Antiviral cellular therapy for enhancing T-cell reconstitution before or after hematopoietic stem cell transplantation (ACES): a two-arm, open label phase II interventional trial of pediatric patients with risk factor assessment.

Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.

Congenital CMV infection in a Brazilian neonatal intensive care unit: high prevalence among twin newborns.

BACKGROUND: Cytomegalovirus (CMV) is one of the most important pathogens associated with congenital infection worldwide. Most congenital CMV-infected infants are asymptomatic at birth; however, some can develop delayed sequelae, especially hearing loss. METHODS: This study aimed to investigate the prevalence of congenital CMV infection in a neonatal intensive care unit in a low-income region of Brazil. The objectives extended to identifying associated factors, assessing the clinical status of infected newborns, and undertaking a two-year follow-up to discern potential long-term consequences in the affected infants. This cross-sectional prospective study enrolled newborns up to three weeks of life requiring intensive medical care. We employed a convenience sampling method to include 498 newborns and 477 mothers in the study. Categorical variables underwent analysis employing Fisher's exact test, whereas the examination of continuous variables involved the Mann‒Whitney test. RESULTS: CMV DNA was detected in saliva/urine samples from 6 newborns (1.21%), confirming congenital infection. We noted a significantly greater incidence (OR: 11.48; 95% CI: 2.519-52.33; p = 0.0094) of congenital infection among twins (7.14%) than among nontwins (0.66%). The twin patients exhibited discordant infection statuses, suggesting that only one of the babies tested positive for CMV. Most of the infected children were born to mothers who initiated sexual activity at a younger age (p = 0.0269). Only three out of the six newborns diagnosed with CMV infection underwent comprehensive clinical assessments and received continuous follow-up until they reached two years of age. Only one of the children had weight and height measurements below the norm for their age, coupled with developmental delays. CONCLUSIONS: The prevalence of congenital CMV infection among newborns admitted to the NICU was low and similar to that in the general population. However, we found a significantly greater incidence of congenital CMV infection in twins than in singletons. Interestingly, the twin-infected patients exhibited discordant infection statuses, suggesting that CMV was present in only one of the babies. We also found that most of the infected children were born to mothers who initiated sexual activity at a younger age. Diagnostic accessibility and comprehensive surveillance programs are imperative for effectively managing and preventing congenital CMV infections.

Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia.

Severe combined immunodeficiency (SCID) is characterized by a severe deficiency in T cell numbers. We analyzed data collected (n = 307) for PHA-based T cell proliferation from the PIDTC SCID protocol 6901, using either a radioactive or flow cytometry method. In comparing the two groups, a smaller number of the patients tested by flow cytometry had <10% of the lower limit of normal proliferation as compared to the radioactive method (p = 0.02). Further, in patients with CD3+ T cell counts between 51 and 300 cells/µL, there was a higher proliferative response with the PHA flow assay compared to the 3H-T assay (p < 0.0001), suggesting that the method of analysis influences the resolution and interpretation of PHA results. Importantly, we observed many SCID patients with profound T cell lymphopenia having normal T cell proliferation when assessed by flow cytometry. We recommend this test be considered only as supportive in the diagnosis of typical SCID.

Avaliação da implantação da Rede de Cuidados à Pessoa com Deficiência no Sistema Único de Saúde: um estudo de casos múltiplos / Evaluation of the implementation of the Care Network for People with Disabilities in the Brazilian Unified National Health System: a multiple case study / Evaluación de la implementación de la Red de Atención a Personas con Discapacidad en el Sistema Único de Salud: un estudio de caso múltiple

