Evaluation of three commercial enzyme immunoassays compared with the 13C urea breath test for detection of Helicobacter pylori infection.

The diagnostic significance of the serological detection of antibodies to Helicobacter pylori has been established by numerous investigators. Reports of the clinical reliabilities of commercial enzyme immunoassay (EIA) kits available for this purpose vary as a result of the different H. pylori antigen sources and reference methods used. The 13C urea breath test (UBT) has been shown to be an extremely accurate and reliable method of detecting H. pylori infection. We used the 13C urea breath test as the confirmatory method for H. pylori status to evaluate three commercially available EIA kits designed to detect immunoglobulin G antibodies to H. pylori. These kits were the HM-CAP EIA kit (Enteric Products, Inc.), the PYLORI STAT EIA kit (BioWhittaker, Inc.), and the G.A.P. kit (Bio-Rad Laboratories/Biomerica, Inc.). The evaluations were performed in a double-blind manner with samples from 473 clinically characterized patients. This group included patients with symptomatic gastrointestinal disorders as well as nonsymptomatic volunteers. The sensitivities of the kits were as follows: HM-CAP, 98.4%; PYLORI STAT, 99.2%; and G.A.P., 100%. The specificities were as follows: HM-CAP, 96.4%; PYLORI STAT, 90.1%; and G.A.P., 26.0%. Although the HM-CAP and PYLORI STAT kits performed comparably, the G.A.P. test yielded significantly more false-positive results and an unacceptably high number of indeterminate results.     

Validation of the string test for the recovery of Helicobacter pylori from gastric secretions and correlation of its results with urea breath test results, serology, and gastric pH levels

The efficacy of the string culture test to isolate Helicobacter pylori from gastric secretions of 28 volunteers was studied. With the urea breath test (UBT) as the "gold standard," the string culture test showed a sensitivity of 75% and a specificity of 100%. The results of string culture did not correlate with the UBT results, with serum antibody levels, or with the pH levels of gastric secretions.     

Reliability of cytological grading of prostatic carcinoma compared with histological grading

Thirty prostatic carcinomas diagnosed simultaneously by fine needle aspiration cytology and punch biopsy were graded into three groups according to their cytological and histological appearances. Among them 23 carcinomas were graded equally by both methods. Cytological examination appears to be suitable for grading prostatic carcinoma, thus being a possible indicator of the patient's prognosis and response to hormonal therapy. In addition to the results obtained here, fine needle aspiration cytology has many advantages such as simplicity, quickness and safety. With increase of attention of urologists and pathologists to fine needle aspiration cytology, this evaluation will become comparable to the histological examination not only as a routine diagnostic tool.     

Evaluation of [13C]urea breath test and Helicobacter pylori stool antigen test for diagnosis of H. pylori infection in children from a developing country

The [(13)C]urea breath test ((13)C-UBT) and Helicobacter pylori stool antigen test (HpSA) for the diagnosis of H. pylori infection in children were validated. The sensitivity, specificity, and positive and negative predictive values were 93.8, 99.1, 97.8, and 98.0%, respectively, for the (13)C-UBT and 96.9, 100, 100, and 98.0%, respectively, for HpSA. Both tests are appropriate for diagnosing H. pylori infection in children.     

Diagnosis of Helicobacter pylori infection in children: comparison of a salivary immunoglobulin G antibody test with the [(13)C]urea breath test

The prevalence of Helicobacter pylori infection in a population-based sample of 477 children (mean age plus minus standard deviation, 5.8 plus minus 0.5 years) determined by the [(13)C]urea breath test ([(13)C]UBT) was 10.7% (95% confidence interval [CI], 8.1 to 13.8%), and that determined by salivary enzyme-linked immunosorbent assay (ELISA) was 11.9% (95% CI, 9.2 to 15.2%). Compared to the [(13)C]UBT, the sensitivity and specificity of the salivary ELISA were 80.9% (95% CI, 66.3 to 90.4%) and 95.3% (95% CI, 92.7 to 97.1%), respectively.     

Estimation of prevalence of Helicobacter pylori infection in an asymptomatic elderly population comparing [14C] urea breath test and serology.

