严重爆炸致烧伤患者感染病原微生物的特征及宏基因组学第二代测序技术在病原微生物检测中的应用

罗汝斌; 黄曼; 胡行; 张嵘; 韩春茂

doi:10.3760/cma.j.cn501120-20201017-00440

严重爆炸致烧伤患者感染病原微生物的特征及宏基因组学第二代测序技术在病原微生物检测中的应用

doi: 10.3760/cma.j.cn501120-20201017-00440

1.
浙江大学医学院附属第二医院外科重症医学科，杭州　　310009
2.
浙江大学医学院附属第二医院综合ICU，杭州　　310009
3.
浙江大学医学院附属第二医院烧伤与创面修复科，浙江省严重创伤与烧伤诊治重点实验室，杭州　　310009
4.
浙江大学医学院附属第二医院检验科，杭州　　310009

基金项目:

国家自然科学基金青年科学基金项目 81601687

详细信息

通讯作者:
韩春茂，Email：zrssk@zju.edu.cn

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出版历程
- 收稿日期: 2020-10-17

Microbiological characteristics of patients with severe burns caused by blast and application of meta- genomics next-generation sequencing in the detection of pathogenic microorganisms

1.
Department of Surgery Intensive Care Unit, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
2.
Department of General Intensive Care Unit, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
3.
Department of Burn and Wound Repair, the Second Affiliated Hospital of Zhejiang University School of Medicine, Key Laboratory of the Diagnosis and Treatment of Severe Trauma and Burn of Zhejiang Province, Hangzhou 310009, China
4.
Department of Laboratory, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China

Funds:

