Evaluation of the KPC K-SeT® immunochromatographic assay for the rapid detection of KPC carbapenemase producers from positive blood cultures

Evaluation of the KPC K-SeT® immunochromatographic assay for the rapid detection of KPC... Sir, The global dissemination of carbapenemase-producing Gram-negative pathogens is a matter of major concern, given their very complex MDR phenotypes and the high morbidity and mortality associated with invasive infections caused by these strains.1,2 When dealing with these pathogens, rapid detection and identification of the carbapenemase type is a crucial step for antimicrobial stewardship and also for the implementation of adequate infection control practices, thus improving patient outcomes and contrasting the diffusion of carbapenem-resistant strains.3 The KPC K-SeT® assay (Coris BioConcept, Gembloux, Belgium) is a CE-marked in vitro diagnostic test, based on fast immunochromatography, able to rapidly detect KPC-type carbapenemases from bacterial cultures.4 The system exploits monoclonal antibodies specific for KPC-type enzymes and is able to provide a result in <15 min, requiring only a minimal hands-on time and no additional instrumentation.5 In this work, we evaluated the performance of the KPC K-SeT® assay for the detection of KPC-type carbapenemases directly from positive blood cultures. The evaluation was performed at the Clinical Microbiology and Virology Unit of the Careggi University Hospital of Florence (central Italy), a setting of relatively high endemicity for KPC-producing Klebsiella pneumoniae.6 Analysis was carried out on 110 positive blood cultures from different patients, in which the presence of Gram-negative bacilli was detected by Gram staining. To enrich for the presence of positive specimens, 10 of these cases were selected after rapid detection of KPC-positive K. pneumoniae by molecular testing with the Verigene Gram-Negative Blood Culture Test (BC-GN) (Nanosphere, Northbrook, IL, USA). All specimens were also processed by the standard protocol employed in the laboratory, including isolation on Chocolate Agar PolyViteX plates (bioMérieux, Marcy-l’Étoile, France) and CHROMagar Orientation Medium plates (BD, Franklin Lakes, NJ, USA), identification by MALDI-TOF MS (Vitek®MS, bioMérieux) and antimicrobial susceptibility testing by commercial dehydrated 96-well Sensititre ITGNEGF panels (Thermo Fisher Scientific, Cleveland, OH, USA) or Vitek®2 cards (AST-N202) (bioMérieux). In all carbapenem-non-susceptible strains, the presence of carbapenemase genes, including blaKPC, blaVIM, blaNDM and blaOXA-48-like, was also investigated by means of a real-time PCR protocol as described previously.7 Testing with the K-SeT® system was carried out with positive blood cultures within a mean time of 20.0 h (median 16.3 h; range 0.2–71.3 h) from culture positivity reported by the blood culture system (BD BACTEC™ FX blood culture system), following the protocol proposed by the manufacturer with some modifications. Briefly, 40 μL of the positive blood culture was gently mixed with 10 drops of LY-A buffer, provided by the manufacturer, and then 3 drops of this mixture were dispensed into the well of the immunochromatographic cassette and incubated at room temperature for a maximum of 15 min. The results obtained with the 110 positive blood cultures investigated in this work are detailed in Table 1. Overall, 24 cultures were positive for a carbapenem-non-susceptible strain and 18 of them were positive for a KPC-producing strain, including K. pneumoniae (n = 17) and Proteus mirabilis (n = 1). The KPC K-SeT® assay detected the presence of a KPC-producing microorganism in all the confirmed KPC-positive blood cultures (sensitivity, 100%). Only in one case, positive for Escherichia coli, did the KPC K-SeT® assay repeatedly yield a positive result which, because this was not confirmed by routine method, was considered to be a false positive (specificity, 98.9%). In this case, the KPC K-SeT® assay was also repeated for the isolated strain following the manufacturer’s instructions, and yielded a negative result, which was also confirmed by the real-time PCR assay performed from total DNA extracted directly from positive blood culture with Nuclisens easyMag® (bioMérieux). Table 1 Results of real-time PCR for blaKPC, of testing with KPC K-SeT® and of carbapenem susceptibility for the 110 non-replicate blood cultures positive for Gram-negative bacilli investigated in this study Bacterial isolates  blaKPC-positive strain (n)  blaKPC-negative strain (n)  KPC K-SeT® positivea (n)  Carbapenem susceptibleb (n)  Carbapenem non- susceptibleb (n)  Total (n)  Acinetobacter spp.  0  5  0  2  3  5  E. coli  0  47  1  47  0  47  K. pneumoniae  17  10  17  10  17  27  