【作者】 王浦；

【导师】 姜泊； 陈烨；

【作者基本信息】 南方医科大学 ， 内科学， 2014， 博士

【摘要】 艰难梭菌(Clostridium difficile, C. difficile)是一种严格厌氧生长的革兰阳性梭状芽孢杆菌,为人类肠道中的正常菌群。艰难梭菌感染(Clostridium difficile infection, CDI)是指艰难梭菌产毒菌株所致的感染,具备相应临床症状(通常是腹泻),并且实验室检测到艰难梭菌毒素或者分离出产毒菌株(粪便培养产毒艰难梭菌阳性,细胞培养细胞毒试验阳性,结肠镜检或组织病理学检查显示假膜性肠炎)即可诊断。1978以来,人们逐渐认识到艰难梭菌与抗生素相关性腹泻(Antibacterial-associated diarrhea, AAD)有关,目前认为25%的AAD由艰难梭菌引发。CDI可导致患者住院时间延长,基础疾病严重性和死亡率增加及医疗负担加重。发病机制艰难梭菌主要在人与人之间通过粪口途径传播并定植于肠道。正常肠道菌群可有效抑制定植艰难梭菌的繁殖；抗生素等因素破坏肠道菌群平衡后可导致该菌过度生长,释放毒素A和毒素B作用于肠道屏障。毒素A为肠毒素,通过改变细胞内的肌动蛋白直接作用于结肠细胞,引起炎症反应。毒素B是细胞毒素,可导致细胞内染色质浓缩,诱使肠细胞凋亡。此外,艰难梭菌与索氏梭菌有交叉抗原,可被毒素中和,直接损伤肠壁细胞,造成伪膜性肠炎。临床表现大部分患者感染艰难梭菌后无临床症状,有临床症状者可表现为轻至重度水样腹泻、爆发性肠炎甚至中毒性巨结肠、肠梗阻及伪膜性肠炎(Pseudomembranous colitis, PMC)。约半数患者腹泻出现于应用抗生素过程中或停用抗生素后短期内,偶有停药数月后发病者。重症患者可出现消耗性腹泻(次数可达20～30次厌),同时伴有发热、恶心、厌食、腹胀、腹痛等全身或局部症状。此外,有系统性疾病的患者常发生CDI。辅助检查CDI患者CT扫描可表现为结肠壁增厚。腹部X线检查可见麻痹性肠梗阻及“拇指纹”症。内镜及活检可见结肠特征性改变,即散在黄色或白色椭圆形斑块,边界清楚,与黏膜粘连牢固,冲洗不易脱落。斑块中央出现点状灰白色或棕褐色伪膜,继而融合并隆起成数毫米至1厘米大小斑块,周围常绕以红晕。病灶间黏膜常无明显炎症,重者可融合成片状甚至成管型。实验室检查可发现白蛋白水平下降,白细胞、血尿素氮、肌酐、血清转铁蛋白水平升高,粪便菌群失调及粪便白细胞等。诊断及治疗根据广谱抗生素应用史、严重腹泻或便血以及结肠镜下特征性表现(水肿、充血、伪膜和非特异性小溃疡或糜烂),可作出CDI临床诊断,确诊有赖于细菌培养和毒素检测结果。CDI实验室诊断多基于腹泻粪便中检测其毒素A或者B。研究发现艰难梭菌产毒菌株大部分同时产生A毒素及B毒素,也有少部分产毒菌株仅仅产生B毒素。因此,实验室及临床常常通过检测B毒素来确诊CDI。目前实验室检测手段多种多样,细胞培养细胞毒试验(Cell culture cytotoxicity assay and toxigenic culture)是检测艰难梭菌产毒菌株的标准方法,但是标准检测方法耗时费力,并且需要专业的研究人员。因此,一些微生物实验室使用酶联免疫分析法,谷氨酸脱氢酶(Glutamate dehydro genase,GDH)抗原测试,以及PCR技术等快速检测粪便中艰难梭菌。甲硝唑为目前治疗CDI的首选药物,口服治疗7-14天(200-250mg,4次/天)。其次为万古霉素(0.125g,4次厌),经甲硝唑治疗无效或病情严重危及生命者服用该药7-14天。对于肠梗阻严重患者,药物可通过胃管、肛管注入结肠,必要时可考虑肠切除。其他治疗药物有杆菌肽(25500U,4次/天)、替考拉宁(200mg,1次厌)、夫西地酸(500mg,1次厌)、考来烯胺(4g,3-4次/天)。国外非达霉素治疗CDI已经进入临床应用,且实践表明非达霉素经济实惠,对万古霉素治疗失败的CDI也有很好的治疗效果,是目前治疗CDI最有效的药物。艰难梭菌相关性腹泻(Clostridium difficile associated diarrhea,CDAD)多在治愈后7-14天复发,尚无针对复发的标准治疗方案。粪便艰难梭菌毒素阳性患者增加10-14天疗程的甲硝唑或万古霉素(125mg,给药间隔时间逐渐延长),可降低复发率。多次复发者应考虑在上述治疗基础上加用其他疗法,如甲硝唑与万古霉素联合治疗、考来烯胺联合万古霉素综合治疗、益生菌生物治疗和静脉滴注人免疫球蛋白等。近年来,对反复发作的难治性CDI,粪便移植法是研究热点。从健康的供者(经严格的传染性病原体筛查)选择粪便,通过灌肠,鼻饲以及内镜植入等方式,种植到CDI反复发作患者结肠中,以恢复患者肠道菌群稳态。大量临床研究表明该法对反复发作性及难治性CDI具有很好的治疗效果,是目前认为最有前途的治疗方法。流行病学变化CDAD相关研究一直是热点。过去20年,艰难梭菌的流行病学发生了很大变化。随着广谱抗菌药物的广泛使用,在全球范围内,CDAD发病率不断提高。美国密歇根的一项研究表明,2002年到20086年,CDI发生率增加了一倍。近些年艰难梭菌来出现暴发流行,患者病死率及感染复发率升高,引起医学界的极大重视。CDI发生率增加及暴发流行的原因可能与高毒力菌株BI/NAP1/027(限制性内切酶分型BI,脉冲场凝胶电泳分型NAP1, PCR-核糖体分型027)有关,该027菌株毒素调节基因tcdC变异,产生二元毒素(Binary toxin)以及较传统产毒菌株15-20倍的毒素A和B。该菌株对非达霉素反应率低,并且具有高复发率、喹诺酮类耐药,死亡率高(高达7%)等特点。自北美首发以来,欧美时常出现BI/NAP1/027暴发流行。澳大利亚以及亚洲的日本、韩国、新加坡及中国香港等地也有该菌株的报道。此外,过去认为是低危险因素的群体,如围产期妇女和儿童等,以及社区获得性艰难梭菌的感染率也呈增加趋势。研究背景CDI在在欧美广泛报道并监测、研究。亚洲对艰难梭菌的研究相对较少,主要集中于一些发达国家或地区,如日本、韩国、台湾等地。中国大陆各级医疗机构中,艰难梭菌并不是一个需申报的感染。此外,限于技术及重视程度等原因,目前只有北京上海等部分大城市的医院或者研究机构有检测及研究。总的来说,艰难梭菌在中国流行情况目前尚不清楚,基础研究也较为薄弱。研究目的和意义据我们近些年的临床及科研经验表明,国内CDI可能呈增加趋势。究其原因,可能与近些年中国抗生素广泛使用及老龄化社会来临有关,也可能与受关注程度的增加以及内镜等检测技术的普及有关。鉴于此,从上世纪90年代起,我们机构对CDAD做了大量的研究工作。本次研究目的主要在：①了解南方医院艰难梭菌的流行情况,分析国内CDI致病的危险因素并探寻能反映CDI病情变化的替代标志物(Surrogate marker),为合理使用抗生素,预防CDI,提高诊疗效率并控制CDI流行提供参考。②分离鉴定艰难梭菌临床菌株,并对其进行毒素分类(Toxinotyping)及核糖分型(Ribo typing),建立艰难梭菌菌种库,为进一步研究艰难梭菌生物学特性、致病性和致病机制、研制有效疫苗打下基础。③探索艰难梭菌快速有效的分子生物学检测技术,以期快速便捷准确的用于临床筛查,检测并鉴定艰难梭菌,达到提高CDI诊疗效率,减缓细菌耐药,控制院内感染播散,减轻医疗压力的目的。