摘要
目的
探讨靶序列富集多重PCR在儿童肺炎多种细菌性病原联合检测中的临床应用价值,了解本地区儿童肺炎的细菌性病原分布,期望建立针对儿童肺炎常见细菌性病原快速、敏感、特异的诊断方法,指导临床进行肺炎的病因学诊断并合理使用抗生素。
方法
确诊为社区获得性肺炎的患儿188例,在入院当天采集深部呼吸道吸引物,用普通培养基和肺炎链球菌、流感嗜血杆菌选择性培养基进行细菌培养,然后提取深部呼吸道吸引物中病原体的DNA,采用荧光定量单PCR的方法检测肺炎支原体,并对同一标本采用靶序列富集多重PCR(Tem-PCR)技术同时扩增肺炎链球菌、流感嗜血杆菌、金黄色葡萄球菌、肺炎克雷伯菌、大肠杆菌、嗜肺军团菌、铜绿假单胞菌、鲍曼氏不动杆菌、脑膜炎奈瑟氏菌、阴沟肠杆菌、奇异变形杆菌、化脓链球菌、粪肠球菌及屎肠球菌14种呼吸道病原菌和肺炎支原体的靶基因,扩增产物用Luminex100多功能悬浮点阵仪检测。以培养和单PCR为参考,比较Tem-PCR对细菌和肺炎支原体检测的敏感性和特异性;分析细菌培养和病原特异性DNA联合检测对肺炎细菌学病原的影响以及肺炎支原体与细菌混合感染的情况。
结果
(1)在188例呼吸道标本中,细菌培养共分离出114株病原菌,其中3例同时培养出2种病原菌,这些病原菌分别是流感嗜血杆菌24株,大肠杆菌16株,粘质沙雷菌14株,肺炎克雷伯菌12株,副流感嗜血杆菌11株,金黄色葡萄球菌9株,肺炎链球菌7株,铜绿假单胞菌3株,鲍曼氏不动杆菌2株,阴沟肠杆菌2株,其他少见细菌9种共14株,培养的阳性率为59.1%(111/188)。
(2)经Tem-PCR技术扩增后,188例标本在Luminex100多功能悬浮点阵仪中有75例呈阳性,共检测出93株病原菌的靶基因,分别是流感嗜血杆菌40株,肺炎链球菌36株,鲍曼氏不动杆菌10株,铜绿假单胞菌4株,金黄色葡萄球菌3株,另外9种Tem-PCR已设计的细菌包括肺炎克雷伯菌、大肠杆菌、阴沟肠杆菌等均未检出,Tem-PCR的阳性率是39.9%(75/188)。
(3)以细菌培养作为参考标准,Tem-PCR对14种病原菌总的敏感性为51.0%,特异性为68.0%,符合率为58.3%,对肺炎链球菌、流感嗜血杆菌、金黄色葡萄球菌、鲍曼氏不动杆菌、铜绿假单胞菌的敏感性分别为100%、41.7%、11.1%、50.0%、66.7%,特异性分别为84.0%、81.7%、98.9%、95.2%、98.9%。Tem-PCR对肺炎链球菌的检出率36/188(19.1%)明显高于培养7/188(3.7%),P＜0.01;对流感嗜血杆菌的检出率40/188(21.3%)与培养24/188(12.8%)之间无明显差异,P＞0.05。
(4)以细菌培养或Tem-PCR任一阳性为标准,联合检测对细菌性病原的总检出率为78.2%(147/188),依次是流感嗜血杆菌36.7%(54/147)、肺炎链球菌24.5%(36/147)、大肠杆菌10.9%(16/147)、肺炎克雷伯菌8.2%(12/147)、金黄色葡萄球菌7.5%(11/147)、鲍曼氏不动杆菌7.5%(11/147)、绿脓杆菌3.4%(5/147)和阴沟肠杆菌1.4%(2/147)。联合检测能提高流感嗜血杆菌、肺炎链球菌、金黄色葡萄球菌、绿脓杆菌和鲍曼氏不动杆菌的检出例数。
(5)在这188例呼吸道标本中,荧光定量单PCR共检出80例肺炎支原体阳性标本,阳性率为42.6%(80/188);Tem-PCR检出51例肺炎支原体阳性标本,阳性率为27.1%(51/188),两者之间无显著差异,P＞0.05;以荧光定量单PCR为参考标准,Tem-PCR对肺炎支原体的敏感性、特异性、符合率分别为30.0%、75.0%、55.9%。
(6)以Tem-PCR检测结果为准,肺炎支原体与细菌的混合感染共16例,混合感染率为8.5%(16/188),其中最常见的是肺炎支原体与流感嗜血杆菌的混合感染,共有8例,占混合感染的50.0%(8/16)。
结论
靶序列富集多重PCR技术对肺炎链球菌有很高的敏感性,但对其他细菌和肺炎支原体还有待改进。细菌培养和病原特异性DNA联合检测应用能显著提高儿童肺炎细菌性病原的检出率,可能更真实地反映肺炎的细菌病原学情况。应用靶序列富集多重PCR技术可以及时明确CAP病原中肺炎支原体与细菌的混合感染。
Objective
To approach the clinic value of Target enriched multiplex PCR for the pneumonic bacterial etiologic diagnosis in children, estabalish the rapid、sensitive、specific diagnostic method for common bacterial pathogen, direct etiologic diagnosis and antibiotic rational use, and understand the distribution of bacterial etiology.