Klebsiella oxytoca  0  3  0  3  0  3  Enterobacter spp.  0  8  0  8  0  8  P. mirabilis  1  7  1  7  1  8  Pseudomonas aeruginosa  0  10  0  9  1  10  Other Gram-negatives  0  2  0  0  2  2  Total (n)  18  92  19  86  24  110  Bacterial isolates  blaKPC-positive strain (n)  blaKPC-negative strain (n)  KPC K-SeT® positivea (n)  Carbapenem susceptibleb (n)  Carbapenem non- susceptibleb (n)  Total (n)  Acinetobacter spp.  0  5  0  2  3  5  E. coli  0  47  1  47  0  47  K. pneumoniae  17  10  17  10  17  27  Klebsiella oxytoca  0  3  0  3  0  3  Enterobacter spp.  0  8  0  8  0  8  P. mirabilis  1  7  1  7  1  8  Pseudomonas aeruginosa  0  10  0  9  1  10  Other Gram-negatives  0  2  0  0  2  2  Total (n)  18  92  19  86  24  110  a Test performed on blood culture. b Based on MIC of meropenem according to EUCAST breakpoint (i.e. MIC ≤2 mg/L). All tested samples were scored as valid due to the positivity of the internal control within 15 min. The positivity of the KPC K-SeT® assay was always apparent in 2 min, at most. Altogether, results from this study suggested that the KPC K-SeT® assay could directly detect the presence of KPC carbapenemase producers in positive blood cultures, with an excellent sensitivity (100%) and good specificity (98.9%). The high sensitivity is consistent with the limit of detection of KPC K-SeT® assay with cultures of KPC-producing strains (∼106 cfu/mL; P. Bogaerts, S. Evrard, T. D. Huang and Y. Glupczynski, unpublished results), which is a value substantially lower than the bacterial load normally found in positive blood cultures (2 × 107–7 × 109 cfu/mL).8 The use of the KPC K-SeT® assay in this setting could allow the rapid detection of KPC-producers in positive blood cultures, providing valuable information for antimicrobial stewardship. The reliability of the test, its rapid and simple setting-up requiring minimal hands-on time and no expensive equipment, along with the low cost (retail price of ∼€6/test)5 make this product particularly useful in settings with high endemicity of KPC-producing Enterobacteriaceae. Further investigations aimed at evaluating the performance of the K-SeT® assay for the rapid detection of other carbapenemases (i.e. OXA-48 and NDM) from positive blood cultures would be of interest, especially for settings with a different carbapenemase epidemiology. In this perspective, the KPC K-SeT® assay could easily be implemented in the routine workflow of the clinical microbiology laboratory, and unlike other rapid methods imposes only a slight extra burden in terms of personnel and reagent costs. Funding This study was supported by internal funding. Transparency declarations None to declare. References 1 Tzouvelekis LS, Markogiannakis A, Psichogiou M et al.   Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev  2012; 25: 682– 707. Google Scholar CrossRef Search ADS PubMed  2 Munoz-Price LS, Poirel L, Bonomo RA et al.   Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis  2013; 13: 785– 96. Google Scholar CrossRef Search ADS PubMed  3 Banerjee R, Humphries R. Clinical and laboratory considerations for the rapid detection of carbapenem-resistant Enterobacteriaceae. Virulence  2017; 8: 427– 39. Google Scholar CrossRef Search ADS PubMed  4 Meunier D, Vickers A, Pike R et al.   Evaluation of the K-SeT R.E.S.I.S.T. immunochromatographic assay for the rapid detection of KPC and OXA-48-like carbapenemases. J Antimicrob Chemother  2016; 71: 2357– 9. Google Scholar CrossRef Search ADS PubMed  5 Glupczynski Y, Evrard S, Ote I et al.   Evaluation of two new commercial immunochromatographic assays for the rapid detection of OXA-48 and KPC carbapenemases from cultured bacteria. J Antimicrob Chemother  2016; 71: 1217– 22. Google Scholar CrossRef Search ADS PubMed  6 Giani T, Arena F, Vaggelli G et al.   Large nosocomial outbreak of colistin-resistant, carbapenemase-producing Klebsiella pneumoniae traced to clonal expansion of an mgrB deletion mutant. J Clin Microbiol  2015; 53: 3341– 4. Google Scholar CrossRef Search ADS PubMed  7 Antonelli A, Arena F, Giani T et al.   Performance of the BD MAX™ instrument with Check-Direct CPE real-time PCR for the detection of carbapenemase genes from rectal swabs, in a setting with endemic dissemination of carbapenemase-producing Enterobacteriaceae. Diagn Microbiol Infect Dis  2016; 86: 30– 4. Google Scholar CrossRef Search ADS PubMed  8 Christner M, Rohde H, Wolters M et al.   Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. J Clin Microbiol  2010; 48: 1584– 91. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Evaluation of the KPC K-SeT® immunochromatographic assay for the rapid detection of KPC carbapenemase producers from positive blood cultures

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Oxford University Press