研究内容研究对象：收集2009年12月-2013年12月南方医院住院、门诊以及部分社区患者共4456份腹泻粪便(布里斯托分类5-7级)标本进行艰难梭菌分析。排除技术因素等原因丢弃的标本,4132份为适合CDI研究的粪便标本。技术方法：①收集2009年12月～2012年5月间南方医院住院、门诊及部分社区腹泻患者新鲜粪便(排出体外半小时内)标本,提取粪便全基因组DNA,以传统PCR技术检测艰难梭菌B毒素基因tcdB。收集患者临床数据进行分析,了解此期间南方医院CDI的流行病学变化情况,并采用单因素及其基础上的多因素logistic回归分析患者CDI的独立危险因素及探索能反映CDI病情变化的替代标志物。②4132份腹泻粪便中一共有适合培养要求标本1962例。对其进行细胞毒测试(Cytotoxicity assay, CTA),并同时接种CCFA培养基厌氧培养,分离纯化典型菌落,革兰氏染色并1000倍油镜观察。再取典型菌落进行乳酸脱氢酶(Glutamate dehydrogenase, GDH)检测及生化鉴定。提取成功鉴定为艰难梭菌的细菌DNA检测艰难梭菌A、B毒素,二元毒素及核糖分型。最后,所有经鉴定的艰难梭菌菌株统一编号,-80℃保存,并收集患者临床信息,建立南方医院艰难梭菌菌种库。③收集2012年7月至2012年12月之间南方医院临床成人(>18岁)腹泻新鲜粪便标本140例(排除重复标本)。分别采用“金标准”——细胞培养细胞毒试验进行检测及Cepheid GeneXpert(?) Dx (Cepheid, GX-XVI)系统检测(该仪器可以特异性的检测艰难梭菌tcdB基因,二元毒素cdt基因以及tcdC基因nt117位点缺失)。以McNemar及Kappa系数检验比较Cepheid Xpert C.difficile检测粪便艰难梭菌与“金标准”检测结果,了解两种检测技术的阳性率及一致性吻合情况,并采用MedCalc软件评价Xpert C. difficile检测艰难梭菌的灵敏度,特异度等稳定性及可靠性指标,以及约登指数,阳性似然比及阴性似然比等仪器筛查指标,探讨其临床应用可能性及优势。结果2009年12月到2012年5月之间南方医院住院、门诊及部分社区粪便标本共3855份,其中177例重复标本,18份因粪便量太少及不满足新鲜粪便标本要求被排除,最后一共3660份标本纳入研究。在这3660份新鲜标本(对应3660例患者)中,平均年龄为54.9±15.8岁,男性患者73.3%。其中住院患者为3324例(90.8%),重症监护中心患者为24例(0.7%)。对3660份腹泻标本进行艰难梭菌B毒素基因(tcdB)检测,并进行感染危险因素及替代标志物分析。3660份腹泻粪便标本艰难梭菌tcdB基因检出率为15.6%,此期间南方医院CDI率基本呈稳定趋势。多因素logistic回归分析结果显示患者性别、年龄、是否有住院、Horn’s index值、恶性肿瘤、炎性肠病、以及6周内灌肠、皮质醇类药物、抗生素使用、胃肠插管及先前发生过CDI都是CDI的独立危险因素。发热、尿素氮、血肌酐、C-反映蛋白和白蛋白可作为判断CDI的独立标志物。在这些独立危险因素及标志物中,以胃肠插管(P=0.000,OR=5.759),灌肠(P=0.000,OR=3.976),恶性肿瘤(P=0.013,OR=1.668),炎性肠病(P=0.019,OR=1.894),皮质醇类药物(P=0.014,OR=2.223),抗生素(P=0.000,OR=1.851),先前发生过CDI(P=0.000,OR=4.107)发病风险最大,而白蛋白降低(P=0.000,OR=4.947)是有效的替代标志物。572例艰难梭菌阳性病人中,近3个月内有抗生素治疗史为386列(67.5%),而3088例艰难梭菌阴性腹泻标本中,有1624列(52.6%)患者3个月内有抗生素治疗史。进一步对抗生素种类分类研究,结果表明CDI可能与以下几种抗生素有关：二代头孢(17.66%vs13.08%,P=0.000),三代头孢(11.89%vs6.38%,P=0.000),氟喹诺酮(10.31%vs7.67%,P=0.03),克林霉素(6.29%vs4.27%,P=0.03)以及碳青霉烯类药物(3.85%vs1.52%,P=0.000)。分离培养实验中,1962例住院及门诊患者平均年龄为56.46(±15.48),男性1120例(57.08%),女性842例(42.92%)。1962份新鲜粪便标本进行CCFA培养并分离鉴定,最终得到262份艰难梭菌临床分离菌株,其中250例产毒菌株,产毒菌株阳性率为12.74%。入组患者以炎性肠病患者最多,有473例(24.11%),其中溃疡性结肠炎患者198例(41.86%),克罗恩病患者275例(58.14%)。溃疡性结肠炎及克罗恩病患者各有34例有CDI,感染率分别为17.17%及12.36%,UC和CD患者艰难梭菌的感染率差异不显著纰=2.162,P=0.141)。男性炎性肠病患者共303例(占总数64.06%),女性炎性肠病患者共170例(35.94%),炎性肠病中男性和女性患者艰难梭菌的发病率无差异(χ2=0.023,P=0.878)。此外,IBD患者中,艰难梭菌阳性组平均年龄为38.35(±15.35),阴性组平均年龄为37.20(±14.84),两组之间无统计学差别(t=-0.690,P=0.490),说明IBD患者中年龄对艰难梭菌发病率无影响。进一步进行A、B毒素检测及二元毒素检测：A+B+191(76.4%)份,A-B+59(23.6%)份。191份A+B+菌株中,10株二元毒素阳性,其中1株为高毒力型菌株BI/NAP1/027。最后,将分离得到的262株艰难梭菌统一编号,并收集患者姓名,ID,入院时间等人口学资料及临床相关信息,连同毒素分类、核糖分型结果,利用计算机进行管理,建立起南方医院艰难梭菌菌种库。收集2012年7月至2012年12月之间南方医院临床成人(>18岁)腹泻新鲜粪便标本140例,其中最常见的是克罗恩病34例(24.3%),依次为恶性肿瘤28例(20.0%),感染性疾病23例(16.4%),慢性疾病18例(12.9%),溃疡性结肠炎15例(10.7%),术后6例(4.3%)以及自身免疫性疾病4例(2.9%)。140例粪便标本经标准SOP富集培养,细胞毒测试及中和实验,最终得到48例产B毒素的艰难梭菌产毒菌株。经Cepheid GeneXpert(?) Dx系统检测得到46例B毒素阳性标本。Cepheid Xpert C.difficile检测艰难梭菌阳性率和“金标准’役有区别,两者之间的一致性好。该机器检测艰难梭菌的灵敏度及特异度均大于90%,说明该机器发现感染的能力及正确识别不属于感染的能力好,即漏诊和误诊少。阳性似然比为42.17(CI：10.69-166.51),远大于10,说明该仪器能正确诊断艰难梭菌的可能性非常大。约登指数为89.49%,说明该仪器筛查CDI的效果非常好,真实性高。该实验进一步表明了我们实验室建立的艰难梭菌培养法具有很好的可靠性及稳定性,适合作为长期研究的基础培养方法。结论通过对艰难梭菌流行病学调查及危险因素分析,我们了解了南方医院CDI人群的临床特征,流行病学特点,感染相关危险因素及病情标志替代物。为及时监测CDI病情变化,合理使用抗生素,预防CDI,提高诊疗效率并控制医院艰难梭菌流行,降低CDI的发病率及死亡率提供了依据。成功建立南方医院艰难梭菌菌库,为进一步研究艰难梭菌生物学特性、致病性和致病机制、分析CDI的流行特征,疫苗研究及其防治打下了基础。并且首次在大陆分离出高致病性027型菌株(BI/NAP1/027),揭示了未来艰难梭菌在我国有暴发流行的可能,具有重要意义。我们实验室采用的艰难梭菌培养法稳定可靠,对于艰难梭菌的长期研究极为有意义。此外,临床试验证实,与“金标准’细胞培养细胞毒试验相比,Cepheid Xpert C.difficile检测系统具有一致性好,方便快捷实惠可靠,相对清洁卫生,同时具有检测二元毒素及027菌株的独特优点,便于临床筛查CDI及监测其暴发流行。对于预防CDI,指导临床合理选择用药、提高CDI诊疗效率、减缓细菌耐药,控制院内感染播散,减轻医疗压力具有重要意义,可作为临床艰难梭菌筛查的常规仪器。更多还原

