Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens

AIM: To comparatively evaluate PCR and other diagnostic methods (the rapid urease test and / or culture) in order to determine which of the three PCR methods (ureA, glmM and 26-kDa, SSA gene) was most appropriate in the diagnosis of Helicobacter pylori (H pylori ) infection and also to evaluate the detection of a putative virulence marker of H pylori, the cagA gene, by PCR in biopsy specimens. METHODS: One hundred and eighty-nine biopsy specimens were collected from 63 patients (three biopsies each) undergoing upper gastroduodenal endoscopy for various dyspeptic symptoms. The PCR methods used to detect H pylori DNA directly from biopsies were the glmM, 26-kDa, ureA and then cagA was used to compare the culture technique and CLO for urease with the culture technique being used as the gold standard. RESULTS: Thirty-five percent of the biopsies were positive for H pylori DNA using the 3 PCR methods, while 68% of these were positive for the cagA gene. Twenty-four percent of the biopsies were negative for H pylori DNA in all PCR methods screened. The remaining 41% were either positive for ureA gene only, glmM only, 26-kDa only, or ureA + glmM, ureA + 26-kDa, glmM + 26-kDa. Out of the 35% positive biopsies, 41% and 82% were positive by culture and CLO respectively, while all negative biopsies were also negative by culture and cagA. Cag A+ infection was also predominantly found in H pylori DNA of the biopsies irrespective of the clinical diagnosis. CONCLUSION: This method is useful for correctly identifying infections caused by H pylori and can be easily applied in our laboratory for diagnostic purposes.     

Helicobacter pylori and other Helicobacter species DNA in human bile samples from patients with various hepato-biliary diseases

AIM: To investigate the presence of Helicobacter species by nested PCR of 16S rRNA genes followed by the presence of Helicobacter pylori (H pylori) 16S rRNA, ureA, cagA genes in bile obtained at endoscopic retrograde cholangio-pancreatography (ERCP) from 60 Indian subjects. METHODS: Sixty bile samples were obtained from patients diagnosed with various hepato-biliary diseases and control subjects at ERCP. PCR analysis was carried out using primers for Helicobacter genus 16S rRNA gene and H pylori (16S rRNA, ureA and cagA) genes. Gastric H pylori status was also assessed from biopsies obtained at endoscopy from patients with various hepato-biliary diseases and controls. The control group mainly consisted of subjects with gastric disorders. Sequencing analysis was performed to confirm that PCR products with 16S rRNA and cagA primers were derived from H pylori. RESULTS No Helicobacters were grown in culture from the bile samples. Helicobacter DNA was detected in bile of 96.7% and 6.6% of groups I and II respectively. Ten from group I were positive for 16S rRNA and ureA and 9 were positive for cagA gene. In contrast of the 2 from the control, 1 amplified with 16S rRNA, ureA and cagA primers used. The sequences of the 16S rRNA genes and cagA were 99% similar to Helicobacter pylori. CONCLUSION: Helicobacters are associated with the pathogenesis of various hepato-biliary disorders.     

Use of different PCR primers and gastric biopsy tissue from CLO test for the detection of Helicobacter pylori

Four different DNA loci were assessed for the detection of H. pylori by PCR on gastric biopsy specimens. PCR, with a primer specific 860 bp DNA fragment, was the most sensitive, with a detection limit of 0.02 pg H. pylori DNA, corresponding to approximately 10 organisms. Nested-PCR of the 860-bp DNA fragment was 10-fold more sensitive than single-step PCR. The sensitivity and specificity of the four PCR methods, in comparison to the results obtained from histology and the urease test, are as follows: 80.7% and 76% for the hpaA gene; 100% and 76% for the 16S rRNA gene; 84.6% and 80.0% for the 860-bp DNA fragment; 61.5% and 84.0% for the ureC (glmM) gene, respectively. The sensitivity of nested-PCR for the 860-bp DNA fragment was 100%. This nested-PCR gave positive results for eight specimens which were negative by conventional methods. PCR can be performed on gastric biopsy specimens obtained from the CLO test.     

Detection of Helicobacter pylori cagA gene by polymerase chain reaction in faecal samples.

