Cubas-Atienzar Ana I, Williams Christopher T, Karkey Abhilasha, Dongol Sabina, Sulochana Manandhar, Rajendra Shrestha, Hobbs Glyn, Evans Katie, Musicha Patrick, Feasey Nicholas, Cuevas Luis E, Adams Emily R, Edwards Thomas
Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Liverpool, UK.
Oxford Clinical Research Unit, Patan Academy of Health Sciences, Kathmandu, Nepal.
J Glob Antimicrob Resist. 2021 Dec;27:123-131. doi: 10.1016/j.jgar.2021.08.006. Epub 2021 Sep 3.
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) (bla and bla groups 1 and 9) and carbapenemase (bla, bla, bla, bla and bla) 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, QuantStudio 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.
本研究旨在开发并评估一种新型的空气干燥高分辨率熔解曲线（HRM）分析法，用于检测8种主要的超广谱β-内酰胺酶（ESBL）（bla和bla第1组及第9组）以及碳青霉烯酶（bla、bla、bla、bla和bla）基因，这些基因可导致对头孢菌素和碳青霉烯类药物产生耐药性。
使用从尼泊尔、马拉维和英国的细菌分离株中提取的439份DNA样本以及来自尼泊尔的390份已知抗菌药物敏感性的临床分离株对该分析法进行评估。在5种不同的实时定量PCR（qPCR）仪器[Rotor-Gene® Q、QuantStudio 5、CFX96、LightCycler® 480和磁感应循环仪（Mic）]上评估分析法的重现性。还在8个月内的6个时间点，于不同储存温度（6.2±0.9°C、20.4±0.7°C和29.7±1.4°C）下评估分析法的稳定性。
与基于凝胶的PCR和测序参考方法相比，检测ESBL和碳青霉烯酶基因的灵敏度和特异性（95%置信区间）分别为94.7%（92.5 - 96.5%）和99.2%（98.8 - 99.5%）；与原始的HRM湿PCR混合形式相比，分别为98.3%（97.０ - 99.3%）和98.5%（98.0 - 98.9%）。在预测肠杆菌科分离株对头孢噻肟和美罗培南的表型耐药性时，总体一致性为91.1%（90.0 - 92.9%）。我们观察到空气干燥HRM分析法在不同仪器间具有近乎完美的重现性，并且在所有储存条件和时间点下均未出现灵敏度损失。
我们展示了一种即用型空气干燥HRM PCR分析法，它为检测DNA样本中的ESBL和碳青霉烯酶基因提供了一种简便、热稳定、快速且准确的工具，以改进抗菌药物耐药性检测。
