25株艰难梭菌多位点序列分型

目的比较两种对艰难梭菌多位点序列分型(MLST)的方案并探讨不同来源艰难梭菌之间的关系。方法选取来自不同人群的25株艰难梭菌,分别用Lemee方案(aroE,ddl,dutA,tpi,recA,gmk,sodA管家基因)和David方案(adk,atpA,dxr,glyA,recA,soda,tpi管家基因)进行多位点序列分型,用START系统及Bionumerics软件分析这些位点及等位基因谱的特征。结果位点分析结果表明,Lemee方案中有2株菌的ddl位点为无效位点,其余的6个管家基因有3～5个等位基因,等位基因之间有3～11个多态性位点。非同义突变/同义突变(dN/dS)为0.000 0～0.261 1;David方案中的7个管家基因有3～5个等位基因,等位基因之间有2～9个多态性位点。dN/dS为0.000 0～0.280 6。等位基因图谱特征结果表明,Lemee方案出现了13个ST型别(ST1/2/3/6/7/13/31/39/48/A/B/C/D),ST6(5)为优势型别;David方案共出现11个ST型别(ST2/3/15/35/37/54/55/92/99/102/117),其中ST54(6),ST37(5)为优势型别。来源于成人和婴幼儿的菌株并未形成各自特异的序列型。结论对于艰难梭菌多位点序列分型,David的方案更可取,ST与宿主人群的来源没有明显的相关性。     

Rapid analysis of Clostridium difficile strains recovered from hospitalized patients by using the slpA sequence typing system

Clostridium difficile is a nosocomial pathogen that is transmissible between patients via hospital staff and via contaminated environmental surfaces. Recently, a typing system based on the slpA sequence for C. difficile was developed. To elucidate the validity and efficacy of the system in the setting of a local hospital, we carried out typing of C. difficile from patients in our hospital using the system. Twenty-eight stool samples obtained from 17 patients with C. difficile-associated diarrhea were investigated. Twenty-two of the 28 samples were positive for C. difficile by stool culture, and they were able to be classified by slpA sequence typing. The smz-1 and smz-2 strains were revealed to be the predominant types in our hospital, accounting for 73% of all strains. The yok-1, yok-2, t25-1, hr-1, and hj2-2 strains were identified in 1 patient each. The smz-1 strain was identified in all wards except for ward D, while smz-2 was identified in 3 of 4 patients in ward D and was restricted to this ward. Nosocomial infection of smz-1 and smz-2 in our hospital was demonstrated. Distinguishing smz-1 from smz-2 by slpA sequence typing clarified the transmission of C. difficile among patients. In conclusion, slpA sequence typing was useful in the setting of a local hospital and may be a powerful tool for the epidemiological study of C. difficile infection.     

艰难梭菌分子分型技术研究进展

艰难梭菌是一种革兰阳性的专性厌氧产芽孢杆菌。核糖体027型高毒力株在发达国家的多次暴发使得艰难梭菌越来越引起重视,近年来艰难梭菌感染的发生频率已在全世界范围内呈显著上升趋势。目前该菌的分子流行病学研究进展较快,常用的分子分型方法包括限制性酶切分型、PCR核糖体分型、脉冲场凝胶电泳、多位点序列分析、重复序列PCR分型、多位点可变数目串联重复序列分析、毒素分型和全基因组测序分析等。同时,仍有不断改进的新型分子分型方法应用于此,以期更准确、快速和有效地对艰难梭菌感染暴发进行识别与调查,为艰难梭菌的感染防控提供可靠的分子流行病学数据。     

Molecular epidemiological typing of enteropathogenic Escherichia coli strains from Australian patients

Enteropathogenic Escherichia coli (EPEC) are an important cause of diarrhoea worldwide, particularly in children. Sixty-one EPEC strains isolated from stool specimens of symptomatic persons from 2008 to 2011 were characterised for the prevalence of diarrhoea-associated putative virulence genes. Phylogenetic typing, serotyping, multilocus variable-number repeat analysis (MLVA), and multilocus sequence typing (MLST) were also performed. The EPEC isolates were highly heterogeneous, representing all 4 phylogenetic groups and comprising 59 MLVA types, 49 MLST types, and 43 serotypes. This diversity is indicative of the complexity of the human enteric EPEC population, which may be either commensal or pathogenic.     

Antimicrobial-resistant strains of Clostridium difficile from North America

A total of 316 toxigenic Clostridium difficile clinical isolates of known PCR ribotypes from patients in North America were screened for resistance to clindamycin, metronidazole, moxifloxacin, and rifampin. Clindamycin resistance was observed among 16 different ribotypes, with ribotypes 017, 053, and 078 showing the highest proportions of resistance. All isolates were susceptible to metronidazole. Moxifloxacin resistance was present in >90% of PCR-ribotype 027 and 053 isolates but was less common among other ribotypes. Only 7.9% of the C. difficile isolates were resistant to rifampin. Multidrug resistance (clindamycin, moxifloxacin, and rifampin) was present in 27.5% of PCR-ribotype 027 strains but was rare in other ribotypes. These results suggest that antimicrobial resistance in North American isolates of C. difficile varies by strain type and parallels rates of resistance reported from Europe and the Far East.     

