Navegando por Palavras-chave "diagnóstico molecular"

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    Aplicação da técnica de pcr em tempo real na identificação microbiana e detecção de genes de resistência a antimicrobianos em episódios de bacteremia de pacientes pediátricos com câncer
    (Universidade Federal de São Paulo (UNIFESP), 2014-04-11) Carlesse, Fabianne Altruda de Moraes Costa [UNIFESP]; Pignatari, Antonio Carlos Campos Pignatari [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Background: Bloodstream infections (BSI) are major cause of infection-related mortality in patients with cancer. Phenotipic methods are utilized to detect and to identify pathogens in blood stream cultures but with slow time for positivity and final results. Molecular techniques have been utilized for a quick ethiologic diagnosis of blood stream infections allowing the institution of an early adequate therapy improving patients survival rates. Aim: To evaluate if the Real Time PCR method used to identify the main pathogens causing blood stream infections and some important antimicrobial resistance genes causing bloodstream infections could improve the early diagnosis and adequate therapy in oncology pediatric patients. Methods: During March 2010 to March 2012 we conducted a retrospective study at the Oncology Pediatric Institute (IOP-GRAACC-Brazil). The blood stream samples were incubated in the automated Bactec® system and the microbal identification and susceptibility tests were done in the automated Phoenix® system. We included 81 blood positive blood stream samples that were submitted to molecular analysis. Polimicrobial infections were excluded, 69 BSI were analysed in 64 patients. Samples from the blood stream bottles were submitted to molecular tests by Real Time PCR with specific Gram probes, 17 specific gender sequences and antimicrobial resistance genes blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB e mecA.The adequacy of the antimicrobial therapy was evaluated at the time of the Gram stain result from the positive blood bottle (time 1) and in the available final phenotypic result for the assistant physician (time 2). Results: Gram positive bacteria were identified in 61% of the samples, Gram negative bacteria in 32% and fungi in 7%. There was agreement in 82,6% for the initial Gram stain and 78,2% in the final species identification between the phenotipic and molecular methods. The mecA gene was detected in 81,4% of Staphylococcus spp, and was 91,6% concordant with the phenotypic method. Detection of vanA gene was 100% concordant. For Gram negative bacteria the concordance was 71,4% for Enterobacteriacea and 50% for Pseudomonas aeruginosa. Therapy was inadequate particularly in patients who died (5/6). The molecular tests was concordant in 50% of theses cases. Conclusion: Real Time PCR could be useful in the early identification of pathogens and antimicrobial resistance genes in bloodstream infections of pediatric oncologic patients and could contribute to improve the antimicrobial therapy.
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    Comparação e validação de metodologias moleculares para diagnóstico da meningite tuberculosa
    (Universidade Federal de São Paulo (UNIFESP), 2016-02-26) Palomo, Flavia Silva [UNIFESP]; Pignatari, Antonio Carlos Campos [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Tuberculosis meningitis is a severe form of extrapulmonary tuberculosis, because of its high morbidity and mortality. The most common agents of this disease are the mycobacteria of the M. tuberculosis? complex. Definitive diagnosis includes smear that have low sensitivity and culture that delay in diagnosis. The use of molecular methods is promising in the diagnosis of this disease, since they are fast with high, sensitivity and specificity. This study aimed to compare and to validate molecular protocols (PCR) for the diagnosis of tuberculosis meningitis directly from CSF samples including the mycobacterium DNA extraction techniques and targets for PCR amplification. Methods: For comparison and validation of molecular protocols we used positive control group (CSF pools known negative and infected with ATCC M. tuberculosis), negative control group and clinical CSF samples. Four DNA extraction techniques (Phenol-Chloroform-Thiocyanate guanidine, Thiocyanate-Silica guanidine, Resin and Resin with ethanol) were compared. Also, the negative and positive control groups and CSF samples were used to determine the best target (IS6110, and MPB64 hsp65KDa) for diagnosis of tuberculosis meningitis. For statistical analyzes the CSF samples were classified as true positive and negative. For this classification a survey in the site TBweb was done with analysis of laboratory data and medical records of patients. Results: The extraction protocol using the phenol chloroform-thiocyanate guanidine showed the best results in terms of quantification and sensitivity of PCR amplification, presenting up to 10 times more DNA than the second best (silica guanidine thiocyanate). The targets that showed the best amplification results was the the IS6110, with a good agreement (0.64 ?) in conventional PCR and moderate (0.54 ?) in real-time PCR. The sensitivity and specificity were, respectively, 100 % and 79 % for the real-time PCR method and 94 % and 87 % for conventional, when compared to culture. The sample analysis for IS6110 real-time PCR amplification showed a 91% sensitivity, 97% specificity and optimal agreement (0.89 ? ) with the clinical diagnosis . When this analysis was grouped by patient, we showed a sensitivity of 100 % , specificity of 98 % and a very good agreement ( ? 0.96 ) with the clinical diagnosis. Conclusion: A protocol using extraction with phenol chloroform and guanidine thiocyanate, followed by amplification of the IS6110 target for real time PCR in house showed to be suitable for molecular diagnosis of tuberculosis meningitis in our clinical setting.