Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens.

A PCR assay for the detection of Helicobacter pylori in gastric biopsy specimens with specific primers for ureC gene amplification (herein referred to as ureC PCR) was compared with other routine invasive methods (culture, the rapid-urease test, and Giemsa staining of histological sections) with samples from a group of 104 consecutive dyspeptic patients. Bacteria were found in 40 (38.5%), 38 (36.5%), 36 (34.6%), and 35 (33.7%) of the patients by ureC PCR, culture, the rapid-urease test, and Giemsa stain, respectively. Sixty-three patients had negative cultures, negative histological examinations, and negative rapid-urease test results, and 61 of these patients were also negative by ureC PCR. ureC PCR detected H. pylori in two culture-negative patients. In parallel, a PCR-based assay to detect the H. pylori cytotoxin-associated antigen (cagA) gene, a putative virulence gene, was also developed. To assess the likelihood of detection of H. pylori genes directly from gastric biopsy samples and from the corresponding H. pylori isolates, specimens from 31 patients were subjected to PCR with ureC- and cagA-targeting primers. All 31 biopsy specimens and the corresponding H. pylori isolates were positive in the ureC PCR. H. pylori strains that were cagA positive also gave positive cagA PCR fragments with biopsy specimens from the same patients. All ureC PCR-positive patients were examined; biopsy specimens from 10 of 11 (91.7%) duodenal ulcer patients harbored H. pylori cagA-positive strains, whereas 19 of26 (73%) of those from patients with chronic gastritis only were found to be cagA positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence of Helicobacter pylori vacA and cagA genotypes in Ethiopian dyspeptic patients

A total of 300 gastric biopsy samples and 50 Helicobacter pylori isolates were collected from Ethiopian adult dyspeptic patients. The vacA and cagA genes were detected in 90 and 79% of biopsy specimens, respectively, and in 100 and 87% of clinical isolates, respectively. Both genes were detected in 84% of the gastric biopsy samples and in 87% of the clinical isolates. Among vacA genotypes, the s1/m1 genotype was the most common in gastric biopsy samples (48%). The vacA and cagA positive H. pylori strains were detected to a higher degree in patients with chronic active gastritis (71%) than patients with other histopathological findings (29%) (P < 0.05).     

High prevalence of Helicobacter pylori in saliva demonstrated by a novel PCR assay.

AIMS--To investigate the prevalence of Helicobacter pylori in the saliva of patients infected with this bacterium. METHODS--A novel polymerase chain reaction (PCR) assay was developed to detect H pylori in saliva and gastric biopsy specimens from patients undergoing endoscopy. RESULTS--Our PCR assay amplified a 417 base pair fragment of DNA from all 21 DNAs derived from H pylori clinical isolates but did not amplify DNA from 23 non-H pylori strains. Sixty three frozen gastric biopsy and 56 saliva specimens were tested. H pylori specific DNA was detected by PCR in all 39 culture positive biopsy specimens and was also identified from another seven biopsy specimens which were negative by culture but positive by histology. H pylori specific DNA was identified by PCR in saliva specimens from 30 (75%) of 40 patients with H pylori infection demonstrated by culture or histological examination, or both, and in three patients without H pylori infection in the stomach. CONCLUSION--The results indicate that the oral cavity harbours H pylori and may be the source of infection and transmission.     

Correlation of serum antibody titres with invasive methods for rapid detection of Helicobacter pylori infections in symptomatic children

Helicobacter pylori (H. pylori) is causally associated with peptic ulcer disease and gastric carcinoma. Typically, children get infected during the first decade of life, but diseases associated with H. pylori are seen mainly in adults. Multiple diagnostic methods are available for the detection of H. pylori infection. The aim of this study was to evaluate the correlation and diagnostic accuracy of three invasive methods [rapid urease test (RUT), histology and bacterial culture] and one non-invasive method (IgG serology) for diagnosis of H. pylori infection in a prospective cohort study conducted on 50 symptomatic children between two and eighteen years of age. Endoscopies with gastric biopsies were performed for RUT, culture and histopathological examination, respectively. IgG antibodies were measured in patient sera using a commercially available enzyme-linked immunosorbent assay (ELISA). RUT and positive H. pylori IgG antibodies were concordant in 88% (44/50) of patients. Both tests were negative in 32% (16/50), and both were positive in 56% (28/50). Disagreement occurred in 12% (6/50) of the patients: three of them (6%) had positive RUT and negative H. pylori IgG, and another three (6%) had negative RUT and positive H. pylori IgG. A combination of RUT with non-invasive serology constituted the optimum approach to the diagnosis of H. pylori infection in symptomatic children. The non-invasive serological test (ELISA) could not be used alone as the gold standard because it cannot distinguish between active and recently treated infection; and bacterial culture could not be used alone because of its low sensitivity.     