艰难梭菌五种实验室检测方法的评价

目的 比较5种艰难梭菌检测方法并进行评价。方法 包括tcdB基因PCR检测、tcdB基因Real-time PCR检测、酶联免疫法、酶联免疫层析法以及环丝氨酸-头孢西丁-果糖琼脂（CCFA）常规培养法。以CCFA培养的检测结果作为参考,对以上几种方法进行评价。结果 得到上述检测方法的敏感度、特异度、阳性预测值和阴性预测值结果以及其与参考方法的Kappa值。在125例样本中,CCFA培养法检出阳性标本11份,tcdB基因普通PCR检测、tcdB基因Real-time PCR检测、酶联免疫法、酶联免疫层析法分别检出阳性12、12、50和25例,4种方法间检出阳性率差异有统计学意义（2=61.452,P0.000 1）,敏感度分别为63.64%、63.64%、63.64%和54.55%,差异无统计学意义（2=0.288,P=0.962）;特异度为95.61%、95.61%、62.28%和83.33%,差异有统计学意义（2=63.597,P0.000 1）。结论 PCR方法敏感度、特异度最高,成本较低,适用于有实验条件的流行病学调查和临床诊断,两种免疫方法试剂盒适用于临床先期筛查以及辅助诊断。     

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.     

Asymptomatic Clostridium difficile colonization as a reservoir for Clostridium difficile infection

Clostridium difficile (CD) infection (CDI) is the leading cause of healthcare associated diarrhea despite intense hospital infection prevention programs. A substantial proportion of the population is asymptomatically colonized with CD, and evidence is mounting that these individuals serve as a reservoir for CDI. The purpose of this review is to discuss the mechanisms by which individuals may harbor toxigenic CD but remain asymptomatic, the evidence that asymptomatically colonized individuals serve as a source of CDI, and the implications of this potential CD reservoir for healthcare infection prevention.     

艰难梭菌感染实验室诊断方法的研究进展

艰难梭菌是一种专性厌氧革兰阳性芽孢杆菌,一般认为是环境和人类肠道中的正常菌群.长期应用抗生素、免疫抑制剂或化疗药物使耐药的艰难梭菌产毒株过度繁殖并释放毒素是导致艰难梭菌相关性腹泻(CDAD)的主要因素.CDAD的发病率在全球范围内不断上升,尤其是高产毒株在北美地区引起了医院内的暴发流行,引起了世界范围的关注.在此对艰难梭菌的致病机制和实验室诊断方法的研究进展进行阐述,为CDAD的早期诊断和治疗提供新思路.     

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。     

Clostridium difficile control measures: current and future methods for prevention

Introduction: Clostridium difficile is the most common cause of healthcare associated infection, and C. difficile infection (CDI) is associated with significant costs, morbidity, and mortality. One obstacle to preventing CDI is lack of high quality data on interventions to prevent CDI. This has led some to focus on areas, such as method of hand hygiene, unlikely to impact CDI incidence as much as others, such as contact precautions. In addition, existing strategies, although effective, do have limitations. Another challenge is the ability to rapidly, and accurately, diagnose CDI. Given these obstacles, new strategies to effectively prevent CDI are imperative to improve patient outcomes.

Areas covered: Evidence of the interventions recommended by international scientific societies will be reviewed, as well as ongoing research on new strategies, such as screening for asymptomatic C. difficile carriage, microbiota sparing agents, bacteriocins and vaccines.

Expert commentary: Current measures to prevent CDI are effective, but have significant limitations. Contact precautions and antimicrobial stewardship are likely the most effective of current prevention recommendations. Diagnostic assay utilization plays a role as well. New strategies to prevent CDI are needed, and, fortunately, several are being studied. Most likely a combination of approaches will be necessary to optimize CDI prevention.     

Antimicrobial phenotypes and molecular basis in clinical strains of Clostridium difficile

Clostridium difficile remains the leading cause of nosocomial-acquired diarrhea. This study investigated antimicrobial susceptibility patterns of C. difficile over a 3-year period. Three hundred seventeen C. difficile isolates recovered between 2002 and 2004 were analyzed for their susceptibility to erythromycin (ERY), clindamycin (CLI), moxifloxacin (MXF), doxycycline (DOX), vancomycin (VAN), and metronidazole (MTR) by Etest. The molecular basis for resistance was investigated using polymerase chain reaction (PCR) and DNA sequencing. PCR ribotyping was used to differentiate strains. All strains were susceptible to VAN and MTR. Resistance rates to ERY/CLI, MXF, and DOX increased during the study period. Eighty-four (26.5%) strains exhibited resistance against ERY/CLI, MXF, and DOX. Prevalence of resistance genes was as follows: ermB, 83; ermQ, 0; ermFS, 1; tetM, 84; tetP, 0; tetO, 2; and gyrA mutation, 76. These results indicate an increasing trend in the prevalence of combined resistance against macrolide-lincosamide-streptogramin B antibiotics, fluoroquinolones, and tetracycline in C. difficile. The lack of understanding of antibiotic resistance mechanisms in C. difficile and the increased resistant strains warrants further investigations.     