Copyright
© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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0305-7453
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1460-2091
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10.1093/jac/dkx406
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Abstract

Sir, The global dissemination of carbapenemase-producing Gram-negative pathogens is a matter of major concern, given their very complex MDR phenotypes and the high morbidity and mortality associated with invasive infections caused by these strains.1,2 When dealing with these pathogens, rapid detection and identification of the carbapenemase type is a crucial step for antimicrobial stewardship and also for the implementation of adequate infection control practices, thus improving patient outcomes and contrasting the diffusion of carbapenem-resistant strains.3 The KPC K-SeT® assay (Coris BioConcept, Gembloux, Belgium) is a CE-marked in vitro diagnostic test, based on fast immunochromatography, able to rapidly detect KPC-type carbapenemases from bacterial cultures.4 The system exploits monoclonal antibodies specific for KPC-type enzymes and is able to provide a result in <15 min, requiring only a minimal hands-on time and no additional instrumentation.5 In this work, we evaluated the performance of the KPC K-SeT® assay for the detection of KPC-type carbapenemases directly from positive blood cultures. The evaluation was performed at the Clinical Microbiology and Virology Unit of the Careggi University Hospital of Florence (central Italy), a setting of relatively high endemicity for KPC-producing Klebsiella pneumoniae.6 Analysis was carried out on 110 positive blood cultures from different patients, in which the presence of Gram-negative bacilli was detected by Gram staining. To enrich for the presence of positive specimens, 10 of these cases were selected after rapid detection of KPC-positive K. pneumoniae by molecular testing with the Verigene Gram-Negative Blood Culture Test (BC-GN) (Nanosphere, Northbrook, IL, USA). All specimens were also processed by the standard protocol employed in the laboratory, including isolation on Chocolate Agar PolyViteX plates (bioMérieux, Marcy-l’Étoile, France) and CHROMagar Orientation Medium plates (BD, Franklin Lakes, NJ, USA), identification by MALDI-TOF MS (Vitek®MS, bioMérieux) and antimicrobial susceptibility testing by commercial dehydrated 96-well Sensititre ITGNEGF panels (Thermo Fisher Scientific, Cleveland, OH, USA) or Vitek®2 cards (AST-N202) (bioMérieux). In all carbapenem-non-susceptible strains, the presence of carbapenemase genes, including blaKPC, blaVIM, blaNDM and blaOXA-48-like, was also investigated by means of a real-time PCR protocol as described previously.7 Testing with the K-SeT® system was carried out with positive blood cultures within a mean time of 20.0 h (median 16.3 h; range 0.2–71.3 h) from culture positivity reported by the blood culture system (BD BACTEC™ FX blood culture system), following the protocol proposed by the manufacturer with some modifications. Briefly, 40 μL of the positive blood culture was gently mixed with 10 drops of LY-A buffer, provided by the manufacturer, and then 3 drops of this mixture were dispensed into the well of the immunochromatographic cassette and incubated at room temperature for a maximum of 15 min. The results obtained with the 110 positive blood cultures investigated in this work are detailed in Table 1. Overall, 24 cultures were positive for a carbapenem-non-susceptible strain and 18 of them were positive for a KPC-producing strain, including K. pneumoniae (n = 17) and Proteus mirabilis (n = 1). The KPC K-SeT® assay detected the presence of a KPC-producing microorganism in all the confirmed KPC-positive blood cultures (sensitivity, 100%). Only in one case, positive for Escherichia coli, did the KPC K-SeT® assay repeatedly yield a positive result which, because this was not confirmed by routine method, was considered to be a false positive (specificity, 98.9%). In this case, the KPC K-SeT® assay was also repeated for the isolated strain following the manufacturer’s instructions, and yielded a negative result, which was also confirmed by the real-time PCR assay performed from total DNA extracted directly from positive blood culture with Nuclisens easyMag® (bioMérieux). Table 1 Results of real-time PCR for blaKPC, of testing with KPC K-SeT® and of carbapenem susceptibility for the 110 non-replicate blood cultures positive for Gram-negative bacilli investigated in this study Bacterial isolates  blaKPC-positive strain (n)  blaKPC-negative strain (n)  KPC K-SeT® positivea (n)  Carbapenem susceptibleb (n)  Carbapenem non- susceptibleb (n)  