用PCR方法检测难辨梭状芽胞杆菌

目的建立PCR方法检测难辨梭状芽胞杆菌．方法采用PCR方法对难辨梭装芽胞杆菌（以下简称c，d）的毒素B基因和毒素A基因进行扩增，同时和细菌厌氧培养法进行对比．结果36例标本中采用PCR方法有16例扩增出毒素B基因（占46．7％），13例扩增出毒素A基因（占36．1％），用细菌厌氧培养法有7例培养出c，d（占19．2％），而且厌氧培养阳性者，用PCR方法也都扩增出毒素B基因和毒素A基因．结论用PCR方法检测c，d比厌氧培养法检出率明显增高（P＜0．05）而且特异性较好．     

肠粘膜细菌培养诊断抗生素相关性腹泻的价值

探讨内镜下活检肠粘膜细菌培养诊断抗生素相关性腹泻的价值.对32例内镜下表现有伪膜的患者粪便和活检肠粘膜进行常规和厌氧菌培养及毒素的鉴定,结果32例患者粪便和活检肠粘膜的常规细菌培养均为阴性,10例患者粪便培养出难辨梭状芽胞杆菌,阳性率31.3%,而活检肠粘膜有28例培养出难辨梭状芽胞杆菌,阳性率87.5%,研究表明内镜下活检肠粘膜细菌培养的阳性率明显高于粪便培养。     

肠粘膜细菌培养诊断抗生素相关性腹泻的价值

目的：探讨内镜下活检肠粘膜细菌培养诊断抗生素相关性腹泻的价值。方法：对３２例内镜下肠粘膜表现有伪膜的患者粪便和活检肠粘膜进行常规和厌氧菌培养及毒素的鉴定。结果：３２例患者粪便和活检肠粘膜的常规细菌培养均为阴性；１０例患者粪便培养出难辨梭状芽孢杆菌，阳性率３１．３％；活检肠粘膜有２８例培养出难辨梭状芽孢杆菌，阳性率８７．５％，结论：内镜下活检肠粘膜细菌培养的阳性率明显高于粪便培养。     

肠粘膜细菌培养诊断抗生素相关性腹泻的价值

目的 :探讨内镜下活检肠粘膜细菌培养诊断抗生素相关性腹泻的价值。方法 :对 3 2例内镜下肠粘膜表现有伪膜的患者粪便和活检肠粘膜进行常规和厌氧菌培养及毒素的鉴定。结果 :3 2例患者粪便和活检肠粘膜的常规细菌培养均为阴性 ;10例患者粪便培养出难辨梭状芽孢杆菌 ,阳性率 3 1.3 % ;活检肠粘膜有 2 8例培养出难辨梭状芽孢杆菌 ,阳性率 87.5 %。结论 :内镜下活检肠粘膜细菌培养的阳性率明显高于粪便培养     

难辨梭状芽胞杆菌毒素和慢性炎症性肠病症状复发的联系

难辨梭状芽胞杆菌(Cl.difficile)毒素在抗生素所致之腹泻性疾病的病理生理学中的作用,现已较肯定。约95％因抗生素所致的伪膜性结肠炎患者,和20％由抗生素引起但无结肠炎的腹泻患者的大便中可发现有难辨梭状芽胞杆菌毒素。以后的工作发现,这种毒素     

难辨梭状芽胞杆菌性结肠炎

难辨梭状芽胞杆菌(Cd)为一种抗生素相关伪膜性结肠炎的致病菌,近已证实为医院内感染的常见病原菌。该菌为G~+厌氧菌。由于其分离艰难且生长缓慢,故命名为难辨梭状芽胞杆菌。现已明确几乎所有的伪膜性结肠炎和多达20％的抗生素相关性腹泻由该菌感染所致。     