Topographical localisation of cagA positive and cagA negative Helicobacter pylori strains in the gastric mucosa; an in situ hybridisation study

BACKGROUND: The cagA gene is a marker for the presence of the cag pathogenicity island, and the presence of cagA positive strains of Helicobacter pylori can identify individuals with a higher risk of developing gastrointestinal diseases. AIMS: To study the interaction between H. pylori cagA(+) and cagA(-) strains and the gastric mucosa. METHODS: Patients with H. pylori associated gastritis and peptic ulcers were studied. Biopsies were obtained from the antrum, corpus, fundus, and incisura for H pylori culture, and for in situ hybridisation studies. From each biopsy, multiple single H. pylori colonies were isolated and propagated for DNA isolation, and cagA was detected by the polymerase chain reaction (PCR). For in situ detection of H. pylori an oligonucleotide specific for an H. pylori common antigen and an oligonucleotide specific for cagA were used as probes. Biotinylated probes were incubated with biopsy sections, developed with streptavidin-horseradish peroxidase, and amplified with the tyramide system. RESULTS: PCR results for cagA in isolated colonies confirmed the in situ hydridisation studies. In situ hybridisation identified cagA(+) bacteria in patients with cagA(+) isolates; cagA(-) bacteria in patients with cagA(-) isolates, and cagA(+) and cagA (-) bacteria in patients with both cagA(+) and cagA(-) isolates. CagA(-) bacteria usually colonised the mucous gel or the apical epithelial surface, whereas cagA(+) bacteria colonised the immediate vicinity of epithelial cells or the intercellular spaces. CONCLUSIONS: These results document a different in vivo interaction between H. pylori cagA(+) or cagA(-) strains and the gastric mucosa.     

Detection of Helicobacter pylori by using the polymerase chain reaction.

A 1.9-kb cloned fragment of chromosomal DNA randomly selected from a Helicobacter pylori cloned library was evaluated as a potential probe. The probe detected 19 of 19 H. pylori strains and yielded a specificity of 98.7% when tested against 306 other bacterial strains representing 32 different species. False-positive results with non-H. pylori strains were due to the presence of contaminating vector sequences. A polymerase chain reaction (PCR) assay was developed by using 20-base oligonucleotide primers homologous to a portion of the 1.9-kb fragment. The PCR assay amplified a 203-nucleotide-pair product which was analyzed by agarose gel electrophoresis and Southern hybridization by using a third 20-base 32P-labeled oligonucleotide complementary to a region of DNA between the primers. The PCR assay was 100% sensitive, detecting all 35 H. pylori strains tested, and did not amplify sequences in several closely related species. The assay was sensitive for as little as one copy of the cloned plasmid DNA or 100 H. pylori bacterial cells. To evaluate the PCR assay for clinical samples, gastric biopsy and aspirate specimens were tested by PCR, and the results were compared with those of microbiologic culture and histologic examination. In fresh biopsy specimens, H. pylori sequences were detected by PCR in 13 of 14 (93%) positive tissues and 0 of 19 negative tissues. In gastric aspirate specimens, 11 of 13 (85%) positive tissues were positive by PCR. H. pylori DNA was detected in 1 of 14 aspirate specimens negative by culture, histology, and PCR of the accompanying biopsy tissue. PCR is a rapid, accurate, and sensitive method for the detection of H. pylori.     