【Abstract】 Clostridium difficile (C. difficile) is component of the normal human gut flora and a strictly anaerobic Gram-positive bacillus. C. difficile infection (CDI) refers to infections caused by toxin-producing C. difficile, which is diagnosed with the typical clinical symptoms (usually diarrhea), and positive testing results of C. difficile toxin (toxin-producing C. difficile positive in stool culture, colonoscopic or histopathological pseuolomembranous colitis). Since1978, C. difficile has been considered related with antibiotic-associated diarrhea (AAD). At present, it is appreciated that25%of AAD are caused by the C. difficile. CDI may lead to prolonged hospitalization, increased illness severity and mortality, as well as more medical expenses and burden.PathogenesisC. difficile mainly spreads between people through the fecal-oral route and colonizes in the intestine. Normal intestinal flora can inhibit colonization of C. difficile. While dysbiosis caused by antibiotics easily leads to overgrowth of C. difficile and release of toxin A and B. Toxin A, an enterotoxin, can directly trigger inflammation by affecting intracellular actin in colonic cells. Toxin B, a cytotoxin, can significantly induce cellular apoptosis, resulting in chromatin condensation and cell death. Moreover, C. difficile has cross antigenicity with sordellii, thus C. difficile toxins can be neutralized by sordellii toxin, directly damaging intestinal cells and causing pseudomembranous colitis.Clinical manifestationsMost patients with C. difficile infection present no significant clinical symptoms. Clinical symptoms may manifest as mild to severe watery diarrhea, fulminant colitis and toxic megacolon or obstruction and pseudomembranous colitis. Diarrhea in almost half of the patients occurs during usage of antibiotics or short after discontinuation. Sometimes it develops several months after drug discontinuance. Debilitating diarrhea can occur in severe patients (up to20to30times per day), accompanied by fever, nausea, anorexia, abdominal distension, abdominal pain, etc. Moreover, C. difficile infection often occurs in patients with systemic disease.Auxiliary examinationPatients with C. difficile infection shows thicken colon wall by CT scan. Abdominal X-ray shows paralytic ileus and "thumbprint" syndrome. Colonoscopy shows characteristic changes of colon, where yellow or white oval-shaped patches with clear boundary are scattered, which are strongly adhered with mucosa, and difficult to wash off Punctate pseudomembranous, gray or tan, appear in center of the plaque, blend and uplift in size of a few mm to1cm. There is no significant inflammation between