Xpert艰难梭菌检测系统的临床应用评价

目的评估Xpert艰难梭菌检测系统的临床应用价值。方法选取43份腹泻粪便标本,采用厌氧培养法、VIDAS检测A/B毒素法和Xpert艰难梭菌检测系统检测艰难梭菌,以产毒培养法作为金标准对检测方法进行评价,并比较不同检测方法对临床标本检测结果的一致性。通过自配027型标准菌株模拟粪便标本,验证Xpert艰难梭菌检测系统筛选027型流行株的能力。结果以产毒培养法为金标准,Xpert艰难梭菌检测系统的敏感性和特异性分别为90.9%和93.8%,阳性和阴性预测值分别为83.3%和96.8%。与产毒培养法结果一致性检验Kappa值为0.822(P0.05),与VIDAS检测A/B毒素法结果一致性检验Kappa值为0.419(P0.05);027型标准菌株模拟粪便标本检测结果阳性并报告为027型。结论 Xpert艰难梭菌检测系统能够快速准确地检测粪便标本中的艰难梭菌相关基因,且能准确报告027型高产毒力菌株。     

GeneXpert实时荧光定量PCR快速检测艰难梭菌的临床评估

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,P0.01),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。     

实时荧光定量PCR直接检测粪便标本艰难梭菌tcdB基因

目的建立艰难梭菌实时荧光定量PCR检测方法,直接检测粪便标本艰难梭菌tcd B基因,并探讨其应用价值。方法根据艰难梭菌基因组设计tcd B基因特异性引物及探针,用ATCC 43255标准株评价其敏感性,选取艰难梭菌生物学特征相近的临床菌株验证其特异性;收集2015年10-12月临床腹泻患者稀便标本115份,提取基因组总DNA后进行tcd B基因检测,并以产毒培养法为参考方法,探讨该方法的临床诊断价值。结果荧光定量PCR法最低检测限为1×10-3ng,并且仅特异性地扩增产毒艰难梭菌;临床样本评价荧光定量PCR的敏感性、特异性、阳性预测值和阴性预测值分别为75.0%、99.1%、85.7%和98.1%。结论实时荧光定量PCR直接检测稀便标本中的艰难梭菌tcd B基因可用于艰难梭菌相关腹泻的快速诊断。     

艰难梭菌谷氨酸脱氢酶抗原及毒素检测试剂盒的性能评估

目的对新产品艰难梭菌谷氨酸脱氢酶抗原及毒素检测试剂盒(免疫层析法,C.DIFF QUICK CHEK COMPLETE)进行性能评估。方法收集临床腹泻患者粪便标本130份,对腹泻粪便标本同时进行艰难梭菌毒素A/B检测试剂盒、艰难梭菌谷氨酸脱氢酶抗原及毒素检测试剂盒和毒素培养检测,以金标准毒素培养法结果作为参比标准,评价各种检测方法的敏感性、特异性、阳性预测值和阴性预测值,从而对艰难梭菌谷氨酸脱氢酶抗原及毒素检测试剂盒进行性能评估。结果毒素培养法检测艰难梭菌的阳性率为13.8%(18/130)。与金标准方法相比,艰难梭菌谷氨酸脱氢酶抗原及毒素检测试剂盒的敏感性、特异性、阳性预测值和阴性预测值分别为55.6%、98.2%、83.3%和93.2%,艰难梭菌毒素A/B检测试剂盒的敏感性、特异性、阳性预测值和阴性预测值分别为50.0%、95.6%、64.3%和92.1%。结论艰难梭菌谷氨酸脱氢酶抗原及毒素检测试剂盒特异性高,阳性预测值较高,且检测周期短,无需特殊仪器平台,可作为医院门、急诊艰难梭菌感染的初筛试剂盒。     

