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Journal of Clinical Microbiology, September 2008, p. 2879-2883, Vol. 46, No. 9
0095-1137/08/$08.00+0     doi:10.1128/JCM.00661-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Detection of blaKPC Carbapenemase Genes by Real-Time PCR{triangledown}

Musa Hindiyeh,1* Gill Smollen,2 Zehava Grossman,1 Daniela Ram,1 Yehudit Davidson,2 Fernando Mileguir,1 Marina Vax,1 Debbie Ben David,2 Ilana Tal,2 Galia Rahav,2 Ari Shamiss,3 Ella Mendelson,1 and Nathan Keller2

Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center,1 Infectious Disease Unit/Microbiology Laboratory, Tel Aviv University, Sheba Medical Center,2 Sheba Medical Center, Tel Hashomer, Israel3

Received 8 April 2008/ Returned for modification 29 May 2008/ Accepted 29 June 2008


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ABSTRACT
 
Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (blaKPC) enzymes are among the most common β-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%. 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 blaKPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while blaKPC genes were detected in 54 (28.9%) 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 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 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.


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INTRODUCTION
 
Carbapenem resistance among Enterobacteriaceae, in particular among Klebsiella pneumoniae and Escherichia coli, is an emerging problem worldwide (14, 16, 20, 22, 25, 26). Several resistance mechanisms have been reported to circumvent the efficacy of carbapenems, and carbapenemases (carbapenem-hydrolyzing β-lactamases) are the most prominent enzymes that neutralize carbapenems (17, 18). Class A carbapenemases, which include blaKPC, NMC, SME-1 to -3, IMI-1, and GES, have been characterized in several genera of the family Enterobacteriaceae (17). Other carbapenem resistance mechanisms, including porin changes and changes in penicillin-binding proteins, have also been implicated in carbapenem resistance (23, 27, 28). blaKPC enzymes, so called because they have been identified mainly in K. pneumoniae, have been reported in Klebsiella oxytoca, Serratia spp., Enterobacter spp., Salmonella spp., Citrobacter freundii, and Pseudomonas aeruginosa (3, 8, 10, 11, 15, 24, 28). The genes encoding the blaKPC enzymes are usually flanked by transposon-related sequences that have been identified on transferable plasmids, thus giving them the potential to disseminate rapidly. Three different blaKPC genes have been reported to date: blaKPC-2, blaKPC-3, and blaKPC-4; blaKPC-1 and blaKPC-2 are identical (27). blaKPC-producing bacteria are usually resistant to virtually all classes of antibiotics—β-lactam agents, including penicillins, cephalosporins, monobactams, and carbapenems (1, 27, 28)—leaving physicians with limited antibiotic choices for treating infected patients.

In order to control the spread of blaKPC-containing bacteria in hospitalized patients, effective infection control measures and controlled antibiotic usage must be complemented by the utilization of rapid and sensitive blaKPC diagnostic assays (13). The utilization of such diagnostic tools will help in rapidly isolating colonized or infected patients and assigning them to cohorts.

In this report, we describe the development and validation of a real-time PCR (q-PCR) assay for the detection of blaKPC genes. The assay was validated by comparing q-PCR with regular bacterial culturing on MacConkey agar-plus-carbapenem disks of routine surveillance perianal/rectal swabs obtained from the Sheba Medical Center intensive care units during an outbreak of one strain of K. pneumoniae harboring blaKPC-3. Moreover, two automated bacterial DNA extraction methodologies were compared: the bioMérieux NucliSENS easyMAG system and Roche MagNA Pure LC DNA isolation kit III (bacteria, fungi).


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MATERIALS AND METHODS
 
Collection of surveillance samples. Sheba Medical Center is a 1,200-bed tertiary teaching medical center in the center of Israel. As a result of an increasing rate of detection of carbapenem-resistant Enterobacteriaceae (CRE), from less than 1% in 2005 to 18% in 2006 and 30% in the first half of 2007, surveillance perianal/rectal swabs were routinely collected from all patients admitted to the 16-bed surgical intensive care unit and the 4-bed neurosurgical intensive care unit in order to control the spread of CRE. Perianal/rectal swabs were collected upon a patient's admission to the unit and once weekly until the patient was discharged. During the study period from 1 April to 31 July 2007, 187 perianal/rectal swabs were randomly collected in duplicate from 128 patients. One sample was collected on a Copan Amies sterile transport swab (Copan Diagnostics, Corona, CA) and transported to the microbiology laboratory for the detection of CRE. The other sample was collected on a rayon swab, submerged in 1 ml saline, and stored at –20°C pending q-PCR analysis. The study was approved by the Sheba Medical Center Ethics Review Board.