OBJECTIVE: The polymerase chain reaction (PCR) has been extensively and successfully used to detect Helicobacter pylori in gastric juice and gastric biopsies. In contrast, the results obtained using faeces as biological samples for PCR are rather conflicting. This may be due to the presence of faecal inhibitory compounds (polysaccharides) which can inhibit the amplification reaction. The aim of this study was to characterize the H. pylori genotype in faecal samples by using specific primers for the cagA gene. To overcome the problem of contamination by polysaccharides, we used a filter-based extraction technique already applied in a previous study. METHODS: Antral and body biopsies were obtained from 30 symptomatic patients undergoing upper endoscopy. PCR was used to detect the presence of H. pylori organisms in faecal samples by using primers selected for the urease gene A. In addition, H. pylori organisms were characterized both in faecal samples and paraffin-embedded biopsies by PCR with specific primers for the cagA gene. RESULTS: All patients showed a positive CLO test (rapid urease test) and evidence of H. pylori by Warthin-Starry stain. PCR detected the urease A gene in the faecal samples of all patients. The cagA gene was detected in the faecal and biopsy samples of 18 subjects (60%). Duodenal ulcer and/or antral erosions were observed in 15 of the 18 cagA-positive patients (83.3%) and in five of the 12 cagA-negative patients (41.7%). Endoscopic features of normal mucosa or gastritis were observed in three cagA-positive patients (16.7%) and in seven cagA-negative patients (56.3%). cagA-positive status was found to be significantly related to the endoscopic features of duodenal ulceration and/or antral erosions. CONCLUSIONS: Our findings prove that faeces are suitable samples for the detection of cagA status. Moreover, they confirm the existence of a significant relationship between cagA-positive status and duodenal ulcer and/or antral erosions.     

高原地区上消化道疾病幽门螺杆菌菌型的临床研究

目的研究高原地区上消化道疾病幽门螺杆菌（Helicobacter pylori,Hp）菌型。方法应用Hp尿素酶基因（ure）和细胞毒（cagA）基因引物对330例上消道疾病患者通过聚合酶链反应（PCR）技术,检测ure和cagA基因阳性检出率;用免疫印迹法检测血清ure和CagA抗体。结果 Hp菌株ure和cagA基因阳性检出率分别为90.0%和82.4%;Ure和CagA抗体阳性率分别为89.3%和63.6%;ure和cagA基因表达率分别为99.3%和77.2%。结论高原地区消化道疾病患者中HpcagA基因阳性率较高,CagA蛋白,CagA抗体（Ⅰ型Hp）的表达率亦相应增高。     

Analysis of eight different methods for the detection of Helicobacter pylori infection in patients with dyspepsia

BACKGROUND: The present study was designed to compare the accuracy of eight different methods for the detection of Helicobacter pylori (H. pylori) infection in patients with dyspepsia. These tests included culture, histology, rapid urease test (CLO test), serology, saliva IgA, gastric juice IgA, and two in-house methods, namely in-house urease test and Gram stain. METHODS: H. pylori infection was diagnosed prospectively in 200 untreated patients who underwent upper gastrointestinal endoscopy at King Chulalongkorn Memorial Hospital, Bangkok, Thailand, between July 1999 and August 2001. The gold standard for H. pylori infection was based on a positive culture or both a positive histological examination and CLO test. RESULTS: The culture provided a sensitivity of 55.9% whereas saliva IgA and gastric juice IgA had a sensitivity of 26.8% and 22.2%, respectively. In contrast, the other tests provided satisfactory sensitivities ranging between 89.3% and 100% (Gram stain 89.3%, histology 93.5%, serology 96.8%, CLO test 99.0%, in-house urease test 100%). The specificities of the tests ranged between 75% and 100% (culture 100%, CLO test 91.9%, histology 90.4%, in-house urease test 88.9%, Gram stain 93.5% serology 96.8%, gastric juice IgA 91.7% and saliva IgA 75%). CONCLUSIONS: Majority of invasive and non-invasive tests in this study were accurate for the diagnosis of H. pylori infection. However, the secretory IgA-based techniques in saliva and gastric juice seem to be inappropriate for determining H. pylori status in our populations due to their low sensitivities.     