高毒力艰难梭菌的基因型和毒力相关基因检测

目的鉴定莫西沙星耐药的艰难梭菌临床菌株的基因型和毒素相关基因的携带情况。方法 22株莫西沙星耐药的艰难梭菌临床菌株收集自澳大利亚悉尼地区,利用基于荧光毛细管电泳的聚合酶链反应(PCR)-核糖体分型技术对其进行基因分型;利用PCR方法检测其毒素A、B编码基因tcdA、tcdB和二元毒素编码基因cdtA、cdtB;利用PCR-基因测序方法检测毒素调节基因tcdC的基因序列,通过与参考菌株VPI 10463(Genbank收录号:X92982)比对了解其基因突变情况,通过与Genbank中的序列比对确定其序列型。结果 21株菌株为高毒力核糖体型027(RT027),另1株为RT078;22株菌株毒力编码基因均为tcdA+tcdB+cdtA+cdtB+;21株RT027 tcdC序列型为sc1,另1株RT078序列型为WA39,与参考株VPI 10463比较,RT027菌株tcdC序列均出现连续18个核苷酸(nt330-347)的缺失和第117位单核苷酸的缺失,RT078菌株则出现连续39个核苷酸(nt341-379)的缺失和第184位(CT)的突变。结论高毒力RT027是最常见的莫西沙星耐药的艰难梭菌基因型;高毒力RT027和RT078均携带毒素A、B和二元毒素编码基因,且毒素调节基因tcdC均存在基因突变。     

TaqMan实时荧光定量PCR检测艰难梭菌

目的 建立特异敏感的实时荧光定量PCR方法,用于艰难梭菌的快速检测;评价基于纯培养艰难梭菌和粪便中艰难梭菌的2种标准曲线;并应用该荧光定量PCR法对急性艰难梭菌感染（CDI）的小鼠进行粪便含菌量检测评价。方法 针对艰难梭菌基因组中16S rRNA序列设计特异性引物和探针,建立一套快速检测艰难梭菌含量的实时荧光定量PCR方法,验证方法的特异性、灵敏性;绘制艰难梭菌纯菌浓度梯度稀释标准曲线和粪便中同浓度梯度艰难梭菌的标准曲线,比较两者的差异;用艰难梭菌高毒株NAP1/027感染用抗生素处理的C57BL/6小鼠,建立CDI小鼠模型,同时应用该荧光定量PCR和活菌培养法定量检测小鼠粪便中的艰难梭菌含量变化。结果 建立的TaqMan实时荧光定量PCR具有较高的灵敏性和特异性,生成标准曲线的相关系数为0.999 8,斜率为-3.400 4;用纯培养艰难梭菌和粪便中艰难梭菌分别制备标准曲线,结果表明2种标准曲线定量检测结果差异无统计学意义。建立CDI小鼠模型,应用该荧光PCR能有效、准确的检测出粪便中艰难梭菌的含量,可替代费时费力的活菌培养计数法。结论 用纯培养艰难梭菌来制备标准曲线不影响对含菌粪便标本的准确定量检测,荧光定量PCR能准确快捷地检测CDI小鼠粪便中的艰难梭菌含量,比活菌计数更快速和方便,可用于艰难梭菌感染小鼠模型中小鼠肠道内艰难梭菌定植的定量检测。     

2种艰难梭菌感染检测方法的比较与分析

目的比较酶联免疫荧光分析(ELFA)与实时荧光定量PCR(PCR)2种艰难梭菌感染(Clostridium difficile infection,CDI)检测方法的检验性能,从中选择灵敏度高、特异性好且操作简便的CDI检验方法开展临床检验。方法对来自重症监护病房(ICU)、消化内科及感染科等临床科室的126份粪便标本进行艰难梭菌毒素或基因检测。以产毒培养试验(toxigenic culture,TGC)结果作为参考标准,评价ELFA与PCR方法的性能,同时分析我院及各科室的CDI流行情况。结果 TGC检测126份腹泻患者的粪便标本,阳性率为19.0%(24/126),其中ICU、消化内科感染率较高。ELFA和PCR方法的检测敏感性分别为66.7%和91.7%,差异有统计学意义(χ2=4.55,P0.05);特异性、阳性预测值、阴性预测值与准确度差异无统计学意义。结论我院CDI形势较为严峻,以ICU和消化内科最为突出。PCR与ELFA均能够提供更为准确、可靠的检测结果,但前者敏感性较好。     

艰难梭菌毒素基因检测及分子流行病学分析

目的了解腹泻患者艰难梭菌感染现状,并分析分离菌株的核糖体分型情况。方法收集161例住院腹泻患者粪便标本进行艰难梭菌毒素基因PCR检测,同时进行产毒培养法,并比较2种方法的一致性;对分离的艰难梭菌进行核糖体分型。结果艰难梭菌产毒培养法阳性率为9.94%(16/161),粪便艰难梭菌毒素基因阳性率为9.94%(16/161),2种方法结果差异无统计学(P0.05),一致性较好(Kappa0.75)。培养所得18株艰难梭菌中A、B毒素基因均阴性的2株(11.11%),tcdA~+tcdB~+产毒株15株(83.33%),tcdA~-tcdB~+1株(5.56%)。18株艰难梭菌核糖体分型分为16种型别(GS1～GS16),未发现核糖体分型027型菌株。结论艰难梭菌粪便毒素基因PCR检测可用于临床诊断,未发现本院艰难梭菌暴发流行。     

