Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP)

Nakano, Ryuichi; Nakano, Akiyo; Ishii, Yoshikazu; Ubagai, Tsuneyuki; Kikuchi-Ueda, Takane; Kikuchi, Hirotoshi; Tansho-Nagakawa, Shigeru; Kamoshida, Go; Mu, Xiaoqin; Ono, Yasuo

doi:10.1016/j.jiac.2014.11.010

Cited by 50 publications

(41 citation statements)

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“…However, 13 of the non-CPE strains with porin loss were CIM negative, although their MEM MIC was higher than that of the IMP producers ( Table 1). The two noncarbapenemase-producing strains showed false-positive results (one formed no inhibition circle, and the other's circle of inhibition was only 8 mm in diameter); no carbapenemases were detected by carbapenemase-multiplex PCR or loop-mediated isothermal amplification (detecting IMP, VIM, NDM, SPM, AIM, DIM, GIM, SIM, KPC, BIC, OXA-48-like, SME, IMI, and GES) (10)(11)(12).…”

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confidence: 99%

Suitability of Carbapenem Inactivation Method (CIM) for Detection of IMP Metallo-β-Lactamase-Producing Enterobacteriaceae

et al. 2017

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Carbapenem-resistant Enterobacteriaceae (CRE) strains have become globally distributed in the past decade, resulting in concern over the control of hospital infections and antimicrobial therapies (1, 2). The majority of CRE isolates are carbapenemase-producing Enterobacteriaceae (CPE) strains, so early detection of CPE strains is essential for providing optimal antimicrobial therapies and preventing horizontal transmission. In 2015, van der Zwaluw et al. reported the carbapenem inactivation method (CIM), a new method for detecting carbapenemase producers with high sensitivity and specificity (3). However, it was unclear whether CIM can identify IMP producers with a low carbapenem MIC or non-CPE strains that are highly carbapenem resistant. In this study, we evaluated whether CIM can identify CPE strains independently of their carbapenem MICs.Were used 233 CPE and 51 non-CPE strains isolated in general hospitals across Japan from 2012 to 2016 and stocked in our laboratory. The strains were collected from blood, sputum, wounds, urine, and feces. Their antibiotic susceptibilities were determined by the agar dilution method in accordance with the recommendations of the CLSI (4). The CPE strains included 191 IMP, 20 KPC, 17 NDM, and 5 OXA-48-like (OXA-48 and OXA-244) producers. The non-CPE strains included 31 extended-spectrum beta-lactamase (CTX-M, SHV, and TEM) producers and 5 plasmid-mediated AmpC ␤-lactamase (DHA, CMY, and CFE) producers. They were identified by DNA sequence amplification as previously described (5-10). Of the non-CPE strains, 13 highly carbapenem-resistant strains were identified as producing cephalosporinases and lacking porin function by SDS-PAGE, DNA sequencing, and quantitative reverse transcription-PCR (11).The CIM was conducted as previously described (3). The isolates were cultured on Mueller-Hinton agar (MHA) plates. A full 10-l inoculation loop of each strain was suspended in 400 l of sterile distilled water, and a 10-g meropenem (MEM) susceptibilitytesting disk (E-DF85; EIKEN) was immersed in the solution. After incubation at 35°C for 2 h, the disk was removed and placed on an MHA plate inoculated with a 0.5 McFarland standard of Escherichia coli strain ATCC 25922 with a sterile cotton swab. Finally, the plate was incubated overnight at 35°C and the inhibition zone around each disk was measured. Inhibition circles Ͻ10 mm in diameter were judged to indicate CIM positivity.The CIM showed a sensitivity of 100% (233/233) for CPE strains and a specificity of 96.1% (49/51) for non-CPE strains (Table 1). The MICs of MEM for the 191 IMP producers ranged from 0.125 to 32 g/ml (MIC 50 ϭ 0.5 g/ml, MIC 90 ϭ 2 g/ml), and those of imipenem (IPM) ranged from 0.06 to 8 g/ml (MIC 50 ϭ 0.125 g/ml, MIC 90 ϭ 0.5

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confidence: 99%

Suitability of Carbapenem Inactivation Method (CIM) for Detection of IMP Metallo-β-Lactamase-Producing Enterobacteriaceae

et al. 2017

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“…; Nakano et al . ). It works by autocycling strand displacement DNA synthesis, using Bst DNA polymerase and a set of four to eight primers that attaches to various portions of the DNA, thereby increasing its sensitivity and specificity (Liu et al .…”

Section: Genotypic Methodsmentioning

confidence: 97%

“…; Nakano et al . ). The amplification process can be directly observed by measuring the turbidity of the reaction solution with a spectrophotometer or a fluorescent reagent (Liu et al .…”

Section: Genotypic Methodsmentioning

confidence: 97%

Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria

2015

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SummaryThe minimal antibiotic options for carbapenemase-producing Gram-negative bacteria necessitate their rapid detection. A literature review of a variety of phenotypic and genotypic methods is presented. Advances in culture methods and screening media are still subject to long incubation hours. Biochemical methods have shorter turnaround times and higher sensitivities and specificities, but cannot differentiate between various types and variants. Spectrophotometric methods are cheap and efficient, but are uncommon in many clinical settings, while the MALDI-TOF MS is promising for species identification, typing and resistance gene determination. Although next generation sequencing (NGS) technologies provide a better platform to detect, type and characterize carbapenem-resistant bacteria, the different NGS platforms, the large computer memories and space needed to process and store genomic data and the nonuniformity in data analysis platforms are still a challenge. The sensitivities, specificities and turnaround times recorded in the various studies reviewed favours the use of the biochemical tests (Carba NP or Rapid Carb screen tests) for the detection of putative carbapenemaseproducing isolates. MALDI-TOF MS and/or molecular methods like microarray, loop-mediated isothermal amplification and real-time multiplex PCR assays could be used for further characterization in a reference laboratory. NGS may be used for advanced epidemiological and molecular studies.

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“…LAMP was successfully demonstrated to detect human respiratory pathogens, such as Mycobacterium tuberculosis Bentaleb et al 2016;Nagai et al 2016;Sethi et al 2016;Sharma et al 2016;Kaewphinit et al 2017), Streptococcus pneumoniae (Seki et al 2005;Xia et al 2014), Bordetella pertussis (Fujino et al 2015;Brotons et al 2016;Kamachi et al 2017), and carbapenem-resistant Klebsiella pneumoniae (Nakano et al 2015). Furthermore, LAMP was also devised to diagnose pathogens associated with food-borne diseases, for …”

Section: Conventional Lampmentioning

confidence: 99%

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

et al. 2018

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Rapid detection of the Klebsiella pneumoniae carbapenemase (KPC) gene by loop-mediated isothermal amplification (LAMP)

Cited by 50 publications

References 36 publications

Suitability of Carbapenem Inactivation Method (CIM) for Detection of IMP Metallo-β-Lactamase-Producing Enterobacteriaceae

Suitability of Carbapenem Inactivation Method (CIM) for Detection of IMP Metallo-β-Lactamase-Producing Enterobacteriaceae

Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

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