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A novel air-dried multiplex high-resolution melt assay for the detection of extended-spectrum β-lactamase and carbapenemase genes

CubasAtienzar, Ana, Williams, Chris, Karkey, Abhilasha, Dongol, Sabina, Sulochana, Manandhar, Rajendra, Shrestha, Hobbs, Glyn, Evans, Katie, Musicha, Patrick, Feasey, Nicholas ORCID: https://orcid.org/0000-0003-4041-1405, Cuevas, Luis ORCID: https://orcid.org/0000-0002-6581-0587, Adams, Emily ORCID: https://orcid.org/0000-0002-0816-2835 and Edwards, Thomas (2021) 'A novel air-dried multiplex high-resolution melt assay for the detection of extended-spectrum β-lactamase and carbapenemase genes'. Journal of Global Antimicrobial Resistance, Vol 27, pp. 123-131.

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Abstract

Objectives
This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended-spectrum β-lactamase (ESBL) (blaSHV and blaCTX-M groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that confer resistance to cephalosporins and carbapenems.

Methods
The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and the UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility. Assay reproducibility was evaluated across five different real-time quantitative PCR (qPCR) instruments [Rotor-Gene® Q, QuantStudioTM 5, CFX96, LightCycler® 480 and Magnetic Induction Cycler (Mic)]. Assay stability was also assessed under different storage temperatures (6.2 ± 0.9°C, 20.4 ± 0.7°C and 29.7 ± 1.4°C) at six time points over 8 months.

Results
The sensitivity and specificity (with 95% confidence intervals) for detecting ESBL and carbapenemase genes was 94.7% (92.5–96.5%) and 99.2% (98.8–99.5%) compared with the reference gel-based PCR and sequencing and 98.3% (97.0–99.3%) and 98.5% (98.0–98.9%) compared with the original HRM wet PCR mix format. Overall agreement was 91.1% (90.0–92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay, and no loss of sensitivity occurred under all storage conditions and time points.

Conclusion
We present a ready-to-use air-dried HRM PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve antimicrobial resistance detection.

Item Type: Article
Subjects: QV Pharmacology > Anti-Bacterial Agents. Tissue Extracts > QV 350 Anti-bacterial agents (General or not elsewhere classified)
QY Clinical Pathology > QY 4 General works
WB Practice of Medicine > Therapeutics > WB 330 Drug therapy
Faculty: Department: Biological Sciences > Department of Tropical Disease Biology
Clinical Sciences & International Health > Clinical Sciences Department
Digital Object Identifer (DOI): https://doi.org/10.1016/j.jgar.2021.08.006
SWORD Depositor: JISC Pubrouter
Depositing User: JISC Pubrouter
Date Deposited: 30 Sep 2021 09:49
Last Modified: 30 Sep 2021 09:49
URI: https://archive.lstmed.ac.uk/id/eprint/19061

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