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1.
Einstein (Sao Paulo) ; 20: eAO6651, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35476084

RESUMO

OBJECTIVE: To detect and treat cases of viral hepatitis B, C and D in patients seen at the Native American Outpatient Clinic of Universidade Federal de São Paulo. METHODS: This sample comprised 81 indigenous recruited between 2018 and 2020. Volunteers were aged 7 months to 70 years (mean age of 28±20 years), belonged to 26 ethnic groups spanning the Brazilian territory and answered a questionnaire, which was attached to their medical records. Peripheral blood samples (20mL) were collected, transported to the Clinical Laboratory of Hospital Israelita Albert Einstein, processed, and tested for markers of viral hepatitis B, C and D. RESULTS: In this study, 39 (48.1%) individuals were anti-HBs (+) only, 13 (16.0%) individuals were anti-HBs (+) and anti-HBc (+), and 28 (34.6%) individuals were negative for all markers. No anti-HBc IgM+ samples were found. No cases of hepatitis C and D were found. CONCLUSION: This analysis provided evidence of previous infection by the hepatitis B virus. These findings led to prescription of vaccination against hepatitis B to all participants who were negative for all viral hepatitis B markers, given records of prior hepatitis B vaccination were unreliable.


Assuntos
Hepatite B , Adolescente , Adulto , Instituições de Assistência Ambulatorial , Biomarcadores , Brasil/epidemiologia , Criança , Hepatite B/diagnóstico , Hepatite B/epidemiologia , Anticorpos Anti-Hepatite B , Humanos , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Adulto Jovem
2.
Einstein (Sao Paulo) ; 19: eRC6369, 2021.
Artigo em Inglês, Português | MEDLINE | ID: mdl-34909977

RESUMO

During the COVID-19 pandemic, a case of a long-term persistence of SARS-CoV-2 infection (from March 26 to May 20, 2020) was identified at a private hospital in São Paulo, SP, Brazil. The long-term positivity for SARS-CoV-2 in reverse transcriptase polymerase chain reaction tests of a patient diagnosed with COVID-19 suggests, at least part of patients who recovered, may still carry and transmit the SARS-CoV-2 virus. This fact emphasizes the importance of having at least two negative reverse transcriptase polymerase chain reaction test results for SARS-CoV-2. Serological assays were not particularly helpful in the case described, since the patient had very low antibodies titers at the end of the follow-up period. Low viral loads may not be detected by current molecular methods, leading to wrong conclusions regarding viral clearance.


Assuntos
COVID-19 , Brasil , Humanos , Pandemias , SARS-CoV-2 , Testes Sorológicos
3.
Artigo em Inglês | MEDLINE | ID: mdl-34755817

RESUMO

In Brazil, few studies on the molecular aspects of hepatitis B virus (HBV) infection have been conducted in the interior regions of Sao Paulo State. This study aimed to identify HBV genotypes and evaluate strains with resistance mutations for nucleoside analogues in the Administrative Region (AR) of the municipality of Sao Jose do Rio Preto. We performed nested PCRs of 127 samples from the Health Care Services of the AR to amplify, sequence and analyze fragments of the HBV DNA, in order to identify genotypes and resistance mutations. The HBV S/Pol regions of 126 samples were successfully amplified and sequenced. Five different genotypes were found, and the main ones were A, D and F; a greater number of samples contained the subgenotypes A1 (n = 51; 40.5%), D3 (n = 36; 28.6%), A2 (n = 14; 11.1%) and F2a (n = 9; 7.1%). Resistance mutations (rtM204V/I/S) associated or not with compensatory mutations (rtL180M, rtV173L) were identified in 13.9% (5/36) of patients undergoing viral treatment and 1.1% (1/90) of naïve patients. The diversity of genotypes/subgenotypes found is probably due to the intense migration occurring in the region. These data can complement epidemiological and clinical surveillance, and can be used for a more effective management of chronic HBV patients.


Assuntos
Hepatite B Crônica , Hepatite B , Brasil , DNA Viral/genética , Farmacorresistência Viral/genética , Genótipo , Vírus da Hepatite B/genética , Humanos , Mutação , Análise de Sequência de DNA
4.
BMJ Open ; 11(7): e045852, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244259

