Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (bla KPC ) 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 bla KPC genes using TaqMan chemistry. The q-PCR amplification of bla KPC DNA was linear over 7 log dilutions (r 2 ؍ 0.999; slope, 3.54), and the amplification efficiency was 91.6%. 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 bla KPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while bla KPC genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for bla KPC 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 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the bla KPC q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to bla KPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.
, and good agreement were observed among all assays, although the Xpert and Aptima assays, which provided the most similar outputs (estimated mean viral loads of 2.67 log copies/ml [95% confidence interval [CI], 2.50 to 2.84 log copies/ml] and 2.68 log copies/ml [95% CI, 2.49 to 2.86 log copies/ml], respectively), correlated best with the RealTime assay (89.8% concordance, with Pearson r values of 0.97 to 0.98). These three assays exhibited greater precision than the NucliSens v2.0 assay. All assays were equally sensitive for subtype B and AG/G samples and for samples with viral loads of 1.60 to 3.00 log copies/ml. The NucliSens v2.0 assay underestimated A1 samples and those with viral loads of >3.00 log copies/ml. The RealTime assay tended to underquantify subtype C (compared to the Xpert and Aptima assays) and subtype A1 samples. The Xpert and Aptima assays were equally efficient for detection of all subtypes and viral loads, which renders these new assays most suitable for clinical HIV laboratories.
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