Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples.
Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples.
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DOI：
10.3389/fmolb.2021.794961
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发表时间：
2021
影响因子：
5
通讯作者：
McFadden J
中科院分区：
文献类型：
--
作者：
Poirier AC;Kuang D;Siedler BS;Borah K;Mehat JW;Liu J;Tai C;Wang X;van Vliet AHM;Ma W;Jenkins DR;Clark J;La Ragione RM;Qu J;McFadden J
肺炎克雷伯菌是一种重要的致病菌，通常与人类卫生保健和社区获得性感染有关。近年来，肺炎克雷伯菌因其高抗菌素耐药性（AMR）而成为全球公共和兽医卫生的重大威胁。肺炎克雷伯菌感染的早期诊断和任何相关抗菌素耐药性的检测将有助于加速定向治疗并减少出现多重耐药分离株的风险。在这项研究中，我们鉴定了来自中国和欧洲的肺炎克雷伯菌分离株共有的三个靶基因（yhaI、epsL和xcpW），并设计了环介导等温扩增（LAMP）方法检测临床样本中的肺炎克雷伯菌。我们还设计了LAMP法检测五种常见的与肺炎克雷伯菌相关的AMR基因。LAMP检测方法在319种类型参考菌株和不同遗传背景的临床分离株以及40份临床人痰样本上进行了验证，结果表明LAMP检测方法可靠、特异性高、灵敏度高。对于肺炎克雷伯菌特异性LAMP检测，计算灵敏度、特异性和阳性和阴性预测值（与培养和基质辅助激光解吸/电离飞行时间质谱法比较）在临床分离株上均为100%，在临床痰样本上分别为100%、91%和90%，100%，明显快于参考方法。对于bla KPC和其他碳青霉烯酶的LAMP检测，在纯培养物（n = 125）和临床样品（n = 18）中，LAMP结果与参考方法（药敏试验）的一致性为100%。总之，我们建立了高灵敏度和特异性的LAMP检测方法，用于临床鉴定肺炎克雷伯菌和检测碳青霉烯类耐药性。
Klebsiella pneumoniae is an important pathogenic bacterium commonly associated with human healthcare and community-acquired infections. In recent years, K. pneumoniae has become a significant threat to global public and veterinary health, because of its high rates of antimicrobial resistance (AMR). Early diagnosis of K. pneumoniae infection and detection of any associated AMR would help to accelerate directed therapy and reduce the risk of the emergence of multidrug-resistant isolates. In this study, we identified three target genes (yhaI, epsL, and xcpW) common to K. pneumoniae isolates from both China and Europe and designed loop-mediated isothermal amplification (LAMP) assays for the detection of K. pneumoniae in clinical samples. We also designed LAMP assays for the detection of five AMR genes commonly associated with K. pneumoniae. The LAMP assays were validated on a total of 319 type reference strains and clinical isolates of diverse genetic backgrounds, in addition to 40 clinical human sputum samples, and were shown to be reliable, highly specific, and sensitive. For the K. pneumoniae–specific LAMP assay, the calculated sensitivity, specificity, and positive and negative predictive values (comparison with culture and matrix-assisted laser desorption/ionization–time of flight mass spectrometry) were all 100% on clinical isolates and, respectively, of 100%, 91%, and 90%, and 100% when tested on clinical sputum samples, while being significantly faster than the reference methods. For the bla KPC and other carbapenemases’ LAMP assays, the concordance between the LAMP results and the references methods (susceptibility tests) was 100%, on both pure cultures (n = 125) and clinical samples (n = 18). In conclusion, we developed highly sensitive and specific LAMP assays for the clinical identification of K. pneumoniae and detection of carbapenem resistance.
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DOI：
10.1038/nrmicro3432
发表时间：
2015-05
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
Flores-Mireles AL;Walker JN;Caparon M;Hultgren SJ
通讯作者：
Hultgren SJ
影响因子：
38.9
作者：
Garnacho-Montero, J.;Gutierrez-Pizarraya, A.;Marquez-Vacaro, J. A.
通讯作者：
Marquez-Vacaro, J. A.
DOI：
10.1073/pnas.78.12.7458
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
FRANKLIN, FCH;BAGDASARIAN, M;TIMMIS, KN
通讯作者：
TIMMIS, KN
影响因子：
8.8
作者：
Gaieski, David F.;Mikkelsen, Mark E.;Goyal, Munish
通讯作者：
Goyal, Munish
影响因子：
2.6
作者：
Cheng, Cancan;Zheng, Fen;Rui, Yongyu
通讯作者：
Rui, Yongyu