mucosal lesions, but severe lesions might integrate. Decreased albumin levels, elevated levels of white blood cells, blood urea nitrogen, creatinine and serum ferritin levels, dysbiosis of fecal flora and fecal leukocytes may be found.Diagnosis and TreatmentClinical diagnosis of C. difficile-associated diarrhea can be made by the history of broad-spectrum antibiotics, severe diarrhea or hemafecia, and characteristic colonoscopic features (including edema, hyperemia, pseudomembrane, and nonspecific small ulcer or erosion). Confirmed diagnosis depends on results of bacterial culture and toxin test. At present, cytotoxicity assay and toxigenic culture is the standard detection method, but it is time-consuming and requires professional researchers. Therefore, enzyme-linked immunosorbent assay, glutamate dehydrogenase antigen testing and PCR technique are performed in some microbiology laboratories.Metronidazole is the first choice to treat patients with C. difficile-associated diarrhea, taken orally for7to14days (200-250mg,4times per day). The second choice is vancomycin (0.125g,4times per day, for7to14days), available for metronidazole insensitive or critical patients. For patients with severe obstruction, drug can be ingested by the gastric tube or anal tube, and bowel resection may be considered if necessary. Other treatments include bacitracin (25500U,4times per day), teicoplanin (200mg, qd), fusidic acid (500mg, qd), cholestyramine (4g,3-4times per day). Recently, some foreign countries have begun to use fidaxomicin to treat patients who insensitive to vancomycin, and the economic value has been indicated by clinical practice.Relapse of C. difficile-associated diarrhea often occurs7to14days after cure, but there is still no standard treatment for it. Recurrence rate may be reduced by increasing10-14-days’ treatment with metronidazole or vancomycin (125mg, gradually extended dosing interval) in patients with positive C. difficile toxin in stool. For those with multiple recurrences, additional treatment should be considered, such as cholestyramine combined with vancomycin, biological therapy and intravenous infusion of human immunoglobulin.In recent years, more and more attention has been paid to fecal transplantation, as its potential value in recurrent refractory CDI. Faeces are collected from healthy donors (after strict screening for infectious agents), and then transplanted to colon of patients with recurrent CDI in order to restore the patients’ flora homeostasis. The treatment can attenuate the pathological disorders caused by intestinal dysbiosis and is thought to be the most promising treatment. The most commonly used transplantation methods include implantation through enemas, nasal and endoscopic. Numerous studies have shown that it has a good therapeutic effect on recurrent refractory CDI.EpidemiologyOver the past20years, great changes have taken place in the epidemiology of C. difficile. With the wide use of broad-spectrum antimicrobial drugs, incidence of C. difficile-associated diarrhea is keeping rising worldwide. A