Rapid detection of blaKPC carbapenemase genes by real-time PCR.
Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (bla(KPC)) enzymes are among the most common beta-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-plus-carbapenem 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 carbapenem-resistant 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.
Hindiyeh
Musa
M
Central Virology Laboratory, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel. hindiyeh@yahoo.com
Smollen
Gill
G
Grossman
Zehava
Z
Ram
Daniela
D
Davidson
Yehudit
Y
Mileguir
Fernando
F
Vax
Marina
M
Ben David
Debbie
D
Tal
Ilana
I
Rahav
Galia
G
Shamiss
Ari
A
Mendelson
Ella
E
Keller
Nathan
N
D001426:Bacterial Proteins / Q000235:genetics*; D004755:Enterobacteriaceae / Q000235:genetics; D004756:Enterobacteriaceae Infections / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics*
D001426:Bacterial Proteins; D001618:beta-Lactamases; C495220:beta-lactamase KPC-3, Klebsiella pneumoniae; C063912:carbapenemase