Clinical application the loop-mediated isothermal amplification assay for the identification of pathogens in lower respiratory tract infections

Yanyan Qiao; Luyao Han; Zaiqiang Wang; Yanzhen Feng; Chan Li; Yujuan Li; Faguang Jin; Enqing Fu

doi:10.3877/cma.j.issn.1674-6902.2022.04.005

Chinese Journal of Lung Diseases(Electronic Edition) >

2022 , Vol. 15 >Issue 04: 477 - 480

DOI: https://doi.org/10.3877/cma.j.issn.1674-6902.2022.04.005

Original Article

Clinical application the loop-mediated isothermal amplification assay for the identification of pathogens in lower respiratory tract infections

Yanyan Qiao ¹ ,
Luyao Han ² ,
Zaiqiang Wang ¹ ,
Yanzhen Feng ¹ ,
Chan Li ¹ ,
Yujuan Li ¹ ,
Faguang Jin ¹ ,
Enqing Fu ^,¹^,^†

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^1.Department of Respiratory and Critical Care Medicine, The Second Affiliated Hospital of Air Force Medical University, Xi′an 710038, China
^2.Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Air Force Medical University, Xi′an 710032, China

Corresponding author: Fu Enqing, Email: fuenqing@fmmu.edu.cn

Received date: 2021-12-21

Online published: 2022-09-22

Copyright

No content published by the journals of Chinese Medical Association may be reproduced or abridged without authorization. Please do not use or copy the layout and design of the journals without permission.

All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.

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Abstract

Objective

To more accurately evaluate the performance of loop-mediated isothermal amplification (LAMP) assay for the identification of pathogens in lower respiratory tract infections(LRTIs).

Methods

The pathogen identification results of 1092 bronchoalveolar lavage fluid (BALF) samples from presumptive LRTI patients admitted to the Tangdu Hospital from January 2018 to September 2018 were analyzed retrospectively. Taking the bacterial culture as the gold standard, the sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of LAMP for detecting 8 common lower respiratory tract infection pathogens were calculated.

Results

The sensitivity of LAMP for detecting 8 pathogens were quite different, with the highest sensitivity to Staphylococcus aureus (100%) and the lowest sensitivity to Escherichia coli (40%). The specificity of LAMP for detecting 8 pathogens were higher than 94%. The positive predictive value of the LAMP test results for 8 pathogens were lower than 65%, the negative predictive value were higher than 98%, the positive likelihood were higher than 13. The negative likelihood ratio of the LAMP test results for 8 pathogens were quite different, with the lowest negative likelihood ratio to Staphylococcus aureus (0.00) and the highest negative likelihood ratio to Escherichia coli (0.60).

Conclusion

The LAMP has high accuracy in detecting pathogens of lower respiratory tract infections, which is helpful for early diagnosis and precise treatment of lower respiratory tract infections.

Key words： Lower respiratory infections; Loop-mediated isothermal amplification assay; Pathogens; Bacterial culture

Cite this article

Yanyan Qiao , Luyao Han , Zaiqiang Wang , Yanzhen Feng , Chan Li , Yujuan Li , Faguang Jin , Enqing Fu . Clinical application the loop-mediated isothermal amplification assay for the identification of pathogens in lower respiratory tract infections[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2022 , 15(04) : 477 -480 . DOI: 10.3877/cma.j.issn.1674-6902.2022.04.005

下呼吸道感染是全球第五大死因和5岁以下儿童因感染死亡的首要原因^[1,2]。快速准确检测出病原体有助于下呼吸道感染的诊断和治疗，减少抗生素的不当使用^[3]。环介导恒温扩增芯片法(loop-mediated isothermal amplification, LAMP)简便、快速、病原体检出率高，在下呼吸道感染病原体检测中有广阔的应用前景^[4,5,6]。细菌培养是病原体诊断金标准，痰LAMP检测和细菌培养结果一致性较差，导致LAMP推广应用受限^{[7,8,9,10,11]}。痰易受到口腔微生物的污染，以痰培养为金标准造成两种方法检测结果差异增大，一致性降低，低估LAMP检测病原体的准确性^{[12,13,14,15]}。本文对我院收治的1 092例疑似下呼吸道感染患者的支气管肺泡灌洗液同时进行细菌培养和LAMP检测，评价LAMP检测8种常见下呼吸道感染病原体的准确性，报道如下。