RESUMO

INTRODUCTION: Acute viral hepatitis is a disease of great clinical importance. This study proposes actions to better characterise cases of acute hepatitis in Brazil and to provide relevant information to institutionalised health policies within the Unified Health System. Available data on acute hepatitis in Brazil need to be re-evaluated regarding the different hepatotropic agent (hepatitis A to E virus) frequencies, as well as other agents that can cause similar clinical conditions, such as Herpes Simplex Virus 1 and 2(HSV1, HSV2), Varicella Zoster Virus (VZV), Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Herpes Virus 6 and 7 (HHV6, HHV7), arbovirus (yellow fever, dengue, chikungunya, Zika), parvovirus B19, adenovirus, parechovirus, enterovirus, HIV, leptospirosis, toxoplasmosis and syphilis, in addition to autoimmune hepatitis. In this context, the primary aim of this study is the clinical-epidemiological and molecular characterisation of acute viral hepatitis in Brazilian health services from all geographical regions of the country. The present article describes the study protocol. METHODS AND ANALYSIS: This study will evaluate 2280 patients with symptoms and/or signs suggestive of acute liver disease in Brazilian health institutions in all five geographic Brazilian regions. Demographic, epidemiological and clinical data will be collected, as well as blood samples to be analysed at Hospital Israelita Albert Einstein Clinical Laboratory. ETHICS AND DISSEMINATION: Ethics approval was obtained at the national research ethics committee (Conselho Nacional de Ética em Pesquisa- CONEP-CAAE 00952818.4.1001.0071) and at all participating sites. Results will be published in journals and presented at scientific meetings.


Assuntos
Infecções por Vírus Epstein-Barr , Hepatite Viral Humana , Infecção por Zika virus , Zika virus , Brasil/epidemiologia , Serviços de Saúde , Hepatite Viral Humana/epidemiologia , Herpesvirus Humano 4 , Humanos
5.
Blood Cells Mol Dis ; 82: 102413, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32109669

RESUMO

Hereditary Xerocytosis (HX) is an autosomal dominantly inherited congenital hemolytic anemia associated with erythrocyte dehydration due to decreased intracellular potassium content resulting in increased mean corpuscular hemoglobin concentration. The affected members of HX families show compensated anemia with splenomegaly, hemosiderosis, and perinatal edema but are in large part transfusion independent. Functional studies show a link between mutations in mechanosensitive ion channel, encoded by PIEZO1 gene and the HX. We identified new PIEZO1 variants that are likely pathogenic in three phenotypically characterized multi-generational HX Brazilian families. Interestingly, one missense variant of the PIEZO1 gene identified, p.E2494V was associated in trans with the previously reported most frequent pathogenic duplication p.E2496ELE. The three-dimensional structure of the human protein modeled using structural coordinates of the mouse Piezo1 solved by cryo-electron microscopy (Cryo-ME) showed that the two identified variants, p.M2007L and p.T2014I, are localized to an important mechanosensitive transmembrane domain suggesting a conformational mechanism for altered channel's gating. The p.E2496ELE variant identified alters the extension of helix α1 bringing it much closer to the beam affecting the position of it structure at the end of the pore.


Assuntos
Anemia Hemolítica Congênita/genética , Hidropisia Fetal/genética , Canais Iônicos , Mutação de Sentido Incorreto , Adolescente , Adulto , Substituição de Aminoácidos , Animais , Criança , Feminino , Humanos , Recém-Nascido , Canais Iônicos/química , Canais Iônicos/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Conformação Proteica em alfa-Hélice , Domínios Proteicos
6.
BMC Cancer ; 19(1): 324, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30953464

RESUMO

BACKGROUND: We are reporting a rare case of MUTYH-associated polyposis, a colorectal cancer hereditary syndrome, diagnosticated after an intussusception. Colorectal cancer is an important cause of cancer related mortality that can be manifested by an intussusception, a rare occurrence in adults and almost always related to tumors. Approximately 5% of colorectal cancers can be attributed to syndromes known to cause hereditary colorectal cancer, such as MUTYH-associated polyposis, autosomal genetic syndrome associated with this disease. CASE PRESENTATION: We present the case of a 44 years old male, that sought medical consultation with a complaint of abdominal discomfort, that after five days changed its characteristics. The patient was sent to the emergency department were a CT-scan revealed intestinal sub-occlusion by ileocolic invagination. Right colectomy was carried out. The anatomic-pathological examination revealed a moderately differentiated mucinous adenocarcinoma and multiples sessile polyps, which led to the suspicion of a genetic syndrome. In the genetics analysis two mutations were observed in the MUTYH gene, and MUTYH-associated polyposis was diagnosticated. CONCLUSION: This case demonstrates the importance of meticulous analysis of the patient examinations results to identify possible discrete alterations that can lead to improved understanding of disease.


Assuntos
Adenocarcinoma Mucinoso/diagnóstico , Polipose Adenomatosa do Colo/diagnóstico , DNA Glicosilases/genética , Doenças do Íleo/etiologia , Intussuscepção/etiologia , Adenocarcinoma Mucinoso/complicações , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/cirurgia , Polipose Adenomatosa do Colo/complicações , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/cirurgia , Adulto , Colectomia , Colo/diagnóstico por imagem , Colo/patologia , Colo/cirurgia , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Doenças do Íleo/diagnóstico por imagem , Doenças do Íleo/cirurgia , Intussuscepção/diagnóstico por imagem , Intussuscepção/cirurgia , Masculino , Tomografia Computadorizada por Raios X
7.
Rev. bras. hematol. hemoter ; 38(2): 113-120, tab, graf
Artigo em Inglês | LILACS | ID: lil-787665

