Navegando por Palavras-chave "Polymerase chain reaction"

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    Absence of mycobacterial DNA in peripheral blood and artery specimens in patients with Takayasu arteritis
    (Springer London Ltd, 2017) Carvalho, Evanir S. [UNIFESP]; Souza, Alexandre Wagner Silva de [UNIFESP]; Leao, Sylvia Cardoso [UNIFESP]; Levy-Neto, Mauricio; de Oliveira, Rosangela Siqueira; Drake, Wonder; Franco, Marcello Fabiano de [UNIFESP]; Saldiva, Paulo Hilário Nascimento; Gutierrez, Paulo Sampaio; Andrade, Luiz Eduardo Coelho [UNIFESP]
    The objective of this study was to demonstrate the presence of mycobacterial nucleic acid sequences in peripheral blood and arteries from patients with Takayasu arteritis (TA). Polymerase chain reaction was performed to detect mycobacterial DNA from three different nucleic acid sequences including the insertion sequence (IS) 6110, the 65-kDa heat shock protein gene (HSP65), and the 16S ribosomal RNA (rRNA) gene in peripheral blood from 32 TA patients and in arterial specimens from 10 TA patients. Twenty-eight HIV-negative patients with pulmonary tuberculosis prior to therapy were tested for IS6110 in peripheral blood as positive controls, and 24 blood donors were evaluated as healthy controls (HC). All TA patients were negative for the insertion sequence IS6110 and for HSP65 and 16S rRNA genes in blood samples and in arterial specimens. IS6110 sequence was found in peripheral blood from 22 (78.5 %) patients with pulmonary tuberculosis but not in HC. In conclusion, the strategy of mycobacterial-specific nucleic acid amplification in the peripheral blood and arterial specimens of TA patients was unable to lend support to the association between TA and tuberculosis long suggested in the literature.
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    Coilocitose focal em colo uterino e vagina: comparação da histopatologia com a biologia molecular para a pesquisa do papilomavírus humano
    (Universidade Federal de São Paulo (UNIFESP), 1996) Miranda, Simone Madeira Nunes [UNIFESP]; Focchi, José [UNIFESP]
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    Comparação de métodos convencionais e reação em cadeia da polimerase em tempo real na detecção de infecção pelo citomegalovírus in vitro
    (Universidade Federal de São Paulo (UNIFESP), 2009-09-30) Cezar, Amanda Cristina [UNIFESP]; Pacheco-Silva, Alvaro [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Introduction: Clinical isolates of Cytomegalovirus (CMV) are easily spread in vitro resulting in impairment of the monolayer cell where the virus was inoculated, thus evidencing the presence or absence of infection. The cell culture is a classic method for detection of CMV and it was widely used in the past. Antigenemia assay, which detects CMV pp65 antigen, is the method most used currently in clinical practice, because it is faster and specific for detection of the active infection. Recently, the real-time PCR has been used in monitoring of the infection through the quantification of viral load for being a high sensitivity and specificity method to viral DNA. Therefore, the aim of the study was employing tests used in diagnosis and monitoring of infection to the standard CMV strain as a protocol for implantation in experiments in vitro. Methods: Quiescent human fibroblasts in confluent monolayer were inoculated with samples of infected cells by the adapted CMV AD169 strain. The effect of the virus on culture was monitored at 1 hour, 24 hours, 48 hours and 72 hours post infection (h.p.i) by observation of cytopathic effect. The same samples were analyzed by antigenemia being estimate the mean of positive cells in 2x105 cells and by real-time PCR being estimate the mean of copies of viral DNA/Log10 present in samples. Results: Cytopathic effect was first noticed 24 h.p.i, showing that the initiation of morphological changes occurred early. This effect became more intense after 72 h.p.i. Antigenemia assay showed the presence of active infection through pattern of labeling of the pp65 viral antigen found on nucleus of infected cells, while the real-time PCR showed the number of copies of viral DNA in different times of infection. Antigenemia showed an mean of 57 ±56 positive cells 1h.p.i. The peak of the infection was reached 24h.p.i with a significant increase in the mean 2.381 ±168 (P<0.05 versus 1h.p.i) and remained high 48h.p.i, showing an mean of 2.012 ±352 positive cells. However, the mean of antigenemia decrease 72h.p.i to 262 ±5 (P<0.05 versus 48h.p.i). As well as in antigenemia, a significant increase of th viral load was observed of 1h.p.i to 24h.p.i, being the mean of viral DNA detected 11.30 ±0.30 and 11.96 ±0.09, respectively (P<0.05). The levels of viral DNA stayed high 48h.p.i, being detected a mean of 12.33 ±0.26. After this period, viral load decreased significantly to 11.57 ±0.06 (P<0.05 versus 48h.p.i). No correlation was found between the quantitative methods of antigenemia and real-time PCR. Conclusion: The three methods, virus isolation, antigenemia and real-time PCR, showed the success of the CMV infection “in vitro” by cyto-morphological changes, detection of viral antigen specific and viral load by virus DNA detection, respectively. PCR method was more sensitive in detecting virus in relation the other methods. Although sensitive and specific, we consider the need for viral titration in any experimental studies in vitro.