Resumo: A Rede de Cuidados à Pessoa com Deficiência (RCPCD) foi implantada em 2012 como desdobramento das ações do Plano Viver sem Limite, sendo objeto de pesquisas recentes, entretanto, não foram encontrados estudos de avaliação do grau de implantação dessa rede. O objetivo deste artigo foi avaliar o grau de implantação da RCPCD em oito estados nas cinco regiões geográficas brasileiras. Realizou-se estudo de casos múltiplos mediante pesquisa avaliativa do grau de implantação da RCPCD nos estados/casos: Amazonas, Bahia, Espírito Santo, Mato Grosso do Sul, Minas Gerais, Paraíba, Rio Grande do Sul e São Paulo. Para tanto, desenvolveu-se um modelo lógico da política e uma matriz de medidas. O grau de implantação de sete estados foi classificado como moderado, somente o Amazonas obteve grau de implantação incipiente. Foram identificadas diferenças importantes na avaliação de cada fase desse processo, as fases de diagnóstico regional e adesão à rede obtiveram grau de implantação de moderado a avançado na maioria dos estados. Na fase da contratualização dos serviços, nenhum estado alcançou o grau avançado de implantação e a fase de acompanhamento e monitoramento da RCPCD basicamente não ocorreu em todos os estados. A matriz de medidas permite avaliar o grau de implantação da RCPCD. Ainda, o reconhecimento dos resultados do grau de implantação pelos grupos condutores e áreas técnicas estaduais referendou o uso desse instrumento. Salienta-se a necessidade de ações para o aprimoramento dessa implantação, tais como: fortalecer a regionalização, instituir grupos condutores regionais, garantir mecanismos de contratualização e definir critérios para certificação dos pontos de atenção.

Abstract: The Care Network for People with Disabilities (RCPCD) was implemented in 2012 as a consequence of the actions of the Viver sem Limite (Living without Limits) plan and has been the research object of recent studies. However, no published studies address the degree of implementation of this network. This study aimed to evaluate the degree of implementation of the RCPCD in eight states in the five regions of Brazil. This multiple case study performed evaluative research of the degree of implementation of the RCPCD in the states of Amazonas, Bahia, Espírito Santo, Mato Grosso do Sul, Minas Gerais, Paraíba, Rio Grande do Sul, and São Paulo. A logical model of the policy and a measurement matrix were developed. The degree of implementation of seven states was moderate, but Amazonas had an incipient degree of implementation. The evaluation of each stage of the process showed important differences, as the regional diagnosis and network adhesion stages presented moderate to advanced degrees of implementation in most states. In the service contracting stage, no state had an advanced degree of implementation, and the stage of follow-up and monitoring of the RCPCD was not even reached. The measurement matrix helps evaluate the degree of implementation of the RCPCD, and, by recognizing its results, the state steering groups and technical area recommend its use. Actions to improve this implementation, such as strengthening regionalization, establishing regional steering groups, ensuring contracting mechanisms, and defining criteria for certification of the care points, are necessary.

Resumen: La Red de Atención a Personas con Discapacidad (RCPCD) se implementó en 2012 en el Sistema Único de Salud como resultado de las acciones del Plan Vivir sin Límite, y es objeto de una investigación reciente; sin embargo, no hay estudios de evaluación del grado de implementación de esta red. El objetivo de este estudio fue evaluar el grado de implementación de la RCPCD en ocho estados de las cinco regiones geográficas brasileñas. Se realizó un estudio de caso múltiple mediante una investigación evaluativa sobre el grado de implementación de la RCPCD en los estados/casos: Amazonas, Bahia, Espírito Santo, Mato Grosso do Sul, Minas Gerais, Paraíba, Rio Grande do Sul y São Paulo. Para ello, se elaboraron un modelo lógico de la política y una matriz de medidas. El grado de implementación de siete estados se clasificó como moderado, solamente Amazonas obtuvo un grado de implementación incipiente. Se identificaron diferencias importantes en la evaluación de cada fase de este proceso, las fases de diagnóstico regional y adhesión a la red lograron un grado de implementación de moderado a avanzado en la mayoría de los estados. En la fase de contratación de los servicios, ningún estado alcanzó el nivel avanzado de implementación, y la fase de seguimiento y monitoreo de la RCPCD básicamente no se realizó en todos los estados. La matriz de medidas permite evaluar el grado de implementación de la RCPCD, y el reconocimiento de los resultados del grado de implementación por parte de los grupos de dirección y el área técnica de los estados avaló su uso. Son necesarias más acciones para mejorar esta implementación, tales como: fortalecer la regionalización, establecer grupos de dirección regional, garantizar mecanismos de contratación y definir criterios para la certificación de puntos de atención.

