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陈旭, 董丹凤, 韩立中, 倪语星. GeneXpert实时荧光定量PCR快速检测艰难梭菌的临床评估. 检验医学2014, 29(6):590-592
CHEN Xu, DONG Danfeng, HAN Lizhong, NI Yuxing. Clinical evaluation of GeneXpert real-time fluorescence quantitation PCR in rapid detection ofClostridiumdifficile . Labratory Medicine, 2014, 29(6):590-592  
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GeneXpert实时荧光定量PCR快速检测艰难梭菌的临床评估
陈旭1, 董丹凤2, 韩立中1, 倪语星1
1. 上海交通大学医学院附属瑞金医院临床微生物科,上海 200025
2. 上海交通大学医学院附属瑞金医院检验科,上海 200025

通讯作者:韩立中,联系电话:021-64370045-600632

作者简介:陈 旭,男,1988年生, 硕士,主要从事微生物学研究。

基金:卫生公益性行业科研专项(201002021);
摘要
目的

对GeneXpert实时荧光定量聚合酶链反应(PCR)在快速检测临床粪便标本中艰难梭菌的应用进行评估。

方法

采用双拭子蘸取临床未成形粪便标本,一支拭子用于GeneXpert实时荧光定量PCR检测艰难梭菌毒素基因tcdB,另一支用于常规厌氧菌培养检测;对GeneXpert实时荧光定量PCR检测结果与常规厌氧菌培养结果的一致性进行统计学分析,并计算GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值等参数。

结果

临床收集到141例未成形粪便标本,GeneXpert实时荧光定量PCR检出艰难梭菌毒素基因tcdB阳性42例,其中常规厌氧菌培养阳性34例,两者一致性较好(Kappa=0.775 0,P<0.01),GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为87.2%、92.2%、81.0%和94.9%。

结论

GeneXpert实时荧光定量PCR直接检测粪便标本中的艰难梭菌具有检测快速、操作简便等优点,有重要的临床应用价值。

关键词: 艰难梭菌; 快速检测; 实时荧光定量聚合酶链反应
中图分类号:R446.5 文献标志码:A 文章编号:1673-8640(2014)06-0590-03
Clinical evaluation of GeneXpert real-time fluorescence quantitation PCR in rapid detection ofClostridiumdifficile
CHEN Xu1, DONG Danfeng2, HAN Lizhong1, NI Yuxing1
1. Department of Clinical Microbiology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
2. Department of Clinical Laboratory, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
Fund:
Abstract
Objective

To evaluate the clinical application of GeneXpert real-time fluorescence quantitation polymerase chain reaction (PCR) in rapid detection ofClostridiumdifficile from stool.

Methods

Two-head swab was dipped in clinical stool specimens, one-head swab was used for GeneXpert real-time fluorescence quantitation PCR in detecting toxity genetcdB , and the other was used for anaerobic culture. The results of these 2 methods were compared and analyzed statistically, and the sensitivity, specificity, positive predictive value and negative predictive value of GeneXpert real-time fluorescence quantitation PCR were calculated.

Results

Totally 141 clinical stool specimens were collected, and GeneXpert real-time fluorescence quantitation PCR revealed thatClostridiumdifficile was positive in 42 cases, among whichClostridiumdifficile was cultured in 34 cases. GeneXpert real-time fluorescence quantitation PCR was well consistent with the culture method (Kappa=0.775 0,P<0.01), and the sensitivity, specificity, positive predictive value and negative predictive value of GeneXpert real-time fluorescence quantitation PCR were 87.2%, 92.2%,81.0% and 94.9%, respectively.

Conclusions

GeneXpert real-time fluorescence quantitation PCR could detectClostridiumdifficile from stool specimens directly and accurately, and it has a prospective potential for clinical application.

Keyword: Clostridium difficile; Rapid detection; Real-time fluorescence quantitation polymerase chain reaction

艰难梭菌( Clostridium difficile)是医院感染的重要病原体[ 1],随着大量抗菌药物的广泛应用,艰难梭菌引起的抗菌药物相关腹泻逐渐引起国内外医务人员和科研学者的关注[ 1, 2]。由于艰难梭菌专性厌氧,培养技术要求较高,培养周期长,且艰难梭菌的致病力与其携带的毒力因子有关,因此单纯的阳性培养结果并不能充分证明该菌的致病力。GeneXpert采用实时荧光定量聚合酶链反应(polymerase chain reaction, PCR)通过特定引物扩增相应病原体的毒素基因 tcdB进行快速鉴定,且可以对临床标本直接进行处理检测[ 3],可快速确定致病性病原体的存在。

材料和方法
一、标本收集

收集2012年9月至2013年3月瑞金医院住院患者的腹泻标本(未成形粪便)共141例。

二、仪器与试剂

Xpert Clostridium difficile检测试剂盒(Cepheid公司,美国),双拭子(Copan公司,意大利),血琼脂平板、厌氧菌产气袋和RAPID ID 32A 厌氧菌鉴定板条(生物梅里埃公司,法国)。

