phenotypes associated with FG resistance for transgenic Arabidopsis thaliana expressing a class-I chitinase (Chi, a Fusarium-specific recombinant antibody gene (CWP2 and fused Chi-CWP2. Plant disease indices, mycotoxin levels, metabonomic characteristics, and expression levels of several key genes were measured together with their correlations. We found that A. thaliana expressing Chi-CWP2 showed higher FG resistance with much lower disease indices and mycotoxin levels than the wild-type and the plants expressing Chi or CWP2 alone. The combined metabonomic and quantitative RT-PCR analyses revealed that such FG-resistance was closely associated with the promoted biosynthesis of secondary metabolites (phenylpropanoids, alkanoids and organic osmolytes (proline, betaine, glucose, myo-inositol together with enhanced TCA cycle and GABA shunt. These suggest that the concurrently enhanced biosyntheses of the shikimate-mediated secondary metabolites and organic osmolytes be an important strategy for A. thaliana to develop and improve FG resistance. These findings provide essential biochemical information related to FG resistance which is important for developing FG-resistant cereals.

  3. Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation

    DEFF Research Database (Denmark)

    Meerson, Ari; Ploug, Thorkil

    2016-01-01

    of specific miRNAs in different samples varied considerably between the tested extraction methods. Of all kits tested, the QIAGEN miRNeasy kits (Mini and Serum/Plasma kits) and the Macherey-Nagel NucleoSpin kit produced the highest RNA yields. The QIAGEN Exo kit produced lesser yields than what could...... be extracted from the UC fraction using the QIAGEN miRNeasy kits and the Macherey-Nagel NucleoSpin kit. Bioanalyzer results showed an average correlation of R2¼0.8 with endogenous miRNA qRT-PCR results, for sample concentrations >40 pg/ml. The levels of the endogenous miRNAs measured in the two volunteer...... samples were compared with those in a larger group of subjects (n¼10) and found to be typical. Our comparison favors the use of the QIAGEN Serum/Plasma kit and the Macherey-Nagel NucleoSpin kit for plasma miRNA applications. Furthermore, extraction of miRNAs from the UC fraction results in higher yield...

  4. Housekeeping gene expression during fetal brain development in the rat-validation by semi-quantitative RT-PCR.

    Science.gov (United States)

    Al-Bader, Maie Dawoud; Al-Sarraf, Hameed Ali

    2005-04-21

    Mammalian gene expression is usually carried out at the level of mRNA where the amount of mRNA of interest is measured under different conditions such as growth and development. It is therefore important to use a "housekeeping gene", that does not change in relative abundance during the experimental conditions, as a standard or internal control. However, recent data suggest that expression of some housekeeping genes may vary with the extent of cell proliferation, differentiation and under various experimental conditions. In this study, the expression of various housekeeping genes (18S rRNA [18S], glyceraldehydes-3-phosphate dehydrogenase [G3PDH], beta-glucuronidase [BGLU], histone H4 [HH4], ribosomal protein L19 [RPL19] and cyclophilin [CY]) was investigated during fetal rat brain development using semi-quantitative RT-PCR at 16, 19 and 21 days gestation. It was found that all genes studied, with exception to G3PDH, did not show any change in their expression levels during development. G3PDH, on the other hand, showed increased expression with development. These results suggest that the choice of a housekeeping gene is critical to the interpretation of experimental results and should be modified according to the nature of the study.

  5. Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp

    International Nuclear Information System (INIS)

    Liu Wangjing; Chang Yunshiang; Wang Chunghsiung; Kou, Guang-Hsiung; Lo Chufang

    2005-01-01

    Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter

  6. O familiar acompanhante como estimulo comportamental de pacientes internados em terapia intensiva

    Directory of Open Access Journals (Sweden)

    Veronica Lopes Louzada Vidal

    2013-09-01

    Full Text Available O objetivos deste estudo foram levantar as expressões verbais e não verbais presentes no comportamento do paciente antes, durante a inserção e permanência do familiar acompanhante no centro de terapia intensiva, e analisar comparativamente as alterações comportamentais do paciente durante esses momentos da internação. MÉTODO: O cenário foi o centro de terapia intensiva de um hospital no Rio de Janeiro, conveniado com o Sistema Único de Saúde (SUS. A abordagem qualitativa resultou da observação livre sobre o comportamento de cinco pacientes e registros em diário de campo durante três dias consecutivos. Priorizou-se observação do paciente no primeiro dia de internação e após a inserção ininterrupta do familiar acompanhante por três dias consecutivos. RESULTADOS: As mudanças nas reações verbais e não verbais do paciente após os estímulos gerados pela presença do familiar variaram de ausência de fala e movimentos, passividade no agir, a reações de aceitação dos procedimentos, comunicação cinésica ampliada e maior participação no cuidado.

  7. A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

    Science.gov (United States)

    Han, Yang; Hou, Shao-Yang; Ji, Shang-Zhi; Cheng, Juan; Zhang, Meng-Yue; He, Li-Juan; Ye, Xiang-Zhong; Li, Yi-Min; Zhang, Yi-Xuan

    2017-11-15

    A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Real-time onestep RT-PCR for the detection and differentiation of European and North American types of PRRSV in boar semen

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Larsen, Lars Erik

    and safe diagnostic procedure, since cDNA synthesis and PCR is performed sequentially without inbetween opening of the PCR-tubes, thus eliminating a substantial contamination risk. The aim of the present study was to validate a real-time OneStep RT-PCR assay for the simultaneous detection...

  9. Tick-borne encephalitis virus-specific RT-PCR - a rapid test for detection of the pathogen without viral RNA purification

    Czech Academy of Sciences Publication Activity Database

    Rudenko, Natalia; Golovchenko, Maryna; Cihlářová, Věra; Grubhoffer, Libor

    2004-01-01

    Roč. 48, č. 3 (2004), s. 167-171 ISSN 0001-723X R&D Projects: GA ČR GA524/03/1326 Institutional research plan: CEZ:AV0Z6022909 Keywords : Ixodes ricinus * tick-borne encephalitis virus * RT-PCR Subject RIV: GJ - Animal Vermins ; Diseases, Veterinary Medicine Impact factor: 0.605, year: 2004

  10. A comparison of the sensitivities of detection of Plasmodium falciparum gametocytes by magnetic fractionation, thick blood film microscopy, and RT-PCR

    Directory of Open Access Journals (Sweden)

    St-Pierre Tim G

    2009-05-01

    Full Text Available Abstract Background The magnetic properties of Plasmodium-infected erythrocytes have been exploited for different clinical and research purposes. A recent study in a rural clinical setting in Papua New Guinea has demonstrated that Plasmodium falciparum gametocyte detection is facilitated by magnetic deposition microscopy but no study has yet determined the relative sensitivity and limit of detection of a magnetic fractionation technique. The present study compares the detection limit and sensitivity of a technique based on the use of commercially available magnetic fractionation columns with those for thick blood film microscopy and reverse transcriptase polymerase chain reaction (RT-PCR methods. Methods Gametocyte detection in six series of dilutions of cultured P. falciparum parasites with known gametocytaemia was conducted using magnetic fractionation, thick blood film, and RT-PCR techniques. Results The preparations obtained by the magnetic fractionation method were of thin film quality allowing easy gametocyte identification by light microscopy. Magnetic fractionation had a higher sensitivity and approximately two orders of magnitude better limit of detection than thick blood film microscopy. Gametocytes were also more readily detectable on the magnetically fractionated preparations. Magnetic fractionation had a similar limit of detection to that of RT-PCR. Conclusion Magnetic fractionation is a highly sensitive and convenient method for gametocyte detection in comparison with the standard thick blood film and RT-PCR methods, and could readily be adapted to field application.

  11. Foot-and-mouth disease virus: A first inter-laboratory comparison trial to evaluate virus isolation and RT-PCR detection methods.

    NARCIS (Netherlands)

    Ferris, N.P.; King, D.P.; Reid, S.M.; Hutchings, G.H.; Shawa, A.E.; Paton, D.J.; Goris, N.; Haas, B.; Hoffmann, B.; Brocchi, E.; Bugnetti, M.; Dekker, A.; Clerq, De K.

    2006-01-01

    Five European reference laboratories participated in an exercise to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and cell cultures for the detection and isolation of foot-and-mouth disease (FMD) virus. Five identical sets of 20 coded samples were prepared from 10

  12. Development of a novel real-time RT-PCR assay to detect Seneca Valley virus-1 associated with emerging cases of vesicular disease in pigs.

    Science.gov (United States)

    Fowler, Veronica L; Ransburgh, Russell H; Poulsen, Elizabeth G; Wadsworth, Jemma; King, Donald P; Mioulet, Valerie; Knowles, Nick J; Williamson, Susanna; Liu, Xuming; Anderson, Gary A; Fang, Ying; Bai, Jianfa

    2017-01-01

    Seneca Valley virus 1 (SVV-1) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-1-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time (r) reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV-1. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid templates prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID 50 /ml and the limit of detection was equivalent using two different rRT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV-1 in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV-1. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Development of a real-time RT-PCR assay based on primer-probe energy transfer for the detection of all serotypes of bluetongue virus

    DEFF Research Database (Denmark)

    Leblanc, N; Rasmussen, Thomas Bruun; Fernandez, J

    2010-01-01

    A real-time RT-PCR assay based on the primer–probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward...

  14. Selection of reference genes for quantitative real-time RT-PCR studies in tomato fruit of the genotype MT-Rg1

    Directory of Open Access Journals (Sweden)

    Karla L. González-Aguilera

    2016-09-01

    Full Text Available Quantitative real-time RT-PCR (qRT-PCR has become one of the most widely used methods for accurate quantification of gene expression. Since there are no universal reference genes for normalization, the optimal strategy to normalize raw qRT-PCR data is to perform an initial comparison of a set of independent reference genes to assess the most stable ones in each biological model. Normalization of a qRT-PCR experiment helps to ensure that the results are both statistically significant and biologically meaningful. Tomato is the model of choice to study fleshy fruit development. The miniature tomato (Solanum lycopersicum L. cultivar Micro-Tom (MT is considered a model system for tomato genetics and functional genomics. A new genotype, containing the Rg1 allele, improves tomato in vitro regeneration. In this work, we evaluated the expression stability of four tomato reference genes, namely CAC, SAND, Expressed and ACTIN2. We showed that the genes CAC and Exp are the best reference genes of the four we tested during fruit development in the MT-Rg1 genotype. Furthermore, we validated the reference genes by showing that the expression profiles of the transcription factors FRUITFULL1 (FUL1 and APETALA2c (AP2c during fruit development are comparable to previous reports using other tomato cultivars.

  15. Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; de Stricker, K.

    2003-01-01

    Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting...

  16. Analysis of ORF 1 in European porcine reproductive and respiratory syndrome virus by long RT-PCR and restriction fragment length polymorphism (RFLP) analysis

    DEFF Research Database (Denmark)

    Nielsen, H. S.; Storgaard, Torben; Oleksiewicz, M.B.

    2000-01-01

    A rapid method was developed for partial characterization of the replicase-encoding open reading frame 1 (ORF 1) of porcine reproductive and respiratory syndrome virus (PRRSV). It comprised long RT-PCR amplification of 11.1 kb (94%) of ORF 1, followed by restriction fragment length polymorphism a...

  17. Use of a custom RT-PCR array to analyze toxicity pathways at different life stages in Brown Norway Rat Brain following acute Toluene exposure.

    Science.gov (United States)

    To investigate the contribution of different life stages on response to toxicants, we utilized a custom designed RT-PCR array to examine the effects of acute exposure by oral gavage of the volatile organic solvent toluene (0.00, 0.65 or 1.0 glkg) in the brains of ma1e Brown Norwa...

  18. Establishment of a nanoparticle-assisted RT-PCR assay to distinguish field strains and attenuated strains of porcine epidemic diarrhea virus.

    Science.gov (United States)

    Zhu, Yu; Wang, Gui-Hua; Cui, Yu-Dong; Cui, Shang-Jin

    2016-09-01

    Porcine epidemic diarrhea virus (PEDV) can cause serious disease and even death in neonatal piglets, resulting in serious damage to the swine industry worldwide. Open reading frame 3 (ORF3) is the only accessory gene in the PEDV genome. Previous studies have indicated that PEDV vaccine strains have a partial deletion in ORF3. In this study, a nanoparticle-assisted polymerase chain reaction (nanoparticle-assisted RT-PCR) assay targeting the ORF3 of PEDV was developed to distinguish PEDV field strains from attenuated strains by using a specific pair of primers. The PCR products of field strains and attenuated strains were 264 bp and 215 bp in length, respectively. The sensitivity and specificity of this assay were also assessed. The nanoparticle-assisted RT-PCR assay was 10-100 times more sensitive than the conventional RT-PCR assay, with no cross-reactions when amplifying porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (RV), and porcine transmissible gastroenteritis virus (TGEV). The nanoparticle-assisted RT-PCR assay we describe here can be used to distinguish field strains from vaccine strains of PEDV, and it shows promise for reducing economic loss due to PEDV infection.

  19. Diagnósticos de enfermagem identificados em pacientes transplantados renais de um hospital de ensino

    Directory of Open Access Journals (Sweden)

    Michelle Soares Josino da Silva

    2009-06-01

    Full Text Available O transplante de órgãos constitui uma última esperança de sucesso na recuperação de doenças em estágio terminal. AEnfermagem participa de forma atuante desde a captação do órgão até as consultas no ambulatório após o transplante,exigindo pessoal capacitado. Esse trabalho tem por objetivos identificar os principais diagnósticos de enfermagem empacientes transplantados renais, em uma unidade de internação de um hospital de ensino no município de Fortaleza-Ce, epropor intervenções baseadas nas reais necessidades desses pacientes. Estudo descritivo-exploratório, de naturezaqualitativa. A população foi constituída por pacientes transplantados em pós-operatório mediato e com complicações póstransplante,sendo a amostra de doze pacientes. A coleta dos dados ocorreu através da aplicação de um histórico deenfermagem e para a análise foi abordada a seqüência das etapas do PE, utilizando-se a taxonomia II da NANDA. Foramidentificados dezessete diagnósticos de enfermagem, o que permitiu a elaboração de intervenções baseadas nas reaisnecessidades desses pacientes. Acreditamos que, as intervenções sugeridas fundamentadas com referencial teórico, serão desuma importância para a prática dos enfermeiros que trabalham nessa área, e principalmente no hospital em questão.

  20. Mogadon endovenoso em pacientes epilepticos: estudo clinico-eletrencefalografico

    Directory of Open Access Journals (Sweden)

    Roberto Sergio Pires de Oliveira

    1979-03-01

    Full Text Available Foi utilizado o Mogadon injetável para o tratamento de 15 casos de "status epilépticus", 10 casos de crises intermitentes de mal epiléptico, 14 casos de síndrome de Lennox, 5 casos de crises mioclônicas e 6 casos de ausências simples. As doses utilizadas variaram entre 3,3 mg e 10 mg. Nos casos de "status epilépticus" e crises intermitentes de mal epiléptico o medicamento fez desaparecer as crises e as alterações irritativas do EEG. Houve remissão em 10 casos e melhora de outros 4 com síndrome de Lennox. A medicação produziu a abolição da sintomatologia em alguns casos de ausências puras; a melhora foi evidente, embora de duração mais curta do que a melhora nas mioclônicas. O único efeito secundário foi a sonolência, observada em alguns casos.

  1. Prevalência de deficiência de vitamina D em pacientes com úlceras de perna de etiologia venosa

    Directory of Open Access Journals (Sweden)

    Claudine Juliana C Burkievcz

    Full Text Available OBJETIVO: Estudar se a prevalência da deficiência de vitamina D em indivíduos com úlcera de perna de causa venosa é maior do que em população controle. MÉTODOS: Estudaram-se os níveis séricos de 25-OH-vitamina D por quimioluminescência em 27 portadores de úlcera venosa crônica e 58 controles do Hospital Universitário Evangélico de Curitiba. RESULTADOS: Os níveis de 25-OH-vitamina D3 eram inferiores a 8 ng/dl em 11,1% dos pacientes com úlcera e 3,4% dos controles; entre 8 e 20 ng/dl em 46,1% dos pacientes com úlcera e 25,8% dos controles; entre 21 e 30 ng/dl em 22.2% dos pacientes com úlcera e 27,5% dos controles e acima de 30 ng/dl em 43,1% dos controles e 18,5% dos pacientes com úlcera (p=0,04. CONCLUSÃO: Existe aumento de prevalência de deficiência de vitamina D em pacientes com úlceras venosas crônicas de pernas.

  2. Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project.

    Science.gov (United States)

    Letovanec, Igor; Finn, Stephen; Zygoura, Panagiota; Smyth, Paul; Soltermann, Alex; Bubendorf, Lukas; Speel, Ernst-Jan; Marchetti, Antonio; Nonaka, Daisuke; Monkhorst, Kim; Hager, Henrik; Martorell, Miguel; Sejda, Aleksandra; Cheney, Richard; Hernandez-Losa, Javier; Verbeken, Eric; Weder, Walter; Savic, Spasenija; Di Lorito, Alessia; Navarro, Atilio; Felip, Enriqueta; Warth, Arne; Baas, Paul; Meldgaard, Peter; Blackhall, Fiona; Dingemans, Anne-Marie; Dienemann, Hendrik; Dziadziuszko, Rafal; Vansteenkiste, Johan; O'Brien, Cathal; Geiger, Thomas; Sherlock, Jon; Schageman, Jeoffrey; Dafni, Urania; Kammler, Roswitha; Kerr, Keith; Thunnissen, Erik; Stahel, Rolf; Peters, Solange

    2018-03-01

    The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be

  3. Letalidade e internacoes de pacientes em hemodialise em plano de saude

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    Sergio Adriano Loureiro Bersan

    2013-06-01

    Full Text Available OBJETIVO: Analisar desfechos clínicos de pacientes incidentes em hemodiálise vinculados a operadora de plano de saúde. MÉTODOS: Estudo de coorte de incidentes em hemodiálise em Belo Horizonte, MG, de 2004 a 2008, a partir de registros no banco de dados de operadora de planos de saúde. Variáveis independentes: sexo, idade, tempo entre primeira consulta com nefrologista e início da hemodiálise, tipo do primeiro acesso vascular, diabetes mellitus, tempo de permanência hospitalar/ano de tratamento e óbito. Variáveis dependentes: tempo entre início da hemodiálise e óbito e tempo de permanência hospitalar/ano de tratamento > 7,5 dias. Análise estatística: teste Qui-quadrado de Pearson na análise univariada para os desfechos óbito e tempo de permanência hospitalar/ano de tratamento; método de Kaplan-Meier para análise de sobrevida; modelo de Cox e regressão Poisson para risco de óbito e chance de tempo de permanência hospitalar/ano de tratamento > 7,5 dias. Foi utilizada ferramenta de Business Intelligence para extração dos dados e software Stata(r 10.0. RESULTADOS: Estudados 311 indivíduos em hemodiálise, 55,5% homens, média de 62 anos (dp: 16,6 anos. A prevalência aumentou 160% no período estudado. Na análise de sobrevivência a mortalidade foi maior entre os mais idosos, nos que não realizaram consulta com nefrologista, fizeram uso de cateter vascular temporário como primeiro acesso, com diabetes mellitus, nos que foram internados no mesmo mês do início da hemodiálise. No modelo de Cox associaram-se a maior risco para óbito a idade avançada, diabetes mellitus, não realizar consulta prévia com nefrologista e internar-se no primeiro mês de hemodiálise. Maior tempo de permanência hospitalar/ano de tratamento não se associou ao sexo e diabetes. As variáveis não foram significativas na regressão Poisson. CONCLUSÕES: A avaliação pelo especialista antes do início da hemodiálise diminui o risco de

  4. Um Novo Indice de Doppler Tecidual para Prever Morte Cardiaca em Pacientes com Insuficiencia Cardiaca

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    Cristian Mornos

    2014-01-01

    Full Text Available Fundamento: Foi demonstrado que um novo índice de Doppler Tecidual, E/(E'×S', incluindo a proporção entre a velocidade diastólica precoce transmitral e a do anel mitral (E/E', e a velocidade sistólica do anel mitral (S', tem uma boa precisão como preditor da pressão de enchimento do ventrículo esquerdo. Objetivo: Investigar o valor de E/(E'×S' para prever a morte cardíaca em pacientes com insuficiência cardíaca. Métodos: Foi realizado sucessivamente o ecocardiograma em 339 pacientes hospitalizados com insuficiência cardíaca, em ritmo sinusal, após tratamento médico adequado, no momento e um mês depois da alta. O agravamento de E/(E'×S' foi definido como um aumento do valor padrão. O ponto final foi a morte cardíaca. Resultados: Durante o período de acompanhamento (35,2 ± 8,8 meses, ocorreu a morte cardíaca em 51 pacientes (15%. O melhor valor mínimo para E/(E'× S' inicial na previsão da morte cardíaca foi de 2,83 (76% de sensibilidade, 85% de especificidade. No momento da alta, 252 pacientes (74,3% apresentaram E/(E'×S' ≤ 2,83 (grupo I, e 87 (25,7% apresentaram E/(E'×S' > 2,83 (grupo II, respectivamente. A morte cardíaca foi significativamente maior no grupo II em relação ao grupo I (38 mortes, 43,7% contra 13 mortes, 5,15%, p 2,83 no momento da alta e com um agravamento após um mês apresentaram o pior prognóstico (todos p < 0,05. Conclusão: Em pacientes com insuficiência cardíaca a relação E/(E'×S' é um poderoso preditor da morte cardíaca, especialmente quando esta estiver associada com o seu agravamento.

  5. Interacoes medicamentosas potenciais em pacientes com doenca renal cronica

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    Alessandra Batista Marquito

    2014-03-01

    Full Text Available Introdução: Interações medicamentosas (IMs são frequentes na prática clínica e estão diretamente relacionadas a fatores como polifarmácia, idade avançada e deficiência na metabolização e excreção de fármacos. Indivíduos com doença renal crônica (DRC comumente utilizam diversas classes de medicamentos, constituindo um importante grupo de risco para IMs. Objetivo: Identificar potenciais interações entre medicamentos prescritos a renais crônicos em tratamento conservador, e fatores associados a sua ocorrência. Métodos: Estudo observacional transversal, com análise de 558 prescrições. O potencial interativo dos medicamentos foi traçado tendo como suporte a base de dados MICROMEDEX®, software que disponibiliza farmacopeias conhecidas internacionalmente. Resultados: Houve predomínio de indivíduos do sexo masculino (54,7%, idosos (69,4%, no estágio 3 da DRC (47,5%, com sobrepeso e obesos (66,7%. As comorbidades mais prevalentes foram a hipertensão arterial sistêmica (68,5% e o diabetes mellitus (31,9%. IMs potenciais foram detectadas em 74,9% das prescrições. De um total de 1.364 IMs detectadas, 229 (16,8% foram de gravidade maior e 5 (0,4% contraindicadas, com necessidade de intervenção imediata. Interações de gravidade moderada ou menor foram identificadas respectivamente em 1.049 (76,9% e 81 (5,9% das prescrições. Observou-se que a probabilidade de ocorrência de uma IM aumentou em 2,5 vezes para cada medicamento adicional (IC = 2,18-3,03. Obesidade, diabetes, hipertensão e estágio avançado da DRC foram fatores de risco fortemente associados para ocorrência de IM. Conclusão: A associação de medicamentos em indivíduos com DRC relacionou-se com alta prevalência de IMs potencialmente graves, especialmente nos estágios mais avançados da doença.

  6. Ritual de despedida em familiares de pacientes com prognóstico reservado

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    Márcia Lucrecia Lisbôa

    2003-06-01

    Full Text Available Pesquisa qualitativa que teve por objetivo investigar os efeitos terapêuticos do ritual de despedida na iminência da morte, em familiares de pacientes com prognóstico reservado. Foram estudadas oito famílias de pacientes internados em um Hospital Geral. Utilizou-se o método clínico, as técnicas de estudo de caso e entrevistas pós-óbito, dados submetidos à análise de conteúdo. Os resultados mostraram que os familiares se despediram através da comunicação verbal e não-verbal, da religião, da "liberação", do "estar junto", sendo favoráveis à realização do ritual, destacando o aprendizado, o privilégio de participar e a importância da orientação psicológica. São difuculdades: sentimentos de pena, tristeza, ajuda na aceitação da morte, a aproximação entre os familiares e o paciente, abertura da comunicação, melhoria de condilções para elaborar o luto, redefinição dos relacionamentos com a pessoa ainda em vida.

  7. O efeito da hidroterapia na dor, qualidade de vida e funcionalidade em pacientes com fibromialgia

    OpenAIRE

    Rocha, Virgílio

    2016-01-01

    Projeto de Graduação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Licenciado em Fisioterapia Objetivo: Sistematizar a evidência científica existente sobre o papel do efeito da Hidroterapia na Dor, Qualidade de Vida e Funcionalidade em pacientes com Fibromialgia Métodos: Estudo de revisão incluindo estudos randomizados controlados, pesquisados nas bases de dados PubMed, EBSCO, PEDro e Web of Science, com doentes com fibromialgia, cujo tipo de...

  8. RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms.

    Science.gov (United States)

    Green, Clayton B; Cheng, Georgina; Chandra, Jyotsna; Mukherjee, Pranab; Ghannoum, Mahmoud A; Hoyer, Lois L

    2004-02-01

    An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.

  9. High sensitive RNA detection by one-step RT-PCR using the genetically engineered variant of DNA polymerase with reverse transcriptase activity from hyperthermophilies.

    Science.gov (United States)

    Okano, Hiroyuki; Baba, Misato; Kawato, Katsuhiro; Hidese, Ryota; Yanagihara, Itaru; Kojima, Kenji; Takita, Teisuke; Fujiwara, Shinsuke; Yasukawa, Kiyoshi

    2018-03-01

    One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4pol L329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis. Optimization of reaction condition revealed that the activities for cDNA synthesis and PCR of K4pol L329A and RTX were highly affected by the concentrations of MgCl 2 and Mn(OCOCH 3 ) 2 as well as those of K4pol L329A or RTX. Under the optimized condition, 300 copies/μl of target RNA in 10 μl reaction volumes were successfully detected by the one-step RT-PCR with K4pol L329A or RTX, which was almost equally sensitive enough compared with the current RT-PCR condition using retroviral RT and thermostable DNA polymerase. Considering that K4pol L329A and RTX are stable even at 90-100°C, our results suggest that the one-step RT-PCR with K4pol L329A or RTX is more advantageous than the current one. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. Analgesia regional periférica com lidocaína em paciente queimado: relato de caso

    OpenAIRE

    Geier, Karl Otto

    2004-01-01

    JUSTIFICATIVA E OBJETIVOS: A realização de anestesia regional em pacientes queimados é dificultosa pela localização aleatória das lesões térmicas. Elevadas taxas de alfa1 glicoproteína ácida com grande afinidade para drogas alcalinas, especialmente a lidocaína, têm sido observadas nesses pacientes. Este caso relata como o uso intermitente de anestesia e analgesia regional periférica com altas doses de lidocaína podem ser úteis em fornecer efetiva analgesia num paciente com queimaduras de segu...

  11. Adiaspiromicose pulmonar: achado casual em paciente falecido de febre amarela

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    Moraes Mário A.P.

    2001-01-01

    Full Text Available Durante um surto de febre amarela (forma rural da infecção instalado, em fins de 1999, no Estado de Goiás, Brasil, um enfermo, com sintomatologia suspeita, faleceu no Hospital Universitário de Brasília, DF, cinco dias após a admissão. À necropsia, microscopicamente, além das alterações hepáticas características da infecção, encontraram-se nos pulmões e linfonodos hilares, estruturas arredondadas, reconhecidas como adiaconídios de Emmonsia parva var. crescens.

  12. Dermatoses em pacientes com diabetes mellitus Skin lesions in diabetic patients

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    N T Foss

    2005-08-01

    Full Text Available OBJETIVO: Ainda é desconhecida a relação do diabetes com fatores determinantes ou precipitantes de lesões dermatológicas em pacientes diabéticos. Assim, o objetivo do estudo foi investigar a presença de lesões cutâneas, não referidas pelo paciente diabético e sua relação com o controle metabólico da doença. MÉTODOS: Foram examinados 403 pacientes, dos quais 31% eram diabéticos do tipo 1 e 69% do tipo 2. Em ambulatório de um hospital universitário, os pacientes foram atendidos por endocrinologista para a avaliação endócrino-metabólica e por dermatologista para a avaliação dermatológica. O grau de controle metabólico foi documentado em 136 pacientes por meio da dosagem de hemoglobina glicada. RESULTADOS: Houve predomínio de dermatofitoses (82,6%, seguido de grupo de dermatoses como acne e degeneração actínica (66,7%, piodermites (5%, tumores cutâneos (3% e necrobiose lipoídica (1%. Entre as dermatoses mais comuns em diabéticos, foram confirmados com exame histológico: dois diagnósticos de necrobiose (0,4%, cinco de dermopatia diabética (1,2% e três casos de mal perfurante plantar (0,7%. Os valores da hemoglobina glicada foram: 7,2% em pacientes com controle metabólico adequado nos dois tipos de diabetes e de 11,9% e 12,7% nos tipos 1 e 2, respectivamente, com controle inadequado. Nos pacientes com controle metabólico inadequado foi observada freqüência maior de dermatofitoses, em ambos os tipos de diabetes. CONCLUSÕES: Os dados revelaram freqüência elevada de lesão dermatológica nos pacientes diabéticos, especialmente dermatofitoses. Dessa forma, o descontrole metabólico do diabético propicia maior suscetibilidade a infecções cutâneas.OBJECTIVE: It is yet unknown the relationship between diabetes and determinants or triggering factors of skin lesions in diabetic patients. The purpose of the present study was to investigate the presence of unreported skin lesions in diabetic patients and their

  13. Efetividade da fonoterapia em pacientes com paralisia facial pós-parotidectomia

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    Vanessa da Hora Machado Miranda

    2015-06-01

    Full Text Available O objetivo deste estudo foi verificar a efetividade da fonoterapia em pacientes com paralisia facial decorrente da manipulação do VII nervo encefálico realizada durante o tratamento cirúrgico para neoplasia de glândula parótida, assim como, identificar e promover intervenção fonoaudiológica das alterações de sucção, mastigação e deglutição. Trata-se de uma pesquisa qualitativa com análise descritiva. A avaliação constou da análise da face em repouso e em movimento, documentação fotográfica, uso do Paquímetro Digital para quantificação da paralisia facial, além da avaliação das funções estomatognáticas. A fonoterapia foi definida com base nos achados da avaliação e seguiu a necessidade de cada indivíduo. Nos resultados da avaliação pós-fonoterapia em repouso houve melhora em todos os aspectos avaliados nos quatro pacientes. Na avaliação em movimento três pacientes apresentaram movimentação mais clara da pele com aumento do número e profundidade das rugas. No registro fotográfico todos os pacientes obtiveram melhora significante nos movimentos avaliados, sendo possível observar um aumento nas linhas de expressões e maior simetria entre as hemifaces. Os valores da incompetência dos movimentos, mensurados pós-fonoterapia, demonstraram melhora significante em todos os pontos medidos. A fonoterapia proposta para os casos de paralisia facial pós-parotidectomia foi eficiente na melhora da mímica facial, sobretudo para as alterações das funções estomatognáticas. É importante salientar a necessidade de novas pesquisas envolvendo um número maior de participantes para garantir a fidedignidade dos achados.

  14. Interações Medicamentosas Potenciais em Pacientes de Unidades de Terapia Intensiva

    Directory of Open Access Journals (Sweden)

    Liliana Batista Vieira

    2012-08-01

    Full Text Available O objetivo do estudo é determinar a frequência de interações fármaco-fármaco em prescrições de pacientes internados em unidades de terapia intensiva e analisar os fatores associados relativos à farmacoterapia e ao paciente. O estudo descritivo transversal foi realizado em dois hospitais de ensino da região centro oeste do Brasil. As interações medicamentosas potenciais foram identificadas nas prescrições do primeiro dia (24 horas e do quinto dia (120 horas de internação empregando o software Drug Reax. Dos 117 pacientes do estudo, 63,2% apresentaram interações medicamentosas potenciais nas primeiras 24 horas de internação e 68,4% pacientes em 120 horas. Houve associação estatisticamente significativa entre o número de medicamentos prescritos e a ocorrência de interações medicamentosas nos dois momentos de internação. O estudo demonstrou que a prevalência de interações fármaco-fármaco foi elevada nos hospitais investigados e que a chance de interação aumentou com o número de medicamentos prescritos, número de diagnósticos e idade maior que 60 anos. O conhecimento do mecanismo farmacológico e dos fatores de risco para interações medicamentosas potenciais contribuem para aumentar a segurança e efetividade do tratamento. Para ampliar a segurança da farmacoterapia, é essencial implementar estratégias que auxiliem a equipe de saúde a identificar as interações e implementar medidas de prevenção e monitorização de pacientes em riscos de desenvolver interações medicamentosas. A atuação do farmacêutico clínico na unidade de terapia intensiva é uma ferramenta importante para a prevenção, identificação e monitorização das interações medicamentosas.

  15. Anestesia venosa total para timectomia em paciente com Miastenia Gravis: relato de caso

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    Rezer Gabrielle

    2003-01-01

    Full Text Available JUSTIFICATIVA E OBJETIVOS: Pacientes com doenças neuromusculares, como Miastenia Gravis, respondem de maneira anormal aos anestésicos, conforme a técnica e as drogas administradas. O objetivo deste relato é mostrar um caso de paciente portadora de Miastenia Gravis, submetida a timectomia sob anestesia venosa total com propofol e remifentanil. RELATO DO CASO: Paciente feminina, 52 anos, 72 kg, com história de Miastenia Gravis submetida a timectomia transesternal, sob anestesia venosa total, com o uso de propofol em infusão alvo controlada (3 g.ml-1 e remifentanil contínuo (0,3 µg.kg-1.min-1. Para a intubação traqueal foi utilizada succinilcolina (50 mg, sendo realizada sem dificuldade. Com a finalidade de realizar a analgesia pós-operatória, foram utilizados tramadol (50 mg, cetoprofeno (100 mg e dipirona (1 g, no per-operatório. Após a cirurgia, a infusão de propofol e remifentanil foi encerrada e, 15 minutos depois, a paciente foi extubada. A paciente apresentava-se eupnéica, acordada, sem dor, movimentando membros, com freqüência respiratória de 14 mpm e mantendo saturação de oxigênio de 97%. Permaneceu com cateter nasal de O2 a 2 L.min-1 na UTI intermediária, durante 36 horas, e recebeu alta hospitalar no 4º dia do pós-operatório. CONCLUSÕES: A anestesia venosa total, com agentes anestésicos de curta duração e sem metabólitos ativos, favoreceu a recuperação e a extubação precoce da paciente

  16. Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR

    Directory of Open Access Journals (Sweden)

    Nagorsen Dirk

    2003-12-01

    Full Text Available Abstract The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV and Influenza (Flu, were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ, Interleukin-2 (IL-2, Interleukin-4 (IL-4, and Interleukin-10 (IL-10. We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization.

