用PCR快速检测胃活检标本中的幽门螺杆菌

本文用ＰＣＲ对１１６份胃活检标本进行幽门螺杜菌（ＨｅｔｉｃｏｂａｃｔｅｒＰｙｌｏｒｉＨｐ）检测,总阳性率７１．５５％（８３／１１６）,胃溃疡、十二指肠溃疡和胃炎的阳性率分别为８７．５％（１４／１６）、８６．９６％（２０／２３）和６３．５１％（４７／７４）,同时用尿素酶法作参照,其总阳性率为５８．６２％（６８／１１６）,上述疾病分别为４３．７５％（７／１６）、５６．５２％（１３／２３）和６２．１６（４６／７４）。结果表明ＰＣＲ能快速、敏感特异地检出ＨＰ,这对研究ＨＰ在胃、十二指肠疾病中的致病作用、传播途径及疗效观察具有重要意义。     

从胃活检组织中分离幽门螺杆菌

比较了不同培养条件和方法对胃活检组织中幽门螺杆菌检出率的影响,从而建立了一种有效的分离培养方法。临床２０例胃活检组织,１４例组织学检查阳性,１０例快速尿素酶和常规尿素酶试验阳性,８例细菌培养阳性,显示此培养方法敏感性５７．１％。     

幽门螺杆菌感染与血清抗体及滴度的相关性研究

为了解幽门螺杆菌（ＨｅｌｉｃｏｂａｃｔｅｒＰｙｌｏｒｉ,ＨＰ）感染时机体的免疫状况,我们对１３４例上消化道疾病患者的血清免疫球蛋白进行了检测。结果表明,细菌培养阳性患者的三种血清抗体滴度显著高于细菌培养阴性患者。血清ＩｇＧ、ＩｇＡ、ＩｇＭ阳性与细菌培养阳性的符合率分别为７９．１％,７４．６％和４３．２％。活动性胃炎和非活动性胃炎的细菌培养阳性率分别为６６％和４２％。证实ＨＰ菌感染与活动性胃炎的发生密切相关,而与胃炎的严重程度无显著性差异。并证实了抗ＨＰ菌ＩｇＧ、ＩｇＡ检测具有较高的诊断ＨＰ菌感染的敏感性和特异性。     

猪胃螺杆样细菌与幽门螺杆菌的比较研究

我们检测１０例普通猪的胄组织,有８例分离到螺杆菌样细菌（ＨＬＯ）。其菌落、菌体形态和某些生化反应与幽门螺杆菌（ＨＰ）相似,但其尿素酶活性较低,ＨＬＯ全菌蛋白的ＳＤＳ一ｐＡＧＥ图谱也与ＨＰ的不同。本文就ＨＰ和ＨＬＯ及其伴发的人、猪慢性胃炎的特点,作了比较和讨论。     

牙斑幽门螺杆菌与慢性胃炎之间的关系

目的研究牙斑幽门螺杆菌与慢性胃炎之间的关系。方法对胃炎组、胃炎治疗组分别进行牙斑和胃黏膜幽门螺杆菌培养和比较。结果牙斑细菌培养：胃炎组阳性14例,阳性率为12．8％;治疗组阳性11例,阳性率为10．1％。胃黏膜细菌培养：胃炎组阳性47例,阳性率为43．1％;治疗组阳性19例,阳性率为17．4％。治疗前后比较牙斑标本差异无显著性（P〉0．05）,胃黏膜标本差异有非常显著性（P〈0．001）。结论牙斑中确实存在着幽门螺杆菌,而且是胃内反复感染的源泉,以致慢性胃病反复发作,难以治愈。     

氧化酶和尿素酶试验对HP感染的鉴别诊断

目的 探讨氧化酶和尿素酶对幽门螺杆菌（HP）感染的检出率和鉴别诊断。方法 采用氧化酶和尿素酶和涂片对1738例胃病患者进行HP检测。结果 1738例中氧化酶试验、尿素酶试验试验阳性检出率分别为47．41％、89．99％,阳性预测值为89．76％,阴性预测值为95．3％;尿毒酶试验的敏感性为99．72％,特异性为96．02％,阳性预浊值为95．36％,阴性预测值为9．76％,两者相比较各个参数间均无明显差（P＞0．05）。结论 氧化酶试验和尿毒酶试验一样对检测HP快速有效,两者联合应用可提供对HP感染诊断和鉴别诊断的准确性。     