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    Correlation between clinical diagnosis and PCR analysis of serum, aqueous, and vitreous samples in patients with inflammatory eye disease
    (Conselho Brasileiro de Oftalmologia, 2007-02-01) Matos, Kimble [UNIFESP]; Muccioli, Cristina [UNIFESP]; Belfort, Rubens Junior [UNIFESP]; Rizzo, Luiz Vicente; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)
    PURPOSE: To study the applicability (sensitivity, specificity) of polymerase chain reaction (PCR) tests in the detection of cytomegalovirus (CMV), herpes virus (HSV) and varicella zoster (VZV), Epstein-Barr virus (EBV), Mycobacterium sp and Toxoplasma gondii in the diagnosis of patients with or without AIDS, with presumably infectious uveitis, using serum, aqueous humor and vitreous humor samples. METHODS: Twenty individuals with uveitis of presumed infectious origin were evaluated. Sixteen of them had AIDS, four were immunocompetent individuals. We also evaluated 4 normal controls who underwent vitrectomy surgery. Clinical evaluation of the patients was performed together by three clinicians. PCR evaluations of the serum, aqueous, and vitreous humor were performed in a masked fashion by the laboratory staff. RESULTS: Twelve patients had a clinical diagnosis of CMV retinitis. Of these 6 (50%) had a positive PCR for CMV in the vitreous, three (25%) had a positive PCR for CMV in the serum, and none were positive in the aqueous. Five patients had a clinical diagnosis of acute retinal necrosis (ARN). Three (60%) of these had positive PCR for HSV/VZV in the vitreous. One of these patients had a positive PCR reaction for both EBV and HSV/VZV in the vitreous samples. One patient with cutaneous herpes zoster had a positive PCR reaction for HSV/VZV in the serum. Four patients had a presumed diagnosis of ocular toxoplasmosis, one patient (25%) had a positive PCR for Toxoplasma gondii in the serum, 3 (75%) had positive results in the aqueous, and 2 (50%) had positive results in the vitreous. One patient with presumed ocular tuberculosis had a positive PCR reaction both in the serum and in the vitreous samples. Finally, none of the four control individuals revealed any positive PCR reaction. CONCLUSION: PCR is an auxiliary diagnostic procedure that should be evaluated together with ophthalmological aspects of the patient.
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    Desenvolvimento da técnica de quantificação de exons (TQE) para a detecção de grandes deleções e inserções no gene SERPING1 para o diagnóstico de angiodema hereditário
    (Universidade Federal de São Paulo (UNIFESP), 2017) Amorim, Priscila Nicolicht de [UNIFESP]; Pesquero, João Bosco Pesquero [UNIFESP]; http://lattes.cnpq.br/0856630824759511; http://lattes.cnpq.br/2045104800186586; Universidade Federal de São Paulo (UNIFESP)
    Hereditary angioedema (HAE) is a genetic disease caused by mutations in SERPING1 gene encoding the inhibitor of C1 esterase (C1-INH), determining the quantitative and/or qualitative deficiency in C1-INH, largely caused by point mutations and approximately 15 -20%, related to the major gene rearrangements. The intronic gene regions have 17 sequences of repetitive elements represented by Alu, making it prone to deletions and insertions gene/duplications. To enable a semiquantitative molecular diagnosis for SERPING1 gene with the capacity to detect large gene rearrangements, was developed and standardized one technique to quantity the exons (EOT). Through capillary electrophoresis was performed separation and quantification of different sized fragments amplified by Multiplex PCR-technique associated with the use of primers labeled with a fluorescent molecule. It was selected sample of blood from a family with clinical history of HAE, low serum C4 and C1-INH, and who were admitled to molecular analysis of SERPING1 gene by sequencing Sanger in our Center. Samples of this family showed no pathogenic known change in SERPING1 gene by Sanger sequencing technique. So, they were analyzed by TOE confirmed by PCR and MLPA Long Range. Th.[ough TOE was deteded deletion in exon 4 in the patient 12 and confirmed by the MLPA. The deleted fragment was separated by Long Range PCR and subsequently by Sanger sequencing, were found the location and size of the deletion. Therefore, the quantification technique Exons (TOE) proved to be an efficient method for the detection of large deletions or insertions/duplication with power efficiency is simple and fast implementation, in addition to a benefit cost. These data show that SO technique successfully developed for the deletion analysis involving SERPING1 gene, serving as support technique in routine laboratory diagnosis of HAE and may be further standardized and applied to other genetic diseases, contributing to the research and detection major gene rearrangements.