Bacterial stocks. The following reference bacterial strains were obtained from the American Type Culture Collection (ATCC; Manassas, VA) and evaluated for the presence of blaKPC genes: K. pneumoniae ATCC 13883, extended-spectrum-β-lactamase-positive K. pneumoniae ATCC 700603, E. coli ATCC 25922, and β-lactamase-positive E. coli ATCC 35218. In addition, clinical isolates of blaKPC-3-positive K. pneumoniae, AmpC-positive K. pneumoniae, AmpC-positive Escherichia coli, Citrobacter koseri, Enterobacter species, Serratia marcescens, Salmonella species, Shigella species, Proteus mirabilis, multidrug-resistant (MDR) Pseudomonas aeruginosa, Morganella morganii, Providencia species, MDR Acinetobacter baumannii, Aeromonas hydrophila, methicillin-resistant Staphylococcus aureus, methicillin-susceptible Staphylococcus aureus, Streptococcus agalactiae, and Enterococcus species recovered at Sheba Medical Center during the year 2007 were evaluated in the study. A blaKPC-2-positive K. pneumoniae strain was generously provided by Shiri Navon-Venezia, Tel-Aviv Sourasky Medical Center.

Detection of carbapenem-resistant bacteria by culture. Perianal/rectal swabs were streaked onto MacConkey agar plates (Hy-Lab, Rehovot, Israel), ensuring that all sides of the swab touched the initial quadrant. The initial quadrant was plated as a lawn, and a meropenem (10 µg) and an ertapenem (10 µg) disk were placed in this area using sterile forceps (5). The plates were incubated overnight at 35°C in ambient air. The presence of bacterial colonies in the area surrounding either disk was worked up microbiologically. Classic bacteriological methods were used to identify the organisms according to routine protocols based on the 2nd edition of the Clinical Microbiology Procedures Handbook, and susceptibility testing was performed according to the CLSI guidelines (7, 12). During the study period, carbapenem resistance was confirmed by the Kirby-Bauer disk diffusion method on Mueller-Hinton plates (BD Diagnostics, Heidelberg, Germany) using ertapenem (10 µg), meropenem (10 µg), and imipenem (10 µg) (Oxoid, United Kingdom).

DNA extraction. (i) DNA extraction from cultured bacterial colonies. Fresh well-isolated colonies were used for DNA extraction with a QIAamp DNA minikit (Qiagen GmbH, Hilden, Germany) according to the protocol suggested by the manufacturer. Briefly, a 2-McFarland-standard bacterial suspension was prepared in saline, and bacterial DNA was extracted from 200 µl (1.2 x 108 CFU) of the suspension. Extracted bacterial DNA was eluted from the columns in 100 µl elution buffer and stored at –20°C.

(ii) DNA extraction from perianal/rectal swabs using the Roche MagNA Pure LC instrument. The MagNA Pure LC DNA isolation kit III (bacteria, fungi) was used to extract bacterial DNA from well-vortexed perianal/rectal swabs in 1 ml saline according to the manufacturer's suggestions. Briefly, external lysis was performed on the maximum aliquot volume allowed (100 µl) to inactivate the bacteria. This was followed by DNA extraction using the MagNa Pure LC extractor. Extracted bacterial DNA was eluted in 100 µl elution buffer and stored at –20°C.