A non-invasive serological assay devised in this laboratory had a sensitivity and specificity of 100% as determined by culture and confirmed by histology in a group of 47 patients who had undergone endoscopy. The correlation between serology and the non-invasive [14C] breath test was very good. Only one of 24 culture positive patients was, while all 23 culture negative patients were, breath test negative. In a group of 46 healthy elderly persons, however, significant anomalies between serology and breath test were observed. Only 83% of the breath test negative persons were seronegative, while only 68% of the breath test positive persons were seropositive. These results can be explained in terms of age related atrophic gastritis and immune incompetence, causing reduced colonisation and decreased antibody production, respectively. These investigations suggest that non-invasive tests for H pylori infection may not be reliable in the elderly.     

Gastric mucosal inflammation and epithelial cell turnover are associated with gastric cancer in patients with Helicobacter pylori infection

BACKGROUND: Infection with a virulent Helicobacter pylori strain is associated with gastric mucosal damage and the increased risk of gastric cancer. AIMS: To examine the characteristics of host gastric mucosal responses in patients with gastric cancer, histological grade of gastritis, gastric epithelial apoptosis, and proliferation were studied. METHODS: Thirty two patients with early gastric cancer and 32 sex and age matched controls were studied. All subjects were infected with a virulent H pylori strain (vacA s1/m1, cagA positive genotype). Biopsy specimens were taken from the antrum and the corpus of the stomach. The grade of gastritis was assessed according to the updated Sydney system. Apoptotic cells were detected using terminal uridine deoxynucleotidyl nick end labelling, and epithelial cell proliferation was determined by means of the Ki-67 labelling index. RESULTS: In patients with gastric cancer, significantly higher grades were observed when glandular atrophy (p < 0.05) and intestinal metaplasia (p < 0.01) were present in the antrum, and when mononuclear cell infiltration was present in the corpus (p < 0.05). The numbers of apoptotic cells were increased in patients with cancer (p < 0.05) and the apoptotic index correlated significantly with the grade of glandular atrophy. Epithelial cell proliferation was more likely to be increased in mucosa where intestinal metaplasia was present. CONCLUSIONS: Infection with H pylori causes increased gastric epithelial apoptosis, resulting in more severe glandular atrophy in patients with gastric cancer. Increased damage of gastric epithelial DNA and the presence of more severe atrophic gastritis might contribute to the development of gastric cancer.     

Comparison of Five PCR Methods for Detection of Helicobacter pylori DNA in Gastric Tissues

Five different PCR methods for the detection of Helicobacter pylori were evaluated. The results of this study indicate that of the five PCR methods examined, the ureC (glmM) gene PCR is the most sensitive and specific for the detection of H. pylori in gastric biopsy specimens.     

Diagnosis of Helicobacter pylori infection by specific gastric mucosal IgA and IgG pylori antibodies.

AIMS--To investigate the diagnostic value of mucosal IgA and IgG Helicobacter pylori antibodies. METHODS--The study population comprised 209 consecutive patients with severe dyspeptic complaints referred for upper gastrointestinal endoscopy. A positive culture or histological identification of H pylori in gastric biopsy specimens, or both, were used to confirm infection. Specific IgA and IgG H pylori antibodies were determined using a modified ELISA technique. RESULTS--Of the 209 patients, 137 were infected with H pylori. The diagnostic value of systemic IgA and IgG H pylori antibodies was confirmed. Systemic IgA antibodies had a sensitivity of 76.6% (95% confidence interval 69.5-83.7) and a specificity of 94.4% (89.1-99.7). The sensitivity and specificity for systemic IgG antibodies were, respectively, 97.1% (94.3-99.9) and 98.6% (95.9-100). A moderate but clinically important correlation was found between local and systemic IgA and IgG. Mucosal IgA H pylori antibodies had a sensitivity of 98.5% (96.5-100) and a specificity of 91.7% (85.3-98.1), while for IgG these figures were, respectively, 88.3% (82.9-93.7) and 98.6% (95.9-100). As a diagnostic test mucosal IgA H pylori antibodies were comparable with culture and histology. CONCLUSION--Determination of local IgA and IgG H pylori antibody levels is a highly sensitive and specific test for the diagnosis of H pylori infection.     

