Navegando por Palavras-chave "PCR em tempo real"

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    A ação in vitro de pós bióticos da microbiota da pele na inibição do crescimento do Staphylococcus aureus: potencial uso na dermatite atópica
    (Universidade Federal de São Paulo, 2023-07-28) Fuzeti, Deborah Corrêa [UNIFESP]; Lopes, Patricia Santos [UNIFESP]; Batista, Wagner Luiz [UNIFESP]; http://lattes.cnpq.br/4373797404389169; ttp://lattes.cnpq.br/7939687315116927; https://lattes.cnpq.br/8969028749372844
    A dermatite atópica (DA) é uma doença inflamatória de pele crônica recidivante que possui como principais sintomas xerose, prurido e lesões eczematosas, no entanto a sua fisiopatologia ainda não é completamente elucidada. Um dos principais fatores para o desenvolvimento da DA é a disbiose da microbiota da pele, onde o Staphylococcus aureus, é o principal microrganismo relacionado. Um dos principais fatores que levam a infecção são os seus fatores de virulência como os superantígenos e o sistema agr. O sistema agr é um gene regulador do sistema quorum sensing da espécie Staphylococcus, que auxilia em fatores de infecção por promover o aumento da densidade celular e a expressão de fatores de virulência secretados. Nesse contexto foram selecionados dois Staphylococcus comensais da microbiota humana, o Staphylococcus epidermidis, e o Staphylococcus capitis, visando a utilização de seus pós bióticos como possível tratamento. Para avaliar a ação dos pós bióticos perante o S. aureus, realizou-se métodos in vitro para a analisar o perfil de polaridade utilizando a CLAE, inibição de crescimento microbiano com métodos de inibição de crescimento por difusão em ágar e em meio líquido e análise da modulação da expressão gênica com PCR em tempo real. Os resultados mostraram moléculas com comprimento de onda diferente nos três microrganismos estudados, indicando ali um perfil molecular particular para cada microrganismo. No método de inibição de crescimento por difusão em ágar, o uso dos derivados de pós bióticos mostrou ser o mais eficaz, sendo as amostras derivadas do S. capitis com melhor índice inibitório de 33,33%, e no teste de inibição de crescimento em meio líquido também houve inibição do S. aureus com o uso de derivados de pós bióticos, tendo como melhor índice inibitório o extrato de S. capitis acetato de etila com 86,58%. Na análise da expressão gênica por PCR em tempo real ocorreu down-regulation nas amostras S. capitis 25% e S. capitis 50% com o gene agrA, e S. capitis 25% com o gene agrC, nas demais amostras com os genes agrA, agrC e agrD ocorreu upregulation da expressão do agr de S. aureus com o uso dos pós bióticos.
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    Desenvolvimento de técnica molecular para avaliação da carga viral do HGV/GBV-C em pacientes co-infectados pelo HIV-1
    (Universidade Federal de São Paulo (UNIFESP), 2008-01-30) Alves, Viviane Kelly [UNIFESP]; Granato, Celso Francisco Hernandes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Introduction: The HGV/GBV-C infection is frequent in humans and could last for years without evident clinical symptoms. HGV/GBV-C is a member of the Flaviviridae family strongly associated with the hepatitis C virus and composed by a single positive RNA strain. Recent studies suggest that HGV/GBV-C infection in HIV seropositive patients is associated with a delayed progression to AIDS, lower HIV viral load and a higher CD4 T-cell count, however, some studies failed to demonstrate such beneficial effects in these patients. Objectives: The aim of this study was to standardize a real time PCR to estimate the prevalence and HGV/GBV-C viral load among the HIV seropositive patients, comparing HIV monoinfected and co-infected in terms of T CD4 cell count and HIV viral load. Methods: To achieve this purpose, the 5’UTR genomic region of HGV/GBV-C was selected. A molecular cloning was needed in order to obtain a recombinant plasmid and a high production of this plasmid through a bacterial cloning in DH10B strain. The recombinant plasmid was linearized and submitted to in vitro transcription to obtain and to quantify synthetic RNA to construct the absolute quantification curve of the real time PCR system. The patient samples were submitted to the assay and the results were compared to the standard curve in order to obtain the HGV/GBV-C viral load. Results: A serial dilution of the synthetic RNA in factor 10 produced a quantification curve with 9 orders of magnitude, varying from 102 to 1010 genome equivalent/ìL. To the assay, the inclination of the straight line was -3.56, the intercept was 45.56, the Pearson correlation coefficient was r2=0.99 and the efficiency of the reaction was of 90.9%. The reproducibility of the assay was evaluated based on a quadruplicate reaction of the quantification curve with a mean coefficient of variation of 1.2%. 