Total (n)  Acinetobacter spp.  0  5  0  2  3  5  E. coli  0  47  1  47  0  47  K. pneumoniae  17  10  17  10  17  27  Klebsiella oxytoca  0  3  0  3  0  3  Enterobacter spp.  0  8  0  8  0  8  P. mirabilis  1  7  1  7  1  8  Pseudomonas aeruginosa  0  10  0  9  1  10  Other Gram-negatives  0  2  0  0  2  2  Total (n)  18  92  19  86  24  110  Bacterial isolates  blaKPC-positive strain (n)  blaKPC-negative strain (n)  KPC K-SeT® positivea (n)  Carbapenem susceptibleb (n)  Carbapenem non- susceptibleb (n)  Total (n)  Acinetobacter spp.  0  5  0  2  3  5  E. coli  0  47  1  47  0  47  K. pneumoniae  17  10  17  10  17  27  Klebsiella oxytoca  0  3  0  3  0  3  Enterobacter spp.  0  8  0  8  0  8  P. mirabilis  1  7  1  7  1  8  Pseudomonas aeruginosa  0  10  0  9  1  10  Other Gram-negatives  0  2  0  0  2  2  Total (n)  18  92  19  86  24  110  a Test performed on blood culture. b Based on MIC of meropenem according to EUCAST breakpoint (i.e. MIC ≤2 mg/L). All tested samples were scored as valid due to the positivity of the internal control within 15 min. The positivity of the KPC K-SeT® assay was always apparent in 2 min, at most. Altogether, results from this study suggested that the KPC K-SeT® assay could directly detect the presence of KPC carbapenemase producers in positive blood cultures, with an excellent sensitivity (100%) and good specificity (98.9%). The high sensitivity is consistent with the limit of detection of KPC K-SeT® assay with cultures of KPC-producing strains (∼106 cfu/mL; P. Bogaerts, S. Evrard, T. D. Huang and Y. Glupczynski, unpublished results), which is a value substantially lower than the bacterial load normally found in positive blood cultures (2 × 107–7 × 109 cfu/mL).8 The use of the KPC K-SeT® assay in this setting could allow the rapid detection of KPC-producers in positive blood cultures, providing valuable information for antimicrobial stewardship. The reliability of the test, its rapid and simple setting-up requiring minimal hands-on time and no expensive equipment, along with the low cost (retail price of ∼€6/test)5 make this product particularly useful in settings with high endemicity of KPC-producing Enterobacteriaceae. Further investigations aimed at evaluating the performance of the K-SeT® assay for the rapid detection of other carbapenemases (i.e. OXA-48 and NDM) from positive blood cultures would be of interest, especially for settings with a different carbapenemase epidemiology. In this perspective, the KPC K-SeT® assay could easily be implemented in the routine workflow of the clinical microbiology laboratory, and unlike other rapid methods imposes only a slight extra burden in terms of personnel and reagent costs. Funding This study was supported by internal funding. Transparency declarations None to declare. References 1 Tzouvelekis LS, Markogiannakis A, Psichogiou M et al.   Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev  2012; 25: 682– 707. Google Scholar CrossRef Search ADS PubMed  2 Munoz-Price LS, Poirel L, Bonomo RA et al.   Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis  2013; 13: 785– 96. Google Scholar CrossRef Search ADS PubMed  3 Banerjee R, Humphries R. Clinical and laboratory considerations for the rapid detection of carbapenem-resistant Enterobacteriaceae. Virulence  2017; 8: 427– 39. Google Scholar CrossRef Search ADS PubMed  4 Meunier D, Vickers A, Pike R et al.   Evaluation of the K-SeT R.E.S.I.S.T. immunochromatographic assay for the rapid detection of KPC and OXA-48-like carbapenemases. J Antimicrob Chemother  2016; 71: 2357– 9. Google Scholar CrossRef Search ADS PubMed  5 Glupczynski Y, Evrard S, Ote I et al.   Evaluation of two new commercial immunochromatographic assays for the rapid detection of OXA-48 and KPC carbapenemases from cultured bacteria. J Antimicrob Chemother  2016; 71: 1217– 22. Google Scholar CrossRef Search ADS PubMed  6 Giani T, Arena F, Vaggelli G et al.   Large nosocomial outbreak of colistin-resistant, carbapenemase-producing Klebsiella pneumoniae traced to clonal expansion of an mgrB deletion mutant. J Clin Microbiol  2015; 53: 3341– 4. Google Scholar CrossRef Search ADS PubMed  7 Antonelli A, Arena F, Giani T et al.   Performance of the BD MAX™ instrument with Check-Direct CPE real-time PCR for the detection of carbapenemase genes from rectal swabs, in a setting with endemic dissemination of carbapenemase-producing Enterobacteriaceae. Diagn Microbiol Infect Dis  2016; 86: 30– 4. Google Scholar CrossRef Search ADS PubMed  8 Christner M, Rohde H, Wolters M et al.   Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting. J Clin Microbiol  2010; 48: 1584– 91. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Feb 1, 2018

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