对象与方法

一、研究对象

选择2018年1月至2018年9月空军军医大学第二附属医院初步诊断为下呼吸道感染的患者1 092例。其中男682例，女410例，年龄9~97岁，平均年龄(58.2±11.63)岁。纳入标准：(1)胸部X线片或CT具有肺炎或支气管炎的典型特征；(2)至少具有以下一个症状，包括发热>38.5 ℃、呼吸困难、咳嗽咳痰、脓性痰、呼吸音粗糙伴或不伴干/湿性啰音。排除标准：(1)患者未行支气管肺泡灌洗液LAMP检测和常规细菌培养；(2)明确或高度怀疑为非感染性疾病；(3)明确或高度怀疑为非细菌性感染或结核。

二、研究方法

采集患者支气管肺泡灌洗液，对支气管肺泡灌洗液进行常规细菌培养和LAMP检测，包括以下8种常见的下呼吸道感染病原体：肺炎链球菌、金黄色葡萄球菌、大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌、嗜麦芽窄食单胞菌、流感嗜血杆菌。

用4% NaOH将支气管肺泡灌洗液液化30 min，取出1 ml离心，15 000转/min，离心5 min，弃上清，用呼吸道病原菌核酸检测试剂盒提取沉淀DNA，34.5 μl DNA溶液与20 μl恒温扩增试剂混合，取出50 μl混合液加入到微流控碟式芯片中，使用恒温扩增微流控芯片核酸分析仪分析，当检测指标的扩增时间值≤该病原体指标的参考值时判读为阳性。将支气管肺泡灌洗液接种于血琼脂平板、巧克力平板和中国蓝平板细菌培养基上，置于35 ℃、5%二氧化碳恒温培养箱中培养48 h。选取优势菌群，应用全自动微生物鉴定仪进行鉴定，细菌每毫升菌落单位≥10³时判定为病原体。

三、统计学方法

采用SPSS 25.0进行数据管理和分析，计数数据采用[n(%)]表示。

结果

一、LAMP和细菌培养病原体检出情况

LAMP对不同病原体的检出率均高于细菌培养，LAMP和细菌培养病原体检出情况，见表1。

表1 LAMP和细菌培养病原体检出情况[n(%)]

病原体	LAMP		细菌培养
病原体	阳性	阳性率	阳性	阳性率
肺炎链球菌	59	5.40	12	1.10
金黄色葡萄球菌	17	1.56	3	0.27
大肠埃希菌	10	0.92	5	0.46
肺炎克雷伯菌	38	3.48	7	0.64
铜绿假单胞菌	40	3.66	30	2.75
鲍曼不动杆菌	60	5.49	51	4.67
嗜麦芽窄食单胞菌	24	2.20	11	1.01
流感嗜血杆菌	63	5.77	7	0.64

注：LAMP：环介导恒温扩增芯片法

二、LAMP检测8种病原体准确性评价

LAMP对金黄色葡萄球菌检测的灵敏度最高(100%)，对大肠埃希菌的检测灵敏度最低(40%)。LAMP对8种病原体检测特异度均较高，对流感嗜血杆菌检测特异度最低达94.65%。LAMP对铜绿假单胞菌和鲍曼不动杆菌的阳性预测值最高(65%)，其次为嗜麦芽窄食单胞菌(29.17%)，对其余五种病原体的阳性预测值均不高于20%。LAMP对8种病原体的阴性预测值均在98%以上。LAMP检测金黄色葡萄球菌的阳性似然比最高(77.79)，对流感嗜血杆菌的阳性似然比最低(13.36)。金黄色葡萄球菌阴性似然比最低(0.00)，见表2。

表2 LAMP检测病原体准确性评价指标

病原体	LAMP	培养法		灵敏度(%)	特异度(%)	阳性预测值(%)	阴性预测值(%)	阳性似然比	阴性似然比
病原体	LAMP	阳性	阴性	灵敏度(%)	特异度(%)	阳性预测值(%)	阴性预测值(%)	阳性似然比	阴性似然比
肺炎链球菌	阳性	10	49	83.33	95.46	16.95	99.81	18.37	0.17
	阴性	2	1 031
金黄色葡萄球菌	阳性	3	14	100.00	98.71	17.65	100.00	77.79	0.00
	阴性	0	1 075
大肠埃希菌	阳性	2	8	40.00	99.26	20.00	99.72	54.35	0.60
	阴性	3	1 079
肺炎克雷伯菌	阳性	6	32	85.71	97.05	15.79	99.91	29.06	0.15
	阴性	1	1 053
铜绿假单胞菌	阳性	26	14	86.67	98.68	65.00	99.62	65.74	0.14
	阴性	4	1 048
鲍曼不动杆菌	阳性	39	21	76.47	97.98	65.00	98.84	37.91	0.24
	阴性	12	1 020
嗜麦芽窄食单胞菌	阳性	7	17	63.64	98.43	29.17	99.63	40.47	0.37
	阴性	4	1 064
流感嗜血杆菌	阳性	5	58	71.43	94.65	7.94	99.81	13.36	0.30
	阴性	2	1 027