102 patients were included in the study, mean age of 42±9 years and 57.8% of them were man. From the total cohort, 21% were positive to HGV/GBV-C RNA in the plasma, with a mean viral load of 300.455 copies/mL and 26.4% were positive to anti-E2 antibodies. There were no statistical significance in regard to the CD4 T-cell count and HIV viral load when comparing HIV monoinfected patients with that co-infected with HGV/GBV-C (p=0,297)and a weak negative correlation was observed between HIV and HGV/GBV-C viral loads (Spearman’s= -0,237). Conclusions: The standardization of the real time PCR could be done and is in agreement with other published studies. According to the literature data, there is a high prevalence of the HGV/GBV-C infection among HIV seropositive patients. The weak negative correlation between HIV and HGV/GBV-C viral load is in agreement with previous publications, suggesting a beneficial effect regarding to AIDS progression, however, other factors can be associated and future studies are needed to better understand this viral interaction.
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    Detecção Bacteriana e de Genes de Resistência a Antimicrobianos pela Técnica de PCR em Tempo Real em Infecções de Corrente Sanguínea de Pacientes Submetidos a Transplante de Órgãos Sólidos
    (Universidade Federal de São Paulo (UNIFESP), 2010-11-24) Rocchetti, Talita Trevizani [UNIFESP]; Pignatari, Antonio Carlos Campos [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Pacientes transplantados de órgãos sólidos apresentam alto risco para infecção da corrente sanguínea. A instituição da terapia antimicrobiana apropriada guiada por um diagnóstico rápido e preciso das infecções da corrente sangüínea está relacionada a um resultado satisfatório. O objetivo deste estudo foi a detecção de bactérias Grampositivas e Gram-negativas em hemocultura automatizada (Bactec®, Becton Dickinson), com a utilização do multiplex para reação da polimerase em cadeia (PCR) em Tempo Real e detecção de genes de resistência. Métodos: Um total de 185 hemoculturas, 126 positivas e 59 negativas, foram obtidos de 117 pacientes submetidos a transplante de órgãos sólidos, dois centros de transplante na cidade de São Paulo, Brasil, Hospital São Paulo e Hospital do Rim e Hipertensão. DNA das culturas de sangue foi extraído pelo método fenol clorofórmio (Brazol®, LGC, Brasil). A detecção do DNA bacteriano foi realizada utilizando iniciadores universais do gene 16S rRNA. A diferenciação entre as bactérias Gram-positivas e Gram-negativas foi feito por hibridização com sondas específicas por multiplex TaqMan em Tempo Real. Os genes de resistência: blaSHV, blaTEM, blaCTX-M, blaKPC, blaSPM, blaVIM, blaIMP, vanA, vanB e mecA foram detectadas utilizando iniciadores específicos, em Tempo Real sitema SYBR Green. A adequação do tratamento antimicrobiano foi avaliada pela revisão do prontuário dos pacientes. Resultados: Cinqüenta e nove amostras foram positivas para bactérias Gram-positivas e sessenta e sete amostras foram positivas para bactérias Gram-negativas. Todas as amostras (100%) foram concordantes entre hemocultura e PCR. Trinta e duas amostras foram positivas para o gene mecA, cinco para o gene blaCTX-M, uma para o gene blaKPC, uma para o gene blaSHV e uma para blaTEM. Oitenta e oito foram negativas para todos os genes. A detecção de genes de resistência a antimicrobianos favoreceria a adequação da antibioticoterapia, particularmente no descalonamento no tratamento de bacteremias causadas por bactérias Gram positivas e na adequação precoce no tratamento de bacteremias por bacilos Gram negativas multiresistentes em pacienetes tranplantados de órgãos sólidos. Conclusão: A PCR multiplex “in house” para bactérias Gram-positivas e Gram-negativas e detecção de genes de resistência aos antimicrobianos por PCR em Tempo Real poderia ser útil para o diagnóstico rápido da infecção da corrente sangüínea em pacientes submetidos a transplante de órgão sólidos.
