艰难梭菌核酸检测方法应用进展

艰难梭菌是一种广泛存在于人和动物肠道中的厌氧革兰阳性芽孢杆菌。 近年来,由于抗生素滥用艰难梭菌感染人数大量增加,发病率和病死率急剧上升,准确的诊断对于最佳治疗手段和预防措施来说至关重要。 因此高灵敏度与特异度、省时省力的核酸检测方法逐渐引起重视。 本研究就目前最新通过美国食品药品监督管理局批准以及其他经典的实验室自建艰难梭菌核酸检测方法的应用进行介绍和比较,为艰难梭菌的检测提供参考。     

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。     

应重视艰难梭菌感染的检测

艰难梭菌(Clostridium difficile)为革兰阳性厌氧产芽孢杆菌,是人类肠道中的正常菌群之一.艰难梭菌本身没有侵袭性,其中部分细菌(产毒株)可通过分泌毒素A、毒素B和(或)二元毒素从而引起抗生素相关性腹泻、结肠炎甚至致死性伪膜性肠炎,统称为艰难梭菌感染(Clostridium di ficile infection,CDI).艰难梭菌为医院获得感染性腹泻最主要的病原菌,在抗生素相关性腹泻病因中艰难梭菌亦占20％～30％;而伪膜性肠炎则几乎100％由艰难梭菌所致.肠道外CDI如败血症等则极为罕见.不仅仅是抗菌药物,其他影响肠道正常菌群平衡、降低艰难梭菌定植抵抗能力的因素,如老年、胃肠道手术、应用抗肿瘤药物、长期住院和免疫功能缺陷等均为CDI的危险因素.     

艰难梭菌感染病原学检测技术

<正>艰难梭菌感染(Clostridiodes dif?cile infection,CDI)是指艰难梭菌引起的感染性疾病,主要表现为腹泻、腹痛、发热等。近年来,CDI的发病率呈逐年上升趋势,表现为复发率,严重程度及病死率增高^[1-2]。2011年美国疾病控制和预防中心流行病学调查发现,艰难梭菌是医疗保健感染最常见的病原体,达到12.1%^[3]。自2000年以来,CDI死亡率也逐年上升,在地方性流行期间为4.5%～5.7%,在大流行期间为6.9%～16.7%^[4]。儿童CDI的发病率总体呈上升趋势,约为13.8例/100000人,71%来自社区感染^[5]。但目前很多医院对CDI的检测手段十分有限,且诊断率不高,     

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。     

聚合酶链反应鉴定艰难梭菌产毒株

以艰难梭菌Ａ毒素基因非重复片段中的２个寡核苷酸作引物，扩增３０６ｂｐ，用多聚酶链反应技术扩增３１株细菌，结果１９株艰难梭菌产毒株均扩增出单一特异的电泳带，面８株艰难梭菌无毒株，２株索氏梭菌和２株大肠杆菌均无特异带出现，将艰难梭菌产毒株的模板ＤＮＡ从５０ｎｇ稀释至０．５ｎｇ后再进行聚合酶链反应，结果仍可见行异扩增带，说明聚合酶链反应鉴定艰难梭菌产毒株较之细菌分离培养，细胞毒素测定方法具有快速。简便，     

3种不同方法检测艰难梭菌芽孢率的比较

目的了解艰难梭菌检测的芽孢率并进行比较。方法采用平板涂布法、相差显微镜计数法和孔雀绿染色法3种检测方法对艰难梭菌ATCC 43596和VPI 10463经培养96 h和120 h后的芽孢率进行了比较分析。结果显示平板涂布法芽孢检出率最高为(25.3%、15.6%),变异较大(SD=7.1%,4.3%);相差显微镜计数法和孔雀绿染色法结果相近分别为(13.3%、9.4%)、(13.4%、9.8%),变异较小(SD=1.9%、0.8%)、(SD=1.4%、0.9%)。结论在选用平板涂布法检测芽孢率时,建议联合相差显微镜法或者孔雀绿染色法以减少偏移。     

聚合酶链反应鉴定艰难梭菌产毒株

以艰难梭菌Ａ毒素基因非重复片段中的２个寡核苷酸链作引物，扩增３０６ｂｐ，用多聚酶链反应技术扩增３１株细菌，结果１９株艰难梭菌产毒株均扩增出单一特异的电泳带，而８株艰难梭菌无毒株、２株索氏梭菌和２株大肠杆菌均无特异带出现。将艰难梭菌产毒株的模板ＤＮＡ从５０ｎｇ稀释至０．５ｎｇ后再进行聚合酶链反应，结果仍可见特异扩增带。说明聚合酶链反应鉴定艰难梭菌产毒株较之细菌分离培养、细胞毒素测定方法具有快速、简便、特异、敏感的优点，可望用于临床标本的直接检测。     

艰难梭菌感染实验室检测技术及其研究进展

<正>艰难梭菌(CD)是一种专性厌氧的革兰染色阳性芽孢杆菌,它广泛分布于自然环境、动物和人的粪便中,芽孢抵抗力较强,可在外界环境存活数周至数月。CD本身没有侵袭性,部分产毒细菌通过分泌毒素A、毒素B及二元毒素引起抗菌药物相关性腹泻、结肠炎甚至致死性假膜性肠炎,统称为艰难梭菌感染(CDI)。广谱抗菌药物的使用、炎性反应肠病、慢性肝病、     

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。     

GeneXpert实时荧光定量PCR在诊断艰难梭菌感染中的临床应用

目的评价Gene Xpert实时荧光定量PCR法在诊断艰难梭菌感染中的应用价值。方法收集住院腹泻患者非重复粪便标本296份,同时进行Gene Xpert试验和产毒素培养(toxigenic culture,TC)。TC包括厌氧培养、菌株基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)鉴定、PCR扩增艰难梭菌毒素基因。以TC结果为参考,评价Gene Xpert试验的敏感性、特异性、阳性预测值、阴性预测值,并评价Gene Xpert试验和TC 2种方法结果的一致性。结果 Gene Xpert试验的敏感性、特异性、阳性预测值和阴性预测值分别为98.5%、92.6%、91.7%和98.7%。Gene Xpert试验与TC一致性好(Kappa=0.905)。Gene Xpert试验报告2株RT027型高毒力菌株,经核糖体分型确证为RT027型艰难梭菌。结论 Gene Xpert实时荧光定量PCR可快速、准确检测粪便标本中的艰难梭菌毒素基因,且可报告高毒力RT027型菌株,具有重要的临床应用价值。     

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.     

Optimizing the diagnostic testing of Clostridium difficile infection

Introduction: Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and is associated with a considerable health and cost burden. However, there is still not a clear consensus on the best laboratory diagnosis approach and a wide variation of testing methods and strategies can be encountered.

Areas covered: We aim to review the most practical aspects of CDI diagnosis providing our own view on how to optimize CDI diagnosis.

Expert commentary: Laboratory diagnosis in search of C. difficile toxins should be applied to all fecal diarrheic samples reaching the microbiology laboratory in patients > 2 years old, with or without classic risk factors for CDI. Detection of toxins either directly in the fecal sample or in the bacteria isolated in culture confirm CDI in the proper clinical setting. Nuclear Acid Assay techniques (NAAT) allow to speed up the process with epidemiological and therapeutic consequences.     

Real-time cellular analysis for quantitative detection of functional Clostridium difficile toxin in stool

Rapid and accurate diagnosis and monitoring of Clostridium difficile infection (CDI) is critical for patient care and infection control. We will briefly review current laboratory techniques for the diagnosis of CDI and identify aspects needing improvement. We will also introduce a real-time cellular analysis (RTCA) assay developed for the diagnosis and monitoring of CDI using electronic impedance to assess the cell status. The RTCA assay uses impedance measurement to detect minute physiological changes in cells cultured on gold microelectrodes embedded in glass substrates in the bottom of microtiter wells. This assay has been adapted for quantitative detection of C. difficile functional toxin directly from stool specimens. Compared to conventional techniques and molecular assays, the RTCA assay provides a valuable tool for the diagnosis of CDI as well as for the assessment of clinical severity and for monitoring therapeutic efficacies.     

Prevalence of Clostridium difficile colonization at admission to rehabilitation

OBJECTIVES: To assess the prevalence of intestinal colonization with Clostridium difficile (C. difficile) at admission to acute rehabilitation and to identify risk factors associated with colonization. DESIGN: Case-control study. PARTICIPANTS: Consecutive admissions to 2 rehabilitation units (spinal cord injury, brain injury and stroke). SETTING: Free-standing acute rehabilitation facility. INTERVENTIONS: Rectal swabs for culture for C. difficile were obtained at admission and cytotoxin assay performed on all culture positive specimens. Rates of colonization with cytotoxic C. difficile were calculated. Charts were reviewed for medical and demographic factors that may have predisposed patients to colonization, and for possible symptoms at the time of admission. MAIN OUTCOME MEASURES: Percentage of patients with culture and cytotoxin assay positive for C. difficile. Frequency of specific patient characteristics that could predispose to C. difficile colonization. RESULTS: Of admission stool samples, 16.4% tested positive for C. difficile; none of these patients had been identified as colonized before admission. No patients were discordant for C. difficile positivity on culture and presence of a toxigenic strain. No medical or demographic factors were associated with increased risk of colonization, including age (t(52)=-.748, P=.458, not significant [NS]), diarrhea within 24 hours of admission (chi(1)(2) test=.001, P=.973 [NS]), or use of oral or intravenous antibiotics at admission (chi(1)(2) test=.044, P=.834 [NS]). CONCLUSIONS: Patients admitted to acute rehabilitation may have an elevated rate of intestinal colonization with C. difficile without having clinical symptoms. No medical or demographic characteristics were found to be predictive of colonization, however, most of the patients admitted had more than 1 factor that may have increased their susceptibility to infection with this organism. Inadvertent transfer of this organism within the rehabilitation setting may occur because asymptomatic colonization is not recognized.     

Treatment of Clostridioides (Clostridium) difficile infection

Abstract

Clostridioides (formerly: Clostridium) difficile infection (CDI) is a major cause of diarrhoea for inpatients as well as outpatients. Usually, CDI is healthcare-associated but the number of community-acquired infections is increasing. CDI is generally associated with changes in the normal intestinal microbiota caused by administration of antibiotics. Elderly and immunocompromised patients are at greater risk for CDI and CDI recurrence. Recently, the treatment options of CDI have undergone major changes: current recommendations speak against using metronidazole for primary CDI, fidaxomicin and bezlotoxumab have been added to the treatment armamentarium and microbial replacement therapies have emerged. Several other therapies are undergoing clinical trials. In this article, we review current treatment guidelines, present the most recent data on the options to treat CDI and glance towards future developments.

• KEY MESSAGES

• The cornerstones for the treatment of CDI are vancomycin and fidaxomicin. Metronidazole should be used only in mild-to-moderate disease in younger patients who have no or only few risk factors for recurrence.

• In recurrent CDI, bezlotoxumab infusion (a monoclonal antibody against C. difficile toxin B) may be considered as an adjunctive therapeutic strategy in addition to the standard care provided to patients with several risk factors for recurrence.

• Faecal microbiota transplantation (FMT) should be offered to patients with frequently recurring CDI.