艰难梭菌毒素A&B测定在医院腹泻患者中的应用 FREE

目的探讨艰难梭菌毒素A&B测定在医院腹泻患者中的应用价值。方法采用VIDAS 艰难梭菌A&B毒素检测试剂对某院47例住院的腹泻患者粪便标本进行毒素检测,并结合细菌常规培养、临床资料及治疗进行综合分析。结果47例腹泻患者中,7例（14.89%）艰难梭菌毒素检测阳性, 此7例阳性结果均与临床诊断和/或治疗相符。结论该院艰难梭菌感染形势较为严峻,VIDAS 艰难梭菌A&B毒素检测可提供简单、准确、快速的检测结果,值得推广应用。     

艰难梭菌毒素蛋白表达与艰难梭菌感染分级关系的研究

目的分析医院艰难梭菌感染(clostridiumdifficile infection,CDI)患者的临床表现与实验室检查结果,初步研究艰难梭菌毒素蛋白表达与CDI严重程度分级的关系。方法收集2016年3月-2017年5月医院连续采集的860例腹泻便样本,采用Techlab Cdiff Quik Check Complete试剂盒和艰难梭菌毒素基因试剂盒分别检测艰难梭菌谷氨酸脱氢酶抗原(Clostridium difficile glutamate dehydrogenase antigen,GDH)、艰难梭菌毒素蛋白和艰难梭菌毒素基因,并对CDI患者临床资料和实验室结果进行统计分析。结果 860份腹泻样本中,GDH阳性99例占11.51%,其中艰难梭菌毒素蛋白阳性16例占16.16%,毒素基因阳性94例占94.95%;艰难梭菌毒素蛋白阳性患者的外周血白细胞数量、血清肌酐水平、腹泻次数、CDI分级指数及院内CDI患者概率均高于毒素蛋白阴性患者(P0.05)。结论艰难梭菌毒素蛋白阳性患者常伴随严重的艰难梭菌感染临床表现,推测艰难梭菌毒素蛋白可作为一项重度艰难梭菌感染的预测指标,为临床判断CDI分级及合理选择治疗方案提供及时客观的依据。     

江苏地区医院艰难梭菌流行病学研究

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。     

艰难梭菌在高风险科室医院感染流行趋势研究

目的探讨高风险科室住院患者中艰难梭菌感染或定植流行趋势,为艰难梭菌有效预防与控制措施的提出提供理论依据。方法收集2014年9月医院感染高风险科室住院的128例患者肛拭子标本,经厌氧培养,通过VIDAS荧光酶联免疫技术进行艰难梭菌毒素A/B检测,利用多重PCR技术进行艰难梭菌毒素基因检测,并对艰难梭菌阳性患者的临床病理特征进行分析。结果 128例患者中艰难梭菌培养阳性22例,阳性率17.19%;22株艰难梭菌中有21株为A/B毒素表型呈阳性,占95.45%,1株未检出A/B毒素;22例艰难梭菌培养阳性患者均为无症状携带者,其中90.91%的患者近期使用过抗菌药物。结论医院感染高风险科室的产毒艰难梭菌检出率较高,应注重该类患者的艰难梭菌监测和有效预防控制措施的落实。     

艰难梭菌分离培养与病原学分子特征研究

目的：建立艰难梭菌分离培养与鉴定的表型诊断技术,并对分离获得的艰难梭菌进行分子学特征研究。方法采集杭州某医院ICU住院患者肛拭标本,接种厌氧血琼脂平皿,置厌氧培养箱,可疑菌株采用厌氧菌鉴定试剂条鉴定;用VIDAS荧光酶联免疫技术检测A/B毒素;用PCR技术检测艰难梭菌毒素基因和核糖体分型。结果28例 ICU 患者检出艰难梭菌9株,检出率为32.14%。9株艰难梭菌中, A/B 毒素表型阳性7株（77.78%）,毒素基因检测均阳性。9株艰难梭菌可分为3种核糖型,其中R3型为优势株,占77.78%。结论杭州市ICU患者检出艰难梭菌的比例较高,均携带A/B毒素基因,且存在优势型别。     