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    Detecção de resistência a antimicrobiano em amostras de vigilância de doentes renais crônicos: comparação de métodos fenotípicos e moleculares
    (Universidade Federal de São Paulo (UNIFESP), 2016-04-29) Rezende, Thais Freitas Teles [UNIFESP]; Pignatari, Antonio Carlos Campos [UNIFESP]; http://lattes.cnpq.br/9461346610553865; http://lattes.cnpq.br/4149593220799749; Universidade Federal de São Paulo (UNIFESP)
    Background: Multidrug resistant (MDR) bacteria colonization is a serious health care problem that improves the risk for infection and contribute for dissemination of antimicrobial resistance in health care associated environments. Among chronic renal failure patients, where the risk for acquisition and dissemination of these resistant bacteria are higher, surveillance culture is recommended as a component of infection control programs Objective: The aim of this study is to compare the performance of phenotypic methods for surveillance of MDR bacteria with real time PCR (qPCR) in large population of chronic renal failure patients. Methods: We included a total of 546 CRFP from a tertiary and University center specialized in chronic renal failure care. These patients were divided in 3 groups: conservative treatment (129), patients under dialysis (217) and transplanted patients (200). We collected nasal swabs for MRSA detection and rectal swabs for carbapenemase and VRE detection, in two different moments; total of 1092 samples (ESwabTM). For phenotypic screening we used the CHROMagar for KPC, MRSA and VRE. For the molecular analysis we used the qPCR for detection of the genes: blaKPC, blaNDM, blaSPM, blaGES, blaVIM, blaOXA48, vanA, mecA and aur (S. aureus identification). Conventional PCR were also performed when necessary. Results: Among the 1,092 samples we found 13.8% of KPC positives according to chromogenic agar (AC). Only 26 were confirmed as KPC positive according conventional PCR. On the other hand, according to qPCR direct from swab were 31 (2,8%) positives to KPC, 39 (3,6%) for GES and 3 (0,3%) for SPM with a concordance index Kappa of 0,256. For VRE the AC was positive in XXX 16 (1,5%) of the patients and the qPCR positive in 20 (1,8%) of the patients, Kappa 0,135. For MRSA we tested only 298 samples, and we observed 4 (1,3%) of positivity with AC and 15 (5%) according to qPCR. Conclusions: We observed for carbapenemase an increased rate of false positives results using AC (than qPCR) with a low agreement between methods. For VRE we observed a better sensitivity using the AC once qPCR could not detect confirmed strains of VRE. However, for MRSA the molecular method had better performance. We conclude that among this high-risk population molecular methods had better results than the culture for carbapenemases detection and MRSA. Besides, it can provide results faster than cultures allowing the early implementation of control and prevention measures to reduce the dissemination of MDR bacteria among patients and health care environment.