Cacipacore virus as an emergent mosquito-borne Flavivirus

Abstract INTRODUCTION: Cacipacore virus (CPCV), a possible bird-associated flavivirus, has yet to be detected in mosquitoes. Our purpose is examining CPCV in mosquitoes from the Amazon region of Brazil. METHODS: Approximately 3,253 Culicidae (grouped into 264 pools) were collected from the Amazon region during 2002-2006 and analyzed using a Flavivirus genus-specific reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assay and by nucleotide sequencing of amplicons. RESULTS: Nucleotide sequences from five mosquito samples showed high similarity to the those of CPCV originally isolated in the Amazon region. CONCLUSIONS: This is the first report of CPCV-infected mosquitoes which has implications on the arbovirus maintenance in nature and transmission to man.

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

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

A real-time reverse transcriptase polymerase chain reaction for detection and quantification of Vesiculovirus

Vesiculoviruses (VSV) are zoonotic viruses that cause vesicular stomatitis disease in cattle, horses and pigs, as well as sporadic human cases of acute febrile illness. Therefore, diagnosis of VSV infections by reliable laboratory techniques is important to allow a proper case management and implementation of strategies for the containment of virus spread. We show here a sensitive and reproducible real-time reverse transcriptase polymerase chain reaction (RT-PCR) for detection and quantification of VSV. The assay was evaluated with arthropods and serum samples obtained from horses, cattle and patients with acute febrile disease. The real-time RT-PCR amplified the Piry, Carajas, Alagoas and Indiana Vesiculovirus at a melting temperature 81.02 ± 0.8ºC, and the sensitivity of assay was estimated in 10 RNA copies/mL to the Piry Vesiculovirus. The viral genome has been detected in samples of horses and cattle, but not detected in human sera or arthropods. Thus, this assay allows a preliminary differential diagnosis of VSV infections.

Molecular and clinical evaluation of the acute human parvovirus B19 infection: comparison of two cases in children with sickle cell disease and discussion of the literature

Human parvovirus B19 is a well-known cause of severe conditions in patients with sickle cell disease, but the molecular mechanisms of the infection are insufficiently understood. The different clinical outcome of the acute parvovirus B19 infection in two pediatric patients with sickle cell disease has been examined. One of them developed life-threatening condition requiring emergency transfusions, while the other had asymptomatic infection, diagnosed occasionally. Both cases had high viral load and identical subgenotype, indicating that the viral molecular characteristics play a minimal role in the infection outcome.

Tradução e adaptação cultural para a língua portuguesa do questionário scoring of patellofemoral disorders: estudo preliminar / Translation and cross-cultural adaptation of the scoring of patellofemoral disorders into portuguese: preliminary study

OBJETIVO: Traduzir e adaptar culturalmente para língua portuguesa o questionário Scoring of Patellofemoral Disorders. MÉTODOS: 40 participantes foram selecionados entre fisioterapeutas e indivíduos leigos. O procedimento de tradução para língua portuguesa foi baseado em métodos padronizados. A escala original passou por 7 etapas até se obter a versão final em português da Escala de Desordens Patelofemorais. Em cada teste participaram 40 indivíduos, sendo 20 indivíduos leigos e 20 fisioterapeutas. O nível de não compreensão aceitável foi de até 10 por cento dos entrevistados. RESULTADOS: No 1º teste apenas as questões 3 não foram compreendidas por mais de 10 por cento dos participantes entrevistados, ocasionando a reaplicação do questionário. Já no 2° teste, apenas duas questões foram compreendidas por 90 por cento dos entrevistados e as demais questões compreendidas por mais de 90 por cento, não ocorrendo dúvidas entre os fisioterapeutas. Utilizou-se então a 2ª versão em português como versão final para a Escala de Desordens Patelofemorais. CONCLUSÃO: A escala Scoring of Patellofemoral Disorders foi traduzida e a adaptada culturalmente para língua portuguesa, tendo como título em português, Escala de Desordens Patelofemorais. Nível de Evidência II. Estudos diagnósticos, Investigação de um exame para diagnóstico.