嗜热链球菌培养条件的研究

将嗜热链球菌（Ｓｔｒｅｐｔｏｃｏｃｃｕｓｔｈｅｒｍｏｐｈｉｌｕｓ）在６种固体培养基及７种液体培养基中的生长情况进行了比较,结果表明,改良ＩＲＩＥ固体培养基上的溶钙圈大,活菌数最多;在７种液体培养基中亦以改良ＩＲＩＥ的ＯＤ值最高,活菌最多,达８×１０^８／ｍｌ以上。以此培养基做生长曲线,该菌的平衡期为１２～１６ｈ。     

C57BL/6小鼠幽门螺杆菌感染动物模型的建立

目的：建立幽门螺杆菌（Helicobacter pylori,Hp）小鼠感染模型。方法：建立Hp经口感染SPF级小鼠的动物模型,取小鼠胃粘膜组织,利用PCR技术、尿素酶实验、细菌培养等方法检测接种小鼠,对结果进行判定。结果：Hp可感染C57BL／6小鼠并在小鼠胃部定植。     

平菇汤培养基的研究及应用

研究以平菇汤为基础成分,代替动物组织提取物,制备平菇液体培养基,平菇血琼脂培养基。平菇血琼脂培养基对１４０份痰及中段尿标本阳性菌的检出率为３４．３％,传统血琼脂培养基对阳性菌的检出率为３２．１％。平菇液体培养基和普通营养肉汤培养基同时对８０份分泌物标本培养,其检出率分别为３８．８％和３６．３％（ｐ＞０．０５）。平菇汤培养基制备简便,成本低廉,有较好的实用价值。     

聚合酶链反应快速检测胃组织和唾液中的幽门螺杆菌

针对幽门螺杆菌（ＨＰ）尿素酶Ａ基因设计一对引物进行聚合酶链反应,检测１株ＨＰ标准株和７株临床分离株均阳性,而４株其它肠道菌均阴性,特异性１００％。１０倍系列稀释试验表明敏感性达到１００ｐｇＤＮＡ水平。从３５例胃镜检查者取幽门旁组织块进行快速和常规尿素酶试验,细菌培养及ＰＣＲ检测,１５例ＨＰ阳性者ＰＣＲ检测也为阳性,其中７例阳性者有３例唾液ＰＣＲ检测为阳性,表明ＨＰ确存在于口腔中。本研究采用直接热裂解法处理临床标本,取其粗提物行ＰＣＲ,免除复杂的酚一氟仿抽提步骤,该法简便快速,且损失小,成功率高,在临床实验诊断中有推广价值。     

Improving the success of culturing Helicobacter pylori from gastric biopsies

Factors influencing the successful isolation of Helicobacter pylori from human gastric biopsies were studied. Within 24 h, each of the gastric biopsies was inoculated onto chocolate blood agar media and incubated for up to 2 weeks. Among 63 (70%) culture positive cases in 90 patients, 58 (64%) cases were culture positive for both specimens, while five (6%) cases were culture positive in only one biopsy. Of the 63 positive cultures, 51 H. pylori strains (81%) grew on both media with and without antibiotics. Eight strains (13%) grew only on medium without antibiotics, while four isolates (6%) were obtained only from medium with antibiotics. These results support the previous histological observation of patchy colonization of H. pylori in the stomach. The success rate for culture of H. pylori from gastric biopsies increased when two biopsies were taken and inoculated on chocolate blood agar media with and without antibiotics.     

Dual-Priming Oligonucleotide-Based Multiplex PCR for the Detection of Helicobacter pylori and Determination of Clarithromycin Resistance with Gastric Biopsy Specimens

Background:  Assessment of Helicobacter pylori ( H. pylori ) clarithromycin resistance has rarely been performed routinely despite an increasing resistance rate. Our aim was to develop and evaluate the use of dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) to detect point mutations in the 23S rRNA gene responsible for clarithromycin resistance of H. pylori.
Materials and Methods:  Gastric biopsy specimens from 212 untreated patients with dyspepsia were examined by culture, histology, and DPO-based multiplex PCR. A disk diffusion test and E-test were used for performing phenotypic antibiotic susceptibility tests.
Results:  Among the biopsy specimens tested, 22.2% (47/212), 42.5% (90/212), and 41.5% (88/212) of the specimens were classified as H. pylori positive by culture, histology, and DPO-based multiplex PCR, respectively. Among 96 strains identified by either culture or DPO-based multiplex PCR, 80 strains were clarithromycin-susceptible and 16 strains (16.7%) were clarithromycin-resistant. There was 94.1% (32/34) concordance between phenotypic susceptibility tests and DPO-based multiplex PCR. In two patients with discrepant results, only DPO-based multiplex PCR detected clarithromycin-resistant strains. DPO-based multiplex PCR identified additional 49 clarithromycin-resistant or clarithromycin-susceptible H. pylori among 165 culture-negative specimens.
Conclusions:  DPO-based multiplex PCR can be used as a practical method for the detection of H. pylori infection and the determination of clarithromycin susceptibility in addition to phenotypic antimicrobial susceptibility tests.     