Effects of cagA+ and cagA- strains of Helicobacter pylori on the human gastric mucus layer thickness

BACKGROUND: Infection with cytotoxin-associated gene A (cagA) Helicobacter pylori is associated with severe gastric diseases, with contradictory views being expressed concerning the effect of H. pylori on the gastric mucus thickness. The aim of the present study was to differentiate between the effect of cagA+ and cagA- strains on gastric mucus thickness. METHODS: Ninety-nine patients without peptic ulcers who were not on medication were randomly recruited from consecutive endoscopy clinics: six biopsies (five antral, one body) were obtained from each patient. Cryostat sections (18 microm) were cut and stained using the modified periodic acid-Schiff/Alcian blue technique. Mucus thickness was measured using computer-assisted light microscopy. The H. pylori status was assessed by histology, Campylobacter-like organism (CLO)test and culture, and cagA+ status determined by polymerase chain reaction (PCR). RESULTS: There was no significant difference (P = 0.784) in mean mucus thickness between cagA+ (52.7 +/- 1.2 microm, n = 10), cagA- (46.6 +/- 1.1 microm, n = 18) or H. pylori-negative patients (51.3 +/- 1.1 microm, n = 30). In cagA- patients, mucus thickness was significantly reduced with increased H. pylori colonization density, Spearman (r(s)) = -0.805, P < 0.0001. In contrast, in cagA+ patients there was a weak positive, but not significant, association between mucus thickness and H. pylori colonization density, r(s) = 0.333, P = 0.381. CONCLUSIONS: The human gastric mucus thickness is not affected by infection with cagA+ or cagA- strains of H. pylori compared with uninfected. Although a trend of increased mucus thickness with cagA+ infection was observed.     

慢性乙型肝炎患者肝组织中螺杆菌菌属特异性16SrRNA基因及HP特异性基因分析

目的对慢性乙型肝炎患者肝组织进行螺杆菌菌属特异性16SrRNA基因及幽门螺杆菌（HP）特异性基因检测,探讨螺杆菌在慢性乙型肝炎发生、发展中的作用。方法对56例行肝穿刺活检的慢性乙型肝炎患者的肝组织应用针对螺杆菌菌属特异性16SrRNA基因的通用引物进行基因扩增及微需氧分离培养,并对螺杆菌菌属特异性16SrRNA基因阳性者进一步应用HPcagA、vacA和glmM基因特异引物进行扩增。结果对56例慢性乙型肝炎患者的肝组织DNA应用螺杆菌菌属特异性16SrRNA基因通用引物进行扩增,共有35例患者的肝组织中发现了螺杆菌菌属特异性16SrRNA基因,其中肝硬化组（72.7%）和原发性肝癌组（87.5%）的检出率明显高于慢性肝炎组（42.3%,P〈0.05）,而慢性肝炎组中随着炎症评分的增加,检出率亦增加。对这35例螺杆菌菌属特异性16SrRNA基因阳性的肝组织DNA进一步应用HPcagA、vacA和glmM基因特异性引物进行扩增,结果证实有21例为HPDNA。所有肝组织经微需氧分离培养均未发现可疑菌落生长。结论慢性乙型肝炎患者肝组织中除存在HPDNA外,可能还存在其他螺杆菌DNA。螺杆菌在慢性乙型肝炎向肝硬化和肝癌的发展中可能发挥致病作用。     

Virulence and potential pathogenicity of coccoid Helicobacter pylori induced by antibiotics

AIM: To explore the virulence and the potential pathogenicity of coccoid Helicobacter pylori (H. pylori) transformed from spiral form by exposure to antibiotic. METHODS: Three strains of H. pylori, isolated from gastric biopsy specimens of confirmed peptic ulcer, were converted from spiral into coccoid from by exposure to metronidazole. Both spiral and coccoid form of H. pylori were tested for the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells, and the differences of the protein were analysed by SDS-PAGE and Western blot. The mutation of the genes including ureA, ureB,hpaA, vacA and cagA, related with virulence, was detected by means of PCR and PCR-SSCP. RESULTS: In the coccoid H. pylori,the urease activity, the adherence to Hep-2 cells and the vacuolating cytotoxicity to Hela cells all decreased. In strain F44, the rate and index of adherence reduced from 70.0% +/- 5.3% to 33% +/- 5.1% and from 2.6 +/- 0.4 to 0.96 +/- 0.3 (P < 0.01), respectively. The invasion of coccoid H. pylori into Hep-2 cell could be seen under electronmicroscope. SDS-PAGE showed that the content of the protein with the molecular weight over Mr 74000 decreased, and the hybriditional signal in band M(r) 125000 weakened, while the band M(r)110000 and M(r)63000 strengthened in coccoid H.pylori as shown in Western blot. The results of PCR were all positive, and PCR-SSCP indicated that there may exist the point mutation in gene hpaA or vacA. CONCLUSION: The virulence and the proteins with molecular weight over M(r)74000 in coccoid H.pylori decrease, but no deletion exists in amplification fragments from ureA, ureB, hpaA, vacA and cagA genes, suggesting that coccoid H.pylori may have potential pathogenicity.     

Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases

OBJECTIVE: Our aim was to detect Helicobacter pylori (H. pylori) from gastric biopsies of 248 patients using a novel, polymerase chain reaction (PCR)-based methodology, which simultaneously facilitates the determination of H. pylori vacA genotypes and cagA gene. METHODS: A simple methodology for sample preparation was established and PCR was performed with primer systems for the 16S rRNA, vacA, and cagA genes, thus circumventing the need to culture H. pylori and to extract DNA from biopsy samples. RESULTS: Infection with H. pylori was detected in 147 (59.3%) of 248 patients. The vacA signal sequence genotype s1 was present in 104 (81.3%) of 128 H. pylori-positive patients, and 24 (18.8%) patients had the genotype s2. The vacA middle region types m1 and m2 were detected in 46 (35.9%) and 79 (61.7%) patients, respectively. The combinations s1/m2 (43%) and s1/m1 (35.9%) were found more frequently than s2/m2 (18.8%). The cagA gene was detected in 75 (72.1%) of 104 H. pylori-positive biopsies with the vacA genotype s1. All 24 biopsies with the type s2 were cagA negative. Strains of the type vacA s1 were found in 97% of H. pylori-positive patients with peptic ulcer disease and were associated with the presence of the cagA gene, whereas 96% of the strains of the type vacA s2 were detected in patients who only had nonulcer dyspepsia. CONCLUSIONS: Using a novel PCR-based methodology, H. pylori 16S rRNA gene, vacA genotypes, and cagA gene can now be rapidly detected directly in gastric biopsies with high accuracy. These data demonstrate that infection with H. pylori strains of the vacA s1 genotype and the cagA gene are more likely to result in peptic ulcer disease. Determination of vacA genotypes and cagA gene may contribute to the potential clinical identification of patients at different levels of risk.     

Evaluation of a new biopsy urease test: Pronto Dry, for the diagnosis of Helicobacter pylori infection

BACKGROUND: The gastric biopsy urease test is the most frequently used test for the diagnosis of Helicobacter pylori infection in routine gastrointestinal endoscopy practice. In Malaysia up to recently, only one commercial biopsy urease test was available: the CLO test (Ballard Medical Products, Draper, Utah, USA). Large endoscopy units use their own 'homemade' unbuffered ultra rapid urease test for diagnosis of H. pylori infection. OBJECTIVE: To compare the accuracy and reaction time of a new biopsy urease test, Pronto Dry (Medical Instruments Corporation, Solothurn, Switzerland) and the CLO test in the diagnosis of H. pylori infection. METHODS: Consecutive patients presenting with dyspepsia to the endoscopy unit, University of Malaya Medical Centre were recruited for the study. Patients who were previously treated for H. pylori infection or who had received antibiotics, proton pump inhibitors or bismuth compounds in the preceding 4 weeks were excluded. H. pylori diagnosis was made based on the ultra rapid urease test and histological examination of gastric biopsies. Four antral and four corpus biopsies were taken for this purpose from all patients. A diagnosis of H. pylori infection was made when both the ultra rapid urease test and histology were positive in either the antral or corpus biopsies. A negative diagnosis of H. pylori was made when both tests from antral and corpus biopsies were all negative. Another four antral and four corpus biopsies (two each) were taken for the Pronto Dry and CLO tests. The Pronto Dry and CLO tests were stored and performed according to the manufacturer's instruction. RESULTS: Two hundred and eight patients were recruited in the study. Eighty-six of the patients were males and 122 were females. The mean age was 46.3 years with a range of 15-82 years. The results for both the Pronto Dry and the CLO tests were completely concordant with sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of 98.1%, 100%, 100%, 98.1% and 99%, respectively. The Pronto Dry test showed a faster reaction time to positive compared with the CLO test, with 96.2% positive reaction by 30 min versus 70.8% and 100% positive reaction time by 55 min versus 83%. The colorimetric change was also more distinct with the Pronto Dry test compared with the CLO test. CONCLUSIONS: Both the Pronto Dry and the CLO tests were highly accurate for the diagnosis of H. pylori infection. The Pronto Dry test showed a quicker positive reaction time and the positive colour change was more distinct.     

