幽门螺杆菌尿素酶临床应用和疫苗研制进展

尿素酶为幽门螺杆菌在胃内定植和生存所必需,是幽门螺杆菌的主要毒力因子。近年来,对幽门螺杆菌尿素酶分子生物学特性、致病机理的深入研究使人们在幽门螺杆菌的诊断、治疗和疫苗研制方面有了一些新的认识和进展。     

幽门螺杆菌感染可视化诊断的临床研究

目的改革国外对幽门螺杆菌（Ｈｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉ，Ｈｐ）感染可视化诊断中的繁杂方法。方法对３００例经胃镜检查病例，在胃镜直视下采用喷洒ｐＨ５．５的调节水来调整胃黏膜表面的ｐＨ值，再对胃黏膜表面直接喷洒０．５ｍｏｌ／Ｌ尿素０．１％酚红溶液。通过酚红的变红反应，使Ｈｐ的感染及其分布可视化。并对尿素酚红液变色呈阳性反应的病例，在变色部位取活检，对呈阴性反应的不变色病例，在胃窦近幽门２ｃｍ左右的前、后壁、胃窦前庭或胃体大弯分别取活检２～３块，进行病理组织切片的Ｇｉｅｍｓａ染色检查并将两者相对照。结果３００例中１９０例胃镜喷洒尿素酚红溶液后胃黏膜表面变红呈阳性反应，病理检查Ｈｐ呈阳性者１８２例，诊断符合率为９５．８％（１８２／１９０）。１１０例呈阴性反应病例，病理检查Ｈｐ呈阴性者９５例，两者符合率为８６．４％（９５／１１０）。镜下诊断与病理诊断总符合率为９２．３％（２７７／３００）。结论对Ｈｐ感染的可视化诊断，改良的尿素酚红液喷洒为一种简单可行的内镜下诊断方法。     

幽门螺杆菌根除治疗前后快速尿素酶试验诊断的准确性

目的 评价快速尿素酶试验(RUT)在根除治疗前后诊断幽门螺杆菌(Hp)感染的准确性。方法 选择250例接受胃镜检查的患者，123例无Hp根除治疗史，127例为Hp根除治疗后复查患者。每例患者取胃窦和胃体活检标本各3块，分别用于RUT、细菌培养和病理组织学检查。以细菌培养及病理组织学检查结果作为“金标准”，即培养和(或)组织学检查结果阳性者为Hp阳性，而培养和组织学检查结果同时阴性者为HP阴性或HP根除。结果 末行Hp根除治疗的患者RUT正确的诊断了86例Hp阳性中的84例和37例Hp阴性中的34例，其敏感性和特异性分别为97．7％和91.9％。根除治疗后RUT敏感性和特异性分别为64.3％和99.0％。然而，根除治疗后6个月以上复查胃镜，RUT敏感性和特异性均达100％。结论 根除治疗前和根除治疗后6个月以上复查，RUT诊断Hp感染准确性高。     

Immunological rapid urease test using monoclonal antibody for Helicobacter pylori

BACKGROUND AND AIM: The current diagnostic methods for detecting Helicobacter pylori infection include rapid urease test (RUT), urea breath test (UBT), histology, culture, and serum antibody detection. The present study evaluated the efficacy of a novel highly specific test, an immunological RUT (IRUT), that uses a monoclonal antibody against H. pylori urease. METHODS: The clinical evaluation of the IRUT was performed in 100 subjects. Each gastric mucus sample obtained during endoscopic examination was incubated for 15 min with a solid tip coated with monoclonal antibody for H. pylori urease, and then the tip was introduced into a pH-monitoring cell containing urea solution. The change in pH of the solution after the enzymatic reaction (delta pH) was measured. The performance of the IRUT was compared with culture, histology, RUT, and UBT. RESULTS: Of the 47 H. pylori-positive subjects, 43 were IRUT positive (sensitivity, 91.5%), and of the 53 H. pylori-negative subjects, 52 were negative (specificity, 98.1%). Compared with the usual diagnostic methods, IRUT had high sensitivity and specificity for the detection of H. pylori and was no less efficient. CONCLUSIONS: IRUT is a sensitive, specific and very rapid (within 20 min) method of detecting H. pylori infection.     