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    High resolution melting versus sequenciamento genético de nova geração na detecção de mutação do gene KRAS em câncer colorretal
    (Universidade Federal de São Paulo (UNIFESP), 2017-05-30) Miranda, Raelson Rodrigues [UNIFESP]; Forones, Nora Manoukian [UNIFESP]; Silva, Tiago Donizetti da [UNIFESP]; http://lattes.cnpq.br/0950776650887037; http://lattes.cnpq.br/7314943504526739; http://lattes.cnpq.br/5692801803165299; Universidade Federal de São Paulo (UNIFESP)
    Introduction: Frequency of KRAS gene mutations in colorectal cancer is around 34% and confers poor prognosis. The most frequency are on exon 2, 3 and 4. The knowledge about KRAS profile plays crucial role in the management of this disease, because the use of MABs like cetuximab and panitumumab are recommended exclusively for KRAS wild-type tumors. In other words, it has important predictive value. PCR-High-Resolution melting (HRM) analysis is a novel technology able to detect mutations with high sensitivity through of variations in melting temperature of DNA with low cost than sequencing methods. Therefore, could be a good tool for screening of KRAS mutations. Objective: Comparing capacity of HRM to detect exons 2, 3 and 4 KRAS mutations with Next Generation Sequencing. Methods: We performed PCR-HRM runs for 47 patients with confirmed colorectal adenocarcinoma in StepOne plus Real-time PCR (Thermo-Fisher/Life Technology©) and compared the melting temperature and HRM curve between mutated and wild-type groups. We used the Illumina HiSeq platform® as reference methods for the Next Generation Sequencing. The statistical analysis was performed through SPSS software v20.1. Results: For exon 2, the melt curves were different between mutant and wild-type groups. The mean of melting temperature was 78.13 ºC for wild-type and 77.87 ºC for mutant profile (p= 0.001; CI 95%: 0,11-0,41; T-test). Sensitivity was 93.8%, specificity 96.6%, PPV 92.2%, NPV 98.51%, accuracy 91.5% and false positive and negative rates 3,4% for PCR-HRM. In terms of exons 3 and 4, melt curve were not different. Conclusions: In this study, the high resolution melting curve profile proved be a good test for screening of exon 2 KRAS mutations in colorectal cancer with high sensitivity and specificity. However, for exons 3 and 4, HRM was not able to detect mutations.
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    Otimização de técnica de PCR em tempo real para detecção das regiões pol e env dos subtipos B e F de HIV-1 e triagem de seus recombinantes
    (Universidade Federal de São Paulo (UNIFESP), 2009-01-28) Teixeira, Daniela [UNIFESP]; Diaz, Ricardo Sobhie [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Background: The discovery of 42 HIV-1 circulating recombinant forms (CRF) together with the innumerous unique HIV recombinants forms, makes clear the role of genetic recombination for the epidemic. In Brazil, clades B, F, and C co-circulate, with 5 recently described CRFs. Real Time PCR is a rapid and reliable tool capable of detecting different HIV-1 subtypes and recombinant profiles. Objective: The aim of this study was to develop real time PCR systems in order to detect the Brazilian CRF_28 and CRF_29, which are B/F recombinants, as well as detect B/F recombinants generated by in vitro competition assays. In future, these systems should be able to discriminate subtypes in clinical surveys. Methodology: MT-4 cells were separately infected with the viral strains BZ167 (subtype B) and BR020 (subtype F), and supernatant was collected in order to optimizing the real time PCR systems (TaqMan®) developed to detect the subtype profile of different genomic regions, including pol gene (protease, reverse transcriptase, integrase) and env gene (gp120 and gp41). The designed primers should be able to equally amplify the subtype B and F, which should be discriminated by subtype-specific probes. For future validation of these PCR systems, 157 clinical samples from the city of Santos were sequenced and phylogeneticaly analyzed in order to perform the clade assignment with Neighbor-Joining algorithm (Phylip software package v3.5). Results: The designed systems were able to differentiate the utilized viral strains. The estimated efficiencies for each system, for each probe, subtypes B and F separately, were respectively: 80,97 and 85,16% for protease region; 89,80 and 75,09% for reverse transcriptase region; 80,90% and 83,83% for integrase region; 93,49 and 98,93% for gp120 region; and 88,45 and 80,19% for gp41 region. For the co-infected cell culture, the detection of each subtype was performed in the first and fifth passages. Generally, the initial concentration of subtype B appeared to have decreased, some of them becoming undetectable, whereas subtype F seemed to increase with the passages, for protease, reverse transcriptase, gp120 and gp41 regions. The integrase region was an exception, since only the subtype B was detected, with increasing Cycle threshold (Ct) values over time. Sequencing results revealed that 65 out of 157 samples had the subtype profile defined for all regions. 43 out of 71 were defined as B, whereas 3 were F in all regions. 12 samples presented the CRF_28 profile, and 9 samples presented the CRF_29 profile. There were no subtype C samples in any genomic regions analyzed. Conclusion: The use of real time PCR technique for identification of fragments’ subtypes in cell culture and for evaluation of replicative dynamics of recombination in co-infected cultures warrants its potential use in future in vivo surveys. This methodology proved to be efficient, fast, less cumbersome and less expensive than DNA sequencing. The newly designed systems performed for supernatant of competition assays had suggested a divergent distribution of subtypes for the different regions, which reflects the possibility of genetic recombination. Results from clinical samples revealed a high prevalence of CRF_28/29 in this geographic region, thus reflecting the resulting consequence of different co-circulating strains and pointing to the need for a careful surveillance.
