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1.
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0.7 pg/μ1,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-/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+/tcdB+。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。  相似文献   

2.
目的 通过对住院腹泻患者粪便中艰难梭菌的分离培养和毒力基因检测及分型,了解郴州市住院腹泻患者艰难梭菌感染状况。方法 收集2020年10-12月湘南学院附属医院、郴州市第一人民医院和郴州市第三人民医院住院腹泻患者粪便标本306例,厌氧培养法分离艰难梭菌菌株,采用荧光聚合酶链反应(RT-PCR)检测A、B毒素基因tcdA、tcdB及二元毒素基因cdtA、cdtB,并对分离的艰难梭菌菌株进行多位点序列分型(MLST)。结果 郴州市住院腹泻患者产毒艰难梭菌核酸阳性率为8.17%(25/306),>60岁患者感染风险更高(χ2=5.499,P=0.019); 306份标本中分离出17株艰难梭菌,粪便标本荧光PCR检测有25份阳性,二者比较有统计学差异(P=0.008),毒力基因检测均为tcdA2=5.499,P=0.019); 306份标本中分离出17株艰难梭菌,粪便标本荧光PCR检测有25份阳性,二者比较有统计学差异(P=0.008),毒力基因检测均为tcdA+tcdB+tcdB+,未检出二元毒素;选取7株艰难梭菌进行MLST分型,分出5个ST型,ST54型(3株)、ST129型(1株)、ST98型(1株)、ST53(1株)和ST631(1株)。结论 郴州市住院腹泻患者艰难梭菌感染率较低,感染毒素类型为tcdA+,未检出二元毒素;选取7株艰难梭菌进行MLST分型,分出5个ST型,ST54型(3株)、ST129型(1株)、ST98型(1株)、ST53(1株)和ST631(1株)。结论 郴州市住院腹泻患者艰难梭菌感染率较低,感染毒素类型为tcdA+tcdB+tcdB+,呈多种ST流行。  相似文献   

3.
目的对艰难拟梭菌630(Cd630)菌株和致病决定区基因座(PaLoc)敲除突变株(ΔPaLoc)进行转录组测序和分析, 获得Cd630野生型菌株和ΔPaLoc基因的差异性表达谱, 并测定野生型Cd630菌株和ΔPaLoc突变株的细胞毒力, 为构建艰难拟梭菌全菌减毒疫苗奠定基础。方法对Cd630菌株和ΔPaLoc敲除突变株进行转录组测序, 接着对差异表达基因进行筛选, 随后对差异基因进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析。最后在非洲绿猴肾细胞(Vero)和人克隆结肠腺癌细胞株(Caco-2)中进行Cd630野生型菌株和ΔPaLoc敲除突变菌株的细胞毒性验证实验。结果转录组数据表明ΔPaLoc突变株毒素基因tcdA、tcdB未转录。CD63036010、CD630020910、CD63002080和cel等基因分别上调17.92、11.40、8.93和7.55倍;编码高丝氨酸脱氢酶的hom2基因、孢子形成蛋白基因CD63015810、锌结合脱氢酶基因CD6302<...  相似文献   

4.
目的了解中国5个城市艰难梭菌携带毒素基因特征,为中国艰难梭菌感染诊治提供试验依据。方法收集2013年6月-2015年6月中国上海、杭州、宁波、郑州和南京5个城市分离自住院患者和环境表面的艰难梭菌,采用多重PCR的试验方法,对所有菌株进行tcdA/tcdB/cdtA/cdtB4个毒素基因检测,分析所携带的毒素基因特征。结果中国5个城市共成功复苏出艰难梭菌408株,371株来自患者的艰难梭菌经多重PCR检测,tcdA+,tcdB+基因阳性299株,阳性率为80.59%,tcdA-、tcdB-阳性44株,阳性率为11.86%;37株来自环境的艰难梭菌经多重PCR检测,tcdA-、tcdB-阳性15株,阳性率40.54%;tcdA-、tcdB+阳性9株,阳性率24.32%;中国部分城市的艰难梭菌携带毒素基因以A+B+毒素基因为主,产双性毒素基因占1.08%。结论研究所揭示的艰难梭菌毒素基因特征应引起临床医师高度关注,尤其是在选择以检测毒素为主的诊断技术时,应考虑不同方法检测毒素基因的能力。  相似文献   