RESUMO

BACKGROUND: Multiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance. Multiple myeloma differs from other hematologic malignancies due to a high fraction of low proliferating malignant plasma cells and the paucity of plasma cells in bone marrow aspiration samples, making cytogenetic analysis a challenge. An abnormal karyotype is found in only one-third of patients with multiple myeloma and interphase fluorescence in situ hybridization is the most useful test for studying the chromosomal abnormalities present in almost 90% of cases. However, it is necessary to study the genetic abnormalities in plasma cells after their identification or selection by morphology, immunophenotyping or sorting. Other challenges are the selection of the most informative FISH panel and determining cut-off levels for FISH probes. This study reports the validation of interphase fluorescence in situ hybridization using CD138 positive cells, according to proposed guidelines published by the European Myeloma Network (EMN) in 2012. METHOD: Bone marrow samples from patients with multiple myeloma were used to standardize a panel of five probes [1q amplification, 13q14 deletion, 17p deletion, t(4;14), and t(14;16)] in CD138+ cells purified by magnetic cell sorting. RESULTS: This test was validated with a low turnaround time and good reproducibility. Five of six samples showed genetic abnormalities. Monosomy/deletion 13 plus t(4;14) were found in two cases. CONCLUSION: This technique together with magnetic cell sorting is effective and can be used in the routine laboratory practice. In addition, magnetic cell sorting provides a pure plasma cell population that allows other molecular and genomic studies.


Assuntos
Humanos , Citogenética , Hibridização in Situ Fluorescente , Mieloma Múltiplo , Plasma , Plasmocitoma
8.
Rev Bras Hematol Hemoter ; 38(2): 113-20, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27208569

RESUMO

BACKGROUND: Multiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance. Multiple myeloma differs from other hematologic malignancies due to a high fraction of low proliferating malignant plasma cells and the paucity of plasma cells in bone marrow aspiration samples, making cytogenetic analysis a challenge. An abnormal karyotype is found in only one-third of patients with multiple myeloma and interphase fluorescence in situ hybridization is the most useful test for studying the chromosomal abnormalities present in almost 90% of cases. However, it is necessary to study the genetic abnormalities in plasma cells after their identification or selection by morphology, immunophenotyping or sorting. Other challenges are the selection of the most informative FISH panel and determining cut-off levels for FISH probes. This study reports the validation of interphase fluorescence in situ hybridization using CD138 positive cells, according to proposed guidelines published by the European Myeloma Network (EMN) in 2012. METHOD: Bone marrow samples from patients with multiple myeloma were used to standardize a panel of five probes [1q amplification, 13q14 deletion, 17p deletion, t(4;14), and t(14;16)] in CD138(+) cells purified by magnetic cell sorting. RESULTS: This test was validated with a low turnaround time and good reproducibility. Five of six samples showed genetic abnormalities. Monosomy/deletion 13 plus t(4;14) were found in two cases. CONCLUSION: This technique together with magnetic cell sorting is effective and can be used in the routine laboratory practice. In addition, magnetic cell sorting provides a pure plasma cell population that allows other molecular and genomic studies.

9.
PLoS One ; 11(1): e0146258, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26741362

RESUMO

AIM: To analyze the genetic heterogeneity of the Amerindian and admixed population (Mestizos) based on the IL28B (rs12979860, rs8099917) and IFNL4 (rs368234815) haplotypes, and their association with spontaneous clearance (SC) and liver damage in patients with hepatitis C infection from West Mexico. METHODS: A total of 711 subjects from West Mexico (181 Amerindians and 530 Mestizos) were studied for the prevalence of IL28B (rs12979860C/T, rs8099917G/T) and IFNL4 (rs368234815∆G/TT) genotypes. A case-control study was performed in 234 treatment-naïve HCV Mestizos (149 chronic hepatitis C and 85 with SC) for the association of haplotypes with SC and liver damage. A real-time PCR assay was used for genotyping, and transitional elastography staged liver damage. RESULTS: Significant Fst-values indicated differentiation between the studied populations. The frequencies of the protective C, T, TT alleles were significantly lower in the Amerindians than in Mestizos (p<0.05). The r2 measure of linkage disequilibrium was significant for all variants and the T/G/ΔG risk haplotype predominated in Amerindians and secondly in Mestizos. The protective C/T/TT haplotype was associated with SC (OR = 0.46, 95% IC 0.22-0.95, p = 0.03) and less liver damage (OR = 0.32, 95% IC 0.10-0.97, p = 0.04) in chronic patients. The Structure software analysis demonstrated no significant differences in ancestry among SC and chronic patients. CONCLUSIONS: West Mexico's population is genetically heterogeneous at the IL28B/IFNL4 polymorphisms. The T/G/ΔG high-risk haplotype predominated in Amerindians and the beneficial alternative haplotype in Mestizos. The C/T/TT haplotype was associated with SC and less liver damage in chronically infected Mestizo patients.