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    Detecção de poliomavírus no carcinoma espinocelular oral
    (Universidade Federal de São Paulo, 2024-04-05) Gomes, Rafael Tomaz [UNIFESP]; Tomimori, Jane [UNIFESP]; Pimentel Neto, Dalva Regina [UNIFESP]; Sichero, Laura; http://lattes.cnpq.br/7837968419392824; http://lattes.cnpq.br/3814292055967483; http://lattes.cnpq.br/1123390691453566; http://lattes.cnpq.br/9979615848188403
    Introdução: O carcinoma espinocelular (CEC) oral é uma neoplasia maligna do epitélio escamoso de elevada morbidade e mortalidade. Fatores predisponentes para o seu desenvolvimento incluem o consumo de álcool e o tabagismo. Entretanto, outros agentes têm sido investigados na sua etiopatogênese, dentre eles os vírus. O envolvimento dos poliomavírus na carcinogênese oral ainda não foi inteiramente elucidado. Objetivo: Investigar a presença do material genético de quatro diferentes tipos de poliomavírus humanos em amostras de CEC oral provenientes de um centro de referência oncológico na cidade de São Paulo (Brasil) e avaliar a possível correlação entre a positividade destes vírus com fatores clínicos e sociodemográficos dos pacientes. Material e Métodos: Sessenta amostras frescas congeladas obtidas do Biobanco do ICESP, de três diferentes sítios (língua, assoalho de boca e orofaringe), com 20 amostras para cada localização, foram selecionadas retrospectivamente dentro de um período de diagnóstico firmado entre 2015 e 2019. Dados de prontuário como sexo, idade, consumo de álcool, tabagismo, estadiamento tumoral e óbito em menos de 5 anos após o diagnóstico foram coletados, bem como a presença da pesquisa da proteína p16, quando disponível. As amostras foram preparadas e tiveram o DNA extraído para pesquisa do material genético dos poliomavírus MCPyV, BKPyV, JCPyV e TSPyV por meio da técnica de reação de cadeia da polimerase (PCR). Os produtos de PCR foram submetidos a sequenciamento e suas identidades foram confirmadas por comparação àquelas depositadas no GenBank®. Resultados: A investigação da presença do DNA dos poliomavírus mostrou positividade de 5% para MCPyV (n=3), 0% para BKPyV, 60% para JCPyV (n=36) e 0% para TSPyV. A identidade das sequências de DNA geradas foi confirmada por meio do alinhamento com àquelas de referência. Não foi constatada qualquer correlação entre a positividade de um determinado poliomavírus nas amostras de CEC oral com fatores clínicos ou sociodemográficos dos pacientes, nem com determinado sítio anatômico, exceto para a associação entre o óbito em menos de 5 anos após o diagnóstico e a positividade para o JCPyV (p=0,009). Também não foi observado qualquer tipo de associação na positividade simultânea dos diferentes poliomavírus entre si ou com a proteína p16. Conclusões: A positividade para poliomavírus no CEC oral foi baixa para MCPyV, alta para JCPyV e nula para BKPyV e TSPyV. Mais estudos são necessários para se compreender com maior clareza a alta prevalência encontrada de JCPyV no CEC oral.
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    Detection of herpes simplex-1 and-2 and varicella zoster virus by quantitative real-time polymerase chain reaction in corneas from patients with bacterial keratitis
    (Consel Brasil Oftalmologia, 2017) Nascimento, Heloisa [UNIFESP]; Watanabe, Aripuanã Sakurada Aranha [UNIFESP]; Vieira, Ana Carolina Cabreira [UNIFESP]; Pellegrini, Andrea Lucia Torres Amorim [UNIFESP]; Yu, Maria Ceclia [UNIFESP]; Bispo, Paulo José Martins [UNIFESP]; Granato, Celso Francisco Hernandes [UNIFESP]; Hofling-Lima, Ana Luisa; Universidade Federal de São Paulo (UNIFESP)
    Objective: Bacterial keratitis occurs worldwide, and despite recent developments, it remains a potentially blinding condition. This study assesses the presence of herpes simplex virus (HSV-1 and -2) and varicella zoster virus (VZV) by quantitative real-time polymerase chain reaction (qPCR) in corneal scrapings from patients with bacterial keratitis. Methods: A total of 65 patients with clinical diagnoses of infectious corneal ulcers prospectively underwent clinical eye examinations. Corneal scrapings were investigated by Gram staining, Giemsa staining, culture, and qPCR (the study group). Risk factors and epidemiological data were recorded. The control group comprising 25 eyes with typical herpes dendritic keratitis was also analyzed by qPCR. Results: From the study group (n=65), nine patients (13.8%) had negative smears, cultures, and qPCR findings. Fifty-six (86.2%) patients had positive cultures: 51 for bacteria, 4 for fungi, and 1 for amoebae. Of the patients who had positive bacterial cultures, qPCR identified 10 patients who were also positive for virus: one for VZV and nine for HSV-1. Of the 25 patients in the control group, 21 tested positive for HSV-1 by qPCR analysis. Conclusions: Herpes may be present in patients with bacterial corneal ulcers, and qPCR may be useful in its detection.