5.
目的通过对江苏地区两所不同规模医院的高危人群进行艰难梭菌筛查,以了解医院内艰难梭菌的感染和定植,从而为预防艰难梭菌的流行提供参考数据。方法 2015年11月对A医院和B医院高危科室的所有住院患者进行肛门拭子采样,共采集122份标本,所有的标本经厌氧培养,VIDAS荧光酶联免疫技术检测艰难梭菌毒素A/B,同时利用多重PCR检测技术测定其毒素基因。结果 A医院接受检测的104例患者中艰难梭菌培养阳性29例,阳性率为27.88%,其中感染10例、定植19例;B医院的18例患者中艰难梭菌培养阳性9例,阳性率为50.00%,其中感染1例、定植8例;38株艰难梭菌中有25株艰难梭菌A/B毒素检测阳性;多重PCR结果显示,25株为tcdA和tcdB双阳性,4株tcdA阳性tcdB阴性,9株tcdA和tcdB双阴性,38株艰难梭菌均未检测到二元毒素基因。结论江苏地区不同规模医院的高危科室均存在艰难梭菌感染和传播的风险,对高危人群的腹泻患者进行艰难梭菌的检测,有利于病情的确诊和尽快采取有效的治疗。  相似文献   

6.
目的探讨杭州地区医院获得性腹泻患者艰难梭菌的克隆分布及药敏表型特征。方法对2013年1月-2016年12月间浙江大学医学院附属第一医院及邵逸夫医院医院获得性腹泻患者的粪便标本进行艰难梭菌(Clostridium difficile,CD)培养初筛,初筛阳性菌株通过PCR扩增及测序分析16-23SrRNA基因及毒素基因(tcdA及tcdB基因),以明确CD菌种以及是否为产毒菌株;以MLST技术对产毒CD菌株进行同源性分析;并对CD菌株进行药敏敏感性试验。结果共培养CD菌株61株,其中54株为产毒菌株,tcdA及tcdB基因阳性菌株数分别为1株(A+B-)和53株(A-B+),未发现毒素基因双阳性菌株;产毒菌株来源于11个不同ST型别,其中7个ST型别为已知的;各ST型别间亲缘关系较远,ST54型别是最主要流行克隆型别占38.9%(21/54),其次是ST2、ST35和ST37,各占比13.0%(7/54);万古霉素及甲硝唑为敏感性最高的药物,未发现耐药菌株,其次是左氧氟沙星、四环素和环丙沙星,耐药率分别为16.6%、31.4%和38.8%,克林霉素及夫西地酸耐药率最高均为96.3%。结论毒素表型A-B+的ST54型艰难梭菌为本地区主要流行克隆,万古霉素和甲硝唑是有效的治疗CDI的抗菌药物。  相似文献   

7.
目的对2018-2020年昆明地区腹泻患者中艰难梭菌感染特征进行分析, 为后续监测和防治提供数据支持。方法收集2018-2020年云南省4家哨点医院腹泻患者粪便标本共388份, 使用实时荧光定量PCR方法进行艰难梭菌粪便毒素基因检测, 对结果阳性的粪便标本进行菌株的分离, 用基质辅助激光解析电离飞行时间质谱鉴定菌株。提取分离菌株的基因组DNA进行多位点序列分型(MLST)。分析毒素阳性和菌株分离阳性与患者的临床特征以及艰难梭菌阳性与其他病原共感染的情况。结果 388份粪便标本中, 艰难梭菌内参tpi基因阳性标本47份, 总阳性率为12.11%。其中, 非产毒艰难梭菌4份(8.51%), 产毒艰难梭菌43份(91.49%)。47份阳性标本分离得到18株艰难梭菌, 阳性标本的分离率为38.30%。其中tcdA、tcdB、tcdC、tcdR和tcdE基因均为阳性的菌株14株。18株艰难梭菌的二元毒素均为阴性。所有分离菌株的MLST结果共形成10种序列型(ST), 其中ST37型5株(27.78%);ST129、ST3、ST54和ST2型各2株;ST35、ST532、ST48、ST27和ST3...  相似文献   