(iii) DNA extraction from perianal/rectal swabs using the bioMérieux NucliSENS easyMAG system. Bacterial DNA was extracted from well-vortexed perianal/rectal swabs in 1 ml saline according to the manufacturer's suggestions. Briefly, external lysis was performed on the maximum aliquot volume allowed (200 µl) to inactivate the bacteria. This was followed by DNA extraction using the easyMAG extractor. Extracted bacterial DNA was eluted in 110 µl elution buffer and stored at –20°C.

blaKPC detection by q-PCR. The ABI Prism 7700, 7500, and 7000 sequence detection systems (Applied Biosystems, Foster City, CA) were used for the amplification and detection of the blaKPC amplicon (246 bp) by TaqMan technology. The forward primer sequence (5'-GAT ACC ACG TTC CGT CTG G-3') specific for the detection of all blaKPC types was designed in-house. The reverse primer sequence (5'-GCA GGT TCC GGT TTT GTC TC-3') was previously reported by Tenover et al. (22). In this study, the blaKPC-specific probe (6-carboxyfluorescein-5'-AGC GGC AGC AGT TTG TTG ATT G-3'-6-carboxytetramethylrhodamine) was designed in-house and was labeled with 6-carboxyfluorescein at the 5' end and the 6-carboxytetramethylrhodamine quencher at the 3' end. The sensitivity of the TaqMan assay was optimized by evaluating different concentrations of the primers (200, 300, 600, and 900 nM) and probe (100, 200, and 300 nM). The concentrations of the primers and probe used in this study that gave the best detection limits were 300 nM for blaKPC primers and 200 nM for the blaKPC probe. The 25-µl q-PCR mixture contained the q-PCR MasterMix Plus reaction buffer (Eurogentec, Belgium), 5-carboxy-X-rhodamine succinimidyl ester (ROX) as an internal reference dye, HotGoldStar DNA polymerase, deoxynucleoside triphosphates (including dUTP), and uracil-N-glycosylase. q-PCR was performed under the following conditions: 2 min at 50°C, 10 min at 95°C, and 50 cycles of 15 s at 95°C and 1 min at 60°C.

Interpretation of results. A sample was considered positive by q-PCR if it crossed the threshold before a threshold cycle (CT) of 40 and negative if the CT was greater than 45. q-PCR was repeated for all samples with CTs of ≥40 and ≤45, and samples were reported as positive if the multicomponent curve indicated that the CT determined by the SDS software was the result of a true amplification event and if a band of the appropriate size was detected by gel electrophoresis on a 2% agarose gel stained with ethidium bromide. Samples with CTs of ≥40 and ≤45 and with a positive multicomponent curve but lacking an appropriate band by gel electrophoresis would be reported as equivocal, and those with a negative multicomponent curve were reported as negative. Actually, we did not encounter a single sample that was reported as equivocal by q-PCR. To determine the efficiency of the q-PCR, CT values obtained from a series of template DNA dilutions were graphed on the y axis versus the log of the dilution on the x axis. The slope of this line was used in the following efficiency (E) equation: E = 10(–1/slope).


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RESULTS
 
Analytical sensitivity and precision of the blaKPC q-PCR assay. The analytical sensitivity of the blaKPC q-PCR assay was determined after serially diluting known concentrations (1.2 x 106 CFU) of K. pneumoniae blaKPC-3 extracted DNA. The assay's amplification was linear over 7 log dilutions (r2 = 0.999; slope, –3.54), and the amplification efficiency was 91.6% (Fig. 1). The detection limit of the real-time blaKPC assay was 1 CFU. Beyond 1 CFU, results were not reproducible, most likely due to the well-recognized stochastic properties of q-PCR on highly diluted nucleic acids. The analytical sensitivity of the assay did not change upon the utilization of three different K. pneumoniae blaKPC-3-positive strains with different carbapenem sensitivity patterns, K. pneumoniae blaKPC-2-positive strains, or Enterobacter cloacae blaKPC-positive strains. The performance of the K. pneumoniae blaKPC-3-positive control over 20 runs indicated that the q-PCR assay was highly stable and precise. Overall, the aliquoted blaKPC-3-positive DNA control had a mean CT of 29.6, a standard deviation of 0.7, and a coefficient of variation of 2.2%.


Figure 1
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  		FIG. 1. Linear limits of detection of the blaKPC q-PCR assay. Serial (10-fold) dilutions of K. pneumoniae DNA harboring a blaKPC-3 plasmid were prepared and tested by the q-PCR assay. CT values were obtained for each dilution and plotted against the number of CFU per reaction.