黄石地区慢性无症状乙型肝炎病毒携带者的基因型分布

目的：了解湖北黄石地区慢性无症状乙型肝炎病毒（HBV）携带者HBV基因型的分布，及其与病毒复制水平、HBeAg表达的相关性。方法：选择黄石地区168例慢性无症状HBV携带者作为研究对象，HBV基因型采用PCR微板核酸杂交．ELISA方法检测；血清HBVDNA复制水平采用荧光定量PCR检测；HBV—M采用ELISA法检测。结果：168例慢性无症状HBV携带者中HBVDNA阳性者为114例（阳性率为67．9％），其基因型分布为B、C、D型以及这3种基因型组成的混合型，而未发现A、E、F基因型。其中以B型、C型为主，所占比例为62．6％和36．8％，D型及混合型比例均为5．3％。C基因型患者中，HBV DNA呈现高水平复制（10^7～10^8 Copies／ml）的患者比例为26．2％（11／42），B基因型为13．3％（8／60）（P〈0．05）。C基因型患者血清抗-HBe阳性率（40．5％）显著高于B基因型（15．0％）（P〈0．01）。结论：黄石地区存在HBV的B、C、D基因型，以及由它们组成的混合基因型；B基因型为优势基因型；C基因型与HBV高复制水平，以及基因变异相关。     

难辨梭状芽胞杆菌性结肠炎和腹泻

人们越来越认识到难辨梭状芽胞杆菌(C.difficile,Cd)是肠道的重要病原菌。近年流行病学资料指出美国1/4住院病人感染此厌氧菌,许多人出现腹泻症状及结肠炎。本文归纳目前对该菌引起疾病流行病学及发病机制的认识并综述其诊断和治疗。 细菌学 Cd是革兰阳性产芽胞厌氧菌,可在含环丝氨酸和甲氧先锋培养基中生长,产生两种毒素;肠毒素(A)和细胞毒素(B)。该菌只在用抗生     

厌氧菌性呼吸系感染

以前即已明确,梭状芽胞杆菌属的气性坏疽杆菌、破伤风杆菌、肉毒杆菌等厌氧菌,在不良环境下可在体外形成芽胞,由外毒素引起人的严重疾病。但是,无芽胞厌氧菌则是构成人肠道和泌尿生殖器、口腔、上呼吸道正常寄生菌丛的重要细菌.虽作为肺、胸膜感染的病原菌决不少见,但过去下呼吸道厌氧菌感染的报告并不多,此有可能被忽略.Guillemot 等(1904年)最早报告13例厌氧菌性脓胸病人的胸液涂片检查,尽管发现大量细菌,但普通需氧培养几乎均为阴性.近年,由于气袋法和厌氧室法等厌氧菌培养技术已在     

抗生素和难辨梭状芽胞杆菌：病因与治疗

难辨梭状芽胞杆菌是最常见的院内胃肠道感染，疾病谱为引起从腹泻到具严重并发症的假膜性结肠炎（PMC）。这可能是一种严重的疾病，增加了发病率和死亡率。该致病菌产生引起结肠炎症和结肠炎的毒素A和B。根据大便标本中检查出的一种或两种毒素即可做出诊断。病情严重时，诊断中通过结肠镜检查可发现假膜，这些假膜黏着在结肠粘膜上，而且是由纤维蛋白、多形核细胞和碎片组成。难辨梭状芽胞杆菌相关性腹泻（CDAD）通常用口服灭滴灵或万古霉素治疗。大多数病人对治疗产生应答，但仍有10％到20％的病人将进展为再发性CDAD（RCDAD），因此需要再治疗。[第一段]     

Usefulness of immunological detection of both toxin A and toxin B in stool samples for rapid diagnosis of Clostridium difficile-associated diarrhea

Toxin detection from stool specimens is critical for the diagnosis of Clostridium difficile-associated diarrhea (CDAD). In Japan, only two toxin detection kits targeting toxin A alone are commercially available. We evaluated ImmunoCard Toxin A & B (ImmunoCard), based on enzyme immunoassay for the rapid detection of both C. difficile toxins A and B in stool specimens, compared to a toxin A detection kit (Uniquick) and cytotoxin assay. C. difficile was also cultured from stool specimens and the toxin production type of C. difficile isolates was identified by PCR analysis. Compared to cytotoxin assay, ImmunoCard sensitivity was 86.2%, specificity 93.8%, positive predictive value 91.8%, and negative predictive value 89.4% (n = 146). Sensitivity was significantly higher than that of Uniquick (60.0%, p = 0.0016). ImmunoCard detected 90.6% of cytotoxin positive specimens with isolated toxin A-positive, toxin B-positive C. difficile strains (Uniquick; 67.9%, p = 0.008) and 70.0% of these with isolated toxin A-negative, toxin B-positive C. difficile strains. Although ImmunoCard was slightly less sensitive than cytotoxin assay, it requires no special equipment and completes the entire test in up to 20 min. ImmunoCard thus appears very useful in the rapid diagnosis of CDAD in the clinical laboratory. Kits for the detection of both C. difficile toxins A and B should be immediately introduced into Japan to ensure the correct diagnosis of CDAD and infection control.     