8.
目的调查胃肠外科病区分离艰难梭菌毒素基因情况及耐药性,为治疗提供依据。方法采集衢州市中心医院235例腹泻患者粪便标本,进行选择性厌氧培养及鉴定,并对艰难梭菌阳性标本进行毒力基因检测,采用E-test检测药物敏感性。结果艰难梭菌检出率为8.51%(20/235),其中11株为A-B+型,5株为A+B+型,4株为A-B-型;未检出二元毒素(cdt A、cdt B)。所有菌株对甲硝唑和万古霉素均敏感,对克林霉素、美罗培南、红霉素和左氧氟沙星的敏感度分别为80%、15%、80%和25%。16株对克林霉素耐药的艰难梭菌中,erm B阳性菌株检出率为81.25%(13/16)。结论本院胃肠外科病区流行的艰难梭菌产毒株以A-B+型菌株为主,其对克林霉素、红霉素耐药严重,甲硝唑和万古霉素可作为艰难梭菌相关腹泻的治疗首选药物。  相似文献   

9.
目的了解中国部分地区艰难梭菌聚合酶链反应(PCR) 核糖体型别分布及其A、B毒素基因多态性,为建立适宜中国的艰难梭菌分子检测和分子分型技术提供基础数据,同时在基因水平上为艰难梭菌感染导致的复杂临床表现提供依据。方法对中国3个城市(北京、广州、济南)分离的64株艰难梭菌临床株进行PCR 核糖体分型,并对不同型别的26株代表菌株的A、B毒素基因进行扩增测序。结果64株艰难梭菌中,毒素基因型以A+B+型(45株,70.31%)为主,A-B+型19株(29.69%)。共存在9种PCR 核糖体型别,以017型(21株,32.81%)为主要型别,其次为001型(13株,20.31%)、012型(11株,17.19%)。A-B+菌株中,14株(73.68%)是017型,1株是001型。A、B毒素基因呈现一定的多态性,其中有7种A毒素序列型别(TSTA),6种B毒素序列型别(TSTB),8种A、B毒素序列型别组合(TSTG)。结论我国部分地区的艰难梭菌可能以PCR 核糖体017型为主,A、B毒素基因在菌株间存在多态性,且核糖体型别与毒素基因多态性间存在相对应的关联。应进一步扩大菌株数量和范围,探寻适合我国的分子检测和分子分型方法,从而帮助医院更好地预防和控制艰难梭菌感染。  相似文献   

10.
目的 研究神经外科住院患者定植及感染产毒艰难梭菌的分子流行病学特征。方法 采用前瞻性研究方法,选取2018年11月-2019年4月所有徐州医科大学附属医院神经外科新入院的成年患者161例为研究对象。在入院后48 h内、入院后每周及发生腹泻时分别采集粪便标本,本研究的主要临床结局是发生艰难梭菌感染(CDI),未发生CDI的患者随访至出院或死亡。对采集的粪便标本进行艰难梭菌培养及毒素基因检测,对所有的产毒艰难梭菌进行多位点序列分型。结果 从41名患者的粪便中培养分离出产毒艰难梭菌共计50株,其中,30株菌株tcdA及tcdB阳性,占60.00%;17株菌株仅tcdB阳性,占34.00%;3株菌株tcdA、tcdB及cdtA-cdtB均阳性,占6.00%。7名患者发生CDI,CDI发病率为4.35%,其中6名患者在入院时定植产毒艰难梭菌。将分离的50株产毒艰难梭菌进行多位点序列分型,共分析出14个ST分型,其中3株二元毒素阳性菌株均为ST5型。研究期间,患者4与患者31、患者13与患者19检测到相同的ST型,且居住过同一病房或床位。结论 本研究未分离到高毒力菌株ST1/RT027型或ST11/RT078型。研究期间存在产毒艰难梭菌定植患者传播艰难梭菌的可能,但有待进一步采用全基因组测序分型进行验证。  相似文献   

11.
目的 建立以毒素基因A/B为靶基因的产毒艰难梭菌的快速定量检测方法.方法 通过设计艰难梭菌毒素A/B基因的特异引物及探针,建立标准产毒菌株DNA(ng)含量与Ct值的标准曲线.结果 该方法仅对产毒艰难梭菌进行特异性扩增,11种其他常见的致病菌及非产毒艰难梭菌均不能扩增; tcdA和tcdB基因扩增标准曲线线性关系R值分别为0.9975、0.9984,检测低限均为2.5×10-3ng.结论 该研究建立的方法具有快速、灵敏、特异性高等优点,可用于艰难梭菌毒素基因的定量检测.  相似文献   