艾滋病与肝病患者胃窦黏膜活检快速尿素酶检测幽门螺杆菌与病理学染色的比较研究

目的探讨胃窦黏膜活检快速尿素酶试验(RUT)和病理学法检测幽门螺杆菌(Helicobacter pylori,H.pylori)的阳性率及其在艾滋病(AIDS)、慢性肝病和普通慢性胃病患者中的差异。方法选取937例因上腹部不适接受胃镜检查的患者,其中艾滋病组患者61例(艾滋病组),慢性肝病组患者600例(慢性肝病组),非艾滋病非肝病普通慢性胃病组患者276例(普通慢性胃病组)。所有患者均于胃镜检查中胃窦部取活检组织2块,分别进行RUT和病理学检测法检测H.pylori。结果胃窦黏膜活检RUT H.pylori的总体阳性率为47.0%,病理学染色检测法的总体阳性率为35.3%;艾滋病组和普通慢性胃病组病理学检测H.pylori阳性率(45.9%、57.97%)均高于同组RUT检验H.pylori阳性率(37.7%、38.77%,P0.05);慢性肝病组RUT检测H.pylori阳性率(42.83%)高于病理学检测H.pylori阳性率(32.67%)(P0.05);以病理学检测结果为最终诊断,慢性肝病组患者H.pylori感染率小于艾滋病组和普通慢性胃病组(P0.05),艾滋病组患者H.pylori感染率小于普通慢性胃病组,但差异无统计学意义(P0.05)。结论艾滋病、慢性肝病和普通慢性胃病患者胃窦黏膜活检RUT与病理学法检测H.pylori均有显著性差异,临床最终诊断结果仍应以病理学检测结果为准;慢性肝病患者RUT检测H.pylori存在较高假阳性及其H.pylori感染率低于艾滋病患者和普通慢性胃病患者。     

表达幽门螺杆菌尿素酶B亚单位的减毒鼠伤寒杆菌口服疫苗的制备

目的制备抗幽门螺杆菌(Hp)尿素酶B亚单位(UreB)减毒鼠伤寒杆菌活菌疫苗,观察其免疫效果.方法构建表达UreB的原核表达载体PTc01-UreB并转化减毒鼠伤寒杆菌SL3261,得到重组菌SL3261/PTc01-UreB.应用抗Hp菌体蛋白兔血清行Western-blot检测UreB在SL3261中的表达.将SL3261/PTc01-UreB口服免疫Balb/c小鼠,12周后应用ELISA检测肠液和血清中的特异性抗体反应.SL3261/PTc01-UreB在Luria-Bertani培养液中连续传代60代以确定其稳定性.结果成功构建PTc01-UreB原核表达载体.Western-blot显示,其转化减毒鼠伤寒杆菌SL3261后能表达相对分子质量约61×103的蛋白,与HpUreB亚单位相符,具有抗原性.口服免疫小鼠后,在肠液和血清中可分别检测到针对UreB的特异性IgA和IgG抗体.体外连续培养60代未见PTc01-UreB质粒丢失及对宿主细胞毒性.结论表达HpUreB的减毒鼠伤寒杆菌SL3261/PTc01-UreB可用作抗Hp感染口服疫苗.     

Modified rapid urease test for Helicobacter pylori detection in relation to an immunohistochemical stain