  17. Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

    Science.gov (United States)

    Provenzano, Maurizio; Mocellin, Simone; Bonginelli, Paola; Nagorsen, Dirk; Kwon, Seog-Woon; Stroncek, David

    2003-01-01

    The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization. PMID:14675481

  18. Factor VIII (F8) inversions in severe hemophilia A: Male germ cell origin and diagnosis with RT-PCR

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E. [Geneva Medical School (Switzerland)]|[Johns Hopkins School of Medicine, Baltimore, MD (United States); Rossiter, J.P. [Johns Hopkins School of Medicine, Baltimore, MD (United States); Young, M. [Geneva Medical School (Switzerland)] [and others

    1994-09-01

    The Factor VIII (F8) gene, which is defective in hemophilia A, is located in the most telomeric megabase of Xq. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of the cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the inversion process, and that therefore the event originates predominantly in male germ cells. In all 21 informative cases in which the inversion originated in a maternal grandparent, DNA polymorphism analysis using markers within or very closely linked to F8, determined that it occurred in the male germline. In addition, all but one of 56 mothers of sporadic cases due to inversions were carriers. The data indicate that the F8 gene inversions leading to severe hemophilia A occur almost exclusively in male germ cells. The mean age of maternal grandfathers at the birth of their carrier daughters was 29.9 years (13 cases), i.e. not different from the mean paternal age in the general population, supporting the hypothesis that the inversions occur in meiosis. The inversions can be diagnosed by Southern blot analysis. For more rapid diagnosis we have used RT-PCR of RNA ectopically expressed in blood. Oligonucleotides were used to PCR amplify, after the initial RT reaction of RNA samples using random hexamers, either the normal transcript (F8 exons 21 to 24;312 bp product) or the novel abnormal transcript that is generated after the inversion. Both type 1 and 2 inversions can be recognized in affecteds and carriers by the presence of the diagnostic PcR product of 248 bp. Correct diagnoses were made in samples from 6 patients and 2 carriers with type 1 inversions, 2 patients and 2 carriers with type 2 inversions and 5 normal controls.

  19. Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities

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    Martinez Alfredo

    2008-05-01

    Full Text Available Abstract Background A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14 derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZm and ADHZm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. It is suggested that this behavior might be due to lineage differences between E. coli W and C. Results This study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZm and ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions. Conclusion Results suggest that a higher ethanol formation rate, caused by ahigher PDCZm and ADHZm activities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates.

  20. Prevalência da fragilidade entre os pacientes com doença renal crônica em tratamento conservador e em diálise

    Directory of Open Access Journals (Sweden)

    Henrique Novais Mansur

    2012-06-01

    Full Text Available INTRODUÇÃO: Fragilidade é um estado de vulnerabilidade fisiológica do paciente que se associa ao maior número de hospitalização e óbito. OBJETIVOS: Avaliar a prevalência de fragilidade e os fatores a ela associados em pacientes com doença renal crônica (DRC em tratamento conservador (TC, hemodiálise (HD e diálise peritoneal (DP. MÉTODOS: A fragilidade foi avaliada em 146 pacientes (86 em TC, 37 em HD e 23 em DP e caracterizada como fraqueza muscular e exaustão - pelos domínios do aspecto físico e da vitalidade, respectivamente, avaliados pelo questionário SF-36 de qualidade de vida; como inatividade física caso a resposta fosse "nunca" ou "quase nunca" - quando questionado sobre a prática de atividade física; e como perda de peso não intencional (> 4,5 kg por ano. Os pacientes foram divididos em três grupos: não frágeis (NF, pré-frágeis (PF e frágeis (F. As variáveis demográficas, clínicas e laboratoriais foram extraídas dos prontuários dos pacientes. RESULTADOS: A fragilidade foi caracterizada em 36% dos pacientes em TC, 37,8% em HD e 47,8% em DP. Foi diagnosticada em 36,8% dos pacientes com idade entre 20 e 40 anos e 40,3% daqueles entre 41 e 60 anos. A fragilidade associou-se significativamente ao uso de vitamina D (r = 0.16; p = 0.03, hemoglobina (r = -0.14; p = - 0.02 e paratormônio intacto (r = 0.16; p = 0.03. CONCLUSÕES: A fragilidade é frequente entre os pacientes com DRC em tratamento conservador e dialítico, mesmo naqueles não idosos. Nos pacientes estudados, o fenótipo de fragilidade se associou com o não uso de vitamina D, menores níveis séricos de hemoglobina e níveis mais elevados de paratormônio.

  1. Variáveis relacionadas com perda da produtividade no trabalho em pacientes com espondilite anquilosante

    Directory of Open Access Journals (Sweden)

    Renata Frauendorf

    2013-06-01

    Full Text Available A perda da produtividade no trabalho, como resultado da espondilite anquilosante, tem se tornado tema de interesse dado o seu impacto socioeconômico. Além das limitações físicas, outras variáveis parecem interferir na produtividade desses pacientes que muitas vezes são aposentados precocemente do mercado de trabalho. Assim, esse manuscrito de revisão buscou identificar artigos publicados na língua inglesa no período de janeiro de 2001 a dezembro de 2011 que discutissem essas variáveis por meio de estudos realizados com pacientes adultos com diagnóstico de espondilite anquilosante, de ambos os gêneros e que usaram instrumentos padronizados para a avaliação da atividade da doença e da capacidade produtiva no trabalho. Foram identificados 33 artigos atendendo aos critérios de inclusão e observou-se que a perda de produtividade no trabalho em pacientes com espondilite anquilosante é influenciada por variáveis demográficas, emocionais, socioculturais e ocupacionais e hábitos de vida. Compreender esses possíveis fatores de risco pode colaborar para a elaboração de estratégias preventivas para a manutenção de pacientes com espondilite anquilosante no mercado de trabalho.

  2. Consumo alimentar e estresse em pacientes com síndrome coronariana aguda

    Directory of Open Access Journals (Sweden)

    Evelise Helena Fadini Reis Brunori

    2015-10-01

    Full Text Available RESUMOObjetivo:Avaliar se o consumo alimentar de pacientes com síndrome coronariana aguda (SCA segue as recomendações nacionais e internacionais e se existe relação com níveis de estresseMétodo:Estudo transversal com 150 pacientes com SCA que foram entrevistados utilizando-se os Questionários de Frequência Alimentar e Escala de Estresse Percebido-10.Resultados:Consumo diário acima das recomendações: colesterol (92%, fi bras (42,7% e proteínas (68%; consumo abaixo das recomendações: potássio (88% e carboidratos (68,7%; consumo conforme recomendações: sódio (53,3% e lípides (53,3%. A maioria (54% com consumo alimentar inadequado era estressada ou altamente estressada. Houve associação estatisticamente signifi cante entre menor nível de estresse e maior consumo de fibras.Conclusão:Em pacientes com SCA, o consumo alimentar esteve fora do recomendado pelas diretrizes e o menor consumo de fibras ocorre concomitantemente com maior estresse. Esforços educativos podem instrumentalizar os pacientes na adequação do consumo alimentar e no controle do estresse.

  3. Validade do sintoma tosse para o diagnóstico de tuberculose pulmonar em pacientes internados em hospital universitário

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    Berenice das Dores Gonçalves

    Full Text Available OBJETIVO: avaliar a acurácia do sintoma tosse como um critério diagnóstico de tuberculose pulmonar (TBP em pacientes internados em um hospital universitário. MÉTODOS: foi conduzida uma avaliação de teste diagnóstico em pacientes com idade ≥15 anos internados em hospital geral de Niterói-RJ, Brasil, em 2011 e 2012. RESULTADOS: foram incluídos 3.677 pacientes, dos quais 608 apresentaram tosse e 15 foram diagnosticados com TBP; entre 184 pacientes sintomáticos respiratórios, 10 foram diagnosticados com TBP; a acurácia do sintoma tosse para a identificação de pacientes com TB foi de 83,4%; a ausência de tosse revelou-se um bom indicador para o descarte do diagnóstico (valor preditivo negativo de 99,7%; todavia, o valor preditivo positivo foi baixo (0,99. CONCLUSÃO: a tosse produtiva apresentou boa acurácia, embora, isoladamente, não seja um bom sintoma de rastreio da TBP em ambientes hospitalares; torna-se necessário identificar outros sintomas e sinais para rastreio e diagnóstico precoce em hospitais.

  4. Estresse ocupacional em enfermeiros intensivistas que prestam cuidados diretos ao paciente crítico

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    Kelly Cristina Inoue

    2013-10-01

    Full Text Available Realizou-se pesquisa descritivo-exploratória em cinco instituições hospitalares da região Oeste do estado do Paraná, entre maio e julho de 2010, com o objetivo de identificar o nível de estresse em enfermeiros intensivistas que prestam cuidados diretos a pacientes críticos. Participaram 58 enfermeiros intensivistas assistenciais, cujo nível de estresse foi avaliado de acordo com o Domínio D da Escala Bianchi de Estresse. A maioria (65,5% apresentou nível de estresse médio. Os estressores com maior pontuação foram Enfrentar a morte (5,6 pontos; Atender às emergências na unidade (5,1 pontos; Atender os familiares de pacientes críticos ou Orientar familiares de pacientes críticos (ambos com 4,8 pontos. É preciso adotar estratégias para a prevenção / redução de estresse, porque os principais estressores apontados relacionam-se a atribuições específicas dos enfermeiros.

  5. Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

    Science.gov (United States)

    Wu, Yi-Cheng; Chang, Il-Chi; Wang, Chi-Liang; Chen, Tai-Di; Chen, Ya-Ting; Liu, Hui-Ping; Chu, Yen; Chiu, Yu-Ting; Wu, Tzu-Hua; Chou, Li-Hui; Chen, Yi-Rong; Huang, Shiu-Feng

    2013-01-01

    Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

  6. Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

    Directory of Open Access Journals (Sweden)

    Yi-Cheng Wu

    Full Text Available BACKGROUND: Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR, fluorescence in Situ hybridization (FISH and Immunohistochemical (IHC stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. RESULTS: Thirteen of the 312 patients (4.17% had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%, but very good specificity (99.32%. IHC stain had better sensitivity (91.67% than FISH, but lower specificity (79.52%, when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product, were also have high expression of ALK protein (IHC3+, and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80% of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%. Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3% was the most common type in Chinese population, while variant 1 (28/37, 75.7% was most common in Caucasian. CONCLUSIONS: Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

  7. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

    Science.gov (United States)

    Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu

    2017-01-01

    Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high

  8. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

    Directory of Open Access Journals (Sweden)

    Ashutosh Wadhwa

    2017-01-01

    Full Text Available Rabies, resulting from infection by Rabies virus (RABV and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses

  9. Estrongiloidíase em pacientes com pênfigo foliáceo

    OpenAIRE

    Tavares-Neto,José; Alves,Denise Barcelos; Franciscon,José Umberto; Serrano,Carlos Roberto S.

    1991-01-01

    Em 30 pacientes com pênfigo foliáceo, a freqüência da estrongiloidíase foi de 40,0%, através de três exames de Baermann-Moraes. No "Hospital do Pênfigo", em Uberaba, as freqüências da estrongiloidíase nos funcionários (n = 14) e escolares (n = 47) da Escola-creche, anexa, também foram altas, respectivamente 35,7% e 23,4%. Em 7 (58,3 %) das 12 amostras do solo, do jardim/pátio do "Hospital", foram observadas formas de vida livre do Strongyloides stercoralis. O fator ambiental e a predisposição...

  10. Fobia específica: um estudo transversal com 103 pacientes tratados em ambulatório

    OpenAIRE

    Terra,Mauro Barbosa; Garcez,Joana Presser; Noll,Betina

    2007-01-01

    OBJETIVOS: Este estudo tem por objetivo investigar a presença de fobia específica (FE) entre pacientes atendidos em um ambulatório de psiquiatria. MÉTODOS: Foi realizado um estudo transversal, no qual foi aplicado o SCID-I em 103 pacientes, para se examinar a ocorrência de fobia específica. Os dados foram analisados por meio de medidas descritivas e mediante os testes de independência baseados na estatística qui-quadrado de Pearson ou no teste exato de Fisher. RESULTADOS: Foi verificada FE em...

  11. Manobra de recrutamento alveolar e suporte ventilatorio perioperatorio em pacientes obesos submetidos a cirurgia abdominal

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    Luiz Alberto Forgiarini Junior

    2013-12-01

    Full Text Available O desenvolvimento da cirurgia abdominal proporcionou uma alternativa terapêutica para obesos mórbidos; entretanto, os pacientes submetidos a esse procedimento frequentemente apresentam complicações pulmonares pós-operatórias. Uma possível alternativa para a redução dessas complicações é a utilização da manobra de recrutamento alveolar e/ou estratégias ventilatórias perioperatórias, com foco na redução das complicações pulmonares pós-operatórias. Nesta revisão, são descritos os benefícios de estratégias ventilatórias perioperatórias, assim como a realização de manobra de recrutamento alveolar em pacientes obesos submetidos a cirurgia abdominal.

  12. Detecção de podocitúria em pacientes com nefrite lúpica

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    Amelia Rodrigues Pereira Sabino

    2013-12-01

    Full Text Available INTRODUÇÃO: A podocitúria tem sido detectada em doenças glomerulares, tais como em nefrite lúpica (NL, em que a proteinúria é uma manifestação importante, e sua ocorrência parece limitar-se à fase ativa da doença. OBJETIVO: Avaliar a podocitúria por imunofluorescência em pacientes portadores de NL e verificar possível associação com atividade clínica da doença. MÉTODOS: Foram avaliados 56 pacientes com NL. Os pacientes foram divididos em três grupos de acordo com o grau de atividade clínica: Grupo B, sem atividade (n = 17; Grupo C, com atividade discreta (n = 29 e Grupo D, moderada a grave (n = 10. Como grupo controle, foram incluídos 29 indivíduos saudáveis (Grupo A. A podocitúria foi estudada por meio de imunofluorescência indireta, usando-se anticorpos primários antipodocina, nefrina e sinaptopodina, e anticorpo secundário conjugado à FITC. Também foram avaliados os níveis de creatinina sérica e da relação proteína/creatinina (P/C urinária, assim como a presença de hematúria e leucocitúria. RESULTADOS: A podocitúria com antipodocina e com antissinaptopodina correlacionou-se estatisticamente com a relação P/C (p = 0,001 e p = 0,013, respectivamente. Tanto a podocitúria com antipodocina, quanto a relação P/C, apresentaram correlação significante (p < 0,001 com a graduação de atividade da doença na NL, diferentemente do que se observou com os outros dois anticorpos, antinefrina e antissinaptopodina. CONCLUSÃO: Nossos achados sugerem que a pesquisa de podocitúria com anticorpos antipodocina poderia ser útil no acompanhamento de pacientes com NL, fornecendo dados relevantes quanto à atividade da doença.

  13. Incidência de disfunção sexual em pacientes com obesidade e sobrepeso

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    Benedito Martins e Silva

    Full Text Available OBJETIVO: avaliar a prevalência de disfunção sexual em pacientes com obesidade e sobrepeso atendidos no Hospital Universitário Professor Alberto Antunes (HUPAA - UFAL. MÉTODOS: trata-se de um estudo descritivo transversal. A amostra foi constituída por pacientes do sexo feminino com sobrepeso ou obesidade. Foram coletados os dados antropométricos para avaliação do índice de massa corporal (IMC e da circunferência da cintura (CC. Em todos os indivíduos foi realizada a avaliação dos níveis séricos de glicose, colesterol total e triglicerídeos. Aplicou-se a versão validada em português do Índice de Função Sexual Feminina (IFSF, que analisa a resposta sexual quanto a desejo, excitação, lubrificação vaginal, orgasmo, satisfação sexual e dor. O escore total é a soma dos escores para cada domínio multiplicada pelo fator correspondente e pode variar de '2' a '36', considerando risco para disfunção sexual um escore total menor ou igual a '26'. RESULTADOS: foram avaliadas 23 mulheres com média de idade de 44 anos, onde 73,9% eram obesas e 82,6% apresentaram risco muito aumentado para complicações metabólicas (CC e"88cm. O risco aumentado para disfunção sexual esteve presente em 78,3% das entrevistadas, ocasionando prejuízos biopsicossociais. HAS, DM e dislipidemia estavam presentes em 33,3%, 22,2% e 61,1%, respectivamente, das pacientes sob risco para disfunção sexual. CONCLUSÃO: a análise dos resultados demonstra a necessidade de uma melhor investigação e atenção dos médicos para com pacientes com obesidade ou sobrepeso.

  14. Mutação BRAF em pacientes idosos submetidos à tireoidectomia

    Directory of Open Access Journals (Sweden)

    Antonio Augusto T. Bertelli

    Full Text Available OBJETIVO: Avaliar a frequência da mutação V600E do gene BRAF em pacientes com mais de 65 anos de idade submetidos à tireoidectomia, correlacionando sua presença ou ausência com as diferentes lesões histológicas, com as variantes e com fatores prognósticos do carcinoma papilífero. MÉTODOS: Foram avaliados 85 pacientes com mais de 65 anos de idade submetidos à tireoidectomia, analisando a mutação BRAF V600E através de reação de PCR-RT realizada após a extração do DNA dos blocos de parafina. RESULTADOS: Detectou-se ausência ou presença da mutação BRAF V600E em 47 pacientes (55,3%. Entre os 17 carcinomas papilíferos estudados, sete apresentavam a mutação (41,2%. Demonstrou-se associação estatística entre a presença desta mutação e a variante clássica do carcinoma papilífero, além de tendência de associação com o extravasamento tireoideano. CONCLUSÃO: A mutação BRAF nos pacientes idosos também é exclusiva do carcinoma papilífero e tem frequência expressiva. Além disso, está relacionada à variante clássica e, possivelmente, ao extravasamento tireoideano.

  15. Prevalência de sintomas depressivos e ansiosos em pacientes com dor crônica

    Directory of Open Access Journals (Sweden)

    Ricardo Cardoso Pinheiro

    2014-09-01

    Full Text Available Objetivos O presente estudo objetivou avaliar a prevalência de sintomas ansiosos e depressivos e relacioná-los com os diferentes tipos e magnitudes de dor crônica; também objetivou caracterizar a população de casos novos agendados para atendimento no ambulatório de dor crônica, no serviço onde o estudo foi realizado. Métodos Estudo de corte transversal, realizado em um ambulatório docente-assistencial para tratamento de dor crônica, realizado entre maio de 2012 e abril de 2013, com 125 pacientes. Instrumentos utilizados Questionário sociodemográfico, Escala Hospitalar de Ansiedade e Depressão (HAD e Escala Visual Numérica (EVN para aferir a intensidade de dor. Resultados Dor intensa foi referida por 64% (n = 80 dos pacientes. Ansiedade atingiu 65% (N = 82 dos pacientes e a depressão, 48% (N = 60. Houve correlação significante entre os mais altos escores de ansiedade (p < 0,001 e depressão (p < 0,001 com a intensidade de dor. A correlação entre intensidade de dor e sintomas ansiosos e depressivos foi positiva para dor crônica neuropática e mista. Os fatores sociodemográficos associados à intensidade de dor crônica foram: renda e religião para depressão, e tempo de dor para ansiedade. Conclusão O estudo mostrou elevada prevalência de sintomas depressivos e ansiosos em pacientes com dor crônica, assim como relação significante desses sintomas psiquiátricos com alguns tipos e intensidade de dor.

  16. Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions.

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    Dung Tien Le

    Full Text Available Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used.

  17. Descalonamento, adequação antimicrobiana e positividade de culturas em pacientes sépticos: estudo observacional

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    Rafael Barberena Moraes

    Full Text Available RESUMO Objetivo: Avaliar a prevalência de descalonamento antibiótico em pacientes com diagnóstico de sepse grave ou choque séptico em hospital acadêmico, público e terciário, além da adequação antibiótica e da positividade de culturas. Métodos: Foram analisadas prevalência de descalonamento, adequação antibiótica e positividade de culturas entre portadores de sepse grave e choque séptico, entre abril e dezembro de 2013, em uma unidade de terapia intensiva de um hospital universitário terciário. Resultados: Entre os 224 pacientes incluídos, o descalonamento era possível em 29,4% dos casos (66 pacientes, mas foi implementado em 19,6% deles (44 pacientes. Entre os pacientes que receberam descalonamento, metade foi por estreitamento de espectro antimicrobiano. A mortalidade foi de 56,3%, não havendo diferença entre pacientes com ou sem descalonamento (56,8% versus 56,1%; p = 0,999, assim como no tempo de internação. Terapia antimicrobiana empírica foi adequada em 89% dos casos. Houve isolamento de germe em 30% de todas as culturas e em 26,3% das hemoculturas. Conclusão: A taxa de adequação antibiótica empírica foi alta, refletindo ativa política institucional de monitorização do perfil epidemiológico e protocolos institucionais de uso de antimicrobianos. No entanto, o descalonamento antimicrobiano poderia ter sido maior do que o registrado. O descalonamento não impactou mortalidade.

  18. Conventional and real time RT-PCR assays for the detection and differentiation of variant rabbit hemorrhagic disease virus (RHDVb) and its recombinants.

    Science.gov (United States)

    Dalton, K P; Arnal, J L; Benito, A A; Chacón, G; Martín Alonso, J M; Parra, F

    2018-01-01

    Since its emergence, variant RHDV (RHDVb/RHDV2) has spread throughout the Iberian Peninsula aided by the apparent lack of cross protection provided by classic (genogroup 1; G1) strain derived vaccines. In addition to RHDVb, full-length genome sequencing of RHDV strains has recently revealed the circulation of recombinant viruses on the Iberian Peninsula. These recombinant viruses contain the RHDVb structural protein encoding sequences and the non-structural coding regions of either pathogenic RHDV-G1 strains or non-pathogenic (np) rabbit caliciviruses. The aim of the work was twofold: firstly to validate a diagnostic real time RT-PCR developed in 2012 for the detection of RHDVb strains and secondly, to design a conventional RT-PCR for the differentiation of RHDVb strains from RHDVb recombinants by subsequent sequencing of the amplicon. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development

    DEFF Research Database (Denmark)

    Kaczmarczyk, Agnieszka Ewa; Bowra, Steve; Elek, Zoltan

    2012-01-01

    expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging. Results We designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and gamma......-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families...... and its subgroups. More over the results indicate the genotypic specific gene expression. Conclusions Quantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal...

  20. Frequency of depression among patients with neurocysticercosis Depressão em pacientes portadores de neurocisticercose

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    Sergio Monteiro de Almeida

    2010-02-01

    Full Text Available Neurocysticercosis (NCC is a common central nervous system infection caused by Taenia solium metacestodes. OBJECTIVE: To investigate the occurrence of depression in patients with calcified NCC form. The study group consisted of 114 patients subdivided in four groups: NCC with epilepsy, NCC without epilepsy, epilepsy without NCC and chronic headache. METHOD: Depression was evaluated and quantified by the Hamilton Rating Scale for Depression (HRSD-21. RESULTS: Percentage of patients with depression was as follows: group 1 (83%; group 2 (88%; group 3 (92%; group 4 (100%. The majority of patients had moderate depression. CONCLUSION: Incidence of depression in all groups was higher than in the general population. It is possible that, in a general way, patients with chronic diseases would have depression with similar intensity. NCC is associated with the presence of depression.Neurocysticercose (NCC é uma infecção do sistema nervoso central comum causada por metacestodes da Taenia solium. OBJETIVO: investigar a ocorrência de depressão nos pacientes com NCC forma calcificada. O grupo de estudo é formado por 114 pacientes subdivididos em quatro grupos: NCC com epilepsia, NCC sem epilepsia, epilepsia sem NCC e cefaléia crônica. MÉTODO: A presença de depressão foi determinada e quantificada pela Escala de Depressão de Hamilton (HRSD-21. RESULTADOS: A porcentagem de pacientes com depressão foi: grupo 1 (83%; grupo 2 (88%; grupo 3 (92%; grupo 4 (100%. A maioria dos pacientes apresentou depressão moderada. CONCLUSÃO: A incidência da depressão em todos os grupos foi mais elevada do que na população geral, contudo não houve diferença entre os grupos estudados. É possível que, de uma maneira geral, os pacientes portadores de doença crônica apresentarem a depressão em intensidade similar. NCC está associada com a presença de depressão.

  1. Soroprevalência da infecção pelo vírus da imunodeficiência humana em pacientes com tuberculose, em Londrina, Paraná

    OpenAIRE

    Morimoto,Arilson Akira; Bonametti,Ana Maria; Morimoto,Helena Kaminami; Matsuo,Tiemi

    2005-01-01

    OBJETIVO: Estimar a prevalência da soropositividade da infecção pelo vírus da imunodeficiência humana em pacientes com tuberculose ativa, atendidos no serviço de Pneumologia Sanitária da 17ª Regional de Saúde do Estado do Paraná, em Londrina, e comparar os pacientes co-infectados com aqueles que apresentavam somente tuberculose, em relação à baciloscopia de escarro, exame radiológico de tórax, e forma clínica e esquema terapêutico para tratamento da tuberculose. MÉTODOS: Estudo transversal co...

  2. An Evidence-Based Approach to Plum Pox Virus Detection by DASI-ELISA and RT-PCR in Dormant Period

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    Antonio Olmos

    2008-01-01

    Full Text Available An evidence-based approach, such as those developed in clinical and veterinary medicine, was applied to the detection of Plum pox virus (PPV during the dormant period. A standardized methodology was used for the calculation of parameters of the operational capacity of DASI-ELISA and RT-PCR in wintertime. These methods are routinely handled to test the sanitary status of plants in national or international trading and in those cases concerning export-import of plant materials. Diagnosis often has to be performed during the dormant period, when plant material is commercialized. Some guidelines to interpret diagnostic results of wintertime are provided in an attempt to minimize risks associated with the methods and over-reliance on the binary outcome of a single assay. In order to evaluate if a complementary test increased the confidence of PPV diagnosis when discordant results between DASI-ELISA and RT-PCR are obtained, NASBA-FH also was included. Likelihood ratios of each method were estimated based on the sensitivity and specificity obtained in wintertime. Subsequently, a Bayesian approach was performed to calculate post-test probability of PPV infection in spring. Results of evidence-based approach show that different PPV prevalences require different screening tests. Thus, at very low PPV prevalence levels DASI-ELISA should be used as the election method, whilst at the highest PPV prevalence levels RT-PCR should be performed. NASBA-FH could be used at medium prevalences to clarify discordances between DASIELISA and RT-PCR.

  3. Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

    Science.gov (United States)

    Noorani, Md Salik; Awasthi, Prachi; Sharma, Maheshwar Prasad; Ram, Raja; Zaidi, Aijaz Asgar; Hallan, Vipin

    2013-10-01

    A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Development and evaluation of tailored specific real-time RT-PCR assays for detection of foot-and-mouth disease virus serotypes circulating in East Africa.

    Science.gov (United States)

    Bachanek-Bankowska, Katarzyna; Mero, Herieth R; Wadsworth, Jemma; Mioulet, Valerie; Sallu, Raphael; Belsham, Graham J; Kasanga, Christopher J; Knowles, Nick J; King, Donald P

    2016-11-01

    Rapid, reliable and accurate diagnostic methods provide essential support to programmes that monitor and control foot-and-mouth disease (FMD). While pan-specific molecular tests for FMD virus (FMDV) detection are well established and widely used in endemic and FMD-free countries, current serotyping methods mainly rely either on antigen detection ELISAs or nucleotide sequencing approaches. This report describes the development of a panel of serotype-specific real-time RT-PCR assays (rRT-PCR) tailored to detect FMDV lineages currently circulating in East Africa. These assays target sequences within the VP1-coding region that share high intra-lineage identity, but do not cross-react with FMD viruses from other serotypes that circulate in the region. These serotype-specific assays operate with the same thermal profile as the pan-diagnostic tests making it possible to run them in parallel to produce C T values comparable to the pan-diagnostic test detecting the 3D-coding region. These assays were evaluated alongside the established pan-specific molecular test using field samples and virus isolates collected from Tanzania, Kenya and Ethiopia that had been previously characterised by nucleotide sequencing. Samples (n=71) representing serotype A (topotype AFRICA, lineage G-I), serotype O (topotypes EA-2 and EA-4), serotype SAT 1 (topotype I (NWZ)) and serotype SAT2 (topotype IV) were correctly identified with these rRT-PCR assays. Furthermore, FMDV RNA from samples that did not contain infectious virus could still be serotyped using these assays. These serotype-specific real-time RT-PCR assays can detect and characterise FMDVs currently circulating in East Africa and hence improve disease control in this region. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

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    Narender S Maan

    Full Text Available Bluetongue (BT is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT are slow (taking weeks, depend on availability of reference virus-strains or antisera and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2 encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h and reliable RT-PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT-PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and

  6. Comparison of multiplex RT-PCR and real-time HybProbe assay for serotyping of dengue virus using reference strains and clinical samples from India

    Directory of Open Access Journals (Sweden)

    Anita Chakravarti

    2016-01-01

    Full Text Available Background: Dengue virus serotyping is crucial from clinical management and epidemiological point of view. Aims: To compare efficacy of two molecular detection and typing methods, namely, multiplex reverse transcription polymerase chain reaction (RT-PCR and real-time Hybprobe assay using a panel of known dilution of four reference Dengue virus strains and a panel of sera collected from clinically suspected dengue patients. Settings: This study was conducted at a tertiary-care teaching hospital in Delhi, India. Materials and Methods: Dengue serotype specific virus strains were used as prototypes for serotyping assays. Viral load was quantified by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR. Acute phase serum samples were collected from 79 patients with clinically suspected Dengue fever on their first day of presentation during September-October 2012. Viral RNA from serum and cell culture supernatant was extracted. Reverse transcription was carried out. Quantitative detection of DENV RNA from reference strain culture supernatants and each of the 79 patient samples by real-time PCR was performed using light cycler Taqman master mix kit. Serotyping was done by multiplex RT-PCR assay and Hybprobe assay. Results: The multiplex RT-PCR assay, though found to be 100% specific, couldn't serotype either patient or reference strains with viral load less than 1000 RNA copies/ml. The Hybprobe assay was found to have 100% specificity and had a lower limit of serotype detection of merely 3.54 RNA copies/ml. Conclusions: HybProbe assay has an important role especially in situations where serotyping is to be performed in clinical samples with low viral load.

  7. Testagem do 1 - beta - D - Ribofuranosil, 1.2.4 - triazole - 3 - carboxamide em pacientes com hepatite

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    Anastácio Ferreira Morgado

    1976-02-01

    Full Text Available Realizou-se estudo do tipo duplo anonimato em 18 pacientes com hepatite aguda benigna. O gruoo experimental foi testado com uma provável droga de ação antiviral: 1-BETA-D-RIBOFURANOSIL, 1,2,4-TRIAZOLE-3- CARBOXAMIDE. O grupo controle ingeriu um placebo de lactose. Teve-se especial cuidado na seleção de pacientes, incluindo apenas pacientes que preenchessem critérios bem estabelecidos. Os pacientes foram seguidos semanalmente, avaliando-os clínica e laboratorialmente. Os resultados não evidenciaram diferenças significativas entre os dois grupos, sugerindo-se estudos com casuística mais numerosa e em regime de internação hospitalar.

  8. Avaliacao da espessura medio-intimal em pacientes com doenca renal cronica nao dialitica: estudo prospectivo de 24 meses

    Directory of Open Access Journals (Sweden)

    Andrea Gaspar Marcos

    2014-03-01

    Full Text Available Introdução: O aumento da espessura média-intimal (EMI avaliada por ultrassom é um preditor de risco cardiovascular na população geral. Porém, em pacientes com doença renal crônica nos estágios iniciais, essa associação ainda não está bem estabelecida. Objetivo: Avaliar a associação EMI com a ocorrência de eventos cardiovasculares e mortalidade em pacientes nos estágios iniciais da doença renal crônica. Métodos: A análise post hoc de uma coorte de pacientes nos estágios 2-4 da DRC. Foram avaliados dados laboratoriais, ultrassom da artéria carótida e tomografia coronariana no início do estudo e a ocorrência de óbito, em seguimento por 24 meses. Resultados: Um total de 117 pacientes (57 ± 11 anos, 61% sexo masculino foram avaliados. A taxa de filtração glomerular foi 36 ± 17 mL/min, 96% dos pacientes eram hipertensos, 23% diabéticos e 27% obesos. Calcificação arterial coronariana esteve presente em 48% dos pacientes, sendo mais prevalente em pacientes nos estágios mais avançados da DRC (p = 0,02. EMI foi 0,6 mm (0,4-0,7 mm. Comparado aos pacientes com EMI < 0,6mm, aqueles com EMI ≥ 0,6 mm eram mais velhos (p = 0,001, apresentavam maior prevalência do sexo masculino (p = 0,001, menor taxa de filtração glomerular (p = 0,01 e maior proporção de pacientes com calcificação (p = 0,001. Não foi observada relação entre a espessura média-intimal e a ocorrência de evento cardiovascular e óbito. Conclusão: A espessura médio-intimal em pacientes DRC se associou à calcificação coronariana, mas não à ocorrência de eventos cardiovasculares e óbito, em um seguimento de 24 meses.

  9. Fisioterapia respiratoria na pressao intracraniana de pacientes graves internados em unidade de terapia intensiva: revisao sistematica

    Directory of Open Access Journals (Sweden)

    Lucas Lima Ferreira

    2013-12-01

    Full Text Available Objetivo: Analisar os desfechos aumento/diminuição da pressão intracraniana e/ou queda da pressão de perfusão cerebral, proporcionados pela fisioterapia respiratória em pacientes graves assistidos em unidade de terapia intensiva. Métodos: Por meio de uma revisão sistemática da literatura, foram selecionados ensaios clínicos publicados entre 2002 e 2012. A busca envolveu as bases de dados LILACS, SciELO, MedLine e PEDro, usando os descritores "physical therapy", "physiotherapy", "respiratory therapy" e "randomized controlled trials" em cruzamento com o descritor "intracranial pressure". Resultados: Foram incluídos 5 estudos, que somaram 164 pacientes, com média de idade entre 25 e 65 anos, e que indicaram que as manobras de fisioterapia respiratória aumentaram significativamente a pressão intracraniana, sem alterar a pressão de perfusão cerebral. Os artigos abordaram as técnicas de vibração, vibrocompressão, tapotagem, drenagem postural, além da manobra de aspiração intratraqueal. Todos os pacientes estavam sob ventilação mecânica invasiva. Conclusão: A fisioterapia respiratória promove aumento da pressão intracraniana. Os estudos sugerem que não há repercussões hemodinâmicas e respiratórias a curto prazo ou alteração da pressão de perfusão cerebral. Entretanto, não há estudos que avaliem desfechos clínicos e que assegurem a segurança das manobras.

  10. A rapid silica spin column-based method of RNA extraction from fruit trees for RT-PCR detection of viruses.

    Science.gov (United States)

    Yang, Fan; Wang, Guoping; Xu, Wenxing; Hong, Ni

    2017-09-01

    Efficient recovery of high quality RNA is very important for successful RT-PCR detection of plant RNA viruses. High levels of polyphenols and polysaccharides in plant tissues can irreversibly bind to and/or co-precipitate with RNA, which influences RNA isolation. In this study, a silica spin column-based RNA isolation method was developed by using commercially available silica columns combined with the application of a tissue lysis solution, and binding and washing buffers with high concentration guanidinium thiocyanate (GuSCN, 50% w/v), which helps remove plant proteins, polysaccharides and polyphenolic compounds. The method was successfully used to extract high quality RNA from citrus (Citrus aurantifolia), grapevine (Vitis vinifera), peach (Prunus persica), pear (Pyrus spp.), taro (Colocosia esculenta) and tobacco (Nicotiana benthamiana) samples. The method was comparable to conventional CTAB method in RNA isolation efficiency, but it was more sample-adaptable and cost-effective than commercial kits. High quality RNA isolated using silica spin column-based method was successfully used for the RT-PCR and/or multiplex RT-PCR amplification of woody fruit tree viruses and a viroid. The study provided a useful tool for the detection and characterization of plant viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. The current incidence of viral disease in korean sweet potatoes and development of multiplex rt-PCR assays for simultaneous detection of eight sweet potato viruses.