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    Detection of human papillomavirus in epithelial lesions of the conjunctiva
    (Associação Paulista de Medicina - APM, 2000-09-07) Palazzi, Maristela Amaral [UNIFESP]; Erwenne, Clélia Maria [UNIFESP]; Villa, Luísa Lina; Universidade Federal de São Paulo (UNIFESP); University of São Paulo Faculty of Medicine
    CONTEXT: Many factors like exposure to UV radiation, climatic conditions, genetic predisposition, immunological state and, more recently, the presence of HPV have been implicated in the genesis of some lesions of the conjunctiva, especially the carcinoma. OBJECTIVE: To evaluate the presence of HPV DNA in acquired lesions of the conjunctiva and in normal mucosa. TYPE OF STUDY: Cross-sectional study. SETTING: A public university referral center (the Ophthalmology Service of the A.C. Camargo Hospital - A. Prudente Foundation, São Paulo). PARTICIPANTS: Thirty patients with acquired lesions of the conjunctiva and 60 matched controls (by age and sex) were evaluated in this study, from June 1993 to March 1995. PROCEDURES: The detection of HPV DNA in the normal conjunctiva and in acquired lesions was done by the PCR technique and dot blot hybridization. The material was collected by scraping the normal mucosa and the surface of the lesions. A fragment of fresh frozen tissue and paraffin embedded specimens of each lesion were also included. MAIN MEASUREMENTS: The association between the HPV infection and the presence or absence of conjunctival lesions. RESULTS: Sequences of HPV DNA were detected in 4 of the 31 lesions evaluated (12.9%) and in the healthy mucosa of one individual of the control group (1.6%). HPV type 16 was detected in 2 carcinomas and in the normal mucosa of one individual of the control group. HPV type 11 was demonstrated in 2 papillomas of one patient with lesions in both eyes. CONCLUSIONS: The low frequency of HPV DNA found in the lesions of this sample and the detection of the viral genome in the normal mucosa indicate that there is a weak possibility of association between HPV infection and the carcinoma of the conjunctiva.
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    Determinação do fenótipo sexual em uma criança com Mosaicismo 45,X/46,X,Idic(Yp): importância da proporção relativa da linhagem 45,X no tecido gonadal
    (Universidade Federal de São Paulo (UNIFESP), 2006-12-31) Guedes, Alexis Dourado [UNIFESP]; Verreschi, Ieda Therezinha do Nascimento [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    We report here on a girl who, despite her 45,X/46,X,der(Y) karyotype, showed no signs of virilization or physical signs of the Ullrich-Turner syndrome [UTS], except for a reduced growth rate. After prophylactic gonadectomy due to the risk of developing gonadoblastoma, the gonads and peripheral blood samples were analyzed by fluorescence in situ hybridization [FISH] and polymerase chain reaction [PCR] to detect Y-specific sequences. These analyses allowed us to characterize the Yderived chromosome as being an isodicentric Yp chromosome [idic(Yp)] and showed a pronounced difference in the distribution of the 45,X/46,X,idic(Yp) mosaicism between the two analyzed tissues. It was shown that, although in peripheral blood almost all cells (97.5%) belonged to the idic(Yp) line with a duplicated SRY gene, this did not determine any degree of male sexual differentiation in the patient, as in the gonads the predominant cell line was 45,X (60%).
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    Direct fluorescent antibody assay and polymerase chain reaction for the detection of Chlamydia trachomatis in patients with vernal keratoconjunctivitis
    (Faculdade de Medicina / USP, 2011-01-01) Nishiwaki-Dantas, Maria Cristina; Abreu, Mariza Toledo de [UNIFESP]; Melo, Cynthia Mendonça de; Romero, Ivana Lopes; Belfort, Rubens Neto [UNIFESP]; Dantas, Paulo Elias Correa; Santa Casa of São Paulo Corneal and External Disease Service Department of Ophthalmology; Universidade Federal de São Paulo (UNIFESP); Sorocaba Eye Hospital
    OBJECTIVES: To identify Chlamydia trachomatis via polymerase chain reaction and a direct fluorescent antibodyassay in patients with vernal keratoconjunctivitis while comparing the efficacies of both tests for detectingChlamydia trachomatis in these conditions. METHODS: Conjunctival scraping samples were obtained from 177 patients who were divided into two groups: avernal keratoconjunctivitis group (group A) and a control group (group B). The polymerase chain reaction and adirect fluorescent antibody assay were performed. Sensitivity, specificity, receiver operating characteristic curves,and areas under the curve were calculated for both tests in groups A and B. Receiver operating characteristic curveswere plotted using a categorical variable with only two possible outcomes (positive and negative). RESULTS: Statistical analysis revealed a significant association between vernal keratoconjunctivitis and Chlamydia trachomatis infection detected by a direct fluorescent antibody assay with high sensitivity and specificity. Allpatients in group A with positive polymerase chain reactions also presented with positive direct fluorescentantibody assays. CONCLUSION: The association between vernal keratoconjunctivitis and Chlamydia trachomatis infection wasconfirmed by positive direct fluorescent antibody assays in 49.4% of vernal keratoconjunctivitis patients and bypositive polymerase chain reactions in 20% of these patients. The direct fluorescent antibody assay detectedChlamydia trachomatis in a higher number of patients than did the polymerase chain reaction. Although thediagnosis of trachoma is essentially clinical, the disease may not be detected in vernal keratoconjunctivitis patients.Due to the high frequency of chlamydial infection detected in patients with vernal keratoconjunctivitis, we suggestconsidering routine laboratory tests to detect Chlamydia trachomatis in patients with severe and refractory allergicdisease.