study in Michigan showed that from2002to2008, rate of CDI has doubled. Recently, C. difficile often outbroke, causing increasing mortality and infection in patients. The outbreak of CDI may be due to the appearing of highly virulent strains (BI/NAP1/027), which characteristic with genetic variation and increased production of toxins, including binary toxin, toxin A and B. The strain is insensitive to fidaxomicin, resistant to quinolone, and has a high recurrence and mortality rate (up to7%). Since the hypervirulent strain debut in Canada, it has been reported in United States, Europe, Japan, Korea, Hong Kong and Singapore. Moreover, infection rates showed an increasing trend in populations such as perinatal women, children and community residences, which were considered with low risk factors.BackgroundCDI is widely reported and monitored in Europe and the United States, while rarely in Asia, except in some developed countries or regions, such as Japan, Korea, Singapore and Taiwan. Report of CDI is not mandatory in medical institutions in China’s mainland, Moreover, related researches are limited in some big cities like Beijing and Shanghai due to lack of technology and equipment. Thus, the prevalence of C. difficile in China is not yet clear.AimThe purposes of this study are as follows:①Investigating the prevalence of CDI in Nanfang Hospital, its risk factors and effective surrogate markers, in order to promote rational use of antibiotics, prevent CDI, improve the efficiency of diagnosis and treatment.②Clinically isolating and identifying C. difficile strains, to build a library of C. difficile in Nanfang Hospital, in order to lay the foundation for further study of biological characteristics, pathogenic mechanisms and vaccine development of C. difficile.③Exploring effective and rapid detection of C. difficile, which can be introduced in clinical testing and identification.Materials and MethodsSubjects:A total of4456stool samples were collect from patients in Nanfang Hospital from December2009to December2013(5-7levels in Bristol classification) and analyzed for C. difficile.4132samples were suitable for the study of CDI, of which127cases were a single test specimens,75double repeated samples,33triplicate samples and11four repeat samples.Methods:①Genome DNA were extracted from samples collected from December2009to May2012and used to detect C. difficile toxin B by PCR as previously described by Kato. Clinical data of patients were collected, independent risk factors and surrogate markers of CDI were analyzed by univariate and multivariate logistic regression analysis.②A total of1962samples were inoculated on CCFA anaerobic culture medium (SOP culture) and tested for cell toxicity (cytotoxicity assay, CTA). Typical colonies were separated and purificated, followed by Gram staining, lactate dehydrogenase (glutamate dehydrogenase, GDH) test and biochemical identification. Bacterial DNA were then extracted from the successfully identified C. difficile and tested for A, B toxin, binary toxin detection and ribotyping. All of the identified strains were stored at-80℃and patients’information were saved.