Assuntos
Hepacivirus/imunologia , Hepatite C Crônica/genética , Índios Norte-Americanos , Interleucinas/genética , Adulto , Alelos , Estudos de Casos e Controles , Feminino , Expressão Gênica/imunologia , Frequência do Gene , Haplótipos , Hepatite C Crônica/etnologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Interferons , Interleucinas/imunologia , Fígado/imunologia , Fígado/virologia , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Remissão Espontânea
10.
Biomed Res Int ; 2016: 4247908, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28074183

RESUMO

Aims. To develop a fast and robust DNA-based assay to detect insertions and deletions mutations in exon 34 that encodes the PEST domain of NOTCH1 in order to evaluate patients with chronic lymphocytic leukemia (CLL). Methods. We designed a multiplexed allele-specific polymerase chain reaction (PCR) combined with a fragment analysis assay to detect specifically the mutation c.7544_7545delCT and possibly other insertions and deletions in exon 34 of NOTCH1. Results. We evaluated our assay in peripheral blood samples from two cohorts of patients with CLL. The frequency of NOTCH1 mutations was 8.4% in the first cohort of 71 unselected CLL patients. We then evaluated a second cohort of 26 CLL patients with known cytogenetic abnormalities that were enriched for patients with trisomy 12. NOTCH1 mutations were detected in 43.7% of the patients with trisomy 12. Conclusions. We have developed a fast and robust assay combining allele-specific PCR and fragment analysis able to detect NOTCH1 PEST domain insertions and deletions.


Assuntos
Alelos , Mutação INDEL , Leucemia Linfocítica Crônica de Células B/genética , Reação em Cadeia da Polimerase/métodos , Receptor Notch1/genética , Trissomia/genética , Cromossomos Humanos Par 12/genética , Estudos de Coortes , Análise Mutacional de DNA/métodos , Feminino , Humanos , Masculino , Domínios Proteicos
11.
Einstein (Sao Paulo) ; 12(3): 366-73, 2014 Sep.
Artigo em Inglês, Português | MEDLINE | ID: mdl-25295459

RESUMO

Personalized medicine is the use of biomarkers, most of them molecular markers, for detection of specific genetic traits to guide various approaches for preventing and treating different conditions. The identification of several genes related to heredity, oncology and infectious diseases lead to the detection of genetic polymorphisms that are involved not only in different clinical progression of these diseases but also in variations in treatment response. Currently, it is possible to detect these polymorphisms using several methodologies: detection of single nucleotide polymorphisms using polymerase chain reaction methods; nucleic acid microarray detection; and nucleic acid sequencing with automatized DNA sequencers using Sanger-derived methods and new generation sequencing. Personalized medicine assays are directed towards detecting genetic variations that alter interactions of drugs with targets or the metabolic pathways of drugs (upstream and downstream) and can be utilized for the selection of drug formulations and detect different immunogenicities of the drug. Personalized medicine applications have already been described in different areas of Medicine and allow specific treatment approaches to be applied to each patient and pathology according to the results of these assays. The application of such a protocol demands an increasing interaction between the clinical laboratory and the clinical staff. For its implementation, a coordinated team composed of basic researchers and physicians highly specialized in their areas supported by a highly specialized team of clinical analysts particularly trained in molecular biology assays is necessary.


Assuntos
Marcadores Genéticos/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Medicina de Precisão/métodos , Anticoagulantes/farmacologia , Biomarcadores Tumorais/genética , Hepatite C/tratamento farmacológico , Hepatite C/genética , Humanos , Farmacogenética , Inibidores da Agregação Plaquetária/farmacologia
12.
Einstein (Säo Paulo) ; 12(3): 366-373, Jul-Sep/2014.
Artigo em Inglês | LILACS | ID: lil-723923

RESUMO

Personalized medicine is the use of biomarkers, most of them molecular markers, for detection of specific genetic traits to guide various approaches for preventing and treating different conditions. The identification of several genes related to heredity, oncology and infectious diseases lead to the detection of genetic polymorphisms that are involved not only in different clinical progression of these diseases but also in variations in treatment response. Currently, it is possible to detect these polymorphisms using several methodologies: detection of single nucleotide polymorphisms using polymerase chain reaction methods; nucleic acid microarray detection; and nucleic acid sequencing with automatized DNA sequencers using Sanger-derived methods and new generation sequencing. Personalized medicine assays are directed towards detecting genetic variations that alter interactions of drugs with targets or the metabolic pathways of drugs (upstream and downstream) and can be utilized for the selection of drug formulations and detect different immunogenicities of the drug. Personalized medicine applications have already been described in different areas of Medicine and allow specific treatment approaches to be applied to each patient and pathology according to the results of these assays. The application of such a protocol demands an increasing interaction between the clinical laboratory and the clinical staff. For its implementation, a coordinated team composed of basic researchers and physicians highly specialized in their areas supported by a highly specialized team of clinical analysts particularly trained in molecular biology assays is necessary.