    Science.gov (United States)

    Kwak, Hae-Ryun; Kim, Mi-Kyeong; Shin, Jun-Chul; Lee, Ye-Ji; Seo, Jang-Kyun; Lee, Hyeong-Un; Jung, Mi-Nam; Kim, Sun-Hyung; Choi, Hong-Soo

    2014-12-01

    Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

  12. The Current Incidence of Viral Disease in Korean Sweet Potatoes and Development of Multiplex RT-PCR Assays for Simultaneous Detection of Eight Sweet Potato Viruses

    Directory of Open Access Journals (Sweden)

    Hae-Ryun Kwak

    2014-12-01

    Full Text Available Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV and sweet potato virus C (SPVC were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1, Sweet potato virus G (SPVG, Sweet potato leaf curl virus (SPLCV, Sweet potato virus 2 ( SPV2, Sweet potato chlorotic fleck virus (SPCFV, and Sweet potato latent virus (SPLV were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1 in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

  13. Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 Subtype C infected patients in Northern India

    Science.gov (United States)

    Agarwal, Atima; Sankaran, Sumathi; Vajpayee, Madhu; Sreenivas, V; Seth, Pradeep; Dandekar, Satya

    2014-01-01

    Background Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. Objectives The objective of this study was to develop and validate an affordable; one step Real-Time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. Results We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. Conclusions The RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India. PMID:17962068

  14. Detection, quantitation and identification of enteroviruses from surface waters and sponge tissue from the Florida Keys using real-time RT-PCR

    Science.gov (United States)

    Donaldson, K.A.; Griffin, Dale W.; Paul, J.H.

    2002-01-01

    A method was developed for the quantitative detection of pathogenic human enteroviruses from surface waters in the Florida Keys using Taqman (R) one-step Reverse transcription (RT)-PCR with the Model 7700 ABI Prism (R) Sequence Detection System. Viruses were directly extracted from unconcentrated grab samples of seawater, from seawater concentrated by vortex flow filtration using a 100kD filter and from sponge tissue. Total RNA was extracted from the samples, purified and concentrated using spin-column chromatography. A 192-196 base pair portion of the 5??? untranscribed region was amplified from these extracts. Enterovirus concentrations were estimated using real-time RT-PCR technology. Nine of 15 sample sites or 60% were positive for the presence of pathogenic human enteroviruses. Considering only near-shore sites, 69% were positive with viral concentrations ranging from 9.3viruses/ml to 83viruses/g of sponge tissue (uncorrected for extraction efficiency). Certain amplicons were selected for cloning and sequencing for identification. Three strains of waterborne enteroviruses were identified as Coxsackievirus A9, Coxsackievirus A16, and Poliovirus Sabin type 1. Time and cost efficiency of this one-step real-time RT-PCR methodology makes this an ideal technique to detect, quantitate and identify pathogenic enteroviruses in recreational waters. Copyright ?? 2002 Elsevier Science Ltd.

  15. Localisation of Abundant and Organ-Specific Genes Expressed in Rosa hybrida Leaves and Flower Buds by Direct In Situ RT-PCR

    Directory of Open Access Journals (Sweden)

    Agata Jedrzejuk

    2012-01-01

    Full Text Available In situ PCR is a technique that allows specific nucleic acid sequences to be detected in individual cells and tissues. In situ PCR and IS-RT-PCR are elegant techniques that can increase both sensitivity and throughput, but they are, at best, only semiquantitative; therefore, it is desirable first to ascertain the expression pattern by conventional means to establish the suitable conditions for each probe. In plants, in situ RT-PCR is widely used in the expression localisation of specific genes, including MADS-box and other function-specific genes or housekeeping genes in floral buds and other organs. This method is especially useful in small organs or during early developmental stages when the separation of particular parts is impossible. In this paper, we compared three different labelling and immunodetection methods by using in situ RT-PCR in Rosa hybrida flower buds and leaves. As target genes, we used the abundant β-actin and RhFUL gene, which is expressed only in the leaves and petals/sepals of flower buds. We used digoxygenin-11-dUTP, biotin-11-dUTP, and fluorescein-12-dUTP-labelled nucleotides and antidig-AP/ streptavidin-fluorescein-labelled antibodies. All of the used methods gave strong, specific signal and all of them may be used in localization of gene expression on tissue level in rose organs.

  16. [Disappearance of residual disease confirmed by RT-PCR following induction chemotherapy in two hypoplastic leukemia patients with t(8;21)].

    Science.gov (United States)

    Sawada, M; Tsurumi, H; Yamada, T; Hara, T; Oyama, M; Moriwaki, H

    1999-04-01

    Reverse transcriptase-polymerase chain reaction (RT-PCR) methods often detect the AML1/MTG8 fusion transcript even in acute myelogenous leukemia (AML) patients with t(8;21) who have been in long-term remission. We encountered 2 hypoplastic leukemia patients with t(8;21) who achieved cytogenetic remission with short-term conventional chemotherapy. Patient 1 was a 42-year-old woman. Chromosomal analysis detected t(8;21) (q22;q22) and PCR analysis (35 cycles PCR amplification; detection limit 1 x 10(-5) cells) detected the AML1/MTG8 fusion transcript. Complete remission was obtained with 1 course of chemotherapy consisting of low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days). After 2 courses of consolidation chemotherapy consisting of conventional-dose cytarabine and mitoxantrone, the RT-PCR findings were negative for the AML1/MTG8 fusion transcript. Patient 2 was a 67-year-old man. Cytogenetic analysis detected t(8;21) (q22;q22), and was positive for the AML1/MTG8 fusion transcript. After 2 courses of induction chemotherapy comprising low-dose cytarabine (20 mg x 14 days) and etoposide (50 mg x 14 days), and 3 courses of conventional consolidation chemotherapy, RT-PCR analysis confirmed the disappearance of the AML1/MTG8 fusion transcript.

  17. A rapid method of accurate detection and differentiation of Newcastle disease virus pathotypes by demonstrating multiple bands in degenerate primer based nested RT-PCR.

    Science.gov (United States)

    Desingu, P A; Singh, S D; Dhama, K; Kumar, O R Vinodh; Singh, R; Singh, R K

    2015-02-01

    A rapid and accurate method of detection and differentiation of virulent and avirulent Newcastle disease virus (NDV) pathotypes was developed. The NDV detection was carried out for different domestic avian field isolates and pigeon paramyxo virus-1 (25 field isolates and 9 vaccine strains) by using APMV-I "fusion" (F) gene Class II specific external primer A and B (535bp), internal primer C and D (238bp) based reverses transcriptase PCR (RT-PCR). The internal degenerative reverse primer D is specific for F gene cleavage position of virulent strain of NDV. The nested RT-PCR products of avirulent strains showed two bands (535bp and 424bp) while virulent strains showed four bands (535bp, 424bp, 349bp and 238bp) on agar gel electrophoresis. This is the first report regarding development and use of degenerate primer based nested RT-PCR for accurate detection and differentiation of NDV pathotypes by demonstrating multiple PCR band patterns. Being a rapid, simple, and economical test, the developed method could serve as a valuable alternate diagnostic tool for characterizing NDV isolates and carrying out molecular epidemiological surveillance studies for this important pathogen of poultry. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.

    Science.gov (United States)

    Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun

    2016-11-01

    Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

  19. Doença celíaca é super-representada em pacientes com constipação

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    Rolf A. A. Pelleboer

    2012-04-01

    Full Text Available OBJETIVO: Tem sido sugerido que pacientes com constipação sejam triados para doença celíaca. Da mesma forma, recomenda-se a investigação desses pacientes para hipotiroidismo e hipercalcemia. Contudo, nenhuma evidência para essas recomendações está disponível até o momento. Assim, propusemos-nos determinar a prevalência de doença celíaca, hipotiroidismo e hipercalcemia em crianças com constipação. MÉTODOS: Estudo de coorte prospectivo com 370 pacientes consecutivos que preencheram os critérios de Roma III para constipação. Esses pacientes foram encaminhados por um clínico geral a um pediatra devido ao fracasso no tratamento com laxantes. RESULTADOS: A biópsia comprovou doença celíaca em sete desses pacientes. Isso é significativamente mais alto (p < 0,001 do que a prevalência de 1:198 de doença celíaca nos Países Baixos. Dois pacientes tinham tiroidite autoimune. Nenhum paciente tinha hipercalcemia. CONCLUSÕES: Conclui-se que a doença celíaca é significativamente super-representada em pacientes com constipação encaminhados por um clínico geral a um pediatra devido ao fracasso no tratamento com laxantes. Todos esses pacientes devem, portanto, ser triados para doença celíaca.

  20. PESQUISA DE HEMOGLOBINOPATIAS E TALASSEMIAS EM PACIENTES PORTADORES DE LÚPUS ERITEMATOSO SISTÊMICO.

    OpenAIRE

    Castro, Frank Sousa

    2005-01-01

    13 O Lúpus Eritematoso Sistêmico é uma doença tipicamente multigênica e multifatorial, com grande complexidade clínica e fisiopatológica. As causas do LES não são totalmente conhecidas, mas sabe-se que fatores ambientais e genéticos estão envolvidos. As manifestações clínicas observadas em pacientes acometidos pelo LES são diversificadas como fadiga, mal-estar, emagrecimento, artrite, febre, nefrite, vasculite e anemias. Dentre as várias manifestações clínicas observadas ...

  1. Meningite e endocardite infecciosa causada por Rhodotorula mucilaginosa em paciente imunocompetente

    Directory of Open Access Journals (Sweden)

    Sergio Henrique Loss

    2011-12-01

    Full Text Available Os autores relatam o caso de um homem imunocompetente admitido com comprometimento agudo do sistema nervoso, crise hipertensiva e insuficiência renal, vindo a receber diagnóstico de meningite e endocardite infecciosa por Rhodotorula mucilaginosa. Até onde sabemos, esta é a primeira descrição de infecção simultânea das meninges e do endotélio causada por Rhodotorula em um paciente sem comprometimento imunológico.

  2. Tracoma em pacientes com conjuntivite alérgica Trachoma in patients with allergic conjunctivitis

    Directory of Open Access Journals (Sweden)

    Haroldo de Lucena Bezerra

    2010-06-01

    Full Text Available Objetivos: Este trabalho teve o objetivo de identificar possível associação entre conjuntivite alérgica e infecção por Chlamydia trachomatis. Método: Realizamos um estudo prospectivo em 104 olhos de 52 pacientes com hipótese diagnóstica de conjuntivite alérgica primaveril e atópica. Os pacientes foram examinados no Núcleo Especializado em Oftalmologia de João Pessoa - NEO. Foi realizada em todos os 52 pacientes, a citologia conjuntival, uma vez que pode evidenciar a presença de eosinófilos e corpúsculos de inclusões no esfregaço do raspado conjuntival, como também a imunofluorescência direta por ser o exame de escolha para confirmação de infecção por Chlamydia trachomatis. Todos os procedimentos foram aprovados pelo Comitê de Ética em pesquisa. Resultados: Dos 52 pacientes, 41 (78,8% apresentou conjuntivite primaveril e 11 (21,2% conjuntivite atópica. Quarenta e um (78,8% eram do sexo masculino e 11 (21,2% do feminino. As idades variaram de 3 a 19 anos, com uma média de 9,8 anos. Quanto à distribuição racial, 16 (30,8% pacientes eram brancos, 14 (26,9% eram negros e 22 (42,3% eram pardos. Pacientes com doença alérgica sistêmica observou-se que 25 (48,1% pacientes apresentaram asma brônquica, 20 (38,5% rinite alérgica e 5 (9,6% dermatite atópica. Os principais sintomas relatados pelos pacientes foram coriza (59,6%, prurido ocular (98,1%, ardor ou queimação (61,5%, lacrimejamento (65,3% e fotofobia (61,5%. Os principais sinais clínicos foram: hiperemia ocular (100%, bilateralidade (100%, papilas no tarso (92,3% e secreção mucosa (82,7%. A citologia do raspado conjuntival encontrou o eosinófilo em 86,5% dos casos. Apenas 7 pacientes não apresentaram eosinófilos no raspado conjuntival. Observamos que 3 pacientes (5,8% apresentou imunofluorescência positiva para Chlamydia, evidenciando associação entre conjuntivite alérgica e tracoma. Conclusão: É importante determinar a existência simultânea de

  3. Qualidade de vida em pacientes com endometriose: um estudo de revisão

    Directory of Open Access Journals (Sweden)

    Maria Paula Custódio Silva

    2014-09-01

    Full Text Available Objetivo: O presente estudo teve como objetivo mostrar os principais resultados dos estudos que avaliaram qualidade de vida (QV em mulheres com endometriose. Também são apresentadas estratégias para promoção da saúde, visando demonstrar as pacientes com essa condição ginecológica que é possível viver bem mesmo sendo portadora de uma doença crônica. Métodos: Para isso, foi realizada uma pesquisa bibliográfica no PubMed utilizando os descritores quality of life and endometriosis e os seguintes filtros: artigos com abstract disponível, com data de publicação dos últimos 10 anos, em humanos do sexo feminino, idioma inglês/português e cujos termos constavam no título/abstract. A busca foi realizada em dezembro de 2013, resultou em 147 artigos e de acordo com os critérios de inclusão, 12 foram selecionados para leitura. Resultados: Todos os artigos evidenciaram que as mulheres com endometriose apresentaram uma redução na QV e cinco estudos associaram nível socioeconômico, intensidade da dor e hábitos saudáveis como fatores que influenciam a QV dessa população. No entanto, nenhum dos estudos propõe as pacientes com a doença estratégias para a melhora da QV. O presente estudo mostra os benefícios da prática regular de atividade física em elevar a QV e, portanto, estimula sua realização como uma maneira de minimizar os sintomas da endometriose, em especial a dor e os sintomas psicológicos. Conclusão: Considerando que a endometriose é uma doença complexa e prevalente, o ideal seria o fornecimento de um atendimento holístico as pacientes, executado por uma equipe multidisciplinar que atuaria de modo integrado na orientação, acompanhamento e tratamento das mulheres com endometriose.

  4. Achados tomográficos em 1000 pacientes consecutivos com antecedentes de crises epilépticas

    Directory of Open Access Journals (Sweden)

    Trentin Ana Paula

    2002-01-01

    Full Text Available Analizamos retrospectivamente as tomografias computadorizadas (TC de crânio de 1000 pacientes consecutivos, com história clínica de crises epilépticas classificadas do ponto de vista clínico em generalizadas (CG e parciais (CP. Nossos resultados mostraram a presença de CG em 70,7% dos pacientes, sendo 57,1% do sexo masculino e 42,9% do sexo feminino e, CP em 29,3%, sendo 60,0% do sexo maculino e 40,0% do sexo femninino. As faixas etárias de maior incidência foram entre 0 a 10 (31,0% e 11 a 20 (21,8% anos nos pacientes com CG e 0 a 10 (24,5%, 21 a 30 (16,7% e 31 a 40 anos (18,4% nos pacientes com CP. Os resultados tomográficos no grupo com CG foram: normais (48,8% e alterados em 51,2%, sendo esses achados definidos como calcificações/cisticercose (14,0%, neurocisticercose/cistos (9,6% hidrocefalia (4,4%, infarto (4,2%, indefinido (4,0%, tumor (2,5% entre outros (12,5,%. Nos pacientes com CP os exames tomográficos foram normais em 37,4%, alterados em 62,7%, tendo os diagnósticos de neurocisticercose/cistos (12,2%, calcificações/cisticercose (11,2%, tumor (10,5%, indefinido (8,1%, infarto (5,4%, hidrocefalia (3,7% e outros (11,6%. Salientamos a importância da TC em pacientes com epilepsia, particularmente para o diagnóstico de neurocisticercose.

  5. Espectro óculo-aurículo-vertebral em pacientes com defeitos cardíacos congênitos

    Directory of Open Access Journals (Sweden)

    Rafael Fabiano Machado Rosa

    2010-10-01

    Full Text Available FUNDAMENTO: Há poucos estudos avaliando a frequência do espectro óculo-aurículo-vertebral (EOAV nos pacientes com defeitos cardíacos congênitos (DCC. OBJETIVO: Verificar a frequência do EOAV em uma amostra de pacientes com malformações cardíacas graves. MÉTODOS: Avaliamos uma coorte prospectiva de pacientes com DCC, admitidos em uma unidade de terapia intensiva (UTI cardiopediátrica no Brasil. O diagnóstico de EOAV foi feito com base em dados clínicos, considerando critérios padrões. Os pacientes que preenchiam esses critérios foram submetidos ao cariótipo de alta resolução com bandamento GTG e hibridização fluorescente in situ para a microdeleção 22q11.2. O teste exato de Fisher (P < 0,05 foi usado para as análises estatísticas. RESULTADOS: Durante o período de avaliação, 330 pacientes foram hospitalizados pela primeira vez na UTI, mas trinta deles não participaram deste estudo. Dos 300 pacientes que constituíram a amostragem final, o EOAV foi verificado em três casos (1%. Todos apresentaram estudos citogenéticos normais. CONCLUSÃO: O EOAV parece ser uma condição frequente em pacientes com DCC. Contudo, não podemos excluir a possibilidade de que a frequência de EOAV encontrada em nosso estudo possa ter sido subestimada devido à baixa taxa de detecção pré-natal de DCC e ao acesso limitado dos pacientes ao tratamento médico apropriado em nosso meio. Estudos prospectivos futuros com critérios clínicos bem definidos e pacientes com defeitos leves e graves serão importantes para avaliar o papel do EOAV na população em geral de indivíduos com malformações cardíacas.

  6. Diagnosticos de enfermagem em pacientes classificados nos niveis I e II de prioridade do Protocolo Manchester

    Directory of Open Access Journals (Sweden)

    Cristiane Chaves de Souza

    2013-12-01

    Full Text Available O estudo objetivou identificar possíveis diagnósticos de enfermagem em pacientes classificados nos níveis I e II de prioridade do protocolo Manchester. Trata-se de estudo descritivo retrospectivo, cuja amostra foi de 40 prontuários de pacientes classificados nos níveis I e II de prioridade. Para identificação dos diagnósticos de enfermagem dois especialistas analisaram sinais e sintomas registrados nos prontuários dos pacientes no momento da classificação de risco. No nível I de prioridade, os diagnósticos de enfermagem mais frequentes foram: dor aguda (65,0%, padrão respiratório ineficaz (45,0% e troca de gases prejudicada (40,0%. No nível II de prioridade foram: dor aguda (80,0%, náusea (10,0% e risco de desequilíbrio eletrolítico (10,0%. Percebeu-se que a utilização do protocolo de Manchester favorece a identificação de características definidoras e fatores relacionados/fatores de risco que subsidiam a elaboração de diagnósticos de enfermagem na classificação de risco.

  7. Efeito do treinamento resistido em paciente com fibromialgia: estudo de caso

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    Vanessa Zadorosnei Rebutini

    2013-06-01

    Full Text Available A fibromialgia (FM é uma doença idiopática de ordem reumatológica e incurável que causa dores crônicas e possui tratamento complexo e multifatorial. O exercício físico pode fazer parte do tratamento por atenuar parte dos sintomas da FM, porém a literatura ainda carece de informações sobre tipo, intensidade, duração e frequência ótimos contra os sintomas da FM. O objetivo do presente estudo foi verificar o efeito de 12 semanas de treinamento resistido em uma paciente com FM que não fazia tratamento farmacológico. Os resultados indicaram que 12 semanas de treinamento resistido foi capaz de reduzir as dores, melhorar a capacidade funcional, o bem-estar geral e a qualidade de vida da paciente. O treinamento resistido pareceu atenuar os efeitos negativos da FM e parece ser uma ferramenta promissora para pacientes com FM que gostem desse tipo de atividade.

  8. An Endogenous Murine Leukemia Viral Genome Contaminant in a Commercial RT-PCR Kit is Amplified Using Standard Primers for XMRV

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    Miyazawa Takayuki

    2010-12-01

    Full Text Available Abstract During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV-related virus (XMRV infection in sera from chronic fatigue syndrome (CFS patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV, XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity to a PmERV on chromosome 7 and highly similar (96.9 to 97.6% to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6% to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.

  9. Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

    International Nuclear Information System (INIS)

    Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

    2008-01-01

    Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation

  10. Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

    Directory of Open Access Journals (Sweden)

    Radtke Arnold

    2008-11-01

    Full Text Available Abstract Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR. The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC is presented. Methods Six genes, beta-2-microglobulin (B2M, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hydroxymethyl-bilane synthase (HMBS, hypoxanthine phosphoribosyl-transferase 1 (HPRT1, succinate dehydrogenase complex, subunit A (SDHA and ubiquitin C (UBC, with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the

  11. Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

    Science.gov (United States)

    Cicinnati, Vito R; Shen, Qingli; Sotiropoulos, Georgios C; Radtke, Arnold; Gerken, Guido; Beckebaum, Susanne

    2008-01-01

    Background Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented. Methods Six genes, beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyl-transferase 1 (HPRT1), succinate dehydrogenase complex, subunit A (SDHA) and ubiquitin C (UBC), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both geNorm and NormFinder software. Results HMBS and GAPDH were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. HMBS, GAPDH and UBC were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of HMBS, B2M, SDHA and GAPDH was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances. Conclusion Of six genes studied, HMBS was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues

  12. Detection of circulating tumor cells with CK20 RT-PCR is an independent negative prognostic marker in colon cancer patients - a prospective study.

    Science.gov (United States)

    Hinz, Sebastian; Hendricks, Alexander; Wittig, Amke; Schafmayer, Clemens; Tepel, Jürgen; Kalthoff, Holger; Becker, Thomas; Röder, Christian

    2017-01-13

    Detection of circulating (CTC) or disseminated tumor cells (DTC) has been associated with negative prognosis and outcome in patients with colorectal cancer, though testing for these cells is not yet part of clinical routine. There are several different methodological approaches to detect tumor cells but standardized detection assays are not implemented so far. In this prospective monocentric study 299 patients with colon cancer were included. CTC and DTC were detected using CK20 RT-PCR as well as immunocytochemistry staining with anti-pan-keratin and anti-EpCAM antibodies. The primary endpoints were: Evaluation of CTC and DTC at the time of surgery and correlation with main tumor characteristics and overall (OS) and disease free survival (DFS). Patients with detectable CTC had a 5-year OS rate of 68% compared to a 5-year OS rate of 85% in patients without detectable CTC in the blood (p = 0.002). Detection of DTC in the bone marrow with CK20 RT-PCR was not associated with a worse OS or DFS. Detection of pan-cytokeratin positive DTC in the bone marrow correlated with a significantly reduced 5-year OS rate (p = 0.048), but detection of DTC in the bone marrow with the anti-EpCAM antibody did not significantly influence the 5-year OS rate (p = 0.958). By multivariate analyses only detection of CTC with CK20 RT-PCR in the blood was revealed to be an independent predictor of worse OS (HR1.94; 95% CI 1.0-3.7; p = 0.04) and DFS (HR 1.94; 95% CI 1.1-3.7; p = 0.044). Detection of CTC with CK20 RT-PCR is a highly specific and independent prognostic marker in colon cancer patients. Detection of DTC in the bone marrow with CK20 RT-PCR or immunohistochemistry with anti-EpCAM antibody is not associated with a negative prognostic influence.

  13. Vasculite mesentérica em paciente com lúpus eritematoso sistêmico juvenil

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    Adão F. Albuquerque-Netto

    2013-04-01

    Full Text Available A vasculite mesentérica lúpica (VML é uma rara causa de dor abdominal aguda. Há poucos relatos de caso demonstrando VML em adultos e, particularmente, em crianças e adolescentes. No entanto, para o nosso conhecimento, a prevalência dessa grave vasculite em uma população pediátrica com lúpus ainda não foi estudada. Portanto, dados de 28 anos consecutivos foram revisados e incluídos 5.508 pacientes em seguimento no Hospital da Faculdade de Medicina da Univesidade de São Paulo (FMUSP. Identificamos 279 (5,1% casos que preencheram critérios de classificação diagnóstica do American College of Rheumatology para lúpus eritematoso sistêmico (LES e um (0,4% desses apresentou VML. Este paciente recebeu diagnóstico de LES aos 11 anos de idade. Aos 13 anos foi hospitalizado com dor abdominal difusa e aguda, náuseas, vômitos biliosos, distensão e rigidez abdominal, com descompressão brusca positiva. O paciente foi prontamente submetido à laparotomia exploradora, identificando isquemia intestinal segmentar, com edema de parede intestinal e aderências. Foi realizada ressecção parcial de intestino delgado, com lise das aderências e pulsoterapia com metilprednisolona. A análise histopatológica identificou arterite de vasos mesentéricos. Após 13 dias, apresentou recorrência de dor abdominal difusa intensa, sendo novamente submetido à laparotomia exploradora, identificando obstrução em intestino delgado por aderências, com gangrena intestinal. Nova ressecção intestinal foi realizada, além de pulsoterapia com metilprednisolona e infusão de imunoglobulina. Portanto, VML é uma rara e grave manifestação abdominal na população com lúpus pediátrico, e pode ser a única manifestação de atividade da doença. Além disso, este estudo reforça a importância do diagnóstico precoce e do tratamento imediato.

  14. Duodenopancreatectomia: avaliação dos resultados em 41 pacientes

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    Luiz Carlos Gomes Rocha

    Full Text Available OBJETIVO: Apresentar os resultados do Hospital Israel Pinheiro - IPSEMG em 41 duodenopancreatectomias realizadas para neoplasias. MÉTODO: Foram coletados dados referentes a 41 pacientes submetidos a duodenopancreatectomias entre 1997 e 2004. A principal operação realizada foi a Whipple "clássica" sem preservação do piloro. A anastomose pancreático-jejunal foi realizada por meio de sutura ducto-mucosa. Foram analisadas a mortalidade e as complicações pós-operatórias. Para avaliar se havia diferença de resultados com a maior experiência da equipe, o estudo foi dividido em dois períodos de quatro anos cada: 1997 a 2000 e 2001 a 2004. RESULTADOS: As complicações pós-operatórias ocorreram em 58% dos casos e a mortalidade foi de 22%. As principais complicações foram pneumonia e infecção de ferida operatória. Quatro pacientes (10% evoluíram com fistulas pancreáticas, porém obteve-se sucesso com o tratamento conservador em todos os casos. A necessidade de hemotransfusão no peroperatório relacionou-se a um pior prognóstico. Observou-se uma redução das taxas de morbi-mortalidade no período de 2001 a 2004, entretanto sem significância estatística. Os pacientes apresentaram sobrevida global em cinco anos de 35% e de 26% quando considerados apenas aqueles com adenocarcinoma de cabeça de pâncreas. CONCLUSÕES: A duodenopancreatectomia é um procedimento cirúrgico complexo, com elevada morbi-mortalidade. Entretanto, com o aumento da experiência das equipes cirúrgica, anestésica e de medicina intensiva, observa-se uma redução nas taxas de complicação.

  15. Infecção pelo HIV: descritores de mortalidade em pacientes hospitalizados

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    Luiz Claudio Santos Thuler

    1998-12-01

    Full Text Available OBJETIVO: Estudar os descritores clínico-epidemiológicos da mortalidade em pacientes internados por condições clínicas associadas à infecção pelo HIV. MÉTODO: Estudo retrospectivo de todos os pacientes adultos hospitalizados em 1990, 1992 e 1994 em hospital universitário. Os resultados foram descritos como números absolutos, percentagens e médias, sendo a significância estatística entre as diferenças avaliada pelos testes do qui-quadrado, exato de Fisher ou t de Student, conforme o caso. Um modelo de regressão logística foi elaborado visando a identificar os principais fatores associados ao risco de evolução para o óbito. RESULTADOS: Foram incluídos no estudo 240 pacientes. Entre 1990 e 1994 a idade média dos pacientes aumentou de 35,0 para 36,9 anos, a razão entre os sexos masculino e feminino caiu de 9,8 para 2,0, a proporção de não brancos cresceu de 18,5 para 41,3 e registrou-se um aumento do tempo médio entre a descoberta da infecção pelo HIV e a hospitalização de 0,7 para 2,5 anos. Observou-se a redução do número médio de dias de hospitalização de 31,3 para 25,3 e aumento da proporção de pacientes em acompanhamento ambulatorial de 47,8 para 83,3%. As infecções respiratórias representaram a principal causa de hospitalização (58% e as infecções oportunistas apresentadas com maior freqüência foram: candidíase oral (27,1%, tuberculose (18,3%, pneumonia por Pneumocystis carinii (15,4% e neurotoxoplasmose (10,4%. Na análise multivariada, apenas o tempo de hospitalização menor ou igual a 7 dias (Odds Ratio [OR]=3,88; p=0,02 e a ausência de acompanhamento ambulatorial (OR=3,29; p=0,01 mostraram-se associados a um maior risco de evolução para óbito. CONCLUSÃO: O conhecimento dos fatores associados a um risco aumentado de morte pode ser útil na tomada de decisão frente a pacientes hospitalizados com infecção pelo HIV.

  16. Musical identity of patients with multiple sclerosis Identidade musical em pacientes com esclerose múltipla

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    Shirlene Vianna Moreira

    2009-03-01

    Full Text Available Musical autobiographies consist of a powerful therapeutic tool by which individuals define themselves. The use of this technique may help (reconstruction personal identities and improve quality of life of patients with multiple sclerosis (MS. Eight adult patients on treatment at CIEM Multiple Sclerosis Investigation Center after selecting 10 to 15 pieces of music most significant in their lives were interviewed. The data collected were classified according to Even Rudd categories, which reveal how a person expresses his personal, social, temporal and transpersonal identities. We observed that recall of musical history makes MS patients get better perception both of their feelings and body awareness, as well as provide them with an alternative way to express themselves, activate and contextualize affective memories, and achieving a sense of life continuity in spite of the disease.Autobiografias musicais constituem um recurso terapêutico pelo qual indivíduos definem a si mesmos, auxiliando a (reconstrução da identidade e contribuindo para a melhora da qualidade de vida de portadores de esclerose múltipla (EM. Oito pacientes adultos sob acompanhamento no Centro de Investigação em esclerose múltipla (CIEM da UFMG, selecionaram entre 10 e 15 músicas significativas em sua vida, a respeito das quais discorreram em entrevista aberta. Os dados foram analisados qualitativamente segundo categorias criadas por Even Rudd, que visam revelar como o indivíduo expressa suas identidades pessoal, social, temporal e transpessoal. Submetidos a tratamento quantitativo, os dados demonstraram que, através da sua história musical, os pacientes aumentaram a percepção dos sentimentos e sensações corporais, expressaram-se de maneiras alternativas e ativaram memórias afetivas, contextualizando-as e adquirindo um senso de continuidade da vida.

  17. Anestesia em paciente com Distrofia Muscular de Duchenne: relato de caso

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    Tonelli Deoclécio

    2003-01-01

    Full Text Available JUSTIFICATIVA E OBJETIVOS: A distrofia muscular de Duchenne é uma afecção recessiva ligada ao cromossomo X, geralmente diagnosticada na infância, acentuando-se progressivamente até agravar a função respiratória. O objetivo deste relato é apresentar um caso de um paciente com distrofia muscular de Duchenne diagnosticada há 2 anos, submetido à postectomia, sob anestesia geral com cetamina S. RELATO DO CASO: Paciente com 9 anos de idade com Distrofia Muscular de Duchenne diagnosticada há 2 anos, submetido à anestesia geral com levo-cetamina (1,5 mg.kg-1, por via venosa, sob ventilação espontânea assistida manualmente por sistema de Baraka (Mapleson A e bloqueio peniano com bupivacaína a 0,5% (25 mg. Foram usados monitores de pressão arterial não invasiva, oximetria de pulso, cardioscopia e temperatura esofagiana. No decorrer da cirurgia, o caso evoluiu sem intercorrências, sendo que no período pós-operatório o paciente apresentou alguns episódios de vômitos sem outras alterações significativas. Permaneceu internado por 24 horas, tendo alta hospitalar assintomático. CONCLUSÕES: A avaliação pré-anestésica cuidadosa, o uso de monitorização adequada e medicações que não predisponham o aparecimento de complicações tornam seguro o procedimento em pacientes portadores de Distrofia Muscular de Duchenne e seu pós-operatório.

  18. Perfil Sistêmico e Manifestações Bucais em Pacientes com Hipotireoidismo

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    Graziele Beanes da Silva Santos

    2012-06-01

    Full Text Available O hipotireoidismo é uma desordem endócrina, de natureza sistêmica caracterizada pela disfunção na glândula tireóide. Os hormônios tireoidianos T3 e T4 são necessários para suprir funções orgânicas normais do organismo. Desta forma, distúrbios na secreção de T3 e T4 podem estar associados a alterações no sistema estomatognático. Dentre essas alterações encontram-se hipoplasia condilar, atresia maxilar ou mandibular, hipodesenvolvimento da mandíbula, prognatismo maxilar, hipoplasia de esmalte e dentina, taurodontia e retardo na erupção dentária e no desenvolvimento radicular. Desta forma é imprescindível que o cirurgião dentista tenha conhecimento sobre as manifestações locais ou sistêmicas associadas a esta desordem endócrina. O objetivo deste artigo é abordar as alterações sistêmicas e bucais em pacientes acometidos pelo hipotireoidismo, bem como os cuidados e condutas terapêutica que podem ser adotados durante o tratamento odontológico. A coleta de dados foi realizada através da seleção de artigos publicados a cerca das manifestações bucais em pacientes acometidos por hipotireoidismo nos principais bancos de dados, Bireme, Medline e Scielo. O hipotireoidismo é uma patologia sistêmica comum, que ocasionalmente apresenta manifestações bucais e necessitam de cuidados especiais durante tratamentos odontológicos. A interação medicamentosa frente à pacientes em tratamento do hipotireoidismo também deve ser considerada, uma vez que medicações comumente prescritas na odontologia podem promover reações adversas. Desta forma, é imprescindível que o cirurgião-dentista tenha conhecimento sobre os procedimentos adequados na terapêutica de pacientes com essa síndrome.

  19. Esquema de Desamparo em Paciente com Dor Crônica e Sintomas Depressivos

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    Elisangela Ferreira Santana

    2015-12-01

    Full Text Available A fibromialgia caracteriza-se por dor generalizada e crônica, de modo geral, associada à incapacidade funcional e quadros depressivos. Por suas repercussões, esta síndrome dolorosa pode ativar e/ou potencializar esquemas de desamparo e desvalor, o que por sua vez pode contribuir para o surgimento de sintomas depressivos. Neste artigo, analisa-se o processo de manejo da dor por meio da identificação de crenças de incapacidade e da alteração de humor numa paciente com dor crônica e sintomas depressivos. Para tanto, são descritas as estratégias e técnicas utilizadas em seu processo terapêutico, realizado em 13 sessões, que teve como embasamento a terapia cognitiva. Além disto, apresenta-se a conceituação cognitiva referente ao esquema de desamparo da paciente. Embora Laura não tenha apresentado mudanças significativas na percepção álgica, os sintomas depressivos tiveram uma remissão importante, o que contribuiu para aquisição de comportamentos mais funcionais para manejo da dor, como solicitar ajuda e evitar o esforço físico.

  20. Efeitos da sondagem nasogástrica em pacientes com acidente cerebrovascular e disfagia

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    Tahissa Frota Cavalcante

    2014-10-01

    Full Text Available O estudo teve por objetivo analisar os efeitos da sondagem gástrica em pacientes com acidente vascular cerebral e disfagia. Revisão sistemática da literatura, realizada em seis bases de dados, com os descritores stroke e intubation, gastrointestinal. Foram encontrados 120 estudos e selecionados três ensaios clínicos. Os resultados apontaram diferentes desfechos, entre os quais: aumento do nível sérico de albumina (gastrostomia, prognóstico ruim e risco de morte (gastrostomia, aumento das falhas no tratamento devido a bloqueio, deslocamento e reinserção da sonda nasogástrica, e aumento da incidência de hemorragia gastrointestinal (sonda nasogástrica. A partir dos resultados obtidos nesta revisão sistemática, ressaltam-se as seguintes evidências: a sondagem nasogástrica deve ser adotada precocemente como um método de alimentação enteral; as falhas do tratamento são mais comuns naqueles que utilizam a sonda nasogástrica como método de alimentação; os resultados relacionados à melhora do estado funcional dos pacientes foram semelhantes, independente do método de terapia nutricional empregado.

