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

Ming Wei  1 , Xi Chen  1 , Jun Liu  1 , Tianmeng Li  1 , Peng Wang  1 , Shuai Wang  1 , Jing Wang  2   3 , Li Gu  1
Affiliations

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 et al. Infect Drug Resist. .

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.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Standard curve for the multi-qPCR assay for detection of (A) blaIMP, y = −3.317x+45.48 (R2 = 0.9953), (B) blaOXA-48-like, y = −3.467x+45.56 (R2 = 0.9978), (C) blaKPC, y = −3.625x+46.24 (R2 = 0.9977), (D) blaVIM, y = −3.598x+46.45 (R2 = 0.9964), and (E) blaNDM genes, y = −3.616x+46.38 (R = 0.9977).
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