OBJECTIVE: The aim of this study was to translate and culturally adapt the questionnaire Scoring of Patellofemoral Disorders for the Portuguese language.METHODS: 40 participants were selected, including physiotherapists and lay individuals. The process of translating the questionnaire into Portuguese was based on standardized methods. The original scale passed through seven stages, before reaching the final version in Portuguese. 40 subjects took part in each test: 20 lay individuals and 20 physiotherapists. The level acceptable of non-comprehension was up to 10% of the interviewees.RESULTS: In the first test, only three questions were not understood by more than 10% of the subjects interviewed, leading to a reapplication of the questionnaire. In the second test, only two questions were understood by 90% of the interviewees, while the remaining questions were understood by more than 90% of the interviewees, and there were no doubts among the physiotherapists. The 2nd version of the test was therefore selected as the final Portuguese version of Scoring of Patellofemoral Disorders.CONCLUSION: The Scoring of Patellofemoral Disorders scale was translated and adapted culturally for the Portuguese language, with title, in Portuguese, of Escala de Desordens Patelofemorais. Level of Evidence: Level II, development of diagnostic criteria on consecutive patients.

Padronização e uso de um método imunoenzimático que utiliza células infectadas como antígeno no diagnóstico rotineiro do dengue / Standardization and use of an immunoenzymatic method using infected cells as antigens in routine diagnosing of dengue

INTRODUÇÃO: Este trabalho mostra a padronização e o uso do método imunoenzimático utilizando células infectadas como antígeno (EIA-ICC) no diagnóstico sorológico rotineiro do dengue. MÉTODOS: Na otimização do teste, com a dose de 1.000 TCID50 de vírus do dengue tipo 3 (DENV-3), foram utilizadas 100.000 células C636 infectadas 1000 TCID50 (DENV-3). RESULTADOS: Os resultados obtidos com EIA-ICC foram comparados com o kit comercial de dengue HUMAN. Os resultados foram altamente coincidentes; o EIA-ICC mostrou-se moderadamente sensível e com alta especificidade. O teste foi usado no diagnóstico sorológico de 1.797 amostras sorológicas de casos suspeitos de dengue durante a epidemia de Ribeirão Preto, em 2006. Na avaliação sorológica, 228 amostras foram positivas para IgM contra DENV-3, e 235 amostras foram positivas para IgG contra DEV-3, e em 35 amostras detectou-se positividade para IgM e IgG. CONCLUSÕES: O EIA-ICC mostrou-se confiável e simples sendo adequado ao diagnóstico sorológico do dengue.

INTRODUCTION: This paper show the standardization and use of the immunoenzymatic method using infected cells as antigens (EIA-ICC) for routine serological diagnosing of dengue. METHODS: In optimizing the test, a dose of 1,000 TCID50 of dengue type 3 virus (DENV-3) was used, and 100,000 C636 cells infected with 1,000 TCID50 (DENV-3) were used. RESULTS: The results obtained with EIA-ICC were compared with the HUMAN commercial dengue kit. The results were highly concordant. The EIA-ICC showed moderate sensitivity and high specificity. The test was used for serologically diagnosing 1,797 blood samples from suspected dengue cases during the 2006 epidemic in Ribeirao Preto. From the serological evaluation, 228 samples were positive for IgM against DENV-3; 235 samples were positive for IgG against DENV-3; and 35 samples were positive for both IgG and IgM. CONCLUSIONS: EIA-ICC was shown to be reliable and simple, and suitable for serologically diagnosing dengue.

Conhecimento de fisioterapeutas sobre a atuação em suporte básico de vida / Physical therapists' knowledge on basic life support