BACKGROUND: The rapid urease test and touch cytology have been used for the rapid detection of Helicobacter pylori infection. Recently, a modified rapid urease (MRU) test, which provides results in 20 min has been available on a commercial basis. To date, few reports have evaluated the accuracy of this test. This study evaluated the sensitivity, specificity, and accuracy of the MRU test and touch cytology to detect H. pylori in relation to the density of H. pylori infection determined semi-quantitatively by using immunohistochemical stains. METHODS: Biopsy specimens obtained from a total of 60 patients who underwent endoscopy for evaluation of gastroduodenal diseases were studied by using the MRU test, Giemsa stain for touch smear tissue and histological methods. An immunohistochemical stain was used as a standard, and the density of H. pylori infection was graded according to the number of individual bacteria seen as follows: grade 0 = 0; grade 1+ = 1-9; grade 2+ = 10-29; grade 3+ = 30-99; grade 4+ > or = 100. The severity of gastritis was evaluated histologically in each specimen and compared with the density of H. pylori infection. RESULTS: The MRU test had an overall sensitivity of 73%, specificity of 100% and accuracy of 85%. The Giemsa stain had a sensitivity of 91%, specificity of 100% and accuracy of 95%.The sensitivities of the MRU test and Giemsa stain decreased in mild H. pylori infection. In the MRU test, the sensitivity was 47% when the density of H. pylori infection was 1+, while 80-100% sensitivities were obtained when the densities of infection were > or = 2+. With the Giemsa stain, the sensitivity was 80% when the density was 1+, while the sensitivity increased to 100% when the densities were > or = 2+. The severity of gastritis determined by the Rauws scores showed a positive correlation with the density of H. pylori infection as evaluated by immunohistochemical staining. CONCLUSIONS: The MRU test had high sensitivity and specificity for moderate to severe H. pylori infection, but it may result in false-negative results in tests for mild infection. As the MRU test has the advantages of shorter incubation times and low cost, a combination of the MRU test and the Giemsa stain for touch cytology may be the most time- and cost-efficient tests in a clinical setting for the diagnosis of H. pylori infection.     

~(14)C-呼气试验检测幽门螺杆菌感染

本文应用国产~(14)C-呼气试验(~(14)C-UBT)试剂检测幽门螺杆菌(Hp)。同时应用病理组织染色、尿素酶试验、细菌培养和血清 ELISA 技术等方法与之进行比较。117例病人中 Hp 阳性率为47.9%(56/117),~(14)C-UBT的敏感性与特异性分别为94.6%和93.4%。~(14)C-UBT 由于相对于胃镜依赖的方法而言创伤性大大降低,是目前检测 Hp 感染,尤其是药物治疗后复查时的首选方法。     

Clinical assessment of the bacterial load of Helicobacter pylori on gastric mucosa by a new multi-scaled rapid urease test

The present study tests the efficacy of the multi-scaled urease test (MUT) in detecting Helicobacter pylori infection and determines whether the MUT can predict the bacterial density on histology. A total of 111 sets of gastric specimens were obtained from patients with dyspepsia but without recent bleeding. Two biopsies were taken as closely as possible in each set. One sample was used for the MUT (Hp fast; GI Supply, Camp Hill, PA, USA), while the other was used to determine the histological density of H. pylori by modified Giemsa stain (grade 0–5). The results of MUT were interpreted as negative if the colour was yellow or bright green (reaction score 0) and positive if the colour was green, light blue, or blue (reaction score 1, 2 and 3, respectively). The reaction scores of MUT were recorded sequentially at 15 and 30 min and 1, 4 and 24 h. On the basis of histological confirmation, MUT had a sensitivity of 89.6%, a specificity of 88.2%, a positive predictive value of 94.5% and a negative predictive value of 78.9%. Focusing on specimens with the presence of bacteria under histology, 77 specimens were divided into five subgroups by grades of density of H. pylori (HPD1–5). The reaction scores had become sequentially elevated from 30 min through to 24 h in each subgroup. For subgroups HPD4 and 5, the positive rates of MUT were 70.6 and 66.6%, respectively, as early as 30 min and progressed to 100% within 4 h. In contrast, the positive rate for the HPD1 subgroup was 16.6% at 4 h and increased to only 62.5% at 24 h. In subgroups HPD 2 and 3, the positive rates were less than 30% at 30 min, but became more than 66.6% at 4 h and were 100% at 24 h. The early (i.e. mean value of reaction scores before 4 h) and late (24 h) mean reaction scores disclosed two elevated trends as the density of H. pylori increased (early: 0.2, 0.7, 0.8, 1.5, 1.2; late: 1.4, 2.3, 2.6, 3.0, 3.0; P < 0.05). In conclusion, MUT is a reliable method for the diagnosis of H. pylori infection. It can also indirectly predict the density of H. pylori on histology.     