临床患者粪便标本中艰难梭菌感染状况研究

目的 探讨医院艰难梭菌带菌及感染状况,分析艰难梭菌相关性腹泻(CDAD)的临床特征,为临床提高艰难梭菌分离率以及采取积极有效的防控措施奠定基础.方法随机收集20例要求做粪便培养的患者标本,采用艰难梭菌毒素A/B检测试剂盒进行毒素检测,对标本进行厌氧培养与分离,并进行耐氧实验,用API 20A生化条进行确证.结果 20例患者经艰难梭菌选择性培养基培养后,8株疑似菌落进行涂片镜检和耐氧实验,6株革兰阳性杆菌耐氧实验阳性,再经API 20A鉴定,4株为艰难梭菌,占所有患者的20%;毒素阳性1例,占5%.结论艰难梭菌的感染与某些内科基础性疾病的存在相关;水样的粪便标本更易于培养出艰难梭菌.     

昆明地区2018-2020年腹泻患者中艰难梭菌感染特征分析

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...     

多重聚合酶链反应方法检测艰难梭菌毒素基因研究

目的探讨同时检测艰难梭菌毒素A、B基因和二元毒素基因的多重聚合酶链反应(PCR)方法。方法收集2014年1-9月医院120例住院腹泻患者的粪便标本,用艰难梭菌选择性培养基CCFA分离培养艰难梭菌;采用法国生物梅里埃公司厌氧菌鉴定试剂盒鉴定;采用酶免夹心与终点荧光检测相结合技术(ELFA),检测艰难梭菌毒素A、B基因,并分析其毒素特征;将艰难梭菌纯培养出的菌落用多重PCR方法测定艰难梭菌的毒素A、B基因和二元毒素基因。结果 120例腹泻患者送检粪便标本中,分离培养出19株艰难梭菌,艰难梭菌分离率达15.8%;应用免疫学方法检测毒素检出率为52.63%;用多重PCR方法艰难梭菌A、B毒素基因检出率为94.7%;15例患者送检粪便标本检测艰难梭菌A、B毒素阳性中,10株分离培养并鉴定出艰难梭菌;105例患者送检粪便标本检测艰难梭菌A、B毒素阴性中,有9例分离培养并鉴定出艰难梭菌。结论利用多重PCR检测艰难梭菌毒素A、B基因和二元毒素基因,较传统的免疫学方法敏感性高,能够准确地区分艰难梭菌毒素基因的类型,多重PCR是一种快速、特异的一步筛选艰难梭菌毒素基因的方法。     

艰难梭菌感染现状与趋势分析

目的通过对某三甲医院进行艰难梭菌及毒素检测,分析目前艰难梭菌感染(CDI)的情况,为临床治疗CDI提供实验室支持依据。方法采用GeneXpert仪器检测粪便标本中艰难梭菌及其毒素基因。结果各科室及各年龄段间阳性率比较,差异均无统计学意义(P0.05)。148例艰难梭菌阳性患者中溃疡性结肠炎患者占比最高,为35.81%(53/148)。艰难梭菌阳性住院患者中住院时间4周患者占比最高,为41.91%(57/136),不同住院时间阳性率比较,差异有统计学意义(P0.01)。不同抗生素使用情况阳性率比较,差异无统计学意义(P0.05)。结论艰难梭菌感染形势较为严峻,临床对腹泻患者需加强艰难梭菌检测,并规范使用抗菌药物,预防和控制CDI发生。     