幽门螺杆菌oipA基因的开关状态及其与胃疾病的关系

目的:了解幽门螺杆菌(Hp)oipA基因的开关状态并探讨其与胃疾病的关系.方法:收集活检胃组织标本360份,对组织快速尿素酶实验阳性和部分阴性的活检标本进行Hp分离培养,分离菌经革兰染色镜检、尿素酶实验和16S rRNA PCR鉴定后,PCR扩增其oipA基因,测序分析oipA信号区的开关状态,分析功能性oipA与临床疾病之间的关联性.结果:组织标本分离培养获得106株Hp,其中72株cagA基因阳性,87株oipA信号区基因阳性.经测序分析80株oipA基因信号区呈开放状态,为功能性oipA.87株Hp中有1株oipA基因信号区呈关闭状态,6株开关状态不能确定.在胃溃疡和萎缩性胃炎中,功能性oipA的检出率[分别为12/12和78.26%(18/23)]均显著高于cagA[分别为58.33%switch status和39.13%switch status].结论:功能性oipA与萎缩性胃炎和胃溃疡关系密切.     

Polymerase chain reaction assay for the detection of Helicobacter pylori in gastric biopsy specimens: comparison with culture,rapid urease test,and histopathological tests.

Ulcer recurrence is probably related to residual Helicobacter pylori (H pylori). Histological examination and culture are considered to be the most specific tests. CLO test is a rapid but less specific test, which is usually used as an alternative test to culture. The aim of this study was to investigate the efficiency of a simplified polymerase chain reaction (PCR) assay as a procedure for the diagnosis of gastric H pylori infection of patients. Biopsy specimens were obtained from antral mucosa of 58 patients at endoscopy and submitted to four tests for detection of H pylori. The bacteria were found in 53%, 43%, 48%, and 50% of patients according to the results of PCR, CLO test, culture, and histological examination. Twenty three patients had both negative histology and negative culture and PCR was negative in all of these. Thirteen patients were not classified because only histology or culture was positive and 10 of these had a positive PCR test. When the diagnosis of H pylori was established by agreement with both histology and culture or three positive tests out of four, 29 patients were H pylori positive (28 having had three positive tests and one displaying positive histology and culture), and 26 were negative, and three undetermined. PCR proved the most sensitive and specific test. These results suggest the simplified PCR assay may be a valuable test for the detection of H pylori.     

Indistinguishable cellular changes in gastric mucosa between Helicobacter pylori infected asymptomatic tribal and duodenal ulcer patients