Role of corpus gastritis and cagA-positive Helicobacter pylori infection in reflux esophagitis

Considering that the role of Helicobacter pylori infection in gastroesophageal reflux and reflux esophagitis (GERD) is still controversial and that the role of virulence markers of the bacterium has not been evaluated in most studies of GERD, we investigated the association among H. pylori infection with cagA-positive and -negative strains, corpus gastritis, and GERD in a large group of patients by controlling for confounding factors. We studied prospectively 281 consecutive adult patients: 93 with GERD and 188 controls. H. pylori infection status was diagnosed by culture, by the preformed urease test, with a carbolfuchsin-stained smear, and by histology. The cagA status was determined by PCR of H. pylori isolates and gastric biopsy specimens. H. pylori infection was diagnosed in 191 (68.0%) of 281 patients. Among the 93 patients with GERD, 84 presented with mild or moderate esophagitis and 9 presented with severe esophagitis. In the multivariate analysis, the age of the patients and the degree of oxyntic gastritis were associated with GERD. Among the strains isolated from patients with GERD and from the control group, 24.4 and 66.9%, respectively, were positive for cagA (P < 0.001). Compared to infection with cagA-negative strains, infection with cagA-positive H. pylori strains was associated with a more intense gastritis in the corpus (P = 0.001). cagA status (odds ratio [OR] = 0.16, 95% confidence interval [CI] = 0.07 to 0.40), gastritis of the corpus (OR = 0.69, 95% CI = 0.48 to 0.99), and age (OR = 1.04, 95% CI = 1.01 to 1.07) were associated with GERD. In conclusion, the study provides evidence supporting the independent protective roles of cagA-positive H. pylori strains and the degree of corpus gastritis against GERD.     

Rapid colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA in gastric biopsy specimens.

A very simple, practical, sensitive, and specific colorimetric hybridization assay for detecting amplified Helicobacter pylori DNA is described. This assay, which combines a sensitive sandwich DNA hybridization reaction and a colorimetric protocol similar to those used in conventional enzyme immunoassays, was shown to be suitable for detecting H. pylori-infected gastric biopsy specimens and for monitoring the eradication of the pathogen after treatment. The specificity and sensitivity of the colorimetric hybridization assay were tested by assaying 27 H. pylori strains (4 reference and 23 clinical isolates), 9 strains of other Helicobacter spp. or Campylobacter spp., and 11 clinical isolates of other urease-positive bacteria. The likelihood of H. pylori detection in gastric biopsy specimens by the colorimetric hybridization assay was evaluated with 23 H. pylori-positive and 41 H. pylori-negative biopsy specimens on the basis of positive and negative results, respectively, of culture, rapid urease test, histological examination, and PCR. Biopsy specimens from 33 treated patients, endoscopied 4 to 8 weeks after the end of treatment, were also tested. All H. pylori strains showed positive results in the colorimetric hybridization assay, presenting optical densities at 450 nm (OD450S) of > or = 3.0. None of the other Helicobacter spp., Campylobacter spp., or the clinical isolates of other urease-positive bacteria showed OD450S equal to or greater than the cutoff (mean OD450 cutoff, 0.208). The colorimetric hybridization assay detected all 23 H. pylori-positive biopsy specimens (mean OD450, 2.910 +/- 0.295), while none of the H. pylori-negative biopsy specimens was shown to be positive in the assay (mean OD450, 0.108 +/- 0.025). H. pylori was considered to be not eradicated from three of the posttreatment biopsy specimens by culture, rapid urease test, histological examination, and PCR. They were all positive by the colorimetric hybridization assay, and their OD450S were > or = 3.0. The colorimetric hybridization assay also detected two other H. pylori-positive patients. Specimens from these two patients had negative culture, rapid urease test, and histology results, and a specimen from one of them also tested negative by PCR. These results indicate that the colorimetric hybridization assay is a suitable method both for the diagnosis of H. pylori in biopsy specimens and for the follow-up of patients after the end of treatment.     

Heterogeneity of cag genotypes in Helicobacter pylori isolates from human biopsy specimens