Field evaluation of a rapid, simple and inexpensive urease test for the detection of Helicobacter pylori

A modified rapid urease test (MRU test) for the detection of Helicobacter pylori was evaluated under field conditions during an endoscopic survey in rural India and compared with a commercially available urease test (CLO test) and with histology. Of 195 consecutive subjects who underwent upper gastrointestinal endoscopy, 153 (78.5%) were positive for Helicobacter pylori when tested by the CLO test and/or histology. The sensitivity and specificity of the MRU test relative to this was 97.4 and 95.2%, respectively when the test was read over a 3 h period. The MRU test was positive in 77.4, 89.0, 93.8 and 96.6% of cases at 1, 5, 20 and 60 min, respectively, compared with 2.7, 14.4, 48.6 and 71.2% of cases for the CLO test at the same time. The accuracy of the MRU test was thus similar to that of other methods for the detection of Helicobacter pylori. Furthermore, it gave a positive diagnosis more rapidly than other tests, in most cases before the subject had left the endoscopy suite. The MRU test is extremely simple to prepare and read and costs less than 0.05 pounds per test compared with 2.26 pounds for a CLO test. It is suitable for use in clinical or epidemiological work and especially where cost factors are critical.     

The effect of test duration on the sensitivity and specificity of ultra-rapid urease test for the detection of Helicobacter pylori infection

Background: 'Home made' ultra-rapid urease tests are used extensively in the Asia Pacific region. Data on the reliability of these 'home made' tests are limited.
Aims: To evaluate the effect of test duration on the sensitivity and specificity of a self-prepared biopsy urease test for the detection of Helicobacter pylori.
Methods: Using histology as the 'gold standard', the effect of test duration on the sensitivity and specificity of a self prepared urease test for the detection of H.pylori was evaluated in 411 consecutive patients undergoing upper gastrointestinal endoscopy.
Results: Histology was positive for H.pylori in 217 of the 411 patients (52.8%). Within 24 hours of retrieving the specimen, 189 (87.1%) of the histology positives and 174 (89.7%) of the histology negatives were correctly identified by the urease test. Of the H.pylori positives, 72.0%, 81.0%, 89.9% and 100% were detected by the urease test within one minute, five minutes, three hours and 24 hours respectively. Thus, sensitivities of the urease test at one minute, five minutes, three hours and 24 hours were 62.7%, 70.5%, 78.3% and 87.1% respectively. Corresponding figures for the specificity were 93.8%, 93.3%, 92.3% and 89.7% respectively. Using a receiver-operating characteristic curve, an optimal combination of sensitivity and specificity was obtained when the urease test was read at 24 hours.
Conclusions: While the biopsy urease test was positive in most cases within a minute, better results could be obtained if the test continued to be read over a 24 hour period.     

尿素酶疫苗在防治幽门螺杆菌感染的动物实验研究

目的利用人类幽门螺杆菌（Ｈｐ）感染的小鼠模型研究Ｈｐ粗抗原及纯化尿素酶在预防与治疗Ｈｐ感染的作用。方法超声粉碎制备Ｈｐ全菌抗原,化学提纯制备Ｈｐ尿素酶,霍乱毒素（ＣＴ）为免疫佐剂。实验分为预防与治疗两部分。预防部分把实验动物无特定致病菌（Ｓ．Ｐ．Ｆ．）ＢＡＬＢ／ｃ小白鼠分成５组,分别通过灌胃方法给予尿素酶（２５０μｇ）加ＣＴ（２μｇ）、Ｈｐ粗抗原（１ｍｇ）加ＣＴ（２μｇ）、生理盐水、单纯尿素酶（２５０μｇ）、单纯ＣＴ（２μｇ）,每周１次,共４次。２周后再用活Ｈｐ灌胃,再４周后处死动物。治疗部分把已感染Ｈｐ的小白鼠分成５组,分组与治疗方法同预防部分,治疗结束后４周处死动物,取胃粘膜行尿素酶试验与改良Ｇｉｅｍｓａ染色检查Ｈｐ情况。结果预防实验的保护率分别为,尿素酶加ＣＴ７０％（７／１０）,Ｈｐ粗抗原＋ＣＴ８０％（８／１０）,生理盐水、单纯尿素酶、单纯ＣＴ保护率均为０。治疗实验各组Ｈｐ根除率分别为：尿素酶加ＣＴ６３．６％（７／１１）,Ｈｐ粗抗原加ＣＴ４４．４％（４／９）,生理盐水组、单纯尿素酶、单纯ＣＴ组均无治疗作用。结论由尿素酶加免疫佐剂组成的口服疫苗,不仅有预防Ｈｐ感染的作用,同时也有根除已感染的Ｈｐ的作用。     