  1. Fungus ball in HIV-infected patients Bola fúngica em pacientes HIV-infectados

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    Luciana Silva Guazzelli

    2009-12-01

    Full Text Available Aspergillus is a phagocyte opportunistic fungus that causes aspergillosis, an unusual disease in patients with AIDS. Six cases of fungal ball in patients with AIDS are reported here. In this group, all patients had hemoptysis and tuberculosis as the underlying lung disease. The diagnosis of pulmonary fungus ball was based on the clinical and radiographic feature, combined with serological and mycological evidence of Aspergillus fumigatus.Os fungos filamentosos são oportunistas de fagócitos, motivo pelo qual aspergilose é incomum em pacientes com Aids. A apresentação clínica depende do estado imune, tamanho do inóculo fúngico e doença de base. São relatados neste trabalho seis casos de bola fúngica em pacientes com Aids. Neste grupo, todos tiveram tuberculose como doença de base e hemoptise foi o principal sintoma. O diagnóstico da bola fúngica foi através da apresentação clínica, achados radiológicos combinados com imunodifusão radial dupla, exame micológico direto e cultivo do material do trato respiratório, sendo A. fumigatus o agente isolado

  2. Lipossarcoma periorbital em paciente pediátrico: relato de caso

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    Fernanda Marcio

    2013-08-01

    Full Text Available O objetivo desse estudo é descrever uma criança com lipossarcoma periorbital, caracterizando seus aspectos clínico-epidemiológicos e terapêuticos. Menina de 6 meses de idade com tumoração crescente há dois meses em região fronto-zigomática direita, a qual foi submetida à exérese e cujas análises anatomopatológica (AP e imuno-histoquímica (IH observaram achados típicos de lipoblastoma. Após isso, apresentou mais três recidivas tumorais com diagnósticos similares. Um ano depois da última cirurgia, houve nova recorrência, porém, dessa vez, o resultado dos exames análises anatomopatológica e imuno-histoquímica foi de lipossarcoma, sendo, então, encaminhada para complementar o tratamento com radio e quimioterapia, sem novas lesões até o momento. Devido a sua raridade, geralmente o lipossarcoma não entra no diagnóstico diferencial em pacientes com massas orbitais, porém, por ser localmente agressivo, torna-se vital a pronta identificação e tratamento de forma a oferecer melhores resultados terapêuticos e influência sobre a qualidade de vida do paciente.

  3. MANIFESTAÇÕES PSICOLÓGICAS DE FAMILIARES COM PACIENTES EM MORTE ENCEFÁLICA

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    Juliana Carneiro Torres

    2014-02-01

    Full Text Available Este estudo originou-se a partir de uma monografia aprovada para graduação de curso de psicologia. Apresenta como objetivo compreender as manifestações psicológicas de familiares com paciente em Morte Encefálica (ME, identificando as possíveis intervenções psicológicas nas diferentes fases vivenciadas: notícia do quadro clínico, verificação de ME, diagnóstico de ME e processo de doação de órgãos. Trata-se de uma revisão bibliográfica de artigos científicos referentes aos últimos dez anos, utilizando como referencial teórico, para análise das manifestações psicológicas, literatura acerca do Luto e da Abordagem Sistêmica Familiar, além de legislação referente ao tema. Nos resultados, foram identificados que os familiares apresentam vivências peculiares que podem repercutir no processo de luto, como a preocupação com o prognóstico do paciente na internação, a incompreensão da possibilidade e do diagnóstico da ME, podendo apresentar uma experiência de perda ambígua e dificuldade de vivência de reações de luto na abordagem para doação de órgãos, devido o tempo restrito para decisão. Dessa forma, conclui-se que é imprescindível o acompanhamento psicológico aos familiares do paciente em Morte Encefálica durante todo o processo.

  4. Prevalência de talassemias e hemoglobinas variantes em pacientes com anemia não ferropênica

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    Wagner,Sandrine C.; Silvestri,Matheus C.; Bittar,Christina M.; Friedrisch,João R.; Silla,Lúcia M. R.

    2005-01-01

    Para estabelecer a freqüência de hemoglobinopatias e talassemias em pacientes com anemia não ferropênica foram estudados 58 casos de pacientes comprovadamente com anemia não ferropênica e 235 controles obtidos de pessoas sem anemia. Todas as amostras foram obtidas do Hospital de Clínicas de Porto Alegre (HCPA), RS, Brasil. As técnicas realizadas foram eletroforese em acetato de celulose, pH alcalino, pesquisa citológica de Hb H, HPLC, hemograma e ferritina. A análise dos dados realizada no gr...

  5. Técnicas cirúrgicas para correção da presbiopia em pacientes fácicos

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    Ferraz Caroline Amaral

    2004-01-01

    Full Text Available A presbiopia é a afecção ocular mais freqüente e é definida como a perda progressiva da amplitude acomodativa relacionada à idade. Atualmente, tentativas de correção cirúrgica da presbiopia em paciente fácicos e afácicos têm recebido considerável atenção, sendo campo vasto de pesquisa. O objetivo deste artigo é discutir as técnicas cirúrgicas propostas para correção da presbiopia em pacientes fácicos publicadas na literatura até o presente momento.

  6. Complicações respiratórias em pacientes com paralisia cerebral submetidos à anestesia geral

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    Mello, Sérgio Silva de; Marques, Ronaldo Soares; Saraiva, Renato Ângelo

    2007-01-01

    JUSTIFICATIVA E OBJETIVOS: Anestesia em pacientes com paralisia cerebral (PC) pode representar um desafio para o anestesiologista. Este estudo prospectivo teve como objetivo determinar a prevalência e o risco de complicações respiratórias em crianças com PC submetidas à anestesia geral inalatória (AGI) para tomografia computadorizada (TC). MÉTODO: Participaram do estudo pacientes com idades entre 1 e 17 anos, estado físico ASA I a III, submetidos a AGI com sevoflurano e máscara laríngea para ...

  7. Study of dermatoses in kidney transplant patients Estudo das dermatoses em pacientes transplantados renais

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    Alexandre Moretti de Lima

    2013-06-01

    Full Text Available BACKGROUND: The increasing in the number of kidney transplant recipients has favored, more frequently than before, the emergence of dermatoses and warranted their study through subsequent publications. OBJECTIVES: to evaluate the frequency of dermatoses in kidney transplant recipients. METHODS: kidney transplant recipients with suspected dermatoses between March 1st 2009 and June 30th 2010. RESULTS: 53 patients (28 males and 25 females, aged between 22 and 69 (mean age = 45 years were evaluated. Most of them came from the cities of Ceilândia, Samambaia and São Sebastião/DF, and had already been transplanted for 5 to 10 years before (37.7%; 62.3% were recipients of living donors and 83% were prednisone-treated. The most prevalent dermatoses were of fungal (45.3% and viral (39.6% etiologies. Among the non-melanoma malignant neoplasms, the basal cell carcinoma prevailed (six cases, in spite of the low incidence. Concerning fungal dermatoses, 12 cases of onychomycosis, five of pityriasis versicolor and four of pityrosporum folliculitis were reported. For diagnosis, in most cases (64.2%, laboratory examinations (mycological and histopathological were performed. CONCLUSION: cutaneous manifestations in kidney transplant recipients are generally secondary to immunosuppression. The infectious dermatoses, especially those of fungal origin, are frequently found in kidney transplant recipients and their occurrence increases progressively according to the time elapsed from the transplantation, which makes follow-up important. FUNDAMENTOS: o crescente aumento do número dos transplantados renais tem favorecido o aparecimento mais frequente das dermatoses e permitido o estudo em sucessivos trabalhos. OBJETIVOS: avaliar a frequência das dermatoses em pacientes transplantados renais. MÉTODOS: captação de pacientes transplantados renais durante o período de 1° de março de 2009 a 30 de junho de 2010 com suspeita de dermatoses. RESULTADOS : foram

  8. Cultura e clima organizacional para segurança do paciente em Unidades de Terapia Intensiva

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    Thaiana Helena Roma Santiago

    2015-12-01

    Full Text Available RESUMO Objetivo Avaliar a percepção dos profissionais de saúde sobre o clima e a cultura de segurança do paciente em Unidades de Terapia Intensiva (UTI e a relação entre os instrumentos Hospital Survey on Patient Safety Culture (HSOPSC e o Safety Attitudes Questionnaire (SAQ. Método Estudo transversal realizado em hospital de ensino no interior do estado de São Paulo, Brasil, em março/abril de 2014. Aplicaram-se o HSOPSC, o SAQ e um instrumento para levantamento das informações sociodemográficas e profissionais aos funcionários das UTI adulto, pediátrica e neonatal. A análise utilizou a estatística descritiva. Resultados As escalas apresentaram boa confiabilidade. Maiores fragilidades para a segurança do paciente foram observadas nos domínios “condições de trabalho” e “percepções da gerência” do SAQ e “resposta não punitiva aos erros” do HSOPSC. As fortalezas no SAQ foram o “clima de trabalho em equipe” e a “satisfação no trabalho” e para o HSOPSC “expectativas e ações de promoção de segurança supervisores/gerentes” e “aprendizado organizacional e melhoria mútua”. Na UTI Neonatal houve maior satisfação no trabalho do que nas demais UTI. A UTI Adulto apresentou menores pontuações para a maioria dos domínios do SAQ e HSOPSC. A correlação entre as escalas foi de força moderada (r=0,66. Conclusão Há diferenças de percepções quanto à segurança do paciente entre as UTI, o que corrobora com a existência de microculturas locais. O estudo não demonstra que o SAQ e o HSOPSC sejam equivalentes.

  9. Perfil lipídico em pacientes adultos com artrite idiopática juvenil

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    Thelma Larocca Skare

    2013-08-01

    Full Text Available O processo inflamatório articular de uma criança com artrite idiopática juvenil (AIJ pode persistir na vida adulta. A inflamação tem sido ligada a distorções do perfil lipídico e à aterogênese acelerada. Neste trabalho, estudou-se o perfil lipídico de indivíduos adultos com AIJ, comparando-o ao de pessoas saudáveis. Foram recrutados 54 adultos com AIJ (57,3% poliarticular, 37% oligoarticular, 1,9% do subtipo entesítica e 3,7% do subtipo sistêmica e 54 indivíduos saudáveis para comparação de perfil lipídico. Nos adultos AIJ, coletaram-se dados sobre sexo, idade e idade de início da doença, presença de fator reumatoide (FR e fator antinuclear (FAN, HAQ (Health Assessment Questionnaire e tempo de doença. Observou-se que hipercolesterolemia, aumento de LDL e diminuição de HDL foram mais comuns em pacientes com AIJ do que nos controles (com P = 0,016; P < 0,0001 e P = 0,0008, respectivamente. Alterações no colesterol total (CT e no LDL foram mais comuns em indivíduos com início de doença mais tardio (P = 0,0017 para CT e P = 0,023 para LDL. Em todo o grupo de pacientes com AIJ nenhuma outra variável pode ser ligada à dislipidemia tal como presença de FR, FAN, duração de doença e HAQ (P = ns. Concluiu-se que pacientes adultos com AIJ apresentam um perfil lipídico com aumento de CT e de LDL, e diminuição de HDL em relação aos controles. Nenhuma característica clínica pode ser ligada a essa alteração, exceto idade de início da doença.

  10. Púrpura em paciente com estrongiloidíase disseminada Purpura in patient with disseminated strongiloidiasis

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    Luciano C. Ribeiro

    2005-05-01

    Full Text Available A infecção pelo Strongyloides stercoralis em associação com imunosupressão pode manifestar-se com lesões em múltiplos órgãos e sistemas, caracterizando a forma disseminada da doença. Lesões cutâneas não são freqüentemente relatadas e, se presentes, manifestam-se como rash e petéquias. Púrpuras bem definidas são pouco descritas. No presente trabalho é descrito um caso de estrongiloidíase disseminada, com acometimento cutâneo em forma de púrpura, que se desenvolveu em um paciente timectomizado e usuário crônico de corticosteróide devido à miastenia gravis.The association of systemic corticosteroid therapy and dissemination of Strongyloides stercoralis has been increasingly documented in the literature. Skin involvement in disseminated strongyloidiasis has been reported and the most commonly described cutaneous manifestations are rash and petechial eruptions. We present a case of an immunosuppressed man that developed disseminated strongyloidiasis with extensive purpura.

  11. Níveis de PCR são maiores em pacientes com síndrome coronariana aguda e supradesnivelamento do segmento ST do que em pacientes sem supradesnivelamento do segmento ST

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    Syed Shahid Habib

    2011-01-01

    Full Text Available FUNDAMENTO: Há grande interesse no uso de proteína C-reativa de alta sensibilidade (PCR-as para avaliação de risco. Altos níveis de PCR-as no início da síndrome coronária aguda (SCA, antes da necrose tecidual, pode ser um marcador substituto para comorbidades cardiovasculares. OBJETIVO: Dessa forma, nosso objetivo foi estudar diferentes medidas de seguimento de níveis de PCR-as em pacientes com SCA e comparar as diferenças entre infarto do miocárdio sem elevação do segmento ST (NSTEMI com pacientes apresentando elevação do segmento ST (STEMI. MÉTODOS: Este é um estudo observacional. Dos 89 pacientes recrutados, 60 apresentavam infarto agudo do miocárdio (IAM. Três níveis seriados de PCR-us, a nível basal na hospitalização antes de 12 horas após inicio dos sintomas, níveis de pico 36-48 horas após hospitalização e níveis de acompanhamento após 4 a 6 semanas foram analisados e comparados entre pacientes com (IAMCSST e sem supradesnivelamento do segmento ST (IAMSSST. RESULTADOS: Pacientes com IAMCSST tinham IMC significantemente mais alta quando comparados com pacientes IAMSSST. Os níveis de creatino quinase fração MB (CK-MB e aspartato aminotransferase (AST eram significantemente mais altos em pacientes com IAMCSST quando comparados com pacientes com IAMSSST (p<0,05. Os níveis de PCR a nível basal e no acompanhamento não diferiram de forma significante entre os dois grupos (p=0,2152 e p=0,4686 respectivamente. Houve uma diferença significante nos níveis de pico de PCR entre os dois grupos. No grupo de pacientes com IAMCSST os níveis foram significantemente mais altos quando comparados aos pacientes com IAMSSST (p=0,0464. CONCLUSÃO: Pacientes com IAMCSST apresentam picos significantemente mais elevados de PCR quando comparados a pacientes IAMSSST. Esses dados sugerem que o processo inflamatório tem um papel independente na patogênese do infarto do miocárdio. Dessa forma, os níveis de PCR podem ajudar na

  12. Perda auditiva sensorioneural em pacientes com acromegalia em tratamento Sensorineural hearing loss in acromegalic patients under treatment

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    Marcelo Alexandre Carvalho

    2012-08-01

    Full Text Available Acromegalia é uma doença endócrina rara. Poucos estudos avaliaram sua associação com deficiência auditiva (DA e os resultados são conflitantes. OBJETIVOS: Avaliar a prevalência e características da DA em um grupo de pacientes com acromegalia em tratamento. Analisar a transmissão auditiva central e periférica. MATERIAL E MÉTODOS: Estudo transversal. Um grupo de 34 pacientes com acromegalia submeteu-se à avaliação metabólica, audiometria tonal e potenciais evocados auditivos de tronco encefálico (PEATE. Considerou-se DA quando a média dos tons puros foi > 25 DBNA para baixas frequências (250, 500, 1000 e 2000 Hz ou altas frequências (3000, 4000, 6000 e 8000 Hz. Os pacientes foram divididos em grupo A (com DA e B (sem DA. RESULTADOS: Doze pacientes (35,3% mostraram DA sensorioneural (grupo A, sendo oito bilateral e quatro unilateral. Nenhum apresentou DA mista ou condutiva. A prevalência de diabetes/intolerância à glicose de jejum foi similar entre os grupos. As frequências de 3000, 4000, 6000 e 8000 Hz foram as mais afetadas e com padrão similar em ambos os lados. CONCLUSÃO: DA sensorioneural esteve presente em 35,3% dos casos. Não foram notadas diferenças clínicas ou metabólicas significativas entre os grupos, bem como na transmissão neural auditiva periférica e central.Acromegaly is a rare endocrine disease. Few studies have evaluated its association with hearing loss (HL and the results are conflicting. AIM: To evaluate the prevalence and features of HL in a group of patients being treated for acromegaly. To analyze peripheral and central auditory transmission. METHODS: Cross-sectional study. A group of 34 patients with acromegaly were submitted to metabolic evaluation, tonal audiometry and brainstem auditory evoked potentials. HL was considered when pure tone average was > 25 DBHL for low frequencies (250, 500, 1000 and 2000 Hz or high frequencies (3000, 4000, 6000 and 8000 Hz. The patients were divided in group

  13. Sarcoma de Kaposi em paciente transplantada renal em uso de Fk-506 Kaposi's Sarcoma in a renal transplant patient receiving Fk-506

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    Jorge David Rocha Zanol

    2002-12-01

    Full Text Available O Sarcoma de Kaposi (SK é neoplasia maligna multicêntrica, cutânea e extracutânea, que tem sido descrita em pacientes transplantados renais que recebem terapia imunossupressora clássica. Este estudo descreve um caso de sarcoma de Kaposi em paciente transplantada renal recebendo FK-506, que surgiu 10 meses após o transplante.Kaposi's sarcoma is a cutaneous and extra cutaneous multicentric malignancy that has been widely described in renal-transplant patients under classic immunosuppressive therapy. This study describes a renal-transplant patient under immunosuppressive therapy with FK-506 who presented Kaposi's sarcoma 10 months after the transplantation.

  14. Privacidade em unidades de terapia intensiva: direitos do paciente e implicações para a enfermagem

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    Maria Aparecida Baggio

    2011-02-01

    Full Text Available O objetivo deste estudo foi compreender as experiências vivenciadas pelos profissionais da enfermagem sobre a privacidade do paciente internado em UTI e suas implicações. Este estudo qualitativo, mediante análise de conteúdo, originou as categorias: Expondo o corpo e a intimidade dos pacientes: atitudes e ações dos profissionais; A intimidade: dificuldades vividas pelos profissionais e reações dos pacientes; Desproteção da intimidade do paciente: contradições que emergem da prática. Os resultados apontam a necessidade dos profissionais repensarem o seu fazer em relação à proteção da privacidade do paciente, por meio de atos/atitudes que transmitam segurança nas ações do cuidado. A preservação da privacidade é direito do paciente e compromisso ético do profissional, conferindo dignidade às pessoas no processo de cuidado na UTI

  15. Avaliação angular do equilíbrio sagital em pacientes obesos

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    Alynson Larocca Kulcheski

    2013-01-01

    Full Text Available OBJETIVO: Tendo em vista a pouca quantidade de informações a respeito das alterações degenerativas e patológicas da coluna vertebral realizou-se este estudo com objetivo de avaliar e quantificar os valores angulares do equilíbrio sagital na população obesa. MÉTODOS: Foram medidos os ângulos de incidência pélvica (IP, versão pélvica (VP e declive sacral (DS. RESULTADOS: Foram avaliados 30 pacientes, sendo 19 do sexo feminino e 11 do sexo masculino, no ano de 2012 que aguardavam realização de cirurgia bariátrica. A média de idade foi de 33 anos. O IMC médio foi de 41 kg/m², sendo que o valor médio para os homens foi de 43 kg/m² e das mulheres 39 kg/m². O IP médio foi de 57°. O VP foi de 19°. O DS foi de 38°. O valor da incidência pélvica e da versão pélvica ficou em média acima do encontrado na população com índice de massa corporal normal. Já o valor do declive sacral encontra-se na média da população. CONCLUSÕES: Cada vez mais o equilíbrio sagital vem ganhando espaço na avaliação da coluna vertebral. Faz-se necessário literatura mais vasta e aumento do conhecimento médico das patologias e comorbidades que podem alterar o equilíbrio sagital em seus pacientes, como a obesidade.

  16. Microbiota conjuntival em pacientes com alergia ocular Conjunctival microbiota in patients with ocular allergy

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    Alexandre Mattoso Libório

    2005-12-01

    Full Text Available OBJETIVO: Avaliar a presença de microbiota aeróbia da conjuntiva de portadores de alergia ocular e comparar a um grupo controle. MÉTODOS: Foram examinados 133 pacientes no período de abril a junho de 2001 divididos em 2 grupos. O grupo A foi composto de 63 portadores de conjuntivite alérgica (sem uso de medicação e o grupo B de 70 pacientes do ambulatório geral (controle. Foram coletadas amostras do fundo de saco conjuntival do olho direito de todos os pacientes e o material foi semeado em meios sólidos de cultura (ágar sangue, chocolate e Sabouraud. RESULTADOS: No grupo A, 30 culturas (47,7% foram positivas e no grupo B, 6 (8,6%. Sete bactérias foram isoladas no grupo A e 4 no B. A análise estatística revelou associação significante entre a positividade dos cultivos e conjuntivite alérgica. CONCLUSÃO: Microbiota bacteriana foi mais freqüentemente encontrada nos pacientes com alergia ocular.PURPOSE: To evaluate de presence of conjunctival aerobic microbiota in patients with ocular allergy as compared to a control group. METHODS: One hundred and thirty-three patients were evaluated from April to June 2001 and divided into 2 groups. Sixty-three patients with allergic conjunctivitis (without medication were in group A and 70 patients from the general outpatient clinic were in group B (control group. Samples from the conjunctival sac of the right eye were collected and cultured in solid media (blood, chocolate and Sabouraud agar. RESULTS: In group A, 30 cultures (47.7% were positive and 6 (8.6% in group B. Seven bacteria were isolated from group A and 4 from group B. Statistical analysis revealed significant association between positive cultures and allergic conjunctivitis. CONCLUSION: Bacterial microbiota was more frequently found in patients with ocular allergy.

  17. Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR method

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    FIGUEIREDO Luiz Tadeu Moraes

    1997-01-01

    Full Text Available We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

  18. Evaluation of 793/B-like and Mass-like vaccine strain kinetics in experimental and field conditions by real-time RT-PCR quantification.

    Science.gov (United States)

    Tucciarone, C M; Franzo, G; Berto, G; Drigo, M; Ramon, G; Koutoulis, K C; Catelli, E; Cecchinato, M

    2018-01-01

    Infectious bronchitis virus (IBV) is a great economic burden both for productive losses and costs of the control strategies. Many different vaccination protocols are applied in the same region and even in consecutive cycles on the same farm in order to find the perfect balance between costs and benefits. In Northern Italy, the usual second vaccination is more and more often moved up to the chick's first d of life. The second strain administration together with the common Mass priming by spray at the hatchery allows saving money and time and reducing animal stress. The present work compared the different vaccine strains (Mass-like or B48, and 1/96) kinetics both in field conditions and in a 21-day-long experimental trial in broilers, monitoring the viral replication by upper respiratory tract swabbing and vaccine specific real time reverse transcription PCR (RT-PCR) quantification. In both field and experimental conditions, titers for all the vaccines showed an increasing trend in the first 2 wk and then a decrease, though still remaining detectable during the whole monitored period. IBV field strain and avian Metapneumovirus (aMPV) presence also was also investigated by RT-PCR and sequencing, and by multiplex real-time RT-PCR, respectively, revealing a consistency in the pathogen introduction timing at around 30 d, in correspondence with the vaccine titer's main decrease. These findings suggest the need for an accurate knowledge of live vaccine kinetics, whose replication can compete with the other pathogen one, providing additional protection to be added to what is conferred by the adaptive immune response. © 2017 Poultry Science Association Inc.

  19. Construction of an adult barnacle (Balanus amphitrite cDNA library and selection of reference genes for quantitative RT-PCR studies

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    Burgess J Grant

    2009-06-01

    Full Text Available Abstract Background Balanus amphitrite is a barnacle commonly used in biofouling research. Although many aspects of its biology have been elucidated, the lack of genetic information is impeding a molecular understanding of its life cycle. As part of a wider multidisciplinary approach to reveal the biogenic cues influencing barnacle settlement and metamorphosis, we have sequenced and annotated the first cDNA library for B. amphitrite. We also present a systematic validation of potential reference genes for normalization of quantitative real-time PCR (qRT-PCR data obtained from different developmental stages of this animal. Results We generated a cDNA library containing expressed sequence tags (ESTs from adult B. amphitrite. A total of 609 unique sequences (comprising 79 assembled clusters and 530 singlets were derived from 905 reliable unidirectionally sequenced ESTs. Bioinformatics tools such as BLAST, HMMer and InterPro were employed to allow functional annotation of the ESTs. Based on these analyses, we selected 11 genes to study their ability to normalize qRT-PCR data. Total RNA extracted from 7 developmental stages was reverse transcribed and the expression stability of the selected genes was compared using geNorm, BestKeeper and NormFinder. These software programs produced highly comparable results, with the most stable gene being mt-cyb, while tuba, tubb and cp1 were clearly unsuitable for data normalization. Conclusion The collection of B. amphitrite ESTs and their annotation has been made publically available representing an important resource for both basic and applied research on this species. We developed a qRT-PCR assay to determine the most reliable reference genes. Transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, although other genes also performed well and could prove useful to normalize gene expression studies.

  20. Selection and validation of reference genes for gene expression analysis in switchgrass (Panicum virgatum using quantitative real-time RT-PCR.

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    Jacinta Gimeno

    Full Text Available Switchgrass (Panicum virgatum has received a lot of attention as a forage and bioenergy crop during the past few years. Gene expression studies are in progress to improve new traits and develop new cultivars. Quantitative real time PCR (qRT-PCR has emerged as an important technique to study gene expression analysis. For accurate and reliable results, normalization of data with reference genes is essential. In this work, we evaluate the stability of expression of genes to use as reference for qRT-PCR in the grass P. virgatum. Eleven candidate reference genes, including eEF-1α, UBQ6, ACT12, TUB6, eIF-4a, GAPDH, SAMDC, TUA6, CYP5, U2AF, and FTSH4, were validated for qRT-PCR normalization in different plant tissues and under different stress conditions. The expression stability of these genes was verified by the use of two distinct algorithms, geNorm and NormFinder. Differences were observed after comparison of the ranking of the candidate reference genes identified by both programs but eEF-1α, eIF-4a, CYP5 and U2AF are ranked as the most stable genes in the samples sets under study. Both programs discard the use of SAMDC and TUA6 for normalization. Validation of the reference genes proposed by geNorm and NormFinder were performed by normalization of transcript abundance of a group of target genes in different samples. Results show similar expression patterns when the best reference genes selected by both programs were used but differences were detected in the transcript abundance of the target genes. Based on the above research, we recommend the use of different statistical algorithms to identify the best reference genes for expression data normalization. The best genes selected in this study will help to improve the quality of gene expression data in a wide variety of samples in switchgrass.

  1. Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR.

    Science.gov (United States)

    Arif, Muhammad; Ali, Murad; Rehman, Anayatur; Fahim, Muhammad

    2014-01-01

    The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in Nicotiana debneyi plants grown in putative viruliferious soils from 20/26 locations. The results were confirmed by back inoculation of sap from both roots and leaves of bait plant on indicator hosts (N. debneyi, Nicotiana benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26 locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566bp were amplified from coat protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait plant bioassay, infectivity assay, ELISA and RT-PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea and tubers of 10 potato cultivars commercially grown in the region. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Early diagnosis of dengue in travelers: comparison of a novel real-time RT-PCR, NS1 antigen detection and serology.

    Science.gov (United States)

    Huhtamo, Eili; Hasu, Essi; Uzcátegui, Nathalie Y; Erra, Elina; Nikkari, Simo; Kantele, Anu; Vapalahti, Olli; Piiparinen, Heli

    2010-01-01

    The increased traveling to dengue endemic regions and the numerous epidemics have led to a rise in imported dengue. The laboratory diagnosis of acute dengue requires several types of tests and often paired samples are needed for obtaining reliable results. Although several diagnostic methods are available, proper comparative data on their performance are lacking. To compare the performance of novel methods including a novel pan-DENV real-time RT-PCR and a commercially available NS1 capture-EIA in regard to IgM detection for optimizing the early diagnosis of DENV in travelers. A panel of 99 selected early phase serum samples of dengue patients was studied by real-time RT-PCR, NS1 antigen ELISA, IgM-EIA, IgG-IFA and cell culture virus isolation. The novel real-time RT-PCR was shown specific and sensitive for detection of DENV-1-4 RNA and suitable for diagnostic use. The diagnostic rate using combination of RNA and IgM detection was 99% and using NS1 and IgM detection 95.9%. The results of RNA and NS1 antigen detection disagreed in 15.5% of samples that had only RNA or NS1 antigen detected. The diagnostic rates of early samples are higher when either RNA or NS1 antigen detection is combined with IgM detection. Besides the differences in the RNA and NS1 detection assays, the observed discrepancy of results could suggest individual variation or differences in timing of these markers in patient serum. Copyright (c) 2009 Elsevier B.V. All rights reserved.

  3. A family-wide RT-PCR assay for detection of paramyxoviruses and application to a large-scale surveillance study.

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    Sander van Boheemen

    Full Text Available Family-wide molecular diagnostic assays are valuable tools for initial identification of viruses during outbreaks and to limit costs of surveillance studies. Recent discoveries of paramyxoviruses have called for such assay that is able to detect all known and unknown paramyxoviruses in one round of PCR amplification. We have developed a RT-PCR assay consisting of a single degenerate primer set, able to detect all members of the Paramyxoviridae family including all virus genera within the subfamilies Paramyxovirinae and Pneumovirinae. Primers anneal to domain III of the polymerase gene, with the 3' end of the reverse primer annealing to the conserved motif GDNQ, which is proposed to be the active site for nucleotide polymerization. The assay was fully optimized and was shown to indeed detect all available paramyxoviruses tested. Clinical specimens from hospitalized patients that tested positive for known paramyxoviruses in conventional assays were also detected with the novel family-wide test. A high-throughput fluorescence-based RT-PCR version of the assay was developed for screening large numbers of specimens. A large number of samples collected from wild birds was tested, resulting in the detection of avian paramyxoviruses type 1 in both barnacle and white-fronted geese, and type 8 in barnacle geese. Avian metapneumovirus type C was found for the first time in Europe in mallards, greylag geese and common gulls. The single round family-wide RT-PCR assay described here is a useful tool for the detection of known and unknown paramyxoviruses, and screening of large sample collections from humans and animals.

  4. Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

    Science.gov (United States)

    Qiu, Reng; Sun, Boguang; Fang, Shasha; Sun, Li; Liu, Xiao

    2013-03-01

    Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

  5. Doenças cerebrovasculares em pacientes entre 15 e 40 anos: achados neuropatológicos em 47 casos

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    Maggio, Everton M.; Montemór-Netto, Mário R; Gasparetto, Emerson L.; Reis-Filho, Jorge S.; Tironi, Fábio A.; Torres, Luiz F. Bleggi

    2001-01-01

    As doenças cerebrovasculares em pacientes entre 15 e 40 anos são pouco estudadas na literatura latino americana, principalmente no que tange aos achados neuropatológicos. Analisamos 47 encéfalos provenientes de necrópsias completas realizadas no período de 1987 a 1997 selecionados com base na faixa etária e alteração neuropatológica básica envolvendo fenômenos vasculares. Destes 47 casos analisados, 26 eram (55,3%) do sexo feminino. Quanto à distribuição etária, 12,8% (n=06) acometeram pacien...

  6. Estresse e estratégias de enfrentamento em pacientes que serão submetidos à cirurgia de colecistectomia

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    André Faro Santos

    2006-06-01

    Full Text Available A presente pesquisa objetivou comparar o estresse em pacientes no pré-operatório da cirurgia de colecistectomia em relação aos pacientes submetidos ao tratamento clínico de gastrite, buscando também delinear as estratégias de enfrentamento utilizadas por esses pacientes. A amostra foi composta por dois grupos, sendo 15 pacientes cirúrgicos e 10 pacientes do tratamento clínico. Para a detecção do estresse utilizou-se o Inventário de Sintomas de Stress para Adultos de Lipp e para o conhecimento das estratégias de enfrentamento, aplicou-se o Inventário de Estratégias de Coping de Folkman e Lazarus. Nos resultados, constatou-se que a maioria dos pacientes cirúrgicos teve estresse (73,3%, enquanto no grupo do tratamento clínico, somente 10% deles apresentaram. Predominaram pacientes na fase de resistência (72,7% e com sintomas físicos (63,3%. Houve relação estatisticamente significativa entre o sexo e o diagnóstico de estresse, indicando que o sexo feminino apresentou maior ocorrência de estresse na amostra pesquisada (p<0,05. Acredita-se que a cirurgia caracterizou-se como um fator estressante, ressaltando a necessidade da implementação de ações que visem o manejo adequado do estresse, em vista da redução da tensão no pré-cirúrgico e uma melhor recuperação no pós-cirúrgico.   Palavras-chave: estresse; cirurgia; enfrentamento.

  7. Desnutrição em paciente com doença pulmonar obstrutiva crônica

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    Andreia Biondo

    2011-10-01

    Full Text Available A doença pulmonar obstrutiva crônica (DPOC é uma patologia responsável por muitas mortes em todo o mundo, causada principalmente pelo tabagismo. Apresenta como característica associada o estado nutricional deficiente. O caso relatado é de uma paciente de 63 anos ex-tabagista, que internou em um hospital do interior do RS por broncopneumonia e diagnóstico de DPOC há 3 anos. Permaneceu internada por 15 dias, com negativa evolução do estado nutricional mesmo superando a ingestão energética estimada. Pacientes com DPOC apresentam taxa de metabolismo basal aumentada e infecções associadas podem acelerar ainda mais o metabolismo e gerar anorexia. A terapia nutricional é muito importante para a recuperação desses pacientes.

  8. Procedimentos cirúrgicos em pacientes proctológicos selecionados, sob anestesia local: estudo de 150 casos

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    HENRIQUES Alexandre Cruz

    2000-01-01

    Full Text Available Experiência no tratamento de 150 pacientes portadores de afecções anorretais e da região sacrococcígea operados sob anestesia local no Hospital de Ensino da Faculdade de Medicina do ABC, São Bernardo do Campo, SP, no período de março de 1995 a março de 1998. Descrevem a técnica anestésica empregada, operações realizadas e a tolerância ao procedimento. A morbidade intra-operatória foi de 10,6% (16 pacientes e a pós-operatória de 6% (9 pacientes. A idade dos pacientes variou entre 15 e 92 anos de idade, com média de 42 anos; 58% eram do sexo masculino e 42% feminino. O tempo médio de cirurgia foi de 45 minutos e o de permanência hospitalar foi de 8 horas. Todos os pacientes foram orientados sobre a técnica anestésica, suas vantagens e desvantagens, e somente após sua aprovação, a cirurgia era programada. Internação hospitalar foi necessária em cinco pacientes (3,3%. Em nenhum caso foi necessário modificar a técnica anestésica. Interrogados, 96,7% dos pacientes afirmaram não sentir dor durante a operação e que repetiriam o procedimento. Conclui-se que o tratamento cirúrgico das afecções orificiais e da região sacrococcígea com anestesia local além de viável e seguro, é bem aceito pelos pacientes.

  9. Immunocapture RT-PCR detection of Bean common mosaic virus and strain blackeye cowpea mosaic in common bean and black gram in India

    DEFF Research Database (Denmark)

    Udayashankar, A.C.; Nayaka, S. Chandra; Niranjana, S.R.

    2012-01-01

    The strains of Bean common mosaic virus (BCMV) and blackeye cowpea mosaic (BICM), genus Potyvirus, were detected from 25 common bean and 14 black gram seeds among 142 seed samples collected from different legume-growing regions of India. The samples were subjected to a growing-on test, an indicator...... plant test, an electron microscopic observations, an enzyme linked immunosorbent assay and an immunocapture RT-PCR. The incidence of the two tested viruses in common bean and black gram seed samples was 1–6% and 0.5–3.5%, respectively in growing-on test evaluations. Electron microscopic observations...

  10. Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

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    Aman Kamboj

    2014-01-01

    Full Text Available Crimean-Congo hemorrhagic fever (CCHF is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV. The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF.

  11. Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions

    DEFF Research Database (Denmark)

    Svingen, Terje; Letting, Heidi; Hadrup, Niels

    2015-01-01

    In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or...... ‘housekeeping’) gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a...

  12. DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7

    DEFF Research Database (Denmark)

    Yi, Sun; Dhumpa, Raghuram; Bang, Dang Duong

    2011-01-01

    of RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal....... In this article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification...