注：LAMP：环介导恒温扩增芯片法

讨论

LAMP是一种新型病原体检测方法，广泛应用于临床病原体检测^[16]。LAMP与高通量测序技术检测结果一致性较高，且快捷、经济、便携，不需要昂贵设备和复杂流程，可一步完成对多种病原体的检测，能满足医疗机构特别是基层医疗机构对病原学诊断的需求，有助于下呼吸道感染的早期诊断和治疗，具有较高的实用价值和社会意义^{[17,18,19,20]}。

LAMP和细菌培养检测病原体原理不同，LAMP是对病原体靶基因的扩增和检测，无论病原体是否存活，基因数量达到设定的限值检测结果为阳性，细菌培养法要求病原体必须是活细菌，且只能培养出优势菌群^[21,22]。口腔中存在数百种微生物，LAMP与细菌培养对病原体分布检测结果存在差异，可能痰标本受到了口腔污染^{[23,24,25,26]}。下呼吸道与上呼吸道相比是相对无菌的环境，使用上呼吸道标本代替下呼吸道标本研究下呼吸道的微生物构成是不恰当的^[14,27]。以痰培养为金标准评价LAMP，会低估LAMP的准确性。本文以细菌培养为金标准，评价LAMP对8种常见下呼吸道感染病原体的检测效能。

灵敏度和特异度是反映诊断试验真实性的指标。LAMP对8种病原体的灵敏度差异较大，对金黄色葡萄球菌检测灵敏度最高(100%)，表明金黄色葡萄球菌导致的下呼吸道感染，LAMP检测一般为阳性，患者被漏诊机率很低。LAMP对大肠埃希菌的灵敏度最低(40%)，表明LAMP对大肠埃希菌漏诊的可能性较高，如要确诊病原体是大肠埃希菌，需联合其他试验方法进一步检测。LAMP检测8种病原体的特异度均高于94%，表明如果不是某病原体导致的下呼吸道感染，则LAMP检测结果通常是阴性，误诊率较低。

临床更想知道LAMP检测病原体阳性时就诊者患病的概率和LAMP检测阴性时就诊者未患病的概率，而灵敏度和特异度无法根据LAMP检测结果估计就诊者患病概率^[28,29]。阳性预测值和阴性预测值是评价诊断试验预测准确性的指标。LAMP对8种病原体阳性预测值不高，最高值仅为65%，即使病原体LAMP阳性，为假阳性的可能性较高，临床意义不大。LAMP阳性预测值较低与不同病原体下呼吸道感染患病率很低有关。患病率很低时，即使LAMP特异度很高，仍有很多正常人被LAMP检测为阳性，导致LAMP检测结果阳性预测值降低。LAMP对8种病原体阴性预测值较高(98.84%)，表明我院病原体检测结果为阴性时，该病原体引起下呼吸道感染的可能性低于2%，可暂不考虑该病原体导致的感染。诊断试验阳性预测值和阴性预测值与患病率有关，随着患病率降低，阳性预测值降低，阴性预测值升高^[29,30]。只有不同病原体下呼吸道感染患病率无明显差异的地区，预测值才能借鉴使用。不同地区、不同级别的医院、普通病房与重症病房之间，不同病原体下呼吸道感染的患病率很可能存在差异，如盲目借用或外延，预测结果可能产生错误。

阳性似然比是真阳性率与假阳性率之比，阴性似然比是假阴性率与真阴性率之比，两者是评价诊断试验真实性的重要指标，不受患病率的影响。获得就诊者支气管肺泡灌洗液LAMP检测结果后，依据当地就诊者不同病原体下呼吸道感染的患病率，利用本文似然比结果，通过Fagans列线图获得不同病原体下呼吸道感染的验后概率，估计就诊者患病概率^[31]。

LAMP检测8种常见下呼吸道感染病原体有较高的特异度、阴性预测值和阳性似然比，利于下呼吸道感染的早期诊断和精准治疗，具有临床意义。

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