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
Arquivos
Data
2010-11-24
Autores
Rocchetti, Talita Trevizani 
Orientadores
Pignatari, Antonio Carlos Campos
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
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.
Solid organ transplanted patients are at high risk for blood stream infection. The institution of appropriate antimicrobial therapy guided by a rapid and accurate microbiological diagnosis of blood stream infections is related to a successful outcome. The aim of this study was the detection of Gram-positive and Gram-negative bacteria from automated blood cultures (Bactec®, Becton Dickinson) with the use of the multiplex real-time polymerase chain reaction (PCR) and detection of antimicrobial resistance genes. Methods: A total of 185 blood cultures, 126 positive and 59 negative, were obtained of 117 patients submitted to solid organ transplant at two transplant centers in the city of São Paulo, Brazil, Hospital São Paulo and Hospital do Rim e Hipertensão. DNA from the blood cultures was extracted by phenol chloroform method (Brazol®, LGC, Brazil). The detection of bacterial DNA was performed using universal primers of 16S rRNA gene. The differentiation between Gram-positive and Gram-negative bacteria was done by hybridization with Gram-specific probes by multiplex real-time TaqMan. The resistance genes: blaSHV, blaTEM, blaCTX-M, blaKPC, blaSPM, blaVIM, blaIMP, vanA, vanB and mecA were detected using specific primers by real-time SYBR Green. The adequacy of antimicrobial treatment was evaluated by reviewing the records of patients. Results: Fifty nine samples were positive for Grampositive and sixty seven samples were positive for Gram-negative. All samples (100%) were concordant between blood culture and PCR. Thirty two samples were positive for mecA gene, five for blaCTX-M gene, one for blaKPC gene, one for blaSHV gene and one for blaTEM. Eighty eight were negative for all genes. The detection of antimicrobial resistance genes could enhance the appropriateness of antibiotic therapy, particularly in descalation the treatment of bacteremia caused by Gram positive and early adequacy in the treatment of Gram negative bacteremia of solid organ transplanted patients. Conclusion: The in house multiplex PCR for Grampositive/ Gram-negative bacteria and detection of antimicrobial resistance genes by Real Time PCR could be useful for rapid diagnosis of bloodstream infection in patients undergoing solid organ transplant.
Solid organ transplanted patients are at high risk for blood stream infection. The institution of appropriate antimicrobial therapy guided by a rapid and accurate microbiological diagnosis of blood stream infections is related to a successful outcome. The aim of this study was the detection of Gram-positive and Gram-negative bacteria from automated blood cultures (Bactec®, Becton Dickinson) with the use of the multiplex real-time polymerase chain reaction (PCR) and detection of antimicrobial resistance genes. Methods: A total of 185 blood cultures, 126 positive and 59 negative, were obtained of 117 patients submitted to solid organ transplant at two transplant centers in the city of São Paulo, Brazil, Hospital São Paulo and Hospital do Rim e Hipertensão. DNA from the blood cultures was extracted by phenol chloroform method (Brazol®, LGC, Brazil). The detection of bacterial DNA was performed using universal primers of 16S rRNA gene. The differentiation between Gram-positive and Gram-negative bacteria was done by hybridization with Gram-specific probes by multiplex real-time TaqMan. The resistance genes: blaSHV, blaTEM, blaCTX-M, blaKPC, blaSPM, blaVIM, blaIMP, vanA, vanB and mecA were detected using specific primers by real-time SYBR Green. The adequacy of antimicrobial treatment was evaluated by reviewing the records of patients. Results: Fifty nine samples were positive for Grampositive and sixty seven samples were positive for Gram-negative. All samples (100%) were concordant between blood culture and PCR. Thirty two samples were positive for mecA gene, five for blaCTX-M gene, one for blaKPC gene, one for blaSHV gene and one for blaTEM. Eighty eight were negative for all genes. The detection of antimicrobial resistance genes could enhance the appropriateness of antibiotic therapy, particularly in descalation the treatment of bacteremia caused by Gram positive and early adequacy in the treatment of Gram negative bacteremia of solid organ transplanted patients. Conclusion: The in house multiplex PCR for Grampositive/ Gram-negative bacteria and detection of antimicrobial resistance genes by Real Time PCR could be useful for rapid diagnosis of bloodstream infection in patients undergoing solid organ transplant.
Descrição
Citação
ROCCHETTI, Talita Trevizani. 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. 2010. 157 f. Dissertação (Mestrado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2010.