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    Doença por citomegalovírus em transplantados renais: características clínicas e cinética da carga viral
    (Universidade Federal de São Paulo (UNIFESP), 2018-11-28) Cavalcante, Samuel de Alencar [UNIFESP]; Pestana, Jose Osmar Medina De Abreu [UNIFESP]; Silva Junior, Helio Tedesco [UNIFESP]; Torres, Leuridan Cavalcante; http://lattes.cnpq.br/1621797721074970; http://lattes.cnpq.br/7250195328752808; http://lattes.cnpq.br/1551824344150025; Universidade Federal de São Paulo (UNIFESP)
    Cytomegalovirus (CMV) is one of the main responsible for opportunistic infections in renal transplantation, associated with important morbidity and mortality. Knowledge about the particularity of viral kinetics may help in the clinical management of renal transplants and to obtain a better cost-effectiveness in the use of prevention therapies. Therefore, the aim of this study was to evaluate the cytomegalovirus (CMV) viral kinetics in renal transplant patients with and without CMV disease. This is a longitudinal study with follow-up of 90 days after transplantation developed at the general transplantation unit (UGT) and at the AC Hart Translational Research Laboratory in IMIP, from April / 2015 to July / 2016, and included 100 patients who underwent cadaveric or live donor kidney transplantation using thymoglobulin and absence of prevention strategies (prophylaxis or preemptive). Blood samples were collected before transplantation and 14, 21, 30, 45, 60, 75, 90 days after transplantation. The CMV viral load was determined by real-time Polymerase Chain Reaction (PCR), and the results expressed in log 10 and IU / mL. The Receiver Operating Characteristic (ROC) curve was used to calculate the best values of the CMV viral load associated with the occurrence of CMV disease, according to the highest accuracy value obtained. For statistical analysis, the program GraphPad Prism 7.0 was used, and considered significant p<0.05. The frequency of CMV disease was 61%, with a mean initial viral load of log10 5.54 or 346,736 IU/mL, median time to anti-CMV treatment was 35.8 days, and clinical recurrence was 13%. The days 30 and 45 after transplantation were the periods with the highest detection of CMV in the group of patients who develoled CMV disease when compared to those who did not developed (p = 0.001 and p <0.0001, respectively). Highers levels of viral load were observed in patients with disease when compared to those without CMV disease in the 21 (p = 0.01), 30 (p = 0.03), 45 (p = 0.001) and 60 (p = 0.01) days after renal transplantation. Viral load kinetics in patients with CMV disease showed exponential growth, with a higher peak at 45 days. Asymptomatic viremias were present in 36% of the patients, with viral load between log10 3.9 - 4.1 from 30 until 90 days after transplantation. A ROC curve with area of 0.77 (95% CI: 0.66- 0.87; p <0.0001) was obtained. The CMV viral load with log10 4.18 (15,135 IU/mL) showed better sensitivity and specificity (70% and 81%, respectively) for the diagnosis of CMV disease. It was observed that the CMV viral load in the patients using cyclosporin was elevated when compared to the tacrolimus and m-TOR inhibitor (p = 0.01). CMV disease showed a high frequency in transplants using thymoglobulin induction and in the absence of prevention strategies, with no impact on mortality. Stable CMV load levels in asymptomatic viremias in most periods after transplantation suggest that preemptive therapy is unnecessary with qPCR log10 <4.0.
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    Endoftalmite bacteriana: aspectos epidemiológicos e diagnósticos
    (Universidade Federal de São Paulo (UNIFESP), 2011-02-22) Melo, Gustavo Barreto de [UNIFESP]; Farah, Ana Luisa Hofling de lima [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Purpose: To report on the incidence, diagnostic technique, microbiological features of endophthalmitis at a university-setting in Brazil. Methods: All cases of presumed postoperative endophthalmitis from 2002 to 2008 at a teaching-hospital were included. Main data assessed were: number of cataract surgeries performed, incidence of endophthalmitis, microbiological outcome (aqueous and/or vitreous culture and Gram staining), and antimicrobial susceptibility testing of the positive cases. Results: Seventy-three eyes of 73 patients (43 females and 30 males) developed endophthalmitis after 24,590 cataract surgeries. The incidence decreased from 0.49% in 2003 to 0.17% in 2006 and stabilized afterwards. Coagulase negative Staphylococci (CoNS) and Streptococcus viridans (56.5% and 15%, respectively) were the most common bacterial isolates. Culture and Gram stain were negative in 36.9%. CoNS presented susceptibility rates of 80%-sensitivity to oxacillin, 90% to fourth-generation quinolones and 100% to vancomycin. Conclusions: The rate of endophthalmitis, diagnostic ability of conventional laboratory investigation, microbial isolates and antibiotic susceptibility are in accordance with other findings of the literature. Despite using prophylactic antibiotic drops, it was possible to identify organisms from infected cases that were susceptible to the antibiotics topically applied.