Methods
From Feb 2006 to Jun 2006,188 hospitalized children in Shenzhen children' s hospital, were collected deep tracheal aspirate at the time of hospitalization. The respiratory tract secretions were immediately sent for bacterial culture with 3 kinds of medium:ordinary medium, Hemophilus influenzae selective medium, Streptococcus penumoniae selective medium. Then we extracted the total nucleic acids from secretions, and detected Mycoplasma pneumoniae by single fluorescent quantitation PCR. Simultaneously, 14 respiratory tract pathogenic bacterium and Mycoplasma pneumoniae were detected by Target Enriched multiplex PCR(Tem-PCR). Amplification products were identified by the Luminex100 suspension array. Take the culture and single FQ-PCR as the referenced standard, the sensitivity and specificity of Tem-PCR for the bacteria and Mycoplasma pneumoniae were evaluated. The affect for pneumonic bacterial etiologic distribution was analysised by the combined detection with bacterial culture and Tem-PCR assay.
Results
(1) Of the 188 respiratory tract specimens, 111 were positive by bacteria culture, including 3 were simultaneously detected 2 kinds of bacterium, the positive strain were : Hemophilus influenzae 24, Escherichia coli 16, Serratia marcescens 14, Klebsiella pneumoniae 12, Hemophilus parainfluenzae 11, Staphylococcus aureus 9, Streptococcus penumoniae 7, Pseudomonas aeruginosa 3, Acinetobacter baumannii 2, Enterobactor cloacae 2, the other bacterium was 9 kinds, 14 strains. The positive rate of culture was 59.1% (111/188) .
(2) 93 target gene were detected by Tem-PCR from 75 specimen, they were: Hemophilus influenzae 40, Streptococcus penumoniae 36, Acinetobacter baumannii 10, Pseudomonas aeruginosa 4, Staphylococcus aureus 3, the other 9 kinds of bacterium including Escherichia coli、Klebsiella pneumoniae and Enterobactor cloacae were not detected by Tem-PCR, the positive rate of Tem-PCR was 39.9% (75/188) .
(3) For the 14 kinds of bacterium designed by Tem-PCR, compared with the culture, the sensitivity、specificity and coincidence of Tem-PCR is 51.0%, 68.0%, 58.3% respectively. For Streptococcus penumoniae、Hemophilus in0fluenzae、Staphylococcus aureus、Acinetobacter baumannii、Pseudomonas aeruginosa,the sensitivety of Tem-PCR was 100%、41.7 %、11.1%、50.0 %、66.7 % respectively; the specificity was 84.0%、81.7%、98.9%、95.2%、98.9% respectively. Tem-PCR was more sensitive than culture in the detection of Streptococcus penumoniae (36/19.1% vs. 7/3.7%, p<0.01), and there is no significant difference between Tem-PCR and culture for the detection of Hemophilus influenzae (40/21.3% vs. 24/12.8%, p>0.05 ).
(4) The total positive rate by combined detection of culture and Tem-PCR assay was 78.2%(147/188), of which 36.7% (54/147) were Hemophilus influenzae , 24.5% (36/147) were Streptococcus pneumoniae, 10.9% (16/147) were Escherichia coli, 8.2% (12/147) were Pseudomonas aeruginosa, 7.5% (11/147) were Staphylococcus aureus, 7.5% (11/147) were Acinetobacter baumannii, 3.4% (5/147) were Pseudomonas aeruginosa, and 1.4% (2/147) were Enterobacter cloacae. The combined detection may increase the positive case of Staphylococcus aureus, Hemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii.
(5) Of the 188 respiratory tract specimens, 42.6% (80/188) were positive by single fluorescent quantitation PCR for Mycoplasma pneumoniae, 27.1% (51/188) was positive by Tem-PCR. There is no significant difference between Tem-PCR and FQ-PCR(P>0.05 ). Take the single FQ-PCR as the reference standard, the sensitivity、specificity and coincidence of Tem-PCR was 30.0%,74.3%,55.9%, respectively.
(6) The mixed infection with Mycoplasma pneumoniae and bacterium was 16 by Tem-PCR. Of which, the most common mixed infection was Mycoplasma pneumoniae and Hemophilis influenzae, it was 50.0%(8/16).
Conclusions
Tem-PCR is highly sensitive for detection of Streptococcus penumoniae causing pneumonia in children. While sensitivities for the other bacterium and Mycoplasma pneumonia need improvement. The combined detection of bacterial culture and Tem-PCR assay may increase the positive rate of bacterial pathogens in hospitalized children with CAP and can really demonstrate the distribution of bacterial etiology. Tem-PCR can timely indicate the mixed infection with Mycoplasma pneumoniae and bacterium in etiologic agent causing CAP.
引文
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