Cross-reactivity with other bacterial pathogens. The specificities of the primers and probe for the detection of blaKPC genes were evaluated by the BLAST search program, available at www.ncbi.nlm.nih.gov. No matches to the primers and probe sequences were found other than those for the blaKPC genes. In addition, the blaKPC q-PCR assay was negative with DNA extracted from the following bacterial pathogens: K. pneumoniae ATCC 13883, extended-spectrum-β-lactamase-positive K. pneumoniae ATCC 700603, E. coli ATCC 25922, β-lactamase-positive E. coli ATCC 35218, AmpC-positive K. pneumoniae, AmpC-positive E. coli, Citrobacter koseri, Enterobacter species, Serratia marcescens, Salmonella species, Shigella species, Proteus mirabilis, MDR Pseudomonas aeruginosa, Morganella morganii, Providencia species, MDR Acinetobacter baumannii, Aeromonas hydrophila, methicillin-resistant Staphylococcus aureus, methicillin-susceptible Staphylococcus aureus, Streptococcus agalactiae, and Enterococcus spp. Likewise, the assay was negative for an MDR Proteus mirabilis clinical isolate that was resistant to all carbapenems, was positive by the modified Hodge test, and did not encode the blaKPC genes.

Detection of carbapenem-resistant organisms by culture and by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae (blaKPC-3), were cultured from 35 (27.3%) of the 128 patients evaluated during the study period, while blaKPC genes were detected in 40 (31.3%) and 38 (29.7%) of the patient samples extracted with the NucliSENS easyMAG and Roche MagNA Pure LC extractors, respectively. No statistically significant difference between the two DNA extraction methods was noted (P = 0.25 by McNemar's test). None of the patient samples grew more than one type of carbapenem-resistant bacteria.

Upon stratification of the results by the total number of samples analyzed, which in some cases included more than one sample from the same patient depending on the duration of the hospital stay, carbapenem-resistant organisms, all the same strain of K. pneumoniae, were isolated from 47 (25.1%) of the 187 swabs collected, while blaKPC genes were detected by q-PCR from 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Seven samples were positive for blaKPC by q-PCR and negative for carbapenem resistance by culture, while all samples for which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity of the q-PCR assay after extraction by the NucliSENS easyMAG system was 100% (95% confidence interval, 92.4 to 100%), while the specificity was 96.4% (95% confidence interval, 90 to 97.6%) (Table 1). The difference between the number of samples in which blaKPC genes were detected after extraction by the NucliSENS easyMAG system and the number of samples that grew carbapenem-resistant bacteria by culture was statistically significant (P = 0.016 by McNemar's test). It is worth noting that of the blaKPC-positive samples analyzed by q-PCR after NucliSENS easyMAG extraction, 26 (48.1%) gave CT readings less than 30, 25 samples (46.3%) had CTs between 30 and 40, and 3 samples (5.6%) had CTs between 40 and 45.


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  		TABLE 1. Comparison between detection of carbapenem-resistant bacteria in perianal/rectal swabs by culture and detection of blaKPC genes by q-PCR after extraction with the BioMérieux NucliSENS easyMAG or the Roche MagNA Pure LC automated extractor

When bacterial DNA was extracted by the Roche MagNA Pure LC kit, blaKPC genes were detected by q-PCR in 51 (27.3%) patient samples. Five samples were positive for blaKPC genes by q-PCR but negative for carbapenem-resistant bacteria by culture, while one sample was culture positive for carbapenem-resistant bacteria but tested negative by q-PCR. Thus, the sensitivity of the q-PCR assay was 97.9% (95% confidence interval, 88.9 to 99.6%), while the specificity was 96.4% (95% confidence interval, 91.9 to 98.5%) (Table 1). The difference between the detection of carbapenem-resistant bacteria by culture and that by q-PCR after extraction by the Roche MagNA Pure LC kit was not statistically significant (P = 0.22 by McNemar's test). Of the positive samples analyzed by q-PCR after Roche MagNA Pure LC extraction, 20 (39.2%) gave CT readings less than 30, 30 (58.8%) had CTs between 30 and 40, and 1 (2.0%) had a CT between 40 and 45.

