Real-Time Detection of
            <i>bla</i>
            <sub>KPC</sub>
            in Clinical Samples and Surveillance Specimens

Mangold, Kathy A.; Santiano, Kristine; Broekman, Ronit; Krafft, Catherine; Voss, Barbara L.; Wang, Vivien; Hacek, Donna M.; Usacheva, Elena A.; Thomson, Richard B.; Kaul, Karen L.; Peterson, Lance R.

doi:10.1128/jcm.00268-11

Cited by 32 publications

(39 citation statements)

References 6 publications

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“…Real-time PCR methods have been developed for the detection of bla KPC ; most of them need the clinical sample to be either cultured on agar plates to obtain pure cultures or inoculated to enrichment broth or blood culture bottles to maximize the recovery of KPC-positive organisms (2,6), with a time delay from sample reception to diagnosis of at least 24 h.…”

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confidence: 99%

Ultrarapid Detection of bla _KPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR

et al. 2012

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The novel real-time PCR assay developed as described here was able to detect bla KPC1/2-12 (bla KPC-1/2 to bla KPC-12 ) from easily available clinical specimens in less than 2 h. The genotypic assay was highly sensitive (100%) and specific (98%). In some cases, it was able to detect bla KPC 48 h before positive detection by standard phenotypic assay on patients who were monitored daily. The high sensitivity and rapidity of the molecular method make it the method of choice for KPC surveillance and, thus, containment purposes. Bacteria producing Klebsiella pneumoniae carbapenemases (KPCs) have emerged as a cause of multidrug-resistant nosocomial infections worldwide. KPC has been identified in several Enterobacteriaceae species, as well as Pseudomonas aeruginosa and Acinetobacter baumannii; to date, 11 different variants (KPC-1/2 to KPC-12) have been reported (4) (www.lahey.org/Studies/other .asp#table1). Initially, carbapenem-resistant strains were found mainly in intensive care units, but unfortunately, they have now expanded to all hospital wards, probably leading to involvement of the nonhospitalized population, with subsequent dangerous community acquisition (1). Early identification of carbapenemase producers, also at the carriage state, is thus becoming mandatory for prevention and adequate management to contain the further spread of resistance.Real-time PCR methods have been developed for the detection of bla KPC ; most of them need the clinical sample to be either cultured on agar plates to obtain pure cultures or inoculated to enrichment broth or blood culture bottles to maximize the recovery of KPC-positive organisms (2, 6), with a time delay from sample reception to diagnosis of at least 24 h.We have developed an original ultrarapid assay based on fast real-time PCR for the detection of bla KPC1/2-12 (bla KPC-1/2 to bla ) genes. The assay was performed on perirectal and nasal swabs which were processed for analysis without prior culturing of bacterial species, allowing the output of results less than 2 h from the reception of swabs.All 216 clinical swabs (116 perirectal and 100 nasal) were obtained from 125 patients hospitalized in the Padua Teaching Hospital, Italy. The real-time PCR method was validated by comparison with standard diagnostic phenotypic analysis of cultured isolates. For phenotypic analysis, two swabs from the same patient were first streaked on nonselective enrichment agar plates (BBL blood agar or BBL chocolate II agar) and on differential and selective MacConkey II agar plates (all from Becton Dickinson Italia, Milan, Italy) and incubated at 35°C Ϯ 2 for 16 to 18 h; if necessary, single colonies (usually, one or two) of each colony type were further streaked on enrichment agar plates to obtain pure cultures. In cases of noncorrespondence with the results of the molecular method, up to 15 single colonies of the Gram-negative species identified were restreaked on chocolate agar plates to obtain pure cultures and on Oxoid Brilliance CRE agar (Oxoid Ltd., United Kingdom) to check for ...

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Ultrarapid Detection of bla _KPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR

et al. 2012

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“…In general, all phenotypic methods are very time consuming, delivering results often only after 1 or 2 days (24). Faster are the molecular tests, such as real-time PCR assays, allowing for quick identification of KPC genes (3,6,11,13,18,21,24). Nevertheless, these assays often have only a limited multiplexing capability and also cannot distinguish single KPC variants from each other.…”

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confidence: 99%

“…In order to reduce and control the further spread of carbapenem resistance, rapid identification is crucial so that appropriate treatment can be applied (18). Classical microbiological methods are often slow and give results only after additional cultivation for 24 or even 48 h (24,34).…”

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Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

et al. 2012

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; and Microbiology, NHS Lothian, Edinburgh, United Kingdom c Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (bla KPC ) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 ؋ 10 5 CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, on average, for K. pneumoniae (Norgen). This study presents, for the first time, the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship.