National Natural Science Foundation of China for Youth 81601687

More Information

Corresponding author: Han Chunmao, Email: zrssk@zju.edu.cn

摘要

摘要: 目的分析严重爆炸致烧伤患者在不同时间段感染病原微生物的特征，同时探讨宏基因组学第二代测序（mNGS）技术在病原微生物检测中的应用价值。方法采用回顾性观察性研究方法。2020年6月13日—9月13日浙江大学医学院附属第二医院收治符合入选标准的23例严重爆炸致烧伤患者，其中男21例、女2例，年龄（64±5）岁，烧伤总面积（86±14）%体表总面积。统计入院时，患者的简明烧伤严重指数（ABSI）评分、修订Baux评分、急性生理学和慢性健康状况评价Ⅱ（APACHEⅡ）评分和序贯器官衰竭（SOFA）评分。记录患者入院后≤7 d、8~20 d、21~30 d并发症及病原微生物感染来源分布情况，统计病原微生物检出情况并比较微生物培养法与mNGS检测效能的差异。统计患者入院后感染的病原微生物来源分布总体情况并比较微生物培养法与mNGS检测效能差异。对数据行McNemar和Fisher确切概率法检验。结果入院时，患者ABSI评分、修订Baux评分、APACHE Ⅱ评分和SOFA评分分别为（12.6±2.4）、（91±22）、（26±4）和（10.3±2.3）分。入院后≤7 d，患者的并发症主要为吸入性损伤、脓毒症休克和低蛋白血症；患者感染病原微生物的来源主要为创面、血流和肺部。入院后8~20 d，患者脓毒症休克的发生率最高，吸入性损伤的发生率明显低于入院后≤7 d（P<0.01）；病原微生物感染来源仍然以创面、肺部和血流为主，其中创面和血流感染发生率均明显低于入院后≤7 d（P<0.01）。入院后21~30 d，患者仅有多器官功能衰竭和急性呼吸窘迫综合征存在且发生率低，吸入性损伤的发生率明显低于入院后≤7 d（P<0.01），脓毒症休克的发生率明显低于入院后≤7 d（P<0.01）和入院后8~20 d（P<0.01）；仅存在较低的血流感染，创面感染发生率明显低于入院后≤7 d（P<0.01）和入院后8~20 d（P<0.05），肺部和血流感染发生率明显低于入院后≤7 d（P<0.01）。入院后≤7 d，革兰阳性菌以金黄色葡萄球菌为主；革兰阴性菌以肺炎克雷伯菌和嗜麦芽窄食单胞菌为主；真菌中仅含念珠菌。入院后8~20 d，革兰阳性菌仍以金黄色葡萄球菌为主，肠球菌的检出率明显低于入院后≤7 d（P<0.01）；革兰阴性菌以铜绿假单胞菌和鲍曼不动杆菌为主，且其检出率均明显高于入院后≤7 d（P<0.01）；新检出了镰刀菌。入院后21~30 d，革兰阳性菌仍以金黄色葡萄球菌为主，肠球菌和芽孢杆菌的检出率明显低于入院后≤7 d（P<0.01）；革兰阴性菌仍以铜绿假单胞菌和鲍曼不动杆菌为主，且随呈时间依赖性增高趋势，铜绿假单胞菌检出率明显高于入院后≤7 d（P<0.01）和入院后8~20 d（P<0.01），鲍曼不动杆菌的检出率明显高于入院后≤7 d（P<0.01），肺炎克雷伯菌检出率均明显低于入院后≤7 d（P<0.01）和入院后8~20 d（P<0.01）；念珠菌、霉菌、镰刀菌均有检出。在入院后≤7 d和入院后8~20 d，mNGS和微生物培养法检出病原微生物的一致性较高（κ=0.659、0.596）；而在入院后21~30 d，mNGS和微生物培养法检出病原微生物的一致性为中度（κ=0.407）。不同时间段，mNGS的病原微生物阳性检验率是基本恒定的，而微生物培养法呈时间依赖性下降。入院后，创面、血液、痰液和留置导管中共分离出506株病原微生物，以金黄色葡萄球菌、铜绿假单胞菌、鲍曼不动杆菌、肺炎克雷伯菌为主。创面标本以金黄色葡萄球菌和铜绿假单胞菌最为多见，血液标本中以肺炎克雷伯菌多见，在痰液标本中以铜绿假单胞菌和鲍曼不动杆菌为主，留置导管标本中以鲍曼不动杆菌最为多见。铜绿假单胞菌在创面和痰液中的检出率均分别显著高于血液（P<0.05或P<0.01）和留置导管（P<0.01）。mNGS和微生物培养法检测病原微生物总体结果的一致性为中度（κ=0.556），其中血液和留置导管标本的mNGS与微生物培养法检测病原微生物结果的一致性为高度（κ=0.631、0.619）；而痰液和创面检测结果的一致性为中度（κ=0.558、0.528）。结论该研究中的严重爆炸致烧伤患者最常见的感染是创面感染和血流感染。随入院时间的延长，患者感染的病原微生物由金黄色葡萄球菌、肺炎克雷伯菌和嗜麦芽窄食单胞菌为主转为以鲍曼不动杆菌和铜绿假单胞菌为主；mNGS检测与微生物培养法相比，表现出更高的病原微生物阳性检出率。
- 烧伤 /
- 感染 /
- 宏基因组 /
- 爆炸伤 /
- 微生物学特征 /
- 二代测序
Abstract: Objective To analyze the microbiological characteristics of patients with severe burns caused by blast in different periods and explore the application value of metagenomics next-generation sequencing (mNGS) in detecting pathogenic microorganisms. Methods The retrospective observational study was applied. From June 13 to September 13, 2020, twenty-three patients (21 males and 2 females) with severe burns caused by blast who met the inclusion criteria were admitted to the Second Affiliated Hospital of Zhejiang University School of Medicine, with age of (64±5) years and total burn area of (86±14) % total body surface area. Abbreviated burn severity index (ABSI) score, revised Baux score, acute physiology and chronic health status evaluation (APACHE) Ⅱscore, and sequential organ failure assessment (SOFA) score were counted on admission. Within 7, 8-20 and 21-30 d after admission, the complications, infection source and distribution of pathogenic microorganisms in patients were recorded. The detection of pathogenic microorganisms was analyzed, and the difference in detection efficiency between microbial culture method and mNGS was compared. After admission, the infection of overall source distribution of pathogenic microorganisms in patients was analyzed, and the difference in detection efficiency between microbial culture method and mNGS was compared. Data were statistically analyzed with McNemar and Fisher exact probability test. Results On admission, ABSI score, revised Baux score, APACHE Ⅱ score and SOFA score were (12.6±2.4), (91±22), (26±4), and (10.3±2.3) respectively. Within 7 d after admission, the main complications of patients were inhalation injury, septic shock, and hypoproteinemia. Patients were mainly infected with pathogenic microorganism on wound, blood stream, and lung. Within 8-20 d after admission, the incidence of septic shock was the highest. The incidence of inhalation injury was significantly lower than that of ≤7 d after admission (P<0.01), the main source of infection were wound, lung, and blood stream, and the incidence of wound and blood stream infection were significantly lower than that of ≤7 d after admission (P<0.01). Within 21-30 d after admission, the incidences of multiple organ failure and acute respiratory distress syndrome were low, the incidence of inhalation injury was significantly lower than that of ≤7 d after admission (P<0.01), and the incidence of septic shock was significantly lower than that of ≤7 d after admission (P<0.01) and 8-20 d after admission (P<0.01). There were only low bloodstream infections, and the incidence of wound infection was significantly lower than that of ≤7 d after admission (P<0.01) and 8-20 d after admission (P<0.05), and the incidences of lung and blood stream infection were significantly lower than those of ≤7 d after admission (P<0.01). Within ≤7 d after admission, gram-positive bacteria were mainly Staphylococcus aureus. Gram-negative bacteria were mainly Klebsiella pneumoniae and Stenotrophomonas maltophilia. The fungi contained only Candida. Within 8-20 d after admission, Staphylococcus aureus was mainly the gram-positive bacteria, and the detection rate of Enterococcus was significantly lower than that of ≤7 d after admission (P<0.01). Pseudomonas aeruginosa and Acinetobacter baumannii were the main gram-negative bacteria, and their detection rates were significantly lower than those of ≤7 d after admission (P<0.01).There was a new detection of Fusarium. Within 21-30 d after admission, Staphylococcus aureus was the mainly gram-positive bacteria, and the detection rates of Enterococcus and Bacillus were significantly lower than those of ≤7 d after admission (P<0.01). Pseudomonas aeruginosa and Acinetobacter baumannii were still the main gram-negative bacteria, and increased with the extension of time after admission. The detection rate of Pseudomonas aeruginosa was significantly higher than that of ≤7 d after admission (P<0.01) and 8-20 d after admission (P<0.01), and the detection rate of Acinetobacter baumannii was significantly higher than that of ≤7 d after admission (P<0.01). The detection rate of Klebsiella pneumoniae was significantly lower than those of ≤7 d after admission (P<0.01) and 8-20 d after admission (P<0.01). All Candida, Mould, Fusarium were detected. Within ≤7 d and 8-20 d, the consistency between mNGS and bacterial culture was high (κ=0.659, 0.596). Within 21-30 d after admission, the consistency between mNGS and bacterial culture was moderate (κ=0.407). In different time periods, the positive test rate of mNGS was basically constant, while that of microbial culture showed a decline with the extension time after admission. Five hundred and six strains of pathogenic microorganisms were isolated from wound, blood, sputum, and indwelling catheter, and Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae were the main pathogenic microorganisms. Pseudomonas aeruginosa and Acinetobacter baumannii were the most common in the wound samples, Klebsiella pneumoniae was more often seen in blood samples while Pseudomonas aeruginosa and Acinetobacter baumannii in sputum samples, and Acinetobacter baumannii in indwelling catheter samples were the most common. The detection rates of Pseudomonas aeruginosa in wound and sputum were significantly higher than those of blood (P<0.05 or P<0.01) and indwelling catheter (P<0.01), respectively. The consistency between the overall results of mNGS and microbial culture were moderate (κ=0.556). The consistency between mNGS and microbial culture was high in samples of blood and indwelling catheter (κ=0.631, 0.619), but those were moderate in sputum and wound (κ=0.558, 0.528). Conclusions The most common infections of patients with severe burn caused by blast injury were wound infection and blood stream infection. With the extension of time after admission, the main pathogenic bacterial strains of patients changed from Staphylococcus aureus, Klebsiella pneumoniae, and Stenotrophomonas maltophilia to Acinetobacter baumannii and Pseudomonas aeruginosa. mNGS showed a higher positive rate of detecting pathogenic microorganisms than conventional microbial culture.
- Burns /
- Infection /
- Metagenome /
- Blast injury /
- Microbiological characteristics /
- Next-generation sequencing
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表1 入院后不同时间段23例严重爆炸致烧伤患者的并发症发生情况及病原微生物感染来源分布情况 [例（%）]