  13. Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

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    Alok Arun

    Full Text Available Real-time quantitative reverse transcription PCR (qRT-PCR is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae, two developmental stages (pupal and adult and two sexes (male and female, all of which were subjected to two food treatments (food stress and control feeding ad libitum. The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the

  14. Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

    Science.gov (United States)

    Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

    2015-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression

  15. Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets.

    Science.gov (United States)

    Liu, Xin; Guan, Huirui; Song, Min; Fu, Yanping; Han, Xiaomin; Lei, Meng; Ren, Jingyu; Guo, Bin; He, Wei; Wei, Yahui

    2018-01-01

    Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported. In this study, 10 candidate RGs namely, 18S , 60S , CYP , GAPCP1 , GAPDH2 , EF1B , MDH , SAND , TUA1 , and TUA6 , were singled out from the transcriptome database of S. chamaejasme , and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper. Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND , TUA1 and CYP , GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes ( P5CS2 and GI ) further verified that the RGs that we selected were suitable for gene expression normalization. This work is the first attempt to

  16. Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets

    Directory of Open Access Journals (Sweden)

    Xin Liu

    2018-04-01

    Full Text Available Background Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported. Method In this study, 10 candidate RGs namely, 18S, 60S, CYP, GAPCP1, GAPDH2, EF1B, MDH, SAND, TUA1, and TUA6, were singled out from the transcriptome database of S. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH were estimated with the programs geNorm, NormFinder, and BestKeeper. Result Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND, TUA1 and CYP, GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2 and GI further verified that the RGs that we selected were suitable for gene expression normalization. Discussion

  17. Detección y cuantificación del Potato mop-top virus (PMTV en Colombia mediante qRT-PCR

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    Nevar García Bastidas

    2013-04-01

    Full Text Available El Potato mop-top virus (PMTV es uno de los virus re-emergentes en cultivos de papa en Colombia. Es transmitido por Spongospora subterranea, el agente causal de la sarna polvosa. La detección del PMTV presenta dificultades debido a su distribución irregular en las plantas, bajo título y movimiento sistémico como ARN desnudo. Con el fin de ampliar el rango de herramientas disponibles para detectar el PMTV en los programas de certificación de tubérculo-semilla, en este estudio se evaluó la prueba de RT-PCR en tiempo real (qRT-PCR en dos pasos: con los cebadores PMTV-1948F/PMTV-2017R y la sonda Taqman® PMTV-1970, dirigidos al gen CP-RT del ARN2 viral. Se construyó una curva estándar a partir de la transcripción in vitro de un fragmento de 1513 pb de este gen. Posteriormente, se evaluó la utilidad de la técnica a partir de tres tipos de muestras: plantas señuelo de Nicotiana benthamiana y Solanum phureja inoculadas con quistosoros de Sss, raíces de papa con síntomas de sarna polvosa del municipio de La Unión (Antioquia y tubérculos-semilla. Mediante qRT-PCR fue posible detectar el virus en 11 de las 20 muestras de raíz de plantas señuelo, mientras que 14 de las 15 muestras de raíces de papa resultaron positivas, estimándose una concentración entre 4.72 x 10(11 y 7.60 x 10(13 partículas virales/µl. Adicionalmente, en el ensayo de tubérculo-semilla se determinó la presencia del PMTV en una de las 16 muestras. Estos resultados indican la viabilidad de utilizar rutinariamente la técnica de qRT-PCR para la detección de PMTV en Colombia.

  18. Zinco e risco cardiovascular de pacientes nefropatas em tratamento de hemodiálise

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    Ádila da Silva Castro

    2018-02-01

    Full Text Available Objetivo: Investigar a relação entre concentração de zinco e risco cardiovascular em pacientes com insuficiência renal crônica em hemodiálise. Métodos: Estudo analítico, retrospectivo e transversal, realizado em Fortaleza, Ceará, em 2012, do qual participaram 43 adultos com insuficiência renal crônica em hemodiálise (grupo HD e 35 saudáveis (CO - controle. Coletaram-se dados socioeconômicos e bioquímicos (colesterol total - CT, lipoproteína de baixa densidade - LDL, lipoproteína de muito baixa densidade - VLDL, lipoproteína de alta densidade - HDL e triglicerídeos - TG; determinou-se zinco plasmático por espectrometria de emissão óptica com plasma acoplado; coletou-se perfil lipídico do grupo HD do prontuário e do grupo CO por meio de kit Bioclin®. Obteve-se colesterol não-HDL pela fórmula: não-HDL = CT – HDL-c. Calculou-se risco cardiovascular pela razão TG/HDL, considerando risco quando >3,8. Análises por meio de testes t de Student, Pearson ou Spearman. Resultados: O zinco do grupo HD encontrou-se abaixo da referência e menor (p<0,001 em relação ao controle (68,40µg/dL e 85,53µg/dL, respectivamente. HDL no grupo HD mostrou-se abaixo da recomendação (39,64±11,58. VLDL (29,02±14,03mg/dL do grupo HD foi maior (p<0,001 que no CO (15,47±10,65mg/dL. LDL do grupo HD maior que no CO (p=0,05 e o TG no grupo HD (145,14±70,15mg/dL maior (p<0,001 que no CO (77,35±53,25mg/dL. Encontrou-se razão TG/HDL no grupo HD de 4,02±2,60 (p=0,04, indicando maior risco cardiovascular. Pacientes com maior relação TG/HDL apresentaram menores níveis de zinco (p=0,011. Conclusão: Os pacientes em hemodiálise apresentaram deficiência de zinco e possuíam elevado risco cardiovascular, porém sem correlação entre zinco e perfil lipídico.

  19. Transtornos de personalidade em pacientes com fobia social Personality disorders in a sample of social phobics

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    Tito Paes de Barros Neto

    2006-01-01

    Full Text Available O objetivo deste estudo foi investigar se outros transtornos de personalidade, além do transtorno de personalidade esquiva, ocorrem entre fóbicos sociais. Vinte e dois pacientes com diagnóstico de fobia social de acordo com os critérios do DSM-III-R foram avaliados através do Inventário Multifásico Minnesota de Personalidade (MMPI, da Entrevista Estruturada para Distúrbios de Personalidade do DSM-III-R (SIDP-R e do Exame do Estado Psíquico (PSE. Catorze pacientes (64% da amostra receberam pelo menos um diagnóstico de transtorno de personalidade - todos estes receberam o diagnóstico de transtorno de personalidade esquiva. Metade dos pacientes que recebeu o diagnóstico de transtornos de personalidade apresentou dois ou mais transtornos de personalidade. O transtorno de personalidade paranóide foi diagnosticado em seis pacientes (27%. Dez pacientes (46%, avaliados através do MMPI, apresentaram escore patológico na escala paranóia (Pa. Outros traços patológicos de personalidade foram observados nas escalas depressão (D, histeria (Hy e introversão-extroversão (Si do MMPI. Idéias de referência não-delirantes (IR do PSE ocorreram em 19 pacientes. O transtorno de personalidade paranóide foi diagnosticado com freqüência maior neste estudo do que na maioria dos estudos realizados com fóbicos sociais. Isso parece ter ocorrido por sobreposição de critérios diagnósticos pouco específicos e também por auto-referência e traços paranóides de personalidade, como hipersensibilidade e preocupação com a opinião alheia.The aim of this study was to investigate if other comorbid personality disorders, other than avoidant personality disorder, occur among social phobics. Twenty-two patients with a social phobia diagnosis according to DSM-III-R criteria were evaluated by using the Minnesota Multiphasic Personality Inventory (MMPI, the Structured Interview for DSM-III-R Personality Disorders (SIDP-R and the Present State

  20. Acolhimento e sintomas de ansiedade em pacientes no pré-operatório de cirurgia cardíaca

    Directory of Open Access Journals (Sweden)

    Cinthia Calsinski Assis

    2014-06-01

    Full Text Available Trata-se de ensaio clínico randomizado, em que se objetivou comparar a frequência e intensidade de sintomas de ansiedade de pacientes em pré-operatório de cirurgia cardíaca que receberam acolhimento do enfermeiro ou do familiar ou os que não receberam nenhum tipo específico de acolhimento. A amostra foi constituída de 66 pacientes em pré-operatório de cirurgia cardíaca, que foram alocados em três grupos: acolhimento pelo enfermeiro, sem acolhimento específico e acolhimento pelo familiar. A ansiedade foi avaliada em dois momentos: antes e após a intervenção. O instrumento utilizado foi o construído e validado por Suriano, composto por 19 características definidoras do diagnóstico de enfermagem ansiedade. Observou-se que a redução dos sintomas de ansiedade foi maior no grupo que recebeu acolhimento dos familiares quando comparado aos outros dois grupos. Os resultados sugerem que o incentivo à participação de familiares pode contribuir para a redução de sintomas ansiosos em pacientes no pré-operatório de cirurgias cardíacas.

  1. Estado nutricional em pacientes atendidos por um programa de adultos para fibrose cística

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    Bruna Ziegler

    2008-02-01

    Full Text Available Introdução: Na fibrose cística (FC, o estado nutricional está associado com o crescimento, função pulmonar e índices de so-brevida. Objetivo: Avaliar o estado nutricional em adultos com FC e correlacionar com escore clínico, escore radiológico, pressões respiratórias estáticas máximas, capacidade submáxima de exercício e função pulmonar. Metodologia: O estudo realizado foi transver-sal e prospectivo, em pacientes (16 anos ou mais, atendidos em um programa para adultos com FC. Os pacientes foram submetidos a uma avaliação nutricional e clínica, ao teste de caminhada de seis minutos (TC6, à medida das pressões respiratórias máximas, a espirometria e exame radiológico do tórax. Resultados: O estudo incluiu 41 pacientes com media de idade de 23,7 ± 6,5 anos e média de índice de massa corporal (IMC de 20,2 ± 2,2 Kg/m2. Vinte e seis pacientes (63,4% foram classificados como bem nutridos (IMC = 21,7 ± 2,0 Kg/m2, 6 (14,6% como risco nutricional (IMC = 19,4 ± 0,5 Kg/m2 e 9 (22% como desnutridos (IMC = 17,6 ± 0,8 Kg/m2. Não houve correlação significativa entre IMC e a idade (r = 0,24; P = 0,13, idade do diagnóstico (r = -0,04; P = 0,81, escore clínico (r = 0,13; P = 0,40 e radiográfico (r = -0,22; P = 0,17, distância percorrida no TC6 (r = 0,20; P = 0,20, VEF1 % (r = 0,11; P = 0,50 e CVF % (r = 0,06; P = 0,72. Também não houve associação entre o declínio do estado nutricional e essas variáveis. Conclusão: Este estudo mos-trou que a maioria dos pacientes com FC (16 anos ou mais tem um adequado estado nutricional (63,4%, mas uma porcentagem signi-ficante (36,6% tem depleção nutricional. Não houve associação entre o estado nutricional e a função pulmonar, pressões respiratórias máximas, escore clínico e radiográfico e a capacidade submáxima de exercício.

  2. Dermatoses em pacientes com AIDS: estudo de 55 casos. Uberlândia, MG, Brasil

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    GOMIDES MABEL DUARTE ALVES

    2002-01-01

    Full Text Available Foram estudados 55 pacientes com a síndrome da imunodeficiência adquirida (Aids, triados ao serviço de Dermatologia, apresentando doenças cutâneo-mucosas, algumas de caráter oportunista. As dermatoses são freqüentes nestes doentes e, às vezes, de difícil diagnóstico pelo caráter atípico das lesões. OBJETIVOS: Analisar a freqüência e apresentação clínica das dermatoses relacionadas à Aids. MÉTODOS: Cinqüenta e cinco pacientes com Aids e lesões tegumentares foram estudados, de modo transversal, no Serviço de Dermatologia da Universidade Federal de Uberlândia, de 1995 a 1997. Foram realizadas biopsias e culturas diversas para elucidação diagnóstica. RESULTADOS: Foram encontradas 116 dermatoses, com predomínio das fúngicas (78%, seguidas pelas virais (40%, eritêmato-escamosas (27%, pápulo-pruríticas (18%, causadas por drogas (10%, neoplásicas (9% e outras afecções cutâneo-mucosas (7%. A maioria dos pacientes apresentou mais de uma dermatose (67%. CONCLUSÕES: Confirmou-se a maior freqüência de dermatoses fúngicas, seguidas pelas virais, na Aids. Observou-se a necessidade de propedêutica bem elaborada para o diagnóstico preciso das dermatoses, devido à sua apresentação atípica em grande número de doentes. O exame dermatológico e a biopsia das lesões tiveram grande relevância na suspeita do diagnóstico de Aids.

  3. Caracterização dos padrões de dor, sono e alexitimia em pacientes com fibromialgia atendidos em um centro terciário brasileiro

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    Lazslo A. Avila

    2014-10-01

    Full Text Available Objetivos: Fibromialgia (FM é uma síndrome complexa, caracterizada por uma dor musculoesquelética crônica duradoura e difusa, derivada de causas não inflamatórias e classicamente associada à presença de pontos sensíveis específicos. No entanto, estudos destacaram outros sintomas importantes associados à má qualidade de vida (QDV em pacientes com FM, por exemplo, distúrbios do sono e alexitimia. Esse estudo teve por objetivo investigar os padrões de dor, sono e alexitimia de pacientes com FM em um centro terciário brasileiro. Métodos: 20 pacientes com FM acompanhados na clínica ambulatorial de reumatologia de um centro terciário brasileiro (Faculdade de Medicina de São José do Rio Preto - FAMERP, São Paulo, Brasil e 20 pacientes sem FM provenientes de outros serviços ambulatoriais da FAMERP completaram um questionário clínico e sociodemográfico, o Fibromyalgia Impact Questionnaire (FIQ, o Pittsburgh Sleep Quality Index (PSQI, o Toronto Alexithymia Scale (TAS-20 e o SF-36 (WHOQOL. Resultados: Os pacientes com FM tiveram desempenhos piores em todas as dimensões de QDV do SF-36 e escores mais altos no PSQI (P = 0,01 e no TAS-20 (P = 0,02. Pacientes com FM também tiveram escores significativamente mais altos em todos os domínios específicos do PSQI e TAS-20. Discussão: Os presentes dados concordavam com a literatura, evidenciando pior desempenho de pacientes com FM no impacto da dor, queixas de sono e maior presença de alexitimia. Conclusão: Estudos evidenciaram, além das queixas de dor, a presença de sintomas importantes e frequentemente subdiagnosticados, em pacientes com FM, como queixas relativas ao sono e alexitimia. Um conhecimento mais aprofundado desses distúrbios poderia melhorar a abordagem e o tratamento dos pacientes com FM.

  4. O efeito em curto prazo do bypass gástrico sobre pacientes obesos diabéticos

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    Aluisio Stoll

    Full Text Available OBJETIVO: estudar o efeito do bypass gástrico sobre a glicemia e o uso de medicação antidiabética em pacientes obesos portadores de diabetes. MÉTODOS: estudo de coorte retrospectivo com 44 pacientes obesos portadores de DM2, provenientes de 469 pacientes submetidos ao bypass gástrico no período de dezembro de 2001 a março de 2009. Os desfechos primários avaliados foram: glicemia em jejum e a necessidade de medicação antidiabética. RESULTADOS: a população foi composta de dez (22,7% homens e 34 (77,3% mulheres, com média de idade de 45,3 (±8,23 anos e índice de massa corporal de 40,9 (±5,03 kg/m². O tempo médio de evolução do DM2 foi 63,6 (±60,9 meses. Dos 40 pacientes que utilizavam medicação para controle do DM2, 20 (50% tiveram sua medicação suspensa na alta hospitalar e 13 (32,5% até nove meses depois. Em uma paciente não foi possível avaliar o uso de medicação, sendo essa a única exclusão. A insulina foi suspensa nos dez (100% pacientes que a utilizavam, sendo seis (60% na alta hospitalar. Houve redução (P<0,05 da glicemia em jejum, em todo o período estudado, em comparação com o valor pré-operatório, e foram atingidos valores inferiores a 100mg/dl no período de sete a nove meses. CONCLUSÃO: Pacientes obesos portadores de DM2, submetidos ao bypass gástrico, apresentaram melhora do controle glicêmico e redução do uso de hipoglicemiantes em curto prazo.

  5. Development of a one-step RT-PCR assay for detection of pancoronaviruses (α-, β-, γ-, and δ-coronaviruses) using newly designed degenerate primers for porcine and avian `fecal samples.

    Science.gov (United States)

    Hu, Hui; Jung, Kwonil; Wang, Qiuhong; Saif, Linda J; Vlasova, Anastasia N

    2018-06-01

    Coronaviruses (CoVs) are critical human and animal pathogens because of their potential to cause severe epidemics of respiratory or enteric diseases. In pigs, the newly emerged porcine deltacoronavirus (PDCoV) and re-emerged porcine epidemic diarrhea virus (PEDV) reported in the US and Asia, as well as the discovery of novel CoVs in wild bats or birds, has necessitated development of improved detection and control measures for these CoVs. Because the previous pancoronavirus (panCoV) RT-PCR established in our laboratory in 2007-2011 did not detect deltacoronaviruses (δ-CoVs) in swine fecal and serum samples, our goal was to develop a new panCoV RT-PCR assay to detect known human and animal CoVs, including δ-CoVs. In this study, we designed a new primer set to amplify a 668 bp-region within the RNA-dependent RNA polymerase (RdRP) gene that encodes the most conserved protein domain of α-, β-, γ-, and δ-CoVs. We established a one-step panCoV RT-PCR assay and standardized the assay conditions. The newly established panCoV RT-PCR assay was demonstrated to have a high sensitivity and specificity. Using a panel of 60 swine biological samples (feces, intestinal contents, and sera) characterized by PEDV, PDCoV and transmissible gastroenteritis virus-specific RT-PCR assays, we demonstrated that sensitivity and specificity of the newly established panCoV RT-PCR assay were 100%. 400 avian fecal (RNA) samples were further tested simultaneously for CoV by the new panCoV RT-PCR and a one-step RT-PCR assay with the δ-CoV nucleocapsid-specific universal primers. Four of 400 avian samples were positive for CoV, three of which were positive for δ-CoV by the conventional RT-PCR. PanCoV RT-PCR fragments for 3 of the 4 CoVs were sequenced. Phylogenetic analysis revealed the presence of one γ-CoV and two δ-CoV in the sequenced samples. The newly designed panCoV RT-PCR assay should be useful for the detection of currently known CoVs in animal biological samples. Copyright © 2018

  6. Insônia em pacientes com câncer de mama

    OpenAIRE

    Rafihi-Ferreira,Renatha El; Soares,Maria Rita Zoéga

    2012-01-01

    A insônia é uma queixa frequente em pacientes com câncer de mama, e suas consequências podem causar impacto na qualidade de vida e no curso da doença. O objetivo deste estudo foi revisar a literatura especializada sobre a relação entre insônia e câncer de mama, abordando definição, prevalência, fatores etiológicos e as consequências da insônia no contexto da neoplasia mamária, bem como os tratamentos para insônia nessa população. A busca da literatura ocorreu nos bancos de dados Scopus, Web o...

  7. Estudo transversal sobre problemas bucais em pacientes obesos mórbidos

    OpenAIRE

    Joselene Martinelli Yamashita

    2013-01-01

    A prevalência da obesidade tem aumentado em proporções alarmantes, tornando-se um problema de saúde pública. Há relatos de uma possível relação da obesidade com a condição bucal e o objetivo do presente estudo foi identificar os problemas bucais e seu impacto na qualidade de vida de indivíduos obesos mórbidos. A amostra foi composta por 100 pacientes obesos mórbidos (grupo obeso) e 50 indivíduos eutróficos (grupo controle), atendidos no Hospital das Clínicas da Faculdade de Medicina de Ribeir...

  8. Alterações neurometabílicas avaliadas pela espetroscopia de massa em pacientes com esquizofrenia

    Directory of Open Access Journals (Sweden)

    Roberta Costa Vargas

    2015-10-01

    Full Text Available Introdução: Embora sejam observados mudanças do volume cerebral e níveis alterados de metabólitos na esquizofrenia em diversos Centros de Pesquisa (1 (2 (3, poucos estudos foram realizados em pacientes sul-americanos com esquizofrenia por meio do exame espectroscopia de ressonância magnética. Objetivos: investigar de forma abrangente estruturas cerebrais em uma amostra de pacientes que estavam em uso de antipsicóticos comparando-os ao grupo controle, formado por indivíduos saudáveis. Metodologia: foram comparados dois grupos de dez pacientes cada, sendo o primeiro com o diagnóstico de esquizofrenia e o segundo com indivíduos normais. Foram medidos metabólitos da região hipocampal e do núcleo caudado por meio de Ressonância Magnética de Espectroscopia de prótons em um único modelo voxel, utilizando uma aquisição automática da máquina. Os pacientes foram previamente entrevistados sob questionário SCID baseados nos critérios do DSM IV (4 e apenas os pacientes com diagnóstico de esquizofrenia foram incluídos. Eles estavam sob uso de diferentes antipsicóticos atípicos e foram estabilizados pelo menos durante 3 meses. Análises de variância (ANOVA foi conduzido para analisar os dados demográficos e análises de co-variância (ANCOVA foram realizados com nível metabólico como covariável para analisar os níveis de metabólitos no córtex frontal ventral lateral. Resultados: Nenhuma diferença foi encontrada em pacientes em comparação com os indivíduos saudáveis em termos de níveis de metabólitos creatina, fosfocreatina e NAA no hipocampo e núcleo caudado.

  9. Mecanismos e fatores associados aos sintomas gastrointestinais em pacientes com diabetes melito

    Directory of Open Access Journals (Sweden)

    Mônica Loureiro Celino Rodrigues

    2012-02-01

    Full Text Available OBJETIVOS: Apresentar a frequência, os principais fatores causadores dos sintomas gastrointestinais em pacientes portadores do diabetes melito e controvérsias quanto à sua ocorrência em crianças e adolescentes. FONTES DOS DADOS: Revisão não sistemática nas bases de dados MEDLINE/PubMed e SciELO (1983-2011, além de capítulos de livros relevantes. Foram selecionados os artigos mais atuais e representativos do tema. SÍNTESE DOS DADOS: A prevalência do diabetes melito vem aumentando ao longo dos anos em vários países do mundo. No sistema digestório, é conhecida a ocorrência de complicações do diabetes melito, entre elas os sintomas gastrointestinais (náuseas, vômitos, dor abdominal, azia, disfagia, constipação, diarreia e incontinência fecal. A patogênese das alterações das funções gastrointestinais no diabetes melito está ainda sob investigação, e o papel do sistema nervoso entérico e seus neurotransmissores tem ganhado significância. Em decorrência do comprometimento do sistema digestório, com danos ao sistema nervoso entérico, portadores do diabetes melito podem apresentar quadros específicos de distúrbios de motilidade, alguns de grande relevância clínica, como gastroparesia diabética, constipação e diarreia. A disfunção deste sistema contribui para aumentar a morbidade desta doença e piora a qualidade de vida de seus portadores. CONCLUSÕES: O diabetes melito, ao longo dos anos, afeta o sistema digestório. Por ser uma condição que piora a qualidade de vida dos portadores e também pode indicar complicação da doença, deve ser valorizada no acompanhamento e tratamento do paciente com diabetes melito. Na infância e na adolescência, ainda existem poucos estudos que abordam o problema.

  10. Rastreio cognitivo em pacientes com acidente vascular cerebral: um estudo transversal

    Directory of Open Access Journals (Sweden)

    Ana Amália Torres Souza Gandour Dantas

    2014-07-01

    Full Text Available Objetivo O estudo objetivou-se a fazer um rastreio cognitivo nos pacientes com acidente vascular cerebral (AVC, a fim de determinar pontos de corte de acordo com a idade, escolaridade e grau de comprometimento neurológico. Métodos Foi realizado um estudo transversal no qual participaram 109 pacientes ambulatoriais, sendo 61 homens, com média da idade de 59 anos (± 11, média do tempo de escolaridade de 5 anos (± 4 e do tempo de sequela de 16 meses (± 14. Os pacientes foram avaliados por meio do Miniexame do Estado Mental (MEEM e pela National Institute of Health Stroke Scale. Os dados foram analisados pela regressão linear múltipla (stepwise forward. Resultados Verificou-se que as variáveis grau de comprometimento neurológico, idade e escolaridade contribuíram significativamente para o valor global do MEEM e explicaram a variância do estado cognitivo (R2 ajustado = 0,24. Cada aumento do comprometimento neurológico representou diminuição de 0,456 no escore do MEEM. Quanto maior a idade, ocorreu uma diminuição de 0,202 no MEEM, e à medida que diminui o tempo de escolaridade, houve uma diminuição de 0,190 no MEEM. Os pontos de corte variaram de 14 a 22 de acordo com o grau de comprometimento neurológico, idade e escolaridade. Conclusão Os resultados apontaram que, por meio do rastreio positivo de déficit cognitivo, foram encontrados pontos de corte associados ao comprometimento neurológico, necessitando também serem ajustados pela idade e escolaridade, sugerindo que essas associações sejam preferencialmente levadas em consideração na planificação da reabilitação neuropsicológica dos pacientes com AVC.

  11. Pandemic preparedness in Hawaii: a multicenter verification of real-time RT-PCR for the direct detection of influenza virus types A and B.

    Science.gov (United States)

    Whelen, A Christian; Bankowski, Matthew J; Furuya, Glenn; Honda, Stacey; Ueki, Robert; Chan, Amelia; Higa, Karen; Kumashiro, Diane; Moore, Nathaniel; Lee, Roland; Koyamatsu, Terrie; Effler, Paul V

    2010-01-01

    We integrated multicenter, real-time (RTi) reverse transcription polymerase chain reaction (RT-PCR) screening into a statewide laboratory algorithm for influenza surveillance and response. Each of three sites developed its own testing strategy and was challenged with one randomized and blinded panel of 50 specimens previously tested for respiratory viruses. Following testing, each participating laboratory reported its results to the Hawaii State Department of Health, State Laboratories Division for evaluation and possible discrepant analysis. Two of three laboratories reported a 100% sensitivity and specificity, resulting in a 100% positive predictive value and a 100% negative predictive value (NPV) for influenza type A. The third laboratory showed a 71% sensitivity for influenza type A (83% NPV) with 100% specificity. All three laboratories were 100% sensitive and specific for the detection of influenza type B. Discrepant analysis indicated that the lack of sensitivity experienced by the third laboratory may have been due to the analyte-specific reagent probe used by that laboratory. Use of a newer version of the product with a secondary panel of 20 specimens resulted in a sensitivity and specificity of 100%. All three laboratories successfully verified their ability to conduct clinical testing for influenza using diverse nucleic acid extraction and RTi RT-PCR platforms. Successful completion of the verification by all collaborating laboratories paved the way for the integration of those facilities into a statewide laboratory algorithm for influenza surveillance and response.

  12. Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland.

    LENUS (Irish Health Repository)

    Menton, John F

    2007-01-01

    BACKGROUND: Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals. RESULTS: A real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII\\/4 variant of NoV. CONCLUSION: The combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII\\/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection.

  13. Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

    Science.gov (United States)

    Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

    2016-03-01

    HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses

    DEFF Research Database (Denmark)

    Schultz, Anna Charlotte; Vega, Everado; Dalsgaard, Anders

    2011-01-01

    BackgroundCurrent detection and genotyping methods of genogroup (G) I and II noroviruses (NoVs) consist of a 2-step approach including detection of viral RNA by TaqMan realtime RT-PCR (RT-qPCR) followed by conventional RT-PCR and sequencing of partial regions of ORF1 or ORF2. ObjectiveTo develop ......Man RT-qPCR assays for the sensitive detection and direct genotyping of GI and GII NoVs from clinical and environmental matrices...... novel long-template one-step TaqMan assays (L-RT-qPCR) for the rapid detection and direct genotyping of GI and GII NoVs and to evaluate the sensitivity and specificity of the assays. Study designGI and GII-specific broadly reactive L-RT-qPCR assays were developed by combining existing NoV primers...... and probes targeting the open reading frame (ORF)1–ORF2 junction as well as region C at the 5′–ORF2. The assays were validated using GI and GII RNA transcripts and a coded panel of 75 stool samples containing NoV strains representing 9 GI genotypes and 12 GII genotypes, as well as sapoviruses, astroviruses...

  15. Studies on Parameters Influencing the Performance of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR in Detecting Prunus Necrotic Ringpot Virus (PNRSV

    Directory of Open Access Journals (Sweden)

    M. Usta

    2005-08-01

    Full Text Available In order to have a more detailed understanding of the various factors influencing a reverse transcriptase polymerase chain reaction (RT-PCR, a number of important parameters such as Mg+2, primer, enzyme concentration and others were optimized for the detection of Prunus necrotic ringspot virus (PNRSV. Using a PNRSV isolate with a pair of primers, complementary DNA of viral genome as template, and an appropriate enzyme together with magnesium chloride, the following optimal conditions were identified: primer concentration between 0.2 and 0.0002 pmol µl-1 and 0.06–2 units µl-1 for Taq DNA polymerase enzyme for a 50 µl reaction volume when other parameters were optimum; magnesium chloride concentration less than 2.5 mM; dNTP concentration between 1 and 10 mM. The optimum cDNA amount should be ~360 ng for a 50 µl reaction mixture. When these optimized concentrations and/or values of the main PCR parameters were brought together for a new RT-PCR, a clear and a reliable PNRSV detection having no background was performed from both growth-chamber and field-grown PNRSV-infected plants.

  16. Selection and Verification of Candidate Reference Genes for Mature MicroRNA Expression by Quantitative RT-PCR in the Tea Plant (Camellia sinensis

    Directory of Open Access Journals (Sweden)

    Hui Song

    2016-05-01

    Full Text Available Quantitative reverse transcription-polymerase chain reaction (qRT-PCR is a rapid and sensitive method for analyzing microRNA (miRNA expression. However, accurate qRT-PCR results depend on the selection of reliable reference genes as internal positive controls. To date, few studies have identified reliable reference genes for differential expression analysis of miRNAs among tissues, and among experimental conditions in plants. In this study, three miRNAs and four non-coding small RNAs (ncRNA were selected as reference candidates, and the stability of their expression was evaluated among different tissues and under different experimental conditions in the tea plant (Camellia sinensis using the geNorm and NormFinder programs. It was shown that miR159a was the best single reference gene in the bud to the fifth leaf, 5S rRNA was the most suitable gene in different organs, miR6149 was the most stable gene when the leaves were attacked by Ectropis oblique and U4, miR5368n and miR159a were the best genes when the leaves were treated by methyl jasmonate (MeJA, salicylic acid (SA and abscisic acid (ABA, respectively. Our results provide suitable reference genes for future investigations on miRNA functions in tea plants.

  17. A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

    Science.gov (United States)

    Lopez-Jimena, B; Cherif, N; Garcia-Rosado, E; Infante, C; Cano, I; Castro, D; Hammami, S; Borrego, J J; Alonso, M C

    2010-10-01

    To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  18. Are PEI-coated SWCNTs conjugated with hepatitis A virus? A chemical study with SEM, Z-potential, EDXD and RT-PCR

    International Nuclear Information System (INIS)

    Carbone, M; Valentini, F; Palleschi, G; Caminiti, R; Petrinca, A R; Donia, D; Divizia, M

    2010-01-01

    The conjugation between nanotubes, coated with different doses of polyethylene imine (PEI) and hepatitis A virus (HAV) was investigated by scanning electron microscopy, Z-potential, thermogravimetric and differential thermal analysis, transmission electron microscopy, energy dispersive x-ray diffraction (EDXD) and reverse transcript polymerase chain reaction (RT-PCR). For the first time, to our knowledge, evidence is obtained that conjugation between the nanotubes and the HAV occurs and that it has an (at least a partial) electrostatic character. Since all components of the conjugated systems, nanotubes, coating material and virus are characterized by different peak shapes in the selected q range, it was possible to infer that conjugation occurred. RT-PCR measurements confirmed that the conjugation of the coated nanotubes and HAV occurred and the result was stable. This opens up the prospect of probing the coated nanotubes as intra-cellular carriers in transfection processes of the virus. Further biological applications will concern a possible vaccine especially for non-replicative viruses.

  19. Cross-platform evaluation of commercial real-time SYBR green RT-PCR kits for sensitive and rapid detection of European bat Lyssavirus type 1.

    Science.gov (United States)

    Picard-Meyer, Evelyne; Peytavin de Garam, Carine; Schereffer, Jean Luc; Marchal, Clotilde; Robardet, Emmanuelle; Cliquet, Florence

    2015-01-01

    This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R (2) values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

  20. Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1

    Directory of Open Access Journals (Sweden)

    Evelyne Picard-Meyer

    2015-01-01

    Full Text Available This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

  1. Survey of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus incidence in Korea by Duplex RT-PCR

    Directory of Open Access Journals (Sweden)

    Seung-Yeol Lee

    2014-12-01

    Full Text Available The incidence of Cherry necrotic rusty mottle virus (CNRMV and Cherry green ring mottle virus (CGRMV have recently been occurred in Korea, posing a problem for sweet cherry cultivation. Since infected trees have symptomless leaves or ring-like spots on the pericarp, it is difficult to identify a viral infection. In this study, the incidence of CNRMV and CGRMV in sweet cherry in Gyeongbuk province was surveyed using a newly developed duplex reverse transcriptase polymerase chain reaction (RT-PCR method that can detect both viruses in a single reaction. CNRMV and CGRMV co-infection rates were 29.6%, 53.6%, and 17.6%, respectively, in samples collected from three different sites (Daegu, Gyeongju and Gyeongsan in Gyeongbuk province during 2012 and 2013. This duplex RT-PCR method offers a simple, rapid, and effective way of identifying CNRMV and CGRMV simultaneously in sweet cherry trees, which can aid in the management of viral infections that could undermine yield.

  2. Anestesia para cesariana em paciente com síndrome de Guillain-Barré: relato de caso

    Directory of Open Access Journals (Sweden)

    Daniel Volquind

    2013-08-01

    Full Text Available JUSTIFICATIVA E OBJETIVOS: A Síndrome de Guillain-Barré durante a gestação é considerada uma CIRURGIA, Cesárea; complicação neurológica rara e o manejo anestésico para a cesariana nessas pacientes ainda não é consenso na literatura. O objetivo deste artigo é relatar o caso de uma paciente gestante portadora da Síndrome de Guillain-Barré submetida à cesariana. RELATO DO CASO: Paciente feminina, 22 anos, com 35 semanas e cinco dias de idade gestacional, da celularidade. A técnica anestésica empregada foi a anestesia geral, induzida com propofol 1,5 mg.kg-1 e mantida com sevofiurano 2% em oxigênio e fentanil 3 µg.kg-1. O procedimento transcorreu sem complicações, tanto para a gestante quanto para o concepto. A paciente obteve alta no décimo dia de internação, após melhora progressiva do quadro neurológico. CONCLUSÕES: A técnica anestésica a ser empregada em gestantes portadoras da Síndrome de Guillain-Barré que necessitam fazer cesariana permanece como escolha do anestesiologista, que deve ser guiado pelo quadro clínico e pelas comorbidades de cada paciente.

  3. Dengue Virus NS1 Protein as a Diagnostic Marker: Commercially Available ELISA and Comparison to qRT-PCR and Serological Diagnostic Assays Currently Used by the State of Florida

    Directory of Open Access Journals (Sweden)

    Jason H. Ambrose

    2017-01-01

    Full Text Available Background. The proper management of patients infected with dengue virus requires early detection. Here, real-time molecular assays have proven useful but have limitations, whereas ELISAs that detect antibodies are still favored but results are obtained too late to be of clinical value. The production of DENV NS1 peaks early during infection and its detection can combine the advantages of both diagnostic approaches. Methods. This study compared assays currently used for detecting DENV infection at the Florida Department of Health including anti-DENV IgM and IgG ELISAs as well as qRT-PCR, against a commercially available DENV NS1 ELISA. These comparisons were made among a group of 21 human sera. Results. Nine of 14 (64.3% DENV qRT-PCR+ samples were also DENV NS1+. Interestingly, the 5 NS1− samples that were qRT-PCR+ were additionally IgM− and IgG+ suggesting a nonprimary infection. Compared to qRT-PCR, the NS1 assay had a sensitivity of 64.3%, specificity 100%, PPV of 100%, and NPV of 58.3%. Conclusions. The NS1 ELISA performed as expected in known DENV qRT-PCR+ samples; however negative NS1 results for qRT-PCR+ and IgG+ sera seemingly reduced the usefulness of the NS1 ELISA for nonprimary cases. We therefore conclude that diagnosis obtained via DENV NS1 ELISA deserves further investigation.