北京一所教学医院艰难梭菌感染分析

目的研究2014-2016年医院艰难梭菌感染情况。方法 2014年6月-2016年5月,临床标本的毒素检测采用酶联免疫法(EIA);随机选择69例非重复住院患者的毒素送检粪便同时进行EIA、GeneXpert PCR和艰难梭菌培养并比较,结合病历回顾研究培养和/或毒素阳性患者的感染及治疗。结果艰难梭菌培养和毒素检测同时送检的非重复患者640例,85例培养或/和毒素阳性,培养阳性率为8.91%,毒素阳性率为7.97%;培养阳性率>10%的科室有胃肠外科、肾内科、风湿免疫内科、消化内科;毒素阳性率>10%的科室有肾内科、骨肿瘤科、风湿免疫内科、胃肠外科、消化内科。标本送检占比56.88%的血液科,其培养、毒素阳性率仅分别为8.24%、5.49%;随机选择的69例非重复患者标本,有9例属于艰难梭菌感染,阳性率为13.04%,培养的灵敏度、特异度分别为66.67%、98.33%;EIA的灵敏度、特异度分别为44.44%、93.33%;PCR的灵敏度、特异度分别为100%、95.00%;相关因素分析,"腹泻时长≥48h"、"每日腹泻≥3次"在艰难梭菌感染组与非感染组中具有显著性差异;培养阳性或毒素阳性均有阳性提示意义,若伴随腹泻症状,应重点考虑艰难梭菌感染并予以万古霉素、甲硝唑和/或肠道菌群调节药物。结论应规范送检标本,选择合适检验方法,重视艰难梭菌感染,避免其成为我国的公共健康威胁。     

住院腹泻患者艰难梭菌检测与分析

目的通过对住院腹泻患者粪便标本中的艰难梭菌进行筛查和不同时期检出率的比较,了解某院腹泻患者艰难梭菌的感染情况。方法收集该院2009年2—12月和2011年4—7月住院腹泻患者粪便标本106份,进行厌氧培养和API鉴定,对培养鉴定获得的菌株应用聚合酶链反应（PCR）扩增法进行A、B毒素及二元毒素基因检测;酶联荧光免疫法检测毒素A/B。结果 106份标本中,厌氧培养艰难梭菌阳性16株（15.09%）。16株菌经PCR扩增,A、B毒素均阳性,二元毒素均阴性。直接毒素A/B检测阳性率为12.26%（13/106）,与厌氧培养阳性率比较,差异无统计学意义（χ^20.16,P〉0.05）。2009年2—12月和2011年4—7月两个时期的标本厌氧培养艰难梭菌阳性率分别为22.81%（13/57）、6.12%（3/49）,两者比较,差异有统计学意义（χ^25.73,P〈0.05）;毒素A/B检出率分别为17.54%（10/57）、6.12%（3/49）,差异无统计学意义（χ^23.18,P〉0.05）。艰难梭菌检测阳性患者住院期间均使用过头孢类、喹诺酮类、碳青霉烯类、广谱青霉素、克林霉素等其中一种或多种抗菌药物。结论该院艰难梭菌相关性腹泻比较严重,抗菌药物的使用是诱使艰难梭菌感染的重要因素。     

Predicting Clostridium difficile toxin in hospitalized patients with antibiotic-associated diarrhea.

OBJECTIVE: Clostridium difficile infection is implicated in 20%-30% of cases of antibiotic-associated diarrhea. Studying hospitalized patients who received antibiotic therapy and developed diarrhea, our objective was to compare the clinical characteristics of patients who developed C. difficile-associated diarrhea (CDAD) with those of patients with a negative result of a stool assay for C. difficile toxin. METHODS: A prospective study was done with a cohort of 217 hospitalized patients who had received antibiotics and developed diarrhea. Patients with CDAD were defined as patients who had diarrhea and a positive result for C. difficile toxin A/B by an enzyme immunoassay of stool. The variables that yielded a significant difference on univariate analysis between patients with a positive assay result and patients with a negative assay result were entered into a logistic regression model for prediction of C. difficile toxin.Setting. A 900-bed tertiary care medical center. RESULTS: Of 217 patients, 52 (24%) had a positive result of assay for C. difficile toxin A/B in their stool. The logistic regression model included impaired functional capacity, watery diarrhea, use of a proton pump inhibitor, use of a histamine receptor blocker, leukocytosis, and hypoalbuminemia. The area under the receiver operating characteristic curve for the model as a predictor of a positive result for the stool toxin assay was 0.896 (95% confidence interval, 0.661-1.000; P<.001), with 95% specificity and 68% sensitivity. CONCLUSIONS: Our results may help clinicians to predict the risk of CDAD in hospitalized patients with antibiotic-associated diarrhea, to guide careful, specific empirical therapy, and to direct early attention to infection control issues.     