12.
BACKGROUND: Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Between August 2003 and January 2004, we experienced an increase in the incidence of C. difficile-associated disease. We describe the investigation into and management of the outbreak in this article. METHODS: A total of 73 consecutive patients with nosocomial C. difficile-associated diarrhea were identified. C. difficile isolates were characterized using toxin-specific enzyme immunoassays, a tissue-culture fibroblast cytotoxicity assay, polymerase chain reaction (PCR), and antimicrobial susceptibility tests. Rates of recurrence and of C. difficile colitis were recorded. Changes in antibiotic use and infection control policies were documented. RESULTS: The incidence of C. difficile-associated diarrhea peaked at 21 cases per 1,000 patient admissions. Of the C. difficile isolates recovered, 85 (95%) were identical toxin A-negative and toxin B-positive strains, corresponding to toxinotype VIII and PCR ribotype 017. All clonal isolates were resistant to multiple antibiotics, including ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin (minimum inhibitory concentrations [MICs] of greater than 32 micro g/mL) and erythromycin, clarithromycin, and clindamycin (MICs of greater than 256 micro g/mL). Recurrent C. difficile-associated disease occurred in 26 (36%) of the patients. At least 10 (14%) of the patients developed C. difficile colitis. Additional infection control measures introduced included the use of ward memos, a hand-hygiene awareness campaign, increased environmental cleaning, attention to prescribing practices for antibiotics, increased awareness of diarrheal illness, and early isolation of affected patients. Total use of fluoroquinolones did not change throughout the study period. Despite persistence of this toxin-variant strain, the incidence of C. difficile-associated disease in our institution decreased to fewer than 5 cases per 1,000 admissions. CONCLUSIONS: We report on the emergence of a fluoroquinolone- and clindamycin-resistant, toxin A-negative, and toxin B-positive strain of C. difficile associated with an outbreak of C. difficile-associated disease in our institution during a 6-month period. We found that careful attention to improvement of infection control interventions was the most important means of controlling this nosocomial pathogen.  相似文献   

13.
Outbreaks due to Clostridium difficile polymerase chain reaction (PCR) ribotype 027, toxinotype III, were detected in 7 hospitals in the Netherlands from April 2005 to February 2006. One hospital experienced at the same time a second outbreak due to a toxin A-negative C. difficile PCR ribotype 017 toxinotype VIII strain. The outbreaks are difficult to control.  相似文献   

14.
Two hundred and forty-eight patients from shared oncology and general medical wards were prospectively studied over a 6-month period for carriage of Clostridium difficile during an outbreak of clinical disease with an epidemic strain of the organism. Risk factors for infection were assessed. Acute leukaemia and/or its treatment were identified as significantly increasing the risk of infection. The relationship between the type of C. difficile isolated (as defined by a typing system based on the incorporation of [35S]methionine into bacterial proteins followed by gel electrophoresis), the presence of faecal toxins A and B and clinical symptoms were analysed. Carriage of the epidemic strain, type X, had a significant association with symptoms amongst oncology patients, with two thirds of these patients having detectable faecal toxin A and one third detectable faecal toxin B. During an outbreak of C. difficile-associated disease, typing the organism and assaying for both faecal toxins in symptomatic patients may be of benefit in determining which patients require specific, urgent treatment.  相似文献   

15.
Antibodies targeting the Clostridium difficile toxin A and toxin B confer protective immunity to C. difficile associated disease in animal models and provided protection against recurrent C. difficile disease in human subjects. These antibodies are directed against the receptor binding domains (RBD) located in the carboxy-terminal portion of both toxins and inhibit binding of the toxins to their receptors. We have constructed a recombinant fusion protein containing portions of the RBD from both toxin A and toxin B and expressed it in Escherichia coli. The fusion protein induced high levels of serum antibodies to both toxins A and B capable of neutralizing toxin activity both in vitro and in vivo. In a hamster C. difficile infection model, immunization with the fusion protein reduced disease severity and conferred significant protection against a lethal dose of C. difficile spores. Our studies demonstrate the potential of the fusion protein as a vaccine that could provide protection from C. difficile disease in humans.  相似文献   

16.
We compared 30-day case-fatality rates for patients infected with Clostridium difficile possessing genes for toxins A and B without binary toxin (n = 212) with rates for patients infected with C. difficile possessing genes for A, B, and binary toxin. The latter group comprised patients infected with strains of PCR ribotype 027 (CD027, n = 193) or non-027 (CD non-027, n = 72). Patients with binary toxin had higher case-fatality rates than patients without binary toxin, in univariate analysis (relative risk [RR] 1.8, 95% confidence interval [CI] 1.2-2.7) and multivariate analysis after adjustment for age, sex, and geographic region (RR 1.6, 95% CI 1.0-2.4). Similar case-fatality rates (27.8%, 28.0%) were observed for patients infected with CD027 or CD non-027. Binary toxin either is a marker for more virulent C. difficile strains or contributes directly to strain virulence. Efforts to control C. difficile infection should target all virulent strains irrespective of PCR ribotype.  相似文献   

