Abstract
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. 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. 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. 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.
Highlights
Antimicrobial resistance (AMR) is a major cause of death worldwide and the development of new antibiotics is considered a public-health priority [1]
Measures of diagnostic accuracy and agreement of the air-dried high-resolution melt (HRM) assay for detecting individual genes compared with the reference tests are detailed in Table 1 (PCR and whole-genome sequencing) and Table 2
The overall sensitivity and specificity of the air-dried HRM assay for all genetic markers in comparison with the reference gel-based PCR and sequencing were 94.7% [95% confidence interval (CI) 92.5–96.5%] and 99.2% and in comparison with the original 9-plex HRM PCR assay [22] were 98.3% and 98.5%
Summary
Antimicrobial resistance (AMR) is a major cause of death worldwide and the development of new antibiotics is considered a public-health priority [1]. An estimated 700 000 deaths are attributable to AMR globally each year, and this number is predicted. Karkey et al. AMR, increases the duration of hospitalisation, damages the patient microbiota and increases the cost of therapy [7,8,9]. Rapid identification of AMR can enable targeted usage of antibiotics, improved patient outcomes and antimicrobial stewardship [4,6,10,11]
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