中美艰难梭菌A-B+株毒力编码区域转录及B毒素表达的研究

目的研究中国艰难梭菌A-B+型分离株BJ08和美国艰难梭菌A-B+型暴发流行株US1的毒力编码区域PaLoc各基因转录及B毒素的表达,为预防和控制中国可能暴发的艰难梭菌感染提供理论支持。方法每隔3 h提取BJ08与US1艰难梭菌和培养上清,用荧光定量聚合酶链反应（PCR）法测定各时间段两菌株毒力编码区域PaLoc各基因（tcdA、tcdB、tcdC、tcdR、tcdE）的表达;用酶联免疫吸附试验（ELISA）法检测BJ08和US1各时间段的细胞和培养液上清中B毒素的含量。结果US1的生长速率稍快于BJ08,衰退速率显著快于BJ08（P＜0.05）;它们都不表达A毒素,但是都检测到tcdA基因的转录,而且tcdA转录没有明显差异。BJ08的tcdB、tcdC和tcdE基因的转录要比US1早3 h。B毒素在两种菌株的胞内和胞外合成或分泌没有明显差异。结论中国艰难梭菌A-B+型高分离株BJ08与美国艰难梭菌A-B+型暴发流行株US1相比,有相似的毒力表达或更强的基因调控能力,要警惕中国艰难梭菌BJ08暴发流行的可能。     

维生素B6、654-2、碳酸氢钠减轻阿奇霉素胃肠道反应效果的比较

目的：比较分析维生素B6、654-2、碳酸氢钠在减轻阿奇霉素胃肠道反应中的效果。方法：随机选取儿科呼吸道感染的150例患儿,分为A组、B组和C组,每组各50例,在使用阿奇霉素的同时,分别加用维生素B6、654-2、碳酸氢钠以减轻患儿胃肠道反应,比较用药后,三组胃肠道不良反应整体发生情况和发生程度。结果：在恶心、呕吐、腹泻及腹痛几种不良反应症状上,A组较之B组发生率明显降低,差异有统计学意义（P〈0.05）,B组和C组比较差异无统计学意义（P〉0.05）;在不良反应发生程度上,A组较之B组发生程度明显降低,差异有统计学意义（P〈0.05）,B组和C组比较差异无统计学意义（P〉0.05）。整体比较,A组不良反应情况发生率和发生程度均相对更低,差异有统计学意义（P〈0.05）。结论：在减轻阿奇霉素胃肠道反应中,维生素B6较之654-2和碳酸氢钠整体效果相对更佳,更值得临床推广应用。     

Rapid detection of toxigenic Clostridium difficile from stool samples by a nested PCR of toxin B gene

Toxigenic Clostridium difficile is the aetiologic agent of most cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present standard method for C. difficile diagnosis is a cytotoxicity assay, performed on human fibroblast cultures. It is time consuming and requires special facilities. A nested-PCR assay detecting toxin B gene within a few hours was designed. One hundred and two stool samples were collected during four months. All samples were processed for toxin B-PCR, cultured for C. difficile and tested for cytotoxicity. This approach achieved 99% concordance with the cytotoxic assay. The sensitivity and specificity for the new PCR assay were 96.3% and 100% respectively. The procedure described is easy to perform, does not require special equipment and has produced excellent results. It deserves serious consideration for routine clinical microbiology laboratory use.     

Active and passive immunization against Clostridium difficile diarrhea and colitis

Clostridium difficile, a gram-positive bacterium, is the major cause of hospital-acquired infectious diarrhea and colitis in industrialized nations. C. difficile colonization results from antibiotic administration and subsequent loss of protection provided by intestinal flora. C. difficile induced-colitis is caused by the release of two exotoxins, toxin A and B. Host factors including advanced age, pre-existing severe illness and weakened immune defenses predispose individuals to symptomatic infection. The generation of antibody responses to toxin A through natural exposure is associated with protection from disease. In addition, an inability to acquire immunity to toxin A puts individuals at risk for recurrent and/or severe disease. Immunological approaches for the management of this disease are being developed which could reduce the reliance on antibiotics for treatment and allow for re-establishment of the natural barrier provided by an intact commensal flora. An active vaccine and various immunotherapeutic strategies under evaluation may prove to be effective against severe or relapsing C. difficile infection.     