重组减毒沙门菌尿素酶B亚单位和过氧化氢酶疫苗治疗幽门螺杆菌感染的实验研究

目的　利用人类幽门螺杆菌 (Hp)感染的小鼠模型研究重组减毒沙门菌尿素酶B亚单位和过氧化氢酶疫苗在治疗Hp感染中的作用。 方法　将 30只二级C5 7BL/ 6小鼠随机分成 3组 ,通过灌胃方法每只小鼠均用活力良好的Hp菌株隔日攻击 2次。在第二次Hp攻击后 4周 ,分别给予重组减毒沙门菌尿素酶B亚单位疫苗 (A组 )、过氧化氢酶疫苗 (B组 )和生理盐水 (C组 )灌胃 1次 ,4周后处死动物 ,取胃组织分别行尿素酶试验、改良Giemsa染色及定量细菌培养 ,观察Hp定植情况。行HE染色观察胃黏膜组织炎症情况。取脾组织行淋巴细胞增殖试验。结果　C组小鼠Hp定植密度为 1.92× 10 6CFU/g胃组织 ,A组和B组小鼠分别为 1.5 8× 10 5CFU/g和 4 .88× 10 5CFU/g胃组织 ,两个治疗组的定植密度明显降低 (P <0 .0 5 )。治疗组与对照组胃黏膜均无明显炎症反应。治疗组脾淋巴细胞增殖试验阳性。结论　重组减毒沙门菌尿素酶B亚单位疫苗和过氧化氢酶疫苗对Hp感染有治疗作用。     

幽门螺杆菌粪便抗原检测在幽门螺杆菌感染诊断及随访中的价值

目的 对幽门螺杆菌粪便抗原（HpSA）检测诊断hp感染、hp根除治疗后的随访及在儿童中的应用价值,进行临床评价。方法 以快速尿素酶试验=、组织学和细菌培养中的二项阳性或细菌培养一项阳性作为诊断HP的金标准,采用ELISA法检测128例因上消化道症状接受胃镜检查患者HPSA,评价HPSA诊断HP感染的敏感性、特异性;其中79例同时进行^13C-UBT作为对照,评价HPSA诊断HP感染的准确性。结果 在128例中,HPSA检测的敏感性、特异性和准确性分别为95.16%,98.41%和96.80%。根除治疗前HPSA检测和^13C-UBT诊断HP的敏感性为97.96%和95.92%,特异性为96.67%和100.00%,准确性均为97.46%;根除治疗后4周随访HPSA检测和^13C-UBT的敏感性均为100.00%,特异性为91.43%和94.29%,准确性 93.18%和95.45%。以^13C-UBT作标准,HPSA检测29例患儿HP感染敏感性、特异性和准确性分别为91.67%,88.23%和89.66%。结论 HPSA检测是一种简便、准确、非侵入性的诊断HP感染的方法,适用于HP感染的诊断、根除治疗后随访及儿童患者。     

幽门螺杆菌感染诊断方法的比较

338例病人同时进行了快速尿素酶试验(简称RUT)。Warthin-Starry染色(简称W-S染色)、培养法、~(13)C-尿素呼气试验(~(13)C-UBT)、血清IgG、IgM等6种诊断方法中任意3种检查,以同时2种(或以上)检查方法一致的结果作为诊断幽门螺杆菌(Hp)是否存在的标准。对上述6种方法的敏感性、特异性、符合率、阳性预测值、阴性预测值分别给予评价,结果显示:RUT、W-S染色、~(13)C-UBT三法诊断Hp的敏感性、特异性等较高,均接近或高于90％。     

Detection of clarithromycin resistance in Helicobacter pylori following noncryogenic storage of rapid urease tests for 30 days

