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

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

doi:10.3389/fmolb.2021.794961

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

Front Mol Biosci. 2022 Feb 9:8:794961. doi: 10.3389/fmolb.2021.794961. eCollection 2021.

Authors

Aurore C Poirier¹, Dai Kuang², Bianca S Siedler³, Khushboo Borah³, Jai W Mehat^{3

4}, Jialin Liu⁵, Cui Tai⁶, Xiaoli Wang⁵, Arnoud H M van Vliet^{1

4}, Wei Ma⁶, David R Jenkins⁷, John Clark⁸, Roberto M La Ragione^{1

3}, Jieming Qu², Johnjoe McFadden³

Affiliations

¹ Department of Pathology and Infectious Diseases, Faculty of Health and Medical Sciences, School of Veterinary Medicine, University of Surrey, Guildford, United Kingdom.
² Department of Pulmonary and Critical Care Medicine, Ruijin Hospital, School of Medicine, Institute of Respiratory Diseases, Shanghai Jiao Tong University, Shanghai, China.
³ Faculty of Health and Medical Sciences, School of Biosciences and Medicine, University of Surrey, Guildford, United Kingdom.
⁴ Centre for Microbial Genomics and Animal Microbiome Research, Department of Pathology and Infectious Diseases, Faculty of Health and Medical Sciences, School of Veterinary Medicine, University of Surrey, Guildford, United Kingdom.
⁵ Department of Critical Care Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
⁶ State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
⁷ Department of Medical Microbiology, University Hospitals of Leicester NHS Trust, Leicester, United Kingdom.
⁸ Department of Medical Microbiology, Epsom and St Helier University Hospitals NHS Trust, Carshalton, United Kingdom.

Abstract

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.

Keywords: Klebsiella pneumoniae (K. pneumoniae); LAMP (loop mediated isothermal amplification); antimicrobial resistance (AMR); carbapenemases genes; rapid diagnostics.