利用实时荧光定量PCR技术检测毒素基因快速鉴定A/B型肉毒梭菌方法的建立

目的建立针对A/B型肉毒梭菌毒素基因的实时荧光定量PCR（real-time PCR）检测方法,构建标准曲线,评价该方法的特异性、敏感性及检测下限,以提高肉毒梭菌检测的快速性、准确性。方法根据肉毒梭菌A/B型毒素基因设计特异性引物及探针,优化反应条件,建立real-time PCR反应体系。 用25种细菌评价该方法的特异性,使用含有A/B型毒素基因特异性序列的重组质粒标准品构建标准曲线,模拟粪便标本,评价建立方法的灵敏度。结果建立的real-time PCR检测方法特异性好,携带A/B型毒素基因的重组质粒均出现相应特异性扩增曲线,对其他25种细菌进行试验扩增时,均未见出现特异性扩增曲线。 根据重组质粒标准品构建的标准曲线可确定其对肉毒梭菌A/B型毒素基因检测灵敏度分别为5.04×102拷贝/μl和6.91×102拷贝/μl。 模拟粪便标本中含有目的基因重组质粒的检测下限为A型1.71×103拷贝/μl,B型2.14×103拷贝/μl。结论本研究设计了适合检测国内肉毒梭菌 A/B 型的引物和探针,建立 real-time?PCR 快速检测体系,为我国肉毒梭菌 A/B 型菌株的快速鉴定提供一种新的技术手段。     

多重PCR检测毒力型艰难梭菌的方法学研究及应用

目的建立多重PCR方法检测毒力型艰难梭菌。方法设计艰难梭菌种特异tpi引物和毒力基因tcdA、tcdB特异性引物,建立多重PCR方法。利用已知菌株,验证方法的特异性和最低检出限。与厌氧培养法、ELISA法比较其检测临床菌株和其毒素分泌的准确性。结果多重PCR方法检测艰难梭菌的最低检出浓度为0．7pg／IM,特异性为100％。53株厌氧培养法鉴定的艰难梭菌临床分离株,多重PCR方法检测tpi基因均为阳性,其中tcdA＋／tcdB＋为39株．tcdA一／tcdB．为14株。ELISA法检测毒素A／B显示23株为阳性、30株为阴性。23株ELISA法毒素A／B阳性的艰难梭菌多重PCR方法检测结果均为tcdA十／配拈＋。结论多重PCR方法可用于检测毒力型艰难梭菌具有较高的临床应用价值。     

Evaluation of the Xpert Clostridium difficile Assay for the Diagnosis of Clostridium difficile Infection

Infection with Clostridium difficile is a growing concern because of the increasing prevalence and spread of nosocomial infections. Emergence of the hypervirulent 027/NAP1/BI strain is also notable. Existing diagnostic methods have low sensitivity or are time-consuming. Therefore, establishing a rapid and accurate microbiological diagnostic assay is needed. We evaluated the Xpert C. difficile assay (Xpert CD assay; Cepheid, USA) to detect toxigenic C. difficile. This assay is a real-time multiplex PCR assay that can be used to detect toxigenic C. difficile strains and differentiate the C. difficile presumptive 027/NAP1/BI strain. A total of 253 loose stool specimens were collected and toxigenic cultures, VIDAS C. difficile A & B assays (VIDAS CDAB assay; bioMérieux, France), and the Xpert CD assay were performed. In comparison to toxigenic cultures, the sensitivity, specificity, and positive and negative predictive values were 100%, 94.6%, 83.1%, and 100%, respectively, for the Xpert CD assay and 40.8%, 98.0%, 100%, and 88.9%, respectively, for VIDAS CDAB assay. Because of the low prevalence of the PCR ribotype 027 in Korea, the evaluation of the usefulness of the Xpert CD assay for screening for the 027 strain was limited. The Xpert CD assay provides great sensitivity in diagnosing toxigenic C. difficile infection. In addition, this method has excellent usability because it is simple and fast.     