The Helicobacter pylori chromosomal cluster of genes known as the cytotoxin-associated gene (cag) island may have different compositions in infecting strains. In this study, we analyzed 150 single colonies obtained from gastric biopsy specimens from 10 patients infected with cagA-positive H. pylori strains and sweep isolates (isolates harvested with sweep in different points of the plate) from 6 patients infected with cagA-negative strains. Three loci in the cag island (cagA, cagE, and virB11) and the conserved gene glmM (ureC) were investigated by PCR. The levels of anti-H. pylori and anti-CagA antibodies in patient sera were also measured. For subjects infected with cagA-negative strains, all sweep isolates were also negative for cagE and virB11, suggesting the complete absence of the cag island. For subjects infected with cagA-positive strains, most of the isolates were positive for all three genes studied, whereas 24.7% of the isolates had a partial or total deletion of the cag island. cagA, cagE, and virB11 were, respectively, present in 87.3, 77.3, and 90% of the colonies. The deletion of virB11 was always associated with the deletion of cagA and/or cagE. H. pylori colonies with different cag genotypes were isolated within a single gastric biopsy specimen from 3 of the 10 patients and were further characterized by random amplified polymorphic DNA (RAPD) analysis and by sequencing of an arbitrarily selected gene segment. Although the colonies had different cag genotypes, their RAPD profiles were highly similar within each patient, and the nucleotide sequences of the selected gene segment were identical. All of the patients had detectable antibodies against H. pylori, and 9 of 10 had anti-CagA antibodies. In conclusion, we show that a single infecting H. pylori strain may include variable proportions of colony subtypes with different cag genotypes. The extension of our analysis to patients with well-characterized gastric diseases may provide significant information on the relationship between cag genotypes and clinical outcomes of H. pylori infections.     

Detection of Helicobacter pylori in gastric mucosa of patients with gastroduodenal diseases by PCR-restriction analysis using the RNA polymerase gene (rpoB)

A novel PCR restriction analysis method using the RNA polymerase beta-subunit- coding gene (rpoB) was employed to both detect and identify Helicobacter pylori in biopsy specimens and culture isolates. The rpoB DNAs (458 bp) were specifically amplified by PCR with the Helicobacter-specific primers (HF and HR). Based on the determined rpoB sequences of the culture isolates, an H. pylori-specific restriction site, Tru9I, was found. H. pylori can be identified by observing two discernible DNA fragments (288 and 138 bp) after Tru9I digestion and agarose gel electrophoresis. The rpoB PCR and subsequent restriction analysis (PRA) enabled the specific detection and identification of H. pylori in biopsy specimens from patients with gastroduodenal diseases. The rpoB PRA conferred a compatible or a slightly higher positive rate (53.7%) than did the Campylobacter-like organism (CLO) test (50.4%) and glmM PCR (48.8%), suggesting that it is useful for diagnosing an H. pylori infection without culture in the clinical laboratory.     

Consensus and variable region PCR analysis of Helicobacter pylori 3' region of cagA gene in isolates from individuals with or without peptic ulcer

The clinical outcome of Helicobacter pylori infection may be associated with the cagA bacterial genotype. To investigate the cagA status of H. pylori-infected patients and the relationship between cagA and peptic ulcer disease, gastric biopsy specimens from 103 Caucasian patients in Brazil were analyzed by PCR. Since allelic variation in cagA exists and distinct H. pylori subgenotypes may circulate in different regions, PCR using primers for a variable 3' region of the cagA gene according to a Japanese methodology and for a consensus cagA 3' region used in Western methods was used for cagA detection. cagA was present in 53 (71%) of 75 H. pylori-positive cases when analyzed by the consensus region method and was associated with duodenal ulcer disease (P = 0.02), but not with gastric ulcer (P = 0.26), when compared to patients with duodenitis or gastritis. The variable region PCR method was able to detect 43 (57%) cagA-positive cases within the same group of H. pylori-positive patients and showed three subtypes of cagA (A, B/D, and C) that were not associated with clinical outcome. However, in 8 (18%) of the cases, more than one subtype was present, and an association between patients with multiple subtypes and disease outcome was observed when compared to patients with isolated subtypes (P = 0.048). cagA was a marker of H. pylori strains for duodenal ulcer disease in our population, and in spite of the differences in the 3' region of the cagA gene, the Japanese methodology was able to detect the cagA status in most cases. The presence of multiple subgenotypes of cagA was associated with gastric ulcer.     

Effect of transport medium and transportation time on culture of Helicobacter pylori from gastric biopsy specimens.

To determine whether transportation time and use of a low budget transport medium (NaCl 0.9%) would influence culture of Helicobacter pylori from gastric biopsy specimens, upper gastrointestinal endoscopy was performed on 42 patients. The specimens were cultured and examined histologically, and H pylori antibodies were determined using an ELISA technique. Patients were regarded as H pylori positive when culture was positive or when histology or IgG anti-H pylori antibodies indicated H pylori infection. Rapid transportation of gastric biopsy specimens in NaCl 0.9%, at room temperature resulted in a high diagnostic yield (23 H pylori positive cultures in 26 patients with H pylori infection). A 24 hour delay in plating gastric biopsy specimens after transportation in NaCl 0.9%, at room temperature, did not seriously affect results (22 instead of 23 H pylori positive cultures). The culture results after transportation in Cairy-Blair medium were comparable with those after transportation in NaCl 0.9%, but because of availability, low cost, and ease of handling in the endoscopy department, NaCl 0.9% was preferred as transport medium. This study shows that for culture of H pylori from gastric biopsy specimens sterile saline is an adequate medium, and that transportation can be delayed for 24 hours without a significant loss of diagnostic yield.     