时间段（d）	并发症							感染来源
时间段（d）	吸入性损伤	脓毒症休克	MOF	消化道出血	凝血功能障碍	ARDS	低蛋白血症	尿路	留置导管	肺部	创面	血流
≤7	14（60.87）	8（34.78）	0	0	0	0	4（17.39）	0	0	8（34.78）	17（73.91）	16（69.57）
8~20	0^a	7（30.43）	3（13.04）	5（21.74）	3（13.04）	1（4.35）	3（13.04）	1（4.35）	3（13.04）	4（17.39）	6（26.09）^a	4（17.39）^a
21~30	0^a	0^ab	1（4.35）	0	0	1（4.35）	0	0	0	0^a	0^ac	1（4.35）^a
注：MOF为多器官功能衰竭，ARDS为急性呼吸窘迫综合征；与入院<7 d比较，^aP<0.01；与入院后8~20 d比较，^bP<0.01，^cP<0.05

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表2 入院后不同时间段23例严重爆炸致烧伤患者感染病原微生物的检出株[频数（%）]

时间段（d）	菌株数（株）	革兰阳性菌			革兰阴性菌							真菌
时间段（d）	菌株数（株）	金黄色葡萄球菌	肠球菌	芽孢杆菌	铜绿假单胞菌	鲍曼不动杆菌	SM	阴沟肠杆菌	肺炎克雷伯菌	BC	黏质沙雷菌	念珠菌	霉菌	镰刀菌
≤7	192	33（17.19）	15（7.81）	10（5.21）	12（6.25）	14（7.29）	35（18.23）	11（5.73）	38（19.79）	6（3.12）	14（7.29）	4（2.08）	0	0
8~20	150	20（13.33）	2（1.33）^a	3（2.00）	26（17.33）^a	31（20.67）^a	22（14.67）	6（4.00）	24（16.00）	3（2.00）	8（5.33）	2（1.33）	0	3（2.00）
21~30	164	19（11.59）	1（0.61）^a	0^a	55（33.54）^ab	44（26.83）^a	13（7.93）	3（1.83）	9（5.49）^ab	2（1.22）	2（1.22）	3（1.83）	5（3.05）	8（4.88）
P值		0.642	<0.001	<0.001	<0.001	<0.001	0.072	0.412	<0.001	0.621	0.221	0.898	0.346	0.116
注：SM为嗜麦芽窄食单胞菌，BC为洋葱伯克霍尔德菌；念珠菌包括白色念珠菌、热带念珠菌、近平滑念珠菌，霉菌包括根霉菌、毛霉菌、曲霉菌；P值为不同时间段微生物总体比较所得；与入院后≤7 d比较，^aP<0.01；与入院后8~20 d比较，^bP<0.01

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表3 入院后不同时间段23例严重爆炸致烧伤患者感染病原微生物的宏基因组学第二代测序（mNGS）法和微生物培养法结果一致性比较

时间段（d）与微生物培养法结果	mNGS法结果		κ值
时间段（d）与微生物培养法结果	阳性	阴性	κ值
≤7
阳性	140	18	0.659
阴性	11	44	0.659
8~20
阳性	139	18	0.596
阴性	21	50	0.596
21~30
阳性	110	14	0.407
阴性	35	34	0.407
注：3个不同时间段的样本数依次为190、150、164

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表4 入院后23例严重爆炸致烧伤患者不同来源标本的病原微生物分布[株（%）]

标本来源	菌株数（株）	革兰阳性菌			革兰阴性菌							真菌
标本来源	菌株数（株）	金黄色葡萄球菌	肠球菌	芽孢杆菌	铜绿假单胞菌	鲍曼不动杆菌	SM	阴沟肠杆菌	肺炎克雷伯菌	BC	黏质沙雷菌	念珠菌	霉菌属	镰刀菌
创面	150	27（18.00）	7（4.67）	10（6.67）	30（20.00）	7（4.67）	21（14.00）	7（4.67）	18（12.00）	2（1.33）	5（3.33）	3（2.00）	5（3.33）	8（5.33）
血液	58	8（13.79）	0	0	3（5.17）^a	9（15.52）	10（17.24）	2（3.45）	15（25.86）	3（5.17）	5（8.62）	2（3.45）	0	1（1.72）
痰液	269	32（11.90）	11（4.09）	3（1.12）	55（20.45）^b	60（22.30）^ab	38（14.13）	11（4.09）	33（12.27）	6（2.23）	14（5.20）	4（1.49）	0	2（0.74）
留置导管	29	5（17.24）	0	0	5（17.24）^cd	13（44.83）^cd	1（3.45）	0	5（17.24）	0	0	0	0	0
P值		0.572	0.571	0.564	<0.001	<0.001	0.438	0.686	0.386	0.425	0.278	0.519	0.608	0.397
注：SM为嗜麦芽窄食单胞菌，BC为洋葱伯克霍尔德菌；念珠菌包括白色念珠菌、热带念珠菌、近平滑念珠菌，霉菌包括根霉菌、毛霉菌、曲霉菌；P值为各标本来源微生物总体比较所得；与创面标本比较，^aP<0.05，^cP<0.01；与血液标本比较，^bP<0.01；与痰液标本比较，^dP<0.01

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表5 入院后严重爆炸致烧伤患者感染病原微生物的宏基因组学第二代测序（mNGS）法和微生物培养法总体结果一致性比较

微生物培养法结果	mNGS法结果		κ值
微生物培养法结果	阳性	阴性	κ值
阳性	389	50	0.556
阴性	67	128	0.556

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表6 入院后不同来源的严重爆炸致烧伤患者感染病原微生物宏基因组学第二代测序（mNGS）法和微生物培养法结果一致性比较

标本来源与微生物培养法结果	mNGS法结果		κ值
标本来源与微生物培养法结果	阳性	阴性	κ值
创面
阳性	124	16	0.558
阴性	20	39	0.558
血液
阳性	54	0	0.631
阴性	8	9	0.631
痰液
阳性	185	34	0.528
阴性	36	77	0.528
留置导管
阳性	26	0	0.619
阴性	3	3	0.619
注：创面、血液、痰液、留置导管的样本数依次为150、58、269、29

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