检测艰难梭菌感染的五种方法比较

目的 比较分析用于美国医院临床微生物实验室的5种艰难梭菌感染检测方法 ,从中选择1种适于早期诊断艰难梭菌感染的灵敏、特异、简单、快速及经济的实验方案.方法 对174份临床送检的腹泻患者粪便标本同时用TGC、Premier Toxin A&B EIA(A/B-EIA)、C.Diff Quick Chek Complete(D-EIA)、BD GeneOhm Cdiff assay(BD-PCR)和实验室发展的PCR(LD-PCR)方法 进行艰难梭菌的检测.以金标准TGC法作为参比标准,评价各种检测方法 的敏感度、特异度、阳性预测值、阴性预测值.结果 TGC法检测174份腹泻患者大便标本,艰难梭菌感染的阳性率为13.8%(24/174).与TGC法比较,4种检测方法 的敏感度、特异度、阳性预测值、阴性预测值分别为:A/B-EIA 62.5%、99.3%、93.8%、94.3%;D-EIA 66.7%、98.7%、88.9%、94.9%;BD-PCR 83.3%、98.7%、90.9%、97.4%;LD-PCR 79.2%、93.3%、65.5%、96.6%.所有标本中,至少1种方法 检测为阳性的标本共计34份,其中15份标本5种方法 检测结果 均为阳性.D-EIA法检测结果 中,18份标本GDH及毒素A/B均为阳性,23份仅GDH阳性,133份GDH及毒素A/B均为阴性.18份D-EIA检测阳性标本全部与BD-PCR检测结果 一致,16份与TGC法检测结果 一致.24份TGC阳性标本中,有22份包括在41份GDH抗原呈阳性的标本中.D-EIA检测为假阴性的8份标本中,4份BD-PCR检测为阳性.根据本实验结果 ,D-EIA与BD-PCR相结合的二步法检测方案灵敏度、特异度、阳性预测值、阴性预测值分别为83.3%、98.7%、90.9%、97.4%.结论 从技术角度评价,以BD-PCR法为最优.结合D-EIA与BD-PCR的二步法检测方案,是对临床送检标本进行艰难梭菌感染检测的灵敏、特异、简便、快速、经济的检测方案.     

Comparison of five assays for detection of Clostridium difficile toxin

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended.     

溃疡性结肠炎患者粪便中艰难梭菌毒素基因A/B携带情况的研究

目的检测溃疡性结肠炎(UC)患者和健康体检者粪便标本中艰难梭菌毒素基因A/B携带情况,探讨艰难梭菌毒素基因携带情况与UC之间的关系。方法收集53例UC确诊患者(活动期32例,静止期21例)和45例健康体检者的粪便标本,提取粪便总DNA,采用荧光定量PCR方法分别检测粪便中艰难梭菌毒素A(cdtA)基因及艰难梭菌毒素B(cdtB)基因,扩增产物进行琼脂糖凝胶电泳,并对扩增产物进行测序验证。结果 UC组共检出7例cdtA、4例cdtB,其中UC活动期组5例cdtA、2例cdtB;静止期组2例cdtA、2例cdtB;健康对照组共检出5例cdtA、2例cdtB。UC组cdtA和cdtB检出率与健康对照组比较差异无统计学意义(P0.05);活动期组cdtA和cdtB检出率与静止期组比较差异无统计学意义(P0.05)。结论粪便中cdtA/B的携带情况与UC发病、疾病分期无明显相关性。     

快速检测粪便中艰难梭菌的方法研究

目的建立可用于粪便中艰难梭菌快速检测的环介导恒温扩增(LAMP)方法。方法针对艰难梭菌毒素A基因,设计引物。采用LAMP对艰难梭菌和其他腹泻干扰菌及不同浓度艰难梭菌的扩增检测,对其特异性和灵敏度进行评价。同时采用LAMP和荧光定量PCR对100例疑似艰难梭菌感染的临床腹泻患者粪便进行检测,对两种方法进行比较。结果 LAMP对艰难梭菌及6种腹泻干扰菌的检测,只针对艰难梭菌有扩增,显示了较好的特异性。LAMP最低检测限为10CFU/mL,灵敏度较高。对100例临床粪便标本LAMP检测结果与荧光定量PCR比较差异无统计学意义(P>0.05,χ2=0.1429)。结论本研究建立的粪便中艰难梭菌的LAMP快速检测方法特异性好、灵敏度较高、操作简便,适用于艰难梭菌的临床快速检测及现场检测。     