OBJECTIVE: Traditional Helicobacter pylori (H. pylori) eradication therapy has been undermined by increasing antimicrobial, especially clarithromycin, resistance. Susceptibility testing in some areas is difficult to achieve or unavailable. We aimed to determine whether gastric biopsy specimens stored at room temperature for rapid urease test (RUT) were suitable for clarithromycin susceptibility testing of H. pylori. METHODS: After 30 days of storage at room temperature, DNA was extracted from gastric biopsies present in RUTs (Hpfast). H. pylori status and clarithromycin susceptibility were evaluated using H. pylori‐specific polymerase chain reaction (PCR) for ureA, vacA, and allele‐specific primer‐PCR of the 23S rRNA genes. The PCR results were compared with histology, RUT, and culture results. H. pylori positive was defined as RUT and either culture or histology positive; H. pylori negative as RUT, culture and histology negative. RESULTS: Samples from 31 patients were evaluated; 11 were H. pylori positive including 9 by culture; seven of which had allele‐specific primer‐PCR results from the RUT specimen for the detection of mutations of the 23S rRNA gene. When both tests were available, culture and PCR results were concordant in 7 cases. In 15 of the 20 histology, RUT and culture negative patients, three PCR were negative. In one patient, all of the three tests were positive; and in three only the 23S rRNA was positive and in one only ureA was positive. CONCLUSION: Gastric biopsy specimens stored in the gel of RUT for 30 days can be used for molecular testing to confirm the diagnosis of H. pylori infection and test for clarithromycin susceptibility.     

安速快速检测试剂盒诊断幽门螺杆菌的现症感染

目的:评价安速幽门螺杆菌(H pylori)快速检测卡诊断H pylori现症感染的敏感性及特异性.方法:对154例因上消化道症状进行胃镜检查的患者同时进行快速尿素酶试验及Warthin- Starry银染,同时取指血进行安速快速检测试剂盒检测.以快速尿素酶试验和Warthin-Starry银染同时阳性或阴性作为诊断是否H pylori感染的金标准.结果:和金标准相比较,安速快速检测试剂盒敏感性为92.0%(81/88),特异性为94.0%(47/50).准确率为92.8%(128/138);与RUT相比安速快速检测试剂盒敏感性为89.7%(87/97),特异性为91.2%(52/57),准确性为90.3%(139/154).结论:安速快速检测试剂盒在没有进行根除治疗的患者中能够较好的反应现症感染的情况.临床可用于H pylori现症感染的诊断.     

金属螯合亲和层析法对重组幽门螺杆菌尿素酶B亚单位的纯化

背景：幽门螺杆菌（H.pylori)疫苗是近年研究的热点。H.pylori尿素酶B亚单位（UreB)是理想的候选抗原。目的：获得纯化的重组H.pylori UreB(rUreB)，为进一步的H.pylori疫苗制备打下基础。方法：采用金属螯合亲和层析法（MCAC）在变性条件下纯化rUreB,SDS-聚丙烯酰胺凝胶电泳（PAGE）和免疫印迹法鉴定纯化产物的分子量、纯度和抗原性。结果：纯化产物的分子量与预计分子量相符，具有良好的特异性UreB免疫原性，且一步即可达到90％以上的纯度。结论：MCAC是一种简单、高效的rUreB纯化方法。     

A combination of the Helicobacter pylori stool antigen test and urea breath test is useful for clinical evaluation of eradication therapy: a multicenter study

BACKGROUND: Helicobacter pylori stool antigen (HpSA) test is a new tool for evaluating the H. pylori infection. The present study was carried out to investigate the clinical usefulness of the HpSA test in the evaluation of eradication therapy by comparing it with the (13)C-urea breath test (UBT). METHODS: One hundred and five patients received eradication therapy for H. pylori. After more than 8 weeks, the success of the therapy was evaluated by the HpSA test and the UBT. Concordant results were regarded as a final diagnosis, but when the results were discordant, histological examination was carried out. RESULTS: Of the 105 patients receiving eradication therapy for H. pylori, 25 patients were regarded as H. pylori positive by the UBT and and 20 patients were regarded as H. pylori positive by the the HpSA test. Nine patients (8.6%) showed discordant results (seven cases with UBT(+) and HpSA(-), and two with UBT(-) and HpSA(+)). Five cases out of nine were ultimately judged as having a false-positive result of the UBT, and in these cases the UBT values were relatively low (below 10 per thousand). The final diagnostic accuracies of the UBT and the HpSA test were 94.3% (88.0-97.9%; 95% CI) and 97.1% (91.9-99.4%), respectively. When we used the HpSA test in cases with weakly positive UBT values, we were able to diagnose the correct status of H. pylori infection after eradication in 99% of all patients (94.8-100.0%). CONCLUSION: The HpSA test is a useful tool for the evaluation of eradication therapy and a combination of the HpSA test and UBT is clinically recommended.