Resumen
Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (blaKPC) enzymes are among the most common -lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of blaKPC genes using TaqMan chemistry. The q-PCR amplification of blaKPC DNA was linear over 7 log dilutions (r2 0.999; slope, 3.54), and the amplification efficiency was 91.6 percent. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-pluscarbapenem disks and for blaKPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1percent) of the 187 samples collected, while blaKPC genes were detected in 54 (28.9percent) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for blaKPC genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenemresistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100percent and 95percent, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9percent sensitivity and 96.4percent specificity. Overall, the blaKPC q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to blaKPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.(AU)