Prevalence of gastrointestinal disease caused by Clostridium difficile in a university hospital in Hungary

A one-year survey was undertaken to investigate the frequency of diarrhoea caused by Clostridium difficile among patients in a 1200-bed university hospital in Hungary. The VIDAS (bioMérieux) toxin A detection kit was used for screening specimens for the presence of C. difficile toxin. For all other diarrhoeal specimens selected according to special criteria, cytotoxin testing was used to determine the presence of 'free toxin' in the faeces. During the study period, a total of 945 diarrhoeal faecal samples were tested for the presence of C. difficile toxin. Of 375 requested samples, 58 (18.3%) were toxin-A positive. Of the 570 remaining faecal samples selected by the laboratory, 120 (21%) proved to be toxin positive. The results showed that patients from the surgical (33.3%), internal (24%) and haematological (12.8%) wards had the greatest frequency of diarrhoea attributable to C. difficile.     

Clinical features of Clostridium difficile-associated infections and molecular characterization of strains: results of a retrospective study, 2000-2004.

BACKGROUND: Recent outbreaks of severe cases of Clostridium difficile-associated diarrhea (CDAD) reported in North America, the United Kingdom, and The Netherlands have emphasized the importance of an ongoing epidemiological surveillance of CDAD. OBJECTIVE: To determine the epidemiology of CDAD over the years 2000-2004 and the rate of nosocomial transmission of C. difficile. DESIGN: Retrospective survey of inpatients with CDAD and molecular characterization of the strains isolated. SETTING: A 760-bed teaching hospital. METHODS: All CDAD cases diagnosed from January 1, 2000, to December 31, 2004, were reviewed. A CDAD case was defined as diarrhea in a hospitalized patient who had a stool specimen that tested positive for C. difficile cytotoxin or had a positive toxigenic culture result. CDAD was considered to be severe if a patient fulfilled at least 1 of the following 3 criteria: (1) presence of a fever (defined as temperature higher than 38.5 degrees C), abdominal pain, and leukocyte count greater than 10,000 cells/mm(3); (2) endoscopically or histologically proven pseudomembranous colitis; or (3) complications (defined as death with C. difficile infection as the primary or a contributing cause, toxic megacolon, perforation, toxic shock, and/or colectomy). CDAD was considered community-acquired if the diarrhea occurred in the patient within 72 hours after admission and if the patient had no history of hospitalization in the previous month; otherwise, CDAD was considered healthcare-associated. All the strains isolated were serogrouped and were characterized by toxinotyping and PCR ribotyping. Detection of toxin A, toxin B, and binary toxin was performed by PCR. RESULTS: One hundred fifty-one cases of CDAD were diagnosed; 147 clinical records could be reviewed, and 131 strains were studied. The overall incidence of CDAD was 1.1 cases per 1,000 patients admitted, but incidence rates were higher in 2003-2004, compared with 2000-2002 (P=.017). Diarrhea was community acquired in 28 patients (19%). For patients with healthcare-associated CDAD, transmission of the strain from patient to patient (ie, infection with a strain of the same serogroup and PCR ribotype as the strain isolated from another patient hospitalized in the same ward or in a linked ward in the previous 2 months) was demonstrated in 12 cases (10.1%). Eleven percent of strains were positive for binary toxin. Binary toxin-positive strains were associated with more-severe diarrhea (P=.01) and with a higher case-fatality rate (P=.03). A specific clone of C. difficile (serogroup H, PCR ribotype sa026) accounted for 35 (26.7%) of all the strains isolated, but this clone was found both in healthcare-associated and community-acquired cases. Three strains belonged to toxinotype III, but only 1 was related to the hypervirulent clone involved in recent outbreaks. CONCLUSION: The incidence of CDAD is low in our hospital, and cross-infection is limited. These results also suggest that strains with binary toxin might be more virulent.     

中国部分地区艰难梭菌PCR 核糖体分型及毒素基因多态性研究

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。