  4. Exercício físico em pacientes dialisados Physical exercise in dialyzed patients

    Directory of Open Access Journals (Sweden)

    Maycon de Moura Reboredo

    2007-12-01

    Full Text Available Pacientes portadores de doença renal crônica (DRC submetidos a tratamento dialítico apresentam alterações físicas e psicológicas que predispõem ao sedentarismo. Nesta população, a prescrição rotineira de exercícios físicos não é uma prática freqüente, especialmente no nosso país. No entanto, alguns autores têm demonstrado que um programa de exercícios para estes pacientes contribui para o melhor controle da hipertensão arterial, da capacidade funcional, da função cardíaca, da força muscular e, conseqüentemente, da qualidade de vida. Além dos benefícios relacionados ao sistema cardiovascular, a realização do exercício traz benefícios secundários, pois quebra a monotonia do procedimento, melhora aderência e pode aumentar a eficácia da diálise. Na presente revisão, os autores discutem aspectos da realização de exercícios físicos em pacientes portadores de DRC em diálise e apresentam dados iniciais de sua experiência com a aplicação de exercícios supervisionados durante as sessões de hemodiálise.Patients with chronic kidney disease (CKD on dialysis present physical and psychological limitations that induce to a sedentary life stile. The prescription of exercise for this population has not been common, especially in our country. In the last few years though, some authors have shown that exercising for these patients improves hypertension control, cardiac function, muscular strength, functional capacity and, consequently, their quality of life. Besides the benefits related to the cardiovascular system, exercise training during hemodialysis reduces the monotony of the procedure, improves adherence and contributes to the increase of dialysis efficacy. In this review, the authors discuss some aspects of exercise training in patients with CKD on dialysis and present their preliminary data of supervised exercises during hemodialysis sessions.

  5. Transtorno de estresse pós-traumático em pacientes de unidade de terapia intensiva

    OpenAIRE

    Caiuby,Andrea Vannini Santesso; Andreoli,Paola Bruno de Araújo; Andreoli,Sergio Baxter

    2010-01-01

    O transtorno de estresse pós-traumático tem sido descrito em pacientes após tratamento em unidade de terapia intensiva. O objetivo foi revisar estudos sobre os aspectos psicológicos e as intervenções terapêuticas destes pacientes após internação em unidade de terapia intensiva. Trinta e oito artigos foram incluídos. A prevalência de transtorno de estresse pós-traumático variou de 17% a 30% e a incidência de 14% a 24%. Os fatores de risco foram: história prévia de ansiedade, depressão ou pânic...

  6. Tratamento farmacológico e interações medicamentosas em pacientes com aneurisma da aorta abdominal

    Directory of Open Access Journals (Sweden)

    Heverton Alves Peres

    2015-01-01

    Full Text Available O aneurisma da aorta abdominal (AAA ocorre quando há uma dilatação anormal e irreversível da artéria superior a 50% do calibre esperado para o vaso e associa-se a uma mortalidade de 80 a 90%. O controle dos principais fatores de risco como a hipertensão, dislipidemia, tabagismo e doenças trombóticas em aneurismas pequenos é feito por vários medicamentos que evitam o desenvolvimento e ruptura do aneurisma, no entanto, o uso destes associados a outros medicamentos pode desencadear interações medicamentosas relevantes sendo crucial o conhecimento sobre estas. Atualmente, há poucos dados na literatura sobre o tratamento farmacológico e interações medicamentosas em pacientes com AAA, sendo o objetivo desta revisão, descrever a farmacoterapia e interações medicamentosas em pacientes com AAA.

  7. Balanços metabólicos do enxofre em pacientes com leucodistrofia metacromática

    Directory of Open Access Journals (Sweden)

    Horacio M. Canelas

    1968-12-01

    Full Text Available Foram estudados os balanços metabólicos do enxofre em dois pacientes com a forma juvenil da leucodistrofia metacromática. Foi verificado, em ambos os casos, um balanço positivo desse metaloide, aparentemente não influenciado pelo tipo de dieta (geral ou vegetal. Este resultado está de acordo com a atual concepção patogênica dessa moléstia, que consiste, essencialmente, em uma sulfatidose com diminuição da atividade das sulfatases.

  8. Ação terapêutica do mebendazole (R 17635 em pacientes poliparasitados: resultados finais

    Directory of Open Access Journals (Sweden)

    Dirceu Wagner Carvalho de Souza

    1973-08-01

    Full Text Available O mebendazole (R 17635 foi testado no tratamento de pacientes, de ambos os sexos, portadores de helmintíases mistas; o grupo selecionado situava-se na faixa etária de 4 a 14 anos, constituindo-se de 140 pacientes necessariamente residentes em comunidades restritas. Cerca de 70% dos pacientes estavam infetados por pelo menos 3 helmintos (os demais pela associação de dois, dentre ancilostomídeos, Ascaris lumbricoides, Enterobius vermicularis, Taenia sp e Trichuris trichiura. O mebendazóle (R 17635 foi administrado em comprimidos de 100 mg, um 30 minutos antes do desjejum e outro 3 horas após o jantar, por 3 dias consecutivos, independentemente do peso corporal. Não foram observadas quaisquer evidências de reações indesejáveis imediatas ou tardias, que pudessem ser atribuídas à droga. O controle de cura foi efetuado mediante as técnicas de Willis e de Hoffman-Pons & Janer, em exames coprológicos realizados 7, 14 e 21 dias contados a partir do último dia do tratamento; nos portadores de teníase e oxiuríase procedeu-se, também, ao método do "anal-swab" durante 7 dias consecutivos, a partir do sétimo dia após o tratamento. Percentual de 100% de cura foi registrado para oxiuríase, tendo sido de 98% na ascaridíase e na triquiuríase e de 94.5% na ancilostomíase. De 9 pacientes com teníase, 8 apresentaram negativação dos exames; entretanto, os Autores insistem na necessidade de maiores estudos a este respeito, quanto aos aspectos técnico e estatístico. Não obstante, consideram demonstrada a real eficácia do mebendazole (R 17635 como droga anti-helmíntica polivalente.Mebendazole (R 17635 was tested in the treatment of mixed helminthiases in 140 male and female patients aged 4 to 14 years. These patients were necessarily treated by physicians visiting their restricted communities. Approximately 70% of the patients were infected by at least three, and the rest by only two, of the following helminths: hookworm, Ascaris

  9. Comparacao dos criterios RIFLE, AKIN e KDIGO quanto a capacidade de predicao de mortalidade em pacientes graves

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    Talita Machado Levi

    2013-12-01

    Full Text Available Objetivo: A lesão renal aguda é uma complicação comum em pacientes gravemente enfermos, sendo os critérios RIFLE, AKIN e KDIGO utilizados para sua classificação. Esse trabalho teve como objetivo a comparação dos critérios citados quanto à capacidade de predição de mortalidade em pacientes gravemente enfermos. Métodos: Estudo de coorte prospectiva, utilizando como fonte de dados prontuários médicos. Foram incluídos todos os pacientes admitidos na unidade de terapia intensiva. Os critérios de exclusão foram tempo de internamento menor que 24 horas e doença renal crônica dialítica. Os pacientes foram acompanhados até a alta ou óbito Para análise dos dados, foram utilizados os testes t de Student, qui-quadrado, regressão logística multivariada e curva ROC. Resultados: A média de idade foi de 64 anos, com mulheres e afrodescendentes representando maioria. Segundo o RIFLE, a taxa de mortalidade foi de 17,74%, 22,58%, 24,19% e 35,48% para pacientes sem lesão renal aguda e em estágios Risk, Injury e Failure, respectivamente. Quanto ao AKIN, a taxa de mortalidade foi de 17,74%, 29,03%, 12,90% e 40,32% para pacientes sem lesão renal aguda, estágio I, estágio II e estágio III, respectivamente. Considerando o KDIGO 2012, a taxa de mortalidade foi de 17,74%, 29,03%, 11,29% e 41,94% para pacientes sem lesão renal aguda, estágio I, estágio II e estágio III, respectivamente. As três classificações apresentaram resultados de curvas ROC para mortalidade semelhantes. Conclusão: Os critérios RIFLE, AKIN e KDIGO apresentaram-se como boas ferramentas para predição de mortalidade em pacientes graves, não havendo diferença relevante entre os mesmos.

  10. Fatores preditivos da disfunção sexual em pacientes com diagnóstico de epilepsia

    OpenAIRE

    Vieira, Fábio Cavalcanti de Faria

    2015-01-01

    Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Ciências Médicas, Florianópolis, 2015. A disfunção erétil é relatada com frequência por pacientes com epilepsia e sua etiologia provavelmente é multifatorial, incluindo anormalidades do sistema endocrinológico, efeitos colaterais de fármacos antiepilépticos, comorbidades psiquiátricas e dificuldades familiares e sociais do paciente. Neste trabalho avaliamos a função er...

  11. Impacto da hipertensão arterial no remodelamento ventricular, em pacientes com estenose aórtica

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    João Carlos Hueb

    2011-09-01

    Full Text Available FUNDAMENTO: A hipertrofia ventricular esquerda (HVE é comum em pacientes com hipertensão arterial sistêmica (HAS e estenose aórtica (EAo e, com certa frequência, encontramos associação entre estas patologias. Mas, em tal situação, não está clara a importância de cada uma na HVE. OBJETIVO: 1 - Avaliar em pacientes portadores de EAo, submetidos previamente a estudo ecocardiográfico, a magnitude da HVE, nos casos de EAo isolada e associada à HAS; 2 - Avaliar o padrão de remodelamento geométrico nas duas situações. MÉTODOS: Estudo retrospectivo, observacional e transversal, incluindo 298 pacientes consecutivos, com EAo ao ecocardiograma. HVE foi considerada para massa miocárdica > 224g em homens e > 162g em mulheres. Os pacientes foram classificados como portadores de EAo leve (gradiente máximo 50,0 mmHg, além disso, foram separados em dois subgrupos: com e sem HAS. RESULTADOS: Nos três níveis de lesão aórtica, a massa ventricular esquerda foi maior na EAo associada à HAS do que na EAo isolada (EAo leve: 172 ± 45 vs 223 ± 73g, p < 0,0001; EAo moderada: 189 ± 77 vs 245 ± 81g, p = 0,0313; EAo grave: 200 ± 62 vs 252 ± 88g, p = 0,0372. Presença de HAS esteve associada a maior risco de HVE (OR = 2,1,IC95%:1,2-3,6; p = 0,012. Pacientes com EAo grave e HAS apresentaram predomínio de hipertrofia concêntrica, quando comparados com aqueles normotensos (p = 0,013. CONCLUSÃO: Em pacientes com EAo, a presença de HAS foi um fator adicional de aumento da massa ventricular esquerda, interferindo também na geometria ventricular.

  12. NGAL urinária em pacientes sem e com lesão renal aguda em unidade de terapia intensiva

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    Mirian Watanabe

    2014-12-01

    Full Text Available Objetivo: Avaliar a eficácia diagnóstica e prognóstica da lipocalina associada à gelatinase neutrofílica urinária em pacientes de unidade de terapia intensiva. Métodos: Estudo do tipo coorte, prospectivo, longitudinal desenvolvido em uma unidade de terapia intensiva clínica especializada em cardiologia. Os pacientes foram estratificados segundo os grupos sem e com lesão renal aguda, acompanhados a partir das primeiras 24 horas de internação até a alta hospitalar ou óbito. A creatinina sérica, o fluxo urinário e a lipocalina associada à gelatinase neutrofílica urinária foram coletadas em dois períodos: 24 horas e 48 horas de admissão. Resultados: Foram avaliados 83 pacientes clínicos da unidade de terapia intensiva, com predomínio do gênero masculino (57,8%. Os pacientes foram agrupados em sem lesão renal aguda (N=18, com lesão renal aguda (N=28 ou com lesão renal aguda grave (N=37. Entre os pacientes com lesão renal aguda e lesão renal aguda grave, foram prevalentes os portadores de doenças crônicas, em uso de ventilação mecânica e em terapia de substituição renal, além daqueles com maiores taxas de permanência na unidade de terapia intensiva e hospitalar, e maior mortalidade. O grupo com lesão renal aguda não apresentou alteração significativa da creatinina sérica nas primeiras 24 horas na unidade de terapia intensiva, apesar dos níveis elevados de lipocalina associada à gelatinase neutrofilica urinária demonstrados nos grupos com lesão renal aguda e lesão renal aguda grave (p<0,001. Níveis elevados de lipocalina associada à gelatinase neutrofílica urinária na amostra foram associados ao óbito. Conclusão: A elevação nos níveis de lipocalina associada à gelatinase neutrofílica urinária antecede as variações da creatinina sérica em pacientes com lesão renal aguda e pode ser associada ao óbito.

  13. Prevalência de hepatite C em pacientes com lúpus eritematoso sistêmico do HUCCF-UFRJ

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    COSTA CLÁUDIA DE ABREU

    2002-01-01

    Full Text Available Os pacientes com lúpus eritematoso sistêmico (LES apresentam fatores de risco para a aquisição de hepatite C, como hospitalizações e hemotransfusões, e compartilham com os pacientes infectados pelo vírus da hepatite C (HCV várias manifestações clínico-laboratoriais relacionadas a auto-imunidade. OBJETIVOS: O objetivo do presente estudo é determinar a prevalência de hepatite C em uma população com LES acompanhada no ambulatório de programas especiais - Colagenoses - do Hospital Universitário Clementino Fraga Filho (HUCFF-UFRJ. MÉTODOS: Foi realizado um estudo de corte-transversal, incluindo os primeiros 91 pacientes com o diagnóstico de LES provável ou definitivo procedentes do referido ambulatório, no período de abril a setembro de 1997. Todos os doentes foram avaliados, sob o ponto de vista clínico e laboratorial, para determinação de atividade de doença lúpica. Foram realizados o teste de triagem UBI HCV EIA 4.0 e o teste suplementar INNO-LIA HCV AbIII nos 91 pacientes selecionados e , em 32 pacientes escolhidos ao acaso e nos pacientes reativos aos testes imunossorológicos foi realizada a pesquisa do RNA-HCV pelo método da reação de polimerização em cadeia (PCR. RESULTADOS: Observou-se um aumento significativo da prevalência de anti-HCV na amostra, quando comparada à população de doadores de sangue do HUCFF (6,6% versus 1,39% com o teste EIA, valor p=0,02 e intervalo de confiança 95%=5,5 a 13,8, contudo , considerando-se resultados obtidos com os testes confirmatórios imunoblot e PCR aplicados em série ( prevalência de HCV de 2,2%, com intervalo de confiança 95%=0,2 a 7,7 , a prevalência de HCV em pacientes com LES não parece mais alta que em doadores de sangue. CONCLUSÕES: A prevalência de hepatite C na amostra estudada utilizando-se um teste de triagem (EIA foi significativamente maior do que a de uma população de doadores de sangue (6,6% versus 1,39%. Porém, aplicando-se em série os testes

  14. Molecular characterization of two rocio flavivirus strains isolated during the encephalitis epidemic in são paulo state, brazil and the development of a one-step rt-pcr assay for diagnosis Caracterização molecular de duas cepas do flavivírus Rocio, isoladas durante a epidemia de encefalite no Estado de São Paulo, Brasil e desenvolvimento do teste one-step RT-PCR para diagnóstico

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    Terezinha Lisieux Moraes Coimbra

    2008-04-01

    Full Text Available Rocio virus (ROCV was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC viruses, particularly with Ilheus (ILHV, St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007.O vírus Rocio (ROCV foi responsável por uma explosiva epidemia de encefalite que ocorreu nos anos 70 afetando cerca de 1.000 habitantes de 20 municípios litorâneos do Estado de São Paulo, Brasil. ROCV foi isolado em 1975 de cerebelo de caso humano fatal de encefalite. As manifestações clínicas da doença são semelhantes àquelas descritas para encefalite St. Louis. ROCV apresenta intensa reatividade cruzada com os vírus do Complexo da Encefalite Japonesa (JEV, particularmente com o vírus Ilhéus (ILHV e com os vírus das encefalites St. Louis, Murray Valley e West Nile. Neste estudo, relatamos o desenvolvimento de um teste de RT-PCR específico para diagnóstico de ROCV e a caracterização molecular das cepas SPAn37630 e SPH37623. Foi realizada a análise filogenética das seqüências parciais dos genes NS5 e E, de ambas as cepas. Os resultados indicaram que essas cepas são intimamente relacionadas ao complexo JEV, mas formando um subgrupo com o

  15. Avaliação da reação de mitsuda em pacientes virchovianos inativos antes e após imunoterapia

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    Maria Sueli Parreira de Arruda

    1995-09-01

    Full Text Available Neste estudo investigou-se o potencial imunomodulador do levamisole e da mistura BCG/Mycobacterium leprae em pacientes virchovianos inativos, utilizando como parâmetro a reação de Mitsuda. Vinte pacientes, classificados como Mitsuda histologicamente negativos há 10 anos, foram divididos em três grupos: cinco pacientes que foram somente reavaliados frente a mitsudina: oito pacientes que receberam levamisole e, sete que receberam a mistura de BCG vivo mais M. leprae morto. Os resultados mostraram que: 1 o levamisole não alterou a reatividade à mitsudina em nenhum dos casos estudados; 2 as modificações da reatividade verificadas com o uso da mistura (tres casos ou aquelas que ocorreram espontaneamente (tres casos foram sempre de pequena amplitude e refletiram variações próprias de pacientes com algum grau de resistência ao Mycobacterium leprae.

  16. Níveis de zinco sérico em pacientes internados com depressão

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    Camila Saueressig

    Full Text Available RESUMO Objetivo Comparar pacientes internados com depressão e com transtorno de humor bipolar em episódio depressivo quanto aos níveis séricos de zinco. Métodos Foram incluídos 46 pacientes com idade igual ou superior a 19 anos, de ambos os sexos, internados em Unidade de Internação Psiquiátrica de um hospital universitário do sul do Brasil. Os participantes foram divididos em dois grupos: Grupo Depressão (Grupo D e Grupo com Transtorno de Humor Bipolar em episódio depressivo (Grupo THB. A análise do zinco sérico foi realizada por meio de espectrofotometria de absorção atômica. Como valores de referência para normalidade, foram adotados níveis de zinco sérico acima de 59 µg/dL para mulheres e acima de 61 µg/dL para homens. Resultados Os níveis de zinco sérico estavam dentro do padrão de normalidade em 95,7% dos pacientes. A mediana de zinco no Grupo D foi de 88,5 µg/dL e de 81,5 µg/dL no Grupo THB, porém essa diferença não foi estatisticamente significativa. O Grupo THB apresentou valores maiores de índice de massa corporal (IMC, LDL colesterol e mais internações psiquiátricas prévias. Conclusão Os resultados encontrados no presente estudo mostram que os níveis de zinco sérico em pacientes em uso de antidepressivos e outras medicações psiquiátricas, internados por THB em episódio depressivo, quando comparados a pacientes com depressão, não diferiram e estavam dentro da faixa de normalidade. O uso dessas medicações pode ter influência nas concentrações séricas do mineral.

  17. Treinamento muscular respiratório em pacientes em desmame da ventilação mecânica

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    Fernanda dos Santos Pascotini

    2014-04-01

    Full Text Available Introdução: A fraqueza da musculatura respiratória é uma das principais causas da dificuldade e/ou insucesso no desmame. Para minimizar os efeitos da ventilação mecânica (VM prolongada, os fisioterapeutas utilizam o treinamento muscular respiratório, sendo o Threshold IMT® o método mais utilizado. Objetivo: Avaliar a eficácia do treinamento muscular respiratório com o uso do aparelho Threshold IMT®, sobre parâmetros respiratórios de pacientes em desmame da VM. Métodos: Os pacientes foram distribuídos aleatoriamente em grupo controle e grupo experimental (GI e GII. Foram avaliados no primeiro dia do início do desmame quanto à força muscular respiratória: Pressão inspiratória máxima/Pressão Expiratória Máxima (PImáx/PEmáx, volume corrente (VC, frequência respiratória (FR e cardíaca (FC. Diariamente, durante sete dias, o GI recebeu três sessões de fisioterapia convencional e o GII realizou, adicionalmente, treinamento muscular respiratório (TMR com o Threshold IMT®, uma vez ao dia, no período da tarde, conectado à traqueostomia, sendo três séries de dez repetições com carga de 20% da PImáx. Os dados foram tratados estatisticamente, adotando-se o nível de significância α=0,05. Resultados: Observou-se aumento (p=0,02 na FR e redução da PImáx (p=0,04 no GI, demonstrando aumento do trabalho respiratório e perda de força muscular entre o primeiro e sétimo dia de desmame. No GII, as variáveis não sofreram alterações significativas, observando-se a manutenção da função respiratória. Conclusão: Sendo assim, o TMR foi benéfico, garantindo a manutenção dos parâmetros respiratórios, podendo ser um aliado para o desmame.

  18. Manejo nutricional em paciente cirúrgico com adenocarcinoma de pâncreas: Um relato de caso

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    Caroline Otoni da Silva

    2018-03-01

    Full Text Available O objetivo desse relato de caso é demonstrar como o manejo nutricional pode ser realizado em pacientes idosos com diagnóstico de adenocarcinoma de pâncreas e submetidos a cirurgia eletiva. Resultados: A dieta proposta supria a necessidade energética e calórica da paciente, o objetivo de recuperação do estado nutricional e preparo da paciente para o procedimento cirúrgico foi atingido, tendo em vista o prazo em que ocorreram os fatos. Foi ofertado o suplemento com imunonutrientes por um período de 12 dias no pré-operatório, sendo continuada no pós-operatório, conforme recomendado pelo DITEN, 2011. Nota se através desse relato a importância do uso das ferramentas de triagem e diagnóstico nutricional, para detecção precoce dos indivíduos desnutridos ou em risco nutricional, otimizando assim o planejamento para a oferta de calorias e nutrientes específicos a fim de melhorar o estado nutricional e prepara-los para a cirurgia. Descritores: Terapia nutricional; Neoplasias pancreáticas; Dietoterapia.

  19. Rapid diagnosis of avian influenza virus in wild birds: Use of a portable rRT-PCR and freeze-dried reagents in the field

    Science.gov (United States)

    Takekawa, John Y.; Hill, N.J.; Schultz, A.K.; Iverson, S.A.; Cardona, C.J.; Boyce, W.M.; Dudley, J.P.

    2011-01-01

    Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal. Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene. The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey

  20. Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

    Science.gov (United States)

    2014-01-01

    Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level

  1. Depressive symptoms in rheumatoid arthritis Sintomas depressivos em pacientes com artrite reumatoide

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    Lucas Francisco Botequio Mella

    2010-09-01

    Full Text Available OBJECTIVE: To determine the prevalence of depressive and anxiety symptoms in patients with rheumatoid arthritis (a chronic inflammatory disease in comparison to a control group with osteoarthritis (a chronic non-inflammatory degenerative disease and to identify the sociodemographic and clinical variables associated with depressive symptoms in these patients. METHOD: Sixty-two rheumatoid arthritis patients and 60 osteoarthritis patients participated in the study. Sociodemographic and clinical data were collected and the Hospital Anxiety and Depression Scale and the Disability Index of the Health Assessment Questionnaire were applied. RESULTS: The prevalence of depressive symptoms was of 53.2% in rheumatoid arthritis and 28.3% in osteoarthritis (p = 0.005. The prevalence of anxiety symptoms was of 48.4% in rheumatoid arthritis and 50.0% in osteoarthritis (p = 0.859. The mean (and standard deviation scores in the Disability Index of the Health Assessment Questionnaire were 1.4 (0.8 in rheumatoid arthritis and 1.4 (0.6 in osteoarthritis (p = 0.864. Rheumatoid arthritis patients with depressive symptoms had lower education and higher disease activity and functional disability. CONCLUSION: Although these two rheumatic diseases are similar in terms of the pain and functional disability that they cause, a significantly higher prevalence of depressive symptoms was found in rheumatoid arthritis patients. This difference might be explained by the hypothesis of a neuroimmunobiological mechanism related to cytokines in inflammatory diseases, which has been considered as a candidate to the development of depressive symptoms.OBJETIVO: Determinar a prevalência de sintomas depressivos e ansiosos na artrite reumatóide (doença inflamatória crônica em comparação com um grupo controle com osteoartrite (doença crônico-degenerativa não inflamatória. Identificar variáveis sociodemográficas e clínicas associadas a sintomas depressivos nesses pacientes. M

  2. Sedação com sufentanil e clonidina em pacientes submetidos a cateterismo cardíaco

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    Anita Perpetua Carvalho Rocha

    2011-03-01

    Full Text Available FUNDAMENTO: A sedação para a realização de cateterismo cardíaco tem sido alvo de preocupação. Benzodiazepínicos, agonistas alfa-2 adrenérgicos e opioides são utilizados para esse fim, entretanto, cada um destes medicamentos possui vantagens e desvantagens. OBJETIVO: Avaliar a eficácia do sufentanil e da clonidina como sedativos em pacientes submetidos a cateterismo cardíaco, observando o impacto dos mesmos sobre os parâmetros hemodinâmicos e respiratórios, a presença de efeitos colaterais, além da satisfação do paciente e do hemodinamicista com o exame. MÉTODOS: Trata-se de um ensaio clínico prospectivo, duplo-cego, randomizado e controlado, que envolveu 60 pacientes que receberam 0,1 µg/kg de sufentanil ou 0,5 µg/kg de clonidina antes da realização do cateterismo cardíaco. O escore de sedação segundo a escala de Ramsay, a necessidade de utilização de midazolam, os efeitos colaterais, os parâmetros hemodinâmicos e respiratórios foram registrados, sendo os dados analisados em 06 diferentes momentos. RESULTADOS: O comportamento da pressão arterial, da frequência cardíaca e da frequência respiratória foi semelhante nos dois grupos, entretanto, no momento 2, os pacientes do grupo sufentanil (Grupo S apresentaram menor escore de sedação segundo a escala de Ramsay, e a saturação periférica da oxihemoglobina foi menor que o grupo clonidina (Grupo C no momento 6. Os pacientes do Grupo S apresentaram maior incidência de náusea e vômito pós-operatório que os pacientes do Grupo C. A satisfação dos pacientes foi maior no grupo clonidina. Os hemodinamicistas mostraram-se satisfeitos nos dois grupos. CONCLUSÃO: O sufentanil e a clonidina foram efetivos como sedativos em pacientes submetidos a cateterismo cardíaco.

  3. Concomitância de fibromialgia em pacientes portadores de hipotireoidismo e de alterações tireoideanas em pacientes com fibromialgia Fibromyalgia concomitance in patients with hypothyroidism and thyroid disorders in patients with fibromyalgia

    Directory of Open Access Journals (Sweden)

    Marlene Freire

    2006-02-01

    Full Text Available INTRODUÇÃO: a fibromialgia (FM pode ser definida como uma síndrome dolorosa musculoesquelética não-inflamatória de caráter crônico, tendo como sintomas mais comuns a fadiga e o sono não-reparador. Tais manifestações podem não resultar da FM, mas de outras doenças associadas, como o hipotireoidismo. OBJETIVO: descrever a ocorrência de FM em pacientes com hipotireoidismo, e os casos de hipotireoidismo em pacientes com FM, além de determinar os sintomas associados a essas condições clínicas. MÉTODOS: foram avaliados 166 pacientes portadores de FM e 56 com hipotireoidismo. Os voluntários responderam a um questionário para detecção dos principais sinais e sintomas dessas doenças em estudo e foram submetidos às dosagens séricas de hormônio estimulador da tireóide (TSH, tiroxina livre (T4 livre e anticorpo antitireoperoxidase (a-TPO. RESULTADOS: nos 166 pacientes com FM, foram diagnosticados hipotireoidismo clínico ou subclínico em 35 (21,1%. Dos 56 pacientes portadores de hipotireoidismo, em 36 (64,3% foi confirmado o diagnóstico de FM pelos critérios do American College of Rheumatology (ACR. Nos pacientes com hipotireoidismo previamente diagnosticado, a fadiga, o sono não-restaurador e a cefaléia crônica foram estatisticamente mais freqüentes naqueles que apresentavam diagnóstico de FM. A rigidez matinal foi mais prevalente nos pacientes com hipotireoidismo sem FM. CONCLUSÃO: há que se considerar a presença de manifestações clínicas ou laboratoriais de disfunção tireoideana nos pacientes portadores de FM e de FM em pacientes portadores de hipotireoidismo, para uma melhor abordagem diagnóstica e posterior tratamento dos mesmos.INTRODUCTION: Fibromyalgia can be defined as a non-inflammatory musculoskeletal pain syndrome. It is a chronic condition, commonly associated with fatigue and non-restorative sleep. However, such symptoms may result from coexisting diseases, for instance hypothyroidism. OBJECTIVE

  4. Perfil de citocinas e tipificação de HLA em pacientes com polipose nasossinusal tolerantes e intolerantes a aspirina Cytokines profile and HLA typing in tolerant and non-tolerant patients to aspirin with nasossinusal polyposis

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    Helena M. G. Becker

    2003-06-01

    Full Text Available A infiltração eosinofílica do pólipo nasossinusal (PNS associado à intolerância aspirínica (IA é característica relevante. Diversos mediadores participam da migração dos eosinófilos para os tecidos. A IA decorre do aumento da síntese de leucotrienos em indivíduos geneticamente susceptíveis. OBJETIVO: Analisar o perfil de citocinas e a tipificação de HLA-A, B e DR em pacientes com PNS tolerantes e intolerantes à aspirina. FORMA DE ESTUDO: Estudo de coorte transversal. MATERIAL E MÉTODO: selecionando-se 45 pacientes: 15 portadores de PNS eosinofílica tolerantes à aspirina (grupo TA; 15 de PNS eosinofílica associada à intolerância aspirínica, manifestada por broncoespasmo (grupo IA e 15 sem PNS, que apresentavam desvio de septo nasal (grupo controle. O perfil de citocinas (IL-2; IL-4; IL-5; IL-6; IL-8; IL-10; IFN-gama e TNF-alfa foi pesquisado nos fragmentos de pólipo nasal ou de mucosa de concha média (grupo controle através da reação reversa da cadeia de polimerase (RT-PCR. A tipificação de HLA-A, B e DR foi realizada através de teste sorológico de microcitotoxicidade ou por amplificação de DNA pela reação em cadeia da polimerase (PCR. RESULTADOS: A expressão de RNAm para as interleucinas 4, 5, 6, 8, 10, IFN-gama e TNF-alfa foi semelhante nos três grupos. A expressão de RNAm para IL-2 associou-se com a IA. Os pacientes portadores dos antígenos A11, B49, DR15 e DR13 apresentaram uma maior probabilidade de desenvolver polipose nasossinusal não relacionada à IA, enquanto os portadores de DR17 apresentaram uma maior probabilidade de desenvolver polipose nasossinusal associada à intolerância aspirínica (Tríade Aspirínica. CONCLUSÃO: A polipose nasossinusal associada à intolerância aspirínica (Tríade Aspirínica mostrou associação significante com HLA- DR17 e IL-2, sugerindo um perfil de citocinas TH1.The eosinophilic infiltration in the nasosinusal polyp associated with intolerance to aspirin is

  5. Erro médico em pacientes hospitalizados Medical errors in hospitalized patients

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    Manoel de Carvalho

    2002-01-01

    Full Text Available Objetivo: o presente artigo tem por objetivo revisar a literatura e discutir a questão do erro médico em pacientes hospitalizados, enfocando sua conceituação, incidência, fatores predisponentes e mecanismos de prevenção. Aborda, em especial, erros e eventos adversos com drogas envolvendo recém-nascidos e pacientes pediátricos. Métodos: revisão bibliográfica utilizando banco de dados Medline, selecionando-se aqueles com informações atuais e relevantes. Resultados:mesmo assumindo que a notificação do erro médico não ocorre em um grande número de eventos, é importante notar que sua incidência é muito maior do que julgamos. Só nos EUA, cerca de um milhão de pacientes por ano são vítimas de erros médicos e eventos adversos com drogas. Segundo recente metanálise, esta é, hoje, a quarta causa de morte nos EUA. Em unidades de terapia intensiva neonatal e pediátrica, nas quais é grande a complexidade e a freqüência de procedimentos, a ocorrência de erros é maior. Estima-se que quinze por cento das internações em unidades de terapia intensiva neonatal sejam acompanhadas de erro médico. A maioria destes erros acontece durante o período noturno, e envolve administração incorreta de droga (35% e erro na interpretação da prescrição (26%. Fatores ambientais (barulho, calor, psicológicos (tédio, ansiedade, estresse e fisiológicos (fadiga, sono contribuem para a ocorrência de erros. Recente estudo revela que após um plantão de 24 horas, sem dormir, o desempenho psicomotor de um profissional de saúde é semelhante ao de um indivíduo legalmente bêbado (nível sérico alcoólico maior ou igual a 0,08%! Conclusões: mesmo em profissionais conscientes, erros são acompanhantes inevitáveis da condição humana. A prevenção de erros deve basear-se na busca de causas reais, que geralmente incluem erros no sistema de organização e implementação do serviço. Erros devem ser aceitos como evidência de falha no

  6. Analgesia regional periférica com lidocaína em paciente queimado: relato de caso

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    Geier Karl Otto

    2004-01-01

    Full Text Available JUSTIFICATIVA E OBJETIVOS: A realização de anestesia regional em pacientes queimados é dificultosa pela localização aleatória das lesões térmicas. Elevadas taxas de alfa1 glicoproteína ácida com grande afinidade para drogas alcalinas, especialmente a lidocaína, têm sido observadas nesses pacientes. Este caso relata como o uso intermitente de anestesia e analgesia regional periférica com altas doses de lidocaína podem ser úteis em fornecer efetiva analgesia num paciente com queimaduras de segundo grau nos quatro membros, abrangendo, aproximadamente, 20% de área superficial queimada. RELATO DO CASO: Paciente do sexo masculino, 23 anos, 86 kg, estado físico ASA II, com queimadura superficial da face, segundo grau nos quatro membros e elevada taxa sérica de alfa1 glicoproteína ácida (260 mg.dl-1, teve sua dor controlada com 11,6 mg.kg-1 de lidocaína com adrenalina 1:400.000 administrada por cateteres introduzidos e tunelizados para diversos procedimentos - irrigações e troca de curativos, desbridamentos, fisioterapia, enxertos cutâneos e analgesia diária durante 28 dias. CONCLUSÕES: Em pacientes queimados com injúrias térmicas localizadas nas extremidades, a analgesia regional periférica pode ser útil. As elevadas taxas séricas de alfa1 glicoproteína ácida e o local da injeção podem permitir o emprego de altas doses de lidocaína.