③A total of140fresh diarrhea stool samples were collected from adults from2012-07-31to2012-12-19(duplicate samples were excluded) and divided into two parts. One was used for C. difficile standard culture together with biochemical identification and cytotoxicity experiments, and another for Cepheid GeneXpert(?) Dx (Cepheid, GX-XVI) system testing as well. McNemar chi-square was used to compare differences between the above two parts, and parameters including sensitivity, specificity, Youden’s index, positive likelihood ratio etc. were determined as well.ResultsA total of3885samples of diarrhea stool were collected from December2009-May2012in Nanfang Hospital, among which177repeated cases and18cases which did not meet requirement of amount or freshness were excluded, thus a total of3660cases were further used for C. difficile toxin B detection, risk factors and surrogate markers analysis. The mean age of patients was54.9±15.8years old, among which73.3%were male,90.8%inpatients,0.7%ICU patients. Infection rate of C. difficile in Nanfang Hospital was15.6%in patients with diarrhea, and CDI kept in a stable trend. Multivariate logistic regression analysis showed that age, gender, Horn’s index, history of broad-spectrum antibiotics, previous infection, history of cortisol drugs and immunosuppressants, and history of inflammatory bowel disease were all independent risk factors for Clostridium difficile infection, among which, gastric intubation (OR5.759), enema (OR3.976), malignant tumor (OR1.668), antibiotics (OR1.851), cortisol drugs (OR2.223), previous CDI (OR4.107) and inflammatory bowel disease (OR1.894) were more notable. fever (OR1.586) and decreased albumin (OR4.947) can be used as independent markers. In572C. difficile-positive patients,386cases (67.5%) had a history of antibiotic treatment within the past three months. In3088cases of diarrhea specimens without C. difficile, there were1624cases (52.6%) had antibiotic treatment history. Second generation and third generation cephalosporins, clindamycin, fluoroquinolones and carbapenems were all related with the high incidence of CDI.A total of1962fresh stool samples for isolation and identification. The mean age of the corresponding patients was56.46±15.48years old, including1120male patients (57.08%) and842female (42.92%).262samples of C. difficile were obtained, including250toxigenic strains and the positive rate was12.74%. In the included patients,473cases (24.11%) presented with inflammatory bowel disease, including198cases of ulcerative colitis (41.86%) and275cases of Crohn’s disease (58.14%).34cases of CDI occurred in ulcerative colitis and Crohn’s disease groups respectively, and the infection rates were17.17%and12.36%without significant difference (P=0.141). Moreover, no significant difference were observed in gender, including303male cases (64.06%) and170female cases (35.94%)(P=0.878). In addition, in inflammatory bowel disease patients, the average age of C. difficile-positive group was38.35±15.35years old, and