Medicina personalizada é o uso de biomarcadores, em sua maioria marcadores moleculares, para a detecção de traços genéticos específicos, a fim de orientar diversas abordagens para a prevenção e o tratamento de diferentes doenças. A identificação de vários genes relacionados a doenças hereditárias, oncológicas e infecciosas permite a detecção de polimorfismos genéticos que estão envolvidos em diferentes evoluções clínicas dessas doenças, bem como com variações na resposta ao tratamento. Atualmente, já é possível detectar esses polimorfismos utilizando diversas metodologias: a detecção de polimorfismos de nucleotídeo único pela reação de polimerização em cadeia; a detecção de microarranjos de ácidos nucleicos; e o sequenciamento de ácidos nucleicos com sequenciadores de DNA automatizados usando métodos derivados de sequenciamento Sanger ou de nova geração. Os ensaios de medicina personalizada são dirigidos para detectar variações genéticas que alteram interações de fármacos com alvos ou vias metabólicas de fármacos (anabólicas e catabólicas), podendo ser utilizados para a seleção de formulações farmacêuticas e para detectar diferentes imunogenicidades da droga. As aplicações de medicina personalizada já foram descritas em várias áreas da Medicina e permitem que abordagens de tratamento específicas sejam aplicadas para cada paciente e para cada doença, de acordo com os resultados dos ensaios utilizados. A aplicação de um protocolo desse tipo exige uma relação intensa entre o laboratório e o corpo clínico. Para sua execução, é necessária uma equipe coordenada, composta por investigadores de pesquisa básica e médicos altamente especializados em suas áreas, apoiada por um time bastante especializado de analistas clínicos treinados em testes de biologia molecular.


Assuntos
Humanos , Marcadores Genéticos/genética , Medicina de Precisão/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Anticoagulantes/farmacologia , Hepatite C/tratamento farmacológico , Hepatite C/genética , Farmacogenética , Inibidores da Agregação Plaquetária/farmacologia , Biomarcadores Tumorais/genética
13.
Einstein (Sao Paulo) ; 12(2): 191-6, 2014 Apr.
Artigo em Inglês, Português | MEDLINE | ID: mdl-25003925

RESUMO

OBJECTIVE: To test and validate a multiplex real-time polymerase chain reaction method for bloodstream infections, as well as to compare the results with conventional blood culture. METHODS: A total of 114 consecutive patients with clinical evidence of sepsis were submitted to blood culture and LightCycler™ SeptiFast tests. RESULTS: More positive specimens (23; 20.2%) were detected using the LightCycler™ SeptiFast than the blood culture (17; 14.9%), with an agreement of 86.8%. Discordant results were seen in four patients positive only to blood culture, ten positive only to LightCycler™ SeptiFast and one to different pathogens found by each test. Infections with microorganisms detected only using blood culture reassured the need to perform both tests. The mean time to results for blood culture was 5 days for negative and 3.5 days for positive results. LightCycler™ SeptiFast results were achieved in less than 8 hours. CONCLUSION: LightCycler™ SeptiFast showed a high potential as a test to be carried out concomitantly with blood culture for sepsis diagnosis in severely ill patients. This test allowed a faster diagnosis of bacterial and fungal infections that helped to reduce hospital stay and to control the use of antibiotics. LightCycler™ SeptiFast can also eventually detect microorganism and infections that are hardly detected by blood culture, especially Candida non-albicans infections.


Assuntos
Bacteriemia/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Bacteriemia/microbiologia , Brasil , Estado Terminal , DNA Bacteriano , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
14.
Einstein (Säo Paulo) ; 12(2): 191-196, Apr-Jun/2014. tab
Artigo em Inglês | LILACS | ID: lil-713002

RESUMO

Objective To test and validate a multiplex real-time polymerase chain reaction method for bloodstream infections, as well as to compare the results with conventional blood culture. Methods A total of 114 consecutive patients with clinical evidence of sepsis were submitted to blood culture and LightCycler™ SeptiFast tests. Results More positive specimens (23; 20.2%) were detected using the LightCycler™ SeptiFast than the blood culture (17; 14.9%), with an agreement of 86.8%. Discordant results were seen in four patients positive only to blood culture, ten positive only to LightCycler™ SeptiFast and one to different pathogens found by each test. Infections with microorganisms detected only using blood culture reassured the need to perform both tests. The mean time to results for blood culture was 5 days for negative and 3.5 days for positive results. LightCycler™ SeptiFast results were achieved in less than 8 hours. Conclusion LightCycler™ SeptiFast showed a high potential as a test to be carried out concomitantly with blood culture for sepsis diagnosis in severely ill patients. This test allowed a faster diagnosis of bacterial and fungal infections that helped to reduce hospital stay and to control the use of antibiotics. LightCycler™ SeptiFast can also eventually detect microorganism and infections that are hardly detected by blood culture, especially Candida non-albicans infections. .


