Infection and Drug Resistance (Aug 2024)
Development and Validation of a Novel Multiplex Real-Time PCR Assay for Rapid Detection of Carbapenemase Genes in Carbapenem-Resistant Enterobacterales Isolates and Clinical Samples
Abstract
Ming Wei,1 Xi Chen,1 Jun Liu,1 Tianmeng Li,1 Peng Wang,1 Shuai Wang,1 Jing Wang,2,3 Li Gu1 1Department of Infectious Diseases and Clinical Microbiology, Beijing Institute of Respiratory Medicine and Beijing Chao-Yang Hospital, Capital Medical University, Beijing, People’s Republic of China; 2Beijing Key Laboratory of Mental Disorders, National Clinical Research Center for Mental Disorders & National Center for Mental Disorders, Beijing Anding Hospital, Capital Medical University, Beijing, People’s Republic of China; 3Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, People’s Republic of ChinaCorrespondence: Jing Wang, Beijing Anding Hospital, Capital Medical University, 5 Ankang Hutong Road, Xicheng District, Beijing, 100088, People’s Republic of China, Email [email protected] Li Gu, Beijing Institute of Respiratory Medicine and Beijing Chao-Yang Hospital, Capital Medical University, 8 Gongren Tiyuchang Nanlu, Chaoyang District, Beijing, 100020, People’s Republic of China, Email [email protected]: Carbapenem-resistant Enterobacterales (CRE) infection is an urgent threat to human health. This study aimed to develop and validate a novel multiplex real-time PCR (multi-qPCR) assay for the detection of the blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM genes in CRE isolates and clinical samples, as well as to compare it with three phenotypic methods.Methods: The reliability and limit of detection (LOD) of the multi-qPCR assay were evaluated. PCR and DNA sequencing were used as the reference methods to identify carbapenemase genes in CRE isolates and clinical samples. The accuracy of the multi-qPCR assay, modified carbapenem inactivation and EDTA-modified carbapenem inactivation method (mCIMandeCIM), carbapenemase inhibitor-based combined disk test (CDT), and colloidal gold-based immunochromatographic test was compared with the reference methods with 182 isolates of CRE. Furthermore, 112 clinical samples were collected to validate the efficacy of this multi-qPCR assay.Results: The standard deviations (CVs) of intra-assay and inter-assay of the multi-qPCR assay were ≤ 0.53% and ≤ 2.04% for detecting the five major carbapenemase genes, respectively; while the LOD ranged from 2× 102 copies/mL to 8× 102 copies/mL. PCR and DNA sequencing confirmed 168 out of 182 CRE isolates producing carbapenemase(s): KPC (n = 93), NDM (n = 46), IMP (n = 8), OXA-48-like (n = 14), VIM (n = 1), KPC&NDM (n = 5), and KPC&NDM&IMP (n = 1). The accuracy of mCIMandeCIM, CDT, Colloidal Gold, and the multi-qPCR assay was 96.2%, 89.6%, 100%, and 100% respectively for detecting carbapenemase(s) producers. Moreover, the sensitivity and specificity of the multi-qPCR assay were all 100% for the detection of each carbapenemase gene in clinical samples, compared with PCR and sequencing.Conclusion: For clinical isolate detection, the multi-qPCR assay is comparable to Colloidal Gold, and superior to mCIMandeCIM and CDT; while for clinical samples detection, it also shows excellent performance. Therefore, the multi-qPCR assay has great potential for clinical diagnosis.Keywords: carbapenem-resistant Enterobacterales, carbapenemases detection, multiplex real-time PCR assay, mCIM/eCIM, combined disk test, immunochromatographic test