Fingerprinting of Nigerian Helicobacter pylori isolates by plasmid profile and PCR

Plasmid profiling and digestion of amplified PCR product of ureA genes were used to determine genomic variation in 56 strains of Helicobacter pylori isolated from patients with peptic ulcers and subjects with gastritis recruited in Lagos and Ife, Nigeria. Twenty-five (45%) of the strains were found to harbour plasmids ranging in size from 0.9 kb to > 10 kb. The plasmid profile was able to detect differences between the strains, and also to distinguish between different strains isolated from the same patient. The expected amplified ureA gene PCR product was detected in all strains and digestion with the restriction enzyme DdeI did not produce discrimination amongst the strains, however, digestion with MluI produced little discrimination amongst strains. In conclusion, plasmid profiling produced better discrimination amongst H. pylori strains than ureA PCR gene profiling.     

Use of a Gastric Juice-Based PCR Assay To Detect Helicobacter pylori Infection in Culture-Negative Patients

A gastric juice-based PCR assay was compared with culture, microscopy, and a rapid urease test with specimens from 114 subjects. The PCR and conventional tests were positive for 76 and 62% of the subjects, respectively. The prevalence of gastroduodenal disease and seropositivity for anti-Helicobacter pylori immunoglobulin G were similarly high among conventional-test-positive and PCR-only-positive subjects compared to all-negative ones. The PCR assay is recommended to confirm the H. pylori status of culture-negative peptic-ulcer patients.     

Correlation of H. pylori density with grading of chronic gastritis

Histopathological study of endoscopic gastric mucosa obtained from 251 patients with acid peptic diseases were studied in relation to the degrees of inflammation and the degree of H. Pylori density. Haematoxylin-Eosin and modified Giemsa stains were used. Gastritis grading was done according to Warren and Marshall with slight modification and H. Pylori grading according to our criteria. In this study no definite relationship could be observed between histological grading of chronic gastritis with that of H. Pylori density.     

Use of a urea breath test versus invasive methods to determine the prevalence ofHelicobacter pylori in zaire

The prevalence ofHelicobacter pylori infection in Zaire was determined by means of a [¹⁴C] urea breath test in 133 asymptomatic subjects, by culture and histological examination of biopsies in 324 consecutive endoscopy patients with chronic epigastric complaints, and by both the breath test and culture/histology in a subset of 92 patients. Sixty healthy Belgian students or hospital laboratory workers were also included for comparison. The prevalence ofHelicobacter pylori was significantly higher in asymptomatic Zairian subjects (77.4 %) than in the Belgians (30 %; p<10^–6). Infection was also acquired much earlier in life in Africans, 66% of the children aged 5 to 9 years already being infected versus none of the Belgian subjects below the age of 20 years. In Zaire, however, the prevalence ofHelicobacter pylori in patients with gastroduodenal disorders (87.5 %) was similar to that in the group of asymptomatic subjects (77.5 %) after adjustment for age and other epidemiological parameters (gender, place of residency, education level, smoking and drinking habits) in a multivariate analysis. The high rate of acquisition ofHelicobacter pylori infection in Zaire emphasizes the need to consider the baseline prevalence ofHelicobacter pylori in a defined population when studying its association with various diseases.     

TaqMan探针法与尿素呼气试验法检测胃镜活检标本幽门螺旋杆菌的比较

目的:通过比较幽门螺旋杆菌(Helicobacter pylori,Hp)感染的两种检测方法,探讨TaqMan探针法在诊断幽门螺旋杆菌感染中的价值.方法:抽取70例有完整病例资料的胃镜活检标本,进行Hp分型、鉴定检测,并将实验结果与临床的碳14尿素呼气试验和病理结果进行相关性分析.结果:Hp感染的两种检测方法(TaqMan探针法和呼气实验法)一致性程度尚可(Kappa=0.550),两种方法检测的阳性率都随病理炎症程度的加重而增加,而呼气实验法检测Hp的阳性率更高(P<0.05).但TaqMan探针法可以将Hp进行不同毒力类型的鉴定,且TaqMan探针法可区分Hp的阳性强度(1+,2+,3+),其与病理的炎症程度有较好的相关性(γ=0.564,P<0.05).结论:TaqMan探针法可以定性地鉴定Hp,与呼气实验法有较好的一致性.TaqMan探针法可进一步定量Hp的阳性强度,更好地反映临床的病理炎症程度.     