17.
Data from the surveillance system of general outbreaks of infectious intestinal disease and from laboratory reports collated by the Communicable Disease Surveillance Centre (CDSC) and requests for outbreak investigation by the PHLS Anaerobe Reference Unit were used to evaluate the current epidemiology of Clostridium difficile infection in England and Wales. Between January 1992 and December 1996, CDSC received 10,220 laboratory reports of C difficile isolation from patient's faeces and 26,873 of toxin in faeces. Over 75% of all reports were of people aged 64 years and over. The surveillance system captured a minimum data set on 694 hospital outbreaks of infectious intestinal disease. C. difficile was responsible for 109 (15%) outbreaks affecting 1625 people, of whom 1152 were found to have a C. difficile toxin producing strain. The median duration of outbreaks was 11 days. Fingerprinting by Pyrolysis Mass Spectrometry (PMS) was performed by the PHLS Anaerobe Reference Unit in 60 outbreaks, and typing by Polymerase Chain Reaction ribotyping (PCR) in 14.  相似文献   

18.
OBJECTIVE: To describe a nosocomial outbreak of Clostridium difficile-associated disease (CDAD). DESIGN: A traditional outbreak investigation. SETTING: Geriatric department of a tertiary care teaching hospital from March through April 2003. METHODS: The outbreak was detected by the C. difficile surveillance program of the infection control unit. CDAD was diagnosed by stool culture and fecal toxin A detection with a qualitative rapid immunoassay. Isolates of C. difficile were serotyped and genotyped using pulsed-field gel electrophoresis. RESULTS: The incidence of CDAD increased from 27 cases per 100,000 patient-days in the 6-month period before the outbreak to 99 cases per 100,000 patient-days during the outbreak. This outbreak involved 21 of 92 patients in 4 geriatric wards, which were located at 2 geographically distinct sites and staffed by the same medical team. The mean age of patients was 83 years (range, 71-100 years). Five (24%) of the 21 patients had community-acquired diarrhea, and secondary hospital transmission resulted in 3 clusters involving 16 patients. Serotyping and genotyping were performed on isolates in stool specimens from 19 different patients; 16 of these isolates were serotype A1, whereas 3 displayed profiles different from the outbreak strain. Management of this outbreak consisted in reinforcement of contact isolation precautions for patients with diarrhea, cohorting of infected patients in the same ward, and promotion of hand hygiene. Relapses occurred in 6 (29%) of 21 patients. CONCLUSION: Control of this rapidly developing outbreak of CDAD was obtained with early implementation of cohorting and ward closure and reinforcement of environmental disinfection, hand hygiene, and enteric isolation precautions.  相似文献   

19.
目的 了解2008年北京地区肠道病毒71型(EV71)分离株的全基因序列特点.方法 收集北京地区手足口病患儿的咽拭子样本12份,对其中1份样品08YM-3,经Vero细胞分离培养,提取病毒RNA.利用RT-PCR和5'、3'RACE扩增EV71全长基因.对PCR产物克隆和测序.利用DNAStar软件包的MegAlign进行核苷酸序列分析,构建系统进化树.结果 分离的EV71病毒株经过扩增后克隆得到3个涵盖EV71全基因组的阳性质粒,测序后拼接为EV71全基因组,命名为BJ08株.BJ08的5'非编码区(UTR)、P1、P2、P3、3'非编码区(UTR)和全基因组的核苷酸与C4亚型的参考序列同源性均最高,分别为95.6%~96.7%、88.3%~96.1%、78.1%~94.0%、90.8%~94.6%、85.9%~94.1%和90.9%~93.9%.BJ08株与其他亚型各区段的核酸同源性均低于90%.在全基因序列与VP1区构建的系统进化树中,BJ08株均与C4亚型在同一分支.BJ08株与C4亚型参考株VP1区的6个亚型相关氨基酸序列一致,VP1抗原性表位(92~107aa)无变异.结论 北京BJ08 EV71病毒分离株为C4业型.  相似文献   

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