O estudo visou avaliar o conhecimento de fisioterapeutas e graduandos em Fisioterapia sobre diagnóstico e atendimento de urgência à parada cardiorrespiratória. A amostra foi composta de 72 estudantes e 108 fisioterapeutas, dos quais 64 atuam no ambiente extra-hospitalar e 44 no ambiente hospitalar. Foi aplicado aos participantes um questionário sobre ressuscitação cardiopulmonar (RCP) baseado nas diretrizes da American Heart Association de 2005. As respostas foram analisadas estatisticamente. Quanto ao diagnóstico da parada cardíaca, os grupos comportaram-se de maneira semelhante, optando pela avaliação da “presença de pulso e respiração”. Quanto à seqüência de atendimento da RCP, a seqüência preconizada foi corretamente indicada por 52,8% do grupo estudante, 65,9% do subgrupo hospitalar e 40,6% do subgrupo extra-hospitalar. Quanto à relação compressão/ventilação, apenas 4,1% do grupo estudante, ninguém do extra-hospitalar e 25% do subgrupo hospitalar indicaram a relação preconizada atualmente. Quase todos (94%) os participantes reconheceram a importância do conhecimento em RCP para o fisioterapeuta. Assim, a maioria dos atuais e futuros fisioterapeutas reconhecem a importância da RCP para sua atuação profissional, mas têm conhecimento insuficiente sobre o tema e apenas uma pequena parcela busca atualizar-se...

This study aimed at assessing undergraduates' and physical therapists' knowledge on diagnosing and emergency treating cardiopulmonary arrest. Subjects were 72 students and 108 physical therapists – of which 64 were active in non-hospital environment and 44 in hospitals – who answered a questionnaire on cardiopulmonary resuscitation (CPR) based on the American Heart Association 2005 guidelines. Answers were statistically analysed. As to diagnosing cardiopulmonary arrest, groups behaved similarly, having chosen the option “absence of consciousness, pulse and breath”. Concerning the pattern of CPR assistance, the recommended ABCD sequence was recognized by 52.8% of the students, 65.9% of the hospital subgroup, and 40.6% of the non-hospital subgroup; nobody of the latter, only 4.1% of the students, and 25% of hospital professionals indicated the currently recommended compression/ventilation ratio. Almost all (94%) participants acknowledged the importance of CPR knowledge in physical therapy practice. Thus, though most current and future physical therapists recognize the importance of CPR for their professional role, they have insufficient knowledge about the subject and only few of them seek to update their knowledge...

The use of qualitative and quantitative polymerase chain reactions for diagnosis of cytomegalovirus infections in bone marrow and kidney transplant recipients

O objetivo deste trabalho foi testar uma PCR qualitativa e uma PCR semiquantitativa para CMV para determinar a carga de CMV nos leucócitos de pacientes transplantados de medula óssea e transplantados de rim. Trinta e três pacientes TMO e 35 TR participaram deste estudo. O DNA foi testado pela PCR qualitativa utilizando primers que amplificam parte do gene gB de CMV. As cargas de CMV das amostras positivas foram determinadas pela PCR semi-quantitativa utilizando como controle plasmídios quantificáveis inseridos com parte do gene gB de CMV. A sensibilidade do teste foi de 867 plamídios/µg DNA. Cargas de CMV entre 2.118 e 72.443 copias/µg DNA foram observadas em 12,1 por cento dos TMO entre 1,246 e 58,613 cópias/µg DNA foram observadas em 22,9 por cento dos TR. Futuros estudos, com maiores casuísticas são necessários para confirmar a utilidade desta PCR semiquantitativa para CMV em pacientes transplantados.

Desenvolvimento de uma metodologia de PCR semiquantitativa utilizando plasmídio clonado com parte do gene gB de citomegalovírus / Development of a semi-quantitative PCR method using a plasmid cloned with part of the gB gene of cytomegalovirus

A infecçäo por citomegalovírus é disseminada em nosso meio e costuma acometer, com significante morbimortalidade, indivíduos imunodeprimidos, especialmente, transplantados de medula óssea e de rim bem como pacientes com AIDS. Neste trabalho, descrevemos o desenvolvimento de uma PCR semiquantitativa para detectar e quantificar cargas de CMV, presentes em materiais clínicos. Para tanto, inserimos em plasmídios PCR II (Invitrogen, USA), o fragmento com 296 pares-base do gene da glicoproteína B do CMV. Os plasmídios com inserto foram transfectados em Escherichia coli, multiplicados, verificados quanto à presença do inserto por seqüenciamento nucleotídico, purificados, e quantificados. Os plasmídios com inserto foram titulados em diluições decimais e as mesmas foram submetidas à PCR descrita anteriormente, que foi, também, utilizada nos testes semiquantitativos, permitindo determinar a sensibilidade da técnica, 867 cópias de CMV / mg de DNA. Com base nas densidades das bandas eletroforéticas dos amplicons de amostras clínicas, comparadas às da titulaçäo de plasmídios contendo inserto de glicoproteína B do CMV, obtivemos as cargas virais. A técnica de PCR semiquantitativa, por nós padronizada, tem, como vantagens, o baixo custo e o fácil manuseio após sua padronizaçäo, podendo ser testada na detecçäo da carga de CMV em diferentes tipos de pacientes com suspeita de citomegalovirose