The discrepancies for the seven samples positive for blaKPC by q-PCR but negative for carbapenem-resistant bacteria by culture were resolved by repeating the extraction from the original tubes using both extraction systems and repeating the q-PCR assay for both new and old extractions. All samples that were initially blaKPC positive by q-PCR tested positive upon repetition, and the negative samples remained negative. In addition, q-PCR-positive samples (from both extraction methods) were also confirmed positive by agarose gel electrophoresis, and PCR amplicons of appropriate sizes were sequenced using the ABI Prism Dye Deoxy Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) as previously described (19). Sequence analysis revealed that all seven positive samples extracted by the NucliSENS easyMAG system and the five positive samples extracted by the Roche MagNA Pure LC instrument were blaKPC genes. The sample that was positive by culture for carbapenem-resistant bacteria but negative by q-PCR for blaKPC genes after Roche MagNA Pure LC extraction was low positive (CT, 41) for blaKPC after extraction with the NucliSENS easyMAG system. This sample was considered false negative after the Roche MagNA Pure LC extraction.


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DISCUSSION
 
Rapid detection of blaKPC genes is of the utmost importance, since these MDR organisms have the potential to spread rapidly in hospital environments and cause nosocomial infections with high mortality rates (4, 20). During a recent K. pneumoniae blaKPC-3 outbreak at Sheba Medical Center, the mortality rate of bacteremic patients was 40% (unpublished data). A similar mortality rate (47%) was reported by Bratu et al. for bacteremic patients from New York City (4).

Detection of carbapenem-resistant organisms has been problematic, because some isolates express low levels of resistance that may not be detected by conventional automated and nonautomated methods (2, 6, 13). To circumvent this problem, it has been recommended to use ertapenem to screen for these organisms. Ertapenem has been shown to be the most sensitive indicator of the presence of blaKPC enzymes (2, 5). In our experience, ertapenem has also proved to be the most sensitive antibiotic for the detection of blaKPC-positive organisms. Currently, ertapenem is not on the panel of most conventional automated and nonautomated methods.

Molecular techniques have been used to rapidly detect blaKPC resistance genes from patients' original samples (22). In addition, in an outbreak setting similar to that witnessed at Sheba Medical Center, screening for colonized but asymptomatic patients by molecular techniques was critical for controlling the outbreak. Indeed, after Israel's Ministry of Health established guidelines, based on CDC recommendations, for assigning patients colonized or infected with blaKPC-positive organisms to cohorts, the incidence of blaKPC strains declined from 0.8/1,000 patient days to 0.4/1,000 patient days (21).

In this study we validated a rapid, sensitive, and specific q-PCR assay for the detection of blaKPC genes from perianal/rectal surveillance samples after extraction by the NucliSENS easyMAG or the Roche MagNA Pure LC automated extractor. Regardless of the automated extraction system utilized, the assay can be performed in less than 4 h, which will allow for rapid assignment of colonized patients to cohorts, thus reducing the chance of spreading the organism in the hospital setting. This was in contrast to the time-consuming, less sensitive, and nonstandardized bacterial culture method, which can take more than 24 h to detect carbapenem-resistant bacteria (13). q-PCR eliminates both the chance of post-PCR contamination and all the time-consuming post-PCR processes such as gel electrophoresis (9). In addition, we have shown that the assay was highly stable and precise, as evidenced by the performance of the aliquoted blaKPC DNA-positive control, which was stable over 20 consecutive runs (mean CT, 29.6; standard deviation, 0.7; coefficient of variation, 2.2%). The performance of the blaKPC-positive control was not affected by the type of ABI instrument utilized (model 7700, 7500, or 7000); all gave similar results. However, we predict that the utilization of a real-time PCR instrument that allows for analysis of bacterial DNA extracted from patient samples upon receipt (i.e., SmartCycler) will be an asset to the laboratory, eliminating dependence on batching patient samples. Such instrumentation would improve the efficiency of assignment of patients colonized with blaKPC-containing bacteria to cohorts.