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.     

Association between TNF-alpha promoter polymorphism and Helicobacter pylori cagA subtype infection

AIMS: To assess the importance of tumour necrosis factor alpha (TNF-alpha) promoter polymorphism in relation to infection with the cytotoxin associated gene A (cagA) subtype of Helicobacter pylori within a dyspeptic Korean population. METHODS: Eighty three patients with gastric disease and 113 healthy controls were studied. The DNA from gastric biopsy specimens was analysed by H pylori specific and cagA specific polymerase chain reaction (PCR). To characterise TNF-alpha polymorphism at positions -308 and -238, PCR based restriction fragment length polymorphism analysis was performed. RESULTS: Helicobacter pylori infection was closely correlated with G to A transition at position -308 of the TNF-alpha promoter when compared with healthy controls (odds ratio (OR), 2.912; 95% confidence interval (CI), 1.082 to 7.836; p = 0.034). Although TNF-alpha -308 polymorphism in patients with H pylori was not significantly different from that in patients without H pylori, the -308A polymorphism was strongly associated with H pylori cagA subtype infection when compared with the polymorphism in cagA negative H pylori infection (OR, 8.757; 95% CI, 1.413 to 54.262; p = 0.019) and healthy controls (OR, 3.683; 95% CI, 1.343 to 10.101; p = 0.011). G to A genetic change at position -238 of the TNF-alpha gene was not significantly associated with H pylori cagA subtype infection. In addition, genetic polymorphisms at both sites of the TNF-alpha promoter in patients with H pylori infection did not correlate with the severity of disease. CONCLUSION: TNF-alpha -308A polymorphism was significantly related to infection with the H pylori cagA subtype in Korean patients with gastric disease.     

cagA基因阳性幽门螺杆菌培养滤液对人胃粘膜上皮细胞系GES-1生长的影响

目的：研究cagA^ 幽门螺杆菌（Hp）培养滤液对人胃粘膜上皮细胞（GES－1）的作用及机制。方法：制备Hp培养滤液，PCR鉴定cagA基因。采用倒置显微镜、电镜、细胞生长曲线、克隆形成实验、单细胞微凝胶电泳及流式细胞仪等，观察Hp (cagA^ ）培养滤液对GES－1细胞的作用。结果：经Hp(cagA^ )培养滤液处理GES－1细胞，细胞核增大、畸形、核染色质变粗、核仁肥大、核分裂。生长曲线可见细胞增生活跃，增殖率195％。克隆形成试验显示细胞克隆形成能力增强，增殖率达到337．5％。流式细胞仪S期细胞比率显著高于对照组。Hp(cagA^ ）培养滤液可使GES－1细胞形成彗星现象。结论：Hp(cagA^ )培养滤液可以导致GES－1细胞的生长特性改变，呈现肿瘤细胞的形态学及生长特征。DNA损伤可能是cagA诱导GES－1细胞生长特性改变的机制之一。     

Detection of Helicobacter pylori gastritis by PCR: correlation with inflammation scores and immunohistochemical and CLOtest findings

We developed a polymerase chain reaction (PCR) assay to detect Helicobacter pylori in gastric and/or gastroesophageal biopsy specimens of adults with dyspepsia, compared the method with immunohistochemical analysis and CLOtest (Ballard Medical Products, Draper, UT), and correlated the results of each test with the histologic features of infection. H pylori was identified in 36 (60%) of 60 patients irrespective of biopsy site and testing method. In the gastric biopsy specimens, PCR detected H pylori in 29 (52%) of 56 cases, including 11 (100%) of 11 immunohistochemically and/or CLOtest-positive cases. PCR-positive gastric biopsy specimens correlated with a higher average cumulative inflammatory score compared with PCR-negative specimens (P = .001). In gastroesophageal biopsy specimens, PCR detected H pylori in 15 (34%) of 44 cases, including 1 (20%) of 5 immunohistochemically positive specimens. PCR-positive gastroesophageal junction biopsy specimens did not correlate with a higher average cumulative inflammatory score. Overall, PCR detected an additional 23 cases negative by immunohistochemical analysis and/or CLOtest. This PCR assay identified a significant number of H pylori infections that would not be detected by immunohistochemical analysis and/or CLOtest.     