Diagnosis of cytomegalovirus infections by qualitative and quantitative PCR in HIV infected patients

A high incidence of cytomegalovirus (CMV) infections is observed in Brazil. These viruses are causatives of significant morbidity and mortality among patients with advanced human immunodeficiency virus (HIV) infection. This work, shows the application of a PCR on determination of CMV load in the buffy coat and plasma. We analyzed the samples of 247 HIV infected patients in order to diagnose CMV infection and disease. We developed a semi-quantitative PCR that amplifies part of the glycoprotein B (gB) gene of CMV. The semi-quantitative PCR was carried out only in positive clinical samples in a qualitative PCR confirmed by a nested-PCR. CD4 lymphocyte count, HIV viral load and CMV disease symptom were correlated with CMV load. CMV genome was detected in the buffy coat of 82 of 237 (34.6 percent) patients, in 10 of these the CMV load was determined varying between 928 and 332 880 viral copies/mug DNA. None of these 237 patients developed any suggestive manifestation of CMV disease. For the other 10 HIV infected patients selected based on the suspicion of CMV disease, CMV genome was detected in only one case. This patient presented a high CMV load, 8 000 000 copies/mug DNA, and developed a disseminated form of CMV disease including hepatitis and retinitis. Our results were greatly influenced by the impact of the highly active antiretroviral therapy that reduced incidence of CMV viremia and occurrence of CMV disease in the HIV infected patients

Amplificación por PCR de una secuencia genomica de 272-pb que permite la diferenciación entre Trypanosoma cruzi y Trypanosoma rangeli / Annual reports 1994 / PCR amplification of a 272-bp genome sequence differentiates between Trypanosoma cruzi and Trypanosoma rangeli

La reacción en cadena de la polimerasa (PCR) fue empleada para amplificar un fragmento de 272-pb presente en el genoma del Trypanosoma cruzi, pero no detectado en el genoma del Trypanosoma rangeli. Para la amplificación por PCR se emplearon los oligonucleótidos TC4-1 y TC4-2 de 25-pb cada uno. Con el fin de aumentar la sensibilidad de la detección de los productos de amplificación en muestras biológicas, se preparó una sonda clonada en el vector pCR II y luego secuenciando el fragmento de 272-pb obtenido por amplificación del ADN de la cepa Tulahuen. Hemos confirmado por hibridación con la técnica Southern blot la no amplificación de ADN de T. rangeli con los cebadores TC4-1 y TC4-2. Estos resultados sugieren que esta técnica podría ser empleada para diferenciar infecciones por T. rangeli y T. cruzi

Detección de secuenciación del ADN del VHB en el torrente sanguíneo de pasientes positivos para HbsAg. Informe Preliminar / Annual Reports 1991 / Detection of DNA sequences in bloodstream of patients positive for HBsAG. Prelininar Report

The plasmid pSP65 which has cloned the complete HBV sequence, was amplified by transformation of coli (DH5). The HBV sequence was then labeled for its utilization as a probe in the HBV DNA detection in serum of patients with HBsAg positive, with no evident physical symptoms of the disease. The probe was labeled using digoxigenin, an enzymatic method. Sixteen samples were tested. Each sample was deproteinated by extraction with phenol-chorofrom in order to obtain Dane particles and then tested by dot bot. All samples yielded negative. This results suggest that the patients positive for HBsAg and who do not present acute symptoms nor high GOT and GPT levels in sera, lack HBV sequences in bloodstream