The analytical sensitivity of the blaKPC q-PCR assay was 1 CFU, with a linear dynamic range from 1 CFU to 1 x 106 CFU, regardless of the imipenem MIC (8 to >32 µg/ml) for the carbapenem-resistant K. pneumoniae strains evaluated. This analytical sensitivity was better than that reported by Landman et al., who showed that as little as 2.7 CFU/ml of K. pneumoniae could be detected for isolates with high imipenem MICs (≥32 µg/ml) by culture. The q-PCR sensitivity was even higher, by about 6 log units, when a carbapenem-resistant strain with a low imipenem MIC (1 to 8 µg/ml) was evaluated (13). A drawback of the protocols described by Landman et al. is that they are time-consuming, requiring on average 48 h to confirm an isolate as blaKPC positive (13).

The excellent analytical sensitivity of the q-PCR assay described was complemented by excellent clinical sensitivity after extraction with the NucliSENS easyMAG (100%) or Roche MagNA Pure LC (97.9%) automated extractor. Overall, the results from the NucliSENS easyMAG system were 2 to 3 CTs better than those from the Roche MagNA Pure LC extractor. The difference is likely related to the maximum sample volume allowed by the extraction method: 100 µl for the MagNA Pure LC compared to 200 µl for the NucliSENS easyMAG extractor. The MagNA Pure LC extraction of bacterial DNA required longer sample manipulation (20 to 30 min) before loading than the NucliSENS easyMAG method (10 min). One of the drawbacks of utilizing either automated extraction system is that every eight samples must be batched in order to reduce sample extraction costs. However, this downside is minimized in large laboratories, where large numbers of samples are extracted.

As a result of the detection of seven blaKPC-positive samples by q-PCR that were negative for carbapenem-resistant bacteria by culture, enrichment broth with a meropenem disk (10 µg) was added to the blaKPC culture protocol as previously described (13). The addition of enrichment broth has been reported to increase the sensitivity of the culture-based detection of carbapenem-resistant bacteria; however, an extra 24 h are needed for the detection of carbapenem-resistant bacteria, thus increasing the chance of the spread of these highly drug-resistant organisms in a hospital setting (2, 13).

One of the drawbacks of the q-PCR assay described is that it detects only blaKPC-positive bacteria, while bacterial culture can detect bacterial isolates with both blaKPC and non-blaKPC mechanisms of carbapenem resistance (17). In addition, we have not evaluated blaKPC-4-positive organisms, since we had no access to these strains. Based on the BLAST search program analysis, we predict that the blaKPC q-PCR assay described will be as sensitive for the detection of blaKPC-4, since no reported mutations are present in the primers or probe sites.

Our data support the utilization of q-PCR for screening patients upon admission and routinely during hospitalization in areas with high prevalences of blaKPC-positive Enterobacteriaceae. Efforts should be invested in developing rapid and sensitive diagnostic techniques for laboratories where q-PCR technology is not available.


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FOOTNOTES
 
* Corresponding author. Mailing address: Central Virology Laboratory, Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel. Phone: 972-3-530-2066. Fax: 972-3-530-2457. E-mail: hindiyeh{at}yahoo.com Back