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    Estudo clínico e molecular de duas novas mutações no gene PROP-1(296G>A e 152G>C) em crianças com deficiência de crescimento
    (Universidade Federal de São Paulo (UNIFESP), 2002) Vieira, Teresa Cristina [UNIFESP]; Abucham Filho, Júlio Zaki [UNIFESP]
    PROP-1 (Prophet of Pit-1), um dos fatores de transcricao especificos hipofisarios, esta envolvido na diferenciacao das celulas da hipofise anterior (somatotrofos, lactotrofos, tirotrofos e gonadotrofos) e mutacoes no gene PROP-1 podem prejudicar a diferenciacao destas celulas durante a organogenese da pituitaria, resultando em DefiCiência Combinada de Hormonios Hipofisarios (DCHH). O fenotipo dos pacientes com mutacoes do PROP-1 e variavel, desde quadros de DCHH grave afetando todos os hormonios adenohipofisarios, ate quadros mais leves, com defiCiências hormonais parciais e nao tao severas. Analisamos o gene PROP-1 de duas familias cujos filhos apresentam DCHH. Na primeira familia, os pais sao primos em primeiro grau, a media de suas estaturas e, baixa (-2 DP da curva de Tanner) e ambos os filhos apresentam baixa estatura (BE), que inicialmente estava compativel com sua altura alvo. O acompanhamento auxologico revelou uma diminuicao gradativa da velocidade de crescimento e aparecimento progressivo de defiCiência de hormonio de crescimento e de outras, defiCiências hormonais adenohipofisarias. Na segunda familia os pais nao tem historia de consanguinidade e a media de suas estaturas esta no -1.3 DP da curva de Tanner. O filho mais velho foi levado ao servico medico por BE e crescimento lento. Seu diagnostico inicial foi BE por defiCiência de GH (DGH). Durante a evolucao, outras defiCiências de hormonios adenohipofisarios foram identificadas. Para analise do gene PROP-1, DNA foi isolado de linfocitos...(au)
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    A experiência da auto coleta para rastreio de infecções sexualmente transmissíveis em mulheres acompanhadas em serviço terciário de referência em Ginecologia
    (Universidade Federal de São Paulo, 2024-08-29) Marques, Miriane Borges [UNIFESP]; Speck, Neila Maria de Goís [UNIFESP]; Tso, Fernanda Kesselring [UNIFESP]; http://lattes.cnpq.br/4640267129189904; http://lattes.cnpq.br/8169544398769371; http://lattes.cnpq.br/1603135213462333
    Objetivo: Avaliar a experiência da auto coleta para rastreio de infecções sexualmente transmissíveis (IST) em mulheres acompanhadas em serviço referência em ginecologia. Métodos: Foram realizados dois estudos, ambos prospectivos, descritivos, qualitativos e quantitativos. O primeiro estudo obteve de forma oportuna 47 participantes do sexo feminino, com colo uterino, sexualmente ativas e acima dos 25 anos de idade com diagnóstico ou risco de IST acompanhadas em serviço de referência em ginecologia. Foram divididas em Grupo 1: diagnóstico de lesão intraepitelial de alto grau do colo uterino (n=18); Grupo 2: pessoas que vivem com o vírus da imunodeficiência humana - PVHIV (n=17) e Grupo 3: risco de IST (n=12). Dados sociodemográficos foram aplicados, seguido da auto coleta e coleta realizada por um clínico, para rastreio de Chlamydia trachomatis (CT) e Neisseria gonorrhoeae (NG) através da técnica de Reação de polimerase em cadeia (PCR). A prevalência, adequabilidade das amostras e concordância entre os resultados foram analisados e comparou-se os grupos quanto ao desconforto. No segundo estudo, as mesmas 47 participantes foram convidadas por mensagens eletrônicas via telefone celular para irem até o serviço e realizar o teste de identificação do ácido desoxirribonucleico do papilomavírus humano de alto risco (DNA-HPVar), pela auto coleta vaginal e coleta realizada por um clínico, sendo avaliado a adesão ao teste. A prevalência de DNA-HPVar, a adequabilidade das amostras e a concordância entre os resultados das duas formas de coleta foram analisados. Comparou-se preferência, confiabilidade e conforto entre os dois métodos de coleta, além de avaliar o conhecimento da auto coleta como forma de rastreio de câncer de colo. Os resultados com valores de p<0,05 foram considerados significativos. Resultados: No primeiro estudo foi observado que PVHIV tinham menor média etária para o início da atividade sexual e que mulheres em risco de IST tiveram o maior número de parcerias sexuais em um ano. A prevalência de CT foi 2,1% e NG: 0%. Nenhuma das amostras foi considerada indeterminada. A concordância dos resultados entre os métodos de coleta foi absoluta. A auto coleta foi menos desconfortável em relação à coleta feita por um clínico. No segundo estudo, das 47 participantes, 17 (39,5%) tiveram adesão, não sendo possível estabelecer contato com 4. As participantes mais jovens e com maior escolaridade preferiram a auto coleta e tinham conhecimento dessa estratégia de rastreio. A confiança no resultado dos dois métodos de coleta foi absoluta. A prevalência de DNA-HPVar foi 29% e não houve resultado indeterminado, sendo 94,7% a concordância positiva dos resultados e 100%, negativa. Conclusões: A experiência da auto coleta para rastreio de IST em serviço terciário de referência em ginecologia foi positiva. A auto coleta pode não ser suficiente para otimizar o rastreamento de IST. A associação de outras estratégias, como educação, informação, organização do sistema de saúde, de forma tecnológica, facilitando o acesso aos kits de coletas e sua devolução bem como orientar seguimento, podem otimizar a adesão à prevenção de IST e influenciar a experiência da auto coleta em serviços de referência.