Clinical and laboratory comparison of botulism from toxin types A, B, and E in the United States, 1975-1988.

Cases of adult botulism (n = 309) were studied to identify clinical differences between toxin types and to evaluate the sensitivity of diagnostic laboratory testing. Patients with illness from type E toxin had the shortest incubation periods. Sporadic case-patients were more severely ill: 85% required intubation compared with only 42% in multiperson outbreaks. Of patients with type A botulism, 67% required intubation compared with 52% with type B and 39% with type E. Toxin testing was positive for 40%-44% of serum and stool specimens obtained within 3 days of toxin ingestion and for 15%-23% of specimens obtained thereafter, while 37% of stool specimens obtained > 3 days after toxin ingestion were positive by culture. Patients with type A botulism have more severe illness. In general, specimens obtained early are more likely to be positive by toxin assay, and stool cultures are more sensitive than toxin detection for specimens obtained later in the illness.     

Clostridium difficile infections in HIV-positive patients

The prevalence of Clostridium difficile infections in HIV-positive patients with regard to the presence of its enterotoxin was investigated. Enzyme immunoassay (EIA, Meridian Diagnostic Inc) was used for the detection of C. difficile enterotoxin in stool specimens collected from 201 HIV-positive and 271 HIV-negative diarrheal patients. Culture was performed on cycloserine cefoxitin fructose agar. Chromosomal DNA types of C. difficile isolates were determined by pulsed-field gel electrophoresis (PFGE). In the HIV-positive group, C. difficile enterotoxin was found in 58.8% and 12.6% of diarrheal and non-diarrheal patients, repectively, whereas this toxin was found in 36.5% of HIV-negative-diarrheal patients. However, 13.6% of stool samples were negative by toxin assay, but were positive for C. difficile by culture and latex agglutination test. Among 11 isolates from both HIV-positive and HIV-negative patients, 6 patterns of PFGE type were observed: A, B, C, D, E and F.     

Detection of Clostridium difficile toxin A from stool specimens by an enzyme immunoassay kit]

Toxin detection from stool specimens is prerequisite for Clostridium difficile-associated diarrhea and colitis. However, in Japan only one toxin detection kit is commercially available, which requires computerized VIDAS fluorescence reader. In this study we evaluated ImmunoCard Toxin A, which is an enzyme immunoassay with a format of individual cassette and needs no special equipment to perform, by comparing with the VIDAS CDA kit. Of 61 stool specimens 12 were positive and 39 were negative by both assays, 7 were VIDAS positive-ImmunoCard negative, 2 were VIDAS invalid-ImmunoCard negative, and 1 was invalid by both assays. Stool specimens which gave inconsistent results between two assays were subjected to cell culture assay to detect toxin B and culture for C. difficile followed by testing of toxin producibility of isolates. Of 7 VIDAS positive-ImmunoCard negative specimens 5 were negative for cell culture assay and toxigenic C. difficile culture and 2 were positive for cell culture assay. By omitting 3 VIDAS invalid specimens and counting 5 specimens, which were cell culture negative but VIDAS positive, as negative and 2, which were cell culture positive and VIDAS positive, as positive, 12 of the 14 VIDAS positive specimens were ImmunoCard positive (sensitivity, 85.7%) and all 44 VIDAS negative were ImmunoCard negative (specificity, 100%). These results indicate that although in comparison with VIDAS CDA, ImmunoCard Toxin A is slightly less sensitive, ImmunoCard is a rapid, simple, and reliable C. difficile toxin A detection kit, which has the advantage of requiring no special equipment to perform and no centrifuge step, as small as 25 muL of a specimen, and as short as approximately 15 min of time to complete the whole testing.     