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    Pesquisa de Polymavirus-BK em transplantes renais: incidência e correlação com parâmetros clínicos e laboratoriais
    (Universidade Federal de São Paulo (UNIFESP), 2010-08-25) Svicero, Bianca Silva [UNIFESP]; Granato, Celso Francisco Hernandes [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Introduction: The BK virus (BKV) belongs to the Polyomaviridae family, is a non enveloped virus with double-stranded DNA, may be associated with hemorrhagic cystitis, ureteral stenosis, and in some cases, cancer. The polyomaviruses are found in about 80% of the general population, however its clinical manifestations are rare and limited to individuals with weakened immune system. The BKV is a virus with the potential to establish latency and reactivate. Asymptomatic reactivation and low level of replication are observed in approximately 5% of the healthy population. In the patients of renal transplant. Reactivation becomes relevant, since 10-40% of patients with BKV active infection can progress to graft loss. The diagnosis of BKV infection can be accomplished by “decoy” cells in urinary sediment and / or viral load evaluation by polymerase chain reaction in Real Time (qPCR). Treatment is not defined by antiviral drugs but by reducing the dose of immunosuppressive drugs. Objectives: The aim of this study was to standardize Real Time PCR to estimate the BKV viral load in plasma and urine and the presence of “decoy” cells in urine of patients undergoing renal transplantation in the period between november/2008 and may/2010, since pre transplant until 12 months after transplantation. Analysis of epidemiological aspects related to infection and the correlation with the course of the disease were evaluated as well. Methods: Analysis of urine sediment by Papanicolaou staining for the presence of "decoy" cells by electron microscopy. For standardization of Real-Time PCR in plasma and urine a commercial BKV DNA with a viral load set at 1.6 x105cópias / mL was used for the construction of standard curve; betaglobin was used as an internal control. Based in the techinique publishe in 2004 by Randhawa and colleagues. Results: The study included 81 patients followed prospectively over 12 months, where 70.3% were male with median age of 39 years at the time of transplantation. The urine cytology provided a positive result in 1.6% of the samples. The standard curve was performed by five serial dilutions of positive control (1-104), the slope of the curve was -3.60, the Pearson correlation coefficient was r2 = 0.99 and efficiency of the reaction was 91 6%. Plasma samples showed variations in viral load, 39.1% from the 1.08 x104 9.51 x104 copies / mL and 26.3% with viral load greater than or equal to 1x105 copies / mL and 34.6% with viral load below 1x104 copies / mL. We demonstrated an incidence of 29 cases by 1000 patients. The clinical features did not correlate with laboratory findings found, since there was no description of symptoms related to infection with BKV in patients supposedly with viral load indicative of infection during the 12 months analyzed. Conclusions: The standardization of real-time PCR was achieved and can be compared with previous studies..We didn’t consider the PCR results indicative of disease because a reanl biopsy was not performed. The result of decoy cells research was confirmed by another analyst who was unaware of the survey data. We couldn’t find define another laboratory parameters as predictors of infection by BKV.