Objetivo Testar e validar um método molecular multiplex para detecção de infecções sanguíneas, além de comparar os resultados com os obtidos pela hemocultura convencional. Métodos Os testes de hemocultura e o LightCycler® SeptiFast foram realizados em 114 pacientes consecutivos com evidência clínica de sepse. Resultados Mais amostras positivas (23; 20,2%) foram detectadas pelo LightCycler® SeptiFast do que pela hemocultura (17; 14,9%), mostrando concordância de 86,8%. Os resultados discordantes foram de quatro pacientes positivos apenas para hemocultura, dez positivos apenas para LightCycler® SeptiFast e um com patógenos diferentes encontrados em cada método. Infecções por micro-organismos não reconhecidos pelo LightCycler® SeptiFast e detectados apelas pela hemocultura confirmam a necessidade da realização dos dois métodos. O tempo médio para os resultados da hemocultura foi de 5 dias para amostras negativas e de 3,5 dias para as positivas. Os resultados pelo LightCycler® SeptiFast foram obtidos em menos de 8 horas. Conclusão O LightCycler® SeptiFast mostrou ser um teste de grande potencial para ser realizado simultaneamente à hemocultura para diagnóstico de sepse em doentes graves, permitindo um diagnóstico mais rápido de infecções por bactérias e fungos e, dessa forma, auxiliando a redução do tempo de hospitalização e racionalização do uso de antibióticos. Eventualmente, o LightCycler® SeptiFast pode detectar inclusive infecções por micro-organismos dificilmente detectáveis via hemocultura, especialmente aquelas causadas por Candida não albicans. .


Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Bacteriemia/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Brasil , Bacteriemia/microbiologia , Estado Terminal , DNA Bacteriano , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
15.
BMC Infect Dis ; 13: 487, 2013 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-24138798

RESUMO

BACKGROUND: Enterovirus and herpes simplex viruses are common causes of lymphocytic meningitis. The purpose of this study was to analyse the impact of the use molecular testing for Enteroviruses and Herpes simplex viruses I and II in all suspected cases of viral meningitis. METHODS: From November 18, 2008 to November 17, 2009 (phase II, intervention), all patients admitted with suspected viral meningitis (with pleocytosis) had a CSF sample tested using a nucleic acid amplification test (NAAT). Data collected during this period were compared to those from the previous one-year period, i.e. November 18, 2007 to November 17, 2008 (phase I, observational), when such tests were available but not routinely used. RESULTS: In total, 2,536 CSF samples were assessed, of which 1,264 were from phase I, and 1,272 from phase II. Of this total, a NAAT for Enterovirus was ordered in 123 cases during phase I (9.7% of the total phase I sample) and in 221 cases in phase II (17.4% of the total phase II sample). From these, Enterovirus was confirmed in 35 (28.5%, 35/123) patients during phase I and 71 (32.1%, 71/221) patients during phase II (p = 0.107). The rate of diagnosis of meningitis by HSV I and II did not differ between the groups (13 patients, 6.5% in phase I and 13, 4.7% in phase II) (p = 1.0), from 200 cases in phase I and 274 cases in phase II. CONCLUSIONS: The number of cases diagnosed with enteroviral meningitis increased during the course of this study, leading us to believe that the strategy of performing NAAT for Enterovirus on every CSF sample with pleocytosis is fully justified.


Assuntos
Infecções por Enterovirus/virologia , Enterovirus/isolamento & purificação , Herpes Simples/virologia , Meningite Viral/virologia , Adolescente , Adulto , Criança , Pré-Escolar , Infecções por Enterovirus/diagnóstico , Feminino , Herpes Simples/diagnóstico , Hospitalização , Humanos , Lactente , Masculino , Meningite Viral/diagnóstico , Simplexvirus/isolamento & purificação , Atenção Terciária à Saúde , Adulto Jovem
16.
BMC Res Notes ; 6: 423, 2013 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-24139701

RESUMO

BACKGROUND: HBV genotype F is primarily found in indigenous populations from South America and is classified in four subgenotypes (F1 to F4). Subgenotype F2a is the most common in Brazil among genotype F cases. The aim of this study was to characterize HBV genotype F2a circulating in 16 patients from São Paulo, Brazil. Samples were collected between 2006 and 2012 and sent to Hospital Israelita Albert Einstein. A fragment of 1306 bp partially comprising HBsAg and DNA polymerase coding regions was amplified and sequenced. Viral sequences were genotyped by phylogenetic analysis using reference sequences from GenBank (n=198), including 80 classified as subgenotype F2a. Bayesian Markov chain Monte Carlo simulation implemented in BEAST v.1.5.4 was applied to obtain the best possible estimates using the model of nucleotide substitutions GTR+G+I. FINDINGS: It were identified three groups of sequences of subgenotype F2a: 1) 10 sequences from São Paulo state; 2) 3 sequences from Rio de Janeiro and one from São Paulo states; 3) 8 sequences from the West Amazon Basin. CONCLUSIONS: These results showing for the first time the distribution of F2a subgenotype in Brazil. The spreading and the dynamic of subgenotype F2a in Brazil requires the study of a higher number of samples from different regions as it is unfold in almost all Brazilian populations studied so far. We cannot infer with certainty the origin of these different groups due to the lack of available sequences. Nevertheless, our data suggest that the common origin of these groups probably occurred a long time ago.