  7. Preditores de internação hospitalar em pacientes com síncope atendidos em hospital cardiológico

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    Leonardo Marques Fischer

    2013-12-01

    Full Text Available FUNDAMENTO: No atendimento ao episódio sincopal é necessário estratificar o risco para melhor diferenciar pacientes que necessitam de internação hospitalar daqueles que podem ser liberados. Os critérios utilizados pelos médicos avaliadores desses pacientes em emergências cardiológicas em nosso meio são desconhecidos. OBJETIVOS: Analisar quais os critérios adotados para internação hospitalar, diferenciá-los dos utilizados nos pacientes liberados e compará-los com os preditores de alto risco definidos pelo escore de OESIL já validado para esse fim. MÉTODOS: Estudo transversal em pacientes diagnosticados com síncope na emergência em nossa instituição no ano de 2011. RESULTADOS: Dos 46.476 atendimentos realizados naquele ano, 216 foram descritos como síncope. Dos 216 pacientes analisados, 39% foram internados, sendo que as principais variáveis associadas à admissão foram síncope prévia, doença cardíaca conhecida, história negativa para acidente vascular encefálico no passado, ECG alterado e possuir plano de súde. Na comparação internação contra não internação, os escores OESIL 0-1 foram associados a maior chance de liberação hospitalar; os escores 2-3 apresentaram maior associação com internação. Um escore OESIL >2 demonstrou razão de chances 7,8 vezes maior de internação comparado com o escore 0 (p < 0,001; IC95%: 4,03-15,11. Aproximadamente 39% dos pacientes não tiveram definição etiológica e em 18% foi identificada uma causa cardiológica. CONCLUSÕES: Fatores como doença cardiovascular conhecida, história sincopal prévia, ausência de AVC prévio, possuir seguro de saúde e eletrocardiograma alterado foram os critérios utilizados pelos médicos em emergência para indicar internação hospitalar. Houve boa correlação entre os critérios clínicos e os critérios de risco do OESIL descritos na literatura.

  8. Epidemiological, clinical and laboratory profile of scrub typhus cases detected by serology and RT-PCR in Kumaon, Uttarakhand: a hospital-based study.

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    Rawat, Vinita; Singh, Rajesh Kumar; Kumar, Ashok; Saxena, Sandip R; Varshney, Umesh; Kumar, Mukesh

    2018-04-01

    We analysed the epidemiology, clinical and laboratory data of the 168 scrub typhus cases confirmed by a combination of any one of the following: real time polymerase chain reaction (RT-PCR) and/or immunofluorescence assay (IFA) (IgM and/or IgG). The peak season for scrub typhus was from July to October. By multivariate binary logistic regression analysis, the risk of scrub typhus was about four times in those working in occupation related to forest work. Major clinical manifestations were fever (100%), myalgia (65%), cough (51%) and vomiting (46%); major complications were meningitis/meningoencephatilitis (12.5%) and multi-organ failure (MOF) and pneumonia (5.3% each). Laboratory investigations revealed raised aminotranferase levels and thrombocytopenia in most confirmed cases. We conclude that scrub typhus is an important cause of febrile illness in the Kumaon hills of Uttarakhand where this disease had not previously been considered to exist.

  9. Detection and confirmation of PPR virus antigen in sheep and goats by sandwich-ELISA and RT-PCR in Andhra Pradesh, India

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    G. Saritha

    2015-06-01

    Full Text Available Peste des petits ruminants (PPR is a highly contagious disease of domestic and wild small ruminants. Rapid and accurate laboratory assay are essential to enable the implementation of appropriate control strategies to restrict the spread of PPR. The present study was designed to detect the PPR virus (PPRV antigen (N-gene in nasal swabs and tissue samples. A total of 195 samples comprising of 138 nasal swabs from PPR suspected sheep (n=72 and goats (n=66, and 57 tissue samples comprising of lymph nodes from dead sheep (n=39 and goats (n=18 were collected from certain parts of Andhra Pradesh. The samples were subjected to sandwich-ELISA followed by RT-PCR for confirmatory diagnosis. In this study, PPRV could be detected in 27.53% (n=38/138 nasal swabs and 49.12% (n=28/57 tissue samples. Data showed that PPRV infection is widespread in the Andhra Pradesh, India.

  10. Simultaneous Detection of CDC Category "A" DNA and RNA Bioterrorism Agents by Use of Multiplex PCR & RT-PCR Enzyme Hybridization Assays

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    Kelly J. Henrickson

    2009-10-01

    Full Text Available Assays to simultaneously detect multiple potential agents of bioterrorism are limited. Two multiplex PCR and RT-PCR enzyme hybridization assays (mPCR-EHA, mRT-PCR-EHA were developed to simultaneously detect many of the CDC category “A” bioterrorism agents. The “Bio T” DNA assay was developed to detect: Variola major (VM, Bacillus anthracis (BA, Yersinia pestis (YP, Francisella tularensis (FT and Varicella zoster virus (VZV. The “Bio T” RNA assay (mRT-PCR-EHA was developed to detect: Ebola virus (Ebola, Lassa fever virus (Lassa, Rift Valley fever (RVF, Hantavirus Sin Nombre species (HSN and dengue virus (serotypes 1-4. Sensitivity and specificity of the 2 assays were tested by using genomic DNA, recombinant plasmid positive controls, RNA transcripts controls, surrogate (spiked clinical samples and common respiratory pathogens. The analytical sensitivity (limit of detection (LOD of the DNA asssay for genomic DNA was 1×100~1×102 copies/mL for BA, FT and YP. The LOD for VZV whole organism was 1×10-2 TCID50/mL. The LOD for recombinant controls ranged from 1×102~1×103copies/mL for BA, FT, YP and VM. The RNA assay demonstrated LOD for RNA transcript controls of 1×104~1×106 copies/mL without extraction and 1×105~1×106 copies/mL with extraction for Ebola, RVF, Lassa and HSN. The LOD for dengue whole organisms was ~1×10-4 dilution for dengue 1 and 2, 1×104 LD50/mL and 1×102 LD50/mL for dengue 3 and 4. The LOD without extraction for recombinant plasmid DNA controls was ~1×103 copies/mL (1.5 input copies/reaction for Ebola, RVF, Lassa and HSN. No cross-reactivity of primers and probes used in both assays was detected with common respiratory pathogens or between targeted analytes. Clinical sensitivity was estimated using 264 surrogate clinical samples tested with the BioT DNA assay and 549 samples tested with the BioT RNA assay. The clinical specificity is 99.6% and 99.8% for BioT DNA assay and BioT RNA assay, respectively. The

  11. Reverse transcription-polymerase chain reaction (RT-PCR based detection and economic impact of foot-and-mouth disease in District Faisalabad, Pakistan during the year 2015

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    W. Ali

    2017-06-01

    Full Text Available The aim of this study was to evaluate the economic impact of the disease by using milk production records and to determine the serotypes circulating in the region during 2015. Sampling was done from different outbreaks initially on the basis of clinical signs and later reverse transcriptase-polymerase chain reaction (RT-PCR was employed for the conformation of FMDV genome. Out of total 88 samples, 73 were found positive which were then serotyped into type O (n=44, Asia1 (n=18 and A (n=06. The economic impact was analyzed by recording milk loss at four affected farms. Their average milk yield was observed 9.2 liters before the onset of disease that decreased dramatically after the disease. Milk loss of 225 and 195 liters was recorded for buffalo and cattle respectively, during 70 days of the study period.

  12. COMPARISON OF TWO TOTAL RNA EXTRACTION PROTOCOLS FROM CHO-K1 CELLS FOR RT-PCR: CUT-OFF COST FOR RESEARCHERS

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    Vasila Packeer Mohamed

    2014-05-01

    Full Text Available ABSTRACT: Various methods have been described to extract RNA from adherent mammalian cells. RNA isolation in conjunction with reverse transcription polymerase chain reaction (RT-PCR is a valuable tool used to study gene expression profiling. This approach is now being used in mammalian cell bioprocessing to help understand and improve the system. The objective of this study was to compare and determine the most suitable RNA extraction method for CHO-K1 cells in a setting where a relatively large amount of samples was involved. Total RNA was extracted using Total RNA purification kit (without DNase treatment; Norgen, Canada and RNeasy mini kit (with DNase treatment; Qiagen, USA respectively. The extracted RNA was then reverse transcribed, and the cDNA was subjected to PCR-amplifying 18S. Yield from RNeasy kit was significantly higher (0.316 ± 0.033 µg/µl; p=0.004 than Total RNA purification kit (0.177 ± 0.0243 µg/µl. However, RNA purity for both methods was close to 2.0 and there was no significant difference between the methods. Total RNA purification kit is less expensive than RNeasy kit. Since there is no DNase treatment step in the former, extraction time for RNA is shorter. When the extracted RNA was subjected to RT-PCR, both methods were able to show detection of 18S at 219 bp.   Therefore, this study demonstrates that both protocols are suitable for RNA extraction for CHO-K1 cells. RNeasy mini kit (Qiagen is recommended if higher yields is the primary concern and Total RNA Purification kit (Norgen is recommended if time and cost are concerned. ABSTRAK: Pelbagai kaedah telah digunakan untuk mengekstrak RNA daripada sel mamalia lekat.  Pemencilan RNA dengan menggunakan reaksi rantai polimerase transkripsi berbalik (RT-PCR merupakan kaedah penting yang digunakan dalam mengkaji pernyataan gen berprofil.  Pendekatan ini kini digunakan dalam pemprosesan bio sel mamalia untuk memahami dan menambah baik sistem.  Tujuan kajian dijalankan

  13. Qualidade de vida em pacientes submetidos à cirurgia ortognática: saúde bucal e autoestima

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    Rubens Guimarães Filho

    Full Text Available A cirurgia ortognática é o ramo da cirurgia bucomaxilofacial que trata das deformidades dentofaciais, buscando a correção da oclusão e melhora articular, respiratória e fonética, que geram benefícios funcionais e harmonizam a face, o que acarreta aspectos positivos em relação à estética e ao fator psicossocial do paciente. Objetivou-se avaliar a qualidade de vida desses pacientes, através dos aspectos funcionais, estéticos e psicossociais. O presente estudo foi realizado em clínica odontológica particular, na cidade de Taubaté. Foram avaliados 33 pacientes, 12 do gênero masculino e 21 do gênero feminino, com idades entre 18 a 45 anos, com tratamento ortocirúrgico indicado ou já realizado, utilizando-se os questionários Escala de Autoestima de Rosenberg/UNIFESP-EPM, Oral Health Impact Profile-14 - OHIP14 e PIDAQ-Brasil/UNIFESP e o Teste não Paramétrico de Mann-Whitney. Os resultados evidenciaram que a cirurgia ortognática é um procedimento capaz de oferecer melhora da qualidade de vida para pacientes portadores de deformidades dentofaciais, por meio dos aspectos funcionais, estéticos e psicossociais.

  14. Aspectos clínicos e epidemiológicos de pacientes queimados internados em um Hospital de Ensino

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    Samanta Flor Montes

    2011-04-01

    Full Text Available Os objetivos foram: caracterizar os pacientes queimados segundo as variáveis epidemiológicas e clínicas e identificar os tratamentos, procedimentos invasivos e as complicações. Trata-se de um estudo retrospectivo, descritivo e quantitativo. A amostra constituiu-se de 138 pacientes queimados internados em um hospital de ensino, no período de janeiro de 2003 a dezembro de 2007, de Uberaba-MG. Dos 138 pacientes internados, 98 (71,0% eram do gênero masculino e a média de idade foi de 26,1 anos. O tempo médio de internação foi de 16,2 dias; 93 (67,4% eram de natureza acidental e a principal causa 68 (49,3% foi a chama aberta. A superfície corporal queimada média foi de 20,8% e a maioria 122 (88,4% apresentou queimadura de 2° grau. A terapia tópica mais utilizada 93 (67,4% foi a sulfadiazina de prata. A sondagem vesical de demora foi instalada em 47 (34,0% pacientes; 30 (21,7% foram submetidos à enxertia e 28 (20,3% ao desbridamento; 14 (10,1% apresentaram infecção da lesão.

  15. Uso de medicamentos, glicemia capilar e índice de massa corpórea em pacientes com diabetes mellitus

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    Márcio Flávio Moura de Araújo

    2013-10-01

    Full Text Available Objetivou-se relacionar o uso de medicamentos com a glicemia capilar e o índice de massa corpórea em pacientes com diabetes mellitus tipo 2. Estudo transversal realizado entre janeiro e julho de 2009, nos domicílios de 437 pacientes de doze centros de saúde de Fortaleza-CE. Utilizou-se um formulário para o registro das variáveis sociodemográficas e clinicas, glicemia capilar, índice de massa corpórea e uso de medicamentos. Dos pacientes com glicemia capilar normal, 93% não utilizavam antidepressivos / ansiolíticos (p = 0,02. Aproximadamente 99% dos que apresentavam a glicemia capilar elevada não utilizavam cálcio diariamente (p = 0,04. Entre aqueles com índice de massa corpórea normal, 45,5% tomavam diariamente anti-hipertensivos da classe inibidor da ECA (p = 0,03. O uso diário de drogas inibidoras da ECA, antidepressivos / ansiolíticos e cálcio apresentou associação com a glicemia capilar e o índice de massa corpórea, respectivamente. É importante que o enfermeiro avalie o peso corporal e a glicemia capilar dos pacientes com diabetes, em particular daqueles que fazem uso contínuo de medicamentos.

  16. Prevalência de aloimunização eritrocitária em pacientes portadores de anemia falciforme

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    Alexandre Gomes Vizzoni

    2017-04-01

    Full Text Available Embora as transfusões de concentrado de hemácias sejam importantes para o tratamento de pacientes com anemia falciforme, elas acarretam riscos imunológicos tais como a aloimunização a antígenos eritrocitários. Aproximadamente 50% dos pacientes de anemia falciforme recebem transfusões no decorrer da vida, e entre 5 e 10% desses pacientes são submetidos a um programa de transfusão crônica. A aloimunização eritrocitária é uma complicação relativamente comum, podendo até mesmo levar a reações transfusionais hemolíticas tardias, contribuindo para aumentar as comorbidades da doença. Importantes medidas para prevenção dessas complicações nesses pacientes são o uso de hemácias previamente fenotipadas, além da fenotipagem do próprio receptor de concentrado de hemácias, determinando seu correto perfil fenotípico e possibilitando a escolha de concentrado de hemácias com antígenos correspondentes ao do paciente a ser transfundido. Extensa genotipagem eritrocitária profilática para selecionar doadores para pacientes que receberão repetidas transfusões durante um longo período é uma aplicação atraente de tipagem de sangue baseado em DNA. Isso é particularmente relevante para pacientes com doença falciforme, nos quais a taxa de aloimunização é elevada.

  17. O estudo da prova calórica em pacientes idoso The study of caloric testing in senior patients

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    Débora Cristina de Oliveira Bezerra

    2008-01-01

    Full Text Available OBJETIVO: realizar o estudo da prova calórica em pacientes idosos com queixa de tontura, acompanhada ou não de zumbido com finalidade de traçar um perfil dos idosos quanto os sintomas vestibulares. MÉTODOS: fizeram parte da amostra 143 pacientes, com faixa etária variando de 60 a 90 anos de idade; todos realizaram o exame otoneurológico. Foram excluídos pacientes idosos com queixa de desequilíbrio e aqueles que apresentaram a queixa de zumbido desacompanhado da tontura. RESULTADOS: o estudo demonstrou que a normorreflexia, em valores absolutos, encontra-se entre 82,3% a 91,2% e os valores relativos são de 93%. Referente aos dados colhidos na anamnese, o que chamou a atenção, foi que do total de pacientes analisados, encontrou-se a tontura acompanhada de zumbido em 69% dos casos e o elevado uso de medicação em pacientes nesta faixa etária. CONCLUSÃO: na população estudada houve um predomínio da tontura acompanhada de zumbido. Em relação à prova calórica, o trabalho demonstrou a alta prevalência de pacientes com valores absolutos e relativos normais.PURPOSE: to accomplish the study of caloric testing in senior patients with dizziness, accompanied or not of with buzzing. METHODS: 143 patients were analyzed with age varying from 60 to 90 years; all of them did an otoneurologic evaluation. Senior patients with unbalance complaints and with buzzing unaccompanied with dizziness were excluded from this study. RESULTS: 82.3% to 91.2% of the patients obtained normoreflexia in absolute values and over 93% obtained normal results in relative values. Referring to the data obtained in the anamnesis, what got attention was 69% of dizziness accompanied with buzzing. And the high medication of the patients in this age group. CONCLUSION: there was a prevalence of dizziness accompanied with buzzing in the studied population. Concerning the caloric testing, the study demonstrated the high prevalence of patients with normal absolute and

  18. A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas

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    Ming-An Tsai

    2017-09-01

    Full Text Available Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4 with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

  19. Validation of reference genes for quantitative RT-PCR normalization in Suaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy

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    Jing Cao

    2016-02-01

    Full Text Available Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR is a powerful analytical technique for the measurement of gene expression, which depends on the stability of the reference gene used for data normalization. Suaeda aralocaspica, an annual halophyte with heteromorphic seeds and possessing C4 photosynthesis pathway without Kranz anatomy, is an ideal plant species to identify stress tolerance-related genes and compare relative expression at transcriptional level. So far, no molecular information is available for this species. In the present study, six traditionally used reference genes were selected and their expression stability in two types of seeds of S. aralocaspica under different experimental conditions was evaluated. Three analytical programs, geNorm, NormFinder and BestKeeper, were used to assess and rank the stability of reference gene expression. Results revealed that although some reference genes may display different transcriptional profiles between the two types of seeds, β-TUB and GAPDH appeared to be the most suitable references under different developmental stages and tissues. GAPDH was the appropriate reference gene under different germination time points and salt stress conditions, and ACTIN was suitable for various abiotic stress treatments for the two types of seeds. For all the sample pools, β-TUB served as the most stable reference gene, whereas 18S rRNA and 28S rRNA performed poorly and presented as the least stable genes in our study. UBQ seemed to be unsuitable as internal control under different salt treatments. In addition, the expression of a photosynthesis-related gene (PPDK of C4 pathway and a salt tolerance-related gene (SAT of S. aralocaspica were used to validate the best performance reference genes. This is the first systematic comparison of reference gene selection for qRT-PCR work in S. aralocaspica and these data will facilitate further studies on gene expression in this species

  20. A plastome primer set for comprehensive quantitative real time RT-PCR analysis of Zea mays: a starter primer set for other Poaceae species

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    Dunn Sade N

    2008-06-01

    Full Text Available Abstract Background Quantitative Real Time RT-PCR (q2(RTPCR is a maturing technique which gives researchers the ability to quantify and compare very small amounts of nucleic acids. Primer design and optimization is an essential yet time consuming aspect of using q2(RTPCR. In this paper we describe the design and empirical optimization of primers to amplify and quantify plastid RNAs from Zea mays that are robust enough to use with other closely related species. Results Primers were designed and successfully optimized for 57 of the 104 reported genes in the maize plastome plus two nuclear genes. All 59 primer pairs produced single amplicons after end-point reverse transcriptase polymerase chain reactions (RT-PCR as visualized on agarose gels and subsequently verified by q2(RTPCR. Primer pairs were divided into several categories based on the optimization requirements or the uniqueness of the target gene. An in silico test suggested the majority of the primer sets should work with other members of the Poaceae family. An in vitro test of the primer set on two unsequenced species (Panicum virgatum and Miscanthus sinensis supported this assumption by successfully producing single amplicons for each primer pair. Conclusion Due to the highly conserved chloroplast genome in plant families it is possible to utilize primer pairs designed against one genomic sequence to detect the presence and abundance of plastid genes or transcripts from genomes that have yet to be sequenced. Analysis of steady state transcription of vital system genes is a necessary requirement to comprehensively elucidate gene expression in any organism. The primer pairs reported in this paper were designed for q2(RTPCR of maize chloroplast genes but should be useful for other members of the Poaceae family. Both in silico and in vitro data are presented to support this assumption.

  1. Development of a real-time RT-PCR assay for the simultaneous identification, quantitation and differentiation of avian metapneumovirus subtypes A and B.

    Science.gov (United States)

    Cecchinato, Mattia; Lupini, Caterina; Munoz Pogoreltseva, Olga Svetlana; Listorti, Valeria; Mondin, Alessandra; Drigo, Michele; Catelli, Elena

    2013-01-01

    In recent years, special attention has been paid to real-time polymerase chain reaction (PCR) for avian metapneumovirus (AMPV) diagnosis, due to its numerous advantages over classical PCR. A new multiplex quantitative real-time reverse transcription-PCR (qRT-PCR) with molecular beacon probe assay, designed to target the SH gene, was developed. The test was evaluated in terms of specificity, sensitivity and repeatability, and compared with conventional RT nested-PCR based on the G gene. All of the AMPV subtype A and B strains tested were amplified and specifically detected while no amplification occurred with other non-target bird respiratory pathogens. The detection limit of the assay was 10(-0.41) median infectious dose/ml and 10(1.15) median infectious dose/ml when the AMPV-B strain IT/Ty/B/Vr240/87 and the AMPV-A strain IT/Ty/A/259-01/03 were used, respectively, as templates. In all cases, the amplification efficiency was approximately 2 and the error values were 0.9375) between crossing point values and virus quantities, making the assay herein designed reliable for quantification. When the newly developed qRT-PCR was compared with a conventional RT nested-PCR, it showed greater sensitivity with RNA extracted from both positive controls and from experimentally infected birds. This assay can be effectively used for the detection, identification, differentiation and quantitation of AMPV subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity.

  2. Pathway and network-based analysis of genome-wide association studies and RT-PCR validation in polycystic ovary syndrome.

    Science.gov (United States)

    Shen, Haoran; Liang, Zhou; Zheng, Saihua; Li, Xuelian

    2017-11-01

    The purpose of this study was to identify promising candidate genes and pathways in polycystic ovary syndrome (PCOS). Microarray dataset GSE345269 obtained from the Gene Expression Omnibus database includes 7 granulosa cell samples from PCOS patients, and 3 normal granulosa cell samples. Differentially expressed genes (DEGs) were screened between PCOS and normal samples. Pathway enrichment analysis was conducted for DEGs using ClueGO and CluePedia plugin of Cytoscape. A Reactome functional interaction (FI) network of the DEGs was built using ReactomeFIViz, and then network modules were extracted, followed by pathway enrichment analysis for the modules. Expression of DEGs in granulosa cell samples was measured using quantitative RT-PCR. A total of 674 DEGs were retained, which were significantly enriched with inflammation and immune-related pathways. Eight modules were extracted from the Reactome FI network. Pathway enrichment analysis revealed significant pathways of each module: module 0, Regulation of RhoA activity and Signaling by Rho GTPases pathways shared ARHGAP4 and ARHGAP9; module 2, GlycoProtein VI-mediated activation cascade pathway was enriched with RHOG; module 3, Thromboxane A2 receptor signaling, Chemokine signaling pathway, CXCR4-mediated signaling events pathways were enriched with LYN, the hub gene of module 3. Results of RT-PCR confirmed the finding of the bioinformatic analysis that ARHGAP4, ARHGAP9, RHOG and LYN were significantly upregulated in PCOS. RhoA-related pathways, GlycoProtein VI-mediated activation cascade pathway, ARHGAP4, ARHGAP9, RHOG and LYN may be involved in the pathogenesis of PCOS.

  3. Jejum pré-operatório em pacientes hospitalizados = Preoperative fasting in hospitalized patients

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    Flores, Patrícia da Fonseca

    2013-01-01

    Full Text Available Introdução: A resposta metabólica ao trauma cirúrgico é potencializada pelo jejum pré-operatório prolongado. Estudos evidenciam que a redução do tempo de jejum com a ingestão de líquidos com carboidratos, no pré-operatório, não resulta em aumento do risco de morbidade associada à anestesia. Objetivo: Revisar sobre as atuais recomendações da literatura acerca do jejum pré-operatório em pacientes hospitalizados. Materiais e Métodos: Realizou-se uma revisão de literatura a partir das bases de dados Medline/Pubmed, Lilacs e Embase, publicados no período de 2002 até 2012, com os termos de indexação: jejum, inanição, período pré-operatório e cuidado pré-operatório. Resultados: Foram incluídos 20 estudos na revisão. Foram encontrados resultados referentes às consequências da abreviação do jejum pré-operatório, através do uso de uma solução de líquido enriquecida com carboidrato. Os resultados demonstraram que o uso da solução determina maior satisfação, menor irritabilidade, aumento do pH gástrico e principalmente, reduz a resposta catabólica ao estresse cirúrgico, com consequente melhora da recuperação pós-operatória. Conclusão: Os resultados de trabalhos clínicos consistentes têm feito com que velhos paradigmas sejam questionados dando lugar a práticas mais modernas. Soluções que contenham carboidratos e outros nutrientes são promissoras quanto à melhora na resposta endócrino-metabólica ao trauma. A satisfação do paciente também deve ser considerada, e é maior quando se empregam períodos mais curtos de jejum no pré-operatório

  4. Vacinação em pacientes da Coorte Brasília de artrite reumatoide inicial

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    Luciana Feitosa Muniz

    2014-10-01

    Full Text Available Introdução: Os pacientes com diagnóstico de artrite reumatoide (AR apresentam risco aumentado de infecções. A vacinação é uma medida preventiva recomendada. Não há estudos avaliando a prática da vacinação nos pacientes com AR inicial. Objetivos: Avaliar a frequência de vacinação e a orientação (feita pelo médico sobre vacinas entre os pacientes com diagnóstico de AR inicial. Métodos: Estudo transversal incluindo pacientes da coorte Brasília de AR inicial. Foram analisados dados demográficos, índice de atividade da doença (Disease Activity Score 28 - DAS28, incapacidade funcional (Health Assessment Questionnaire - HAQ, dados sobre tratamento e vacinação após o diagnóstico da AR. Resultados: Foram avaliados 68 pacientes, sendo 94,1% mulheres, com idade média de 50,7 ± 13,2 anos. O DAS28 foi de 3,65 ± 1,64, e o HAQ de 0,70. A maioria dos pacientes (63% possuía cartão vacinal. Apenas cinco pacientes (7,3% foram orientados pelo médico sobre uso das vacinas. Os pacientes foram vacinados para tríplice viral (8,8%, tétano (44%, febre amarela (44%, hepatite B (22%, gripe (42%, influenza H1N1 (61,76%, pneumonia (1,4%, meningite (1,4% e varicela (1,4%. Todos os pacientes vacinados com vírus vivo atenuado estavam em uso de imunossupressores e receberam as vacinas de forma inadvertida, sem orientação médica. Não houve associação entre o uso de nenhuma vacina e atividade da doença, incapacidade funcional, anos de escolaridade, hábitos de vida, comorbidades. Conclusão: Os pacientes foram pouco orientados pelo médico com relação ao uso das vacinas, com elevada frequência de vacinação inadvertida com componente vivo atenuado, enquanto a imunização com vírus mortos ficou aquém do recomendado.

  5. Anestesia subaracnóidea para cesariana em paciente portadora de esclerose múltipla: relato de caso

    OpenAIRE

    Barbosa, Fabiano Timbó; Bernardo, Ronaldson Correia; Cunha, Rafael Martins da; Pedrosa, Maria do Socorro Melo

    2007-01-01

    JUSTIFICATIVA E OBJETIVOS: A esclerose múltipla é uma doença adquirida que se caracteriza por áreas desmielinizadas no encéfalo e na medula espinal. O quadro clínico depende das áreas anatômicas acometidas. As principais causas de morte são infecção, falência respiratória e estado de mal epiléptico. Ocorre em pacientes geneticamente predispostos após contato com fatores ambientais, principalmente os vírus. O objetivo desse relato foi apresentar a técnica anestésica adotada em paciente com esc...

  6. Candidíase oral como marcador de prognóstico em pacientes portadores do HIV

    OpenAIRE

    Cavassani Valdinês Gonçalves dos Santos; Andrade Sobrinho Jozias de; Homem Maria da Graça Naclério; Rapoport Abrão

    2002-01-01

    Introdução: A candidíase oral é uma das doenças oportunistas mais fortemente associadas à infecção pelo Vírus da Imunodeficiência Humana (HIV). Vários relatos epidemiológicos enfatizam a prevalência da candidíase em pacientes HIV positivos e ressaltam a sua importância como marcador da progressão da doença e preditivo para o aumento da imunodepressão. Objetivo: Verificar as alterações estomatológicas em pacientes portadores do HIV tratados no Hospital Heliópolis - São Paulo, Brasil e comparar...

  7. Infecção hospitalar em pacientes cirúrgicos de Hospital Universitário

    Directory of Open Access Journals (Sweden)

    Medeiros Aldo da Cunha

    2003-01-01

    Full Text Available OBJETIVO: Analisar fatores intercorrentes e a incidência da infecção em pacientes operados no Hospital Universitário da UFRN. MÉTODOS: Foram estudados, através de protocolo previamente estabelecido, 3.120 pacientes internados que se submeteram a procedimentos cirúrgicos no período de janeiro de 1999 a outubro de 2002. RESULTADOS: O índice de infecção hospitalar foi de 5,9%, e a topografia de maior incidência foi a ferida operatória (3,7%. Infecção respiratória ocorreu em 1,2%, urinária em 0,6% e bacteremia em 0,1%. O índice de infecção comunitária foi de 9,2%, predominando infecção urinária (5% e respiratória (2,1%. Quanto ao grau de contaminação das feridas operatórias, as feridas limpas (1479 apresentaram infecção em 2,9%, as feridas limpas-contaminadas (1277 em 6,0% dos casos, as feridas contaminadas (270 em 15,1%, e as ferida infectadas (94 resultaram em infecção em 30,75% dos casos. CONCLUSÃO: Concluiu-se que a incidência de infecção cirúrgica foi compatível com os índices na literatura mundial. A partir desses dados, ratifica-se a importância de medidas de controle de infecção hospitalar de forma sistemática, como vem sendo realizado no hospital onde o estudo foi realizado.

  8. Avaliação da qualidade de vida em pacientes submetidos à cirurgia de catarata, com implantes de lentes monofocais bifocais e multifocais

    Directory of Open Access Journals (Sweden)

    Beogival Wagner Lucas Santos

    2014-04-01

    Full Text Available Objetivo: Fornecer uma avaliação objetiva e subjetiva da qualidade de vida e o nível de satisfação de pacientes que se submeteram à cirurgia de catarata, com implante de lentes bifocais, multifocais e monofocais. Métodos: Lentes bifocais foram implantadas em 72 pacientes; lentes multifocais asférica em 16 pacientes; lentes multifocais apodizada em 20 pacientes e lentes monofocais em 63 pacientes. Foi aplicado um questionário de 47 perguntas cujos resultados foram avaliados e analisados. Resultados: O nível de satisfação dos pacientes implantados com lentes bifocais e multifocais foi significantemente mais alto do que aqueles com lentes monofocais (teste Kruskal-Wallis p<0,01, seguido do teste Dunn, p<0,05. Quanto à comparação de lentes monofocais com lentes bifocais e multifocais, a visão para perto sem correção foi consideravelmente mais baixa em pacientes com lentes monofocais (teste Z, p<0,001. Os pacientes com lentes monofocais sem correção apresentaram uma menor capacidade de ler jornais ou livros e no que diz respeito a trabalhos manuais do que aqueles com lentes bifocais e multifocais (teste Z, p<0,001. Conclusão: Os resultados deste estudo mostram que os pacientes que receberam as lentes bifocais e multifocais tiveram uma melhora notável em sua acuidade visual para perto, estando satisfeitos com sua visão geral, sem óculos. A satisfação foi atribuída ao fato de conseguirem ler livros e jornais, preencher cheques, preencher formulários, praticar esportes, se barbear ou se maquiar sem correção.

  9. Oxidative stress in patients with type 2 diabetes mellitus treated with metformin = Estresse oxidativo em pacientes com diabetes mellitus tipo 2 em tratamento com metformina

    Directory of Open Access Journals (Sweden)

    Dries, Samuel Selbach

    2017-01-01

    RESULTADOS: A amostra inicial foi composta por 49 pacientes com idade de 59±9 anos e índice de massa corporal de 29,8±5,1 kg/m2 , com diabetes por uma mediana de tempo de 36 (intervalo interquartil 1-240 meses e em uso de metformina há uma mediana de 36 (intervalo interquartil 1-180 meses. Vinte e cinco pacientes deixaram o estudo entre a segunda e a quarta reunião. Os níveis de malondialdeído diferiram entre antes e após o acompanhamento farmacoterapêutico, correlacionando-se positivamente com glicemia, glicohemoglobina e triglicerídeos e negativamente com metformina e superóxido dismutase. Encontrou-se elevação da glicemia, glicohemoglobina e malondialdeído, e diminuição da metformina no grupo com complicações diabéticas, e foi identificada correlação entre malondialdeído e o número de complicações diabéticas por paciente. CONCLUSÕES: Nesta amostra de pacientes com diabetes mellitus tipo 2 em tratamento com metformina, o estresse oxidativo foi mais pronunciado nos que apresentavam pior controle glicêmico e complicações diabéticas

  10. Síndrome hemolítico-urêmica causada por gencitabina em uma paciente jovem com colangiocarcinoma

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    John Fredy Nieto-Ríos

    2016-06-01

    Full Text Available Resumo A gencitabina é um fármaco utilizado no tratamento de vários tipos de neoplasias malignas. Há poucas descrições de associação entre a droga e a síndrome hemolítico-urêmica (SHU, apesar de os pacientes em questão terem ido a óbito em pelo menos 50% dos casos. O presente artigo relata o caso de uma paciente com 25 anos de idade em remissão diagnosticada com colangiocarcinoma que apresentou anemia hemolítica microangiopática acompanhada de insuficiência renal aguda anúrica após cinco ciclos de quimioterapia com gencitabina; as manifestações eram condizentes com SHU causada pelos efeitos colaterais do medicamento. A administração de gencitabina foi interrompida, e a paciente foi tratada com hemodiálise, transfusões de sangue, trocas de plasma, corticosteroides, doxiciclina e rituximabe. Foi atingido um desfecho favorável; mais especificamente, a hemólise foi controlada e a função renal foi plenamente restabelecida.

  11. Força máxima da língua em pacientes com bruxismo

    OpenAIRE

    Lis Meirelles

    2014-01-01

    Resumo: O bruxismo é assunto de intensa investigação na área odontológica e está correlacionado a fatores emocionais e a eventos de estresse experimentado pelos indivíduos. Uma vez que o hábito de ranger de dentes pode levar a edentações na língua, este estudo avaliou a força da língua em pacientes bruxistas antes e após a terapia por meio de placas oclusais estabilizadoras. Para tanto, voluntários foram selecionados na Faculdade de Odontologia de Piracicaba - UNICAMP e separados em dois grup...

  12. Hanseníase: um estudo sobre a experiência da enfermidade de pacientes em Salvador-Bahia

    OpenAIRE

    Martins, Patrícia Vieira

    2013-01-01

    A pesquisa que originou esta tese foi realizada com indivíduos residentes no Estado da Bahia, que buscaram tratamento em um hospital de referência na cidade de Salvador, e teve como objetivo responder, a partir de uma perspectiva socioantropológica, a uma série de indagações, tais como: de que maneira os pacientes construíram a experiência da enfermidade vivenciada por eles; qual foi a repercussão social da doença em suas vidas; quais os significados atribuídos a ela; quais as estratégias de ...

  13. Utilização da albumina na citologia esfoliativa em pacientes com conjuntivite alérgica

    OpenAIRE

    Bezerra Haroldo de Lucena; Rizzo Luiz Vicente; Yu Maria Cecília Zorat; Freitas Denise de

    2003-01-01

    OBJETIVO: Avaliar a albumina como fixador de material obtido de raspado conjuntival para citologia esfoliativa. MÉTODOS: Foi realizada citologia esfoliativa conjuntival por meio de estudo prospectivo em quarenta e seis olhos de vinte e três pacientes com conjuntivite alérgica. Foram realizadas três colheitas: (1) na fase aguda, (2) após uso de corticosteróides (tratamento A) e (3) após uso de estabilizador de membrana de mastócitos (tratamento B); em dois tipos de lâminas, com e sem albumina,...