三、方法

1. 艰难梭菌培养 采用双拭子蘸取未成形粪便,一支拭子放入环丝氨酸头孢西丁甘露醇(CCMB-TAL)与胆盐肉汤中进行厌氧菌培养,35 ℃ 48 h,如培养基未变色,则说明无艰难梭菌生长,此标本判定为艰难梭菌培养阴性;如培养基变色,则吸取CCMB-TAL培养基至头孢西丁丝氨酸果糖琼脂平板上厌氧培养,如有疑似菌落生长,则进行耐氧试验及厌氧菌鉴定板条确认[ 1, 4]。

2. 艰难梭菌 tcdB基因快速检测 另一支拭子用Xpert Clostridium difficile检测试剂盒进行GeneXpert实时荧光定量PCR,操作按照公司说明书进行,操作过程如下:(1)拭子放入含洗脱液的管子,折断拭子;(2)含洗脱液管子盖上盖子后,震荡;(3)打开试剂盒的盖子,把洗脱液全部加至试剂盒的标本室;(4)关上试剂盒的盖子,放入检测仪器中,GeneXpert仪器检测结果由仪器系统判读。

四、统计学方法

采用四格表分别统计GeneXpert实时荧光定量PCR在快速检测临床粪便标本中艰难梭菌毒素基因 tcdB和常规培养结果,采用SAS 8.2软件对两者一致性进行 Kappa分析,并计算GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值等参数。

结果

141例腹泻标本中,GeneXpert实时荧光定量PCR检出艰难梭菌阳性42例,其中常规厌氧菌培养阳性34例,见表1。GeneXpert实时荧光定量PCR检测艰难梭菌的阳性曲线图与阴性曲线图见图1。 Kappa分析两者的一致性, Kappa值为0 .775 0( P<0.01)。以常规厌氧菌培养作为参考方法,GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为87.2%、92.2%、81.0%和94.9%。

表1 GeneXpert实时荧光定量PCR与常规厌氧菌培养法检测艰难梭菌的结果比较

图1 GeneXpert实时荧光定量PCR检测艰难梭菌毒素基因 tcdB阳性与阴性曲线图

讨论

艰难梭菌是引起住院患者抗菌药物相关性腹泻的主要病原体之一[ 1],近年来艰难梭菌日益受到关注,尤其在欧美地区,艰难梭菌感染的发病率较高[ 5]。因此,对艰难梭菌进行检测具有重要的临床意义。但目前国内尚无标准的方案对其进行培养,且厌氧培养操作相对繁琐、耗时较长,阳性检出率不高;其他检测方法有酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测艰难梭菌共同抗原、酶免疫分析(enzyme-immunoassay,EIA)或ELISA检测毒素及实时荧光定量PCR或普通PCR等[ 1, 6]。

艰难梭菌可以产生毒素A和毒素B 2种毒素,这2种毒素分别由 tcdA和 tcdB编码。毒素A为一种肠毒素,毒素B为一种细胞毒素,研究表明毒素B在艰难梭菌致病性方面起主要作用[ 1, 4]。在本研究中,Xpert Clostridium difficile试剂盒基于实时荧光定量PCR技术,直接检测临床粪便标本中的艰难梭菌 tcdB基因,可以判断待检标本中有无致病性的艰难梭菌。然而在本研究收集的腹泻标本,以培养出艰难梭菌作为参考方法,并未从菌株毒素表型进行确认,具有一定的局限性。通过比较GeneXpert实时荧光定量PCR与常规厌氧菌培养法在艰难梭菌检测方面的结果,显示两者具有较好的一致性( Kappa=0 .775 0, P<0.01),且GeneXpert实时荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为87.2%、92.2%、81.0%和94.9%,表明该方法操作简便、快速,结果灵敏、准确,在临床上具有较好的应用前景。

The authors have declared that no competing interests exist.

参考文献
[1] 章黎华, 董丹凤, 江 岑, 等. 院内感染艰难梭菌的毒素检测及核糖体分型研究[J]. 上海交通大学学报(医学版), 2012, 32(11): 1436-1439. [本文引用:6]
[2] Faires MC, Pearl DL, Berke O, et al. The identification and epidemiology of meticillin-resistant Staphylococcus aureus and Clostridium difficile in patient rooms and the ward environment[J]. BMC Infect Dis, 2013, 13: 342. [本文引用:1] [JCR: 3.025]
[3] Hernández-Rocha C, Barra-Carrasco J, Álvarez-Lobos M, et al. Prospective comparison of a commercial multiplex real-time polymerase chain reaction and an enzyme immunoassay with toxigenic culture in the diagnosis of Clostridium difficile-associated infections[J]. Diagn Microbiol Infect Dis, 2013, 75(4): 361-365. [本文引用:1] [JCR: 2.26]
[4] Dong D, Zhang L, Chen X, et al. Antimicrobial susceptibility and resistance mechanisms of clinical Clostridium difficile from a Chinese tertiary hospital[J]. Int J Antimicrob Agents, 2013, 41(1): 80-84. [本文引用:2] [JCR: 4.415]
[5] Bauer MP, Notermans DW, van Benthem BH, et al. Clostridium difficile infection in Europe: a hospital-based survey[J]. Lancet, 2011, 377(9759): 63-73. [本文引用:1] [JCR: 39.06]
[6] Riley TV. Nosocomial diarrhoea due to Clostridium difficile[J]. Curr Opin Infect Dis, 2004, 17(4): 323-327. [本文引用:1] [JCR: 4.87]