{triangledown} Published ahead of print on 9 July 2008. Back


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REFERENCES
 
    1
  1. Alba, J., Y. Ishii, K. Thomson, E. S. Moland, and K. Yamaguchi. 2005. Kinetics study of KPC-3, a plasmid-encoded class A carbapenem-hydrolyzing beta-lactamase. Antimicrob. Agents Chemother. 49:4760-4762.[Abstract/Free Full Text]
    2. 2
  2. Anderson, K. F., D. R. Lonsway, J. K. Rasheed, J. Biddle, B. Jensen, L. K. McDougal, R. B. Carey, A. Thompson, S. Stocker, B. Limbago, and J. B. Patel. 2007. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J. Clin. Microbiol. 45:2723-2725.[Abstract/Free Full Text]
    3. 3
  3. Bratu, S., D. Landman, M. Alam, E. Tolentino, and J. Quale. 2005. Detection of KPC carbapenem-hydrolyzing enzymes in Enterobacter spp. from Brooklyn, New York. Antimicrob. Agents Chemother. 49:776-778.[Abstract/Free Full Text]
    4. 4
  4. Bratu, S., D. Landman, R. Haag, R. Recco, A. Eramo, M. Alam, and J. Quale. 2005. Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium. Arch. Intern. Med. 165:1430-1435.[Abstract/Free Full Text]
    5. 5
  5. Bratu, S., M. Mooty, S. Nichani, D. Landman, C. Gullans, B. Pettinato, U. Karumudi, P. Tolaney, and J. Quale. 2005. Emergence of KPC-possessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection. Antimicrob. Agents Chemother. 49:3018-3020.[Abstract/Free Full Text]
    6. 6
  6. Chiang, T., N. Mariano, C. Urban, R. Colon-Urban, L. Grenner, R. H. Eng, D. Huang, H. Dholakia, and J. J. Rahal. 2007. Identification of carbapenem-resistant Klebsiella pneumoniae harboring KPC enzymes in New Jersey. Microb. Drug Resist. 13:235-240.[CrossRef][Medline]
    7. 7
  7. CLSI. 2006. Performance standards for antimicrobial susceptibility testing. The Clinical and Laboratory Standards Institute sixteenth informational supplement. CLSI, Wayne, PA.
    8. 8
  8. Deshpande, L. M., P. R. Rhomberg, H. S. Sader, and R. N. Jones. 2006. Emergence of serine carbapenemases (KPC and SME) among clinical strains of Enterobacteriaceae isolated in the United States Medical Centers: report from the MYSTIC Program (1999-2005). Diagn. Microbiol. Infect. Dis. 56:367-372.[CrossRef][Medline]
    9. 9
  9. Hindiyeh, M., V. Levy, R. Azar, N. Varsano, L. Regev, Y. Shalev, Z. Grossman, and E. Mendelson. 2005. Evaluation of a multiplex real-time reverse transcriptase PCR assay for detection and differentiation of influenza viruses A and B during the 2001-2002 influenza season in Israel. J. Clin. Microbiol. 43:589-595.[Abstract/Free Full Text]
    10. 10
  10. Hong, T., E. S. Moland, B. Abdalhamid, N. D. Hanson, J. Wang, C. Sloan, D. Fabian, A. Farajallah, J. Levine, and K. S. Thomson. 2005. Escherichia coli: development of carbapenem resistance during therapy. Clin. Infect. Dis. 40:e84-e86.[CrossRef][Medline]
    11. 11
  11. Hossain, A., M. J. Ferraro, R. M. Pino, R. B. Dew III, E. S. Moland, T. J. Lockhart, K. S. Thomson, R. V. Goering, and N. D. Hanson. 2004. Plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 in an Enterobacter sp. Antimicrob. Agents Chemother. 48:4438-4440.[Abstract/Free Full Text]
    12. 12
  12. Isenberg, H. D. (ed.). 2004. Clinical microbiology procedures handbook, 2nd ed. ASM Press, Washington, DC.
    13. 13
  13. Landman, D., J. K. Salvani, S. Bratu, and J. Quale. 2005. Evaluation of techniques for detection of carbapenem-resistant Klebsiella pneumoniae in stool surveillance cultures. J. Clin. Microbiol. 43:5639-5641.[Abstract/Free Full Text]
    14. 14
  14. Leavitt, A., S. Navon-Venezia, I. Chmelnitsky, M. J. Schwaber, and Y. Carmeli. 2007. Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae in an Israeli hospital. Antimicrob. Agents Chemother. 51:3026-3029.[Abstract/Free Full Text]
    15. 15