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    Expression of heparanase in basal cell carcinoma and squamous cell carcinoma
    (Soc Brasileira Dermatologia, 2016) Silva Pinhal, Maria Aparecida [UNIFESP]; Leal Almeida, Maria Carolina [UNIFESP]; Costa, Alessandra Scorse [UNIFESP]; Theodoro, Therese Rachell [UNIFESP]; Serrano, Rodrigo Lorenzetti [UNIFESP]; Santos Machado Filho, Carlos D'Apparecida [UNIFESP]
    Background: Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives: Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods: Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results: The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion: The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment.
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    High occurrence of Shiga toxin-producing Escherichia coli (STEC) in healthy cattle in Rio de janeiro State, Brazil
    (Elsevier B.V., 1999-10-01) Cerqueira, Aloysio de Mello Figueiredo [UNIFESP]; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Joaquim, Rogério Marques; Andrade, João Ramos da Costa; Universidade Federal Fluminense (UFF); Universidade Federal de São Paulo (UNIFESP); Universidade do Estado do Rio de Janeiro (UERJ)
    In order to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains, 197 fecal samples of healthy cattle from 10 dairy farms, four beef farms and one slaughterhouse at Rio de Janeiro State, Brazil, were examined for Shiga toxin (Stx) gene sequences by polymerase chain reaction (PCR). for presumptive isolation of O157:H7 E. coli, the Cefixime-potassium tellurite-sorbitol MacConkey Agar (CT-SMAC) was used. A high occurrence (71%) of Stx was detected, and was more frequently found among dairy cattle (82% vs. 53% in beef cattle), in which no differences were observed regarding the age of the animals. Dot blot hybridization with stx1 and stx2 probes revealed that the predominant STEC type was one that had the genes for both stx1 and stx2 in dairy cattle and one that had only the stx1 gene for beef cattle. Three (1.5%) O157:H7 E. coli strains were isolated from one beef and two dairy animals by the use of CT-SMAC. To our knowledge, this is the first report of O157:H7 isolation in Brazil. A PCR-based STEC detection protocol led to the isolation of STEC in 12 of 16 randomly selected PCR-positive stool samples. A total of 15 STEC strains belonging to 11 serotypes were isolated, and most of them (60%) had both stx1 and stx2 gene sequences. Cytotoxicity assays with HeLa and Vero cells revealed that all strains except two of serotype O157:H7 expressed Stx. the data point to the high prevalence of STEC in our environment and suggest the need for good control strategies for the prevention of contamination of animal products. (C) 1999 Elsevier Science B.V. All rights reserved.
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    Investigation of pseudogenes RHD Psi and RHD -CE-D hybrid gene in D-negative blood donors by the real time PCR method
    (Elsevier B.V., 2012-12-01) Szulman, Alexandre [UNIFESP]; Machado Nardozza, Luciano Marcondes [UNIFESP]; Barreto, Jose Augusto [UNIFESP]; Araujo, Edward [UNIFESP]; Moron, Antonio Fernandes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Introduction: the Rh system is the most polymorphic and immunogenic of all systems of blood groups. Currently more than 49 antigens were identified with five major antigens D, C, c, E. e. Knowledge of the molecular basis of the Rh system permitted the understanding of both the mechanism of Rh phenotype on the antigen variants of RHD and RHCE in Caucasians the primary mechanism of D-negative phenotype is the complete deletion of RHD gene, while the black Africans is the presence of pseudogene and gene hybrid RHD-CE (3-7)-D.Objective: To determine the prevalence gene pseudogene and hybrid gene and standardization of molecular techniques in method of Taqman on real-time PCR for RHD genotyping. Patients and methods: 203 samples of D-negative donor were used to establish and validate the effectiveness of RHD genotyping in real-time PCR using Taqman technology. the extraction was performed using a commercial kit QIAmp DNA mini kit. Samples exon 10 and 7 positive were submitted to amplification of exon 5, confirming the pseudogene RHD Psi, whereas exon 10 + exon 7 - for the hybrid gene (C) cdes and mutation C733G (Leu245Val) of the RHCE gene.Results: Twenty-five (12.3%) samples were positive, 14 amplified for both exons 10 and 7 while in 11 only for the exon 10. When extended the screening using exon 10, 7 and 5, only 06 amplified. the pseudogene was present in 07 samples (3.5%) and the hybrid RHD-CE (3-7) in 04 (1.97%), while in 177 (87.2%) of Rh negative donors were RHD gene deletion. in 07 samples not amplified for exon 3 had mutated and the mutation C733G antigen.Conclusion: the prevalence of pseudogene was 3.5% and the gene hybrid RHD-CE of 1.9%. This approach for real-time PCR as a complementary tool is technically feasible and the results of this study helped develop a new strategy for RHD genotyping. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.