  14. Morbidade febril puerperal em pacientes infectadas pelo HIV Puerperal morbidity in HIV-positive women

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    Andrea De Marcos

    2003-04-01

    Full Text Available OBJETIVO: avaliar as taxas de morbidade febril puerperal em pacientes infectadas pelo HIV e sua correlação com a via de parto, duração do trabalho de parto, tempo de rotura de membranas, número de células CD4+ e carga viral do HIV periparto. MÉTODOS: foram incluídas 207 gestantes infectadas pelo HIV, com seguimento pré-natal e parto entre maio de 1997 e dezembro de 2001, sendo 32 submetidas a parto vaginal e 175 a cesárea. Do total de pacientes, 62,8% foram submetidas a cesárea eletiva. A idade média no grupo analisado foi de 27,4 anos, 25,6% eram nulíparas e 26% primíparas, com idade gestacional média de 37,8 semanas no momento do parto. A contagem média de células CD4+ foi de 481 células /mm³ e da carga viral do HIV de 49.100 cópias/mL, ambas no final da gestação. RESULTADOS: a morbidade febril puerperal ocorreu em 34 pacientes, sendo 33 pós-cesárea e 1 pós-parto vaginal. O tipo mais comum de intercorrência infecciosa pós-cesárea foi infecção de cicatriz cirúrgica (13% dos casos de infecção. Os fatores analisados, como duração do trabalho de parto, tempo de rotura de membranas, contagem de células CD4+ ou carga viral do HIV periparto, não interferiram na taxa de morbidade febril puerperal. CONCLUSÕES: A incidência de morbidade febril puerperal foi de 16,8%, sendo mais freqüente pós-cesárea (18,9% que pós-parto vaginal (3,1%. Os demais fatores não mostraram relação significativa com a taxa de morbidade febril puerperal.PURPOSE: the morbidity in HIV-positive patients due to puerperal fever was studied and correlated to the method and duration of labor, the duration of premature rupture of the membranes, CD4+ cell count and the viral load (VL at peridelivery. METHODS: a total of 207 HIV-positive women with prenatal examinations and deliveries between May 1997 and December 2001 were enrolled. Of these, 32 had natural childbirth and 175 had a cesarean section. Of the total of enrolled patients, 62

  15. PERFIL LÍPIDICO DOS PACIENTES HIV POSITIVOS EM USO DA TERAPIA ANTIRRETROVIRAL

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    Maria Roseane dos Santos

    2017-07-01

    Full Text Available Corresponde a estudo descritivo de corte transversal, buscou-se evidenciar o impacto da TARV sobre o perfil lipídico. Realizada com 37 pacientes atendidos no ambulatório de Doenças Infecciosas e Parasitárias do centro de referência para HIV do Hospital Universitário Oswaldo Cruz em Recife-PE/Brasil. Através da realização de entrevistas, coleta de informações sobre terapia antirretroviral, curva de CD4 e carga viral obtidas nos prontuários. Tendo como aprovação no comitê de ética e pesquisa CAAE de nº 11365112.8.0000.5192. Revela o impacto no perfil lipídico associado ao uso contínuo da TARV, demostrando aumento do TG 54%, CT 49%, VLDL (41%, LDL-c 19% e diminuição do HDL-c 24%.   As associações de esquemas de ITRN e IRNN apresentaram o maior número de alterações. As drogas da TARV induzem distúrbios metabólicos graves e associados com outros fatores encontrados como HAS 19%, tabagismo 35% e sedentarismo 81% constituem uma forte predisposição de aterosclerose e consequentemente riscos cardiovasculares.

  16. Insuficiência renal aguda em paciente tratada com ATRA e anfotericina B: relato de caso

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    Gelcimar Moresco

    2011-06-01

    Full Text Available O presente relato apresenta o caso clínico de uma paciente com leucemia promie-locítica aguda tratada com ácido todo-transretinoico (ATRA, que apresentou suspeita de síndrome do ácido transreti-noico (síndrome de ATRA. Com a ocor-rência de leucopenia febril inespecífica, foram associados ao tratamento antimi-crobianos e antifúngicos. A diminuição da função renal, observada inicialmente, contribuiu para a suspeita de síndrome de ATRA, que foi agravada pelos antifúngi-cos. Assim, o uso de ATRA foi suspenso, mas somente 8 dias depois foi caracteriza-da pneumonia e descartada a hipótese de síndrome de ATRA. Nesse contexto, foi discutida a nefrotoxicidade do ATRA e a potencialização desse efeito adverso pelo uso de antifúngicos nefrotóxicos, em par-ticular da anfotericina B, assim como a im-portância do diagnóstico diferencial entre síndrome de ATRA e doença infecciosa.

  17. Infecção pulmonar tripla em paciente gravemente imunocomprometido por AIDS: relato de caso

    Directory of Open Access Journals (Sweden)

    Faucz Rafael Artigas

    2006-01-01

    Full Text Available Os autores relatam um caso de paciente do sexo masculino, 38 anos de idade, motorista, soropositivo para HIV há oito anos, sem acompanhamento, com quadro de tosse produtiva com secreção acinzentada e episódios intermitentes de dispnéia há 15 dias. Informava dois episódios pregressos de tuberculose pulmonar (1983 e 2001 tratados. A radiografia de tórax evidenciou áreas de hipotransparência nodular e broncogramas aéreos bilateralmente. A tomografia computadorizada de tórax evidenciou vários achados inespecíficos, dentre eles áreas esparsas de consolidação, cavitação, bronquiectasia, opacidade em vidro fosco, espessamento intersticial e broncogramas aéreos. A lavagem broncoalveolar evidenciou numerosas hifas com raros septos bifurcados sugestivos de Aspergillus sp. e a cultura foi positiva para Nocardia sp. e Mycobacterium tuberculosis. Foi instituída terapia com anfotericina B, sulfametoxazol-trimetoprim e anti-retrovirais. Após 20 dias, recebeu alta sem queixas pulmonares. Decorridos 15 dias, retornou com diarréia, febre, disfagia e emagrecimento importante. Foi a óbito após cinco dias, por sepse estafilocócica.

  18. Psicoterapia em grupo de pacientes com transtorno afetivo bipolar Group psychotherapy for bipolar disorder patients

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    Bernardo Carramão Gomes

    2007-01-01

    Full Text Available CONTEXTO: Vem crescendo nos últimos anos o número de estudos com abordagens psicoterápicas no tratamento de pacientes com transtorno afetivo bipolar. Contudo, pouco ainda se sabe sobre o efeito que tem estas abordagens nestes pacientes. OBJETIVO: Avaliar a efetividade da terapia de grupo no tratamento do transtorno afetivo bipolar. MÉTODO: Levantamento bibliográfico no Medline, Lilacs, PubMed e ISI de artigos publicados em língua inglesa no período de 1975 a 2005 e busca manual com base na bibliografia dos artigos selecionados. Foram incluídos apenas os estudos que utilizaram grupo controle, randomizados, com casuística superior a 20 pacientes. RESULTADOS: Foram encontrados somente cinco trabalhos, três deles publicados nos últimos cinco anos. Três utilizaram modelos de psicoeducação, encontrando aumento na adesão ao tratamento farmacológico. Um destes verificou redução no número de hospitalizações e recaídas. Dois estudos combinaram psicoeducação e abordagens psicoterápicas variadas. Destes, um mostrou aumento no período de remissão e redução de sintomas para episódios maníacos. CONCLUSÕES: O interesse por intervenções psicoterápicas baseadas em evidência, no tratamento do TAB, vem aumentando nos últimos anos. Este fato contrasta com o baixo número de estudos dedicados à modalidade de atendimento em grupo, que poderia ser muito útil em instituições onde grande número de pacientes é atendido. A complexidade clínica dessa doença, a presença de diversas comorbidades e os diferentes graus de adesão à farmacoterapia demandam a criação de opções terapêuticas variadas que atendam às necessidades de cada indivíduo. Os estudos indicam que a terapia de grupo pode ser uma opção eficaz de tratamento que merece ser mais bem investigado para permitir sua aplicação na prática clínica.BACKGROUND: In the last years there has been a growing number of studies using psychotherapy approaches in the

  19. Dosimetria de pacientes e médicos em intervenções coronárias percutâneas em Recife, Pernambuco, Brasil

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    Maria do Socorro Rocha da Silva

    2011-04-01

    Full Text Available OBJETIVO: Este trabalho teve como objetivo estimar os valores de doses de radiação recebidas por médicos e pacientes em procedimentos intervencionistas cardíacos realizados em um hospital público na cidade de Recife, Pernambuco. MATERIAIS E MÉTODOS: As medidas foram determinadas em 31 pacientes adultos, dos quais 22 tiveram acompanhamento clínico após o procedimento, e em dois cardiologistas com mais de dez anos de experiência. Parâmetros de irradiação para cada procedimento foram registrados. RESULTADOS: Os valores obtidos para a dose absorvida máxima na pele do paciente variaram entre 612 e 8.642 mGy, sendo que 53% foram maiores que 2.000 mGy, valores estes que podem causar efeitos determinísticos. Com relação aos médicos, a dose efetiva média por procedimento foi de 11 µSv e os valores médios do equivalente de dose nas extremidades, mais altos, foram: 923 µSv no pé esquerdo, 514 µSv no pé direito, 382 µSv na mão esquerda e 150 µSv no olho esquerdo. Dependendo do número de procedimentos, as doses recebidas pelos médicos podem exceder os valores limites de doses estabelecidos pelas normas nacionais e internacionais. CONCLUSÃO: Os resultados obtidos sinalizam a necessidade de adoção de estratégias para otimização da proteção radiológica tanto de pacientes quanto de médicos.

  20. Intervenção educacional de enfermagem para redução da hiperfosfatemia em pacientes em hemodiálise

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    Eniva Miladi Fernandes Stumm

    Full Text Available RESUMO Objetivo: avaliar a efetividade de uma intervenção educacional de enfermagem para redução da hiperfosfatemia em pacientes renais crônicos em hemodiálise. Método: estudo quase experimental realizado com 63 pacientes hiperfosfatêmicos em hemodiálise. A intervenção se constituiu em orientar e disponibilizar um manual impresso e ilustrado aos pacientes, contendo informações sobre o controle da doença. Foi solicitado o preenchimento de um checklist diário, com finalidade recordatória dos aspectos abordados no manual. Os exames laboratoriais e a intensidade do prurido foram analisados no início do estudo, aos 30 e 60 dias após a intervenção educacional. Resultados: a média de idade dos participantes foi 58±13,1 anos e tempo de tratamento de 51,1±44,7 meses. Houve redução dos valores séricos do fósforo de 7,06±1,43 para 5,80±1,53 (p<0,001 e da intensidade do prurido após a intervenção. Conclusão: a Intervenção Educacional de Enfermagem foi efetiva para redução do fosfato e diminuição do prurido nos pacientes hiperfosfatêmicos.

  1. Potenciais interações medicamentosas em pacientes com artrite reumatoide Potential drug interactions in patients with rheumatoid arthritis

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    Fabíola Bagatini

    2011-02-01

    Full Text Available INTRODUÇÃO: O termo polifarmácia, ou seja, a utilização concomitante de múltiplos fármacos pelo mesmo indivíduo vem sendo amplamente associado a pacientes institucionalizados e idosos, no entanto pode ocorrer em grupos de pacientes portadores de doenças crônicas como artrite reumatoide (AR. OBJETIVO: Quantificar a polifarmácia em um grupo de pacientes com AR e realizar um levantamento sobre o risco de potenciais interações indesejáveis entre os medicamentos utilizados no manejo dessa doença e os fármacos utilizados em enfermidades não crônicas. MÉTODOS: Realizou-se um estudo de coorte com 103 pacientes portadores de AR, atendidos no Componente Especializado da Assistência Farmacêutica/MS, Florianópolis/SC. Os pacientes foram acompanhados mensalmente, por meio de fichas. As interações medicamentosas foram identificadas pelo Drugdex System - Thomson Micromedex® - Interactions. RESULTADOS: Observou-se a presença de polifarmácia em 95,1% dos pacientes e de 19 potenciais interações indesejáveis entre os medicamentos utilizados por 74 pacientes, em média 3,0 ± 1,2 interações/paciente. Todas as potenciais interações estavam relacionadas a metotrexato. Omeprazol foi o principal representante, correspondendo a 29,3% delas, seguido por diclofenaco sódico (17,6% e dipirona sódica (13,2%. CONCLUSÃO: Considerando que este estudo confirma que a polifarmácia é uma prática comum na terapêutica dos pacientes portadores de AR, deve haver maior vigilância acerca de efeitos adversos ou de redução da efetividade de determinados fármacos devido às suas interações farmacológicasINTRODUCTION: The term polypharmacy, meaning the concomitant use of multiple medications by one individual, has been widely reported in institutionalized or elderly patients. It can, however, occur in patients with chronic diseases, such as rheumatoid arthritis (RA. OBJECTIVE: To quantify polypharmacy in a group of RA patients and to assess the

  2. Alterações retinianas apresentadas em pacientes portadores de hemoglobinopatia falciforme atendidos em um Serviço Universitário de Oftalmologia

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    Luiz Guilherme Azevedo de Freitas

    2011-10-01

    Full Text Available OBJETIVOS: Identificar os principais achados fundoscópicos em pacientes portadores de anemia falciforme encaminhados a um Serviço Oftalmológico de Referência em Goiânia (GO. MÉTODOS: Foram realizados exames oftalmológicos em 50 pacientes (100 olhos portadores de hemoglobinopatia falciforme para observar quais as alterações retinianas mais comuns nesse grupo. RESULTADOS: O tipo de hemoglobinopatia mais encontrado foi o SS, seguido pelas hemoglobinopatias SC, AS e Sthal. Dentro da amostra estudada, 22% apresentaram alterações retinianas. Destes 73% eram do sexo masculino. A alteração retinianas encontradas foram: "sea fan", "black sunburst", hemorragia vítrea e descolamento de retina. Em relação à classificação da retinopatia, 73% apresentaram a forma proliferativa, sendo vista nos tipos AS e SC e 27% apresentaram a forma não-proliferativa da retinopatia, sendo vista nos portadores do tipo SS. CONCLUSÃO: Foi observado elevado número de pacientes com alterações retinianas na amostra estudada, sendo o maior número em portadores da hemoglobinopatia SC, seguido dos grupos AS e SS. As alterações proliferativas foram as mais observadas. Hemorragia vítrea e descolamento de retina foram as manifestações proliferativas de maior prevalência e mostraram ser mais frequente em portadores da hemoglobinopatia SC na população estudada.

  3. Alterações dos hábitos de eliminação intestinal em pacientes hospitalizados

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    Lieselotte Marianne Harder

    1984-06-01

    Full Text Available O presente trabalho procurou estudar as possíveís alterações dos hábitos de eliminação intestinal, em pacientes hospitalizados nas unidades de Clínica Médica, de três hospitais gerais do Município de São Paulo. Procurou-se verificar os tipos de alterações apresentados e as causas que os pacientes atribuem às modificações ocorridas. A população do estudo constou de 200 pacientes, adultos, ambulantes, de ambos os sexos. Conseguiu-se obter os dados do estudo através de entrevista com aplicação de formulário. Os resultados obtidos, levaram às seguintes conclusões: a maioria dos pacientes entrevistados referiu alterações dos hábitos de eliminação intestinal após a internação hospitalar. As causas apontadas pelos pacientes como responsáveis pelas alterações dos hábitos de eliminação intestinal foram agrupadas em: fisiológicas, psicossociais, terapêuticas e ambientais. As fisiológicas e psicossociais foram as que mais se destacaram. A alimentação foi o fator mais apontado como causa fisiológica. Dos fatores psicossociais foram salientados o nervosismo, a ansiedade e a preocupação. Apenas a variável independente sexo, influenciou nas alterações dos hábitos de eliminação intestinal, na hospitalização, conforme resultado estatístico.

  4. Valor diagnóstico da cintilografia miocárdica em pacientes com doença coronariana multiarterial

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    Maria Eduarda Menezes de Siqueira

    2011-09-01

    Full Text Available FUNDAMENTO: A cintilografia de perfusão miocárdica (CPM é um método não invasivo extremamente útil na avaliação de isquemia em portadores de doença coronária. Entretanto, persiste o conceito de que não seria um bom método para os portadores de doença coronária multiarterial. OBJETIVO: Avaliar o valor da CPM com gated-SPECT na identificação de isquemia induzida por estresse farmacológico em pacientes com diagnóstico de doença coronária obstrutiva multiarterial. MÉTODOS: Foram incluídos 68 pacientes com diagnóstico de doença coronária obstrutiva multiarterial pela cineangiocoronariografia (cine para realização de CPM sob estímulo farmacológico com dipiridamol. Os exames foram analisados por dois médicos nucleares sem prévio conhecimento do resultado da cine. RESULTADOS: Dos pacientes, 64 (92,8% apresentaram alteração nas imagens de perfusão e 4 (7,2% apresentaram perfusão normal, sendo que, destes, três apresentaram alterações funcionais na análise do gated-SPECT. CONCLUSÃO: A CPM mostrou-se de grande valor na identificação de pacientes portadores de doença coronária multiarterial, já que a maioria dos pacientes apresentou alterações perfusionais sugestivas de isquemia.

  5. Estudio comparativo entre una prueba rápida y RT-PCR tiempo real en el diagnóstico de influenza AH1N1 2009 Comparative study of a rapid testing with real time RT-PCR for diagnosis of influenza AH1N1 2009

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    Luz Araceli Castro-Cárdenas

    2011-08-01

    Full Text Available OBJETIVO: Comparar la prueba QuickVue Influenza A+B empleando como estándar la RT-PCR tiempo real para influenza AH1N1 2009. MATERIAL Y MÉTODOS: Estudio retrospectivo-comparativo de 135 muestras de vías respiratorias de individuos sintomáticos para influenza procesadas de mayo 2009 a octubre 2010.Las pruebas citadas se realizaron simultáneamente. Se utilizó el software Confidence Interval Analysis 2000. RESULTADOS: Sensibilidad 62.96; especificidad 94.44; valor predictivo negativo 62.9; valor predictivo positivo 94.44; razón de probabilidad positiva 11.33 y razón de probabilidad negativa 0.39. Se calcularon intervalos de confianza a 95. DISCUSIÓN: Los valores obtenidos concuerdan con otros estudios donde la sensibilidad fluctúa de 50 a 70 y especificidad entre 90 y 95 por ciento. La prueba QuickVue Influenza A+B es rápida, simple y de menor costo que el RT-PCR tiempo real, útil para identificar el tipo de virus en brotes de influenza de una población determinadaOBJECTIVE: Compare QuickVue Influenza A+B test with real-time RT-PCR for the diagnosis of influenza AH1N1 2009. MATERIAL AND METHODS: Retrospective-comparative study of 135 respiratory specimens from individuals with symptoms of influenza processed from May 2009 to October 2010.The above mentioned tests were performed simultaneously. For statistic analysisthe softwareof Confidence IntervalAnalysis 2000 was used. RESULTS: The parameters obtained were: sensitivity 62.96; specificity 94.44; negative predictive value 62.9; positive predictive value 94.44; positive likelihood ratio 11.33; negative likelihood ratio 0.39. Confidence intervals to 95,were calculated to all of the above data. DISCUSSION: The test QuickVue InfluenzaA+B is a rapid,simple test,with lower cost than real-time RT-PCR useful for identifying the type of virus outbreaks of influenza in a given population.It correlates well with more specific test and similar reports.

  6. Baixa resposta da vacinação intradérmica contra hepatite B em pacientes incidentes em hemodiálise

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    Regina H. Medeiros

    2011-03-01

    Full Text Available INTRODUÇÃO: A hepatite B pode evoluir para cirrose e hepatocarcinoma. Sua prevalência estimada é de 3,2% em pacientes em hemodiálise (HD. A vacina para hepatite B (HB, quando aplicada por via intramuscular (IM em pacientes com insuficiência renal crônica fase V, frequentemente não induz produção adequada de anticorpos. A injeção intradérmica (ID foi sugerida como sendo o método de inoculação mais eficiente. OBJETIVO: Comparar a resposta imune à injeção IM ou ID da vacina em indivíduos em HD. PACIENTES E MÉTODOS: Trinta e um pacientes incidentes em HD foram randomizados alternativamente para vacinação contra HB via IM ou ID. Dezesseis foram designados aleatoriamente para receber vacina IM (40 mg/dose e 15 ID (4mg /dose. Os níveis de anticorpos de superfície do vírus da hepatite B, parâmetros hematimétricos, ureia sérica, e Kt/V foram avaliados mensalmente. Proteína-C reativa, paratormônio, ferritina, aminotransferases e albumina foram avaliados antes da inoculação inicial e seis meses após a mesma. RESULTADOS: Os níveis de uréia foram maiores no grupo ID (P(1 = 0,031; os níveis de ferritina foram mais elevados no IM (P(2 = 0,037. Houve tendência a aumento nos níveis de proteína C reativa no grupo ID. A avaliação do Comitê de Monitoramento de Segurança dos indivíduos expostos recomendou a suspensão do estudo já que a inoculação por via IM converteu 62,5% e a ID converteu apenas 13,3% dos pacientes expostos. CONCLUSÃO: Com a metodologia utilizada, os resultados da vacina contra HB aplicada por via ID foi inferior à inoculação IM. Tais resultados podem ser decorrentes das doses inoculadas ou de outros fatores, como inflamação.

  7. Retinocoroidite toxoplásmica em pacientes com AIDS e neurotoxoplasmose Toxoplasmic retinochoroiditis in patients with AIDS and neurotoxoplasmosis

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    Jane Mary Alves

    2010-04-01

    Full Text Available OBJETIVO: A neurotoxoplasmose é a alteração do sistema nervoso central mais frequente observada em pacientes com AIDS. A ocorrência de toxoplasmose ocular em neurotoxoplasmose ainda é pouco estudada. O objetivo deste estudo foi de investigar a ocorrência de retinocoroidite toxoplásmica, típica ou provável, em pacientes com AIDS e neurotoxoplasmose. MÉTODOS: Foi desenvolvido estudo prospectivo, tipo série de casos incluindo 70 pacientes, de ambos os sexos, com idade variando de 20 a 63 anos, internados nas enfermarias de três hospitais públicos da cidade do Recife, Pernambuco, com tais diagnósticos firmados segundo os critérios do CDC (1992, no período de janeiro a outubro de 2008. Os pacientes caracterizavam-se por: primeiro episódio de neurotoxoplasmose (65; 92,9% ou recidiva (5; 7,1%; desconhecimento de ter AIDS (23; 32,9%, contagem média de linfócitos T CD4 de 139,8 ± 3,04 células/mm³ e carga viral média igual a 137.080 ± 39.380 cópias/mL. Todos os pacientes foram submetidos a exame oftalmológico, consistindo de: inspeção ocular; aferição da acuidade visual; investigação da função muscular extrínseca ocular e fundoscopia, empregando oftalmoscópio indireto binocular (modelo OHN 3.5 (Eyetec® e lente externa de 20 dioptrias (Volk®. RESULTADOS: Foram diagnosticados 4 (5,7% pacientes com lesões cicatriciais de retinocoroidite, características de toxoplasmose ocular, sendo típica em 3 (75% pacientes e bilateral em 1. Não houve qualquer caso de retinocoroidite ativa, típica ou provável. As lesões oculares ativas foram raras comparadas às lesões cicatriciais, as quais se associam à presença de cistos na retina. CONCLUSÃO: Recomenda-se que mesmo lesões cicatriciais sejam valorizadas em pacientes com AIDS.PURPOSE: Neurotoxoplasmosis is the most common central nervous system disorder in patients with AIDS. The occurrence of ocular toxoplasmosis in neurotoxoplasmosis is not well studied. The objective of

  8. Escore de cálcio na avaliação da aterosclerose em pacientes com HIV/AIDS

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    Verônica Soares Monteiro

    2011-11-01

    Full Text Available FUNDAMENTO: A terapia antirretroviral aumentou drasticamente a expectativa de vida em pacientes com HIV/AIDS, embora a aterosclerose esteja associada a uma terapia de longo prazo. OBJETIVO: Investigar a prevalência de aterosclerose em pacientes com AIDS submetidos à terapia antirretroviral e a influência de tratamentos de diferentes regimes e durações. MÉTODOS: Pacientes com HIV/AIDS foram abordados durante consultas de rotina. Aqueles que estiveram em terapia antirretroviral por, pelo menos, dois anos tiveram o sangue coletado para análise do perfil lipídico e da glicemia em jejum e foram submetidos à tomografia computadorizada cardíaca para quantificação do escore de cálcio dentro de seis dias, no máximo. A aterosclerose foi definida como escore de cálcio maior que zero (CAC > 0. Fatores de risco tradicionais, síndrome metabólica e o escore de Framingham foram analisados. RESULTADOS: Cinquenta e três pacientes realizaram tomografia computadorizada cardíaca: 50,94% eram do sexo masculino, com idade média de 43,4 anos; 20% tinham hipertensão; 3,77% tinham diabetes; 67,92% tinham hipercolesterolemia; 37,74% tinham hipertrigliceridemia; 47,17% tinham HDL baixo; 24,53% atenderam aos critérios para síndrome metabólica; 96,23% foram classificados no escore de Framingham como "baixo risco"; e 18,87% eram tabagistas. A duração média do tratamento antirretroviral foi de 58,98 meses. A aterosclerose coronária ocorreu em 11 pacientes (20,75%. A duração da terapia antirretroviral não se relacionou à aterosclerose (p = 0,41, e não houve diferenças significativas entre os diferentes esquemas antirretrovirais (p = 0,71. Entre os fatores de risco tradicionais, o tabagismo (OR = 27,20; p = 0,023 e a idade (OR = 20,59; p = 0,033 foram significativos na presença de aterosclerose. Havia tendência para uma associação positiva da aterosclerose com a hipercolesterolemia (OR = 8,30; p = 0,0668. CONCLUSÃO: Os fatores associados

  9. Estudo clínico e padrão de herança em pacientes com retinose pigmentar

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    Ana Cristina Cotta de Queiroz

    2013-02-01

    Full Text Available OBJETIVO: Realizar análise epidemiológica de pacientes com retinose pigmentar (RP, caracterizando aspectos clínicos da doença e o padrão de herança encontrado em nosso meio, de acordo com a presença ou não de síndrome de Usher. MÉTODOS: Foram estudados 155 pacientes com RP, tendo sido a amostra dividida em 2 grupos: grupo 1 (n=130, com pacientes diagnosticados com RP clássica, sem associação com alterações sistêmicas; e grupo 2 (n=25, com pacientes diagnosticados com Síndrome de Usher (USH. Foram caracterizados aspectos clínicos da doença (sexo, idade, sintomas oculares, acuidade visual, alterações do segmento anterior e posterior e alterações em exames complementares e o padrão de herança encontrado. Os dados foram obtidos através de anamnese, exame oftalmológico completo e exames subsidiários (campo visual manual, eletrorretinograma, retinografia simples e fluorescente, no período de fevereiro de 2003 a dezembro de 2009. Foi utilizado o programa SPSS versão 13.0 para análise dos dados estatísticos. RESULTADOS: A herança autossômica recessiva foi a forma mais comumente encontrada (76,2% no grupo 1, mas em proporção maior do que a de outros trabalhos da literatura. Um menor número de casos com padrão recessivo ligado ao X (1,5% também foi notado no grupo 1. Não houve diferença estatisticamente significante entre as características clínicas entre os dois grupos. CONCLUSÃO: O padrão de herança encontrado nos pacientes com RP clássica foi similar ao encontrado em outros trabalhos. As características clínicas foram semelhantes nos dois grupos estudados.

  10. Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

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    Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji

    2015-07-01

    We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.

  11. Uso do ultra-som para punção venosa central em paciente obeso com adenomegalia cervical

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    Jaderson Wollmeister

    2008-08-01

    Full Text Available JUSTIFICATICA E OBJETIVOS: As técnicas clássicas para punção venosa central são realizadas com base em referências anatômicas de superfície e conhecimento da anatomia vascular da região em que se realizará a punção. O uso do ultra-som permite a realização da punção sob visão direta das estruturas vasculares, peri-vasculares e da agulha de punção. O objetivo deste relato foi descrever o uso do ultra-som no auxílio de acesso venoso central em paciente obeso e com adenomegalias. RELATO DO CASO: Paciente do sexo masculino, branco, 28 anos, 1,70 m, 120 kg, com diagnóstico de linfoma de Hodgkin esclerose nodular. Solicitado ao Serviço de Anestesiologia do Hospital Governador Celso Ramos, punção de veia jugular interna direita guiada por ultra-som devido à presença de gânglio supraclavicular que prejudicava a referência anatômica de punção e à obesidade do paciente. Após a obtenção da melhor imagem a veia jugular interna esquerda foi puncionada e colocado um cateter venoso de triplo lúmen. A punção foi única, com progressão fácil do cateter e realizada sem complicações. CONCLUSÕES: O uso da ultra-sonografia para punção venosa central pode evitar complicações tornando o procedimento mais seguro para o paciente.

  12. Incidência de infecções por enterobactérias resistentes a carbapenêmicos em pacientes previamente colonizados

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    Paula Lüttjohann Rodrigues

    2015-09-01

    Full Text Available Introdução: Nos últimos anos, a incidência mundial de enterobactérias resistentes a carbapenêmicos (ERC tem aumentado. A disseminação de ERC é preocupante considerando as limitações terapêuticas e a alta mortalidade de infecções causadas por essas bactérias. O objetivo deste estudo foi determinar a incidência de infecções por ERC em pacientes previamente colonizados. Métodos: Estudo de coorte, retrospectivo, realizado em hospital universitário de Porto Alegre, no período de janeiro a dezembro de 2014. Foram incluídos todos os pacientes com coleta de swab retal para pesquisa de ERC. Foram considerados infectados pacientes com isolado de ERC em amostra clínica que apresentaram swab retal prévio positivo para a mesma espécie. A mortalidade bruta foi analisada em 30 dias após o isolamento de ERC. Resultados: Foram coletados 2.733 swabs retais, sendo 78 (2,85% destes positivos para ERC (pacientes colonizados. Um total de 33% (26/78 dos pacientes colonizados também apresentaram ERC em amostra clínica. O tempo médio entre o isolamento de ERC em swab (colonização e em material clínico (infecção foi de 14,9 dias (± 13,6. A maior parte das amostras clínicas positivas para ERC foi em urina (46,1%; 12/26. A mortalidade foi de 61% (16/26 nos pacientes colonizados e com infecção versus 46% (24/52 nos pacientes colonizados que não desenvolveram infecção. Conclusão: Nossos dados demonstram que 1/3 de pacientes colonizados desenvolveram infecção por ERC. Foi possível observar uma mortalidade maior em pacientes infectados em relação a pacientes apenas colonizados por ERC.

  13. Hearing loss in patients with diabetes mellitus Perdas auditivas em pacientes portadores de diabetes melito

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    Thiago Hernandes Diniz

    2009-08-01

    Full Text Available The relationship between diabetes mellitus and hearing loss is described as 'controversial' in the literature, given that in the clinical realm many patients present dysacusis while others do not. AIM: this study aims to investigate the relationship between hearing loss and diabetes mellitus and add to the knowledge being developed in this area. STUDY DESIGN: cross-sectional clinical trial. MATERIALS AND METHOD: in our study we analyzed the medical charts of 50 adult patients of both genders, aged above 45 years, and diagnosed with diabetes mellitus, seen in our institution in 2006 and 2007, and compared them to the charts of 50 metabolic disease-free individuals. RESULTS: this study found statistically significant worse audiometric thresholds among patients with diabetes mellitus when compared to patients in the control group. CONCLUSION: the results identified in this study point to a correlation between hearing loss and diabetes mellitus. This possibility should therefore be further investigated by health care workers providing care to patients with diabetes mellitus, in the form of closer follow-up on the auditory health of this patient group.A literatura especializada descreve que a relação causa e efeito entre diabetes melito e perda auditiva é bastante controversa, visto que, na prática clínica, muitos pacientes apresentam disacusia e outros não. OBJETIVO: Investigar a relação entre perda auditiva e diabetes melito, a fim de contribuir para resultados mais precisos sobre o assunto em pauta. FORMA DE ESTUDO: Estudo clínico com coorte transversal. MATERIAL E MÉTODO: No presente estudo foram analisados os prontuários de 50 indivíduos adultos atendidos nos anos de 2006 e 2007, de ambos os sexos, com idade superior a 45 anos, portadores do diabetes melito, e 50 prontuários de indivíduos que não apresentaram doenças metabólicas. RESULTADOS: A presente pesquisa identificou uma piora dos limiares audiométricos dos pacientes com

  14. Development of tailored real-time RT-PCR assays for the detection and differentiation of serotype O, A and Asia-1 foot-and-mouth disease virus lineages circulating in the Middle East

    DEFF Research Database (Denmark)

    Reid, Scott M.; Mioulet, Valerie; Knowles, Nick J.

    2014-01-01

    transcription polymerase chain reaction (rRT-PCR) assays using primer/probe sets designed from the VP1 coding region of the virus genomes were developed for the specific detection of serotype O, A and Asia-1 FMD viruses (FMDVs) circulating in the Middle East. These assays were evaluated using representative...... by the generic rRT-PCR diagnostic assays but negative by virus isolation and antigen-detection ELISA which would otherwise have to be serotyped by nucleotide sequencing. A similar approach could be used to develop serotyping assays for FMDV strains circulating in other regions of the world....

  15. Significados e práticas da espiritualidade no contexto dos cuidados paliativos em pacientes oncológicos adultos

    Directory of Open Access Journals (Sweden)

    Denis Iaros Silva da Silva

    2011-10-01

    Full Text Available Objetivou-se conhecer os significados e práticas da espiritualidade para os pacientes oncológicos adultos, enfermeiros e família no contexto dos cuidados paliativos. Elaborou-se uma revisão integrativa nas bases de dados CINAHL, SciELO, LILACS e MEDLINE, com os descritores palliative care, spirituality e nursing, no período de novembro de 2007 a novembro de 2009. A amostra constituiu-se de 11 artigos. Dentre os significados que se destacaram, para os pacientes e enfermeiros evidenciou-se fonte de conforto e para a família fonte de cura e manutenção da saúde. Dentre as práticas evidenciadas encontram-se, para os pacientes, ir a igreja, para os enfermeiros, estar presente e, para a família, exercício da caridade. Diferentes significados e práticas reveladas neste estudo evidenciam uma gama de possibilidades que poderão ser aplicadas na prática clínica. Conclui-se que a espiritualidade pode ajudar pacientes, familiares, enfermeiros e demais profissionais a enfrentarem melhor as situações de iminência de fim da vida ou mesmo a morte em si.

  16. Ensino odontológico para pacientes especiais e gestão em saúde.

    Directory of Open Access Journals (Sweden)

    Sérgio Spezzia

    2017-08-01

    Full Text Available As pessoas com necessidades especiais constituem uma população heterogênea portadora de grande variedade de deficiências físicas, mentais, neurológicas ou sociais. Sob o ponto de vista odontológico são aqueles indivíduos que necessitam de cuidados especiais por tempo indeterminado ou por parte de sua vida. São todos aqueles que apresentam condições debilitantes, que acarretam uma atenção maior por parte do cirurgião dentista. Muito se questiona sobre a forma adequada de manejo destes indivíduos e poucos são os profissionais interessados em suprir as suas carências odontológicas. O objetivo deste artigo foi verificar como procede a ministração da disciplina de pacientes especiais nos cursos de graduação em Odontologia e averiguar como estão sendo feitas as adequações no Sistema Único de Saúde (SUS, no intuito de viabilizar os atendimentos odontológicos prestados a esse público de pacientes através de capacitação profissional. Medida posta em prática para resolução dos problemas de insuficiência profissional no atendimento dos pacientes especiais pelos cirurgiões dentistas, foi a de promover a Capacitação de Profissionais da Odontologia Brasileira Vinculados ao SUS para a Atenção e o Cuidado da Saúde Bucal da Pessoa com Deficiência. Concluiu-se que o aprimoramento das grades curriculares dos cursos de graduação em Odontologia, no que tange a oferta da disciplina de pacientes especiais possui condições de propiciar meios para cobertura da demanda por atendimentos, ao mesmo tempo que pode possibilitar menores gastos e custos públicos voltados para capacitação dos cirurgiões dentistas atuantes em unidades assistenciais públicas, viabilizando a gestão pública em saúde realizada no SUS com menores esforços.

  17. In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

    International Nuclear Information System (INIS)

    Haupt, Larisa M; Thompson, Erik W; Trezise, Ann EO; Irving, Rachel E; Irving, Michael G; Griffiths, Lyn R

    2006-01-01

    Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) meth