Uptake of Helicobacter pylori Outer Membrane Vesicles by Gastric Epithelial Cells

Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA⁺ strain and OMV from a VacA⁻ isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA⁺ strain was inhibited by H. pylori LPS (58% inhibition with 50 μg/ml LPS), while uptake of OMV from the VacA⁻ mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA⁻ mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA⁺ strain was less affected (25% inhibition with 15 μg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization.Infection with the gastric pathogen Helicobacter pylori results in chronic gastritis (13) and is associated with increased risk for the development of peptic ulcer disease (35), gastric carcinoma (41, 57), and gastric lymphoma (5, 60). H. pylori persistence, in an environment where peristalsis and sloughing of cells are continually occurring, is mediated by a variety of adhesins present on the bacterial surface (14, 21, 36, 40). However, despite the ability to adhere to the gastric epithelium, the majority of organisms remain unattached to surface cells (32), leading to speculation that lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV) shed by these bacteria (15, 19, 26) contribute to H. pylori pathogenesis via the persistent delivery of bacterial virulence factors (including the vacuolating cytotoxin VacA) and antigens to the gastric mucosa (26, 27). Observations that H. pylori OMV modulate gastric epithelial cell proliferation (22), induce apoptosis (3), stimulate secretion of the proinflammatory cytokine interleukin-8 (22), increase micronucleus formation (8), and are at the luminal surface (15, 26) and within cells of the gastric epithelium (15) support this hypothesis.OMV shedding by Gram-negative bacteria is well described in the literature (reviewed by Kuehn and Kesty [33]), yet little is known of the mechanisms of vesicle adherence to and internalization within mammalian host cells. The adherence of enterotoxigenic Escherichia coli (ETEC) OMV to host cells is mediated via a heat-labile enterotoxin (LT) associated with these OMV (31), whereas leukotoxin, associated with OMV shed by Actinobacillus actinomycetemcomitans, is not involved in vesicle binding (12). The internalization of ETEC, Porphyromonas gingivalis, and Pseudomonas aeruginosa OMV has been shown to involve cholesterol-rich lipid rafts (6, 16, 31), and recently, Kaparakis and colleagues (25) reported that uptake of H. pylori OMV is also lipid raft dependent. This is in contrast to the uptake of Shigella flexneri OMV, which occurs via phagocytosis, with the proposed subsequent fusion of OMV with the phagosomal membrane and the release of vesicle contents into the cell cytoplasm (24).In this study, we sought to examine whether VacA cytotoxin associated with H. pylori OMV was involved in vesicle binding. We also examined the rate of OMV internalization and the involvement of LPS, cholesterol, and clathrin-mediated endocytosis in vesicle uptake by AGS gastric epithelial cells. We report that within 20 min essentially all VacA⁺ OMV associated with AGS cells were intracellular and that uptake was enhanced by the presence of vesicle-associated cytotoxin. Excess H. pylori LPS reduced vesicle uptake, having a more significant effect on VacA⁺ OMV than VacA⁻ vesicle uptake. Uptake of both VacA⁺ and VacA⁻ OMV did not require cholesterol. However, a reduction in clathrin-mediated endocytosis inhibited VacA⁻ OMV uptake and to a lesser extent VacA⁺ OMV internalization.     

Prevalence of Helicobacter pylori in Sri Lanka as determined by PCR

Fifty-seven Sinhalese patients were investigated for the presence of Helicobacter pylori by PCR. A prevalence of 70.1%, with 47.5% positive for cagA, was demonstrated. The most common vacA allele was s1am1. There was no significant association between either the s1 allele or the cagA allele and severe gastroduodenal disease. There was an association between the s1 allele and the cagA locus.