Determination of Helicobacter pylori virulence by simple gene analysis of the cag pathogenicity island

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.     

The efficacy of laboratory diagnosis of Helicobacter pylori infections in gastric biopsy specimens is related to bacterial density and vacA, cagA, and iceA genotypes

A total of 500 consecutive patients undergoing upper endoscopy were biopsied and tested for H. pylori infection by the Campylobacter-like organism (CLO) test, culture, histology, and PCR. Serum samples were tested by two different serological assays. Patients were considered H. pylori positive if at least two of the four biopsy specimen-based methods yielded positive results. PCR had the highest diagnostic sensitivity (99.4%), followed by histology (92.2%), culture (89.5%), and the CLO test (89.0%). The specificities of all methods were higher than 98%. Of the organisms from the 181 PCR-positive patients, the vacA (s and m regions), cagA, and iceA genotypes were determined by reverse hybridization (line probe assay) or an allele-specific PCR. Organisms that were detected by PCR but that remained undetected by the CLO test were significantly more often vacA s1 (P = 0.006), m1 (P = 0.028), and cagA positive (P = 0.029) than vacA s2, m2, and cagA negative, respectively. Organisms that were detected by PCR but that remained undetected by culture or histology more often contained iceA1 (P = 0. 034 and P = 0.029, respectively) than iceA2. Higher H. pylori density was associated with vacA s2 (P = 0.024), vacA m2 (P = 0.050), and cagA-negative (P = 0.035) genotypes. Also, the diagnostic results of the CLO test (P = 0.001) and culture (P = 0.031) but not those of the PCR (P = 0.130) were significantly associated with the H. pylori density. The rate of detection by the four biopsy specimen-based tests was lower for patients who used proton pump inhibitors, but this was independent of the H. pylori genotypes. These observations may be explained by different bacterial densities, as established by the distinct genotypes of H. pylori, and confirm that the biologies of strains with such genotypes are considerably different.     

Quantitative culture of Helicobacter pylori from gastric juice: the potential for transmission

The transmission of Helicobacter pylori may occur by spread of organisms from gastric juice which has been introduced into the mouth by gastro-oesophageal reflux. The aim of this study was to quantify the load of H. pylori present in gastric juice available for transmission. Gastric antral biopsy and gastric juice samples were collected from 108 adult dyspeptic patients undergoing routine upper gastroscopy and the presence of H. pylori was determined. In all, 54 (50%) of 108 patients gave positive results in the gastric antral biopsy rapid urease test and for H. pylori histology. The gastric juice of 40 (37%) of patients gave positive results for the urease A gene by PCR assay; 34 (31%) of patients were positive by these three tests and H. pylori was cultured from the gastric juice of 13 (38%) of these patients. The median count of H. pylori in gastric juice was 1.75 x 10(1) cfu/ml. Viable organisms in gastric juice may lead to transmission of H. pylori when refluxed or vomited into the mouth.     

Identification of Helicobacter pylori DNA in the mouths and stomachs of patients with gastritis using PCR.

AIMS--To determine the prevalence of Helicobacter pylori colonisation in the mouths of patients with H pylori gastritis. METHODS--A nested polymerase chain reaction test for the 16S ribosomal RNA gene of H pylori was used on saliva, dental plaque, gastric juice and gastric biopsy specimens from patients attending a dyspepsia clinic. RESULTS--Thirteen patients had histologically confirmed Helicobacter associated gastritis. Twelve of these had positive gastric aspirates by PCR. Five had at least one positive oral specimen. Eight patients with normal gastric biopsy specimens had no PCR positive oral specimens or gastric aspirates. All, however, had PCR positive gastric biopsy specimens. In an attempt to determine the origin of these positive results in normal patients, it was shown that biopsy forceps could contaminate specimens with DNA from previous patients. CONCLUSION--The demonstration of the organism in the mouths of a substantial proportion of dyspeptic patients has major implications for the spread of H pylori and identifies a potential source for reinfection following eradication of the organism from the stomach.