Detection of Clostridium difficile toxins by enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) for the rapid diagnosis of antibiotic-associated colitis (AAC) is presented. Commercially available antisera to Clostridium difficile toxins contain antibodies to other antigens found in non-toxigenic C. difficile and other bacteria. Removal of these unwanted antibodies by absorption increased the specificity of ELISA for detection of C. difficile toxins. Specimens tested included 40 faecal extracts positive for cytotoxicity from cases of AAC, 30 diarrhoeic and 30 well-formed stools negative for cytotoxicity and 50 culture filtrates of toxigenic and non-toxigenic C. difficile and other clostridial species. Use of absorbed sera reduced false-positive reactions observed with faecal specimens from 23 to 8%. About 90% of specimens that were positive by the tissue culture cytotoxicity test were positive by ELISA using the absorbed sera. The relative merits of ELISA and other methods for the rapid diagnosis of AAC are discussed.     

Taqman实时定量PCR检测产毒艰难梭菌方法的建立

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.     

Toxicity assessment of Clostridium difficile toxins in rodent models and protection of vaccination

Clostridium difficile is the leading cause of hospital-acquired diarrhea, also known as C. difficile associated diarrhea. The two major toxins, toxin A and toxin B are produced by most C. difficile bacteria, but some strains, such as BI/NAP1/027 isolates, produce a third toxin called binary toxin. The precise biological role of binary toxin is not clear but it has been shown to be a cytotoxin for Vero cells. We evaluated the toxicity of these toxins in mice and hamsters and found that binary toxin causes death in both animals similar to toxins A and B. Furthermore, immunization of mice with mutant toxoids of all three toxins provided protection upon challenge with native toxins. These results support the concept that binary toxin contributes to the pathogenicity of C. difficile and provide a method for monitoring the toxicity of binary toxin components in vaccines.     

Clostridium difficile

Clostridium difficile is the leading cause of healthcare‐associated infectious diarrhea. Although C difficile is part of normal flora in some healthy individuals, patients with selective risk factors are often vulnerable to the toxigenic potential of this virulent healthcare pathogen. The spectrum of C difficile infection (CDI) is highly variable, ranging from mild to severe illness, presenting with single to multiple disease recurrences. Current approaches to treatment are based on severity of illness, number of recurrences, and clinical presentation. Oral vancomycin and metronidazole have formed the foundation for treatment of CDI, but therapeutic failures are commonly reported, especially involving hypervirulent clones. Alternative therapies, including newer antimicrobials, probiotics, immunotherapy, and fecal transplantation, have all met with varying degrees of efficacy. Although toxigenic culture (TC) testing from anaerobic culture remains the gold standard, newer technologies, including enzyme immunoassay, common antigen (glutamate dehydrogenase) testing, and real‐time polymerase chain reaction (PCR) are less time‐consuming and rapid. However, TC and PCR have reported high specificity and sensitivity when compared with other laboratory tests. Because of the significant morbidity and mortality associated with CDI, a high index of suspicion is warranted. Prevention and eradication of CDI require a multidisciplinary approach, including early disease recognition through appropriate surveillance, implementation of effective contact isolation strategies, adherence to environmental controls, judicious hand hygiene, evidence‐based treatment, and management that includes antibiotic stewardship, continuous education of healthcare workers, and administrative support.     

Clostridium difficile.

Seventy-five meconium samples were examined for the presence of Cl. difficile; 3 strains were isolated. Additionally 45 laboratory animal faeces specimens were tested for the same purpose, a further 2 cases were isolated. These five suspicious strains were identified as Cl. difficle according to the tests mentioned in the previous paragraphs. The organisms isolated here showed the same characteristics as five of the strains received and also as the organisms isolated from the inoculated animals with the crude cultures of Cl. difficile. These organisms were variable in size, roughly 2-9 XO.3-0. 8u, Gram positive rods, motile, capsulated, flagellated, most probably peritrichous, possessing non-bulging spores located terminally or subterminally, free spores were rarely detectable. Cell arrangements: singly or in pairs and occasionally in short chains. On longer incubation the organisms slightly shifted to become Gram variable and longer in size. Colonies on ordinary agar and solid blood agar appeared to be punctiform and rough. On the other hand the colony appearance on the rest of the solid media which are mentioned previously are as follows: 1-3 mm in diameter, greenish, smooth, non-haemolytic, entire some showing slight irregularities of their edges. Colonies slightly raised, butyrous and semi opaque to opaque. This organism does not liquify the serum of Loeffler medium and also does not cause any changes of this medium. The metachromatic granules are readily seen by Albert's staining. Neither proteolytic nor lipolytic activities are possessed by this organism. Sensitivity to antibiotics showed the same pattern as mentioned about the strains received.     

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。     

Detection of serum antibodies in human Hymenolepis infection by enzyme immunoassay.

Although hymenolepiasis is the commonest cestode infection of man, there are no data available on the human immune response to this parasite. Thus, in order to determine if infection induces antibodies against Hymenolepis nana antigens, sera from 52 infected children were initially studied on Ouchterlony plates and then by enzyme-linked immunosorbent assay (ELISA), using a crude antigenic extract prepared from scolex and neck regions of adult worms. In addition, sera from persons with cysticercosis, taeniasis and other parasitoses, and normal human sera, were studied. Only one serum from the Hymenolepis group showed precipitin antibodies against H. nana antigen, while several were positive by ELISA. The sensitivity of the ELISA was 84.62% and its specificity was 100%. Very high cross-reactivity rates were obtained with taeniasis (70.6%) and cysticercosis (75%) sera. These results show that Hymenolepis infection in man induces a low but detectable humoral immune response. Although not useful for diagnostic purposes, this may be relevant to the serodiagnosis of other tissue cestode infections of man, since antibodies detected in serological tests used for the diagnosis of cysticercosis, and probably hydatidosis, could be induced by H. nana instead of Taenia solium or Echinococcus larvae.     

Detection of chicken antibodies to mosquito salivary gland antigens by enzyme immunoassay

Sentinel chickens are used to detect western equine encephalomyelitis, St. Louis encephalitis, and West Nile virus activity. Flocks that receive high mosquito exposure will be most effective for surveillance purposes. However, mosquito population indices at the flock sites may only provide an indirect measure of potential exposure. Therefore, we developed an indirect enzyme immunoassay to detect chicken antibodies to salivary gland antigens (SGAs) from Culex tarsalis, the primary arbovirus vector in California. Chickens fed upon by Cx. tarsalis developed an antibody response that was proportional to the amount of exposure. Cross-reactivity between sera from Cx. tarsalis-exposed chickens and SGAs from Culex pipiens quinquefasciatus, Culex pipiens pipiens, Ochlerotatus melanimon, and Ochlerotatus sierrensis was likely due to shared SGAs among these species. This serologic assay for mosquito exposure could be used to evaluate the sensitivity of sentinel flocks for detecting arboviral activity.     

使用Real-Time PCR粪便中检测艰难梭状芽孢杆菌

目的：探讨Real-TimePCR对粪便检测艰难梭菌的可行性。方法：以组织培养细胞毒素试验为标准，参考艰难梭菌的毒素A／B基因相应的引物和分子信标探针，对标准菌株和粪便提取的DNA实行Real-TimePCR扩增。结果：Real-TimePCR的敏感度为96．9％；特异性为100％；阳性预测值为100％；阴性预测值为98．3％。结论：Real—TimePCR与传统诊断方法相比具有快速、敏感、特异等优点，可以提高艰难梭菌的检测水平。     

Epidemiology of Clostridium difficile infection

Pseudomembranous colitis (PMC), antibiotic-associated diarrhoea (AAD), and colitis (AAC) caused by Clostridium difficile are recognized as complications of antibiotic treatment (cephalosporins, penicillins, clindamycin and others). Two groups are particularly at risk: older and immunocompromised patients. In recent years C. difficile has been recognized as a common nosocomial pathogen. To understand the epidemiology of the C. difficile infection, many outbreaks have been investigated by various methods. In the paper we reviewed different methods of C. difficile typing and discussed the epidemiology of C. difficile-associated infections in light of recent publications.     

Relapsing infections with Clostridium difficile

Two women, aged 78 and 85 years, presented with watery diarrhoea and fever after a course of antibiotic therapy. Pseudo-membranous colitis was diagnosed, which was adequately treated. In both patients the C. difficile colitis relapsed, which was successfully treated with a pulse and tapering scheme of vancomycin. C. difficile infection is a frequent cause of antibiotic-associated diarrhoea. Clinical presentation can vary in severity. Cytotoxin testing, immunoassay and endoscopy are important tools in diagnosing C. difficile colitis. Like the first infection, the first relapse must be treated with metronidazole or vancomycin. To treat a second relapse, a pulse and tapering dose of vancomycin has been recommended. Nevertheless, multiple recurrences may occur, which are difficult to treat.     

Diminished intestinal colonization by Clostridium difficile and immune response in mice after mucosal immunization with surface proteins of Clostridium difficile

Clostridium difficile pathogenesis is mainly due to toxins A and B. However, the first step of pathogenesis is the colonization process. We evaluated C. difficile surface proteins as vaccine antigens to diminish intestinal colonization in a human flora-associated mouse model. First, we used the flagellar cap protein FliD of C. difficile, in order to test several immunization routes: intranasal, rectal, and intragastric. The rectal route, which is the most efficient, was used to vaccine groups of mice with different antigen combinations. After immunizations, the mice were challenged with the toxigenic C. difficile and a significant statistical difference between the control group and the immunized groups was observed in the colonization levels of C. difficile.     

Detection of the schistosome circulating cathodic antigen by enzyme immunoassay using biotinylated monoclonal antibodies

We have developed an enzyme immunoassay (ELISA) for the quantification of the schistosome circulating cathodic antigen (CCA), a glycoprotein associated with the syncitium lining the gut of the parasite. A mouse monoclonal antibody of IgG3 isotype was used as coating (antigen-capture) antibody, while a biotinylated mouse monoclonal IgM was used as second (antigen-detecting) antibody. Streptavidin-alkaline phosphatase was used as enzyme label. The lower detection limit of the assay was 1.0 ng of the trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, which corresponds to approximately 0.2 ng CCA per ml. The ELISA showed a linear range from 1.0 to 62.5 ng AWA-TCA per ml. Serum and urine samples of 16 individuals infected with Schistosoma mansoni (egg counts ranging from 5 to 4820 eggs per gram of faeces) were tested in the assay. Antigen titres ranged from less than 4-8192. This assay represents a considerable advantage in diagnosis of Schistosoma infections as it allows the detection and quantification of CCA in serum and urine in even lightly infected individuals.     

Detection of antimicrofilarial ES antigen-antibody in immune complexes in Bancroftian filariasis by enzyme immunoassay

An enzyme immunoassay using penicillinase conjugated to Wuchereria bancrofti microfilarial ES antigen has been developed to detect specific antibody in circulating immune complexes in Bancroftian filariasis. Immune complexes were prepared by 3% polyethylene glycol (PEG) precipitation. 44 sera belonging to different groups were tested. 16 of 19 clinical filarial and two of 16 endemic normal sera but none of the non-endemic normal sera showed the presence of antimicrofilarial ES antigen-antibody in immune complexes.