  15. Miriagou, V., L. S. Tzouvelekis, S. Rossiter, E. Tzelepi, F. J. Angulo, and J. M. Whichard. 2003. Imipenem resistance in a Salmonella clinical strain due to plasmid-mediated class A carbapenemase KPC-2. Antimicrob. Agents Chemother. 47:1297-1300.[Abstract/Free Full Text]
    16. 16
  16. Naas, T., P. Nordmann, G. Vedel, and C. Poyart. 2005. Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC in a Klebsiella pneumoniae isolate from France. Antimicrob. Agents Chemother. 49:4423-4424.[Free Full Text]
    17. 17
  17. Nordmann, P., and L. Poirel. 2002. Emerging carbapenemases in Gram-negative aerobes. Clin. Microbiol. Infect. 8:321-331.[CrossRef][Medline]
    18. 18
  18. Queenan, A. M., and K. Bush. 2007. Carbapenemases: the versatile beta-lactamases. Clin. Microbiol. Rev. 20:440-458.[Abstract/Free Full Text]
    19. 19
  19. Regev, L., M. Hindiyeh, L. M. Shulman, A. Barak, V. Levy, R. Azar, Y. Shalev, Z. Grossman, and E. Mendelson. 2006. Characterization of human metapneumovirus infections in Israel. J. Clin. Microbiol. 44:1484-1489.[Abstract/Free Full Text]
    20. 20
  20. Samra, Z., O. Ofir, Y. Lishtzinsky, L. Madar-Shapiro, and J. Bishara. 2007. Outbreak of carbapenem-resistant Klebsiella pneumoniae producing KPC-3 in a tertiary medical centre in Israel. Int. J. Antimicrob. Agents 30:525-529.[CrossRef][Medline]
    21. 21
  21. Siegel, J. D., E. Rhinehart, M. Jackson, and L. Chiarello, for the Healthcare Infection Control Practices Advisory Committee. 2006. Management of multidrug-resistant organisms in healthcare settings. CDC, Atlanta, GA. http://www.cdc.gov/ncidod/dhqp/pdf/ar/mdroGuideline2006.pdf.
    22. 22
  22. Tenover, F. C., R. K. Kalsi, P. P. Williams, R. B. Carey, S. Stocker, D. Lonsway, J. K. Rasheed, J. W. Biddle, J. E. McGowan, Jr., and B. Hanna. 2006. Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing. Emerg. Infect. Dis. 12:1209-1213.[Medline]
    23. 23
  23. Villar, H. E., F. Danel, and D. M. Livermore. 1997. Permeability to carbapenems of Proteus mirabilis mutants selected for resistance to imipenem or other beta-lactams. J. Antimicrob. Chemother. 40:365-370.[Abstract/Free Full Text]
    24. 24
  24. Villegas, M. V., K. Lolans, A. Correa, J. N. Kattan, J. A. Lopez, and J. P. Quinn. 2007. First identification of Pseudomonas aeruginosa isolates producing a KPC-type carbapenem-hydrolyzing beta-lactamase. Antimicrob. Agents Chemother. 51:1553-1555.[Abstract/Free Full Text]
    25. 25
  25. Villegas, M. V., K. Lolans, A. Correa, C. J. Suarez, J. A. Lopez, M. Vallejo, and J. P. Quinn. 2006. First detection of the plasmid-mediated class A carbapenemase KPC-2 in clinical isolates of Klebsiella pneumoniae from South America. Antimicrob. Agents Chemother. 50:2880-2882.[Abstract/Free Full Text]
    26. 26
  26. Wei, Z. Q., X. X. Du, Y. S. Yu, P. Shen, Y. G. Chen, and L. J. Li. 2007. Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China. Antimicrob. Agents Chemother. 51:763-765.[Abstract/Free Full Text]
    27. 27
  27. Yigit, H., A. M. Queenan, G. J. Anderson, A. Domenech-Sanchez, J. W. Biddle, C. D. Steward, S. Alberti, K. Bush, and F. C. Tenover. 2001. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45:1151-1161.[Abstract/Free Full Text]
    28. 28
  28. Yigit, H., A. M. Queenan, J. K. Rasheed, J. W. Biddle, A. Domenech-Sanchez, S. Alberti, K. Bush, and F. C. Tenover. 2003. Carbapenem-resistant strain of Klebsiella oxytoca harboring carbapenem-hydrolyzing beta-lactamase KPC-2. Antimicrob. Agents Chemother. 47:3881-3889.[Abstract/Free Full Text]

Journal of Clinical Microbiology, September 2008, p. 2879-2883, Vol. 46, No. 9
0095-1137/08/$08.00+0     doi:10.1128/JCM.00661-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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