A nosocomial outbreak of diarrhea caused by toxin A-negative, toxin B-positive Clostridium difficile in a cancer center hospital

Between February and July 2001, 15 patients were diagnosed as Clostridium difficile-associated diarrhea in a ward of hematological neoplasm and lung cancer in a cancer center hospital. Of these 15 patients, 10 had malignant lymphoma, and 12 and 11 had exposure to antimicrobial agents and cancer chemotherapy, respectively, before the onset of diarrhea. Toxin A-positive, toxin B-positive (A+ B+) C. difficile was recovered from five patients and the remaining 10 patients suffered from diarrhea caused by toxin A-negative, toxin B-positive (A- B+) strains. All of the 10A- B+ isolates represented an identical banding pattern by PCR ribotyping and classified into one type (two subtypes) by pulsed field gel electrophoresis typing, indicating that a nosocomial outbreak of diarrhea caused by A- B+ C. difficile occurred among the patients hospitalized on this ward. Detection of toxin A in stool specimens by a toxin A detection kit was performed on 14 patients. Although two patients who carried A+ B+ strains were positive for toxin A assay, toxin A detection test was negative in 12 patients including 10 patients with A- B+ C. difficile infection. Diagnosis of C. difficile-associated diarrhea by combination of toxin A assay in feces and culture of C. difficile could successfully lead to recognition of an outbreak caused by A- B+ C. difficile in a cancer center hospital.     

Detection of enteroviruses in faeces by polymerase chain reaction.

A polymerase chain reaction (PCR) technique for the detection of human enteroviruses in stool specimens was developed. The test was based on the synthesis of cDNA, followed by PCR and slot blot hybridization. The primers used were selected from a highly conserved sequence in the 5'non-coding region of the enteroviral genome. By this method 27 different enterovirus serotypes (15 echo, 6 coxsackie A, 4 coxsackie B, poliovirus type 2 and enterovirus 71) from 89 patients could be detected. Using positive virus culture as reference, the sensitivity of PCR was 69% after 30 cycles of amplification, 91% using 30 + 10 cycles and 100% following 2 rounds of amplification with ensuing hybridization. None of 23 stool samples from healthy individuals or patients with meningitis of proven non-enteroviral etiology were positive by the PCR. By contrast, 13/26 culture-negative, randomly chosen stool samples from patients with suspected enteroviral disease were positive by the test. These findings demonstrate a high sensitivity and an apparently high specificity of PCR for detection of enteroviruses in stool samples. Therefore, the methodology may be useful in the laboratory diagnosis of enterovirus infections.     

Laboratory Approaches to the Diagnosis of Adenovirus Infection Depending on Clinical Manifestations

Abstract Background: While commercial enzyme immunoassays (EIA) intended for the detection of adenovirus in fecal specimens are widely used, there are no rapid, convenient, and sensitive commercial tests available for the detection of adenoviruses in respiratory and conjunctival specimens. The applicability of EIA for the detection of adenovirus in stool and throat samples was investigated. One-day rapid culture assay (RCA) for the detection of adenovirus in respiratory and conjunctival specimens was developed and evaluated. Patients and Methods: Stool samples from patients with gastroenteritis were tested by adenovirus EIA and by cell culture using human embryonic lung cells (HEL) and Graham 293 cells. Blood and stool samples from two BMT patients were also tested for adenovirus by PCR for at least 6 months. Throat specimens from patients with respiratory infections and conjunctival specimens were used for the evaluation of 1-day RCA compared with conventional adenovirus isolation in Graham 293 cells. Results: A total of 3,860 stool samples were tested by EIA Ridascreen^?, 8,169 by Novitec™, and 2,218 by ProSpectT^? yielding 135 (3.5%), 308 (3.7%), and 77 (3.5%) positive results, respectively. From 305 Ridascreen^?- and 340 Novitec™-negative stool samples, adenoviruses were isolated in three (0.9%) and eight (2.4%) cases, respectively, including two patients undergoing BMT. Multiple sequential stool samples from one BMT patient were repeatedly negative by EIA, but positive by PCR and cell culture. Graham 293 cells were better suited for isolation of adenovirus than HEL. EIA proved unreliable for detecting adenovirus in throat swabs. The sensitivity and specificity of RCA in throat swabs were 90% (37/41) and 100% (64/64), respectively, and 76% (16/21) and 100% (132/132) in conjunctival specimens, respectively. Conclusions: Generally, EIA is sufficiently sensitive for the diagnosis of adenovirus-associated diarrhea. However, it may not be sensitive enough to detect adenovirus in immunocompromised patients undergoing BMT and shedding very few viral particles in stools. Thus, in such cases, a more sensitive assay, such as PCR, is recommended. Furthermore, EIA is not sufficiently sensitive for the reliable detection of adenoviruses in throat swabs. One-day RCA may be useful for the detection of adenoviruses in respiratory and conjunctival specimens.     

Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection

AIM:To develop a real-time polymerase chain reaction(PCR) method to detect and quantify Campylobacter jejuni(C.jejuni) from stool specimens.METHODS:Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C.jejuni.The specificity of the primers and probe were tested against a set of Campylobacter spp.and other enteric pathogens.The optimal PCR conditions were determined by testing a series of conditions with standard a C.jejuni template.The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen.Two hundred and forty-two specimens were analyzed for the presence of C.jejuni by direct bacterial culture and real-time PCR.RESULTS:The optimal PCR system was determined using reference DNA templates,1 × uracil-DNA glycosylase,3.5 mmol/L MgCl 2,1.25 U platinum Taq polymerase,0.4 mmol/L PCR nucleotide mix,0.48 μmol/L of each primer,0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL.The PCR reaction was carried as follows:95 ℃ for 4 min,followed by 45 cycles of 10 s at 95 ℃ and 30 s at 59 ℃.The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10 3 CFU/g using DNA from stool specimens.Twenty(8.3%,20/242) C.jejuni strains were isolated from bacterial culture,while 41(16.9%,41/242) samples were found to be positive by realtime PCR.DNA sequencing of the PCR product indicated the presence of C.jejuni in the specimen.One mixed infection of C.jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.CONCLUSION:The sensitivity of detection of C.jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.     

PCR法与细菌培养法在急性腹泻患者沙门氏菌检测中的应用比较

目的通过与传统细菌培养法比较,明确聚合酶链反应(PCR)法能否提高沙门氏菌检出率。方法分别应用传统细菌培养法(含生化、血清学鉴定)及普通PCR方法,对300例急性腹泻患者粪标本进行检测,将两种方法检测的阳性率进行比较。结果300例急性腹泻患者粪标本,传统法分离培养出沙门氏菌29例,阳性率9.67%;普通PCR检测沙门氏菌高侵袭性位点A(hyperinvasive locus A,hilA)阳性65例,阳性率为21.67%,两者差异有统计学意义(P<0.01)。其中传统法分离培养阳性的标本,PCR检测均为阳性。PCR法扩增阳性的65例粪标本hilA基因测序结果与基因库标准菌株序列一致率为100%。结论与传统细菌培养生化法相比,PCR法可提高腹泻患者沙门氏菌的检出率,缩短检出时间,适于临床快速诊断。     

Enzyme-linked immunosorbent assay for the detection of Giardia lamblia in fecal specimens

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Giardia lamblia in human feces. The assay could detect between 37 and 375 trophozoites from cultures and between 12.5 and 125 cysts purified from human stool. Stool specimens were positive by ELISA in 36 (92%) of 39 patients with giardiasis; negative specimens came from patients with low numbers of parasites in their stool. In 10 patients stools became negative by ELISA after successful treatment. Stool specimens were positive by ELISA in three (2%) of 128 patients without demonstrable Giardia organisms in their stools; one positive specimen was from a child with IgA deficiency and diarrhea who responded clinically to metronidazole. The ELISA is a simple, sensitive, and specific diagnostic test for G lamblia that will be useful in diagnosis, in follow-up treatment, and in large-scale studies directed at defining the epidemiology and pathophysiology of G lamblia infection.