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“…These enzymes are associated most often with Klebsiella pneumoniae but have been identified in other genera of Enterobacteriaceae, such as Escherichia coli and Enterobacter spp., as well as Pseudomonas aeruginosa and Acinetobacter baumannii (2). Infections with KPC-producing organisms are associated with mortality rates ranging from 27.5 to 57% (3,4,17). These high mortality rates may be due in part to inappropriate or inadequate antimicrobial therapy due to the inability to detect carbapenem resistance in these strains using current susceptibility methods.…”

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confidence: 99%

“…There are molecular assays for the detection of bla KPC available, but these often require specialized equipment and expensive reagents (3,(6)(7)(8). Moreover, no existing assay is able to differentiate between bla KPC-2-like (including alleles 2, 4, 5, 6, 11, and 12) and bla KPC-3-like (including alleles 7, 8, 9, 10, and 13) genes without direct sequencing or digestion of the amplified product with restriction enzymes (7).…”

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Rapid Detection and Statistical Differentiation of KPC Gene Variants in Gram-Negative Pathogens by Use of High-Resolution Melting and ScreenClust Analyses

Roth¹,

Hanson²

2013

J Clin Microbiol

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In the United States, the production of the Klebsiella pneumoniae carbapenemase (KPC) is an important mechanism of carbapenem resistance in Gram-negative pathogens. Infections with KPC-producing organisms are associated with increased morbidity and mortality; therefore, the rapid detection of KPC-producing pathogens is critical in patient care and infection control. We developed a real-time PCR assay complemented with traditional high-resolution melting (HRM) analysis, as well as statistically based genotyping, using the Rotor-Gene ScreenClust HRM software to both detect the presence of bla KPC and differentiate between KPC-2-like and KPC-3-like alleles. A total of 166 clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with various ␤-lactamase susceptibility patterns were tested in the validation of this assay; 66 of these organisms were known to produce the KPC ␤-lactamase. The real-time PCR assay was able to detect the presence of bla KPC in all 66 of these clinical isolates (100% sensitivity and specificity). HRM analysis demonstrated that 26 had KPC-2-like melting peak temperatures, while 40 had KPC-3-like melting peak temperatures. Sequencing of 21 amplified products confirmed the melting peak results, with 9 isolates carrying bla KPC-2 and 12 isolates carrying bla KPC-3 . This PCR/HRM assay can identify KPC-producing Gram-negative pathogens in as little as 3 h after isolation of pure colonies and does not require post-PCR sample manipulation for HRM analysis, and ScreenClust analysis easily distinguishes bla KPC-2-like and bla KPC-3-like alleles. Therefore, this assay is a rapid method to identify the presence of bla KPC enzymes in Gram-negative pathogens that can be easily integrated into busy clinical microbiology laboratories.

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Real-Time Detection of bla _KPC in Clinical Samples and Surveillance Specimens

Cited by 32 publications

References 6 publications

Ultrarapid Detection of bla _KPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR

Ultrarapid Detection of bla _KPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

Rapid Detection and Statistical Differentiation of KPC Gene Variants in Gram-Negative Pathogens by Use of High-Resolution Melting and ScreenClust Analyses

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Real-Time Detection of bla KPC in Clinical Samples and Surveillance Specimens

Cited by 32 publications

References 6 publications

Ultrarapid Detection of bla KPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR

Ultrarapid Detection of bla KPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR

Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test

Rapid Detection and Statistical Differentiation of KPC Gene Variants in Gram-Negative Pathogens by Use of High-Resolution Melting and ScreenClust Analyses

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Real-Time Detection of bla _KPC in Clinical Samples and Surveillance Specimens

Ultrarapid Detection of bla _KPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR

Ultrarapid Detection of bla _KPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR