AUTHOR=Poirier Aurore C. , Kuang Dai , Siedler Bianca S. , Borah Khushboo , Mehat Jai W. , Liu Jialin , Tai Cui , Wang Xiaoli , van Vliet Arnoud H. M. , Ma Wei , Jenkins David R. , Clark John , La Ragione Roberto M. , Qu Jieming , McFadden Johnjoe 

TITLE=Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=8

YEAR=2022

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2021.794961

DOI=10.3389/fmolb.2021.794961

ISSN=2296-889X

ABSTRACT=<p><italic>Klebsiella pneumoniae</italic> is an important pathogenic bacterium commonly associated with human healthcare and community-acquired infections. In recent years, <italic>K. pneumoniae</italic> has become a significant threat to global public and veterinary health, because of its high rates of antimicrobial resistance (AMR). Early diagnosis of <italic>K. pneumoniae</italic> 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 (<italic>yhaI</italic>, <italic>epsL</italic>, and <italic>xcpW</italic>) common to <italic>K. pneumoniae</italic> isolates from both China and Europe and designed loop-mediated isothermal amplification (LAMP) assays for the detection of <italic>K. pneumoniae</italic> in clinical samples. We also designed LAMP assays for the detection of five AMR genes commonly associated with <italic>K. pneumoniae</italic>. 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 <italic>K. pneumoniae</italic>–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 <italic>bla</italic><sub><italic>KPC</italic></sub> and other carbapenemases’ LAMP assays, the concordance between the LAMP results and the references methods (susceptibility tests) was 100%, on both pure cultures (<italic>n</italic> = 125) and clinical samples (<italic>n</italic> = 18). In conclusion, we developed highly sensitive and specific LAMP assays for the clinical identification of <italic>K. pneumoniae</italic> and detection of carbapenem resistance.</p>