Assuntos
DNA Viral/classificação , Vírus da Hepatite B/classificação , Hepatite B Crônica/virologia , Filogenia , Teorema de Bayes , Brasil/epidemiologia , DNA Viral/genética , Feminino , Genótipo , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B Crônica/epidemiologia , Humanos , Masculino , Tipagem Molecular , Método de Monte Carlo , Análise de Sequência de DNA
17.
Einstein (Säo Paulo) ; 10(3): 286-291, jul.-set. 2012. ilus, tab
Artigo em Português | LILACS | ID: lil-654336

RESUMO

OBJETIVO: Descrever a metodologia para detecção de mutações nos éxons 8 e 17 do gene KIT em pacientes portadores de leucemia mieloide aguda, para implementação desse teste no laboratório clínico do Hospital Israelita Albert Einstein. MÉTODOS: Extração do DNA genômico de 54 amostras de sangue periférico ou medula óssea de pacientes com leucemia mieloide aguda para amplificação, por reação em cadeia da polimerase, sequenciamento e análise de fragmentos. RESULTADOS: Dentre as amostras analisadas, quatro apresentaram mutação no éxon 8, duas no éxon 17 e uma amostra apresentou mutação nos dois éxons. CONCLUSÃO: A pesquisa de mutação nos éxons 8 e 17 do gene KIT foi padronizada com sucesso e o teste está em processo de inclusão no menu de exames do laboratório clínico do Hospital Israelita Albert Einstein.


OBJECTIVE: This study describes a new method used in the clinical laboratory at Hospital Israelita Albert Einstein to detect mutations in exons 8 and 17 of the KIT gene in patients with acute myeloid leukemia. METHODS: Genomic DNA extraction was performed on 54 samples of peripheral blood or bone marrow from patients with acute myeloid leukemia. The extracted DNA was amplified by polymerase chain reaction and sequenced, and the fragments were analyzed. RESULTS: Within the analyzed samples, we detected four mutations in exon 8, two mutations in exon 17, and mutations or a double mutation in one sample. CONCLUSION: The tests detecting mutations in exon 8 and 17 on the KIT gene were successfully standardized. The test is now included among the routine diagnostics employed for patients at Hospital Israelita Albert Einstein clinical laboratory.


Assuntos
Fatores de Ligação ao Core , Expressão Gênica , Leucemia Mieloide Aguda , Receptores Proteína Tirosina Quinases
18.
Infect Genet Evol ; 12(6): 1295-304, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22538208

RESUMO

Southern Brazil is considered an area of low Hepatitis B endemicity, but some areas of higher endemicity have been described in the Southwest of Paraná and Santa Catarina states. The aim of this study was to evaluate viral genotypes circulating throughout Paraná state. PCR amplification and partial sequencing of the S gene was carried out in 228 samples from HBsAg positive candidate blood donors. Samples have been collected in seven different counties (Cascavel, Curitiba, Foz do Iguaçu, Francisco Beltrão, Maringá, Londrina and Paranaguá). The most common HBV genotype in Paraná state was D (82.9%; 189/228), followed by A (14.1%; 32/228). Genotypes F (1.3%; 3/228), C (1.3%; 3/228) and H (0.4%; 1/228) were also found. Distribution of genotypes was different in the studied counties, but genotype D was the most frequent in all of them. In Francisco Beltrão, all studied samples belonged to genotype D. The high prevalence of HBV genotype D in South of Brazil is explained by the intense migration of settlers from Europeans countries. Subgenotypes A1 and A2 were identified circulating in all cities where HBV/A was found. As observed in other areas of Brazil, HBV/A1 is more frequent than the HBV/A2 in Paraná state and its presence was significantly larger in black and mulatto individuals. Genotype C was found only in individuals with Asian ancestry from Londrina and Maringá. Most HBV/F sequences identified in this study were classified as subgenotype F2a that was previously described in Brazil. The sole case of subgenotype F4 was from Foz do Iguaçu city, near to Northern Argentina, where F4 is highly prevalent. The single genotype H sample was from Curitiba. This is the first case of this genotype described in Brazil. Further studies should be carried out to determine if more genotype H samples can be found in other populations from Brazil.