The results of Helicobacter pylori eradication on repeated bleeding in patients with stomach ulcer

The triple therapy of Helicobacter pylori eradication prevents repeated bleeding from stomach ulcer. The aim of this one-way blind prospective study was to evaluate the efficiency of the two-week triple therapy for Helicobacter pylori eradication in preventing renewed bleeding in patients with stomach ulcer within one year. This research included 60 hospitalized patients with bleeding stomach ulcer and positive Helicobacter pylori infection, 34 men and 26 women (average age 59.7 years). The patients were given therapeutic scheme of omeprazol--amoxicilin--metrodinazol (OAM) eradication for 14 days. Eradication of H. pylori infection was defined as lack of proof of the infection one month or several months after therapy suspension. By applying triple OAM therapy within two weeks the eradication was successful in 72%. In the group of 17 H. pylori positive patients there were 8 patients (47.6%) with repeated stomach ulcer and 3 patients (18%) with bleeding. Within the group of 43 H. pylori negative patients there were only 2 patients (4.65%) with repeated stomach ulcer and 1 patient (2%) with bleeding, during the observed period of 12 months. This research confirms the hypothesis about the necessity of eradication of Helicobacter pylori infection in patients with bleeding stomach ulcer as prevention of repeated bleeding.     

Helicobacter pylori stool antigen test in patients with bleeding peptic ulcers

BACKGROUND: Helicobacter pylori has been linked to chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Invasive tests are less sensitive than noninvasive tests in diagnosing H. pylori infection in patients with bleeding peptic ulcers. The H. pylori stool antigen test has been useful in diagnosing H. pylori in patients with peptic ulcers before and after eradication of H. pylori. The aim of this study was to evaluate the H. pylori stool antigen test in patients with bleeding peptic ulcers. METHODS: Patients with bleeding and nonbleeding peptic ulcers underwent a rapid urease test, histology, bacterial culture and H. pylori stool antigen test. Positive H. pylori infection was defined as a positive culture or both a positive histology and a positive rapid urease test. Helicobacter pylori stool antigen was assessed with a commercial kit (Diagnostec H. pylori antigen EIA Kit, Hong Kong). RESULTS: Between October 2000 and April 2002, 93 patients with bleeding peptic ulcers (men/women: 78/15, gastric ulcer/duodenal ulcer: 58/35) and 59 patients with nonbleeding peptic ulcers (men/women: 47/12, gastric ulcer/duodenal ulcer: 30/29) were enrolled in this study. Forty-seven (50.5%) patients with bleeding peptic ulcers and 30 (50.8%) patients with nonbleeding peptic ulcers, were found to be infected with H. pylori (p > .1). Helicobacter pylori stool antigen tests were positive in 54 (58.1%) and 30 (50.8%) patients with bleeding peptic ulcers and nonbleeding peptic ulcers, respectively (p > .1). The sensitivity (82% vs. 93%), specificity (68% vs. 93%), positive predictive value (74% vs. 93%), negative predictive value (77% vs. 93%) and diagnostic accuracy (75% vs. 93%) were all lower in patients with bleeding vs. nonbleeding peptic ulcers. The specificity, positive predictive value, and diagnostic accuracy of the H. pylori stool antigen test in patients with bleeding peptic ulcers were significantly lower than those in patients with nonbleeding peptic ulcers (p = .01, p = .02 and p = .003, respectively). CONCLUSION: The H. pylori stool antigen test is not reliable for diagnosing H. pylori infection in patients with bleeding peptic ulcers.     