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    Laboratory results in ocular viral diseases: implications in clinical-laboratory correlation
    (Conselho Brasileiro de Oftalmologia, 2007-03-01) Marangon, Fabiana Bogossian [UNIFESP]; Miller, Darlene; Alfonso, Eduardo; Universidade Federal de São Paulo (UNIFESP); Universidade de Miami Bascom Palmer Eye Institute
    PURPOSE: To document etiology and predictive value of clinical diagnosis in laboratory confirmed viral diseases. METHODS: Reports of culture-positive cases of samples collected from patients presenting from January 1987 - December 2001 were evaluated. RESULTS: One thousand nine hundred and sixty-four (1964) cultures were submitted during 1987-2001. Twenty-six percent were positive (514). Human herpesvirus 1 was the most frequent agent isolated from all positive culture (56%). Adenovirus was the most common virus isolated from conjunctiva (66%), human herpesvirus 1 from lid and cornea (76%, 88%) and cytomegalovirus from vitreous (27%). Some unusual pathogens were recovered from conjunctiva as cytomegalovirus and from cornea as adenovirus, enterovirus and cytomegalovirus. Recognition of common viral syndromes was human herpesvirus 1 (88%), epidemic keratoconjunctivitis (88%), acute hemorrhagic conjunctivitis (70%) and varicella zoster virus (100%). However, some misdiagnosed cases were observed. Thirteen percent of conjunctivitis thought to be caused by herpes were due to adenovirus, 3.2% to Enterovirus, 3.2% to varicella zoster virus and 3.2% to human cytomegalovirus. Also, 5% of cases with a clinical diagnosis of herpes keratitis were caused by adenovirus and 2.7% by enterovirus. Finally, 4.8% of cases thought to be adenovirus conjunctivitis were herpes conjunctivitis. CONCLUSIONS: Human herpesvirus 1 remains the most frequently isolated virus from ocular sites in general (56%). Nonherpetic corneal isolates were in decreasing order: adenovirus, enterovirus and cytomegalovirus. Clinical and laboratory correlation was less than 90%. The most misdiagnosed cases were herpes conjunctivitis and keratitis, some cases of adenovirus conjunctivitis some cases of acute hemorrhagic conjunctivitis. It is essential that a rapid and specific diagnosis is offered under atypical viral presentation for the institution of specific antiviral therapy and to avoid complications that can be a result of misdiagnosis and inappropriate treatment. Also it is important to do viral testing in order to confirm clinical diagnosis, report emerging infections, resistance and change in the epidemiology.
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    Leishmaniose tegumentar americana: apresentação pouco comum
    (Soc Brasileira Dermatologia, 2008-09-01) Guedes, Antonio Carlos Martins [UNIFESP]; Carvalho, Maria de Lourdes Ribeiro de; Melo, Maria Norma; Universidade Federal de Minas Gerais (UFMG); Universidade Federal de São Paulo (UNIFESP)
    We report the case of a patient with American cutaneous leishmaniasis and prominent lesions on the face. Diagnostic criteria included clinical and epidemiological data, Montenegro's skin test. identification of Leishmania by means of polymerase chain reaction and clinical response to treatment. Our report is important to call attention to an unusual presentation of American cutaneous leishmaniasis.
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    Molecular biology applied to the laboratory diagnosis of bacterial endophthalmitis
    (Conselho Brasileiro de Oftalmologia, 2009-10-01) Bispo, Paulo José Martins [UNIFESP]; Hofling-Lima, Ana Luisa [UNIFESP]; Pignatari, Antonio Carlos Campos [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Bacterial endophthalmitis is a serious but uncommon intraocular infection which frequently results in vision loss. Early diagnosis and appropriate therapy are associated with better visual outcome. Conventional microbiological methods are currently used for microbiological characterization of eyes with suspected endophthalmitis. However, the sensitivity of bacterial detection from aqueous and vitreous humor using microbiology techniques is poor, and time-consuming to confirm the results. The application of molecular methods enhances significantly laboratory confirmation of bacterial endophthalmitis, demanding a shorter time to draw a definitive result and thereby promoting the early initiation of a more specific therapy to limit the empirical or unnecessary use of broad-spectrum antibiotics. PCR-based techniques, including post-PCR methods such RFLP, DNA probe hybridization and DNA sequencing have been successfully used for the diagnostic elucidation of clinically suspected bacterial endophthalmitis cases, showing promising application in the routine practice of ocular microbiology laboratories.