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

Ming Wei; Xi Chen; Jun Liu; Tianmeng Li; Peng Wang; Shuai Wang; Jing Wang; Li Gu

doi:10.2147/IDR.S475630

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

Infect Drug Resist. 2024 Aug 9:17:3451-3462. doi: 10.2147/IDR.S475630. eCollection 2024.

Authors

Ming Wei¹, Xi Chen¹, Jun Liu¹, Tianmeng Li¹, Peng Wang¹, Shuai Wang¹, Jing Wang^{2

3}, Li Gu¹

Affiliations

¹ Department 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.
² Beijing 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.
³ Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing, People's Republic of China.

Abstract

Purpose: 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; combined disk test; immunochromatographic test; mCIM/eCIM; multiplex real-time PCR assay.

Grants and funding

This research was funded by the National Natural Science Foundation of China (Grant No. 82302551); Beijing Natural Science Foundation (Grant No. 7234369); Beijing Chao-Yang Hospital Science and Technology Innovation Fund (Grant No. 22kcjjyb-19); and the Reform and Development Program of Beijing Institute of Respiratory Medicine (Grant No. Ggyfz202425 and Ggyfz202419).