艰难梭菌实验室不同检测方法的比较

目的比较不同方法检测艰难梭菌(CD)的临床可行性。方法选取2016年疑似抗菌药物相关腹泻患者大便标本,采用酶联免疫吸附试验(ELISA)和鉴定培养基培养法进行检测,比较两种检测方法的灵敏性、特异性和一致性。结果 ELISA检出抗原阳性标本29例,其中毒素阳性25例,毒素阴性4例;鉴定培养基培养法检出可疑菌株29例,经质谱仪鉴定,其结果为CD28例,1例未检出种属;ELISA和鉴定培养基培养法的灵敏度和特异度分别为97%、100%和93%、95%,两者有极好的一致性(Kappa=0.92)。结论 ELISA具有快速、高效、省时、操作简单、判读容易等特点,可快速准确地筛检CD相关性腹泻病患;培养基培养法特异性好,可以快速得到感染株;二者联合使用可大大提高CD检出率,并对后期治疗有很大帮助。     

Detection of toxigenic Clostridium difficile in pediatric stool samples: an evaluation of Quik Check Complete Antigen assay, BD GeneOhm Cdiff PCR, and ProGastro Cd PCR assays

The performance of C. Diff Quik Chek Complete (QCC), BD GeneOhm Cdiff PCR (BD), and ProGastro Cd PCR (PG) assays was evaluated in detecting Clostridium difficile infection (CDI) in children using 200 frozen stool specimens. The results of the tests were compared to the toxigenic culture (TC) as ‘gold standard.’ The sensitivity, specificity, positive predictive value, and negative predictive value were as follows. QCC antigen (GDH + Toxin-A/B) = 70.8%, 97.4%, 89.5%, and 91.4%; BD PCR = 89.6%, 96.7%, 89.6%, and 96.7%; PG PCR = 100%, 93.4%, 82.8%, and 100%. Polymerase chain reaction (PCR) assays detected an additional 11 positives missed by TC, 7 of which were confirmed positive by an alternate tcdB gene PCR assay. However, retrospective clinical chart review indicated CDI in only 3 of the 11 patients in whom C. difficile was detected by PCR only. A 2-step algorithm utilizing QCC antigen test as a screening test followed by confirmation of GDH-positive and toxin-negative samples with either BD or PG PCR assay will provide rapid and accurate results for majority of the samples and reduce laboratory testing cost.     

三种婴幼儿A组轮状病毒检测方法的方法学评价

目的比较婴幼儿A组轮状病毒三种不同检测方法的优劣性。方法选择318例婴幼儿粪便或肛拭子标本分别行酶联免疫吸附(ELISA)、聚丙烯酰胺凝胶电泳(PAGE)、胶体金法(GICA)、荧光定量逆转录聚合酶链式反应(RT-PCR)检测A组轮状病毒,并以荧光定量RT-PCR为标准,对ELISA、PAGE、GICA进行方法学评价。结果四种检测方法阳性率:ELISA 55.0%、PAGE 43.1%,GICA 53.2%,RT-PCR 58.5%;以RT-PCR为对照,灵敏度(91.4%)、约登指数(0.876)、符合率(93.4%)、阴性预测值(95.5%)以ELISA最高,特异度(99.2%)、阳性预测值(99.3%)以PAGE最高。结论 ELISA、GICA检测A组轮状病毒操作简单,方法学评价指标良好,适合在基层医院推广应用。     

High prevalence of toxin A-negative toxin B-positive Clostridium difficile in hospitalized patients with gastrointestinal disease

The incidence of Clostridium difficile toxin A-negative toxin B-positive in hospitalized patients with severe gastrointestinal disease was evaluated. Of 530 stool specimens tested in parallel by two immunoassay tests, Tox A and Tox A/B (TechLab, Inc., Blacksburg, VA), 422 produced negative results on both tests. One hundred eight specimens (20.4%) tested positive by Tox A/B assay, and only 47 of them were also positive by Tox A. The 61 specimens with discrepant results were confirmed to be positive for toxin B by tissue culture cytotoxicity assay. Furthermore, 3 of the 422 specimens that were negative by enzyme immunoassay tested positive by cytotoxicity assay. The sensitivity and specificity of the Tox A/B test were 95.3% and 100%, respectively, with negative and positive predictive values of 99.3% and 100%, respectively, and a correlation rate of 99.4%. The high prevalence (56.5%) of specimens from symptomatic patients having detectable toxin B, but undetectable toxin A emphasizes the importance of using diagnostic tests that include toxin B. Furthermore, our data support the potential role of toxin B in the pathogenesis of toxigenic Clostridium difficile.