negative37.20±14.84years old, without significant difference (P=0.490), indicating that age had no effect on the incidence of C. difficile in inflammatory bowel patients. Our results showed that191samples were A+B+(76.4%) and59A-B+(23.6%). Ten binary toxin positive cases were found in the A+B+strains, and one case of C. difficile027type (BI/NAP1/027) were finally identified. The isolated262cases of C. difficile were numbered, and demographic information of patients including name, ID, time of admission as well as toxin classification, ribotyping results were collected and saved to establish C. difficile library of Nanfang HospitalA total of140fresh diarrhea stool samples were collected in adult patients (>18years) between2012-07-31to2012-12-19, among whom the most common diagnosis was Crohn’s disease (34patients,24.3%), followed by malignant tumors (28patients,20.0%), infectious diseases (23samples,16.4%), chronic disease (18patients,12.9%), ulcerative colitis (15patients,10.7%), post operation (6patients,4.3%), and autoimmune diseases (4patients,2.9%). Forty-eight cases of C. difficile toxin B producing toxigenic strains were obtained by standard SOP culture, cytotoxicity test and neutralization test. Forty-six cases of toxin B-positive were obtained using Cepheid GeneXpert (?) Dx System, without significant difference with golden standard detection. McNemar test showed that sensitivity and specificity of detection using Cepheid GeneXpert (?) Dx System were both more than90%, indicating capacity of identifying infection from uninfection with little missed diagnosis and misdiagnosis. The positive and negative likelihood ratio was42.17and0.085respectively, indicating large possibility of correct detection of C. difficile or identifying uninfected patients. The Youden’s index was89.49, indicating efficiency of CDI detection and higher reliability.ConclusionWe outlined the clinical features, epidemiology and risk factors of C. difficile infection in Nanfang Hospital by epidemiological investigation and risk factor analysis. We concluded that albumin might be the best surrogate marker reflecting changes in CDI progression. Our results also provide important conclusion for how to rationally use antibiotics, prevent CDI, efficiently improve diagnosis and treatment, control hospital epidemic and reduce the incidence and mortality. We successfully established C. difficile bacteria library, which is the foundation of clearly understanding the biological characteristics and pathogenic mechanism of C. difficile, and developing an effective vaccine. More importantly, for the first time, we isolated the highly pathogenic strain027(BI/NAP1/027) in China’s mainland, revealing the potential outbreak of C. difficile. In addition, our study confirmed that Cepheid Xpert is convenient, rapid and reliable to detect C.difficile. And more importantly, the Cepheid Xpert C.difficile can facilitate binary toxin and027strains detection, and monitor outbreaks of C. difficile. Therefore, Cepheid Xpert can be routinely used in clinical screen of C. difficile.更多还原