Assuntos
Vírus da Hepatite B/genética , Hepatite B/virologia , Adulto , Análise de Variância , Doadores de Sangue , Brasil/epidemiologia , Doenças Endêmicas , Europa (Continente)/epidemiologia , Feminino , Genótipo , Hepatite B/epidemiologia , Humanos , Masculino , Filogenia
19.
Einstein (Sao Paulo) ; 10(3): 286-91, 2012.
Artigo em Inglês, Português | MEDLINE | ID: mdl-23386005

RESUMO

OBJECTIVE: This study describes a new method used in the clinical laboratory at Hospital Israelita Albert Einstein to detect mutations in exons 8 and 17 of the KIT gene in patients with acute myeloid leukemia. METHODS: Genomic DNA extraction was performed on 54 samples of peripheral blood or bone marrow from patients with acute myeloid leukemia. The extracted DNA was amplified by polymerase chain reaction and sequenced, and the fragments were analyzed. RESULTS: Within the analyzed samples, we detected four mutations in exon 8, two mutations in exon 17, and mutations or a double mutation in one sample. CONCLUSION: The tests detecting mutations in exon 8 and 17 on the KIT gene were successfully standardized. The test is now included among the routine diagnostics employed for patients at Hospital Israelita Albert Einstein clinical laboratory.


Assuntos
Éxons/genética , Leucemia Mieloide Aguda/genética , Mutação/genética , Proteínas Proto-Oncogênicas c-kit/genética , Eletroforese Capilar , Humanos , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA
20.
Einstein (Säo Paulo) ; 9(4)out.-dec. 2011. graf
Artigo em Inglês, Português | LILACS | ID: lil-612023

RESUMO

Objective: Functional in vitro studies are fundamental to understand the role of microRNAs, small non coding RNA molecules that function as post-transcriptional regulators, in cancer. The objective of this study was to determine the applicability of head and neck squamous cell carcinoma cell lines and human oral keratinocytes as models for functional studies on microRNAs previously identified as deregulated in head and neck squamous cell carcinomas. Methods: The expression level of four microRNAs was assessed in cell lines and in primary cultures of oral keratinocytes using specific real-time polymerase chain reactions. The identity of oral squamous cell carcinoma cell lines was confirmed by means of STR (short tandem repeats) profiling. The possible impact of feeder-layer gene expression in global microRNA expression results from keratinocyte primary culture was also evaluated. Results: Significant differences in microRNA gene expression were observed among squamous cell carcinoma cell lines, particularly among cells lines from distinct subsites, as well as between primary culture of human keratinocytes and immortalized keratinocyte cell lines. Conclusions: Primary cultures of human keratinocytes and diverse tumor cell lines are relatively easy to obtain. However, each cell model possesses a characteristic phenotype; whereas one may be useful for a specific study, it may be inappropriate for another. Therefore, it is imperative that suitable cell lines are cautiously selected for functional studies in cancer.


Objetivo: Estudos funcionais in vitro são essenciais para a compreensão do papel de microRNAs, pequenas moléculas de RNA que desempenham papel importante na regulação gênica, no câncer. Neste estudo, analisamos a viabilidade de linhagens celulares derivadas de carcinoma epidermoide de cabeça e pescoço, queratinócitos orais provenientes de culturas primárias e queratinócitos imortalizados, como modelos para estudos funcionais de microRNAs previamente identificados como desregulados nesse tipo de carcinoma. Métodos: Avaliamos a expressão de quatro microRNAs em linhagens celulares e em cultura primária de queratinócitos orais por meio de reações em cadeia da polimerase em tempo real específica. As linhagens celulares de carcinoma epidermoide de boca foram previamente caracterizadas quanto ao seu perfil de sequências de DNA do tipo STR (do inglês short tandem repeats ou repetições curtas em sequência) com o objetivo de confirmar a identidade da linhagem. Avaliamos ainda a possível influência da expressão gênica detectada na camada de sustentação usada no cultivo de queratinócitos no resultado global obtido. Resultados: Nossos resultados apontam diferenças significativas na expressão dos microRNAs entre linhagens celulares passíveis de serem utilizadas como modelos para estudos funcionais em carcinoma epidermoide de cabeça e pescoço. Ressaltam-se diferenças entre linhagens de carcinoma de língua e de faringe, bem como diferenças expressivas entre a linhagem de queratinócitos orais imortalizados e queratinócitos orais normais provenientes de culturas primárias. Conclusão: Culturas primárias de queratinócitos orais bem como linhagens tumorais são obtidas de forma relativamente simples. Entretanto, cada modelo celular possui características particulares que os tornam mais ou menos adequados para um determinado estudo. Conclui-se que a seleção cuidadosa das linhagens é fundamental para estudos funcionais sobre câncer.


Assuntos
Carcinoma de Células Escamosas , Expressão Gênica , Neoplasias de Cabeça e Pescoço , Queratinócitos
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