碳青霉烯类耐药肠杆菌科细菌分子流行病学分析

目的研究西部战区总医院碳青霉烯类耐药肠杆菌科细菌(CRE)的流行病学特征及耐药机制。方法收集2015年1月-2018年12月临床各种标本分离出的CRE 121株,采用VITEK 2-Compact全自动微生物分析仪进行鉴定和体外药敏试验,聚合酶链反应(PCR)法及测序检测碳青霉烯酶、ESBL酶、AmpC酶和膜孔蛋白基因;肠杆菌科基因间重复一致序列PCR(ERIC-PCR)进行同源性分析。结果121株CRE中碳青霉烯酶阳性96株,其中65株产blaKPC、25株产blaNDM-1、7株产blaOXA-48、2株产blaVIM,有2株同时产blaKPC和blaNDM-1,1株同时产blaKPC和blaVIM;未检测到blaIMP、blaGES基因;blaSHV、blaTEM和blaDHA检出率分别为46.3%、47.1%和37.2%;OMPF、OMPC缺失/突变率分别为48.1%、84.6%,OMPK35、OMPK36缺失/突变率分别为35.7%、91.1%。药敏结果分析发现121株CRE对青霉素、头孢菌素类抗生素耐药率高,对阿米卡星耐药率最低。ERIC-PCR结果显示56株碳青霉烯类耐药克雷伯菌有5种基因型;26株碳青霉烯类耐药大肠埃希菌有3种基因型;23株碳青霉烯类耐药阴沟肠杆菌有3种基因型。结论该院临床分离的CRE以blaKPC为主要耐药基因,其次为blaNDM-1;部分CRE既携带碳青霉烯酶基因,同时合并膜孔蛋白基因缺失/突变。从分型结果得出,部分菌株同源性非常高,存在克隆传播现象。     

Policy development for Clostridium difficile

The Advisory Committee on Antimicrobial Resistance and Healthcare Associated Infection (ARHAI) was created at the height of the incidence of Clostridium difficile infection (CDI). This article describes the role of ARHAI in the evaluation of laboratory testing for CDI, a related consultation on the legal requirements for manufacturers of in vitro diagnostic medical devices, a CDI healthcare bundle and surveillance of CDI in children.     

肉毒梭菌及其毒素分型方法概述

自1897年van Ermengem分离并报道了可形成孢子的肉毒梭菌导致人类中毒以来,研究人员通过血清中和方法发现自然界中存在7种不同型别的肉毒梭菌。 随后,再次应用血清学方法又发现了可产生双价毒素Ab、Ba、AB、Af和Bf等肉毒梭菌。 随着基因测序技术的逐渐成熟和完善,通过提取肉毒梭菌基因进行全基因组测序,不仅可识别不同血清型肉毒毒素的基因组成,也可开展核酸或蛋白质分子水平的分型研究。 相比血清中和分型方法,这些新涌现的分型技术和方法不仅可解开血清学分类中遇到的难题,更可快速准确地鉴别肉毒梭菌导致的食物或药物中毒,并追踪源头及应用相应药物治疗。 此外,不同分型方法对于肉毒梭菌菌种鉴定和使用,新型毒素的研发都提供了新的工具和思路。     

Molecular techniques for diagnosis of Clostridium difficile infection: systematic review and meta-analysis

ObjectiveTo assess the usefulness of 2 rapid molecular diagnostic techniques, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP), in Clostridium difficile infection (CDI).MethodsWe conducted a systematic review and meta-analysis to evaluate the accuracy of PCR and LAMP in diagnosis of CDI, including studies that used toxigenic culture or cytotoxicity assay as reference standard.ResultsA search of PubMed and CinAHL medical databases yielded 25 PCR studies, including 11,801 samples that met inclusion criteria and 6 heterogeneous studies that evaluated LAMP. With toxigenic culture as a standard, pooled sensitivity was 0.92 (95% confidence interval [CI], 0.91-0.94); specificity, 0.94 (95% CI, 0.94-0.95); and diagnostic odds ratio, 378 (95% CI, 260-547). With cytotoxicity as a standard, pooled sensitivity was 0.87 (95% CI, 0.84-0.90); specificity, 0.97 (95% CI, 0.97-0.98); and diagnostic odds ratio, 370 (95% CI, 226-606).ConclusionPolymerase chain reaction is a highly accurate test for identifying CDI. Heterogeneity in LAMP studies did not allow meta-analysis; however, further research into this promising method is warranted.