AIM： To investigate the changing pattern of different histological parameters occurring in the stomach tissue of Helicobacter pylori （H pylori） infected tribal populations and duodenal ulcer patients among ethnic Bengalis and correlation of the genotypes of H pylori with different histological parameters. METHODS： One hundred and twelve adult individuals were enrolled into this study between 2002 and 2004. Among them, 72 had clinical features of duodenal ulcer （DU） from ethnic Bengali population and 40 were asymptomatic ethnic tribals. Endoscopic gastric biopsy samples were processed for histology, genotyping and rapid urease test. Histologically, haematoxylin and eosin staining was applied to assess the pathomorphological changes and a modified Giemsa staining was used for better detection of Hpylori. For intestinal metaplasia, special stainings, i.e. Alcian blue periodic acid-Schiff and high iron diamine-Alcian blue staining, were performed. PCR was performed on bacterial DNA to characterize the presence or absence of virulence-associated genes, like cagA, and distribution of different alleles of vacA and iceA. RESULTS： Intraglandular neutrophil infiltration, a hallmark of activity of gastritis, was present in 34 （94%） of tribals （TRs） and 42 （84%） of DU individuals infected with H pylori. Lymphoid follicles and aggregates, which are important landmarks in H pylori infection, were positive amongst 15 （41%） of TRs and 20 （40%） of DU subjects. Atrophic changes were observed in 60% and 27.7%, respectively, among DU cases and tribals （P 〉 0.003）. Metaplastic changes were detected in low numbers in both groups. Moderate to severe density distribution of Hpylori in the gastric mucosa was 63% among TRs, whereas it was 62% in DU subjects. There were no significant differences in the distribution of virulence-associated genes like cagA, vacA and iceA of H pylori strains carried by these two populations. CONCLUSION： Our study showed almost similar distribution of inflammatory cells among asymptomatic tribals and DU Bengali patients. Interestingly, the tribal population are free from any clinical symptoms despite evidence of active histologic gastritis and infection with Hpylori strains carrying similar virulence markers as of strains isolated from patients with DU. There was an increased cellular response, especially in terms of neutrophil infiltration, but much lower risk of developing atrophy and metaplastic changes among the tribal population.     

Genotypes of Helicobacter pylori in children with upper abdominal pain

BACKGROUND: Studies related to genotypes of Helicobacter pylori infection and upper abdominal pain (UAP) in children are scarce all over the world. We prospectively analyzed the association between H. pylori infection and UAP in our study group of children and evaluated the vacA genotypes associated with this disorder. METHODS: We investigated 34 children with UAP (group I) and another 110 children as controls without UAP (group II) for H. pylori infection, using antral biopsies by culture, rapid urease tests, histopathology and ureA polymerase chain reaction (PCR). Genotyping was performed using specific cagA and vacA primers on 52 H. pylori strains (group I = 21; 18 boys, mean age 10.33 +/- 2.47 years; group II = 31; boys 21, mean age 9.63 +/- 2.51 years). RESULTS: A significant association between H. pylori infection and UAP was observed when compared to controls (21/34; 61.8%vs 31/110; 28.2%; P = 0.0004). cagA positive H. pylori strains were detected in 20 (95.2%) children with UAP and in 28 (90.3%) controls. Mixed infection was detected in 25% of children with no significant difference between the groups. On univariate regression analysis, s1a, m1 alleles and s1a/m1 genotype of vacA had significant associations with UAP (P = 0.018, 0.015 and 0.007, respectively), while s2 and m2 alleles and the s2/m2 genotype were significantly more frequent in controls (P = 0.034, 0.001 and 0.034, respectively). CONCLUSIONS: H. pylori infection is strongly associated with UAP in children and a significantly higher proportion have s1a, m1 alleles and s1a/m1 genotype. The negative associations of vacA s2, m2 alleles and s2/m2 genotype with UAP indicate that they are unlikely to have an important role in this disorder.     

Higher frequency of cagA EPIYA-C Phosphorylation Sites in H. pylori strains from first-degree relatives of gastric cancer patients

ABSTRACT: BACKGROUND: To evaluate the prevalence of more virulent H. pylori genotypes in relatives of gastric cancer patients and in patients without family histories of gastric cancer. METHODS: We evaluated prospectively the prevalence of the infection by more virulent H. pylori strains in 60 relatives of gastric cancer patients comparing the results with those obtained from 49 patients without family histories of gastric cancer. H. pylori status was determined by the urease test, histology and presence of H. pylori ureA. The cytotoxin associated gene (cagA), the cagA-EPIYA and vacuolating cytotoxin gene (vacA) were typed by PCR and the cagA EPIYA typing was confirmed by sequencing. RESULTS: The gastric cancer relatives were significant and independently more frequently colonized by H. pylori strains with higher numbers of CagA-EPIYA-C segments (OR = 4.23, 95%CI = 1.53--11.69) and with the most virulent s1m1 vacA genotype (OR = 2.80, 95%CI = 1.04--7.51). Higher numbers of EPIYA-C segments were associated with increased gastric corpus inflammation, foveolar hyperplasia and atrophy. Infection by s1m1 vacA genotype was associated with increased antral and corpus gastritis. CONCLUSIONS: We demonstrated that relatives of gastric cancer patients are more frequently colonized by the most virulent H. pylori cagA and vacA genotypes, which may contribute to increase the risk of gastric cancer.     

Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene, CagA protein in Helicobacter pylori isolates and its antibody in sera of patients

AIM: To construct a prokaryotic expression system of a Helicobacter pylori (H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA(+) H pylori) isolates cause diseases. METHODS: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed, and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively. Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients' sera, and the correlations between infection with CagA(+) H pylori and gastritis as well as peptic ulcer were analyzed. RESULTS: Of all the clinical specimens obtained, 80.8% (126/156) were found to have H pylori isolates and 97.2% of the isolates (106/109) were positive for cagA gene. In comparison with the reported data, the cloned cagA1 fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences, respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein. rCagA1 was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109) expressed CagA and 88.1% of the patients' serum samples (96/109) were CagA antibody-positive. The percentage of CagA(+) H pylori strains (97.9%) isolated from the biopsy specimens of peptic ulcer appeared to be higher than that from gastritis (88.5%), but the difference was not statistically significant (chi (2)=3.48, P>0.05). CONCLUSION: rCagA1 produced by the prokaryotic expression system constructed in this study possesses good immunoreactivity and antigenicity, and the established ELISAs can be used to detect CagA of H pylori and its antibody. H pylori isolates show high frequencies of cagA gene and CagA expression, but the infections by CagA(+) H pylori strains are not the most decisive factors to cause gastric diseases.     

Analysis of Helicobacter pylori antimicrobial susceptibility and virulence genes in gastric mucosal biopsies in the United Arab Emirates

AIM: To determine the virulence attributes (presence of cagA and vacA genes) of Helicobacter pylori , and presence of clarithromycin resistance genes in gastric mucosal biopsy samples obtained in the United Arab Emirates. METHODS: DNA was extracted from antral gastric biopsy samples from 91 dyspeptic patients. Real-time PCR and melting curve analysis were used to identify patients infected with H. pylori and to further identify strains containing the A(2142/43)G or the A(2142)C mutations that are associated with clarithromycin resistance. PCR was also used to identify cagA - and vacA -positive strains. RESULTS: Real-time PCR analysis detected the presence of H. pylori in 55 (60%) samples. Thirty-six pathogen-positive samples contained at least one of three point mutations associated with clarithromycin resistance. The vacA gene was present in 40 (72.7%) and cagA was present in 41 (74.5%) of the positive samples. Both genes were present in 36 (65%) of the positive samples. The presence of each clarithromycin-inducing mutation was largely independent of the others. Mutation at one position, A(2142/43)G, was strongly associated with the presence of both the vacA gene and the cagA gene. CONCLUSIONS: A high proportion of gastric mucosal biopsies obtained in the UAE is positive for genes associated with clarithromycin resistance. This may have implications for treatment of the infection.     

胃粘膜石蜡切片PCR法检测幽门螺杆菌

目的建立从石蜡包埋的胃粘膜标本中扩增ＨｐＤＮＡ的聚合酶链反应（ＰＣＲ）．方法以一对Ｈｐ特异的寡核苷酸为引物，采用双扩增ＰＣＲ扩增Ｈｐ１６ＳｒＲＮＡ基因，并与常规检测方法进行了比较．结果双扩增ＰＣＲ检出的最小ＨｐＤＮＡ量为００１ｐｇ．用该方法检测十二指肠溃疡患者２０例石蜡包埋的胃粘膜标本，并与尿素酶试验、细菌培养、Ｗａｒｔｈｉｎ＿Ｓｔａｒｒｙ银染色及ＰＣＲ对新鲜胃粘膜标本的检测作比较，结果１８例患者上述五项检测结果一致，２例尿毒酶试验、银染色及ＰＣＲ对新鲜胃粘膜标本的检测呈阳性的患者，采用双扩增ＰＣＲ对石蜡包埋标本的检测未发现ＨｐＤＮＡ．ＰＣＲ对石蜡包埋标本及新鲜胃粘膜标本的检测有显著相关性．结论双扩增ＰＣＲ为分子水平上Ｈｐ感染的回顾性研究提供一有利工具．