环介导恒温扩增技术在呼吸道感染常见细菌临床检测中的应用评价

Application evaluation of loop-mediated isothermal amplification in clinical detection of common bacteria in respiratory tract infection

摘要:

【背景】传统的细菌培养为下呼吸道感染诊断的金标准，但其检验周期长且敏感性低。环介导恒温扩增(loop-mediated isothermal amplification，LAMP)技术方法简单、检测快速，可应用于临床呼吸道感染常见细菌的快速检测。【目的】评估环介导恒温扩增技术对呼吸道感染常见7种细菌的检测能力。【方法】设计呼吸道感染常见的7种病原菌肺炎克雷伯菌(Klebsiella pneumoniae，KP)、鲍曼不动杆菌(Acinetobacter baumannii，AB)、金黄色葡萄球菌(Staphylococcus aureus，SA)、铜绿假单胞菌(Pseudomonas aeruginosa，PA)、肺炎链球菌(Streptococcus pneumoniae，SP)、卡他莫拉菌(Moraxella catarrhalis，MC)和流感嗜血杆菌(Haemophilus influenzae，HI)的环介导恒温扩增特异性引物，建立检测7种病原菌的LAMP方法。通过梯度稀释的方法检测敏感性，交叉反应实验检测特异性。回顾性分析2019年11月–2021年3月北京大学人民医院的240例疑似下呼吸道感染患者的呼吸道标本(痰和肺泡灌洗液)。利用自动化核酸提取仪提取DNA后，利用特异性引物进行环介导恒温扩增检测7种病原菌。检测结果与常规细菌培养结果进行比较，分析环介导恒温扩增的灵敏度和特异性，评估优化不同菌种的阳性判断值(C_t值)，探讨环介导恒温扩增技术在临床上的应用价值。【结果】LAMP法检测结果与模板浓度具有较好的线性相关性，特异性实验结果表明LAMP体系的特异性较好，与其他菌株无交叉反应。共检测了218例痰和22例肺泡灌洗液样本，其中178例样本培养出7种目标菌，采用环介导恒温扩增法检测出7种目标菌的样本共176例。从提取核酸到环介导恒温扩增结果检出的平均检测时间为2–3 h，可同时检测多个样本。7种常见呼吸道病原菌流感嗜血杆菌、肺炎克雷伯菌、鲍曼不动杆菌、卡他莫拉菌、金黄色葡萄球菌、铜绿假单胞菌和肺炎链球菌的最佳C_t值分别为18.5、20.0、20.0、15.0、25.0、19.0和18.0。检测灵敏度和特异性分别为肺炎克雷伯菌90.7%和94.1%，鲍曼不动杆菌84.0%和94.2%，铜绿假单胞菌90.8%和89.1%，卡他莫拉菌75.0%和99.2%，金黄色葡萄球菌81.8%和97.4%，流感嗜血杆菌75.0%和90.3%，肺炎链球菌33.3%和95.4%。肺炎克雷伯菌、鲍曼不动杆菌和铜绿假单胞菌的环介导恒温扩增技术检测结果与痰培养半定量检测结果具有较好的符合率，R²值分别是0.855 7、0.804 4和0.924 3。【结论】环介导恒温扩增技术操作简单、检测快速。与培养方法相比，环介导恒温扩增检测具有较高的特异性(89.1%–99.2%)，其中针对肺炎克雷伯菌、鲍曼不动杆菌、金黄色葡萄球菌和铜绿假单胞菌的检测具有较高灵敏度(81.8%−90.8%)，可用于临床呼吸道感染病原菌的快速诊断。环介导恒温扩增技术的检测结果与痰培养半定量的检测结果符合率较好。流感嗜血杆菌和肺炎链球菌由于培养阳性标本量较少，有待进一步评估。

Abstract:

[Background] The traditional method of bacterial culture is the gold standard for the diagnosis of lower respiratory tract infection. However, bacterial culture needs a long cycle and has low sensitivity. Loop-mediated isothermal amplification (LAMP) as a simple and rapid method can be used for rapid detection of common bacteria in clinical respiratory tract infection. [Objective] To evaluate the detection capability of LAMP for seven common bacterial species of respiratory tract infection. [Methods] Specific primers were designed for the LAMP of seven common pathogens of respiratory tract infections, including Klebsiella pneumoniae (KP), Acinetobacter baumannii (AB), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), Streptococcus pneumoniae (SP), Moraxella catarrhalis (MC), and Haemophilus influenzae (HI). The sensitivity and specificity of this LAMP method were evaluated by gradient dilution method and cross-reaction experiment, respectively. We then retrospectively analyzed the residual tract specimens (sputum and bronchoalveolar lavage fluid) from 240 patients with suspected lower respiratory tract infection in Peking University Peoples Hospital from November 2019 to March 2021. After DNA extraction by the automatic nucleic acid extraction system, we employed LAMP to detect the seven common pathogens. Further, we compared the results obtained from LAMP and bacterial culture to evaluate the sensitivity and specificity of LAMP. The cut-off value (C_t value) of different species were optimized, and the application value of LAMP in clinical practice were discussed. [Results] The LAMP method had a good linear correlation with the template concentration. The LAMP system had good specificity as there was no cross reaction with other strains. A total of 218 sputum specimens and 22 bronchoalveolar lavage fluid specimens were detected in this study. The seven pathogens were detected in 178 specimens based on the bacterial culture method and in 176 specimens based on the LAMP method. The LAMP method generally consumed 2–3 h for detection and could simultaneously detect multiple specimens. The optimal C_t values of HI, KP, AB, MC, SA, PA, and SP were 18.5, 20, 20, 15, 25, 19, and 18, respectively. The sensitivity and specificity were as follows: KP (90.7%, 94.1%), AB (84.0%, 94.2%), PA (90.8%, 89.1%), MC (75.0%, 99.2%), SA (81.8%, 97.4%), HI (75.0%, 90.3%), and SP (33.3%, 95.4%). The LAMP results for KP, AB, and PA showed good coincidence with the semi-quantitative results of sputum culture, and the R² values were 0.855 7, 0.804 4, and 0.924 3, respectively. [Conclusion] LAMP had low requirements for operating personnel, short cycle, and high specificity (89.1%–99.2%) compared with the culture method. It has high sensitivity (81.8%–90.8%) for the detection of K. pneumoniae, A. baumannii, S. aureus, and P. aeruginosa, and thus can be used for the rapid detection of these bacteria in clinical respiratory tract infection. The results of LAMP were in good agreement with the semi-quantitative results of sputum culture. The detection of H. influenzae and S. pneumoniae needs to be further evaluated due to the lack of positive culture specimens.