RESUMEN
Rapid detection of drug-resistant bacteria in clinical samples plays an instrumental role in patients' infection management and in implementing effective infection control policies. In the study described in this report, we validated a multiplex TaqMan real-time quantitative PCR (qPCR) assay for the detection of bla(KPC) genes and the human RNase P gene in Bactec blood culture bottles. The MagNA Pure LC (version 2.0) instrument was utilized to extract nucleic acids from the inoculated broth, while bovine serum albumin (BSA) was utilized as the PCR inhibitor reliever. The multiplex assay, which was specific for the detection of bla(KPC) genes, had a limit of detection of 19 CFU per reaction mixture with human blood-spiked Bactec bottles. Of the 323 Bactec blood culture sets evaluated, the same 55 (17%) blood cultures positive for carbapenem-resistant bacteria by culture were also positive by the validated qPCR assay. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the qPCR assay compared to the results of culture were all 100%. bla(KPC) genes were also detected from the same Bactec bottle broth after manual extraction with a QIAamp DNA minikit; however, there was an average 3-threshold-cycle delay in the qPCR readings. With the limited therapeutic options available, the accurate and rapid detection of bla(KPC)-possessing bacteria by the described bla(KPC)/RNase P assay will be a crucial first step in ensuring optimal clinical outcomes and infection control.
Asunto(s)
Proteínas Bacterianas/genética , Técnicas Bacteriológicas/métodos , Enterobacteriaceae/enzimología , Enterobacteriaceae/genética , Reacción en Cadena de la Polimerasa/métodos , beta-Lactamasas/genética , Sangre/microbiología , Enterobacteriaceae/crecimiento & desarrollo , Humanos , Valor Predictivo de las Pruebas , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The emergence of oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus highlights the need for rapid oseltamivir resistance screening. We report the development and validation of high-throughput real-time reverse transcriptase PCR assays for the detection of the H275Y substitution in the neuraminidase 1 gene that can be accomplished in 3 to 4 h.