H.pylori抗体鸡蛋制品对H.pylori感染BALB/c小鼠的实验研究

目的通过幽门螺杆菌(Helicobacter pylori,H.pylori)标准株的接种,建立BALB/c小鼠感染H.pylori胃炎动物模型,评价H.pylori抗体鸡蛋制品对小鼠感染性胃炎的预防效果。方法将灭活的H.pylori国际标准菌株(NCTC 11637)作为抗原,对产蛋鸡进行免疫。免疫后收集鸡蛋,对达到效价的鸡蛋,无菌采集卵黄。BALB/c小鼠60只,适应性喂养1周后,随机分为5组,Ⅰ组为胃炎模型组,Ⅱ组为生理盐水组,Ⅲ、IV、V组分别为低剂量、中剂量和高剂量卵黄抗体组,每组12只。Ⅰ组予H.pylori菌液灌胃造模,Ⅱ组先予生理盐水灌胃后再予H.pylori菌液灌胃对照,Ⅲ、IV、V组分别用不同剂量抗H.pylori卵黄抗体灌胃后再予H.pylori菌液灌胃造模。小鼠均于距最后一次灌胃后8周全部处死,用微需氧细菌培养检测H.pylori定植;HE染色观察小鼠胃黏膜病理组织学改变。结果在接种H.pylori后第8周,Ⅰ组和Ⅱ组小鼠胃内均有大量H.pylori定植,感染率为91.7%,Ⅲ组的感染率是58.3%,IV和V组的感染率均小于30%。结论 H.pylori抗体鸡蛋制品可以抑制BALB/c小鼠感染H.pylori,抗体的保护作用与抗体的剂量呈正相关。     

Detection of Helicobacter in the liver of patients with chronic cholestatic liver diseases.

Helicobacter species were identified in human liver tissues by PCR. Biopsies were obtained from patients with primary sclerosing cholangitis, primary biliary cirrhosis and noncholestatic liver cirrhosis. One set of Helicobacter genus-specific primers and two different primer sets for Helicobacter pylori were used in the PCR-assays. Using Helicobacter genus-specific primers 80% (8/10) of patients with primary sclerosing cholangitis and 90% (9/10) of patients with primary biliary cirrhosis were positive. Seven of these 17 samples were positive using two different primers for H. pylori and Southern blot hybridization. Among the non-cholestatic liver cirrhosis controls, only one sample was positive in the Helicobacter genus-specific PCR-assay. Significantly higher values of alkaline phosphatases and prothrombin complex was found for the patients positive for Helicobacter genus. In conclusion, gene sequences of Helicobacter species and H. pylori were detected in human liver tissue using PCR and DNA hybridization in patients with a cholestatic liver disease, but rarely in noncholestatic liver cirrhosis.     

Development of a plating medium for selection of Helicobacter pylori from water samples

The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media. Three organisms (Acinetobacter, E. coli, and H. pylori) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (10(6) CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37 degrees C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.     

Helicobacter species colonizing pig stomach: molecular characterization and determination of prevalence.

The infection rate of 60 pigs (10 pigs from each of six farms) by Helicobacter species was studied by two techniques. Histological examination of the cardiac area of the stomach yielded a 58% positive result versus an 80% positive result by PCR with genus-specific primers. Eighty percent of the 16S rRNA gene was amplified, classified in four groups by PCR-restriction fragment length polymorphism, and sequenced. Isolates from all farms except one (farm C) were identified as Helicobacter heilmannii type 1, while those from farm C were identified as H. heilmannii type 2. Attempts to culture this organism in vitro failed. Helicobacter pylori was not found in these animals.     

Use of HP selective medium to detect Helicobacter pylori associated with other enteric bacteria in seawater and marine molluscs

AIMS: This project investigated the utility of HP selective medium to isolate H. pylori cells from seawater and from marine molluscs. METHODS AND RESULTS: Nested-PCR was performed to reveal the presence of Helicobacter genus. All samples were cultured in HP selective medium and 16 cultures were initially selected as putative Helicobacter. Helicobacter spp. DNA were detected in 9/16 cultures and three of them had 99-100% homology to H. pylori based on 16S RNA gene sequence. Helicobacter pylori isolation was unsuccessful. On the basis of 16S RNA gene sequences the contaminating organisms were shown to be Proteus mirabilis and Vibrio cholerae. CONCLUSIONS: These results indicate the coexistence of three predominant bacterial genera in the cultures and that HP selective medium can grow other enteric bacteria besides Helicobacter. Additional assays will improve the HP selective medium formulation for marine samples avoiding P. mirabilis and V. cholerae interferents. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows the effectiveness of the selective HP medium for the Helicobacter culture from marine samples.